Proceedings of the 28th EBC Congress

BUDAPEST 2001

ISBN 90-70143-21-6
Contents
A. Contributions 28th EBC Congress, Budapest, 2001
B. General information on the European Brewery Convention

A. Contributions 28th EBC Congress, Budapest , 2001

Index of Authors
Index of Descriptors / Deskriptorenindex / Index de Descripteurs
Contributions:

Beer and health

1. Promoting healthy moderate drinking
Hubert Sacy (CDN)

2. Update on alcohol research in 2001

Mack C. Mitchell (USA)

3. Beer: drink and food

Renton Righelato (UK)

4. Folate in beer and the prevention of cardiovascular disease

Caroline J. Walker, Darash Patel, Caroline Wolfe, Anthony Wright & Paul Finglas
(UK)

5. Antioxidant and radical-scavenging potential of phenolic constituents of beer

A. Gamal-Eldeen, A. Alt, C. Gerhuser, I. Neumann, N. Frank, H. Chmiel,
H. Bartsch & H. Becker (D)

6. Cancer chemopreventive potential of xanthohumol (XN), a prenylated chalcone

from hops
C. Gerhuser, A. Alt, E. Heiss, A. Gamal-Eldeen, K. Klimo, J. Knauft, I. Neumann,
H. Scherf, N. Frank, H. Bartsch & H. Becker (D)

7. Prenylated hop flavonoids are key agents in relation to health-beneficial properties

of beer
Denis De Keukeleire (B), Stuart R. Milligan, Jogen C. Kalita, Victoria Pocock
(UK), Luc De Cooman, Arne Heyerick, Haojing Rong & Frederik Roelens (B)

8. Healthy drinks?- beer and cider antioxidants

Caroline J. Walker, Louise Bolshaw & Sachin Chandra (UK)

9. Beer and health research

E. Denise Baxter & Caroline J. Walker (UK)

Raw materials
10. The barley and malt factors underlying lautering performance: identification and use
in barley breeding programs
Christopher M. Ford & Evan Evans (AUS)
11. Molecular markers for fermentability - a novel selection tool for barley breeding
M. Voetz, J. Gunkel & F. Rath (D)

12. Characterisation and detection of the gushing factors produced by fungi

T. Kleemola, T. Nakari-Setl, M. Linder, M. Penttil, E. Kotaviita, J. Olkku &
A. Haikara (FIN)

13. Evolution of oxygen and water up-take during steeping in a lab scale malting unit
Ahmed Hariri, Patrick Boivin, Ivan Marc & Michel Fick (F)

14. High-throughput assay for identification of the barley varieties of individual malt
kernels with the use of microsatellite DNA polymorphism
Kanta Sakamoto, Kazutaka Ozaki & Yasuyuki Ohtake (J)

15. Investigations into flavour and flavour stability of dark beer in dependence on
malting and kilning parameters
Thomas Preu (D) , Clemens Forster (CDN), Bernhard Thum & Werner Back (D)

16. What is it about antioxidative characteristics of hops?

A. Forster, K. Simon, R. Schmidt & D. Kaltner (D)

17. Investigations of the influence of hop products on the microbiological stability of

beer
D. Kaltner, I. Bohak, A. Forster, A. Gahr & W. Back (D)

18. Chemical properties of the naturally dried hop plant and its influence on the quality
of beer
Majda Virant (SI)

Processing
19. A new industrial tool for monitoring the malting process
Nathalie Allosio-Ouarnier, Paul Robert, Dominique Bertrand & Patrick Boivin (F)

20. Industrial kilning technologies and their influence on organoleptic quality of malt
Michel Dumoulin & Patrick Boivin (F)

21. Genetic difference of -amylase thermostability among barley varieties during beer
production process
Makoto Kihara, Takashi Asakura, Takafumi Kaneko & Kazutoshi Ito (J)

22. Vibrating membrane filtration of mash for the beer production

Jan Schneider, Martin Krottenthaler, Werner Back & Horst Weisser (D)

23. The influence of polysaccharides from yeast and bacteria on the filterability of wort
and beer
S. Kreisz, F. Wagner & W. Back (D)

24. Continuous wort boiling and hot trub separation

Bruno Bonacchelli, Frdrique Harmegnies & Rafal Tigel Gil (B)
25. Non-Newtonian behaviour of beer in laminar flow through porous media a
possible explanation for a sudden increase of pressure difference during beer
filtration
Martyn A. Drost & Erich J. Windhab (CH)

26. Special filteraids for the optimisation of the kieselguhr filtration and for the
reduction of the pollution
J. Cardelli (S), M. Crestini & T. Zangrando (I)

27. Use of RVF technology to achieve rough beer clarafication and cold sterilisation of
beer
L. Fillaudeau, S. Ermolaev, N. Jitariouk & A. Gourdon (F)

28. Collection of operational data in the brewery as a tool for process optimisation
C. Eger, F. Spillmer, W. Neske, N. Abraham, S. Pauls, T. Isenberg, H. Wehrenberg
& F. Bechmann (D)

Yeast and fermentation

29. Propagation of brewery yeast modelling and simulation
T. Kurz, T. Becker & A. Delgado (D)

30. The optimization of yeast transfer timing in a propagation process

Yuji Nishida, Nobuyuki Fukui, Atsushi Fujita, Fumihiko Omura & Akira Isoe (J)

31. Use of corn steep liquor to increase the yield of brewing yeast obtained from
propagation
Behnam Taidi, H. Gwendoline Mazike & Jeff A. Hodgson (UK)

32. The management of brewing yeast stress

Katherine A. Smart (UK)

33. Does osmotic pressure affect yeast performance in high gravity fermentation?
John Hammond, Daniel Davis, Mark Lee & Kim Storey (UK)

34. The response of brewers yeast to a defined shear field for differing exposure times
Robert A. Stafford (SA), Thomas Stoupis & Graham G. Stewart (UK)

35. Yeast Membrane potential and carbohydrate utilisation

S.M. Van Zandycke, R. Siddique & K.A. Smart (UK)

36. Fermentation of high gravity worts - its influence on yeast metabolism and
morphology
Graham G. Stewart (UK)

37. Daily changes in maltopermease and maltase activities during normal and high
gravity fermentations by ale and lager strains
J. Rautio, T. Markkula (FIN), M. Lee , J.R.M. Hammond, W. Lancashire (UK) &
J. Londesborough (FIN)

38. Yeast management and process control by flow cytometric analysis

K.-J. Hutter & C. Lange (D)
39. Novel method for evaluation of yeast vitality and its application to yeast handling
technology
T. Imai (J), W. Back (D), Y. Yasuda, H. Arimura, T. Hori, M. Abe & T.Takeuchi (J)

40. Impact of wort composition and serial repitching on lager brewing yeast
fermentation performance and organelle integrity
Cheryl Jenkins, Alan Kennedy, Pat Thurston, Jeff Hodgson &
Katherine Smart (UK)

41. Why warm cropping is best!

David Quain, Chris Powell, Alisdair Hamilton, David Ruddlesden & Wendy Box
(UK)

42. The use of concanavalin A to investigate the onset and mechanism of flocculation
by a brewing yeast strain
Franciska Mochaba, Ian Cantrell & Waha Vundla (SA)

43. Construction of brewers yeast harbouring killer plasmids and investigation of the
zymocin production under various fermentation conditions
Gabriella Farkas, Anna Marz, Judit Rezessy-Szab & goston Hoschke (H)

44. Amino acid uptake and sensing in yeast

Helge A. Andersen, (DK) Richard F. Gaber (USA), Peter L. Madsen,
Peter S. Nielsen, Kim Ottow & Morten C. Kielland-Brandt (DK)

45. Insulin-like anabolic and mitogenic activities of a yeast peptide complex on brewing
yeast
M. Dillemans, L. Van Nedervelde & A. Debourg (B)

46. Evaluation of antioxidant properties of Saccharomyces cerevisiae

P. Bastin, L. Van Nedervelde, M. Dillemans (B), P. Boivin (F) & A. Debourg (B)

47. Development of brewers yeast with increased carbonyl reducing activity

Maija-Leena Vehkomki, Virve Vidgren, Arvi Vilpola, Hannele Virtanen (FIN),
Beatriz Snchez, Ivn Galindo-Castro (YV), Merja Penttil (FIN) & Rafael Rangel-
Aldao (YV)

48. Brewers yeast oxidoreductase with activity on Maillard reaction intermediates of

beer
Beatriz Snchez, Lorena Reverol, Ivn Galindo-Castro, Adriana Bravo,
Jos Luis Ramrez & Rafael Rangel-Aldao (YV)

49. Controlled beer fermentation with continuous one-stage immobilized yeast reactor
Esko Pajunen, Kaisa Tapani, Harry Berg, Bo Ranta (FIN), Joe Bergin (IRL),
Heikki Lommi (DK) & Tapio Viljava (FIN)

50. Continuous beer fermentation using polyvinyl alcohol entrapped yeast

Daniela mogroviov, Zoltn Dmny, Marin Navrtil (SK) & Petr Dvok (CZ)

51. A new way for immobilized yeast systems: secondary fermentation without heat
treatment
Frank Nitzsche, Gerrit Hhn, R. Meyer-Pittroff, Svend Berger &
Rainer Pommersheim (D)
52. Application of genome-wide transcriptional analysis to identify genetic markers
useful in industrial fermentations
Vincent J. Higgins, Anthony D. Oliver, Rachel E. Day, Ian W. Dawes &
Peter J. Rogers (AUS)

53. Metabolic engineering approaches - opportunities for brewing

Merja Penttil (FIN)

Flavour and stability

54. Influence of the acrospire of malted barley on the flavour stability and other quality
parameters of beer
Achim Zrcher, M. Krottenthaler & W. Back (D)

55. Development of novel malt evaluation method for improving beer flavor stability
T. Ueda, K. Sasaki, K. Inomoto, K. Kono, N. Kagami, K. Shibata & M. Eto (J)

56. Improvement of flavour stability by reduction of trans-2-nonenal a case study

Olav Vind Larsen, Sten Aastrup, Henning Nielsen & Anne Cathrine Lillelund (DK)

57. Linoleic acid hydroperoxides, trans-2-nonenal and nonenal potential during the
brewing process: evolution and relationship
Rgis Fournier, Michel Dumoulin & Patrick Boivin (F)

58. Evaluation of the addition of gallotannins in the brewing liquor for the improvement
of the flavour stability of beer
Guido Aerts, Luc De Cooman, Gert De Rouck, Zoltan Pnzes, Annemie De Buck,
Roger Mussche (B) & Joseph van Waesberghe (NL)

59. The role of polyphenols and oxidation processes in brewhouse on beer quality
A. Mikyka, D. Hakov, M. Hrabk, J. rogl & T. Hork (CZ)

60. Comparative study of the stability of iso--acids, dihydroiso--acids and

tetrahydroiso--acids during beer ageing
Luc De Cooman, Guido Aerts, An Witters, Marjan De Ridder, Annick Boeykens,
Koen Goiris & Denis De Keukeleire (B)

61. A new technology for beer flavor stabilization: control of key intermediates of the
Maillard reaction
Rafael Rangel-Aldao, Adriana Bravo, Ivn Galindo-Castro, Beatriz Snchez,
Lorena Reverol, Erika Scherer, Jorge Madrid, Jos Lus Ramrez, Julio Herrera
(YV), Merja Penttil, Maija-Leena Vehkomki, Virve Vidgren, Hannele Virtanen &
Silja Home (FIN)

62. Impact of different parameters on the absorption integral

Ulrich Peters, Thomas Fgen & Frank-Jrgen Methner (D)

63. Highly sensitive chemical indices of beer aging

Adriana Bravo, Beatrz Snchez, Erika Scherer & Rafael Rangel-Aldao (YV)

64. Identification of -dicarbonylic compounds in aged beers: their role in beer aging
process
Adriana Bravo, Erika Scherer, Jorge Madrid, Julio Herrera (YV), Hannele Virtanen
(FIN) & Rafael Rangel-Aldao (YV)
65. pH dependence of radical scavenging activity of polyphenols, phenolic acid and
sulfite
Takashi Nakamura, Oliver Franz & Werner Back (D)

66. DPPH-scavenging activity of beer and polyphenols measured by ESR

Oliver Franz & W. Back (D)

Foam
67. Generation of foaming proteins along the malting and brewing processes
Didier Marion, Sandrine Jgou, Jean-Paul Douliez, Thrse Gaborit &
Patrick Boivin (F)

68. Interfacial mechanisms underlying lipid damage of beer foam

Peter J. Wilde, Fiona A. Husband, Daniel Cooper, Alan R. Mackie,
A. Patrick Gunning, Victor J. Morris, Nicola Woodward & Clare Mills (UK)

69. The background of the adherence of beer foam to the glass

Helene C. M. Mocking-Bode, Piet S. Peereboom, Emile T.M.J. Martynowicz &
Anneke C. Douma (NL)

70. Trihydroxyoctadecenoic acids having negative effects on beer foam are produced by
enzymatic factors present in malt
Hisao Kuroda, Naoyuki Kobayashi, Hirotaka Kaneda, Masachika Takashio &
Ken Shinotsuka (J)

71. Monitoring barley lipid transfer protein levels in barley, malting and brewing
Lance T. Lusk, Henry Goldstein, Kelly Watts, Alfonso Navarro & David Ryder
(USA)

Analysis
72. Better analytical techniques for beer and brewing raw materials analysis
F. Richard Sharpe (UK)

73. A new test for measuring endosperm structure and quality

L.K. Wheaton, R.E. Muller, C.D. Booer, & S. Chandra (UK)

74. Development of a biosensor allowing reliable and fast dectection of mycotoxins

Bram van der Gaag, Edwin Stigter, Sabine Spath, Sjaak van Veen &
Maarten Schans (NL)

75. Development of a process control system for the optimisation of the cytolytic,
proteolytic and amylolytic degradation during mashing
G. Fischer, M. Mitzscherling, T. Becker, A. Delgado, T. Dickel,
M. Krottenthaler & W. Back (D)

76. From seeds to beer: development of a quantitative detection method of transgenic

maize
Pierre Brignon, Marc Vaitilingom, Luc Didierjean, Estelle Carniel, Aurlie Brelin &
Christophe Bastien (F)

77. Development and validation of a rapid wort and beer filterability test
Chris Ford, Doug Stewart, Martin Barski & Evan Evans (AUS)
78. A novel and rapid method for the automatic and simultaneous determination of total
and viable cell concentration in pitching yeast slurries
Chris A. Boulton, Wendy G. Box, John Carvell & Kirsty Turner (UK)

79. Real time monitoring of viable yeast concentration in production fermenters using
novel RF impedance probes
John Carvell, Robert Todd, Stephen Cunningham (UK) & Takeshi Yonezawa
(J/UK)

80. The use of GC-olfactometry to assess hop aromatic quality

Guillaume Lermusieau & Sonia Collin (B)

81. Analytical device for measuring the ethanol concentration in beer based on NIR
absorption
Gerald Zanker & Roman Bene (A)

82. Solid Phase Microextraction the new alternative for the determination of the
vicinal diketones in beer
T. Hork, V. Kellner, J. ulk, M. Jurkov & P. ejka (CZ)

83. Recovery of volatiles from gasses of fermenting wort

M. Arnould (B), M. Bes (F), F. Harmegnies, F. Delvaux (B), J.-L. Escudier (F) &
G. Derdelinckx (B)

84. Application of voltametry in beer analysis

A. Barros, J. Rodrigues, P. Almeida, L. Guido, P. Rodrigues, J. Santos,
J.M.Machado Cruz & A.A. Ferreira (P)

85. Electrochemical determination of heavy metals in beer

Pavel Dostlek, Jaroslav epika, Jan Enge, Richard Koplk & Eva urdov (CZ)

86. The effect of pH on protein-polyphenol particle size

Karl J. Siebert & P.Y. Lynn (USA)

87. Applications of chemometrics in brewing

Karl J. Siebert (USA)

88. Development of image analysis technology for breweries

Gearid Cahill, Padraig K. Walsh & Dan Donnelly (IRL)

89. Sensory analysis - a bridge with the consumer. The use of an external expert sensory
panel
Paul Hegarty, Janice Chilver & Lee Threapleton (UK)

Risk management
90. Consumer perception of risk
Joachim Scholderer (DK)

91. Mycotoxins in the total chain from barley to beer

Liisa Vanne & Auli Haikara (FIN)

92. Using HACCP to prevent engineering related product safety and quality risks
Alan I. Kennedy (UK)
93. Disinfectant testing against brewery-related biofilms
E. Storgrds, M. Nrhi & G. Wirtanen (FIN)

Packaging
94. Designing packaging for brand growth
A. Ray Morgan (USA)

95. Beer in plastic bottles highlights EBC Symposium November 2000

David R. Wiggins (UK)

96. Validation of PET-bottles for the filling of beer

Christian Drr & Horst Weisser (D)

97. Development of a standardised stained beer bottle for testing bottle washers
Karl Wackerbauer, Hartmut Evers & Knut Soltau (D)

98. Seeing the light using innovative instrumental analysis to assess new packaging
Peter G. Hill, Stefan Lustig & Hans-Jrgen Barklage (D)

99. Standard interface between the field and production level of filling and packaging
lines
Tobias Voigt, Thomas Rdler, & Horst Weisser (D)

100. Heineken Filled Bottle Inspector (FBI) - from pilot to operation

B.C.J. Landman (NL)

101. Flexible packaging strategies

Knud-Erik Pedersen (DK)

102. Supply chain management information system development in a just in time

environment with driver sales operations
Eduardo Bezares (E)

Environment
103. Implementing environmental management systems
Tom Woollard (UK)

104. Production-integrated environmental protection in the brewing industry

M. Kaschek, H. Chmiel, V. Mavrov & H. Janke (D)

105. Environmentally sustainable alternative uses for brewery by-products

P.J.M. Bruijn, T.R. Noordman, P.C. Deurinck (NL) & S. Grass (CH)

106. Enhancing the value of spent yeast and brewers spent grain
Peter J. Rogers, Michael Pecar, Aldo Lentini, Adrian Gardner &
Joseph Kulandai (AUS)

107. Use of spent grains

Werner Kepplinger & Gerald Zanker

108. The role of environmental biotechnology for the brewing industry

T.L.F.M. Vereijken & W.J.B.M. Driessen (NL)
109. State detection and control of overloads in the anaerobic treatment of brewery waste
water using fuzzy logic
E. Murnleitner, T. Becker & A. Delgado (D)

Poster Development and demonstration of polymerase chain reaction based methods for
process control in breweries

Poster The New International Calibration Standards (ICS) for the HPLC Analysis of
Isomerized and Reduced Isomerized -Acids

Curriculum Vitae of (co-)Authors

Participants Exhibition Scientific Institutes & Brewing Schools
List of Participants in the Congress

B. General information on the European Brewery Convention

Poster EBC as presented at the Congress

Committees and Groups:
EBC Analysis Committee
- biennial report
- poster presented at the Congress
EBC Barley & Malt Committee
- biennial report
- poster presented at the Congress
EBC Brewing Science Group
- biennial report
- poster presented at the Congress
List of EBC Publications
List of EBC Member Organisations
List of EBC Congresses
Index of authors
(The numbers refer to the contributions)

Aastrup, S. 56 Cahill, G. 88
Abe, M. 39 Cantrell, I. 42
Abraham, N. 28 Cardelli, J. 26
Aerts, G. 58, 60 Carniel, E. 76
Allosio-Ouarnier, N. 19 Carvell, J. 78, 79
Almeida, P. 84 ejka, P. 82
Alt, A. 5, 6 epika, J. 85
Andersen, H.A. 44 Chandra, S. 8, 73
Arimura, H. 39 Chilver, J. 89
Arnould, M. 83 Chmiel, H. 5, 104
Asakura, T. 21 Collin, S. 80
Cooman, L., de 7, 58, 60
Back, W. 15, 17, 22, 23, Cooper, D. 68
39, 54, 65, 66, Crestini, M. 26
75 ulk, J. 82
Barklage, H.-J. 98 Cunningham, S. 79
Barros, A. 84 urdov, E. 85
Barski, M. 77
Bartsch, H. 5, 6 Davis, D. 33
Bastien, C. 76 Dawes, I.W. 52
Bastin, P. 46 Day, R.E. 52
Baxter, E.D. 9 Debourg, A. 45, 46
Bechmann, F 28 Delgado, A. 29, 75, 109
Becker, H. 5, 6 Delvaux, F. 83
Becker, T. 29, 75, 109 Derdelinckx, G. 83
Bene, R. 81 Deurinck, P.C. 105
Berg, H. 49 Dickel, T. 75
Berger, S. 51 Didierjean, L. 76
Bergin, J. 49 Dillemans, M. 45, 46
Bertrand, D. 19 Dmny, Z. 50
Bes, M. 83 Donnelly, D. 88
Bezares Carretero, E. 102 Drr, C. 96
Boeykens, A. 60 Dostlek, P. 85
Bohak, I. 17 Douliez, J.-P. 67
Boivin, P. 13, 19, 20, 46, Douma, A.C. 69
57, 67 Driessen, W.J.B.M. 108
Bolshaw, L. 8 Drost, M.A. 25
Bonacchelli, B. 24 Dumoulin, M. 20, 57
Booer, C.D. 73 Dvok, P. 50
Boulton, C.A. 78
Box, W.G. 41, 78 Eger, C. 28
Bravo, A. 48, 61, 63, 64 Enge, J. 85
Brelin, A. 76 Ermolaev, S. 27
Brignon, P. 76 Escudier, J.-L. 83
Bruijn, P.J.M. 105 Eto, M. 55
Buck, A., de 58 Evans, E. 10, 77
Evers, H. 97 Hoschke, . 43
Farkas, G. 43 Hrabk, M. 59
Ferreira, A.A. 84 Husband, F.A. 68
Fick, M. 13 Hutter, K.-J. 38
Fillaudeau, L. 27
Finglas, P. 4 Imai, T. 39
Fischer, G. 75 Inomoto, K. 55
Fgen, T. 62 Isenberg, T. 28
Ford, C.M. 10, 77 Isoe, A. 30
Forster, A. 16, 17 Ito, K. 21
Forster, C. 15
Fournier, R. 57 Janke, H. 104
Frank, N. 5, 6 Jgou, S. 67
Franz, O. 65, 66 Jenkins, C. 40
Fujita, A. 30 Jitariouk, N. 27
Fukui, N. 30 Jurkov, M. 82

Gaag, B., van der 74 Kagami, N. 55

Gaber, R.F. 44 Kalita, J.C. 7
Gaborit, T. 67 Kaltner, D. 16, 17
Gahr, A. 17 Kaneda, H. 70
Galindo-Castro, I. 47, 48, 61 Kaneko, T. 21
Gamal-Eldeen, A. 5, 6 Kaschek, M. 104
Gardner, A. 106 Kellner, V. 82
Gerhuser, C. 5, 6 Kennedy, A.I. 40, 92
Goiris, K. 60 Kepplinger, W. 107
Goldstein, H. 71 Keukeleire, D., de 7, 60
Gourdon, A. 27 Kielland-Brandt, M.C. 44
Grass, S. 105 Kihara, M. 21
Guido, L. 84 Kleemola, T. 12
Gunkel, J. 11 Klimo, K. 6
Gunning, A.P. 68 Knauft, J. 6
Kobayashi, N. 70
Haikara, A. 12, 91 Kono, K. 55
Hamilton, A.J. 41 Koplk, R. 85
Hammond, J.R.M. 33, 37 Kotaviita, E. 12
Hariri, A. 13 Kreisz, S. 23
Harmegnies, F. 24, 83 Krottenthaler, M. 22, 54, 75
Hakov, D. 59 Kulandai, J. 106
Hegarty, P. 89 Kuroda, H. 70
Heiss, E. 6 Kurz, T. 29
Herrera, J. 61, 64
Heyerick, A. 7 Lancashire, W. 37
Higgins, V.J. 52 Landman, B.C.J. 100
Hill, P.G. 98 Lange, C. 38
Hodgson, J.A. 31, 40 Larsen, O.V. 56
Hhn, G. 51 Lee, M. 33, 37
Home, S. 61 Lentini, A. 106
Hork, T. 59, 82 Lermusieau, G. 80
Hori, T. 39 Lillelund, A.C. 56

2
Linder, M. 12 Omura, F. 30
Lommi, H. 49 Ottow, K. 44
Londesborough, J. 37 Ozaki, K. 14
Lusk, L.T. 71
Lustig, S. 98 Pajunen, E. 49
Lynn, P.Y. 86 Patel, D. 4
Pauls, S. 28
Machado Cruz, J.M. 84 Pecar, M. 106
Mackie, A.R. 68 Pedersen, K.-E. 101
Madrid, J. 61, 64 Peereboom, P.S. 69
Madsen, P.L. 44 Penttil, M. 12, 47, 53, 61
Marz, A. 43 Pnzes, Z. 58
Marc, I. 13 Peters, U. 62
Marion, D. 67 Pocock, V. 7
Markkula, T. 37 Pommersheim, R. 51
Martynowicz, E.T.M.J. 69 Powell, C.D. 41
Mavrov, V. 104 Preu, Th. 15
Mazike, H.G. 31
Methner, F.-J. 62 Quain, D.E. 41
Meyer-Pittroff, R. 51
Mikyka, A. 59 Rdler, T. 99
Milligan, S.R. 7 Ramrez, J.L. 48, 61
Mills, C. 68 Rangel-Aldao, R. 47, 48, 61, 63,
Mitchell, M.C. 2 64
Mitzscherling, M. 75 Ranta, B. 49
Mochaba, F. 42 Rath, F. 11
Mocking-Bode, H.C.M. 69 Rautio, J. 37
Morgan, A.R. 94 Reverol, L. 48, 61
Morris, V.J. 68 Rezessy-Szab, J. 43
Muller, R.E. 73 Ridder, M., de 60
Murnleitner, E. 109 Righelato, R. 3
Mussche, R. 58 Robert, P. 19
Rodrigues, J. 84
Nakamura, T. 65 Rodrigues, P. 84
Nakari-Setl, T. 12 Roelens, F. 7
Nrhi, M. 93 Rogers, P.J. 52, 106
Navarro, A. 71 Rong, H. 7
Navrtil, M. 50 Rouck, G., de 58
Nedervelde, L., van 45, 46 Ruddlesden, J.D. 41
Neske, W. 28 Ryder, D. 71
Neumann, I. 5, 6
Nielsen, H. 56 Sacy, H. 1
Nielsen, P.S. 44 Sakamoto, K. 14
Nishida, Y. 30 Snchez, B. 47, 48, 61, 63
Nitzsche, F. 51 Santos, J. 84
Noordman, T.R. 105 Sasaki, K. 55
Schans, M. 74
Ohtake, Y. 14 Scherer, E. 61, 63, 64
Oliver, A.D. 52 Scherf, H. 6
Olkku, J. 12 Schmidt, R. 16

3
Schneider, J. 22 Veen, S., van 74
Scholderer, J. 90 Vehkomki, M.-L. 47, 61
Sharpe, F.R. 72 Vereijken, T.L.F.M. 108
Shinotsuka, K. 70 Vidgren, V. 47, 61
Shibata, K. 55 Viljava, T. 49
Siddique, R. 35 Vilpola, A. 47
Siebert, K.J. 86 Virant, M. 18
Siebert, K.J. 87 Virtanen, H. 47, 61, 64
Simon, K. 16 Voetz , M. 11
Smart, K.A. 32, 35, 40 Voigt, T. 99
mogroviov, D. 50 Vundla, W. 42
Soltau, K. 97
Spath, S. 74 Wackerbauer, K. 97
Spillmer, F. 28 Waesberghe, J., van 58
rogl, J. 59 Wagner, F. 23
Stafford, R.A. 34 Walker, C.J. 4, 8, 9
Stewart, D. 77 Walsh, P.K. 88
Stewart, G.G. 34, 36 Watts, K. 71
Stigter, E. 74 Wehrenberg, H. 28
Storey, K. 33 Weisser, H. 22, 96, 99
Storgrds, E. 93 Wheaton, L.K. 73
Stoupis, T. 34 Wiggins, D.R. 95
Wilde, P.J. 68
Taidi, B. 31 Windhab, E.J. 25
Takashio, M. 70 Wirtanen, G. 93
Takeuchi, T. 39 Witters, A. 60
Tapani, K. 49 Wolfe, C. 4
Threapleton, L. 89 Woodward, N. 68
Thum, B. 15 Woollard, T. 103
Thurston, P. 40 Wright, A. 4
Tigel Gil, R. 24
Todd, R. 79 Yasuda, Y. 39
Turner, K. 78 Yonezawa, T. 79

Ueda, T. 55 Zandycke, S.M., van 35

Zangrando, T. 26
Vaitilingom, M. 76 Zanker, G. 81, 107
Vanne, L. 91 Zrcher, A. 54

4
Index of Descriptors
(The numbers refer to the contributions)

Accelerated method, 77 , 78 Bioreactor, 109

-Acids, 18 Biotechnology, 108
Acrospire, 54 Bitter substances, 16
Agriculture, 106 Bitterness, 60
Air pollution, 107 Bottle colour, 98
Albumin, 67 Bottle washing, 97
Alcohol free beer, 83 Bottling line, 101
Alcohol percentage, 81 Breeding, 10, 11, 21
Alcoholic beverage, 1, 2 Brewers' yeast, 29, 32, 36, 37, 40, 42,
Aldehyde, 84 43, 44, 45, 47, 48, 53
Amino acid, 44 Brewery, 28
-Amylase, 11, 21 Brewing industry, 90, 95, 102, 103,
Anaerobic effluent treatment, 104, 109 104, 105, 108
Analysis method for beer, 81, 82, 84, Brewing liquor, 58
85 By-product disposal, 105, 106, 107
Analysis method for brewing, 87
Analysis method for cereals, 76 Cancer chemoprevention, 6
Analysis method, 72 Carbohydrate, 35
Anthocyanogen, 18 Carbon dioxide, 83
Antioxidant, 4, 5, 8, 16, 20, 46, 58, 65, Carbonyl compound, 47, 48, 59, 61, 64
66, 72 Cell cycle analysis, 38
Aromatic compound, 80, 83 Cell damage, 34
Automatic analysis equipment, 78 Cell morphology, 36
Automation, 28 Cell wall, 35
Chalcone, 6
Barley, 11, 21, 73, 91 Chemical and physical analysis
Barley constituent, 71 method, 87
Barley quality, 10, 14, 74 Chemical composition, 67
Barrier technology, 95 Chemistry, 63
Beer, 3, 8, 77 Cider, 8
Beer analysis result, 62 Cling, 69
Beer aroma, 80 Combustion, 105, 107
Beer colour, 18 Comparison, 93
Beer constituent, 5, 9 Consumer research, 1, 90, 94, 95
Beer consumption, 4, 7, 9 Contamination, 85, 91, 92
Beer filtration, 23, 25, 26, 27 Continuous fermentation, 49, 50
Beer quality, 18, 20, 27, 49, 50, 60, Continuous process, 24
68, 89, 96 Cylindro-conical tank, 79
Beer spoilage organism, 17, 93
Beer storage conditions, 63, 64 Dark beer, 15
Beer treatment, 62, 66 Data processing, 28, 87, 99
Beverage industry, 94 Deoxyribonucleic acid, 14
Biochemistry, 69 Deterioration, 32
Biological analysis method, 74 Determination of filterability, 77
Biological stability, 17 Diacetyl, 82, 84
Biomass, 31 Dimethylsulphide, 20
Disinfectant, 93 Genetics, 11, 21, 32, 53, 76, 90
Drinking habits, 1, 2 Gliadin, 86
-Glucan, 10
Ecology, 108 Glucose, 45
Efficiency, 93, 97, 99, 100, 108 Glycogen, 88
Electrode, 85 Glycol, 92
Endosperm, 19 Grist, 22, 54
Energy policy, 105 Growth inhibitor, 43
Engineering, 92, 101 Gushing, 12
Environmental protection, 26, 103,
104, 108 HACCP, 92
Enzymic activity, 11, 21, 37, 45, 47, Haze measurement, 86
48, 51, 70, 75 Health, 1, 3, 4, 7, 8, 9
Enzymic reaction, 61 Health hazard, 90
Equipment, 79 Heat exchanger, 92
Essential oil, 18 Heavy metal, 85
Ester, 36 High gravity brewing, 33, 36, 37
Ethanol, 33 High-capacity equipment, 100
Evaluation, 97 Homogeneity, 72, 73
Extract yield, 72 Hop analysis result, 16
Extraction, 72, 82 Hop constituent, 6, 7, 80
Hop product, 17
Feed, 105 Hops, 18
Fermentability, 11, 21 Hot break, 24
Fermentation by-product, 83 HPLC, 63
Fermentation defect, 52 Humulone, 17
Fermentation inhibitor, 33 5-Hydroxymethylfurfural, 63, 64
Fermentation speed, 37
Fermentation, 30, 36, 38, 41, 42, 44 Identification, 14
Fermentative ability, 39, 40, 43 Image analysis, 88
Fermentative power, 34, 35, 41, 45 Immobilised yeast, 49, 50, 51
Filled bottle inspector, 100 Immunology, 12, 71
Filler, 99 Information management, 28, 102
Filter aid, 26 Infrared radiation, 19, 81
Filterability, 23, 25 Iso--acid, 17, 58, 60
Finance, 101
Flavonoid, 7 Kieselguhr filter, 26
Flavour formation, 15, 20, 44, 49, 57 Killer factor, 43
Flavour profile, 50 Kilning, 15, 20
Flavouring agent, 83
Flocculation, 41, 42, 79 Laboratory equipment, 69, 75, 81
Flow cytometry, 38, 39 Lautering, 10
Flow measurement, 25 Lectin, 42
Foam stability, 67, 68, 70, 71 Legislation, 76, 91
Folic acid, 4 Lethality, 93
Fungi, 12 Lightstruck flavour, 98
Fungicide, 106 Linoleic acid, 57
Lipid, 68
Gas chromatography, 98 Lipoxygenase, 54, 70
Genetic marker, 14, 52 Logistics, 102

2
Lubricant, 104 Pitching yeast, 78
Planning, 102
Maize, 76 Plastic bottle, 95, 96
Malt, 42 Polyethylene terephthalate, 96
Malt constituent, 55, 70 Polypeptide, 68
Malt modification, 67, 72, 73 Polyphenol, 4, 5, 16, 18, 59, 65, 66
Malt quality, 13, 20, 74, 77 Polysaccharide, 23
Maltase, 37 Prediction, 12, 35
Malting, 19, 71 Process control, 29, 30, 32, 38, 42, 49,
Maltose, 30 75, 87, 109
Malty flavour, 15 Process supervision, 28
Management, 103 Product safety, 92
Marketing, 94 Protein, 12, 44, 71
Mash filtration, 22 Protoplast fusion, 43
Mash separation time, 10 Psychology, 90
Mashing conditions, 75 Public relations, 1
Medicine, 2, 5, 6, 7, 9 Purity, 85
Membrane filtration, 22, 104
Metabolism, 3, 30, 37, 45, 53 Quality analysis, 73, 74
Microorganism, 17, 23 Quality control, 19, 89
Milling, 54 Quantitative analysis, 76
Model simulation, 13, 29, 109
Moistening, 13 Radical scavenging, 4, 5
Moisture content, 91 Raw material, 9
Multipack, 101 Reaction mechanism, 6, 68, 69
Mycotoxin, 74, 91 Real-time operation, 79
Refractometry, 73
Non-enzymic activity, 48 Research, 2, 9
Non-enzymic browning, 47, 61, 63 Returnable bottle, 95
2-Nonenal, 55, 56, 57
Nutritive value, 3 Saving, 24
Secondary fermentation, 50, 51
Oleic acid, 70 Sensor, 74
Optimization, 99 Sensory analysis, 89
Osmosis, yeast, 33 Sensory analysis result, 80
Oxidation, 54, 59, 60 Shear stress, 34
Oxygen, 6 Shelf life, 95, 96
Sodium hydroxide, 104
Packaging material, 94, 101 Sparging liquor, 56
Particle size, 86 Spectroscopy, 19, 66
Peptide, 45 Spent grains, 105, 106, 107
Permeability, 96 Stabilisation, 5, 61
Peroxidase, 70 Staining, 88
Peroxide, 57 Staling, 20, 47, 48, 55, 58, 60, 61, 62,
pH, 56, 65, 86 63, 64, 84
Phenolic acids, 65, 66 Standardisation, 97
Physiology, 32, 34, 35, 38, 40 Steep water, 31
Phyto-oestrogens, 7 Steeping, 13
Pilot plant trial, 49 Sterile filtration, 27
Pitching, 40 Stirring, 34

3
Sulphites, 65 Waste water, 104
Sulphur compound, 72 Waste yeast, 106
Sulphur dioxide, 84 Wine yeast, 53
Supply, 102 Wine, 8
Surface activity, 68 Wort, 77
Wort boiling, 24, 57
Tannic acid, 58, 86 Wort composition, 40
Taste panel, 89 Wort filtration, 23
Taste stability, 15, 46, 54, 55, 56, 58,
59, 62, 65 Xanthohumol, 6
Total resin, 18
Yeast, 23, 35
Ultra-violet radiation, 98 Yeast cell, 34, 36, 39
Yeast extract, 46
Variety, 18 Yeast growth, 31, 38, 79
Viability, 34, 35, 36, 39, 41, 78, 79 Yeast propagation, 29, 30, 31, 32, 88
Vibration, 22 Yeast strain, 43
Vicinal diketones, 82 Yeast technology, 41, 52, 53, 88
Viscosity, 25 Yeast treatment, 34
Vitality, 88 Yield, 24, 27, 31
Volatile compound, 83
Voltametry, 84 Zinc, 52

4
Deskriptorenindex
(Die Zahlen beziehen sich auf die Nummern der Beitrge)

Abfallhefe, 106 Bierhefe, 29, 32, 36, 37, 40, 42, 43, 44,
Ablutern (Luterbottich), 10 45, 47, 48, 53
Abnormale Grerscheinung, 52 Bierqualitt, 18, 20, 27, 49, 50, 60, 68,
Albumin, 67 89, 96
Aldehyd, 84 Bierschdliche Organismen, 17, 93
Alkoholfreies Bier, 83 Bierverbrauch, 4, 7, 9
Alkoholgehalt, 81 Bildanalyse, 88
Alkoholisches Getrnk, 1, 2 Biochemie, 69
Altgeschmackbildung, 20, 47, 48, 55, Biologische Stabilitt, 17
58, 60, 61, 62, 63, 64, 84 Biomasse, 31
Aminosure, 44 Bioreaktor, 109
-Amylase, 11, 21 Biotechnologie, 108
Anaerobe Abwasserklrung, 104, 109 Bittere, 60
Analysemethode, 72 Bitterstoffe, 16
Analysenmethode fr Bier, 81, 82, 84, Blattkeim, 54
85 Brauen mit hohem Stammwrzegehalt,
Analysenmethode fr Getreide, 76 33, 36, 37
Analysenmethode zur Bierherstellung, Brauerei, 28
87 Brauindustrie, 90, 95, 102, 103, 104,
Anlage, 79 105, 108
Anstellen, 40 Brauwasser, 58
Anstellhefe, 78
Anthocyanogen, 18 Carbonylverbindung, 47, 48, 59, 61, 64
Antioxidantium, 4, 5, 8, 16, 20, 46, 58, Chalcon, 6
65, 66, 72 Chemie, 63
Apfelwein, 8 Chemische Zusammensetzung, 67
Aromabildung, 15, 20, 44, 49, 57 Cylindrokonischer Tank, 79
Aromatische Verbindung, 80, 83
thanol, 33 Darren, 15, 20
therisches l, 18 Datenverarbeitung, 28, 87, 99
Ausbeute, 24, 27, 31 Desinfektionsmittel, 93
Ausrstung zur automatischen Desoxyribonucleinsure, 14
Analyse, 78 Diacetyl, 82, 84
Automation, 28 Dimethylsulfid, 20
Dunkles Bier, 15
Barriertechnologie, 95 Durchflucytometrie, 39
Befeuchten, 13 Durchflumessung, 25
Beschaffung, 102 Durchlssigkeit, 96
Bestimmung der Filtrierbarkeit, 77
Beurteilung, 97 Einsparung, 24
Bier, 3, 8, 77 Elektrode, 85
Bieraroma, 80 Endosperm, 19
Bierbehandlung, 62, 66 Energiewirtschaft, 105
Bierbestandteil, 5, 9 Enzymaktivitt, 11, 21, 37, 45, 47, 48,
Bierfarbe, 18 51, 70, 75
Bierfiltration, 23, 25, 26, 27 Enzymreaktion, 61
Ergebnis von Bieranalysen, 62 Glucose, 45
Ergebnis von Hopfenanalysen, 16 Glykogen, 88
Ergebnis von sensorischen Analysen, Glykol, 92
80 Gteregelung, 19, 89
Ester, 36
Extraktausbeute, 72 HACCP, 92
Extraktion, 72, 82 Halbtechnischer Versuch, 49
Haltbarkeitsdauer, 95, 96
Frbung (Biologie), 88 Hefe, 23, 33, 35
Filterhilfsmittel, 26 Hefebehandlung, 34
Filtrierbarkeit, 23, 25 Hefeextrakt, 46
Finanzierung, 101 Hefestamm, 43
Flaschenfarbe, 98 Hefetechnologie, 41, 52, 53, 88
Flaschenflllinie, 101 Hefevermehrung, 29, 30, 31, 32, 88
Flaschenreinigung, 97 Hefewachstum, 31, 38, 79
Flavonoid, 7 Hefezelle, 34, 36, 39
Flavour nach Malz, 15 Hochleistungsanlage, 100
Flockung, 41, 42, 79 Homogenitt, 72, 73
Flchtige Substanz, 83 Hopfen, 18
Fluzytometrie, 38 Hopfenbestandteil, 6, 7, 80
Folsure, 4 Hopfenprparat, 17
Forschung, 2, 9 HPLC, 63
Fller, 99 Humulon, 17
Fungi, 12 5-Hydroxymethylfurfural, 63, 64
Fungizid, 106
Futtermittel, 105 Immunologie, 12, 71
Informationswesen, 28, 102
Grfhigkeit, 39, 40, 43 Infrarote Strahlung, 19, 81
Grgeschwindigkeit, 37 Iso--Sure, 17, 58, 60
Grkraft (Triebkraf), 34, 35, 41, 45
Grkraft, 11, 21 Kieselgurfilter, 26
Grung, 30, 36, 38, 41, 42, 44 "Killer factor", 43
Grungsinhibitor, 33 Kochtrub, 24
Grungsnebenprodukt, 83 Kohlendioxide, 83
Gaschromatographie, 98 Kohlenhydrat, 35
Genetik, 11, 21, 32, 53, 76, 90 Kontamination, 85, 91, 92
Gerbsure, 58, 86 Kontinuierliche Grung, 49, 50
Gerste, 11, 21, 73, 91 Kontinuierlicher Proze, 24
Gerstenbestandteil, 71 Korngre, 86
Gerstenqualitt, 10, 74 Krebs-chemoprvention, 6
Gerstensorte, 14 Kunststoffflasche, 95, 96
Gesamtharz, 18
Geschmacksstabilitt, 15, 46, 54, 55, Laboratoriumausstattung, 69, 75, 81
56, 58, 59, 62, 65 Lagerkellerfhrung, 63, 64
Geschmacksstoff, 83 Landwirtschaft, 106
Gesundheit, 1, 3, 4, 7, 8, 9 Luterdauer, 10
Gesundheitsrisiko, 90 Lebensfhigkeit, 34, 35, 36, 39, 41, 78,
Getrnkeindustrie, 94 79
Gliadin, 86 Lektin, 42
-Glucan, 10 Letalitt, 93

2
Lichtgeschmack, 98 Peptid, 45
Linolsure, 57 Peroxid, 57
Lipid, 68 Peroxidase, 70
Lipoxygenase, 54, 70 pH, 56 , 65, 86
Logistik, 102 Phenolsuren, 65, 66
Luftverschmutzung, 107 Physiologie, 32, 34, 35, 38, 40
Phyto-strogen, 7
Mais, 76 Planung, 102
Maischefiltration, 22 Polythylenterephthalat, 96
Maischefhrung, 75 Polypeptid, 68
Maltase, 37 Polyphenol, 4, 5, 16, 18, 59, 65, 66
Maltose, 30 Polysaccharid, 23
Malz, 42 Produktsicherung, 92
Malzbestandteil, 55, 70 Protein, 12, 44, 71
Malzherstellung, 19, 71 Protoplastenfusion, 43
Malzlsung, 67, 72, 73 Prozesteuerung, 29, 30, 32, 38, 42,
Malzqualitt, 13, 20, 74, 77 49, 75, 87, 109
Management, 103 Prozeberwachung, 28
Marketing, 94 Psychologie, 90
Markierungsgen, 14, 52 Public relations, 1
Medizin, 2, 5, 6, 7, 9
Mehrwegflasche, 95 Qualittskontrolle, 73, 74
Membranfiltration, 22, 104
Mengenmssige Analyse, 76 Radikalfnger, 4, 5
Methode zur biologischen Analyse, 74 Reaktionsmechanismus, 6, 68, 69
Methode zur chemischen und Recht, 76, 91
physikalischen Analyse, 87 Rechtzeitbetrieb, 79
Mikroorganismen, 17, 23 Refraktometrie, 73
Modellbildung, 13, 29, 109 Reinheit, 85
"Multipack", 101 Rohstoff, 9
Mykotoxin, 74, 91 Rhren, 34

Nachgrung, 50, 51 Sauerstoff, 6

Nachweis (Identifizierung), 14 -Suren, 18
Nhrwert, 3 Schaumbestndigkeit, 67, 68, 70, 71
Natriumhydroxid, 104 Schaumhaftung, 69
Nebenproduktbeseitigung, 105, 106, Scherbeanspruchung, 34
107 Schmier-mittel, 104
Nichtenzymatische Brunung, 47, 48, Schmutzwasser, 104
61, 63 Schnellverfahren, 77, 78
Nonenal-2, 55, 56, 57 Schrot, 22, 54
Schroten, 54
Oberflchenaktivitt, 68 Schwefeldioxid, 84
kologie, 108 Schwefelverbindung, 72
Oleinsure, 70 Schwermetall, 85
Optimierung, 99 Sensor, 74
Organoleptisches Profil, 50 Sensorische Analyse, 89
Osmose, 33 Spektroskopie, 19, 66
Oxidation, 54, 59, 60 Stabilisierung, 5, 61
Standardisierung, 97

3
Sterielfiltration, 27 Zink, 52
Stoffwechsel, 3, 30, 37, 45, 53 Zchtung, 10, 11, 21
Sulfite, 65

Technik, 92, 101

Trgergebundene Hefe, 49, 50, 51
Treber, 105, 106, 107
Trinkverhalten, 1, 2
Trbungsmessung, 86

berschwnzwasser,56
Ultraviolette Strahlung, 98
Umweltschmutz, 104
Umweltschutz, 103, 108, 126

Variett, 18
Verbraucherforschung, 1, 90, 94, 95
Verbrennung, 105, 107
Verderb, 32
Vergleich, 93
Verkostergremium, 89
Verpackungsmaterial, 94, 101
Vibration, 22
Vicinale Diketone, 82
Viskositt, 25
Vitalitt, 88
Vollgutausleuchter, 100
Voltametrie, 84
Vorhersage, 12, 35

Wachstumsinhibitor, 43
Wrmeaustauscher, 92
Wassergehalt, 91
Weichen, 13
Weichwasser, 31
Wein, 8
Weinhefe, 53
Wildwerden (Bier), 12
Wirkungsgrad, 93, 97, 99, 100, 108
Wrze, 77
Wrzefiltration, 23
Wrzekochen, 24, 57
Wrzezusammensetzung, 40

Xanthohumol, 6

Zellmorphologie, 36
Zellschdigung, 34
Zellwand, 35
Zellzyklusanalysen, 38

4
Index de Descripteurs
(Les chiffres se rapportent aux numros des contributions)

Acide amin, 44 Brunissement non-enzymatique, 47,

Acide dsoxyribonuclique, 14 48, 61, 63
Acide folique, 4
Acide iso-, 58, 60 Capteur, 74
Acide linoligue, 57 Cassure chaud, 24
Acide olique, 70 Cellule de levure, 34, 36, 39
Acide tannique, 58, 86 Chalcone, 6
Acides , 18 Chimie, 63
Acides phnoliques, 65, 66 Chimio-prvention, 6
Acquisition, 102 Chromatographie en phase gaseuze, 98
Activation du mtabolisme, 45 Cidre, 8
Activit enzymatique, 11, 21, 37, 47, Coloration (biologie), 88
48, 51, 70, 75 Combustion, 105, 107
Adhrance de mousse, 69 Commande de procd, 29, 30, 32, 38,
Adjuvant de filtration, 26 42, 49, 75, 87, 109
Agitation mcanique, 34 Comparaison, 93
Agriculture, 106 Compos aromatique, 80, 83
Albumine, 67 Compos carbonyl, 47, 48, 59, 61, 64
Aldhyde, 84 Compos chimique, 67
Aliment pour animaux, 105 Compos soufr, 72
Amertume, 60 Compos volatil, 83
-Amylase, 11, 21 Composition du mot, 40
Analyse de qualit, 73, 74 Conditions de brassage, 75
Analyse d'image, 88 Conditions de garde, 63, 64
Analyse du cycle cellulaire, 38 Consommation de bire, 4, 7, 9
Analyse quantitative, 76 Constituant de la bire, 5, 9
Analyse sensorielle, 89 Constituant de l'orge, 71
Anthocyanogne, 18 Constituant du houblon, 6, 7, 80
Antioxydant, 4, 5, 8, 16, 20, 46, 58, 65, Constituant du malt, 55, 70
66, 72 Contamination, 85, 91, 92
Aromatisant, 83 Contrle de procd, 28
Arme de la bire, 80 Contrle de qualit, 19, 89
Automatisation, 28 Couleur de bouteille, 98
Couleur de la bire, 18
Bire, 3, 8, 77 Croissance de la levure, 31, 38, 79
Bire brune, 15 Cuisson du mot, 24, 57
Bire sans alcool, 83 Cytomtrie de debit, 38
Biochimie, 69 Cytomtrie fluidique, 39
Biomasse, 31
Bioracteur, 109 Dfaut de fermentation, 52
Biotechnologie, 108 Dsagrgation du malt, 67, 72, 73
Boisson alcoolique, 1, 2 Dsinfectant, 93
Bouteille consigne, 95 Dtrioration, 32
Bouteille en plastique, 95, 96 Diactyle, 82, 84
Brasserie, 28 Dictones vicinales, 82
Dimension des particules, 86
Dimthylsulfure, 20 Formation de la flaveur, 15, 20, 44, 49,
Dioxyde de carbone, 83 57
Dioxyde de soufre, 84 Formation du got dvent, 20, 47,
Drche, 105, 106, 107 48, 55, 58, 60, 61, 62, 63, 64, 84
Dure de conservation, 95, 96 Fungi, 12
Dure de sparation de la maische, 10 Fusion de protoplaste, 43

Eau de brassage, 58 Gntique, 11, 21, 32, 53, 76, 90

Eau de lavage des drches, 56 Gestion de linformation, 28
Eau de trempage, 31 Gestion de l'nergie, 105
Echangeur de chaleur plateaux, 92 Gestion de l'entreprise, 103
Ecologie, 108 Gestion de l'information, 102
Effet antiradicaux (libres), 4, 5 Gestion des eaux, 104
Efficacit, 93, 97, 99, 100, 108 Gestion financire, 101
Electrode, 85 Giclage, 12
Endosperme, 19 Gliadine, 86
Ensemencement, 40 -Glucane, 10
Epargne, 24 Glycogne, 88
Essai en installation pilote, 49 Glycol, 92
Ester, 36 Got de lumire, 98
Ethanol, 33 Got de malt, 15
Etude du march (consommation), 1, Groupe d'embouteillage, 101
90, 94, 95
Evaluation, 97 Habitude en matire de consommation
Extraction, 72, 82 de boissons, 1, 2
Extrait de levure, 46 HACCP, 92
Homognit, 72, 73
Fabrication de bire haute densit, Houblon, 18
33, 36, 37 HPLC, 63
Facteur "killer", 43 Huile essentielle, 18
Fermentabilit (de la levure), 39, 40, Humidification, 13
43 Humulone, 17
Fermentabilit, 11, 21 Hydrate de carbone, 35
Fermentation anarobie, 104 Hydroxymthyl-5 furfural, 63, 64
Fermentation continue, 49, 50
Fermentation secondaire, 50, 51 Identification, 14
Fermentation, 30, 36, 38, 41, 42, 44 Immunologie, 12, 71
Filtrabilit, 23, 25 Industrie brassicole, 90, 95, 102, 103,
Filtration de la bire, 23, 25, 26, 27 105, 108
Filtration de la maische, 22 Industrie des boissons, 94
Filtration du mot, 23 Ingnirie, 92, 101
Filtration strile, 27 Inhibiteur de croissance, 43
Filtration sur membrane, 22 Inhibiteur de fermentation, 33
Filtre kieselgur, 26 Installation haute capacit, 100
Flavonode, 7 Insuline, 45
Floculation, 41, 42, 79
Fonction logistique, 102 Jury de dgustation, 89
Fongicide, 106
Force de cisaillement, 34 Lavage de bouteilles, 97
Lectine, 42

2
Lgislation, 76, 91 Moture, 54
Lsions cellulaires, 34 Multipack, 101
Ltalit, 93 Mycotoxine, 74, 91
Levain, 78
Levure de brasserie, 29, 32, 36, 37, 40, Nonnal-2, 55, 56, 57
42, 43, 44, 47, 48, 53
Levure de vin, 53 Obtention (culture), 10, 11, 21
Levure immobilise, 49, 50, 51 Optimisation, 99
Levure rsiduelle, 106 Orge, 11, 21, 73, 91
Levure, 23, 33, 35, 45 Osmose, 33
Lipide, 68 Oxydation, 54, 59, 60
Lipoxygnase, 54, 70 Oxygne, 6
Lupulone, 17
Paroi cellulaire, 35
Mais, 76 Performance en fermentation, 45
Malt, 42 Permabilit, 96
Maltage, 19, 71 Peroxydase, 70
Maltase mtabolisme, 37 Peroxyde, 57
Maltose, 30 pH, 56, 65, 86
Marketing, 94 Physiologie, 32, 34, 35, 38, 40
Marqueur gntique, 14, 52 Phyto-estrognes, 7
Matriau d'emballage, 94, 101 Planification, 102
Matriel danalyse automatique, 78 Plumule, 54
Matriel de laboratoire, 69, 75, 81 Pollution de l'air, 107
Matriel, 79 Polyethylne terephthalate, 96
Matires premires, 9 Polypeptide, 68
Mcanisme de raction, 6, 68, 69 Polyphnol, 4, 5, 16, 18, 59, 65, 66
Mdecine, 2, 5, 6, 7, 9 Polysaccharide, 23
Mesure du dbit, 25 Pouvoir fermentaire, 34, 35, 41
Mesure du trouble, 86 Prdiction, 12, 35
Mtabolisme, 3, 30, 53 Procd acclr, 77, 78
Mtal lourd, 85 Procd continu, 24
Mthode danalyse biologique, 74 Procds membranes, 104
Mthode danalyse de crales, 76 Profil de flaveur, 50
Mthode danalyse de la bire, 81, 82, Propagation de la levure, 29, 30, 31,
84, 85 32, 88
Mthode danalyse pour la fabrication Protection de lenvironnement, 26,
de la bire, 87 103, 108
Mthode d'analyse, 72 Protine, 12, 44, 71
Mthode de dtermination de la Psychologie, 90
filtrabilit, 77 Puret, 85
Mthode physico-chimique danalyse,
87 Qualit de lorge, 10, 74
Microorganisme contaminant, 93 Qualit de la bire, 18, 20, 27, 49, 50,
Microorganisme, 23 60, 68, 89, 96
Mireuse de bouteilles pleines, 100 Qualit du malt, 13, 20, 74, 77
Modlisation, 13, 29, 109
Morphologie cellulaire, 36 Radiation infrarouge, 19, 81
Mot, 77 Radiation ultraviolette, 98
Moture (versement), 22, 54 Raction enzymatique, 61

3
Recherche, 2, 9 Varit, 18
Rcupration des lessives alcalines, Viabilit, 34, 35, 36, 39, 41, 78, 79
104 Vibration, 22
Recyclage des vapeurs, 104 Vin, 8
Rfractomtrie, 73 Viscosit, 25
Rejet de sous-produits, 105, 106, 107 Vitalit, 88
Relations publiques, 1 Vitesse de fermentation, 37
Rendement en extrait, 72 Voltamtrie, 84
Rendement, 24, 27, 31
Rsines totales, 18 Xanthohumol, 6
Rsultat danalyse de bire, 62
Rsultat danalyse du houblon, 16 Zinc, 52
Rsultat d'analyse sensorielle, 80
Risque pour la sant, 90

Sant, 1, 3, 4, 7, 8, 9
Scurit des produits, 92
Sparation de la maische en cuve filtre,
10
Souche de levure, 43
Sous-produit de fermentation, 83
Soutireuse, 99
Spectroscopie, 19, 66
Stabilisation, 5, 61
Stabilit de la mousse, 67, 68, 70, 71
Stabilit microbiologique, 17
Stabilit organoleptique, 15, 46, 54,
55, 56, 58, 59, 62, 65
Standardisation, 97
Substances amres, 16
Sulfites, 65

Tank cylindroconique, 79
Technologie de la levure, 41, 52, 53,
88
Technologie d'tanchit, 95
Teneur en alcool, 81
Teneur en eau, 91
Tensio-activit, 68
Touraillage, 15, 20
Traitement anarobie des effluents,
109
Traitement de la bire, 62, 66
Traitement de la levure, 34
Traitement des donnes, 28, 87, 99
Traitement en temps rel, 79
Trempage, 13

Valeur nutritive, 3
Varit dorge, 14

4
1

Promoting healthy moderate drinking

Hubert Sacy
Educalcool, 606, rue Cathcart, bureau 700, Montral (Qubec) H3B 1K9, Canada
(www.educalcool.qc.ca, e-mail: [email protected])

Descriptors
Alcoholic beverage, consumer research, drinking habits, health, public relations

Deskriptoren
Alkoholisches Getrnk, Gesundheit, public relations, Trinkverhalten, Verbraucherforschung

Descripteurs
Boisson alcoolique, tude du march (consommation), habitude en matire de consommation
de boissons, relations publiques, sant
I have always believed that, in the same country, peoples relation to alcohol was the
same. Until the day I found myself talking with my 16 year old nephew about
drinking in moderation. He listened to me politely, then looked at me as if I was an
alien from an unknown planet and wondered aloud: "But why drink if not to get
drunk?".

Imagine my reaction! I learned at a very young age that the first miracle of Jesus
Christ was to turn water into wine. As far back as I can remember, I have seen my
family and friends enjoying alcoholic beverages and behaving normally. And while I
knew that some people were getting drunk every now and then, and even that a few of
them were alcoholics, I always thought those people were the exception. Until that
moment, I never even imagined that anyone in my own family could ask such a
question. I supposed I suffered what might be called "extreme culture shock".

The only answer I could come up with was: "Because it's good. I like the taste of it".
But he had the last word: "Nah. It all tastes the same".

I understand now that peoples relation to alcohol is culturally determined. That

relationship can be healthy or unhealthy; and what matters most to us, alcohol
educators, is how we can help people be healthy drinkers. And the key means to
achieving this is to reach people at the level of their "drinking culture". We have to
draw people away from the culture of drunkenness and towards the culture of taste. In
the culture of drunkenness, the goal is effect: you drink to get drunk. In the culture of
taste, you drink because you enjoy the taste. But when youre drunk, you cant taste
things properly. So its important not to lose that sense of taste. What we have to do is
make people drink better.

What does drinking better mean? It means:

Drinking less on each occasion: not more than 5 drinks.

Drinking for the right reason: not to drown your problems.
Drinking in the right context: not as the main activity.
Drinking better quality products.
Sometimes not drinking at all.
Respecting those who choose not to drink.

So our mission is to promote "Better Drinking": a balanced and sensible approach that
is positive, not repressive or curative or filled with dire warnings. In fact, the slogan
"La modration a bien meilleur got / Moderation is always in good taste", even
conveys a sense of the pleasure related to drinking. As we believe that sensible
drinking is based mainly on cultural values, we are convinced that prevention
campaigns must not be only about limiting excessive drinking, but must also take into
account and promote the moderate consumption of alcohol.

I can already hear you asking: "So what do you mean by moderation?".

The nice thing about the concept is that it is realistic and, therefore, flexible.
Moderation can mean different things depending on the person and, for the same
person, on the circumstances. It is not the same for men and women. It depends on
how alcohol affects the drinker. It depends on the moment, on the particular

2
conditions and situation. Moderation will mean something different for pregnant
women. And it will not be the same at 7:00 in the morning as it is at 7:00 in the
evening.

This is why our main goal is to debunk the myths and prejudices surrounding alcohol
and ensure its rightful place in normal human activity, which, in most cases, relates to
conviviality, relaxation and good taste. As you will see in the examples that follow,
our message is conveyed at every available opportunity, and is adapted appropriately
to the environment in which it is delivered. I would even go so far as to say that our
message is inserted wherever possible in an attempt to achieve optimum efficiency.

Thus, we promote the culture of taste versus the culture of drunkenness. We

recognise, however, that there is often a very thin line separating the pleasures of
moderate consumption and the problems of excessive drinking. That is why we also
denounce alcohol abuse, while always avoiding the admonishing approach or shock
and horror campaigns.

One last remark: It is possible to implement a culture of taste with regard to drinking
because, unlike tobacco and illegal drugs, our society recognises that there IS such a
thing as a safe level of alcohol consumption. So drinking can be socially accepted and
promoted because it has a cultural value. This is why we always separate alcohol from
tobacco and illicit drugs in all our communications; and that separation is a condition
of every one of our partnerships.

Alcohol and health

For several years now, not a month has gone by without new research on alcohol
and health. The results are frequently contradictory.
According to our own studies, 58% of Quebecers believe that, taken in
moderation, alcohol is good for your health. Five years ago, the figure was 53%.
A full 70% want to know more about alcohol and health.
The subject is already of public interest and a topic of public debate.

Protective process
Lifestyle has an impact.
Protective effect seen with 11 drinks/week for women and 17 drinks for men.
Drinking must be regular (well spaced) and moderate.
All things considered, two drinks a day generally protect against coronary
disease.

Disseminating the information

Its not enough to simply have conclusions; they must be processed.
Information transmitted must be complete.
Data must be made understandable to the general public.
Results must be disseminated widely.
Avoid generalizing, even when simplifying.

3
 Exercise great prudence when disseminating information.
Be sure to remind people that the research is constantly changing.
Ensure mechanisms for feedback and response.

Highlights
Moderation is important: two drinks a day may be good for you but four drinks
will NOT double the benefits.
Regularity is the key: there is a world of difference between having two drinks a
day over seven days and having seven drinks a day over two days.
Time is of the essence: a pre-dinner cocktail or a glass of wine with a meal is not
equivalent to having a drink on an empty stomach at seven in the morning.
There's more to it than drinking: you have to do more than just have two drinks a
day. Eating well, not smoking and exercising regularly are also important in
reducing risk.
You don't have to drink: if you choose not to drink, nobody will recommend that
you start drinking because it's good for you. After all people drink for pleasure,
not for medical reasons.

Conclusion on alcohol and health

It is essential to put principles into practice.
We must measure results and continually question the validity of our own actions.
We must leave the door open for adjustment and change.

Implementing the culture of taste

Implementing the culture of taste is not an easy task and requires long-term strategies.
We have to start early, maintain our presence, make sure we reach all target groups
and measure our results.

So we started going to school, so to speak. And we introduced ourselves to children

who are well below the legal drinking age. There were two reasons for this:

1. Because attitudes, beliefs and behaviours are developed at a very early age.

2. Because official statistics show clearly that nearly 25% of students under the age
of 12 have already started drinking. Not on a regular basis of course, but they
have tried the product on several occasions.

By presenting young people with an enlightened perspective on their initial

experience with drinking and their relationship to alcohol, we help them establish a
healthier attitude towards drinking for the rest of their lives.

Our " toi de juger / You be the judge" programme helps students 9 to 13 make value
judgements about drinking and how they approach alcohol. Our goal is to have them
start drinking as late as possible and, when they choose to do so, to be aware of what
theyre doing. We want them to discover the pleasures of drinking, not the effects of

4
drunkenness. We believe it is important to make the distinction between the way
alcohol is consumed and alcoholic beverages themselves, and specify clearly that
alcohol in itself is neither good nor bad.

This programme is made available to all Quebec teachers. It provides them with a
general framework for the course and proposes various learning activities adapted to
all grades, according to the specific objectives of the Quebec Department of
Education.

More than 87% of the students targeted by this programme have been reached during
this school year. Teacher evaluations show a remarkable level of implementation and
success.

In secondary school, we deal with students 13 to 16, but we do not try to teach them
anything. They generally feel they have things to teach others. So thats what we ask
them to do: deliver the message to other students themselves. With that in mind, we
sponsor an advertising contest open to all. Contestants are required to create a poster
on a subject related to the culture of taste and denouncing the culture of drunkenness.
In other words, promoting moderation. duc'alcool has given out several scholarships
to finalists and the creative concept of the winning entry is used in a real bus billboard
for one year. This ongoing programme is heavily advertised in Quebec schools. Its
spokesperson is always a young, talented and very popular celebrity. It gives our
organisation and more importantly our message, strong media visibility and
awareness. This year, we received entries from more than 3,500 students.

Among the college and university crowd, we strongly denounce the culture of
drunkenness. Our approach towards this group is based on statistics that show they are
the most at-risk in our society. So our message against excessive drinking is tougher,
more direct and cruder than the message designed for adults. We try to take the
glamour out of getting drunk and we point out that in the mating game, being drunk
can lead to unpleasant surprises.

Three times a year, we run poster campaigns, distribute handy promotional items and
submit articles for student publications (presentation of the campaign themes).

Programmes for adults

I would now like to highlight a few of our programmes that target an adult audience.

Pregnancy and alcohol

In our concern to provide complete information about what "moderation" means
during pregnancy, we joined with the Quebec College of Physicians to publish
"Pregnancy and drinking: your questions answered," a brochure for women who are
pregnant or planning to have children. In simple, straightforward language, the
brochure answers all the questions women tend to ask, and it is now part of the
"pregnancy kit" that all Quebec doctors give to their pregnant patients. It deals with
drinking during pregnancy, the risk of foetal alcohol syndrome, the reasons why
drinking is not advised during pregnancy, and alcohol and the nursing mother.

5
However, it's important to put this publication in context, since a few years ago, the
Department of Health considered requiring all bottles of alcoholic beverages to bear a
label warning pregnant women of the risks of drinking.

We opposed this proposed policy for all the reasons on earth. However, we never take
a strictly "anti" position, and always seek a credible alternative to propose. Believing,
as we do, that foetal alcohol syndrome deserves to be discussed seriously and at
length, in quiet surroundings, we created a committee of experts that arrived at a
consensus on the issue, and then we produced the booklet.

After the first version, which was "tougher" and more conservative, we came out with
a more "liberal" version, since a few women were feeling guilty because they had
drunk a couple of glasses here and there and asked for abortions because they were
overly frightened by the possible consequences. This was a graphic demonstration of
how important it is to avoid scare, shock and horror tactics that, at the end of the day,
defeat the purpose.

The Minister of Health announced that warning labels would not be required on
alcoholic beverages. Our programme is still in force. And just a few months ago, the
Department of Health asked us if we would accept them as a partner and co-publisher
of the next edition of our booklet. But the debate on warning labels has strated once
again lately.

Alcochoix and P.CRA

The third adult-targeted programme is in the field of secondary prevention:
"Alcochoix" P.CRA translates as "Alcochoice/Reasonable Alcohol Consumption
Programme". It is designed to help people who have difficulty controlling their
drinking, but who are not alcoholics and do not want to stop drinking completely. The
results have been fantastic: more than 75% of those who followed the seven-course
programme had their drinking perfectly under control one year after their last session.
The programme has proved to be another way to promote the culture of taste, rather
than the culture of abstinence.

Bars and restaurants customers

To help fight the culture of drunkenness in bars and restaurants, we have created
ACES (Aware, Competent, Efficient Servers). This programme is designed to inform
the employees of licensed establishments of their legal and social obligations to their
clients. But it also gives them tips on how to recognise heavy drinkers, how to help
customers avoid drinking too much, how to deal with those who insist on having one
too many, and how to avoid potential trouble. Research has shown that servers can
play a key role in promoting the culture of taste; moreover, they can prevent traffic
injuries by up to 25% in some areas.

Hunters and fishers

The hunting and fishing crowd is a high-risk group, to say the least. Some
psychologists think that many in these groups consider fishing or hunting parties a
substitute for the old-time taverns where men used to go and drink far from their

6
wives' watchful eyes (women were not allowed in those men-only taverns, which
have since been outlawed). We therefore address this audience directly through
hunting and fishing magazines, writing humorous articles and "food for thought"
items in the publications where our ads appear. The message is direct. It suggests that
they'd better drink in moderation.

Mass communication
Communication and advertising campaigns designed to enlighten the general public
about drinking are another important aspect of our integrated approach to promoting
the culture of taste versus the culture of drunkenness. The campaigns add to the
general body of information. The message of balance and moderation is conveyed to
large segments of the population through radio, television, public appearances and
articles in large-circulation publications, as well as through billboards and posters.

The message, which is never preachy, reminds people that the real pleasure to be had
from drinking comes through moderation. All the communications convey a sense of
joie de vivre and balanced living, equating enjoyment with an enlightened approach to
drinking. On the other hand they also try to demonstrate that drunkenness can lead to
unpleasant consequences. In this way, the primary objective --- to teach young and
adult Quebecers how to incorporate responsible drinking and the culture of taste in
their lifestyle --- is achieved gradually.

Our latest ad campaign does just that with its three radio spots. The first is about a
client in a bar ordering "one too many". The two others show what can happen when
effect obliterates taste. All three are created with a sense of humour.

The magazine and TV campaigns are about stages and degrees in the drinking
process. They show how there is pleasure in moderation and how embarrassing
situations arise from alcohol abuse. All are in set in a "social" context.

And the results?

Your next question is probably: "Does it work?".

And the answer is: Yes. We believe we are on the right track. Our conviction comes
from surveys by independent research companies showing that Quebecers'
relationship to alcohol is growing healthier all the time. The duc'alcool slogan has
also reached an incredible peak of awareness: 98% of people in Quebec are familiar
with it --- thats more than any common proverb. Our impact has also been eloquently
confirmed by a study done by the Canadian Centre for Health Information and the
Addiction Research Foundation.

It shows that despite the fact that Quebecers are notorious bons vivants, that Quebec
has the most lenient liquor laws in all of Canada, that alcohol can be purchased in
hundreds upon hundreds of points of sale, seven days a week, and that young people
and adults are openly educated about the joys of moderate drinking, Quebecers have
fewer alcohol-related problems than people in any other province. And that's a fact,

7
according to an index that takes into account data on drinking, Highway Code
infractions, hospitalisation time and mortality rates.

Moreover, the most recent study, done among more than 7,800 students in 18
universities, has proved that even if Quebec students drink more overall and more of
them are drinking, they drink better and get less drunk than students elsewhere in
Canada. In terms of numbers, Quebec students rank first in Canada: 92.2% of them
drink. But they rank last in terms of the number of occasions in the last 6 months on
which they had more than 5 drinks: only 3.6.

Far be it from us to take all the credit for this state of affairs. At the very least,
however, we might admit that an approach promoting the culture of taste as opposed
to the culture of drunkenness actually works.

I could tell you so much more. But I think I'll take my own advice and end my
presentation here. With speeches as with drinking, moderation is always in good taste.

8
2

Update on alcohol research in 2001

Mack C. Mitchell

Alcoholic Beverage Medical Research Foundation, Carolinas Medical Center, Department of

Internal Medicine, P.O. Box 32861, Charlotte, NC 28232-2861, U.S.A. (e-mail:
[email protected])

Descriptors
Alcoholic beverage, drinking habits, medicine, research

SUMMARY
The last 20 years of research in the alcohol field has not only expanded our understanding of
the problems related to the excessive use of alcohol, but also has pointed out the potential
benefits associated with the moderate use of alcoholic beverages. The cardiovascular health
benefits of moderate consumption are no longer a subject of controversy within the medical
research community. They are an accepted fact. Most of the current research in the field of
moderate drinking is aimed at elucidating the mechanism underlying the observed effects or
at finding effects in other less common conditions such as diabetes, osteoporosis and stroke.
Although each of these is a major health problem for our aging population, the incidence of
each of these chronic diseases is much lower than that of coronary heart disease which
remains the leading cause of death in all Western countries.
With the last few years, several studies have shown that moderate drinkers are less likely to
develop type 2 diabetes mellitus (adult onset diabetes). This decrease in incidence persists
even after adjustment for many other variables. Since diabetes is a major risk factor for the
development not only of coronary heart disease, but also strokes and peripheral vascular
disease, this observation offers yet another potential mechanism by which moderate alcohol
consumption exerts its health benefits.
While the majority of those who drink alcoholic beverages enjoy them in moderation, a small
percentage develop problems related to consumption. Basic research using animal models and
clinical studies in those with and without alcohol problems are helping us understand why
these individuals differ from the majority of drinkers in the population. It is now clear that
many substances including alcohol trigger common neurochemical pathways in the brain.
Some of these pathways, such as the dopamine pathway, are responsible for the pleasurable
effects of alcohol. What remains a mystery is how the various pathways interact to produce a
modest degree of pleasure in most people, yet in others produce an insatiable desire for
amounts of alcohol that are potentially harmful. Within the last few years, scientists have
found clues as to which pathways are important and how to pursue the understanding of these
mechanisms.
Although there are no magic cures for problem drinking yet, many researchers feel that our
improved understanding of this area will lead to effective pharmacological therapy within the
next decade. This exciting possibility offers hope to those with problem drinking and also
helps the majority of moderate drinkers continue to enjoy the potential health benefits.
Neuester Bericht zur Alkoholforschung in 2001

Deskriptoren
Alkoholisches Getrnk, Forschung, Medizin, Trinkverhalten

ZUSAMMENFASSUNG
In den vergangenen 20 Jahren hat die Forschung im Bereich des Alkoholkonsums nicht nur
unsere Kenntnisse ber die Probleme in Zusammenhang mit dem bermigen Konsum von
Alkohol vergrert, sondern auch auf die potentiellen Vorteile eines gemigten Konsums
von alkoholischen Getrnken hingewiesen. Die Vorteile eines gemigten Alkoholkonsums
fr die Gesundheit von Herz und Kreislauf werden in der medizinischen Forschungs-
gemeinschaft heute nicht mehr bestritten. Sie sind akzeptierte Fakten. Der grte Teil der
laufenden Studien im Bereich des gemigten Alkoholkonsums widmet sich der Erforschung
des Mechanismus, der den observierten Folgen zugrunde liegt, oder der Suche nach
Auswirkungen auf andere, weniger hufig auftretende Leiden wie Diabetes, Osteoporose und
Schlaganfall. Obwohl jedes einzelnen von ihnen fr unsere lter werdende Bevlkerung ein
groes Gesundheitsproblem darstellt, ist die Hufigkeit, mit der diese chronischen
Erkrankungen auftreten um vieles geringer als die Verbreitung von koronaren
Herzkrankheiten, die in allen Lndern der westlichen Welt auch heute noch die hufigste
Todesursache darstellen.
In den letzten Jahren haben verschiedene Studien gezeigt, dass Diabetes mellitus Typ II
(Manifestation im Erwachsenenalter) bei gemigten Trinkern seltener auftritt. Diese
verringerte Hufigkeit besteht auch noch nach der Korrektur der Forschungsdaten hinsichtlich
zahlreicher anderer Variablen. Da Diabetes nicht nur im Hinblick auf das Entstehen einer
koronaren Herzkrankheit, sondern auch im Hinblick auf Schlaganflle und periphere
Geferkrankungen als Hauptrisikofaktor zu betrachten ist, lassen diese Observationen noch
einen weiteren potentiellen Mechanismus, durch den ein gemigter Alkoholkonsum
gesundheitliche Vorteile bewirkt, erkennen.
Whrend der grte Teil der Menschen, die alkoholische Getrnke zu sich nehmen, diese in
Maen genieen, entwickelt ein kleiner Prozentsatz Probleme in Zusammenhang mit dem
Konsum. Grundlagenforschung mit Versuchstieren und klinische Studien mit Personen mit
und ohne Alkoholproblemen helfen uns, zu verstehen, warum sich diese Personen von der
Mehrheit der Konsumenten innerhalb der Gesellschaft unterscheiden. Heute ist es klar, dass
zahlreiche Substanzen einschlielich Alkohol in zahlreichen neurochemischen Leitungs-
bahnen im Gehirn Impulse auslsen. Einige dieser Bahnen, wie beispielsweise die
Dopaminbahn, sind fr die angenehmen Folgen des Alkoholkonsums verantwortlich. Noch
nicht beantwortet ist die Frage, wie die Interaktionen zwischen den verschiedenen
Leitungsbahnen erfolgen, die bei den meisten Menschen ein leichtes Wohlbehagen bewirken,
jedoch bei einigen anderen einen unersttlichen Wunsch nach dem Konsum potentiell
schdlicher Alkoholmengen hervorrufen. In den letzten Jahren haben Wissenschaftler
Hinweise entdeckt, die erkennen lassen, welche Leitungsbahnen hier von Bedeutung sind und
wie das Verstndnis dieser Mechanismen vergrert werden kann.
Obwohl es fr problematischen Alkoholkonsum noch keine magischen Kuren gibt, sind
viele Wissenschaftler davon berzeugt, dass sich auf der Grundlage unseres gesteigerten
Wissens in diesem Bereich schon innerhalb des nchsten Jahrzehnts wirksame
Pharmakotherapien entwickeln lassen. Diese erfreuliche Mglichkeit bietet allen, die ein
Alkoholproblem haben, Hoffnung und hilft der groen Mehrheit der gemigten Trinker, sich
der potentiellen Gesundheitsvorteile zu erfreuen.

2
Mise jour des tudes sur l'alcool ralises au cours de l'anne 2001

Descripteurs
Boisson alcoolique, habitude en matire de consommation de boissons, mdecine, recherche

RESUME
Les tudes sur l'alcool effectues au cours de ces 20 dernires annes nous ont non seulement
permis de mieux comprendre les problmes lis la consommation excessive d'alcool, mais
nous ont galement aid relever les avantages potentiels associs un usage modr de
boissons alcoolises. Les avantages au niveau cardiovasculaire engendrs par une
consommation modre d'alcool ne sont plus un sujet controvers dans le secteur de la
recherche mdicale; ils sont devenus un fait admis de tous. La majeure partie des tudes en
cours sur la consommation modre d'alcool vise lucider le mcanisme qui est la base des
diffrents effets observs ou dcouvrir d'autres effets dans le cas de maladies moins
frquentes, telles que le diabte, l'ostoporose et les attaques. Bien que ces maladies
constituent un problme de sant de premier ordre pour notre population vieillissante, ces
affections chroniques sont nettement moins frquentes que les maladies coronariennes,
lesquelles restent l'une des principales causes de dcs dans tous les pays occidentaux.
Au cours de ces dernires annes, de nombreuses tudes ont permis de dmontrer que les
consommateurs modrs d'alcool ont moins de chances de dvelopper du diabte sucr de
type 2 (diabte non-insulinodpendant). Cette baisse de frquence persiste mme aprs
l'adaptation de plusieurs autres variables. Dans la mesure o le diabte est un facteur de risque
majeur pouvant non seulement engendrer des maladies coronariennes mais aussi des attaques
et des affections cardiovasculaires, cette observation met en vidence un mcanisme
supplmentaire dmontrant les effets bnfiques d'une consommation modre d'alcool.
Si la majeure partie des personnes buvant de l'alcool le fait avec modration, un faible
pourcentage connat certains problmes de consommation excessive. Les tudes de base
ralises sur des animaux et les tudes cliniques impliquant aussi bien des alcooliques que des
personnes non alcooliques nous aident mieux comprendre les raisons pour lesquelles ces
individus diffrent de la majeure partie des buveurs dans la population. Il est clair que bon
nombre de substances, dont l'alcool, dclenchent des transmissions neurochimiques standard
dans le cerveau. Certaines de ces transmissions, telles que celle de la dopamine, sont
responsables des effets agrables suscits par l'alcool. Mais ce que nous ne savons toujours
pas, c'est la manire dont ces diffrentes transmissions interagissent et provoquent chez la
plupart une agrable sensation de plaisir et chez certains une envie insatiable d'alcool, laquelle
peut s'avrer nocive pour la sant. Au cours de ces dernires annes, les scientifiques ont
dcouvert des indices, lesquels leur ont permis de connatre les transmissions les plus
importantes et de dterminer la manire dont ils vont poursuivre leur comprhension de ces
mcanismes.
Bien qu' ce jour il n'y ait aucun remde miracle contre les problmes d'alcoolisme, bon
nombre de chercheurs pensent qu'une meilleure intelligence du problme nous permettra de
trouver une thrapie pharmacologique efficace dans les dix annes venir. Cette nouvelle
redonne de l'espoir ceux qui souffrent d'alcoolisme et permet la majorit des buveurs
modrs de continuer leur consommation sans aucune crainte pour leur sant.

3
Update on Alcohol Research in
2001

Mack C. Mitchell, M.D.

The Alcoholic Beverage
Medical Research Foundation

Alcoholic Beverage
Medical Research Foundation

The largest, independent, non-profit

foundation in North America devoted
solely to supporting research on:
Effects of alcohol on health and behavior
Prevention of alcohol-related problems

Page
 Long Range Goal

To influence alcohol research to shift

from problem-oriented approach to
examination of how the whole
spectrum of use affects health and
behavior

All-Cause Mortality Physicians Health

Survey 2 n=22,071
1.8
1.6
1.4
1.2
RR 1
0.8
0.6
0.4
0.2
0
<1 1 2-4 5-6 7-13 14+
Drinks Per Week

Page
 PHS - Category of Mortality
2.2
n=22,071
2 Cancer
1.8
1.6
1.4
1.2
RR 1
0.8
Other
0.6
0.4
CVD
0.2
0
<1 1 2-4 5-6 7-13 14+
Drinks Per Week

Mechanisms of Effects of Alcohol

on Lower Risk of CAD

Alcohol increases serum level of HDL

Alcohol decreases serum fibrinogen
Alcohol increases serum TPA
Alcohol decreases platelet adhesion
Possible effects of antioxidants or other
minor components of beverages

Page
 Type 2 Diabetes and Vascular
Diseases

Prevalence of type 2 diabetes (adult onset) is

increasing in Western countries
Diabetes is a major risk factor for coronary
heart disease, stroke, peripheral vascular
disease, kidney failure and blindness
The mechanism by which diabetes
contributes to vascular disease is unproven,
but may be related to high levels of glucose
or insulin in type 2 diabetes

Incidence of Type 2 Diabetes in

Moderate Drinkers

Health Professionals Follow-up Study

reported RR of 0.61 (95% CI, 0.44-0.91) for
development of DM in moderate (1-2
drinks/day) drinkers (BMJ 310:560, 1995)
Nurses Health Study also found a lower risk
of type 2 DM in moderate drinkers (Am J
Epidemiology 128:549, 1988)
British Regional Heart Study reported a U-
shaped curve for risk of diabetes in 7577 men
followed for 12.8 years with RR of 0.64 for
moderate drinkers (BMJ 310:560, 1995)

Page
 Alcohol Consumption and
Risk of Type 2 Diabetes
1.2
1
Relative Risk

0.8
0.6
0.4
0.2
0
None <1 1 2--4 5--6 >7
Drinks/week

Physicians Health Study, Arch Int Med 160:1025 2000

Alcohol Intake and Type 2

Diabetes in Men
3
2.5
Odds Ratio

2
1.5
1
0.5
0
None 1--62 62--123 123--276 > 276
Alcohol intake (gm/wk)

Wei et al. Diabetes Care 23:18, 2000

Page
 Alcohol and Diabetes in 2000

Several studies confirmed the finding of a

lower incidence of type 2 diabetes in
moderate drinkers.
While the mechanism remains uncertain,
moderate alcohol intake may improve
sensitivity to insulin which in turn reduces
demand on the pancreas.
Lower insulin levels may be beneficial in
reducing risks of cardiovascular disease.

Alcohol Intake and Risk of Stroke

Cerebrovascular accidents (stroke) are

dividied into two broad categories: ischemic
(thrombotic) and hemorrhagic
Previous studies have indicated a decrease
in the risk of ischemic stroke in moderate
drinkers
Some studies have suggested that the risk
of hemorrhagic stroke may be higher in
heavy drinkers

Page
 Alcohol Intake and Risk of Stroke

1.4
1.2
Relative Risk

1
Total
0.8
Ischemic
0.6
Hemorrhagic
0.4
0.2
0
<1 1 2--4 5--6 >7
Drinks/week

Physicians Health Study, NEJM 341:1557,

1999

Alcohol Intake and Risk of Stroke

In Young Women
Case control study of stroke in women 15-44
years of age
Alcohol intake up to 24 gm/day was
associated with a reduced risk of ischemic
stroke, Odds ratio .38 (95% CI 0.17-0.86)

Malarcher, et al. Stroke 32:77, 2001

Page
 Variance of Risk for Alcoholism

Environment
40%

Genetics
60%

(Heterogeneous and Polygenic)

Page
 Harmful Alcohol Drinking
140,000 deaths annually
44% fatal traffic; 15-63% fall fatalities
~50% violence incidents
Medical, psychological costs
Prevalence: 10-15% lifetime alcohol
abuse/dependence
Question: Why do some people continue to
drink at hazardous levels? What factors
determine the development of alcohol craving
and dependence?

Overall population estimates for

binge drinking

Dawson (1999): Frequency (past year)

of 5+ drinks/occasion (adults):

Never 44%
1-2 times 15.1%
3-11 times 14.7%
1-3x/month 13.5%
>1x/week 12.3%

Page
 Early Stages of Heavy
Social Drinking
Emerges in late teens/early 20s
Binge: 44% college students:
52% of men and 31% of women
~2/3 of problem drinkers mature out during
their 20s
Hasin (1990): Alcohol Abuse follow-up
47% remit
24% Alcohol Abuse
30% Alcohol Dependence

Prevailing Theories on Risk for

Harmful Drinking

Predominant results/theory:
**Low level response to alcohol in FH+
10-year follow-up: low response in lab study
predicted alcohol problems in adulthood
(Schuckit, 1994)
Meta-analysis (11 studies): sons of alcoholics
less sensitivity to alcohol during rising &
declining phase of blood alcohol concentration
(Pollock, 1992)

Page
 Sensitization

Alcohol
intake
Tolerance

Differentiator Model -- response to alcohol in

At-Risk (FH+) persons over the BAC curve

Molecular Mechanisms in Alcohol

Dependence
Dependence develops slowly over years
At-risk individuals appear to experience
sensitization at low levels of alcohol that
lead to uncontrolled use followed by the
development of tolerance requiring greater
amounts of alcohol to produce a desired
effect
Dopamine, a pleasure producing neuro-
transmitter, is released in response to alcohol
and may contribute to craving in alcoholics

Page
 Effects of Alcohol on
Neurotransmitter Systems
Many neurotransmitter systems are
affected by alcohol
Glutamate and GABA are involved in
sensitization, whereas the rewarding
properties of alcohol may be mediated
through dopamine
Serotonin plays a role in mediating stress
in CNS
Endogenous opiates are thought to
provide reinforcement

Mesocorticolimbic Dopamine System

Dopamine
GABA

Hippo

Caudate
MPC

VTA
NAcc VP AMG

Page
 Ethanol Specifically
Targets the GABA
Receptor-Chloride
Channel Complex

Microdialysis in Nucleus Accumbens

Cannula Placement and Headstage

Page
Enhanced GABAA Receptor Function by
Pharmacological Inhibition of PKC

300
(% above muscimol) 250 *
Cl uptake

200
150 Not Developmental
Compensation
-

100
36

50
0
C Sap V SV

No Ethanol-Induced Dopamine Release in N.

Accumbens of PKC Knockout Mice
Dopamine (% Baseline)

Basal Saline EtOH (2 g/kg)

*
300

200 * Wildtype
Knockout

100

0
0 100 200 300 400
Time (min)

Page
Potential Mechanisms for Lack of
DA Release
Altered DA Transporter Function
Rapid reuptake?

Inability to Release DA

PKCe Modulates Alcohol Sensitivity,

Self-administration, and Relapse via
GABAergic and Dopaminergic
Mechanisms

Supersensitive
GABAA Receptor
Blocked DA
Absence Reduced Alcohol
Release
of PKC Self-Admin
after Ethanol
Elevated Taurine

Page
 Chronic alcohol use
Stress
Sensitization
(mediated by
serotonin)
Changes in
neurotransmitters
Initial (opiates, dopamine, Prolonged
abstinence glutamate, GABA) abstinence

Reward memory
Withdrawal
Craving

Relapse

Anton,R. Alcohol Research and Health 1999

Page
 ABMRF Achievements

Deeper insights into why most people are

able to drink alcohol in moderation and why
others drink to excess
Understanding many factors influencing the
development of alcohol-use problems in
youth
Continued exploration of the health and
behavioral effects of moderate alcohol use

A Unique Partnership:
Industry and Academia
ABMRF was established in 1982 with
support of the brewing industries of US
and Canada
Over 500 scientists have received ABMRF
support in the last 20 years
The potential for greater international
cooperation in funding alcohol research is
substantial

Page
 Alcohols Mixed Messages

In the scientific world, the debate

centers on whether moderate
consumption is good or bad for your
health. But in the world of conflicting
business, social and political
interests, the crucial issue is who will
get to say what about alcohol.
Shari Roan, Los Angeles Times June 15, 1993

Page
PUBLICATIONS

1. Mitchell MC, Williams GM. Platelet mixed agglutination: A sensitive method

for detecting HL-A and non HL-A antigens. Surgical Forum 25:194-196, 1974.

2. Mitchell MC, Arregui A, Boitnott J, Maddrey WC. Granulomatous hepatitis

with carbamazepine therapy. Amer. J. Med. 71:733-735, 1981.

3. Mitchell MC, Mezey E, Maddrey WC. Effects of variation in dietary protein

and ethanol on hepatic microsomal drug metabolism in the rat. Hepatology
1:336-340, 1981.

4. Mitchell MC, Schenker S, Avant GR, Speeg KV. Cimetidine protects against
acetaminophen hepatotoxicity in the rat. Gastroenterology 81:1052-1060, 1981.

5. Speeg KV, Patwardhan RV, Avant GR, Mitchell MC, Schenker S. Inhibition of
microsomal drug metabolism by histamine H2-receptor antagonists studied in
vivo and in vitro in rodents. Gastroenterology 82:89-96, 1982.

6. Mitchell MC, Hoyumpa A, Schenker S, Patwardhan RV. Differential effects of

chronic ethanol feeding on cytochrome P-448 and P450 mediated drug
metabolism in the rat. Biochem. Pharmacol. 31:695-699, 1982.

7. Mitchell MC, Boitnott J, Kauffman S, Cameron JL, Maddrey WC. Hepatic vein
occlusion: The Budd-Chiari syndrome. Medicine 61:199-218, 1982.

8. Patwardhan RV, Mitchell MC, Johnson RF, Schenker S. Differential effects of

oral contraceptive steroids on the metabolism of benzodiazepines. Hepatology
3:248-253, 1983.

9. Mitchell MC, Hoyumpa AM, Schenker S, Johnson RF, Nichols S, Patwardhan

RV. Inhibition of caffeine elimination by short-term ethanol administration. J.
Lab. Clin. Med. 101:826-834, 1983.

10. Mitchell MC, Hainew T, Meredith CM, Schenker S. Increased plasma

elimination of acetaminophen in women receiving oral contraceptive steroids.
Clin. Pharmacol. Ther. 34:48-53, 1983.

11. Mitchell MC, Schenker S, Speeg KV Jr. Selective inhibition of acetaminophen

oxidation and toxicity by cimetidine and other Histamine H2-receptor
antagonists in vivo and in vitro in the rat and in man. J. Clin. Invest. 73:383-
391, 1984.

12. Morton S, Mitchell MC. The effects of chronic ethanol feeding on glutathione
turnover in the rat . Biochem. Pharmacol. 34:1559-1563, 1985.

13. Mitchell MC. Alcohol-induced impairment of central nervous system function:

behavioral skills involved in driving. J. Stud. Alcohol Supplement 10:S109-
S116, 1985.

4
14. Speeg KV, Mitchell MC, Maldonado AL. Additive protection of cimetidine
and N-acetylcysteine treatment against acetaminophen induced hepatic necrosis
in the rat. J. Pharmacol. Exp. Ther. 234:550-554, 1985.

15. Pierson J, Mitchell MC. Increased hepatic efflux of glutathione after chronic
ethanol feeding. Biochem. Pharmacol. 35:1533-1537, 1986.

16. Mitchell MC, Herlong HF. Alcohol and nutrition: Caloric value, bioenergetics,
and relationship to liver damage. Ann. Rev. Nutr. 6:457-474, 1986.

17. Diehl AM, Mitchell MC, Herlong HF, Potter JJ, Wacker LS, Mezey E.
Changes in plasma amino acids during sobriety in alcoholic patients with and
without liver disease. Am. J. Clin. Nutr. 44:453-460, 1986.

18. Callans DJ, Wacker LS, Mitchell MC. Effects of ethanol feeding and
withdrawal on plasma glutathione elimination in the rat. Hepatology 7:496-501,
1987.

19. Scott R, Mitchell MC. Alcohol and aging. J. Am. Geriatric Soc. 36:255-265,
1988.

20. Mezey E, Kolman CJ, Diehl AM, Mitchell MC, Herlong HF. Alcohol and
dietary intake in the development of chronic pancreatitis and liver disease in
alcoholism. Am. J. Clin. Nutr. 48:148-151, 1988.

21. Mitchell MC, Hall SD, Schenker S, Branch RA. Impaired hepatic elimination
of paranitrophenol and its metabolites in the rat following chronic ethanol
pretreatment. Alcoholism: Clin. Exp. Res. 13:264-270, 1989.

22. Ambrosio G, Jacobus WE, Mitchell MC, Litt MR, Becker LC. Effects of ATP
precursor administration on ATP and "free" ADP content and on recovery of
contractility in post-ischemic stunned hearts. Am. J. Physiol. 256:H560-6,
1989.

23. Mitchell MC, Hamilton R, Wacker LS, Branch RA. Zonal distribution of
paracetamol glucuronidation in the isolated perfused rat liver. Xenobiotica
19:389-400, 1989.

24. Burdick JF, Colombani PM, Pitt HA, Perler BA, Merritt WT, Crandall BC,
Mitchell MC, Herlong HF and Williams GM. Overcoming early cyclosporine
nephrotoxitity after liver transplantation. Transplantation Proceedings 21:
2236-2237, 1989.

25. Sitzman JV, Bulkley G, Mitchell MC, Campbell K. Role of prostacyclin in the
splanchnic hyperemia contributing to portal hypertension. Ann. Surg. 209322-
7, 1989.

26. Litt M, Potter JJ, Mezey E, Mitchell MC. Analysis of pyridine dinucleotides in
cultured rat hepatocytes by high-performance liquid chromatography. Anal.
Biochem. 179:34-6, 1989.

5
27. Mullin GE, Greenson JK, Mitchell MC. Fulminant hepatic failure after
ingestion of sustained release nicotinic acid. Ann. Int. Med. 111:253-255,
1989.

28. Felver ME, Mezey E, McGuire M, Mitchell MC, Herlong HF, Veech GA,
Veech RL. Plasma tumor necrosis factor predicts decreased long-term survival
in severe alcoholic hepatitis. Alcoholism: Clin. & Exp. Res. 14:255-59, 1990.

29. Mitchell MC, Mallat A, Lipsky JL. Cephalosporin-induced alteration in hepatic

glutathione redox state. A potential mechanism for inhibition of hepatic
reduction of vitamin K1, 2,3-epoxide in the rat. J. Clin. Invest. 86:1589-1594,
1990.

30. Buescher PC, Pearse DB, Pilla RP, Litt MR, Mitchell MC, Sylvester JT.
Energy state and vasomotor tone in hypoxic pig lungs. J. Appl. Physiol.
70:1874-1881, 1991.

31. Raiford DS, Sciuto AM, Mitchell MC. Effects of hormones and protein kinase
C agonists/antagonists on efflux of glutathione and permeability of tight
junctions in the perfused rat liver. Am. J. Physiol. 261: G578-84, 1991.

32. Mezey E, Caballeria J, Mitchell MC, Pares A, Herlong HF, Rodes J. Effect of
parenteral amino acid supplementation on short-term and long-term outcomes in
severe alcoholic hepatitis: A randomized controlled trial. Hepatology 14:1090-
96, 1991.

33. Blumenthal RS, Flinn IW, Proske O, Jackson DG, Tena RG, Mitchell MC and
Feldman AM. Effects of chronic ethanol exposure on cardiac receptor-adenyl
cyclase coupling: studies in cultured chick myocytes and ethanol fed rats.
Alcoholism: Clin. Exp. Res. 15:1077-1083, 1991.

34. Klein AS, Savader S, Burdick JF, Fair J, Mitchell M, Colombani P, Perler B,
Osterman F and Williams GM. Reduction of morbidity and mortality from
biliary complications after liver transplantation. Hepatology 14:818-23, 1991.

35. Westra WH, Hruban RH, Bauthman KL, Olson JL, Porterfield JK, Mitchell
MC and Hutchins GM. Progressive hemochromatotic cardiomyopathy despite
reversal of iron depostion after liver transplantation. Am J Clin Path 99:39-44,
1993.

36. Risby TH, Maley W, Scott RPW, Bulkley GB, Kazui M, Sehnert SS, Schwarz
KB, Potter J, Mezey E, Klein AS, Comombani P, Fair J, Merritt WT, Beattie C,
Mitchell MC, Williams GM, Perler BA, Donham RT and Burdick JF. Evidence
for free radical-mediated lipid peroxidation at reperfusion of human orthotopic
liver transplants. Surgery 115:94-101, 1994.

6
37. Venbrux AC, Mitchell SE, Savader SJ, Lund GB, Trerotola SO, Newman JS,
Klein AS, Mitchell MC, Rosch J, Uchida BT and Osterman FA, Jr. Long-term
results with the use of metallic stents in the inferior vena cava for treatment of
Budd-Chiari syndrome. J. Vascular Interventional Radiology 5:411-416, 1994.

38. Dienstag JL, Schiff ER, Mitchell MC., et al. Extended lamivudine retreatment
for chronic hepatitis B: Maintenance of viral suppression after discontinuation
of therapy. Hepatology 30 (4): 1082-87, 1999.

7
3

Beer: food and drink?

Renton Righelato
Ashbourne Biosciences, 63 Hamilton Road, Reading RG1 5RA, United Kingdom (e-mail:
[email protected])

Descriptors
Beer, health, metabolism, nutritive value

SUMMARY
Most of us eat and drink primarily for pleasure, giving little thought to the quantity and balance of
nutrients in our diets, particularly so when drinking. Nonetheless, beers, more so than most other
alcoholic drinks, can contribute significantly to consumption of a wide range of micronutrients
and to dietary energy. The metabolisable energy of beers, usually largely as ethanol, can be a
significant contribution to total dietary energy. The micronutrients include many of the B
vitamins and essential minerals. In addition, there are many phytochemicals arising from malt and
hops with antioxidant, oestrogen-mimic and other properties whose dietary significance is still
speculative. This paper will review the nutritional properties of beers and consider the
contribution of moderate consumption patterns to dietary intakes in the context of the diet as a
whole.

Bier: Essen und Trinken?

Deskriptoren
Bier, Gesundheit, Stoffwechsel, Nhrwert

ZUSAMMENFASSUNG
Die meisten von uns essen und trinken in erster Linie zum Vergngen. Wir denken wenig ber
die Menge und die ausgeglichene Zusammensetzung der Nhrstoffe in unserer Kost nach. Ebenso
verhlt es sich mit den Getrnken, die wir zu uns nehmen. Dennoch kann Bier, mehr als andere
alkoholische Getrnke, zur Deckung einer Vielzahl von Spurenelementen und dem tglichen
Energiebedarf beitragen. Die verwertbare Energie des Bieres, grtenteils aus dem Ethanol
stammend, kann einen bedeutenden Anteil der Nahrungsenergie ausmachen. Die Spurenelemente
beinhalten viele der B-Vitamine und essenzielle Mineralstoffe. Bier enthlt zustzlichviele
Pflanzeninhaltsstoffe aus dem Malz und dem Hopfen mit anti-oxidaktiven und strogenhnlichen
Eigenschaften. Deren ernhrungsphysiologische Eigenschaften sind spekulativ. Diese
Verffentlichung gibt einen berblick ber die ernhrungs-physiologische Beschaffenheit des
Bieres. Dabei wird ein moderater Bierkonsum im Zusammenhang mit der gesamten Ernhrung
betrachtet.
La bire : boisson et aliment?

Descripteurs
Bire, mtabolisme, sant, valeur nutritive

RESUME
Dans nos pays, la plupart des gens mangent et boivent essentiellement pour le plaisir et pensent
peu la qualit et l'quilibre des nutriments dans l'alimentation, surtout dans la boisson. Or les
bires, plus que la plupart des autres boissons alcoolises, peuvent contribuer significativement
la consommation d'un large ventail de micronutriments et l'nergie dittique. L'nergie
mtabolisable des bires, qui vaut largement celle de l'thanol, peut representer une part
significative de l'nergie dittique totale. Les micronutriments sont plusieurs vitamines du
groupe B et des sels minraux. La bire contient en outre de nombreux composs
phytochimiques, issus du malt et des houblons, dont les proprits antioxydantes,
estrognomimtiques et autres restent largement tudier. Cette confrence fera le point sur les
proprits nutritionnelles des bires et voquera le rle possible d'une consommation modre
dans le contexte gnral d'une nutrition quilibre.

2
On the whole, in Europe and the richer western world, most of us eat and drink for
pleasure, not for food at least consciously. This pleasure though, has nutritional and
health consequences. Some arise from the consumption of alcohol, the main energy
source in beer, which, like most nutrients can beneficial in moderation but is damaging in
excess. Leaving aside the alcohol issues, this paper discusses beer in the context of what
we eat as a whole, looking at it as a food, competing with others for our mouths and
stomachs and interacting with others to create good or bad diets.

Most beers are fundamentally extracts of malted barley and contain most of the nutrients
that can be solubilised with hot water and the malt enzymes (Walker & Baxter 2000). So
beers contain most of the micronutrients and energy of the grain.

DIETARY ENERGY
Low in protein and virtually fat-free, the macronutrients in beer are predominantly
sources of energy (table 1). The energy comes mainly from the alcohol: ascribing a value
of 28 kJ/g (7 kcal/g), ethanol contributes two thirds of the energy in a normal beer.
Under current European labeling regulations, there is no provision for alcohol in the
macronutrient declaration. The carbohydrate contributes most of the remainder to
calculation of energy on the label.

Table 1: Nutritional label information for a typical beer

per litre per 25 cl
Energy 1600 MJ/380 kcal 400kJ/95 kcal
Protein 4g 1g
Carbohydrate 27g 7g
of which sugars 4g 1g
Ethanol 37g 9g
Fat Trace Trace
Fibre 3g 0.7g
Sodium 0.04g 0.01g

Imbalances in dietary energy, are not rare: obesity is arguably the most serious diet-
related problem in the western world, because it is associated with many disease states.
Quite small imbalances between consumption and expenditure of energy can result in
becoming overweight. 1.6 MJ (the caloric value of 1 litre of beer) above the energy need
of an individual could result in weight gain of around a kilogram per month. However,
although obesity is common, the majority of people do not become overweight, hence the
interest in the mechanisms of appetite control and caloric compensation.

The majority of studies suggest that additional calories from alcohol are not compensated
by reduction in other food intake (reviewed by Westerterp-Plantenga & Verwegen, 1999).
These authors found that the same amounts of food were consumed at lunch following
aperitifs of alcohol (beer or wine) or water, ie the energy from the alcohol was not
compensated for by eating less at the meal. By contrast, aperitif drinks with carbohydrate,

3
protein or fat as their main energy source did result in a compensatory reduction of food
eaten at the meal.

Paradoxically, there is little evidence for an epidemiological correlation between body

mass index and alcohol consumption (Gruchow et al, 1985; Colditz et al, 1991). The
failure of short term eating behaviour studies to reflect this may be due to the short
observation periods (hours or days), compared with the timescales of other factors, social
and physiological, which determine eating behaviour, or to other changes in the
macronutrient composition of the diet (Colditz et al 1991), or to changes in body
composition (Kromhout, 1983). BMI may be an inappropriate measure, as there is some
evidence from the Zutphen study that body fatness is correlated with alcohol
consumption (Kromhout, 1983).

EMPTY CALORIES?
No single food contains all the nutrients we need we eat and drink a mixture of foods
that provide, we hope, a balanced diet, in which the deficiencies of specific nutrients in
one food are made up for by surpluses in others. Because the amount of energy we can
consume without incurring the health risks of obesity fixes the safe limit of total food, the
amount of energy we have to consume to get the other nutrients we need is a useful
measure of the quality of our diet. Hence the concept of empty calories sometimes
applied to foods or drinks providing little other than energy.

The content of minerals and vitamins in beers varies widely and for tables 2, 3, 4 median
values were taken, combining data from several sources (Piendl, 1981; Piendl,1990;
Piendl, 1992; Devreux 1986, Walker and Baxter 2000). Beer is rich in magnesium
compared with other sources of food and a good source of potassium, but deficient in
iron, calcium and zinc (table 2). It may also be a significant contributor of other essential
minerals whose role is less clear, for example silicon (Walker & Baxter, 2000). However,
alcohol is a diuretic and as such can cause a loss of minerals.

Beer is a good source of many of the water-soluble vitamins, notably pyridoxin,

riboflavin, niacin, folate and pantothenate (table 3) and, as a vitamin source, beer
compares well in nutrient density with cereals and meat. However, the fat-soluble
vitamins are virtually absent, those present in the malt remaining with the spent grains.

Table 2: Minerals density of selected foods (mg/MJ) *

RNI/MJ Beer Cereals Meat Fruit &Veg
Potassium 350 300 136 310 850
Magnesium 30 59 29 22 53
Iron 0.9 0.3 2.0 1.1 1.8
Calcium 70 23 80 26 62
Zinc 1 0.3 0.8 2.1 1.0
Consumption MJ 8.4 1.6(1 litre) 2.8 1.1 1.1
*National Food Survey 1999 (UK)

4
Table 3: Vitamin densities of selected foods (mg or g per MJ)
PRI/ Beer Cereals Meat Fruits &
MJ Vegetables

Consumption MJ 8.4 1.6(1 litre) 2.8 1.1 1.1

B1: thiamin mg 0.09 0.02 0.23 0.20 0.35

B6: pyridoxine mg 0.14 0.28 0.17 0.40 0.81
Niacin mg 1.8 3.44 2.36 9.1 2.9
B2: riboflavin mg 0.13 0.14 0.14 0.23 0.14
Folate g 20 31 29 13 93
B12: cyanocobalamin g 0.15 0 0.08 1.4 0
Biotin mg 0.02 0.01
Pantothenate mg 0.60 0.81
C: ascorbic acid mg 4.0 0.71 Tr 52
E: -tocopherol mg 0.60 0 0.43 0.36 2.4
A:retinal equivalent mg 0.07 0 0.021 0.15 0.24
D: g 0.60 0 0.21 0.59 0.02

Table 4: UK consumption of micronutrients % PRI

Micronutrient % PRI in UK average diet2 Contribution of 1 litre beer: % PRI 1

Potassium 81 13
Magnesium 84 29
Zinc 85 1
A: retinol equivalent 121 0
Iron 122 1
Calcium 127 5
Folate 131 25
B2: riboflavin 141 17
B6: pyridoxine 150 32
Niacin 157 31
B1: thiamin 162 3
C: ascorbic acid 170 0
E: -tocopherol 178 0
B12: cyanocobalamin 500 0
Biotin N/a 0.07
Pantothenate N/a 0.22
1
1 litre beer contributes about 17% of energy for average male consumer
2
National Food Survey 1999, UK household consumption plus an allowance of 10%
additional intake from food taken outside the home.

The potential value to a healthy diet from the hypothetical litre of beer depends on how
much of these micronutrients is consumed from other sources. In the average UK diet the
minerals in table 4 are at or below the reference nutrient intake (RNI) and some of the

5
vitamins are close to the population reference intake (PRI): the UK RNI (UK National
Food Survey, 1999) and European PRI (Mathioudakis, 2001) are the amounts of a
nutrient that will meet the needs of 95% of the population; the figure used here is that for
adult males. In these cases the contribution from beers could be significant, particularly
of magnesium, folate, pyridoxine and riboflavin.

This analysis takes no account of differences in bio-availability from different foods that
could favour assimilation of vitamins from dissolved nutrients from drinks and cooked
foods. The data suggest firstly that deficiencies in the vitamins and minerals in the UK
are unusual, secondly, that beer, like bread or meat, is poor source of some vitamins and
minerals, but a good source of many others and hence should not be regarded as empty
calories.

ANTIOXIDANT ACTIVITIES
The antioxidant properties of plant foods have been linked to the strong epidemiological
evidence of the protective effects of fruit and vegetables against cancers and heart
disease. Attention has focussed on the lipid soluble antioxidants, eg vitamin E and the
carotenoids (eg Zeigler 1991), and, more recently, the polyphenols (eg Hertog et al 1995).

Although beer is low in the antioxidant vitamins C and E, it has high levels of other
antioxidants. Beers are good sources of polyphenols, containing up to around 1g/l, levels
comparable with fruit juices, teas and wines (Vinson et al 1999; Gorinstein et al 2000).
Many of the beer polyphenols are much more potent as antioxidants in tests as inhibitors
of oxidation of LDL+VLDL lipoproteins than the well-known antioxidant vitamins:
ascorbic acid (C), -tocopherol (E) and -carotene (Vinson et al 1995). However,
preparations of beer polyphenols were not as effective as teas and wines in another
model, looking at lipoprotein-bound activity (Vinson et al 1999).

These in vitro tests may be construed as supportive of a physiological role, but in vivo
demonstration is still awaited. There have been some clinical studies that give further
support. A study by a team at Guys Hospital in London and Brewing research
International (Walker and Baxter 2000; Bourne et al 2000) showed rapid, high bio-
availability of ferulic acid and other phenolic antioxidants from beer.

This study used excretion in urine as indicating bioavailability of the antioxidants. There
is little evidence of increased circulating antioxidant levels. Ghiselli et al (2000) saw an
increase in total antioxidant activity in the blood after drinking 500 ml lager, falling back
to baseline after two hours. Van der Gaag et al (2000) found no increase in antioxidants
after three weeks of moderate consumption of beers, wines or spirits (40 g/day alcohol).

With the present state of knowledge, it is difficult to draw clear conclusions on the
relative merits of phenolic antioxidants from different foods and drinks. One can perhaps
conclude that beer, along with other plant-derived drinks, provides a range of compounds
that are readily bioavailable and have potent antioxidant properties. It is reasonable to
speculate that these are protective, though direct evidence is yet to be demonstrated.

6
DIETARY FIBRE
There is a wide range of benefits ascribed to fibre in the diet, both soluble and insoluble
and it is generally accepted that levels are suboptimal in most western diets (for review
see Krichevsky and Bonfield 1995).

Beer contains a complex mixture of carbohydrates, arising mostly from malt, that are not
fermented by the yeast. These include higher malto-oligosaccharides (Schur and Piendl,
1977; Piendl 1981). Piendl (1981) reported an average of 27 g/l of sugars (up to DP 17)
for a range of Venezuelan lagers. Piendl and co-workers reported dietary fibre levels in
German beers up to 6 g/l (Gromes et al 2000) and carbohydrate averaging 27 g/l (Piendl,
1981). The composition of the dietary fibre is not clear, but it probably comprises some
of the limit dextrins, -glucans, arabinoxylans and traces of yeast cell wall-derived
carbohydrates. Han and Schwartz (1996) found the major non-starch polysaccharides in
beers to be arabinoxylans at 3 g/l.

The contribution of non-starch polysaccharide (or dietary fibre) to the diet from a litre of
beer would be between around 2 g (average of data of Gromes et al) and 4 g/l (Han &
Schwartz, 1996), equivalent to between 17 and 30% of the UK daily intake (13 g) (UK
National Food Survey, 1999). Since fibre consumption in many countries falls well short
of the minimum intake (dietary reference value) of 18 g recommended by the UK
government advisory committee (Department of Health Report). Fruit and vegetables are
the main source of dietary fibre. Surprisingly, though, based on the published data, beer
compares well with cereal products in fibre content (table 5), so the possible fibre
contribution of beers (and malt drinks) may be worth examining further.

Table 5: Contribution of foods to fibre consumption (NFS)

Total RDA Beer Cereals Meat Fruits &
diet (1 litre) Vegetables
Fibre g/MJ 1.3 1.8 3 2.1 trace 5.7
Consumption MJ 10 1.6 2.8 1.1 1.1

Dietary fibre in beer is soluble fibre; probably largely the partially hydrolysed cell wall
polysaccharides of barley. These, together with some of limit dextrins remaining in the
beer, are likely to escape absorption in the small intestine and become available as
nutrients for the bacteria in the large bowel. The importance of the large bowel microflora
to gut function and health has become increasingly evident over the last decade as a result
of work on probiotics and prebiotics.

Probiotics are micro-organisms, usually lactobacilli and bifidobacteria, added to the diet
that grow in the large bowel. Probiotic bacteria are added to foods such as yoghurts and
fruit juices and have found substantial markets (Young, 1998). It has usually been found
that regular doses of the probiotic are needed to maintain their presence in the bowel.

Prebiotics are nutrients that favour the growth of the desired large bowel bacteria. They
are oligosaccharides that are not absorbed in the small intestine and so available to the
large bowel flora and may selectively favour the growth of the organisms thought to be

7
beneficial (Roberfroid, 2001; Gibson, 1999). Some oligosaccharides, in addition, exclude
pathogenic organisms by interfering with their binding to the gut epithelia directly
(Pustzai et al, 1990) or by stimulating the growth of probiotic organisms.

Although most recent research has been on the commercial fructo-oligosaccharide

products, many other carbohydrates have been shown to have prebiotic effects (Rebuk et
al 1995). Prebiotics are sometimes, though not necessarily, used in conjunction with
probiotic inocula.

There is no published work on the prebiotic function of oligosaccharides in beer, though

the flatulence that many people experience after drinking beer may be circumstantial
evidence of an effect. Given the health benefits associated with prebiotics and the public
interest in the subject, this may be an area for future research. In vitro models have been
developed that can be used to screen the effects of sugars added to simulated gut streams
and in vivo tests of the effects of different foods and drinks on human gut flora are
available (eg Gibson 1999; Menne et al, 2000).

CONCLUSIONS
As a food, beers, at quite moderate levels of consumption, can be significant sources of
energy. Although there appears not to be a short-term reduction of other food
consumption to compensate for beer consumption, there must be longer-term control as a
correlation between alcohol consumption and body mass is not generally observed.

Beer is undoubtedly a good source of many vitamins and minerals and can enrich the diet
in several of the B vitamins and folic acid. Although beer does not contain the classical
antioxidant vitamins, it is an excellent source of bioavailable phenolic antioxidants.
Whether these are important physiologically remains to be established.

Lastly, it beer may be a significant source of soluble dietary fibre. It may be speculated
that the oligosaccharides and non starch polysaccharides in beers would stimulate the
development of a beneficial bowel microflora.

Are these issues important? People do not drink a beer as a medicine, they drink it
because they like it. But there is a strong and growing interest in nutrition from
increasingly well-informed consumers, and such knowledge conditions liking. So, the
physiological effects of the huge range of compounds from malt and hops, interests many
consumers. And in our enthusiasm, perhaps sometimes we are guilty of treating some of
these compounds as elixirs or we invoke them to explain tenuous differences in health
effects that are sometimes reported for different types of fermented drinks.

I believe that it is important to understand the nutritional effects of our foods and drinks
but we should beware magic bullet hypotheses. They may make for good press, but not
necessarily for good nutrition. We simply should regard, beer as most other foods, food,
as something that is enjoyable and that, drunk sensibly, can contribute to a sound diet.

8
REFERENCES
1. Bourne L, Paganga, G., Baxter, D., Hughes, P. & Rice Evans, C., Free Radical Research,
2000, 32, 273-280.
2. Department of Health Report on Health and Social Subjects 41: Dietary Reference Values
for Food Energy and Nutrients for the United Kingdom. HMSO ISBN No 0-11-321397-2).
3. Colditz, G.A., Giovannucci, E., Rimm, E.B., Stampfer, M.J., Rosner, B. Speizer, F.E.,
Gordis, E. & Willett, W.C., American Journal of Clinical Nutrition, 1991, 54, 49-55.
4. Devreux, A., 1986, Birra e Malto, 29
5. Ghiselli, A., Natella, F., Guidi, A., Montanari, L., Fantozzi, P. & Scaccini, C., Journal of
Nutritional Biochemistry, 2000, 11, 76-80
6. Gibson, G.R., Journal of Nutrition, 1999, 129, 1438S-41S
7. Gorinstein, S., Caspi, A., Zemser, M. & Trakhtenberg, S., Nutrition Research, 2000, 20,
131-139
8. Gromes, R., Zeuch, M. and Piendl, A, 2000, Brauwelt International, 18, 24-27
9. Gruchow, H.W., Sobocinski, K.A., Barboriac, J.J. & Sheller, J.G., American Journal of
Clinical Nutrition, 1985, 42, 289-295.
10. Han, J.Y., & Schwarz, P.B., J.American.Society of Brewing Chemists, 1996, 54, 216-220.
11. Hertog, M.G.L., Kromhout, D., Aravanis C., Blackburn, H., Buzina, R., Fidanza, F.,
Giampaoli, S., Jansen, A., Menotti, A., Nedeljkovic, S., Pekkarinen, M., Simic, B.S.,
Toshima, H., Feskens, E.J.M., Hollman, P.C.H., Katan, M.B., Archives International
Medicine, 1995, 155, 381-386.
12. Krichevsky, D. & Bonfield, C., eds: Dietary Fiber in Health and Disease, 1995, St Paul,
Minnesota, USA, Eagan Press
13. Kromhout, D., American Journal of Clinical Nutrition, 1983, 37, 295-299.
14. Mathioudakis, B., in Walter, P, Hornig, D & Moser, U, eds: Functions of Vitamins beyond
Recommended Dietary Allowances, Karger, Basel, 2001, 14-21.
15. Menne, E., Guggenbuhl, N. & Roberfroid, M., Journal of Nutrition, 2000, 130, 1197-1199.
16. National Food Survey 1999, ww.maff.gov.uk/esg/work_htm/publications/cf/nfs.htm
17. Piendl A., Brewers Digest, January 1981, 32-45
18. Piendl A., Brauindustrie, 1990, 12, 1383-85
19. Piendl A., Brauindustrie, 1992, 10, 949-51.
20. Pustzai, A., Hinton, M.H. & Mulder R.W.A.W. eds: 10. The attachment of bacteria to the
gut FLAIR No. 6; DLO, ISBN 90-71463-64-8
21. Rebuk, K., Righelato, R.C. & Young J., Future Opportunities for Functional Foods, 1995,
Leatherhead Food Research Association, Leatherhead, UK (ISBN 1900184001)
22. Roberfroid, M.B., American Journal of Clinical Nutrition, 2001, 73, 406S-409S
23. Schur., F & Piendl, A., Brauwissenschaft, 1977, 30: 46
24. Van der Gaag, M.S., van den Berg, R., van den Berg, H., Schaafsma, G. & Hendiks, H.F.J.,
European Journal of Clinical Nutrition, 2000, 54, 586-591.
25. Vinson, J.A., Dabbagh, Y.A., Serry, M.M & Jang, J., Journal of Agriculture and Food
Chemistry, 1995, 43, 2800-2802
26. Vinson, J.A., Jang, J., Yang, J., Dabbagh, Y., Liang, X., Serry, M., Proch, J., Cai, S.,
Journal of Agriculture and Food Chemistry, 1999, 47, 2502-2504
27. Walker C, Baxter E.D., 2000, MBAA TQ, 301-5
28. Westerterp-Plantenga, M.S., & Verwegen C.R. American Journal of Clinical Nutrition,
1999, 205-212.
29. Young J., British Journal of Nutrition, 1998, 80, S231-3
30. Zeigler, R.G., American Journal of Clinical Nutrition, 1991, 53, 251S-9S

9
4

Folate in beer and the prevention of

cardiovascular disease
Caroline J. Walker1, Darash Patel1, Caroline Wolfe2, Anthony
Wright2 & Paul Finglas2
1
Brewing Research International, Lyttel Hall, Nutfield, Surrey RH1 4HY, United Kingdom
(e-mail: [email protected])
2
Institute of Food Research, Colney, Norwich NR4 7UA, United Kingdom

Descriptors
Antioxidant, beer consumption, folic acid, health, polyphenol, radical scavenging

SUMMARY
Folate is one of the vitamins most likely to be lacking in the diet - which is unfortunate since
it may protect us against cardiovascular disease and cancer. Beer is relatively rich in folate
and could potentially be a useful dietary source. As a part of an EU-funded project, we are
looking at the folate content of beer and how levels are related to malt content, adjunct usage
and processing. Folate content has also been determined in reduced-alcohol beers, to allow
selection of a suitable beer for a clinical trial which addresses how beer drinking may affect
the risk of cardiovascular disease.

Folat in Bier und die Prvention von kardiovaskulren Erkrankungen

Deskriptoren
Antioxidantium, Bierverbrauch, Folsure, Gesundheit, Polyphenol, Radikalfnger

ZUSAMMENFASSUNG
Folat gehrt zu den Vitaminen, die hufig in unserer Ernhrung fehlen. Das ist
bedauernswert, da Folat den Menschen vor kardiovaskulren Erkrankungen und Krebs
schtzen kann. Bier ist relativ reich an Folat und knnte aus diesem Grund eine ntzliche
Quelle hierfr sein. Im Rahmen eines von der EU untersttzten Projekts wurde die
Korrelation zwischen dem Folatgehalt und der Malzgabe bzw. der Verarbeitung
untersucht. Der Folatgehalt von alkoholreduzierten Bier bestimmte die Entscheidung zur
Auswahl von geeigneten Bieren zur klinischen Anwendung. Mit Hilfe dieser Biere sollte
im Rahmen eines klinischen Versuches die Beeinflussung des Biergenusses auf das
Risiko von kardiovaskulren Erkrankungen erforscht werden.
Acide folique de bire and prvention des maladies cardiovasculaires

Descripteurs
Acide folique, antioxydant, consommation de bire, effet antiradicaux (libres), polyphnol,
sant

RESUME
L'acide folique (folate) est l'une des vitamines les plus susceptibles de manquer dans
l'alimentation ce qui est regrettable car il peut avoir un effet protecteur contre les maladies
cardiovasculaire et le cancer. La bire est relativement riche en folate et pourrait en tre une
source alimentaire utile. Dans le cadre d'un projet financ par l'UE, nous tudions la teneur en
folate de la bire et les relations entre le taux de folate et la teneur en malt, l'emploi d'additifs
et le procd de fabrication. La teneur en folate a galement t dtermine dans des bires
pauvres en alcool, afin de permettre la slection d'une bire pour un essai clinique tudiant
l'influence de la bire sur les risques de maladie cardiovasculaire.

2
INTRODUCTION
Folates are involved in many basic functions in the cell, participating as methyl donors
in a variety of fundamental processes. Their basic role is precisely why folates are so
important in our diet. Generally, when basic cell functions are perturbed, diseases such
as cancer are likely to arise. Indeed the last decade or so, many studies have shown
that a low intake of folate may increase the risk of developing cancers e.g. colon and
cervical cancer.
There is also a suggested connection between cardiovascular disease (CVD) and
folates. Research has indicated that high levels of the sulphydryl amino acid
homocysteine (Hcy), which is formed during the metabolism of the essential amino
acid methionine, are associated with an increased risk of CVD. In fact, Hcy levels are
now often routinely measured in patients to assess their risk of CVD, much in the way
that cholesterol levels are monitored. Currently, the most effective intervention to
lower plasma Hcy is to increase the amount of folate in the diet (1,3). The
biochemical rationale for this treatment can be seen in figure 1, which illustrates the
metabolism of Hcy. Although Hcy can be metabolized to cysteine, much Hcy is
methylated to form methionine, in a reaction catalyzed by the enzyme methionine
synthase; the methyl donor for this reaction is 5-methyl tetrahydrofolate (5MeTHF). In
turn, methionine donates the methyl group in at least 100 important methylation
reactions thus reforming Hcy. If folate levels are low, and 5MeTHF is limiting, then
conversion of Hcy to methionine will be reduced and consequently plasma Hcy levels
will rise. Conversely, a high folate intake will increase the rate of conversion of Hcy to
methionine, and therefore lower the plasma Hcy levels.

folate

THF methionine

1 B12

5 MeTHF homocysteine

B6
1 methionine synthase
THF tetrahydrofolate cysteine
B12 vitamin B12
B6 vitamin B6

Figure 1: Homocysteine metabolism

Folate is currently an area of focus in the EU, because of its health benefits and
because it is one of the vitamins most likely to be lacking in the diet. Strategies for
increasing the amount of folates in our diet are therefore a key public health issue and
are being explored in the EU project Folate: from food to functionality and optimal
health. This project aims to look at a variety of high folate foods across Europe, and
establish what contribution they make to our diet. For example, in Finland rye bread is
being tested, and in Spain it is gazpacho. In the UK we are testing beer, in a

3
collaboration between BRI and the Institute of Food Research at Norwich. In this
project, BRIs role is to:
follow folate through the brewhouse, to identify the points at which this vitamin
may be lost;
evaluate the potential for increasing the level of folates in beer;
collaborate with the Institute of Food Research on a human study assessing the
consumption of a high-folate beer and its potential to (a) increase the body folate
stores and (b) decrease plasma Hcy which is currently hypothesized to be a risk
factor for CVD.

MATERIALS AND METHODS

Folate analysis
Cereals are a good source of folates, and since folates are both water soluble and
relatively heat stable it is hardly surprising that folates have been reported in beer
(4,6). There are a few published values which vary widely, with up to 150 micrograms
per litre being reported in some beers! Each report has used different methods of
analysis, making it difficult to compare values. A method of folate analysis that is
optimised for beer has therefore been developed as a part of this project that can be
used to give accurate values for comparing beer types.
Folates are found in a variety of forms. In cereals there are typically 5 forms, varying
in the position and degree of substitution on the basic pteridine ring (figure 2). There
are several methods currently being used for folate analysis. The oldest, gold
standard technique, is a microbiological assay which measures total folate content
i.e. the total of all the various forms of folate.

The basis of this assay is the use of a Lactobacillus strain, which will only grow in the
presence of folate. Briefly, this folate-dependant bacterium is added to samples
containing either a known amount of folate or a beer extract. After overnight
incubation, cell growth (measured by optical density) is plotted against the folate
content of the standards. The standard curve generated gives the concentration of total
folate in the beer extracts.

The extraction of folates from beer, cereals and brewhouse samples was adapted from
the trienzyme treatment of Pfeiffer et al (5).

Malting timecourse
Barley was malted on a 300 g scale with a 8 h wet; 16 h air rest; 24 h wet steeping
schedule. Steeping and germination were at 16C. Samples were removed daily and
dried for 8 h at 45C followed by 16 h at 65C. Other malts analysed were obtained
from commercial maltings.

Laboratory mashing
Mashing was carried out using a BRI mashing bath, using standard IOB or EBC
protocols.

4
 OH
10
*N5 *
CH NH
2 CO-glutamic acid(n)
N

Coenzyme Substituent at
H2N N N N5 N10
5-formylTHF -HCO -H
5,10-methyleneTHF =CH+
5-MethylTHF -CH3 -H
THF -H -H
10-formylTHF -H -HCO

Figure 2: Structure of folates

RESULTS AND DISCUSSION

Human study on the effects of beer on plasma Hcy concentration, a putative risk
factor for CVD
As mentioned above, one of the aims of the EU project is to conduct a human study on
the effects of beer drinking on plasma Hcy, a possible CVD risk factor. This study will
be carried out at the Institute of Food Research (IFR), and will involve approximately
90 healthy male volunteers. The volunteers will consume a litre of alcohol-free beer
every day for 4 weeks. Blood samples taken at the start and finish of the study will
indicate whether the volunteers Hcy levels have been reduced.

The decision to use an alcohol-free beer, rather than a regular strength beer, may seem
a little strange at first glance. There are in fact two major reasons for this choice.
Firstly, there is an ethical consideration in terms of asking volunteers to drink alcohol
every day. Although the amount of alcohol would only be moderate, the length of the
trial is of concern for the ethical committee. Second, the study will focus on the non-
alcohol ingredients in beer i.e. the ingredients that distinguish beer from other
alcoholic beverages. By using an alcohol-free beer, the focus of the study will be on
beer, rather than alcohol. The disadvantage of this approach is that the presence of
alcohol itself may affect the absorption of vitamins in our gut either positively or
negatively. Unfortunately, it is not possible to have a perfect study, and the use of an
alcohol-free beer has been considered to be the best compromise.
Calculations based on published work, suggest that an additional intake of 100
micrograms of folate per day would be sufficient to give a statistically significant
decrease in Hcy levels, assuming that the folate is absorbed from the food stuff.
Therefore, a beer with a folate content of at least 100 micrograms/liter was considered
the minimum requirement for this study. Fortunately there were several beers with this
folate level (table 1). The final decision on which beer to use for the trial was based on
the results of a taste panel with members of the public. The beer selected (Lager B)
was more bitter than some of the others, and was more compatible with the typical
British Ale drinkers palate.

The human study started in March 2001, and will continue for the next two years.

5
 Beer (country) Total folate Country of
(ug/litre) Origin
Lager A 115 5 D
Lager B 103 1 D
Lager C 89 15 D
Lager D 88 11 D
Lager E 80 15 D
Lager F 75 3 D
Lager G 57 12 SF
Lager H 57 5 D
Ale A 104 12 UK
Ale B 47 4 UK
Weibier 125 10 D
Table 1: The folate content of low alcohol and alcohol-free beers

Folates and malting

In general, seed germination is accompanied by vitamin synthesis in the embryo and
malted cereals therefore tend to have a higher vitamin content than unmalted cereals
(2). In a small scale malting trial, we observed that in the first 3 days of germination,
the folate content approximately doubled (figure 3).

2.5
folate (mg/kg dry

2
wt.)

1.5

0.5
0 1 2 3 4
Days germination

Figure 3: The folate content of barley increases on malting

The increase in folate content will depend on many diverse factors such as malting
conditions, malt type, kilning conditions and barley variety. These factors will be
investigated in the future. At this stage, our data suggest that malts have a higher folate
content than adjuncts such as rice and maize and some highly kilned or roasted cereals
(table 2). Beers made with a higher proportion of malt, will therefore have a higher
folate content in the raw materials.

6
 Cereal (variety) Folate content (mg/kg)
Ale malt (Fanfare) 4.0 0.1
Lager malt (Fanfare) 4.0 0.2
Wheat malt (Atlantis) 3.1 0.1
Crystal malt (Optic) 1.6 0.3
Crystal wheat malt (Atlantis) 1.9 0.1
Roasted barley 1.0 0.1
Broken rice 1.4 0.1
Maize grits 1.7 0.1
Barley (Opal) 1.0 0.1
Table 2: The folate content of a range of malted and unmalted cereals

Recovery of folate through the brewhouse

The relative importance of raw materials and processing in determining the folate
content of beer depends on knowledge of the recovery of folates through the various
stages of brewing. In terms of maximizing the folate content of beer, it is essential to
identify the key steps during processing where folate is lost; these can then be studied
in detail and optimized.

It was most practical to follow the recovery of folates in mashing using a laboratory
mashing bath. Here 50 g samples of 100% malt grist could be used and folate
extraction could be monitored at time intervals during mashing. Samples of lager and
ale malts were mashed with both standard EBC and IOB mashing regimes. In an EBC
mash, at the lower temperatures (45C), there was an increased extraction of folates
from the grist; however, at higher temperatures (70C) the folates were broken down
(figure 4). The value at zero time represents the amount of folate which was
extracted from brief stirring with liquor at 45C, then subsequent filtration through a
fluted filter paper. The high value at this time suggests that at least half of the folate in
the grist is very readily extractable.

In IOB mashes, which are isothermal at 65C, the folate concentrations in the extract
decreased steadily during mashing. Typical recoveries of folate from the grist in IOB
mashes was 20-30%, with slightly higher recoveries (40%) for EBC mashes. Clearly,
the loss of folate in this step is rather high. Generally, folates are unstable in the
presence of oxygen and heat, and unfortunately a mashing bath provides both of these!
Therefore a priority will be to check the recovery of folates during larger scale mashes,
where the vessel dimensions may reduce the level of oxygenation. Also, such
parameters as the redox conditions during mashing can be manipulated to stabilise the
folate and thus increase recovery.

Subsequent steps in brewing were analyzed by taking samples from standard brews in
the pilot brewery at BRI. Samples from lager, ale and stout brews showed that folate
losses during boiling and trub separation were low - typically less than 10% for each
step. Fermentation also did not appear to make either a positive or negative
contribution to folate content. After post fermentation conditioning and filtration for
both lager and ale brews, the folate content was approximately the same as in the
pitching wort. This is not necessarily unexpected, since yeast does not require folate
for growth. The yeast itself makes folate, and analysis of fermentation samples with
suspended yeast gave very high folate contents. However, after yeast removal, there

7
was no apparent contribution of folate from the yeast to the beer. Therefore, overall
fermentation had no impact on folate content.

200 90
folate (ug/litre)

temp (C)
150 60

100 30

50 0
0 50 100
folate
mashing time (min) temp (C)

Figure 4: Extraction of folate during mashing of a lager malt

The concentration of folate in the bright beers were high compared to those seen in
commercial beers, which suggested that there are some losses in-pack. In the BRI pilot
brewery, beer is packaged in crown capped brown glass bottles, pasteurized in a
tunnel-type system, and is stored at 4C. After packaging, the folate content of both
the ale and lager had decreased by approximately 50%. Preliminary results indicate
that this was not due to the pasteurization process, but rather losses during packaging
and storage (in this case 2 weeks). By comparison of folate levels in other commercial
beers, these losses are probably typical. On a positive note, it is likely that any
improvements developed for the stability of folates could also yield benefits in terms
of flavor stability!

CONCLUSIONS
The variability in folate levels seen in commercial beers suggests that it may be
possible to develop products with improved vitamin content if the brewer so desires.
We have identified key areas that may have an impact on the folate content of beers
mashing, packaging and choice of raw materials. In the next phase of the project, these
steps will be looked at in more detail with a view to optimization and providing the
industry with guidelines on how to achieve a high folate beer.
In the second part of this EU project, the human studies will be important in
establishing the effectiveness of the non-alcohol ingredients of beer in reducing
plasma Hcy, a currently suggested risk factor for CVD. Hopefully this study will bring
public attention to the vitamins and antioxidants that are important, but often
overlooked, healthy ingredients in beer.

This work has been funded in part QLRT-1999-00576. For further information please
see www.ifr.bbsrc.ac.uk/folate

REFERENCES
1. Eikelboom, J.W., Lonn, E., Genest Jr, J., Hankey, G. & Yusef, S., Annals of
Internal Medicine, 1999, 131, 363-375

8
2. Finney, P.L., Recent Advances in Phytochemistry, 1982, 17, 229-305
3. McDowell, I.F.W. & Lang, D., Journal of Nutrition, 2000, 130, 369S-372S
4. Piendl, A., Brauwelt, 1993, 133, 2198-2203
5. Pfeiffer, C.M., Rogers, L.M. & Gregory, J.F. III, Journal of Agricultural and Food
Chemistry, 1997, 45, 407-413
6. Savage, D., Alcohol Clinical and Experimental Research, 1995, 19, 1596

9
5

Antioxidant and radical-scavenging

potential of phenolic constituents of
beer
A. Gamal-Eldeen1, A. Alt2, C. Gerhuser1, I. Neumann1, N.
Frank1, H. Chmiel3, H. Bartsch1 & H. Becker2
1
Deutsches Krebsforschungszentrum DKFZ, Im Neuenheimer Feld 280, D-69120 Heidelberg,
Germany (e-mail: [email protected])
2
Universitt des Saarlandes, Saarbrcken, Germany
3
Gesellschaft fr Umweltkompatible Prozesstechnik, Saarbrcken, Germany

Descriptors
Antioxidant, beer constituent, medicine, polyphenol, radical scavenging, stabilisation

SUMMARY
We have initialized a study to investigate the anti-oxidant potential of beer and of a residue R
removed from beer during the stabilization process. Using physiologically relevant reactive
oxygen species, about 40 polyphenolic constituents were evaluated. Catechins and flavonoids
were more potent than acetophenones and derivatives of benzoic and cinnamic acid.
Generally, for all compounds, radical scavenging capacity was hydroxyl > peroxyl >
superoxide anion radicals and correlated with the number and position of free OH-groups.
These findings might indicate a possible application in cancer prevention and promote the
development of a less stabilized beer with a higher content in polyphenols.

Antioxidative Kapazitt von Polyphenolen aus Bier

Deskriptoren
Antioxidantium, Bierbestandteil, Medizin, Polyphenol, Radikalfnger, Stabilisierung

ZUSAMMENFASSUNG
Inhaltsstoffe von Bier sowie von Polyphenol-Rckstnden, die bei der Bier-Stabilisierung
anfallen, wurden auf antioxidative Aktivitt getestet. Unter Verwendung von physiologisch
relevanten reaktiven Sauerstoffspezies wurden ca. 40 verschiedene Polyphenole analysiert.
Catechine und Flavonoide waren strker antioxidativ als Acetophenone sowie Benzoe- und
Zimtsure-Derivate. Im allgemeinen wurden von diesen Substanzen Hydroxylradikale besser
als Peroxyl- und Superoxidanionradikale abgefangen. Die antioxidative Kapazitt korrelierte
mit der Zahl und der Stellung freier OH-Gruppen. Diese Ergebnisse knnten einerseits in der
Krebs-Chemoprvention Anwendung finden, andererseits knnten sie als Grundlage zur
Entwicklung eines polyphenolreicheren Bieres dienen.
Les constituants phnoliques de la bire ont une capacit antioxydante et pigent les
radicaux oxygns

Descripteurs
Antioxydant, constituant de la bire, effet antiradicaux (libres), mdecine, polyphnol,
stabilisation

RESUME
Nous avons analys le potentiel antioxydant de bire et d'un rsidu R limin de bire pendant
sa stabilisation. Environ 40 composants poly-phnoliques diffrents ont t valus pour leur
capacit rduire des espces de radicaux oxygns. Les catchines et flavonodes etaient
plus puissantes que des actophnones et des drivs de l'acide benzoque et cinnamique. En
gnral, ces composs pigeaient prfrentiellement des radicaux hydroxyles puis peroxyles
et enfin superoxydes, et cette capacit correspondait au nombre et la position des
groupements OH libres. Ces rsultats pourraient trouver une application dans la prvention du
cancer et favoriser le dveloppement d'une bire moins stabilise contenant un pourcentage
plus lev de polyphnoles.

2
6

Cancer chemopreventive potential of

xanthohumol (XN), a prenylated
chalcone from hops
C. Gerhuser1, A. Alt2, E. Heiss1, A. Gamal-Eldeen1, K. Klimo1,
J. Knauft1, I. Neumann1, H. Scherf1, N. Frank1, H. Bartsch1 & H.
Becker2
1
Deutsches Krebsforschungszentrum DKFZ, Im Neuenheimer Feld 280, D-69120 Heidelberg,
Germany (e-mail: [email protected])
2
Universitt des Saarlandes, Saarbrcken, Germany

Descriptors
Cancer chemoprevention, chalcone, hop constituent, medicine, oxygen, reaction
mechanism, xanthohumol

SUMMARY
Identification of dietary components as cancer chemopreventive agents has become an
important issue in current cancer-related research. Xanthohumol (XN) was tested in a battery
of cell- and enzyme-based test systems relevant for inhibition of carcinogenesis in humans.
XN was able to scavenge various reactive oxygen species and was identified as an anti-
inflammatory agent. Anti-proliferative mechanisms included anti-estrogenic properties,
inhibition of DNA polymerase and induction of cell differentiation. Finally, chemopreventive
activity was demonstrated in mammary organ culture. These data might open new markets for
XN or hop products, but further demonstration of efficacy and safety in humans is required.

Krebs-chemoprventive Eigenschaften von Xanthohumol, ein prenyliertes Chalcon aus

Hopfen

Deskriptoren
Chalcon, Hopfenbestandteil, Krebs-chemoprvention, Medizin, Sauerstoff, Reaktions-
mechanismus, Xanthohumol

ZUSAMMENFASSUNG
Eine bedeutende Sparte in der gegenwrtigen Krebsforschung ist die Untersuchung von
Nahrungsbestandteilen auf krebsprventive Wirkung. Deshalb untersuchten wir Xanthohumol
(XN) in einer Reihe von Testsystemen auf zellulrer oder enzymatischer Ebene, die fr die
Krebsentstehung beim Menschen relevant sind. XN wurde als ein starker Radikalfnger und
Entzndungshemmer identifiziert. Zellwachstumshemmende Wirkung konnte anti-strogenen
Eigenschaften, der Hemmung der DNA Polymerase und einer Induktion der Zell-
differenzierung zugeschrieben werden. Chemoprventive Wirksamkeit von XN wurde in
einem Brustdrsen Organkulturmodell verifiziert. Diese Ergebnisse knnten einen neuen
Markt fr XN oder Hopfenprodukte ffnen, wenn die Wirksamkeit auch am Menschen
nachgewiesen sein wird.

Effet chimio-prventif du Xanthohumol (XN), une chalcone isol partir du houblon

Descripteurs
Chalcone, chimio-prvention, constituant du houblon, mcanisme de raction, mdecine,
oxygne, xanthohumol

RESUME
L'identification des composants dittiques comme agents chimio-prventifs du cancer est
devenue une voie explorer dans la recherche actuelle contre le cancer. Nous avons analys
l'activit du Xanthohumol (XN) dans une srie d'expriences faisant intervenir des cellules et
des enzymes impliqus dans l'inhibition de la carcinogense chez lhomme. Le XN a t
identifi comme un agent anti-inflammatoire. Il est capable de piger diffrentes espces de
radicaux oxygns ractifs. Les mcanismes anti-prolifratifs ont inclus des proprits anti-
oestrogniques, l'inhibition de DNA polymrase et l'induction de la diffrenciation cellulaire.
De plus, l'activit chimio-prventive a t dmontre dans la culture d'organe mammaire. Ces
donnes pourraient ouvrir des marchs nouveaux pour XN ou des produits dhoublon, mais
des tudes complmentaires d'efficacit et de tolrance s'avrent ncessaires chez lhomme.

2
7

Prenylated hop flavonoids are key

agents in relation to health-beneficial
properties of beer
Denis De Keukeleire1, Stuart R. Milligan2, Jogen C. Kalita2,
Victoria Pocock2, Luc De Cooman1, Arne Heyerick1, Haojing
Rong1 & Frederik Roelens1
1
Ghent University, Faculty of Pharmaceutical Sciences, Laboratory of Pharmacognosy and
Phytochemistry, Harelbekestraat 72, 9000 Ghent, Belgium (e-mail:
[email protected])
2
Kings College, School of Biomedical Sciences, Endocrinology and Reproduction Research
Group, Guys Campus, London SE1 1UL, United Kingdom

Descriptors
Beer consumption, flavonoid, health, hop constituent, medicine, phyto-oestrogens

SUMMARY
The interest in health-beneficial aspects of beer is being focussed on bioactive constituents
derived from hops, as hop polyphenols, in particular prenylated flavonoids, exhibit varying
biological properties. A targeted study based on in vitro and in vivo bioassays has led to the
identification of 8-prenylnaringenin (hopein) from hops as a most potent phytoestrogen.
Current investigations are aimed at deciphering positive contributions of prenylated hop
flavonoids to health including activities against cardiovascular diseases, hormone-dependent
cancers, and climacteric symptoms. The presence of hopein and related compounds in beer
highlights the key role of hops in improving health on moderate beer consumption.

Prenylisierte Hopfenflavonoide sind Schlsselwirkstoffe in Bezug auf die gesundheits-

frdernde Wirkung des Bieres

Deskriptoren
Bierverbrauch, Flavonoid, Gesundheit, Hopfenbestandteil, Medizin, Phyto-strogen

ZUSAMMENFASSUNG
Das Interesse der gesundheitsfrdernden Wirkung des Bieres ist auf die bioaktiven
Bestandteile, die sich vom Hopfen herleiten, gerichtet. Es handelt sich dabei um
Hopfenphenole, einige prenylisierte Flavonoide, die verschiedene biologische Wirkungen
zeigen. Eine in vitro und in vivo durchgefhrte Untersuchung fhrte zur Identifikation des
wirksamsten Phyto-strogens in Hopfen, dem 8-Prenylnaringenin (Hopein). Laufende
Studien richten ihre Untersuchungen auf die Entschlsselung der gesundheitsfrdernden
Wirkung der prenylisierten Hopfenflavonoide in Hinsicht auf kardiovaskulre Erkrankungen,
hormon-verursachtem Krebs und klimakterischen Symptomen. Die Inhaltsstoffe Hopein und
verwandte Stoffe im Bier unterstreichen die Schlsselrolle des Hopfens bei der
gesundheitsfrdernden Wirkung eines moderaten Biergenusses.

Les flavonodes prnyls du houblon sont les principaux agents des effets bnfiques de
la bire sur la sant

Descripteurs
Consommation de bire, constituant du houblon, flavonode, mdecine, phyto-estrognes,
sant

RESUME
L'intrt pour les constituants de la bire utiles la sant se porte actuellement sur les
composs bio-actifs drivs du houblon, comme les polyphnols, en particulier les
flavonodes prnyls, prsentant diverses proprits biologiques. Une tude base sur les
dosages biologiques in vitro et in vivo a permis d'identifier dans le houblon un trs puissant
phyto-estrogne, la 8-prnylaringnine (hopine). Les tudes actuelles visent dterminer les
effets positifs des flavonodes prnyls du houblon sur la sant, notamment leur activit
contre les maladies cardiovasculaires, les cancers hormonodpendants et les symptmes de la
mnopause. La prsence de hopine et de composs apparents dans la bire souligne le rle
cl du houblon dans l'amlioration de la sant, lorsque la consommation de bire est modre.

2
INTRODUCTION
As a number of studies have suggested that moderate consumption of any alcoholic
beverage lowers the rates of cardiovascular diseases, a remarkably vivid interest is
currently being noted in nutritional and health benefits associated to drinks in
accordance with a healthy lifestyle. Given the health problems due to overindulging in
drinking alcohol, acceptance of the validity of these health-positive effects was not
easily garnered and endorsement of this attitude is definitely not a permissive licence
to drink in excess. On the other hand, sales of so-called functional drinks featuring
health improvement or nutritional upgrading have skyrocketed during recent years.
Meta analyses evaluating benefits of different alcoholic beverages indicate that
findings of superiority of one beverage over another are equally distributed among
beer, wine, and spirits6,23. The persuasive consistency of the data suggests that the
majority of the cardioprotective effect is caused by alcohol (ethanol). Furthermore, in
recent studies, it was shown that moderate alcohol consumption has positive effects
on bone mineral density in elderly women, which appear to be mediated by a decrease
in bone remodeling19.
However, part of the beneficial effects of alcoholic beverages could be caused by non-
alcohol components. Beer contains more proteins and vitamins B than wine and the
nutritional value of beer can contribute substantially to the diet. A variety of
biologically active compounds are present in alcoholic beverages and, in particular,
polyphenols represent a class of high health-related interest. The antioxidant content
of beer is equivalent to that of wine, but the specific antioxidants are different,
because raw materials, mainly barley and hops applied in beer brewing, possess
flavonoids, a subclass of polyphenols, different from those in grapes used for the
production of wine6.
Beer is the only beverage containing hops (Humulus lupulus L.; plant family of the
Cannabaceae) and any specific hop-associated health benefit could be exploited to
advocate an evidence-based preference of beer over other alcoholic drinks. Indeed,
hops have been used since ancient times for the treatment of a variety of diseases.
Brewers have learnt to appreciate the characteristics of hops already during the early
middle ages and, nowadays, hops contribute pivotally to the quality of beer. They
afford bitterness, aroma, enhanced foam potential, and antimicrobial protection.
Clearly, most recent developments regarding bioactivities of hop constituents add a
significant value and a novel asset to the health-beneficial properties of beers.

PRENYLATED FLAVONOIDS
Of the various flavonoids (over 4,000) known to accumulate in plants, the occurrence
of prenylated flavonoids as natural plant constituents came to be recognized fairly
recently. The distribution is restricted to a relatively small number of plant families,
yet the structural diversity is great1. Prenyl transferases catalyze the transfer of a
prenyl residue to flavonoid acceptors. Generally, most flavonoids are C-prenylated, in
particular on ring A at positions 6 and 8 in flavanones and flavones or at positions 3
and 5 in chalcones (scheme 1). The more frequent type of prenylation is represented
by the 3,3-dimethylallyl side chain, although 1,1-dimethylallyl, geranyl, and
lavandulyl derivatives are known as well. Extensive modification of the terpenic side
chains may occur by further oxidation, reduction, dehydration, and/or cyclization.
Prenylated flavanones are the most abundant subclass with a rich variety of structures,
almost half of which occur in the (2S)-configuration. It is interesting to notice that

3
cannabigerol, a precursor of cannabinoids in Cannabis sativa L. (plant family of the
Cannabaceae), shows biosynthetic similarity to prenylated flavonoids present in hops.

8 1 B (OH)x 8 1 B (OH)x
O O
(HO) y A 2S (HO) y A
6 6

O O
FLAVANONES 3 FLAVONES

1
5' (OH)x
OH 3,3-DIMETHYLALLYL
(HO) y A = PRENYL
1'
3'
1
O
1
CHALCONES

1,1-DIMETHYLALLYL GERANYL LAVANDULYL

Scheme 1

BIOACTIVE CONSTITUENTS OF HOPS AND PRENYLATED HOP

FLAVONOIDS
The diversity of natural hop constituents undoubtedly accounts for the varied and rich
panoply of bioactivities hitherto reported: sedative, antistress, and soporific activities,
estrogenicity, treatment of complaints related to the menopause, anticancer properties
(in particular inhibition of hormone-dependent cancers of breast, uterus, and prostate),
bacteriostatic activity, antiinflammatory action, stimulation of the digestive tract (hops
belong to the amara or bitters), diureticum and agent against bladder complaints,
diaphoretic and perspiration-stimulating effects, and anaphrodisiacum4,5.
All interesting compounds are accumulated in the so-called lupulin powder, which is
composed of microscopic glandular plant structures profusely present at the
appendages of the bracteoles (modified leaves that are part of the hop inflorescence)
with the cone stig. In fact, only near the end of the hop growing period the lupulin
glands ripen to function as a rich source of bioactive molecules (so-called secondary
metabolites) aimed at protecting the vulnerable hop plant against adverse ecological
interactions (pests, diseases, moulds, aphids). Not only precursors (-acids) to beer
bitter agents (iso--acids) and constituents of the essential oil are present in the
lupulin glands, but also polyphenols and, certainly, prenylated flavonoids.
In addition to proanthocyanidins and flavonols, both in free form and as glycosides,
characteristic flavonoid profiles are observed on HPLC (high performance liquid
chromatography) separation of polyphenolic hop extracts. Xanthohumol (scheme 2), a
chalcone, is clearly the principal prenylated flavonoid (80-90% of the total)28, while
more than 20 other prenylated flavonoids have been identified until now31.
Xanthohumol is the precursor of the flavanone isoxanthohumol, while
desmethylxanthohumol gives rise to the flavanones 6-prenylnaringenin and 8-
prenylnaringenin by an intramolecular Michael type reaction (scheme 2). It is not

4
clear whether or not the enzyme chalcone isomerase effects the reaction. We have
found that 8-prenylnaringenin is present as a racemate in lupulin glands and this
observation suggests thermal formation of the heterocyclic ring. The contents of
prenylated flavonoids in hops range from 0.2% to 0.6% depending on storage time
and conditions29.

OH OH
R3
HO OH HO O

R2
OR O OR 1 O
R = Me: XANTHOHUMOL R1 = Me, R2 = H, R 3 = prenyl: ISOXANTHOHUMOL
R = H: DESMETHYLXANTHOHUMOL R1 = R 3 = H, R 2 = prenyl: 6-PRENYLNARINGENIN

1.2 nm
1.1 nm OH OH
H H
HO O
H

HO Me
OH O
8-PRENYLNARINGENIN -ESTRADIOL
17
HOPEIN

Scheme 2

The biological activities of some of the prenylated flavonoids have been examined by
in-vitro techniques. Proliferation of human breast cancer cells (MCF-7), colon cancer
cells (HT-29), and ovarian cancer cells (A2780) was inhibited by 6 prenylated hop
flavonoids tested and xanthohumol proved to be the most cytotoxic compound in the
micromolar range14. Since normal cells were not affected, some prenylated hop
flavonoids may have preferential growth-inhibitory effects on tumor cells not caused
by general toxicity. Further findings related to inhibition of the activation of
cytochrome P450 enzymes that mediate metabolic conversion of procarcinogens to
carcinogens confirmed the chemopreventive power of xanthohumol and related
prenylated hop flavonoids10,14.
Xanthohumol, isoxanthohumol, 6-prenylnaringenin, and 8-prenylnaringenin showed
antimicrobial activity against a number of fungi and comparison with naringenin
indicated that the presence of a prenyl group is required for activity18. It is, however,
not clear whether prenylated flavonoids offer protection against fungal attack of the
hop inflorescences. Xanthohumol has a strong inhibitory effect on
diacylglyceroltransferase (in rat liver microsomes), which converts diacylglycerol into
triacylglycerol, and, thus, may favorably effect atherosclerosis32. Metabolites of
xanthohumol produced by rat liver microsomes have recently been identified36.
Xanthohumol was also most active in the oxidation of low-density lipoprotein16.

5
ESTROGENIC ACTIVITY OF 8-PRENYLNARINGENIN
Circumstantial evidence over many years has linked hops with potential estrogenic
activity in women and, as menstrual disturbances were reportedly common among
hop pickers, xanthohumol was considered the culprit35. The literature contains a
number of reports on the possible estrogenic activity of hops ranging from
suggestions that they may be one of the richest sources of plant estrogens to the
failure to find any evidence of estrogenic activity. Our strategy to identify the
estrogenic activity in hops was based on two highly selective and very sensitive in-
vitro estrogenic screens, an estrogen-inducible yeast bioassay (Saccharomyces
cerevisiae) expressing the human estrogen receptor and a bioassay based on a human
endometrial adenocarcinoma cell line, called Ishikawa Var-I12.
We found that the alleged hop hormone, xanthohumol, was devoid of any estrogenic
activity2 and, furthermore, we confirmed that hop--acids, hop--acids, and iso--
acids are not estrogenic. On the other hand, polyphenolic extracts of a large number of
different hop varieties showed strong estrogenic activity with dose-response curves
that paralleled that of the endogenous female hormone, 17-estradiol3. We then
carried out activity-guided fractionation of the polyphenolic hop extracts and
progressive refining of the fractions containing the activity allowed to conclude that
the endocrine activity of hops and hop products is mainly due to the potent estrogenic
properties of 8-prenylnaringenin. Some estrogenicity is present in a few other
prenylated flavonoids including isoxanthohumol and 6-prenylnaringenin, but the
activity is very weak12 (figure 1). There was no evidence of androgenic or
progestogenic activity in either polyphenolic hop extracts or pure prenylated
flavonoids. Like some other phytoestrogens, 8-prenylnaringenin also shows strong
affinity for the estrogen receptor (profusely present in breast, ovaria, uterus, and
blood vessels), but also to the estrogen receptor (mainly in prostate, bladder, bone,
and brain), suggesting that 8-prenylnaringenin may have many potential sites of
action within the body 13. In fact, 8-prenylnaringenin shows higher activity than any
of the established phytoestrogens (plant constituents that exhibit estrogenic activity)
such as coumestrol (from red clover) or genistein and daidzein (from soy)13 (figure 2).

Figure 1: Comparison of the in-vitro Figure 2: Comparison of the in-vitro

estrogenic activity of 8-prenylnaringenin with estrogenic activity of 8-prenylnaringenin
17-estradiol, isoxanthohumol, 6-prenylnarin- with 17-estradiol, coumestrol, genistein,
genin, and xanthohumol (Ishikawa Var-I). and daidzein (Ishikawa Var-I).

6
Table 1 contains relevant features of the estrogenic activities of 8-prenylnaringenin
with respect to the endogenous female hormone, 17-estradiol, and the known
phytoestrogens coumestrol, genistein, and daidzein. We can - with a high degree of
confidence - deduce from our data that 8-prenylnaringenin is the most potent
phytoestrogen identified so far and this remarkable property has been confirmed by
others8,17. It should, therefore, be straightforward to develop 8-prenylnaringenin as a
phytotherapeuticum with extremely high estrogenic potency and investigations
towards achieving this goal are in progress in our laboratory.

Table 1: EC50-values* (nM) for 17-estradiol, 8-prenylnaringenin, and established

phytoestrogens, and relative binding affinities to the estrogen receptor.

17-ESTRADIOL 8-PRENYLNARINGENIN COUMESTROL GENISTEIN DAIDZEIN

EC50 (nM) for recombinant yeast bioassay
0.29 44 72 1,170 2,215
EC50 (nM) for Ishikawa Var-I bioassay
0.82 4.2 30 194 1,452
Relative binding affinity to the estrogen receptor
1 0.023 0.008 0.003 -
* Effective concentration to produce 50% of maximum estrogenic activity.

We are, furthermore, gaining increasingly detailed insight into the features

determining the estrogenic character of 8-prenylnaringenin. First, structural
similarities between 17-estradiol and 8-prenylnaringenin are obvious. A preeminent
requirement for binding to the estrogen receptor is the presence of 2 hydroxyl groups
separated by a well-defined distance, which keep the ligand attached to the binding
pocket of the receptor via hydrogen bonds at both ends (Glu353 and Arg394, and
His524, respectively). As indicated in scheme 2, both 17-estradiol (1.1 nm) and 8-
prenylnaringenin (1.2 nm) fulfill this prerequisite21. Lack of structural rigidity of the
scaffold between these hydroxyl groups and incorporation of oxygen atoms account
for less efficient binding of 8-prenylnaringenin when compared to 17-estradiol.
On the other hand, the presence of a prenyl group at C(8) increases the estrogenic
potential of 8-prenylnaringenin relative to that of other phytoestrogens including
coumestrol and genistein. It can, indeed, be readily observed from X-ray data and
modeling that the prenyl group is positioned in close proximity to isoleucine
(Ileu424), lysine (Lys520), and glycine (Gly420 and Gly521) residues of the estrogen
receptor and resulting hydrophobic interactions of the relevant hydrocarbon chains
enhance efficient binding20,21 (figure 3). These data are nicely supported by the very
low estrogenicity of both isoxanthohumol, which does not have the hydroxyl group at
C(5) necessary for hydrogen bonding to the estrogen receptor, and of 6-
prenylnaringenin, which carries the prenyl group at an inappropriate position for
efficient hydrophobic association.
The in-vivo activity of 8-prenylnaringenin was determined using an acute bioassay
based on the ability of estrogens to induce a rapid rise in uterine vascular permeability
in ovariectomized mice (figure 4). The doses of genistein required to produce a
similar effect were at least tenfold higher. Recent findings indicate that 8-
prenylnaringenin is also orally active, since administration to the drinking water (100
g/ml) stimulated epithelial mitoses in the uterus and vagina of ovariectomized mice.
We were, moreover able to show that 8-prenylnaringenin is very effective in slow
aggregation of MCF-7/6 breast cancer cells (figure 5), which indicates that the
compound may inhibit metastasis25. Also, hop extracts in very low concentrations

7
proved to be most efficient growth inhibitors of T-47D estrogen receptor-positive
breast cancer cells37. Clinical trials focussing on these highly interesting features are
in preparation.

R
TO
EP
EC
R
Lys520

EN
Ileu424

G
His524

O
Gly420

TR
ES
O H N
Gly521 N
H
Arg394 NH 2 O
OH
NH 2
O O
H
Hydrophobic residues
HO O

Glu353 8-PRENYLNARINGENIN

Figure 3: Schematic map of the ligand-binding pocket of the estrogen receptor drawn
for the 8-prenylnaringenin/estrogen binding complex.

Figure 5: Stimulation of aggregation of

Figure 4: Dose-response effects of 8-prenyl- MCF-7/6 breast cancer cells in the absence
naringenin on vascular permeability in uteri of (above) and the presence (below) of 8-
ovariectomized mice (E2: 17-estradiol). prenylnaringenin .

Hops proved to be superior to black cohosh root (Cimicifuga racemosa L.) and vitex
berry (Vitex agnus-castus L.), which are currently the most widely used plant
preparations to relieve post-menopausal symptoms7. Additionally, it was shown that
hop extracts quite efficiently suppress hot flushes associated to the menopause9, while
8-prenylnaringenin acts as an estrogen agonist against osteoporosis17. Other studies
are being conducted to evaluate the bioactivity spectrum of 8-prenylnaringenin.
Inhibition of human aromatase and 5-reductase, which are key enzymes in the
development of prostate cancer.
Inhibition of angiogenisis (formation of new blood vessels important in tumor
growth).

8
 Modulation of the activity of NFB, a key factor in the development of immune
diseases including osteoporosis, as well as in the regulation of apoptosis, which
refers to programmed cel death and killing of cancer cells .
As a result of its promising bioactivities and imminent applications in
phytotherapy, 8-prenylnaringenin has been renamed as hopein (from hops and
hope).

PRENYLATED HOP FLAVONOIDS IN BEER

The only natural exposure that humans are likely to have to the high estrogenic
activity of hops is via beer consumption. What is then the relevance of the estrogenic
properties of hops with respect to beer? Obviously, prenylated hop flavonoids, in
particular 8-prenylnaringenin, exhibit desirable health-beneficial effects, hence their
presence in beer must be considered advantageous for beer consumers.
Our experiments and those of others indicate that the estrogenic fraction of hops,
which is not of isoflavone nature, is to some extent transferred to beer. The total
content of prenylated flavonoids in beers can be up to 4 ppm. We were able to reliably
quantify 8-prenylnaringenin in various beers24,33,34. Significant differences are
observed, not only with respect to the different beer types, but even for a selection of
lager beers. We found concentrations of 8-prenylnaringenin varying between 0 and 21
ppb (ca. 60 nM), while Stevens et al. detected up to 240 ppb in a particular American
porter30. Recently, Promberger et al. determined an activity corresponding to an
average of 43 ng 17-estradiol/l in Austrian beers22, but they were, surprisingly,
unable to identify 8-prenylnaringenin. Isoflavones including genistein an daidzein
have been detected in beer (levels between 1.26 and 29 nM)11,26,27. The origin,
however, is unclear and possible estrogenic activity should be dwarfed by the
presence of 8-prenylnaringenin in beers. The use of particular hop varieties, e.g.,
varieties rich in phytoestrogens, or hop products, e.g., pre-isomerized hop extracts or
even reduced iso--acids, should be decisive as to whether or not hop-derived
estrogens are present in beers.

CONCLUSIONS
It must be emphasized that prenylated hop flavonoids are exclusive for beer as an
alcoholic beverage and the bioactivities associated to these agents are of key
importance to confer health-beneficial properties to beer. In particular, 8-
prenylnaringenin (hopein), the most potent phytoestrogen known to date, may offer
promising prospects for the design of beers with specific health claims targetted at a
particular public. As exploration is still in its initial stages, the significance of the full
bioactivity spectrum of 8-prenylnaringenin will become even more timely as new
scientific and biomedical results will emerge.

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2. De Keukeleire, D., Milligan, S.R., De Cooman, L. & Heyerick, A., Pharmaceutical and
Pharmacological Letters, 1997a, 7, 83-86.
3. De Keukeleire, D., De Cooman, L., Heyerick, A. & Milligan, S.R., Proceedings of the 26th
European Brewery Convention Congress, Maastricht, The Netherlands, 24-29 May 1997, Oxford:
Oxford University Press, 1997b, 239-246.

9
4. De Keukeleire, D., De Cooman, L., Rong, H., Heyerick, A., Milligan, S.R. & Kalita, J., Functional
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9. Goetz, P., Revue de Phytohrapie Pratique, 1990 (4),13-15.
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30, 235-251.
11. Lapcik, O., Hill, M., Hampl, R., Whl, K. & Adlercreutz, H., Steroids, 1998, 63, 14-20.
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Clinical Endocrinology and Metabolism, 1999, 83, 2249-2252.
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J.F. & Deinzer, M., Journal of Clinical Endocrinology and Metabolism, 2000, 85, 4916-4920
14. Miranda, C.L., Stevens, J.F., Helmrich, A., Henderson, M.C., Rodriguez, R.J., Yang, Y.-H.,
Deinzer, M.L., Barnes, D.W. & Buhler, D.R., Food and Chemical Toxicology, 1999, 37, 271-285.
15. Miranda, C.L., Yang, Y.-H., Henderson, M.C., Stevens, J.F., Santana-Rios, G., Deinzer, M.L. &
Buhler D.R., Drug Metabolism and Disposition, 2000a, 28, 1297-1302.
16. Miranda, C.L., Stevens, J.F., Ivanov, V., McCall, M., Frei, B., Deinzer, M.L. & Buhler, D.R.,
Journal of Agricultural and Food Chemistry, 2000b, 48, 3876-3884.
17. Miyamoto, M., Matsushita, Y., Kiyokawa, A., Fukuda, C., Iijima, Y., Sugano, M & Akiyama, T.,
Planta Medica, 1998, 64, 516-519.
18. Mizobuchi, S. & Sato, Y., Agricultural and Biological Chemistry, 1984, 48, 2771-2775.
19. Mukherjee, S. & Sorrell, M.F., American Journal of Clinical Nutrition, 2000, 72, 1073.
20. Oostenbrink, B.C., Pitera, J.W., van Lipzig, M.M.H., Meerman, J.H.N. & van Gunsteren, W.F.,
Journal of Medicinal Chemistry, 2000, 43, 4594-4605.
21. Pike, A.C.W., Brzozowski, A.M., Hubbard, R.E., Bonn, T., Thorsell, A.-G., Engstrm, O.,
Ljunggren, J., Gustafsson, J.-A. & Carlquist, M., The European Molecular Biology Organization
Journal, 1999, 18, 4608-4618.
22. Promberger, A., Dornstauder, E., Frhwirth, C., Schmid, E.E. & Jungbauer, A., Journal of
Agricultural and Food Chemistry, 2001, 49, 633-640.
23. Rimm, E.B., Klatsky, A., Grobbee, D. & Stampfer, M.J., British Medical Journal, 1996, 312, 731-
736.
24. Rong, H., Zhao, Y., Lazou, K., De Keukeleire, D., Milligan, S.R. & Sandra, P., Chromatographia,
2000, 51, 545-552.
25. Rong, H., Boterberg, T., Maubach, J., Depypere, H., Van Slambrouck S., Serreyn, R., De
Keukeleire, D., Mareel, M. & Bracke, M., European Journal of Cell Biology, 2001, in press.
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Experimental Research, 1992, 16, 843-845.
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29. Stevens, J.F., Miranda, C.L., Buhler, D.R. & Deinzer, M.L., Journal of the American Society of
Brewing Chemists, 1998, 56, 136-145.
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M.L., Phytochemistry, 2000, 53, 759-775.
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Agricultural and Food Chemistry 1999, 47, 5059-5063.
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Biomedical Chromatography, 2000, 14, 34-36.
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36. Yilmazer, M., Stevens, J.F., Deinzer, M.L. & Buhler D.R., Drug Metabolism and Disposition,
2001, 29, 223-231.
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378.

10
8

Healthy drinks?- beer and cider

antioxidants
Caroline J. Walker, Louise Bolshaw & Sachin Chandra
Brewing Research International, Lyttel Hall, Nutfield RH1 4HY, United Kingdom (e-mail:
[email protected])

Descriptors
Antioxidant, beer, cider, health, wine

SUMMARY
Antioxidants may help to prevent cancer and cardiovascular disease and are a hot health topic.
The high antioxidant content of red wine is widely recognised (and publicised!), but much less is
known about those in beer and cider. The antioxidant profiles of several popular beers and ciders
have been compared using three different methods of analysis. While the profile of ciders is
similar to that of red wine, beers have a more unique profile. The poster discusses the quality and
quantity of antioxidants in these beverages, and whether beer and cider can make a positive
contribution to our antioxidant intake.

Gesunde Getrnke? Bier- und Cider-Antioxidantien

Deskriptoren
Antioxidantium, Apfelwein, Bier, Gesundheit, Wein

ZUSAMMENFASSUNG
Antioxidantien knnen helfen, Krebs und Herzkranzgef-Krankheit zu verhindern und sind ein
heies Gesundheitsthema. Der hohe Gehalt an Antioxidantien im Rotwein ist weithin bekannt
(und publiziert!), aber weit weniger ist ber die Antioxidantien in Bier und Cider bekannt. Die
Antioxidansprofile einiger populrer Biere und Ciders wurden mit drei unterschiedlichen
Analyseverfahren verglichen. Whrend das Profil des Ciders dem des Rotweins hnlich ist, haben
Biere ein eindeutigeres Profil. Das Poster behandelt die Qualitt und die Quantitt der
Antioxidantien in diesen Getrnken und ob Bier und Cider einen positiven Beitrag zu unserer
Antioxidansaufnahme darstellen knnte.
Des boissons saines? - Teneur de la bire et du cidre en antioxydants

Descripteurs
Antioxydant, bire, cidre, sant, vin

RESUME
Les antioxydants peuvent aider prvenir l'apparition dun cancer et de maladies cardio-
vasculaires et sont un sujet d'actualit brlant. Le taux lev d'antioxydants contenu dans
le vin rouge est largement reconnu (et on en fait mme de la publicit), mais on connat
moins la teneur en antioxydants de la bire et du cidre. Le profil antioxydant de plusieurs
bires et cidres populaires a t compar l'aide de trois mthodes d'analyse diffrentes.
Alors que le profil des cidres est similaire celui du vin rouge, les bires prsentent quant
elles un profil plus particulier. Le poster dcrit la qualit et la quantit d'antioxydants
prsents dans ces boissons et examine si la bire et le cidre peuvent contribuer de manire
positive notre absorption d'antioxydants.

2
INTRODUCTION
A diet high in antioxidants may be protective against cardiovascular disease and cancer,
and there is therefore considerable interest in assessing the antioxidant content of popular
foods and beverages. While the public is very much aware of the antioxidant content of
red wine, much less is known about the antioxidants in beer and cider! Beer and cider are
natural products and could thus make a significant contribution to our daily antioxidant
intake. Cider and beer have a lower ABV than wine or spirits, and because larger
volumes are generally consumed, their contribution to dietary antioxidants could be
significant even although the actual concentration may be modest.

This paper compares methods for assessing quantity and quality of antioxidants in these
popular alcoholic beverages, and discusses the data in relation to standard nutritional
information.

METHODS
Total polyphenol analysis.
Measured according to the EBC method 9.11(3). This is a colorimetric assay, measured at
600nm against a protocatechuic acid standard.

Polyphenol analysis by HPLC

Filtered, degassed samples of beer, cider and red wine were analysed for polyphenols
using a reversed-phase octadecyl silane column, with gradient elution, and fluorescence
detection. Wavelengths of detection are 270 nm (excitation) and 323 nm (emission).

Total antioxidant activity (ABTS)

Th e A B TS R ad ical C ation Q uench in g A ssay

Abs

+ E XTR A CT
[T ro lox ]

1 . G en era tion 2 . A d d ex tract 3 . R ad ical 4 . C om p are to

o f co lou red q u en ch ing a ctiv ity o f
A B T S rad ica l (a n tiox id an t stan d ard
a ctiv ity ) a ntiox id an t
m easured e.g . Tro lox
a s lo ss of
colou r
Figure 1: The ABTS radical cation quenching assay

3
The basis for this assay is shown in figure 1. The method utilises the generation of a long-
lived ABTS [2,2azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] radical cation
chromophore(1). In this assay, AAPH [2,2-azobis(2-methylpropionamidine)
dihydrochloride] is used as a peroxyl radical generator to produce the ABTS radical. The
relative abilities of antioxidants to quench this radical are measured in relation to ascorbic
acid or other relevant antioxidant standards.

HPLC with post-column derivatisation

The concentration of individual antioxidant species within a sample are determined using
a reversed-phase C18 column with isocratic elution. A chemiluminescent assay is
performed on the individual antioxidant species using a post-column reaction pump. The
chemiluminescent signal is generated using luminol with hydrogen peroxide in the
presence of a light enhancer, diethylenetriamine penta acetic acid (DTPA) which emits
blue light. In the presence of compounds with antioxidant activity the chemiluminescence
signal is inhibited, reducing the light output. This results in a negative peak on the post
column chemiluminescence trace. The peaks eluted are dependent on antioxidant activity
and the concentrations of the antioxidant are calculated relative to a standard.

RESULTS AND DISCUSSION

Antioxidant activity varies due to the raw materials used in various beverages and the
processing conditions applied. Antioxidants can be mainly classified as naturally
occurring e.g. vitamins, polyphenols, phenolic acids, or generated by heat e.g.
melanoidins and intermediate compounds formed due to Maillard reactions.

Grapes and apples used in the production of wines and cider are a rich source of
polyphenols. Malts and hops used in the production of beer also contain polyphenolic
antioxidants and, in addition, malts also have heat-induced antioxidants such as
melanoidins that contribute to the total antioxidant activity of beers.

Polyphenols are the major source of antioxidants in beers, ciders and red wine.
Polyphenols can be measured using one of two methods: a total polyphenol assay (EBC
method 9.11), which gives the overall polyphenolic content within a given sample, and an
HPLC fluorescence method of detection which profiles the individual polyphenols within
the beverage. Table 1 shows the range of total polyphenol concentrations found in beer,
cider and red wine. Large variations can be seen in the quantities of polyphenols, with
ciders showing a variation of up to 80%.

Although the total polyphenol assay gives an overview of the polyphenol content, it does
not show the levels of individual polyphenols that may be important.

4
 Sample Polyphenol Portion Polyphenol content
Content (mg/l) Size (mg/portion)
Red wine 1 3182 150ml 477
Red wine 2 3198 150ml 480
Cider 1 996 500ml 498
Cider 2 195 500ml 98
Beer 1 180 500ml 90
Beer 2 126 500ml 63
Beer 3 195 500ml 98
Beer 4 166 500ml 83
Beer 5 182 500ml 91
Beer 6 121 500ml 61
Beer 7 171 500ml 86
Beer 8 130 500ml 65

Table 1: Total Polyphenols measured by EBC method 9.11

A polyphenolic profile (measured by HPLC fluorescence) may be of more use in

highlighting the differences between beverages. Individual polyphenol concentrations are
calculated against an external catechin standard, with the results expressed as catechin
equivalents.

Figure 2 shows the differences between the profiles of beer, cider and red wine. The
profile for beer shows that the polyphenols are more diverse than those found in cider and
red wine, with notable differences in the levels of catechin, epicatechin and coumaric
acid. Coumaric acid, which appears in high concentrations in beer, originates from
cereals and as such would not be expected at significant levels in either cider or red wine.

C a te c h in

4 5 . 0 0

4 0 . 0 0
R e d w in e
3 5 . 0 0

3 0 . 0 0
C o n c e n t r a t io n ( m g / l c a t e c h in e q u i v a l e n t s )

2 5 . 0 0

2 0 . 0 0

1 5 . 0 0

1 0 . 0 0

5 . 0 0

0 . 0 0
1 3 5 7 9 1 1 1 3 1 5 1 7 1 9 2 1 2 3 2 5 2 7 2 9 3 1 3 3

4 5 . 0 0

4 0 . 0 0
C id e r
E p ic a t e c h in
3 5 . 0 0

3 0 . 0 0

2 5 . 0 0

2 0 . 0 0

1 5 . 0 0

1 0 . 0 0

5 . 0 0

0 . 0 0
1 3 5 7 9 1 1 1 3 1 5 1 7 1 9 2 1 2 3 2 5 2 7 2 9 3 1 3 3

4 5 . 0 0

4 0 . 0 0
Beer C o u m a r ic a c id
3 5 . 0 0

3 0 . 0 0

2 5 . 0 0

F e r u lic a c id
2 0 . 0 0

1 5 . 0 0

1 0 . 0 0

5 . 0 0

0 . 0 0
1 3 5 7 9 1 1 1 3 1 5 1 7 1 9 2 1 2 3 2 5 2 7 2 9 3 1 3 3

Peak num ber

Figure 2: Polyphenolic profiles for beer, cider and red wine.

5
The polyphenolic content of a sample has often been used as a guide to antioxidant
content. Figure 3 shows that only a weak correlation exists between polyphenolic content
and antioxidant activity, which indicates that not all polyphenols act as antioxidants.
Antioxidant activity should therefore be measured using more specific methods of
analysis.

150
R2 = 0.5532
140
HPLC (mg/l catechin equivalents)
Total polyphenol concentration by

130

120

110

100

90

80
130.00 150.00 170.00 190.00 210.00 230.00 250.00

Total antioxidant activity (mg/l catechin equivalents)

Figure 3: Correlation between polyphenol concentration and antioxidant activity

Antioxidants can be measured using one of two methods, which measure either the total
antioxidant activity (TAA) or individual antioxidant species (post-column
chemiluminescence HPLC).

TAA is measured utilising a stable and long-lasting ABTS radical cation chromophore,
since the antioxidants present in a sample quench formation of this chromophore (see
figure 1). This method, however, does not highlight the types of antioxidants present.
Typical values for beer, cider and red wine are shown in table 2.

Post-column chemiluminescence HPLC shows the profile of individual antioxidant

species contained within a sample. The presence of antioxidants will suppress the
luminescent light signal proportional to their antioxidant ability. An external calibration
using a catechin standard is used to quantify the species present. Figure 4 shows typical
antioxidant profiles for beer, cider and red wine. The profiles can be categorised into four
groups of compounds: ascorbic acid and related compounds, polyphenolic flavanoids,
catechin and related compounds, and epicatechin and related compounds.

6
 2 0 0 0 0 0

1 5 0 0 0 0 (1:20 dilution)
1 0 0 0 0 0 Epicatechin

5 0 0 0 0
Red wine
0
0 0 : 0 0 0 7 : 1 2 1 4 : 2 4 2 1 : 3 6 2 8 : 4 8 3 6 : 0 0

2 0 0 0 0 0
Ascorbic acid
1 5 0 0 0 0
(1:10 dilution)
Relative Light Units

1 0 0 0 0 0

5 0 0 0 0
Cider
0
0 0 : 0 0 0 7 : 1 2 1 4 : 2 4 2 1 : 3 6 2 8 : 4 8 3 6 : 0 0

2 0 0 0 0 0 Polyphenolic Flavanoids

1 5 0 0 0 0

(1:5 dilution)
1 0 0 0 0 0

5 0 0 0 0
Catechin Beer
0
0 0 : 0 0 0 7 : 1 2 1 4 : 2 4 2 1 : 3 6 2 8 : 4 8 3 6 : 0 0

Time (minutes)

Figure 4: Antioxidant profiles for beer, cider and red wine using HPLC

Although figure 4 would suggest that beer and cider perform less well than red wine, on a
per portion basis cider antioxidants appear in much higher quantities (figure 5).

80.00 Red wine

Cider
Beer
Concentration (mg/portion)

60.00

40.00

20.00

0.00
Ascorbic acid + Polyphenolic Catechin + Epicatechin +
related Flavanoids related related
compounds compounds compounds

Figure 5: Antioxidant group concentrations for beer, cider and red wine on a per portion
basis.

7
Antioxidants and health
One of the advantages of measuring TAA relative to the Trolox standard is that it allows
for ready comparisons between various foodstuffs. The table below, compares the TAA
of beer, cider and red wine with some other foods on a per portion basis (table 2).

Source Portion size T AA activity

(Mol
( T rolox equivalents)
Apple (peeled) 100g 640
Tomato 100g 160
Aubergine 100g 490
Onion 100g 580
W hite wine 150ml 220
Black tea (0.25%) 150ml 1400
Apple Juice 150ml 140
Orange juice 150ml 400
Beer 500ml 910-1340
Cider 500ml 200-5190
Red W ine 150 ml 1340-3400

Table 2: Total antioxidant activities (TAA) of beer, cider and red wine compared to
other common foodstuffs.

Although this table gives a quantitative estimate for the relative TAA values in foods,
there is another equally important consideration in health terms- this is bioavailability, or
the relative ease with which the antioxidants in foods are absorbed. Bioavailability will
be influenced by factors such as the food matrix itself, the type of antioxidants and the
presence of other factors such as alcohol. Generally it would be expected that
antioxidants already in solution, such as in beer, would be more readily and rapidly
absorbed than those in solids. Also, smaller antioxidant molecules may be more readily
absorbed than bulky ones. The relative bioavailability of antioxidants from various foods
can be estimated by using human volunteers. In this type of study, volunteers are asked to
consume a portion of the foodstuff and then the presence of the antioxidant on their blood
or urine is followed over the next 24 hours.

Two such studies on the bioavailability of antioxidants from beer were published last
year. Bourne et al (2) looked specifically at the absorption of the malt-derived antioxidant
ferulic acid. Five volunteers were asked to drink 7 pints of a low alcohol beer over 4
hours. Their urine was then collected and analysed for ferulic acid. Figure 7 shows the
results from one of these volunteers. They found that 100% of the ferulic acid was
absorbed from beer, indicating excellent bioavailability. To put this in context, typical
absorption of ferulic acid from fresh tomatoes is only 11-25%.

8
 10

8
total Ferulic acid (mg)
6

0 500 1000 1500

time (min)

Figure 6: The accumulation of ferulic acid in the urine after drinking beer(2)

In a second study, Ghiselli et al (4) asked their volunteers to drink a more modest 500 ml
of lager and then measured the appearance of antioxidants in the blood (see figure 7).
Overall, after only 1 hour they were able to measure a 17% increase in the TAA in the
volunteers blood. They also found that absorption was more rapid in the presence of
alcohol, indicating that in this case alcohol is increasing the bioavailability of
antioxidants.

2
Caffeic acid
Normalised plasma antioxidant

Sinapic acid
1.8 Vanillic acid
Syringic acid
1.6
concentration

1.4

1.2

0.8
0 0.5 1 1.5 2
Time after beer comsumption (hours)

Figure 7: Increase of Plasma antioxidant concentration after drinking beer (4)

In order to assess the relative value of foods as sources of antioxidants in the diet it is
therefore necessary to have two pieces of information the TAA and the bioavailability.
To date, the bioavailability of antioxidants in beer seems to be quite good which is
probably due to the presence of alcohol and the fact that the antioxidants are already
present in solution.

9
CONCLUSION
Consumers are becoming more focussed on functional foods and are thinking about how
food can make a positive contribution to health. There is currently no recommended daily
intake for antioxidants, but current thinking is that a diet high in antioxidants may be
beneficial to health and that one should try to increase consumption of antioxidant-rich
foods and drinks. It seems that this is therefore an appropriate time to look closely at the
antioxidant content of beer and cider.

The antioxidant profiles of a number of popular beers and ciders have been compared and
the results indicate that, while the profile of ciders is similar to that of red wine, beers
have a unique profile. The polyphenol content of beer and cider does not always correlate
with antioxidant content, suggesting that it is better to measure the antioxidant content
directly to gain accurate information. Total antioxidant activity itself varies widely within
both beverage types reflecting the variety of raw materials used.

Values for TAA are useful in that they allow for comparisons to be made between the
antioxidant content of beer and cider and other foodstuffs. However, HPLC coupled with
an antioxidant-based detection system offers a more detailed profile of the antioxidants
present. It is the quality of the antioxidants present as well as the total amount, which will
be of importance for health, since some antioxidants may be more readily absorbed than
others. We would recommend that both methods should be used together to characterise
beverages.

In conclusion, beer and cider are good sources of antioxidants, and in moderation could
make a positive contribution to the diet. In the future, work with human volunteers will
continue to highlight how well the antioxidants in these beverages are absorbed and what
effect they have on the antioxidant levels in the blood, and by inference, our health.

REFERENCES
1. Araki, S., Kimura, T., Shimizu, C., Furusho, S., Takashio, M. and Shinotsuka, K.
Journal of the American Society of Brewing Chemist 1999. 57(1), 34-37.
2. Bourne, L., Paganga, G., Baxter, D., Hughes, P. & Rice-Evans, C. Free Radical
Research 2000, 32, 273-280.
3. European Brewery Convention, Analytica EBC, Beer polyphenol method, 9.11.
4. Ghiselli, A., Natella, F., Guidi, A., Montanari, L., Fantozzi, P. & Scaccini, C. Journal
of Nutritional Biochemistry 2000, 11, 76-80.

ACKNOWLEDGEMENTS
The authors would like to thank the Chief Executive Officer of BRi for permission to
publish this paper.

10
9

Beer and health research

E. Denise Baxter & Caroline J. Walker
Brewing Research International, Lyttel Hall, Nutfield, Surrey RH1 4HY, United Kingdom (e-
mail: [email protected])

Descriptors
Beer constituent, beer consumption, health, medicine, raw material, research

Deskriptoren
Bierbestandteil, Bierverbrauch, Forschung, Gesundheit, Medizin, Rohstoff

Descripteurs
Consommation de bire, constituant de la bire, matires premires, mdecine, recherche, sant
Beer and Health Research

Hops
In vitro tests suggest that hop compounds may:

be cancer fighting Anti-inflammatory

prevent osteoporosis protect against
be anti-biotic (ulcer prevention) cardiovascular disease
protect against liver
disease
Xanthohumol inhibits growth of
ovarian cancer cells (Miranda, 1999) Research Centres
Oregon State University
120 Buhler & Deinzer
Ghent university, NL
cell growth
80

40
De Keukeleire
0
XN (M)
University of Tokushima, Japan
0 10 20 30 40 Yamamoto & Tobe
Kings College, London
Milligan

Vitamins
2.5
folate (mg/kg dry

The vitamin B9 content of malt 2

wt.)

increases during germination 1.5

beer is rich in B vitamins 0.5

barley 1 2 3 4

high homocysteine levels are a risk Days germination

indicator for CHD

Walker, 2001
folate, vitamin B6 and vitamin B12 can
reduce homocysteine levels

folate
Hom ocysteine m etabolism
Research Centres

Brewing Research International,

THF
m ethionine

UK
1 B 12

5 MeTH F hom o cysteine

IFR, Norwich, UK (Finglas)
B6 Universidad CEU, Spain
1 m ethionine synthase (Moreiras)
THF tetrahydrofolate cysteine
B 12 vitam in B 12
B 6 vitam in B 6

2
Minerals Beer
new research suggests that dietary 500

(umol/fraction)
400
silicon is important for protecting

Urine silica
300
against osteoporosis 200
beer is rich in silicon in the form of silicic 100
acid, which is readily bioavailable 0
0 20 40
studies link diets high in potassium Time (h)
and low in sodium with reduced risk
of stroke
Silicon is readily absorbed from beer
Research Centres
Rayne Institute, London (Powell)
Centre Hospitalier de Toulon, France (Eisinger)
Liverpool University Hospital, UK (Roberts)

Antioxidants
Antioxidants in beer and wine
can protect against free radical ( Paganga, 1999)
damage which causes cancer and moles Trolox
equivalents
cardiovascular disease / drink
2000

1500

mainly found in fruit and vegetables 1000

malt contains natural antioxidants 500

such as ferulic acid and melanoidins 0

hops contain antioxidant flavonoids
Beer White Red
wine
antioxidants in beer can be readily absorbed by the body

10
1.6 Time 0
Ferulic
+ 1 hour
acid
in urine 5
1.4 (mg)
Time
(Minutes)
1.2
0
Whole beer 500ml Alcohol 0 500 1000 1500

Increase in antioxidant capacity of blood Bioavailability of ferulic acid from beer and
after drinking beer tomatoes
Ghiselli, J Nutr. Biochem., 2000 Bourne et al, Free Rad. Res., 2000
Research centres
University of Perugia, Italy (Fantozzi) Guys, Kings & St Thomass
Free Radical Research Group, Rome Hospitals, London (Rice-Evans)
(Ghiselli) IFR, Norwich, UK (Williamson)

3
Alcohol Beer
moderate alcohol consumption moderate consumption of beer
protects against heart disease reduced heart disease
moderate alcohol drinkers were beer and wine were equally
up to 50% less susceptible to effective at reducing heart disease
Alzheimers
a beer a day cut the risk of kidney
moderate drinking is associated stones by 40%
with a decreased risk of non-insulin
dependant diabetes
components in beer counteracted
adverse effects of alcohol on
homocysteine levels in the blood
(see vitamins)
Alcohol consumption and
mortality
Deaths/1000 men

40
Deaths from Plasma homocysteine levels
all causes

30 mol/litre
after drinking
Alcohol related
deaths 14.5
20
Other cancers 14
10
13.5
0
13
0 20 40 60 80
Units alcohol / week 12.5

From Doll, BMJ 1994. 12

Water Beer Wine Spirits

From Van der Gaag, Lancet, 2000.

Malt
Beer is low in sugar and fat
Beer contains a balance of protein,
complex carbohydrates and dietary fibre

Nutritionists advice is to consume less sugar and fat and more complex carbohydrates
such as starch and fibre.

Malted cereals are Overall mortality

the source of most of the
is lower for moderate
vitamins and many minerals in beer Total anti-oxidant
drinkers activity in relation to
Barley cell walls contain the antioxidant ferulic
malt colour
acid Total
Dark malts are rich in melanoidins which are also anti-
oxidant
10
8
antioxidants activity
6
4
2
Research Centres 0
0 1000 2000

Brewing Research International, UK Malt Colour (EBC)

Weihenstephan, Germany
Heasman & Hughes, 1997

4
 Sources of information
Alcohol Research (TNO Nutrition & Food Research, The Netherlands)
an international review journal covering articles on the biomedical and
psychosocial effects of alcohol use and misuse; 6 issues per year
Health Benefits Network (Caroline Walker, BRI)
interpretation and comment on recent relevant articles: bimonthly; e-mail with
annual hard copy.
Alcohol Issues Insights (editor Erik Shepard, New York)
pamphlet summarising current alcohol issues, mainly in the USA, particularly
social; monthly.
Quarterly Review of Alcohol Research (Portman Group, London)
a review with abstracts of published papers on social and medical aspects or
alcohol research
Alcohol in Moderation Magazine (editor Helena Connibear, UK)
articles and commentary of alcohol, legislation and health
www.aim-digest.com
Cerveza y Salud (Dr. Javier Posada, Madrid)
commissions research and articles on beer and health
www.cervezaysalud.org

For further information, contact Caroline Walker

([email protected])

Beer and Health Research;

Published References

Alcohol and heart disease: key early studies

Yano, K. et al, New Eng. J. Med., 1977, 297(8), 405 (The Honolulu Heart study).
Marmot, M.G. et al, Lancet 1981, 8220, 580 (Civil servants, UK).
Jackson, R. et al, BMJ 1991, 303(6796), 211 (Auckland study, New Zealand).
Klatsky, A.L., Alcohol Clin. Exp. Res., 1994, 18, 88-96 (The Kaiser-Permanente study,
USA).
Doll, R. et al, BMJ., 1994, 309, 911 (British Doctors Study, UK).
Gronbaek, M., et al, BMJ, 1994, 308(6924), 302 (The Copenhagen Heart study,
Denmark).
Berberian, K.M. et al, Eur. J. Epidemiol., 1994, 10(5), 587 (Erasmus study, the
Netherlands).
Fuchs, C.S. et al, New Eng. J. Med., 1995, 332(19), 1245 (Nurses Health Study).
Shaper, A.G., et al, Int J Epidemiology, 1997, 26(3), 523 (British Regional Heart
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Yuan, J-M. et al, BMJ, 1997, 314, 18 (Shanghai study, China).

5
Alcohol and heart disease recent studies
Williams, P.T., Am.J.Clin. Nutr., 1997, 66(5), 1197 (National Runners Health study
shows independent effects of exercise and alcohol).
Simons, L. A. et al, MJA, 2000, 173, 121 (The Dubbo study, Australia).
Hendriks, H.F.J. et al, Alcoholism: Clin.Exp. Res., 2001, 25, 563 (Benefits of alcohol in
high and low risk populations).

Biochemical mechanisms for protection against heart disease

Rimm, E.B. et al, BMJ, 1999, 319, 1523 (Meta analysis, increase in HDL and
apolipoprotein A1, decrease in clotting factors).
Nanchahal, K. et al, Int. J. Epidemiology, 2000, 29, 57 (Moderate alcohol consumption
in women decreases BMI, total cholesterol and increases HDL and apolipoprotein A1).
Imhof, A. et al, Lancet 2001, 357 (9258), 763 (Moderate alcohol consumption lowers
CRP, a protein marker for inflammation which is a risk factor for heart disease).
Van Tol, A. and Hendrks, H.F.J., Curr. Opin. Lipidol., 2001, 12, 19 (Review of
protective mechanisms).

Alcohol type
Marques-Vidal, P. et al, Amer. J Epidemiol., 1996, 11, 1089 (ECTIM study compares
wine and beer drinking populations).
Rimm, E.B. et al, BMJ, 1996, 312, 731 (Review).
Keil, U. et al, Epidemiology, 1997, 8(2), 150 (Coronary heart disease in a beer
drinking population).
Brenner, H. et al, Int J Epidemiology, 1997, 26, 85 (Mortality in a beer drinking
population).
Gaziano, J.M. et al, Am J Cardiol., 1999, 83, 52 (Type of beverage and heart attack).
Cleophas, T.J., Biomed. & Pharmacother, 1999, 53, 417 (Review).
Bobak, M. et al, BMJ, 2000, 320, 1378 (Heart attack and beer drinking).
Gronbaek, M. et al, Ann. Int. Med., 2000, 133, 411.

Alcohol and stroke

Wannamethee, S.G. et al, Stroke, 1996, 27(6), 1033 (Heavy drinking increases stroke
risk).
Wannamethee, S.G. et al, Neuroepidemiology, 1998,17, 288 (Review, J-shaped Curve).
Berger, K. et al, New Eng. J. Med., 1999, 341(21), 1557 (US male physicians study,
protective effect of light to moderate drinking).
Sacco, R.L. et al, JAMA, 1999, 281, 53 (Northern Manhattan Stroke study, protective
effects of moderate drinking).

Drinking patterns
Kauhanen, J. et al, BMJ, 1997, 315, 846 (Binge drinking and increased mortality,
Kuopio study, Finland).
Kauhanen, J. et al, Arterioscler. Thromb. Vasc. Biol., 1999, 19, 3001 (Binge drinking
and atherosclerosis, Kuopio study, Finland).
Hart, C.L. et al, BMJ, 1999, 318, 1725 (Risks associated with binge drinking,
Scottish study).
Britton, A. et al, J Epidem. Comm. Health, 2000, 54, 328 (Meta analysis of drinking
patterns and mortality).

6
Alcohol and diabetes
Stampfer, M.J. et al, Am J Epidemiol., 1988, 128(3), 549 (Nurses Health Study, lower
incidence of non-insulin dependent diabetes in moderate drinkers).
Mayer, E.J.M. et al, Circulation, 1993, 88(5), 2190 (Kaiser Permanente Twin Study,
moderate drinking lowers risk factors for non-insulin dependent diabetes).
Rimm, E.B. et al, BMJ, 1995, 310, 555 (Health Professionals Study, moderate alcohol
consumption increases insulin sensitivity and reduces risk of diabetes).
Lazarus, R. et al, Am. J. Epidemiol., 1997, 145(10), 909 (Normative Aging Study, USA:
moderate drinking reduced risk factors for non-insulin dependent diabetes).
Tsumura, K. et al, Diabetes Care, 1999, 22(9), 1432 (Moderate alcohol consumption
reduced risk of non-insulin diabetes in Japanese men).

Alcohol and dementia

Hellenbrand, W. et al, Neurology, 1996, 47(3) 644 (Inverse association between
Parkinsons disease and moderate drinking, Germany).
Orgogozo, J-M. et al, Rev. Neurol (Paris), 1997, 153(3), 185 (Inverse association
between moderate drinking and dementia, France).
Cervilla, J.A. et al, Brit. J. Psychiatry, 2000, 177, 66 (Moderate drinking protects
against age- related deterioration of cognitive function, UK).
Cupples, L.A. et al, Alzheimers Reports, 2000, 3(2), 105 (Regular moderate drinkers
had significantly reduced risk of Alzheimers, MIRAGE study, Boston USA).

Kidney stones and gallstones

Curham, G.C. et al, Ann.Int.Med., 1998, 128(7), 534 (Increased fluid intake reduces
risk of kidney stones in women).
Caroli-Bosc, F-X. et al, Digestive Diseases Sci., 1998, 43(9), 2131 (Moderate alcohol
consumption protects against gallstones, France).
Attili, A.F. et al, Hepatology, 1998, 27(6), 1492 (Moderate alcohol intake protects
against kidney stones in men).
Hirvonen, T. et al, Am.J. Epidemiol., 1999, 150(2), 187 (Magnesium and moderate beer
consumption decreased risk of kidney stones, ATBC study, Finland).
Leitzmann, M.F. et al, Alcoholism, 1999, 23(5) (Regular moderate intake of alcohol
decreases risk of gallstones in men).

Alcohol and ulcers

Aldoori, W.H. et al., Epidemiol., 1997, 8(4), 420 (Small inverse association between
moderate drinking and duodenal ulcers, Health Professionals follow up study, USA).
Brenner, H. et al, Epidemiol., 1999, 149, 571 (Protective effect of moderate drinking for
H. pylori infections, Southern Germany).
Brenner, H. et al, Epidemiol.,1999, 10, 214 (Protective effect of moderate drinking
against H pylori infection, Western Germany).
Ogihara, A. et al, Gastroent. Hepatol., 2000, 15(3), 271 (Protective, dose-dependent
effect of alcohol on H pylori infection).

Osteoporosis
Johnell, O. et al, J Bone Mineral Health, 1995, 10(11), 1802 (Moderate intake of
alcohol protects against fractures in young women).
Naves-Diaz, M. et al, Osteoporosis Int., 1997, 7(1), 65 (Moderate drinking protects
elderly women from vertebral deformity; European Vertebral Osteoporosis Study
Group).

7
Feskanich, D. et al, J Womens Health, 1999, 8(1), 65 (Women who were moderate
drinkers had higher bone density than abstainers).

Antioxidants
Paganga, G. et al, Free Rad.Res., 1999, 30, 153 (Antioxidant content of beer & wine,
Guys Hosp, UK).
Gorinstein, S. et al, Nutr. Res., 2000, 20(10), 131 (Phenolics in beers and wines).
Pignatelli et al, Am. J. Clin. Nutr., 2000, 72, 1150 (Antioxidant flavonoids can inhibit
blood clotting in vitro).
Kampa, M. et al, Nutr. Cancer, 2000, 37(2), 223 (Antioxidant flavonoids can inhibit
cancer cell
proliferation in vitro).
Ghiselli, A. et al, J. Nutr. Biochem., 2000, 11, 76 (Beer increases antioxidant status of
blood).
Bourne, L. et al, Free Rad. Res., 2000, 32(3), 273 (Antioxidant ferulic acid is readily
absorbed into the blood from beer, Guys Hospital and BRi, UK).
Scalbert, A.and Williamson.G., J. Nutr. 2000, 130 (Suppl.8S) 2073S. (Bioavailability of
flavonoids such as quercetin, IFR, UK).

Vitamins
Ubbink, J.B. et al, Atherosclerosis, 1998, 140, 349 (Raised blood homocysteine is a risk
factor for heart disease; dietary folate is protective Caerphilly study, Wales).
Zhang, S. et al, JAMA, 1999, 281(17), 1632 (Dietary folate may protect against breast
cancer, Nurses Health Study, USA).
Verhaar, M.C. et al, Circulation, 1999, 100, 335 (Folate can protect against the damage
to blood vessels associated with atherosclerosis, Netherlands).
Bellamy, M.F.et al, Eur. J. Clin. Invest., 1999, 29(8), 659 (Dietary folate supplements
improve cardiovascular function, Wales, UK).
Van der Gaag, M.S. et al, Lancet, 2000, 355(9214), 1522 (Moderate beer drinking
counters the rise in blood homocysteine caused by alcohol, TNO, Netherlands).
Walker, C., Proc. 28th Congress Eur. Brew. Conv., Budapest, 2001, in press (Folate in
beer, BRI, UK).

Minerals
Potassium
Suter, P.M., Nutr. Rev., 1999, 57(3), 84 (Dietary potassium, magnesium and fibre can
protect against stroke, Switzerland).
Fang, J. et al, Stroke, 2000, 31, 1532 (Dietary potassium may protect against stroke,
NHANES I study, New York, USA).

Silicon
Anderson, I.W. et al, Proc. Eur. Brew. Conv. Brussels, 1995, 543 (Beer is a good source
of bioavailable silicon).
Rico.H. et al, Calcif. Tissue Int., 2000, 66(1), 53 (Dietary silicon may protect against
osteoporosis by improving bone mass, rat study, Spain).

Hops
Tagashira, M. et al, Biosci.Biotech.Biochem., 1995, 59(4), 740 (Radical-scavenging
activity of hop bitter acids, Japan).

8
Tobe, H. et al, Biosci. Biotech.Biochem.,1997, 61(1), 158 (Humulone can inhibit
resorption of bone, Japan).
Honma Y. et al, Leukemia Res., 1998, 22(7), 605 (Humulone inhibits growth and
proliferation of leukemia cells in vitro, Japan).
Miranda, C.L. et al, Toxicol., 1999, 48, 348 (Xanthohumol inhibits growth and
proliferation of human prostate cancer cells in vitro, Oregon, USA).
Henderson, M.C. et al, Toxicol., 1999, 48, 98 (Prenylated flavonoids from hops inhibit
the activity of carcinogens in vitro, Oregon, USA).
Rodriguez, R.J. et al, Toxicol., 1999, 48, 197 (Antioxidant and antihepatotoxic activities
of prenylated flavonoids from hops, Oregon, USA).
Miranda, C.L. et al, Cancer Letters, 2000, 149, 21 (Prenyl flavonoids from hops can
induce quinone reductase, a enzyme which can break down carcinogens, in cultured
cells, Oregon, USA).
Milligan, S.R. et al, J.Clin.Endocrinol.Metab., 2000, 85(12), 4912 (Phytoestrogenic
activity of prenylated flavonoids in hops, Kings College, UK; Ghent, Belgium; and
Oregon, USA).

Malt
Griggs, D.L. et al, Proc. 5th Int.Brew.Conf., Harrogate, 1992, 259 (Reducing agents
from dark malts, UK).
Maillard, M-N, J.Sci.Agric.Food Chem.,1995, 43(7), 1789 (Availability of antioxidant
phenolic acids in malt increases during kilning, France).
Muller, R.M. and Chandra, S., BRI Q., 1998, 6 (Antioxidants in malt and extraction into
beer, BRI, UK).
Goupy, P. et al, J.Sci.Food Agric. Chem., 1999, 79(2), 1625 (Quantification of phenolic
compounds and other antioxidants in barley and malt, IFBM, France).

9
10

The barley and malt factors underlying

lautering performance: identification
and use in barley breeding programs
Christopher M. Ford & Evan Evans
Adelaide University, Barley Biochemistry and Brewing Research Laboratory, Department of
Plant Science, Waite Campus, PMB1, Glen Osmond, SA 5064 South Australia (e-mail:
[email protected])

Descriptors
Barley quality, breeding, -glucan, lautering, mash separation time

SUMMARY
Lautering remains a bottleneck in breweries. Malt factors causing reduced-lautering (run-off
rates and extract recovery) are poorly characterised and breeding of barleys with improved
brewery performance has been largely ignored. A laboratory-scale device of 1 kg malt
capacity was built, replicating grist loading and run-off rates of commercial lauter tuns.
Preliminary results gave discrimination of the lautering performance of several Australian
malts. By operating the device at temperatures used commercially, interference from -
glucans was reduced. Malt characteristics that promote or retard lautering can be targeted
within existing breeding programs to produce barley varieties closely attuned to the needs of
brewers.

Den Gersten- und Malzfaktoren zugrunde liegende Luterleistungen: Erkennung und

deren Verwendung fr die Gerstenzucht

Deskriptoren
Ablutern (Luterbottich), Gerstenqualitt, -Glucan, Luterdauer, Zchtung

ZUSAMMENFASSUNG
Der Luterprozess ist noch immer ein Engpassfaktor in der Brauerei. Malzfaktoren, welche
die Luterleistung (Ablaufraten und Extraktausbeute) verringern, sind unzureichend
charakterisiert. Die Zucht von Gerstensorten, die den Anforderungen der Brauereien gengen,
wurde weitgehend vernachlssigt. Eine Einrichtung im Labormastab, mit dem Fassungs-
vermgen von einem kg Malzschrot, wurde errichtet. Die Schrotbeladung und die Ablauf-
zeiten wurden den grotechnischen Parametern nachempfunden. Erste Ergebnisse spiegelten
Unterschiede der Luterleistung in Abhngigkeit von verschiedenen australischen Malzsorten
wieder. Beim Betrieb der Anlage mit blichen Temperaturen konnten die Probleme durch -
Glucan reduziert werden. Malzcharakteristika, die den Luterprozess beschleunigen oder
verlangsamen, knnen erfasst werden. Nun ist es mglich, im Rahmen von Zuchtprogrammen
Gerstensorten zu zchten, die den Anforderungen der Brauer entsprechen.
Facteurs de l'orge et du malt la base de la filtrabilit en cuve-filtres: identification et
utilisation dans les programmes de culture d'orge

Descripteurs
Dure de sparation de la maische, -glucane, obtention (culture), qualit de lorge,
sparation de la maische en cuve filtre

RESUME
La filtrabilit en cuve-filtres demeure un goulot d'tranglement dans les brasseries. Les
facteurs du malt rduisant cette filtrabilit (taux d'coulement et rcupration de l'extrait) sont
mal caractriss et la culture d'orges aux meilleures performances de brassage a t largement
ignore. Un systme de laboratoire d'une capacit de 1 kg de malt a t construit, reproduisant
le chargement du grain broyer ainsi que le taux d'coulement des cuves de maltage
commerciales. Les rsultats prliminaires ont donn une discrimination des performances de
filtrabilit de plusieurs malts australiens. L'interfrence des -glucanes a t rduite en faisant
fonctionner le systme des tempratures commerciales. Les caractristiques du malt
favorisant ou retardant la filtrabilit peuvent tre vises dans les programmes de culture
existants, pour produire des varits d'orge rpondant exactement aux besoins des brasseurs.

2
INTRODUCTION
The common thread linking each new malting variety released by barley breeders is
an improvement in levels of extract or diastase activity when compared with the
variety to be replaced. Traditionally this is achieved through selection specifically for
these factors, with little or no emphasis placed on the likely performance of the
variety when it is malted and passes into the brew-house. Modern-day brew-houses
may operate at up to 10-12 brews per day, and it is generally accepted that the rate-
limiting step of the processes from milling to chilling lies with the separation of
sweet wort and spent grains following mashing. The means by which this is achieved
has varied with brewing styles and the advent of hydraulically actuated press-filters,
but in each case there is a premium to be had by recovering the maximum extract in
the minimal cycle time.
Of each of the unit processes identified in the brewing process, it may be suggested
that over the history of brewing research, the separation of sweet wort from spent
grains has received the least investigation and scientific enquiry. This may reflect the
relatively straightforward nature of the process, which in essence is simply a gravity-
driven run-off of liquid from a supported solid bed. Alternatively, the complex nature
of the mathematics needed to fully describe fluid dynamics in compressible cakes
may have proved too opaque for any meaningful information to be quickly and
usefully extracted. Whatever the reason, there has been only a scant handful of reports
over the last 30 years or so in which any serious effort has been made to provide a
technical or scientific explanation of the principles underlying the process of wort and
grain separation.
In the unending quest for improved malting barley varieties, there has to the best of
our knowledge been no attempt made to select directly for improved performance
during the wort separation process. We have recently started a research project that
aims for the first time to provide barley breeders with a measurable parameter for the
prediction of potential efficiency in the wort separation process. This will be achieved
by developing an operational understanding of the most common form of wort
separation, namely lautering, and determining the role of individual barley and malt
factors in the process. Subsequently, tests to detect the presence or absence of
desirable or deleterious components will be developed or adopted from existing
analytical protocols, and applied to barley lines undergoing evaluation within our
breeding programs.

Lautering research and development a brief historical perspective

The heyday for lautering research on a laboratory scale seems to have been the period
of 20 years or so between about 1970 and 1990. Both before and since that time, few
if any reports of new developments or understandings in lautering have appeared.
The exceptions to this sorry state of affairs may perhaps be found in two excellent and
comprehensive overviews of lautering and mash separation by Smith (1997) and
Geering (1996). Both articles were published in the trade press rather than the
scientific or technical literature, and are the more usefully informative for the efforts
their authors have made to address their specific readerships.
During the 1970s, several papers were published that described the development of
increasingly sophisticated laboratory-scale lautering vessels. These were often of all-
glass construction, and were designed to allow the assessment of a number of factors
in apparatus that mimicked the salient features of brewery lauter vessels. For instance,
Crabb and Bathgate (1973) and Bathgate et al (1975) reported the construction and
operation of a combined mash and lauter vessel with a capacity of 1 kg malt. The

3
equipment incorporated a host of control units and measurement devices to allow
monitoring of wort run-off rates and differential pressure across the bed of spent
grains, and was capable of discriminating between two malts with quite similar
specifications. Similar, although less complex designs were reported also by Huite
and Westermann, (1974), Webster (1981), Armitt et al, (1984) and Laing and Taylor
(1984).
It is interesting to note that 20 years ago, Webster and Portno (1981) stated that the
lautering performance of malt is a factor of increasing economic significance...any
incidence of poor lautering will inevitably lead to loss of valuable extract and low
brew-house yield; yet it remains largely a matter of serendipity if a new barley
variety performs well during the wort separation process. The outcome of their work
was the derivation of an expression that allowed the prediction of wort run-off times
from a set of malt parameters, namely the grist mean particle size, the sedimentation
value, wort viscosity and the volume of fine particles.
This idea built on the principle first suggested by Bathgate et al (1975), that there was
a need to select parameters which truly reflect performance on the large scale rather
than to make observations on a miniature replica of commercial plant. With regard to
lautering, this had specific relevance to the dimensions of the spent grain bed, which
if merely reproduced in a scaled-down version would present minimal depth of grains
and thereby negligible filter bed capacity.
At least three experimental devices have been developed that specifically target
components of the lautering or wort separation process on a laboratory scale. Barrett
et al (1973) and Bathgate and Palmer (1975) reported a novel type of inverted mash
filter placed in a glass beaker into which a mash was poured. The filter, a Perspex disc
fitted with a wire mesh to prevent blockage by small particles, was beneath the outlet
tube, through which wort was drawn upwards on the establishment of a siphon effect.
The grain bed itself acted as the primary filter for wort run-off, with the wort flow
controlled via a tap in the run-off tubing. Similarly, Moll and Flayeux (1980)
described the Tepral device for mash filtration. This is essentially a pressure vessel
attemperated to ca 78C, the operating temperature of brew-house lauter tuns, into
which mash is transferred prior to filtration through a membrane under the influence
of an applied positive pressure. Whilst few brew-house lauters use positive pressure in
this way, the device has found considerable use in several laboratories. Neither of
these laboratory-scale devices aim to replicate the entire lauter tun, but rather to allow
detailed analysis of specific components or steps of the process. A third means by
which laboratory-scale lautering has been investigated is via the use of Buchner
funnels lined with filter paper (Brown et al, 1990, Sjholm et al, 1994). The flat-
bottomed design of the Buchner funnel suggests comparison with the geometry of a
brew-house lauter, and although as mentioned earlier a proportional scale-down from
full-size will inevitably result in a very shallow grain bed, the results obtained using
Buchner funnels are among the most reliable of all experimental lautering
measurements.

The control of lautering

The outcome of the research summarised in the previous section has been the
determination of a list of factors that in various ways have an impact on lautering
efficiency. The measurement of lautering efficiency has been achieved by several
means, including time to achieve the run-off of a pre-determined wort volume
(Webster and Portno, 1981), differential pressure across the grain bed (Laing and

4
Taylor, 1984) and integration under the curve of differential pressure against filtration
time (Armitt et al, 1984).
In most cases, the parameters controlling lautering efficiency can be split into physical
factors and those derived from barley and malt. The purely physical parameters
include the effects of grain bed geometry (shallow beds filter more quickly than deep
ones), temperature (higher temperature equates to faster run-off, but at the cost of
extraction of lipids and, from the husks, tannins), flow rate (lautering is a combination
of filtering of first worts from spent grain and leaching of soluble sugars into the
sparge water; at excessive flow rates there is insufficient time for adequate leaching to
occur) and the size of the particles that make up the grain bed (smaller particles give
better extraction but a lower flow rate). Successful lautering is therefore a
compromise between these and other factors, superimposed on which are the effects
arising from the mash that is to be separated.
Numerous biological factors have been shown to play important roles in lautering,
including many associated with malt proteins and their breakdown (Moonen et al,
1987). The role of protein oxidation and its effects on the formation of the
impenetrable oberteig layer that forms on the surface of the grain bed during
lautering remains an area of active research in at least one laboratory (Saara Poyri,
VTT Finland, personal communication).
Perhaps the most widely studied malt components with regard to lautering are the
non-starch polysaccharides, namely the -glucans and arabinoxylans. These
molecules, found in the endosperm cell walls and released during germination
(malting) and to some extent mashing, are responsible for large increases in the
viscosity of solutions in which they are dissolved. This can cause innumerable
problems with subsequent filtration processes, and for the -glucans especially,
breeding programs select for varieties in which levels of malt and wort -glucan are
low (which may reflect either low levels of synthesis, high levels of hydrolytic
degrading enzymes or both).
Barrett et al (1973) pointed out that at lautering temperatures (ca 74-78C), -glucan
viscosity is minimal, and that their effects in impeding wort run-off may be due to
formation of cementing material, which causes the grain bed to become increasingly
impervious. Certainly, the common brewery practice of adding -glucanases to the
mash vessel would result in their removal from the wort, but the exact role of other
non-starch polysaccharides, and of intramolecular interactions between -glucans and
arabinoxylans in determining wort run-off and spent grain behaviour demands further
research (Izydorczyk and MacGregor, 2000).

Lautering research for the new millennium

It is clear that although no consensus exists by which effective and efficient behaviour
of malt during lautering may be completely predicted, a considerable body of
knowledge exists that describes specific factors important in the process. Our research
will focus first on gaining an understanding of the particular barley or malt factors
that are associated with efficient lautering. When this has been achieved, we will be
able to provide to the Australian barley breeding community tools by which new
varieties may be selected with improved performance in the brew-house.

5
DESIGN, CONSTRUCTION AND OPERATION OF A SMALL-SCALE
LAUTER TUN (SSLT)
Design of the laboratory-scale lauter tun used in this study was based on several
devices reported in the literature. The main design considerations were the need to
mimic brewery conditions of malt loading, grain column dimensions and run-off flow
rates, while simultaneously measuring meaningful parameters of malt performance
during lautering. It was decided to work with approximately 1 kg malt per trial, using
both free-flow and pumped run-off of worts to determine the best set of experimental
lautering conditions.
Similarly, a number of parameters for measuring grain bed performance were chosen.
These included differential pressure across the grain bed, the volume of wort collected
and the decrease in depth of the grain bed. A glass column 900 mm long with an
internal diameter of 80 mm was cut and fitted with Perspex ends. A false bottom of
commercial manufacture (a kind gift from Briggs of Burton Ltd.) with an aperture of
ca 10% was fitted into a 100 mm diameter Quickfit flask lid and the entire lower
assembly was fastened to the column using wing nuts and bolts. An attemperating
glass jacket was sealed in place and tested for water tightness. Attemperating water
was pumped from a thermostatically controlled water bath, and the complete small-
scale lauter tun (SSLT) mounted onto a trolley to allow secure and easy access (see
figure 1). Malt was milled using a Valley 2-roll home-brew mill, with roll diameters
of 31.75 mm, driven by a cordless electric drill running at 350 rpm. Conditions for
mashing were based on the Small Scale Brewing protocol developed previously in the
laboratory (Stewart et al, 1998), and a grist:liquor ratio of 3:1 was chosen accordingly.

.
Figure 1: The Small-scale lauter apparatus

Lautering performance is assessed by measuring the differential pressure across the

bed of spent grains by way of a water manometer connected beneath the false bottom;
in later developments this has been supplemented with an electronic pressure
transducer connected via an analogue-to-digital converter to a laptop computer. The
height of water in the manometer is compared to the height of liquid within the lauter

6
vessel at regular intervals following mash transfer and the recirculation and run-off
that follows. The differential pressure is then plotted against time, or when comparing
lautering trials at different run-off rates, against the volume of wort passed through
the grain bed. The use of rakes or knives was decided against in the design of the
SSLT; the proportion of the grain bed surface area cut would be much larger than that
occurring in a commercial lauter, thereby causing disproportionate increases in bed
channel to grain bed ratio.

Preliminary trials were promising, with a pumped flow of wort and sparge liquor
proving to be more reliable than allowing flow under gravity alone. Operating
conditions including temperature, flow rates and regimes for mash transfer and grain
bed establishment were critical for consistent lautering performance. A standard
protocol was developed, and was used to determine the sensitivity of the device to
differences in malt quality with malts obtained from local suppliers. It soon became
apparent however that differences in run-off performance, determined as described
above, were very slight for all of the malts tested; moreover, it was not possible to
discriminate any of the malts tested on the basis of their performance in the
experimental lauter. This situation arose from at least three considerations, each of
which is addressed below.
The first factor is that lautering with run-off rates comparable to those used in modern
breweries (between 3.6 and 10 hl m-2 hr-1) causes only a very small increase in
differential pressure across a well-formed grain bed. We saw maximum differential
pressure values of between 250 and 400 mm water across a 30 to 45-cm grain bed
with run-off at 80 to 100 ml/minute (50 cm2 cross-sectional area); these values are
equivalent to between 0.35 and 0.56 psi.
The samples supplied by Australian malting industry collaborators for use in our
research necessarily have been chosen from barleys and malts destined for use in
breweries, and therefore are all of largely similar specifications. It is unlikely
therefore that since they represent varieties that have found favour with brewers, they
will exhibit poor lautering behaviour; thereby making it difficult to discriminate them
under laboratory conditions.
A third explanation for the difficulties seen in discriminating malts by experimental
lautering lies with the design and operation of the SSLT, and more specifically with
the replication of specific and critical components of the brew-house process at a
laboratory scale. This is discussed further below.
Arising from visits to a number of breweries in Australia, two factors have been
identified as critical to the success of laboratory scale lautering. These are grist
composition, and establishment of the initial spent grain bed and the subsequent
management of wort run-off. With regard to grist composition, we are currently
investigating the use of larger diameter and more precisely adjustable rolls in the mill
to provide more consistent and representative crushing of malt, especially from under-
modified samples.
Two alternative strategies for managing wort run-off were identified from
observations of brew-house lauter tun operation in Australian breweries. The first
method uses an essentially constant run-off rate for establishment of the grain bed and
the subsequent run-off of first worts and sparge that follows. This protocol appears to
be widely used in Australian breweries, and results in lauter cycle times of the order
of 2.5 to 3.5 hours. A second approach, whereby establishment of the grain bed is
achieved very quickly with extremely fast initial run-off rates during recirculation, is
used in breweries with a high throughput (ca 18+ runs per day via a twin-stream

7
brew-house). Run-off proceeds at high rates following bed establishment, with
continuous monitoring of differential pressure and flow rate used to control the depth
to which raking is employed. Cycle times in the order of 90 minutes are achieved
using this method.
Two laboratory lautering protocols were devised to reflect the different strategies by
which Australian brewers manage wort run-off:
(i) A constant-rate recirculation, run-off and sparge at relatively low (30-40 ml
min-1 for 50 cm2 false bottom, equivalent to ca 3.6 to 4.8 hl m-2 hr-1) or higher,
80-100 ml min-1 (equivalent to 9.6 to 100 hl m-2 hr-1) flow rates.
(ii) ii) An initial free-flow run-off (ca 600 ml min-1, equivalent to 72 hl m-2 hr-1)
for 15-30 secs followed by recirculation, run-off and sparge at 80-100 ml min-1
(equivalent to 9.6 to 100 hl m-2 hr-1) (see figure 2).
Using a constant run-off rate, a clear difference in differential pressure was seen
for the two rates, but a ca 20% increase in the final depth of spent grains occurred
at the lower rate. The difference in maximum differential pressure across the spent
grains bed between the two run-off rates was only 0.35 psi, suggesting that
accurate discrimination of malts by this protocol would be very difficult.
An initial free-flow run-off with the outlet tube placed 35 cm above the false
bottom gave a higher differential pressure for the same malt than with constant-
rate run-offs. Additionally, the final height of the spent grains bed was lower,
suggesting that bed formation was more complete using this protocol than with a
constant-rate run-off.
At constant run-off rates, differential pressure (DP) across the spent grain bed varied
with flow rate. Maximum DP across the spent grain beds differed by only 0.35 psi
between the two run-off rates (figure 2).

400 65

350
60

300

55
250
Differential pressure (mm H2O)

Height of grain bed (cm)

200
50
DP, low flow rate
Bed height, low flow rate
150 DP, high flow rate
Bed height, high flow rate
45
100

50
40

35
-50

-100 30
0 1000 2000 3000 4000 5000
Volume of wort filtered (ml)

Figure 2: Lautering performance of Sloop malt at two run-off rates.

8
An initial free-flow run-off gave a higher DP than with constant rate run-offs (figure
3). Also, the final height of the spent grain bed was lower, suggesting that bed
formation was more complete than by constant rate run-off.

500 50

400 45

Height of grain bed (cm)

Differential pressure (mm H2O)

40
300

35
200

30

100

Differential pressure
Height of grains bed 25
Depth of fines above surface

Fines (cm)
0 20

10

-100 0
0 10 20 30 40 50 60
Time after mash transfer (minutes)

Figure 3: Lautering performance of Harrington malt using a 30-second free-flow run-

off to initiate grain bed formation.

Reproducibility between duplicate runs using either protocol was low, with
unacceptably high variation in differential pressure and spent grain bed formation.
Further trials are underway in an attempt to improve the reproducibility of the SSLT.

A dual-lauter apparatus has been built for the simultaneous analysis of two malt
samples milled, mashed and lautered under identical physical parameters. This will
allow comparisons to be made between different malts (variety, modification,
treatment during mashing etc) and also between specific malt treatments, for example
the effect of enzyme addition during the mash cycle. Additionally, the use of
electronic pressure transducers beneath the false bottom of each lauter will allow
continuous monitoring of pressure changes during spent grain bed development rather
than the discrete sampling of manometer levels used earlier.

CONCLUSIONS
A laboratory-scale lauter tun was designed and successfully tested that enables the
comparison of malts under conditions that mimic those found in a commercial brew
house. Experimental lautering protocols have been developed that reflect the
conditions used in Australian breweries for establishment of the spent grains bed and
run-off of sweet wort. Only very small differences were seen in the experimental
lautering performance of a number of commercial Australian malts. We are currently
addressing the remaining technical shortcomings with the experimental lautering

9
apparatus and the parameters needed to ensure reliable and consistent data. With the
completion of the preliminary stages of this project, our research efforts will now be
directed towards the precise determination of malt factors influencing lautering
efficiency, and developing assays that will permit their rapid and accurate
measurement, thereby providing breeding programs with a valuable tool in the
continuous search for better malting barley varieties.

ACKNOWLEDGMENTS
This work is supported by the Grains Research and Development Corporation of
Australia. We thank numerous members of the Australian barley breeding, malting
and brewing industries for their assistance during development of the project.

REFERENCES
1. Armitt, J.D.G., Healy, P.J. and Coates, K.D. (1984). Proc. 18th Convention,
Institute of Brewing (Australia and New Zealand Section), Adelaide, 100-108.
2. Barrett, J., Clapperton, J.F., Divers, D.M. and Rennie, H. J. (1973). J. Inst.
Brewing, 79, 407-413.
3. Bathgate, G.N. and Palmer, G.H. (1975). Proceedings of the European Brewery
Convention Congress, Nice, 61-83.
4. Bathgate, G.N., Bennett, H.O. and Crabb, D. (1975). J. Inst. Brewing, 81, 225-
231
5. Brown, A.T., Keay, R. and Pearson, P.L. (1990). Proc. 3rd Aviemore Conf. Malt,
Brew., Distill., Aviemore. Institute of Brewing, London, 313-318.
6. Crabb, D. and Bathgate, G.N. (1973). J. Inst. Brewing, 79, 519-524.
7. Geering, P. (1996). Brewers Guardian, October 1996, 32-36.
8. Huite, N.J. and Westermann, D.H. (1974). Proc. 13th Convention, Institute of
Brewing (Australia and New Zealand Section), Gold Coast, 133-149.
9. Izydorczyk, M.S. and MacGregor, A.W. (2000). Carb. Polymers, 41, 417-420.
10. Laing, H. and Taylor, D.G. (1974). Proc. 18th Convention, Institute of Brewing
(Australia and New Zealand Section), Adelaide, 109-114.
11. Moll, M. and Flayeux, R. (1980). Proceedings of the European Brewery
Convention Symposium on the relationship between malt and beer, Helsinki, 19.
12. Moonen, J.H.E., Graveland, A. and Muts, G.C.J. (1987). J. Inst. Brewing, 93,
125-130.
13. Sjholm, K., Pietil, K., Home, S., Aarts, R. and Jaakola, N. (1994).
Monatschrift fr Brauwissenschaft, 47, 165-171.
14. Smith, P.R. (1997). New Brewer, July/August 1997, 55-59.
15. Stewart, D.C., Hawthorne, D. and Evans, D.E. (1998). J. Inst. Brewing, 104,
321-326.
16. Webster, R.D.J. (1981). J. Inst. Brewing, 87, 52-56.
17. Webster, R.D.J. and Portno, A.D. (1981). Proceedings of the European Brewery
Convention Congress, Copenhagen, 153-160.

10
11

Molecular markers for fermentability -

a novel selection tool for barley
breeding
M. Voetz, J. Gunkel & F. Rath
Versuchs- und Lehranstalt fr Brauerei in Berlin (VLB), Seestrasse 13, D-13353 Berlin,
Germany (e-mail: [email protected])

Descriptors
-Amylase, barley, breeding, enzymic activity, fermentability, genetics

SUMMARY
The main goal of this study was to identify the genetic basis for different levels of
fermentability in different genotypes. It could be shown that the content of fermentable sugars
in the wort and the apparent attenuation limit are strongly influenced by the genotype
dependent thermostability of the -amylase. Comparative DNA sequence analyses revealed
that the thermostability of the enzyme is dependent on the structure of the -amylase gene.
Based on DNA polymorphisms between the alleles, PCR primers could be designed serving
as markers for thermostability properties. These primers offer new prospects in barley
breeding on the basis of marker assisted selection of genotypes well adapted to high
temperatures in the industrial mashing process.

Molekulare Marker fr Vergrungseigenschaften - neuartige Selektionsmglichkeiten

fr die Braugerstenzchtung

Deskriptoren
-Amylase, Enzymaktivitt, Grkraft, Genetik, Gerste, Zchtung

ZUSAMMENFASSUNG
Ziel der Forschungsarbeit war die Aufklrung der molekularen Ursachen unterschiedlicher
Vergrungseigenschaften in verschiedenen Gerstengenotypen. In Versuchen wurde nach-
gewiesen, da die Zuckerzusammensetzung der Wrze und der erreichte Endvergrungsgrad
mageblich von der sorten-unterschiedlichen Thermostabilitt der -Amylase bestimmt wird.
Vergleichende DNA-Sequenz-analysen beweisen die Abhngigkeit der Thermostabilitt des
Enzyms von der Struktur des -Amylase-Gens. DNA-Polymorphismen in den Gensequenzen
wurden zur Entwicklung spezifischer PCR-Marker genutzt, die eine eindeutige
Charakterisierung der Thermostabilittseigenschaften eines Genotyps erlauben. Diese PCR-
Primer erffnen der Braugerstenzchtung neue Perspektiven fr die markergesttzte Selektion
solcher Genotypen, die an hohe Temperaturen moderner industrieller Maischprozesse optimal
angepat sind.
Les marqueurs molculaires de la fermentabilit - un nouvel outil de slection pour la
culture des orges

Descripteurs
Activit enzymatique, -amylase, fermentabilit, gntique, obtention (culture), orge

RESUME
Le but de cette tude tait dclaircir les causes molculaires des qualits dattnuation
diffrentes dans les gnotypes dorge varis. Les expriences ont prouv que la composition
en sucres du mot et lattnuation limite atteinte sont dtermines par la stabilit thermique de
la -amylase. Les analyses compares de squence ADN prouvent que la stabilit thermique
de lenzyme dpend de la structure du gne de la -amylase. Les polymorphismes dADN
dans les squences gntiques ont t utiliss pour le dveloppement des indicateurs
spcifiques PCR, qui permettent une caractrisation claire des qualits de stabilit thermique
dun gnotype. Ces primer PCR ouvrent de nouvelles perspectives la culture dorge de
brasserie par la slection des indicateurs de tels gnotypes idalement adapts aux hautes
tempratures des mthodes de brassage modernes et industrielles.

2
INTRODUCTION
The intensive breeding development work in recent decades has led to a considerable
improvement in malt modification and in the extract yield. The extract yields which
are now attained with the top quality malting barley varieties are at the limit of what is
physiologically possible considering the structure and chemical composition of the
grain. There is a danger that attempts to enhance the extract yield further would be
coupled with disadvantages in other areas, e.g. an increased susceptibility to fungal
attack caused by split husks.
Efforts to improve the malt yield should therefore be concentrated in the direction of
increasing the fermentability of the extract. The high heritability of this characteristic
supplies a favourable precondition for successful breeding work. However, this
complex characteristic must first be unravelled into its component parts. We aimed to
investigate -amylase - one of the major components determining the fermentability -
in particular concentrating on the elucidation of the genetic basis for the genotype-
dependent influence of -amylase activity on the content of fermentable sugars in the
wort.

RESULTS
Sugar content of the wort
The investigations were performed on barley varieties selected on account of their
attenuation characteristics. Cultivars with high fermentability - Alexis, Barke and
Scarlett - were compared to ones with poor fermentability Derkado, Korinna and
Renata.

Table 1: Carbohydrate composition of the wort and selected malt parameters for
barley varieties grouped according to apparent attenuation limit levels

Variety
LSD .05 [Scheff]
Group A Group B
Derkado
Dimension

Korinna
Scarlett

Renata
Alexis

Barke

Mean

Mean

Extract % dm 82,5 83,3 83,2 83,0 83,0 82,5 83,0 82,8 1,2

App. Final Attenuation % 84,4 84,9 83,4 84,2 80,9 81,2 82,7 81,6 1,6

Glucose + Fructose g/l 17,0 16,6 17,3 17,0 17,1 16,6 17,9 17,2 1,7

Maltose g/l 55,4 55,9 54,0 55,1 51,5 52,7 51,9 52,0 2,0

Maltotriose g/l 12,0 12,1 12,6 12,2 13,5 11,7 13,0 12,7 0,8

Non-Fermentable Sugar g/l 25,5 25,4 26,3 25,7 29,0 28,6 24,4 27,3 3,2

Total Carbohydrates g/l 109,9 110,1 110,1 110,0 111,0 109,5 107,2 109,2 3,9

Starch % dm 56,7 58,6 54,7 56,7 57,4 57,1 55,8 56,8 -

Amylose % 28,3 27,7 28,1 28,0 27,7 28,0 29,2 28,3 1,5

-Amylase BU/g dm 862 920 684 822 471 824 705 667 132

3
In order to evaluate the genetic and environmental influences, attenuation trials were
carried out together with the corresponding barley, malt and wort analyses on material
from six different locations over two harvest years.
Table 1 shows a selection of results obtained by comprehensive barley and malt
analyses performed according to EBC and MEBAK rules. The starch content of the
malts and the amylose fraction of the total starch were determined using Megazyme
Kits (Amylose/Amylopectin Assay Kit and Total starch assay procedure, respectively)
following manufacturers instructions. The carbohydrates of the worts were separated
using HPLC and subsequently quantified by refraction index detection.
-amylase activities in malt extracts were determined with a kit purchased from
Megazyme (Wicklow, Ireland) according to the instructions of the manufacturer.

Observations:
! Different levels of the apparent attenuation limit (AAL) in various barley geno-
types are mainly due to differences in the availability of fermentable sugars in the
wort (r = 0.9**).
! Varieties showing high levels of AAL Alexis, Barke and Scarlett contain signi-
ficantly higher concentrations of maltose in the wort than those varieties with
lower AAL levels Derkado, Korinna and Renata.
! Genotyes with poor fermentability properties are characterised by a considerable
shift in the sugar spectrum of the wort towards maltotetroses, maltopentoses and
other highly polymerised non-fermentable sugars.
! Genotype-dependent differences in the supply of fermentable sugars cannot be
traced back to the chemical composition of the barley grain. In particular, no
correlation could be found between the concentration of fermentable sugars and
either the starch content or the ratio of amylose to amylopectin.
! There is no clear correlation between the activity of individual amylolytic
enzymes and the content of sugars in the wort. No direct influence on the maltose
concentration in the wort could even be demonstrated for -amylase - the key en-
zyme of starch degradation.

Thermostability of -amylase
The determination of the -amylase activity in malt extracts using conventional
methods occurs at 40C with little thermal pressure on the enzyme. This contrasts
strongly to the higher temperatures involved in the mashing process before examining
the composition of sugars in the wort and attenuation properties. These different
temperature conditions explain the apparent lack of correlation between the malts
-amylase activity and the maltose concentation in the wort as shown in table 1.
With reference to the work of Kihara et al. (1998), the behaviour of the -amylase
under increasing temperature was investigated for the different cultivars.
The analyses were carried out on very dilute malt extracts with comparable protein
contents thus avoiding any falsification of the results due to the formation of
protective colloids. Equal amounts of malt extracts were filled into thin-walled 0.2 ml
tubes, transferred to a thermocycler and heated over a 150 seconds period to the
temperatures indicated in figure 1. The entire solution was tested after heating for its
remaining -amylase activity using the Betamyl Method (Megazyme). To control the
reproducability of the results the experiments were repeated twice for each cultivar
tested.

4
 100,0

80,0
Enzyme Activity [ % ]

60,0

Alexis
Barke
40,0
Derkado
Korinna
Renata
20,0
Europ. Landrace

0,0
40 45 48 50 52 54 55 56

Temperature [ C ]

Figure 1: Thermostability of -amylase - Genotype specific loss of enzyme activity

with increasing temperature

Observations:
! In the range between 40C and 60C significant differences in the -amylase
thermostabilities can be detected for the various genotypes.
! The -amylases of the cultivars Alexis, Barke and Scarlett show a higher degree of
thermostability (Alexis-type) than the -amylases of Derkado, Korinna and
Renata (Derkado-type).
! The curves indicating the decrease of -amylase activity as a response to in-
creasing temperature show almost identical features within each group (Alexis-
type vs Derkado-type).
! A European landrace could be identified with a -amylase whose thermostability
clearly exceeds that of the Alexis-type and apparently corresponds to the
Haruna-type described by Kihara et al. (1998).

Molecular markers for fermentability

The reason for the different temperature sensitivities of the -amylases from the
genotypes examined (figure 1) are to be found in the structures of the corresponding
-amylase genes (Eglinton et al., 1998; Kihara et al., 1998). Relevant DNA sections
of this gene were amplified from the genotypes and subsequently compared. The aim
was to develop specific PCR primers on the basis of interallelic polymorphisms to be
used to identify genotypes with good attenuation properties. The codominant marker
system shown in figure 2 is based on the characteristic insertion/deletion feature of the
-amylase genes intron III described by Erkkil et al. (1998). A selection of all
cultivars tested is shown in figure 2. Genotypes carrying the allele for the more
thermostable -amylase exhibit an over 100 bp shorter PCR product than the ones
with the thermolabile enzyme. Various breeders lines were analysed in order to

5
 cathode

Pasadena
Derkado

Korinna

Viskosa
Scarlett

Extract

Riviera
Renata
Aspen
Barke
Alexis

Optic
0.6 kbp
0.5 kbp

+ + + - - - + + + - - -
anode

Figure 2: PCR assisted identification of -amylase alleles in selected varieties

responsible for the different thermostabilities (+, high / -, low) of the
enzymes. The arrow indicates the direction of nucleic acid migration in the
agarose gel.

investigate the interaction between thermostability of -amylase and cytolytic

properties of a genotype. Lines with different -amylase thermostabilities and varied
cytolytic properties were chosen for comparison. Representatives for different
combinations of both independent traits are shown in figure 3.

Observations:
! The genotypes with -amylase of differing thermostability (figure 1) have differ-
ing alleles of the -amylase gene. Variations in relevant amino acid positions can
explain this phenomenon.
! Specific PCR primers, on the basis of interallelic polymorphisms, can identify the
Alexis-type which has better attenuation properties than the Derkado-type due
to the activity of a more thermostable -amylase.
! Using a different PCR primer set the -amylase gene encoding the very thermo-
stable enzyme (landrace allele) can be discriminated from both the Alexis and the
Derkado alleles.
! The genotyping of numerous European malting barley varieties (representatives in
figure 2) reveals that in the actual gene pool, the undesirable Derkado allele is also
widespread alongside the Alexis allele.
! The Alexis allele coding for the thermostable -amylase is an important
prerequisite for good fermentability properties of a barley genotype. Nevertheless,
genotypes with poor cytolytic properties are unable to realize this genetic potential
(figure 3). The presence of the Derkado allele generally excludes the possibility of
good fermentability (figure 3).

6
 + + + + + + - - -

9 5 ,0 8 1 ,0

9 0 ,0 8 0 ,0

App. Final Attenuation [ % ]

Friability [ % ]

8 5 ,0 7 9 ,0

8 0 ,0 7 8 ,0

7 5 ,0 7 7 ,0

7 0 ,0 7 6 ,0

6 5 ,0 7 5 ,0
A le xis B arke S train A S train B S train C S train D S train E S train F S train G

F riab ility 9 0 ,0 8 7 ,7 9 6 ,0 7 1 ,0 7 3 ,0 7 3 ,0 9 2 ,3 9 1 ,0 8 9 ,0
A p p . F inal A tt. 8 0 ,5 8 0 ,5 8 0 ,2 7 8 ,0 7 6 ,7 7 7 ,0 7 8 ,0 7 7 ,8 7 6 ,9

Figure 3: Different AAL levels of genotypes as a result of the interaction between the
thermostability of the -amylase and malt cytolytic modification

CONCLUSIONS
In this study the relationship between (i) -amylase gene/allele, (ii) thermostability of
the expressed enzyme, (iii) content of fermentable sugars in the wort and (iv) apparent
attenuation limit could be enlightened for the first time as a complete physiological
chain without any black box.
In particular the correlation between the genetic basis and the substrates for
fermentation is of practical relevance for breeders, maltsters and brewers. The data
presented are the basis for a novel and highly focussed marker assisted selection
targetted upon a distinct quality trait for beer production. This will enable breeders to
eliminate the thermolabile Derkado-type -amylase from their selections. Further
improvements to the apparent attenuation limits are to be expected by the crossing in
of the highly thermostable landrace-type enzyme.
Using a PCR assisted approach it is possible to select for a thermostable enzyme in
early breeding generations without extensive micromalting, thus achieving a
substantial reduction in time and costs spent on breeding optimised barley varieties.
PCR analyses performed upon green plantlets will give information about the
thermostability of the -amylase which will be synthesised in later stages of
development and thereby about the potential for producing high concentrations of
fermentable sugars.
In this way, genotypes bred with thermostable amylolytic enzymes are better suited to
the demands of modern high-temperature mashing and allow a further optimisation of
the brewing process.

7
REFERENCES
Eglinton et al. (1998), J. Cereal Sci, 28: 301-309.
Erkkil et al. (1998), Plant Physiol, 117: 679-685.
Kihara et al. (1998), Plant Breeding, 117: 425-428.

8
12

Characterisation and detection of the

gushing factors produced by fungi
T. Kleemola1, T. Nakari-Setl1, M. Linder1, M. Penttil1, E.
Kotaviita2, J. Olkku3 & A. Haikara1
1
VTT Biotechnology, P.O.Box 1500, FIN-02044 VTT, Espoo, Finland (e-mail:
[email protected])
2
Raisio Group, Raisio Malt, P.O.Box 101, FIN-21201 Raisio, Finland
3
LP Research Centre Ltd., P.O.Box 22, FIN-15141 Lahti, Finland

Descriptors
Fungi, gushing, immunology, prediction, protein

SUMMARY
Gushing factors produced by the strains of the genera Fusarium, Nigrospora and
Trichoderma were isolated and purified. The partial N-terminal sequences of these small
proteins were analysed and identified as hydrophobins. Addition of an amount as low as 1 g
of hydro-phobins into bottled beer repeatedly caused gushing. An immunological ELISA test
for hydrophobins in barley and malt was developed and the results of the method were
compared to other predictive gushing tests. This novel method can be used in quality control
for predicting the gushing activity of barley and malt.

Charakterisierung und Detektion von Gushing-Faktoren, die von Pilzen stammen

Deskriptoren
Fungi, Immunologie, Protein, Vorhersage, Wildwerden (Bier)

ZUSAMMENFASSUNG
Gushing-Faktoren, die von genera Fusarium, Nirospora und Trichoderma produziert werden,
wurden isoliert und gereinigt. Die partiale N-terminal Sequenz dieser kleinen Proteine wurde
analysiert und als Hydrophobe identifiziert. Die Zugabe von weniger als 1 m dieser
Hydrophoben in Flaschenbier, verursachte wiederholt Gushing. Nun wurde ein
immunologischer ELISA-Test fr Hydrophobe in Gerste und Malz entwickelt und mit
anderen vorhersagenden Gushing-Tests verglichen. Diese ungewhnliche Methode kann in
der Qualittssicherung dazu verwendet werden, die Gushing-Aktivitt vorherzusagen.
Caractrisation et dtection des facteurs de giclage d'origine fongique

Descripteurs
Fungi, giclage, immunologie, prdiction, protine

RESUME
Les facteurs de giclage (gushing) produits par les souches de champignons des genres
Fusarium, Nigrospora et Trichoderma ont t isols et purifis. Les squences N-terminales
partielles de ces petites protines ont t analyses, rvlant qu'il s'agit d'hydrophobines.
L'addition d'une quantit aussi faible que 1 g d'hydrophobines dans de la bire en bouteille a
constamment provoqu le jaillissement de la bire. Un test ELISA pour l'identification des
hydrophobines dans l'orge et le malt a t dvelopp et les rsultats de la mthode ont t
compars ceux d'autres tests prdictifs de ce jaillissement. Cette nouvelle mthode peut tre
utilise en contrle de qualit pour prvoir l'activit jaillissante de l'orge et du malt.

2
INTRODUCTION
Fungal infection of barley and malt, in particular by strains of the genus Fusarium,
have been known to cause gushing of beer (Gjertsen et al. 1965, Haikara 1983, Munar
and Sebree 1997, Schwarz et al. 1996). Gushing factors may be produced by fungi in
the field or during malting. Amaha et. al (1973) studied the gushing factors of
Nigrospora sp., Penicillium sp., Stemphylium sp. and Fusarium graminearum. They
showed that the gushing factor of Nigrospora sp. was a polypeptide having a high
level of hydrophobic amino acid residues. The factor was observed to have an
estimated molecular weight of 15 kDa and to induce gushing at the concentration of
about 0.05 ppm. The gushing factor of Stemphylium sp. was found to be a
peptidoglycan inducing gushing at the concetration of 4 ppm. Fusarium graminearum
was reported to produce two different gushing factors one of which appeared to be a
peptide-containing substance. The gushing activity of gushing factors were
demonstrated to be retained at acid and neutral pH values after heating for two hours
at 100C (Amaha et. al 1973).
The gushing potential of barley and malt can be predicted by a cultivation method
where the precentage of kernels contaminated with Fusarium spp. is determined
(Abildgren et al. 1987, EBC Analytica Microbiologica 1992). Immunological
methods for quantifying Fusarium antigens have also been developed (Manke and
Raath 1997, Vaag 1991). Moreover, the gushing tendency of malt can be predicted by
the rocking test, in which an aqueous extract of malt is added to bottled beer and the
bottles are rocked for three days (Vaag et al. 1993).
Hydrophobins are small (100 25 amino acids), moderately hydrophobic proteins
secreted by fungi. A characteristic feature of these proteins is their eight cysteine
residues, ordered in a conserved pattern (Wessels 1996). Hydrophobins self-assemble
at hydrophilic/hydrophobic interfaces, forming amphipathic membranes. This
property allows them to fulfil a broad spectrum of functions in fungal development.
They are involved in formation of aerial structures, in aerial dispersion of spores, and
they line air channels within fruiting bodies with a hydrophobic coating, probably
serving gas exchange. Hydrophobins also mediate hyphal attachment to hydrophobic
surfaces such as those of plants. By self-assembly, hydrophobins are able to stabilise
air bubbles and oil droplets in water, decrease the surface tension of water and change
the nature of a surface from hydrophilic to hydrophobic or vice versa (Wessels 1996).
Hydrophobins are divided into two classes, I and II, based on their hydropathy
patterns and biophysical properties (Wessels 1994). Hydrophobins, especially class I
ones, form highly insoluble aggregates and are extremely stable. Our recent studies
have indicated that hydrophobins act as gushing factors of beer (Haikara et al. 2000).
The aim of this study was to develop a fast, sensitive and reliable method, e.g.
immunological test kit, for quality control of barley and malt.

MATERIALS AND METHODS

Hydrophobins were isolated as described by Nakari-Setl (1995) using the sequential
extraction of mycelium or by bubbling the culture medium of the fungal strains of
Fusarium poae VTT D-82182 (D182), Nigrospora sp. VTT D-79122 (D122) and
Trichoderma reesei VTT D-74075 (D75). Hydrophobins were detected using SDS-
PAGE performed in 17.5 % or 20 % gels. Hydrophobin preparates were added to beer
or mineral water, and the bottles were rocked in a horizontally rotating shaker with
rate of 50 rpm for three days. After opening the bottles the amount of gushing beer

3
was measured (Vaag et al. 1993). The proteins were purified by reversed phase
chromatography on a Vydac C-4 column using the kta Explorer system (Pharmacia
Biotech). Elution was performed with a linear gradient of acetonitrile in 0.1 %
trifluoroacetic acid. Partial N-terminal amino acid sequences of the purified proteins
were determined in the Protein Chemistry Laboratory of the Institute of
Biotechnology, Finland. Polyclonal antibodies against a protein isolated from F. poae
and identified as hydrophobin were raised in rabbits. An immunological competitive
ELISA-test for detection of hydrophobins in barley and malt was developed (table 1).

Table 1: Procedure of the competitive ELISA-test for detection of hydrophobins in

barley and malt.
Step Description
number
1 Immunoplates were coated with hydrophobin antigen.
2 Barley or malt flour was stirred with buffer for 60 min and centrifuged.
3 Antibody against hydrophobin was mixed with samples.
4 Antibody-sample mixtures were transfered to the wells of coated
immunoplates. Incubation for 2 hours at 37C. Washing.
5 Goat anti-rabbit IgG - alkalic phosphatase conjugate was added to the
plates. Incubation for 2 hours at 37C. Washing.
6 Addition of substrate: p-nitrophenyl phosphate in diethanolamine +
MgCl2 buffer. Incubation for 30 min at room temperature.
7 Absorbance was read at 405 nm.

Barley was artificially inoculated with F. culmorum VTT D-80148 (D148), F.

graminearum (telem. Gibberella zeae) VTT D-95470 (D470) and F. poae D182, in
the field in order to produce gushing active material for the hydrophobin ELISA-test
described above. Barley samples were malted in laboratory scale of 1 kg. Barley was
steeped for two days at 12C, germinated six days at 14C and kilned 21 hours up to
85C. Barley and malt samples containing naturally high Fusarium contamination
were also tested. The number of the samples were 17 and 26, respectively. Gushing
positive and negative control malt were obtained from Carlsberg Research
Laboratory, Denmark. The gushing propensity of the malt samples was determined
according to Vaag et al. (1993) and the results were compared with the hydrophobin
ELISA-test.

RESULTS
Hydrophobin-like proteins were isolated from the gushing active fungal strains, F.
poae D182 and Nigrospora sp. D122, and from the reference strain T. reesei D75
known to produce hydrophobins. The size of the proteins was found to be about 10
kDa when analysed by SDS-PAGE (figure 1). The proteins were purified by reversed
phase chromatography and their partial N-terminal sequences were determined. The
typical conserved cysteine pattern of hydrophobin was found in the analysed
sequences (figure 2).

4
 1. 2. 3. 4. 5.

94 kDa
67 kDa
43 kDa
30 kDa
20 kDa
14 kDa

Figure 1: 20 % SDS-PAGE analysis of the hydrophobin samples isolated from

mycelium or culture medium of the following fungi: F. poae D182 (1), Nigrospora
sp. D122 (2) and T. reesei D75 (purified 3 and unpurified 4). The molecular weight
markers are shown on the right (5).

General formula of hydrophobins:

X2-38 - C - X5-9 - C - C - X11-39 - C - X8-23 - C - X5-9 - C - C - X6-18 - C - X2-13
F. poae D182:
X18 - C - X9 - C-C ....
Nigrospora sp. D122:
X11 - C - X11 - C-C - X11 - C ...

where C = cysteine,
X = any other amino acid

Figure 2: Cysteine spacing in the fungal hydrophobin according to Wessels (1996)

compared to the spacing in the hydrophobins of F. poae D182 and Nigrospora sp.
D122.

All isolated hydrophobins repeatedly induced gushing of beer. The gushing was more
pronounced in beer than in mineral water (table 2). In beer more than 50 % of the
content could be lost in the overfoaming. Addition of an amount as low as 1 g of the
hydrophobin of T. reesei into bottled beer (0.33 l) was sufficient to cause gushing
(data not shown).

5
Table 2: Gushing of beer and mineral water induced by hydrophobins isolated from
the strains T. reesei D75, F. poae D182 and Nigrospora sp. D122.
Hydrophobins Gushing (g)
isolated from beer mineral water
T. reesei (raw extract) 168 106
F. poae (raw extract) 80 49
T. reesei (purified) 190 nd
F. poae (purified) 27 nd
Nigrospora sp. (purified) 162 nd
nd: not determined

The immunological detection method for hydrophobins was developed using the
antibodies raised against the hydrophobin of F. poae D182. Barley and malt samples,
both artificially and naturally contaminated with Fusarium, were analysed with the
ELISA-test and the results were compared to the gushing test. The developed
competitive ELISA -test was able to detect hydrophobins from the gushing positive
samples, which was indicated by a lower absorbance value of the samples with
gushing propensity (figure 3).

1.2
-
-
1

0.8
+-
+ +
Absorbance

Barley
0.6
Malt

0.4

0.2

0
Control 1 F. culmorum F. graminearum F. poae Control 2

Figure 3: Results of the hydrophobin ELISA-test of barley artificially inoculated

with F. culmorum D148, F. graminearum D470 and F. poae D182, and
corresponding malt. Gushing potentials of the malt samples are shown above the
columns: + = gushing positive, +- = variable gushing, - = gushing negative.

In order to be able to compare the results of ELISA-tests of samples analysed at

different times the absorbance value of the gushing positive control barley or malt was
subtracted from the values of the other tested samples and a tentative gushing limit
value was specified. Relative absorbance values of both the naturally contaminated

6
barley and corresponding malt samples were observed to correlate with the gushing
potential of the malts; the lower the value, the higher the gushing propensity of the
sample (figures 4 and 5). Three samples (A, F and O) out of 26 tested malts gave
conflicting ELISA-results compared to the results of the gushing test. The relative
absorbance value of the malt F was above the tentative gushing limit value although it
induced gushing (figure 5). However, only gushing in a low volume of 2 g was
detected predicting a low gushing risk of the sample. On the other hand, the relative
absorbance values of the malts A and O were found to be under the limit gushing
value, but no gushing or only a foam cap in a case of the sample A was observed
(figure 5). However, all these samples, A, F and O, were cosidered to have a risk of
gushing on the basis of Fusarium contamination.

0.25

-
0.2
-
-
Relative absorbance value of barley

0.15
-
-
0.1
(+) + / ++
+
0.05
+++ ++
++ ++
++
0

F. culmorum inoculated
F

O
J
A

N
B

M
I

Non-inoculated
D

-0.05
++
+ ++
-0.1

-0.15
++

-0.2

Figure 4: Relative absorbance values of naturally contaminated barley samples

compared to the results of the gushing test of malt. - = no gushing, (+) = a foam cap, +
= gushing of 1-2 g, ++ = gushing of 30-90g. ,a tentative limit value for gushing.

7
 0.25
-
0.2
- - - -
0.15
+
Relative absorbance value of malt

0.1
- + ++ ++ ++ ++ - ++
0.05
+++ +++
(+) ++ - +++
0

Negative control
A

R
D1
D2

Non-inoculated
F. culmorum inoculated
F

P
B

Positive control
I
G
H

Q
E1
E2

J
-0.05
+++ +++
++
-0.1

-0.15
+

-0.2
+++
-0.25 +++

-0.3

Figure 5: Relative absorbance values of malt samples prepared from naturally

contaminated barley compared to the results of the gushing test of malt. - = no
gushing, (+) = a foam cap, + = gushing of 1-2 g, ++ = gushing of 30-90g, +++ =
gushing > 100 g. , a tentative limit value for gushing.

DISCUSSION
The gushing factors isolated and purified in this study were found to be hydrophobins
with a molecular weight about 10 kDa, one of which induced gushing at the
concetration of about 0.003 ppm. Amaha et al. (1973) isolated a gushing-inducing
factor of Nigrospora sp. (NGF), which was identified as a hydrophobic protein with a
molecular weight about 15 kDa. The factor was observed to induce gushing at the
concetration of about 0.05 ppm. The amino acid analysis of NGF showed that the
protein consisted of 166 amino acid residues and had a high half-cystine content,
approximately 16 residues per mole of NFG (Kitabatake and Amaha 1977). If NGF
was in a dimeric form then the amount of total amino acid and cysteine residues of the
monomeric protein correlate with those of hydrophobins.
The risk of producing gushing active malt from barley depends on the composition
and extent of Fusarium contamination in the barley, on the activity and viability of
gushing active Fusarium spp. present in the sample, and on the malting process. Thus
the examination of the gushing potential by quantifying total Fusarium contamination
in barley can not be regarded as sufficiently reliable. The developed competitive
ELISA test, which directly detects hydrophobins i.e. gushing factors in barley, was in
this study shown to predict the gushing potential of corresponding malt when the
rocking test was used as a reference method. Some of the false positive and negative
results obtained might be caused by the rocking test itself because of its poor
repeatability and reproducibility. Barley samples giving values very near the set
gushing limit could be micromalted in order to get a picture of the production of
hydrophobins during malting. This would increase the reliability of the ELISA-test

8
giving more predictable results of the gushing risk of barley and corresponding malt.
However, more samples must be analysed before a precise gushing limit value for
barley and malt can be set.

CONCLUSIONS
Hydrophobins were isolated from gushing active fungi F. poae D182, Nigrospora sp.
D122, and T. reesei D75. The hydrophobins were demonstrated to induce gushing of
beer and mineral water. An immunological competitive ELISA-test for detection of
hydrophobins in barley and malt samples was developed. This test was able to detect
hydrophobins from the gushing positive samples. The method is simple enough to be
used in quality control of barley and malt.

ACKNOWLEDGEMENT
We thank the personnel of VTT Biotechnology, especially Tarja Nordenstedt for her
skillful assistance. We also thank Nisse Kalkinen and his co-workers in the Protein
Chemistry Laboratory of the Institute of Biotechnology, Finland for N-terminal
sequence analyses. Financial support from the Finnish Malting and Brewing Industry,
the National Technology Agency (TEKES) and VTT Biotechnology is gratefully
acknowledged.

REFERENCES
1. Abildgren, M.P., Lund, F., Thrane, U. and Elmholt, S., Czapex-Dox agar
containing iprodione and dichloran as a selective medium for isolation of
Fusarium species, Lett. Appl. Microbiol., 5 (1987) 83-86.
2. Amaha, M., Kitabatake, K., Nakagava, A., Yoshida, J. and Harada, T., Gushing
inducers produced by some moulds strains, Proc. Eur. Brew. Conv. 14th
Congress, Salzburg 1973, 381-398.
3. EBC Analytica Microbiologica II, 1992.
4. Gjertsen, P., Trolle, B. and Andersen, K., Studies on gushing. II. Gushing
caused by micro-organisms specially Fusarium species, Proc. Eur. Brew. Conv.
10th Congress, Stockholm 1965, 428-438.
5. Haikara, A., Malt and beer from barley artificially contaminated with Fusarium
in the field, Proc. Eur. Brew. Conv. 19th Congress, London 1983, IRL Press,
401-408.
6. Haikara, A., Sarlin, T., Nakari-Setl, T. and Penttil, M., Method for
determining a gushing factor for a beverage, Pantent application
PCT/FI99/00305 (Patent FI 104390 / 2000), 2000.
7. Kitabatake, K. and Amaha, M, Effect of chemical modifications on the gushing-
inducing activity of a hydrophobic protein produced by a Nigrospora sp., Agric.
Biol. Chem., 41 (6) (1977) 1011-1019.
8. Manke, W. and Rath, F., Rapid test for Fusarium as a practical tool for HACCP
in malting, Eur. Brew. Conv. Monogr. 26 - Symposium Quality Issues &
HACCP, 1997, Verlag Hans Carl, 27-33.
9. Munar, M.J. and Sebree B., Gushing - A maltster's view, J. Am. Soc. Brew.
Chem., 55 (3) (1997) 119-122.

9
10. Nakari-Setl, T., Highly expressed Trichoderma reesei genes. Cloning,
characterization and use in protein production on glucose-containing media,
VTT Publications nro 254, 1995, 94 p. + app. 40 p..
11. Schwarz, P., Beattie, S. and Casper, H., Relationship between Fusarium
infestation of barley and the gushing potential of malt, J. Inst. Brew., 102 (1996)
93-96.
12. Vaag, P., Immunological detection of Fusarium in barley and malt, Proc. Eur.
Brew. Conv. 23rd Congress, Lissabon 1991, 553-560.
13. Vaag, P., Riis, P., Knudsen, A.-D., Pedersen, S. and Meiling, E., A simple and
rapid test for gushing tendency in brewing materials, Proc. Eur. Brew. Conv.
24th Congress, Oslo 1993, IRL Press, 155-162.
14. Wessels, J.G.H., Developmental regulation of fungal cell wall formation, Annu.
Rev. Phytopathol., 32 (1994) 413-437.
15. Wessels, J.G.H., Fungal hydrofobins: proteins that function at an interface,
Trends in plant science, 1 (1996) 9-15.

10
13

Evolution of oxygen and water up-take

during steeping in a lab scale malting
unit
Ahmed Hariri1, Patrick Boivin2, Ivan Marc1 & Michel Fick1
1
Laboratoire des Sciences du Gnie Chimique - Ecole Nationale Suprieure dAgronomie et
des Industries Alimentaires (ENSAIA), 2, avenue de la Fort de Haye, F-54505 Vandoeuvre
Cedex, France
2
Institut Franais des Boissons de la Brasserie Malterie, Ple Technologique de Brabois, 7 rue
du Bois de la Champelle, B.P. 267, F-54512 Vandoeuvre, France

Descriptors
Malt quality, model simulation, moistening, steeping

SUMMARY
Steeping is the most important operation during the malting process. Unfortunately, there is a
lack of knowledge to appreciate the optimal steeping scheme. The objectives of this study
were to measure the evolution of a lot of variables during an industrial steeping in order to
correlate steeping phenomena to water and oxygen up-take and endosperm hydration and to
conceive a laboratory scale steeping vessel to test different steeping diagrams on the
laboratory unit and to evaluate the effect of steeping management on the malt quality. The
first results lead to a better knowledge of the principal mechanisms of air and water
absorption and the impact of steeping on final malt quality.

Modellierung von Weichprozessen im Industriemastab

Deskriptoren
Befeuchten, Malzqualitt, Modellbildung, Weichen

ZUSAMMENFASSUNG
Weichen ist der wichtigste Vorgang whrend des Mlzungsprozesses. Leider gibt es nur
wenig Erkenntnisse zur Entwicklung eines optimalen Weichprogramms. Das Ziel dieser
Arbeit ist es, die Vernderungen vieler Variablen whrend des industriellen Weichens zu
erfassen, in der Hoffnung, die Weichphnomene in Hinsicht auf Wasser- und
Sauerstoffaufnahme und Hydratation des Endosperms besser zu verstehen. Aus diesen
Kenntnissen wurde ein Weichbehlter entwickelt, um verschiedene Weichdiagramme im
Labormastab zu testen und die Auswirkungen des Weichmanagements auf die Malzqualitt
zu untersuchen. Die ersten Ergebnisse fhren zu einem besseren Wissen ber die
Hauptmechanismen der Luft- und Wasseraufnahme und die Wirkung des Weichens auf die
Malzqualitt.
Modlisation de la macration industrielle

Descripteurs
Humidification, modlisation, qualit du malt, trempage

RESUME
La trempe est ltape la plus importante en malterie, malheureusement, il y a un manque de
connaissances pour optimiser le diagramme de trempe. Les objectifs de cette tude taient de
mesurer diffrents paramtres au cours du maltage industriel pour corrler les phnomnes de
la trempe avec ladsorption deau et doxygne et lhydratation de lendosperme; de
construire un pilote pour valuer leffet des diagrammes de trempe sur la qualit du malt. Les
premiers rsultats obtenus ont permis de mieux comprendre les mcanismes dadsorption
deau et doxygne et limportance du diagramme de trempe pour la qualit finale du malt.

2
INTRODUCTION
In the production process of brewing malt, the first step consists of the steeping.
Steeping conditions (Axcell et al, 1983; Chapon, 1960) and duration of germination
have been identified as crucial quality factors of malted grains.
The main objective of the malting process is the synthesis and activation of hydrolytic
enzymes, which occur mainly during the germination period and the first hours of the
kilning phase. The energy for these biochemical process is probably provided by the
respiratory activity of the grain. Partially during malting but mainly during brewing
the barley starch is converted into fermentable sugars by the action of these enzymes.
Process conditions determine the metabolic activity of the grain and the progress of
germination. As a consequence, the final malt qualities are correlated with the process
conditions during malting .

The rapid hydration of the barley grain that occurs during steeping, in conjunction
with the leakage of nutrients into the steep, results in the rapid proliferation of
bacteria, yeasts and mycelial fungi (Kelly and Briggs, 1992a, 1992b; Van
Campenhout et al, 1999).

Barley grain carries a numerous, variable, and complex microbial population that
mainly consists of bacteria, yeasts, and filamentous fungi (Noots et al, 1999). The
extent and the activity of this microflora are determined by the altering state of the
grain and the environmental conditions in the malt production chain.

A sufficient absorption of water and oxygen by barley and hydration of endosperm

leads to a malt having good brewing qualities (Powell et al, 1993).

So, on a pilot steeping plant, conceived from an interpolation of an industrial unit, the
objectives of this study are :to test two steeping diagrams in order to
evaluate the effect of steeping management on the malt quality;
to model the rate of oxygen consumption to estimate the anoxic degree during the
wet rests.

MATERIALS AND METHODS

Malting
The steeping step was conducted according to the first phase of an industrial malting
process. Barley showing a normal germination vigour was used. Batch of 5 kg grain
were immersed in 8 l of sterile water for 8 h. This operation was followed by an air
rest of 16 h and a second immersion phase of 5 h in a removed batch of sterile water
and this operation was followed by an second air rest of 10 h. The temperature during
steeping was maintained at 14 C in the first wet rest and 20 C in the second wet rest.
For the present series two experiments were used.

For the first experiment, pure air at 12 liters/h was introduced into the grain.
For the second experiment, the steeping was carried out without airing.
Each experiment was conducted two times.

After the steeping phase, the grain was allowed to germinate for 5 days in a micro-
malting unit. The temperature is maintained at 16 C.

3
Analytical methods
The pH and the concentration of dissolved oxygen are measured during the first and
the second wet rests.
During steeping and germination, samples of barley and of water were analysed .
The cells concentration on grain and in water were evaluated (bacteria, yeasts and
moulds) by counting of cells under media Nutrient Agar 1,5 for bacteria and PDA
(Potato Dextrose Agar) for yeasts and moulds.
The absorption of water by grain was evaluated by drying grains in infra-red weighing
machine.
For the steeping index determination, grains from each variety were plunged into
boiling water during 30 sec, using the method of Chapon (Chapon, 1960).
The % of germinated grain was evaluated by counting the germinated grains on 100
barley grains.
The organic acids and ethanol were analysed by HPLC with an UV detection for
ethanol and infrared detection for organic acids using the following conditions:
eluent H2SO4 0,04 N, flow 0,9 ml/mn, temperature 65C, column polypore H 25 cm *
7 mm, injection 10 l, sensibility is 64 and time of analysis 14 mn.
The malt was tested by using two methods: Friability (EBC 4.15 method) and
Filtration performances (Tepral method).

RESULTS
During each series of experiments, there were no significant modification of pH
which was stabilised between 7 and 8 (data not shown).
Figures 1 and 2 describe the evolution of oxygen during the two types of tests.

Steeping without airing ( second wet-rest )

Steeping without airing ( First wet-rest )
100 100

80 80
% of air saturation
% of air saturation

60
60

40
40

20
20

0
0 0 1 2 3 4 5
0 1 2 3 4 5 6 7 8 Time (hours)
Time ( hours)

Figures 1: Evolution of percentage of dissolved oxygen during the experiments

without airing

Steeping with airing ( first wet-rest ) Steeping with airing ( second wet-rest )
140
100
120
80
% of air saturation

100
% of air saturation

80
60
60
40 40

20
20
0
0 1 2 3 4 5
0 Time (hours)
0 1 2 3 4 5 6 7 8
Time (hours)

Figures 2: Evolution of percentage of dissolved oxygen during the experiments with

airing

4
Without airing, the evolution of dissolved oxygen in water showed a rapid decrease
less than 20% after 45 hours during the first wet rest and 1 hour during the second
wet rest. With airing, oxygen did not vary significantly during the two wet rests.
Table 1 compares the evolution of microbial cells during the two processes between
the beginning of the 1st wet rest (1WRbegin), the end of the 1st wet rest (1WRend) and
the end of the 2nd wet rest (2WRend).

Bacteria Yeasts Moulds

Ufc / gr or ml ufc / gr or ml ufc / gr or ml
on grain in water on grain in water on grain in water
Without airing
1st.W.R.b 2,3.107 5,2.107 1,1.104 5,5.102 4.102 0
1nd.W.R.e 5.108 4,3.107 1,2.104 3,2.103 3.103 0
2nd.W.R.e 2.108 3.106 1,5.105 2.103 0 0
With airing
1st.W.R.b 1.107 1,4.107 8.103 8.104 1,6.103 2,3.104
1nd.W.R.e 1,8.107 1,66.109 1.104 1,2.104 5,8.102 2.103
2nd.W.R.e 2,6.1010 5,8.1010 4.106 4.106 2,4.103 2.103
Table 1: Evolution of cells concentrations on grain or in water (units forming
colonyper gram of grain or per ml of water) during different phases of steeping

Without airing:
Bacteria reached a maximum of 5.107 UFC/g of grain at the end of the first rest; in
water the maximum concentration corresponds to the initial value (5,2.107 UFC/ml).
For yeasts, the maximal value on grain was obtained at the end of the 2nd wet rest
(1,5.105 UFC/g); in water the same value was observed at the end of the two wet rests
( 2-3.103 UFC/ml).
Moulds were present on the grain at a level of 3.103 UFC/g before completely
disappearing. No moulds were observed in water.

With airing:
When steeping was aerated, a general growth was observed between the beginning of
the 1st wet rest and the end of the 2nd wet rest (around 107-108 to 1010 for bacteria, 103-
104 to 106 for yeasts); for moulds data showed stabilised concentrations on grain and
in water.

The measurements of organic acids (acetic, butyric and lactic) and ethanol did not
indicate any difference between the two types of process. These molecules were
produced at a low level with ethanol at 0,2 g/l and organic acids at less than 0,1 g/l
(data not shown).

Figures 3 show that the water absorption evolved from 11 12 % to around 40% with
similar kinetics during the two kinds of process.

5
 50 50

40 40
% of water absorption

% of water absorption
30 30

20 20

10 10

0 0
Barley 1.W.R.b 1.W.R.e 1.A.R.e 2.W.R.e 2.A.R.e Barley 1.w.r.b 1.w.r.e 1.a.r.e 2.w.r.e 2.a.r.e
Sample

without airing with airing

Figures 3: Evolution of grain moisture during the different phases of steeping for the
two kind of process

The steeping indexes were decreasing between the end of the 2nd wet rest and the end
of the 2nd air rest without significant differences between the two kinds of steeping
(figures 4).

400 350
350 300
% of water destribution

% of water destribution

300
250
250
200
200
150
150
100
100
50 50

0 0
2nd wet rest end 2nd air rest end 2nd wet rest end 2nd air rest end
Sample

without airing with airing

Figures 4: Steeping indexes at the end of the 2 wet rest and the end of the 2nd air nd

rest for the two types of process.

100 100

80 80
% of grain germinated

% of grain germinated

60 60

40 40

20 20

0 0
Barley 1.w.r.b 1.w.r.e 1.a.r.e 2.w.r.e 2.a.r.e Barley 1.w.r.b 1.w.r.e 1.a.r.e 2.w.r.e 2.a.r.e
Sampe

without airing with airing

Figures 5: Evolution of percentage of germinated grains during the different steps of
steeping for the two processes.

The germinated grains appeared at the end of the 1st rest without airing and only
during the 2nd wet rest when aerating ; at least, the percentages of germinated grains at

6
the end of the second air rest were similar in the two ways of processing (between 90
and 92%) (figures 5).

The different malts produced gave close friabilities: 84% with airing and 88% without
airing.
The Tepral method was applied in order to value the performances of filtration of
these malts: table 2 shows the results.

mass, time, density, yield, Viscosity slope b slope c

g mn DMA % MS (cp)
Without airing 402 11,19 11,03 81,6 1,59 54 57
With airing 402 10,52 10,80 80,2 1,58 59 65
Table 2: Comparison of the parameters of Tepral method applied to the malts issued
from the two ways of steeping

The times of filtration were good and quite similar (10,511,2 mn); the wort
viscosities were the same (1,58-1,59 cp) and the yields were between 80,2% and
81,6%.

DISCUSSION
With a continuous aeration, the dissolved oxygen is stabilised around 85 90%. Pure
aerobic conditions are favoured in this type of steeping process. This explains the
general growth of yeasts and bacteria all along the process. But, because of the
constant values of dissolved oxygen concentrations, it is difficult to work these data.
For the non aired process, oxygen is quickly decreasing indicating possible anaerobic
zones around the grains during the wet rests. Because of these close to anoxic
conditions the growths are slowed down. Nevertheless, when bacteria are decreasing,
yeasts continue to grow or are stabilised: this indicates a better adaptation of yeasts to
these anoxic conditions.
In order to estimate the modification of microbial aerobic behaviour a modelling
approach is proposed. The evolution of dissolved oxygen concentration with time can
be simulated by a polynomial model; the result is shown in figure 6.
concentration (mg/l)
Dissolved oxygen

8
6
4
2
0
0 200 400 600
Time (mn)

Figure 6: Evolution of dissolved oxygen concentration with time: diamonds represent

the measured concentrations, the straight line represents the model.

7
In this case, the resulting equation representing the evolution of dissolved oxygen
concentration (DOC) with time is:
DOC = -10 8 .t 3 + 4 .10 -5
.t 2
-0,026.t +6,68
The rate of oxygen consumption r(mg of oxgen/l.mn) is given, at each time,
approximately by the absolute value of the derivative of the previous equation. So,
-8
r = 3.10 .t 2 8.10 5 . t + 0,026
By dividing this rate by the amount of microbial cells, one obtains the specific rate of
oxygen consumption.
For each significant cellular type (yeasts and bacteria), the amount of cells is
calculated by adding the cells present in water and on the grain. In order to work with
the same reference, each concentration Xg or Xw is expressed by grain dry matter.

For the cells on the grains:

If xg is the cell concentration on grain (UFC/g grain) and H, the grain moisture (g
water/g grain):
xG
XG =
1- H
For the cells in water:
If xw is the cell concentration in water (UFC/ml water), the porosity of the grain bed
(ml water/(ml water+ml grains)),, the volumetric mass of grain(g grain/ml grain):

x w .
Xw =
(1 - )..(1 H)

The total amount of cells expressed by grain dry matter, Xt, is:

Xt = Xg + Xw
And so the specific rate of oxygen consumption , can be calculated:
r' ' '
=
Xt

The different values of are related in table 3.

Bacteria Yeasts
mg/L/mn/(ufc/gDM) mg/L/mn/(ufc/gDM)
1st Wet Rest Begin 1,70 . 10 10 1,70 . 10 6
1st Wet Rest End 7,60 . 10 13 2,60 . 10 8
2nd Wet Rest End 1,80 . 10 11 2,50 . 10 8
Table 3: Evolution of the specific rate of oxygen consumption

The analysis of the specific oxygen consumption rates shows a decrease of the
respiratory activity between the beginning of the first wet rest and the end of the
second wet rest.
The ratio of respiratory activity decrease reaches a maximum of around 102 for
bacteria and yeasts at the end of the first wet rest.

8
The lack of oxygen in the medium adjusts the microbial behaviour to aero-anaerobic
conditions of growth.
For all the other parameters (organic acids, ethanol, moisture, steeping index, malt
quality) there are no significant differences between the two ways of processing. All
these results show that totally non-oxygenated wet rests during steeping have not any
effect on macroscopic quality of malt.
During totally aerated wet rests, oxygen is essentially used for microbial cellular
growth.
It seems that oxygen is not a limiting factor during the wet rests for the growth of
embryo. Two explanations are possible:
the different air rests are sufficient to supply oxygen to the grain
low concentrations of oxygen during the wet rests are not contrary to a good malt
development
The prospects of this study are:
in similar conditions, to measure other malt quality properties like specific
enzymatic activities (Chinyere et al, 1997; Sim and Berry, 1996) or Kirin test to
assess the specific oxygen demand of the grain;
to extrapolate our results to an industrial scale in order to optimize steeping with
regard to oxygen transfer.

The authors thank the associations Malteurs de France and Malteurs de Belgique
for financial and technical supports.

REFERENCES
1. Axcell, B, Jonkovsky, D and Morell, P. (1983). Steeping: the crucial factor in
determining malt quality, Brewers Digest, August, (58): 20-23.
2. Chinyere, I., Iwuoha and Aina, J.O. (1997) Effects of steeping condition and
germination time on the alpha amylase activity, phenolics content and malting
loss of nigerian local red and hybrid short kaura sorghum malts, Food Chemistry,
(58), 4: 289 - 295.
3. Kelly, L. and Briggs D.E. (1992a). The influence of the grain microflora on the
germinative physiology of barley, J. Inst. Brew., September - October, (98): 395 -
400.
4. Kelly, L. and Briggs D.E. (1992b). Barley maturity and the effects of steep
aeration on malting, J. Inst. Brew., July - August, (98): 329 - 334.
5. Powell, G.E., Chumbley, J. D. and Cole, N. W. (1993). The effect of oxygen and
carbon dioxide levels on enzyme development in malting, IOBC & SA: 45-54.
6. Sim, G.B. and Berry, D.R. (1996). Malted barley enzyme activity under optimum
and process conditions from the scotch malt whisky industry, Enzyme and
microbial technology (19): 26 - 31.
7. Van Campenhout, L., Shen, H.-Y., Iserentant, D. and Verachtert, H. (1999). The
gaz environment of germinating barley in various microbial states during malting,
Process Biochemistry: October (34): 929 - 937.

9
14

High-throughput assay for identification

of the barley varieties of individual malt
kernels with the use of microsatellite
DNA polymorphism
Kanta Sakamoto, Kazutaka Ozaki & Yasuyuki Ohtake
Asahi Breweries Ltd., Foods & Pharmaceuticals Research & Development Laboratory, 1-21,
Midori 1-chome, Moriya-machi, Kitasoma-gun, Ibaraki 302-0106, Japan (e-mail:
[email protected])

Descriptors
Barley variety, deoxyribonucleic acid, genetic marker, identification

SUMMARY
Assuring varietal purity in barley is of great importance for quality control in malting and
brewing. Recently DNA markers for barley have been developed. Among them, microsatellite
DNAs are very useful because of their extraordinary polymorphism. Combining six
microsatellite DNAs, we developed a new method which identified each of approximately
forty European barley varieties including malting and feed varieties. Furthermore, we
developed a high-throughput assay in which the barley varieties of ninety-six malt kernels
were identified individually. This method enabled us to detect the contamination of foreign
variety(ies) and estimate its ratio at the same time in one test.

Hoher Probendurchsatz zur Identifikation von Gerstensorten anhand einzelner

Malzkrner mittels microsatellite DNA polymorphism

Deskriptoren
Desoxyribonucleinsure, Gerstensorte, Markierungsgen, Nachweis (Identifizierung)

ZUSAMMENFASSUNG
Die Sicherstellung der Sortenreinheit ist eines der wichtigsten Kriterien fr Qualittskontrolle
in der Mlzerei und Brauerei. Diese microsatellite DNAs sind darber hinaus aufgrund ihrer
Polymorphie auergewhnlich hilfreich. Vor kurzem wurden DNA-Marker fr Gerste
entwickelt. Wir entwickelten mit Hilfe der Kombination von sechs microsatellite DNAs eine
neue Methode, welche die ungefhr 40 europischen Gerstensorten, einschlielich deren
Malze, identifiziert. Darber hinaus entwickelten wir einen Test mit hohem Probendurchsatz,
mit dem man die Gerstensorten von 96 Malzkrnern einzeln identifizieren kann. Diese
Methode gibt uns die Mglichkeit, kontaminierte Sorten zu erkennen und den Fremdanteil
abzuschtzen.
Dosage haut rendement pour l'identification des varits d'orge de grains de malt
individuels par analyse du polymorphisme de l'ADN microsatellite

Descripteurs
Acide dsoxyribonuclique, identification, marqueur gntique, varit dorge

RESUME
Assurer la puret des varits d'orge de brasserie est primordial pour le contrle de qualit en
malterie et brasserie. Rcemment, des marqueurs ADN ont t dvelopps pour l'orge. Parmi
eux, les ADN microsatellites sont trs utiles en raison de leur extraordinaire polymorphisme.
En combinant six ADN microsatellites, nous avons mis au point une nouvelle mthode qui a
permis d'identifier chacune des varits d'une quarantaine d'orges europennes, dont des
varits servant au maltage et l'alimentation. Par ailleurs, nous avons dvelopp un dosage
haut rendement dans lequel les varits d'orge de quatre-vingt seize grains de malt ont t
identifies individuellement. Cette mthode nous a permis, en un seul test, de dtecter la
contamination de varits trangres et d'estimer sa proportion simultanment.

2
INTRODUCTION
The conventional and most popular identification method for barley variety has been
based on their morphological characteristics. Currently electrophoretic analysis of
barley protein is also applicable (1)-(10). However those methods are not sufficient to
distinguish varieties of malted barley because the morphological characteristics and
protein patterns change during malting process.
On the other hand, the identification methods based on DNA have been developed
recently (11)-(24). Although they are very effective even for malt as well as barley,
the number of varieties which are able to distinguish is still limited in many cases.
Quantification of the mixture of a few varieties of malt was tried in some reports, but
it was necessary to be informed the variety of contamination prior to quantification.
In this study, we aimed to develop the more practical and rapid method with high
resolution that are able to detect and quantify the contaminated variety(ies) in one test.

MATERIALS AND METHODS

Barley varieties
39 European barley varieties including both malting and feed varieties were obtained
from institutes / laboratories or barley breeders in Europe (table 1).

Spring barley Winter barley

No. Variety Country row usage No. Variety Country row Usage
1 Akcent Czech 2 malting 21 Angora U.K. 2 Malting
2 Alexis U.K. 2 malting 22 Clarine France 2 Malting
3 Astoria France 2 malting 23 Fanfare U.K. 2 Malting
4 Chalice U.K. 2 malting 24 Gleam U.K. 2 Malting
5 Chariot U.K. 2 malting 25 Halcyon U.K. 2 Malting
6 Cheri U.K. 2 malting 26 Melanie U.K. 2 Malting
7 Cooper U.K. 2 malting 27 Regina U.K. 2 Malting
8 Kompakt Slovakia 2 malting 28 Tiffany Germany 2 Malting
9 Nevada France 2 malting 29 Esterel France 6 Malting
10 Optic U.K. 2 malting 30 Hanna U.K. 2 Feed
11 Prisma U.K. 2 malting 31 Intro U.K. 2 Feed
12 Reggae U.K. 2 malting 32 Jewel U.K. 2 Feed
13 Scarlett U.K. 2 malting 33 Marinka U.K. 2 Feed
14 Amulet Czech 2 feed 34 Pastral U.K. 2 Feed
15 Baronesse U.K. 2 feed 35 Krimhild U.K. 6 Feed
16 Cork U.K. 2 feed 36 Manitou U.K. 6 Feed
17 Dandy U.K. 2 feed 37 Muscat U.K. 6 Feed
18 Henni Germany 2 feed 38 Theresa France 6 Feed
19 Jubilant Slovakia 2 feed 39 Venus U.K. 6 Feed
20 Tankard U.K. 2 feed
Table 1: List of 39 European barley varieties.

DNA extraction
Single kernel of barley or malt was crushed by 1,500 rpm for 20 to 40 seconds with
the Multi-Beads Shocker MB301S (Yasui Kikai Ltd., Osaka, Japan) in a sterile plastic
tube. Genomic DNA was extracted with the Qiagen DNeasy Plant Kit (Qiagen

3
GmbH, Hilden, Germany).

DNA markers and PCR primers

Six microsatellite DNA markers reported by Liu et al. (24) were employed in this
study. PCR conditions and the primers were also according to Liu et al.
Oligonucleotides sequences of the primers were shown in table 2. Forward primers
were labeled with different fluorescent dyes at their 5-end. The reactions for HVM3,
4, 70 or HVM 27, 40, 68 were performed in the same tube.

Microsatellite Primer sequences* (5' to 3') Fluorescent PCR*

dye
HVM3 forward Acaccttcccaggacaatccattg 6-FAM(Blue) (3)
reverse Agcacgcagagcaccgaaaaagtc
HVM4 forward Agagcaactaccagtccaatggca HEX(Green) (3)
reverse Gtcgaaggagaagcggccctggta
HVM70 forward Ccgccgatgaccttctc NED(Yellow) (3)
reverse Acccacgacctatggcac
HVM27 forward Ggtcggttcccggtagtg HEX(Green) (1)
reverse Tcctgatccagagccacc
HVM40 forward Cgattccccttttcccac 6-FAM(Blue) (1)
reverse Attctccgccgtccactc
HVM68 forward Aggaccggatgttcataacg NED(Yellow) (1)
reverse Caaatcttccagcgaggct
* Primer sequences and PCR condition according to Liu et al.(24).
Table 2: Primer sequences for the microsatellite DNA markers.

Electrophoresis
The PCR products mixed with a molecular weight marker GS400HD (Applied
Biosystems Japan, Tokyo, Japan) were denatured by heating at 95C for 2 minutes in
the presence of formamide and separated on a 36 cm long urea-polyacrylamide gel
with ABI Prism 377-96 (Applied Biosystems, USA).

Analysis
The size of DNA bands of the PCR products was determined with GeneScan
(Applied Biosystems, USA). Genotyping of the microsatellite DNA markers of each
sample was performed with Genotyper (Applied Biosystems, USA).

RESULTS
Polymorphism of the microsatellite DNA markers and genotypes of the
European barley varieties
Most of the six microsatellite DNA markers were highly polymorphic among the 39
European varieties. HVM3 had eleven alleles which ranged from 165 to 220 bases.
HVM4 had eleven alleles ranging from 195 to 270 bases. HVM70 had 2 alleles
ranging from 145 to 160 bases. HVM68 had nine alleles ranging from 180 to 220
bases. HVM40 had four alleles ranging from 131 to 161 bases. HVM27 had three
alleles ranging from 179 to 195 bases.
Unique pattern of the combination of genotypes of the six markers was obtained for
each variety (table 3).

4
 Marker HVM3 HVM4 HVM70 HVM68 HVM40 HVM27
No. of alleles 11 11 2 9 4 3
Variety Genotype
Chariot D B B A B A
Alexis F B A A A B
Scarlett C, F, M A A A C A
Prisma C B A,B A A B
Optic F B B A C A
Nevada F B A D A B
Esterel J B B H A,C B
Cork E B B A A B
Baronesse F B A A A A
Amulet I B A G A B
. . . . . . .
. . . . . . .
. . . . . . .

Table 3: Polymorphism of the microsatellite DNA markers and the genotype pattern
of each varieties. Genotype of each microsatellite DNA marker was expressed
alphabetically. Chariot, for example, had the genotype of D, B, B, A, B, A for HVM3,
4, 70, 27, 40, 68 respectively. In some markers, a few alleles were observed in one
variety (HVM3 in Scarlett, HVM70 in Prisma and HVM40 in Esterel).The genotype
pattern for each variety was unique.

Development of a high-throughput assay for the variety identification of 96

individual malt kernels
For rapid and precise variety identification, we developed a high-throughput assay. It
included apparatus applied for 96 samples, such as the Multi-Beads Shocker MB301S
(Yasui Kikai), the DNeasy 96 Plant Kit (QIAGEN), the PCR machine for 96 tubes
and the ABI Prism 377-96 (Applied Biosystems). Multiplex PCR was employed for
the reaction of HVM 3, 4, 70 as well as for that of HVM 27, 40, 68. Mixture of the
PCR products of all of the six markers were separated in one run of electrophoresis
because different dyes were labeled for the markers whose alleles overlapped those of
the other markers in their band size. GeneScan and Genotyper, computer assisted
analysis programs gave us very precise data rapidly. Those apparatus and programs in
this assay enabled one operator to complete identification of the varieties of two sets
of 96 malt kernels individually within two days.

Sample trial
A number of malt samples purchased commercially were analyzed with this assay.
The results of somesamples were shown in table 4. The electrophoresis image and the
result of variety identification of the individual 96 malt kernels of sample no.2 were
shown in figure 1 and table 5, respectively.

5
Sample Maltster Shipping data Result
no.
1 A Chariot100% Chariot100%
2 B Scarlett100% Scarlett92%, Nevada5%, Cork1%, N/A*2%
3 C Scarlett50%, Scarlett33%, Prisma56%, Cheri2%, Esterel2%,
Prisma50% Reggae2%, Henni1%, Regina1%, N/A*2%
4 D Scarlett65%, Scarlett73%, Alexis0%, Optic22%, Cork2%, Prisma1%,
Alexis35% N/A*2%
* N/A = not available.
Table 4: Variety identification of the commercially purchased malt samples.

Figure 1: The electrophoresis image of the sample no.2. The PCR products from one
kernel were separated in one lane of the ge1. The red bands were from the size
standard markers (GS400HD, Applied Biosystems).

6
Kernel no. HVM Variety Kernel no. HVM Variety
3 4 70 27 40 68 3 4 70 27 40 68
1 C A A A C A Scarlett 49 C A A A C A Scarlett
2 F A A A C A Scarlett 50 F A A A C A Scarlett
3 F B A B A D Nevada 51 C A A A C A Scarlett
4 C A A A C A Scarlett 52 C A A A C A Scarlett
5 C A A A C A Scarlett 53 F A A A C A Scarlett
6 C A A A C A Scarlett 54 C A A A C A Scarlett
7 C A A A C A Scarlett 55 C A A A C A Scarlett
8 C A A A C A Scarlett 56 C A A A C A Scarlett
9 F B A B A D Nevada 57 C A A A C A Scarlett
10 C A A A C A Scarlett 58 F B A B A D Nevada
11 M A A A C A Scarlett 59 C A A A C A Scarlett
12 C A A A C A Scarlett 60 C A A A C A Scarlett
13 F B A B A D Nevada 61 C A A A C A Scarlett
14 C A A A C A Scarlett 62 F A A A C A Scarlett
15 C A A A C A Scarlett 63 C A A A C A Scarlett
16 C A A A C A Scarlett 64 C B A B D D N/A*
17 C A A A C A Scarlett 65 C A A A C A Scarlett
18 C A A A C A Scarlett 66 F A A A C A Scarlett
19 C A A A C A Scarlett 67 F A A A C A Scarlett
20 C A A A C A Scarlett 68 F A A A C A Scarlett
21 C A A A C A Scarlett 69 C A A A C A Scarlett
22 F A A A C A Scarlett 70 C A A A C A Scarlett
23 C A A A C A Scarlett 71 C A A A C A Scarlett
24 C A A A C A Scarlett 72 F A A A C A Scarlett
25 E B B B A A Cork 73 C A A A C A Scarlett
26 C A A A C A Scarlett 74 F B A B A D Nevada
27 F A A A C A Scarlett 75 C A A A C A Scarlett
28 C A A A C A Scarlett 76 C B A B A D N/A*
29 C A A A C A Scarlett 77 C A A A C A Scarlett
30 C A A A C A Scarlett 78 C A A A C A Scarlett
31 C A A A C A Scarlett 79 C A A A C A Scarlett
32 F A A A C A Scarlett 80 C A A A C A Scarlett
33 F A A A C A Scarlett 81 C A A A C A Scarlett
34 C A A A C A Scarlett 82 C A A A C A Scarlett
35 C A A A C A Scarlett 83 C A A A C A Scarlett
36 F A A A C A Scarlett 84 C A A A C A Scarlett
37 F A A A C A Scarlett 85 C A A A C A Scarlett
38 C A A A C A Scarlett 86 C A A A C A Scarlett
39 C A A A C A Scarlett 87 C A A A C A Scarlett
40 F A A A C A Scarlett 88 C A A A C A Scarlett
41 F A A A C A Scarlett 89 C A A A C A Scarlett
42 F A A A C A Scarlett 90 C A A A C A Scarlett
43 C A A A C A Scarlett 91 F A A A C A Scarlett
44 F A A A C A Scarlett 92 F A A A C A Scarlett
45 C A A A C A Scarlett 93 F A A A C A Scarlett
46 C A A A C A Scarlett 94 F A A A C A Scarlett
47 C A A A C A Scarlett 95 F A A A C A Scarlett
48 C A A A C A Scarlett 96 C A A A C A Scarlett
* N/A = not available.

Table 5: Variety identification of the individual 96 malt kernels of the sample no.2.
The genotype pattern for each kernel was shown. Comparing those patterns to the
microsatellite DNA database constructed in this study, the variety of each kernel was
determined.

7
CONCLUSIONS
Most of the microsatellite DNA markers studied in this report had high
polymorphism. So the combination of genotypes of only six DNA markers enabled us
to identify 39 European barley varieties.
We developed a high-throughput assay of variety identification of the individual 96
malt kernels. This assay enabled us not only to detect the contamination of foreign
variety(ies) but also to estimate its ratio at the same time in one test.
Sample trials revealed that there were actually contamination of foreign varieties
including feed varieties in the commercially purchased malt samples.
This assay is useful to assuring varietal purity of malt or barley in breweries and
maltsters.

ACKNOWLEDGEMENT
We appreciate to Dr. Kazuhiro Sato (Research Institute for Bioresources, Okayama
University, Japan) for giving us his precious advice and discussion, and to Dr. Saghai
Maroof (Virginia Polytechnic Institute and State University, USA) for giving us kind
permission to use their PCR-primers.

REFERENCES
(1) Baxter et al., J. Inst. Brew., 87(3):173-176, 1981
(2) Guenzel, Brauwelt, 121 (36):1296-1297, 1981
(3) Guenzel, Brauwelt, 122 (7):262-267, 1982
(4) Montembault, J., Inst. Brew., 89(4):299-302, 1983
(5) Burbidge et al., Brauwelt, 125 (8):282-286, 1985
(6) Marchylo et al., J. ASBC, 43 (1):29-35, 1985
(7) Gebre et al., Crop Science, 26:454-460, 1986
(8) Huston et al., J. ASBC, 46 (1):18-20, 1988
(9) Jones et al., J. ASBC, 49:93-98, 1991
(10) HSAM, Zeller et al., Monatsschr.Brauwiss., 46 (3):86-94, 1993
(11) Hofstra et al., Proceeding of E.B.C., 1989, 179-185
(12) Chee et al., J. ASBC, 51:93-96, 1993
(13) M.A. Saghai Maroof et al., Proc. Natl. Acad. Sci. USA., 91:5466-5470, 1994
(14) Ko et al., J. Inst. Brew., 100:405-407, 1994
(15) Tsuchiya et al., Proceeding of E.B.C., 1995, 109-116
(16) Vogeser et al., Brauwelt, 135 (42):2077-2079, 1995
(17) Hoffman et al., J. ASBC, 54 (3):172-176, 1996
(18) Lauer et al., Proceeding of E.B.C, 1997, :45-51
(19) William et al., J. ASBC, 55 (3):107-111, 1997
(20) Schuhbeck et al., Proceeding of E.B.C., 1997, 37-43
(21) Schuhbeck et al., Brauwelt, 138 (37)1680-1684, 1999
(22) Habernicht et al., J. ASBC, 57 (2):64-71, 1999
(23) Hang et al., J. ASBC, 58 (4):147-151, 2000
(24) Liu, Z.W., Biyashev, R.M., Saghai Maroof, M.A., Development of simple
sequence repeat DNA markers and their integration into a barley linkage map,
Theor. Appl. Genet., 93:869-876, 1996

8
15

Investigations into flavour and flavour

stability of dark beer in dependence on
malting and kilning parameters
Thomas Preu1, Clemens Forster2, Bernhard Thum3 & Werner
Back1
1
TU-Mnchen, Lehrstuhl fr Technologie der Brauerei I, Weihenstephaner Steig 20,
D-85350 Freising-Weihenstephan, Germany (www.wzw.tum.de, e-mail:
[email protected])
2
Labatt Brewing Company Ltd., 197 Richmond Street, London, ON N6A 4M3,
Canada
3
Stdtische Berufsschule fr Zahntechnik, Chemie-, Biologie- und Drogerieberufe,
Orleanstrae 46, D-81667 Mnchen, Germany

Descriptors
Dark beer, flavour formation, kilning, malty flavour, taste stability

SUMMARY
The most important flavour compounds which cause the significant malty flavour notes of
dark beers are formed during the Maillard reaction and the Strecker degradation in the malting
process. Kilning trials were carried out in a state-of-the-art computer controlled pilot kiln to
investigate the formation of different for dark beer characteristic flavour notes and to
achieve very high flavour stability in dark beer. The applied analytical methods allow an
explicit assessment of the flavour characteristics as well as flavour stability of dark beers in
conjunction with a specifically trained sensory panel. In dependence on the parameters
applied during the kilning of dark malt it is possible to obtain sweet, caramellike, nutty or
even roasty flavour notes in darks beers. Applying a specially emphasized withering process
during the kilning of dark malt, dark beers with extraordinary high flavour stability can be
produced.

Einflu der Darrtechnologie dunkler Malze auf das Malzaroma und die Geschmacks-
stabilitt dunkler Biere

Deskriptoren
Aromabildung, Darren, dunkles Bier, Flavour nach Malz, Geschmacksstabilitt

ZUSAMMENFASSUNG
Die wichtigen Aromastoffe fr das Dunkelmalzaroma dunkler Biere werden berwiegend im
Rahmen der Maillardreaktion und des Streckerabbaus whrend der Malzherstellung gebildet.
In einer prozegesteuerten Pilotdarre wurden Darrversuche durchgefhrt, um die Bildung
verschiedener charakteristischer Aromastoffe und die Geschmacksstabilitt der resultierenden
Biere gezielt zu beeinflussen. Die Biere wurden anhand geeigneter analytischer und
sensorischer Methoden zur Beschreibung des Aromas und der Geschmacksstabilitt unter-
sucht und bewertet. Es zeigte sich, da durch die Darrtechnologie dunkler Malze gezielt
sliche, karamelartige, nussige oder rstige Aromanoten in dunklen Bieren erzielt werden
knnen. Aus dunklen Malzen, die mit intensiverer Schwelkarbeit hergestellt werden,
resultieren uerst geschmacksstabile dunkle Biere.

Investigations sur l'arme et la stabilit de l'arme de la bire brune en fonction des

paramtres de maltage et de touraillage

Descripteurs
Bire brune, formation de la flaveur, got de malt, stabilit organoleptique, touraillage

RESUME
Les principaux composs aromatiques donnant les notes aromatiques maltes typiques des
bires brunes se forment pendant la raction de Maillard et la dgradation de Stecker au cours
du maltage. Des essais de touraillage ont t conduits dans un four pilote contrl par
ordinateur de dernire gnration pour explorer la formation de diffrentes notes aromatiques
caractristiques de la bire brune et obtenir une trs haute stabilit des armes dans la
bire brune. Les mthodes analytiques appliques permettent une valuation explicite des
caractristiques aromatiques ainsi que de la stabilit de l'arme des bires brunes, en
conjonction avec un panel de goteurs spcialement forms. En liaison avec les paramtres
utiliss pendant le touraillage du malt brun, il est possible d'obtenir des notes aromatiques
sucres, de caramel, de noisette, voire de grill dans les bires brunes. En appliquant un
procd de desschement spcialement soulign pendant le touraillage du malt brun, il est
possible de produire des bires brunes d'une extraordinaire stabilit aromatique.

2
INTRODUCTION
Preceding extensive analytical experiments helped to investigate the key flavour
compounds as well as staling compounds of dark beers by applying sophisticated
analytical methods like the combination of high vacuum transfer technique with
aroma extract dilution assay and mass-spectrometry/olfactometry.
The knowledge of these key flavour compounds enables to influence systematically
the flavour and flavour stability of dark beer. The most important flavour compounds
which cause the significant malty flavour notes of dark beers are formed during the
Maillard reaction and the Strecker degradation in the malting process, espacially
during the kilning of malt.
The flavour stability of dark beer is characterized by a loss of the malt aroma5).
The quantity of Maillard products formed during the kilning process depends on the
thermal charge, expressed as kilning temperature and kilning time2). However, the
odour quality is strongly influenced by the ratio of amino acids and reducing
sugars3,6). A surplus of reducing sugars results in a higher level of Maillard products4).
Based on these findings kilning trials were carried out in a state-of-the art computer
controlled pilot kiln to investigate the formation of different - for dark beer
characteristic - flavour notes and to achieve very high flavour stability in dark beer.

MATERIAL & METHODS

Kilning experiments
The technological trials were performed under the following exactly defined
conditions:

A 2-row malting barley variety of one homogenous batch was steeped and germinated
to obtain standardized green malt which was kilned in a state-of-the-art computer
controlled single floor high performance pilot kiln on a 20 kg scale. On this occasion,
only one parameter was modified at each experiment. All produced dark malts were
brewed under defined conditions in a ratio of 3:1 with pale malt in a pilot brew stand
(60 l scale). The flavour and flavour stability in the fresh and aged dark beers were
evaluated by specific sensory and analytical methods.

Sensory analysis
The dark beers were evaluated by a specifically trained sensory panel using the
following sensory analysis:

- sensory test by DLG (German Agricultural Society)

- specific sensory taste pattern for aged beer1)
- evaluation of the intensity and quality of the malt flavour in odour and taste
- characterization of the odour perception.

The staling properties of the dark beer were evaluated every five weeks over a period
of at least 25 weeks of shelf-life (naturally aging of the beer at 20 C).

Flavour analysis
The different flavour compounds were determined by the following analytical
methods:

3
- Determination of flavour compounds in beer by solid phase extraction7)
Quantitative determination of flavour compounds of beer by solid phase extraction
(polar mechanism) at beer pH. This allows in conjunction with very moderate
conditions during sample preparation to determine even polar and labile
compounds which are relavant for flavour perception.

- Strecker aldehydes in beer

Quantitative determination of relevant aldehydes by solid phase extraction (C18)
after derivatisation of the carbonyl group with O-2,3,4,5,6-(pentafluoro-benzyl-
)hydroxylamine. Analysis of volatiles in the organic phase by GC/MS with
negative chemical ionisation7).
In addition to this, Methionale is determined by the quantitative masses 178, 197,
205, 229, 232, 249, 279 (internal standard: Pentanal)

- Other methods
Determination of volatile fermentation by-products (acetaldehyde, higher alcohols,
ester; improved headspace GC/FID7))

The developed analytical methods allow an explicit assessment of the flavour

characteristics as well as flavour stability of dark beers in conjunction with a specially
trained sensory panel.

RESULTS
Variation of the kilning temperature
After defined malting and kilning profile the final kilning temperature was adjusted
for 4,5 hours at 95, 100, 105, 110 and 115 C respectively. Green malt modification
was at 45% for each trial.

3000
2500
R2 = 0,9946
Conc. [g/l]

2000
1500
1000
500
0
90 95 100 105 110 115 120
Kilning temperature [C]

Figure 1: Concentration of 2,5-dimethyl-4-hydroxy-3-(2H)-furanone in fresh dark

beer produced with dark malts at different kilning temperatures.

4
 5

Intensity of malty flavou 4

0
95 100 105 110 115
Kilning temperature [C]

Figure 2: Intensity of the malt flavour in fresh dark beers produced with dark malts at
different kilning temperatures.

Higher kilning temperatures during the malting process result in more caramel, sweet
and roasty flavour notes in fresh dark beer. The steady increase of the caramel like
smelling 2,5-dimethyl-4-hydroxy-3-(2H)-furanone (see figure 1), a key odorant for
malty flavour, corresponds with this flavour perception.
The intensity of the malty flavour rises significantly between a kilning temperature of
100 C and 105 C (see figure 2).

5000

4000 T95
Conc. [g/l]

3000 T100
T105
2000
T110
1000 T115

0
0 3 6 9
Storage [month]

Figure 3: Concentration of 2,5-dimethyl-4-hydroxy-3-(2H)-furanone in the dark beers

produced with dark malts at different kilning temperatures (T95 T115)
during storage.

5
The concentration of some important flavour compounds, e.g. 2,5-dimethyl-4-
hydroxy-3-(2H)-furanone, which cause the significant malty flavour notes of dark
beer are very stabil within 6 months - during staling (see figure 3).
The fruity, flowery and honey like flavour notes are getting more intense during the
storage within the first six month. The flavour perception corresponds with the
increase of fruity esters (e.g. ethylacetate, 2-methylpropylacetate, ethylbutanoate, 3-
methylbutylacetate) and Strecker aldehydes (isobutyraldehyde, 2-methylbutyral-
dehyde, 3-methylthiopropionaldehyde) in this period.
The flavour stability of dark beer is increasing with the final kilning temperature that
is applied during the drying process of the dark malt.

Variation of the kilning period

After a defined malting and kilning profile, the final kilning temperature of 100 C
was held for 2, 4,5 and 7 hours respectively. Green malt modification was at 45% for
each trial.

1800
1600
Conc. [g/l]

1400
1200
1000
800
600
0 2 4 6 8
Kilning period [h]

Figure 4: Concentration of 2,5-dimethyl-4-hydroxy-3-(2H)-furanone in fresh dark

beer produced with dark malts which was held at the final kilning
temperature for different periods

Extended periods of kilning at higher temperatures have a similar effect on the flavour
changes in the fresh dark beers as increasing kilning temperatures.
The caramel like and roasty flavour notes become more intense and the malty flavour
increases slightly.

6
 4,5

Intensity of malty flavour

4,0

3,5
Z 4,5
3,0 Z7

2,5

2,0
0 5 10 15 20 25
Storage [month]

Figure 5: Intensity of the malt flavour in the dark beers produced with dark malts
which was held at the final kilning temperature for different periods (Z4,5,
Z7) during storage.

The intensity of the malt flavour shows a strong decreases after a period of 15 weeks
of storage for the dark beer with a short kilning period (max. 4,5h / Z4,5). On the
other hand, the malt flavour of the beer with 7 h kilning period increases slightly (see
figure 5). This corresponds with the changes in the evaluation (DLG) of the dark beers
during storage.
The malt held for the longest period at the final kilning temperature obtained the best
sensory evaluation.

Variation of the green malt modification

In order to form great amounts of precursors for the subsequent Maillard reaction,
green malt at very high modification was produced.

Green malt modification

Volatile compound [g/l] 45% 48% 51%
Isobutyraldehyde 20,3 22,9 23,8
2-methylbutyraldehyde 6,1 7,3 9,7
3-methylbutyraldehyde 18,4 21,3 26,9
3-methylthiopropionaldehyde 3,8 3,6 5,0
2,5-dimethyl-4-hydroxy-3-(2H)- 2278 2537 2924
furanone

Table 1: Concentration of certain volatiles in fresh dark beers made with dark malts at
different degrees of green malt modification.

Higher green malt modification results in higher concentrations of most volatiles,

especially in a significant rise of 2,5-dimethyl-4-hydroxy-3-(2H)-furanone and the
Strecker aldehydes (see table 1). Honey, nutty and caramel like notes were also more
intense in the dark beers with higher modification of the green malt. The intensity of
the malt flavour increases with the degree of the green malt modification.

7
The intensity of the malt flavour slightly increases during the first 25 weeks of shelf
life. After six months the malty flavour notes reach the same level as in the fresh beer
again.
This corresponds with the change of the concentration of Strecker aldehydes during
natural aging of dark beer.

Variation of the 65 C rest during kilning

In order to change the ratio of sugars and amino acids for the subsequent Maillard
reaction, the rest at 65 C during the first phase of the drying process of malt was
extendend (no rest, 1, 2 and 3 hours rest respectively).

Thereby, an increased amount of reducing sugars compared to amino acids result in

more roasty, nutty and bread like aroma notes (trial with 3 hours rest) together with
sweet and malty notes in dark beer. The fresh dark beer of this series of experiments
were rated lower as the beers of the previous series.

2000
Conc. [g/l]

1500 R0
R1
1000
R2
500 R3

0
0 3 6
Storage [month]

Figure 6: Concentration of 2,5-dimethyl-4-hydroxy-3-(2H)-furanone in the dark beers

produced with dark malts which was held at the rest at 65 C for different
periods (R0 / 0 hour rest R3 / 3 hour rest) during storage.

An extended 65 C rest during kilning results in a better flavour stability in the first
weeks (at least 25 weeks) of shelf life.
During storage the concentration of 2,5-dimethyl-4-hydroxy-3-(2H)-furanone shows a
decrease when the rest at 65 C is held not longer than 2 hours. On the other hand, the
concentration decreases after 6 months when the 65 C rest lasts 3 hours (see figure
6).
After 5 to 10 weeks of shelf life at 20 C, the dark beers produced with a 2 and 3 hour
rest at 65 C during kilning are rated better than in fresh condition (see figure 7). On
the other hand, the acceptance of the beers produced with malt without or even 1 hour
rest at 65 C decreases with extended storage.

8
 5,0

4,5
Sensory score by DLG R0
4,0
R1
3,5 R2
3,0 R3

2,5

2,0
0 5 10 15 20 25
Weeks

Figure 7: Evaluation by DLG of the dark beers produced with dark malts which was
held at the rest at 65 C for different periods (R0 / 0 hour rest R3 / 3 hour
rest) during storage.

CONCLUSIONS
Depending on the parameters applied during the kilning of dark malt, it is possible to
create sweet, caramel like, nutty or even roasty flavour notes.
The application of a specially emphasized withering process (i.e. the first phase of the
drying process of malt) with an extended 65 C rest during the kiling of dark malt,
results in dark beers with extraordinary high flavour stability.
Conditions during malting which favour Maillard reaction in the kilning process,
generally cause a more intense malt flavour and higher flavour stability of dark beer.

REFERENCES
1. Eichhorn, P., TU Mnchen-Weihenstephan, Doctoral Thesis, 1991.
2. Forster, C., TU Mnchen-Weihenstephan, Doctoral Thesis, 1996.
3. Nursten, H.E., Food Chemistry, 1981, 6, 263-277.
4. OBrian, J.& Morrissey, P.A., Critical Review on Food Chemistry, 1989, 24,
847-852.
5. Preu, Th., TU Mnchen-Weihenstephan, Doctoral Thesis, 2001.
6. Shibamoto, T. & Bernhard, R.A., Journal of Agriculture and Food Chemistry,
1976, 24, 847-852.
7. Thum, B., Proceedings of the European Brewery Convention Congress, Cannes,
999, 45-52.

We are indepted to the Wissenschafsfrderung der Deutschen Brauwirtschaft e.V. for the
benefit for that project.

9
16

What is it about antioxidative

characteristics of hops?
A. Forster, K. Simon, R. Schmidt & D. Kaltner
NATECO2 GmbH & Co. KG, Auenstrasse 18-20, D-85283 Wolnzach, Germany (e-mail:
[email protected])

Descriptors
Antioxidant, bitter substances,hop analysis result, polyphenol

SUMMARY
In the literature different methods to describe possible antioxidative effects of hops are used
with contradictory results. Here the rancimat method and the fluoro-scan procedure have been
applied for systematical investigations on substances, substance groups and different extracts
of hops. Only if the bitter components are removed from hop samples, the rancimat method
can describe reliable polyphenols with antioxidative effects.The fluoro-scan procedure does
not confirm any antioxidative characteristics of hop samples from which bitterness was
removed or of extracts soluable in water, but clearly of extracts of specific bitter acid
character. This antioxidative potential obviously is lost in isomerised hop extract.

Die antioxidativen Eigenschaften von Hopfen Ursachenforschung

Deskriptoren
Antioxidantium, Bitterstoffe, Ergebnis von Hopfenanalysen, Polyphenol

ZUSAMMENFASSUNG
In der Literatur werden verschiedene Methoden zur Beurteilung der antioxidativen
Auswirkungen von Hopfen mit gegenstzlichen Ergebnissen genannt. Hierzu wurden die
Rancimat- und Fluoro-Scan-Methode eingesetzt, um Substanzen, Substanzgruppen und
verschiedene Hopfenextrakte systematisch zu untersuchen. Nur wenn Bitterkomponenten von
den Hopfenproben entfernt werden, kann ber die Rancimat-Methode eine zuverlssige
Aussage zu den antioxidativen Auswirkungen von Polyphenolen gemacht werden. Die
Fluoro-Scan-Methode besttigt keine antioxidativen Eigenschaften von Hopfenproben, bei
denen die Bitterstoffe entfernt wurden oder von wasserlslichen Extrakten, aber deutlich von
Extrakten mit spezifischen Bittersuren. Dieses antioxidative Potenzial geht offensichtlich in
isomerisierten Hopfenextrakten verloren.
Le point sur les caractristiques anti-oxydantes des houblons

Descripteurs
Antioxydant, polyphnol, rsultat danalyse du houblon, substances amres

RESUME
Dans la littrature, diffrentes mthodes sont utilises pour dcrire les ventuels effets anti-
oxydants des houblons, avec des rsultats contradictoires. Ici, la mthode rancimat et la
technique fluoroscan ont t utilises pour des investigations systmatiques sur les substances,
groupes de substances et diffrents extraits de houblon. C'est uniquement si les chantillons
de houblon sont dbarrasss de leurs composants amers que la mthode rancimat est en
mesure de dcrire fiablement des polyphnols dots d'effets anti-oxydants. La technique
fluoroscan ne confirme aucune caractristique anti-oxydante des chantillons de houblon
dbarrasss des composants amers ni des extraits hydrosolubles, mais en rvle partir
d'extraits d'un caractre acide-amer spcifique. A l'vidence, ce potentiel anti-oxydant est
perdu dans les extraits de houblon isomriss.

2
INTRODUCTION
There are mainly two reasons for the interest in antioxidative characteristics of plants,
plant constituents or food-stuffs:
Antioxidants protect food-stuffs against spoilage caused by oxygen.
Antioxidants may act as interceptors of radicals in the cell. They are additionally
assumed to protect humans against heart diseases and cancer.
There are innumerable methods describing antioxidative characteristics, which are
based on completely different mechanisms and therefore often provide diverging
results.
Hops have already been investigated on various occasions. The two examples we
selected represent the different methods used. Van Waesberghe (3) used the Swift
Test, which measures oxidation products resulting from the ageing of maize oil.
With this method it is possible to distinguish between prooxidative and antioxidative
characteristics of additives. Contrary to expectations, prooxidative characteristics
were found in nearly all hop samples. Tageshira et al (2) obtained completely
contradictory results with 2 different cell test methods - one system that catches
radicals and one fat oxidation system. The authors found high antioxidative activities
with and -acids, which are within the range of usual antioxidants like -tocopherol
or ascorbic acids.
Due to the contradictory results, we decided to initially pursue both directions. Finally
we selected the Rancimat method of the company Metrohm, an automated and
modern apparatus, which can be compared to a certain degree with the Swift Test and
the Fluoro Scan procedure, a cell test described by Mayer et al (1).

METHODS
The Rancimat works according to the following principle: Lard is heated with and
without a test substance to 110 C and air is constantly blown into the mixture. After
all components with antioxidative effects have been consumed, easily volatile
substances are formed from the lard. They are expelled with the airstream and
collected in bidistilled water, where they increase the conductance according to the
quantity added. The time of increase in conductance is registered. If the time
decreases with an additive, there is a prooxidative effect, and if the time increases, an
antioxidative effect is present. Figure 1 shows the measuring arrangement.

Air
Volatile degra-
dation products

Indicator:
Reaction vessel Conductance
of solution
Heating block
Electrode

Lard with sample

Bidistilled
Absorption vessel water

Figure 1: Measuring arrangement of the Rancimat

3
Figure 2 demonstrates a print of 4 parallel measurements. The antioxidation activity
index (AI) is calculated from the measured induction times according to the following
formula:

Induction' time of lard with additive AI < 1 = prooxidative effect

AI = ;
Induction' time of pure lard AI > 1 = antioxidative effect

prooxidative

antioxidative

Figure 2: Printing from the Rancimat with 4 cells

a) Control: pure lard
b) Sample with prooxidative effect AI < 1
c) Sample with antioxidative effect AI > 1
d) CO2-extract without lard

When working exactly, this method offers a simple, efficient and automated
measuring principle . Table 1 shows the AI values of some typical antioxidants and of
some polyphenols, which can also be found in hops. Many components have
antioxidative effects, but some are ineffective according to this method.

Substance AI
Tertiary butylated hydroxyanisole (BHA) 3.0
Butyl hydroxytoluene (BHT) 2.0
Ascorbic acid (vitamin C) 2.5
Trolox 4.0
Catechine/Epicatechine 2.0 - 3.0
Gallic acid >5
Ferulic acid and p-cumaric acid 1.3 - 1.5
4-hydroxybenzoic acid, flavonoids 1.0
Xanthohumole 1.0

Table 1: Antioxidation activity index (AI) of various antioxidants and

polyphenols (0.02 % addition)

4
In the Fluoro Scan procedure, test substances are added to a endothel cell culture with
a fluorescent colour. Intracellular radicals oxidise the colour and characteristic
fluorescent emissions result, which can be measured at 530 nm. Comparing untreated
cells with those to which an additive has been given, a relative fluorescence results,
this being a measure of the antioxidative capacity of a test substance. Furthermore, by
adding hydrogen peroxide, an oxidative stress can be induced. The method is time-
intensive and can only be realised in special laboratories. It is of advantage that under
physiological conditions, free radicals (and not secondary reaction products) are being
tested directly. Figure 3 shows the temporary process of a fluorometry. Control
specimen without additive, additives with prooxidative and antioxidative effects are
discernible. The latter delay the relative fluorescence due to the restricted activity of
intracellular radicals.

5.0

4.5
b
4.0
a
3.5
rel. fluorescence

3.0
c
2.5

2.0

1.5

1.0

0.5

0
0 30 60 90 120 150 time (min)

Figure 3: Temporary process of a fluorometry

(Cells after induction of an oxidative stress with H2O2)
a) Control without addition
b) Addition without an antioxidative effect
c) Addition with an antioxidative effect

Besides the relative fluorescence of a sample within the scope of the control
specimen, a comparison with the effective antioxidant Trolox, (a vitamin E analogue)
is also possible:

relative' fluorescence of a sample

TEAC = Trolox equivalent antioxidant capacity =
relative' fluorescence of Trolox

TEAC <0 : prooxidative effect

TEAC from 0 to 1 : antioxidative effect from low to Trolox
TEAC >1 : antioxidative effect stronger than Trolox

5
TEST RESULTS
Rancimat method
Table 2 shows the AI values of some pure bitter and aroma components added
directly or in the same concentration as aqueous alcoholic solution. The results do not
change even with ethanol/water as a solubilizer. This also applies to all subsequent
test results. In no case can an antioxidative activity be established.

Substance AI direct AI as ethanol-water mixture

-acids 0.8 0.8
Iso--acids 0.2 - 0.5 0.3 - 0.5
Rho-iso--acids 0.4 - 0.8 0.4 - 0.8
Tetrahydro-iso--acids 0.6 - 1.2 0.7 - 1.1
Myrcene, Humulene, 0.9 - 1.0 ---
Linalool and others

Table 2: Antioxidation activity index (AI) of various bitter and aroma components
(0.2 % addition)

In table 3 typical extracts are listed which tend to show prooxidative effects. Trials,
however, showed that with this test arrangement even without lard components
were set free from the bitter substances, which caused an increase of conductivity (see
figure 2, sample d). It can therefore be concluded that this measuring arrangement is
not suitable for investigating hops or usual hop extracts. This finding can also be
applied to the Swift Test and explains the obtained illogical results.

Type of sample AI
CO2-extracts normal 0.2 0.8
CO2-extracts poor in oil 0.2 0.8
CO2-extracts rich in oil 0.1 0.4
Ethanol-extracts 0.3 0.9
Base-extracts (-acids and hop oil) 0.1 0.3
Hop oils 0.3 0.7
Hot water extracts 0.6 1.1

Table 3: Antioxidation activity index (AI) of various hop extracts (0.2 % addition)

The AI values of typical hop samples (0.1 to 0.3), which are shown in figure 4, also
confirm these problems. As it was to be expected that the bitter substances would
impair the usefulness of the method, hops were investigated after CO2-extraction
(spent hops) in the next step. The result was the detection of an antioxidative capacity
(AI 1.6 to 2.0) of these samples (see also figure 4).

6
 2,5

2,0

1,5

1,0

0,5

0,0
Tradition Perle Magnum Taurus
hop sample spent hops

Figure 4: AI of various hop samples and spent hops (Hallertau varieties)

Mixing a sample free from bitter substances with CO2-extract produces constantly
decreasing AI values as shown in figure 5. This confirms once again that bitter acids
disturb the measuring principle.

2,0

1,5

AI 1,0

0,5

0,0
0% 5% 10 % 20 % 40 %
Proportion CO2-extract

Figure 5: AI-values of mixtures from CO2-spent hops and CO2-extracts

measurements at 110 C with 2 % dosage

When selecting a suitable sample, the Rancimat test turns out to be an appropriate
method for describing antioxidative activities of hops.

Fluoro Scan procedure

The Trolox equivalents of some hop samples are demonstrated in figure 6. No
antioxidative capacity can be found in aqueous hop extracts, samples free from bitter
substances (spent hops), hop oils and all isomerised -acids (iso-, rho-iso-,
tetrahydro-iso-). Lupulin enriched hop powders and ethanol extracts show moderate
antioxidative effects, while -acids and CO2-extacts clearly have an antioxidative
potential, which exceeds the effect of Trolox.

7
 Trolox
-2 -1,5 -1 -0,5 0 0,5 1 1,5 2 2,5

Hot water extract

Spent hops

Iso-alpha-acids

Hop oil

Ethanol extract

Hop powder Type 45

Alpha-acids

CO2-extracts

TEAC normal TEAC H2O2-induced

Figure 6: Trolox equivalent antioxidant capacity (TEAC) from the Fluoro

Scan test of various hop samples

CONCLUSIONS
Hops, hop extracts and individual substances were examined by means of two
completely different methods for their antioxidative capacity. The Rancimat registers
the more or less fast oxidation of lard, the Fluoro Scan procedure covers the potential
available for intercepting radicals in cell cultures.
The 2 methods provide controversial results. This implies that every method for
demonstrating antioxidative characteristics has to be carefully examined. The
Rancimat test like the Swift Test shows the seemingly prooxidative effect of hop
extracts. This, however, is exclusively due to the test procedure. Degradation products
of the bitter acids falsify the results. Clear antioxidative characteristics can be found
when using suitable samples without bitter substances. The Rancimat thus reflects the
composition of hop polyphenols. Additionally, a differentiation can be made between
more or less effective individual components.
The Fluoro Scan procedure elucidates high antioxidative effectiveness with hop
extracts of bitter substance character, which is confirmed by the results obtained by
Japanese researchers. Extracts of polyphenolic character, however, react neutrally.
The Fluoro Scan method opens up possibilities for using hop bitter substances as
antioxidants also outside of brewing technology. As radical interceptors in a cell
system, they seem to be considerably better than polyphenols.
The Rancimat is best suited for describing the antioxidative potential based on
polyphenols. The influence of the hop variety, storage and production procedures still
has to be investigated in detail in order to be able to draw the utmost benefit for the
brewing technology. According to the results obtained from the Fluoro Scan method,
it cannot be ruled out that remaining -acids are also among the oxygen consumers in
beer.

8
To avoid misinterpretation by applying unsuitable methods or so as not to succumb to
the temptation to look for the right method to make an assertion, it is recommended
to select different methods simultaneously. In future, the Rancimat may provide the
brewing technologist with interesting information; the Fluoro Scan test, however, may
additionally furnish new ideas for the use of hop extracts beyond the world of beer.
Further investigations are necessary in order to ascertain the influence which hop
constituents have on the flavour stability of beer.

LITERATURE
1. Mayer, D.H., Bernhardt, J., Heinrich, F., Biesalski, H.K., European Journal of
Medical Research, 6, 1-8, 2001
2. Tageshira, M., Watanabe, M., Uemitsu, N., Biosci. Biotech. Biochem., 59 (4),
740-742, 1995
3. van Waesberghe J., MBAA Technical Quarterly, Volume 33 (2), 96-101, 1996

Expression of thanks
Many thanks to the BioTeSys GmbH, Schelztorstrae 54-56, 73728 Esslingen for the
precise execution of all trials with the Fluoro Scan procedure.

9
17

Investigations of the influence of hop

products on the microbiological stability
of beer
D. Kaltner1, I. Bohak2, A. Forster1, A. Gahr1 & W. Back2
1
Hopfenveredlung HVG Barth, Raiser GmbH & Co. KG, D-93358 St. Johann/
Hallertau, Germany (e-mail: [email protected])
2
TU Mnchen, Lehrstuhl fr Technologie der Brauerei I, D-85350 Freising-Weihenstephan,
Germany

Descriptors
Beer spoilage organism, biological stability, hop product, Humulone, iso--acid,
microorganism

SUMMARY
Beer-spoiling micro-organisms are influenced to various degrees in their growth in beer when
different hop products are added during the brewing process. The micro-organisms of the
species Lactobacillus and Pediococcus show specific sensitivity against these hop products.
Experiments demonstrated that -acids are about 3 to 4 times more effective in their
antimicrobiological potential than iso--acids. However, the influence of -acids, tetrahydro-,
hexahydro- and rhohydroiso--acids on the growth of beer-spoiling micro-organisms is still
unknown. The aim of this work is to systematically examine the influence of special hop
products on the microbiological stability of beer.

Untersuchungen des Einflusses von Hopfenprodukten auf die mikrobiologische

Stabilitt von Bier

Deskriptoren
Bierschdliche Organismen, biologische Stabilitt, Hopfenprparat, Humulon, Iso--Sure,
Mikroorganismen

ZUSAMMENFASSUNG
Bierschdliche Mikroorganismen werden in ihrem Wachstum unterschiedlich stark
beeinflut, wenn verschiedene Hopfenprodukte whrend des Brauprozesses zugegeben
werden. Mikroorganismen der Gattungen Lactobacillus und Pediococcus zeigen spezifische
Empfindlichkeiten gegen diese Hopfenprodukte. Untersuchungen haben gezeigt, da -
Suren um den Faktor 3 bis 4 antimikrobiell wirksamer sind als iso--Suren. Der Einflu
von -Suren, Tetrahydro-, Hexahydro- und Rhohydroiso--Suren auf das Wachstum
bierschdlicher Mikroorganismen ist bislang jedoch unbekannt. Ziel dieser Arbeit ist die
systematische Untersuchung zum Einflu von speziellen Hopfenprodukten auf die
mikrobiologische Stabilitt des Bieres.

Etudes sur l'influence des produits du houblon sur la stabilit microbiologique de la

bire

Descripteurs
Humulone, lupulone, stabilit microbiologique,

RESUME
Dans la bire, la croissance des micro-organismes responsables du vieillissement et de
l'altration est influence divers degrs par des produits du houblon ajouts pendant le
brassage. Les bactries des espces Lactobacillus et Pediococcus montrent une sensibilit
spcifique l'gard de ces produits du houblon. Des expriences ont montr que les -acides
possdent un potentiel antimicrobien environ 3 4 fois suprieur celui des iso--acides.
Toutefois, l'influence des -acides, des ttrahydro-, hexahydro- et rhohydro-iso--acides sur
la croissance des micro-organismes pourrisseurs de bire reste indtermine. Ce travail a pour
but d'tudier de manire systmatique l'influence de produits du houblon spciaux sur la
stabilit microbiologique de la bire.

2
INTRODUCTION
Iso--acids at concentrations of 10 mg/l in beer show an anti-microbiological effect
on beer-spoiling micro-organisms. However, the influence of -acids, -acids,
tetrahydro-, hexahydro- and rho-iso--acids on the growth of beer-spoiling micro-
organisms is still unknown.

An application of -acids outside of the brewing industry is employed in the sugar

industry to inhibitate gram-(+)-micro-organisms (3). An addition to beer might be
interesting but is not practible because of the acid character of beer and subsequent
very low solubility.

Objective of this work

Systematic investigation of the influence of special hop products on the
microbiological stability of beer.

MATERIALS AND METHODS

Technological experiments were carried out in the Research Brewery of St. Johann to
a 200 l scale (see figures1 and 2).

Unhopped, nonstabilized (basic) beer was produced with pure culture yeast (table 1).
The pitching yeast was enriched several times in unhopped wort. The necessary
dosage for each hop product had been worked out in previous experiments. The best
results were obtained when adding the products in solution against the beer stream
during filtration (figure 2). The concentration of the products was controlled by
HPLC-DAD.

At the Institute of Brewery Technology I - Weihenstephan, beer-spoiling micro-

organisms of the genera lactobacillus and pediococcus were added to the beer and
controlled by assessing the turbidity and carrying out a final microscopical control.

Filter

Dosing unit
Brewhouse

Figures1 and 2: Pictures of the Research Brewery of St. Johann

3
Tetrahydroiso-, hexahydroiso- , rhoiso- and iso--acids are in an aqueous alkaline
solution. Hexahydroiso--acids are only available as a mixture of hexa- and
tetrahydro-iso--acids (1:1) in a product. Alpha-acids are used in an ethanolic
solution for a better utilisation in beer. There was no success in solubilizing -acids in
beers with a pH of 4.4 to 4.6.

Figure 3 gives an overview of the chemical structure of various hop compounds.

-acid iso--acid

rho-iso--acid tetrahydro-iso-- hexahydro-iso--

acid acid

Figure 3: Structure of hop bittering compounds

Incubation conditions
The germs which are part of the specific culture collection of the Institute of Brewery
Technology I tested in these investigations were bred in beer and adapted to the
selective factors of beer.

The micro-organisms of the genera lactobacillus and pediococcus were added to the
test beers in two different amounts (concentration A and B; see table 2).

The beers were incubated at 25 - 28 C in darkness under anaerobic conditions over a

period of 6 weeks.

4
Table 1: Specifications of beers produced with different hop products

Content of hop
bittering compounds
EBC-BU PH [ppm]
Unhopped beer 1 4.47 ---
Alpha-acids 11 4.45 9.6
-acids
Iso- 9 4.59 10.5
Tetrahydro-iso--
10 4.55 10.5
acids
-acids
Rho-iso- 8 4.46 9.2
Hexahydro- 4.6
+tetrahydro-iso- 10 4.42 + 5.4
-acids

Table 2: Concentration of micro-organisms added to beer

Colony forming units CFU per 330 ml beer

Strains Concentration A Concentration B
Lactobacillus L2 1.2*10 E3 1.3* E5
lindneri
Lactobacillus L 940 1.8*E-1 1.8*E2
lindneri
Lactobacillus L 771 1.3*E3 1.3*E5
lindneri
Lactobacillus L7 1.6*E5 1.6*E7
lindneri
Lactobacillus L 23 3.9*E2 3.9*E4
lindneri
Lactobacillus L 32 6.0*E-3 6.0*E2
brevis
Lactobacillus L 43 4.3*E0 4.0*E3
brevisimilis
Pediococcus P 91 1.5*E-1 1.5*E2
damnosus
Pediococcus SaT 2.0*E-3 2.0*E2
damnosus

RESULTS
Tables 3 and 4 show the time micro-organisms in the two concentrations A and B
needed to cause visible turbidity. Table 4 shows the result of the test series
inoculated with a higher amount of germs. Turbidity caused by micro-organisms,
product or chemical-physical stability was additionally controlled by microscope
at the end of the test to confirm the results.

5
 As elucidated in tables 4 and 5, the most efficient hop components are -acids
followed by hexahydro-, tetrahydro-, rho- and iso--acids. The last two
components are comparable. In comparison to the other hop products, -acids
monitored the best inhibition efficiency against the tested strains of l. brevis,
l.brevisimilis and most strains of l. lindneri, whereas tested strains of pediococcus
damnosus were very effectively inhibitated by all hop products.

Strains of l. lindneri (L 2, L 23, L 7, L 771) demonstrated -with the exception of

-acids -a high resistance against all hop products.

The hydrogenated iso--acids (hexahydro- and tetrahydro-iso--acids) were more

effective than iso--acids and their reduced forms (rho-iso-acids). The different
chemical structures of hop compounds (see figure 3) obviously cause various
effects on the growth of beer-spoiling bacteria of the genera lactobacillus and
pediococcus.

There was a higher than average inhibition time at the lower concentration (A) of
micro-organisms. The order of inhibition efficiency was similar to that of all hop
bitter compounds in both concentrations (A and B). The microbiological stability
of the beer with -acids was almost the same in both concentrations A and B,
whereas the others showed a significant difference.

Table 3: Time of visible turbidity in beer with micro-organisms at concentration A (in

weeks)

-
unhopped Iso- Rho-iso- Tetrahydro- Hexahydro- Alpha-
beer acids -acids -acids + tetrahydro-
iso- acids
-acids
iso-
Strains
L2 2 2 2 4 5 7
L 940 2 7 7 7 7 7
L 771 2 2 2 5 7 6
L7 2 2 2 4 7 7
L 23 2 3 4 4 3 7
L 32 2 4 7 7 7 7
L 42 2 7 7 7 7 7
P 91 6 7 7 7 7 7
SaT 3 7 7 7 7 7
weeks 2.6 4.6 5.0 5.8 6.3 6.9

poor good

6
Table 4: Time of visible turbidity in beer with micro-organisms at concentration B
(in weeks)

-
unhopped Iso- Rho-iso- Tetrahydro- Hexahydro- Alpha-
beer acids -acids -acids + tetrahydro-
iso- acids
-acids
iso-
Strains
L2 2 2 2 2 2 3
L 940 2 2 2 4 6 7
L 771 2 2 2 2 3 5
L7 2 2 2 2 2 7
L 23 2 2 2 2 2 7
L 32 2 2 3 7 7 7
L 43 2 7 7 4 7 7
P 91 3 7 7 6 7 7
SaT 2 7 7 7 7 7
weeks 2.1 3.7 3.8 4.0 4.8 6.3

poor good

CONCLUSIONS
Alpha-acids show the best anti-microbiological effect against the growth of strains
of lactobacillus and pediococcus followed by hexahydro-, tetrahydro-, rho- and
iso--acids.
Especially strains of lactobacillus lindneri demonstrate - with the exception of -
acids - a high resistance against all other hop bitter compounds.
The different chemical structures of hop compounds obviously cause various
effects on the growth of beer-spoiling bacteria.
It is conventionally possible to improve the microbiological quality / stability
when adding hops at the end of boiling or in the whirlpool, because of obtaining a
higher amount of -acids in the beer. At the same time, the beer foam (2), the
flavour stability and the intensity of hop flavour are increased (1).
In general, a dosage of -acids to beer is possible in an ethanolic solution. We are
still working on application proposals for the brewing industry in this field.

LITERATURE
1. Kaltner, D., Forster, C., Thum, B., Back, W., Untersuchungen zur Ausbildung
des Hopfenaromas whrend des Brauprozesses, Proceedings of the 27th EBC
Congress 1999, Cannes, 63-70.
2. Narzi, L., Abri der Bierbrauerei, Enke Verlag, Stuttgart, 319-320.
3. Pollach, G., Hein, W., Hollaus, F., Use of hop products as bacteriostaticum in
the sugar industry, Zuckerindustrie 121 (1996), Nr. 12, 919-926.

7
18

Chemical properties of the naturally dried

hop plant and its influence on the quality
of beer
Majda Virant
Institute of Hop Research and Brewing alec, Cesta alskega tabora 2, SI-3310 alec,
Slovenia (e-mail: [email protected])

Descriptors
-acids, anthocyanogen, beer colour, beer quality, essential oil, hops, polyphenol, total resin,
variety

SUMMARY
In the past five years we have been monitoring the chemical composition of two hop cultivars
Aurora and Bobek, harvested during the time of technical maturity and naturally dried on the
hop plant in the hop garden. The hop was harvested at the end of November or at the
beginning of December. The influence of chemical changes on hop resins, the quantity of
essential oil and the composition of the oils was measured in beer, brewed in the pilot
brewery. The results show that late harvest of so-called "Ice Hop" affects the colour of wort
and beer (more intensive), the content of polyphenols and anthocyanogenes is reduced and the
utilisation of -acids is better. It also affects the sensory estimates of beer.

Chemische Eigenschaften von natrlich getrockneten Hopfenpflanzen und deren

Einfluss auf die Bierqualitt

Deskriptoren
Anthocyanogen, therisches l, Bierfarbe, Bierqualitt, Gesamtharz, Hopfen, Polyphenol, -
Suren, Variett

ZUSAMMENFASSUNG
In den letzten fnf Jahren haben wir die chemische Zusammensetzung zweier Hopfensorten
(Aurora und Bobek) beobachtet. Die Proben wurden zum einen whrend der technischen
Reife und zum andern an natrlich getrockneten Hopfenpflanzen im Hopfengarten
genommen. Der Hopfen wurde entweder Ende November oder Anfang Dezember geerntet.
Der Einfluss der chemischen Vernderungen der Hopfenharze, der Qualitt der therischen
Hopfenle und die Zusammensetzung der le wurde im Bier analysiert, das in einer
Pilotanlage gebraut wurde. Die Ergebnisse zeigen, dass der spt geerntete Hopfen, auch
Eishopfen genannt, die Farbe sowohl in der Wrze als auch im Bier intensiviert. Weiter
wird der Gehalt an Polyphenolen und Anthocyanogenen reduziert und die Ausbeute der a-
Suren steigt. Zudem wird die sensorische Beurteilung des Bieres beeinflusst.
Proprits chimiques du houblon sch naturellement et son influence sur la qualit de
la bire

Descripteurs
Acides , anthocyanogne, couleur de la bire, houblon, huile essentielle, polyphnol, qualit
de la bire, rsines totales, varit

RESUME
Au cours des cinq dernires annes, nous avons suivi la composition chimique de deux
varits de houblon, Aurora et Bobek, rcoltes pendant leur maturit et sches
naturellement sur le plant dans le carr de culture du houblon. Le houblon a t rcolt fin
novembre ou dbut dcembre. L'influence des modifications chimiques sur les rsines de
houblon, la quantit d'huiles essentielles ainsi que la composition des huiles a t mesure
dans la bire provenant d'une brasserie pilote. Les rsultats montrent que le "houblon des
neiges", de rcolte tardive, influe sur la couleur du mot et de la bire (plus intense), rduit la
teneur en polyphnols et en anthocyanognes et amliore l'utilisation des -acides. Il influe
galement sur les paramtres organoleptiques de la bire.

2
INTRODUCTION
The composition of hops depends on hop variety, growing district, growing
conditions, crop, harvesting time and on drying and storage consitions (1). Hops are
picked when fully ripe, i.e. in the time of its "technological ripeness". The contents of
bittering substances as well as that of -acids depends on the ripening stage of hops.
Early harvesting can mean up to 20 % lower values, late harvesting can lower them
for as much as 10 % (1). The cohumulone fraction is about 25 % in the time of
optimal ripeness and can drop to 20 % because of too early or too late harvesting (2).
The contents of essential oil and its composition depend on the hop variety and also
on crop and harvesting time or stage of ripeness respectively (3, 4, 6). The formation
of the essential oil begins in the stage of closing of the cones and increases all the way
to the stage of full ripeness. The synthesis of essential oil takes longer than the
synthesis of -acids. In ripe cones the increase of the essential oil content in some
cultivars is due to increase of the content of myrcene, while the content of
sesquiterpenes (with the exception of farnesene) declines. The contents of linalool and
2-methylisobutirate also increase. Beer containing more than 20 l of linalool/l has a
pronounced hoppy aroma (10). Oxidation products of -humulene and -
caryophyllene have been reported to be present to varying degrees in hops. These
compounds are not likely products of the plant biosynthetis apparatus, but are
oxidation products that vary with the post harvest age of the hops (9). The content and
the composition of the polyphenols depends on hop variety and origin and even more
on storing time and conditions. During the storing, oxidation processes take place and
they result in loss of bittering substances, essential oil and polyphenols. Their
oxidation products have a significant impact on beer taste and this reduces the
brewing value of such hops (5). In the brewing process, the contents of polyphenols
(and that of some proteins) are reduced using PVPP filtration (7) in order to increase
physical and chemical stability of the final product. A high content of polyphenols
does not necessarily mean a lower colloid stability of beer. The right raw materials
(e.g. fresh aromatic hops) or/and middle or shorter boiling time of the wort can have a
positive influence on taste and stability of beer (8).
The aim of our investigation was to follow the changes in hop composition during the
technological ripeness as well as during the physiological ripeness when hops was
naturally dried on the plants and was exposed to natural oxidation and ageing
processes with the goal to test the brewing quality of such hops.

MATERIALS AND METHODS

Hops
The experiment was performed in the 1995 to 2000 period with hops from a hop
garden where Aurora and Bobek (two aromatic Slovenian hop varieties) are grown.
In the time of technological ripeness, depending on variety and crop, the hops was
picked mechanically in August or September and dried in the usual way. Twenty
plants of each variety were left in the hop garden to study physiological ripening. Two
plants were manually picked each month, they were dried in a pilot plant, except for
the hops of the last picking which has been performed after the outdoor temperarture
has been below 0 C for one week. This hops did not need any additional drying since
the natural drying on the plant was sufficient.

3
Pilot brewing
The pilot brewing was performed in a pilot Ziemann brewery (capaciy 35 l) on the
Institute of Hop Reserch and Brewing alec. The wort (12 % of extract) was hopped
with hop cones of varieties Aurora and Bobek picked in the time of technological
ripeness and with hops naturally dried on the plants ("ice hops"). The calculation of
the necessary amount of hops to be added to the wort was calculated on the basis of
the -acid content in air-dry hops (LCV method). The -acid amount was 8.0 g of -
acids per 100 l of wort for all the experiments. The hopping was performed in three
equal parts added in the following way: the first one at the beginning of the boiling,
the second one after 60 minutes of boiling and the third one 10 minutes before the end
of boiling. The boiling lasted 90 minutes altogether. The only variable in the entire
technological process was the hop variety, which has been picked at different time
intervals. The wort has been analyized before adding of yeast (strain W 34). The
ageing of the beer took place for three weeks at the temperature 0 C to +1 C and
after filtering it through pilot kiselguhr sheet filter it was bottled. It has been neither
pasteurized or stabilized.

Analytical parameters
The following analyses of hops were performed: -acid content by the LCV method,
hop resins by Wllmer and HPLC analyses, essential oil content and composition by
steam distillation and GC analysis.
To establishing the differences in essential oil composition between the
technologically ripe and the physiologically ripe hops the ANOVA statistical method
was used, namely the analysis of variance, where the repetitions were represented by
years (n = 6). The null hypothesis (H0), claiming that there are no statistically
significant differences between the different dates, was tested by the F-test at a
standard confidence level p = 0.95.
Wort and beer were analysed according to Analytica-EBC, 1998 and MEBAK
Band II, 1993 methods. The analyses of the components of the essential oil were not
performed, as the method is not routinely used in our laboratory. The foam of the beer
was tested only visually, as a considerable uncertainty can be expected for the results
of a pilot brewing experiment.
The sensory evaluation of beer has been performed according to the tasting test DLG
(MEBAK Band II, 1993, p. 80.). On the scale from 1 to 5 points the following
parameters have been evaluated: the intensity of the hop aroma (1 not perceivable
5 perfumy, intensive), the quality of the hop aroma (1 unpleasant 5 pleasant), the
intesity of bitterness (1 very weak 5 very strong) and the quality of the bitterness
(1 unpleasant 5 pleasant).

Marked as A a B b C c D d E e F f
Picking date 30. 8. 7. 11. 3. 9. 6. 12. 4. 9. 4. 11. 29. 8. 2. 11. 6. 9. 29.10 22. 8. 1. 11.
Crop 1995 1995 1996 1996 1997 1997 1998 1998 1999 1999 2000 2000

Table 1: List of the experiments of pilot brewing using hop cultivars Aurora and
Bobek picked at different stages of ripeness

RESULTS AND DISCUSSION

LCV Lead Conductance Value, Wllmer and HPLC methods (all according to
Analytica-EBC) were used to monitor the contents of -acids and hop resins in the

4
six-years period (1995-2000) in the technologically ripe hops (A, B, C, D, E, F) and in
physiologically ripe hops (a, b, c, d, e, f). The results are too numerous to be presented
here in full.

-Acid content
The -acid content in technologically ripe cultivar Aurora varies from 6.7 to 12.6 %
(on d.m. basis) and from 4.9 to 10.0 % in cultivar Bobek. (It is well known that the
results vary owing to growing conditions as well as from year to year). In
physiologically ripe samples the -acid content (LCV method) is somewhat lower
compared to the technologically ripe samples (5.5 - 11.4 %).
The loss of alpha acids (relative %)

10
Aurora Bobek
5

-5

-10

-15

-20

-25
1995 1996 1997 1998 1999 2000 1995 1996 1997 1998 1999 2000
LCV Method HPLC Method

Figure 1: The loss of alpha acids due to physiological ripening.

The loss of -acids varies from year to year (from 2.6 relative % in 1996 up to 19.9
relative % in 1998 (figure 1), the average loss being 12.6 (LCV method) or 12.8
(HPLC method) relative %. The -acid content in physiologically ripe cultivar Bobek
is somewhat lower in 1997 though, the results were 0.2 % higher if compared with the
results of technologically ripe samples (LCV method) and only 0.1 % lower if HPLC
method is considered. The average loss of -acids for the entire period being 10.4
relative % (LCV method) or 9.2 relative % (HPLC method) (figure 1). The average
loss of -acids in physiologically ripe hops for cultivars Aurora (12.6 %) and Bobek
(10,4 %) cannot be compared with the storage stability which is determined for
technologically ripe, dried and stored hops, since these data are not yet available. It is
known that cultivar Aurora has a very good storage stability, while the storage
stability of Bobek is considered good (11).

The total resin content and the content of cohumulone

The average loss in total resin content for cultivar Aurora was 7.6 relative % (the
results for the 1999 crop, where the value for the physiologically ripe hops was even
higher compared to the technologically ripe hops were not considered). The average
loss in total resin content for cultivar Bobek was 4.8 relative % (the results for the
1995 and 1996 crops, where the values for the physiologically ripe hops were even
higher compared to the technologically ripe hops were not considered). The average
loss in cohumulone was 11.2 relative % for cultivar Aurora and 7.1 relative % for
cultivar Bobek (figure 2).

5
 The loss of total resins
and cohumulone (relative %)
5
Aurora Bobek

-5

-10

-15

-20

-25

-30
1995 1996 1997 1998 1999 2000 1995 1996 1997 1998 1999 2000

Total resins Cohumulone

Figure 2: The loss of total resins and cohumulone.

Essential oil and its components

The results of determing the essential oil content (by steam distillation) clearly
indicate that its contents increases from the stage of technological ripeness to the stage
of physiological ripeness and was for cultivar Aurora the highest in 1999
(2.99 ml/100 g), and for cultivar Bobek in 1997 (5.49 ml/100 g) (figure 3). The results
confirm that the essential oil content depends on the harvesting time i.e. on the
ripening stage. The statistical analysis of the results on the contents of individual
components of the essential oil (figures 4 and 5) showed a significant difference in the
composition of the essential oil between technologically and physiologically ripe hops
for cultivar Bobek but nor for cultivar Aurora.
In the essential oil of cultivar Bobek the relative percentage if myrcene is significantly
lower (P = 0,05 and those of -selinene, humulene epoxyde-1 and humulene
epoxyde-2 are significantly higher (P = 0.05). The relative percentages of -humulene
and -caryophyllene are also higher (P = 0,05).

Hop oil (mL/100g)

steam distillatin, 1995 - 2000

6
Aurora Bobek

0
A a B b C c D d E e F f A a B b C c D d E e F f

Figure 3: Hop oil content in cultivars Aurora and Bobek (see table 1 for details).

6
 The components of the essential oil
cultivar Aurora, 1955 - 2000

% Total oil

70
60
50
40
30
20
10
myrcene
0 alpha-humulene
beta-caryophyllene
A a B b C farnesene
alpha-selinene
c D d beta-selinene
E e F linalool
f

Figure 4: The oil components in cultivar Aurora (see table 1 for details).

The components of the essential oil

cultivar Bobek, 1995 - 2000

%Total oil

80
70
60
50
40
30
20
10 myrcene
alpha-humulene
0 beta-caryophyllene
A a B farnesene
b C alpha-selinene
c D beta-selinene
d E e F f linalool

Figure 5: The oil components in cultivar Bobek (see table 1 for details).

The analyses of beer obtained by pilot brewing

The contents of total polyphenols and anthocyanogens in beers where hopping was
performed with cultivars Aurora and Bobek picked at the time of physiological
ripeness was lower compared to those where technologically ripe hops were used. On
the average, the total contents of polyphenols was as much as 9.1 relative % lower,
while that for anthocyanogens was as much as 12,2 relative % lower, the values being
19.2 and 18.8 relative % for cultivar Bobek (figure 6). Hopping with the same amount
of -acids (8 g/100 l of wort) gave a higher bitterness if physiologically ripe hops was
used (either for cultivar Aurora of for cultivar Bobek). This is evident from the values
of the -acid utilisation. The utilisation of -acid is 27.3 % in the case of
technologically ripe cultivar Aurora, compared to the 29.7 % for the physiologically
ripe cultivar Aurora, the values being 28.1 and 31.2 relative % for cultivar Bobek
(figures 7, 8). Late picking of both cultivars influenced the colour of the wort and beer
(figures 7, 8). In all the cases, physiologically ripe hops gave worts and beers of more
intense colour compared to technologically ripe hops. The average differences in wort
and beer colour hopped with physiologically and technologically ripe for cultivar
Aurora being 1.4 EBC units for wort and 0.7 EBC units for beer, the values being 1.1
and 0.6 EBC units for cultivar Bobek (figure 8).

7
 The loss of the total polyphenols and
anthocyanogens (relative %) in beer

Aurora Bobek
10

-10

-20

-30

-40
1995 1996 1997 1998 1999 2000 1995 1996 1997 1998 1999 2000

Total Polyphenols Anthocyanogens

Figure 6: The loss of total polyphenols and anthocyanogenes in beer.

Chemical analysis of beer

Cultivar Aurora
Bitterness (BU) Iso alpha acids (mg/L)
40 40

30 30

20 20

10 10

0 0
A a B b C c D d E e F f A a B b C c D d E e F f

Utilisation alpha acid (%) Colour (EBC units)

40 40

30 30

20 20

10 10

0 0
A a B b C c D d E e F f A a B b C c D d E e F f

Figure 7: Chemical analyses of beer, hopping with cultivar Aurora

Chemical analysis of beer

Cultivar Bobek
Bitterness (BU) Iso alpha acids (mg/L)
40 40

30 30

20 20

10 10

0 0
A a B b C c D d E e F f A a B b C c D d E e F f

Utilisation alpha acid (%) Colour (EBC units)

40 40

30 30

20 20

10 10

0 0
A a B b C c D d E e F f A a B b C c D d E e F f

Figure 8: Chemical analyses of beer, hopping with cultivar Bobek

Physiologically ripe hops also gave beer of better physico-chemical stability

compared to technologically ripe hops. If physiologically ripe hops were used, the
stability of the beer was for 0.5 warm days superior (forcing test 0/40/0 C). The
average value of the four quality parameters tested in sensory evaluation of beer
obtained from "ice hops" of cultivar Aurora was 3.14 points and of that obtained from

8
technologically ripe cultivar Aurora 3.10 points. This is confirmed also by the
analysis of the composition of the essential oil, where no statistically significant
differences were determined. In the case of cultivar Bobek the values are 3.16 and
2.97 respectively (figures 9, 10). The difference is mainly due to higher scores for
intensity and quality of the aroma for beer obtained with cultivar Bobek picked at the
time of physiological ripeness.
Tasting test DLC
Cultivar Aurora

0
A a B b C c D d E e F f

Hop aroma (intensity) Hop aroma(quality) Bitterness (intensity) Bitterness (quality)

Figure 9: Sensory analysis of beer, hopping with cultivar Aurora.

Tasting test DLG

Cultivar Bobek

3.5

2.5

1.5

1
A a B b C c D d E e F f

Hop aroma (intensity) Hop aroma(quality) Bitterness (intensity) Bitterness (quality)

Figure 10: Sensory analysis of beer, hopping with cultivar Bobek.

CONCLUSIONS
This report is very probably the first one on the potential use of physiologically ripe
hops (which we named "ice hops") in beer brewing. The comparison with the use of
technologically ripe hops enables the following conclusions:
the average loss in the -acid content in phyisiologically ripe hops compared to
technologically ripe hops is 12.5 relative % for cultivar Aurora and 10.4 % for
cultivar Bobek
the direct comparison of the upper values with standard storage stability tests is not
possible yet
the content of the essential oil increased in the period from technological to
physiological ripeness for both cultivars

9
 the changes in the essential oil composition between technologically and
physiologically ripe hops are statistivally significant for cultivar Bobek, while no such
difference was established for cultivar Aurora
the contents of total polyphenols and anthocyanogens in beer were lower if
physiologically ripe hops were used for hopping compared to technologically ripe
hops
the -acid utilisation in beer is higher for both cultivars if physiologically ripe hops
is used
the colour of wort and that of beer are more intense if physiologically ripe hops is
used
in the case of cultivar Bobek, the beer obtained from physiologically ripe hops was
given a higher sensory score, mostly due to higher intensity and better quality of its
aroma.

Further investigations will be needed to evaluate the potential use of physiologically

ripe hops in beer brewing and these should include especially analysis of hops as such
as well as analyses of beer.

REFERENCES
1. Narziss,L., Die Technologie der Wrzebereitung, 6. Auflage, 1985, 63-77.
2. Maier, J., Dtsch. Brauwirtschaft 80, 1971, Spezialausgabe, Mai, 25.
3. Maier, J., Hopfenrundschau 28, 1977, 298
4. Kammhuber, K., Monatschrift fr Brauwissenschaft, Heft 7/8, 2000, 138-142.
5. Hops and Hop Products, EBC Manual of Good Practice, 1997, 25-36.
6. Garden, D. S. J., Monograph XXII, EBC Symposium on Hops, Zoeterwoude,
1994, 114-124.
7. Watzl, B. and Litzman, Hippokrates Verlag, Stuttgart, 1999, 254.
8. Foster, A., Beck, B. and Schmidt, R., Hopfenrundschau International, 1999, 68-
74.
9. Deinzer, M. and Yang, X., Monograph XXII, EBC Symposium on Hops,
Zoeterwoude, 1994, 181-195.
10. Kaltner, D., Thum, B. and Back, W., Brauwelt International, 2001/I, 2001, 40-
45.
11. Kralj, D. and Zupanec, J., Monatsschrift fr Brauwissenschaft, Heft 5, 1992,
165-169.
12. Brautechnische Analysenmethoden, published by MEBAK, Band II, 1993.
13. Analytica-EBC, Verlag Hans Carl, Nrnberg, 1998.

10
19

A new industrial tool for monitoring the

malting process
Nathalie Allosio-Ouarnier1, Paul Robert2, Dominique Bertrand3
& Patrick Boivin1
1
Institut Franais des Boissons de la Brasserie Malterie, Ple technologique de Brabois, 7 rue
du bois de la Champelle, B.P. 267, F-54512 Vandoeuvre Cedex, France (e-mail:
[email protected])
2
Institut National de la Recherche Agronomique, Laboratoire de Physico-chimie des
Macromolcules, P.B. 71627, F-44316 Nantes Cedex 03, France
3
ENITIAA-SMAD, Rue de la Graudire, B.P. 82225, F-44322 Nantes Cedex 03, France

Descriptors
Endosperm, infrared radiation, malting, quality control, spectroscopy

SUMMARY
For producing good malts, the malting process has to be carefully controlled. Analyses have
to be performed during the transformation. Reference analyses are time-consuming and
impossible to achieve daily. Rapid techniques have to be developed. This work was aimed at
evaluating mid infrared spectroscopy. MIR spectra were recorded daily during industrial
maltings. Statistical treatments allowed the sample classification according to the malting
step. This can be explained by an increase of the soluble carbohydrate concentration and a
decrease of ester linkage quantity due to endosperm cell wall degradation. This study showed
that MIR Spectroscopy could be a tool for monitoring the malting process.

Ein neues industrielles Werkzeug zur berwachung des Mlzungsprozesses

Deskriptoren
Endosperm, Gteregelung, infrarote Strahlung, Malzherstellung, Spektroskopie

ZUSAMMENFASSUNG
Zur Herstellung von guten Malzen ist die genaue Beobachtung des Mlzungsprozesses
unerlsslich. Whrend der Umwandlung der Gerste zum Malz mssen Analysen durchgefhrt
werden. Referenzanalysen sind Zeit raubend und daher kaum tglich durchfhrbar. Schnellere
Analysenmethoden mssen daher entwickelt werden. Diese Arbeit zielte auf eine
Untersuchung der Mittelinfrarotspektroskopie hierfr ab. Das MIR-Spektrum wurde tglich
whrend des Herstellungsprozesses aufgenommen. Eine statistische Ausarbeitung erlaubte es,
die untersuchten Malzproben den einzelnen Herstellungsschritten zuzuordnen. Dies kann
durch eine Zunahme der lslichen Kohlenhydrate und eine Abnahme der Menge der
Esterverbindungen bei der Zellwandlsung erklrt werden. Diese Studie zeigte, dass die MIR-
Spektroskopie ein Werkzeug zur berwachung des Mlzungsprozesses sein knnte.
Un nouvel outil industriel pour le monitorage du maltage

Descripteurs
Contrle de qualit, endosperme, maltage, spectroscopie, radiation infrarouge

RESUME
Pour produire des malts de qualit, le maltage doit tre soigneusement contrl. Les analyses
de rfrence sont longues et difficiles raliser pendant la transformation. De nouvelles
techniques rapides doivent tre dveloppes. Le but de cette tude tait d'valuer les
potentialits de la spectroscopie moyen infrarouge. Des spectres ont t enregistrs chaque
jour pendant des maltages industriels. Des traitements statistiques ont permis de classer les
chantillons en fonction de leur stade de transformation. Ceci peut s'expliquer par une
augmentation de la concentration en sucres solubles et une diminution de la quantit de
liaisons ester dues la dgradation des parois cellulaires de l'endosperme. Cette tude a
montr que la spectroscopie moyen infrarouge pourrait tre un outil de contrle du procd de
maltage.

2
INTRODUCTION
The quality of food products now constitutes an essential objective for governments,
manufacturers, and consumers. The beer production and therefore, the fabrication of
its main raw material, malt, are also concerned by this economic context. The malting
process has to be carefully controlled. The maltster has to detect faults before the end
of the process to be able to correct them. Analyses have therefore to be performed
during the transformation of barley into malt. Reference analyses are time consuming
and are impossible to achieve regularly during the process. Rapid techniques have to
be developed to analyse barley at each step of its transformation.
Among available rapid techniques, Near and Mid Infrared spectroscopies appear to be
adapted for the control of the malting process. Mid Infrared (MIR), with the
development of techniques such as Attenuated Total Reflectance, offers advantages
compared with Near Infrared (NIR). Indeed, the observed bands are fundamental ones
whereas bands observed in the Near Infrared region are harmonics and combination
bands. This implies that peaks are thin and well separated in MIR and large and
overlapped in NIR. Moreover, analyses of all kind of linkages are possible by Mid
Infrared Spectroscopy, e.g. C-C and C-O bonds constituting carbohydrate backbone.
Near Infrared Spectroscopy allows only the study of bonds linking a light atom and a
heavy atom, e. g. C-H, N-H and O-H.
The aim of this study was to test the feasibility of Mid Infrared Spectroscopy in the
Attenuated Total Reflectance mode to control industrial malting process. Previous
studies have been done by N. Allosio-Ouarnier et al. at micro1 and pilot2 malting
scales with a commercial spectrophotometer.
In this work, spectra have been recorded in an industrial malting plant during two
months with a MIR prototype specially designed for industrial applications.

MATERIAL AND METHODS

Sample collection
Samples were collected daily from the end of steeping until the end of germination of
39 industrial malting processes. Seven barley varieties and one wheat variety were
studied.
Two sub samples of 20 g were ground during 75 seconds with a laboratory grinder
A10 IKA (Bioblock Scientific, Illkirch, France). Three spectra were recorded per
grinding. Six spectral analyses were performed for each collected sample.

Spectral acquisition
400 800 2500 25000 10000000
nm
cm-1
2500 12500 4000 400 1

X Rays Ultraviolet Micro Radio

Infrared
waves frequencies

Near Mid Far Infrared

Visible
Infrared Infrared

Figure 1: Mid infrared region.

3
Mid infrared region is comprised between 4000 and 400 cm-1 (2500-25000 nm)
(figure 1). The regions 4000-2000 cm-1 and 800-400 cm-1 are not very informative for
barley transformation study.

Spectra were recorded with a prototype specially designed for industrial use. It is a
robust, and easy to use apparatus able to work in dusty atmosphere. A schematic
representation of this prototype is given on figure 2.
Measurements were done in the Attenuated Total Reflectance mode.

Pressure system
Ground green malt
ZnSe crystal
1. 6

1. 4

1. 2

Ab so rb an ce s
0. 8

0. 6

0. 4

0. 2

26 1

25 1

24 1

23 1

22 1

21 1

20 1

19 1

18 1

17 1

16 1

15 1

14 1

13 1

12 1

11 1

10 1

91

81

71

61

51

41

31

21

11

1
Optical system

Figure 2: The MIR prototype.

As it can be seen on the figure, the ground green malt sample is crashed on a ZnSe
crystal thanks to a pressure system to obtain an optimal contact between the sample
and the crystal. The optical system is well protected in a sealed box designed to avoid
the penetration of dusts and water.
The principle of measurement in the ATR mode is represented on figure 3.

Ground green malt

I
I0 ZnSe crystal
Detector

Mid infrared
source

Figure 3: Principle of measurement in the Attenuated Total Reflectance mode.

The sample is lightened with infrared light at different frequencies. For each
frequency, the quantity of energy absorbed is measured thanks to a detector. The
drawing of light absorbed according to frequencies is a spectrum.

Statistical treatment
More than one thousand spectra were recorded. It was impossible to study then one by
one. The spectral collection was studied by performing Principal Component
Analyses (PCA) on two characteristic spectral zones. These regions were chosen from
current knowledge of MIR Infrared spectra interpretation. Spectra of green malt were
represented on figure 4. Main attributions of bands are given. There are four
characteristic regions: 1800-1700 cm-1 where C = O can be studied, two peaks around
1650 cm-1 and 1540 cm-1 characteristic of proteins, 14001200 cm-1 characteristic of

4
C-H vibrations, and the area 1186-956 cm-1 where carbohydrates can be studied.
Considering that the peak centred on 1650 cm-1 is also characteristic of O-H
vibrations and that C-H linkages are present in lots of molecules, it is difficult to
attribute these two zones to a particular barley compound.
The two chosen areas are therefore 1800-1700 cm-1 and 1186-956 cm-1.
Amide I
O-H
Ester bonds Carbohydrates
1.2

1 Amide II
C=O
0.8
absorbance

0.6 C-H
0.4

0.2
C-C and C-O
0
2000 1800 1700 cm-1 1186 956 800

Figure 4: Green malt spectra

Considering modifications according to time, PCA results were not interpreted with
maps but with curves representing scores on one principal component versus malting
steps.

In order to take off spectra modifications due to intensity effects and not to
biochemical changes, signals were previously mathematically transformed by two
procedures called N and Z.
If M is a n (samples) x p (frequencies) matrix containing absorbencies of recorded
spectra, N and Z can be defined as follows:

Procedure N
If mij is the M data corresponding to the absorbance measured for the jth frequency for
the ith sample, N calculates the terms xij of a n x p matrix X :

mij
xij = with ni. = m
2
i.
ni.

Procedure Z
If mij is the M data corresponding to the absorbance measured for the jth frequency for
the ith sample, and ai the lower recorded absorbance of the ith sample spectrum, Z
calculates the terms xij of a n x p matrix X:

xij = mij a i

RESULTS
Reproducibility of the prototype
In order to evaluate the reproducibility of the spectrometer, spectra of an organic
compound called tetradecane were recorded each analysis day. Figure 5 shows the
mean spectrum of all the tetradecane spectra recorded inserted between a spectrum

5
representing mean plus standard deviation and a spectrum representing mean minus
standard deviation. These spectra are obtained by calculating mean and standard
deviation of all the absorbance values recorded at each frequency.
The standard deviations are comprised between 6.57 10-3 and 1.55 10-2 absorbance
units, which correspond to variation coefficients comprised between 2.27 % and
27.13 %.
The reproducibility of the spectrometer is therefore quite good.
0.5

0.45

0.4

0.35

0.3

0.25

0.2

0.15

0.1

0.05

0
2000 1818 1667 1538 1429 1333 1250 1176 1111 1053 1000 952 909
cm-1

mean mean + standard deviation mean - standard deviation

Figure 5: Mean and standard deviation representations.

Spectral Collection
The spectral collection recorded is given figure 6. In order to acquire spectra in 3
minutes, recording was made between 1800 and 1695 cm-1 and between 1203 and 952
cm-1. A little shoulder can be observed at 1744 cm-1. This peak is characteristic of
ester linkages. The area 1203-952 cm-1 has the shape commonly observed for green
malt spectra (see figure 3).
4.5

3.5

3
absorbance

2.5

2 1744
1.5

0.5

0
1786 1176 1111 1053 1000 952
cm-1

Figure 6: Spectral collection.

Application of Principal Component Analyses

Analysis of the area comprised between 1800 and 1700 cm-1.

6
First of all, spectra are pre-treated using procedure N. Before performing PCA on the
considered spectral region, spectra are transformed using procedure Z in order to
correct baseline.

Spectra of samples collected at different malting step seem to be separated on the

second principal component. In order to visualise spectral changes occurring as
malting progresses, means of the 6 scores obtained on the second principal component
for the 6 spectra recorded for each sample are represented according to malting steps
(figure 7). A general decrease of these means is observed as malting progresses.

2.50E-03

2.00E-03

1.50E-03
scores on the 2nd principal componen

1.00E-03

5.00E-04

0.00E+00

-5.00E-04

-1.00E-03

-1.50E-03

-2.00E-03

-2.50E-03
malting step

Figure 7: Means of scores obtained on the second principal component versus

malting steps - PCA performed between 1800 and 1700 cm-1.

The loading corresponding to the second principal component is a large peak centred
on 1744 cm-1 (figure 8). This frequency is characteristic of ester linkage elongation.
This positive peak and the score decrease can be explained by a progressive
disappearance of ester bonds in barley during its transformation into malt.
0.4

0.35

0.3

0.25

0.2

0.15

0.1

0.05

0
1792 1783 1773 1764 1754 1745 1736 1727 1718 1709 1701
-0.05

-0.1

-0.15

-0.2

-0.25

-0.3
cm-1

Figure 8: Loading associated to the second principal component PCA performed

between 1800 and 1700 cm-1.

7
Bamforth et al. (1997)3 have showed that degradations of ester linkages might be
implied in -glucans and pentosans solubilisation. Esters linkages might be present
between ferulic acids and arabinoxylans in barley endosperm cell walls. The observed
decrease of scores on the second principal component might be therefore, due to
barley endosperm cell wall breakdown that is know to occur during malting.
MIR infrared spectroscopy in the ATR mode combined with principal component
analysis between 1800 and 1700 cm-1 could be a useful tool for controlling cell wall
degradation progression during malting.

Analysis of the area comprised between 1186 and 956 cm-1.

First of all, spectra are pre-treated using procedure N. Then PCA is performed.
Spectra of samples collected at different malting steps seem to be separated on the
second principal component. As it was the case in the study of the peak centred on
1744 cm-1, means of the 6 scores obtained on the second principal component for the
6 spectra recorded for each sample are represented according to malting steps (figure
9). A general increase of these means is observed as malting progresses for all the
studied varieties.
0.04

0.03
scores on the 2nd principal component

0.02

0.01

-0.01

-0.02

-0.03
malting step

Figure 9: Means of scores obtained on the second principal component versus

malting steps - PCA performed between 1186 and 956 cm-1.

0.15

0.1

0.05

0
1182 1148 1116 1086 1057 1030 1004 979 956

-0.05

-0.1

-0.15
cm-1

Figure 10: Loading associated to the second principal component PCA performed
between 1186 and 956 cm-1.

8
The associated loading (figure 10) shows that spectral changes correspond to
modifications of intensity in the region comprised between 1148 and 1000 cm-1.
These modifications might be due to vibrations of carbohydrate backbone.

The observed positive peak explained the score increase on the second principal
component by an increase of vibrations of bonds C-O and C-C and therefore by an
increase of carbohydrate concentration in barley during its transformation into malt.
N. Allosio-Ouarnier et al. (2000)4 have shown that low molecular weight
carbohydrates appear during malting because of glucans, arabinoxylans and starch
degradation.

MIR infrared spectroscopy in the ATR mode combined with principal component
analysis between 1186 and 956cm-1 could be a useful tool for controlling the
transformation into malt.

CONCLUSION
The present study shows that Mid Infrared Spectroscopy combined with Principal
Component Analyses allows the control of two important phenomena occurring
during industrial malting: ester linkage disappearance and low molecular weight
carbohydrate appearance.
This work has proved that mid infrared spectroscopy can be considered as a new
objective tool for monitoring the malting process. This tool is easy to use and allows
daily and rapid measurements. It can therefore be a useful help for the maltster to
perform an easy and efficacious control of his process.

ACKNOWLEDGEMENT
The authors thank the Malteurs de France and EEC (fair programme) for the financial
support

REFERENCES
1. Allosio N., Robert P., Bertrand D., Boivin P., Proceedings of the 26th EBC
Congress, Maastricht, 1997, 151-158.
2. Allosio-Ouarnier N., Robert P., Boivin P., Bertrand D., Proceedings of the 27th
EBC Congress, Cannes, 1999, 485-492.
3 Bamforth C.W., Moore J., Mac Killop D., Williamson G., Kroon P.A.,
Proceedings of the 26th EBC Congress, Maastricht, 1997, 75-82.
4. Allosio N., Quemener B., Bertrand D., Boivin P., Journal of The Institute of
Brewing, 2000, 106, 1, 45-52.

9
20

Industrial kilning technologies and their

influence on organoleptic quality of malt
Michel Dumoulin & Patrick Boivin
Institut Franais des Boissons de la Brasserie Malterie, Ple technologique de Brabois, 7 rue du bois
de la Champelle, B.P. 267, F-54512 Vandoeuvre Cedex, France (e-mail: michel.dumoulin@ifbm-
qualtech.com)

Descriptors
Antioxidant, beer quality, dimethylsulphide, flavour formation, kilning, malt quality, staling

SUMMARY
Beer flavour quality and staling are still the most serious problem in the brewing industry. It is well
known that malt has a great impact on the quality of the final beer. DMS-precursors, pro- and anti-
oxidant activities on malt play an important role on the organoleptic quality of beer. These activities
on malt will depend both on barley variety and malting process, particularly kilning process. Accurate
evolution of the organoleptic activities was obtained during kilning in industrial malting plants.
Critical step in kilning was observed for the evolution of these activities. Significant difference on the
evolution and end malt of all these activities was obtained on one flour and two-flours kilning.

Industrielle Darrtechnologien und deren Einflu auf die organoleptische Qualitt des Malzes

Deskriptoren
Altgeschmackbildung, Antioxidantium, Aromabildung, Bierqualitt, Darren, Dimethylsulfid,
Malzqualitt

ZUSAMMENFASSUNG
Geschmacksstabilitt und Bieralterung sind nach wie vor die dringendsten Probleme in der
Brauindustrie. Es ist bekannt, dass das Malz einen groen Einfluss auf das fertige Bier hat. DMS-
Precursor sowie die Pro- und Antioxidantien im Malz spielen eine bedeutende Rolle bei der
organoleptischen Qualitt des Bieres. Diese Malzparameter hngen zum einen von den Malzvarietten,
zum anderen vom Mlzungsprozess, insbesondere dem Darren ab. Die genaue Entwicklung der
organoleptisch interessanten Parameter wurde im Darrprozess industrieller Anlagen beobachtet.
Insbesondere die kritischen Schritte des Darrprozesses wurden in Bezug auf die oben genannten
Parameter beobachtet. An einer Ein-Horden- und einer Zwei-Horden-Darre wurden die Unterschiede
und die Parameter beobachtet.
Les technologies de touraillage industriel et leur influence sur les qualits organoleptiques du
malt

Descripteurs
Antioxydant, dimthylsulfure, formation de la flaveur, formation du got d'vent, qualit de la bire,
qualit du malt, touraillage

RESUME
La qualit organoleptique de la bire est toujours un problme major en brasserie. Il est reconnu que le
malt a un impact important sur la qualit finale de la bire. Les prcurseurs du DMS et les activits
pro-et-antioxydantes du malt influencent la qualit organoleptique de la bire. Ces activits dans le
malt vont dpendre de la varit dorge et du procd de maltage, particulirement du touraillage. Les
activits "organoleptique" ont t suivies au cours de touraillages industriels. Des tapes cls du
touraillage ont t identifies. Des diffrences significatives ont t observes entre les tourailles
simple et double plateaux.

2
INTRODUCTION
For the consumer point of view, the quality of beer can be defined by its taste and its flavours.
The organoleptic characteristics of beer are the result of complex associations between a lot of
volatil compounds.

Up to now, almost 600 volatil compounds can be identified in beer. The carbonyl compounds,
that is to say: aldehydes, ketones, acids and esters, represent 50% of these volatils. Besides
these, sulfur compounds represent 7 to 8% of the volatils that can be found in beer (1).

Organoleptic quality of beer is still the most serious problem encountered by the brewer
industries, because consumers judge their products on the quality of their flavours. It is well
known that malt has a great impact on the organoleptic characteristics of beer (2).

DMS precursors, that is to say DMSO and S-Methyl Methionine (3), pro and anti-oxidant
activities could be very helpfull to determine the organoleptic quality of malt. This parameter
is both a function of barley variety and of the malting process, especially the kilning process.

The goals of this study are to study the involvement of DMS and nonenal precursors during
the malting process and to evaluate the influence of the kilning technology on these flavour
precursors.

MATERIALS AND METHODS

Malting processes
Two industrial processes were considered for this work. The first used an Esterel barley
variety. After 4 days of germination, green malt is divided into two parts. The first part is
kilned in an one floor kiln. The second part is kilned in a two floor kiln. The second used a
Scarlett barley and after 4 days of germination, the green malt is kiln into a continuous kiln.

In a same way, a pilot malting process using 600 kg of barley was studied with Optic and
Alexis barley varieties.

For all the processes, samples were taken to follow the evolution of DMS and nonenal
precursors. Sampling were then perform at barley, the end of steeping, green malt, each
temperature level of the kilning process.

Sulfur compounds analytical method

Three different sulfur compounds are analysed in the samples from barley to malt :
DiMethylSulfOxide (DMSO), free DimethylSulfide (DMSfree) and DMS precursors (pDMS).
DMSO
DMSO is extracted from cereal sample with water. Aqueous extract is filtered and
partitionned with chloroform. After evaporation, dry extract is solubilised into ethyl
alcohol and analysed with GC-MS. Quantification is realised with deuterated DMSO as
internal standard (4).

DMSfree
Free DMS is directly analysed into aqueous sample cereal extract with a gaz
chromatograph equipped with a head space injector and FPD detector specific for sulfur
compounds.

3
 pDMS
DMS precursors is analysed after alcaline hydrolysis of aqueous cereal sample extract.
The quantity of DMS precursors is the difference between total DMS (thus obtained) and
free DMS.

Nonenal precursors analytical methods

To evaluate the potential of malt for trans-2-nonenal formation we use lipoxygenase activity,
hydroperoxides potential and antioxidant power.

Lipoxygenase activity
Lipoxygenase is extract from malt with a phosphate buffer and the enzymatic reaction
with linoleic acid is performed. The enzymatic activity of malt is evaluated from the
kinetic of double isomerization (5).

Hydroperoxides potential
For the hydroperoxides potential evaluation, an equivalent of 40 nkat enzyme activity
reacts with linoleic acid. The hydroperoxides thus obtained are reduced into alcohol and
quantified with HPLC chromatography. Hydroperoxides potential is the quantity of 9 and
13-hydroperoxides obtained from linoleic acid oxidation (2).

Antioxidant power
The antioxidant power is defined to characterized formation of antioxidant compounds.
Antioxidant power is the ratio between lipoxygenase activity and hydroperoxide
potential.

RESULTS AND DISCUSSION

1: Sulfur compounds from barley to beer
1.1: Pilot malting process
In figure 1 we have plotted the involvement of free DMS, DMSO and DMS precursors from
barley to malt in the pilot malting process relative to Alexis variety. For the Optic barley
variety the same graph is obtained with almost the same level. The absence of variety effect
on sulfur compounds involvement should be check with many other varieties.

DMS precursors, that is to say S-Methyl Methionine, is produced during germination step.
During this step, enzyme activation allows the formation of S-Methyl Methionine from
Methionine. This amino acid is very sensitive to temperature and it decomposes itself easily
into DMS and homoserine as kilning begin.

Up to 60C this thermal decomposition is very slow and the absence of DMS in cereal
samples is certainly could be ascribe to evaporation. Above 60C, the thermal degradation of
DMS precursors is running faster and DMS is appearing in cereal samples. At this time, the
DMS level is sufficient to allow its oxidation into DMSO. This could explain the apparition of
DMSO in malt at the same as DMS.

4
 free DMS pDMS DMSO
30

25

20
mg eq.DMS/kg ms

15

10

0
Stp. Green 50C 50C 60C 60C 80C 80C Malt
barley malt (start) (end) (start) (end) (start) (end)

Figure 1: Involvement of sulfur compounds during pilot malting process with

Alexis barley variety.

1.2: Industrial malting processes

a: One floor and two floor kilning technologies
Figures 2 and 3 give the evolution of sulfur compounds into cereal samples during malting
processes using one floor and two floor kilning technologies.

free DMS pDMS DMSO

20
18
16
mg eq.DMS/kg ms

14
12
10
8
6
4
2
0
Barley Green malt 60C 65C 70C 75C Malt

Figure 2: Sulfur compounds involvement during malting process with one floor
kilning technology.

For this two industrial processes, we can notice that sulfur compounds involve in the same
way as the pilot malting process. DMS precursors are produced with germination. Their
thermodegradation is relatively slow up to 70-75C and all the DMS produced is evaporated.
At the end of kilning, the thermodegradation is faster and DMS accumulates itself in malt. Its
oxidation leads to DMSO.

5
The only difference between the two processes lays in the final levels of DMS precursors and
DMSO in malt. With two floor kiln technology, the level of DMS precursors in malt is less
than with the one floor kiln technology. This could be explained by the fact that kilning is
smoother with this technology. As there are less DMS produced, DMSO level in malt is lesser
in two floor kiln than in one floor kiln.

free DMS pDMS DMSO

20
18
16
mg eq.DMS/kg ms

14
12
10
8
6
4
2
0
Barley Green malt 65C 75C Before Malt
transfert

Figure 3: Sulfur compounds involvement during malting process with two floor
kilning technology.

b: Continuous kilning technology

Figure 4 gives the evolution of sulfur compounds during industrial malting process using
continuous kilning technology. Each point in the kilning step is the mean value of three
samples taken at the bottom, the middle and the top of the malt layer.
free DMS pDMS DMSO

14

12

10
(mg eq.DMS/kg)

0
Barley Stp. 1/2 germ. Green 56C 66C 76C 86C Malt
Barley malt

Figure 4: Sulfur compounds involvement during malting process with continuous

kilning technology.

For this process, nothing more can be said about sulfur compounds evolution. The same
relationship links free DMS, DMSO and DMS precursors.

6
2: Nonenal precursors from barley to beer
2.1: Pilot malting process
As for sulfur compounds study, lipoxygenase activity, hydroperoxides potential and
antioxidant power were followed during the pilot malting process using Alexis and Optic
barley. The two varietys of barley exhibit the same evolution for lipoxygenase activity,
hydroperoxides potential and antioxidant power. Evolutions of these parameters are plotted on
figure 5 for Alexis barley variety.

Lox activity ROOH potential Antioxidant power

1000 40

900 35
800
30
700
25
600
(nkat/g)

(A.U)
500 20

400 15
300
10
200
5
100

0 0
Barley Stp. Green 50C 50C 60C 60C 80C 80C Malt
Barley malt

Figure 5: Involvement of lipoxygenase activity, hydroperoxides potential (X10)

and antioxidant power during pilot malting process with Alexis barley variety.

During germination, enzyme activation explain the increase of lipoxygenase activity. In the
same time there is an increase of the hydroperoxides potential. As we used the same quantity
of lipoxygenase activity, this increase could certainly be explained by the production of pro-
oxidant compounds which facilitate hydroperoxides formation.

With kilning, enzyme activity decreases slowly until 60C and then more rapidly above 80C.
In the same time, the antioxidant power increases, which is consistant with the fact that
hydroperoxides potential decreases.

The antioxidant power increases more rapidly at the end of kilning. This could be explained
by the fact, that the high temperature facilitate the formation of antioxidant compounds.

2.2: Industrial malting processes

a: One floor and two floor kilning technologies
Concerning industrial processes, we have the same evolution for lipoxigenase activity,
hydroperoxides potential and antioxidant power as in the pilot process.

For the one floor kiln process (figure 6), during germination and up to 70C of the kilning,
enzyme activation explains the increasing of lipoxygenase activity. After 70C, temperature is
too high and we observe de-activation of lipoxygenase.

7
 Lox activity ROOH potential Antioxidant potential

250 160

140
200
120

150 100
(nkat/g)

(A.U)
80

100
60

40
50
20

0 0
Barley Stp. Barley Green malt 60C 65C 70C 75C Malt

Figure 6: Involvement of lipoxygenase activity, hydroperoxides potential (X10)

and antioxidant power during malting process with one floor kilning technology.

Lox activity ROOH potential Antioxidant potential

700 40

600 35

30
500
25
(nkat/g)

400
(A.U)
20
300
15
200
10

100 5

0 0
Barley Stp. Barley Green malt 65C 75C Before Malt
transfert

Figure 7: Involvement of lipoxygenase activity, hydroperoxides potential (X10)

and antioxidant power during malting process with two floor kilning technology.

As in pilot process, hydroperoxides potential follows the evolution of lipoxygenase activity.

During germination and at the beginning of the kilning process, the increase could be
explained both by enzyme activation and by the presence or the production of pro-oxidant
compounds. At the end of the kilning, the decreasing of hydroperoxides potential is consistent
with the formation of antioxidant compounds. This fact is illustrated by the rapid increasing of
antioxidant power during the last step of the kilning process.

For the two floor kiln process (figure 7), the same observations as for one floor kiln process
can be made. The formation of antioxidant compounds occur during the last step of the
kilning. The level reach by the antioxidant power is practicly one fourth of the level reach

8
with one floor kiln. It seems that one floor kiln process is better for the formation of
antioxidant compounds.

b: Continuous kilning technology

Concerning coninuous kilning process (figure 8), there is not much differences with the one or
two floor kiln processes.

Lox activity ROOH potential Antioxidant potential

1200 30

1000 25

800 20
(nkat/g)

(A.U)
600 15

400 10

200 5

0 0
Barley Stp. Barley 1/2 germ. Green malt 56C 66C 76C 86C

Figure 8: Involvement of lipoxygenase activity, hydroperoxides potential (X10)

and antioxidant power during malting process with continuous kilning technology.

There is production of lipoxygenase activity during germination and degradation of enzyme

during kilning step. In the same way, degradation of hydroperoxides potential can be
associated with formation of antioxidant compounds during the last step of kilning.

CONCLUSIONS
In conclusion, we can observe that sulfur compounds associate with DMS have the same
evolution along the malting process. The DMS precursors are produced during germination
with enzymatic activation. These compounds are thermally instable and lead rapidly to DMS
and DMSO during the kilning steps.
A second point is that we clearly observe a difference between one floor and two floor kiln.
One floor kiln lead to malts with higher level of DMS and DMSO.

Concerning the continuous kiln, our data are insufficient to establish an influence of the
process on the level of sulfur compounds in malt.

More over, this study has given us a lot of crucial informations about nonenal precursors
along the malting process. For all the processes we have tested, lipoxygenase activity
increases with germination and up to 60 or 70C. Above this temperature, degradation of
lipoxygenase occurs and its activity is decreasing.

Hydroperoxides potential has the same evolution as lipoxygenase activity. Its increasing
during germination could be associated with the formation of pro-oxidant compounds. Its
decreasing during kilning is clearly associated with the formation of antioxidant compounds.

9
Concerning the influence of kilning technology, a clear difference can be establish between
one and two floor kiln. The first is leading to the production of more antioxidant compounds
and then would be more convenient to control nonenal formation during the brewing process.

ACKNOWLEDGEMENTS
The authors wish to thank the French Malsters and the French Brewers who support this
work.

REFERENCES
(1) Hanakoa, K., Thse de Doctorat, ENSIA, 2000.
(2) Boivin, P., Malanda, M., Clamagirand, V., Proceedings of the European Brewery
Convention Congress, Cannes, 1999, 397-404.
(3) Leemans, C., Dupire, S., Macron, J.Y., Proceedings of the European Brewery
Convention Congress, Oslo, 1993, 709-716.
(4) Dumoulin, M., Boivin, P., EBC Flavour and Flavour Stability Sub Group Meeting,
France, 1999.
(5) Billaud, C., Garcia, R., Boivin, P., Nicolas, J., Proceedings of the European Brewery
Convention Congress, Maastricht, 1997, 159-166.

10
21

Genetic difference of -amylase

thermostability among barley varieties
during beer production process
Makoto Kihara, Takashi Asakura, Takafumi Kaneko &
Kazutoshi Ito

Sapporo Breweries Ltd., Plant Bioengineering Research Laboratories, 37-1, Kizaki, Nitta,
Gunma, 370-0393, Japan (e-mail: [email protected])

Descriptors
-Amylase, barley, breeding, enzymic activity, fermentability, genetics

SUMMARY
We have reported that the genetic difference of -amylase thermostability in barley grain has
a close relationship to the apparent attenuation limit, which is a major malting quality
parameter for the fermentability. In the present report, therefore, we investigate the -amylase
activities of three malting varieties during the mashing process, and clarify the degrees of
thermostability. This result supports to our previous suggestions, and also indicates an
alternative approach for barley breeding, namely, -amylase thermostability is a useful
marker for the selection of barley with better fermentability without micromalting.

Genetische Unterschiede der Hitzestabilitt von Gersten-b-Amylase whrend der

Bierbereitung

Deskriptoren
-Amylase, Enzymaktivitt, Grkraft, Genetik, Gerste, Zchtung

ZUSAMMENFASSUNG
Wir haben bereits berichtet, dass die Hitzestabilitt der -Amylase in engem Zusammenhang
zu der scheinbaren Abschwchungsgrenze, die einer der Hauptparameter beim Malz fr die
Vergrbarkeit darstellt, besteht. In dem jetzigen Poster werden die Untersuchungen an drei
verschiedenen Malzvarietten whrend des Maischvorgangs dargestellt, sowie deren
Hitzestabilitt. Die Ergebnisse untersttzen unsere bisherigen Annahmen und zeigen, dass die
von der Gerste herrhrende -Amylase-Hitzestabilitt eine alternative Mglichkeit zur
Auswahl einer besser vergrbaren Gerste darstellt und somit auf Kleinmlzungen verzichtet
werden kann.
Diffrence gntique de thermostabilit de la -amylase d'orge pendant la fabrication de
la bire

Descripteurs
Activit enzymatique, -amylase, fermentabilit, gntique, obtention (culture), orge

RESUME
Nous avons indiqu que la diffrence gntique de thermostabilit de la -amylase dans les
drches d'orge prsente une corrlation troite avec la limite d'attnuation apparente, qui est
un paramtre majeur de qualit du maltage pour la fermentabilit. Dans le prsent article,
nous tudions les activits -amylase de trois varits de malt pendant le brassage et
clarifions les degrs de thermostabilit. Ce rsultat taye nos prcdentes suggestions et
indique en outre une approche alternative pour la culture de l'orge, en ce sens que la
thermostabilit de la -amylase est un marqueur utile pour la slection d'orges de meilleure
fermentabilit sans micromaltage.

2
INTRODUCTION
Activities of the various enzymes in barley and malt have a significant effect on the
characteristics of beer (8). Diastatic power (DP), which is an important character in
malting quality, describes the combined activities of -amylase, -amylase, limit
dextrinase and -glucosidase which hydrolyze starch during malt and mash
production into simple sugars including maltose. -Amylase (-1,4-glucan
maltohydrolase; EC 3.2.1.2) catalyzes the liberation of -maltose from the
nonreducing ends of starch, and this simple sugar is utilized by yeast during
fermentation to produce beer (9). The total activity of this enzyme is the most
important factor for the evaluation of diastatic power (DP) (2).
In addition to enzyme activity, thermostability is also very important, because kilning
and mashing processes, which employ high temperatures, reduce enzyme activity in
the barley and malt. In our previous studies, crude enzyme was extracted from barley,
and relative remaining activity of -amylase was investigated after heat treatment
condition (5, 6, 7). Our results indicated that the thermostability of -amylase differed
among barley varieties, and suggested the correlation between thermostability of -
amylase and apparent attenuation limit (AAL), which is a parameter for the
determination of fermentability. Another research group also supported to our
conclusions (1, 3).
Therefore, in the present study, to clarify the difference of -amylase thermostability
during beer production process, the -amylase activity was investigated in barley,
malt and mash of three malting barley varieties.

MATERIALS AND METHODS

Barley and malt samples
Barley and malt of three malting varieties (cvs. Haruna Nijo, Harrington and
Schooner) were used in our experiments. Malting quality (DP and AAL) of these
samples were 318 and 84.6, 331 and 83.5, and 164 and 78.5, respectively.

Investigation of -amylase thermostability in barley and malt

Barley and malt of three malting varieties were crushed, and 50 mg samples were
incubated in 500 microliters of 50 mM acetate buffer (pH 5.5) containing 10 mM
DTT (Dithiothreitol) at 4 centigrade for 3 h with shaking. The extract was centrifuged
at 15,000 rpm for 10 min, and the supernatant was used as the crude enzyme solution
for the determination of beta-amylase activity. Each sample was diluted (100-fold)
with 50mM MOPS (3-[N-Morpholino]propanesulfonic acid) buffer (pH 7.1)
containing 1% Bovine Albumin Fraction V, and 30 microliter samples were incubated
at 57.5 centigrade for 30 min, then the residual enzyme activity was determined.
-Amylase activity was measured at 37 centigrade using a p-nitrophenyl
maltopentaoside kit (Megazyme Co., Ireland). The relative remaining activity was
calculated by dividing activity under treatment conditions by activity under no-
treatment condition (6).

Investigation of -amylase thermostability in mash

Two-hundreds microliters of mash was sampled at 0, 10, 20, 30 - 65 min (2.5 min at
intervals) during the mashing process. The mashing program followed the EBC
congress methods (Analytica-EBC 4.5). The mash sampled was centrifuged at 15,000
rpm for 10 min, and the supernatant was diluted (200-fold) with the same buffer as

3
described for barley and malt, then the enzyme activity was determined.

RESULTS
-Amylase thermostability in barley and malt
For the investigation of -amylase thermostability, crude enzymes were extracted
from barley and malt of three malting varieties, Haruna Nijo, Harrington and
Schooner. These samples were incubated at 57.5 centigrade, and relative remaining
activity of -amylase was examined after heat treatment. This treatment condition
clearly confirmed the difference of thermostability reported previously (6). Our result
indicates that the thermostability in the -amylase from malt shows the same
tendency as in the thermostability from barley, and three varieties tested are clearly
divided into three types based on thermostability in both barley and malt samples
(figure 1). Haruna Nijo showed high themostability (type A), and Harrington and
Schooner showed intermediate (type B) and low thermostability (type C),
respectively.

-Amylase thermostability during mashing process

We investigated the -amylase activity in three malting varieties during mashing
process (figure 2). In the variety Schooner, activity started to decline after 40 min of
mashing time, approximately 55 centigrade, while in Haruna Nijo and Harrington,
activity started to decline after 42.5 min. Until 47.5 min, activity of Harrington was
higher than that of Haruna Nijo, but after 50 min, activity of Haruna Nijo was
higher than that of Harrington.
We calculated the relative remaining activity of -amylase in three malting varieties.
-Amylase activity at 40 min was defined as 100 % activity. As shown in figure 3, the
-amylase of Haruna Nijo is thermostable and that of Schooner is thermolabile
during mashing process. This inactivation pattern shows the same tendency as
described in our previous study (6).

Figure 1: -amylase thermostability in barley and malt

4
 Figure 2: Decline in -amylase activity during mashing process

Figure 3: Relative remaining activity of -amylase during mashing process

We investigated the concentration of fermentable sugars in mash of three malting

varieties. Harune Nijo and Schooner showed the highest and the lowest
concentration of maltose, respectively (data not shown). The high thermostability of
-amylase may promote the generation of maltose during mashing process, which
causes the resulting better fermentability.

CONCLUSIONS
-Amylase is a key enzyme in the mobilization of starch in barley and is the only
enzyme to be highly correlated with DP (2). Therefore, total activity of this enzyme is
very important for the brewing. In addition, thermostability of this enzyme has a
major influence on malting quality. In our previous study, we prepared crude enzyme
from barley, and determined the difference in -amylase thermostability among
barley varieties. We suggested a correlation between its thermostability and AAL,
which is a parameter for the determination of fermentability (5, 6).

5
In the present work, we compared the thermostability of -amylase extracted from
barley and malt in three varieties. Our results indicate that the type of thermostability
in -amylase extracted from malt shows the same tendency as thermostability in
barley. This result indicates that thermostability of -amylese does not change before
and after malting. Furthermore, the difference in -amylase thermostability among
barley varieties was also observed during the mashing process, and the inactivation
pattern of -amylase showed the same tendency as in the -amylase extracted from
barley (6). These facts suggest an alternative approach for breeding barley, because
this character is a useful marker for the selection of barley with higher levels of
fermentability. It is possible to investigate the thermostability of -amylase in a
number of samples at the same time, and the investigation needs only half the amount
of grain (non-embryo portion) and a short time (less than 24 hours from crude
enzyme preparation to determination of thermostability). The embryo portions of
bisected grains may be used for plant production. To improve fermentability in
malting barley breeding, therefore, it may be an efficient strategy to select the
progeny in an early generation which has more thermostable -amylase from crosses
between different thermostability varieties.
Recently, marker assisted selection (MAS) has been developed as an efficient strategy
to select progeny which has the desirable characteristics. This suggests that the
development of a DNA marker for -amylase thermostability could make it easy to
select the progeny which has more thermostable -amylase. In our previous study (4),
the genomic DNA sequences were compared among three malting varieties, Haruna
Nijo(10), Harrington and Schooner. Consequently, we developed sequence
tagged site (STS) marker of the AAL trait based on the different genomic DNA
sequences.
In the world malting barley varieties tested, most of the latest Japanese malting
varieties contain the type A -amylase, although most of malting varieties in other
areas contain the type B or type C -amylase (6). This fact suggests that the
introduction of the type A -amylase is important for the improvement in
fermentability of leading malting barley in other areas. In addition, recently, -
amylase with higher thermostability than type A has been discovered in world
landrace barley (Kaneko et al. in preparation) and in a wild barley species (Hordeum
vulgare ssp. spontaneum) (3). These -amylases might contribute to further
improvements in fermentability.
In the present study, we investigated the effect of -amylase thermostability by using
only the EBC congress method, however, we will confirm its effect using several
different mashing methods in the future.

ACKNOWLEDGMENTS
We thank Prof. K. Takeda, Okayama University, for the useful suggestions. We also
thank to Dr. P.A. Lazzeri, DuPont UK, for the critical reading of the manuscript.
Thanks are finally due to M. Miyata, Y. Yaginuma and C. Shikada for their technical
assistance.

REFERENCES
1. Ahokas, H. & Manninen, M.-L., Hereditas, 2000, 132, 111-118.
2. Erdal, K., Jensen, M.O., Kristensen, M., Krough, J.J., Riis, S. & Vaag, P.,

6
 Proceedings of the European Brewery Convention Congress, Oslo, 1993, 147-
154.
3. Eglinton, J. K., Langridge, P. & Evans, D. E., Journal of Cereal Science, 1998,
28, 301-309.
4. Kaneko, T., Kihara, M. & Ito, K., Plant Breeding, 2000, 119, 197-201.
5. Kihara, M., Kaneko, T., Fukuda, K. & Ito, K., Proceedings of the Australian
Barley Technical Symposium, Goldcoast, 1997, 3, 5-6.
6. Kihara, M., Kaneko, T. & Ito, K., Plant Breeding, 1998, 117, 425-428.
7. Kihara, M., Kaneko, T., Ito, K., Aida, Y. & Takeda, K., Plant Breeding, 1999,
118, 453-455.
8. MacGregor, A.W., Barley Genetics VI, Volume II, Copenhagen, 1992, 969-978.
9. Shinke, R., Mikami, B., Morita, Y., Takeda, Y. & Nanmori, T., Handbook of
amylases and related enzymes, Oxford, 1988, 81-104.
10. Yoshigi, N., Okada, Y., Sahara, H. & Tamaki, T., Bioscience, Biotechnology,
and Biochemistry, 1995, 59, 1991-1993.

7
22

Vibrating membrane filtration of mash

for the beer production
Jan Schneider1, Martin Krottenthaler2, Werner Back2 & Horst
Weisser1
1
TU Mnchen-Weihenstephan, Lehrstuhl fr Brauereianlagen und Lebensmittel-
Verpackungstechnik, D-85350 Freising-Weihenstephan, Germany (e-mail:
[email protected])
2
TU Mnchen-Weihenstephan, Lehrstuhl fr Technologie der Brauerei I, D-85350 Freising-
Weihenstephan, Germany

Descriptors
Grist, mash filtration, membrane filtration, vibration

SUMMARY
A new filtration technique - the Vibrating Membrane Filtration (VMF) - has been studied in
order to apply finely ground grist. The mechanism of the particle rejection on the membrane
surface bases on the oscillation of the whole filter device while the peripheral equipment
matches to conventional systems. By that means it is possible to maintain a constant flux and
transmembrane pressure up to a critical solid concentration. Sensory and analytical quality of
wort and beer do not differ negatively to the products in a lautertun process. The processing
of poorly malted and unmalted cereals bring up remarkable benefits.

Maischefiltration mit einer vibrierenden Membran

Deskriptoren
Maischefiltration, Membranfiltration, Schrot, Vibration

ZUSAMMENFASSUNG
Eine neue Membranfiltrationstechnik, die Vibrating Membrane Filtration (VMF), wurde mit
dem Ziel, feinstvermahlenes Schrot einsetzen zu knnen, untersucht. Die Technik basiert auf
der Oszillation des gesamten Filtermoduls, die das Abtragen der Feststoffe von der Membran
bewirkt. Die Peripherie-ausstattung der VMF entspricht dabei dem einer herkmmlichen
Crossflowfiltrationsanlage. Die VMF ermglicht bis zu einer kritischen Suspensionsviskositt
einen konstanten Transmembrandruck und Permeatfluss. Die sensorische und analytische
Qualitt weist keine negativen Unterschiede zu Vergleichsuden mit einem Luterbottich auf.
Das Verarbeiten von schlecht gelstem Malz oder Rohfrucht wird deutlicht verbessert.
Filtration sur membrane vibrante du brassin pour la production de bire

Descripteurs
Filtration de la maische, filtration sur membrane, moture (versement), vibration

RESUME
Une nouvelle technique de filtration la filtration sur membrane vibrante a t tudie afin
de l'appliquer aux grains finement moulus. Le mcanisme de rejet des particules la surface
de la membrane repose sur les oscillations de l'ensemble du filtre, alors que le matriel
priphrique correspond celui des systmes classiques. Par ce moyen, il est possible de
maintenir un flux constant et une pression transmembranaire jusqu' l'obtention d'une
concentration solide critique. La qualit analytique et organoleptique du mot et de la bire
n'est pas moins bonne que celle des produits en cuves filtre. Le traitement de crales non
maltes ou faiblement maltes apporte des bnfices remarquables.

2
MEMBRANES AS FILTER MEDIA FOR THE MASH SEPARATION
Why should the filter media spent grains be replaced by membranes for the purpose
of lautering? In the past, top priority has been given to combine unit operations with
simple machinery into one production step. Since the transport of even viscous media
(e. g. concentrated mash) is no longer an obstacle the unit operations can separated.
This is advantageous as the unit operations are now able to perform various
specialised techniques. The lautering process specifically can segregate the solid-
fluid-separation from the sparging with the use of dynamic membrane filtration.
Another aim of the membrane filtration of mash is to break the restrictions regarding
the grist fineness. Therefore, the grist fineness must outperform the achievable
fineness of modern mash filters.

In the 80s and 90s a few papers dealt with the dynamic membrane filtration of mash:
crossflow filtration (1, 2) and rotating disk filtration (6, 7). These did not produce
satisfactory results. The reasons were either the need of a preliminary clarification of
the mash or the impact of fouling on the flux (flow rate of permeate).

The object of this work is the development of the Vibrating Membrane Filtration
(VMF) for the purpose of mash filtration with finely ground malt. Three
characteristics of a lautering technique has been studied for an validation of the new
filtration technique:
Capacity: filtration characteristics (flux and transmembrane pressure)
Quality: Analyses of wort and beer
Quantity: extract - conversion yield and sparging efficiency

CONVENTIONAL CROSSFLOW FILTRATION AND VIBRATING

MEMBRANE FILTRATION (VMF)
The VMF is similar to crossflow microfiltration. The basic difference of the VMF is
that the shear rate is created by the oscillation of the filter pack and the inertia of the
suspension in a gap (1,5 mm) between two membranes.
A

Filterpack
Filtermodul
J1,, m1
e
l
m3

Seismic mass
Gegenmasse
J2, m2

Figure 1: Generation of the oscillation by means of a coupled torsional vibration

of two masses

3
The oscillation is maintained in a system of a coupled torsional vibration of two
masses close to the resonance frequency (figure 1), requiring a very low power
consumption. The filter pack consists an assembly of up to 100 disks with PTFE
membranes (pore size 0,45 m) on both sides. The maximum filter area is 40 m per
filter pack unit (figure 2).

Figure 2: VMF membrane filter assembly

Some important characteristics of conventional crossflow filtration and VMF are

schematically shown in figure 3.

retentate
suspension retentate suspension
permeate
permeate
oscillation
Conventional Crossflow Process Vibrating Membrane Filtration
Shear rate depends on pressure conditions shear rate independent of the pressure
Transmembrane pressure 1,5-2 bar transmembrane pressure 0,4-0,8 bar
Cross flow velocity 3-5 m/s cross flow velocity about 0,01 m/s
High power consumption low power consumption

Figure 3: Comparison of the conventional crossflow filtration and VMF

FILTRATION CHRACTERISTICS
In the very first phase of the filtration, a narrowing of the pores of the membrane takes
place (figures 4 and 7). Consequently, the filter resistance R increases for a short
moment and then remains constant (figure 4). This behaviour can be observed in the
cases of new or intensely cleaned membranes. The described first phase is followed

4
by a stage with a stationary flux and transmembrane pressure (pTM) respectively by
a stationary filter resistance (R):
p TM
R=
( Matrix Flux )

Volume (Wort) in l/m 2

0 10 20 30 40 50 60 70
200
10 in m-1

150 the viscosity dominates

the inertia effect: the
Resistance x -12

increasing solid deposit of particles

concentration dominates the reject
100

50 solid concentration = const.

narrowing of the
pores in the very return of permeat (wort) to the
beginning suspension (mash)
0
0 1000 2000 3000 4000 5000 6000
Time in s
Figure 4: Filter resistance versus time: typical filtration characteristics

In the (unrealistic) case that the mash is not thickened (return of permeate to the
suspension) the stationary filter resistance can be maintained. But for the purpose of
real mash filtration, the suspension need to be as concentrated as possible
(extraction efficiency). High concentrations implicate an increase of the mash
viscosity which results in a decline of the reject effect at the membranes surface. Thus
a limit for the stationary state is given by the solid concentration and by the dynamic
viscosity of the suspension (figure 5).
140 200
= 379 s-1 = 340 s-1
10 in m-1

120
10 in m-1

Viscosty (first wort) = 2,35 mPa s 160

100 = 354 s-1
= 379 s-1
Resistance x -12
Resistance x -12

80 120

60
80
40
Viscosity (second wort) = 1,066 mPa s
40
20
Viscosity (last wort) = 1,031 mPa s Viscosity (first wort) = 2,35 mPa s
0 0
5% 7% 9% 11% 13% 15% 0 40 80 120 160
Solid Concentration Viscosity (Mash) in m Pa s
Figure 5: Filter resistance versus time depending on matrix viscosity and
frequency (Measurement of the mash viscosity with a plate-plate
rheometer Rheometrics SR 5000 [3].)

5
The critical viscosity depends on the chemical properties of the filtered product and
on the oscillation frequency (and the amplitude). Beyond the critical conditions an
exponential increase of the filter resistance can be observed. (Measurement of the
mash viscosity with a plate-plate rheometer Rheometrics SR 5000 [3].)

QUALITY OF WORT AND BEER

Influence of grist fineness
A higher degree of grist fineness leads to an acceleration of molecule degrading
proces-ses. The immediate dissolving of already soluble substances is especially
enhanced. But a restriction of the conversion processes by missing out of particular
temperature rests is partly not possible (e. g. protein degradation). The impact of the
grist fineness on the conversion yield depends considerably on the liquor ratio (figure
6). Furthermore, a reduction of the liquor ratio leads to a lower -glucane
concentration [5] and allows the increase of the sparging liquor (better extraction).

90
Liquor Ratio
4,0 3,5
in % anhydrous
Conversion Yie

3,0 2,5
85

80

75
0 100 200 300 400 500 600
x 75,3 in m

Figure 6: Conversion yield versus grist fineness

Influence of the membrane process

In the early phase of the mash filtration suspended particles narrow the pore system of
the membrane in a way that large (dissolved) molecules can not pass through
anymore. The figure indicates, exemplary for -glucan, that in the further processing
of the first wort running the concentration in the permeate as well as the filter
resistance get to a constant level. These curves are also found for protein, dextrin,
total extract and suspended particles.

In fact, the kinetics of the molecular selection and filter resistance correspond with
each other (figure 7), indicating the separation size is depended on the filtration
conditions (e. g. transmembrane pressure and the degree of concentrating).

6
 200 500
180 450

mg/l
-Glucan in Wort (Permeate)
160 400
in m-1 Filter Resistance
140 350
120 300

-Glucanin
100 250
-12
R x 10

80 200
60 150
40 100
20 50
0 0
0 5 10 15 20
V Wort in l
Figure 7: Correspondence of the filter resistance and betaglucan selection in the
first filtration stage

Characteristics of wort and beer in a comparison (Lautertun / VMF)

A definition to dictate the desired separating size for the mash filtration is not
available. Therefore, a direct comparison of the new technology with the conventional
system was performed. 14 Pilot scale brews (15 kg grist) were evaluated. The results
are shown in tables 1 and 2.

Lautertun conf. VMF conf. Standard /

Coarse Interv powder interv. target value
Grist . grist

Maltose g/l 6,5 9,9 50-55

Maltotriose g/l 31,2 41,6 8,5-11
Betaglucane mg/l 240 27 97 15 200->800
Total N mg/l 1256 29 1211 153 900-1200
MgSO4-N ref. To 12 % mg/l 198 10 193 11 200-240
FAN ref. To 12 % mg/l 204 4 194 24 200-250
Zn mg/l 0,17 0,005 0,15 0,005 0,05-0,20
Solid Conc. Heinecken mg/l 89 24 13 6 <100
Turbidity (90) EBC 24 2 10-30
App. Fermentability % 88,5 0,1 86,9 0,4 80-85
Viscosity mPa s 1,731 0,18 1,589 0,035 1,7-1,9
Iodine test, photom. 1 0,11 0,09 0,01 0,05 0,37-0,45
Colour (light beer) EBC 7,7 0,3 10,3 0,9 7-11
PH-value 1 5,78 0,05 5,76 0,05 4,8-5,6

Table 1: Wort analyses

7
 Lautertun conf. VMF conf.
Coarse grist interv. powder grist interv.

Filterability normally g/l 7,7 1,9 10,1 0,5

modified malt (KREISZ [4])
Filterability poorly modified g/l 2,1 0,2 10,0 0,4
malt (KREISZ [4])
Polyphenols (total) g/l 132 8 148 11
Anthocyanogen g/l 73 0,1 73 0,2
Foam (NIBEM) s 123 20 114 12
Betaglucane mg/l 194 24 82 4
Tasting (DLG) 4,07 0,0 4,17 0,2
Tasting forc. (EICHHORN) 1,9 1,8

Table 2: Wort analyses

A triangle sensory test with 65 tasters pointed out that there is no significant
difference in taste between the lautertun beer and the VMF beer.

EXTRACTION EFFICIENCY (SPARGING)

The performance of the sparging in a dynamic filtration process is different to the
sparging in the cake filtration systems (figure 8). The unit operations (separation and
extraction) can be segregated. Homogenous sparging is possible by the means of a
stirred vessel.
The grist fineness accelerates the extract transport from the solid to the matrix fluid
considerably. Figure 8 shows the kinetics of the extract concentration.
25
Spent Grains (Solid Matter)
Extract in % GG

20
Wort (Matrix)

15

10

0
0 20 40 60 80 100
Time in min
Figure 8: Extract concentration in the solid matter and in the matrix fluid versus
time

8
There is only a slight difference between the final concentration in the spent grains
and the wort. The final extract concentration of 0,8 % GG in the wet spent grains (and
4,0 % ref. to dry matter) as demanded by the MEBAK can be achieved without
exceeding a total liquor ratio of 7,2. The moisture of the spent grains is higher than in
lautertun or mashfilter systems (85-87 %).

CONCLUSION
The filtration characteristics of the VMF (constant flux and pressure) supply new
arguments for a change from conventional filtration systems to membrane filtration.
This is displayed by the facts of a low power consumption and little spatial
requirements comparably to the cross flow filtration. In regard to the problem of mash
filtration the inter-actions between the three characteristics (capacity, quality and
quantity) have to be considered. The narrowing of the pores has an impact on removal
of high molecular substances but also on the filter resistance (resp. flux). A finer grist
affects the sparging efficiency as well as the wort quality. A higher degree of
concentration leads to a better sparging efficiency but is of bad influence on the flux.
The important findings are a constant flux and a constant transmembrane pressure, no
remarkable differences in the product quality, a better filterability of the beer and a
extraction yield which fulfils the MEBAK directives.

REFERENCES
1. Bhler, T., Burell, K., Eggars, H.U., Reed, R.J.R.: The application of membranes
for new approach to brewery operations, Proceedings of the 24th EBC Congress
(1993), 691-700
2. Daoud, I.: Crossflow Filtration: An alternative for mash separation, Brewing and
Distilling International, 23 (1985), Nr. 5, 31-32
3. Gtz, J., Schneider, J., Weisser, H.: Korrelationen zwischen der dynamischen
Viskositt und der T2-Relaxation aus NMR-Messungen fr reine Flssigkeiten,
Lsungen und Suspensionen, Chem.-Ing.-Tech., 72 (2000), Nr. 9, 1074-1075
4. Kreisz, S., Back, W.: Neue Aspekte der Filtrierbarkeit von Bier, Proceedings of
the 27th EBC Congress (1999), 779-786
5. Krottenthaler, M., Zrcher, J., Schneider, J., Back, W., Weisser, H.: Grist
fractionating and adjusted mashing to improve the reduction of -glucan,
Proceedings 27th EBC Congress (1999), 603-610
6. Lotz, M.: Neue Maischefiltrationstechnik fr das Verarbeiten von Pulverschrot,
Freising-Weihenstephan, TU Mnchen, Dissertation, 1997
7. Lotz, M., Schneider, J., Weisser, H., Krottenthaler, M., Back, W.: New Mash
Filtration Technique for Processing Powder Grist, Proceedings of the 26th EBC
Congress (1997), 299-305

9
23

The influence of polysaccharides from

yeast and bacteria on the filterability of
wort and beer
S. Kreisz, F. Wagner & W. Back
TU Mnchen, Lehrstuhl fr Technologie der Brauerei I, D-85350 Freising-Weihenstephan,
Germany (e-mail: [email protected])

Descriptors
Beer filtration, filterability, microorganism, polysaccharide, wort filtration, yeast

SUMMARY
The filterability of beer is influenced by a lot of high-molecular compounds. Polysaccharides
are playing an important role in this effort. The polysaccharides of malt and their influence on
the filterability are well investigated. Less attention was paid so far on other sources of
polysaccharides like yeast and micro-organisms. This work shows the impact of the
polysaccharides from yeast and micro-organisms on the filterability of wort and beer.
Furthermore a method to detect the origin of problems in filterability is presented. Therefore
the polysaccharides are released from diatomaceous earth after filtration. Then the
polysaccharides are hydrolysed and assigned to their source.

Der Einfluss von Polysacchariden aus Hefe und Bakterien auf die Filtrierbarkeit von
Wrze und Bier

Deskriptoren
Bierfiltration, Filtrierbarkeit, Hefe, Mikroorganismen, Polysaccharid, Wrzefiltration

ZUSAMMENFASSUNG
Die Filtrierbarkeit von Bier wird von vielen meist hochmolekularen Inhaltsstoffen beeinflusst.
Hierbei spielt die Gruppe der Polysaccharide eine bedeutende Rolle. Die Polysaccharide des
Malzes sind in diesem Zusammenhang bereits gut erforscht. Weitere bisher wenig beachtete
Quellen von Polysacchariden sind Hefen und Mikroorganismen. In dieser Arbeit wird ihr
Einfluss auf die Filtrierbarkeit von Bier dargestellt. Auerdem wird eine Analyse vorgestellt,
die est ermglicht, die Quelle der filtrationshemmenden Polysaccharide und den Ort an dem
sie entstanden sind, zu erfassen. Dafr werden die Polysaccharide nach der Filtration aus der
Kieselgur gelst und nach Hydrolyse ber das mittels HPLC analysierte Zuckerspektrum ihrer
Quelle zugeordnet.
Influence des polysaccharides de levure et de bactries sur la filtrabilit du mot et de la
bire

Descripteurs
Filtrabilit, filtration de la bire, filtration du mot, levure, microorganisme, polysaccharide

RESUME
La filtrabilit de la bire est influence par de nombreux composs haut poids molculaire.
Les polysaccharides jouent un rle important cet gard. Les polysaccharides du malt et leur
influence sur la filtrabilit sont bien tudis. Jusqu'ici, une d'attention moindre a t porte
autres sources de polysaccharides, comme la levure et les micro-organismes. Nous avons
tudi le rle des polysaccharides de levure et de micro-organismes sur la filtrabilit du mot
et de la bire. Par ailleurs, nous prsentons une mthode permettant de dtecter l'origine des
problmes de filtrabilit. Les polysaccharides sont librs du Kieselgur aprs filtration.
Ensuite les polysaccharides sont hydrolyss et attribus leur source.

2
INTRODUCTION
The great number of publications concerning the filterability of wort and beer
indicates that this topic was and is still of interest to the brewing industry. The
filterability describes the attributes of wort or beer to influence the filtration process.
Therefore, quality parameters for the filterability are the pressure rise at the filter
entrance and the turbidity of the filtrate. Several authors [1,2,3,4,5] named mostly
high molecular ingredients influencing the filterability of beer. These include
polysaccharides, proteins, polyphenols, minerals, yeast and bacteria. Not for every of
the above mentioned materials exists a scientific proof that they have a direct impact
on the filterability of wort and beer and there are different statements about the
limiting value and the strongness of the influence. There is a secure perception that
malt polysaccharides -glucane, Pentosane and above all -glucane may cause
substantial difficulties [6,7]. Besides the malt, there can be other sources of
polysaccharides in the brewing process such as yeast and bacteria. Micro-organisms
contain structural and reserve polysaccharides which can be dispensed into the beer.
These sources of polysaccharides have received only a little consideration so far.
Gracey [8] and later Back [9], reported that in the malting and brewing process there
are bacterial contaminations which have a negative impact on the lauter and filter
performance. Walker et al. [10] found that haze in wort and green beer contained dead
bacteria (Enterobacter agglomerans and Gluconbacter frateurii) from barley.
Malcorps et al. [11] published at the EBC congress in Cannes that glycogen released
from the yeast may cause what they called unfilterable haze in beer.
This paper shows the influence of bacterial and yeast polysaccharides on the
filterability of wort and beer. From the great number of bacterial polysaccharides
dextran, gellan and xanthan were chosen to perform the experiment. They were added
to wort and beer in different concentrations and the filterability was measured. The
same procedure was done with the yeast polysaccharides mannan and glycogen, and
with a dried yeast extract however, these were only added to beer.

MATERIAL AND METHODS

Bacterial polysaccharides
Xanthan (SIGMA EEC No 234-394-3).
Xanthan is constructed with a repeating five-member unit of three monosacharides, -
D-glucose, -D-mannose and glucoronic acid in a 2:2:1 ratio. The main chain is a 1,4-
-D-glucan, and the side chains are trisaccharides of - and -D-mannose and
glucuronic acid. Acetyl and pyruvate groups are present [13].

Gellan (ICN GEL-GRO)

Gellan gum is a partly acetylated linear heteropolysaccharide whose repeating unit is
a tetramer of one 1,3--D-glucose, one 1,4--D-glucose, one 1,3--D-glucuronic acid,
and one 1,4--l-rhamnose [13].
Dextran (ICN 9004-54-0)
Dextran is a structurally heterogeneous -D-glucan with 1,6--D, 1,2--D, 1,3--D
and 1,4--D linkages [13].

Yeast polysaccharides
Mannan (SIGMA M-7504)

3
Mannan is a polymer composed of mannose units. The yeast mannan is the
polysaccharide portion of a proteoglycan. It contains a 6-linked D-mannopyranosyl
main chain, extended in the second position by side chains linked by -1,2 and -1,3
bonds [12].
Glycogen (SIGMA G-1508)
Glycogen is a branched polysaccharide of glucose which has chains of 4-O-subsituted
-D-glucopyranosyl units that are occasionally interrupted by 4,6-di-O-substituted
units [12].
Yeast extract (SIGMA Y-0375)

Filter test
The filterability of beer and wort was measured by a filter test based on a method
published by Raible 1990 [14]. Two permanently cooled (0 C) containers were filled
with either water and coarse guhr or green beer and fine guhr. The filter, cooled as
well to 0 C, contained a 15 m wire tissue situated at the bottom to simulate a
powder filtration. After the precoat (1 kg/m2) the green beer was filtered with fine
guhr (80 g/hl). During the filtration process, a constant pressure difference of 100 kPa
was applied while measuring the time required to filtrate a defined quantity of beer.
The results were indicated as specific filtrate volume (Fspez) in hl/m2h [15].

Preparation of the wort

The wort was treated with ethanol (5 % Vol.) and citric acid (pH-level 4.5) followed
by a storage period of one week at 0 C. This treatment simulates agglomeration and
precipitation procedures which normally take place during fermentation and storage.
If poor malt quality or wrong processing in the brewhouse causes problems in
filterability it can already be shown by a poor filterability of the treated wort [15].

Hydrolyse
The hydrolyse was prepared according to the procedure MEBAK II 2.38.2.1 [16].

HPLC
The HPLC was performed according to a modified version of the procedure MEBAK
III 3.2.1 [17]

RESULTS AND DICUSSION

The influence of bacterial polysaccharides on the filterability of wort
The wort was prepared as described previously. Polysaccharides were then added to
the wort in different concentrations. After one week of storage at 0 C, the filterability
was measured. Figure 1 shows the influence of the different polysaccharides on the
filterability of wort. Gellan has the strongest impact but dextran and xanthan also have
a strong influence on the filterability. The level of the filterability was reduced
significantly, from good to critical, with only 5 mg/l of each polysaccharide added to
the wort. Increased levels (10-15 mg/l) create an unfilterable wort. The results
demonstrate that a small amount of bacterial polysaccharides released from malt or
wort contaminants may have a negative impact on the filterability of wort.

4
 Fspez hl/m2h
7,0
6,0 Dextran Xanthan Gellan
5,0
4,0
3,0
2,0
critical
1,0
range
0,0
0 5 10 15 20
Polysaccharide concentration in mg/l

Figure 1: The influence of xanthan, dextran and gellan on the filterability of wort

The influence of bacterial and yeast polysaccharides on the filterability of beer

Bacterial polysaccharides and the yeast polysaccharides mannan and glycogen as well
as yeast extract were added in different concentrations to unfiltered beer and stored
for one week at 0 C. The filterability was then measured using the filter-test as
described in the material and methods section.
Figure 2 shows the influence of dextran on the filterability of beer. The critical level
(0.5 EBC) of haze is surpassed when the dextran concentration rose to 10 mg/l.
However, the Fspez dropped from 8.7 hl/m2h to 2.1 hl/m2h. The critical concentration
of dextran was 30 mg/l. Over 40 mg/l the beer was unfilterable.

Fspez hl/m h
2 Haze EBC
9,0 0,9
8,7
0,8
8,0 0,8
6,7 6,9
7,0 0,7
6,0 5,3
0,6
5,0 0,58
0,53 0,5
4,0
3,0 0,37
0,4
2,1
2,0 0,3
0,22 Fspez Haze
1,0 0,2
0 10 20 30 40 50
Dextran mg/l

Figure 2: The influence of dextran on the filterability of beer

Xanthan (figure 3) showed a stronger impact on the Fspez than dextran. The Fspez had
already decreased from 8.9 to 2.8 hl/m2h with a concentration of 10 mg/l xanthan.

5
After adding 30 mg/l or more of xanthan the haze level rose to the critical point of 0.5
EBC.

Fspez hl/m2h Haze EBC

Fspez Haze
9,0 8,9 0,6
8,0 0,52
0,50
7,0 0,5
6,0
5,0 0,4
4,0 2,8
2,5
3,0 2,0
0,3
0,27 1,5
2,0 0,22 0,24
1,0 0,2
0 10 20 30 40 50
Xanthan mg/l

Figure 3: The influence of xanthan on the filterability of beer

Gellan (figure 4) revealed to have a strong impact on the Fspez.. A gellan concentration
of 10-20 mg/l change the Fspez. from a good filterability to an unfilterable beer. The
haze level increased with a lower concentration of gellan (10 mg/l) to the critical level
of 0.5 EBC. However, if the concentration was increased the haze level remained the
same.

Fspez hlm2h
Haze EBC
9,0 8,9 Fspez Haze 0,8
8,0 0,7
7,0
0,52 0,55 0,6
6,0 0,5
5,0 5,2 0,5
4,0 4,8 0,4
3,0 0,33
3,3 1,5 0,3
2,0
0,22
1,0 0,2
0 10 20 30 40 50
Gellan mg/l

Figure 4: The influence of gellan on the filterability of beer

6
The influence of mannan (figure 5) on the Fspez was negligible. A small decrease of
the Fspez was the result of the addition of mannan, however, the critical value (3.8-4.5
hl/m2h) was still unattainable at concentrations as high as 50 mg/l. The haze level
increased slightly up to the critical area with 50 mg/l of mannan.

2 Haze EBC
Fspez hl/m h
6,0 Fspez Haze 0,6
4,7
5,0 4,7
4,9
0,5
0,5
4,0 4,5 4,5
0,35 0,36
0,4
0,34
3,0 0,31

0,3
2,0

1,0 0,2
0 10 20 30 40 50
Mannan mg/l

Figure 5: The influence of mannan on the filterability of beer

Glycogen (figure 6) had no effect on the Fspez and only a slight influence on the haze,
but the level remained under the critical value of 0.5 EBC.

Fspez hl/m2h Haze EBC

6,0 0,5
4,9 4,7 5,1
5,0 4,8
0,4
0,4
4,0 0,34

3,0
0,34 0,3
0,32
2,0
Fspez Haze
1,0 0,2
0 10 20 30 40 50
Glycogen mg/l

Figure 6: The influence of glycogen on the filterability of beer

Figure 7 shows the influence of yeast extract on the filterability of beer. Like the yeast
polysaccharides mannan and glycogen, yeast extract has no impact on the Fspez .

7
Nevertheless, the haze level rose over the critical level as the concentrations increased
to 10-20 mg/l.

2 Haze EBC
Fspez hl/m h
6,0 0,63 0,67 0,7
4,9 4,7
5,0 4,8 4,8 0,6

4,0 0,5

3,0 0,4
Fspez Haze
2,0 0,3
0,32 0,33

1,0 0,2
0 10 20 30 40 50
Yeast extract mg/l

Figure 7: The influence of yeast extract on the filterability of beer

The identification of the polysaccharides

After the filtration, the filter cake was collected and stored. The polysaccharides were
released from the cake. Most of the bacterial or yeast polysaccharides differed in
structure and contained a variety of monosaccharides. Through hydrolyzing the
polysaccharides and detecting the monosaccharides with high performance liquid
chromatography (HPLC), it was possible to assign the spectrum of monosaccharides
to the source of the polysaccharides. This method will only work if the
polysaccharides consist of a variety of monosacharides other than glucose. For
polysaccharides that are constructed only of -D-glucopyranosyl units, the analyses
have to be extended. In this case specific enzymes could aid in the determination of
their source.

CONCLUSION
Bacterial contamination and yeast may release polysaccharides into the wort and beer.
Bacterial polysaccharides like xanthan, dextran and gellan do have a strong impact on
the filterability of wort. If these polysaccharides are added in the brewhouse by
contamination of the malt or the wort, their negative impact on the filterability of beer
may be already shown by a poor filterability of the wort. Bacterial and yeast
polysaccharides like mannan and glycogen have an influence on the filterability of
beer. Bacterial polysaccharides cause a significant decrease of the Fspez and an
increase of haze. The amount of the bacterial polysaccharides (10-50 mg/l) was very
small compared to the high content of malt polysaccharides, for example the -
Glucan (app. 200-500 mg/l), required to have a significant influence on the
filterability. Yeast polysaccharides do not change the Fspez but can increase the haze
of the filtered beer as reported from Malcorps [11]. It is possible to detect the source
of polysaccharides by releasing them from the filter cake and comparing their
monosaccharide spectrums. For the future, this method should be amplified with

8
enzymatic analysis to detect glucans consisting only of glucose, like dextran or
glycogen. Furthermore, the solution of polysaccharides should by substituted with
real contamination of the malt and yeast. In doing so, the magnitude of influence of
the contamination could be identified throughout the production process.

ACKNOWLEDGMENT
This work was supported by the Wissenschaftliche Station fr Brauerei Mnchen e.V.

LITERATURE
1. Schimpf, F.W. et al.: Untersuchungen ber die Filtrierbarkeit des Bieres,
Monatschrift fr Brauwissenschaft, 1969, Nr. 12, Berlin, 353-361.
2. Hug, H., Pfenninger, H.: Filtrationsschwierigkeiten - Ursachen und
Mglichkeiten zur Behebung, Schweizer Brauerei Rundschau, 85, 7 / Juli 1974,
133-152.
3. Narzi, L.: ber die Filtrierbarkeit des Bieres, Brauwelt, 115, 45 / Nov. 1975,
1491-1496.
4. Schur, F.: Bierfiltration und Einflsse auf die Bierfiltrierbarkeit, EBC
Proceedings of the 22th Congress, Zrich, 1989, 371-383.
5. Annemller, G.: ber die Filtrierbarkeit des Bieres, Monatsschrift fr
Brauwissenschaft, 2 / 1991, 64-71.
6. Wagner, N., Krger, E: Bedeutung von Beta-Glucan-Gel fr die Filtrierbarkeit
von Bier, Brauwelt, 12 / 1991, 426-434.
7. Narzi, L.: Beta-Glucan und Filtrierbarkeit, Brauwelt, 37 / 1992, 1696-1704.
8. Gracey, D.E.F., Barker, R.L.: A new perspective on the composition of beer
haze, EBC Proceedings of the 18th congress, Copenhagen, 1981, 471-478.
9. Back, W.: Filtrationsprobleme durch Kontaminationen mit Bakterien, Brauwelt,
1995, Nr.7, 270.
10. Walker, M.D. et al.: The influence of malt derived bacteria on the haze and
filterability of wort and beer, EBC Proceedings of the 26th congress,
Maastricht, 1997, 193.
11. Malcorps, Ph. et al.: Glycogen released by the yeast as a cause of unfilterable
haze in the beer, EBC Proceedings of the 27th congress, Cannes, 1999, 831-840.
12. Gorin, P.A.J., Barreto-Bergter, E.: The chemistry of polysaccharides of fungi
and lichens. Hrsg.: Aspinall G. O.: The polysaccharides, Vol. 2. Toronto 1982.
Academic press, 366-400.
13. Walter, R.H.: Polysaccharide Dispersions, New York, Academic press, 1998.
14. Niemsch, K., Heinrich, Th.: Raible-test for evaluation of filtration properties,
Journal of the Institute of Brewing, 2000, 106, No. 5, 277-285.
15. Kreisz, S., Back, W.: Neue Aspekte der Filtrierbarkeit von Bier, EBC
Proceedings of the 27th congress, Cannes, 1999, 779-786.
16. Pfenninger, H. et al.: Hydrolyse. MEBAK Brautechnische Analysenmethoden
Band II, 3. Aufl. Weihenstephan, 1994, 246-247.
17. Pfenninger, H. et al.: vergrbare Zucker in Wrze und Bier. MEBAK
Brautechnische Analysenmethoden Band III, 2. Aufl. Weihenstephan, 1996, 68-
74.

9
24

Continuous wort boiling and hot trub

separation
Bruno Bonacchelli, Frdrique Harmegnies & Rafal Tigel Gil
Meura Technologies, Voie Minckelers 1, B-1348 Louvain-la-Neuve, Belgium (e-mail :
[email protected])

Descriptors
Continuous process, hot break, saving, wort boiling, yield

SUMMARY
Classical wort boiling and trub removal imply high evaporation rates, mainly for the
elimination of unwanted volatiles. We have developed a continuous concept of wort boiling
and hot trub separation. The wort heated at 100 C is sent at a constant flowrate to
successively a closed vessel, a cylindro-conical settling tank and a stripping column. This
continuous boiling system is environmental friendly and allows energy costs savings, a high
productivity and a an easy automation. This paper presents the concept, the results obtained
f'rom trials with a pilot installation of 4 hl/h and the extrapolation of an industrial plant.

Kontinuierliche Wrzekochung und Heitrubabscheidung

Deskriptoren
Ausbeute, Einsparung, Kochtrub, kontinuierlicher Proze, Wrzekochen

ZUSAMMENFASSUNG
Klassische Wrzekochung und Trubabscheidung bringen hohe Verdampfungsziffern
hauptschlich zur Entfernung von unerwnschten flchtigen Bestandteilen mit sich. Wir
entwickelten ein kontinuierliches Konzept zur Wrzekochung und Heitrubabscheidung. Die
bei 100 C kochende Wrze wird mit einem konstanten Volumenstrom nacheinander durch
einen geschlossenen Kessel, einen zylindrokonischen Absetztank und eine Strippingkolonne
gefrdert. Dieses kontinuierliche Kochungssystem ist umweltfreundlich, ermglicht Energie-
kosteneinsparung, eine hohe Produktivitt und eine einfache Automation. Diese Arbeit stellt
ein Konzept vor, das Ergebnisse eines Versuches im Pilotmastab (4 hl/h) und die
Extrapolation auf industriellen Anlagen beinhaltet.
Ebullition continu du mot et sparation chaud du trouble

Descripteurs
Cassure chaud, cuisson du mot, pargne, procd continu, rendement

RESUME
L'bullition classique du mot et l'limination du trouble induisent des pourcentages
d'vaporation levs, essentiellement pour l'limination des volatiles indsirables. Nous avons
dvelopp un concept continu d'bullition du mot et de cassure chaud. Le mot port 100
C est envoy dbit constant vers successivement une cuve close, un dcanteur cylindro-
conique et une colonne de rtention. Ce systme d'bullition en continu est respectueux de
l'environnement et permet la rduction des cots nergtiques, une haute productivit et une
automation aise. Cet article prsente le concept, les rsultats obtenus avec un quipement
pilote de 4 hl/h ainsi que l'extrapolation vers une installation industrielle.

2
INTRODUCTION
Wort boiling is an important step of the brewing process where numerous chemical,
physical and biochemical reactions occur.
Its main functions can be summarised as follows:
- evaporation of unwanted aroma compounds
- extraction and isomerisation of hop bitter substances
- sterilisation of the wort
- formation of reducing and aromatic molecules
- deactivation of enzymes
- coagulation of proteins
- evaporation of water to achieve the desired wort concentration

In conventional systems, wort boiling is achieved in a batch operation inducing high

evaporation rates from 7 to 10%.

In recent years, increased pressures of environmental legislations and energy economy

have led to focus efforts and to develop new technologies for reducing energy
consumption.

Nowadays we dispose of arguments and technologies enough to proceed an efficient

wort boiling without evaporation and without affecting wort quality.

Firstly, main of the listed reactions do not need an evaporation since they are only
time and temperature dependent. Only the elimination of unwanted volatile molecules
and the coagulation of proteins are respectively linked to the evaporation or the
agitation.

Moreover the use of modern technologies in the brewhouse such as the 2001 filter
makes unnecessary the evaporation as a way of concentration.

Until soon, the elimination of undesirable aroma compounds was then the only reason
justifying the need of an important evaporation during wort boiling.
Recently, new systems called strippers have been developed to remove unwanted
volatile with an optimised evaporation surface.
Among them, the use of a wortstripping column between wort clarification and wort
cooling allows the elimination of unwanted volatile more efficiently than a classical
evaporation and complies with environmental and energy saving expectations
(vapours rich in unwanted volatile are condensed, hot water is recovered, CO2
emissions are reduced due to energy gain, ).
This technology was presented by Interbrew at the EBC Congress in 1997 (3).

With all these considerations, mainly the fact that a long period of strong evaporation
is no longer needed, we have developed a concept of continuous wort boiling and hot
trub separation.

CONCEPT
Wort boiling and clarification are so closely linked that they have to be considered as
one system. The quality of clarification depends strongly on the quality of the boiling
and both steps have high significance to the quality of the product.

The motivation to introduce a continuous concept of wort boiling and trub separation
is based on a number of potential benefits, mainly a reduction of the equipment size, a
better process rentability and energy savings.

3
This concept is of course made possible considering some technical aspects:

- To assume a correct break formation, the wort kettle must be designed and
equipped in such a way that each particle of wort will get sufficient and gentle
heat treatment, with shear stresses as low as possible. For this, the formation tank
is equipped with an adapted agitator and heated with a direct steam injection. The
use of direct steam is here of great importance because at the opposite of a double
jacket, it ensures a constant heating power independent of the useful duration.
- Wort boiling must not have a role of concentration; so the wort density have to be
correct before boiling.
- The system must be equipped with a wortstripper placed between wort
clarification and wort cooling to eliminate the unwanted volatile

1. Buffer tank 2. Formation tank 3. Decanter 4. Wortstripper

5. Condenser 6. Wortcooler

Figure 1: Process layout of the continuous wort boiling / trub separation process

The filtered wort and sparging waters are collected in a buffer tank where the
temperature is raised from 78C to 100C .
This 100C wort is then transferred continuously in a closed tank called "formation
tank" (this name refers to the reactions occuring in it and to the different compounds
resulting) .
Hop is continuously dosed on line between these two tanks.
The formation tank is equipped with an agitator and a direct steam injector.
The wort stays approximately 45 minutes there between 100C and 104C before to
overflowing to the decantor.
This decantor is a cylindro-conical tank with a wort inlet located above the cone level.
It is equipped with a static device helping the break decantation.
The hot break is continuously removed from the bottom of the decantor. This trub can
be easily recovered and sent to the brewhouse since it was never been in contact with
air nor damaged by mechanical shear forces.
The wort at 100c stays another 45 minutes in the decantor before overflowing to the
stripping column and then to the wort cooler.
Finally the total residence time of the wort in the continuous boiling - decantation
system is of about 90 minutes and is performed without any evaporation.
From the decanter outlet, the clarified wort runs continuously to the stripping column
where the unwanted volatiles are eliminated efficiently. This is done with a minimum
energy consumption and without any atmospheric dumping (vapours are condensed).

4
MATERIAL AND METHODS
The laboratory equipment for boiling and trub separation consists of:
- a 10 l opened recipient stirred with an agitator of variable speed
- a cylindric shaped decantor, thermostatically controlled and equipped with
sampling valves at different levels (h 700 mm; d 108 mm)
- a turbidimeter
- Imhoff vials of 1 l.

The pilot installation for boiling, decantation and stripping consists in:
- a buffer tank of 12 hl capacity, equipped with a bottom agitator and heated by a
double jacket and/or a direct steam injector
- a formation tank of 4 hl capacity, equipped with a bottom agitator and a direct
steam injection
- a cylindro-conical decanter of 4 hl capacity, equipped with a peristaltic pump for
trub removal
- a stripping column
- 2 transfer pumps
- a heat plate exchanger.

Figure 2: Pilot continuous boiling/trub Figure 3: Pilot wortstripping

decantation system column

Once the wort is heated at 100C in the buffer tank it runs continuously via a
volumetric pump to the formation tank, the decanter and then the stripping column at
a flowrate of 5 hl/h.
The hot trub is removed from the bottom of the decanter via a peristaltic pump and
sent in the mash before filtration.
The casting wort is recovered in the bottom part of the stripping column and
transferred at the same flowrate to the heat plate exchanger and then to fermentation.
At the top of the stripping column, vapours (rich in unwanted volatile) are condensed.

5
RESULTS AND DISCUSSIONS
Laboratory experiments
Considerations on break formation are complex since any process or technical
variations have a direct impact on wort composition and beer characteristics.
Assuming that the formation tank is designed and equipped to provide a gentle and
homogeneous heat treatment to each wort particle, the main concern is the necessary
time to achieve the desired protein coagulation.
Preliminary lab trials have been firstly carried out to study trub settling .
These tests allowed us to compare the trub decantation for different boiling times and
also to determine the residual trub that can not be removed by decantation, at least for
a fixed boiling intensity.

700
tuurbidity ( EBC )

600
30 min boiling
500
400 60 min boiling
300 120 min boiling
200
100
0
0 500 1000 1500 2000
settling time ( sec )

Figure 4: Wort turbidity related to settling time

200 1000
180 900
Wort turbidity (EBC)

160 800
Settling time ( sec )

140 700
120 600
100 500
80 400
60 300
turbidity (EBC)
40 200
20 settling time (sec) 100
0 0
30 60 120
Boiling time ( min )

Figure 5: Wort turbidity and settling time related to boiling duration

The longer the boiling time the better the final turbidity of the clarified wort. A
minimal final turbidity of 62 EBC is achieved with a boiling time of 120 min.
This value represents the residual trub that can not be eliminated by decantation,
at least for a fixed boiling intensity (5% per hour in our case). It would be
interesting to continue this study with higher evaporation rates and a stronger
mechanical agitation to observe the effect on the final wort clarity.

6
 For each boiling time, stable values of the turbidity are obtained after a settling
time of approximatly 800 sec. This corresponds to a settling velocity of 0.06
cm/sec.
Longer settling times would not improve the decanter efficiency. This first value
will be taken into account for the first pilot trials.
Optimisation of the wort flowrate at pilot scale
Assuming that the formation tank is designed and equipped to provide a gentle and
homogeneous treatment to each wort particle, the optimal functionment of the
continuous process is determined by the maximum wort velocity providing a correct
trub formation and a good clarification efficiency. A sedimentable matter content
below 5 ml/l or a turbidity below 35 EBC is usually considered as a standard.
Pilot trials have been carried out by measuring the clarified wort turbidity at different
flowrates. For these tests, a classical wort boiling was achieved to ensure a correct and
reproducible break formation and then only study break settling.
However foam concerns led us to limit the evaporation rate at 5%, corresponding to a
boiling time of 80 minutes.

100
Turbidity (EBC)

80
60
Turbidity of the clarified
40 wort ( after 8 h )
20
0
0 0,05 0,1 0,15 0,2
Wort flowrate ( hl/h)
Figure 6: Turbidity of clarified wort related to wort flowrate

10
1,4
sedimentable matters

sedimentable matters ( ml/L)

pressure ( bars )

8 pressure ( bars ) 1,2

1
( ml/L )

6
0,8
4 0,6
0,4
2
0,2
0 0
0,05 0,09 0,11 0,14
wort ascentional velocity ( cm/sec )
Figure 7: Sedimentable matters and pressure related to wort velocity

The turbidity of the clarified wort is constant in the tested flow range: 70 EBC
measured with a turbidimeter at 20C and 4 ml/l of sedimentable matters
measured with Imhoff vials.
These values are similar to what was measured after a settling time of 8h and
correlates with the residual turbidity measured at lab scale.
Better results would be obtained by improving break formation, for instance with
a higher evaporation rate.

7
 The maximum wort velocity tested (0,14 cm/sec) is not a limiting value regarding
the decanter efficiency. It is the maximum technically allowed velocity with our
pilot configuration since the pressure increases in the system with the flow.
Continuous pilot trial
Once the operating conditions determined, assuming a correct hot break formation
and an efficient trub separation, some pilot tests have been realised to validate the
complete system.
The table below gives an overview of some wort analyses of continuous pilot trials
carried out at a flowrate of 9 l/min (0,05 cm/sec ).

Inlet of the boiling Outlet of the

system stripping column

Extract (P) 16.4 15.4

Colour (EBC) 12.4 23.5
PH 5.4 5.2

Sugars (g/100 ml)

- Sucrose 0.25 0.35
- glucose 1.19 1.36
- saccharose 0.07 0.04
- maltose 4.38 4.86
- maltotriose 1.05 1.41
- Dp4 0.16 0.12
Nitrogen compounds
- soluble nitrogen matters (g/l) 9 8.9
- free amino nitrogen (mg/l) 237 207

Lipids (mg/l)
- palmitic acid 14.87 3.19
- stearic acid 0.91 0.55
- oleic acid 3.39 0.8
- linoleic acid 19.85 0.39
- linolenic acid 3.43 0.93

Table 1: Wort analyses before and after the continuous process (test at 0,05 cm/sec)

The wort gravity decreases of 3%, due to the dilution by steam injection in the
formation tank and in the stripping column. This value should be reduced in an
industrial insulated system since the only target of the steam is to keep the wort at
100C and drive off the volatile in the stripping column.
Wort coloration increasing and pH lowering are comparable to the values
observed with a classical wort boiling system.
There is no significant modification of the sugars and nitrogen compounds.
Lipids are strongly reduced due to their removing with the trub.
The trub, measured with Imhoff vials, is 2.4 ml/l of sedimentable matters.

Figure 8 shows the reduction of the DMS level in the wortstripping column, for 2 tests
realized at different global evaporation rates (4,5% for brew 1 and 3% for brew 2).

8
 250
200

DMS ( ppb )
150
100
50
0
Brew 1 Brew 2

DMS content before wortstripping

DMS content after wortstripping
Figure 8: DMS content before and after stripping for brews realized in
different operating conditions

Worstripping efficiency is 93,5% in the first test and 88,3% in the second one.
DMS contents are low and similar to what would be obtained with a classical process
equipped with a wortstripping column.

INDUSTRIAL EXTRAPOLATION
The table below gives a comparison of the necessary boiling equipment for a classical
system equipped with a wortstripping column and the continuous boiling / decantation
process. The calculation is done considering a brewhouse equipped with a 2001 filter
of 10.5 tons throw and a wort flow of 300 hl/h.

Equipment Continuous system Batch system

(with a stripping column)
Buffer tanks 2 x 600 hl 1 x 650 hl
Formation tank 150 hl 840 hl
Decanter 80 hl 650 hl
Stripping column 300 hl/h 600 hl/h
Wort cooler 300 hl/h 600 hl/h

Table 2: Comparison between equipment needed for the continuous process and a
classical system (with a wortstripper)

The advantage of the continuous process is clearly an important reduction of the

equipment sizes. As in the classical process with a wortstripping column, energetic
consumption is significantly reduced, with the additional benefit to decrease high
steam consumption periods.

CONCLUSIONS
First labscale experiments allowed us to determine the range of settling times related
to the boiling duration and the residual trub that can not be removed by decantation in
fixed conditions.
Pilot tests have demonstrated the feasibility of the continuous wort boiling and trub
separation process. The efficiency of the continuous cylindro-conical decanter has
been confirmed. Next step of our study will aim at optimising proteins coagulation
without strong boiling.

9
Compared to a classical boiling process equipped with a wortstripping column, the
continuous system allows to improve the equipment productivity (5 to 7 times), and to
avoid periods of high energetic consumption.

REFERENCES
1. Narzi, L., Miedaner, H., Schneider, F., Brauwelt International, 1992, IV, 346-
355.
2. Reed, R.J.R. and Jordan, G., Proceedings of the European Brewery Convention
Congress, Lisbon, 1991, 673-680.
3. Seldeslacht , D., Van den Eynde, E., Degelin, L., Proceedings of the European
Brewery Convention Congress, Maastricht, 1997, 323-332.
4. Van Haecht, J-L., De Brackeleire, C., Dufour, J-P. and Devreux, A., European
Brewery Convention Symposium on separation processes, Leuven, 1990, 96-111.
5. Weinzierl, M. et al., Brauwelt International, 2000, I, 40-42

10
25

Non-Newtonian behaviour of beer in

laminar flow through porous media a
possible explanation for a sudden
increase of pressure difference during
beer filtration
Martyn A. Drost1 & Erich J. Windhab2
1
Feldschlsschen-Getrnkegruppe, Brauerei Feldschlsschen, CH-4310 Rheinfelden,
Switzerland (www.feldschloesschen.com, www.carlsberg.com, e-mail: [email protected])
2
Institut fr Lebensmittelwissenschaft der Eidgenssischen Technischen Hochschule (ETH)
Zrich, Laboratorium fr Lebensmittelverfahrenstechnik, CH-8092 Zrich, Switzerland

Descriptors
Beer filtration, filterability, flow measurement, viscosity

SUMMARY
Although beer is being considered as a Newtonian fluid, it can be shown that mechanical
stress in the laminar flow through porous media1 can evoke a distinct non-Newtonian flow
behaviour. This phenomenon is coupled with partly irreversible changes of the colloidal
structure of beer. The flow through fine pores can thereby cause elongations of colloidal beer
compounds. The results of this study lead to the conclusion that filterability of beer is
dependent not only on shear viscosity but strongly on elongational viscosity.
1
Packed beds of glass spheres were used to simulate the porous structure of kieselguhr.

Nicht-Newtonsches Fliessverhalten von Bier beim laminaren Strmen durch porse

Medien eineplausible Erklrung fr einen pltzlichen Anstieg des Durchstrmungs-
widerstandes im Kieselgurfilter

Deskriptoren
Bierfiltration, Durchflumessung, Filtrierbarkeit, Viskositt

ZUSAMMENFASSUNG
Bier wird in der Regel als newtonsches Fluid bezeichnet. Es kann jedoch gezeigt werden, dass
Bier, ausgelst durch mechanische Belastungen beim laminaren Strmen durch porsen
Medien1, ein ausgeprgtes nicht-Newtonsches Fliessverhalten aufweisen kann. Dieses
Phnomen geht einher mit teils irreversiblen Vernderungen der kolloidalen Struktur von
Bier. Die in entsprechenden Poren auftretenden Dehnkrfte knnen zur Auffaltung von
knuelfrmigen Makromoleklstrukturen fhren. Daraus lsst sich schliessen, dass die Bier-
Filtrierbarkeit neben der Scherviskositt auch wesentlich von der Dehnviskositt des Bieres
abhngt.
1
Feinkrnige Glaskugelpackungen wurden verwendet, um die porse Struktur von Kieselgur
zu simulieren.

Comportement non-newtonien de la bire sous flux laminaire travers des milieux

poreux explication possible de l'augmentation soudaine de la diffrence de pression
pendant la filtration de la bire

Descripteurs
Filtrabilit, filtration de la bire, mesure du dbit, viscosit

RESUME
Bien que la bire soit considre comme un liquide newtonien, il est possible de montrer que
les contraintes mcaniques sous flux laminaire travers des milieux poreux1 peuvent
dclencher un comportement de flux clairement non newtonien. Ce phnomne est coupl
des changements partiellement irrversibles de la structure collodale de la bire.
L'coulement au travers de pores trs fins peut donc entraner une longation des composs de
la bire collodale. Cette tude permet de conclure que la filtrabilit de la bire dpend non
seulement de la viscosit de cisaillement, mais aussi fortement de la viscosit d'longation.
1
Des lits tasss de billes de verre ont t utiliss pour simuler la structure poreuse de la terre
diatomes.

2
INTRODUCTION
Although beer is usually considered as being a classical Newtonian fluid, it can be
shown that mechanical stress in the flow through porous media can evoce a distinct
non-Newtonian flow behaviour. (This statement implies a complete novelty in the
history of beer rheology!). In the flow through porous media the colloids in beer are
subject to elongational stress. Above a certain volume flow rate this elongational
stress can cause a drastic increase of flow resistance.
The filtration of beer with low filterability is often accompanied by a sudden increase
of the pressure difference inside the filtration process unit. The mechanisms which
lead to a low filterability are not fully understood. In the thesis of Drost (1999) it
could be shown, that the elongation of beer compounds during the flow through the
pores of a filter-like porous media can lead to a distinctive increase of the pressure
difference and to a likewise distinctive change of the colloidal structure of beer (see
picture 1).

colloidal
structure of
beer
ion
at
rel

aff
al

ec
on

ts
cti
fun

mechanical
flow
stress in
resistance in
porous
filters
affects media

Picture 1 : Functional relations between elongational stress during flow through

porous media, the pressure difference and changes of the colloidal
structure of beer.

EXPERIMENTAL
This pressure increase phenomenon was found through experiments with filtered beer
flowing laminarily through the pores of different packed beds of super-fine glass
spheres (see picture 2), so as to simulate the flow of beer through a packed bed of
kieselguhr as it is used for industrial beer filtration. Three different sphere diameters
were used: 400 m, 730 m and 1000 m.

3
 120 mm

25 mm
beer beer

Picture 2: Cylindrical packed bed of super-fine glass spheres (simplified

drawing).

During these experiments the pressure difference p appeared to be a non-linear

function of the volume flow rate dV/dt. To describe the flow through the packed beds
of spheres the dimension-free flow resistance number and the dimension-free
Reynolds number Re were used:

p d p 3 v dp
= Re = [1]
v L (1 ) 2 (1 )

p stands for pressure difference over the packed bed, dp for the glass sphere
diameter, for porosity, for the shear viscosity of beer, L for the length of the
packed bed and v for the flow velocity (before and after the packed bed of glass
spheres).

RESULTS 1

100'000

Ergun : = 185 + 1.75 Re

10'000 Wasser dp = 435 m

Wasser dp = 730 m

Wasser dp = 1000 m
1'000

100
0.01 0.1 1 10 100 1000
Re [-]

Picture 3: -Re-relation for the flow of water through packed beds with different
glass spheres diameters.

For a classical Newtonian fluid like water the function (Re) is independent of shear
viscosity and of the diameters dp of the glass spheres and always obeys to the

4
empirical equation [3] of Ergun (Durst 1987) as shown in picture 3:

= 182 + 1.75 Re [2]

In the case of beer flowing through the packed bed of spheres the -Re-relation is
very different from the one for Newtonian fluids (as seen in picture 3) and
furthermore it is dependent on the glass sphere diameters dp. Picture 4 shows a
distinctive increase of over a certain range of Re for different sphere diameters dp
which implies the mentioned non-Newtonian behaviour of beer.

100'000 Ergun Bier; dp = 435 m

Bier; dp = 730 m Bier; dp = 1000 m

10'000
I II III IV

1'000

100
0.01 0.1 1 10 100 1000
Re [-]

Picture 4: -Re-relation for the flow of beer through packed beds with different
glass sphere diameters dp.

DISCUSSION 1
The increase of (respectively the pressure difference p) has its origin in the
stretching of coil molecules within the pores of the packed beds. This is a cause of
elongational flows which are present in the narrowing entrances of each of the
numberous pores (Debus, 1997). Picture 5 shows the difference of between shear flow
and elongational flow.

y y
vx
vx
x x

Picture 5: Velocity fields of shear flow (left) and of elongational flow (right).

It is known that coil molecules in elongational flow can be stretched and even be
unraveled under certain conditions, whereas this is not likely to ever happen in shear

5
flow. Picture 6 shows a model for the unraveling and stretching of a coil molecule in
elongational flow according to Pahl et al. (1991).

l0

a)

b)

Picture 6: Model for the deformation of coil molecules in elongational flow:

a ) coil at rest b) elongated coil.

The increase of represents a loss of energy resulting from intramolecular friction

between adjacent molecule chain segments during a deformation. This is called a
viscous deformation. From a rheological point of view, furthermore, the increase of
due to elongational flow can be interpreted as an increase of the effective elongational
viscosity D of beer.

RESULTS 2
The tendency of the molecules to aggregate during the flow through the packed beds
of glass spheres was described with EBC-haze measurements as shown in picture 7.

100000 0.6

of beer
of water
10000
EBC haze

EBC-haze
[]

0.5

1000

100 0.4
0.01 0.1 1 10 100 1000 10000
Re [-]

Picture 7: The flow resistance number during the flow and the resulting EBC-
haze value of the beer while coming out of the bed of glass spheres
(dp = 435 m) as a function of Re.

Picture 7 shows (Re) and the EBC-haze value that resulted as a cause of the

6
unraveling of some coil molecules. Re, again, represents the flow velocity of beer
inside the packed bed.

DISCUSSION 2
An unraveled coil molecule in beer is very likely to aggregate with other (unraveled
or not unraveled) coil molecules in its close neighbourhood, because in the
unraveled state the inner parts of a coil molecule are exposed and no longer
protected from interacting with other molecules. Besides the unraveled molecule
has a bigger extension than a randomly coiled molecule and is therefore more likely to
overlap with other molecules.
Especially the parallel course of and the resulting EBC-haze value in picture 7 in
the areas I to III (see also picture 4), gave way to the presumption, that the increase of
the flow resistance is coupled to the deformation of colloidal beer compounds.
Within the area IV the increase of the EBC-haze value is caused by some turbulent
flow zones within the bed of glass spheres.
Remember: All of the shown experiments were done with filtered beer! This is a clear
sign that the very small, non-filterable particles (< 500 nm) can strongly influence the
flow of beer through porous media and probably through kieselguhr as well.

CONCLUSION
The results of this study lead to the conclusion that filterability of beer is not only
dependent on shear viscosity but also decisively on elongational viscosity.
Furthermore, it can be shown that flow resistance in porous media can be coupled to
the deformation of colloidal beer compounds. This offers a promising perspective for
a better understanding of beer filtration processes in the future.

REFERENCES
1. Debus, K. (1997): Numerische Untersuchung der Kugelhaufendurchstrmung
Anstze zur Berechnung strmungsbedingter Deformation verformbarer Krper,
Diss. TU Mnchen, Fakultt Brauwesen, Lebensmitteltechnologie und
Milchwissenschaften.
2. Drost. M. (1999): Methoden zur Untersuchung der Auswirkungen mechanischer
Belastung auf kolloidale Struktur, Rheologie und Filtrationsverhalten von Bier,
Diss. ETH Nr. 13218, Zrich.
3. Durst, F., Haas, R., Interthal, W. (1987): The nature of flows through porous
media. J. Non-Newtonian Fluid. Mech. 22, 169-189.
4. Pahl, M., Gleissle, W., Laun, H.-M. (1991): Praktische Rheologie der Kunststoffe
und Elastomere (Ed.: Kunststofftechnik,VDI-Gesellschaft) Dsseldorf, VDI-
Verlag, 327-329.

ACKNOWLEDGEMENTS
This research project was kindly supported by the Foundation for Scientific Research
of the Swiss Brewers Association (SBV) and the Komission fr Technologie und
Innovation (KTI) des Departementes fr Volkswirtschaft.

7
26

Special filteraids for the optimisation

of the kieselguhr filtration and for
the reduction of the pollution
J. Cardelli1, M. Crestini2 & T. Zangrando3
1
Spindal Europe Nord AB, Brodasvagen 1, Hus B, S-43338 Partille, Sweden
2
AEB SpA, Brescia, Italy
3
Consultant, Via Trento 79, I-32034 Pedavena, Italy (e-mail: [email protected])

Descriptors
Beer filtration, environmental protection, filter aid, kieselguhr filter

SUMMARY
The filtration is the last step to characterise the quality of the beer. Usually the filtration is
controlled by varying composition and dosage of kieselguhr. Better qualitative and
quantitative-economical results have however been obtained utilising for the precoat various
types of special filteraids: longer filter runs, larger filtration volumes, more filtrations/year,
reduced kieselguhr and energy consumption, reduced beer losses, much lower germ counts,
brighter finished beer. The poster will discuss the advantages and show some of the important
results obtained with different filters and types of beer, with special regard to the reduction of
effluent volumes and filtration costs.

Spezielle Filterhilfsmittel zur Optimierung der Kieselgurfiltration und zur Herab-

setzung des Abwasseranfalls

Deskriptoren
Bierfiltration, Filterhilfsmittel, Kieselgurfilter, Umweltschutz

ZUSAMMENFASSUNG
Die Filtration ist der letzte Schritt um die Qualitaet des Bieres zu charakterisieren.
Gewhnlich wird sie durch die Auswahl der Zusammensetzung und der Menge der Kieselgur
geregelt. Bessere qualitative und quantitativ-wirtschaftliche Ergebnisse sind jedoch durch die
Bildung der Voranschwemmung mit verschiedenen speziellen Filterhilfsmitteln erzielt
worden. Durch diese Optimierung sind u.a. folgende Ergebnisse erzielt worden: Verlngerung
der Filterstandzeit, Erhhung des Durchsatzes und der Anzahl der jhrlich mglichen
Filtrationen, Herabsetzung des Schwandes, bedeutende Verminderung des Kieselgur- und
Energieverbrauchs, beachtenswerte Verbesserung des mikrobiologischen Zustandes des
Filtrates, Erhhung der Glanzfeinheit des Bieres. Der Poster beschreibt die Vorteile und zeigt
einige der wichtigen mit verschiedenen Filtertypen und Biersorten erzielten Ergebnisse, mit
besonderer Bercksichtigung der Herabsetzung der Abwassermenge und der Filtrations-
kosten.
Filtres spciaux pour loptimisation de la filtration sur kieselgur et la reduction de la
pollution

Descripteurs
Adjuvant de filtration, filtration de la bire, filtre kieselgur, protection de lenvironnement

RESUME
La filtration est la dernire tape pour caractriser la qualit de la bire. Gnralement elle est
controle en variant la composition et la quantit de kieselgur. Les meilleurs rsultats
qualitatifs, quantitatifs et economiques ont t obtenus en utilisant une pr-couche compose
de differents types de coadjuvants de filtration spciaux. Cette optimisation a permis dobtenir
des cycles de filtration plus longs, des quantits de bire filtres plus importantes, plus de
filtrations dans lanne, des freintes rduites, une diminution importante de la consommation
de kieselgur et dnergie, une rduction significative de la population microbienne travers
de filtre et une bire plus brillante. Le poster rapporte les avantages et montre quelques
rsultats importants obtenus en utilisant des filtres et des bires diffrentes, avec une attention
particulire pour la rduction du volume des effluents et du cot de filtration.

2
INTRODUCTION
The filtration is the latest step that characterises the quality of the beer. Its
optimisation is one of the most important goals of every brewmaster. The objectives
of the filtration are both quantitative and qualitative.

Quantitative objectives
1. Long filtration cycles (hl/cycle)
2. High number of cycles per year
3. Low kieselguhr consumption
4. Low cold and hot water consumption
5. Low beer losses
6. Low energy consumption

Qualitative objectives
1. Low cell count in the bright beer
2. Haze within specifications (usually < 0,5 EBC)

In many breweries, in order to attain the above objectives, different technological

measures are taken (as e.g. centrifugation or enzymatic treatment) in order to reduce
the turbidity of the unfiltered beer and it's yeast content. Often negative side-results
are obtained in this way: e.g. a too low yeast count means more filtration problems
due to the fact that the mechanical separation effect is decreased. Hence the
kieselguhr dosing is increased, which means higher consumption of filter aid and
early exhaustion of the filter.

The correct building of the precoat is of definitive importance during the entire
filtration. Usually two different precoats are carried out. The first one, with 500-1000
g/square meter coarse kieselguhr, has a permeability of about 1 darcy. Its main task is
to produce an elastic support structure preventing the filter aid from entering into the
filter elements. On the first precoat, a second one (500-800 g/square meter) is applied:
its permeability is usually between 0,09 and 0,2 darcy. The filter aid mixture used for
the second precoat, is the same as that of the body feed and the clarifying effect relies
on it.

The exclusive use of kieselguhr for the formation of the first precoat building has
several disadvantages. The most important one is the fact that the kieselguhr particles
form "unstable bridges", bound to break down at any slightest change of pressure,
allowing haze particles to pass trough the filter.

For this reason we have developed and successfully applied in many breweries all
around the world complex precoat filter aids which have allowed us to overcome the
above mentioned problems.

CHARACTERISTICS OF THE PRECOAT FILTER AIDS AND THEIR

FUNCTION
The special precoat filter aids are composed of fibres (celluloses and cotton),
kieselguhr and perlites of optimum particle size, distribution and permeability. Their
different components exhibit various important actions:

3
*cotton (figure1): the length
and the consistency of the 1
fibres give to the compound a
structure which is highly
resistant to pressure
hammers and also acts as a
support;

*celluloses (figures 2-3): they

confer to the structure a high
adsorbing power (resulting
from the fibres being
charged both positively and 2 3
negatively). Furthermore,
through the nature of the
fibres, their consistency,
structure and composition,
they give elasticity to the
coat;

*kieselguhr (figure 4) and

perlites (figure 5): due to the 4
4 5
nature and consistency given
to the precoat, they enable to
carry out a strong in depth
action.

The new complex filter aid has the following main features:
1. it is perfectly homogeneous;
2. it has a constant alveolus;
3. it detaches easily from the filter screen at the end of the filtration;
4. it resists well to pressure hammers because it is elastic;
5. due to its adsorbing power it withholds very fine haze particles with different
electric charges;
6. it is completely insoluble in beer, and hence it complies with the "German purity
law";
7. it can be formulated with different permeabilities, to meet the requirements of
different kinds of beer.

RESULTS
The results shown here have been obtained in a number of breweries with outputs
between 180.000 and 11.000.000 hectoliters per year. Everywhere we have
substituted the kieselguhr precoat with our compounded precoat, at a rate of 500-1000
grams per square meter.

In order to better meet the requirements of different brands, in some breweries we

have used the type "F10"- precoat (permeability 185-215 l/square meter/min), in
others the type "F20"- precoat (permeability 135-165 l/square meter/min).

4
Differential pressure

The maximum differential pressure is reached in the traditional filtration much earlier
than when using the compounded precoat.

This is due to the fact that using our compounded precoat it is possible to optimise the
composition of the kieselguhr used for the body feed, further increasing the brightness
of the beer.

Haze
The compounded precoat is
able to resist much better to
variations of the pressure at
the filter inlet, without any
increase of the haze value.

Cell count
The positive effect of the
compounded precoat is
particularly evident at the
beginning of filtration.

Filter performance
The use of the compounded precoat increases the length of the filter run and allows
considerable savings of kieselguhr, as shown by the following figures (average data of
one year in two different breweries).

5
POLLUTION
It is obvious that longer filter runs result in a reduced number of filtrations needed to
filter in a given period of time the same volume of beer.

In average the number of filtrations is reduced by approx. 30%.

This means a saving of beer losses, of cold and hot water consumption, of detergents,
of labour and of costs for the disposal of spent kieselguhr and for the treatment of
waste water.

Together with the reduction of the kieselguhr consumption, all the above elements
allow a reduction of the filtration costs which we have found to be between 0,02 and
0,06 Euro per hectoliter in all the breweries using our compounded filter aid.

CONCLUSIONS
The precoat is an essential factor for a correct and economic filtration of the beer.
Unfortunately it is often neglected both by the brewmasters and by the manufacturers
of filtration equipment.
A compounded precoat has been developed which allows to optimise the filtration in
respect of the quality of the filtered beer and of the performance of the filter. Another
important advantage derived from the use of our compounded precoat is the
considerable reduction of pollution and of costs.

6
27

Use of RVF technology to achieve rough

beer clarafication and cold sterilisation
of beer
L. Fillaudeau1, S. Ermolaev1,2, N. Jitariouk2 & A. Gourdon2
1
Institut National de la Recherche Agronomique (INRA), Laboratoire de Gnie des Procds
et Technologie Alimentaire (LGPTA), 369, rue Jules Guesde, B.P. 39, F-59651 Villeneuve
dAscq Cedex, France (e-mail: [email protected])
2
ProFiltra SARL, 78, rue dAguesseau, F-92100 Boulogne-Billancourt, France

Descriptors
Beer filtration, beer quality, sterile filtration, yield

SUMMARY
Over the last two years, RVF technology (Rotating and Vibrating Filtration) has been studied
for rough beer clarification and cold sterilisation of beer. RVF laboratory module is divided
into parallel cells including flat disc membrane fixed onto porous substrate which drain the
permeate and impeller shaped rotating bodies attached by a central shaft. The membranes
used were metalo-ceramic flat discs with mean pore diameters in microfiltration. The results
show the quantitative (flux, hydraulic resistance, retention) and qualitative (pH, haze, colour,
bitterness, polyphenol, protein, dry matter and yeast concentration) performances. The
conclusions demonstrate an interesting potential for rough beer clarification and cold
sterilisation of beer

RVT-Technologie zur Bierfiltration und Kaltsterilisation

Deskriptoren
Ausbeute, Bierfiltration, Bierqualitt, Sterielfiltration

ZUSAMMENFASSUNG
In den letzten beiden Jahren wurde die RVT-Technologie (Rotating and Vibrating Filtration)
zur Bierfiltration und Kaltsterilisation von Bier untersucht. Das RVT-Labormodul ist in
parallele Zellen unterteilt. Die flachen Scheibenmembranen, die an durchlssiges Material
fixiert sind, sorgen fr den Ablauf des Permeats. Der wie ein Laufrad geformte rotierende
Krper ist an einer Welle befestigt. Die verwendeten Membranen bestehen aus flachen,
metallkeramischen Scheiben; der durchschnittliche Porendurchmesser ermglicht eine
Mikrofiltration. Die Ergebnisse zeigen die quantitative (Durchfluss, hydraulischer
Widerstand, Retention) und qualitative (pH, Trbung, Farbe, Bitterkeit, Polyphenole,
Proteine, Trockensubstanz und Hefekonzentration) Leistung. Die Ergebnisse zeigen das
Potenzial der RVT-Technologie fr die Bierfiltration und die Kaltsterilisation von Bier.
Utilisation de la technologie RVF pour obtenir la clarification de la bire brute et la
strilisation froid de la bire

Descripteurs
Filtration de la bire, filtration strile, qualit de la bire, rendement

RESUME
Au cours des deux dernires annes, la technologie RVF (Rotating and Vibrating Filtration) a
t tudie pour clarifier la bire de garde et striliser froid la bire. Le module RVF se
compose de deux cellules parallles comprenant des membranes planes fixes sur un support
poreux permettant de collecter le permat, et dun systme dagitation fix sur un axe central.
Les membranes utilises taient des disques mtallo-cramique avec des pores de diamtre
moyen en microfiltration. Les rsultats prsentent les performances quantitative (flux,
rtention) et qualitative (pH, trouble, couleur, amertume, polyphnol, protine, matire sche
et concentration en levure). Les conclusions dmontrent la potentialit de cette technologie
pour clarifier et striliser la bire.

2
INTRODUCTION
Membrane separation technology has become widely used in the food processing
industry [5]. Important developments have taken place in the dairy industry, in
drinkable water production as well as in wastewater treatment. In the cross-flow
microfiltration (CFMF) of fermented food products (beer, wine), the fouling
mechanisms and local phenomenology associated with fouling are largely unknown
and still unidentified. In consequence, industrial applications of CFMF encounter two
main problems: (i) the control of fouling mechanisms and (ii) the enhancement of
permeate quality.
In the brewing industry, clarification and pasteurisation are among the most important
operations. At the final step of beer processing, beer clarification (i.e. elimination of
yeast cells, permanent and chill hazes after beer maturation) is currently obtained by
conventional dead-end filtration with filter-press using filter-aids (Kieselguhr).
Pasteurisation is then necessary to ensure microbiological stability of the final
product. Brewers are very concerned that the finishing techniques they use are the
best, in terms of product quality and cost effectiveness. Considerations of the brewing
process indicate two areas where MF might play useful roles [10]: (i) loss reduction
in the brewing process, and (ii) as a technological alternative to the conventional
solid-liquid separations. The aim of this work is closely related to the clarification of
rough beer and the pasteurisation of clarified beer, hence a rapid description of these
operations and their objectives.

Clarification of rough beer

Beer clarification is probably one of the most important operations: rough beer is
filtered in order to eliminate yeast and colloidal particles responsible for haze (chill
and permanent hazes); in addition, this operation should also ensure the biological
stability of the beer. This operation should comply with the haze specification of a
lager beer in order to produce a clear bright beer (European Brewery Convention
norm). Standard filtration consists in the retention of solid particles (yeast cells,
macro-colloids, suspended matter) and solutes responsible for haze. The variety of
compounds (chemical diversity, large size range) to be retained makes this operation
one of the most difficult to control. The use of MF is to provide an alternative to
conventional dead-end filtration with filter-aids such as diatomaceous earth. However
this operation must satisfy the same economic and qualitative criteria [16, 19]. MF
should be able: (i) to produce a clear and bright beer with similar quality to a
Kieselguhr filtered beer, (ii) to perform separation in a single-step without additives,
(iii) to operate at low temperature (0C) and (iv) to achieve economic flux.

Cold sterilisation of clarified beer

Clarification is usually followed by pasteurisation (with a plate heat exchanger).
Pasteurisation is necessary to ensure the microbiological stability of the final product.
A stability of 3 to 6 months can be ensured when the retention of beer spoilage
organisms (bacteria, yeast) is obtained. The level of one yeast cell per millilitre
represents an admissible standard with common filtration techniques [1]. Currently,
heat treatment is mainly performed by flash pasteurisation before conditioning or
tunnel-pasteurisation of bottles. Sterile filtration (by CFMF) appears interesting to
replace pasteurisation by heat treatment and allows the elimination of the organoleptic
problems induced by thermal processing [13, 14]. CFMF is examined in order to (i)
produce a microbial free beer without deterioration in beer quality by operating at low
temperature (close to 0C), (ii) ensure beer stability (biological, colloidal, colour,

3
aroma and flavour, foam stability), (iii) achieve economical flux and (iv) indicate the
viability of MF as a commercial alternative to pasteurisation and dead-end filtration
with cartridges. Cold-sterile filtered beer (draught beer or bottled beer) corresponds to
a strong demand from consumers for quality and natural products. The objective of
eliminating thermal treatment of the finished product is achieved with membrane
cartridge systems (dead-end filtration) installed directly upstream of the filling system
[8, 9]. However, cold-sterilisation by cross-flow membrane is under study and appears
feasible [18].

For both these operations, the choice of filtration process (dead-end, cross-flow or
dynamic filtration) and membranes (chemical nature, mean pore diameter) are
essential. Over the last two years, a new filtration process call RVF technology
(Rotating and Vibrating Filtration patent nFR-97-14825) has been studied with
microfiltration flat disc membranes. Clarification of rough beer and cold-sterilisation
of clarified beer were performed at pilot scale under severe and well-controled
operating conditions (temperature close to 0C, product under CO2 pressure) in order
to satisfy product quality and industrial process requirements. The results show the
quantitative (flux, hydraulic resistance, retention) and qualitative (pH, haze, colour,
bitterness, polyphenol, protein, dry matter and yeast concentration) performances of
the process. Most of these analyses correspond to EBC methods which are widely
used in the brewing industry. Analytical profiles of conventional dead-end filtration
(brewery reference) and permeate (RVF technology) were compared. The
experimental results provided invaluable indications about permeate quality,
membrane selection and the most effective operating conditions through a
hydrodynamic study of the technology. The main conclusions highlight that RVF
technology may be a satisfactory method of rough beer clarification and cold-
sterilisation of beer for accurate membrane operating condition choices.

EXPERIMENTS
Pilot plant
Experiments were carried out using a pilot plant composed of two major parts: the
feed (retentate) and the permeate loops. The retentate loop and feed tank had
respectively a tubular heat exchanger and a double-jacketed vessel including
temperature regulation (1C 1C). The installation was kept under CO2 pressure
(under 150 kPa) in order to maintain identical conditions to industrial process. The
pressure could reach 300 kPa inside the apparatus and the retentate flow rate 3.0m3.h-1
in maximum values. Measurements of data were made with the following sensors:
flow-meters, temperature gauges, relative and differential pressure sensors as shown
in figure 1. After calibration, the precision of measurements was better than 1%.

Filtration module and membranes

RVF laboratory module (filtration area 0.048 m2) is divided into parallel cells including
flat disc membrane fixed onto porous substrate which drain the permeate, and impeller shaped
rotating bodies attached to a central shaft. This simple mechanical device runs
continuously and maintains a high shear rate as well as a hydrodynamic perturbation
at the membrane surface. The impeller is made of three blades (ext = 142 mm,
thickness = 8 mm) included in a horizontal plane between two membranes. The gap
between the blade and the membrane is small and equal to 3 mm. Transmembrane

4
pressure (up to 300 kPa) and rotation frequency (up to 50 Hz = 50rd/sec) can be
adjusted to reach the optimal conditions for cross-flow filtration. The two separated
cells makes it possible to work with two different membranes in similar conditions.
TP
T PP

PERMEATE MEMBRANE
TM2 TM1

TER
T
QP1 QP2

F
QPR
F
F
IMPELLER
PRE

Motor

TSR
DP T

QBR, QP1 and QP2 : flow-meter

TER, TSR, TP : Temperature gauge
PRE and PP : Relative pressure sensor
DP, TM1 and TM2 : Differential pressure sensor RETENTATE
F : Tachometer

Figure 1: Schematic flow diagram of the RVF technology and instrumentation.

We worked with stainless membranes (coarse membranes) with a ceramic selective

layer (TiO2, ZrO2 or SiO2). The membranes used were flat discs with an external
diameter of 142 mm and a thickness of 0.25 mm. Filtration area per membrane is
equal to 0.012 m2 and forms a crown shape with an internal diameter of 62.25 mm
and an external diameter of 135.5 mm (including corrections due to gasket). In this
work, a large range of pore diameters (0.15 m, 0,20 m, 0.40 m, 0.60 m, 1.10 m
and 1.50 m) used in microfiltration were investigated.

Experimental protocol and measurements

All filtrations were carried out at between 0C and 2C to ensure that the beer was
representative of that being transferred from cold conditioning to the filter line. A
transmembrane pressure of between 20 and 200 kPa and an impeller rotational speed
between 10 and 50 Hz were investigated during different trials. During the
experiment, the permeate was recycled through the feed tank to maintain constant
inlet concentration. In this work, three trials were dedicated to the cold-sterilization of
clarified beer (MFT n1, 2 and 3) and four trials to the clarification of rough beer
(MFT n4, 5, 6 and 7). The experiments were made up of two parts. The first part was
classic cross-flow microfiltration under constant operating conditions, lasting 3 to 4
hours in order to reach the quasi steady state flux. In the second part, we investigated
other conditions (pressure, velocity). In each case we waited for the quasi steady-state
flux whatever the time required (5 to 40 min).

Fluids and analyses

Experimental fluids
A local brewery (Terken GBM, Roubaix, France) supplied concentrated rough and
clarified beers (Lager beer type, equivalent to 15Plato) and a new beer was used for

5
each experiment. Rough beer (RB) was taken from the maturation tank and clarified
beer (CB) came from the downstream side of a Kieselguhr sheet filter. Specific
attention should be paid to experimental fluids. Rough and clarified beers are real
products; consequently a large and inevitable variability of product quality is
noticeable (from brand to brand, from batch to batch and even within a tank due to
stratification) [2, 11, 12, 17]. Table 1 shows the average analysis of the different beers
used for the microfiltration runs. The beer composition variability is evident but
variations in dry matter, protein and polyphenol concentrations between rough beers
and clarified beers are no greater than the differences observed between the rough
beers. Thus, some trends can be drawn from the comparison of all the filtration run
results, even if the effect of the composition of beers [4] used for different runs can
not be eliminated.
Rough beer Clarified beer
Mean Mean
Protein (Bradford) [mg/l] 270 20 215 30
Protein (Lowry) [g/l] 6.5 0.4 6.0 0.4
Dry matter [%] 5.0 0.2 4.9 0.1
Colour [EBC] / / 10.5 0.2
Carbohydrate [g/l] 40.1 3.5 40.0 4.4
Polyphenol [mg/l] 219 21 202 17
Bitterness [EBC] 24.3 3.1 22.2 1.3
Haze [EBC] 17.8 3.7 0.44 0.04
PH [/] 4.4 0.3 4.2 0.1
Yeast cells [cell/ml] 1.0E+6 5.1E+5 / /
Table 1: Mean composition of rough and clarified beers used for filtration runs: mean values
and standard deviation ().

Physical and chemical analyses

The physical properties (density and viscosity) versus temperature have been
characterised. Several analyses were carried out to characterise rough and clarified
beers before each filtration according to the European Brewery Convention methods.
Analyses were achieved by collecting permeate and retentate samples during
experiments. Most of these analyses corresponded to EBC methods which are widely
used in the brewing industry (pH, dry matter, haze, polyphenols). Proteins and their
derivatives affect much of the brewing process and the quality of beer. As a
consequence samples were analysed using two colorimetric methods: the Bradford
and Lowry methods. Bradfords assay is based upon the binding of Coomassie
Brillant Blue and has been reported to be optimal for beer protein analysis. This
method enables the variation in concentration of high molecular weight nitrogen
compounds (MW>5000) to be followed and displays minimal interference. The
Lowry method is based on the Biuret reaction and this method may be considered as
being complementary to the Bradford one, in that it is sensitive to nitrogen-containing
compounds of lower molecular weight.

RESULTS & DISCUSSIONS

Hydrodynamic study of the RVF module
Dynamic or high-shear cross-flow filtration consists in creating a relative motion
between the membrane and the impeller in order to produce high shear rates at
membrane surface. The main advantage of this system is that it allows operation at a

6
low transmembrane pressure and high shear rates, a combination that limits the
growth and the compression of a cake on the membrane.
One of the widely used theory for modelling flux in pressure independent, mass-
transfer-controlled systems is the film theory. Dimensional analysis for mass transfer
(analogy to heat transfer) lead to establish a general correlation between the Sherwood
(Sh), Schmidt (Sc) and Reynolds (Re) numbers. It makes possible an evaluation of the
mass-transfer coefficient, K and provides insight into how membrane geometry and
fluid flow conditions can be specified to optimise flux. In laminar flow condition,
Lvque's solution may be used to evaluate the mass-transfer coefficient where the
laminar-parabolic velocity profile is assumed. It shows that flux (mass transfer
coefficient) may be increase by increasing the velocity or by decreasing the cross-
section. In more general terms, any fluid management technique which increases
shear rate at the membrane surface will increase flux.
However, RVF technology exhibits a complex hydrodynamic system and the
modelling of shear-rate appears difficult. The lack of an accurate model to describe
this module drives us to make the following comparison: RVF technology may be
defined as (i) the flow of a Newtonian liquid in a duct with a complex shape [7] and
as (ii) a stirred vessel with a continuous feed and extract [6]. In the first assumption,
we do not have the criteria necessary to choose the characteristic geometrical
dimension (dh, e, w, L) and to define the impact of rotational velocity of the impeller.
In the second assumption, we should take into account the retentate flow-rate and to
integrate the shear-rate all along the blades and versus time. Both these analogies are
defined by dimensionless numbers relating to fluid flow and mixing (theoretical
system) in laminar flow. In each case, experimental measurements (pressure drop,
power consumption) enable a dimensionless geometrical parameter (, Kp) to be
determine, which will allow the mean shear rate to be quantified.
Firstly, we assume a complex shape duct (table 2) in which we assume a laminar
regime. Figure 2 shows the evolution of pressure drop divided by viscosity versus
retentate flow-rate for different rotational velocities. The linear evolution
characterises a laminar regime. It enables us to evaluate a slope equivalent to a
general geometrical parameter but it does not permit the values of dh, or L to be
identified in order to calculate a Reynolds number.

Dimensionless relation: Dimensional relation:

f Re = P 4 L
2 = 2 Q
dh S
f p P d h
= , p = In laminar flow
2 U 2
4 L
1
U dh &= Q
Re = , = p dh S
&
Table 2: Relationship between dimensionless numbers and experimental parameters in a
complex shape duct.
Secondly, we assume a linear model describing the geometrical parameter versus
rotational speed (F) as shown in figure 3. As a consequence, we propose a linear
relationship between the shear rate versus operating condition (retentate flow-rate,
rotational velocity). This value may be considered as an estimation of the axial shear-
rate but radial shear-rate need to be identified.

7
 1.2E+07
Linear increase DP vs Flow rate

Pressure Drop / Viscosity - DP/ [1/s]

=> Laminar regim
1.0E+07

F=50Hz
8.0E+06 F=40Hz
F=30Hz
6.0E+06 F=25Hz
F=20Hz
4.0E+06 F=10Hz

2.0E+06 Slope for F=10Hz

P [1/m3]

0.0E+00
0.0E+00 2.0E-05 4.0E-05 6.0E-05 8.0E-05 1.0E-04 1.2E-04 1.4E-04
3
Flow-rate [m /s]

Figure 2: Evolution of Retentate pressure drop divided by viscosity versus retentate flow-
rate for different rotational speeds.
2.5E+11 5.0
P 4 L
Modelling of reduced shear-rate
4.5 = 2 Q
dh S
versus frenquency (F) for a given flow
2.0E+11
rate (Q)
4.0
3.5
4L
(DP/DP0)=(/0)

with 2 = A + B F
Slope [1/m ]
3

1.5E+11 Slope : P vs F 3.0

Reduced shear-rate [/]

dh S
2.5

1.0E+11 2.0

d
then &tot = (A + B F ) Q h
1.5
Slope vs F [Hz]
5.0E+10 1.0

4 L
y = 1.56E+09x + 2.01E+10
R2 = 9.86E-01 0.5

0.0E+00 0.0
0 10 20 30 40 50 60
Frequency F [Hz]

Figure 3: Evolution of the geometrical parameter versus rotational speeds and modelling
of reduced shear-rate.

Clarification of rough beer

The quantitative performances of CFMF of rough beer correspond to the experimental
steady-state flux versus operating conditions and mean pore diameter. The flux values
confirm traditional levels of performances according to the operational variables. We
can summarise them as follows: (i) flux is closely correlated to mean pore size, (ii)
flux increases in a non-linear fashion with transmembrane pressure (figure 5) and with
shear-rate (figure 4). We can clearly distinguish the membrane with a high mean pore
diameter (superior to 1 m) which exhibits a flux value of above 100 l.h-1.m-2 whereas
small mean pore diameters remain inferior to 80 l.h-1.m-2. Flux values appear to be
close to those mentioned in existing literature [3, 12, 15].
The chemical analyses and retention rate were determined for the following elements:
(i) beer haze, (ii) colour, (iii) bitterness, (iv) polyphenols, (v) carbohydrates dry
matter, (vi) pH and (vii) proteins. Tables 3 and 4 sum up the mean composition of
permeate and retention rate versus mean pore diameter. We note that the permeate
quality and retention rate evolve with the nominal pore size. Permeate quality
becomes acceptable with 1.10 m and 1.50 m membranes even for haze. In this case
the global retention rate is inferior to 10% which means that there is not an excessive
loss of essential compounds. Yeast cell retention is close to 100% and residual
concentration is inferior to 10 cell/ml.

8
 300 300
MFT n4 : 0.60m, F=50Hz
MFT n4 : 0.80m, F=50Hz
250 250 MFT n6 : 1.10m, F=50Hz
MFT n7 : 1.10m, F=50Hz

Steady-state flux [l.h .m ]

-2
MFT n5: 1.10m, TMP=1400mBar MFT n6 : 1.50m, F=50Hz
200 200

-1
MFT n5: 1.50m, TMP=1400mBar MFT n7 : 1.50m, F=50Hz
Flux [l.h .m ]
2

MFT n4 : 0.60m, TMP=2750mBar

-1

150 150
MFT n4 : 0.80m, TMP=2900mBar

100 100

50 50

0 0
0.0E+00 2.0E+06 4.0E+06 6.0E+06 8.0E+06 1.0E+07 1.2E+07 0 500 1000 1500 2000 2500 3000 3500

(A+B.N).Q [1/s] Transmembrane pressure [mbar]

Figure 4: Steady-state flux versus Figure 5: Steady-state flux versus

4 L transmembrane pressure and mean pore
& and mean pore diameter. diameter with F=50Hz.
d h

Cold-sterilisation of beer
Qualitative performances during cold-sterilisation of clarified beer indicate the same
level of performances than for rough beer. With 0.60 m membrane, flux is slightly
superior to rough beer (common value inferior to 100l.h-1.m-2). We observed a regular
increase of flow rate with an increase in mean pore diameter for similar operating
conditions. This remark is also valid for qualitative and retention rates as shown in
tables 3 and 4. A critical pore size close to 0.50 m appears. Below 0.50 m, the
retention rates are too great which will induce a downgraded beer. In addition we
check whether high molecular weight nitrogen compounds (MW>5000) suffer from a
high retention rate with 0.15 m and 0.20 m membranes.

Cold-sterilisation of CB Clarification of RB
Mean pore diameter [m] 0.15 0.20 0.40 0.60 0.60 0.80 1.10 1.50
Protein (Brad.) [mg/l] 99 89 163 160 192 212 251 252
Protein (Lowry) [g/l] 3.69 4.05 4.73 5.02 5.78 5.87 5.92 5.96
Dry matter [%] 3.07 3.19 4.24 4.28 4.10 4.29 4.61 4.59
Colour [EBC] 6.7 6.8 8.7 9.1 8.4 8.8 9.8 9.7
Carbohydrate [g/l] 24 26 32 34 35 35 39 38
Polyphenol [mg/l] 140 146 181 180 170 185 184 190
Bitterness [EBC] 17.5 18 20 20.5 20 20.5 21 20.6
Haze [EBC] 0.26 0.23 0.33 0.30 0.35 0.38 0.59 0.55
PH [/] 4.24 4.29 4.29 4.25 4.38 4.37 4.36 4.37
Yeast cells [cell/ml] / / / / 0 0 1-10 1-10
Table 3: Mean composition of permeate during (i) the cold-sterilisation of clarified beer
and (ii) the clarification of rough beer versus mean pore diameter.
Cold-sterilisation of CB Clarification of RB
Mean pore diameter [m] 0.15 0.20 0.40 0.60 0.60 0.80 1.10 1.50
Protein (Brad.) [mg/l] 56.2 60.6 15.5 14.4 24.7 16.9 5.8 5.4
Protein (Lowry) [g/l] 39.2 33.3 14.5 10.9 15.7 14.4 6.5 5.9
Dry matter [%] 37.0 34.6 13.4 12.3 19.4 15.7 4.2 4.7
Colour [EBC] 35.4 34 15.5 12.9 / / / /
Carbohydrate [g/l] 34.8 27.9 11.9 10.3 10.3 10.3 7.9 8.1
Polyphenol [mg/l] 33.5 30.6 11.3 10.7 30.9 24.8 16.4 13.5
Bitterness [EBC] 19.5 17.2 11.1 9.9 20.0 18.0 5.7 7.3
Table 4: Mean retention rate composition of permeate during (i) the cold-sterilisation of
clarified beer and (ii) the clarification of rough beer versus mean pore diameter.

9
CONCLUSIONS
Potential applications of microfiltration in the beer industry are clarification
(elimination of yeast cells and suspended matter) and cold-sterilisation. If the
objective of the filtration is clarification, large pore membranes (superior to 1 m)
should be used because of the higher permeate flow rates and the low retention of
essential beer compounds. We observed that 1.10 m and 1.50 m may achieve these
criteria in presence of a high shear rate due to the rotational velocity of impeller. If the
objective of the filtration is pasteurisation, this study shows that no membrane can
satisfy flux requirement and beer quality criteria at the same time. Moreover, the
permeate flux obtained with these membranes is still too low to make this technique
economically viable. The cake layer erosion due to hydrodynamic perturbation seems
to improve membrane selectivity in regard with retention rate versus mean pore
diameter. Finally, we should understand and model the hydrodynamics of this system
in relation to permeate flux and quality. The knowledge of the average shear-rate will
permit to appreciate its impact on filtration performances.

NOMENCLATURE
A Geometrical constant, [m-3] TMP Transmembrane pressure, [Pa] or [mbar]
B Constante, [s.m-3] U mean velocity, [m/s]
dh Hydraulic diameter, [m] S cross section, [m2]
f Fanning friction coefficient, [/] Sh Sherwood number, [/]
F Rotation frequency, [Hz] or [rd/s] Greek letters
L Length, [m] dimensionless geometrical parameter, [/]
P Power consumption, [W] viscosity, [Pa.s]
Q flow rate, [m3.s-1] P pressure drop, [Pa]
Re Reynolds number, [/] & shear rate, [s-1]
p wall shear stress, [Pa]

REFERENCES
1. Blanpain P., Fillaudeau L., Lalande M., Trans IchemE, 77, Part C, (1999), 75-89.
2. Burrell K., Gill C., McKetchnie M., Murray J., MBAA Technical Quarterly, 31, (1994), 42-50.
3. Burrell K.J., Reed R.J.R., Filtration & Separation, 31 (4), (1994), 399-405.
4. Czekaj P., Lpez F., Gell C., J. Membrane Sci., 166 (2), (2000), 199-212.
5. Daufin G., Ren F, Aimar P., Ed. Lavoisier Tech & Doc, Paris (France), ISBN 2-7430-0228-X,
(1998), 592p.
6. Delaplace G., PhD thesis, Universit de Nancy I (France), (1998), 148p.
7. Delplace F., Delaplace G., Lefebvre S. and Leuliet J.C., 7th International Congress on Engineering
and Food, Brighton (UK), (1997), 36-39.
8. Dickmann H., Neradt F., Brewer's Guardian, June 1995, (1995), 27-30.
9. Dunn A.E., Leeder G.I., Molloy F., Wall R., Ferment, 9 (3), (1996), 155-161.
10. Fillaudeau L., Brauwelt International, 17 (5), (1999), 414-421.
11. Fillaudeau L., PhD thesis, Universit de Compigne (France), (1998), 150p.
12. Gan Q., Field R.W., Bird M.R., England R., Howell J.A., McKetchnie M.T., O'Shaughnessy C.L.,
TransIChemE, 75, Part A, (1997), 3-8.
13. Gaub R., Brauwelt International, (5), (1993), 448-457.
14. Leeder G., Brauwelt International, (4), (1993), 372-373.
15. McKetchnie M.T., Burrell K.J., Gill C., Kotzian R., O'Sullivan P., Food Process Engineering,
University of Bath (UK), (1994), 47-53.
16. OReilly S.M.G., Lummis D.J., Scott J., Molzahn S.W., Proceeding of the 21st EBC Congress,
Madrid (Spain), (1987), 639-647.
17. O'Shaughnessy C.L., Durosinmi-Etti O., Proceeding of the 26th European Brewery Convention,
Masstricht (The Netherlands), (1997), 681-690.
18. Stewart D.C., Hawthorne D., Evans D.E., J. Inst.Brewing, 104 (6), (1998), 321-326.
19. Wackerbauer K., Evers H., Brauwelt International, (2), (1993), 128-133.

10
28

Collection of operational data in the

brewery as a tool for process optimisation
C. Eger1, F. Spillmer1, W. Neske1, N. Abraham1, S. Pauls1, T.
Isenberg2, H. Wehrenberg1 & F. Bechmann3
1
Brauerei Beck & Co, Am Deich 18/19, D-28199 Bremen, Germany (e-mail: [email protected])
2
Werum GmbH
3
Gesellschaft fr Informationstechnik Weihenstephan

Descriptors
Automation, brewery, data processing, information management, process supervision

SUMMARY
In 1991, Beck & Co started the development of the PROLAB-System to manually collect
production and laboratory data. The goal was for data, which had previously been collected
manually, to be automatically transferred into the system. Data from different parts of the
brewery, such as the energy, logistics, brewing and filling areas, would all be linked in one
system. The central gathering of information allowed the correlation and relationships between
processes in different areas of the brewery to be investigated and improvements in the efficiency
of the brewerey to be made.

Betriebsdfatenerfassung in der Brauerei als Werkzeug fr Prozessoptimierungen

Deskriptoren
Automation, Brauerei, Datenverarbeitung, Informationswesen, Prozeberwachung

ZUSAMMENFASSUNG
1991 begann Beck & Co mit dem PROLAB-System zur manuellen Erfassung von Produktions-
und Labordaten. Ziel des neuen Projektes war es, bisher manuell erfasste Daten automatisch in
ein bergeordnetes Betriebsdatenerfassungssystem zu transferieren. Daten aus verschiedenen
Bereichen einer Brauerei, z.B. Braubetrieb, Abfllung, Energiezentrale, Logistik, sollten in ein
gemeinsames System gebracht und mit einem vorhandenen SAP R3-System verknpft werden.
Durch die zentrale Datenerfassung ist es mglich Daten aus den verschiedenen Bereichen
miteinander zu vergleichen und Prozesse zu optimieren. Die Mglichkeiten dieses Ansatzes zur
Prozessoptimierung sind anhand verschiedener Beispiele dargestellt.
La collecte des donnes operationnelles en brasserie comme outil d'optimisation des
mthodes

Descripteurs
Automatisation, brasserie, gestion de linformation, contrle de procd, traitement des donnes

RESUME
En 1991, Beck & Co ont lanc le dveloppement du systme Prolab destin recueillir
manuellement des donnes de production et de laboratoire. L'objectif tait de transfrer
automatiquement dans le systme les donnes qui taient prcdemment collectes manuellement.
Les donnes des diffrentes parties de la brasserie, comme l'nergie, la logistique, les zones de
brassage et de conditionnement, seraient toutes regroupes en un seul systme. La collecte
centralise de l'information a permis d'tudier la corrlation et les liens entre les processus dans
les diffrents secteurs et d'amliorer l'efficacit du brassage.

2
INTRODUCTION AND STRUCTURE OF NETWORK AND DATABASES
In 1991 Beck & Co started its PROLAB system to collect manual data during the
production process and in the laboratory. The aim was to reduce paper-based
documentation and make traceability during the production process easier.
In 1997 Becks started a new project for automatic data collection in several production
areas.
The aims were the optimisation of processes, visualisation of production parameters
during the process, visualisation of the consumption of different media and the reduction
of resource losses.
Manual collection and input of data was to be replaced by automatic data collection in
order to release manpower for other tasks.
With the installed software, a system which could be applied to the whole brewery was
planned. The optimised data collection system would allow data from all parts of the
brewery to be collected. This central gathering of information would then allow the
correlation and relationships between processes in different areas of the brewery to be
investigated.
S5 S7

Industrial Ethernet

BDE-System Power Optimisation Industrial Ethernet

Industrial Ethernet
S5
S5 Central BDE -Server PROLAB-Server

S7 BDE-System Energy Supply

SAP-Server S7

BDE-System Filling Plant

Token-Ring

S7
BDE-System Filtration Plant BDE-Terminals in the Beck
BDE-Terminals
& Co-Web in the Beck & Co-Web
Braumat-CIS-Server
BDE-System Brewhouses
SH1/SH3/KH/Filtration
Mobile BDE-Terminals with radio connection
Industrial Ethernet Ethernet Industrial Ethernet
to the Beck & Co-Web Industrial Ethernet

ET 200 L ET 200 L
ET 200 L ET 200 L

Braumat Filtration Plant Braumat Brewhouses

Siemens PLC - BRAUMAT Siemens PLC - BRAUMAT

Figure 1: Network structure in the brewery

Existing databases (such as PROLAB and SAP) were to be connected with the new
databases of the data collection system. As an example, the connection of the brewhouse
data collection system to PROLAB and SAP is shown.
Between the warehouse data collection system and SAP another connection was
established.

3
BATCH RECORDING AND REPORTING OPTIONS
In two brewhouses and the wort-cooling the BRAUMAT systems are connected to the
data collection system. Recipe and batch-based data are collected from the BRAUMAT
batch record and summarised in the data collection system (figure 2)

Figure 2: Concentrated batch record

REPORTS
The summarised batch record is sent to the PROLAB-System (figure 3) where the
connection to the fermentation batches and laboratory data is established. Additionally
there are different reports in different report levels available, which are based on the data
in the several databases like production losses (figure 4) or capacity of brewhouses.

Figure 3: PROLAB report Figure 4: Report for production losses

4
For special purposes a free access report can be used to receive summarised data over
specified periods, recipes or other possible influences. So for every problem a special
view can be created.
For example, the influence of changes in the malt and mashing recipe parameters on
analytical parameters in bright beer (figure 5); the change of technological parameters on
parameters in the bright beer (figure 6). Or for maintenance of valve switches of a valve
block versus valve failures (figure 7).

Figure 5: Parameters in bright beer Figure 6: Parameters in bright beer

(malt change) (technological change)

Figure 7: maintenance report for valve switches and failures

COLLECTION AND PRESENTATION OF DATA - A TOOL FOR PROCESS

OPTIMISATION
Cross correlation of all data collected from the batch, e. g. measuring devices, valves or
signals, is possible to obtain an overview of the running processes. Compared to other
data collection systems it does not matter from which source the data is derived because
all data of the different systems can be linked together. Processes which are running in
different areas can easily be linked and influences to processes in in other areas can be
detected.

5
All existing data sources are identified in the Central BDE-Server and can be assigned to
any area in the PLS-Hierarchy as shown in figure 8. An example comparing different
brewhouses is shown in figure 9.

Figure 8: PLS hierarchy

Figure 9: Turbidity and temperatures of three brewhouses

6
STEAM AND ENERGY PRODUCTION AND BREWHOUSE BATCHES
Together with Steinecker, Beck & Co started a simulation to forecast the steam
consumption of three brewhouses. The target was to get continuous steam consumption
and as a result less fluctuations in energy production.
With the existing steam consumption data and batch reports (figures 10 and 11), a model
was created to change the starting time of mashing in and the starting time of wort
cooking in order to close gaps that derive in slight changes of production periods.

Figure 10: Steam Consumption of the brewhouses

24
18
BDE
12
Dampf [t/h]
6
0
24
18
Simulation
12
Dampf [t/h]
6
0
24
18
Simulation
optimiert 12
Dampf [t/h]
6
0

BDE
Dampf
binr

Simulation
Dampf
binr

Simulation
optimiert
Dampf
binr

06:00 09:00 12:00 15:00 18:00 21:00 00:00 03:00 06:00

03.07. 03.07. 03.07. 03.07. 03.07. 03.07. 04.07. 04.07. 04.07.

Figure 11: Results of the simulation of steam

7
WHIRLPOOL STAND TIME
For Beck & Co it is of great importance to reduce thermal influences to the wort after
cooking, to reduce staling components.
For this reason the stand time of the whirlpools is controlled. The stand time was set at a
Maximum level of 25 min. Every brew with a standing time over 25 min is recorded as a
failure and reported in a failure review.
A failure review is shown in figure 12. The efforts before and after optimisation of the
process can be seen in figure 13.

Figure 12: Failure Report of whirlpool stand Figure 13: Stand time failures
time before and after optimisation

REPORTS IN THE FILLING PLANT

With the automated reporting system in two filling lines, Beck & Co started data
collection in the filling plant. Automated reports for the efficiency of bottling lines and
special reports of failures of the different machines could be created. In figure 14 an
overview failure report is shown for a filler and in figure 15 a detailed report for the
group external failure is shown.

Figure 14: Overview failure report Figure 15: Detail report for external failure

8
STANDARD TOOL FOR OPTIMISATION
For the optimisation of processes a standard tool (figure 16) is available to combine any
valve or measuring device. With a zoom option (figure 17), every part of the chart can be
set to a detail view to obtain, for example, exact information of valve switches and related
parameters.

Figure 16: Example for process optimisation

Figure 17: Detailed view

9
CONCLUSION
A system applied at Beck & Co for the automatic data collection has been demonstrated.
The flexybility of the reporting of all data can be used for process optimisation for all
related processes.

The system was installed by Werum, Nord IT and Beck & Co, the simulation for the
steam consumption was created in co-operation with Steinecker, based on the original
data from the data collection system.

10
29

Propagation of brewery yeast

modelling and simulation
T. Kurz, T. Becker & A. Delgado
1
TU Mnchen, Lehrstuhl fr Fluidmechanik und Prozeautomation, Weihenstephaner Steig
23, D-85354 Freising-Weihenstephan, Germany (e-mail: [email protected])

Descriptors
Brewers' yeast, model simulation, process control, yeast propagation

SUMMARY
A brewer requires a specific amount of yeast cells at the proper time for a consistent
fermentation. Therefore, the propagation process must be adapted to different propagation
scenarios. Active control in this regard is not yet realised because of a lack of process-
knowledge. A comprehensive model of brewing yeast propagation is introduced, which
allows an evaluation of control strategies and a precise adaptation of the propagation strategy
to the varying demands in practice. Case scenarios can be created, which allow the evaluation
of the current settings of the manipulated variables and their adaptation to the variations of the
production plan.

Herfhrung von Brauereihefe Modell und Simulation

Deskriptoren
Bierhefe, Hefevermehrung, Modellbildung, Prozesteuerung

ZUSAMMENFASSUNG
Fr eine gleichbleibende Grung wird eine definierte Zellzahl zu definierten Zeitpunkten
bentigt. Deshalb muss der Propagationsprozess an verschiedene Herfhrungsszenarien
angepasst werden. Eine aktive Prozessfhrung konnte aufgrund von mangelndem Prozess-
wissen bisher nicht realisiert werden. In dieser Arbeit wird ein umfassendes Modell fr das
Hefewachstum vorgestellt, das eine Bewertung von Prozessfhrungsstrategien und eine
genaue Anpassung der Herfhrungsstrategie an die variierenden Anforderungen in der Praxis
erlaubt. Szenarien knnen geschaffen werden, die eine Bewertung der aktuellen Einstellung
der Regelgren und eine Anpassung dieser an die Variationen im Produktionsplan erlauben.
Prolifration de la levure de bire modlisation et simulation

Descripteurs
Commande de procd, levure de brasserie, modlisation, propagation de la levure

RESUME
Un brassin ncessite une quantit spcifique de cellules de levure, au bon moment, pour une
fermentation homogne. Par consquent, le processus de propagation doit tre adapt
diffrents scnarios. A cet gard, il n'existe pas encore de contrle actif en raison du manque
de connaissance du processus. Nous prsentons ici un modle dtaill de propagation des
levures en brasserie, permettant une valuation des stratgies de contrle et une adaptation
prcise de la stratgie de propagation aux diverses exigences dans la pratique. On peut crer
des scnarios de cas, permettant d'valuer les rglages actuels des paramtres manipuls et de
les adapter aux variations du plan de fabrication.

2
INTRODUCTION
The state of the propagated brewing yeast before pitching exerts a relevant influence
on the performance of the following beer fermentation concerning fermentation time
and quality of the resulting beer. The pitching yeast should be taken out of the
exponential (log-) state (figure 1) of the propagation, because the highest fermentation
activity is associated to this state. Therefore, in practice for the brewer one specific
question is of substance:
Is it possible to make available a defined amount of brewery yeast in desired
quality using a practice relevant system, in particular if variations in the
production plan or the propagation process occur?
An active control in this regard has so far not been realised because of a lack of
methodical knowledge about the process behaviour. Prerequisite of an active control
on the one hand is the observability of the propagation process including online
measurement of relevant quantities, e.g. gravity, pH-value, turbidity, oxygen and
temperature, which can be taken as granted. On the other hand a comprehensive
knowledge about manipulated variables (e.g. temperature and dissolved oxygen
concentration) and their influence on the process behaviour abstracted in a process
model is necessary. Figure 1 shows a model based simulation of a propagation
process of bakers yeast Saccharomyces cerevisiae, realized by Barford (4) in 1990.
Shown are measurement and simulation data. It can be seen, that a high accuracy of
the simulation has been achieved.
100

Exponential State
80
Concentration [mmol/l]

Ethanol
60

40 Substrate

20

Biomass
0
0 2 4 6 8 10 12
Time [h]
Figure 1: Propagation Process of S. cerevisiae. Measurement and simulation data by Barford
(1990). Distinguished is the exponential state of the propagation. Before (left) the Lag- and
later (right) the stationary state of the propagation appear. Additionally measurement (dotted)
and simulation data (lines) of biomass, substrate and ethanol are shown in the graph.

In the literature numerous structured (5) or unstructured (11,12) modelling approaches

for the bakers yeast propagation can be found. A comprehensive and reliable model
for the propagation of brewery yeast however is not existent. Approaches can be
found concerning biochemical problems, yeast fermentation activity (6,7), proper
aeration (1) and experimental considerations to the propagation process (1,3,9). Here
the motivation for the development of a comprehensive process model for the brewery
yeast propagation was found. In the following the developed modelling approach for
the propagation of brewery yeast is described. Model based simulation runs of
propagations at different temperatures are verified with experimental data. The
relevant parameters for parameter fitting are mentioned, in particular the influence of

3
temperature on the yeast growth is discussed. Feasibility experiments are presented to
show the transferability of the solution to different propagation plants and case
scenarios were realized under practical conditions to prove the potential of the
modelling approach as a basis for an active process control.

MODELLING
Biotechnological processes as the yeast propagation are too complex to comprehend
all details. Thus, modelling, prognosis, diagnosis and optimization of biotechnological
processes is one major research field of the Chair of Fluid Mechanics and Process
Automation (8,10). The present contribution focusses on the kinetic process
modelling of the yeast propagation process. A model is a simplification of reality and
is therefore useful for a better understanding and predictability of different aspects of
the real process. The potential of process modelling lies in an improvement of the
process understanding concerning the influence of the manipulated variables on the
process behaviour. Hence it generally can be useful
for a better process understanding,
for the planning of experiments,
for the description and interpretation of experimental results and
as a simulation and optimization tool to develop and evaluate precise control
strategies.
A wide range of possible applications in biotechnology from measurement models to
comprehensive process models can be found in the field of biotechnology but are
generally not applied in the practice of brewing technology.
The modelling approaches derived from the field of bakers yeast can not be
transferred to brewery yeast without adaptations concerning the growth medium and
the characteristics of the yeast type. The propagation medium in breweries is the beer
wort. The main differences to growth media in biotechnology lie in a lack of zinc and
amino acids, in the concentration of the main substrate (low concentration glucose
compared to high concentrations of maltose and maltotriose) and in the resulting
regulation mechanisms of the yeast metabolism (e.g. crabtree effect). The different
yeast types (saccharomyces cerevisiae, saccharomyces carlsbergensis) differ in the
stoichiometry and the kinetics concerning the oxygen and substrate uptake (2,4).
Additionally, most of the specific kinetic parameters for the brewers yeast strains are
not examined. At the Chair for Fluid Mechanics and Process Automation two
different modelling approaches were developed. In this contribution a black box
model is introduced. In figure 2 the structure of the model is described schematically.
Only substances were taken into account, which are interacting with the surrounding
medium. Considered were uptake and formation rates for the main substrate
(formulated as glucose), biomass, nitrogen source, oxygen, carbon dioxide, water and
ethanol. The composition of biomass is assumed to be constant (table 1). The
elements S and P were neglected. Concerning the substrate uptake the underlying
kinetics (Monod kinetics) include substrate limitation, nitrogen limitation, ethanol
inhibition, lag-time and the influence of temperature. In table 1 the applied
stoichiometric and kinetic parameters are quantified.

4
 Substrate Nitrogen Dissolved
e.g. Glucose source Oxygen

Biomass /
Yeast cells
Black-Box H2O
CO2

Product
e.g. ethanol
Figure 2: Schematic description of the modelling approach. Considered are only the uptake
and formation rates of main substrate (as glucose), biomass, nitrogen source, oxygen, carbon
dioxide, water and ethanol.

Stoichiometry Parameter Value Unit

Oxidative Yield YX/Sox 3,527 mol/mol
Fermentative Yield YX/Sf 0,3599 mol/mol
Biomass composition HX 1,79 mol/mol
CHHXOOXNNX OX 0,57 mol/mol
NX 0,15 mol/mol
Kinetics
Max. substrate uptake rate qS,max 0,486 mmol/(mmolh)
Max. oxygen uptake rate qO2,max 0,234 mmol/(mmolh)
Half saturation constants for mmol/l
Substrate limitation KS 2,8 mmol/l
Oxygen limitation KO 0,0031 mmol/l
Ethanol inhibition Ki,eth 500 mmol/l
Nitrogen limitation KN 2 mmol/l
Ethanol inhibition (oxygen uptake) Ki,eth,o 1000 mmol/l
Table 1: Stoichiometric and kinetic parameters applied in the modelling approach.

The actual specific substrate uptake rate qs can be described with the following
equation:
S K i ,eth N
q S = qS ,max f temp Lt
S + K s K i ,eth + E N + K n
with ftemp (temperature coefficient) and a sigmoid lag-time function with
1
Lt =
1 + e ( t tl ) .
The maximum specific substrate uptake rate qs,max is referring to 20C. To simulate
kinetics at other temperature levels the temperature coefficient ftemp is introduced to
adapt the actual substrate uptake rate qs.
Oxygen limitation and ethanol inhibition is considered for the oxygen uptake. The
specific oxygen uptake rate qO2 is limited by the maximum oxygen uptake rate qO2,max.
If the concentration of the main substrate (glucose) is sufficient, then qO2 reaches
qO2,lim, which represents the oxidative bottleneck.

5
 O K i ,eth ,o
qO 2 qO 2,max = qO 2,lim
O + K O K i ,eth ,o + E

Three different states can be distinguished for the degradation of sugars in the yeast
cells. The oxidative degradation occurs with low substrate concentrations and
presence of dissolved oxygen. The demanded oxygen uptake rate is lower than qO2,lim
and the whole substrate can be degrade using the oxidative pathway. If the substrate
uptake rate increases, e.g. due to higher substrate concentrations, the demanded
oxygen uptake rate is higher than qO2,lim. As the oxygen uptake rate is limited to
qO2,lim, the overflow is degraded using the fermentative pathway (Overflow
metabolism). In absence of oxygen all substrate is degraded using the latter way
(Fermentative degradation). The regulation mechanism of the overflow metabolism
is known as the Crabtree-effect. Although this explanation for the Crabtree-effect is
biochemically not correct, Pham (11) was able to show, that it can be used in a good
approximation. In the Crabtree effect the reason can be found, why the yeast is always
fermenting even if oxygen is available. A surplus of oxygen wont cause an increase
of the resulting yield. The energy demand of the yeast cell for maintenance is
formulated by a full oxidation of glucose (aerobic) or via ATP-production in the
glycolysis (anaerobic). For the maintenance energy of the yeast cells an additional
substrate uptake correlating to a constant energy demand of 0.01 mol ATP/C-mol
biomass is taken into account.

MATERIAL AND METHODS

Propagator
A 150 litre (net volume) propagator with an integrated continuous aerator and a
defoamer unit was used. A similar system was described by Methner (9) in 1999. The
propagator was equipped with online measurement devices for turbidity, gravity, pH-
value, temperature, pressure and air flow as well as for dissolved oxygen. Underlying
control loops guaranteed an accurate progress of the manipulated variables
temperature (+/- 0.1C) and dissolved oxygen (+/- 0.1 ppm).

Yeast strain and medium

The Saccharomyces carlsbergensis yeast strain W34 was used for the experiments.
For the propagator experiments yeast suspension from own propagations was
harvested. Industrial wheat beer wort was used as medium. 0.2 ppm zinc was added to
avoid a limitation effect. The concentration of free amino acids was nearly constant
over all trials with about 220 ppm.

Experiments
Experiments were made in the relevant range of manipulated variables. In particular
experiments on the influence of temperature during the yeast propagation were carried
out at 10, 15, 20 and 25C with an dissolved oxygen concentration of 0.5 ppm. The
influence of dissolved oxygen was examined at concentrations of 0.1, 0.3, 0.5 and 0.8
ppm at a temperature of 15C. In order to prove the potential of modelling for the
evaluation of control strategies case scenarios were also realised. Here the
propagation temperature (20C is reduced after 5 hours to 10C and increased during
the next 15 hours back to 20C. All propagation runs were carried out 3 times, in
order to gain reproducible information.

6
Software / simulation
For convenience, the modelling and simulation was carried out by using the software
package Aquasim 2.0. Aquasim allows the identification and simulation of aqueous
systems. For each simulation the starting conditions including concentrations of
biomass, gravity, nitrogen, ethanol and dissolved oxygen in the wort as well as the
progression of the manipulated variables were required.

RESULTS AND DISCUSSION

Experiments and simulation runs
With the developed process model (Parameters s. table 1) it was possible to simulate
the measurement data of the realized propagation runs with a high accuracy. In figure
3 progressions of propagations with temperatures of 10, 15, 20 and 25C with a
constant concentration of dissolved oxygen (0.5 ppm) are shown. The belonging
experiments were carried out in the described 150 litre propagator with continuous
aeration.

500
Concentration [mmol/l]

Substrate
400

300 25C 20C 15C 10C

200

Biomass
100

0
0 10 20 Time [h] 30 40 50

Figure 3: Simulation runs (lines) and experimental data (dotted) of propagations at 10, 15, 20
and 25C. Plotted are the progressions of substrate and biomass concentrations. Experiments
were made with the 150 l propagator with continuous aeration.

Presented are substrate and biomass progressions and the belonging offline reference
values. For the presented simulation runs the parameters were kept constant except for
three variable parameters. To fit the basic model to the particular propagation run the
temperature coefficient (influence of temperature), the maximum oxygen uptake rate
as an important aspect concerning the Crabtree effect and the lag time, where no
exponential growth of biomass can be observed, where fitted. In order to show the
feasibility of a transfer of the model to different propagation plants, data of an
industrial propagator with a conventional discontinuous aeration system were
simulated as well. Similar to figure 3 progressions are plotted in figure 4. After fitting
the variable parameters the propagation data also could be described very well by the
model. However these data are not reproduced and have to be verified in further trials.

7
 700

Substrate
600

Concentration [mmol/l]
500

400

300

200
Ethanol
100
Biomass
0
0 10 20 30 40 50 60 70
Time [h]
Figure 4: Simulation runs (lines) and experimental data (dotted) of propagations at 10, 15, 20
and 25C. Plotted are the progressions of substrate, ethanol and biomass concentrations over
the time. Experiments were made with an industrial propagator with discontinuous aeration.

Influence of temperature
The analysis of the experimental data brought important specific information about
the relationship between temperature and oxygen content during the yeast
propagation, substrate uptake and biomass formation respectively. The influence of
temperature on the biomass growth could be modelled with an Arrhenius approach. In
figure 5 a half logarithmic plot of the growth rate and the inverse temperature is
shown. It can be seen, that a significant relationship (Arrhenius) exists between the
process temperature and the specific growth rate. This cognition could be integrated in
the process model, whereby a reduction of the variable parameters needed for fitting
to only two was achieved. In the modelling approach the specific substrate uptake rate
at 20C is multiplied with by the temperature coefficient ftemp, which is following the
mentioned Arrhenius approach similar to the specific growth rate shown in figure 5.

1
Specific growth rate

25C

15C
0.1 20C

10C

0.01

Temperature 1/T
Figure 5: Specific growth rate and the temperature for several experiments in the continuous
aerated propagator system (dotted). A significant relationship between the two quantities
could be detected with an exponential Arrhenius approach (line).

Herewith a further prerequisite for the realization of an active process control and
evaluation tool for control strategies could be established.

8
Influence of dissolved oxygen
The results of the experiments are not shown. The data were gained with the
continuous aerated propagator system at a temperature of 15C. In these experiments
a maximum specific growth rate was found with an dissolved oxygen content between
0.3 and 0.5 ppm. This comes along with data published by Hartmeier (7) in 1972. The
influence of a manipulation of the dissolved oxygen concentration however appeared
to be negligible compared to the influence of a manipulation of the process
temperature. Actually, further trials are in progress to verify these results.

Case scenario
In order to show this potential, case scenarios were carried out, which e.g simulate a
delay in the production in the brewhouse. Therefore the yeast propagation has to be
decelerated by manipulating the temperature. Figure 6 shows the progression of
biomass and substrate during a propagation where a temperature profile was applied
for an active control of the yeast propagation process. The temperature was decreased
from 20 to 10C very fast. After a holding time the temperature was increased slowly
back to 20C that the process model represents the experimental data very well. Given
are the progressions of a 20C experiments with similar starting conditions (dotted
line) and the progressions following the temperature profile. The experiments were
run with an dissolved oxygen concentration of 0.5 ppm. It can be seen, that the data
from the model based simulation runs (lines) represents the reference data points
(dotted) very well.
600

20C
Substrate
Concentration [mmol/l]

400

200

10C Biomass
0
0 5 10
Time [h]15 20 25

Figure 6: Progressions of biomass and substrate during a propagation applying a temperature

profile for an active control of the yeast propagation process. The temperature was decreased
from 20 to 10C. After a holding time the temperature was increased slowly back to 20C.
Shown are the progressions resulting from the temperature profile of the simulation (lines)
and the results of the experiment (dotted). The experiment was run with an dissolved oxygen
concentration of 0.5 ppm.

CONCLUSION
A reasonable modelling approach for the propagation of brewery yeast was
developed. Model based simulations matched experimental data very well. With only
three variable parameters an easy manageable simulation tool is provided for the
brewer. The expected Arrhenius approach for the temperature influence on the yeast
growth could be found both in the experimental data and the modelling approach. A

9
feasibility test showed, that a transfer to other plants and propagation methods is
possible. In current experiments on the one hand the influence of the oxygen
concentration on the growth of S. carlsbergensis is examined further. On the other
hand additional factors concerning the growth process are considered. For Zinc
concentration and pH-value in the wort is has to be checked, whether these quantities
have to be taken into account in the modelling approach. Collateral experiments deal
with the optimization of the determination of the amount of yeast cells using turbidity
measurement or/and further information, e.g. pH-value). Additionally examinations to
the transferability and the scale up of the model validity are started using experimental
methods as well as a combination of numerical simulation of fluid dynamics in a
propagator and the presented kinetic modelling approach. The modelling of the yeast
proliferation state and the vitality is another subject of current research. The benefit
for the brewer, delivered with this research project, lies in a gain of process
knowledge. Process control strategies can be simulated and evaluated. The
propagation strategy can be planned more efficient than before. Mistakes in the
process strategies can be avoided and an active yeast can be provided for pitching
even considering scenarios in practice. This results in trouble-free propagation and
fermentation runs and a constant beer quality.

ACKNOWLEDGEMENT
The German Brewers Association is acknowledged for the financial support. We
thank the Bitburger Brauerei, Bitburg and the Staatsbrauerei Weihenstephan, Freising
for the technical support.

REFERENCES
1. Annemller, G., Manger, H.-J., Brauwelt, 1999, 21/22, 9931007.
2. Annemller, G., Manger, H.-J., Brauwelt, 1998, 49/50, 24782481.
3. Back, W., Imai, T., Forster, C. Narzi, L., Monatsschrift fr Brauwissenschaft,
1998, 11/12, 189195.
4. Barford, J. P., Biotechnology and Bioengeneering, 1990, Vol. 35, 907929.
5. Hejinen, J. J., Black Box Description of Microbial Growth, in: BODL-Advanced
Course Microbial Physiology and Fermentation Technology, Delft, 1996
6. Hutter, K.-J., Mller, S., Monatsschrift fr Brauwissenschaft, 1996, 7/8, 234-239.
7. Imai, T., Ohno, T., Applied and Environmental Microbiology, 1995, 10, 3604
3608.
8. Kurz, T., Becker, T., Fellner, Delgado, A., Journal of the Institute of Brewing,
2000, accepted.
9. Methner, F.J., Proceedings of the European Brewery Convention Congress,
Cannes, 1999, 637-646.
10. Murnleitner, E., Becker, T., Delgado, A., Water Research, 2001, accepted.
11. Pham, H. T. B., Larsson, G., Enfors, S.-O., Biotechnology and Bioengeneering,
1998, Vol. 60, No. 4.
12. Sonnleitner, B., Kppeli, O., Biotechnology and Bioengineering, 1986, Vol. 28,
927-937.

10
30

The optimization of yeast transfer

timing in a propagation process
Yuji Nishida1, Nobuyuki Fukui2, Atsushi Fujita3, Fumihiko
Omura2 & Akira Isoe1
1
Suntory Ltd., Research Institute for New Product Development, 1-1-1,Wakayamadai,
Shimamoto-cho, Mishima-gun, Osaka 618-8503, Japan (e-mail: [email protected])
2
Suntory Ltd., Institute for Fundamental Research
3
Suntory Ltd., Kyoto Brewery

Descriptors
Fermentation, maltose, metabolism, process control, yeast propagation

SUMMARY
The control of maltose assimilation is important during beer fermentation. As an important
index of fermentation performance, we established novel analytical methods of maltose
uptake activity and maltose transport protein content of yeast. Maltose uptake ability of yeast
increased at the early stage of fermentation, and it decreased after it reached to the maximum
level. The change of maltose transport protein content was also similar performance to the
maltose uptake ability during beer fermentation. Using the index of maltose uptake ability, we
optimized yeast transfer timing from a propagation tank to a fermentation tank.

Die Optimierung der Hefeverweilzeit im Propagationsprozess

Deskriptoren
Grung, Hefevermehrung, Maltose, Prozesteuerung, Stoffwechsel

ZUSAMMENFASSUNG
Die Kontrolle der Maltoseassimilation ist ein wichtiger Parameter whren der Grung. Wir
entwickelten neue Methoden zur Bestimmung der Maltoseaufnahmeaktivitt und des
Maltosetransport-Protein-Gehaltes der Hefe. Das Maltoseaufnahmevermgen der Hefe steigt
in der frhen Phase der Grung und sinkt erst wieder nachdem das Maximum erreicht wurde.
Die nderung des Gehaltes an Maltosetransportproteinen korrelierte mit dem Maltose-
aufnahmevermgen whrend der Grung. Mit Hilfe des Maltoseaufnahmevermgens
optimierten wir den Zeitpunkt der berfhrung der Hefe vom Propagator in das Grgef.
Optimisation du timing du transfert de levure dans un procd de propagation

Descripteurs
Commande de procd, fermentation, maltose, mtabolisme, propagation de la levure

RESUME
Le contrle de l'assimilation du maltose est important pendant la fermentation de la bire.
Comme indice important des performances de fermentation, nous avons labor de nouvelles
mthodes analytiques pour mesurer l'activit de capture du maltose et la teneur en protine de
transport du maltose dans la levure. La capacit de capture du maltose de la levure augmente
au stade prcoce de la fermentation et diminue aprs avoir atteint son maximum. Les
variations de teneur en protine de transport du maltose taient galement un niveau
similaire pendant la fermentation de la bire. Au moyen de l'indice de capacit de capture du
maltose, nous avons optimis le droulement du transfert des levures de la cuve de
propagation la cuve de fermentation.

2
INTRODUCTION
Among the several fermentable sugars contained in wort glucose, fructose, sucrose,
maltose and maltotriose the most important for brewing is maltose. Generally,
glucose, fructose and sucrose are first assimilated, and then maltose and maltotriose
are assimilated consecutively. The primary factor determining the rate of beer
fermentation is the assimilation rate of maltose, making the control of maltose
assimilation very important during this process. Maltose is taken into yeast cells by
the maltose transporter and is then hydrolyzed to glucose by maltase. Many papers
about maltose transporter and maltase have already been published (1, 2), but the
change in maltose transporter during beer fermentation have rarely been reported. If
the change in the maltose transporter during fermentation can be determined, it may
be useful in improving the fermentation rate.
In this study, we established a novel analytical method for indexing maltose uptake
ability, and found out the change in this ability during beer fermentation. Next, we
used an assimilation method (3), and optimized yeast transfer timing from
propagation tank to fermentation tank in order to improve the fermentation rate using
a pitching yeast with higher maltose uptake ability.

MATERIALS AND METHODS

The determination of maltose uptake ability

5 ml pH4.5 HEPES 30oC , 20 minutes

buffer with 6% maltose in shaking bath
Certain
yeast cells
Maltose concentration (%)

6.5
Maltose Uptake Ability (unit)
6 = reduction rate of maltose (%/min) Sample 1ml of solution
every 5 minutes
5.5
Yeast cells were
5
removed by filtration
4.5
0 5 10 15 20 Determination of
Time (min) maltose content

Figure 1: Analytical procedure for maltose uptake ability.

The maltose uptake ability of yeast cells was measured as shown figure 1.
The fermenting wort at each stage of fermentation was harvested, and yeast cells were
collected via centrifuge. About 1 g of the wet yeast cells was washed in distilled
water, and certain yeast cells were incubated in 5 ml pH 4.5 HEPES buffer with 6%
maltose at 30oC for 20 minutes. 1 ml of the solution was then withdrawn at 5-minute
intervals and the yeast cells were removed by filtration. The maltose concentration in

3
the filtered solution was measured. We defined maltose uptake ability as the reduction
rate in the amount of maltose concentration per minute per certain yeast cells.
Yeast strain
Saccharomyces cerevisiae from our stock culture were used.
Propagation conditions
All experiments of propagation were carried out using 400 ml of 11oP adjunct wort in
a 1 l fermentor and incubated at 15oC for 72 hrs with constant aeration.
Fermentation conditions
All fermentation experiments were carried out using 2 l of 11oP adjunct wort in an
EBC tube at 12oC for 10 days.

RESULTS AND DISCUSSION

The change in maltose uptake ability during beer fermentation
Figure 2 shows the change in maltose uptake ability of harvested yeast cells from
fermenting wort at each stage, time course of apparent extract and time course of
yeast growth during beer fermentation. Maltose uptake ability increased at the early
stage of fermentation and began to decline before yeast growth reached at its peak.
Next, we investigated the relationship between maltose uptake ability and
fermentation rate, expressed as the amount of alcohol production in an hour by a yeast
cell (figure 3). As this figure shows, it was obvious that the maltose uptake ability of
yeast cells closely relates to specific alcohol production velocity. Using this index, we
researched in further detail the changes during beer fermentation at the yeast growth
phase.

100 12
Maltose uptake ability
A-Ex 10
80
Suspending yeast (10 cells/ml)
Maltose Uptake Ability (unit)

yeast growth
Apparent Extract (%)

8
60
6

6
40
4

20
2

0 0
0 17 41 65 89 113 137 161 185
Fermention period (hr)

Figure 2: Change in Maltose uptake ability, time course of apparent extract

and time course yeast growth during beer fermentation.

4
 140

Maltose Uptake Ability (unit)

120
2
100 R = 0.9398
80
60
40
20
0
0 5 10 15 20
Specific alcohol production velocity
-12
10
i g/cell/hr)

Figure 3: Relationship between maltose uptake ability and specific alcohol

production velocity during beer fermentation.

Figure 4 shows the change in maltose uptake ability of yeast cells at the growth phase
of fermentation. Maltose uptake ability increased, then began to decline before the
yeast growth reached at its peak. We investigated why maltose uptake ability stops
increasing before the yeast growth has peaked. Figure 5 shows the relationship
between maltose uptake ability and amino acid concentration in the fermenting wort.
When lysine, a Group A amino acid (arginine, lysine, aspartate, asparagine,
glutamate, glutamine, serine, threonine; absorbed immediately), had almost
completely disappeared, maltose uptake ability began to decline. When further
concentrations of Group A amino acid increased in adjunct wort, we found that
maltose uptake ability was not decreased at the growth phase of fermentation (data not
shown).
80
Maltose Uptake Ability
70
Yeast growth
Maltose Uptake Ability (unit)
Suspending yeast(10 cells/ml)

60

50
6

40

30

20

10

0
0 5 10 15 20 25 30 35 40 45 50
Fermentation period (hr)

Figure 4: Change in maltose uptake ability of yeast cells at the growth

phase of fermentation.

5
 80 0.6 Maltose Uptake Ability
70 Lysine
0.5 Valine
Maltose Uptake Ability (unit)

60
Phenylalanine
0.4
50

Amino Acid (mM)

40 0.3

30
0.2
20
0.1
10

0 0
0 10 20 30 40 50
Fermentation period (hr)

Figure 5: Change of maltose uptake ability and the assimilation of amino

acid at the growth phase of fermentation.

We investigated the influence of the maltose uptake ability of pitching yeast on

fermentation rate (figure 6). As the maltose uptake ability of pitching yeast was
higher, the fermentation rate was faster. It is important to obtain pitching yeast with
higher uptake ability for the improvement of the fermentation rate.

Pitching yeast with low uptake ability

Pitching yeast with high uptake ability
140 12

120 10
Maltose Uptake Ability (unit)

100
Apparent Extract (%)

8
80
6
60
A-Ex
4
40

20 2

0 0
0 24 48 72 96 120 144 168
Fermention period (hr)
Figure 6: Influence of maltose uptake ability of pitching yeast on
fermentation rate.

6
The optimization of yeast transfer timing from propagation tank to fermentation
There were various methods, which increased maltose uptake ability of the pitching
yeast. We used an assimilation method as one of them, and investigated to optimize
yeast propagation by controlling parameters such as temperature, aeration, and timing
of yeast transfer. In the next experiment, we described the optimization of yeast
transfer timing from propagation tank to fermentation tank using the index of maltose
uptake ability.
Figure 7 shows the change in maltose uptake ability and the time course of yeast
growth during propagation. Maltose uptake ability during propagation increased at the
yeast growth phase, and began to fall before the yeast growth reached at its peak,
which was similar to the change during fermentation. We evaluated yeast, which were
harvested 40 hours and 55 hours after the beginning of propagation. The harvested
yeasts were labeled as following: after 40 hours -timing A; after 55 hours -timing
B. The timing of the harvested yeast in propagation, and time course of glucose and
maltose concentration in propagation wort are shown in figure 8. The yeast of timing
A was harvested at the logarithmic growth phase. Its maltose uptake ability was
maximum during propagation. Glucose had almost disappeared in the propagation
wort and maltose just began to assimilate. The yeast of timing B was harvested at the
late logarithmic growth phase. At that time, its maltose uptake ability just began to
decline. Glucose was already lost and maltose was assimilated in the propagation
wort. Figure 9 shows the time course of apparent extract during fermentation using
yeast harvested at timing A and B. In this experiment, the pitching rate of both was
the same. The fermentation rate of yeast harvested at timing B was more sluggish in
comparison with the yeast harvested at timing A. It was known that yeast transfer
timing from propagation tank to fermentation tank is better as timing B, where
glucose is already lost and maltose is assimilated in the propagation wort. In this
experiment, Timing B was the time when Group A amino acid was already lost
and maltose uptake ability began to decline in propagation (figure 10). We thought
that Timing A was the optimal yeast transfer timing from propagation tank to
fermentation tank, because maltose uptake ability was maximum in this point during
propagation, where Group A amino acid had almost completely disappeared.
When Group A amino acid had almost completely disappeared, maltose uptake
ability was maximum, which was similar to the change during fermentation.

140
Maltose Uptake Ability
Suspending Yeast (10 cells/ml)
Maltose Uptake Ability (unit)

120
Yeast growth
100
6

80
60
40
20
0
0 10 20 30 40 50 60
propagation period (hr)
Figure 7: Change in maltose uptake ability of yeast cells and time course
of yeast growth during propagation.

7
 Maltose Uptake Ability Glucose
Maltose
Yeast growth
Suspending Yeast (10 cells/ml) 160 1.5 7
Maltose Uptake Ability (unit)

A (b)

Glucose concentration (%)

Maltose concentration(%)
140 (a) 6
B
120
6

5
1.0
100
4
80
3
60 0.5
2
40 A B
20 1

0 0.0 0
0 20 40 60 0 20 40 60
propagation period (hr) propagation period (hr)

Figure 8: (a) Change in maltose uptake ability during propagation and

timing of the harvested yeast in propagation.
(b) Time course of glucose and maltose concentration in
propagation wort.

12

10
Apparent extract(%)

8 Tim ing A
Tim ing B
6

0
0 50 100 150 200
Ferm entation period (hr)

Figure 9: Influence of propagation yeast on the change of the apparent

extract during fermentation.

8
 Maltose Uptake Ability
Group A amino acid
160 1.4

Amino acid concentration (ppm)

Maltose Uptake Ability (unit)
A
140 1.2
120 B
1

100
0.8
80
0.6
60
40 0.4
20 0.2
0 0
0 20 40 60
propagation period (hr)
Figure 10: Time course of Group A amino acid concentration in
propagation wort and timing of the harvested yeast in
propagation.

CONCLUSIONS
1) We established a novel analytical method as an index of maltose uptake ability.
2) The maltose uptake ability was closely related to specific alcohol production
velocity during beer fermentation, and as the maltose uptake ability of pitching
yeast was higher, the fermentation rate was faster.
3) In order to improve the fermentation rate using pitching yeast with higher
maltose uptake ability, we optimized yeast transfer timing from propagation tank
to fermentation tank. It was obvious that the optimal timing was the peak point of
maltose uptake ability, where Group A amino acid had almost completely
disappeared.

REFERENCES
1. Stewart, G.G., Some observation on maltose, fructose, and glucose metabolism
in Saccharomyces cerevisiae, Amer. Soc. Brew. Chem. Proc.1973, 8
2. Kodama, Y, Fukui, N, Ashikari, T and Shibano, Y., Improvement of maltose
fermentation efficiency: Constitutive expression of MAL genes in brewing
yeasts, Amer. Soc. Brew. Chem. 53 (1): 24-29,1995
3. Back, W, Bohak, I and Ackermann, T., Optimierte Hefewirtschaft, Brauwelt nr.
39, 1993

9
31

Use of corn steep liquor to increase the

yield of brewing yeast obtained from
propagation
Behnam Taidi1, H. Gwendoline Mazike2 & Jeff A. Hodgson1
1
Scottish Courage Brewing Ltd., Technical Centre, 160 Canongate, Edinburgh EH8 8DD,
United Kingdom (e-mail: [email protected])
2
Strathclyde University, Department of Biosciences and Biotechnology, 204 George Street, Glasgow
G1 1XW, United Kingdom

Descriptors
Biomass, steep water, yeast growth, yeast propagation, yield

SUMMARY
Yeast propagation in the brewing industry relies predominantly on fermentative yeast growth
and is an inefficient process. This results in an insufficient amount of newly propagated yeast
to pitch a full fermenter and often leads to an aberrant first fermentation. The yield of yeast
was increased by 50% at laboratory scale through nutritional supplementation of wort and
optimisation of yeast growth conditions. This was confirmed at 5 hl pilot scale where the
biomass increased by 50% and the yeast cell concentration doubled. The findings of this work
could be applied to current yeast-propagation vessels without the need for considerable plant
modifications.

Die Verwendung von Weichwasser zur Verbesserung der Hefeausbeute bei der
Propagation

Deskriptoren
Ausbeute, Biomasse, Hefevermehrung, Hefewachstum, Weichwasser

ZUSAMMENFASSUNG
Die Hefepropagation in der Brauindustrie zielt vorwiegend auf das fermentative Hefe-
wachstum ab. Dies ist ein ineffizienter Prozess. Das Ergebnis ist eine unzureichende Menge
neu propagierter Anstellhefe und fhrt oft zu einer ungleichmigen Grung. Die Ausbeute
der Hefe wurde durch Zugabe von Ernhrungsergnzungsstoffen der Wrze und der
Optimierung der Voraussetzungen des Hefewachstums um 50% im Labormastab gesteigert.
Die Ergebnisse wurden im 5 hl-Pilotmastab besttigt, hier wurde die Biomassenausbeute um
50% gesteigert und die Zellkonzentration verdoppelt. Die Erkenntnisse dieser Arbeit knnen
mit heutigen Hefepropagatoren ohne erhebliche Umbauten umgesetzt werden.
Utilisation du jus de macration du bl pour accrotre le rendement de la levure de bire
obtenue par propagation

Descripteurs
Biomasse, croissance de la levure, eau de trempage, propagation de la levure, rendement

RESUME
La propagation des levures dans lindustrie brassicole seffectue en majorit lors dune
croissance fermentative - un procd qui nest pas efficace. Il en rsulte une quantit
insuffisante de levure frachement propage pour ensemencer la totalit dun fermenteur. De
plus, cette premire fermentation est souvent aberrante. Le rendement de levure a t
augment de 50 % lchelle du laboratoire par laddition de nutriments dans le mot et
loptimisation des conditions de croissance pour les levures. Ceci a t confirm lchelle
pilote 5 hl o la biomasse a augment de 50 % et la concentration des cellules de levures a
doubl. Les conclusions de ce travail pourraient tre appliques aux cuves de propagation
habituelles, sans quil soit ncessaire de modifier considrablement le site.

2
INTRODUCTION
The objective of yeast propagation is to produce a sufficient quantity of high viability
brewing yeast, free of contaminating micro-organisms, in a short period of time. The
physiological state of the yeast culture thus produced is also of importance. Newly
propagated yeast has to be fermentatively active and able to produce the correct range
of by-products responsible for the final products distinctive flavour and other quality
attributes.
The yeast propagation process normally commences with either a newly isolated yeast
culture from the brewery or more commonly with a master culture from the
companys culture collection. Increasingly the master culture is kept in liquid nitrogen
storage. The master culture is normally used to produce yeast slopes that can be stored
for short periods or distributed to brewing sites.
Laboratory yeast propagation from starter cultures or slopes takes place through
incrementally increasing volumes of liquid culture, normally involving a high level of
aeration or oxygenation. The lower volume cultures are often in laboratory complex-
media but as the volume increases, brewery wort is used as the growth medium. The
final volume of cultures obtained in the laboratory is usually in the range of 50-60 l.
The yeast obtained from laboratory culture is then introduced into culture vessels,
which vary greatly in size and shape. These are normally located in a specific section
of the brewery and receive wort from the brewhouse. Usually a first vessel with a
volume of 10-20 hl and a second vessel with a volume of 50-100 hl are employed1.
At this stage the aeration or oxygenation and also the propagation temperature are
gradually decreased in order to acclimatise the yeast to brewing conditions.
The yeast culture from the culture plant is often only sufficient to a half-filled
fermentation vessel. The conditions at this stage are brewing conditions, however, the
products of the first fermentations are often atypical and are blended into the product
mainstream. This first fermentation is also often slow and aberrant in nature.
The process described above relies predominantly on fermentative yeast growth and is
an inefficient process for biomass generation. The high concentrations of fermentable
carbohydrate present in wort prevents respirative growth of yeast (Crabtree effect)
even in the presence of high concentrations of dissolved oxygen (DO). The aberrant
nature of the first fermentation, at least in part, can be attributed to the insufficient
amount of yeast produced in the culture plant and the low pitching rate of the first
fermentation.
Provision of DO during yeast propagation encourages some respirative growth but the
main mode of carbohydrate utilisation in wort remains through the fermentative route
of glycolysis followed by ethanol production. Fermentation apart from generating
much less energy also diverts a considerable portion of the carbon present in
fermentable carbohydrates towards ethanol production rather than cell synthesis.
The industries supplying yeast to bakers, and dry yeast to wine makers, have for many
years employed fed-batch fermentation to produce large quantities of respiratively
grown yeast. This truly aerobic method of yeast culture relies on maintaining a
continuously low concentration of fermentable carbohydrates in the growth medium
which is usually based on sugar molasses. This allows carbohydrate metabolism in the
presence of a high DO through respiration (Pasteur effect) which results in a high
yield of yeast with little or no ethanol production.
The propagation of brewing yeast strains through aerobic fed-batch culture is possible
but is not without its disadvantages. Fed-batch propagations require specialised
knowledge which is currently absent from most brewing sites. These propagations
have a very high requirement for oxygen supply and involve critical control of the

3
feeding regime2. This would require major capital expenditure and construction of
new culturing plants. Finally, aerobically propagated yeast would be in a considerably
different physiological state than the yeast obtained from current culture plants. This
would still be likely to result in aberrant first fermentations.
The work described in this paper attempted at increasing the yield of yeast from
current culture plants through nutritional supplementation of wort and optimisation of
growth conditions. The ultimate aim of this work was to significantly increase the
amount of yeast produced in current culture plants without the need for extensive
capital expenditure.

EXPERIMENTAL
Production ale and lager yeasts were used throughout this work in conjunction with
brewery lager wort. Laboratory fermentations were performed at 100 ml scale in 250
ml conical flasks incubated at 27C in a shaking incubator (150 rpm).
The pilot brewery trial was performed at 5 hl scale in the conical section of a 40 hl
vessel. Wort (40% maize grits adjunct) was produced in the pilot brewery in the
normal fashion and was sterilised in the fermenting vessels by boiling. Wort was
always supplemented with additional zinc to a concentration of 0.5 ppm immediately
prior to pitching.
In laboratory experiments, corn steep liquor (CSL) was added to wort, the pH of the
wort was adjusted to 5.5 and the wort was pasteurised (60C;1 h). For the pilot
brewery study, the pH of CSL (10 kg) was adjusted to 5.5 prior to addition to the
wort. The wort was then brought to boil and cooled down to 20C. All pH
adjustments were made using 30% (w/v) NaOH.
In the pilot brewery study wort (10 hl) was divided equally into two vessels. Both
vessels were treated equally except that one vessel received CSL and the other vessel
acted as control. The OG of wort was 14P and a laboratory culture (8 l) of lager yeast
was used to pitch each vessel. Continuous aeration was used throughout the
propagation until cooling was applied so that the yeast could be collected and
measured.
Yeast viability was measured using methylene blue staining. Cell concentrations were
determined by haemocytometry. FAN was measured using a LECO FP428
instrument. Fermentable carbohydrates and glycerol were measured using HPLC.
ABV was measured using a FOSS NIR system. OG and PG measurements were made
using a DMA 55 Anton Paar density meter.

RESULTS AND DISCUSSION

The time course of yeast propagation was determined in shake-flask culture (figures 1
& 2). A batch of wort was inoculated and divided into 8 shake flasks. At intervals
individual cultures were removed from the incubator and analysed. At 27C both
lager and ale propagations were complete within 28 h (figure 1). At this time the PG
of the cultures had reached their minimum value, the yield of yeast in the cultures had
reached its maximum (figure 1) and the viability of both yeast strains was at 97-99%
(figure 2). The budding index of the ale yeast peaked at 22 h but the budding index of
the lager yeast did not appear to peak at any stage of the experiment although it is
possible that a peak had occurred between analysis points.

4
After 28 h incubations both the viability and the yield of the ale and lager yeast strains
decreased. The PG of the cultures rose towards the end of the incubation period,
which could be explained by the decrease in ABV brought on by yeast ethanol
utilisation (data not shown).
Under the culture conditions tested the optimal time for transfer of cultures to the next
stage of propagation was 22-28 h. In this time period the maximum yield of yeast with
highest viability and budding index was reached. The optimal time for culture transfer
to a larger culture volume is thought to be prior to the release of daughter cells.

Figure 1: PG and yeast production profiles during ale and lager yeast propagations

Ale PG Lager PG Ale-yeast yield Lager-yeast yield

15 12

(g dry yeast / l)
10

Yeast yield
10 8
PG (P)

6
5 4
2
0 0
0 20 40 60 80 100

Time (h)

Figure 2: Viability and budding index profiles during ale and lager yeast
propagations

Ale viability Lager viability

Ale budding index Lager budding index

100 80
Budding index
Viability (%)

80 60
60
(%)

40
40
20 20

0 0
0 20 40 60 80 100

Time (h)

The influence wort OG on yeast propagation was investigated in the OG range of 10-
17.5P (table 1). All propagations were performed in duplicate and the cultures were
harvested after 24 h incubation. The yield of both ale and lager yeast strains increased
with increasing OG. The increase in biomass production was accompanied by an
increase in ethanol and glycerol production. The increased biomass production at

5
higher OG values was not due to an increase in the efficiency of the conversion of
fermentable carbohydrates to yeast biomass as the yeast yield-coefficient, calculated
on the basis of fermentable carbohydrates utilisation, was quite constant at all OG
values tested. The increased yeast biomass formation was a result of faster
carbohydrate utilisation at higher OG values. At higher OG values the PG decrease
was greater during the course of the propagations. This could have been due to faster
carbohydrate uptake at higher extracellular concentrations of carbohydrate. An
increase in biomass produced per unit time with increasing OG has been reported
previously3, however, a loss of viability with higher wort OG was reported by the
authors.
The ale yeast FAN utilisation efficiency increased with increasing OG and peaked at
an OG of 15P then decrease slightly at the OG of 17.5P. The FAN utilisation
efficiency for the lager yeast was less clear. The increased biomass production at
higher wort OG values is probably due to the increased nutrient concentration in wort
with a higher OG. A slight decrease in total nitrogen utilisation at higher wort OG has
been reported4.

Table 1: Influence of OG on ale and lager yeast propagations

Yeast OG Final Final Final Yield Fermentable FAN
strain PG ABV glycerol carbohydrate yeast-
yeast-yield yield
coefficient coefficient
P P % g/l g dry g dry yeast per g dry yeast
yeast g carbohydrate per g FAN
per l used used
wort
Ale 10.0 2.75 3.5 0.9 5.9 0.08 61
Ale 12.5 3.75 4.6 1.2 7.6 0.08 61
Ale 15.0 4.25 5.1 1.3 8.7 0.09 66
Ale 17.5 5.25 6.2 1.8 10.7 0.09 64

Lager 10.0 3.00 3.6 0.6 5.8 0.08 60

Lager 12.5 4.25 4.4 0.9 7.1 0.08 57
Lager 15.0 4.75 4.8 1.2 7.9 0.08 59
Lager 17.5 6.75 5.7 1.6 9.1 0.08 56

Corn steep liquor (CSL) is a natural by-product of the sugar-syrup production

industry. The grain, maize or wheat, is steeped in water containing natural endemic
microflora to soften and hydrate the grain prior to chemical treatment. A component
of the microflora is lactic acid bacteria and CST contains lactic acid and other organic
acids. CSL contains many nutrients and is used by the pharmaceutical industry as a
nutritional supplement for fermentation. The batch of CSL used in this work has a
FAN concentration of 10 g/kg.
The effect of CSL on the yield of yeast was tested in laboratory propagations. CSL is
acidic due to the presence of organic acids, therefore the wort pH was always adjusted
to 5.5 after addition of CSL. The effective titration of the organic acids (weak acids)
with NaOH (strong base) considerably increased the buffering capacity of the wort.
This buffering capacity was directly related to the amount of supplement added to the
wort.

6
The addition of CSL to wort significantly increased the yield of yeast (table 2). Ale
yeast production increased by 53% upon supplementation of wort with 19 g/l of CSL.
There was no further yield benefit by increasing the CSL concentration beyond this
concentration. The lager yeast-yield on the other hand was raised by 48% with the
addition of CSL to 19 g/l but increased even further with higher concentrations of
CSL. A 69% improvement in biomass was observed with a CSL concentration of 76
g/l. Yeast viability was high at the end of all propagations. It was noted that
propagation of both yeast strains in the presence of CSL dramatically increased the
flocculence of the yeast.
The increase in biomass production when wort was supplemented with CSL was
caused by better carbohydrate utilisation evident from the fermentable carbohydrate
yeast-yield coefficient data (table 2). Once again ethanol production increased in step
with biomass production indicating that the balance between fementative and
respirative metabolism had not been changed. FAN utlilisation efficiency was lower
when the yeast were supplemented with CSL even though CSL contains relatively
high amounts of FAN. At least part of the stimulatory effect of CSL on yeast biomass
production was due to the increased buffering capacity of wort. Fermented wort pH
values were considerably higher for worts supplemented with CSL. A higher pH
would have allowed a faster and more extensive carbohydrate utilisation.
Supplementation with CSL also demonstrated that the relative proportion of carbon
which is diverted by the yeast cell into biomass generation can be controlled through
nutritional means.

Table 2: Stimulation of the yeast growth through supplementation of wort with CSL
Yeast Initial Final Final FAN Yield Fermentable FAN
strain CSL pH ABV used carbohydrate yeast-
yeast-yield yield
coefficient coefficient
g/l % g/l g dry g dry yeast per g dry yeast
yeast g carbohydrate per g FAN
per l used used
wort
Ale 0 4.0 3.5 0.18 8.3 0.10 45
Ale 19 4.6 5.2 0.33 12.7 0.14 38
Ale 38 4.8 5.8 0.34 12.9 0.12 38
Ale 76 5.1 4.8 0.34 12.1 0.13 36

Lager 0 3.9 4.6 0.18 8.4 0.09 46

Lager 19 4.5 6.1 0.33 12.4 0.12 37
Lager 38 4.9 5.9 0.35 13.6 0.12 39
Lager 76 5.1 6.5 0.38 14.2 0.15 38

The stimulatory effect of CSL on biomass production was tested at pilot scale. A
batch (10 hl) of wort was divided equally between two fermenting vessels. The trial
propagation was supplemented with pH adjusted CSL, both worts were brought to
boil, allowed to cool to 20C and inoculated with the same amount of starter culture
of lager yeast. The cultures were aerated continuously throughout the propagation.
After 67 h, the suspended cell concentration and yeast solids contents of the cultures
were determined. The aeration was then stopped and the cultures were cooled to 2C.

7
After a further 48 h from the time when cooling was initiated, the settled yeast was
separated and the amount of yeast solids in the yeast cone determined.
The trial culture containing CSL yielded 50% more yeast solids and twice as many
yeast cells (table 3). Furthermore, due to the increased flocculation of the yeast in the
presence of CSL it was possible to separate the yeast crop much more effectively and
2.6 times more yeast biomass was obtained from the trial fermentation as was
obtained from the control fermentation. The viability of the yeast was 98-99% for the
two cultures.

Table 3: Pilot scale propagation of yeast using CSL supplementation

Fermentation Final Final Maximum Maximum cell Final yeast
PG pH yeast solids concentration crop collected
P % v/v Million cells Kg wet yeast
per ml solids
Control 1.28 3.3 4 140 6.3
without CSL
Trial with CSL 0.78 4.1 6 290 16.6

At this point in time CSL is not approved for food use. In future, more work will be
carried out to determine the mechanism in which CSL increases biomass production.
Also, the suitability of the yeast, propagated in the presence of CSL, for beer
production remains to be tested too. The increased flocculation characteristics being
of particular importance.

CONCLUSIONS
During batch yeast-propagations at 27C, biomass generation is complete within
22-28 h.
In batch yeast-propagations, ethanol and glycerol production is directly linked to
biomass formation.
In the OG range 10-17.5P, higher biomass concentrations are generated at higher
OG values.
Supplementation of wort with CSL increases yeast biomass production by 50%.
The increase in biomass production is in part due to a more efficient carbohydrate
utilisation and in part due to increased culture-pH stability.
At 5 hl scale supplementation of wort with CSL resulted in a biomass increase of
50%, the doubling of the yeast cell concentration and recovery of 2.6 times more
wet yeast biomass from the culture vessel.

ACKNOWLEDGEMENT
The authors wish to thank the directors of Scottish Courage Brewing Ltd for
permission to publish this article.

REFERENCES
1. Kennedy, A.I., Yeast handling in the brewery. Brewing Yeast Fermentation
Performance. Ed. Katherine Smart, Blackwell Science, 2000, 129-134.

8
2. Masschelein, C.A., Borremans, E. and van de Winkel, L., Application of
exponentially fed batch cultures to the propagation of brewing yeast, Proc. Inst.
Brew. (Asia Pacific Conv.), 23, 104-108.
3. Cahill, G., Murray, D.M., Walsh, P.K. and Donnelly, D., Effect of the
concentration of propagation wort on yeast cell volume and fermentation
performance, J. Am. Soc. Brew. Chem., 2000, 58 (1), 14-20.
4. Takahashi, S., Yoshioka, K., Hashimoto, N. and Kimura, Y., Effect of wort plato
and fermentation temperature on sugar and nitrogen compound uptake and
volatile compound formation, MBAA TQ, 1997, 34 (3), 156-163.

9
32

The management of brewing yeast stress

Katherine A. Smart
Oxford Brookes University, School of Biological and Molecular Sciences, Headington,
Oxford OX3 0BP, United Kingdom (e-mail: [email protected])

Descriptors
Brewers' yeast, deterioration, genetics, physiology, process control, yeast propagation

SUMMARY
Brewing yeast is subjected to several biological, chemical and physical stresses during
propagation and handling. It is therefore suggested that process developments require an
understanding of yeast responses to changing environments. Exposure to stresses that affect
yeast quality permits the identification of potential biomarkers of cellular deterioration. The
applications of the methods used to assess these characteristics as diagnostic, process
management and quality assurance tools is considered. In addition, the importance of new
techniques such as DNA microarray expression profiling in the understanding of the yeast
stress response will be discussed.

Der Umgang mit Hefestress

Deskriptoren
Bierhefe, Genetik, Hefevermehrung, Physiologie, Prozesteuerung, Verderb

ZUSAMMENFASSUNG
Brauhefe ist verschiedenen biologischen, chemischen und physikalischen Belastungen
ausgesetzt. Es ist ratsam, die Erkenntnisse aus dem Verhalten der Hefe auf vernderte
Umgebungen in die Prozessentwicklung einflieen zu lassen. Belastungen, welche die
Hefequalitt beeinflussen, ermglichen die Erkennung von zellulren Vernderungen. Es
werden die Diagnostikmethoden, die auf diesen Prinzipien beruhen, vorgestellt. Weiterhin
werden Anwendungen fr das Prozessmanagement und die Qualittssicherung behandelt.
Zustzlich wird die Bedeutung neuer Techniken, wie die DNA-Microarray-Expression, fr
das Verstndnis des Bierhefeverhaltens unter Belastung diskutiert.

1
Comment grer les stress s'exerant sur la levure de bire

Descripteurs
Commande de procd, dtrioration, gntique, levure de brasserie, physiologie,
propagation de la levure

RESUME
La levure de bire est soumise de nombreux stress physiques, chimiques et biologiques
pendant sa propagation et son traitement. Nous suggrons donc que le dveloppement des
mthodes exige une bonne comprhension des ractions de la levure un environnement
toujours changeant. L'exposition des stress affectant la qualit des levures permet
d'identifier des biomarqueurs potentiels de la dgradation cellulaire. Les applications des
mthodes utilises pour valuer ces caractristiques, comme le diagnostic, la gestion des
mthodes et l'assurance qualit, sont considres. Cette confrence discute en outre de
l'importance des nouvelles techniques (comme le profilage d'expression des microrseaux
d'ADN) pour la comprhension des ractions de la levure aux stress.

2
INTRODUCTION
It is accepted that repeated exposure to stress during yeast handling and fermentation
often results in the occurrence of deteriorated and dying cells, however, the processes
involved in the transition from life to death have not been fully elucidated (16). One
reason for this is that although individual cell deaths regularly occur they are often
masked by the replication of surviving cells or represent a minimal reduction in
overall population viability (14). Thus brewing yeast slurries are considered to exhibit
a resilience to yeast handling stresses, but in reality cell death is a common
occurrence.
Mortality in yeast involves two primary pathways known as senescence and necrosis
(7,13) which result in the transition from live and healthy to dead cells (figure 1).
Senescence occurs in the absence of stress and involves the deterioration of the cell
following a predetermined number of divisions known as the Hayflick Limit (5). For
brewing yeast the Hayflick limit is strain dependent within the range of 9-33 divisions
(7,8) with chain-forming strains exhibiting reduced replicative longevities (Powell,
Quain and Smart, unpublished data). Biomarkers of increasing replicative age include
an increase in mother cell size and the accumulation of bud scars and wrinkles. On
completion of the final division senescence occurs in which mother cells lose
replicative capacity exhibiting a non-culturable but viable phenotype which leads to
cell lysis (figure 2).
Completion of brewing yeast replicative lifespan may be temporarily hindered or
permanently prevented by the occurrence of physiological stress (table 1). Exposure
to yeast handling stresses may lead to necrosis involving the deterioration of cellular
components. Initially this deterioration is reversible but continued exposure to stress
may cause irreparable damage to cellular components leading to the irreversible loss
of replicative capacity (figure 3). In this case as with senescence a viable but non-
culturable state is achieved in which replication capacity is permanently lost leading
eventually to cell death and lysis.

Fermentation Cropping and Storage Vessel Acid Pitching

Transfer Washing

Ethanol Cold Shock Shear PH Osmotic

Starvation Shear Cold Shock Cold Free
Lethal Anaerobiosis Starvation Shock Radicals
DNA Anaerobiosis
Damage
(Suicide
Mutations)
Hydrostatic
Pressure
CO2
Table 1: Summary of stresses that may lead to necrosis during brewery yeast
handling (Smart, 16).

3
 Healthy
HealthyLive
LiveCell

Senescenc
Senescence Necrosis

Dead Cell
Dead Dead Cell

Figure 1: Transitional Pathways From Figure 2: The Replicative Lifespan in

Life to Death Brewing Yeast. Numbers indicate
divisional age. A indicates lysed
senescent cell and B its terminal
daughter.

Cell Replication Stress Stress Response

G0 Entry

Lifespan Deteriorated
Progression

Senescence Viable Non-

Non - culturable culturable

Death

Figure 3: Schematic demonstrating the major phenotypes associated with the

transition from life to death through the senescence and necrotic pathways.

MATERIALS AND METHODS

Yeast strains and growth conditions
Strain Type Morphology Source
W303a Haploid Discrete Oxford Brookes University Culture
Collection
NCYC Ale Discrete National Collection of Yeast Cultures,
2593 Norwich, UK
Table 2: Haploid and Brewing Yeast Strains utilised or cited

4
Stock cultures were maintained on standard agar slopes consisting of 1% yeast
extract, 0.5% neutralised bacteriological peptone, and 1 2 % glucose solidified with
1.5% agar (w/v). All media were autoclaved immediately after preparation at 121C
and 15 psi for 15 min. Yeast cell populations were grown aerobically at 25C by
shaking in an Erlenmeyer flask (250 ml) at 120 rpm.

Lifespan potential and age synchronised populations

Yeast replicative lifespan was determined according to the method of Barker and
Smart (2).

Scanning electron microscopy

Cell surface topography was determined using scanning electron microscopy
according to the method of Barker and Smart (2).

Viability and replicative potential

Viability was monitored using citrate buffered methylene violet according to the
method of Smart et al (18). Replicative potential was determined using standard plate
counts on YPD.

Vacuole fragmentation
Vacuole fragmentation was determined using the Safranin O Methylene Blue double
staining technique according a modified method (9).

Surface charge
Cell surface charge was measured using either the amine-modified latex bead assay,
according to the method of Rhymes and Smart (11) or the modified alcian blue dye
retention assay according to the method of Rhymes and Smart (10) as appropriate.

Surface hydrophobicity
Cell surface hydrophobicity was measured using either the solvent partition assay
according to the method of Smart et al (17) or the hydrophobic latex microsphere
attachment assay, according to the method of Rhymes and Smart (11).

RESULTS AND DISCUSSION

Starvation stress and reversible cellular deterioration
In the event of exposure to starvation stress the preliminary response is the immediate
decrease in cyclic AMP levels which result in cell cycle progression arrest in G1 and
entry into G0. This response is fully reversible on exposure to nutrients. In G0
stationary phase metabolism is initiated and involves the utilisation of intracellular
carbohydrates (12) which can be indirectly measured using the modified acidification
power test in which the passive proton efflux is determined. During this initial phase
of starvation stress cell wall carbohydrates are also utilised as indirectly demonstrated
by the modifications in cell surface physical properties (10, 11, 12, 16) including a
reduction in cell wall thickness.

Starvation stress and irreversible deterioration

The duration of starvation stress which culminates in the development of irreversibly
deteriorated phenotypes is strain dependent. However, for the lager strain KS1 this
was achieved within only 24 hours of nutrient depletion and was indicated by an

5
irreversible reduction in flocculation capacity (11). The impaired performance was
retained by the yeast slurry through several serial fermentations indicating phenotypic
inheritance of wall characteristics. In addition to this observation it has recently
been demonstrated that although starvation may temporarily interrupt progression
through the cell cycle (13, 14) the transistion from the necrotic pathway to the
senescent pathway may not be fully reversible (figure 3). Ashrafi et al, (1)
demonstrated that long term starvation followed by re-exposure to nutrients may lead
to an impaired replicative longevity (table 3).

Duration of Starvation (Days) Hayflick Limit (Divisions)

1 27
5 22.5
13 20
25 18
33 16
Table 3: The impact of starvation stress on longevity potential in Saccharomyces
cerevisiae (adapted from Ashrafi et al, 1).

Stress and the viable but non-culturable phenotype

In addition to impaired longevity potential, long term starvation can lead to the viable
but non-culturable phenotype in which cells exhibit a profusely wrinkled cell surface
(figure 4) and cannot re-enter the cell cycle to divide (figure 5). Loss of replicative
capacity in yeast without loss in intracellular reducing power as demonstrated using
citrate buffered methylene violet has not been previously reported in yeast but is also
demonstrated by senescent cells. The commonality in the near death phenotypes
from both the senescent and necrotic pathways implies a partially shared death
metabolism. In addition, the impact of a viable but non-culturable phenotype on
brewery fermentations is not known, however, current methods employed to measure
pitch viability would not differentiate between cells exhibiting this physiology and
those which were capable of division.

Cold shock and the yeast stress response

The viable but non-culturable phenotype may also be induced by exposure to cold
shock during incubation on YPD plates at 4C. However, in this case, the viable but
non-culturable phenotype has only been observed for newly formed daughter or virgin
cells and not mother cells irrespective of divisional age (unpublished observation
Smart, Barker, Van Zandycke, Powell and Maskell). Cold shock induced virgin cell
deterioration has been observed in several brewing and laboratory yeast strains and
appears to be a universal phenomenon. The reason for this response is not known but
implies that the stress tolerance of yeast may be a function of replicative age.
It is suggested that the occasional occurrence of small non-viable cells in yeast
slurries may be due to virgin cell death following exposure to cold shock during slurry
storage.
In addition to the induction of a viable but non-culturable phenotype, cold shock
results in several other phenotypic modifications to the outer cell envelope and
internal membranes. Increased polyunsaturated membrane fatty acids, a decline in
membrane sterols and impaired membrane fluidity occur leading to impaired
membrane function. The vacuolar membrane (tonoplast) is rapidly affected leading to
the formation of pro-vacuoles through a process known as vacuolar fragmentation.

6
Incubation at 4C promotes pro-vacuole formation in the brewing ale strain NCYC
2593 (figure 6) supporting the observations of Fargher and Smart (4). Whilst this
fragmentation is reversible, it can be initially exacerbated during cold shock recovery
by exposure to oxygen. In the brewing context potentially cold shocked slurry is
pitched into oxygenated wort initiating an oxidative burst and the generation of
reactive oxygen species including the hydroxyl radical. Hydroxyl radicals (OH) also
damage membranes through a process known as lipid peroxidation (figure 7)
promoting membrane damage and vacuolar fragmentation (15).
Finally cold shock induces modifications in cell wall composition and architecture as
demonstrated by the impact of incubation temperature during storage on cell surface
charge and hydrophobicity (figure 8) following 24 and 48 hours incubation
respectively. Although these modifications were reversible the reasons for the
changes observed are not known. However, cold shock induces both global (STRE
activated) and specific (non-STRE activated) stress genes including a cluster encoding
serine rich proteins (SRPs) (3,6). These are considerably shorter than flocculation
lectins (table 4) and exhibit no sequence homology FLO genes. Their role is largely
undefined yet it appears that they interact with the cell wall B glucan and it is
suggested that their role may involve the anchoring of the cell wall to cold shock
impaired plasma membrane.
Furthermore it is suggested that the incorporation of these serine rich peptides into the
cell wall promotes modified cell wall turnover resulting in the modifications to the
yeast cell wall surface characteristics observed.

TIP1 FLO1
Serine Rich Repeats Serine Threonine Rich Repeats
Transmembrane GPI Anchor Transmembrane GPI Anchor
Function: B Glucan Binding to Function: Flocculation Lectin
Membrane (?)
254 amino acids 1537 amino acids
Table 4: Cell wall protein function

CONCLUSIONS
Brewing yeast exhibit mortality either as a result of necrosis or in the absence of
stress as a result of senescence. Immediately prior to death cells exhibit a viable but
non-culturable phenotype in which they cannot re-enter the cell cycle but are able to
reduce bright-field stains. During necrosis this phenotype and other reversible stress
phenotypes can be induced following starvation and cold shock, though the extent of
deterioration incurred may be related to replicative age.

7
 A B

Figure 4: The impact of extreme starvation stress on the wall phenotypic

characteristics of the ale brewing yeast strain NCYC 2593.

100

80
Viability (%)

60 CMV
40 S PC

20

0
0 1 2 3 4 5 6 7 8 9 10 11 12
Time (Months)

Figure 5: The effect of extreme starvation stress on the replicative potential of the
brewing ale yeast strain NCYC 2593. Demonstration of the viable but non-
culturable phenotype.

A B
Figure 6: Vacuole integrity following incubation at (A) 10 and (B) 4 for the ale
brewing yeast strain NCYC 2593.

8
 OH
O Impaired Membrane Function
Decreased Fluidity
Peroxyl Radical
Inactivation of Membrane
H Bound Receptors and Proteins

Lipid
Peroxidation

Figure 7: Lipid Peroxidation and Vacuolar Fragmentation (adapted from Smart,15)

30 80
70
25
60
20 50
15 4
40
10
10 30
25 20
5
10
0
0 24 48 72 0
0 24 48 72
Time (hours)
Time (hours)

A B
Figure 8: The impact of cold shock on the cell surface physical properties (A) Charge
and (B) Hydrophobicity of NCYC 2593. Cell populations were incubated at
4, 10 and 25C. Adapted from Rhymes and Smart (11).

STRE TIP1, TIP2 NSR1

TIR1, TIR2 (SRPs)

Trehalose Oxidative Stress Heat Shock Serine Ribosome

Metabolism Response Rich Biogenesis
Peptide

Membrane CTT HSP12 Wall Function (?)

Stability UB14 HSP104
DDR2

Figure 9: The cold shock stress response.

9
ACKNOWLEGMENTS
The author gratefully acknowledges those whose work she has cited and in particular,
Maureen Rhymes, Gordon Barker, Sylvie Van Zandycke, Chris Powell and Dawn
Maskell. The author is the Scottish Courage Reader in Brewing Science and a Royal
Society Industrial Fellow and gratefully acknowledges the support of Scottish
Courage Breweries Limited and the Royal Society accordingly.

REFERENCES
1. Ashrafi, K., Sinclair, D., Gordon, J. and Guarente, L., Proceedings of the
National Academy of Sciences, USA, 1999, 96, 9100-9105.
2. Barker, M.G. and Smart, K.A., Journal of the American Society of Brewing
Chemists, 1996, 54 (2),121-126.
3. Donzeau, M., Molecular Microbiology, 1996, 20, 449-459.
4. Fargher, J. and Smith, N.A., Proceedings of the European Brewing Convention,
Brussels, 1995, 345-352.
5. Hayflick, L., Experimental and Cellular Research, 1965, 37, 614-636.
6. Kowalski, L.R., Molecular Microbiology, 1995, 15, 341-353.
7. Powell, C.D., Van Zandycke, S.M., Quain, D.E. and Smart, K.A., Microbiology,
2000, 146, 1023-1034.
8. Powell, C.D., Quain, D.E. and Smart, K.A., In Brewing Yeast Fermentation
Performance pp 114-118. Edited by K.A. Smart, Blackwell Science Limited,
2000.
9. Rhymes, M.R., PhD Thesis, 1999.
10. Rhymes, M.R. and Smart, K.A., Journal of the American Society of Brewing
Chemists, 1996, 54 (1), 50-56.
11. Rhymes, M.R. and Smart, K.A., Journal of the American Society of Brewing
Chemists, 2001, 59, 32-38.
12. Smart, K.A., Monograph EBC Symposium on Brewers Yeast Immobilisation,
1995, 24, 146-155.
13. Smart, K.A., Brewers Guardian, 1999, 128 (2), 19-25.
14. Smart, K.A., In Brewing Yeast Fermentation Performance pp 105-113. Edited
by K.A. Smart, Blackwell Science Limited, 2000.
15. Smart, K.A., Proceedings of the Jean De Clerck Chair, 2000.
16. Smart, K.A., Proceedings of the Institute and Guild of Brewing African Congress,
in press.
17. Smart, K.A., Boulton, C.A., Hinchliffe, E. and Molzahn, S., Journal of the
American Society of Brewing Chemists, 1995, 53 (1): 33-38.
18. Smart, K.A., Chambers, K.M., Lambert, I., Jenkins, C. and Smart, C.A., Journal
of the American Society of Brewing Chemists, 1999, 57, 18-23.

10
33

Does osmotic pressure affect yeast

performance in high gravity
fermentation?
John Hammond, Daniel Davis, Mark Lee & Kim Storey
Brewing Research International, Lyttel Hall, Nutfield RH1 4HY, United Kingdom (e-mail:
[email protected])

Descriptors
Ethanol, fermentation inhibitor, high gravity brewing, osmosis, yeast

SUMMARY
Current demands for increasing fermenter productivity have stimulated interest in using worts
of high gravities which increase the osmotic pressure to which yeast is subjected during
fermentation. This has been reported to have a negative effect on yeast fermentation
performance. Our studies indicate that osmotic pressure does not have significant inhibitory
effects on fermentation but that the effects observed are due to the accumulation of inhibitory
metabolites, especially ethanol. Consequently future efforts to develop very high gravity
fermentation processes must concentrate on the nutritional status of wort and the likely
protective effects against ethanol toxicity which such nutrients provide.

Hat der osmotische Druck Einfluss auf die Hefe bei der High-Gravity-Grung?

Deskriptoren
thanol, Brauen mit hohem Stammwrzegehalt, Grungsinhibitor, Hefe, Osmose

ZUSAMMENFASSUNG
Laufende Forderungen zur Verbesserung der Grbehlterauslastung haben das Interesse an
High-Gravity-Wrzen geweckt, die den osmotischen Druck auf die Hefe erhhen. Es wird
behauptet, dass dies einen negativen Einfluss auf die Hefefermentation hat. Unsere
Untersuchungen zeigen, dass der osmotische Druck keinen signifikant hemmenden Einfluss
auf die Grung hat, jedoch auf die Anreichung von Metaboliten, besonders Ethanol.
Zuknftige Untersuchungen zur Entwicklung sehr starker High-Gravity-Vergrungen mssen
sich auf den Nahrungsstatus der Wrze und die mglichen schtzenden Effekte von
Nahrungsbestandteilen gegen die Alkoholtoxizitt konzentrieren.
La pression osmotique influence-t-elle les performances de la levure dans la
fermentation haute?

Descripteurs
Ethanol, fabrication de bire haute densit, inhibiteur de fermentation, levure,
osmose

RESUME
Les exigences rcentes d'une meilleure productivit des fermenteurs ont stimul l'intrt pour
les mots de haute densit qui augmentent la pression osmotique laquelle la levure est
soumise pendant la fermentation. Cela exerce un effet ngatif sur les performances de
fermentation de la levure. Nos tudes montrent que la pression osmotique n'a aucun effet
inhibiteur significatif sur la fermentation, mais que les effets observs proviennent de
l'accumulation de mtabolites inhibiteurs, notamment d'thanol. En consquence, les futurs
travaux visant dvelopper des procds de fermentation trs haute gravit doivent se
concentrer sur l'tat nutritionnel du mot et les ventuels effets protecteurs contre la toxicit
thanolique qu'apporte de tels nutriments.

2
INTRODUCTION
Osmotic pressure is the force present when two solutions of different molar
concentrations are separated by a semi-permeable membrane. Thus yeast suspended in
wort is subjected to an osmotic pressure, due to the different concentrations of
intracellular and wort solutes. However, as the cell membrane is freely permeable to
ethanol, beer does not exert as great an osmotic pressure on yeast cells, since the
concentrations of intra- and extra-cellular ethanol are equivalent. High concentrations
of ethanol do however affect water activity (aw) which is a measure of water
availability (5). Solute molecules of a low molecular weight such as ethanol are
energetically attracted to water. The strength of this attraction determines how
available water is to the cell. Thus high concentrations of both sugars and ethanol can
increase the water stress exerted on yeast cells suspended in wort and beer.

Current demand to intensify the brewing process and increase fermenter productivity
has stimulated interest in using worts of higher gravities. A consequence of increasing
wort specific gravity is to increase the initial osmotic pressure and to reduce the final
water activities to which yeast is subjected during fermentation.

There are many reports concerned with the negative effects of high sugar
concentrations on yeast fermentation performance. A number of authors (1,4,6,7,8)
have concluded that high sugar concentrations inhibit yeast fermentation performance
due to the high osmotic pressures exerted. However, the levels of sugars required to
induce such inhibition are too high (typically 30-40P) to be commercially significant.
In this study the effects of sugar concentration on yeast growth rate and fermentation
rate have been assessed to examine the relevance of osmotic pressure and reduced
water activity as potential inhibitors of fermentation. Direct additions of ethanol and
sorbitol to a fermenting culture of yeast have also been used as a model system to
compare the effects of osmotic pressure and ethanol toxicity.

MATERIALS AND METHODS

Yeast strain
The lager strain NCYC 1324 and the ale yeast strain NCYC 1681 were used.
Permanent stocks were kept at -196C in liquid nitrogen, with working cultures
maintained on MYGP (0.3 g/l malt extract, 0.3 g/l yeast extract, 1 g/l glucose, 0.5 g/l
peptone) agar slopes at 4C.

Fermentation media
Standard 16P lager wort (92% malt extract, 8% very high maltose syrup adjunct,
12.5 g/hl liquid CO2 hop extract, 20 g/hl hop pellets, 0.22 mg/l zinc sulphate)
produced in the BRI pilot brewery, and a synthetic medium (100-250 g/l maltose; 13.4
g/l yeast nitrogen base; 6.62 g/l ammonium phosphate dibasic; 0.215 g/l CaSO4; 4.2g/l
citric acid pH 5.2) were used.

Fermentation culture preparation

A loopful of yeast was used to inoculate 10 ml MYPG broth. The yeast was grown
overnight with shaking at 25C and was then transferred into the appropriate
fermentation medium and again grown overnight with shaking (150 rpm) at 25C.
This culture was finally transferred into 900 ml of the appropriate fermentation

3
medium, and grown static at 25C overnight. All flasks used for propagation were
stoppered with cotton wool bungs. Cultures were harvested by centrifugation (3000
rpm, 15 min, 4C) and resuspended in wort or synthetic medium as appropriate to
give the desired pitching rate.

Fermentation
2 l bioreactors (FT Applikon, Tewkesbury, UK) were used. Media were oxygen-
saturated prior to pitch. Fermentations were carried out at 18C, and stirred at 200
rpm unless otherwise stated. Samples were taken at regular intervals and measured for
specific gravity (SG), cell count, viability, and dry weight. pH and dissolved oxygen
concentration were measured on-line. CO2 evolution was measured using a Sierra
Instruments Series 820 Top-TrakTM flow monitor, calibrated for a 0-50 ml/min CO2
flow. SG was measured using a Kyoto Instruments DA-300 Specific Gravity Meter.
Cell counts and viability measurements were performed as described in the Institute
of Brewing Methods of Analysis. Yeast dry weight was measured using 5 ml samples
of centrifuged yeast (3000 rpm, 5 min, 4C), washed in deionised water and placed at
105C until a constant weight was achieved.

Analysis
Higher alcohols and esters were measured using a 60 m x 0.25 mm internal diameter
CP Wax S7-CB (WCOT) fused silica column in a Perkin Elmer Autosystem XL Gas
Chromatograph fitted with an HS40 Headspace Analyser and a flame ionisation
detector (FID). Ethanol was measured using a 15 m x 0.53 mm internal diameter HP-
Wax cross-linked polyethylene glycol column in a Hewlett Packard 5890A Gas
Chromatograph fitted with an HP6890 Series Injector and an FID. Glycerol was
measured using a Waters HPLC system fitted with an Aminex HPX-87H+ (300mm x
7.8 mm internal diameter) column (BioRad Laboratories) operating at 60C. The
mobile phase was 0.01 M H2SO4 pumped through the column at 0.6 ml/min.
Detection was by refractive index.

RESULTS
Effects of increasing maltose concentration on yeast performance
Fermentations using the ale yeast NCYC 1681 were carried out in a synthetic medium
at 18C. Four fermentations were performed, with maltose concentrations of 100 g/l,
150 g/l, 200 g/l, and 250 g/l. Figures 1, 2 and 3 show respectively the SG, biomass
and ethanol production profiles at each maltose concentration.

The SG profiles (figure 1) show very similar fermentation rates during the initial
phases of fermentation (up to ca. 70 hrs). The average specific sugar uptake rates
(measured over the first 70 hrs of fermentation) were similar in all four fermentations
(table 1). Essentially the same results were obtained with lager yeast NCYC 1324
(table 1). Biomass (figure 2) and ethanol (figure 3) production profiles also showed
little difference in rates of production during the initial phase of fermentation. These
data indicate that the initial sugar concentration does not significantly affect the initial
rates of fermentation.

4
 25
100g/l maltose
150g/l maltose
20
200g/l maltose
250g/l maltose
15
SG (P)

10

0
0 50 100 150 200

Time (hrs)

Figure 1: Fermentation profiles of yeast NCYC 1681 in media containing a range of

maltose concentrations

Yeast strain Average specific sugar uptake rate

(g/g/hr) over initial 70hrs of fermentation
100 g/l 150 g/l 200 g/l 250 g/l
maltose maltose maltose maltose
NCYC 1324 0.49 0.55 0.55 0.50
NCYC 1681 0.31 0.33 0.31 0.32

Table 1: Specific sugar uptake rates measured in fermentations containing different

concentrations of maltose over the first 70 hrs of fermentation.

12
100g/l maltose

10 150g/l maltose
200g/l maltose
Dry weight (g/l)

8 250g/l maltose

0
0 50 100 150 200

Time (hrs)

Figure 2: Biomass production by yeast NCYC 1681 during fermentation of media

containing a range of maltose concentrations.

5
 12
100g/l maltose
10 150g/l maltose
200g/l maltose
8
Ethanol (%v/v)

250g/l maltose

0
0 50 100 150 200 250
Time (hrs)

Figure 3: Ethanol production by yeast NCYC 1681 during fermentation of media

containing a range of maltose concentrations.

Since osmotically stressed Saccharomyces cerevisiae cells produce glycerol to

provide protection against inhibitory environmental conditions, glycerol production
throughout fermentation was also analysed. Figure 4 shows the glycerol production
profile in NCYC 1681 trial fermentations. At all maltose concentrations, the glycerol
production rates were identical for the first 70 hrs of the fermentation, and only
differed in final concentration, presumably due to the excess produced as a by-product
of glycolytic metabolism. This profile again suggests that there was no significant
osmotic stress exerted on yeast fermenting at high maltose concentrations.

7
100g/l maltose
6 150g/l maltose
200g/l maltose
5
250g/l maltose
Glycerol (g/l)

0
0 50 100 150 200 250
Time (hrs)

Figure 4: Glycerol production by yeast NCYC 1681 during fermentation of media

containing a range of maltose concentrations.

This was further supported by analysis for a number of key flavour compounds.
Figures 5 and 6 show the production profiles in fermentations using NCYC 1681 of
ethyl acetate and isoamyl alcohol, respectively. Again the same pattern emerged, with

6
no differences in production patterns at different initial maltose concentrations during
the first 70 hours of fermentation. If initial sugar concentration exerted an osmotic
pressure related inhibition effect on yeast metabolism, one would expect this
inhibition to be evident in the production of primary and secondary metabolites. Our
data suggest that the rates of yeast metabolite production rate were not significantly
inhibited by the initial sugar concentration.

35
100g/l maltose
30 150g/l maltose
Ethyl acetate (mg/l)

25 200g/l maltose
250g/l maltose
20

15

10

0
0 50 100 150 200 250
Time (hrs)

Figure 5: Ethyl acetate production by yeast NCYC 1681 during fermentation of

media containing a range of maltose concentrations.

180
100g/l maltose
160
150g/l maltose
140
Isoamyl alcohol (mg/l)

200g/l maltose
120 250g/l maltose
100
80

60

40
20

0
0 50 100 150 200 250

Time (hrs)

Figure 6: Isoamyl alcohol production by yeast NCYC 1681 during fermentation of

media containing a range of maltose concentrations.

Inhibition of fermentation rates was noted towards the end of fermentation of the
higher maltose concentrations. This was probably due to the presence of high
concentrations of ethanol in the culture at this stage. The growth and fermentation
rates of the yeast strains used in this study are inhibited by ethanol concentrations in
excess of ca. 8% v/v. Although ethanol was the most likely cause of inhibition of
growth and fermentation, other yeast metabolites could have been responsible.
Therefore the effects of secondary metabolites on the growth of NCYC 1324 were

7
examined by plating on MYGP agar containing the following concentrations of yeast
metabolites:
Isoamyl alcohol 0 450 mg/l
Isoamyl acetate 0 6 mg/l
Iso-butanol 0 120 mg/l
Ethyl acetate 0 120 mg/l
Acetaldehyde 0 150 mg/l

The higher concentrations tested were 5 10 fold greater than the levels normally
produced by this yeast strain during conventional lager fermentation and cover the
concentration ranges found in the fermentations described here. No effect on yeast
growth was noted.

Comparison of the effects of addition of ethanol or sorbitol on fermentation rate

Two 16P wort fermentations were carried out using the lager yeast NCYC 1324.
The pitching yeast cultures originated from the same propagation. The fermentations
were maintained at 12C, and stirred at 200 rpm until steady CO2 evolution was
observed. Up to this point, both fermentations had followed identical profile patterns
for SG drop, CO2 output and yeast dry weight.

CO2 (mls/min)
10

Sorbitol
8 addition

Ethanol
addition
2

0
55 60 65 70 75 80 85
Time (hrs)

Figure 7: Comparison of the effects on CO2 production of adding ethanol or sorbitol

to a fermenting culture of yeast NCYC 1324. Times of addition are indicated by the
arrows.

At this time, additions of 20 ml ethanol (fermenter 1) or 40 g sorbitol (fermenter 2)

were made hourly for 8 hours, and the effect on CO2 production rate monitored.
Figure 7 shows the CO2 production profiles in both fermenters following additions of
ethanol or sorbitol. The response of the fermenting cultures to ethanol was different
from the response to sorbitol. Ethanol additions resulted in a characteristic response.
CO2 production rates fell in a step-wise manner in immediate response to ethanol
addition and when 16% ethanol had been added no CO2 production could be
measured. Sorbitol addition resulted in a different CO2 production profile. Over the
addition period of 8 hours, some reduction in CO2 production rate was observed for
the first 3 hours. However, CO2 production rate subsequently recovered to the

8
previously measured maximum rate, indicating that the yeast had acclimatised to the
presence of high concentrations of sorbitol over a short period, and that the osmotic
pressure did not represent a significant long-term inhibitor. It is clear from these
results that the effect of sorbitol (and thus osmotic pressure) inhibition was relatively
small and different from the inhibition induced by ethanol.

DISCUSSION
The deleterious effects of increasing wort sugar concentrations on brewing yeast
performance have been discussed for many years. This study has demonstrated that
only minor inhibitory effects on yeast metabolism are caused by an increase in
medium sugar concentration up to 250 g/l maltose. In general, inhibition of
fermentation rates was only observed in the later stages of fermentation when ethanol
concentrations had risen to levels that can account for the inhibition seen.

A comparison of the effects of ethanol and sorbitol on CO2 production rate have
highlighted that, unlike ethanol toxicity, osmotic pressure exerted by sorbitol does not
markedly affect yeast fermentation rate over an extended period of time. Analysis of
the production profiles of key flavour compounds has also demonstrated little effect
of sugar concentration on yeast secondary metabolism. Crucially the glycerol
production profiles (figure 4) show that the rate of production of this key marker
solute for osmotic stress does not vary with sugar concentration during the initial
phases of fermentation. Greater final glycerol concentrations at higher sugar
concentrations are probably due to higher fermentable sugar concentrations ultimately
leading to the production of greater concentrations of all glycolytic end products,
including glycerol.

Our data differ from other reports indicating effects of osmotic pressure on
fermentation rate (6,7,8) possibly because of the medium used. We chose a synthetic
medium in order to manipulate sugar concentration without altering the concentration
of other medium nutrients. Most studies into the effects of high sugar concentrations
in brewing fermentations have used wort supplemented with sugar syrups. This has
the effect of reducing the concentration of other wort components such as minerals,
vitamins and free amino nitrogen and so introduces additional variables into the
experimental design. A key difference between the synthetic medium used here and
brewers wort is the concentration of free amino nitrogen. Levels were very high
(ca.1.2 g/l) compared to levels found in wort (ca. 250 mg/l in our 16P worts). This
concentration was used to ensure that nutritional deficiencies did not lead to inhibition
that could be mistaken for osmotic pressure effects. This may further explain our
results since others (2,3,8) have previously reported that very high gravity
fermentation is possible, provided yeast is supplied with sufficient nutrients.

CONCLUSIONS
Investigations into the effects of solute concentration on yeast growth and
fermentation rate have been carried out. Growth and fermentation rates were not
significantly affected by initial maltose concentrations over the range 100250 g/l.
Similarly, initial rates of production of glycerol and secondary metabolites were not
significantly different in fermentations at the four experimental maltose

9
concentrations. This indicates that the yeast strains used were not inhibited by either
the osmotic pressure or by the reduced water availability caused by high sugar levels.
Sorbitol additions to fermenting cultures had only transient effects on CO2 evolution,
whereas ethanol addition significantly reduced CO2 production rate. We propose that
fermentation and growth inhibition in very high gravity fermentations is not due to
high osmotic pressures induced by fermentable sugar, but is due to the build-up of
high concentrations of inhibitory metabolites, most likely ethanol which is known to
inhibit growth and fermentation rate at concentrations as low as 2% v/v.

Future efforts to develop high gravity fermentation processes will benefit from
investigating the nutritional status of the wort and the likely protective effects against
ethanol toxicity which such nutrients provide. Most high gravity processes involve the
addition of significant proportions of sugar adjunct, which has the effect of reducing
the concentration of other already limited wort nutrients. High gravity fermentation
will be facilitated by a thorough understanding of the effects of nutrient supply and/or
the development of ethanol-tolerant yeast strains, rather than by focussing on what
appear to be less significant osmotic stress inhibition effects elicited by high sugar
concentrations.

ACKNOWLEDGEMENT
The authors thank the Chief Executive Officer of Brewing Research International for
permission to publish.

REFERENCES
1. Casey, G.P. & Ingledew, W.M., Journal of the American Society of Brewing
Chemists, 1983, 41, 148-152.
2. Casey, G.P., Magnus, C.A. & Ingledew, W.M., Applied and Environmental
Microbiology, 1984, 48, 639-646.
3. Casey, G.P., Chen, E.C.H. & Ingledew, W.M., Journal of the American Society
of Brewing Chemists, 1985, 43, 179-182.
4. DAmore, T., Celotto, G. & Stewart, G.G., Proceedings of the European Brewery
Convention Congress, Lisbon, 1991, 337-344.
5. Hallsworth, J.E., Journal of Fermentation & Bioengineering, 1998, 85, 125-137.
6. Jakobsen, M. & Piper, J.U., Technical Quarterly of the Master Brewers
Association of the Americas, 1989, 26, 56-61.
7. Majara, M., OConnor-Cox, E.S.C. and Axcell, B.C., Journal of the American
Society of Brewing Chemists, 1996, 54, 149-154.
8. Stewart, G.G., DAmore, T., Panchal, C.J. & Russell, I., Technical Quarterly of
the Master Brewers Association of the Americas, 1988, 25, 47-53.

10
34

The response of brewers yeast to a

defined shear field for differing
exposure times
Robert A. Stafford1, Thomas Stoupis2 & Graham G. Stewart2
1
The South African Breweries Ltd, 65 Park Lane, P.O. Box 782178, Sandton 2146, South
Africa (e-mail: [email protected])
2
Heriot-Watt University, International Centre for Brewing and Distilling, Riccarton,
Edinburgh EH14 4AS, Scotland, United Kingdom

Descriptors
Cell damage, fermentative power, physiology, shear stress, stirring, viability, yeast cell, yeast
treatment

SUMMARY
The work reported extends the limited knowledge of the effects of shear stress on brewers
yeast by addressing aspects of fluid mechanics, which have been proposed as the root cause of
cell damage during agitation. Production lager and ale slurries were exposed to defined shear
mixing in an agitated tank. Increases in slurry pH, protease concentration, supernatant haze
and decreases in cell viability were observed. These reductions in yeast quality manifested
themselves to differing degrees in fermentation performance and beer quality. The practical
relevance of the results is discussed through the concept of agitator performance criteria,
which include a rigorous account of yeast damage.

Das Verhalten der Bierhefe in einem definierten Scherfeld bei verschiedenen

Versuchszeiten

Deskriptoren
Grkraft (Triebkraft), Hefebehandlung, Hefezelle, Lebensfhigkeit, Physiologie, Rhren,
Scherbeanspruchung, Zellschdigung

ZUSAMMENFASSUNG
Diese Arbeit erweitert das eingeschrnkte Wissen ber die Auswirkungen von
Scherbeanspruchung auf die Bierhefe. Die Scherbeanspruchung gilt als Hauptgrund fr die
Zellschdigung von Bierhefe whrend der Grung. In einem Rhrtank wurden untergrige
und obergrige Hefen einer definierten Rhrbeanspruchung ausgesetzt. Dabei stiegen der pH-
Wert, die Proteasekonzentration und die Trbung an und die Zellaktivitt verschlechterte sich.
Die Verschlechterung der Hefequalitt machte sich zudem durch eine schlechtere Grleistung
und eine verminderte Bierqualitt bemerkbar. Der Bezug zur Praxis wird durch die
Diskussion der Rhrerleistungen in Hinsicht auf die Zellschdigung erbracht.
Rponse de la levure de bire un champ de cisaillement donn pour diffrents temps
d'exposition

Descripteurs
Agitation mcanique, cellule de levure, force de cisaillement, lsions cellulaires, physiologie,
pouvoir fermentaire, traitement de la levure, viabilit

RESUME
Les travaux dcrits ici tendent la connaissance limite des effets d'une force de cisaillement
sur la levure de bire en abordant certains aspects de la mcanique des fluides qui seraient la
cause fondamentale des lsions cellulaires pendant l'agitation. Des mlanges de bire de
production ont t exposs des forces de cisaillement donnes dans une cuve sous agitation.
Nous avons observ des augmentations du pH du mlange, de la concentration en protases et
du trouble du surnageant, ainsi que des diminutions de la viabilit cellulaire. Ces rductions
de la qualit de la levure se sont manifestes diffrents degrs dans les rsultats de
fermentation et dans la qualit de la bire. La signification pratique de ces rsultats est
discute du point de vue des critres de performance de l'agitateur, qui comprennent une prise
en compte rigoureuse des lsions aux cellules de levure.

2
INTRODUCTION
Brewing is unique among other major industrial fermentation processes, since the
cropped yeast is normally recycled (16). Therefore, the necessary yeast handling
circuit, which is nowadays a common practice in breweries worldwide, has become a
vital link in the process and essential to the success of brewing. This circuit includes
processes that, unless carefully designed and operated, could exert different kinds of
stress on yeast cells such as thermal, osmotic, mechanical and hydrodynamic shear
stress (12).
The latter two cases of stress have been the least studied, although equipment that
might induce them is unavoidable. Operations such as mechanical agitation, pumping,
flow through extensive pipework and heat exchangers, and centrifugation are very
common and their effect on yeast and beer quality might have been underestimated,
since equipment design and specifications may largely be based on experience rather
than knowledge of such effects (2).
It is generally accepted that cropped yeast is more resistant to environmental stresses
(1). Cropped yeast is in a metabolically resting state, during which they exhibit high
stress tolerance, mainly due to their ability to form protective compounds like
trehalose or shock specific proteins. However, such conclusions have exclusively
come from studying the effect of stresses other than shear stress.
Very sparse information can be found in the literature about the effects of shear stress,
but the existing reports offer enough evidence that the role of mechanical and
hydrodynamic shear stress in the brewing industry is not insignificant. Agitation and
prolonged yeast handling have been shown to cause loss in viability and glycogen
content, damage to the cell wall and cell membrane. Consequently, fermentations
pitched with mechanically agitated yeast can display longer lag phases and poor
attenuation. In addition, material from the cell wall (mannan & glucan), intracellular
enzymes and malfunction of the cell membrane can deteriorate beer clarity, head
retention and headspace profile (7, 8, 11).
Information on yeast slurry rheology and yeast vessel engineering is not abundant. A
few references indicate that yeast slurries are non-Newtonian fluids, mostly described
as Bingham plastic fluids, the rheological pattern depending strongly on type of yeast
(ale, lager), concentration and flocculence (14).
In this paper we report on shearing trails in which we investigated the effects of shear
fields on yeast strain (ale and lager) and brewing generation (early and mid).

MATERIALS & METHODS

Yeast slurry
The yeast slurries (both lager and ale) were obtained from a production brewery and
taken directly from yeast collection vessels. The slurries were intercepted at two
brewing cycles or generations from the same original propagated cultures, namely: G2
and G9 for the ale strain; and G2 and G5 for the lager.
All slurries were 5-5.3% abv and the yeast concentration is expressed in % wet weight
as measured after centrifugation at 4000 g for 5 minutes. Slurries were adjusted to a
concentration between 47-52% wet weight by removing or adding beer, which came
from the same batch of yeast slurry.

3
Yeast agitation
Agitation of the yeast was carried out in a 2 litre flat-bottomed cylindrical vessel
(figure 1), filled with 1.5 litres of yeast slurry. The vessel was baffled and a dual
Rushton-turbine (6-blade) was used to mix the slurry at two speeds: 550 rpm (1.44
m/s tip speed) and 900 rpm (2.36 m/s). The vessel was placed in a refrigerated glycol-
water bath and was controlled at 4-4.5oC

80
50
1.5L Level

30

85
130
11

13 10

116

Figure 1: Schematic of agitated vessel with 4 baffles. All dimensions in mm.

Shear rate estimation

The average shear rate of the shear field generated within the agitated vessel was
estimated using the correlation of Metzner et al. (9):

= kN

Where is the average shear rate (s-1); k is an empirical constant equal to 11.6 for
the agitator configuration used here; and N is the rotational speed of the agitator (rps).
Hence agitator speeds of 500 and 900rpm generated an average shear rate of 97 and
174 s-1 respectively.

Assays
The methylene violet stain (3RAX) was employed to measure yeast viability with an
Improved Neubauer counting chamber.
The protease assay was based on the method by Mochaba et al. (10), using
modifications suggested by Hulse (5). Yeast slurry was centrifuged at 4000 g for 5
minutes, the supernatant was filtered through a 0.45 m cellulose acetate filter
(Nalgene) and frozen until measured. Yeast Proteinase-A (Sigma) was employed as a
standard enzyme to calibrate the assay between 0-14 g/ml.
The pH of the slurry supernatant was measured after centrifugation at 4000 g for 5
minutes.

Rheology measurements
Yeast slurry rheology was measured in a Carri Med CSL 100 Rheometer (Cone and
plate, Cone: diameter 6cm, 159). A 1 min equilibration period at 5oC was used

4
 followed by a 1 min ascending shear rate to a preset shear stress value and a 1 min
descending shear rate (constantly at 5oC).

RESULTS & DISCUSSION

Figures 2 and 3 show the drop in viability caused by continuous agitation at 500 and
900 rpm respectively. The drop in viability at 500 rpm was between 0.8 and 1.8 % for
all slurries and there is no obvious distinction between strain or brewing generation.
At 900 rpm however the drop in viability is greater increasing to 3.5% for the G9 ale
slurry. Loss in viability during yeast handling has been reported by several other
researchers that investigated mechanical agitation and centrifugation of yeast slurries
(3, 7, 11) but only the Harrison et al. (3) reports increased loss in viability with
increased brewing generations. Serial repitching has also been reported (6) to generate
physiological changes as brewing generation increases, particularly with regard to
trehalose concentration and flocculation, which can explain the sensitivity of the ale
G9 slurry.

Ale G9 Ale G2 Lager G2 Lager G5

Ale G9 Ale G2 Lager G2 Lager G5

2 4
1.8 3.5
% Viability

1.6
3
% Viability

1.4
1.2 2.5
1 2
0.8
0.6 1.5
0.4 1
0.2 0.5
0
-0.2 0 0
10 20 30
0 10 20 30
Shear Exposure Time (hr)
Shear Exposure Time (hr)
Figures 2 & 3: The influence of shear exposure time on drop in % viability for
agitator speeds of 500 and 900 rpm respectively.

The increase in slurry supernatant pH is shown in figures 4 & 5 for 500 and 900 rpm
agitation respectively. The first point to be made about these graphs is the fact that, as
in figures 2 & 3, the degree of agitation affects the condition of the slurry, since in all
four slurries the increase in pH was higher at 900 rpm. At 500 rpm, the ale slurries
generate a significantly higher pH than the lager slurries. The deacidifying action of
the slurries increases with the increased agitator speeds, with the G9 ale slurry
responding most significantly in keeping with its significant drop in viability (figure
3).
The different response between the lager and the ale strain, namely the pronounced
response of ale G9, does not necessarily constitute a trend, since only two strains were
examined. Generally, for other stress conditions (e.g. high gravity) ale strains appear
more susceptible to damage (16) but overall their main differences are hydrophobicity
of the cell wall and flocculation behaviour (4). Since the cell wall has been reported to
be susceptible to shear environments (7), it can only be assumed that this might be
one of the reasons behind the different responses to shear stress.

5
 Ale G9 Ale G2 Lager G2 Lager G5 Ale G9 Ale G2 Lager G2 Lager G5
0.2 0.5
0.18 0.45
0.16 0.4
Slurry pH

0.14 0.35

Slurry pH
0.12 0.3
0.1 0.25
0.08 0.2
0.06 0.15
0.04 0.1
0.02 0.05
0 0
0 10 20 30 -0.05 0 10 20 30
Shear Exposure Time (hr) Shear Exposure Time (hr)

Figures 4 & 5: The influence of shear exposure time on drop in slurry pH for agitator
speed of 500 and 900 rpm respectively.

Once more, with the exception of lager G5, the higher the shear stress, the higher the
concentration of extracellular protease, which enhances the idea that increase in shear
rate and/or shear stress, deteriorates the general condition of the yeast. Although a
considerable amount of protease might be a result of cell lysis, there is a poor
correlation between loss of viability and protease release, particularly for the lager G5
slurry which showed the lowest viability loss but at the same time the highest protease
release. Therefore it can be suggested that damage or malfunction of the cell
membrane is taking place, something that can be also supported by the literature (8,
11).

Ale G9 Ale G2 Lager G2 Lager G5 Ale G9 Ale G2 Lager G2 Lager G5

Equiv. Proteinase A

5.00 3.50
Equiv. Proteinase A

4.00 3.00
2.50
g/ml)

3.00
g/ml)

2.00
2.00
1.50
(

1.00
1.00
0.00 0.50
-1.00 0 10 20 30 0.00
Shear Exposure Time (hr) 0 10 20 30
Shear Exposure Time (hr)

Figures 6 & 7: The influence of shear exposure time on increase in extracellular

Proteinase A for agitator speed of 500 and 900 rpm respectively.

Figures 8 & 9 show the rheological behaviour of the lager G5 yeast slurry. One
practical implication of these graphs is the very rapid changes that happen up to 40-50
s-1, which means that small changes in shear rate cause large changes in shear stress
and viscosity. Therefore if yeast handling procedures (transport by pumps, mixing

6
etc.) operate at this region the accurate estimation of shear fields might be very
crucial.
It is important to note that agitation significantly affects the rheological profile of the
slurry. Although figure 9 shows a small but consistent decrease in viscosity, the
dramatic difference of the rheological profile is evident on figure 8. This can be
associated with the elastic floc model that has been reported to describe yeast slurry
rheology (14). This model connects rheology with the agglomeration or flocculation
forces between cells and basically the difference highlighted on figure 8, indicates
that due to agitation, the forces required to separate yeast cells are lower than before
agitation.
Flocculation and cell wall characteristics have been reported to change during storage
of yeast slurries. Prolonged starvation of yeast has resulted in reduction of cell surface
charge and flocculation (13). On the other hand intense agitation (15) and
centrifugation (3) have been shown to result in loss of flocculation, increase in surface
charge and decrease in hydrophobicity. According to Speers et al. (14), flocculence of
yeast is a determining factor for yeast slurry rheology, therefore, the change in
rheology that we observed (figures 8 & 9) seems to be justified.

Agitated Unmixed
Agitated Unmixed

1000
Shear Stress (dynes/cm 2)

250
Apparent Viscosity (P)

200 100

150
10
100
1
50

0 0.1
0 100 200 300 400 500 0 100 200 300 400 500
-1 -1
Shear Rate (s ) Shear Rate (s )

Figures 8 & 9: The influence of 24 hours of agitation (500 rpm) on the rheological
behaviour of the G5 lager yeast.

CONCLUSIONS
Although only two strains (ale and lager) were considered, the ale strains appeared to
be more susceptible to shear stresses than the lager as indicated by a drop in cell
viability and increase in slurry pH. Furthermore, the older generation ale strain
appeared to be most susceptible of all. This trend was not reflected by the leakage of
proteinase A.
Significant changes in slurry rheology were noted after 24 hours of agitation which
suggests changes in cell surface properties as noted elsewhere.

ACKNOWLEDGEMENTS
Dr Benham Taidi, Scottish Courage, Edinburgh for provision of the yeast slurries and
Gavin Hulse, SAB for assistance with the protease assay.

7
REFERENCES
1. Axcell, B.C. & OConnor-Cox, E.S.C., The Institute of Brewing (Proceedings
Asia & Pacific Section), 1996, 64-71.
2. Harrison, S.T.L., Basson, L., Robinson, A., Godfrey, T.A., OConnor-Cox,
E.S.C. & Axcell, B.C., The Institute of Brewing (Proceedings C & SA Section)
1997, 55-60.
3. Harrison, S.T.L. & Robinson, A., The Institute of Brewing (Proceedings C &
SA Section), 2001 (in print).
4. Hinchliffe, E., Box, W.G., Walton, E.F. & Appleby, M., Proceedings of the
European Brewery Convention Congress, Helsinki, 1985, 323-330.
5. Hulse, G. (South African Breweries). Personal communication.
6. Jenkins, C., Kennedy, A., Thurston, P., Hodgson, J. & Smart, K., The European
Brewery Convention Congress, Budapest, 2001 (in print).
7. Lewis, M.J. & Poerwantaro, W.M., The Journal of the American Society of
Brewing Chemists, 1991, 49(2): 43-46.
8. McCaig, R. & Bendiak, D.S., The Journal of the American Society of Brewing
Chemists, 1985, 43(2): 114-118.
9. Metzner, A.B., Feehs, R.H., Ramos, H.L., Otto, R.E. & Tuthill, J.D., The
American Institute of Chemical Engineers Journal, 1961, 7(1): 3-9.
10. Mochaba, F., Torline, P.A. & Axcell, B., Proceedings of the European Brewery
Convention Congress, Oslo, 1993, 533-537.
11. Pickerell, A.T.W., Hwang, A. & Axcell, B.C., The Journal of the American
Society of Brewing Chemists, 1991, 49(2): 87-92.
12. Quilliam, W.R., The Institute of Brewing (Proceedings C & SA Section), 1997,
61-66.
13. Smart, K.A, Boulton, C.A., Hinchliffe, E. & Molzahn, S., The Journal of the
American Society of Brewing Chemists, 1995, 53(1): 33-38.
14. Speers, R.A., Durance, T.D., Tung, M.A. & Tou, J., Biotechnology Progress,
1993, 9: 267-272.
15. Stewart, G.G., Garrison, I.F., Goring, T.E., Meleg, M., Pipasts, P. & Russell, I.,
Kemia-Kemi, 1976, 10: 58-72.
16. Stewart, G.G., & Russell, I., An introduction to brewing science and technology
Brewers yeast (Series III). The Institute of Brewing., 1998.

8
35

Yeast membrane potential and

carbohydrate utilisation
S.M. Van Zandycke, R. Siddique & K.A. Smart
Oxford Brookes University, School of Biological Sciences, Headington, Oxford OX3 0BP,
United Kingdom (e-mail: [email protected])

Descriptors
Carbohydrate, cell wall, fermentative power, physiology, prediction, viability, yeast

SUMMARY
The efficiency of beer fermentation is predominantly influenced by the quality of the yeast.
Yeast vitality represents the physiological state of a viable cell population. Assessing yeast
vitality by proton efflux using the acidification power test has been demonstrated to predict
fermentation performance. Although the acidification power test represents a predictive assay
for fermentation performance, the basis of the test involves membrane function and
carbohydrate uptake. The relationship between this assay and such parameters as glycogen,
trehalose, sterol content and membrane potential may lead to the identification of a predictive
test for fermentation performance.

Das Potenzial der Hefezelle und die Verwertung von Kohlenhydraten

Deskriptoren
Grkraft (Triebkraft), Hefe, Kohlenhydrat, Lebensfhigkeit, Physiologie, Vorhersage,
Zellwand

ZUSAMMENFASSUNG
Die Effizienz der Grung ist hauptschlich von der Qualitt der Hefe abhngig. Die
Hefevitalitt spiegelt die physiologische Verfassung der Zellpopulation wieder. Die
Bewertung der Hefevitalitt durch den Protonenfluss in Verbindung mit dem Suerungs-
krafttest hat gezeigt, dass damit die Grleistung vorhergesagt werden kann. Die Basis dieses
Tests ist der Zusammenhang von Membranfunktion und Kohlenhydrataufnahme. Die
Beziehung der Parameter Glykogen, Trehalose, Sterol-Gehalt und dem Membranpotenzial
knnte dabei helfen einen Test zu entwickeln, der die Grleistung vorhersagt.
Potentiel de membrane de la levure et utilisation des sucres

Descripteurs
Hydrate de carbone, levure, paroi cellulaire, physiologie, pouvoir fermentaire, prdiction,
viabilit

RESUME
L'efficience de la fermentation de la bire est surtout influence par la qualit de la levure. La
vitalit de la levure reprsente l'tat physiologique d'une population cellulaire viable. Il est
tabli qu'en valuant la vitalit de la levure par efflux de protons (test de pouvoir acidifiant), il
est possible de prvoir les rsultats de la fermentation. Bien que ce test constitue un essai
prdictif des performances de fermentation, il est bas sur la fonction membranaire et la
capture des sucres. La relation entre cet essai et des paramtres tels que le glycogne, le
trhalose, la teneur en strols et le potentiel de membrane peut conduire l'laboration d'un
test prvisionnel des performances de fermentation.

2
INTRODUCTION
Yeast vitality may be defined as the physiological state of a viable cell population.
Assessing yeast vitality by proton efflux using the acidification power test (APT) has
been first reported by Opekarova and Sigler (1). The assay has since been used to
predict fermentation performance (2,3) and demonstrate the detrimental effect of
increasing storage temperature (4) and acid washing (5) on yeast vitality. The
acidification power test is based on the ability of a yeast population to acidify the
surrounding medium before and after suspension in glucose. When yeast cells are
resuspended in water, they passively extrude protons in order to reach equilibrium
with the pH of the outer media. This represents the passive proton efflux, which has
been postulated to depend on endogenous substrates such as glucose and trehalose (3).
When a metabolisable substrate is added to the yeast suspension, proton efflux is
induced and relies on both endogenous and exogenous substrate (2).
Since the test determines the extent of proton efflux from the cell, it reflects the action
of the plasma membrane ATPase. This H+ translocating enzyme uses ATP to generate
an electrochemical proton gradient, which together with the membrane potential
provides the driving force for the uptake of solutes (6). H+ATPase can constitute up to
50% of the total membrane protein and it controls cell pH, nutrient transport, ion
transport and cell growth (7). Intracellular pH is mostly constant due to the activity of
the cell membrane H+ATPase; however, extracellular pH varies following the uptake
of solutes. Although the electrogenic proton-pumping ATPase creates most of the
extracellular acidification, dissociation of CO2 into H+ and HCO3- and production of
organic acids can also play a part in the overall acidification (8).
Recent developments of the assay have significantly improved the reproducibility of
the test and it has been suggested that the Glucose Induced Proton Efflux (GIPE)
represents the true vitality of a yeast cell population (9). GIPE (Glucose Induced
Proton Efflux) involves the measurement of the water induced proton efflux for 20
minutes (WAP). This control value is then deducted from the glucose induced proton
efflux (GAP) (9).
The relationship between GIPE, carbohydrate uptake and membrane potential is
investigated.

MATERIALS AND METHODS

Yeast strains and growth conditions
The following strains were used in this study. Lager strain KS1 (formerly BB5) was
obtained from Bass Brewers; Burton-on-Trent, UK; ale strain NCYC 2593 was
obtained from the National Collection of Yeast Cultures, Norwich, UK, lager strain
SVZ1 was obtained from the Oxford Brookes University Yeast Collection, haploid
wild type strain EG103 and sod1-sod2 mutant (EG133) derived from EG103 were
obtained from Dr E. Gralla, UCLA, USA.
Strains were stored on beads at 80C in YPD containing 20% glycerol (w/v). Stock
cultures were maintained on YPD consisting of 1% yeast extract (w/v), 0.5%
bacteriological peptone (w/v) and 1% glucose (w/v); solidified with 1.5% agar (w/v).
All media were autoclaved at 121C and 15 psi for 15 min.
Yeast were grown aerobically to the required cell density at 25C by shaking in an
erlenmeyer flask (250 ml) at 120 rpm. Cell growth was monitored and measured by
optical density at 600 nm using a spectrophotometer to achieve exponential and
stationary physiological states.

3
Acidification power test
Acidification power test, was conducted according to the method of Siddique and
Smart (9) and a modified version of Kara et al (2). In a universal bottle, sterile
deionised water (15 ml) was continuously stirred at 150 rpm at room temperature for
5 min to allow for pH probe equilibration. The passive proton efflux was monitored
for 10 min (AP10) prior to the addition of glucose (5 ml of 20.2% (w/v)). The glucose
induced proton efflux (GAP 20) was then monitored for a further 10 min. Glucose
acidification power (GAP) was calculated by adding the passive proton efflux to
glucose induced proton efflux (AP10 + GAP20). The water acidification power
(WAP) was performed according to the same method described for GAP; however,
glucose was replaced by sterile deionised water (5 ml) and WAP was obtained by
adding AP10 to WAP20. The GIPE was calculated by subtracting WAP20 from
GAP20.

Glycogen determination
Glycogen and trehalose were determined using the method of Parrou et al (10) and
expressed as g glucose per 109 cells.

Viability
Yeast viability was determined using 8-anilino-1-naphtalene sulfonic acid (Mg-ANS)
as described in McCaig (11).

RESULTS AND DISCUSSION

Impact of water pH on the acidification power test
The passive (spontaneous) proton efflux was observed to be less reproducible than the
induced proton efflux. The impact of initial water pH on passive (AP10) and induced
(GAP20) proton efflux was assessed using an initial water pH ranging from 5.8 to 9.
Unlike the induced proton efflux, the passive proton efflux was observed to be highly
dependent on the initial pH of the water prior to addition of the cells (figure 1).
However, it is suggested that the 10-minute incubation period before glucose addition
is crucial allowing the cells to reach an equilibrium pH.

4 AP10
3.5 GAP20
3 GAP
2.5
pH

2
1.5
1
0.5
0
5 6 7 8 9 10
Water pH

Figure 1: Effect of initial water pH on passive (AP10) and induced proton efflux
(GAP20) for SVZ1 cells exhibiting exponential phase of growth.

4
Carbohydrate utilisation
Although glucose has always been utilised to generate proton efflux in the past (1-5),
the main carbohydrate in wort is not glucose but maltose (12); fructose could also be
present if sucrose has been added to the wort. Therefore, it is suggested that maltose
or fructose may represent an appropriate alternative measure of brewing yeast vitality.
This was investigated for an ale strain (2593) in exponential and stationary phase.
In both exponential and stationary phase, glucose and fructose were observed to
induce proton efflux to a greater extent than maltose (figures 2a and 2b). This can
readily be explained by the fact that fructose and glucose are monosaccharides and
enter the cell by facilitated diffusion; which is an energy-independent system
mediated by protein carriers. Whereas maltose requires a proton symport, or active
energy-dependent ATPase mediated proton motive force, to enter the cell and
therefore necessitates the uptake of protons from the surrounding media (13).
Consequently, the pH of the external media will decrease to a lesser extent with
maltose than glucose and fructose. The extent of proton efflux induced by glucose,
fructose and maltose was reduced in stationary phase (figure 2b) compared to
exponential phase populations (figure 2a). It is postulated that this is due to a decline
in the activity of the ATPase enzyme (mainly encoded by the PMA1 gene) in
stationary phase, resulting in reduced acidification of the outer media (14).

7
6
pH

Glucose
5 Maltose
Fructose
4
Water
3
0 5 10 15 20
Time (min)

a
Glucose
7 Maltose
Fructose
6 Water
pH

3
0 5 10 15 20
Time (min)

Figure 2: The impact of glucose, fructose, maltose and water as substrates on the
induced proton efflux of 2593 cells grown on YPglucose to exponential (a) and
stationary (b) phase. Data represents the mean of three independent experiments. The
standard deviation is indicated.

5
Impact of growth on different carbon sources on glucose induced proton efflux
Although glucose has always been utilised to grow yeast cells prior to the test (1-5),
the main carbohydrate in wort is maltose and fructose may only be present
occasionally. Thus, brewing yeast from production environments is not predominantly
pregrown on glucose and the use of alternative carbon sources to pre-grow laboratory
populations of yeast may be more appropriate as a model system for the brewery
environment. Therefore, the effect of growth on glucose, fructose and maltose on
glucose proton efflux was investigated for an ale (2593) and lager (KS1) strain
exhibiting stationary phase.
Growth on fructose and maltose compared to glucose did not significantly change the
extent of induced proton efflux for the lager strain KS1 (figure 3); however, it was
significantly lower for maltose compared to glucose and fructose (p<0.05) for the ale
strain 2593 (figure 3). Cells grown on maltose and fructose are derepressed and can
theoretically start using glucose immediately, and therefore the use of these
alternative carbon source should not have an impact on the glucose induced proton
efflux. Although, for 2593, growth on maltose caused a reduction in proton efflux.
The reasons for this are not know; however, levels of glycogen were observed to be
lower for 2593 cells pre-grown on maltose compared to glucose and fructose (data not
shown).

1.50 2593
KS1
1.00
GIPE

0.50

0.00
Glucose Fructose Maltose

Figure 3: The impact of growth on glucose, maltose and fructose on glucose induced
proton efflux of 2593 and KS1 cells grown to stationary phase. Data represents the
mean of three independent experiments. The standard deviation is indicated.

Impact of ethanol on induced proton efflux

A major effect of ethanol is to disorder or fluidise the membrane, although the extent
of this effect is strain dependent and may be modified by membrane lipid composition
(15). The deleterious effect of ethanol is of importance when you consider the
extensive use of high gravity brewing. Increased levels of ethanol have been
demonstrated to adversely affect yeast quality (16). The extent of the detrimental
effect of ethanol on the membrane was investigated using ale (2593) and lager (KS1)
yeast cells grown to stationary phase and resuspended for a 6 hour period in
increasing concentrations of ethanol.
Both strains were adversely affected by increasing the concentration of ethanol. A
decrease in the extent of proton efflux was observed when cells where resuspended in
4% ethanol compared to the control (water) (p<0.05), this effect was observed to be
greater in 8% ethanol (p<0.05) (figure 4). The extent of proton efflux appears to be
stabilised for ethanol concentrations from 12 to 20% (figure 4). Yeast viability

6
remained higher than 95% for ethanol concentrations up to 12% and decreased below
50% in 20% ethanol (data not shown).
It is suggested that the decrease in proton efflux with increasing concentration of
ethanol is due to an accumulation of unsaturated fatty acids, which results in a
decrease in membrane fluidity (17). Cells are therefore likely to accumulate greater
levels of intracellular ethanol and hence are subject to greater toxicity. Moreover,
PMA1 activity was observed to be reduced in the presence of sublethal concentrations
of ethanol (18).

1
0.8 KS1
0.6 2593
GIPE

0.4
0.2
0
-0.2 0 5 10 15 20
Ethanol concentration (%)

Figure 4: Effect of ethanol concentration on stationary phase cells of lager (KS1) and
ale (2593) strains after 6 hours. Data represents the mean of three independent
experiments. The standard deviation is indicated.

Relationship between passive and glucose induced proton efflux and levels of
intracellular carbohydrate
Glycogen and trehalose are the two major reserve carbohydrates in Saccharomyces
cerevisiae (19). Glycogen represents a reserve of energy, which can be utilised during
periods of starvation commencing with entry into diauxic shift. Glycogen levels are
crucial for sterol synthesis, consequently it is important to be able to determine
intracellular glycogen levels prior to pitching; low levels of glycogen may result in
insufficient yeast sterols, which may negatively affect fermentation performance (20).
Trehalose is believed to serve as a stress protectant and increased levels have been
observed following heat shock, desiccation and reduced water activity (21). It has
been postulated that increased intracellular levels of trehalose may be indicative of
stressed yeast populations (22).
A positive correlation was established (p<0.05) (r = 0.6908) between the passive
proton efflux of yeast cells resuspended for 10 min in water and glycogen levels
(figure 5). This result confirms that passive proton efflux represents a biomarker for
the glycogen content of cells (23). However, no correlation could be established
between passive proton efflux and trehalose levels.
A negative correlation was observed (p<0.05) (r = -0.7313) between GIPE and
intracellular levels of trehalose (figure 6), indicating a potential role of induced proton
efflux in detecting stressed yeast populations. However, no correlation could be
established between GIPE and glycogen levels.

7
 300

Glycogen (ug/109
250
200

cells)
150
100
50
0
-0.20 0.00 0.20 0.40 0.60 0.80 1.00 1.20
Passive proton efflux (AP10)

Figure 5: Correlation between passive proton efflux (AP10) and glycogen content of
exponential, stationary and starved cells of ale strain 2593. Data represents the mean
of 6 (AP10) and 3 (glycogen levels) independent experiments.

350
Trehalose (ug/109

300
250
cells)

200
150
100
50
-
0.6 0.8 1 1.2 1.4 1.6
Glucose induced proton efflux (GIPE)

Figure 6: Correlation between glucose induced proton efflux (GIPE) and trehalose
content of exponential, stationary and starved cells of ale strain 2593. Data represents
the mean of 3 independent experiments.

The impact of proton efflux on yeast mutant strains impaired for membrane
function
Superoxide dismutase enzymes are involved in oxidative stress defence. There are
two types present in the cytosol (CuZnSOD) and in the mitochondria (MnSOD),
encoded by SOD1 and SOD2 respectively. It has been demonstrated that a deletion
mutant lacking these enzymes accumulates reactive oxygen species due to impaired
oxidative defence and is therefore more susceptible to free radical damage. Free
radicals damage cellular membranes via a process known as lipid peroxidation (24).
When this happens, cell membrane function is impaired. Traditionally, this can only
be determined using complex lipid peroxidation biochemical analysis. Given that
impaired membrane function is likely to adversely affect ATPase activity and
therefore modify proton efflux, the GIPE 20 of a sod1-sod2 mutant and corresponding
wild type strain was measured for stationary phase populations.
It was observed that the extent of proton efflux from the mutant strain was less than
that of the corresponding wild type (p<0.05) (figure 7), indicating a lack of membrane
fluidity for the mutant strain and impaired ATPase function. To ascertain that the
decrease in membrane fluidity was the result of an increase in lipid peroxidation,
measurement of by products of lipid peroxidation will be the subject of future
investigation.

8
 0.8
0.7
0.6
0.5

GIPE
0.4
0.3
0.2
0.1
0

sod1-sod2 Wild Type

Figure 7: Induced proton efflux for wild type (EG103) and corresponding sod1-sod2
mutant (EG133). Data represents the mean of three independent experiments. The
standard deviation is indicated.

CONCLUSIONS
The glucose induced proton efflux (GIPE) has been observed to inversely correlate
with intracellular trehalose levels, confirming its potential for detecting stressed yeast
populations. The passive proton efflux was confirmed to correlate with the levels of
intracellular glycogen and could be used to indicate the quality of pitching yeast and
particularly its ability to perform well and synthesise sterols.
GIPE has successfully demonstrated the detrimental effect of ethanol at
concentrations as low as 4%, indicating that ethanol stress results in changes in
membrane composition and consequently reduced proton efflux. Similarly, GIPE
demonstrated that accumulation of free radicals impairs membrane function resulting
in decreased proton efflux.
Measurement of membrane fluidity, membrane potential, lipid peroxidation by-
products and sterol content should clarify some of the phenomenon impairing
membrane function and may lead to the identification of a predictive test for
fermentation performance.

ACKNOWLEDGMENTS
Sylvie Van Zandycke is a postdoctoral researcher funded by Smart Brewing Services.
Rukhsana Siddique is supported by Gilbert Vandenput. Katherine Smart is grateful for
the support of Scottish Courage Brewing Limited and the Royal Society.

REFERENCES
1. Opekarova, M. & Sigler, K., Folia Microbiologica, 1982, 27, 395-403.
2. Kara, B.V., Simpson, W.J. & Hammond, J.R.M., Journal of the Institute of
Brewing, 1988, 94, 153-158.
3. Mathieu, C., Van der Berg, L. & Iserentant, D., Proceedings of the European
Brewery Convention Congress, 1991, 23, 273-278.
4. Fernndez, S., Gonzlez, G. & Sierra, A., MBAA Technical Quarterly, 1991,
28, 89-95.
5. Fernndez, S., Gonzlez, G. & Sierra, A., MBAA Technical Quarterly, 1993,
30, 1-8.

9
6. Goffeau, A. & Slayman, C.W., Biochimica et Biophysica Acta, 1981, 639, 197-
223.
7. Serrano, R., Annual Reviews of Plant Physiology and Plant Molecular Biology,
1989, 40, 61-94.
8. Sigler, K. & Hffer, M., Biochimica et Biophysica Acta, 1991, 1071, 375-391.
9. Siddique, R. & Smart, K.A., Brewing Yeast Fermentation Performance, Oxford:
Blackwell Science, 2000, 46-53
10. Parrou, J., Teste, M. & Francois, J., Microbiology, 1997, 143, 1891-1900.
11. McCaig, R., Journal of the American Society of brewing Chemists, 1990, 48,
22-25.
12. Stewart, G.F. & Russel, I., MBAA Technical Quarterly, 1993, 30, 150-168.
13. Walker, G., Yeast Physiology and Biotechnology, Chichester: John Wiley &
Sons Ltd, 1998, Chapter 3.
14. Panaretou, B. & Piper, W., European Journal of Biochemistry, 1992, 206, 635-
640.
15. Chin, J.H. & Goldstein, D.B., Molecular Pharmacology, 1977, 19, 435-441.
16. Odumeru, J.A., DAmore, T., Russel, I. & Stewart, G., Journal of Industrial
Microbiology, 1992, 9, 229-234.
17. Thomas, D.S. & Rose, A.H., Archives in Microbiology, 1979, 122, 49-55.
18. Monteiro, G.A., Supply, P., Goffeau, A. & Sa-Correia, I., Yeast, 1994, 10,
1439-1446.
19. Lillie, S.H. & Pringle, J.R., Journal of Bacteriology, 1980, 143, 1384-1394.
20. Quain, D.E., Journal of the Institute of Brewing, 1988, 95, 315-323.
21. Wiemken, A., Antonie van Leeuwenhoek, 1990, 58, 209-217.
22. Boulton, C., Brewing Yeast Fermentation Performance, Oxford: Blackwell
Science, 2000, 10-19.
23. Quain, D.E., Thurston, P.A. & Tubb, R.S., Journal of the Institute of Brewing,
1981, 87, 108-111.
24. Bucala, R. Redox Report, 1996, 2, 291-307.

10
36

Fermentation of high gravity worts - its

influence on yeast metabolism and
morphology
Graham G. Stewart
Heriot-Watt University, International Centre for Brewing and Distilling, Riccarton, Edinburgh
EH14 4AS, United Kingdom (e-mail: [email protected])

Descriptors
Brewers' yeast, cell morphology, ester, fermentation, high gravity brewing, viability, yeast cell

SUMMARY
The negative effect of high gravity brewing on yeast quality and performance is still a concern.
Specifically, three areas of concern are: disproportionate synthesis of esters (particularly ethyl
acetate and isoamylacetate) excretion of elevated levels of proteinase A into the fermenting
medium with a negative effect on beer foam stability and changes in yeast cell morphology.
Changes in wort sugar spectrum can be employed to reduce ester levels. The reasons for
increased proteinase A excretion are not well understood and at this time solutions cannot be
proposed for reducing this excretion. However, reduction in mean cell volume and enlargement of
the vacuole may be part of the reason.

Der Einfluss von High -Gravity-Wrze auf den Stoffwechsel und die Morphologie der Hefe

Deskriptoren
Bierhefe, Brauen mit hohem Stammwrzegehalt, Ester, Grung, Hefezelle, Lebensfhigkeit,
Zellmorphologie

ZUSAMMENFASSUNG
Die negativen Einflsse von High -Gravity-Verfahren auf die Hefequalitt und Hefeleistung sind
nach wie vor ein Thema. Insbesondere sind dabei drei Effekte zu nennen: unverhltnismige
Synthese von Estern (insbesondere Ethylacetat und Isoamylacetat), eine erhhte Ausschttung
von Proteinase A in das Grmedium, was zu einer Verschlechterung der Schaumstabilitt fhrt,
und die Vernderung der Hefezellenmorphologie. Das vernderte Zuckerspektrum in der Wrze
knnte fr die vernderten Esterkonzentrationen verantwortlich gemacht werden. Der Grund,
warum mehr Proteinase A ausgeschttet wird, ist noch nicht gut genug erforscht. Aus diesem
Grund knnen im Moment noch keine Lsungsvorschlge erbracht werden. Eine Verringerung
des durchschnittlichen Zellvolumens und die Vergrerung der Vakuolen knnte eine mgliche
Antwort sein.

Fermentation des mots de haute densit - influence sur la morphologie et le mtabolisme

des levures

Descripteurs
Cellule de levure, ester, fermentation, fabrication de bire haute densit, levure de brasserie,
morphologie cellulaire, viabilit

RESUME
L'effet ngatif du brassage haute densit sur la qualit et les performances de la levure reste
problmatique. Plus prcisment, trois aspects sont proccupants: la synthse dis-proportionne
d'esters (en particulier d'actate d'thyle et d'isoamylactate), l'excrtion de fortes concentrations
de protase A dans le milieu de fermentation avec un effet ngatif sur la stabilit de la mousse de
bire, et les variations morphologiques des cellules de levure. Les modifications du spectre des
carbohydrates du mot peuvent tre mises profit pour rduire les taux d'esters. Les causes de
l'excrtion accrue de protase A sont mal comprises et, pour l'instant, on ne peut proposer aucune
solution pour rduire cette excrtion. Toutefois, la rduction du volume moyen des cellules et le
grossissement de la vacuole pourraient faire partie de ces causes.

2
INTRODUCTION
The benefits of high gravity brewing are well documented (8,9,13) and have been
discussed at many previous Congresses of this Convention (4,6,11,15). However, many
of the negative consequences of this brewing method are still of concern (5,10,14). For
example, high gravity worts can negatively influence yeast performance with effects
upon fermentation, yeast and beer quality and stability and flocculation. The negative
effects of employing worts of 16Plato and higher concentrations are largely due to
increased osmotic pressure and ethanol. Specifically, the effects of high gravity worts on
yeast are:
disproportionate decrease in yeast growth
decrease in yeast viability and vitality necessitating a reduced number of yeast
generations (cycles)
the production of disproportionately higher levels of esters
greater leakage (secretion/excretion) of specific intracellular enzymes (for example,
proteinase A)
alternations in yeast vacuolar size, cell volume and cell surface morphology.

In this paper three areas will be considered, namely:

the influence of wort sugar spectrum and gravity on ester formation during
fermentation
the effect of proteinase A secretion and gravity on beer foam stability
yeast morphological and structural changes induced by high gravity worts.

METHODS AND MATERIALS

Yeast strains
The yeast strains employed in this study were representative production Sacchaccomyes
ceravesiae ale and lager strains. The yeast cultures were stored in 1ml ampoules at -70C
and subcultured on peptone yeast extract glucose nutrient agar slopes prior to being
used for fermentation experiments.

Fermentations
Wort employed in this study was produced in the Centres 2 hl pilot brewery. Yeast
biomass was obtained by culturing a single colony in 5 ml of all malt 10 or 12Plato wort
for 24 h at 25C before pitching this 5 ml into 300 ml of similar medium for a further 24
h in shaking flasks. This volume of culture medium was then scaled up to 2 l prior to use
in static fermenters. Fermentation was either conducted in the pilot brewerys 2 hl
stainless steel vertical unitanks or in custom built tall-tube fermenters (1586 mm x 50
mm, sample port at 55 mm, Scotia Glass Technology, Stirling, UK). Each fermenter
contained 1.5 l of medium at a constant controlled temperature of 13C (lager) or 20C
(ale). Yeast was pitched at a rate of 0.28 g/100 ml for 10Plato wort, 0.35 g/100 ml for
12Plato wort and 0.65 g/100 ml for 20Plato wort. Wort was oxygenated to a level of 1
mg/l per degree Plato using a model 3650 Micro 02 loger (Orbisphose U.K. Ltd.,
Chesterfield, U.K.). For the foam stability studies 10Plato and 20Plato worts containing

3
30% maltose syrup (MS) were employed. For the ester formation studies three worts
were studied: 12Plato and 20Plato worts containing 30% MS syrup and 20Plato wort
containing 30% very high maltose syrup (VHMS). Syrups were obtained from Cerestar
(Manchester, UK Ltd) and were added directly to the kettle immediately prior to the boil.
The typical carbohydrate profile of the syrups is:
MS VHMS
Glucose 15% 5%
Maltose 55% 70%
Maltotriose 10% 10%
Dextrins 20% 15%

Analytical methods
Samples were removed at specified times throughout the fermentation cycle in order to
assay, wort gravity, cell viability, biomass, hydrophobic polypeptide levels (3), proteinase
A activity (7) and beer foam stability (1,12). Wort sugar uptake was monitored using
HPLC while ethanol production was measured with a gas chromatograph. The formation
of the esters ethyl acetate and isoamyl acetate was followed using Hewlet Packard 5890
Series II GC with split/splitless injector with FID. Details of the temperature profile, etc.
are discussed by Younis and Stewart (15).

Yeast morphology and structure

Fermentations were conducted in 300 ml volumes in 500 ml Erlenmeyer flasks at 13C
and 20C for lager and ale yeast strains, respectively, and shaken at 150 rpm. All
fermentations were conducted in triplicate. Samples for microscopic examination were
taken at time zero and then every 24 h until 120 h.

For scanning electron microscopy (SEM) the yeast samples were filtered on a Millipore
0.22 micron filter and washed with deionised water. A small portion of the filtrate was
transferred onto an aluminium stub and rapidly frozen under liquid nitrogen until required
for use.

For fluorescence microscopy, 1 ml of 7M 4-64 fluorescent dye was added to 1 ml of

diluted yeast sample and allowed to perfuse for 1h. The sample was filtered and
transferred to a glass slide and examined under a Zeiss Axiophot microscope. The images
were captured using a JVC KY-755B colour video camera.

RESULTS
It has previously been reported (2) that worts of gravity greater than 16Plato produce
disproportionately higher levels of esters (particularly ethyl acetate and isoamyl acetate)
when compared to conventional gravity worts less than 12Plato. However, other
publications have reported that the increase in ester levels with high gravity worts is
proportional to the wort gravity (11). Nevertheless, it is generally agreed that a reduction
in ethyl acetate and isoamyl acetate from high gravity brewed beers would be welcome.

4
The influence of wort sugar spectrum on ester formation has been the subject of much
debate. The influence of maltose and glucose levels in two 20Plato worts has been
studied. One wort contained 30% maltose 55% syrup (MS) and the other contained 30%
very high maltose 70% syrup (VHMS). In addition, a 12Plato wort containing 30% MS
was employed as the control. The sugar spectrum of these three worts is shown in figure
1.
200
Su
ga 180

r 160

co 140
nc 120
en
tra 100

tio 80

n 60
(g/ 40
L)
20

12Plato (30% maltose 20Plato (30% maltose 20Plato (30% VHM)

syrup) syrup) syrup

Figure 1: Wort sugar profiles. Glucose plus fructose and maltose plus maltotriose
The maltose, plus maltotriose concentration in the 20Plato (VHMS) wort had increased
compared to the 20Plato (MS) wort with a corresponding decrease in the concentration
of glucose plus fructose. The three worts were fermented in the Centres 2 hl pilot
brewery with a lager yeast strain at 13C and the concentration of ethyl acetate and
isoamyl acetate determined throughout the fermentations (figures 2 and 3). The profiles
were similar with both esters, and the concentration of ethyl acetate was approximately
50-fold greater than isoamyl acetate. The concentration of both esters in the 20Plato
(MS) wort was greater than twice that in the 12Plato (VHMS) wort - which contained
elevated levels of maltose was approximately 35% reduced compared to 20Plato (MS)
wort. A number of ale and lager yeast strains have been studied and similar trends
obtained to the above results (16). In addition, yeast fermenting worts (high and low
gravity) with elevated concentrations of maltose had high levels of viable cells and
overall increased vitality when compared to similar gravity worts with lower maltose
concentrations.
40

30

20

10

0
0 40 80 120 160 200 240

F erm entatio n T im e (ho urs)

Figure 2: Ethyl acetate concentration in fermenting worts of

differing gravities and sugar composition.
12Plato (30% MS) , 20Plato (30% MS) and 20Plato (30% VHMS)

5
 1

0.8

0.6

0.4

0.2

0
0 40 80 120 160 200 240

Fermentation Time (hours)

Figure 3: Isoamyl acetate concentration in worts of differing gravities and sugar composition.
(Symbols as per figure 2).

It has been well documented (4,6,11,13) that beers brewed at higher gravities have poorer
head (foam) stability when compared to similar beers brewed at lower gravities. It is
known that specific polypeptides play an important role in foam formation and stability
(3), also important are isoalpha acids, metal ions and melanoidins. Perhaps the single
most important factor in foam stability is the hydrophobic character of the polypeptides
(3). The level of hydrophobic polypeptides has been determined throughout the brewing
and fermentation of high gravity (20Plato) and low gravity (10Plato), 70% barley malt
and 30% syrup beers. During brewing there is a proportionately greater loss of
hydrophobic polypeptides with the 20Plato wort than the 10Plato counterpart (4). When
the high gravity beer was diluted to 4.5% alcohol by volume, equivalent to the low
gravity beer, it had a level of hydrophobic polypeptide that was less than 50% of the low
gravity produced beer (figure 4).

120

100
140
80 120
100
60
80
40 60
40
20 20
0
0
Rudin value Sigma value
Ferm. Start Ferm. End Final Beer
(seconds)

Final beer high gravity diluted to 4.5% abv High gravity diluted to 4.5% abv

Figure 4: Changes in hydrophobic polypeptide Figure 5: Comparison of foam tests

levels during fermentation. for high and low gravity beers.
Low gravity , high gravity Low gravity ,high gravity

6
The head retention of the diluted high gravity brewed beer was less than that of the low
gravity brewed beer (figure 5). This difference was very apparent when both beers were
poured into 100 ml measuring cylinders and the time taken for foam collapse determined.
Two minutes after pouring, there was very little foam or cling with the diluted high
gravity beer sample, but substantial foam and cling remained on the low gravity beer
sample.

In addition to greater losses of hydrophobic polypeptides during mashing, wort separation

and boiling, the fermentation stage is a key stage where these polypeptides are lost.
Detailed analysis of this stage has shown that high gravity wort fermentation has a more
negative effect on hydrophobic peptide levels than low gravity fermentations (figure 4).
Two factors would account for this loss of hydrophobic polypeptides during
fermentation. Firstly, fermenter foaming (particularly in cylindroconical vessels) is
known to be responsible for the loss of a large amount of foam active substances and that
this problem is exacerbated during the fermentation of high gravity worts. Secondly,
yeast secretes proteolytic enzymes into the fermenting wort and these enzymes will have
a negative effect on the foam stability of finished beer through polypeptide degradation
that will occur during fermentation and storage. Proteinase A increased throughout the
fermentation (figure 6) with the highest enzyme activity occurring at the end. Higher
amounts of proteinase A were released during the 20Plato fermentations compared to the
10Plato wort fermentations. During high gravity brewing, increased stress on the yeast
in the form of elevated osmotic pressure and ethanol concentrations, appears to have
stimulated the secretion of proteinase A into the wort during fermentation.

9
8
7
6
5
4
3
2
1
0
1 2 3 4 5 6 7 8 9 10 11 12

Fermentation Time (days)

Figure 6: The effect of wort gravity on yeast proteinase A release

during fermentation. 10Plato wort () and 20Plato wort (()

Effects on overall yeast performance when high gravity worts are employed have been
the subject of this paper thus far. In addition, studies in this laboratory are focussing on
the effect of high gravity worts on yeast cell surface changes, cell volume and vacuolar
morphology. Fluorescence microscopy has shown that at the end of fermentation, yeast
cells cultured in 20Plato wort contained much larger vacuoles compared to yeast cells
from fermentations conducted 12Plato wort (figure 7). Also, yeast cells examined with a
Scanning Electron Microscope, showed that cell surface changes were more extensive in
cells cultured in high gravity worts (figure 8). These results are preliminary and further

7
details regarding the influences of high gravity worts on yeast morphology will hopefully
be published in due course.

Figure 7: Effect of wort gravity on vacuole size in a brewers yeast strain.

Fluorescent stain employed FM 4-64 dye.
Low gravity cultured cells High gravity cultured cells

Figure 8: Effect of wort gravity on cell surface morphology of

brewers yeast cells. Scanning electron micrographs.
Low gravity cultured cells High gravity cultured cells

CONCLUSION
The benefits of high gravity brewing are well documented. There are also a number of
negative effects associated with this procedure and three of them have been discussed in
this paper. Esters (particularly ethyl acetate and isoamyl acetate) are disproportionately
increased in fermented high gravity worts. In beer from high gravity worts containing
elevated levels of maltose, ester levels were reduced. Yeast fermented in high maltose
worts has increased viability and vitality. Yeast fermented in high gravity wort excretes
elevated levels of proteinase A into the medium which has a negative effect on beer foam
stability. Yeast cell morphology is influenced by fermentation in high gravity wort. This

8
is illustrated by changes in cell surface topography and enlargement of intracellular
vacuoles.

ACKNOWLEDGEMENTS
The author is grateful to James Bryce, Omar Younis, Dan Cooper, Graham McKernan,
Stephan Bray, Patrica Pratt-Marshall, James Buckman and Margaret Stobie for advice,
support and assistance.

Gratitude is also due to the International Centre for Brewing and Distilling, Suntory
Limited, Scottish Courage Brewing Limited and Heineken International for financial
assistance in support of various aspects of the research being reported in the presentation.

REFERENCES
1. American Society of Brewing Chemists, Methods of Analysis, 1992, 8th revised edition.
2. Anderson, R.G. & Kirsop, B.H., The Journal of the Institute of Brewing, 1975, 81, 286-301.
3. Bamforth, C.W., The Journal of the Institute of Brewing, 1985, 91, 370-383.
4. Bryce, J.H., Cooper, D. & Stewart, G.G., Proceedings of the European Brewery Convention
Congress, Maastricht, 1997, 357-365.
5. Cooper, D.J., Bryce, J.H. & Stewart, G.G., The Journal of the Institute of Brewing, 1998,
104, 83-87.
6. DAmore, T., Celotto, G. & Stewart G.G., Proceedings of the European Brewery Convention
Congress, Lisbon, 1975, 255-267.
7. Kondo, H., Yono, H., Furukubo, S., Kawasabi, Y. & Nakatani, K., Proceedings of the 25th
Convention of the Asia Pacific Section of the Institute of Brewing, Perth, 1998, 119-124.
8. Murray, C.R. & Stewart, G.G., Birra Malto, 1991, 44, 52-66.
9. OConnor-Cox, E.S.C. & Ingledew, W.M., Journal of the American Society of Brewing
Chemists, 1990, 48, 26-32.
10. OConnor-Cox, G.S.C, Majara, M.M., Locholo, G.J., Mochaba, E.M. & Axcell, B.C.,
Ferment, 1996, 9, 321-328.
11. Pfisterer, E.A. & Stewart, G.G., Proceedings of the European Brewery Convention Congress,
Nice, 1975, 255-267.
12. Rudin, A.D., The Journal of the Institute of Brewing, 1957, 44, 52-64.
13. Stewart, G.G., Bothwick R., Bryce, J.H., Cooper, D., Cunningham, S., Hart, C. & Rees, E.,
Technical Quarterly of the Master Brewers Association of the Americas, 1997, 34, 264-270.
14. Stewart, G.G., Bryce, J., Cooper, D., Monagas, M. & Younis, O., Proceedings of the 7th
Brewing Convention of the Africa Section of the Institute of Brewing, Nairobi, 1999, 100-
110.
15. Younis, O.S. & Stewart, G.G., Proceedings of the European Brewery Convention Congress,
Cannes, 1999, 37-44.
16. Younis, O.S. & Stewart, G.G., Journal of the American Society of Brewing Chemists,
1999, 57, 39-45.

9
37

Daily changes in maltopermease and

maltase activities during normal and
high gravity fermentations by ale and
lager strains
J. Rautio1, T. Markkula1, M. Lee2 , J.R.M. Hammond2, W.
Lancashire2 & J. Londesborough1
1
VTT Biotechnology, P.O. Box 1500, FIN-02044 VTT, Espoo, Finland (e-mail:
[email protected])
2
Brewing Research International, Lyttel Hall, Nutfield RH1 4HY, United Kingdom

Descriptors
Brewersyeast, enzymic activity, fermentation speed, high gravity brewing, maltase,
metabolism

SUMMARY
Glucose-induced inactivation of maltose transport is thought to slow brewing fermentations.
In yeast collected daily from fermentations the zero-trans-maltose uptake rate (the initial
transport rate at zero intracellular maltose) under standard conditions dropped on Day1, but
recovered while glucose was still present (20 g/l). The Day1 decrease was smaller in a fast
mutant. The zero-trans-maltose uptake rate for each day's yeast measured in each day's wort
labelled with 14C-maltose agreed closely with the maltose utilization rate. Thus, transport has
high control over maltose utilization and greater specific fermentation rates require higher
maltose transport capacity.

Tgliche Vernderungen in der Maltopermease- und Maltaseaktivitt whrend

normaler und High-Gravity-Grung bei untergrigen und obergrigen Hefestmmen

Deskriptoren
Bierhefe, Brauen mit hohem Stammwrzegehalt, Enzymaktivitt, Grgeschwindigkeit,
Maltase, Stoffwechsel

ZUSAMMENFASSUNG
Es gibt berlegungen, dass die glucoseinduzierte Inaktivierung des Maltosetransports die
Grung verlangsamt. Anhand von tglich genommenen Hefeproben aus der Grung wurde
festgestellt, dass die zero-trans-maltose-Aufnahmerate (Anfangstransportrate, bei der noch
keine Maltose in der Zelle vorhanden ist) am ersten Tag sinkt. Am zweiten Tag stieg die
Aufnahmerate wieder im Beisein von Glucose (20 g/l) an. Am ersten Tag zeigten schnelle
Mutanten eine schwchere Verringerung. Die zero-trans-maltose-Aufnahmerate wurde mit
Hilfe von 14C-Maltose gemessen. Die zero-trans-maltose-Aufnahmerate korrelierte genau
mit der Maltoseverwertungsrate. Das zeigt, dass der Transport einen hohen Einfluss auf die
Maltoseverwertungsrate hat. Somit bentigen hhere spezifische Grungsleistungen hhere
Maltosetransportleistungen.

Variations quotidiennes des activits de maltopermase et maltase pendant des

fermentations basses et hautes par des souches de levure de bire lgre et forte (ale &
lager)

Descripteurs
Activit enzymatique, fabrication de bire haute densit, levure de brasserie, maltase
mtabolisme, vitesse de fermentation

RESUME
D'aprs les connaissances actuelles, l'inactivation du transport du maltose induite par le
glucose ralentirait les fermentations de brasserie. Dans des levures prleves quotidiennement
de fermenteurs, le taux de capture du zro-trans-maltose (vitesse de transport initiale
concentration intracellulaire nulle de maltose) dans les conditions standard a chut J1, mais
est remont pendant la prsence du glucose (20 g/l). La diminution J1 tait plus faible avec
un mutant rapide. Le taux de capture de zro-trans-maltose pour la levure de chaque jour,
mesur dans le mot de chaque jour marqu au 14C-maltose tait trs proche du taux
d'utilisation du maltose. Ainsi, le transport contrle fortement l'utilisation du maltose et une
plus grande capacit de transport du maltose est ncessaire pour que les taux de fermentation
spcifique soient plus levs.

2
INTRODUCTION
It has been suggested that brewery fermentations would be faster if glucose and
maltose were used simultaneously. Faster fermentations have been achieved by
overexpressing maltose transporter using the MAL6T gene (6). Jesperson et al (5)
screened 5 ale yeasts and 25 lager yeasts. All contained MALxT genes and all but one
contained AGT1 genes, so most brewer's yeasts should contain two kinds of maltose
transporter. Glucose, maltose and maltotriose can interfere with each others' use (8).
Glucose represses the genes coding maltose transporters and maltases. It also destroys
already synthesised maltose transporter (glucose-induced inactivation; 2, 3). The
AGT1 gene encodes a transporter that carries maltose and other -glucosides (4).
Maltose and maltotriose inhibit each other's transport by this transporter. In some
strains maltose and glucose also inhibit each other's transport although they have
different transporters. These effects mean that the amount of maltose transporter
present in yeast changes during fermentations and that also the specific activity of the
transporter changes as wort sugars, ethanol, pH and temperature change. We
measured how maltose permease and maltase activities of three related ale strains and
a lager strain change during fermentations of wort and compared the daily maltose
consumption to the maximum maltose transport activity possible by each day's yeast
in each day's wort.

METHODS
Yeasts, worts and fermentations. Three related ale strains (A179, an ale yeast;
A180, an ethanol-tolerant mutant of A179 and A181, a "derepressed" mutant of
A180) and a lager yeast (A24) from VTT's collection were used. 24 P wort was made
at VTT with barley syrup as adjunct (40 %), and was diluted to 16 and 11 P with
oxygen-free water.Yeast was pitched into 24, 16 and 11 P worts at 8, 5 and 3.5 g
fresh yeast/l and 2.0 l portions fermented in stainless steel tall tubes.
Maltose transport assays. Wort samples containing about 1 g fresh yeast were
withdrawn and centrifuged. The clear wort was collected (and sugars assayed by
HPLC) and the pelleted yeast washed twice with ice-cold distilled water. For standard
assays the washed yeast was suspended as 0.1 M Tris/tartrate pH 4.2 to 200 mg/ml
and assayed immediately as described (3) at 5 mM 14C-maltose, 20 C with 20 second
uptake times. For assays in each day's wort the yeast was suspended in distilled water
to 500 mg fresh yeast/ml. The wort was labelled by adding Amersham 14C-maltose.
15 l portions of yeast were added to 60 l portions of labelled wort at the
fermentation temperature and the reaction quenched after 10 to 40 sec (at 20 C) or 20
to 120 sec (at 10 C) while uptake was still linear. Wort was thus diluted about 20 %
for the assay.

RESULTS AND DISCUSSION

Kinetic properties of the transporters
The ale and lager yeast both showed high affinity (Km of 1-2 mM maltose) and low
affinity (Km 15 to 50 mM) maltose transport systems and little pH dependence (< 30
% change between pH 4 and 5, optimum at pH 4.2). Whereas maltose transport by the
lager strain was not inhibited by other sugars (suggesting a MALxT-encoded
transporter) transport by the ale strains was competitively inhibited by -glucosides

3
(suggesting an AGT1-encoded transporter) and glucose (table 1). Crumplen et al (3)
also found that maltose transport was inhibited by glucose in an ale strain but not in a
larger strain. However, even for the ale strains, inhibitions by glucose and sucrose are
not practically important in wort, because while glucose and sucrose are still present,
maltose concentrations are high enough (150 to 250 mM) to overcome these
competitive inhibitions. Competition between maltose and maltotriose is probably
important for the ale strains in late fermentation stages.

Table 1: Inhibition of maltose transport by other sugars

Inhibitions were measured by varying the concentrations of other sugars at 5 mM
maltose. K1/2 is the sugar concentration causing 50 % inhibition. Not Sig: inhibition
observed was not significant (< 10 %) up to sugar concentrations found in HG worts
(or 100 mM for trehalose).

Glucose Fructose Maltotriose Sucrose Trehalose

Concentration 200 mM 25 mM 20 mM 7 mM (traces)
in 16 oP wort
Ale strain K1/2=150 Not Sig. K1/2 = 15 K1/2 = 10 K1/2 = 15
MM MM mM mM
Lager strain Not Sig. Not Sig. Not Sig. Not Sig. Not Sig.

For both yeasts, instantaneous inhibition by ethanol was modest, reaching about 25 %
at 100 g/l, but preliminary results suggested that pre-incubation of the yeasts for a few
minutes at 20 C with more than 100 g ethanol/l caused a stronger inhibition (70 % at
150 g/l), suggesting that ethanol may irreversibly inactivate maltose transport during
the final stages of VHG fermentations.
For the ale strain A179 the apparent Km (calculated from data between 1 and 15 mM
maltose) was 3 1 mM between 0 and 30 C. The Vmax changed more than 100-fold,
most of this change (> 20-fold) occuring between 0 and 10 C. The unusally large
change in Vmax below 10 C presumably results from changes in membrane structure
that interfere with the transport process.

Glucose-induced inactivation
Glucose-induced inactivation was studied with the ale strains in several media, in the
presence and absence of a nitrogen source. The yeasts were grown in YP(yeast
extract, peptone)/maltose (to induce maltose transport and maltase) washed,
suspended in medium and incubated at 30 C with and without addition of glucose or
ethanol. For A179 in YP medium alone the transport activity steadily declined, with
half the activity being lost in about 200 min (figure 1). Addition of glucose (40 g/l)
accelerated this loss, causing over 95 % inactivation in 80 min. For the "derepressed"
mutant, A181, glucose-induced inactivation was much smaller, with 33 % of activity
still present after 160 min. Thus, the difference between A181 and A179 (and A180)
is connected to the stability of maltose transporter, but we do not yet know the
mechanism. For both strains, activity recovered to the control level when glucose was
consumed. Recovery was also seen when the glucose-induced inactivation was carried
out in 0.1 M MES buffer with no nitrogen source (not shown). This contrasts with the
irreversible inactivation reported for laboratory yeast (7). Figure 1 also shows that
ethanol stabilised maltose transport. This ethanol concentration is less than that (100
g/l) reported above to inactivate maltose transport. Lucero et al, (7) reported that
ethanol inhibits glucose-induced inactivation of laboratory strains in N-free media.

4
The activity of maltase did not change in any of these glucose-induced inactivation
experiments. This agrees with the literature: maltase is repressed by glucose, but
glucose-induced inactivation of pre-existing maltase has not been reported.
A179

2.0 40
Transport (mmol/h/g

1.5 30

Glucose (g/l)
DY)

1.0 20

0.5 10

0.0 0
0 100 200 300 400

A181
2.0 40
Transport (mmol/h/g

1.5 30

Glucose (g/100 ml)

DY)

1.0 20

0.5 10

0.0 0
0 100 200 300 400
Time (min)

Glucose Ethanol
2.0 50

0.0 0
0 50 100 150 200 250 300 350 400

control Remaining glucose

Figure 1: Glucose-induced inactivation of the maltose transporter. Glucose (40 g/l) or

ethanol (30 g/l) were added as shown to suspensions of A179 or A181 in YP medium.

Maltose transport activity and sugar consumption during wort fermentations

The ethanol-tolerant A180 mutant fermented 16 and 22 P worts a little slower than
did the parent, A179, strain, but the "derepressed A181 mutant fermented faster and to
a higher attenuation (table 2). A181 clearly consumed maltose faster in early stages
and maltotriose much faster in later stages (figure 2). Under these conditions (pitch at
10 oC, shift to 20 C after 46 or 72 h) there was no marked difference between the
rates of glucose consumption (figure 2), but if fermentations continued at 10 C the
faster maltose consumption by A181 was approximately compensated by a slower
glucose consumption so that there was little or no gain in overall fermentation speed.
Changes in standard maltose transport activity are qualitatively the same for all

5
 Glucose
60
Glc (g/l)
40

20

0
0 20 40 60 80 100 120 140 160 180 200 220

Maltose

100
80
Mal (g/l)

60
40
20
0
0 20 40 60 80 100 120 140 160 180 200 220

Maltotriose
25
20
Mal-3 (g/l)

15
10
5
0
0 20 40 60 80 100 120 140 160 180 200 220
Fermentation Time (h)

A179, 16P A180, 16P A181, 16P

A179, 22P A180, 22P A181, 22P

Figure 2: Sugar consumption during the fermentations of 16 P and 22 P worts by

the ale yeasts. See table 2 for details.

three strains (critical points shown in table 2) and the same pattern was seen with the
lager strain (not shown). During the first 20 h activity drops, presumably due to
glucose-induced inactivation. Activity then recovers, starting while glucose levels are
still high (30 to 40 g/l: enough to trigger inactivation and to cause complete repression
with laboratory strains). In normal gravity worts activity is still high at the end (not
shown), but with HG worts activity decreases rapidly during the final stages, after
most of the yeast has sedimented. This is one reason why early cropping is important
in HG fermentations. Quantitatively (table 2), maltose transport activity of A181 was
higher at pitching and, especially, at 20 h, consistent with the partial resistance of
A181 to glucose-induced inactivation (figure 1). Maltase activity was fairly constant
throughout the fermentations. A relatively small decrease in the first 20 h

6
Table 2: Fermentation speed and standard maltose transport activities during
fermentations by the ale strains.
Fermentations were started at 10 C and raised to 20 C after 46 h (16 P) or 72 h (22
P). The apparent attenuations of the wort and standard maltose transport activities of
the yeasts are shown at selected fermentations times. Results are averages from
duplicate fermentations. ND, not determined

Time (h) 16 P wort 22 P wort

A179 A180 A181 A179 A180 A181
Apparent attenuation (%)
92 80.0 77.3 81.8 42.1 45.4 47.2
163 85.7 82.6 87.7 77.5 76.4 81.9
211 ND ND ND 79.0 77.7 82.9
Standard maltose transport (mol/h/g dry yeast)
0 380 260 610 380 260 610
20 170 100 280 170 200 380
66 560 650 480 250 460 450
120 190 340 ND 400 390 560
163 ND ND ND 130 ND 150

14
(molmin/g dry yeast)

12
Maltose transport

10
8
6
4
2
0
0 50 100 150 200 250

60

50
Maltose uptake (g/l)

40

30

20

10

0
0 50 100 150 200 250
F e rm e n t a tio n T im e (h )

A 1 8 0 , N o a d d itio n A 1 8 0 , G lc a t 2 0 h A 1 8 0 , G lc a t 6 8 h
A 1 8 1 , N o a d d itio n A 1 8 1 , G lc a t 2 0 h A 1 8 1 , G lc a t 6 8 h

Figure 3: Darauflassen fermentations. Glucose-rich wort was added at 20 or 68 h to

fermenting 16 P wort to give increases in glucose of 80 g/l and the temperature
simultaneously raised from 10 to 20 C. Changes in standard maltose transport
activity and maltose consumption are shown.

7
 A
500

400

300

200

100

0
0 10 19 31 42 55 68 80 92 104 116 140 163 187

6 0 0
Rates (umol/h/g dry yeast)

5 0 0

4 0 0

3 0 0

2 0 0

1 0 0

0
0 1 9 4 2 6 8 9 2 1 1 6 1 6 3

1000

800

600

400

200

0
0 11 21 34 48 58 68

Fermentation Time (h)

Figure 4: Comparison of zero-trans maltose uptake rates measured in each day's wort
(dark columns) with maltose consumption rates (light columns) for fermentations of
(A) 16 P wort at 13 C, (B) 11 P wort at 13 C, (C) 16 P wort at 20 C by the lager
strain A24. Error bars in C show the ranges of duplicate or triplicate assays.

presumably represents yeast growth while maltase is repressed. By 70 h the pitching

value was regained and maintained to the end.

8
Figure 3 shows darauflassen experiments where glucose-rich worts (1/3 the original
volume of 320 g glucose/l of 16 P wort) were added at 20 or 68 h to fermentations of
16 P wort. The temperature was raised from the 10 to 20 C at the addition. Maltose
transport was rapidly inactivated by the glucose addition, and in contrast to the
behaviour at pitching (table 2) did not recover. Maltose consumption increased
temporarily after the additions (because of the temperature rise) but then slowed to a
halt for strain A179, leaving unfermented maltose. For A181, residual transport
activity was greater than for the A179 and maltose was fermented out.

Comparison of zero-trans maltose uptake rates of each day's yeast in each day's
wort with maltose consumption.
The standard transport activities shown in table 2 and figure 3 are measured at 5 mM
14
C-maltose, 20 C and pH 4.2 in Tris/tartrate buffer. They show that the amount of
maltose transporter in the yeast changes during fermentation, but do not tell the actual
activity of the transporter at the concentration of maltose, other sugars, ethanol and
pH of each day's wort. In figure 4 the zero-trans maltose uptake rates (which are the
fastest rates at which maltose can enter the yeast before intracellular accumulation
starts to slow the uptake) measured for each day's (lager) yeast in each day's wort are
compared with the daily consumption of maltose. Errors are high at the start of each
fermentation because then (1) maltose consumption is calculated from small
differences between large maltose concentrations and (2) the blank radioactivity in
transport experiments is high. However, a consistent pattern is seen in figure 4. In
early and mid fermentation, the maltose consumption rate during each about 24 h
period was close to the average of the zero-trans uptake rates at the start and end of
each period. However, in late fermentation, the zero-trans rates were about twice the
actual rates of maltose consumption. Similar results were also obtained with the ale
strain A181 in 16 P wort at 10 C. We conclude that transport has almost complete
control over the maltose fermentation rate in early and mid fermentation whereas in
later stages other factor(s) also become important.

PRACTICAL CONCLUSIONS
1. The capacity of maltose transporter(s) is the main factor limiting the rate of
maltose fermentation by ale and lager yeasts in brewery conditions. Thus, figure
4 shows that the rate of maltose consumption in early and mid fermentation is
close to the zero-trans maltose uptake rate of each day's yeast in each day's wort,
whereas figure 2 shows faster and more complete fermentations by a spontaneous
mutant with higher maltose-transport activity.

2. If darauflassen (fed-batch) is used with high-glucose worts, there is a risk that the
maltose transporter will be inactivated and not recover, so that maltose (and
maltotriose) are not fermented out (figure 3). Viability of the cropped yeast will
also be poor (not shown). These problems can be alleviated by finding a mutant
like A181 with decreased sensitivity to glucose-induced inactivation. Or, more
simply, by using maltose-rich rather than glucose- or sucrose-rich adjuncts in
darauflassen.

3. Results in table 2 provide another reason for early cropping. Maltose transport
activity is still high when the fermentations are almost complete and most yeast
has already sedimented (not shown) but drops rapidly during the slow final

9
 stages. Transport activity in recycled yeast is much higher if it is cropped as soon
as enough has sedimented.

ACKNOWLEDGEMENT
We thank Helena Simolin for HPLC analyses, Aila Siltala and Eero Matila for
technical assistance and the Technology Development Centre, Finland (TEKES) and
Oy Panimolaboratorio for financial support.

REFERENCES
1. Benito, B. & Lagunas, R., J. Bacteriol., 1992, 174, 3065-3069
2. Busturia, A. & Lagunas, R., Biochim. Biophys. Acta., 1985, 820, 324-326.
3. Crumplen, R.M., Slaughter, J.C. & Stewart, G.G., Letts. Appl. Microbiol., 1996,
23, 448-452
4. Han, E.-K., Cotty, F., Sottas, C., Jiang, H. & Michels, C.A., Mol. Microbiol.,
1995, 17, 1093-1107
5. Jespersen, L., Cesar, L.B., Meaden, P. & Jakobsen, M., Appl. Environ.
Microbiol., 1999, 65, 450-456.
6. Kodama, Y., Fukui, N., Ashikari, T. & Shibano, Y., J. Am. Soc. Brew. Chem.,
1995, 563, 24-29.
7. Lucero, P., Pealver, ., Moreno, E. & Lagunas, R., Appl. Environ. Microbiol.,
1997, 63, 3831-3836
8. Spencer-Martins, I., Anjos, J., Goncalves, P., Rodrigues de Sousa, H & Salema-
Oom, M., EBC Symposium: Yeast physiology - a new era of opportunity,
Nutfield, UK, 1999.

10
38

Yeast management and process control

by flow cytometric analysis
K.-J. Hutter & C. Lange
Eichbaum Brauereien AG, Kfertaler Strasse 176, D-68179 Mannheim, Germany

Descriptors
Cell cycle analysis, fermentation, flow cytometry, physiology, process control, yeast growth

SUMMARY
Due to ultra-rapid production of beer in closed cylindroconical tanks, the control of the
fermentation process - from the beginning of yeast inoculation to filtration - requires more
efficient on-line methods (i.e. flow cytometry, image analysis) today. Particularly flow
cytometry became an efficient tool for controlling food processes. Cell cycle analysis (DNA
content/yeast cell) gives detailed information about the actual growth phase of millions of
yeast cells in the population in a few minutes. Analysis of glycogen or neutral lipid content
reflects precisely the physiological status of the yeast. Resuls of our yeast management and
process control by flow cytometric analysis are demonstrated. Controlling of running
fermentation processes by these methods means technological and economic advantages.

Hefemanagement und Prozekontrolle mit Hilfe fluzytometrischer Analysen

Deskriptoren
Fluzytometrie, Grung, Hefewachstum, Physiologie, Prozesteuerung, Zellzyklusanalysen

ZUSAMMENFASSUNG
Die beraus schnelle Produktion von Bier in zylindrokonischen Gefen - von der
Herfhrung der Reinkultur bis zur Filtration - verlangt heute effiziente on-line Methodes
(Fluzytometrie und/oder Bildanalyse) zur Kontrolle und Optimierung des Prozesses.
Besonders die Fluzytometrie wurde in den letzten Jahren zu einer wertvollen Methode fr
die Lebensmittel-Prozesskontrolle entwickelt. Hierbei liefert die Zellzyklusanalyse innerhalb
von Minuten detaillierte Informationen ber den aktuellen Wachstumzustand von Millionen
von Hefezellen in der Population. Der Glykogen- und/oder Neutrallipidgehalt gibt Auskunft
ber den zellphysiologischen Zustand der Hefe. Die Ergebnisse unseres Hefemanagement und
der Prozekontrolle mit Hilfe der Fluzytometrie werden demonstriert. Laufende Gr-
prozesse mit dieser Methode zu kontrollieren hat technologische und konomische Vorteile.
Traitement des levures et contrle des mthodes par cytomtrie de flux

Descripteurs
Analyse du cycle cellulaire, commande de procd, croissance de la levure, cytomtrie de
debit, fermentation, physiologie

RESUME
En raison de la production ultrarapide de bire dans des cuves cylindro-coniques fermes, le
contrle de la fermentation - depuis l'inoculation de la levure jusqu' la filtration - ncessite
aujourd'hui des mthodes en ligne plus efficaces (cytomtrie de flux, analyse d'images). La
cytomtrie de flux, en particulier, est devenue un outil efficace de contrle des mthodes dans
l'industrie alimentaire. L'analyse du cycle cellulaire (teneur en ADN/cellule de levure) donne
en quelques minutes des informations dtailles sur la croissance relle de millions de cellules
de levure dans le milieu. L'analyse de la teneur en glycogne ou en lipides neutres reflte avec
prcision l'tat physiologique de la levure. Les rsultats de notre contrle des mthodes et du
traitement des levures par cytomtrie de flux sont prsents. Le contrle de la fermentation en
cours par ces mthodes apporte des avantages technologiques et conomiques.

2
INTRODUCTION
Control of fermentation processes from beginning of pure yeast strain cultivation to
cylindroconical fermentation is performed today by direct and indirect methods. In
former times open tub fermentation was controlled empirically by studying yeast
cover. Changes of yeast cover corresponds to growth phases of the population.
These empiric controlling has been taken over more or less by closed cylindroconical
fermentation at the beginning of the eighties.
All conventional data like temperature measurements, pH-value determinations,
extract-, diacetyl-, ethanol- and CO2-content analysis contributes little to the cell cycle
or cell physiological state of yeast cells.
In normal case a reaction or modellation of process can be realized after fermentation
is finished.
Since 1992 we at Eichbaum breweries have performed a new yeast management
called biomonitoring (Hutter et al., 1995) including fluorescence optical analysis with
incorporation of following parameters:
1. DNA content of yeast cells
2. esterase activity of yeast cells
3. glycogen content of yeast cells.
These parameters are analysed by flow cytometry. Rapid flow cytometric assay will
be useful to monitor yeast cells in industrial process. Improvements in the optical,
fluidic and electronic sections of flow cytometers, cell preparation, staining
procedures and application of new specific fluorochromes and fluorophores
respectively resulted in optimal working conditions for obtaining data of different
cellular constituents of yeasts in process.
The object of our biomonitoring assay was to make fermenting processes transparent.

MATERIALS AND METHODS

Flow cytometric instrumentation
We used a flow device, PAS, from Partec GmbH, Mnster, with the possibilities of
excitation of two light sources, a laser (argon-ion laser, excitation at 488 nm) and a
mercury lamp (HBO 100, excitation at 360 nm) (Ghde & Dittric, 1969). With this
excitation arrangements it is possible to use fluorochromes like DAPI, bisbenzimides
or Calcofluor which are excitable at UV-light and dyes like propidium iodide or
fluorophores like FDA.excited with blue light at 488 nm.

Staining procedures
a) DNA content of yeast cells
Yeast cells were fixed in 70% ethanol. After adequate fixation time cells were
incubated in 0.1% RNase solution at 37 C for 1 hour in a water bath. DNA was
fluorochromized in a propidium iodide-solution 3 x 10-6 g PI/ml Tris buffer, pH 7.5
and 70 mM MgCl2 6 H2O. The cells were allowed to stain for 20 minutes on ice
(Hutter & Eipel, 1978).

b) Esterase activity of yeast cells

Yeast cells were stained according to protocol of Rotman & Papermaster (1966).

3
c) Glycogen content of yeast cells
Glycogen content was carried out of procedure decribed in Diploma-Thesis of M.
Remor (1998).

RESULTS AND DISCUSSION

Cell cycle differentiation of lager and ale yeasts
a) Lager yeasts
Industrial lager yeasts have predominantly a diploid DNA content. Corresponding to
the cell cycle of eucaryotes 2n yeasts cell cycle is divided into G0/G1-, S-, and G2+M-
phase. The DNA histograms show characteristic frequency distributions consisting of
a first peak (= G0/G1-phase cells) with yeast cells of similar diploid DNA content. A
fraction of DNA replicating cells. These cells are located between first and second
peak of histogram and represents S-phase-cells. The third fraction of cells consists of
tetraploid cells. These are budding or G2+M-phase yeast cells.

b) Ale yeasts
Cell cycle of ale yeasts runs essentially corresponding to cell cycle of lager yeasts.
But ale yeasts have in contrast to lager yeasts a polyploid (aneuploid) DNA content.
Ale yeasts consist of two or more than two clones with different DNA contents. Each
clone of this inhomogeneous population proliferates individually as mentioned in
diploid cell cycle of lager yeasts. It is difficult to differentiate a common cell cycle of
an ale yeast population, because of their clonality and diauxistic growth. Diauxie
means one clone of the population starts with proliferation while the other clones stay
in a quiescent phase. The next clone starts cell cycling when conditions of wort have
changed and become optimal for these cells.
The genomic constitution of ale yeasts causes very difficult frequency distributions
with different peaks which are superimposed corresponding to different cell cycles of
several clones.

Cultivation of pitching yeast

Figure 1: Flow cytometric and conventional control of cultivation of industrial

yeast strain. After 8 hours cultivation population is ready for pitching into
cylindroconical tank. At this time yeast cells are in G2 + M-phase and the amount
of cells is 60 mios/ml. That means a time saving of 17 hours.

4
Figure 1 shows cultivation of cropped yeasts over 25 hours. After 25 hours these cells
routinely were pitched into a tank of 10 hl. The result of controlling this cultivation
time shows that the population after 6 to 7 hours is ready for pitching because most of
the cells of the population are in budding phase and cell number amounts of 30
million cells/ml. That means a time saving of 18 hours.

Propagation of lager yeast

The main item of our biomonitoring practice is propagation of yeast population and
controlling growth phases by flow cytometry. That means the development of distinct
growth phases are optimal for aerob and anaerob proliferation.
Examples of figures 2 and 3 show the advantage of an optimal yeast management.
First we started conventially without any propagation. Cropped population was
inoculated when most of the cells are in quiescent growth phase. It lasts six hours
when cells came to first step of propagation. After again 2 hours cells reach
exponentially phase. The adaptation to environmental conditions took eight hours
time. All further steps of aerob cell growth and anaerob fermentation show a
retardation of further several hours.
The population of second fermenting process was propagated by aeration, pumping
and without temperature oscillation. Flow cytometric analysis indicated when cells
were in budding phase. At this time we inoculated the population in cylindroconical
tank. Yeasts continued in their aerob growth directly after inoculation. There was no
retardation by adaptation. A successful yeast management in consideration of cell
cycle analysis means a time saving of 14 hours at this fermentation.

Figure 2: Pitching without propagation means a retardation of fermentation

process of 14 hours. Cropped yeast cells have a long adaptation phase.

5
 Figure 3: Propagated cells can pitched in budding phase into cylindroconical
tank and continue immediately with aerob proliferation.

Viable/dead analysis
The cell physiological state of cropped yeast cells is often unknown. The ratio alive to
dead cells is an important criterion to consider if yeasts are suited for repitching.

a) Esterase activity
We analysed viability of cropped yeasts by simultaneously staining with FDA and PI.
This is a biochemical determination. FDA molecules penetrate into the cells with
intact membranes. Viable cells contain esterases which splits fluorescein from FDA
molecules and show a green fluorescence of yeast cells.
Dead cells were stained simultaneously by PI. This intercalating dye only can
permeate through damaged membranes of yeasts and stains ribonucleic acids red after
blue light excitation. Green and red fluorescence light can succesfully be separated by
flow cytometry.

b) Second application to analyse cell physiological state of yeast cells is

determination of the amount of apoptotic cells of the population.
Before yeast cells are eliminated by autolysis they start a suizid program (apoptose).
That means the apoptotic cell never can start a new cell cycle. Such cells absolutely
have to die. Endonucleases destroy DNA so that the nucleus is divided into several
micronuclei with inhomogeneous size (figure 4). After apoptosis dying process is
continued by autolysis. When flow cytometric analysis show a result of apoptotic
yeasts (histogram) yeast population will not be used for pitching again.

6
 Figure 4: One criterion of cell physiological state of a yeast population is the
amount of apoptotic cells. That means yeast cell never can start a new cell cycle.
Endonucleases destroy DNA so that the nucleus is divided in several micronuclei
with inhomogeneous size.

c) Glycogen content
Besides live/dead analysis glycogen is an application for functionality testing of
cropped yeast cells. Glycogen synthesis takes an oszillating cours during
fermentation. Yeasts at the end of anaerob proliferation accumulate glycogen to
overcome disadvantageous nutritional conditions at low temperature, acidic pH,
ethanol and diacetyl content etc.
As soon as yeast cells enter exponential growth phase glycogen content is reduced.
While during anaerob growth phase an increase of glycogen content again is
noticeable. This is due to nutrient limitation, high cell rate and production of
metabolic substances which force yeast to accumulate glycogen.
An extremely low fluorescence signal of flow cytometric glycogen content analysis of
yeasts points to exhausted yeast cells. This populations should not be pitched again.

CONCLUSIONS
Results of yeast management with flow cytometric analysis show several advantages:
1. fermentation process becomes transparent;
2. growth of yeast population is well known in a closed tank during running
process at each point of time;
3. control of fermentation by flow cytometric analysis guarantees time saving
processes.

ACKNOWLEDGEMENT
We thank Wissenschaftsfrderung of Deutscher Brauerbund for supports of B 60 b.

LITERATURE
1. Ghde, W. & Dittrich, W., Z. Naturforsch., 24b, 360-361, 1969
2. Hutter, K.-J. & Eipel, H.E., FEMS Microbiology Lett., 3, 35-38, 1978
3. Hutter, K.-J., Herber, M. & Lindemann, B., Brauwissenschaft, 48, 5/6, 184-190, 1995
4. Remor, M., Diploma Thesis, Fachhochschule Mannheim, 1999
5. Rotman, B. & Papermaster, B.W., Proc. Natl. Acad. Sci. U.S.A., 55, 134-141, 1966

7
39

Novel method for evaluation of yeast

vitality and its application to yeast
handling technology
T. Imai1, W. Back2, Y. Yasuda1, H. Arimura1, T. Hori1,
M. Abe1 & T. Takeuchi3
1
Kirin Brewery Co. Ltd., Technology Development Department., Production Division, 1-17-
1, Namamugi, Tsurumi-ku, Yokohama 230-8628, Japan (e-mail: [email protected])
2
TU Mnchen, Lehrstuhl fr Technologie der Brauerei I, D-85350 Freising-Weihenstephan,
Germany
3
Kirin Brewery Co. Ltd., Tochigi Brewery, Production Division, 147 Hanaoka, Takanezawa-
cho, Shioya-gun, Tochigi 329-1291, Japan

Descriptors
Fermentative ability, flow cytometry, viability, yeast cell

SUMMARY
Numerous methods for the determination of yeast vitality have been developed in the 20th
century. However, all of the conventional methods had the potential defect that they
measured the only "mean" vitality of "cell population". We have developed the new method,
which enables us to measure the distribution of individual cell vitality highly sensitively and
instantly by using the modified "intracellular pH" method and the newly developed
flowcytometrical technique. Using this method we have first clarified the relationship
between the distribution of individual cell and the fermentation performance and its
usefulness for the yeast handling technology.

Neuartige Methoden zur Beurteilung der Hefevitalitt und ihre Anwendungen in der
Hefebehandlung

Deskriptoren
Durchflucytometrie, Grfhigkeit, Hefezelle, Lebensfhigkeit

ZUSAMMENFASSUNG
Im 20. Jahrhundert wurden zahlreiche Methoden zur Bestimmung der Hefevitalitt
entwickelt. Alle diese Methoden haben jedoch den Nachteil, dass sie immer die
durchschnittliche Vitalitt der Population messen. Wir entwickelten eine neue Methode, die
uns erlaubt, die Verteilung der einzelnen Zellen genau und unmittelbar zu messen. Die
Methode bedient sich der modifizierten intrazellulren pH-Methode und der krzlich
entwickelten Durchflusszytometrie-Technik. Durch Anwendung dieser Methode war es uns
mglich, Rckschlsse auf die Beziehung zwischen der Verteilung der einzelnen Zellen und
der Grleistung zu ziehen und somit die Zweckmigkeit fr die Hefebehandlung zu
demonstrieren.

Nouvelle mthode d'valuation de la vitalit des levures et application la technologie

de traitement des levures

Descripteurs
Cellule de levure, cytomtrie fluidique, fermentabilit (de la levure), viabilit

RESUME
De nombreuses mthodes de dtermination de la vitalit de la levure ont t dveloppes au
20me sicle. Toutefois, toutes les mthodes conventionnelles avaient le dfaut potentiel de
mesurer uniquement la vitalit "moyenne" d'une "population cellulaire". Nous avons mis au
point une nouvelle mthode, qui nous permet de mesurer la distribution de la vitalit de
cellules individuelles, instantanment et avec une haute sensibilit, en utilisant la mthode du
"pH intracellulaire" modifie et la technique de cytomtrie de flux nouvellement dveloppe.
Avec cette mthode, nous avons d'abord clair la relation entre la distribution des cellules
individuelles et la qualit de la fermentation, ainsi que son utilit pour la technologie de
manipulation des levures.

2
INTRODUCTION
The L. Pasteurs finding 124 years ago led to the advent of the revolutionary
technology, so called pure cultivation by E. Ch. Hansen, on which todays brewing
had been laid. Since 100 years ago the yeast quality has been focussed on because the
physiological state of the yeast cell plays an important role on the fermentation
performance and the resulting beer quality. Yeast quality has been defined in terms of
viability and/or vitality. Yeast viability is a measure of the number of dead or living
cells and yeast vitality is a measure of the vigor or physiological state of the living
cell (figure 1 (13)).
Owing to the valuable efforts for 100 years, various methods for the determination of
yeast viability and vitality have been developed, including the staining methods, the
cell replication methods, methods based on the physiological parameter
(polysaccharides, ATP, CO2 evolution rate, oxygen uptake rate, acidification power,
protease, Mg release, etc). We came to obtain some information of yeast vitality by
these methods. As the yeast vitality in the actual brewing circumstances is very
subtle, the much more sensitive method than these methods had been needed. In this
situation the ICP method (intracellular pH method) had been developed (1, 7, 8, 9,
10, 11, 12, 13) and this method is now used practically (1, 2, 3, 5, 15, 17).
However, all of the conventional methods above had the potential defect. It was the
only mean vitality of the cell mass (cell population) that the conventional methods
measured. It goes without saying that the only mean value of the population is not
enough to characterize the population. Not only the mean value but also the
distribution of vitality in the population is originally important (this is similar to the
estimation of malt quality (that is, not only modification but also homogeneity is
important)). The difference of the distribution of the pitching yeast can influence the
fermentation performance of the yeast and the quality of the finished beer.
Therefore we have developed the new method, which enables us to measure the true
vitality with the distribution of individual cell vitality highly sensitively and instantly,
and we have applied this new method to optimize the yeast handling.

Living cell
Vitality

Difference of Viability

Dead cell

Figure 1: Difference between viability and vitality (13).

3
MATERIALS AND METHODS
The measurement of the low pH by the flowcytometer
The fluorescent probe 5(6)-carboxyfluorescein was used for the determination of the
low intracellular pH. To make the stable calibration for the determination of the
brewing yeast intracellular pH, the following particle was newly used because there
had not been found the suitable protonophore for the yeast cells. Non-fluorescent
particle (SuperdexTM 200, Amersham Pharmacia Biotech AG) was equivalent with
the indicated pH buffer (50 mM citrate/disodium hydrogen phosphate buffer; pH 3.0-
7.0) containing 5(6)-carboxyfluorescein. The two fluorescent intensities at 525 nm
and 575 nm were measured by the flowcytometer (Beckman-Coulter EPICS Elite)
with the simple argon laser (488 nm).

The measurement of the individual yeast cell vitality

The measurement of the individual yeast cell vitality was performed by the modified
ICP method (1) combined with the new flowcytometrical method described above.

RESULTS AND DISCUSSION

The breakthrough to measure the low intracellular pH by flowcytometer for the
application of the highly sensitive ICP method
To develop the novel method, we applied the principle of the ICP method because the
intracellular pH is the highly sensitive indicator of the yeast physiological condition
(11). The ICP method measures the intracellular pH under low external pH, which
indicates the in situ activity of plasma membrane ATPase of yeast (8). Today there
are two major technologies to analyze the individual cell, the microscopic image
processing technology (11, 12) and the flowcytometrical technology (6, 14). The best
way to measure the individual cell vitality (intracellular pH) accurately and instantly
is to use the flowcytometer because the flowcytometer can process 10,000 cells pro 1
minute. In the field of mammalian cells, the technology to measure intracellular pH
has been developed. However, this method has the potential limitation in the
measurable pH range (pH 6.5-7.5) (14).
Therefore a new flowcytometrical method must have been developed to solve the
potential problem described in the Introduction. The devise of the fluorescent dye
(5,6- carboxyfluorescein) and fluorescent wavelengths (525 nm, 575 nm) enabled us
to measure the intracellular pH (pH 3.0-7.0) beyond the range of 6.5-7.5 (figure2).
This method has another practical merit, that is, this new method requires the simple
flowcytometer with only one standard argon-laser.

4
 1000

900

I 525nm/ I575nm
800

700

600

500

400

300
3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0
pH

Figure 2: The new relationship between the fluorescent ratio and the pH beyond
the range of 6.5-7.5 using the flowcytometer

The fluctuation of yeast vitality during the storage.

The fluctuation of yeast vitality was observed by this new method. Figures 3-6
showed one example during a deterioration process of yeast. Though the intracellular
pH (ICP) decreased according to the storage period as shown in these figures and the
manuscripts (1, 2, 5, 7, 8, 9, 10, 11, 13, 15, 16, 17), these figures also showed that the
vitality (ICP) of this yeast population fluctuated homogenously under this storage
condition. As the homogeneousness of pitching yeast vitality also plays an important
role to brew the aimed high quality beer constantly, this storage condition is thought
to be optimized from this viewpoint.

100 100
80 80
Cell Number

Cell Number

60 60
40 40
20 20
0 0
11 0
0
00
00
00
00
00
00
00
00

10 0
.0
.0
0

11 0
0
00
00
00
00
00
00
00
00

10 0
1.
2.
3.
4.
5.
6.
7.
8.
9.

.0
.0
0
1.
2.
3.
4.
5.
6.
7.
8.
9.

Intracellular pH (ICP) Intracellular pH (ICP)

Figure 3: Storage period: 0 day Figure 4: Storage period: 5 days

100 100
80 80
Cell Number

Cell Number

60 60
40 40
20 20
0 0
00
00
00
00
00
00
00
00

10 0
11 0
0

11 0
0
0
.0
.0

00
00
00
00
00
00
00
00

10 0
.0
.0
1.
2.
3.
4.
5.
6.
7.
8.
9.

0
1.
2.
3.
4.
5.
6.
7.
8.
9.

Intracellular pH (ICP) Intracellular pH (ICP)

Figure 5: Storage period: 8 days Figure 6: Storage period: 12 days

5
The relationship between the distribution of yeast vitality and young beer
quality
The fermentations were examined using five types of yeast cells with different
distribution of yeast (figures 7-11). The young beer quality was also analyzed in table
1. The distribution of yeast cell vitality affected the beer quality (the fermentation
speed / diacetyl / the protease activity (16) / the middle chain fatty acids / pH).
Especially yeast D, E, which contained the less vital cells, affected the fermentation
performance and the beer quality.

60 50
50 40
Cell Number

Cell Number
40 30
30
20 20
10 10
0 0
0
0
0
0
0
0
0
0
0

0
0
0
0
0
0
0
0
0
.00
.00

.00
.00
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0

1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10
11

10
11
Intracellular pH (ICP) Intracellular pH (ICP)

Figure 7: Yeast A Figure 8: Yeast B

50 50
40 40
Cell Number

Cell Number

30 30
20 20
10 10
0 0
0
0
0
0
0
0
0
0
0
.00
.00
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0

0
0
0
0
0
0
0
0
0
.00
.00
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10
11

10
11

Intracellular pH (ICP) Intracellular pH (ICP)

Figure 9: Yeast C Figure 10: Yeast D

80
70
60
Cell Number

50
40
30
20
10
0
0
0
0
0
0
0
0
0
0
.00
.00
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10
11

Intracellular pH (ICP)

Figure 11: Yeast E

6
Table 1: Comparison of the young beers using the yeasts with different distribution
of yeast vitality
Yeast A B C D E
Fermentation Speed 1.55 1.49 1.43 1.35 1.25
( P/day)
Diacetyl(ppm) 0.23 0.30 0.29 0.34 0.37
Protease(U) 10.5 11.1 10.6 15.4 16.7
Middle Chain Fatty 5.87 5.88 6.39 6.92 6.59
Acids(ppm)
pH 4.36 4.41 4.42 4.46 4.49

The application to the yeast handling technology

The effect of yeast handling technology on the distribution of yeast vitality (ICP) was
also observed. As one example, two types of the yeast technology were compared in
figures 12 and 13. The results of the conventional ICP method (using
spectrofluorophotometer) were shown in table 2. According to the conventional ICP
method there was no difference between these two technologies. However, by using
this new method it became clear that technology B described in (1,2) was superior to
A because more vital cells remained after 7 days by the technology B.

80 80
Cell Number

Cell Number

60 60
40 40
20 20
0 0
0
0
0
0
0
0
0
0
0

0
0
0
0
0
0
0
0
0
.00
.00

.00
.00
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0

1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10
11

10
11

Intracellular pH (ICP) Intracellular pH (ICP)

Figure 12: Fluctuation of yeast vitality using the yeast technology A

The left figure shows the yeast vitality (ICP) at the beginning of the yeast storage.
The right figure shows the distribution of yeast vitality (ICP) after 7 days.

80 80
Cell Number

Cell Number

60 60
40 40
20 20
0 0
0
0
0
0
0
0
0
0
0

0
0
0
0
0
0
0
0
0
.00
.00

.00
.00
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0

1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10
11

10
11

Intracellular pH (ICP) Intracellular pH (ICP)

Figure 13: Fluctuation of yeast vitality using the yeast technology B

The left figure shows the yeast vitality (ICP) at the beginning of the yeast storage.
The right figure shows the distribution of yeast vitality after 7 days.

7
Table 2: The comparison between two different yeast technologies A, B by
the conventional ICP method (using spectrofluorophotometer)

Storage Period 1 day 7 days

Yeast Technology A 5.93 5.45
Yeast Technology B 5.94 5.46

CONCLUSION
Owing to the valuable efforts for more than 100 years, the novel and practically
useful method to assess the individual yeast vitality and to improve the yeast handling
condition in the actual brewing circumstances could be developed.
This method could make it possible to measure the individual yeast vitality with high
sensitivity and instance; moreover, this method enabled us to obtain the distribution
of the individual yeast vitality precisely.
We believe this method and/or the viewpoint of this method mark a new epoch in the
field of the assessment of the yeast physiology in the 21st centurys modern beer
brewing.

REFERENCES
1. Back, W., Imai, T., Forster, C. & Narzi, L., Monats. Brauwissen., 51, 189,
1998.
2. Back, W., 32. Technologischen Seminar Weihenstephan, 1999.
3. Bracy, D., Holyoak, C.D. & Coote, P.J., J. Appl. Microbiol., 85, 1056, 1998.
4. Eils, H.-G., Eidtmann, A. & Back, W., Brauwelt, 140, 590, 2000.
5. Hori, T., Imai, T., Yasuda, Y., Takeuchi, T. & Ohkochi, M., Tech. Quart., 37, 31,
2000.
6. Hutter, K.J., Remor, M., Klein, K. & Lange, C., Proceedings of the European
Brewery Convention Congress, Cannes, 735-742, 1999.
7. Imai, T., The Institute of Brewing, Asia Pacific Section, Proceeding of the 24th
Convention, Singapore. 60, 1996.
8. Imai, T., Iwamatsu, A. & Back, W., Microbial Response to Stress: what's new
and how can it be applied?, Sesimbra, Portugal, 31, 1997.
9. Imai, T., Nakajima, I. & Ohno, T., J. Amer. Soc. Brew. Chem., 52, 5, 1994.
10. Imai, T., Nakajima, I. & Ohno, T., Tech. Q. Master Brew. Assoc. Am., 30, 109,
1993.
11. Imai, T. & Ohno, T., Appl. Environ. Microbiol., 61, 3604, 1995.
12. Imai, T. & Ohno, T., J. Biotechnol., 38, 165, 1995.
13. Imai, T., Brewers Guardian, 128, 20, 1999.
14. Methods in Cell Biology, 41, 135, 1994.
15. Nakajima, I., Imai, T. & Takeuchi, T., Proc. Master Brew. Assoc. Am., Anaheim .
1993.
16. Stamm, M., Dissertation, Technische Universitt Mnchen-Weihenstephan, 2000.
17. Viegas, C.A., Aleida, P.E., Cavaco, M. & Sa-Correia, I., Appl. Environ. Microbiol.,
64, 779, 1998.

8
40

Impact of wort composition and serial

repitching on lager brewing yeast
fermentation performance and
organelle integrity
Cheryl Jenkins1, Alan Kennedy2, Pat Thurston3, Jeff Hodgson2
& Katherine Smart1
1
Oxford Brookes University, School of Biological and Molecular Sciences, Headington,
Oxford OX3 0BP, United Kingdom (e-mail: [email protected])
2
Scottish Courage Brewing Ltd., Technical Centre, Sugarhouse Close, 160 Canongate,
Edinburgh EH8 8DD, Scotland, United Kingdom
3
Scottish Courage Brewing Ltd., Berkshire Brewery, Imperial Way, Reading RG2 0PN,
United Kingdom

Descriptors
Brewersyeast, fermentative ability, physiology, pitching, wort composition

SUMMARY
The impact of serial repitching on lager brewing yeast physiology and fermentation
performance has not been previously reported. Biomarkers have been identified which permit
yeast condition and more specifically organelle integrity during serial repitching to be
investigated. The relationship between variations in wort composition, serial repitching,
fermentation performance and organelle integrity of lager brewing yeast was investigated. It
is suggested that serial repitching affects fermentation performance, mitochondrial function,
cell membrane integrity and cell wall physiology.

Der Einfluss der Wrzezusammensetzung und der mehrfachen Hefewiederverwendung

auf die untergrige Fermentation und die organelle Integritt

Deskriptoren
Anstellen, Bierhefe, Grfhigkeit, Physiologie, Wrzezusammensetzung

ZUSAMMENFASSUNG
Vom Einfluss der mehrmaligen Hefeverwendung auf untergrige Hefe und die Grleistung
wurde bis jetzt noch nicht berichtet. Es wurden Biomarker entdeckt, die es erlauben, den
Hefezustand und die organelle Integritt whrend der mehrfachen Hefeverwendung zu
erforschen. Der Zusammenhang zwischen verschiedenen Wrzebestandteilen, Hefewieder-
verwendung, Grleistung und organeller Integritt wurde bei untergriger Hefe erforscht. Es
zeigte sich, dass mehrfache Hefewiederverwendung die Grleistung, die mitochondrialen
Funktionen, die Zellmembranintegritt und die Zellwandphysiologie beeinflussen.

Impact de la composition du mot et du re-ensemencement en srie sur l'activit

fermentante et l'integrit des organites de la levure

Descripteurs
Composition du mot, ensemencement, fermentabilit (de la levure), levure de brasserie,
physiologie

RESUME
L'impact d'un re-ensemencement en srie sur la physiologie et les performances de
fermentation de la levure de bire forte n'a fait l'objet d'aucune analyse ce jour. Nous avons
identifi des biomarqueurs permettant d'tudier l'tat de la levure, et plus spcialement
l'intgrit des organites, pendant un re-ensemencement en srie. Les relations entre les
variations de la composition du mot, le re-ensemencement en srie, les performances de
fermentation et l'intgrit des organites de la levure de bire ont t tudies. Il est suggr
que le re-ensemencement en srie affecte les performances de fermentation, la fonction
mitochondriale, l'intgrit des membranes cellulaires et la physiologie de la paroi cellulaire.

2
INTRODUCTION
During serial repitching the yeast cell is transferred through several physiological
states (36, 33), from periods of active growth and division during propagation and the
early stages of fermentation, to stationary phase during storage. The number of
generations for which a brewing yeast slurry may be serially repitched will depend on
the apparent robustness of the strain, microbial stability of the slurry and company
policy.
It is believed that the performance of brewing production strains following serial
repitching begins to degenerate after 10 generations (2,33). Despite this the number of
serial repitchings utilised may exceed this in accordance with company policy rather
than the tolerance of the individual yeast strain (2).
Yeast physiological state prior to pitching determines the success of the fermentation
(27, 3). Although slurry storage is an essential part of the serial repitching cycle, this
practice exposes the yeast to a number of stresses which may result in cellular
deterioration including anaerobiosis (2), nutrient limitation (3), low pH (27), ethanol
stress (24) and cold shock (11). As a result of these combined stresses together with
variations in fermentation conditions, wort composition and chance mutations (42),
yeast quality can be compromised.
Theoretically the yeast slurry is immortal (33) but stresses to which the yeast is
exposed over successive generations can result in deterioration of physiological state
(36). Good yeast handling can maintain the physiological state and ultimately increase
the repitching longevity of the yeast slurry, while achieving consistent fermentations
that result in the production of a quality beer (27).
The effect of serial repitching on yeast physiological state and organelle integrity was
investigated in order to identify possible biomarkers, which indicate the onset of yeast
cell deterioration, and requirement for replacement with newly propagated fresh yeast.

MATERIALS AND METHODS

2.1 Yeast strain
A proprietary lager brewing yeast (SCB3) was obtained from Scottish Courage
Brewing Ltd Berkshire Brewery.

2.1.1 Methylene violet

Methylene violet (Sigma Chemical Company) was dissolved in sodium citrate to a
concentration of 0.01% [w/v] and utilised according to the method of Smart et al (34).

2.1.2 Intracellular glycogen and trehalose

Intracellular concentrations of glycogen and trehalose were determined enzymatically
utilising a modified method of Parrou et al (28).

2.1.3 Budding index

The replicative capacity was determined by assessing the budding index. A final
concentration of 1.6 x 10 7 cells/ml-1 was achieved in wort (OG 1065.8) and incubated
with continuous shaking at 120rpm for 6 hours. The percentage of budding cells was
calculated, a minimum of 100 cells were enumerated.

3
2.1.4 MgANS
MgANS (Sigma Chemical Company) was dissolved in 2 ml absolute ethanol and
diluted with 98 ml of sterile water to achieve a final concentration of 0.3 % and
utilised according to the method of McCaig (23).

2.1.5 Flocculation
Flocculation capacity was assessed utilising the modified helms test according to the
method of DHautcourt and Smart (8).

2.1.6 Cell surface charge

Cell surface charge was measured using a modification of the alcian blue dye
retention method of Rapoport and Beker (29).

2.1.7 Hydrophobicity
The hydrophobicity was determined using the latex microsphere attachment assay
according to the method of Rhymes and Smart (31).

2.1.8 Propensity to form petites

Petite formation was induced using a modified method of Rickwood et al, (30). Cells
were inoculated in NO media (2% glucose, 1% yeast extract, 1% bacteriological
peptone, buffered with 50 mM phosphate buffer pH 6.24) to give a final concentration
of 1 x 10 5 Cells/ml-1. Cultures were exposed to 100, 20, 10, 1, 0.1, 0.01, 0 g
concentrations of ethidium bromide for 24 hours at 25 C on an orbital shaker at 120
rpm.

2.1.9 Vicinal diketone uptake

Vicinal diketone uptake was assessed according to the method of Boulton et al (4).

RESULTS AND DISCUSSION

Impact of serial repitching on yeast quality
In order to determine the effects of serial repitching on the quality of the lager
brewing yeast SCB3, a freshly propagated yeast slurry sample was collected from
Scottish Courage Brewing Ltd, Berkshire Brewery. This slurry was monitored
through the repitching cycle using samples cropped from fermentation vessel.
Yeast quality during serial repitching was monitored using characteristics, which
reflect physiological state. These included viability, replicative capacity and
intracellular glycogen content. Figure 1a demonstrates the viability and replication
capacity of SCB3 crop for 6 successive fermentations. It was observed that although
the viability fluctuated it did not significantly decline. The replicative capacity of
SCB3 during serial repitching as determined using the budding index was also
relatively stable (between 40-50%) during the entire sequence of serial repitching. It
is suggested that the variations in yeast quality observed were due to batch to batch
variations in wort composition and fermentation parameters. Figure 1b illustrates the
intracellular glycogen content during serial repitching of SCB3.

4
 120
100
80
Methylene Violet
60
Budding Index
40
20
0
G0 G1 G2 G3 G4 G5 G6
Generation Number

1a
Mg Glucose Equivalent

600
500
400
300 Gly cogen
200
100
0
G0 G1 G2 G3 G4 G5 G6
Generation Number

1b

Figure 1a: Percentage viability determined by the methylene violet dye reduction assay and
percentage of budding cells of serially repitched SCB3; Figure 1b: Intracellular glycogen
content of serially repitched SCB3. Values expressed are the mean of three replicates with the
standard deviation displayed as error bars.

The intracellular glycogen increased from generation two but did not exceed that
observed for propagation slurries. In a previous study (20) a comparison of
propagated and final serial repitch crop demonstrated that the glycogen content also
increased. The accumulation of glycogen as a function of generation number from
generation 2 to generation 6 has not been previously reported and cannot be readily
explained. This phenomenon merits further investigation.

Impact of serial repitching on brewing yeast cell wall physiology

It has been demonstrated that extensive serial repitching results in a progressive
modification in flocculation capacity and cell wall surface charge (39, 36).
The flocculation of SCB3 progressively increased during serial repitching until
generation 4 where it reached a plateau (figure 2). Tiexera et al (39) and Smart and
Whisker (36) also observed this initial increase in flocculation with serial repitching.
Potential reasons for the increased flocculation capacity include repeated exposure to
wort and the gradual accumulation of calcium and magnesium ions (38); exposure to
ethanol, which can promote flocculation in some strains (8); the acquisition of
flocculation competence as a result of previous physiological history (35); or the
selection of more flocculent cells due to the cropping regimes applied (33).
Modifications in cell surface charge are also evident and a positive correlation existed
between the two parameters, supporting the suggestion that cell surface physical
properties play a key role in flocculation (32). The positive correlation observed in a

5
lager yeast is the converse of the findings of Smart and Whisker (36) where an inverse
correlation was demonstrated between cell surface charge and flocculation in an ale
yeast. Surface charge is governed by phosphate groups in lager yeast and carboxyl
groups in ale yeast (32) and this may explain the differences observed.

The hydrophobicity also a function of cell wall, was monitored during serial
repitching of SCB3, but remained unchanged, supporting the observations of Smart
and Whisker, (36). It is therefore suggested that cell surface charge and not
hydrophobicity, represents a biomarker for flocculation performance.

100
80
60 Flocculation
40 Surface Charge
20
0
G0 G1 G2 G3 G4 G5 G6
Generation Number

Figure 2: Flocculation and cell surface charge of serially repitched SCB3. Values expressed
are the mean of three replicates with the standard deviation displayed as error bars.

Impact of serial repitching on the integrity of lager brewing yeast plasma

membrane
The yeast plasma membrane is a dynamic interface between the cell and its
immediate environment. This is the site of nutrient transport and removal of
metabolites from the yeast cell (10). The various stresses to which the yeast slurry is
exposed during serial repitching may result in modifications of membrane
composition, including sterol concentration, ratio of saturated and unsaturated fatty
acids and membrane fluidity. Any such modifications in composition may in turn
affect the specific biological functions related to sugar and amino acid transport (17).
Elucidating any relationship, which may exist between stress, membrane composition
and yeast metabolism may allow for adaptations in process conditions to be
implemented in order to improve yeast performance during serial repitching (21).
The fluorescent microscopy dye 1-Anilino-8-Napthalene Sulphonic acid (MgANS)
can be utilised to assess yeast cell viability (23) and membrane integrity, since the
mechanism of action for this particular dye involves membrane exclusion (7).
MgANS was employed to assess the membrane integrity of SCB3 during serial
repitching.
Figure 3a illustrates the viability and membrane integrity demonstrated by MgANS.
The results indicate variations in membrane exclusion potential. Highlighting the
variability of the condition of the plasma membrane during the serial repitching cycle.
To further investigate the impact of serial repitching on membrane integrity
alternative biomarkers were examined.
Trehalose is as a stress protectant, which is accumulated on both sides of the plasma
membrane in order to confer stability. Its accumulation has been linked to ethanol
toxicity (9), freezing (22), dessication (6) and exposure to toxic chemicals (1). It has
also been suggested that trehalose protects dehydrated cellular membranes (5),

6
increases the thermal stability of proteins (18), and may also act as an osmoprotectant
(25).

100
% Viability

95
MgANS
90

85
G0 G1 G2 G3 G4 G5 G6
Generation Number

3a
Mg Glucose Equivalent

250
200
150
Trehalose
100
50
0
G0 G1 G2 G3 G4 G5 G6
Generation Number

3b

Figure 3a: Percentage viability of serially repitched SCB3, determined using MgANS. Figure
3b: Intracellular trehalose of serially repitched SCB3. Values represent the mean of three
replicates and the standard deviation is displayed by error bars

The intracellular trehalose concentration of SCB3 increased as a function of

increasing generation number during serial repitching (figure 3b). It is suggested that
the yeast cell was indeed exposed to stress during serial repitching resulting in the
induction of the trehalose synthase complex in order to confer membrane stability.
These data support the hypothesis of Smart (33) that serial repitching results in a
repetitive stress injury response and it is further postulated that the main site of
deterioration may be the plasma membrane. However the protection which trehalose
confers to the plasma membrane is sufficient to maintain the integrity despite the
accumulation of stress deterioration as demonstrated by MgANS exclusion.
Vicinal diketones (VDK) are the product of amino acid metabolism, the most
important being diacetyl due to its low organoleptic detection threshold (14). Upon
completion of fermentation both the beer and yeast may remain in residence in the
fermentation vessel to ensure that diacetyl concentrations reach specification. It has
been suggested that the ability of the yeast cell to assimilate diacetyl mirrors the
pattern of sterol synthesis. The positive correlation that exists between sterol
concentration and membrane function suggests that the VDK assay demonstrates the
ability of the yeast cell to assimilate diacetyl rather than its reduction (4) and therefore
reflects membrane integrity.

7
Generation 0 SCB3 demonstrated a lower total VDK uptake than later generations
during the repitching cycle. The ability of the later generations to assimilate diacetyl
did not significantly alter as a function of generation number. Although freshly
propagated cells are highly oxygenated and therefore sterol rich, they are also exposed
to oxidative stress leading to lipid peroxidation. On balance it is suggested that
propagated cells may exhibit a reversibly deteriorated membrane integrity and
therefore a reduced VDK uptake.

100
% VDK Uptake

80
60
VDK Uptake
40
20
0
G0 G1 G2 G3 G4 G5 G6
Generation Number

Figure 4: Percentage VDK uptake of serially repitched SCB3. Values represent the mean of
three replicates and the standard deviation of displayed by error bars.

Impact of serial repitching on mitochondrial function and the propensity for

petite formation
Petite or respiratory deficiency is the most frequently identified spontaneous mutation
in brewing yeast. It is usual for petite mutation to occur at an incidence of
approximately 1% of the population (13). Petite mutations can be induced by ethanol
(19), leucine and lysine limitation (16), vitamin and micronutrient deficiencies (26).
These mutations result in an inability to metabolise lactate, glycerol and ethanol, and
phenotypic differences such as modifications in sugar uptake (37), flocculation (15),
cell wall and plasma membrane structure (41). A high incidence of petite mutation
may account for aberrant fermentations and modification in yeast physiology.
During the serial repitching of SCB3 the incidence of petite mutation was relatively
low. It was not until generation 5 that petites were detected in the yeast slurry (table
1).

Generation Number 0 1 2 3 4 5 6
% Petites 0 0 0 0 0 2 7
Table 1: Incidence of petite mutation during serial repitching of lager brewing yeast SCB3.

In order to determine whether increased incidence of petite mutation was a function of

generation number and not wort composition variation, propagation and generation 8
slurries were exposed to ethidium bromide (section 2.2.8), a chemical known to
induce petite mutation. It was observed that generation 8 cultures displayed a greater
propensity to form respiratory deficient mutants when exposed to varying
concentrations of ethidium bromide (figure 5). The reasons for this observation are
not known. It is suggested that repeated exposure to stress results in an increased
frequency of DNA damage which exceeds the rate of repair leading to the
accumulation of mutations. Thus the older generations exhibit mitochondrial and
nuclear genetic drift leading to the formation of petites and variants respectively.

8
 120
100
80
% Petites
Propagation
60
Generation 8
40
20
0
100 10 1 0.1 0.01 0

micrograms ethidium bromide

Figure 5: Percentage of freshly propagated and generation 8 populations to form respiratory

deficient mutants upon exposure to ethidium bromide. Values expressed are the mean of three
replicate samples with the standard deviation displayed as error bars.

CONCLUSIONS
Serial repitching permits the accumulation of macromolecular deterioration through
repetitive stress injury. The impact of extended serial repitching on the physiology
and fermentation performance of lager brewing yeast remains the subject of further
investigation. Future work will involve the elucidation of the stress responses of lager
brewing yeast during slurry handling to determine the impact on yeast cell
physiology, organelle integrity and fermentation performance.

REFERENCES
1. Attfield, P.V., FEBS Letters, 1983, 225, 259-263
2. Boulton, C., Brewers Guardian, 1991, 4, 25-29
3. Boulton, C., Proceedings of the second brewing yeast and fermentation symposium,
2000, 10-18
4. Boulton, C., Box, W.G., Quain, D.E., & Molzhan, S.W., Proceedings of the 27th
European Brewery Convention Congress, 1999, 687-694
5. Crowe, J.H., Crowe, L.M. & Chapman, D., Science, 1984, 223, 701-703
6. DAmore, T., Crumplen, R. & Stewart, G.G., Journal of Industrial Microbiology, 1991,
7, 191-196
7. Deere, D., Shen., G., Vessey, P., Bell. P., Bissinger, P. & Veal, D., Yeast, 1998, 14, 147-
160
8. DHautcourt, O. & Smart, K.A., Journal of the American Society of Brewing Chemists,
1999, 57 (4), 123-128
9. Eleutherio, E.C.A., Araujo, P.S. and Panek, A.D., Biochimica et Biophysica Acta, 1993,
1156, 263-266
10. Entian, K.D., Journal of Brewing Biotechnology, 1996, 21 (4), 43-47
11. Fargher, A. & Smith, N.A., Proceedings of the European Brewery Convention, 1995,
345-357
12. George, E., The influence of brewing yeast physiology on cell surface physical
characteristics, PhD Thesis, 1996, Oxford Brookes University
13. Good, L., Dowhanick, T.M., Ernandes, J.E., Russell, I., & Stewart, G.G., Journal of the
American Society of Brewing Chemists, 1993, 51 (1), 35-39
14. Hammond, J.R.M., The Yeasts Rose, A. H. & Harrison, J. S., 1993, Academic press
15. Hinrichs, J., Stahl. U., & Esser, K., Applied Microbial Biotechnology, 1998, 29, 48-54
16. Heidenreich, E. & Wintersberger, U, Current Genetics, 1997, 31, 408-413

9
17. Henschke, P.A. & Rose, A.H., The Yeasts Volume 4 Cytology, Harrison, J.S. & Rose, A.
H. Eds, 1991, Academic Press
18. Hottinger, T., De Virgillio, C., Hall, M.N., Boller, T. & Wiemken, European Journal of
Biochemistry, 1994, 219, 187-193
19. Ibeas, J.I. & Jimenez, J., Applied Environmental Microbiology, 1997, 63, 7-12
20. Jenkins, C.L., Kennedy, A.I., Thurston, P., Hodgson, J. & Smart, K.A., World Brewing
Congress, 2000
21. Lentini, A. & Rogers P., Proceedings of the second brewing yeast and fermentation
symposium, 2000, 160.
22. Lewis, J.G., Learmonth, R.P. & Watson, K., Microbiology, 1995, 141, 687-694
23. McCaig, R., Journal of the American Society of Brewing Chemists, 1990, 43, 114-118
24. McCaig, R. & Bendiak, D., Journal of the American Society of Brewing Chemists, 1985,
43, 114-118
25. Mackenzie, K.F., Singh, K.K. & Grown, A.D., Journal of General Microbiology, 1988,
134, 1661-1666
26. Nagai, S., Mutation Research, 1969, 8, 557-564
27. OConnor-Cox, E., Brewers Guardian, 1998, 126, (12), 26-34
28. Parrou, J.L., Teste, M. & Francois, J., Microbiology, 1997, 143, 1891-1900
29. Rapoport, A. and Beker, M., Mikrobiologiya, 1985, 52, 259-261
30. Rickwood, R., Dujon, B., & Darley-Usmar, M., Yeast mitochondria, in Yeast a practical
approach, Campbell, I., Duffus J. H. Eds,. 1991, IRL Press, Oxford.
31. Rhymes, M. & Smart, K.A., Journal of American Society of Brewing Chemists, 1996, 54
(1), 50-56, 1996
32. Smart, in press, Institute of Brewing, South Africa Section meeting.
33. Smart, K.A., Brewers Guardian, 1999, 128: (2) 19-24
34. Smart, K.A., Chambers, K.M., Lambert, I., Jenkins, C.L. & Smart, C.A., Journal of the
American Society of Brewing Chemists, 1999, 57 (1), 18-23
35. Smart, K.A., Boulton, C.A., Hinchcliffe, E. & Molzahn, S., Journals of the American
Society of Brewing Chemists, 1995, 53, 33-38
36. Smart K.A. & Whisker, S., Journal of the American Society of Brewing Chemists, 1996,
54, 14-44
37. Spencer, J.F.T., Spencer, D.M. & Miller, R., Zur Naturforsch, 1983, 5/6 405-407
38. Stewart, G.G. & Russell, I., European Brewing Convention Monograph XII, 1987, 53-68
39. Tiexera, J.M., Tiexera, J.A., Mota, M., Manuela, M., Guerra, B., Machado Cruz, J. M. &
Sa Almeida, A.M., Proceedings of the European Brewing Convention, 1991, 23, 241-248
40. Wilkie, D. & Evans, I., Trends in Biochemistry Sci. Pers., 1982, Ed 7, 147-151
41. Young, T.E., Priest, F.G., Campbell, I., Eds., 1987, Elsevier Applied Science Publishers
Ltd, London.

ACKNOWLEDGEMENT
The authors would like to thank the Directors of Scottish Courage Brewing Limited
for permission to publish this paper.

10
41

Why warm cropping is best!

David Quain1, Chris Powell2, Alisdair Hamilton1, David
Ruddlesden3 & Wendy Box1
1
Bass Brewers Ltd, Technical Centre, P.O. Box 12, Cross Street, Burton-on-Trent DE14 1XH,
United Kingdom (e-mail: [email protected])
2
Oxford Brookes University, School of Biological and Molecular Sciences, Headington,
Oxford OX3 0BP, United Kingdom
3
Bass Brewers Ltd, Burton Brewery, 74/75 Station Street, Burton-on-Trent DE14 1XH,
United Kingdom

Descriptors
Fermentation, fermentative power, flocculation, viability, yeast technology

SUMMARY
A previous study from production scale trials described the benefits of early warm cropping
of yeast from fermenter. The work reported here extends these observations by monitoring
crop parameters and fermentation performance of cuts taken during early warm and
conventional yeast cropping from production fermentations. Although marked differences
were observed during both processes, warm cropped yeast had better process capability
(viability, fermentation performance) than conventionally cropped yeast. Other work has
focussed on process optimisation, understanding and development. Specifically we have
explored the requirements for yeast flocculation and the selective advantage of enrichment of
older mother cells in warm yeast crops.

Warum die warme Hefeernte am besten ist!

Deskriptoren
Flockung, Grkraft (Triebkraft), Grung, Hefetechnologie, Lebensfhigkeit

ZUSAMMENFASSUNG
Vorausgehende Untersuchungen im Produktionsmastab versuchen die Vorteile einer frhen
Warmernte der Hefe aus dem Grbehlter zu beschreiben. Die hier vorgestellte Arbeit
erweitert diese Beobachtung durch die berwachung der Ernteparameter und Grleistung aus
Proben, die whrend der frhen Warmernte und der konventionellen Hefeernte aus
Produktionsgrbehltern genommen wurden. Es zeigten sich auffllige Unterschiede
zwischen den beiden Prozessen. Warm geerntete Hefe zeigte bessere Prozesseigenschaften
(Lebensfhigkeit, Grleistung) als die konventionell geerntete Hefe. Eine andere Arbeit
befasste sich mit der Prozessoptimierung, dem Verstndnis und der Weiterentwicklung.
Zudem wurden die Voraussetzungen der Hefeflockung und die selektiven Vorteile der
Anreicherung von lteren Mutterzellen bei der warmen Hefeernte untersucht.
De la supriorit de la rcolte chaud

Descripteurs
Fermentation, floculation, pouvoir fermentaire, technologie de la levulre, viabilit

RESUME
Une prcdente tude l'chelle de production a dcrit les bnfices d'une rcolte prcoce
chaud des levures dans le fermenteur. Les travaux rapports ici tendent ces observations en
surveillant les paramtres de la rcolte et les rsultats de fermentation d'chantillons prlevs
pendant une rcolte chaud et une rcolte classique dans des fermenteurs de production.
Bien que ces deux procds prsentent des diffrences marques, les levures rcoltes chaud
avaient une meilleure capacit (viabilit, rsultats de fermentation) que celles rcoltes par un
procd classique. D'autres travaux ont t consacrs l'optimisation et au dveloppement des
mthodes. Plus prcisment, nous avons tudi les exigences de floculation de la levure et
l'avantage slectif d'un enrichissement de cellules mres plus vieilles dans les rcoltes
chaud de levures.

2
INTRODUCTION
In the last 30 or so years, understanding what makes yeast tick has loomed large in
brewing laboratories worldwide. Today, driven by key issues such as product quality,
consistency, efficiency and planning, brewers and brewing technologists have the
tools to optimise brewery fermentations and implement agreed best practice for the
management and handling of yeast.
In terms of brewing yeast research activity, there have perhaps been 10 big themes -
pitching rate, oxygen, wort quality, acid washing, storage, "vitality", propagation,
genetic stability, immobilised systems and cropping. Of these, yeast cropping has
received comparatively little attention. For example, a search of the excellent BRi
Brew database (19) on "yeast cropping" recovers a mere 7 references compared to 32
for "yeast vitality" and 53 for "yeast viability". This is somewhat surprising given the
central importance and significance of yeast cropping from the cones of
cylindroconical vessels to the process.
Arguably, this relative lack of activity may reflect the belief that the cropping process
is relatively simple. The process outputs are straightforward - sufficient yeast of
suitable physiological and microbiological quality to enable pitching-on to two or
more fermenters. Fundamental to this is the balance between yeast growth and
flocculation characteristics, which ensure sufficient yeast, can be cropped together
with relatively little yeast going forward from fermenter in green beer. Unfortunately,
this apparent simplicity is the root of the problem! Led by OConnor-Coxs (11)
excellent review there is growing recognition that cone cropping requires more
stringent management to minimise what can only be the damaging effects of "storage"
in the yeast cone. Indeed, appreciation that storage commences with entry into the
vessel cone has refocused effort in this area. Inevitably, yeast storage anywhere in the
process (but particularly in the cone) can only be deleterious to yeast quality and,
subsequently, fermentation performance/product quality.
The guidelines for yeast cropping from cylindro-conical vessel (CCV) cones have
been broadly limited to cropping post cooling and to discarding the first trub-rich cut
(2,10,11,14,15) and, occasionally, the uppermost cut which is considered to be rich in
"less flocculent" yeast (9,11). The concept of warm cropping - when the fermentation
is complete but diacetyl remains to be reduced - has been recognised (10,11,17,18).
However, with exception of South African Breweries (11) and, on a trial basis,
ourselves (8) warm cropping appears to have found little application in the "real
world" of production brewing. An alternative view is that given the low profile of
yeast "cropping" it may be that little has been published of warm cropping and other
approaches to yeast recovery.
Not unexpectedly, warm cropping "adds value" by removing yeast early from the
vessel and, consequently, minimising cell damage triggered by the environment
within compacted yeast in the cone. Cell vitality and, ultimately viability are
compromised by the twin impact of localised "hotspots" of temperature and ethanol.
Although difficult to model, temperature hotspots of 5-8C higher than the vessel
temperatures have been reported (2,4,18). Accordingly, early removal from such an
environment makes eminent physiological sense!
Protracted production trials with warm cropping (8) had a major impact on process
performance. Vessel residence time was shorter, more consistent and yeast viability
improved dramatically. However, a SWOT (strengths, weaknesses, opportunities, and
threats) analysis of the process posed some questions that the work reported here goes
some way to answering. The three ambitions of this work were (i) a better
understanding of the warm cropping process, (ii) process implications of cropping

3
older/bigger cells and (iii) benchmarking of the process with a non-flocculent and
very flocculent yeast.

METHODS
"Warm" and "cold" cropping of S. cerevisiae BB11 was evaluated in commercial
fermentations of lager wort (15P) in cylindroconical vessels of 2000 hl. Vessel
dimensions were 17 m x 4.24 m with cooled cones at a 60 included angle. Pilot Plant
fermentations were performed with three lager strains (BB6, 10, 11) in vessels with
dimensions of 2.4 m x 0.91 m with cones at a 70 included angle. Stirred min-
laboratory fermentations were as described previously (13). Yeast viability was
determined using the methylene method yeast solids were determined after
laboratory centrifugation. Yeast flocculation was determined using the method of
DHautcourt & Smart (5) [figure 7] or Goodger et al., (7).

RESULTS
Ambition 1 "a better understanding of the warm cropping process". To better
characterise the warm cropping process, various yeast and barm ale parameters were
monitored during warm (!) and cold (") cropping of two 2000 hl lager
fermentations. In figures 1-7, the "x axis" is graduated in kg of yeast slurry rundown
from CCV.

Temperature
Figure 1 (right) shows the
temperature of the yeast cropped 20
warm was about 15C in-line with
15
the fermentation temperature.
10
Correspondingly, the temperature
of yeast cropped cold ranged 5

between 1 and 8C against the 0

0
1000
2000
2600
4000
4500
5100
5178
6000
7000
8500
9500
10750

nominal temperature of 4C.

pH
Figure 2 (right) shows that the pH
of the barm ale was higher in the 6
early fractions irrespective whether
5.5
the process was cold or warm.
Seemingly the higher pH was 5

associated with higher yeast solids 4.5

found in the bottom third of the 4
0

1500

2600

4100

5100

5500

7000

9000

10750

cone.

4
Suspended solids
Figure 3 (right) reports the crop
solids for the two approaches and 60
shows that the warm crop and the 50
cold crop were broadly similar 40
30
although the cut-off from high to 20
thin solids was more abrupt with 10
the smaller warm crop. Whether 0

0
1000
2000
2600
4000
4500
5100
5178
6000
7000
8500
9500
10750
this is a function of cone geometry
remains is not clear.

Abv
Figure 4 (right) shows the abv of
the barm ale, which showed a 9
similar pattern to that of the yeast 8.5
8
solids reaching as high as 8.5% 7.5
(v/v) in the fractions greater than 7
40% solids. There was little 6.5
distinction between the abv of 6
0
1000
2000
2600
4000
4500
5100
5178
6000
7000
8500
9500
10750
barm ale from the warm or cold-
cropped yeast.

ABV and yeast solids

Figure 5 (right) shows the
relationship between yeast-wet 9
solids (figure 3) and barm abv
(figure 4). It is suggested that the 8
ABV

additional ethanol is derived from 7

glycogen breakdown and from a
6
"forced fermentation" effect of
0 20 40 60
high yeast solids. Evidence for the
Solids (%)
later comes from the PG gradient
from the bottom of the vessel cone
(PG < 6) to the top (PG > 8.5).

Yeast viability
Figure 6 (right) provides the most
revealing insight into the success 100

of warm cropping, which came 95

from tracking viability across the 90

fractions during the process. 85

Given the principle behind warm 80

cropping, it is no surprise to find 75

0

1500

2600

4100

5100

5500

7000

9000

10750

the viability of the entire warm

cropped fractions to be
significantly higher (91.1 1.6%)
than the control cold crop (83.4
3.3%).

5
Flocculation
Contrary to expectation, figure 7 (below) shows that - irrespective of warm (1st cut- !,
2nd cut - ) or cold (") cropping yeast flocculation (as a %) exhibits a peak that
loosely correlates with the final fraction of warm cropping and the mid-point of cold
cropping. This is in stark contrast to the widely held view that there is a gradient of
flocculence across the cone with yeast at the base being heavily flocculent.

90
88
86
84
82
80
78
76
74
72
70
0 2000 4000 6000 8000 10000 12000

Ambition 2 - "process implications of cropping older/bigger cells". Here, our

concern was built around the observations of Deans and co-workers (6) who
monitored "lager yeast industrial fermentations" and through analysis of five hl cuts
showed an age gradient across the vessel cone. From the perspective of warm
cropping, Deans et al., clearly showed that the cells in the middle and upper half of
the yeast cone were of superior quality to the yeast at the base of the cone. In terms of
cell age (see 12, 16 for a review), warm cropping would be expected to select older,
bigger cells, which might be expected to perform poorly when compared directly with
younger, smaller cells.
To test this hypothesis, brewery yeast ex storage vessel was fractionated using sucrose
gradients into (unbudded) "virgin" () and "mother cells" (!). The fermentation
performance of these fractions together with the "mixed" (") is reported in figure 8
below. (Note the below data was sourced from mini-fermenter experiments based
on weight loss (y-axis) against time (x-axis) the data is directional and,
intentionally, is not clouded by detail).

Contrary to Deans et al (6) the above results would suggest that older (and inevitably
"bigger") cells outperform the younger/smaller cells.

6
Ambition 3 "benchmarking of the process with a non-flocculent and very
flocculent yeast". Intuitively it would be assumed that warm cropping would only
work with flocculent brewing yeasts and would struggle with powdery non-flocculent
strains that crop poorly. To test this we compared and contrasted the performance of
three lager yeasts of differing flocculence normal (BB11), heavy (BB6) and
powdery (BB10). To facilitate comparison we moved away from production-scale
fermentations to the Pilot Plant, using common worts and matched 8 hl CCV vessels.
This approach although more convenient cannot fully replicate production
conditions but clearly provides insight which can be extrapolated to the "real world".
As warm cropping can only be sustained if sufficient yeast can be recovered, the
performance measure was to quantify the warm crop as a proportion of the total yeast
crop (see figure 9 below).

100
% warm of total crop

80

60

40

20

0
0 20 40 60 80 100
flocculation (%)

Not surprisingly, flocculation is a key parameter in the success or failure of warm

cropping. With two flocculent strains, warm cropping accounted for 70-80% of the
total yeast crop whereas with the non-flocculent yeast, warm cropping represented a
paltry 45%. However, it is noteworthy that in the production trials described in figures
1-8 the warm crop accounts for 62% (1928 kg centrifuged yeast) of the total crop
(3107 kg). This reinforces the earlier point about scale-up of trials, together with the
balance between yeast growth and yeast cropped. Suffice to say that this and our
previous work (8) suggest that a yield of 60% is sufficient to maintain warm
cropping as a viable commercial process. In conclusion, although warm cropping can
only be used with flocculent yeasts, it might be argued that this process can be used
with any yeast that is cropped conventionally in the vessel cone.

DISCUSSION
Previous work (8) clearly demonstrated the process benefits of warm cropping of
yeast from fermenter. Although early removal of yeast from fermenter would be
anticipated to minimise the impact of a physiologically damaging environment, we
have sought in this work to build on previous observations and provide further insight
into the warm cropping process.
Although no surprise, much of the data reported here from production fermentations
confirms what might be anticipated intuitively. The data for temperature (figure 1)
clearly demonstrates - in the yeast cropped "cold" - the presence of hotspots, which
together with high abv result in cell damage and, ultimately death. Conversely, the
data for yeast viability (figure 6) presents perhaps the most cohesive case for warm

7
cropping. There can be no debate that warm cropping results in yeast of consistently
higher viability than "conventional" cold cropping. Armed with this, it is
incomprehensible that warm cropping has not had a wider take-up. However, as
previously noted (8) warm cropping creates process complexity through the need to
crop twice warm and conventionally cold (post diacetyl reduction). This added
complexity presents the biggest barrier to routine application of the process.
Despite the obviousness of much of the data from these trials there are numerous
observations that are insightful and, in some cases, unexpected. The impact of yeast
solids on pH (figure 2), abv (figure 4) and PG provides welcome insight into the
stratification and heterogeneity of yeast in the fermenter cone. There is no doubt that
that the environment within the cone is far more complex and organised than
previously understood. Parameters such as abv, solids, pH and viability increase
toward the base of the cone whereas PG increases toward the top of the cone. Perhaps
the most interesting observation is that captured in figure 7, where yeast flocculence
(irrespective of cold or warm cropping) peaks at the mid-point of the cone as opposed
to the vessel bottom. Consequently, any search for more flocculent variants of
brewing yeast should focus on mid-cut as opposed to early runnings from a CCV.
Realistically the "jury is still out" as to the importance of cell age/size on fermentation
performance. However, the protracted production trials reported previously (8)
together with the argument that the work of Deans et al., requires to be overlaid with
viability data suggests that selection of older/bigger cells is by no means a
showstopper!

ACKNOWLEDGEMENTS
Bob Shieldon and Dave Greening are thanked for making the Pilot Plant experiments
happen. Dr Stuart Molzahn is thanked for his support and guidance. The Directors of
Bass Brewers are thanked for permission to publish.

REFERENCES
1. Boulton, C & Quain, D., Brewing Yeast and Fermentation, Blackwell Science,
Oxford, 2001, 456-464
2. Boughton, R., The Brewer, 1983, July, 260-264
3. Boughton, R., Technical Quarterly of the Master Brewers Association of the
Americas, 1987, 24, 133-136
4. Cahill, G., Walsh, P.K & Donnelly, D., Proceedings of the 27th Congress of the
European Brewery Convention, 1999, 695-702
5. DHautcourt, O & Smart, K.A., Journal of the American Society of Brewing
Chemists, 1999, 57, 123-128
6. Deans, K., Pinder, A., Catley, B.J & Hodgson, J.A., Proceedings of the 26th
Congress of the European Brewery Convention, 1997, 469-476
7. Goodger, A., Box, W.G & Quain, D.E., in preparation
8. Loveridge, D., Ruddlesden, J.D., Noble, C.S & Quain, D.E., Proceedings of the
Seventh Convention of the African Section of the Institute of Brewing, 1999,
95-99
9. Matilla, F., Garayblas, E., Moreno, M & Vazquez, L., Proceedings of the 16th
Convention of the Australian and New Zealand Section of the Institute of
Brewing, 1980, 143-154

8
10. Noble, S., The Brewer, 1997, June, 253-261
11. OConnor-Cox, E., Brewers Guardian, 1997, December, 26-34
12. Powell, C.D., van Zandycke, S.M., Quain, D.E & Smart, K.A., Microbiology,
2000, 146, 1023-1034
13. Quain, D.E., Box, W.G & Walton, E.F., Laboratory Practice, 1985, 34, 84
14. Quain, D.E., Proceedings of the Third Aviemore Conference on Malting,
Brewing & Distilling, 1990, 74-83
15. Quilliam, W.R., Proceedings of the Sixth Convention of the Central and
Southern African Section of the Institute of Brewing, 1997, 61-66
16. Smart, K., Brewers Guardian, 1999, February 19-24
17. Takis, S., Lentini, A., Wheatcroft, R., Pecar, M., Hawthorne, D.B & Kavanagh,
T.E., Proceedings of the 24th Convention of the Asia Pacific Section of the
Institute of Brewing, 1996, 245-246
18. Wheatcroft, R., Lentini, A., Tai, L., Shaw, R., Hawthorne, D.B & Kavanagh,
T.E., Proceedings of the Fourth Convention of the Central and Southern African
Section of the Institute of Brewing, 1993, 153-165
19. www.brewingresearch.co.uk

9
42

The use of concanavalin A to investigate

the onset and mechanism of flocculation
by a brewing yeast strain
Franciska Mochaba, Ian Cantrell & Waha Vundla
The South African Breweries Ltd, Brewing Research & Development, P.O. Box 782178,
Sandton, 2146, Gauteng, South Africa (e-mail: [email protected])

Descriptors
Brewers' yeast, fermentation, flocculation, lectin, malt, process control

SUMMARY
Inconsistent yeast flocculation patterns in production fermentations have negative cost
implications. Better understanding of the mechanism of flocculation and the flocculation
characteristics of our production brewing strain was achieved using concanavalin A. This made it
possible to
- predict the onset of flocculation
- identify factors contributing to delayed or premature flocculation in industrial fermentations
- take corrective action to control flocculation
A rapid screen for the selection of malts and yeast slurries free from the adverse flocculation
factors was developed.

Untersuchungen mit Concanavalin A zur Erforschung des Beginns und des Mechanismus
der Flockung von Bierhefen

Deskriptoren
Bierhefe, Flockung, Grung, Lektin, Malz, Prozesteuerung

ZUSAMMENFASSUNG
Vernderliche Verhaltensmuster bei der Flockung von Bierhefe ziehen erhhte Kosten nach sich.
Ein besseres Verstndnis des Flockungsmechanismus und der Flockungscharakteristika unserer
Hefestmme wurde durch Versuche mit Verwendung von Concanavalin A erreicht. Dies
ermglichte:
- den Beginn der Flockung vorauszusagen;
- die Faktoren, welche die Flockung verzgern oder beschleunigen zu ermitteln
- und durch geeignete Manahmen die Flockung zu steuern.
Ein Schnelltest zur Bestimmung von Malzen und Hefen mit ungnstigen Flockungsfaktoren
wurde entwickelt.

Utilisation de la concanavaline A pour tudier le dclenchement et le mcanisme de

floculation d'une souche de levure de bire

Descripteurs
Commande de procd, fermentation, floculation, lectine, levure de brasserie, malt

RESUME
L'irrgularit des profils de floculation de la levure dans les fermentations de production a des
rpercussions ngatives sur les cots. L'utilisation de la concanavaline A permis de mieux
comprendre le mcanisme et les caractristiques de floculation de notre souche de levure de
production. Cela a permis:
- de prvoir le dclenchement de la floculation
- d'identifier les facteurs contribuant retarder ou dclencher prmaturment la floculation en
fermentation industrielle
- de prendre des mesures correctives pour contrler la floculation
Un test rapide pour slectionner les malts et les mlanges de levure dnus de facteurs de
floculation indsirables a t mis au point.

2
INTRODUCTION
Yeast flocculation is important to the brewing industry because the extent of fermentation
and flavour of beer are greatly affected by the extent and timing of flocculation.
Numerous studies have therefore been carried out to elucidate the mechanism and the
factors that influence flocculation (2,8,9,10,11,14,15,17,20). Among them are studies
using the lectin concanavalin A (3,16,18,21). Its use has led to the definite identification
of yeast flocculation receptors.
Flocculation occurs when specific proteins (lectins) present on cell walls bind to mannan
receptors on neighbouring cell walls ( 20,8). The lectins, also known as adhesins, are
reportedly present only on flocculent cells while the receptors are present on both
flocculent and non-flocculent cells. Tkacz et al. (21), and Miki et al. (8), using
concanavalin A, were able to show that yeast cells lacking mannan in their walls lacked
flocculation receptors. Stratford (19), also used this lectin to study the onset of
flocculation and concluded that the receptors on the surface of cells were present
throughout growth as evidenced by agglutination of cells by concanavalin A from the
early stages of growth. However, flocculation only occurred naturally when cellular
lectins had been expressed and activated by some unknown mechanism.
This paper describes the use of concanavalin A to study the onset and mechanism of
flocculation in our industrial fermentations. This has led us to a better understanding of
premature flocculation associated with some malts.

MATERIALS AND METHODS

Yeast strain
The lager brewing strain SAB 5 was used throughout the study. The inocula for
fermentations both at laboratory scale and industrial scale were obtained from a Brewery
Yeast Collection Vessel.

Fermentations
Fermentations were carried out in 16 oPlato (70%:30%) malt:adjunct wort obtained from
a commercial brewery. Both the laboratory scale fermentations and the industrial
fermentations were inoculated at 1.0 kg/hl and oxygenated to give final oxygen
concentrations of 16 mg/l.

Flocculation assays
The flocculation ability of the cells was monitored using the method of Smit et al., (12).
Cells were harvested by centrifugation at 6000 x g, washed in ice cold 0.05 mM sodium
acetate buffer, pH 4.5, then resuspended in the same buffer plus 0.1% (w/v) calcium
chloride to A620nm = 2.5. (The cell count at this optical density was equivalent to 35 x 107
cells/ml). The cell suspension was left to attemperate at room temperature for 30 min
after which 550 l were dispensed into a 1.0 ml cuvette and 100 l of different
concentrations of concanavalin A or control (buffer) were added. The cuvette was placed
on a vortex mixer at maximum speed for 20 seconds, inverted 5 times and immediately
placed in a Beckman DU640 spectrophotometer. The decline in optical density was
monitored over 2 minutes.

3
Effect of concanavalin A on yeast slurries of different quality
Slurries with different pH values, reported to be indicative of quality (7), were obtained
from two production plants. Their flocculating abilities with and without concanavalin A
were measured as described above.

Effect of malt extracts on flocculation

Malt samples (45 g) were washed in 75 ml 0.05 mM sodium acetate buffer pH 4.5 for 1
hour. The filtrate was obtained by filtration through Whatman No. 1 filter paper then
filtered through a 0.45 m acetate filter. The filtrates were added at 100 l to cell
suspensions and flocculation assays carried out as described above.

RESULTS AND DISCUSSION

Results obtained at the laboratory scale were similar to those obtained with industrial
fermentations. Therefore, for clarity only results obtained at the industrial scale will be
discussed. The ability of cells to flocculate was measured from immediately after tank
filling (T0) and at 24, 48, 72 and 96 hours into fermentation (T24,T48,T72 and T96
respectively). The results in figure 1 show that the cells ability to flocculate varied with
time in fermenter. Thus at T0 the cells showed considerable flocculating ability. This
ability was reduced at T24 and then was seen to increase both at T48 and T72 where the
cells showed the highest flocculating ability. Subsequently, for the 96 hour sample (T96),
a reduction in flocculating ability was observed.

36
CELL COUNTS (x 107 cells/ml)

30

24

18

T0
12
T24
6 T48
T72
0
0 30 60 90 120 T96
TIME (SECONDS)

Figure 1: Flocculation of cells harvested at T0, T24, T48, T72 and T96

An understanding of why the flocculating ability of the cells varied with fermentation
time was gained from experiments involving the addition of varying concentrations of
concanavalin A. Thus cells harvested at T0, although fully dispersed in the wort showed
considerable flocculating ability in the assay buffer. However, this ability to flocculate
changed with increasing concentrations of concanavalin A as shown in figure 2 .

4
 36
CELL COUNTS (x 107 cells/ml)

30

24

18

12
CONTROL
6 5mg/ml con.A
0.5mg/ml con.A
0
0 30 60 90 120 0.01mg/ml con.A

TIME (SECONDS)
Figure 2: Flocculation of cells harvested at T0 concanavalin A

Here, the addition of low concentrations of concanavalin A (0.01 mg/ml) increased the
quantity of cells flocculated 60 seconds into the assay while higher concentrations (0.5
mg/ml) delayed floc formation. The highest concentration (5 mg/ml) led to complete
dispersal of the cells. It has been established that the ability of the cells to form flocs
when placed in a buffer medium containing calcium ions was indicative of their intrinsic
flocculating ability at this time (1). This means that the lectins and receptors involved in
floc formation were available on the T0 cells. In order to explain our observations we
developed a model based on published reports on the mechanism of flocculation
(8,18,20).
R Ca2+ R
L L R L
R L
2+
Ca

2+
Ca
R
L L Ca
2+
R
R L L
R
Flocculation model
L= lectin
R= receptor and
Binding between lectins and receptors on
adjacent cells

Figure 3: Flocculation model. When cells are ready to flocculate the lectins
assume the correct conformation stabilized by calcium ions. Aggregates are
formed when lectins bind receptors on adjacent cells.

5
The reactions to low concentrations of concanavalin A indicated that the ratio of
receptors to lectins favoured receptors (figure 4 A). Since the lectin concanavalin A
L R R
R L R L (A)
R L R
C

L R R
L R L (B)
R
L R
R

L R RC
CR L CR C L
RC L RC
(C)
C= concanavalin A

Figure 4(A-C): Use of model to explain flocculation observations (see text)

binds to the same receptors as cellular lectins additional cells were aggregated by it
(figure 4B) (18). Increasing the concentration of concanavalin A to 0.5 mg/ml caused a
slight delay in floc formation. This was possibly due to initial competition between
cellular lectins and concanavalin A for available receptors but this was relieved as
stronger cellular lectin-receptor bonds were formed in favour of the weaker concanavalin
A receptor bonds (18). The complete dispersal of cells by 5 mg/ml concanavalin A was
due to supersaturation of receptors by the lectin (figure 4 C) thus preventing aggregation
of cells as previously observed by Stratford (19).
The reaction of cells harvested at T24 to varying concentrations of concanavalin A
further confirmed the differences in the surface structures of cells at the different times
during fermentation as shown in figure 5.

36
CELL COUNTS (x 107 cells/ml)

30

24

18

12
CONTROL
6 5mg/ml con.A
0.5mg/ml con.A
0
0 30 60 90 120 0.01mg/ml con.A
TIME (SECONDS)

Figure 5: Flocculation of cells harvested at T24 concanavalin A

6
The cells harvested at T24 showed the least flocculating ability and the addition of low
concentrations (0.01 mg/ml) concanavalin A had no effect. Higher concentrations, (0.5
mg/ml) slightly increased the final quantity of flocculated cells. The inability of the cells
to flocculate strongly at this time was due to dilution of the initial inoculum as new buds
were produced at this time and the nonflocculent nature of the newly produced buds (13).
It is more likely though that there was active mannan synthesis at this time. Lectins are
not permanently present on the surface of the cells (2), but are only synthesized during
growth whereas the mannan receptors are reportedly present throughout (19).
Furthermore, Masschelein et al., (6) reported that the period of maximum deflocculation
coincided with the period of maximum mannan synthesis. However the fact that a few
cells flocculated indicated that there were few lectins available. According to our model
these cells had the surface structure:
R R R
R L R
R R

Higher concentrations of concanavalin A were required to aggregate extra cells, thus

confirming the excess mannan receptors. Stratford, (18) postulated that there are more
concanavalin A receptor bonds to only a few cellular lectin receptor bonds for floc
formation. The highest concentration of concanavalin A (5 mg/ml) again completely
dispersed the cells due to receptor saturation.
The effect of concanavalin A on the flocculating ability of cells harvested at T72, which
showed the highest natural flocculating ability, (figure 1 above) was different as shown in
figure 4.

36
CELL COUNTS (x 107 cells/ml)

30

24

18

12
CONTROL
6 5mg/ml con.A
0.5mg/ml con.A
0
0 30 60 90 120 0.01mg/ml con.A
TIME (SECONDS)

Figure 6: Flocculation of cells harvested at T72 concanavalin A

Low concentrations of concanavalin A (0.01 mg/ml) delayed floc formation although the
final quantity of flocculated cells was the same as in the control while the higher
concentration (0.5 mg/ml) delayed floc formation even further but the final quantity of
flocculated cells was slightly higher. These observations were possibly due to the fact
that at this time the lectins were fully exposed and activated at the surface of the cells and

7
the floc forming ability of the cells was at a maximum. The ratio of receptors to lectins
was therefore close to one:

R L R L R L

The addition of low concentrations of concanavalin A confirmed the above hypothesis.

There was initial competition between the lectin and cellular lectins which was more
pronounced at higher concentrations. However this was later relieved as shown by the
fact that at 60 seconds the quantity of flocculated cells was the same as that in the control.
The slightly higher quantity of cells flocculated with (0.5 mg/ml) concanavalin A was
due to the lectin aggregating a few cells remaining in suspension after the initial cellular
lectinreceptor binding. Again the highest concentration (5 mg/ml) completely dispersed
the cells due to saturation of the available receptors.
The use of concanavalin A clearly showed how the surface properties of fermenting yeast
cells changed throughout the growth period confirming earlier studies (1,2,6,13,19). The
reaction of the cells to low concentrations of the lectin also confirmed reports that
mannan receptors are present on the surface of the cells throughout growth while the
amount of lectins increases during growth. The availability of lectins and their proper
activation determines the onset and extent of flocculation.

Screening of malts for the presence of a premature flocculation factor

The reaction of the cells to concanavalin A led to the postulate that the premature
flocculation factor present in some malts (4) behaved in a similar manner to concanavalin
A. Extracts obtained by washing the malts in buffer and added to flocculent cells
(pitching yeast slurry) showed interesting results as shown in figure 7.

36
CELL COUNTS (x 107 cells/ml)

30

24

18

12
CONTROL
6 + MALT 1 extract
+ MALT 2 extract
0
0 30 60 90 120 + MALT 3 extract
TIME (SECONDS)
Figure 7: Effect of malt extracts on flocculation.

Malt 1 repeatedly gave rise to premature flocculation in laboratory scale fermentations.

Furthermore high concentrations of the partially purified extract from Malt 1 showed
similar effects to high concentrations of concanavalin A in that when this extract was
added to wort, premature flocculation occurred but slightly later than usual. Future work

8
will involve screening more malts in this manner and subsequently preparing wort for
fermentations. Further purification of the extract from this malt is in progress and future
work will involve competition studies between the factor and concanavalin A to further
elucidate the mechanism by which this factor causes premature flocculation.

Predictive flocculation test for yeast slurries

The reaction of fermenting cells to varying concentrations of the lectin concanavalin A
further led to the possibility of using the response to the lectin as an assay for selection of
pitching yeast slurries for consistent fermentation performance. Preliminary results
shown in figure 8 indicated that there is potential for this assay as a predictive tool in
selecting suitable slurries for inoculation.

36
CELL COUNTS (x 107 cells/ml)

30

24

18

12
SLURRY 1
6 SLURRY 1 + con.A
SLURRY 2
0
0 30 60 90 120 SLURRY 2 + con.A
TIME (SECONDS)

Figure 8: Effect of 1.0 mg/ml concanavalin A on slurries of different quality

Although both slurries showed similar flocculation behaviour, their reaction to 1.0 mg/ml
concanavalin A was different. The cells of Slurry 1 which showed a high pH value,
indicative of deterioration (7), possibly had reduced mannan receptors due to long storage
(5) and, as shown earlier for cells harvested at T72 concanavalin A, delayed flocculation
due to competition between itself and cellular lectins for the available receptors. The cells
in Slurry 2 with a lower pH were affected to a much lesser extent by concanavalin A.
Future work will involve inoculating slurries subjected to the test into fermentations and
the correlation of this behaviour with other yeast quality measures.

CONCLUSIONS
The use of the lectin concanavalin A led to a determination of the changes in cell surface
structure with fermentation time during industrial fermentations and confirmed other
reports in the literature which mention that onset of flocculation is determined by
availability or exposure of lectins. Furthermore it was possible using the lectin to
postulate that the premature flocculation factor associated with some malts may act in a
similar manner to concanavalin A by forming loose aggregates which then formed

9
stronger bonds through cellular lectin receptor binding. The fact that cells showed
appreciable flocculating ability even as early as 48 hours made this a strong possibility.

REFERENCES
1. Baker, D.A. & Kirsop, B.H., Journal of the Institute of Brewing, 1972, 78, 454-458.
2. Bony, M., Barre, P. & Blondin, P., Yeast, 1998,14, 25-35.
3. Evans, I.H., Diala, E.S., Earl, A. & Wilkie, D., Biochimic et Biophysica Acta, 1980,
602, 201-206.
4. Herrera, V.E. & Axcell, B.C., Journal of the American Society of Brewing Chemists,
1989, 47(2), 29-34.
5. Lyons, J.P. & Hough, J.S., Journal of the Institute of Brewing, 1970, 76, 564-571.
6. Masschelein, C.A., Jeunehomme-Ramos, C., Castiau, C. & Devereux, A., Journal of
the Institute of Brewing, 1963, 69, 332-337.
7. Mochaba, F.M., OConnor-Cox, E.S.C. & Axcell, B.C., Journal of the American
Society of Brewing Chemists, 1998, 56(1), 1-6.
8. Miki, B.L.A., Poon, N.H., James, A.P. & Seligy, V.L., Journal of Bacteriology,
1982, 150, 878-889.
9. Nishihara, H. & Toraya, T., Agricultural Biological Chemistry, 1987, 51(10) 2721-
2726.
10. Smart, K.A. & Whisker, S., Journal of the American Society of Brewing Chemists,
1996, 54(1) 41-44.
11. Smart, K.A., Boulton, C.A., Hinchliffe, E. & Molzahn, S., Journal of the American
Society of Brewing Chemists, 1995, 53(1), 33-38.
12. Smit, G., Straver, M.H., Lugtenberg, B.J.J. & Kijne, J.W., Applied and
Environmental Microbiology, 1992, 58(11), 3709-3714.
13. Soares, E.V. & Mota, M., Canadian Journal of Microbiology, 1996, 42, 539-547.
14. Speers, R.A., Tung, M.A., Durance, T.D. & Stewart, G.G., Journal of the Institute of
Brewing, 1992, 98, 239-300.
15. Stewart, G.G. & Russell, I., European Brewery Convention Monograph-XII,
Symposium on Brewers Yeast, Helsinki, Finland, Nurnberg:Verlag Hans Carl, 1987,
53-70.
16. Stratford, M & Assinder, S., Yeast, 1991, 7, 559-574.
17. Stratford, M., FEMS Microbiology Letters, 1996, 136, 13-18.
18. Stratford, M., Yeast, 1992, 8, 635-645.
19. Stratford, M., Yeast, 1993, 9, 85-94.
20. Taylor, N.W. & Norton, W., The Journal of the Institute of Brewing, 1978, 84, 113-
114.
21. Tkacz, J.S., Cybulska, E.B. & Lampern, J.O., Journal of Bacteriology, 1971, 105, 1-
5

10
43

Construction of brewers yeast

harbouring killer plasmids and
investigation of the zymocin production
under various fermentation conditions
Gabriella Farkas1, Anna Marz2, Judit Rezessy-Szab1 &
goston Hoschke1
Szent Istvn University, Budapest, Hungary
1
Department of Brewing and Distilling, Mnesi t 45, H-1118 Budapest (e-mail:
[email protected])
2
Department of Microbiology and Biotechnology, Somli t 14-16, H-1118 Budapest

Descriptors
Brewersyeast, fermentative ability, growth inhibitor, killer factor, protoplast fusion, yeast
strain

SUMMARY
Introduction of zymocin producing ability to brewers yeast would enable the yeast to protect
itself from invading wild yeast strains, which may result in extra protection during continuous
fermentation. In our work a brewers yeast strain with killer character has been constructed by
protoplast fusion of a widely used brewers strain and a K1 killer yeast strain. Study of the
zymocin production showed that it had inhibitory effect on the growth of sensitive yeast
strains under various fermentation conditions. In model fermentation there was no significant
difference between fermentation ability of the killer producing and the traditional brewers
yeast strains.

Vernderung von Bierhefe durch Einschleusung eines Killerplasmids und

Untersuchungen ber die Produktion von Zymocin

Deskriptoren
Bierhefe, Grfhigkeit, Hefestamm, Killer factor, Protoplastenfusion, Wachstumsinhibitor

ZUSAMMENFASSUNG
Durch die Produktion von Zymocin wre die Bierhefe in der Lage, sich gegen eindringende
Wildhefen zu schtzen. Das knnte ein Schutzmechanismus fr die kontinuierliche Grung
sein. In unserer Arbeit erzeugten wir den Bierhefestamm mit Killereigenschaften durch eine
Fusion der beiden Protoplasten einer herkmmlichen Bierhefe und einer K1 Killerhefe. Die
Beobachtungen der Zymocinproduktion zeigen einen hemmenden Effekt auf das Wachstum
von empfindlichen Hefestmmen unter verschiedenen Grungsvoraussetzungen. Bei
Grungen im Labormastab zeigte die Hefe mit den Killereigenschaften die selben
Greigenschaften wie die herkmmliche Bierhefe.

Construction d'une souche de levure de bire portant des plasmides "killer" et tude de
la production de zymocine dans diffrentes conditions de fermentation

Descripteurs
Facteur "killer", fermentabilit (de la levure), fusion de protoplaste, inhibiteur de croissance,
levure de brasserie, souche de levure

RESUME
L'adjonction d'une capacit de production de zymocine la levure de bire permettrait celle-
ci de se protger des souches sauvages envahissantes, ce qui serait susceptible de lui procurer
une protection supplmentaire pendant une fermentation continue. Dans notre travail, nous
avons construit une souche de levure de bire de phnotype "tueur" par fusion de protoplaste
dans une souche de levure largement utilise et une souche de levure "killer" K1. L'tude de
la production de zymocine a montr qu'elle avait un effet inhibiteur sur la croissance de
souches de levure, dans diffrentes conditions de fermentation. Dans une fermentation
modle, nous n'avons observ aucune diffrence significative entre la capacit de
fermentation de la souche "killer" et celle de la levure de bire traditionnelle.

2
INTRODUCTION
Microbiological quality of the product is a key issue in every brewery. The procedure
of wort boiling that lasts minimally 90 minute is capable to eliminate most
contaminating agent. But in the following steps it is the best interest of the brewery to
keep out any kind of contamination from the hopped wort. Although it is a general
opinion that a good, strong yeast in the proper concentration is able to suppress
pathogenic bacteria or a wild yeast infection, perhaps it is better to play safe.
Nowadays, genetics and genetic engineering have become part of our daily life, one
can rightfully expect it that their influence is becoming stronger in the breweries, as
well. Genetic improvement of brewers yeast can give a new meaning to the term
good, strong yeast.
Toxin producing ability of yeasts is not rare in the nature e.g. among wine yeast
strains. Exotoxins that are able to kill susceptible cells belonging to the same or
cogeneric species have been defined as killer toxins or zymocins. Killer yeasts are
toxin-producing fungi that are immune to the activity of their own killer toxins
(Magliani et al., 1997). However, brewers yeast strains do not possess the plasmids
responsible for that. When new fermentation techniques are getting introduced and
applied in breweries, i.e. continuous fermentation or immobilised yeast cells, it can be
an extra safety measure to use a killer brewers yeast strain that has to have, of
course, the same fermentative ability as the parental brewers yeast strain. The method
of protoplast fusion can be used to introduce the plasmids into the yeast cells (Marz
et al, 1994). The same result can be achieved by the technique of rare mating (Young,
1981).
Of the three types of toxin, produced by Saccharomyces yeasts (K1, K2, K28), the
production, secretion and effect of K1 type is the best known, it is thoroughly
investigated (Magliani et al., 1997), and it is harmless for humans (Pfeiffer et al.,
1988).

In our work a brewers yeast strain was constructed by protoplast fusion that carried
the plasmids responsible for K1 type killer toxin (zymocin) production. Cybrids were
isolated, because they carried the plasmids, but their chromosomal pattern was like
that of the parental brewers yeast. Zymocin production was investigated under
various conditions. The fermentative ability of the newly constructed yeast strain was
studied at low temperature and compared to that of the parental brewers yeast strain.
Immobilised cells and continuous system were used for the investigation, because
nutrient limitation and physical boundaries limit the growth of immobilised cells and
this enhances the stability of genetically engineered micro-organisms (Hahn-
Hgerdal, 1990; Willaert et al, 1996).

MICROORGANISMS
Brewers yeast strains
Saccharomyces cerevisiae WS 34/70
Saccharomyces cerevisiae NCAIM Y01223 (syn. S. logos)
Saccharomyces cerevisiae NCAIM Y00846
Saccharomyces pastorianus NCAIM Y00805
Saccharomyces cerevisiae Uvaferm dried yeast
Killer yeast strain
Saccharomyces cerevisiae K7 [Mata arg9 (L-A) (M-1)] (K1 type)]

3
Supersensitive test strain
Saccharomyces cerevisiae S6
Marker strain (used in kariotype analysis)
Saccharomyces cerevisiae S 288c

METHODS
Selection of brewers yeast strains for protoplast fusion
Suspension of each brewers yeast and the supersensitive strain were spread onto a
medium containing methylene blue. Holes ( 7 mm) were cut in the agar plates.
The killer yeasts were inoculated in YEPD liquid medium and incubated at 23 C in a
rotary shaker for 36 hours to allow toxin production. Cells were centrifuged
(3000 rpm, 10 min), and 200 l of the supernatants were pipetted in the holes. Plates
were incubated at 23C for 48 hours.

Protoplast fusion
Protoplasts were produced with the aid of cell-wall-lysing enzyme lyticase (Sigma) in
0.6 mol KCl at 37C, in approx. 30 minutes. Osmotically stabilised protoplasts in 0.3
mol CaCl2 were mixed and treated with 25% polyethylene glycol (PEG) 4000 solution
in the presence of calcium ions to promote fusion. Fusion products were regenerated
in isotonic minimal medium (pH 4.3).

Molecular analysis of the fusion products

dsRNA of the cells originating from the selected colonies were isolated and separated
by gel electrophoresis, to prove that the plasmids responsible for the toxin production
were present in them.
Kariotype analysis was performed to check whether the chromosomal patterns of the
bred yeasts were identical with that of the brewers yeast strains.

Immobilisation
2.5% Na-alginate (Sigma A-7128) was added to yeast suspension (0.5-1*109 cells/ml).
The standard dripping procedure was used: the alginate-yeast suspension was pumped
through a 0.6 mm diameter needle into 2% CaCl2 solution. The diameter of alginate
beads was 1.5-2.0 mm. The beads were washed and stored in 0.9% CaCl2 solution,
and used within 1 or 2 days.

Detection of zymocin
Suspension of the supersensitive strain was spread onto a medium containing
methylene blue. Holes ( 7 mm) were cut in the agar plates. Yeast cell suspension
was centrifuged (3000 rpm, 10 min), and 200 l of the supernatants were pipetted in
the holes. Plates were incubated at 23C for 48 hours.

Primary fermentation
Fermentation was carried out at low temperature (4C) in order to limit yeast
metabolism.
The fermentation unit included: a jacketed packed bed column, a peristaltic pump, a
wort feed tank, a sample collecting bottle and a thermostat to ensure the desired
temperature.

4
The column was filled up with 160 ml bead, and positioned in a 45 degree angle.
(This way the CO2 formed could not push up beads to block the tube leading to the
collecting bottle. A filter was also fitted to the end of the column for the same reason.)
The flow direction of the wort was upward, the flow rate (60-120 ml/hour) was
controlled by a peristaltic pump.
The wort originated from two breweries. The higher gravity wort (13.5 B) was the
kind support of Dreher Brewery, Budapest, and the 10 B wort was produced in the
pilot brewery of the Department. To get 5 B wort, the 10 B wort was diluted with
sterile water. All the worts were sterilized by autoclaving at 121.2C for 15 minutes,
prior to fermentation.

ANALYTICAL METHODS
Samples were taken twice a day. Volume of samples and the elapsed time was
recorded. The product was centrifuged (3000 rpm, 10 min.), 100 ml supernatant was
deep-frozen for further analysis.
Optical density and pH were measured immediately.
Classical fermentation parameters, i.e. gravity, ethanol concentration, original
extract etc. were measured by a CENTEC Beer Analyser. The method suggested
for the evaluation of data from non-alcoholic beer was selected.
Volatile compounds were determined by a Perkin-Elmer AUTOSYSTEM Gas
Chromatograph. A CP-Wax 52 C.B., 50m X 0.32 mm capillary column was used.
Total diacetyl and 2,3-pentandione concentrations were determined after a 90-
minute heat treatment at 60C, with an electron capture detector (ECD)
Esters and higher alcohols were measured by headspace capillary gas
chromatography, with a flame ionising detector (FID).
Assimilable sugars were determined by Waters HPLC with a SUPELCOGEL Ca,
30 cm X 7.8 mm ID column. Waters 410 differential refractometer was used as
detector. Distilled water was the eluent, the flow rate was 1.0 ml/min, and the
column temperature was 80C. Millennium 2010 integrated software controlled
the equipment, and evaluated the chromatograms

RESULTS & DISCUSSION

Selection of brewers yeast strains for protoplast fusion
Two of the five brewers yeast strains were not sensitive to the toxin produced by the
K7 (K1 type) killer yeast. Sensitivity was used as a selection marker in the later steps
during protoplast fusion, thus it was important for the brewers yeast to have this
feature.

Tested strains S. cerevisiae K7 (K1 type killer)

S. cerevisiae WS 34/70 Sensitive
S. cerevisiae NCAIM Y01223 Resistant
S. cerevisiae NCAIM Y00846 Sensitive
S. pastorianus NCAIM Y00805 Sensitive
S. cerevisiae (UVAFERM ) Resistant
Table 1: Interaction between the killer and brewers yeast strains

5
Protoplast fusion
Based on these results of killer sensitivity, protoplast fusion of the following pairs
were carried out:
A) S. cerevisiae WS 34/70 S. cerevisiae K7
B) S. pastorianus NCAIM Y00805 S. cerevisiae K7
C) S. cerevisiae NCAIM Y00846 S. cerevisiae K7
Regenerated progenies were gained from two protoplast fusions: A and B
Eleven colonies from the fusion A, and 10 colonies from fusion B were isolated and
transferred onto minimal medium, where all of them were capable to grow. It meant
that they could be either fusion products or regenerated brewers parental strains,
because the killer parental strain would have not been able to grow on this medium (it
was arginine auxotroph).
The regenerated colonies were replicated onto methylene blue medium that was
previously inoculated with the supersensitive strain, S6. From the 11 colonies among
fusion A, 9 proved to be toxin producers, they made clear inhibition zone, killing off
the supersensitive yeast cells around them. In the case of the fusion B, 7 of the 10
colonies produced the toxin.

dsRNA plasmid detection

Result of the electrophoretic separation of the isolated nucleic acids showed that the
fusion products carried both the L and M plasmids originated from the K7 killer yeast
(figure 1). The parental brewers yeast on the other hand did not carry the plasmids.
This also verified that the protoplast fusion was successful.

Kariotype analysis
It was necessary to perform this analysis to distinguish hybrid from cybrid fusion
products. We wanted to select cybrids because in this case the fermentation ability
was supposed to be similar to that of the brewers yeast parent. Although the large
chromosomes did not separate perfectly, the karyograms showed that chromosomal
patterns of the killer yeast strain was different from that of the brewers yeast and the
fusion products. On the other hand chromosomal patterns of the original brewers
yeast strains and the selected fusion products were similar.
WS 34/70
WS-K7/4
WS-K7/5
WS-K7/6
N-K7/2
N-K7/3
marker
N 805

K7

DNA N 805: S. p. NCAIM Y 805,

L dsRNA
M dsRNA N-K7/1, N-K7/2, N-K7/3: fusion
products of N 805 and K7
WS 34/70: Weihenstephan 34/70,

rRNA WS K7/4, WS K7/5, WS K7/6:

fusion products of WS and K7

Figure 1: Detection of dsRNAs in the fusion products and parental strains

6
 WS 34/70
WS-K7/4
WS-K7/5
N-K7/1
N-K2/2
marker
N 805

K7

Figure 2: Karyograms of the fusion products and parental strains

Based on the results of these analyses it can be stated that the fusion products were
cybrids.

Immobilisation of yeast cells

Cells were immobilised in 0.5-1*109 cells/ml concentration. Figure 3 shows the SEM
of the killer (WS-K7/5) and the traditional (WS 34/70) brewers yeast strains
immobilised in alginate gel.

A B

Figure 3: Immobilised killer (A) and traditional (B) brewers yeast cells

7
Fermentation with immobilised yeast cells: a comparison of the fermentative
abilities of traditional and genetically improved brewers yeast strains
! Ethanol production: Both the parental and the fusion yeast strains had low ethanol
production, which was virtually independent from flow rate in the tested range
(60-120 ml/h) or from the gravity of the wort. The ethanol concentration was
below 1.2 % V/V.
! Carbohydrate utilisation: The conversion rate was low, but difference between the
two strains was negligible, which is attributable to that fermentation conditions
and state of immobilised cells are different from experiment to experiment.
However, conversion rate could be increased by reducing the flow rate.
! Formation of volatile aroma compounds: Concentration of compounds was
generally low, it was below the minimal values given by the literature both for
beer as a finished product (Pollock, 1981), and for unmaturated (green) beer
(Andersen et al, 1999).

Traditional Green beer

Tested volatile Killer Finished beer
brewers (Andersen et
compounds brewers yeast (Pollock, 1981)
yeast al, 1999)
Acetaldehyde (ppm) 1.7-15.1 1.4-12.7 6.2-7.1 1.2-24.4
DMS (ppb) 15-343 19.33-226 n.a. 18-144
Ethyl acetate (ppm) 0.5-1.1 0.3-0.7 27.4-28.5 8.2-45.8
Propanol (ppm) 0.9-3.3 0.8-4.0 30.7-31.8 7.5-16.7
Isobutanol (ppm) 0.4-0.8 0.4-1.0 29.0-31.3 4-23
Isoamyl acetate (ppm) 0 0 0.25-0.41 0.8-6.6
Isoamyl alcohol (ppm) 2.4-4.7 3.5-6.1 n.a. 27-62
Diacetyl (ppb) 64-248 64-122 330-420 <100
Pentandione (ppb) 16-98 15-52 54-68 <50
n.a.: not available

Table 2: Concentration of volatile compounds after primary fermentation with

traditional and killer brewers yeast compared to values given by the
literature

INVESTIGATION OF KILLER TOXIN (ZYMOCIN) PRODUCTION

Molecular analyses showed that the yeast cells, constructed by protoplast fusion,
carried the dsRNAs encoding the toxin.
In the following experiments the toxin production, toxin secretion and the killer effect
were investigated. Common in all experiments that the presence of the toxin was
detected by the agar diffusion method involving agar plates containing methylene
blue dye. In case the toxin is present (in proper concentration) a cleared-up zone
appeared around the holes. Also, every time a test was performed by inoculating a
streak of yeast cell onto the same type of agar plates, and in case the cells produced
toxin, a cleared-up zone appeared again.
1. In the first set of experiments killer toxin producing brewers yeasts were
inoculated in glucose-maltose medium in 106-107 cells/ml concentration. Yeast
suspensions were not shaken during incubation that was carried out at six different
temperatures: 2C, 5C, 10C, 20C, 23C, 25C, and 30C.
2. The second experiment was conducted on 23-25C and this time an orbital shaker
was used. Besides the usual testing, supernatant was centrifuged in a special

8
 centrifuge tube filter (Whatman, VectraSpin Micro, Polysulphone, 30MWCO).
Both the retentate and the permeate were tested.
3. In the third set of experiments sensitive yeast cells were introduced to the system
in three different killer to sensitive ratios: 1:100, 1:10 and 1:1, respectively. Cell
concentrations were set between 106 and 108 cells/ml, incubation was at 23C, in
an orbital shaker. The change of cell count and ratio of the dead cells were tested
daily.

In the first set of experiments positive results were achieved at 10C, 23C and
25C. At 2C and 5C even cell multiplication was limited to a very low level, cell
metabolism was minimal thus zymocin production was most probably stopped. At
30C the negative result can be attributed to heat sensitivity of the toxin.
However, when the yeast cells themselves were tested, it showed that neither
temperature affected the ability of the cells to produce and secrete zymocin.
In the second experiment there was a cleared-up zone around the hole into which the
retentate was pipetted. It suggests that the toxin molecules could not be passed
through the filter membrane, albeit according to the literature the size of the toxin
protein as approximately 19 kD (Magliani et al, 1997).
When the killer effect was investigated in a system containing both killer and
sensitive yeast cells the following was found. Even if the concentration of killer yeast
cells was only one tenth of the sensitive yeast cells, the zymocin produced was
capable to eliminate 75 % of the cells. The majority of the dead cells were sensitive
yeast cells that were morphologically different from brewers yeast cells. At the 1:1
ratio, as it is expected, similar result was achieved (figure 4). At the 1:100 (killer:
sensitive) ratio, the zymocin concentration was too low to detect any killing effect on
the sensitive yeast cells. Less than 10 % of the sensitive yeast cells died when they
were cultivated in pure culture, under the same conditions.

80

70

60

50
Dead cell %

1:10 ratio
40 1:1 ratio
S6

30

20

10

0
0 24 48 72 96 120
Incubation time (hour)

Figure 4: Percentage of dead cells in killer yeast-sensitive yeast systems at different

ratios

9
CONCLUSION
Brewers yeast strain harbouring killer plasmids was constructed by protoplast fusion,
and the strain remained genetically stable, carrying the plasmids, and producing and
secreting zymocin. Under tested circumstances in an immobilised continuous system
at low temperature, fermenting ability of the newly constructed strain was similar to
that of its parental brewers yeast strain. Newly constructed brewers yeast cells with
killer character were able to kill sensitive yeast cells under defined conditions.

REFERENCES
1. Andersen, K., Bergin, J., Ranta, B., Viljava, T., Proceedings of the European
Brewing Convention, Cannes, 1999, 771-778.
2. Hanh-Hagerdahl, B., Physiology of Immobilized Cells. Proceedings of an
International Symposium held at Wageningen, the Netherlands, 10-13 December,
1989, 1990, 481-486.
3. Magliani, W., Conti, S., Gerloni, M., Bertolotti, D. & Polonelli, L., Clinical
Microbiology Reviews, 1997, 10, 369-400.
4. Marz A., Zkny F., Lovenyk M., Hungarian Agricultural Research, 1994, 3,
34-41.
5. Pfeiffer, P., Radler, F., Caspritz, G. & Hnel, H., Applied and Environmental
Microbiology, 1988, 54, 1068-1069.
6. Pollock, J.R.A.., Brewing Science, Academic Press, 1981, Volume 2, Chapter
3&4.
7. Willeart, R.G., Baron, G.V., de Backer, L. (ed.), Immobilized living cell systems
Modelling and experimental methods. John Wiley & Sons, England, 1996, 1-17.
8. Young, T.W., Journal of the Institute of Brewing, 1981, 87, 292-295.

ACKNOWLEDGEMENT
The authors would like to thank the kind help of the Department of Quality Assurance
of the Borsodi Brewery, Bcs, Hungary for performing GC analyses.
Brewers yeast strains with NCAIM numbers were kindly provided by the National
Collection of Agricultural and Industrial Microorganisms, Budapest, Hungary.
This research was supported by the Hungarian Ministry of Education through the
project No. FKFP 0096/1999.

10
44

Amino acid uptake and sensing in yeast

Helge A. Andersen, Richard F. Gaber*, Peter L. Madsen, Peter
S. Nielsen, Kim Ottow & Morten C. Kielland-Brandt
Carlsberg Laboratory, Department of Yeast Genetics, Gamle Carlsberg Vej 10, DK-2500
Copenhagen Valby, Denmark (e-mail: [email protected])
* On sabbatical leave from Northwestern University, Evanston, Illinois 60208, USA

Descriptors
Amino acid, brewers' yeast, fermentation, flavour formation, protein

SUMMARY
Amino acids are a principal source of nitrogen for the yeast in brewery fermentations.
Furthermore, carbon skeletons of several amino acids make part of aroma compounds formed
by the yeast. An increased understanding of the control of amino acid uptake in yeast
therefore gives opportunities for influencing brewery fermentations in several respects, in
particular the aroma profile. We will describe new details of a system of proteins that act in
the response of yeast to the presence of amino acids. One of the proteins has structural
similarity to amino acid permeases and is a candidate for sensing extracellular amino acids.

Aminosureaufnahme und Bestimmung in der Hefe

Deskriptoren
Aminosure, Aromabildung, Bierhefe, Grung, Protein

ZUSAMMENFASSUNG
Aminosuren sind fr die Hefe die Hauptquelle fr Stickstoff im Brauprozess. Darber hinaus
wandelt die Hefe die Karbongerste mancher Aminosuren zu Aromastoffen um. Ein besseres
Verstndnis des Mechanismus der Aufnahme von Aminosuren ermglicht eine bessere
Steuerung des Grprozesses und des Aromaprofils. Es werden neue Erkenntnisse ber ein
Proteinsystem vorgestellt, das beim Regelmechanismus in Anwesenheit von Aminosuren
eine Rolle spielt. Eines der Proteine hat eine Struktur, welche der Aminosure-Permease
hnlich ist und knnte somit zur Aufsprung extrazellulrer Aminosuren dienen.
Dtection et capture des acides amins par la levure

Descripteurs
Acide amin, fermentation, formation de la flaveur, levure de brasserie, protine

RESUME
Les acides amins sont la principale source d'azote pour les cellules de levure en fermentation
dans l'industrie brassicole. Par ailleurs, les squelettes carbons de plusieurs acides amins font
partie des composs aromatiques forms par la levure. Une meilleure comprhension du
contrle de la capture des acides amins par la levure donne donc, plusieurs gards, des
occasions d'influencer les fermentations brassicoles, en particulier le profil des armes. Nous
apportons de nouveaux dtails sur un systme de protines agissant sur la rponse des levures
la prsence d'acides amins. L'une de ces protines est structurellement similaire aux
permases des acides amines et pourrait tre la protine assurant la dtection des acides
amins extracellulaires.

2
INTRODUCTION
Brewing yeast strains belong to species of the genus Saccharomyces. Like all other
living cells, they need nitrogen as a nutrient to be assimilated into protein, RNA, DNA
and other compounds. Like green plants, and contrary to animals, yeast is able to
synthesise all the building blocks of their nitrogenous macromolecules using
inorganic nitrogen in the form of ammonium. However, the nitrogen that yeast can
assimilate from brewers wort is also, and mostly, present as amino acids, the building
blocks of proteins, and to some extent also as low-molecular-weight peptides. The
first step in the assimilation is uptake through the lipid bilayer surrounding the yeast
cell, the plasma membrane. The lipid membrane itself is impermeable to all the
mentioned nutrients, and dedicated proteins embedded in the membrane, so-called
permeases, carry out the transport. The permeases are highly regulated by other
proteins.

Yeast genetics
The identities and transport capabilities of the permeases have to a large extent been
determined by isolating mutants defective in transport and through gene technology,
genomics and physiology. The same array of methods has also yielded much
information on how the permeases and their transport are regulated, but the picture of
regulation is still very far from complete. Genetic studies of uptake of nitrogen-
containing nutrients in Saccharomyces yeasts have been carried out in genetic
reference strains of Saccharomyces cerevisiae. Determination of the full sequence of
the 12 million building blocks (nucleotide pairs) of the genetic material (DNA) of this
yeast was completed in 1996, and it was found that the DNA could be subdivided into
approximately 6000 genes. Except for the minority of the genes that specify RNA
structures, each gene contains the information for specifying the sequence of the 20
different kinds of building blocks of proteins (amino acids), thereby determining the
structure and properties of a particular protein. On the basis of the amino acid
sequences, proteins can be grouped in families even if their functions are unknown. A
yeast gene with a known or suggested function is given a name consisting of three
letters and a number. Gene names are written in italics; a functional version of the
gene, typically the so-called wild type, is written in CAPITALS, whereas a gene that
has undergone a change (mutation) leading to a loss of function is typically written in
lower case. Thus, as will be described in more detail below, the gene encoding an
important permease transporting the amino acids isoleucine, leucine and valine is
called BAP2 (for branched-chain amino acid permease, gene no. 2), whereas an
inactive mutant version of the gene is called bap2. Following a common terminology,
the permease itself may be called Bap2p (written in roman), the p referring to the
protein encoded by the indicated gene. Lager brewing yeast is a species hybrid with
half its genome being S. cerevisiae, and the fundamental properties of the transport
systems and their regulation are expected to be the same as those of the well-
characterised genetic reference strains of S. cerevisiae, although important differences
in behaviour of the lager yeast certainly exist. Because of this hybrid nature, most
proteins known in S. cerevisiae are expected to be present in two closely related
versions in lager yeast.

3
UPTAKE OF NITROGEN-CONTAINING NUTRIENTS
Ammonium
Ammonium ions are imported into the yeast cell by three permeases, Mep1p, Mep2p
and Mep3p (Marini et al. 1997). All three permease genes are subject to repression by
easily assimilated nitrogen sources such as glutamine. This may explain why
ammonium is not among the nutrients first taken up in beer fermentation (Jones and
Pierce 1964; Palmqvist and yrp 1969; Jin et al. 1996).

Amino acids
Each of the 20 amino acids that are building blocks of proteins can be taken up by
yeast. In a beer fermentation, some are taken up before others, and some are taken up
inefficiently (Jones and Pierce 1964; Palmqvist and yrp 1969; Jin et al. 1996)
thus proline is not taken up to any appreciable extent.
Amino acids can be taken up against a concentration gradient; i.e. their uptake is
coupled to energy consumption. The general energy currency in the metabolism of
living cells is adenosine triphosphate (ATP), which delivers its energy when it is
hydrolysed. ATP delivers energy to amino acid uptake in yeast in an indirect way, as
follows. An essential protein, Pma1p, embedded in the plasma membrane, uses ATP
to pump protons out of the cell, generating and maintaining both an electrical
potential difference (positive outside) and a pH difference (acid outside) over the
membrane. The energy coupling of amino acid uptake is accomplished by a
peculiarity of the amino acid permeases; they must take up protons together with
amino acid molecules. In other words, the energy of the ATP is first converted into
electrical energy and a higher concentration of protons outside the cell than inside,
and the protons then help drive the amino acids into the cell through individual
permeases.
All amino acid permeases in S. cerevisiae belong to a single family of proteins with
similarities in their amino acid sequences. The family comprises 24 members; most
but not all of them transport amino acids. The permeases and their regulation will be
dealt with in detail below.

Peptides
S. cerevisiae has a permease, Ptr2p (Perry et al. 1994), for the uptake of dipeptides
and tripeptides. Once inside the cell, the peptides are cleaved to amino acids. The
gene encoding this permease is controlled by at least three systems; stimulation by
amino acids (Island et al. 1987) through the Ssy1p system (Didion et al. 1998,
described below) repression by easily accessible nitrogen, and derepression by
intracellular small peptides (Byrd et al. 1998).

AMINO ACIDS CENTRAL BUILDING BLOCKS

Metabolism
As already mentioned, S. cerevisiae is able to synthesise all the amino acids that go
into protein synthesis. If one amino acid is available in the medium in excess and no
other nitrogen source is available, yeast can in most cases (exceptions are histidine
and lysine) use the amino acid as sole nitrogen source. A central process in this is
transamination, by which intracellular pools of amino acids and keto acids participate
in constant exchange of nitrogen.

4
Hydrophobic amino acids
The carbon skeleton of the keto acid that has been left from an amino acid serving as
nitrogen source can often also be assimilated; however, in the cases of isoleucine,
leucine, phenylalanine and valine, the keto acid undergoes decarboxylation
(Dickinson et al. 1997, 1998, 2000) and reduction to yield a higher alcohol. For
example, valine yields isobutanol. Except for acetylation of part of each of the higher
alcohols, these are not further metabolised and leave the cells. The higher alcohols are
also formed as side products in the synthesis of isoleucine, leucine, phenylalanine and
valine, a fact that is compatible with the idea that formation of higher alcohols and/or
their esters has important functions in yeast ecology. Perhaps, like ethanol, the higher
alcohols inhibit competing microorganisms and attract insects that may spread the
yeast. The brewer knows these volatiles as important flavour compounds.

Diacetyl
A generally unwanted flavour compound in beer is diacetyl, an early side product of
valine biosynthesis, which has its characteristic, butter-like flavour. It is formed non-
enzymatically from -acetolactate that leaks out of the yeast cells. During late stages
of fermentation and maturation, diacetyl is reduced to acetoin and butanediol by the
yeast, and the time it takes to convert -acetolactate through diacetyl to its reduced
products can be a limiting factor in production rate. This has been approached in
various ways. For example, there is a full-scale procedure of heating the beer to speed
up the conversion of -acetolactate and subsequently removing diacetyl with
immobilised yeast (Pajunen et al. 1991; Pajunen and Jskelinen 1993). Also,
increased fermentation temperatures or addition of a commercial bacterial
decarboxylase are being used. A GMO-based procedure has been devised (see e.g.
Suihko et al. 1989) in which the yeast is engineered to express the bacterial
acetolactate decarboxylase in the cytoplasm. This approach prevents leakage of -
acetolactate from the cell without interfering with valine synthesis, which takes place
in the mitochondria. There is also a non-GMO approach to obtaining strains with
lower diacetyl potential; C. Gjermansen (unpublished, see Kielland-Brandt et al.
1989) devised a procedure for isolation of mutants with lower activity of acetolactate
synthase; this enzyme is encoded by ILV2. However, he found that interference with
acetolactate synthase activity causes growth retardation due to inefficient uptake of
valine from wort or other complex media. This finding initiated our interest in amino
acid uptake.

Bap2p, a permease for isoleucine, leucine and valine

Grauslund et al. (1995) isolated a gene that, when overexpressed, stimulated the
uptake of isoleucine. By sequence determination, they found that this gene, designated
BAP2, encodes a member of the amino acid permease family. They further found that
the Bap2p permease is quite specific for isoleucine, leucine and valine when many
amino acids are present. Subsequently, more sensitive tests in the presence of single
amino acids (Dring-Olsen et al. 1999; Regenberg et al. 1999) showed that Bap2p is
also capable of importing some other neutral amino acids. Grauslund et al. (1995) also
made the rather surprising observation that a truncation of the gene, resulting in
removal of the last 29 amino acids of the protein, led to increased uptake of all three
amino acids. Omura et al. (2001) have found that the shorter form of the permease is
more stable than the wild-type protein with regard to turnover. Thus, the truncated
mutant form of the permease is present in higher amounts in the plasma membrane,
explaining the higher uptake capacity.

5
The amino acid permease family
The complete nucleotide sequence of the S. cerevisiae genome revealed that the
amino acid permease family comprises 24 members (Paulsen et al. 1998), of which
two Met permeases have been considered to fall outside the family sensu stricto
(Isnard et al. 1996), while other, even more distantly related, members have other
functions than transport of L--amino acids. The three-dimensional structures of the
permeases, probably very similar to each other, are unknown, but the topology of one
of them, the so-called general amino acid permease, Gap1p, has been determined by
Gilstring and Ljungdahl (2000); the polypeptide chain traverses the membrane 12
times, and the termini are both inside. By deletion (Regenberg et al. 1998) and over-
expression (Regenberg et al. 1999), we studied uptake capabilities and substrate
preferences of members of this family for which a function had not yet been defined.
Regenberg et al. (1998) found Dip5p to be an efficient transporter for glutamate and
aspartate, and Regenberg et al. (1999) found Alp1p to be specific for arginine, while
Regenberg et al. (1999) and Iraqui et al. (1999) independently found that Agp1p is an
important broad-specificity permease expressed on non-preferred nitrogen sources.
Regenberg et al. (1999) studied uptake of all 20 protein amino acids and, together
with the identification of SAM3 and MMP1 as permeases for the non-protein L--
amino acids S-adenosyl methionine and S-methyl methionine, respectively (Rouillon
et al. 1999), Regenberg et al. (1999) presented a comprehensive compilation of the
capabilities of the yeast amino acid permeases. They concluded that all known amino
acid uptake capability by the yeast cell could be accounted for by activities of this
family. As further described below, a member of the family is an essential part of a
sensing system.

Yeast can taste amino acids in the medium

Grauslund et al. (1995) noted that the promoter (the region controlling transcription)
of the BAP2 gene contains sequence elements characteristic of binding the
transcription factors Gcn4p and Leu3p, and the removal of these elements (Didion et
al. 1996) had consequences for the activity of the promoter as measured by -
galactosidase activity generated by fusion constructs. Most noteworthy, Didion et al.
(1996) found a strong transcriptional induction of BAP2 upon the addition of certain
amino acids to the medium. This induction was seen independently of the presence of
the Gcn4p and Leu3p binding sites, but as further studied by Nielsen et al. (2001), the
Leu3p binding site is important for the level of transcription. Didion et al. (1996)
found that many amino acids were active in the induction, but leucine was particularly
potent; as little as 10 M gave a five-fold induction in one of the fusion constructs.
The inducing effect of leucine was also observed in a leu3 strain, supporting the
notion that Leu3p is not an essential player in the induction. Consistent with this,
other hydrophobic amino acids are also potent inducers of BAP2 transcription. De
Boer et al. (1998) Didion et al. (1998), Klasson et al. (1999), Regenberg et al. (1999)
and Iraqui et al. (1999) have studied the transcriptional induction of several other
amino acid permease genes by external amino acids, and it was noted at an early point
(Island et al., 1987) that low concentrations of amino acids can induce uptake of
dipeptides, later found to take place through a particular peptide transporter, Ptr2p
(Perry et al. 1994).

A system for sensing extracellular amino acids

The mechanism by which extracellular amino acids induce amino acid permeases and
the peptide permease is unknown. However, several parts of the system have been

6
identified. Thus, Jrgensen et al. (1997) found that STP1 is necessary for efficient
induction of transcription from the BAP2 promoter and proposed that Stp1p is a
transcription factor, a hypothesis consistent with the presence of sequences
characteristic of zinc fingers, as well as the nuclear localisation of Stp1p (Wang et al.,
1992). De Boer et al. (1998) found that STP1 is also needed for the similar induction
of BAP3. Nielsen et al. (2001) confirmed and analysed the binding of Stp1p and the
similar protein Stp2p to the BAP2 promoter. They found that the two proteins have
redundant functions as transcriptional activators and that the sequence element
ACGGCGACACGGC upstream of the coding region of BAP2 is needed for Stp1p
binding and for transcriptional activation.
Of particular interest is the finding by Didion et al. (1998), Jrgensen et al. (1998)
and independently by Iraqui et al. (1999) and Klasson et al. (1999) that SSY1,
encoding a member of the amino acid permease family, is necessary for the
transcriptional induction of the permease genes that are induced by extracellular
amino acids. We hypothesise that the amino acids interact with Ssy1p at the cell
surface, and that this interaction in some way leads to the induction. Despite its strong
homology to the other members of the amino acid permease family, Ssy1p is not itself
a permease with detectable efficiency (Didion et al. 1998), but it is possible that the
sensing is associated with a level of transport too low to be detected by conventional
criteria. Other putative parts of the sensing system are proteins encoded by PTR3 and
SSY5 (Jrgensen et al. 1998; Forsberg and Ljungdahl 2001). PTR3 was found by its
role in transcription of the peptide transporter by Island et al. (1991) and Barnes et al.
(1998) and was found independently by Jrgensenet al. (1998) (SSY3) and Klasson et
al. (1999) by its importance for transcription of amino acid permease genes.
Importantly, Klasson et al. (1999) and Forsberg and Ljungdahl (2001) found Ptr3p
and Ssy5p to be associated with the plasma membrane and with Ssy1p, but the
mechanism of their putative role in sensing and signal transduction is unknown.
Deletions of SSY1, PTR3 and SSY5 have so far indistinguishable phenotypes.
Other proteins are important for transcription of the amino acid permease genes. They
include Grr1p (Iraqui et al., 1999), which is involved in protein ubiquitination, Rts1p
(Larsson et al. 2000), which is a subunit of one of the isoforms of protein phosphatase
2A, and Tup1p (Nielsen et al. 2001), which is involved in chromatin structure.

Improving uptake capacity in a lager brewing yeast

Different lager brewing yeast strains growing in complex media are to various extents
sensitive to inhibitors of acetolactate synthase (C. Gjermansen and P.S. Nielsen,
unpublished). Metsulfuron methyl is one of these inhibitors belonging to the sulfonyl
urea herbicides. This sensitivity is presumably due to the blocked valine biosynthesis
combined with the fact that some lager brewing strains take up valine less efficiently
than does S. cerevisiae (Kielland-Brandt et al. 1990). We furthermore speculate that
2-oxobutyrate, which piles up in the parallel pathway to isoleucine when the inhibitor
is present, might compete for 2-oxoisovalerate in the first committed step of leucine
biosynthesis. In this way, the concentration of the product of the latter step, -
isopropyl malate, which via Leu3p is an inducer of BAP2, would drop. If this is so,
metsulfuron methyl not only inhibits valine biosynthesis but also diminishes valine
uptake through BAP2. In figure 1, small amounts of cells of fifteen yeast colonies
have been stamped onto complex medium (1% yeast extract, 2% peptone and 2% D-
glucose, 1.5% agar) containing metsulfuron methyl (100 mg/l); the lower colony is a
lager brewing yeast, M204, which is seen to grow poorly on the plate.

7
Figure 1: Lager brewing yeast M204
(lower colony) and 14 derivatives
containing one or more copies of an
extra piece of DNA with TAT1 and a
hyperactive, truncated BAP2 gene
(the two rows). Small amounts of
cells were stamped onto the shown
plate of complex medium with an
inhibitor of acetolactate synthase, and
tolerance to the inhibitor was tested
by allowing growth. The colonies that
grow well contain at least two copies
of the introduced DNA.

An interesting fact about the BAP2 gene is that it happens to be a neighbour (on
chromosome II, right arm) to TAT1, the gene encoding the second-most important
permease for the branched-chain amino acids. This prompted us to take a piece of
DNA (5,809 nucleotide pairs long) containing both TAT1 and the truncated,
hyperactive version of BAP2 and introduce the piece into brewing strain M204. This
was carried out by first inserting the piece into vector pYC050 (J. Hansen et al., in
preparation), a derivative of the vector series described by Olesen et al. (2000), and
then cutting the resulting circular plasmid open with a sequence-specific nuclease
called StuI. Cells of lager yeast M204 were allowed to take up the plasmid DNA, and
genetic transformants were selected that contained one or more copies of the plasmid
integrated by homologous recombination on chromosome II together with the BAP2
and TAT1 gene copies already there. The yeast cells in the two rows on the upper part
of the metsulfuron methyl-containing plate in figure 1 contain one or several
integrated copies of the plasmid. Molecular analysis by so-called Southern
hybridisation revealed that the transformant cells that grow poorly on the plate only
contain one copy, while the cells that have become resistant to the inhibitor contain
two or more copies of the plasmid. Now we need to determine if uptake has been or
can be improved to useful extents. This could be to control higher alcohol and/or ester
formation or to allow mutations in ILV2 that completely block diacetyl formation.

CONCLUSIONS
Gene technology has allowed an improved uptake of valine in a lager brewing yeast
by introduction of extra copies of wild-type and mutant genes for amino acid
permeases. This paper also reviews the current understanding of amino acid uptake
and the transcriptional induction of amino acid permease genes by external amino
acids.

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10
45

Insulin-like anabolic and mitogenic

activities of a yeast peptide complex on
brewing yeast
M. Dillemans, L. Van Nedervelde & A. Debourg
Institut Meurice, Department of Brewing Sciences, 1 Avenue E. Gryson, B-1070 Brussels,
Belgium (e-mail: [email protected])

Descriptors
Brewers' yeast, enzymatic activity, fermentative power, glucose, metabolism, peptide

SUMMARY
Yeast performances and vitality are not constant parameters during successive fermentations,
particularly on high gravity wort. The results show that a yeast peptide complex (YPC)
obtained by alcoholic extraction of yeast stimulates yeast metabolism and increases the
energetic level by stimulation of glucose metabolism. The addition of YPC has a activation
effect on many enzymes, starting from pyruvate, the key intermediate in anabolic process and
other important metabolic enzymes like citrate synthase, acetylCoA carboxylase and the
ATPase. Results also demonstrated that YPC exhibits an insulin-mimetic activity not only on
glycolysis but also on the overall metabolism.

Insulinhnliche und mitogene Aktivitt eines Hefepeptidekomplexes bei Bierhefe

Deskriptoren
Bierhefe, Enzymaktivitt, Grkraft (Triebkraf), Glucose, Stoffwechsel, Peptid

ZUSAMMENFASSUNG
Hefeleistung und Vitalitt sind keine konstanten Parameter bei aufeinander folgenden
Grungen, insbesondere bei High-Gravity-Wrzen. Die Ergebnisse zeigen, dass ein Hefe-
Peptid-Komplex (YPC), der durch alkoholische Extraktion aus Hefe gewonnen wird, den
Hefemetabolismus stimuliert und das Energieniveau durch die Anregung des Glucose-
stoffwechsels erhht. Zustzlich lst YPC einen Aktivierungseffekt bei vielen Enzymen aus.
Hier ist das Pyruvat, das Schlsselzwischenprodukt im anabolischen Prozess zu nennen, die
Zitratsyntase, Acetyl-CoA-Carboxylase und die ATPase. Die Ergebnisse zeigen zustzlich,
dass YPC eine insulinhnliche Aktivitt besitzt, die nicht nur die Glykolyse, sondern den
gesamten Stoffwechsel anspricht.
Anabolisant insulinomimtique et activits mitogniques dun complexe peptidique de
levure sur la levure de bire

Descripteurs
Activation du mtabolisme, insuline, levure, performance en fermentation

RESUME
Au cours de fermentations successives, et en particulier sur mot haute densit, les
performances de la levure ainsi que sa vitalit sont altres. Les rsultats indiquent quun
complexe peptidique (YPC) obtenu par extraction alcoolique de la levure stimule le
mtabolisme de la levure et augmente le niveau nergtique par activation du mtabolisme du
glucose. Laddition de YPC active diffrents enzymes impliqus dans le mtabolisme du
pyruvate, le mtabolite cl dans les voies anaboliques, ainsi que dautres enzymes
importantes comme la citrate synthase, lactylCoA carboxylase et lATPase. Les rsultats
montrent galement que le YPC exerce une activit similaire celle de linsuline sur la
glycolyse et le mtabolisme cellulaire.

2
INTRODUCTION
Yeast performances and vitality are not constant parameters during successive
fermentations, particularly on high gravity wort. Indeed, yeast is being exposed to
many stresses like osmotic and ethanol shocks that have a negative effect on yeast
performances.
Recently the first results concerning the partial purification of a yeast factor (termed
yeast peptide complex or YPC) that has a positive effect on yeast metabolism have
been presented. The purified fraction isolated from brewers yeast by a four steps
procedure was characterized as a low molecular weight water-soluble substance (6).
Previous works indicate that YPC is able to stimulate the fermentation rate and to
improve the attenuation of the wort and that such influence is persisting during
successive fermentations of high gravity worts (6).
Moreover, it has also been shown that YPC increases the energetic level by
stimulation of glucose metabolism as well as glycogen, lipids and protein intracellular
concentration during high gravity wort fermentation (7).
Looking to all these properties, YPC factor seems to have mitogenic and anabolic
insulin-like effects. It is therefore interesting to point out that the purified fraction has
a structure very similar to insulin mediators, low molecular weight water-soluble
peptides, isolated from treated insulin-sensitive mammalian cells (9). Indeed, it has
been reported that insulin has an effect on fructose-2,6-biphosphate level in human
fibroblasts (8) and that glucose metabolism in yeast is stimulated by human insulin.
Moreover, insulin has been shown to be active in brewing yeast strains (10). Recent
findings suggest that an insulin-like signal transduction cascade is somehow involved
in the regulation of metabolic biosynthetic pathways in yeast (13).
Therefore, to characterize the potential similarities between insulin biosynthetic and
mitogenic activities via its signal transduction pathway and YPC stimulating activities
in yeast, we have examined in this work the effects of YPC on a set of insulin-
sensitive key anabolic enzymes and metabolic events in comparison with the well-
known insulin second messenger, diacylglycerol (DAG).

MATERIALS AND METHODS

Yeast strains
The strains used in this study were Saccharomyces cerevisiae ale strain n391 for
fermentation experiments and laboratory diploid strain YF for enzymatic assays, both
from the collection of the Department of Brewing Sciences and Fermentation
Technology of the Institute Meurice.

Determination of enzyme activities

Cells were grown to stationary phase on minimal glucose medium then harvested by
centrifugation, and washed with 0.1M potassium phosphate buffer pH 6.4. The yeast
was depleted of endogenous substrates by shaking in this buffer at room temperature
for 1 hour. The cells (2x107 cells/ml) were then centrifuged and resuspended in fresh
buffer containing glucose 27 mM in presence of YPC (1 mg/ml) or DAG (0.4 mg/ml).
After 2 hours of anaerobic incubation, the yeast suspension was centrifuged, the pellet
was resuspended in 0.5 M potassium phosphate buffer pH 6.4 and crushed in a French
pressure cell. The extract was centrifugated at 12000 rpm for 20 min. and the
supernatant was collected.
Citrate synthase was assayed based on the method of Bergmeyer (2) by measuring the
amount of oxaloacetate transformed in function of time. The transformation of
oxaloacetate can be monitored by the rate of NAD+ reduction. In presence of an
excess of malate dehydrogenase and malate, the removal of oxaloacetate is balanced
by regeneration of oxaloacetate and a concomitant reduction of NAD+. So, the
determination is based on the formation of NADH measured by the increase in
absorbance at 340 nm. The composition of the 1 ml reaction mixture was: 100 mM
Tris/HCl buffer (pH 8), L-Malate 9.3 mM, NAD+ 1 mM and malate dehydrogenase 13

3
U /ml. The reaction carried out at 25C was started by addition of acetyl-CoA at a
final concentration of 0.18 mM and followed spectrophotometrically at 340 nm.
Specific activities are expressed as nmol oxaloacetate transformed per min. and per
mg protein and are the mean of 3 reproducible and independant assays.
Pyruvate dehydrogenase was assayed by the method of Wenzel et al. (16) with slight
modifications following the production of NADH. In this assay the NADH produced
by the action of pyruvate dehydrogenase is linked to the reduction of the dye p-
iodonitrotetrazolium violet (INT). The absorbance at 500 nm was recorded using a
double bean spectrophotometer. The reaction mixture, in a final volume of 1 ml of
buffer 50 mM Tris/HCl, 0.5 mM EDTA, and 0.2 % (wt/vol) Triton X-100 (pH 7.8),
contained 2.5 mM NAD+, 0.1 mM CoA, 1mM MgCl2, 0.1 mM oxalate, 1 mg bovine
serum albumin, 0.6 mM INT, 6.5 M phenazine methosulphate, 0.2 mM thiamine
pyrophosphate (TPP), and 5 mM pyruvate. The reference cuvette contained all
constituents except pyruvate. After addition of TPP and extract (0.4 - 0.5 mg protein)
to both cuvettes and when a stable baseline is obtained, the reaction was started by
adding puruvate. The absorbance of the reaction mixture at 500 nm was recorded to
follow the reduction of INT. The pyruvate dehydrogenase specific activity is
expressed as nmol NADH oxidized per min. and per mg protein and is the mean of 3
reproducible and independant assays.
Acetyl-CoA carboxylase activity was monitored by the determination of malonyl-CoA
produced by the enzyme according to Bergmeyer (3).
Pyruvate carboxylase was assayed by the method of Cazzulo and Stoppani (5) with
slight modifications measuring oxaloacetate formation. The reaction mixture of a final
volume of 1 ml contained: Tris/HCl buffer 100 mM pH8, malate dehydrogenase 0.93
U/ml, KHCO3 40 mM, pyruvate 10 mM, NADH 0.4 mM, acetylCoA 23 M, MgCl2
0.5 mM and ATP 2 mM. The reaction was started by addition of cell extract and
followed spectrophotometrically at 340 nm. Specific activities are expressed as nmol
oxidized cofactor per min. and per mg protein and are the mean of 3 reproducible and
independant assays.

Glycerol determination
Cells were grown in glucose minimal medium until the end of exponential phase,
harvested and resuspended (cell density was 75 mg wet weight per ml) in glucose-
minimal medium and incubated anaerobically for 2 hours with or without the two
specific substances YPC (1 mg/ml) and DAG (0.4 mg/ml).
Glycerol production was determined by HPLC using a Waters 600E system equipped
with a Waters 410 refractometer detector (34C) and fitted with a Shodex Ionpack
KC-810 P precolumn and two Shodex Ionpack KC-811 columns (60C). A solution of
perchloric acid (2 ml 60%/l of milliQ water) was used as eluent at a flow rate of 0.6
ml/min.

Acidification power test

Cells were grown in glucose minimal medium until the end of exponential phase,
harvested and resuspended in 0.5 M potassium phosphate buffer - glucose 1 %, pH
6.4 for 1 hour with or without addition of YPC (1 mg/ml) or DAG (0.4 mg/ml).
Washed cells were then resuspended in water at 30C for the determination of
extracellular acidification. The external pH was measured with an Orion pH-meter
model 720 A. Before the addition of glucose (100 mM), the pH of the cell suspension,
pre-incubated or not with specific substances was adjusted to about 7 and a base line
was established for 10 min. H+-efflux from the cells after addition of glucose was
measured for another 10 min. at 30C.

Farnesol-induced growth inhibition

Cells were grown in glucose minimal medium until the end of exponential phase, and
were inoculated into fresh glucose minimal medium containing 150 M farnesol and
tested substances. The growth was followed by measuring the optical density at 660
nm as a function of time.

4
RESULTS
Influence of YPC and DAG on anabolic enzyme activities
It is today well-established that many processes required for cell synthesis take place
in the mitochondria or require mitochondrial function. Moreover, previous work has
already shown that YPC has a positive effect on different cellular synthesis like lipids,
proteins and glycogen (7).
Most of the tricarboxylic acid (TCA) cycle as well as some enzymes involved in sterol
biosynthesis and amino acid synthesis are localized inside the mitochondria as
illustrated in figure 1.

Sugars Glycolysis Pyruvate

Pyruvate
carboxylase
Pi ADP ATP BIOSYNTHESIS
CO 2
ATP/ADP
translocator Pyruvate
Pyruvate
deshydrogenase

ATP BIOSYNTHESIS Citrate OAA

synthase
Citrate Citrate
OAA Citrate lyase

Acetyl CoA
ATP-ase Acetyl CoA
carboxylase
Fatty acid
H+ solutes synthase
Lipids

Figure 1: Subcellular localisation of some key anabolic enzymes.

Pyruvate is a key intermediate in anabolic processes. Its carboxylation to oxaloacetate

catalyzed by pyruvate carboxylase is an anaplerotic process for the generation of
tricarboxylic acid cycle intermediates.
Pyruvate dehydrogenase complex has a role in amino acid synthesis; this is further
supported by a partial leucine requirement of mutants lacking pyruvate dehydrogenase
activity (16).
Citrate synthase is a key enzyme of the TCA cycle catalysing the condensation of
oxaloacetate and acetyl-CoA to produce citrate. Citrate carries acetyl groups from
mitochondria to the cytosol for fatty acid synthesis. Indeed, acetyl CoA formed in
mitochondria must be transferred to the cytosol, but mitochondria are not readily
permeable to acetyl CoA. When present at high levels, citrate is transported to the
cytosol, where it is cleaved by citrate lyase and transformed in acetyl CoA and
oxaloacetate.
The biosynthesis of long-chain fatty acids requires four enzymatic systems among
which acetyl CoA carboxylase has been shown to be the rate limiting step (15).
Therefore, the influence of YPC on the activity of some key anabolic enzymes was
tested under fementation conditions and compared with the effect of DAG, the well-
known insulin mediator.
As illustrated in table 1, the addition of YPC or DAG to intact Saccharomyces
cerevisiae cells caused respectively a 50 to 90 % increase of key anabolic enzymes
after 2 hours of anaerobic incubation in phosphate buffer. These increased enzymatic
activities correlate well with the enhancement of macromolecular biosynthesis
observed on wort fermentation in the presence of YPC.
Moreover, it has also to be mentioned that insulin is know to activate pyruvate
dehydrogenase and acetylCoA carboxylase in mammalian cells (1).

5
 Control + YPC (1 mg/ml) + DAG (0.4 mg/ml)
Pyruvate carboxylase 2.7 4.6 5.1
Pyruvate dehydrogenase 5.2 8.1 n.d.
Citrate synthase 71.0 110.0 130.0
Acetyl CoA carboxylase 8.2 12.7 n.d.

Table 1: Influence of YPC (1 mg/ml) and DAG (0.4 mg/ml) on anabolic enzyme
activities. Enzymatic activities are expressed as nmol/min./mg protein.
n.d. not determined.

Influence of YPC and DAG on glycerol production

Furthermore, it is known that under anaerobic conditions, the surplus of reducing
equivalents formed in anabolic reactions is balanced by the formation of glycerol. As
previous results have shown that YPC has a positive effect on cellular synthesis, it is
not surprising that in presence of YPC or DAG about 8 fold more glycerol was
produced during anaerobic incubation in glucose minimal medium compared to the
control as illustrated in table 2.

Control + YPC (1 mg/ml) + DAG (0.4 mg/ml)

Glycerol (mg/ml) 0.023 0.200 0.175

Table 2: Influence of YPC (1 mg/ml) and DAG (0.4 mg/ml) on glycerol

production during anaerobic incubation in glucose minimal medium.

Growth-inhibitory effect of farnesol

ligands, hormones, growth factors

Tyrosine-kinase receptors

PLASMA MEMBRANE
PIP2 DAG

PIK PKC
PP PP
PLC
IP3
RAS
RAS
G
T
p

? LMW ++
Ca
CA++
insulin-mediators
Calmodulin

MAP calmodulin-kinases kinases C

Kinases

CELLULAR ACTIVITIES & MITOGENESIS

Cascade of protein-protein interactions

DNA transcription & replication
mitogenic effects
transports
anabolic processes
stress tolerance

Figure 2: Insulin transduction pathways.

6
The biological action of insulin is initiated by the binding of the hormone to its
receptor tyrosine kinase on the plasma membrane. Three pathways have been
identified and are thought to mediate different biological functions of the hormone as
summarized on figure 2.
Among them, the activation of the specific phospholipase C that hydrolyses
glycosylphosphatidylinositol lipids (PIP2) in plasma membrane plays an important
role. Indeed, this activity generates two second messengers: inositol 1,4,5-
triphosphate (IP3) and 1,2-diacylglycerol (DAG). Indeed, hydrolysis of
phosphatidylinositol-2-phosphate in Saccharomyces cerevisiae is required for a
number of nutritional and stress related responses.
Farnesol, an isoprenoid alcohol, induces in yeast and mammalian cell a significant
decrease in the cellular DAG level that leads to a reduction of cell growth. In
mammalian like in yeast cells, the farnesol-induced inhibition of cell proliferation
could be restored with exogenously added DAG (11).
Therefore, the influence of YPC, compare to DAG, on the growth-inhibitory effect of
farnesol was investigated.

Growth (OD 660nm)

6
control
5
control+FOH
4
YPC+FOH
3 DAG+FOH

2
1
0
0 10 20 30
time (hour)

Figure 3: Influence of YPC (1 mg/ml) and DAG (0.4 mg/ml)on the growth-
inhibitory effect of farnesol.

The exogenous addition of YPC was effective, like DAG, in preventing the growth-
inhibitory effect of farnesol as illustrated in figure 3. This finding confirms that YPC
mode of action may be involved in cellular signal transduction.

Influence of YPC on the yeast plasma membrane H+-ATPase

The plasma membrane H+-ATPase is the major system responsible for cellular proton
excretion in yeast. An application of the measurement of this activity is the
acidification power test used in the brewing industry (12).
The mechanism by which the ATPase is activated is known as a cascade of reactions
involving phospholipase C which generates diacylglycerol from phosphatidylinositol
4,5-biphosphate, the DAG then activating protein kinase C which phosphorylates and
activates the H+-ATPase (4).
Moreover, the important role of the plasma membrane H+-ATPase in wort
fermentation has been illustrated in previous works. Indeed, the inhibition of plasma
membrane H+-ATPase by diethylstilbestrol drastically reduced yeast wort
fermentation (14).

7
 equiv.H+/gDW
2,5
control (aerobic) 2,26
2 YPC (aerobic)
control (anaerobic)
YPC (anaerobic) 1,59
1,5
DAG (anaerobic) 1,45

1 0,95
0,85
0,5

0
0 5 10 15 20 25
time (min)

Figure 4: Influence of YPC (1 mg/ml) and DAG (0.4 mg/ml) on the yeast plasma
membrane H+-ATPase activity.

So, as it has been shown that YPC increases the energetic level, the effect of YPC on
glucose induced activation of the H+-ATPase has been evaluated.
Therefore, the release by yeast cells of H+ in water after addition of 100 mM glucose
has been followed as done for the acidification power test measurements.
As illustrated in figure 4, preincubations with YPC or DAG cause a 1,5-2,5 fold
increase in glucose-induced activation of the plasma membrane H+-ATPase whether
the preincubation was done aerobically or anaerobically.
These results confirm that YPC is directly involved in controlling the overall
metabolism by phosphorylation transduction cascade.

CONCLUSION
This work presents the first results concerning the mode of action of a yeast factor that
has anabolic and mitogenic effects. The results of this work indicate that YPC mimics
a broad spectrum of metabolic insulin actions on yeast cells and it raised the
possibility that YPC acts through an insulin-like transduction pathway.
Indeed, the presented results indicate that YPC and DAG have similar effects on
overall yeast metabolism like anabolic enzymes activation, H+-ATPase activation and
reduction of growth-inhibitory effect of farnesol.
To elucidated the physiological relevance of the insulin-like signal transduction
cascade of YPC, the initial step has to be identified. Therefore, the potential effect of
YPC on tyrosine kinase, phosphatidylinositol phospholipase C and protein kinase C
activities will be evaluated. Moreover, production of second messengers
diacylglycerol and IP3 will also be measured. Indeed, all these enzymatic activities
and metabolite concentrations are known to be modulated by insulin.

8
REFERENCES
1. Belsham G.J., Brownsey, R.W., Hughes, W.A. and Denton, R.M., Anti-insulin
receptor antibodies mimic the effects of insulin on the activities of pyruvate
dehydrogenase and acetylCoA carboxylase and on specific protein
phosphorylation in rat epididymal fat cells, Diabetologia, 1980, 18, 307-12.
2. Bergmeyer H.U., Methods of Enzymatic Analysis, third edition, Weinheim:
VCH, 1985, vol. IV, 353-358.
3. Bergmeyer H.U., Methods of Enzymatic Analysis, third edition, Weinheim:
VCH, 1985, vol. VII, 225-230.
4. Brandao, R.L., de Magalhaes-Rocha, N.M., Alijo, R., Ramos, J. and Thevelein,
J.M., Possible involvement of a phosphatidylinositol-type signaling pathway in
glucose-induced activation of plasma membrane H(+)-ATPase and cellular
proton extrusion in yeast Saccharomyces cerevisiae, Biochim. Biophys. Acta,
1994, 1223, 117-24.
5. Cazzulo, J.J. and Stoppani, A.O., Effects of adenosine phosphates and
nicotinamide nucleotides on pyruvate carboxylase from baker's yeast, Biochem.
Journal, 1969, 112,755-762.
6. Dillemans, M., Van Nedervelde, L., and Debourg, A., Characterization of a
novel yeast factor increasing yeast brewing performances, Proceedings of the
European Brewery Convention Congress, Cannes, 1999, 711-718.
7. Dillemans, M., Van Nedervelde, L., and Debourg, A., An approach to the mode
of action of a novel yeast factor increasing yeast brewing performances, J. Am.
Soc. Brew. Chem., 2001, under press.
8. Farnararo, M., Vasta, V., Bruni, P., and D'Alessandro, A., The effect of insulin
on Fru-2,6-P2 levels in human fibroblasts, FEBS Letters, 171:117-20, 1984.
9. Gottschalk, W.K., Macaulay, S.L., Macaulay, J.O., Kelly, K., Smith, J.A., and
Jarett, L., Characterization of mediators of insulin action, Ann. NY Acad. Sci.,
1986, 488, 385-405.
10. Lodolo, E.J., OConnor-Cox, E.S.C., and Axcell, B.C., Novel application of
glucagon and insulin to alter yeast glycogen concentrations, J. Am. Soc. Brew.
Chem., 1995, 53, 145-151.
11. Machida, K., Tanaka, T., Yano, Y., Otani, S. and Taniguchi, M., Farnesol-
induced growth inhibition in Saccharomyces cerevisiae by a cell cycle
mechanism, Microbiology, 1999, 145, 293-299.
12. Mathieu Ch., van den Berg, L. and Iserentant, D., Prediction of yeast
fermentation performance using the acidification power test, Proceedings of the
European Brewery Convention Congress, Lisbon, 1991, 273-280.
13. Mller, G., Grey, S., Jung, C., and Bandlow ,W., Insulin-like signaling in yeast :
modulation of protein phosphatase 2A, protein kinase A, cAMP-specific
phosphodiesterase, and glycisyl-phosphatidylinositol-specific phospholipase C
activities, Biochemistry, 2000, 39, 1475-1488.
14. OConnor-Cox, E.S.C., Lodolo, E.J., and Axcell, B.C., Role of oxygen in high
gravity fermentations in absence of unsaturated lipid biosynthesis, J. Am. Soc.
Brew. Chem., 1993, 51, 97-107.
15. Schneiter R., Hitomi, M., Ivessa, A.S., Fasch, E., Kohlwein, S.D. and Tartakoff,
A.M., A yeast acetyl Coenzyme A carboxylase mutant links very-long chain
fatty acid synthesis to the structure and function of the nuclear membrane-pore
complex, Molecular and Cellular Biology, 1996, 16,161-72.
16. Wenzel, T.J., Van Den Berg, M.A., Visser, W., Van Den Berg, J.A. and de
Steensma, H.Y., Characterization of Saccharomyces cerevisiae mutants
lacking the E1 a subunit of the pyruvate dehydrogenase complex, Eur. J.
Biochem., 1992, 209, 697-705.

9
46

Evaluation of antioxidant properties of

Saccharomyces cerevisiae
P. Bastin1, L. Van Nedervelde1, M. Dillemans1, P. Boivin2 &
A. Debourg1
1
Institut Meurice, Department of Brewing Sciences and Fermentation Technology, Avenue E.
Gryson 1, B-1070 Brussels, Belgium (e-mail : [email protected])
2
Institut Franais des Boissons de la Brasserie Malterie, Ple Technologique de Nancy-
Brabois, 7 rue du Bois de la Champelle, B.P. 267, F-54512 Vandoeuvre Cedex, France

Descriptors
Antioxidant, taste stability, yeast extract

SUMMARY
In order to improve quality and organoleptical stability of food products and beverages, it is
often necessary to control oxidation reactions responsible of stalling. This study specifies the
composition of antioxidant compounds in yeast extracts susceptible to contribute to beer
stability. A convenient procedure allowing to coextract in a single fraction with a high degree
of purity, multiple antioxidants from yeast is presented. Among four different extraction
techniques, yeast hydrolysate after alcoholic extraction presents the best antioxidant activities.
The results indicate that low molecular weight compounds, around 1500, ninhydrin positive
are both good lipoxygenase inhibitors and radical scavengers.

Bewertung der antioxidativen Eigenschaften von Saccharomyces cerevisiae

Deskriptoren
Antioxidantium, Geschmacksstabilitt, Hefeextrakt

ZUSAMMENFASSUNG
Bei dem Versuch, die Qualitt und die organoleptische Qualitt von Lebensmitteln und
Getrnken zu verbessern, ist es oft notwendig, die oxidativen Reaktionen, welche Alterung
verursachen, zu beobachten. Diese Arbeit spezifiziert die Zusammensetzung der oxidativen
Verbindungen in Hefeextrakten, welche die Bierstabilitt beeinflussen knnen. Es wird ein
praktisches Verfahren vorgestellt, das es erlaubt verschiedene Antioxidantien mit hoher
Reinheit in eine einzige Fraktion zu extrahieren. Von den vier verschiedenen Extraktions-
verfahren zeigen Hefehydrolysate, die durch alkoholische Extraktion gewonnen wurden, die
besten antioxidativen Eigenschaften. Die Ergebnisse zeigen, dass ninhydrinpositive
Verbindungen mit niedrigen Molekulargewichten (ca. 1500) gute Inhibitoren fr
Lipoxigenasen und gute Radikalenfnger sind.
Evaluation des propriets anti-oxydantes de Saccharomyces cerevisiae

Descripteurs
Antioxydant, extrait de levure, stabilit organoleptique

RESUME
Afin damliorer la qualit et la stabilit organoleptique des produits alimentaires et des
boissons, il est souvent ncessaire de contrler les ractions doxydation responsables du
vieillissement. Ce travail prcise la prsence dans la levure de nombreux composs
antioxydants susceptibles damliorer la stabilit organoleptique de la bire. Une methode
permettant la coextraction de plusieurs substances antioxydantes dans une mme fraction est
prsente. Parmi quatre techniques dextraction diffrentes, lhydrolysat de levure aprs
extraction alcoolique prsente les meilleures proprits antioxydantes. Les rsultats montrent
que des composs de faible poids molculaire, aux environs de 1500 daltons et positifs la
ninhydrine, sont la fois de bons inhibiteurs de la lipoxygnase et dexcellents piges
radicaux libres.

2
INTRODUCTION
In order to improve quality and organoleptical stability of many food products and
beverages, it is often necessary to control oxidation reactions responsible of staling.
Indeed, one of the most serious problems in deterioration of food materials or
products is the oxidation of food ingredients during processing, storage and
distribution. In order to prevent oxidation, synthetic antioxidants have been generally
used for many years but the today tendancy is to find natural antioxidants (1).
In the brewing industry, it is today well established that lipid oxidation during wort
production is directly responsible for production of stale off-flavor during beer storage
(5). The enzymatic or auto-oxidation of the unsaturated fatty acids results in the
formation of their hydroperoxydes which are unstable and degraded into low
molecular weight compounds such as aldehydes, ketones, and acids. The production
of hydroperoxydes during mashing increased with lipoxygenase activity in malt.
Addition of an inhibitor of lipoxygenase to mash significantly inhibed the production
of the hydroperoxyde (4). Many natural antioxidants commonly used today are
derived from plants and micro-organisms. Different studies have shown the presence
in barley and malt of different antioxidant compounds (3). Indeed, many papers have
described the importance of antioxidative enzymatic components of Saccharomyces
cerevisiae such as superoxide dismutase, catalase or GSH reductase (8). Moreover,
yeast is also particularly rich in nonenzymatic antioxidants like GSH, ubiquinone,
hydroquinone, sulfhydryl amino-acid or mineral ions. The isolation of new
antioxidants from Saccharomyces cerevisiae appears of potential importance for food
technology.
The objective of this study is to specify the composition of antioxidant compounds in
yeast extracts susceptible to contribute to beer stability and to develop a convenient
procedure allowing to coextract in a single fraction with a high degree of purity,
multiple antioxidants from yeast. The main advantage of a pool rather than a single
antioxidant consists in potential synergism of the different compounds together with
recycling effect and complementary mode of action.

MATERIALS AND METHODS

The different yeast extracts
The brewing yeast was harvested at the end of fermentation (7 days) and stored at -
80C before use. The different fractions obtained are:
A French Press Extract: The yeast was collected by centrifugation (4 000 rpm for 5
min. at 4C) and the pellet was resuspended in distilled water and crushed in a French
pressure cell. The extract was centrifugated at 17 000 x g for 20 min. and the
supernatant was collected. Proteins are eliminated by centrifugation after boiling
during 10 min. The extraction yield expressed in dry weight percentage is of 10,6%.
An Alcoholic Extract: The yeast was extracted twice with methanol/water mixture
(7:3, V/V) under agitation. The extract was collected by filtration and concentrated by
evaporation under vacuum. The yield is about 10,9%.
An Acid Hydrolysis Extract: The dry alcoholic extract was resuspended in water and
hydrolysed in 10 parts of sulfuric acid 4 N during 18 hours under boiling and
agitation. The sulfate ions were eliminated by addition of barium hydroxyde. The
barium sulfate formed was eliminated by filtration and washing 3 times with boiling
water. The filtrate was dessicated by evaporation under vacuum and resuspended in

3
hot water. After precipitation at 4C for a few days, the Acid Hydrolysis Extract was
collected by centrifugation. The yield is about 7,8%.
A Direct Acid Hydrolysis Extract: The yeast was directly hydrolysed in 10 parts of
sulfuric acid during 18 hours under boiling and agitation. The hydrolysate was treated
as described above. The yield is about 26,2 %.

Measurement of antioxidant activities

As many oxidation processes are existing, different methods have been used to
evaluate the antioxidant properties.
Inhibition of lipoxygenase activity: In this test, the production of conjugated diene
hydroperoxide by oxidation of linoleic acid in an aqueous dispersion is monitored at
234 nm (7). This production is of course reduced in presence of potent antioxidants.
DPPH free radical scavenging: This method determines the antiradical power of the
antioxidant compounds by using the stable free radical DPPH (1,1-diphenyl-2-
picrylhydrazyl) and the reaction is monitored at 515 nm (2). The antioxidant activity
is expressed in mol of radical DPPH scavenged / h.
Co-oxidation of -carotene in linoleate model system: The -carotene oxidation by
free radicals produced by heating linoleic acid at 50C is monitored at 470 nm (6).

RESULTS
Evaluation of antioxidant properties of different yeast extracts
Using the different extracts of yeast in the different antioxidant assays, we were able
to analyse the antioxidant capacity of these different fractions and to evaluate their
mode of action.

Inhibition of lipoxygenase
As described earlier, this test will evaluate the inhibition of lipoxygenase linoleic acid
oxidation by the different yeast fractions. As illustrated in figure 1, the enzymatic
oxidation of linoleic acid by lipoxygenase in function of time is reduced in presence
of increasing concentration of the Acid Hydrolysis Extract.

Lipoxygenase activity
120
(moles P/mg protein)

100
no inhibitor
80
0.06 mg D.W./ml
0.08 mg D.W./ml
60
0.12 mg D.W./ml
0.24 mg D.W./ml
40

20

0
0 2 4 6 8 10 12 14
Time (min)

Figure 1: Influence of yeast Acid Hydrolysis Extract on the inhibition of linoleic acid
oxidation by lipoxygenase.

4
In the same way, the lipoxygenase activity is inhibited by all the different extracts in
different ranges as illustrated in figure 2. The Acid Hydrolysis Extract is the most
efficient inhibitor of lipoxygenase activity with about 80% inhibition at a
concentration of 0,26 mg extract/ml.

Percent inhibition
lipoxygenase
100
A.H.
D.A.H.
80
F.P.
A.E.
60

40

20

0
0 0,05 0,1 0,15 0,2 0,25 0,3
Concentration (mg D.W. extract/ml)

Figure 2: Influence of the different yeast fractions on the inhibition of linoleic acid
oxidation by lipoxygenase.A.H.: Acid Hydrolysis Extract, D.A.H.: Direct Acid
Hydrolysis Extract, F.P.: French Press Extract, A.E.: Alcoholic Extract.

DPPH free radical scavenging activity

Figure 3 shows that all 4 yeast fractions have a free radical scavenging activity, the
Acid Hydrolysis Extract having again the higher effect. Looking to the dose-response
of this extract on DPPH free radical, the scavenging activity is higher than for the
other extracts mainly at low concentration, indicating the efficiency of this fraction.

Scavenging power
1,8
(mole DPPH/h)
1,6 A.H.
D.A.H.
1,4
F.P.
1,2 A.E.

1
0,8
0,6
0,4
0,2
0
0 0,05 0,1 0,15
Concentration (mg D.W. extract/ml)

Figure 3: Free radical scavenging activity of the different yeast fractions.A.H.: Acid
Hydrolysis Extract, D.A.H.: Direct Acid Hydrolysis Extract, F.P.: French Press
Extract, A.E.: Alcoholic Extract.

5
Co-oxidation of -carotene in linoleate model system
Heating linoleic acid at 50C generates potent free radicals that oxidize -carotene
inducing a decrease of optical density at 470 nm as illustrated in figure 4. The -
carotene oxidation is reduced by increasing concentration of Direct Acid Hydrolysis
Extract. The dose-response of the antioxidant power illustrated in figure 5, expressed
as percent of inhibition of -carotene oxidation indicates that all the yeast fractions
present an antioxidant activity, the Direct Acid Hydrolysis Extract having the higher
effect. Moreover, the antioxidant activity of the Acid Hydrolysis Extract, especially at
low concentration, is somewhat lower compared to its efficiency shown in the other
assays.

Abs. 470 nm
0,7 (non oxidized -carotene)
no inhibitor
0.05 mg D.W./ml
0,6 0.10 mg D.W./ml
0.19 mg D.W./ml
0,5 0.38 mg D.W./ml

0,4

0,3

0,2
0 5 10 15 20 25 30 35
Time (min)

Figure 4: Inhibition of -carotene oxidation in presence of different doses of Direct

Acid Hydrolysis Extract.

80 Antioxidant power
(% inhibition) A.H.
70 D.A.H.
F.P.
60
A.E.
50

40
30

20
10

0
0 0,1 0,2 0,3 0,4
Concentration (mg D.W. /ml)

Figure 5: Inhibitory effect of the different yeast fractions on co-oxidation of -

carotene in linoleate model system. A.H.: Acid Hydrolysis Extract, D.A.H.: Direct
Acid Hydrolysis Extract, F.P.: French Press Extract, A.E.:Alcoholic Extract.

6
The summary of the antioxidant efficiency of the different yeast fractions at their
optimal concentration in each assay is illustrated in figure 6.
As each of the three tests are based on different antioxidant properties, it is not
surprising that the percent of efficiency are different for a same fraction. This
illustrates the complexity of oxidation reactions occuring in any process.
Nevertheless, it can be considered that the Acid Hydrolysis Extract is the most potent
antioxidant fraction followed by the Direct Acid Hydrolysis Extract. This last extract
has a much simpler extraction procedure that makes the extract less purified.
Moreover, our results confirm the importance of these nonenzymatic thermostable
fractions in antioxidant defenses of brewing yeast.

% efficiency
90 A.H.
80 D.A.H.
70 F.P.
60 A.E.

50
40
30
20
10
0
% inhibition % DPPH scavenged % inhibition co-
lipoxygenase oxidation
(0.26 mg D.W./ml) (0.13 mg D.W./ml) (0.35 mg D.W./ml)

Figure 6: Antioxidant efficiency of the different yeast fractions in the different assays.
A.H.: Acid Hydrolysis Extract, D.A.H.: Direct Acid Hydrolysis Extract, F.P.:
French Press Extract, A.E.: Alcoholic Extract.

Isolation of components of Acid Hydrolysis Yeast Extract

The Acid Hydrolysis Extract having the higher antioxidant efficiency, we have
separated the antioxidative components into different fractions based on molecular
weight using a Sephadex G25 column. The elution profile was followed by measuring
optical density at 210 nm and 6 fractions were separated, as illustrated in figure 7.
Each of the 6 fractions were concentrated 10 times by evaporation under vacuum in
order to obtain a final volume for each fraction equal to the initial volume of the crude
extract putted on the column. So, each fraction of different molecular weight is
roughly at the same concentration as in the crude extract. The molecular weight of the
different fractions was evaluated using standards. Fraction 1 contains substances
having a molecular weight around 20 000; fraction 2 has components with molecular
weight ranging from over 1500 to less than 5000; fraction 3 consists of components
with molecular weight around 1500 and fractions 4 to 6 are composed of substances
with molecular weight below 1000.

7
 Abs 210 nm MW
1450
90
80
F1 F2 F3 F4 F5 F6
70
60
50
40 MW
30 20000
20
10
0
0 50 100 150 200 250 300
Elution volume (ml)

Figure 7: Elution profile on Sephadex G25 of the Acid Hydrolysis Extract.

Characterisation of the antioxidant activity of the Acid Hydrolysis Extract

fractions
Antioxidant activity of the 6 fractions was evaluated using the same three methods
already described in this work.
The percent efficiency of the crude Acid Hydrolysis Extract and the different fractions
is presented in the following figure 8. The last column (F1-F6) represents the
percent inhibition of the 6 fractions polled and 6 times concentrated and indicates that
all active compounds of the crude extract are collected after fractionation.

% e ff ic ie n c y
120

100 A .H . E x tr a c t

F1
80
F2

60 F3

F4
40
F5

20 F6

0
F1-F6
L ip o x y g e n a s e S c a v e n g in g C o - o x i d a tio n
i n h i b i ti o n power i n h ib i ti o n

Figure 8: Influence of the six Acid Hydrolysis Extract fractions on the inhibition of
linoleic acid oxidation by lipoxygenase, the DPPH free radical scavenging activity
and the co-oxidation of -carotene in linoleate model system.

The active compounds as far as inhibition of the lipoxygenase activity is concerned

are mainly present in fraction 2 and 3, with respectively 47% and 52% of the total
lipoxygenase inhibition efficiency of the Acid Hydrolysis crude extract.

The essay of DPPH free radical scavenging activity shows that fraction 3 represents
more than 55% of the antioxidant activity of the crude extract.

8
On the other hand, the fractions 4, 5 and 6 do not have any scavenging activity,
indicating that the low molecular weight compounds present in these fractions seems
not to be involved in this antioxidant capacity.
As measured by the assay of co-oxidation of -carotene in linoleate model system, the
fractions 2 and 3 contain the most efficient antioxidant. Nevertheless, fraction 6
shows some important antioxidant activity taking into account its low dry weight
content.
Comparing the antioxidant efficiency of the different fractions obtained by
fractionation of the Acid Hydrolysis Extract expressed in percent compared to the
crude Acid Hydrolysis Extract it clearly appears that fraction 3, containing component
with molecular weight around 1500, predomines.

Comparison of antioxidative activity of Acid Hydrolysis yeast extract with pure

substances
The antioxidant activity of pure substances has been compared to the Acid Hydrolysis
Extract in 2 methods.
The essay of co-oxidation of -carotene in linoleate model system shows that the Acid
Hydrolysis crude extract has the same antioxidant activity as the pure (+)-catechin
used at a 10 times lower concentration. Moreover, the free radical scavenging activity
of the Acid Hydrolysis crude extract has also been compared to some pure substances
used as antioxidants. The kinetic pattern is much more close to the effect of glutathion
than to the one of ascorbic acid, which is known to be a reductor and thereby involved
in the direct chemical reaction.

Impact of antioxidants on the formation of carbonyl compounds responsible of

stale off-flavor during brewing

2-Thiobarbituric acid (TBA) test

2-Thiobarbituric acid was used to measure the degree of beer staling. The reaction of
TBA with beer or wort components at 60 C yield to a predominant yellow pigment
monitoring at 445 nm. This TBA test is used to measure the oxidative changes during
processing. As illustrated in figure 9, the addition during brewing of antioxidant and
yeast extracts obtained in this work decrease the intensity of this pigment. In order to
specify the interest of this test, an addition of linoleic acid show an increasing of the
intensity of this pigment.

D.O. at 445 nm
1.2

1 Control
0.8 40 ppm E1
0.6 80 ppm E1
0.4 80 ppm F3
0.2 2,5 ppm (+) -Catchine
0 10 ppm Linoleic acid
Wort Wort after 6 days at
60 C

Figure 9: Effect of antioxidant on the TBA reactives substances formation

9
Inhibitory effect on the formation of trans-2-nonenal
The nonenal potential is a kind of forcing test to determine the potential of a wort to
form trans-2-nonenal under beer conditions. The level of trans-2-nonenal formed is
determined by GC-MS. The first results show a decreasing of the concentration of the
trans-2-nonenal after an addition of yeast extract or (+)-catechine (unpublished). In
order to confirm these encouraging results, others analyses have to be done.
The level of hydroxyperoxydes 9 and 13 that are responsible of the formation of trans-
2-nonenal is actually determined in wort by HPLC-UV after an addition of
antioxidant during the process.

CONCLUSIONS
In order to improve quality and organoleptical stability of many food products and
beverages, it is often necessary to control oxidation reactions responsible of staling.
Many antioxidant could be used, but the today tendancy is to find natural antioxidants.
This work is dedicated on the study of antioxidant properties of Saccharomyces
cerevisiae. Different methods have been used to evaluate the antioxidant efficiency
due to the complexity of oxidation reactions occuring in any process.
All the tests indicate that yeast collected at the end of fermentation contains different
antioxidant fractions. An yeast acid hydrolysate presents the best antioxidant activity:
its a very good free radical scavenger and also inhibits the lipoxygenase activity.
A partial purification based on molecular weight has allowed to identify antioxidant
compounds with a molecular weight between 1000 and 5000.
First results also demonstrated that yeast extracts, added during wort production,
could decrease the oxidative deterioration of wort.

REFERENCES
1. Boers, J., Antioxydants Naturels ou Synthtiques? Process, 2000, 1156, 32-34.
2. Brand-Williams, W., Cuvelier, M.E. & Berset, C., Lebensm-Wiss. u.-Technol,
1995, 28, 25-30.
3. Goupy, P., Hugues, M., Boivin, P., Amiot, M.J., Antioxidant compounds of
barley (Hordeum vulgare) and malt extracts, Proceedings of the 27th EBC
Congress, Cannes, 1999, 445-451.
4. Kobayashi, N., Kaneda, H., Kano, Y. & Koshino, S., Lipid oxidation during wort
production, Proceedings of the 25th EBC Congress, Brussels, 1995, 405-412.
5. Ligeois, C., Lermusieau, G. & Collin, S., Measuring Antioxidant Efficiency of
Wort, Malt, and Hops against the 2,2-Azobis(2-amidinopropane)
Dihydrochloride-Induced Oxidation of an Aqueous Dispersion of Linoleic Acid,
J. Agric. Food Chem., 2000, 48, 1129-1134.
6. Pratt, D.E. & Hudson, B.J.F., In Food antioxidants Hudson B.J.F., Edition
Elsevier, 1990, 171-191.
7. Richard-Forget, F., Gauillard, F., Hugues, M., Thiry, J.M., Boivin, P. & Nicolas,
J., J. Food Sci, 1995, 60, 1325-1329.
8. Santiago, L. & Mori, A., Antioxidant Defenses of Bakers Yeast against Free
Radicals and Lipid Peroxides in Rat Brain, Archives of Biochemistry and
Biophysics, 1993, 306, 16-21.

10
47

Development of brewers yeast with

increased carbonyl reducing activity
Maija-Leena Vehkomki1, Virve Vidgren1, Arvi Vilpola1,
Hannele Virtanen1, Beatriz Snchez2, Ivn Galindo-Castro2,
Merja Penttil1 & Rafael Rangel-Aldao2
1
VTT Biotechnology, P.O. Box 1500, FIN-02044 VTT, Espoo, Finland
2
Empresas Polar, Polar Technology Center, Caracas, Venezuela

Descriptors
Brewersyeast, carbonyl compound, enzymic activity, non-enzymic browning, staling

SUMMARY
To lower the initial load of dicarbonyls that accumulate in beer upon aging, we attempted
their reduction during fermentation by overexpressing in brewers yeast the enzyme OYE2, or
the transcription factor yAP-1 that regulates it and other reductases. Modified yeasts showed
resistance to methylglyoxal, and specific increases of mRNA levels (12 to 16 fold) and up to
17 fold rise in the specific activities of NADPH-dependent reductases; as well as a lower
production of diacetyl and pentanedione (50%) during fermentation. Strecker aldehydes were
down 30 to 40%, opening the way to use modified yeasts to study the role of Maillard
reaction dicarbonyls on beer aging.

Entwicklung einer Brauhefe mit vergrerter Aktivitt Carbonyle zu reduzieren

Deskriptoren
Altgeschmacksbildung, Bierhefe, Carbonylverbindung, Enzymaktivitt, nichtenzymatische
Brunung

ZUSAMMENFASSUNG
Wir versuchten die Dicarbonylanfangskonzentration, die whrend der Alterung noch
anwchst, whrend der Grung zu verringern. Dies wurde whrend der Grung durch die
verstrkte Expression des Enzyms OYE2, oder der Transkription des yAP-1-Faktors, der
OYE2 und andere Reduktasen reguliert, bewerkstelligt. Vernderte Hefen zeigten eine
Widerstandsfhigkeit gegen Methylgyoxal, eine spezifische Vergrerung des mRNA-
Spiegels, einen Anstieg der spezifischen Aktivitt der NADPH-abhnigen Reduktase,
genauso wie eine verringerte Bildung von Diacetyl und Pentandion (50%) whrend der
Grung. Eine Verringerung der Streckeraldehyde um 30 40 % zeigt die Mglichkeiten auf,
mit Hilfe von modifizierter Hefe den Einfluss, den die Maillard-Carbonyle auf die
Bieralterung haben, zu erforschen.
Dveloppement d'une levure de bire suractive dans la rduction des carbonyles

Descripteurs
Activit enzymatique, brunissement non-enzymatique, compos carbonyl, formation du got
dvent, levure de brasserie

RESUME
Afin de diminuer la charge initiale de dicarbonyles s'accumulant dans la bire vieillissante,
nous avons procd leur rduction pendant la fermentation, en surexprimant dans la levure
de bire l'enzyme OTE2 ou le facteur de transcription yAP-1 qui la rgule et rgule d'autres
rductases. Les levures modifies se sont avres rsistantes au mthylglyoxal et ont montr
des augmentations spcifiques des taux d'ARNm (12 16 fois) et de l'activit spcifique des
rductases NADPH-dpendantes (jusqu' 17 fois). Elles produisaient galement moins de
diactyle et de pentanedione (50 %) pendant la fermentation. Les aldhydes de Strecker
avaient diminu de 30 40 %, ouvrant la voie l'emploi de levures modifies pour tudier le
rle des dicarbonyles de la raction de Maillard dans le vieillissement de la bire.

2
INTRODUCTION
In a previous work (1) we have identified and cloned a brewer's yeast oxidoreductase,
Old Yellow Enzyme (Oye2, EC 1.6.99.1), capable to reduce 3-deoxy-2-hexosulose (3-
DH), methylglyoxal, diacetyl and other dicarbonyls that are present in wort and beer.
We have also shown that these compounds may play an important role in beer flavor
deterioration (2). DeRisi and colleagues (3) have demonstrated that transcription of
OYE2 and other reductase genes are induced in yeast cells overproducing the
transcriptional activator Yap1, thereby connecting this group of enzymes with the
oxidative stress response of yeast. Taken all together, this information opened up the
possibility to attempt a novel approach by overexpressing OYE2 and YAP1 in our
brewer's yeast with the expectation that the improved reducing capacity should lessen
the aldehyde and dicarbonyl levels in wort during the fermentation. In this work, we
describe the production of brewer's yeast with increased reductase activities by
following two genetic strategies: i) overexpression of OYE2 under the control of
modified sequences of the yeast ADH1 promoter (monoenzymatic approach) and; ii)
overexpression of the stress tolerance transcriptional activator YAP1 under its own
promoter to activate OYE2 and other reductases (multienzymatic approach). The
effects of such modifications on brewing performance, carbonyl contents and
organoleptic characteristics of fresh and aged beers are discussed.

MATERIALS AND METHODS

Yeast strains
In-house lager brewing yeast was obtained from our corporate microbiological
facilities.
Yeast transformation
YPD medium or wort were used for cell culture. Yeast transformation was
accomplished by the LiAc-method described by Ito et al. (4). NEPRA medium
supplemented with 0.6 mM CuSO4 (5) or YPD medium containing 5 g/ml Cerulenin
were used for selection of the OYE2 and YAP1 transformants, respectively.
RNA extraction and Northern blot analysis
Brewing yeast cells were collected from culture flasks or fermentation tanks by
centrifugation at different time points and the total RNA was isolated following
standard procedures. Northern blot analysis were performed according to Sambrook et
al. (6).
Pilot fermentations
Pilot scale fermentations were carried out in the VTT pilot brewery, using commercial
wort with 16.1 % (m/m) original gravity. The wort batch was dosaged into 50 l tanks
and pitched with the genetically modified yeasts (252 g centrifuged yeast/50 l). Wort
was oxygenated up to a soluble oxygen content of 11-14 mg/l. The main fermentation
was carried out at 10 C. When the apparent extract did not change on the successive
days, flocculated yeast was removed and the wort transferred to the secondary
fermentation, where it was kept until diacetyl content was reduced below 0.035 mg/l.
Temperature was then decreased to -1.5 C, and secondary fermentation continued for
3 more days. Finally, the brewed wort was filtered and the gravity was adjusted with
deoxygenated water to 12 % (m/m). Carbon dioxide content in beers was adjusted to 5
g/l.

3
Expression plasmid constructs
The amplification of OYE2 protein coding region with PCR was accomplished as
described in a previous work (1). Three expression cassettes containing the ADH1
promoter fragments, the OYE2 protein coding region and the ADH1 terminator were
constructed and ligated to the expression vector pET13.1. The plasmids obtained
(pETOYE2L, pETOYE2M, pETOYE2S) have three versions of the yeast ADH1
promoter as described by Ruohonen et al. (7, 8) and they were further used to
transform a lager brewer's yeast strain. The plasmid pETYAP1 carrying wild type
YAP1 gene (GenBank Accession Number: X58693) was constructed from the
plasmid pCR2, obtained from Dr. S. Harashima (Osaka University, Japan). The latter
plasmid has an incomplete YAP1 gene regulated by its own promoter and lacks
nucleotides coding for amino acids from 556 to 650 at the 3' end of the gene. The
gene was completed with the corresponding YAP1 sequence from our brewing yeast
by using PCR procedures and then ligated to the vector pET13.1. The plasmid pCR4
contains the truncated form of YAP1 in opposite orientation compared to pCR2.
Organoleptic evaluation of beer
A trained tasting panel evaluated by sensory analysis of 37 attributes, the aroma and
taste profiles of the produced alcoholic beverages after brewing (fresh beer). To force
flavor aging, beer was exposed to 28 C for up to 6 weeks in a dark place, and
evaluated every two weeks. A group of beers was kept at 5 C as a reference for all
evaluations.

RESULTS AND DISCUSSION

Our initial attempt to diminish the -dicarbonyl contents during fermentation resulted
in the purification and cloning of OYE2 (1), an NADPH-dependent oxidoreductase
capable to reduce ,-unsaturated carbonyls present in beer (1), as well as in wort
(data not shown). This enzyme, as many other reductases, is associated with the
oxidative stress response in yeast, a process regulated by the transcriptional activator
Yap1 (3). From these findings, we first verified the normal levels of Oye2 during the
brewing process and found that its mRNA was most abundant during the first three

Fermentation time [days]

1 2 3 4 5 6 7

OYE2 mRNA

26 S
18 S

Figure 1: Levels of OYE2 mRNA in an unmodified brewers yeast strain during a

standard fermentation

4
days of fermentation (figure 1). These results suggested that yeast cells were sensing
fresh wort as a highly oxidative environment. This is in agreement with the results of
Martin and colleagues (7) showing that wort is more stressing to yeast than the
defined growth medium YPD, as indicated by the levels of glutathion.

Late log phase Stationary phase

pET13.1 1.4 1.84

pETOYE2L 4.22 (3-fold) 4.12 (2-fold)
pETOYE2M 9.70 (7-fold) 30.53 (17-fold)
pETOYE2S ND 2.21

Table I: Overall oxidoreductase activity of modified yeast. Oxidoreductase activity is

reported as nkat/g of protein using oxygen as substrate.

Overexpression of OYE2 was accomplished by transforming lager brewer's yeast with

three plasmid constructs pETOYE2L, pETOYE2M and pETOYE2S, each carrying a
modified version of the yeast ADH1 promoter (8, 9), which conferred three different
transcription patterns during fermentation. Table I shows that in all cases transformed
yeast cells displayed higher oxidoreductase activity than the control cells (carrying
pET13.1). There were no remarkable differences in fermentation rate or in yeast
growth (figure 2) among the different recombinant strains or in comparison with the
control strain. All strains showed a similar flocculation ability. However, the lower
diacetyl content during the fermentation is noteworthy in the yeast transformed with
pETOYE2M, although the reduction of diketones was efficient and almost similar in
all strains at the end of primary fermentation (figure 3).
The overexpression of OYE2 was also studied at the mRNA level from flask
Fermentation time [days]
A 17
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

15 POLAR
Extract ,%

pETOYE2M
13 pCR4
11 pET13:1
9
7
5
3
17

B 15
pET13:1
Extract ,%

13 pETOYE2L
11 pETOYE2M
9 pETOYE2S
7
5
3
0 24 72 120 168 216 264 312 360

Fermentation time [hours]

Figure 2: Apparent extract during primary fermentation of two trials (A and B) with
different modified brewers yeasts.

5
cultivations. Figure 4 shows that the pETOYE2M was highly transcribed at both time
points studied. The long version of the ADH1 promoter yielded normal (active) sized
OYE2 mRNA at late log phase together with a larger non-functional transcript. At the
2,0

1,8 POLAR
Total diacetyl contents ( mg/l )
pETOYE2M
1,6 pCR4
pET13:1
1,4

1,2

1,0

0,8
End of primary
fermentation
0,6

0,4

0,2
Taste threshold 0.032 mg/l
0,0
0 7 14 21 28
Fermentation time [days]

Figure 3: Diacetyl contents during primary and secondary fermentation

stationary phase, the normal sized mRNA had almost disappeared and the larger
transcript dominated. Under the regulation of the short promoter, OYE2 mRNA
started to become more abundant in the stationary phase. In all cases, the three
expression cassettes overexpressed OYE2 in comparison with the control yeast. These
cells, as expected, exhibited a clear resistant phenotype when grown under the
presence of toxic levels of dicarbonyls such as methylglyoxal (figure 5).

A Late log phase Stationary phase

1 2 3 4 1 2 3 4

Late log phase Stationary phase

B
1. pETOYE2L 1200000 600000
2. pETOYE2M 5100000 4500000
3. pETOYE2S 400000 500000
4. pET13.1 100000 50000

Figure 4: Levels of OYE2 mRNA in modified yeast strains. A. Northern blot

corresponding to samples taken at late log and stat growth phases.
B. Signal quantity of OYE2 mRNA in arbitrary units after normalization
with rRNA.
The YAP1 gene encodes a basic region-leucine zipper transcriptional activator of 650
amino acids, a key determinant regulator of the oxidative stress tolerance in yeast
(10). This protein contains a cysteine rich domain (CRD) which is a signal required

6
for nuclear localization of the protein when the oxidative stress response is
experienced by the cell. We therefore prepared two forms of YAP1 to be

mol/min/mg)
YPD
Wort
0.8
Enzymatic activity (

0.6

0.4

0.2

0.0
Control pETOYE2S pETOYE2M pETOYE2L pCR2 pCR4 pETYAP1

Figure 5: Methylglyoxal reductase activity of modified yeast in different culture

media
overexpressed in our brewer's yeast, one encoding the wild type regulated protein, and
the other, encoding a shortened aminoacid sequence (YAP1CRD) which behaves as
a constitutive transcriptional activator (11,12). In flask cultivations the mRNA levels
of OYE2 (figure 6) as well as other known stress responsive genes such as TRR1,
AAD3, AAD15 and YPR1, were also higher in yeast strains overexpressing either the
wild type or the shortened form of YAP1 compared to the control strain.
Unexpectedly, the strain expressing the wild type YAP1 showed constitutively higher
level of OYE2 mRNA while the other strains behaved similarly to the control.
However, the overall reductase activity was slightly higher in those yeast strains
carrying the truncated version of the transcriptional factor. In pilot scale brewing trials
Yap1 overproduction was not seen to particularly overinduce OYE2 transcription even
though a slight increase in overall reductase activity was observed.
The carbonyl contents were analyzed daily from the fermenting wort. It was found
OYE2 mRNA amount(arbitrary units)

250000

200000

150000

Late log phase

100000
Stationary phase

50000

0
Control pCR2 pCR4 pETYAP1

Figure 6: Levels of OYE2 mRNA in YAP1 overproducing yeast strains

during a flask cultivation

7
that the sum of Strecker aldehydes in the transformed yeasts samples after primary
and secondary fermentation was 30 to 40% lower compared to the control yeast
(figure 7).

20
Concentration [g/l]

10

0
Control pETOYE2L pETOYE2M pETOYE2S Control pETOYE2L pETOYE2M pETOYE2S

End of primary fermentation End of secondary fermentation

Me-Propanal 3-Me-Butanal Phenylacetaldehyde Furfural Methional

Figure 7: Concentration of some Strecker aldehydes at the end of primary and

secondary fermentation with modified yeasts 

Due to the fact that Oye2 is not capable to reduce Strecker aldehydes (1), these
results suggest that this enzyme is either reducing -dicarbonyl compounds (which
are known precursors of Strecker aldehydes) or facilitating the action of other
reductases by decreasing the contents of , unsaturated carbonyls in the fermenting
wort.
Estery Estery
A 3 B 3
2 2
Bitter Malty + Malty +
1 Bitter 1
Bready Bready
0 0
-1 -1

Sweet + Sweet +
Papery Papery
Caramel Caramel

Burnt Burnt

Control yeast pETOYE2M pCR4(YAP1)

Figure 8: Sensory analysis of fresh (A) and after 2 weeks at 28 C (B) of beer
produced by a yeast transformed with OYE2 or shortened YAP1. It is
represented the mean value of two fermentation trials.

Sensory evaluation of fresh beers made with modified yeasts revealed less intense
malty and bready notes, which is in agreement with the lower contents of Strecker
aldehydes (figure 8). However, after two weeks at 28 C this initial difference

8
vanished and it seems that beer prepared with pETOYE2M-transformed yeast
developed a slightly less intense sweet and caramel notes. These positive effects of
OYE2 overproduction was more pronounced in beers kept for 4 weeks at 5C, as
shown in figure 9. In this diagram we can see that control beer developed a more
intense sweet-caramel and malty-bready notes, flavors commonly associated with
beer aging. Contrary to our expectations, beers prepared with the YAP1 modified
yeast strain did not show any favorable effect on beer flavor stability .

Estery
4
3
2 Malty +
Bitter
1 Bready
0
-1

Sweet + Papery
Caramel

Burnt
Control yeast pETOYE2M pCR4(YAP1)

Figure 9: Sensory analysis after 4 weeks at 5 C of beer produced by a yeast

transformed with OYE2 or shortened YAP1. It is represented the mean
value of two fermentation trials.

CONCLUSION
In this work we described the use of two genetic strategies to obtain a set of modified
brewer's yeast strains with homologous genes that increased the carbonyl reductase
activity of the parental yeast strain during the fermentation. These approaches opened
up the possibility to evaluate the role of Maillard reaction intermediates on beer aging.

REFERENCES
1. Snchez, B., Reverol, L., Galindo-Castro, I., Bravo, A., Ramrez, J. & Rangel-
Aldao, R., Proceedings of the European Brewery Convention Congress, 2001,
Budapest, in press.
2. Bravo, A., Rangel-Aldao, R., Scherer, E., Madrid, J., Herrera, J. & Virtanen, H.,
Proceedings of the European Brewery Convention Congress, Budapest, 2001, in
press.
3. DeRisi, J., Lyer, V. & Brown, P., Science, 1997, 278, 680-685
4. Ito, H., Fukuda, Y., Murata, K. & Kimura, A., J.Bacteriol.,1983, 153, 163-168
5. Henderson, R., Cox, B. & Tubb, R., Curr.Genet., 1985, 9, 133-138.

9
6. Sambrook, J., Fritsch, E. & Maniatis, T., 1989, 2nd ed., p. 11.39. Cold Spring
Harbor Laboratory, Cold Spring Harbor, N.Y
7. Martin, V., Quain, D. & Smart, K., Proceedings of the European Brewery
Convention Congres, Cannes, 1999, 679-686.
8. Ruohonen, L, Penttila, M. & Keranen, S., Yeast, 1991, 7, 337-346
9. Ruohonen, L, Aalto, M. & Keranen, S., J. Biotech., 1995, 39, 193-203.
10. Fernandes, L., Rodrigues-Pousada, C. & Struhl, K., Molecular and Cellular
Biology, 1997, 17, 6982-6993.
11. Kuge, S., Jones, N. & Nomoto, A., 1997, The EMBO J., 16, 1710-1720.
12. Kuge, S., Toda, T., Iizuka, N. & Nomoto, A., Gene to Cells, 998, 3, 521-523.

10
48

Brewers yeast oxidoreductase with

activity on Maillard reaction
intermediates of beer
Beatriz Snchez1, Lorena Reverol1, Ivn Galindo-Castro1,
Adriana Bravo1, Jos Luis Ramrez2 & Rafael Rangel-Aldao1
1
Empresas Polar, Polar Technology Center, Caracas, Venezuela (e-mail:
[email protected])
2
Institute of Advanced Studies (IDEA), Caracas, Venezuela

Descriptors
Brewersyeast, carbonyl compound, enzymic activity, non-enzymic activity, staling

SUMMARY
We purified to homogeneity, a cytosolic NADPH-dependent reductase from brewers yeast
that displayed catalytic activity towards wort dicarbonyls, including a highly sensitive
chemical indicator of beer aging. Its partial amino acid sequence was 96 % similar to Old
Yellow Enzyme (OYE; EC 1.6.99.1) and its addition to fresh beer reduced the area of
chemical indicators during storage at 28 C. This enzyme may be a useful tool to reduce
dicarbonyls during fermentation by preparing recombinant yeasts overexpressing OYE.

Oxidoreduktase der Bierhefe mit Wirkung auf die Zwischenprodukte der Maillard-
reaktion

Deskriptoren
Altgeschmacksbildung, Bierhefe, Carbonylverbindung, Enzymaktivitt, nichtenzymatische
Brunung

ZUSAMMENFASSUNG
Wir reinigten eine cytosolische NADPH-abhngige Reduktase aus der Bierhefe, die
katalytische Wirkung gegenber Wrzedicarbonylen zeigt und die zudem ein sensitiver
chemischer Indikator der Alterung ist. Ihre partiale Aminosuresequenz ist zu 96% identisch
mit dem Old Yellow Enzym (OYE; EC 1.6.99.1). Die Zugabe zu frischem Bier reduzierte den
Bereich der chemischen Indikatoren whrend der Lagerung bei 28 C. Durch Rekombination
von Hefe, die stark OYE expremiert, knnte dieses Enzym ein ntzliches Werkzeug zur
Reduzierung von Dicarbonylen darstellen.
Une oxydo-rductase de levure de bire active sur les intermdiaires de la raction de
Maillard de la bire

Descripteurs
Activit enzymatique, brunissement non-enzymatique, compos carbonyl, formation du got
dvent, levure de brasserie

RESUME
Nous avons purifi jusqu' homognit une rductase NADPH-dpendante cytosolique de
levure de bire prsentant une activit catalytique sur les dicarbonyles du mot, dont un
indicateur chimique extrmement sensible du vieillissement de la bire. Sa squence partielle
tait similaire 96 % celle de l'Old Yellow Enzyme (OYE; EC 1.6.99.1) et son addition de
la bire frache a rduit l'aire des indicateurs chimiques pendant le stockage 28 C. Cette
enzyme peut tre un outil utile pour la rduction des dicarbonyles pendant la fermentation, en
prparant des levures recombinantes surexprimant OYE.

2
INTRODUCTION
Maillard reaction intermediates such as 3-deoxy-2-hexosulose (3-DH) and 1-deoxy-
2,3-hexodioluse (1-DH) are the most abundant -dicarbonyl species in wort and beer
(1). Results from our laboratory suggested that these intermediates may play an
important role as precursors of carbonylic compounds associated with beer flavor
deterioration (1). Even though a few yeast reductases have been described to reduce
monocarbonyls and some dicarbonyls (2,3), no enzymatic activity in yeast has yet
been reported towards 3-DH or any other Maillard reaction dicarbonyl intermediates.
Based on these facts, we searched for brewers yeast enzymes capable to reduce -
dicarbonyl compounds to allow us to assess their impact on beer flavor deterioration.
In this work, we isolated, purified, cloned, and expressed in bacteria a singular
NADPH-dependent oxidoreductase (out of more than 140 enzymes constituting the
superfamily of yeast reductases and dehydrogenases genes) that could play a
significant role in the stabilization of fresh beer flavor.

MATERIALS AND METHODS

Bacterial strains and DNA manipulations
Plasmid pProEx-HT (Life Technologies, USA) was used for cloning and expression
of OYE2 in E. coli strain JM109 (endA1, recA1, gyrA96, thi, hsdR17, (rK-, mK+),
relA1, supE44, -, (lac-proAB), [F, traD36, proAB, lacIqZM15]). Recombinant
plasmid screening and DNA purification were done according to standard procedures
(4).
Purification of native OYE and its amino acid sequence determination
In house pitching brewing yeast cells were washed twice with 25 mM potassium
phosphate buffer, pH 7.5, and disrupted by glass beads in a cell disintegrator unit. The
clarified homogenate was chromatographed through a DEAE-Sepharose column, and
the non-retained fraction was precipitated with ammonium sulfate. The 50 % - 90 %
saturation fraction was passed through a CM-Sephadex column equilibrated with 5
mM potassium phosphate buffer, pH 6.5. The reductase activity towards the products
of a glycine-glucose reaction system (see below), contained within the flow through,
was then absorbed to a Cibacron Blue column previously equilibrated with 25 mM
potassium phosphate, pH 7.0. The enzyme was eluted with the same buffer containing
400 mM KCl. The fractions containing the enzyme activity were pooled, dialyzed and
applied to second Cibacron Blue column and the activity eluted with a linear gradient
of 100-500 mM KCl. The dialyzed eluate was applied to a Red Sepharose column
where it eluted within a linear gradient of 0-1 M KCl. For partial aminoacid
sequencing, we used a preparative reverse-phase ProRPC column equilibrated in 5%
acetonitrile, 0.1% TFA coupled to an HPLC. Reductase activity eluted at 56 %
acetonitrile and 0.06% TFA. The relative molecular mass of the reductase was
determined by gel filtration chromatography and by SDS-PAGE. The amino-terminal
sequence of the purified reductase was determined with the aid of a ProSequencer
unit.
Primer design, PCR amplification, and gene cloning of OYE2
Based on the reported DNA sequence of OYE2 (GenBank Accession Number:
L06124), we designed a set of specific PCR primers: (sense primer) 5-GGA ATT
CAT GCC ATT TGT TAA GGA C-3 and (antisense primer) 5-CTC TAG ATT
AGA GCT TCT TCG TAC G-3, which included at their 5-termini the recognition
sites for the restriction enzymes Eco RI and Xba I to facilitate the gene cloning. PCR

3
reaction was performed in 100 l of a mixture containing: 1 mM of each dNTP; 200
M of each primer; 2 units of Taq DNA polymerase in 1X reaction buffer (10 mM
Tris HCl, pH 8.3, 1.5 mM MgCl2). The thermal cycler was set with the following
conditions: one incubation step of 5 min. at 95 C; a cycle of 45 sec at 54 C, 45 sec at
72 C and 45 sec at 94 C (repeated 35 times), and a last extension step of 10 min. at
72 C to improve the yield of complete amplification products. The 1,194 bp long
PCR fragment containing OYE2 coding sequence was cloned into the pProEx-HTa. E.
coli JM109 cells transformed with the plasmid (pProExOYE2) were grown in Terrific
Broth (5), supplemented with 100 g/ml Ampicilin. Expression of recombinant OYE2
was induced by addition of 0.6 mM isopropyl-1--D-galactopyronoside (IPTG) to the
culture medium. Harvested bacterial cells were lysed by a single passage through a
French press at 20 Kpsi, and the clarified lysate affinity-absorbed through a Red
Sepharose column equilibrated with 50 mM potassium phosphate. Recombinant
oxidoreductase eluted with 500 mM KCl in the same buffer.
Enzyme assay
Enzyme activity was assayed by measuring the rate of pyrimidine nucleotide
oxidation at 340 nm (6). The assays were performed between 25 C and 35C in 50
mM potassium phosphate buffer, pH 6.5 containing 100 mM -NADPH, 9 mM
methylglyoxal and a suitable amount of enzyme. Specific activity was expressed as
mol of the oxidized cofactor per min-1 per mg-1 of protein.
Maillard reaction model system
For the preparation of a Maillard reaction model system we made a solution of 1 M
glucose and 0.5 M glycine, and heated it at 90C for 3 hours (7).
Analysis of chemical indices LC18 and HMF
Chemical indices LC-18 and 5-HMF of beer were detected by HPLC as described by
Bravo et al. (8).

RESULTS AND DISCUSSION

After the DEAE sepharose chromatography of brewers yeast crude extract we
detected two methylglyoxal reductase activities, Pool I and Pool II (see figure 1),
0.05 3 0.30
reductase activity (mol/ min/ ml)

Pool I Pool II
Methylglioxal reductase activity

0.04 0.25
Maillard-intermediate

0.20
(mol/ min/ ml)
Absorbance280 nm

0.03 2
0.15
0.02
0.10
0.01 1
0.05

0.00
0.00

0
0 10 20 30
Fraction Number

Figure 1: DEAE Sepharose Chromatographic elution of yeast soluble protein extract.

Reductase activity of main protein fractions were assayed in the presence
of methylglyoxal (circles) and products of the Maillard reaction model
system (triangles). 

4
however higher reductase activity towards Maillard reaction intermediates was
contained only in Pool I, and eluted within the flow throw of the column.
Additionally, Pool I, as can be seen in figure 2, was also capable of reducing peak
LC18 in beer, a dehydration product of 3-DH (1); whereas Pool II did not show
activity on this compound. Successive chromatographic steps were then carried out

75%reduction 0%reduction

IA LC 18 II
B LC 18
Control beer Control beer

Beer + Pool II
Beer + Pool I

Figure 2: HPLC chromatograms of fresh beer incubated with the yeast reductases.
Pool I and II, eluted from DEAE Sepharose, were assayed to follow
their effects on LC-18 peak in fresh beer. Panel A, LC-18 peak after
addition of Pool I.; Panel B, LC-18 peak after addition of Pool II. 

with Pool I due to its ability of reducing peak LC18 as well as compounds present in
the Maillard reaction model system.
The yeast reductase of Maillard reaction intermediates was purified to homogeneity
as a single protein band on SDS-PAGE with a relative molecular mass (Mr) of 86,000
determined by gel filtration chromatography (results not shown). Under denaturing
conditions, the reductase gave a protein band of Mr 44,000 (figure 3), suggesting that
the enzymes native structure may correspond to a dimer.
1 2 3 4 5 6 7 8

70,000
50,000
40,000 44,000

30,000

Figure 3: SDS-PAGE (12.5%) of Pool I protein fractions during purification

steps. 1.-Molecular weight markers, 2.- Crude extract (30 mg), 3.- Pool
I DEAE (7 mg.), 4.-Ammonium sulfate pellet (15 mg), 5.- Pool CM
Sephadex (40 mg), 6.- Pool Cibacron Blue I (10 mg), 7.- Pool CB II (5
mg), 8.- Pool Red Sepharose (5 mg).

5
When the terminal amino acid sequence of the purified reductase was obtained, it
showed a 96% identity with Oye1 and Oye2, and 64% with Oye3. These genes
encode for isozymes of the FMN-flavoprotein known as Old Yellow Enzyme, an
NADPH-dependent dehydrogenase (EC 1.6.99.1) described initially by Warburg and
Christian in 1933 (9), being the first enzyme found to contain flavin as prosthetic
group (10). Despite of the extensive knowledge of the physical and biochemical
properties of Oye (11), its actual physiological role remains an enigma.
Figure 4 shows the ability of the enzyme to reduce the dicarbonyl moiety of peak
LC18 in beer and how this reduction positively correlated with the amount of enzyme
A C
0,09 0,09
0,08 5-HMF 0,08 5-HMF
0,07 0,07
LC -18 0,06 LC -18
0,06 Control Control

AU
0,05
AU

0,05
0,04 0,04
0,03 0,03
0,02 0,02
0,01 Beer + enzyme 0,01
0,00 Beer + enzyme
0,00
- 0,01 - 0,01
22,00 23,00 24,00 25,00 22,00 23,00 24,00 25,00
Minutes Minutes
30
B 0,09 D
0,08 5-HMF
25
0,07
0,06 Control LC -18 20
% of LC18
AU

0,05
0,04 15
0,03
0,02 10
0,01
0,00 Beer + enzyme
5
- 0,01
22,00 23,00 24,00 25,00
0
Minutes 0.5 1.0 2.0
( mol/ min/ ml)
Figure 4: Effect of Pool I on peak LC-18 present in beer. A, B and C) HPLC
Chromatograms showing peaks corresponding to wort treated with 0.5,
1.0 and 2.0 reductase enzymatic units after Red Sepharose
chromatography. D) Peak LC-18 levels (%) remaining after beer
treatment with the same amount of enzyme units. 

units added to beer. This reductase showed reactivity on dicarbonyls, and no activity
was detected when using Streckers aldehydes, alcohols, carbohydrates or fructosyl
aminoacids as substrates. From all tested substrates, three -dicarbonyls, namely, 2,3-
hexanodione, methylglyoxal, and 2,3-butanodione showed the highest reactivity. The
Oye reduction mechanism of the -dicarbonyl was explained by the group of Massey
O O O OH -NADPH O OH
C C C C C CH
CH3 CH2 OYE CH3
H3C H3C H3C

2,3-butanodione

Figure 5: Reduction of 2,3-butanodione by OYE. The alfa-diketone structure of

2,3-butanodione is in equilibrium with an ,-unsaturated carbonyl,
which is then reduced by the Oye to produce 3-hydroxibutanone.
(11) by virtue of the keto-enol equilibrium as can be seen in figure 5. The reductase

6
Oye then catalyzes the NADPH-dependent reduction of the olephinic bond of these
,-unsaturated carbonylic compounds (11, 12). The preference of Oye for
compounds containing the ,-unsaturated carbonyl moiety is noteworthy, specially
when comparing the reactivity of o-nitrobenzaldehyde (0 %) to p-nitrobenzaldehyde
(80 %).
The recombinant expression of OYE2 in E. coli (see figure 6) allowed us to obtain

1 2 3
Mr x 10-3

97.4
66.2
55.0
42.7
40.0

31.0

21.5

14.4

Figure 6: 12.5 % SDS-PAGE analysis of the recombinant expression of OYE2 in

E. coli. 1) Molecular weight standards. 2) IPTG-induced crude E.coli
extract. 3) Affinity purified Oye2

HMF

Control beer at 28C

OYE2-treated beer at 28C

Control beer at 5C

Figure 7: Effect of Oye2 on forced beer aging. HPLC chromatograms

corresponding to experimental beers treated and untreated with Oye2
after storage at 28 C for two weeks.

7
high protein yields after a simplified purification procedure. The recombinant Oye2
was added to beer to study its effects in forced-aging experiments. Similarly to the
native enzyme, and after beer storage at 28 C for two weeks (figure 7), the
recombinant Oye2 was capable of reducing peak LC18 from beer (data not shown),
and therefore preventing HMF formation. This result paved the way to the use of the
reductase Oye2 to investigate the possible effects of Maillard reaction -dicarbonyl
intermediates on the generation of stale flavor compounds during beer aging, since
they may be selectively reduced from the wort by Oye2.
Although the physiological function of Oye2 remains to be elucidated, its ability to
reduce ,-unsaturated carbonylic compounds could be playing an important
protective role in yeast cells growing in stressful media such as wort (13). These
findings are also supported by the possible involvement of this enzyme in the
oxidative stress response of yeast as it was recently demonstrated by genomic
experiments with DNA microarrays (14).

CONCLUSION
We purified, cloned, and expressed in E. coli a singular NADPH-dependent yeast
reductase, from more than 140 enzymes constituting the reductase/dehydrogenase
gene superfamily. This enzyme was capable to reduce 3-DH, methylglyoxal, diacetyl
and other -dicarbonyls from wort and beer, making Oye2 a useful tool to evaluate
the role of -dicarbonyls in beer aging by preparing overexpressing recombinant
yeasts with such homologous genes.

REFERENCES
1. Bravo, A., Scherer, E., Madrid, J., Herrera, J., Virtanen, H. & Rangel-Aldao,
R., Proceedings of the European Brewery Convention Congress, Budapest,
2001, in press.
2. Debourg, A., Laurent, M., Goosens, E., Borremans, Van De Winkel, L. &
Masschelein, C., Am. Soc. Brew. Chem., 1994, 52: 100-106.
3. Heidlas, J. & Tressl, R., Eur. J. Biochem., 1990, 188: 165-174.
4. Sambrook, J., Fritsch, E. & Maniatis, T., 1989, 2nd ed., p. 11.39. Cold Spring
Harbor Laboratory, Cold Spring Harbor, N.Y
5. Tartoff, K. & Hobbs, C., 1987, BRL Focus 9, 2-12.
6. Liang, Z-Q., Hayase, F. & Kato, H., 1991, Eur. J. Biochem. 197, 373-379.
7. Olsson, K., Pernemalm, P. & Theander, O., 1981, In Progress in Food and
Nutrition Science series 5, 47-55. Pergamon Press, C. Eriksson editor.
8. Bravo, A., Snchez, B., Scherer, E. & Rangel-Aldao, R., Proceedings of the
European Brewery Convention Congress, Budapest, 2001, in press.
9. Warburg, O. & Christian, W., Biochem. Z., 1933, 266: 377-411.
10. Theorell, H., Biochem. Z. ,1935, 275: 344-346.
11. Stott, K., Saito, K., Thiele, D. & Massey, V., 1993, J. Biol. Chem. 268, 6097-
6106
12. Vaz, A., Chakraborty, S. & Massey, V., 1995, Biochem. 34, 4246-4256
13. Martin, V., Quain, D. & Smart, K., Proceedings of the European Brewery
Convention Congress, 1999, Cannes, 679-686.
14. DeRisi, J., Lyer, V. & Brown, P., 1997, Science 278, 680-685.

8
ACKNOWLEDGEMENT
We would like to thank Drs. Timoteo Otamendi and Lourival Possani, from the
Institute of Biotechnology (Cuernavaca, Mexico) for helping us to determine the
amino acid sequence of Oye.

9
49

Controlled beer fermentation with

continuous one-stage immobilized yeast
reactor
Esko Pajunen1, Kaisa Tapani1, Harry Berg1, Bo Ranta1, Joe
Bergin2, Heikki Lommi3 & Tapio Viljava4
1
Oy Sinebrychoff Ab, P.O. Box 87, FIN-04201 Kerava, Finland (e-mail:
[email protected])
2
Guinness R&D, Ireland
3
GEA Liquid Processing Scandinavia, Denmark
4
Danisco Sugar and Sweeteners Development, Finland

Descriptors
Beer quality, continuous fermentation, flavour formation, immobilized yeast, pilot plant trial,
process control

SUMMARY
The new continuous one-stage immobilized yeast beer fermentation system has further been
tested in pilot scale of 12 hl/day to establish the process parameters for high quality beer and
for scaling up. Good quality beer can be produced in 20-30 h time with more constant flavour
compared with batch to batch process. Fermentation process and flavour formation could
readily be controlled by feed, circulation and fermentation temperature, giving possibilities to
rapid on line control of beer fermentation and flavour. In addition to improved controllability
also cost reductions can be achieved.

Kontrollierte Vergrung mit der kontinuierlichen einstufigen Fermentation mit

immobilisierter Hefe

Deskriptoren
Aromabildung, Bierqualitt, halbtechnischer Versuch, kontinuierliche Grung, Proze-
steuerung, trgergebundene Hefe

ZUSAMMENFASSUNG
Das neue kontinuierliche einstufige Bierfermentationssystem wurde im Pilotmastab (12 hl/d)
getestet, um die Parameter fr eine bessere Bierqualitt und ein scale-up zu ermglichen. In
20 30 Stunden kann eine gute Bierqualitt erreicht werden, und im Vergleich zum
Chargenbetrieb liefert das kontinuierliche System konstanteren Geschmack. Der Grprozess
und die Geschmacksbildung knnen durch die Beschickung gesteuert werden. Durch Online-
Steuerung der Zirkulation und Grtemperatur besteht die Mglichkeit, den Grprozess zu
beschleunigen und die Geschmacksbildung zu steuern. Durch die verbesserte Steuerung des
Grprozesses kann zustzlich eine Kostenreduzierung erzielt werde.

Fermentation contrle de la bire avec un bioracteur en circuit continu levures

immobilises

Descripteurs
Commande de procd, essai en installation pilote, fermentation continue, formation de la
flaveur, levure immobilise, qualit de la bire

RESUME
Le nouveau systme de fermentation continue en une seule tape avec levures immobilises a
t test nouveau l'chelle pilote de 12 hl/jour pour dfinir les paramtres
mthodologiques pour une bire de haute qualit et un passage l'chelle industrielle. Il est
possible de produire une bire de bonne qualit en 20 30 h, avec un arme plus constant par
rapport au procd de fabrication par lots. Le procd de fermentation et la formation de
l'arme pourraient facilement tre contrls par l'alimentation, la circulation et la temprature
de fermentation, donnant des possibilits de contrle rapide en ligne de la fermentation et de
l'arme de la bire. Outre une meilleure capacit de contrle, cela permet galement de
rduire les cots.

2
INTRODUCTION
Applications of immobilized yeast systems have been thoroughly discussed and
documented in the monograph of EBC symposium on "Immobilized yeast
applications in the brewing industry" (1) highlighting the worldwide situation in 1995.
The summary of that symposium refers to technical opportunities of producing sound
flavour and good quality beer continuously by applying immobilized yeast. It was
emphasized, however, that results are based on laboratory or small pilot scale only
and more work is needed in pilot or small industrial scale to verify the technical and
economical feasibility of immobilized yeast system. Also the need of low cost carrier
material was expressed in the same context.

Since the immobilized yeast symposium some work has been performed having the
technical applicability of immobilized yeast systems in large scale operations as a
starting point (2). The major technical challenges are fast enough a process with high
volumetric productivity to create an attractive investment alternative to traditional
process, good control possibilities for flavour formation, opportunities for flexible
operation and more practical targets of proper CO2 removal as well as good
temperature control. Usually these challenges are hardly recognisable in laboratory
scale. Part of the technical challenges arises from the requirement of fast process; all
the phenomena are expected to take place in about one day instead of 7 to 10 days.
Especially this is problematic for temperature control and CO2 removal where existing
practices should be intensified close to ten fold.

In addition to above there are some operational challenges to match the proper flavour
formation. Basically these are to reach and maintain a real steady state in the process,
where, traditionally, fermentation environment and yeast physiological state is
changing continuously. To have a controlled steady state gives possibilities for a
controlled flavour formation.

BASIC PROCESS SCHEME

Starting from the above challenges the development of the new immobilized yeast
fermentation process has proceeded (3). The central idea was to create conditions for
a real steady state, by abandoning of the commonly used two-stage process scheme,
which mimics the traditional beer fermentation with various physiological stages of
the yeast life span. Instead, the new process is operating in one stage only
corresponding the late primary fermentation stage in the traditional batch fermentation
process. At this stage the yeast is operating in its stationary phase with yeast
population remaining at constant level and where the specific fermentation rate of
yeast is very steady (4). The constant rate of the fermentation and flavour compound
formation in the yeast stationary phase means that yeast growth is limited and the
flavour formation is solely derived from the slow growth during stationary phase and
from the yeast metabolism of alcohol fermentation. It has been reported that under
certain specific immobilized systems with growth limited conditions the yeast
multiplication doubling time can be up to 2000 h (5).

The above mentioned steady state conditions can simply be reached when normal
wort is allowed to ferment to desired level and when reaching that level, instead of

3
finishing the fermentation, fresh wort feeding is started simultaneously with
withdrawal of the readily fermented beer to keep the steady state conditions (figure 1).

Wort feed
started
Yeast

Higher alcohols

Esters

Alcohol

Extract

Yeast

Fermented beer

Figure 1: Basis for immobilized continuous fermentation

At this stage the specific fermentation rate remains fairly constant, the yeast is
consuming maltose and maltotriose at a constant rate and the limiting factor for
fermentation rate is most probably the maltose and maltotriose transport to yeast cells.
In batch fermentations the decline of fermentation rate is mainly due to the yeast
sedimentation towards the end of fermentation cycle. Similarly the formation of
flavour compounds, higher alcohol and esters, takes place at linear rate until the
fermentation slows down. To reach the rapid fermentation the yeast concentration is
kept high enough by immobilizing yeast in the solid matrix instead of letting yeast
float free in the beer. Finally the beer with wort is led through the immobilized yeast
giving much better contact of sugars to yeast.

The practical arrangement includes a circulation loop, where the fully fermented beer
is circulating and to which beer the wort feed is added (figure 2).

WORT

BEER COOLING

IMMOBILIZED
CO2
YEAST
REMOVAL
REACTOR

FERMENTED
BEER
OPTIONAL YEAST
REMOVAL

Figure 2: Basic flow scheme

4
Fermentable sugars in the feed are fermented in the immobilized yeast reactor where
the yeast is immobilized in wood chip carrier with good flow properties. During the
fermentation, the carbon dioxide formed is kept dissolved to avoid CO2 bubbles to
replace the liquid volume and to reduce the effective surface contact area of yeast and
sugars in solution. However, the carbon dioxide formed and the requirement to keep it
dissolved is giving one limitation to fermentation. The maximum amount for sugars to
be fermented is limited by the CO2 solubility at the given temperature.

The fully fermented beer leaving the immobilized yeast reactor contains very few
yeast cells leaking out of reactor, so there is no reason for a yeast separation step to be
included. The beer is led to a carbon dioxide removal unit to remove the carbon
dioxide formed during the fermentation. Critical steps in CO2 removal is to avoid
excessive beer foaming and collapsed foam to safeguard the final beer foam
properties. After CO2 removal the heat formed during fermentation is removed by
cooling the beer in a plate heat exchanger. After cooling the beer is ready for feed and
back to loop to be fermented. The readily fermented beer withdrawn from the system
is clear enough and have a low yeast cell concentration and can be led as such to an
immobilized maturation plant for final maturation and beer treatment. The capacity of
the pilot installation is 12 hl/d with an immobilized yeast reactor volume of 10 hl
(figures 3 and 4).

BUFFER
TANKS

WORT
CO2
WORT FILTER PASTEURIZER
WORT
BEER
BUFFER
PLATE HEAT TANK
YEAST
CIP PROPAGATOR
EXCHANGER
CO2
REMOVAL
UNIT

IMMOBILIZED
YEAST REACTOR

FERMENTED
DISC-STACK BEER
CENTRIFUGE

Figure 3: Pilot plant key units

Carrier material Aspen chips 1 x 1 x 2 mm

Yeast concentration 108 - 109 cells/g carrier
Reactor volume 1000 l
Circulation rate 1000 - 2000 l/h
Feed 25 - 50 l/h
Beer circulation 20 - 40 times
Yeast leakage 103 - 105 cells/ml

Figure 4: Pilot environment

5
PROCESS CONDITIONS
The immobilized yeast fermentation process is started when yeast slurry is loaded into
the reactor. The reactor and the beer loop is filled with aerated wort which is
circulated until the final attenuation of the beer in the loop. By this stage the free yeast
cells in the circulation has practically all been immobilized or trapped in the reactor,
maltose and maltotriose have started to be fermented and alcohol is produced; the
yeast has reached the stationary growth phase. Most probably the yeast has also
induced all the stress protection means to be able to ferment the sugars available and
to survive the quite stressful conditions of relatively high alcohol concentration as
well as high carbon dioxide content with corresponding high pressure involved. When
wort feed starts at this stages, yeast, used to maltose and maltotriose, has also
additionally some glucose available. Most probably there is also excess of all the
other nutrients available for yeast metabolism with exception of oxygen, which, of
course, limits the yeast growth. So there are strong growth limited conditions with
excess of nutrients available.

The process conditions during the fermentation are quite severe, as described in figure
5.

Fermentable extract available 0.5 - 1.0 oP

No oxygen available < 0.02 ppm
Other nutrients < 5 % wort
High ethanol concentration 5 - 9 vol %
High CO2 concentration 3.5 - 5.0 g/l
High pressure 1.5 - 3.5 bar
High temperature 16 - 24 0C
Wort gravity 14 - 20 0 P

Figure 5: Fermentation conditions

Yeast is continuously under several stress factors like relatively high temperature,
high alcohol content, high CO2 content and high pressure. The conditions are,
however, quite similar to those at the end of primary fermentation of high gravity
wort in batch fermentation, but here there is a continuous state of stress with plenty of
nutrients available. The stress is not to be relieved after a short while of fermentation,
when all the nutrients are depleted like in batch process. Most probably the solid
carrier in the immobilized yeast reactor gives some protection against continuous
stress during immobilized fermentation. However the normal production yeast strain
was used, which seems to be quite robust and suitable also for immobilized primary
fermentation, in addition to immobilized maturation process.

RESULTS & DISCUSSION

Fermentation
The process described above has been tested in pilot scale of 50 l/h. The immobilized
yeast reactor volume was 1000 l. Several gravities, temperature and process
conditions and wort compositions were tested.

6
In general the fermentations in the immobilized yeast systems proceeded well.
(Practically the only problems in tests were occasional secondary contaminations due
to equipment failures of small scale equipment.) When starting the initial circulation
loop the yeast immobilization was not complete from the very beginning and the yeast
number leaking out from the reactor was in the order of 106-107 consisting of both
single and budding cells. Before the end of initial loop circulation, however, all the
yeast was immobilized or trapped the total number being 108-109 cells/g carrier. The
immobilized yeast reactor seems to retain the yeast extremely well at the tested flow
rates, and number of leaking cells is quite low, 103-105 only. The wood chip carrier is
economically attractive and had also good flow characteristics, no excessive pressure
build up during the long runs due to low yeast growth in the reactor and no reactor
clogging took place after the runs of several weeks.

As reported earlier (3,6) the average fermentation time varied between 20-30 h
depending on the fermentation conditions and the yeast strain. The results also
showed a more constant fermentation pattern as the traditional batch fermentation as
regards the usual beer parameters followed.

0,5
0,4
0,3 delta alcohol (%V/V)
0,2 delta extract (%)
0,1
0
7 10 11 18 21 25 27 33 35
Time (days)

Figure 6: Extract consumption and alcohol production in a single pass

Also the extract consumption and alcohol production over a single pass in the reactor
was quite constant over a lengthy period of time in given conditions (figure 6).
However, over a lengthy period of time some accumulation of maltotriose and
maltose took place, indicating too high a feed rate of too high gravity. The yeast
fermentative power and/or yeast vitality is decreasing to some extent during the
constant stressful fermentation conditions during long running periods. However, the
longest run with good fermentation and flavour performance has been 8 weeks run so
far. Usually much shorter periods were employed to test the various process
conditions.

Yeast growth
Yeast growth in the immobilized yeast reactor was very limited; almost no growth
was calculated when the yeast balance was produced. This could be seen in the low
number of yeast leaking out from the reactor as well as from the increased pH of the
readily fermented beer. Most prominently this was, however, seen in the free amino
nitrogen (FAN) analysis which showed that FAN consumption in total was only 10-20
mg/l compared to more than 100 mg/l FAN consumed in the normal batch
fermentation. This also means that there are significant high amounts of residual

7
amino acids left in the final beer giving their impact on beer pH and to some extent to
beer flavour as well. The problem has been approach by wort pH adjustments as well
as by use of high proportion of brewery adjuncts, but still the pH seems to remain at
higher level than in normal beer.

VICINAL DIKETONES
Low yeast growth also means that strongly growth related metabolic phenomena are
less pronounced than in normal batch fermentation. Especially this is the case with
nitrogen metabolism where less proteins and amino acids are needed for new cell
mass production. On the contrary there is excess of amino acids and other wort
nutrients available. The situation can clearly be seen in the case with low production
of vicinal diketones (VDK), diacetyl and pentadione, whose precursors are strongly
produced during the exponential yeast cell growth phase in normal batch
fermentation. In immobilized yeast system there is no similar yeast growth phase, so
VDK values should be low, deriving from the fermentative metabolism only. This
was shown to be the situation regarding diacetyl, the concentrations being normally
close 0.1 ppm and quite constant through out the fermentation. However, 2,3-
pentadione concentrations, which normally in batch fermentations are lower than
diacetyl showed concentrations 2-4 fold compared to diacetyl (figure 7).

1,2
Concentration (ppm)

1,0
0,8
diacetyl
0,6 pentanedione
0,4
0,2
0,0
7 10 11 14 18 21 25 27 33 35
Time (days)

Figure 7: VDK formation

The pentanedione pathway seems to be more active in immobilized yeast system in

low/no growth situation with excess of available amino acids and continuous sugar
feed. Sugar feed seems to essential for the production of the precursors of diacetyl and
2,3-pentane dione; -acetolactase and -hydroxybuturate, since the concentrations are
reduced considerably when sugar feed is reduced with excess of amino acids still
available.
When closing the wort feed and continuing the beer circulation, it is possible to
reduce both vicinal diketone concentrations below the taste threshold values
indicating that immobilized yeast process gives too little time for complete oxidative
decarboxylation of VDK precursors. On the other hand, although pentadione
concentrations are higher, correspondingly the taste threshold value is ten folds to that
of diacetyl.

8
FLAVOUR COMPOUNDS
It is usually referred that flavour compound formation, especially the higher alcohol
production, is closely related to nitrogen metabolism and amino acid synthesis and
hence closely related to yeast growth phase, although the formation of higher alcohols
take place throughout the whole fermentation cycle, even when the yeast is in the
stationary growth phase. Furthermore the higher alcohol formation is to some extent
determined by the free amino nitrogen level of wort, because higher alcohols are by-
products of yeast nitrogen metabolism. The usual pattern is that at higher nitrogen
levels the higher alcohol production is suppressed indicating that biosynthetic
pathway of amino acids is used less.

Higher alcohols
In case of immobilized yeast, the yeast growth is very limited and hence low levels of
amino acids are used for yeast growth and there is excess of available free amino
nitrogen available (figure 8).
Average Range Batch Flavour
threshold
Acetaldehyde 15.2 8.5 - 23.7 16.0 25

Ethyl acetate 26.4 20.1 - 39.8 21.5 33

3-methylbutylacetate 1.0 0.3 - 1.9 1.5 1.6

Propanol 24.9 15.7 - 50.5 10.0 800

2-methyl propanol 8.4 7.5 - 10.8 8.2 200

Butanol 0.9 0.3 - 2.1 0.1 450

3-methyl butyl alcohol 47.0 30.0 - 59.5 51.0 70

Ethyl hexanoate 0.2 0.1 - 0.5 0.4 0.2

Ethyl octanoate 0.6 0.2 - 1.2 1.4 0.9

Figure 8: Beer flavour

Low levels of higher alcohols and keto-acid derived flavour compounds are to be
expected. However, the results show that the higher alcohol levels are mostly at the
comparable level to the batch fermentation and regarding some of the alcohols even at
a higher level. Especially this is true with propanol, which is overproduced compared
to batch fermentation. Interesting enough the high propanol production has been
reported earlier in connection to immobilized yeast systems (7, 8). Propanol
overproduction might be connected in the relative high 2,3-pentanedione
concentration and thus to -ketobutyrate mediated process. However the propanol
formed is far beyond the taste threshold value as well as 2,3-pentanedione values are
also below the taste limits. Although there is limited yeast growth in the immobilized
yeast systems, the high level of higher alcohols can be explained by the normal yeast
metabolic activities taken place and the relatively high amount of yeast cells involved.
It seems that -ketobuturate pathway is involved more actively but the mechanism
behind is unclear.

In an earlier paper (3) it was reported that temperature changes can affect also the
higher alcohol production of propanol and 3-methyl butanol reversibly indicating

9
further that changes in yeast metabolic activities can be seen in the flavour profile in
the fermented beer.
Similar reversible effect can be noticed when increasing the gravity of the wort
considerably. The higher alcohols are responding to gravity changes and the level of
3-methyl butanol last longer after adjusting the gravity back to normal again.
However, since the immobilized yeast system with recirculation acts alike a
completely mixed stirred reactor and not like a packed bed reactor, all the changes in
wort gravity or in other wort properties have a quite slow response to changes.

20 60
gravity
18 40
propanol
16 20 3-methyl butyl

14 0
5 10 15 20 25 30
original wort gravity
Time (days)

Figure 9: Formation of higher alcohols

Esters
As to ester formation, which are the most flavour active compounds, the major flavour
esters ethyl acetate and 3-methyl butyl acetates are very much at the same level as in
the batch fermentation, however with a somewhat elevated levels of ethyl acetate. The
pattern is quite understandable in the case of immobilized yeast fermentation with
limited yeast growth and with excess of assimilable nitrogen and fermentable
carbohydrates available leading to the situation where ester production is preferred
instead of lipid synthesis. However, the level of ester synthesis seems to be controlled
to some extent by the continuous addition of sugars in the fermenting system,
similarly to that in batch process. Regarding ester production we have earlier reported
that ethyl acetate levels are reversibly changing according to temperetary changes
similarly to that of propanol and 3-methyl butyl alcohol. The same effects were also
noticed when varying the wort gravity following the pattern of propanol and 3-methyl
butanol but with a slow response. 3-methyl butul acetate behaves a little differently
following more rapidly the actual change in wort gravity change (10).

40 2
Concentration (ppm)

35
1,6
30
25 1,2 ethyl acetate
20 3-methyl butyl
15 0,8 ethyl octanoate
10
0,4
5
0 0
5 10 15 20 25 30
Time (days)

Figure 10: Formation of esters

10
3-methyl butyl acetate was in acceptable level and the temperature changes had not
much effect on that level followed the pattern of 3-methyl butyl acetate, ethyl
hexanoate and ethyl octanoate. The two latter flavour compound seem to be at quite a
low level in immobilized systems but the levels can be affected by increasing
attenuation and ethanol level.

Controllability
In the present study the target has been to find out the process parameters for an one
stage immobilized yeast system with recirculation to be able to control the
fermentation system itself and more specifically the flavour formation in order to
produce the beer of desired flavour and to control the process on line. It has been
shown that the major technical challenges temperature control and carbon dioxide
removal have been solved giving the possibility to accurate temperature control with
desired temperature throughout the whole process.

It has been shown that the fermentation process in the immobilized yeast reactor can
be very constant with fixed amount of sugars fermented and constant amount of
ethanol produced in the single pass. Thus it is possible to control the feed volume and
recirculation right, to match the desired alcohol and attenuation level and control that
continuously on line. This means in practice that the sweetness of beer can be adjusted
on line. Further it has been shown that wort gravity and alcohol content has also an
flavour impact in the immobilized yeast fermentation process and can be used to
control the flavour. On the other hand temperature is also influencing the beer flavour
formation and it has been noticed that even at high temperatures low flavour beers can
be produced with right process conditions.

As to diacetyl and pentadione control, one of the targets were to manage the whole
fermentation process in one stage. The low yeast growth with limited -ketoacid
production gave promising expectations to that direction. However, the -ketoacid
synthesis seems to be high enough and the whole process time too short that there is
simply no time enough for oxidative decarboxylation process of the precursors to take
place, so that the final VDK values are slightly above the taste threshold values.
However this slight drawback can easily be handled with continuous immobilized
maturation (9), which is established technology today.

There is however one serious problem in the immobilized yeast primary fermentation
system with recirculation. This problem is caused by the low yeast growth and it is the
low consumption of free amino nitrogen. The consumption level of FAN is only 10-
20 % of that in normal batch fermentations in some occasions even less. This means
that in the beer there is excess of amino acids available, having possibly a flavour
impact. A second disadvantage is the higher pH value due to excess of amino acids
having impact on beer freshness and vulnerability to microbial contamination.

The problem can be approached by various strategies by using low FAN malt, FAN
dilution by adjuncts, pH adjustments or combinations of those. So far high adjuncts
levels with pH adjustments have been tested with promising results.

11
SUMMARY
The immobilized yeast primary fermentation system with recirculation has been tested
in pilot scale trials to establish the parameters for on-line controllable fermentation
process. The fermentation process itself has shown to be technically sound and the
calculations show the economical feasibility. It has been shown that during the
fermentation all the fermentable carbohydrates are consumed, although there is a
tendency of some accumulation of maltose and maltotriose at the end of long process
runs of several weeks. However, it is possible to choose the process parameters like
wort gravity, wort feed and recirculation rate so that desired attenuation and alcohol
content can be reached. Gravity and alcohol affects also the flavour production as well
as fermentation temperature and it seems to be possible to control the flavour
formation on line.

The low yeast growth rate creates a problem with free amino nitrogen accumulation in
beer, which problem can be approached with various ways but optimal solution is still
pending.

The immobilized yeast is acting in non growth phase under constant but quite severe
conditions being metabolically active as to fermentation performance. However, very
little so far is known of the basic metabolism and physiology of yeast under these
circumstances.

ACKNOWLEDGEMENT
The authors like to thank the National Technology Agency in Finland (Tekes) for
financial support to this project. Furthermore we like to thank Mrs Piia Soininen-
Tengvall and Mrs Johanna Siiril for technical assistant and analysis performed.
Finally we like to thank our companies for allowing this presentation.

REFERENCES
1. Monograph XXIV of European Brewery Convention, Immobilized Yeast Applications in
the Brewing Industry, Verlag Hans Carl Getrnke-Fachverlag, Nrnberg, 1996
2. Andersen, K., Bergin, J., Ranta B. & Viljava T., Proceedings of the 27th EBC Congress,
Cannes, 1999, 771-778
3. Pajunen, E., Ranta, B., Andersen, K., Lommi, H., Viljava, T., Bergin, J., Guercia, H.,
Proceedings of the 26th Convention of Institute of Brewing-Asia Pacific Section, 2000,
91-94 (CD-ROM)
4. Londesborough, J., Personal communications
5. Ryder., D.S., Masschelein C.A., J. American Society of Brewing Chemists, 1985, 43 (2),
66-75
6. Pajunen, E., Lommi, H., Viljava, T., Technical Quartely, 2000, 37 (4), 483-488
7. Virkajrvi, I., Lindborg, K., Kronlf, J., Pajunen, E., Monatsschrift fr Brauwissenschaft,
1999, 52 1/2, 9-12, 25-28
8. Cop, J., Dyon, D., Iserentant, D., Masschelein, C.A., Proceedings of the 22nd EBC
Congress, Zrich, 1989, 315-322
9. Pajunen, E., Grnqvist, A. & Ranta, B., Proceedings of the 23rd EBC Congress, Lisbon,
1991, 361-368

12
50

Continuous beer fermentation using

polyvinyl alcohol entrapped yeast
Daniela mogroviov1, Zoltn Dmny1, Marin Navrtil1 &
Petr Dvok2
1
Slovak University of Technology, Radlinskho 9, SK-81237 Bratislava, Slovak Republic (e-
mail: [email protected])
2
BCS Engineering, a.s., Purkyova 79 a, CZ-657 25 Brno, Czech Republic

Descriptors
Beer quality, continuous fermentation,flavour profile, immobilised yeast, secondary
fermentation

SUMMARY
A continuous immobilised system for rapid fermentation and maturation was developed. The
primary fermentation was carried out in an up-flow gas lift reactor, the maturation in column
reactors with packed-beds. Prior to the maturation a heat treatment of young beer was applied.
Yeast cells entrapped in a matrix based on polyvinyl alcohol were employed for both wort
fermentation and maturation. The system was stable for 2 months by the residence time of 24
to 36 hours. The produced beer was characterised by excellent quality with a composition and
flavour profile similar to that of beer produced by classical batch fermentation.

Kontinuierliche Grung mit Hilfe von an Polyvinylalkohol gebundener Hefe

Deskriptoren
Bierqualitt, kontinuierliche Grung, Nachgrung, organoleptisches Profil, Trgergebundene
Hefe

ZUSAMMENFASSUNG
Es wurde ein schnelles, kontinuierliches Grungs- und Reifungssystem mit immobilisierter
Hefe entwickelt. Die Hauptgrung wurde in einem aufwrts strmenden Gasfrderreaktor und
die Reifung in einem Kolonnenreaktor mit Fllkrpern durchgefhrt. Vor der Reifung wurde
das Jungbier wrmebehandelt. Zur Grung und Reifung wurden Hefezellen verwendet, die in
einer Matrix, auf Polyvinylalkohol basierend, gebunden werden. Das System arbeitete mit
Verweilzeiten von 24 bis 36 Stunden und lief zwei Monate stabil. Das Endprodukt zeichnete
sich durch einen hervorragenden Geschmack aus. Das Geschmacksprofil war dem Bier, das
im herkmmlichem Batch-Verfahren hergestellt wurde, gleich.
Fermentation continue de la bire au moyen de levures piges dans de l'alcool
polyvinylique

Descripteurs
Fermentation continue, fermentation secondaire, levure immobilise, profil de flaveur, qualit
de la bire

RESUME
Nous avons dvelopp un systme continu avec levures immobilises pour une fermentation
et une maturation rapides. La fermentation primaire a t conduite dans un racteur gaz
ascendant, la maturation dans des racteurs en colonne tasses. La bire jeune a t soumise
un traitement thermique avant maturation. Des cellules de levure piges dans une matrice
d'alcool polyvinylique ont t utilises pour la fermentation et la maturation du mot. Le
systme s'est rvl stable pendant 2 mois avec un temps de rsidence de 24 36 heures. La
bire produite se caractrisait par une excellente qualit, avec une composition et un profil
d'armes comparables ceux de la bire produite par fermentation classique en lot.

2
INTRODUCTION
Continuous production of beer has been the subject of research for many years for
both primary and secondary fermentation. The main aim of continuous processes is to
shorten the time of fermentation and at the same time to maintain desirable analytical
and flavour characteristics of the beer produced. Combination of continuous
fermentation and immobilised biomass removes the washout limitation in continuous
operation with free cells and results in a higher productivity (2, 3, 7, 9, 12).
The most widespread cell immobilisation techniques are entrapment in a matrix of
polymer or adherence of cells to the surface of a large number of sintered or porous
materials. The immobilisation into a matrix of polymer allows considerably higher
cell loading compared to immobilisation by adsorption, but application of polymer
gels is limited by their unfavourable diffusional properties. Many carriers, including
calcium alginate and pectate, agar or -carrageenan have been tested, while calcium
pectate appeared to be the most suitable for its good mechanical properties (3, 9, 11,
12).
Immobilised yeast technology is used successfully in industrial scale application
primarily for the maturation of primary fermented beer and for the manufacture of
alcohol-free beer. Several world breweries (i.e. Oy Sinebrychoff, Finland or Miller
Brewing Co., USA) use immobilised cell technology for continuous beer production
(5).
We have developed a continuous immobilised system for both, rapid fermentation and
maturation using a relatively new matrix referred as LentiKats, based on polyvinyl
alcohol. Polyvinyl alcohol as a non-toxic polymer is suitable for beverage
fermentation, LentiKats offer the possibility of entrapping cells under gentle
conditions in stable hydrogels and their lens-shaped form is optimised for minimal
limitations due to undesired diffusion effects (6).

MATERIALS AND METHODS

Microorganism and medium
Saccharomyces cerevisiae, brewing strains for bottom fermentation, were obtained
from local breweries and maintained on the malt-extract agar at 5C.
Cells for immobilisation were grown at 28C under aerobic condition in shake flasks
containing 500 ml of complete medium (11), collected by centrifugation from the
medium, washed and re-suspended in sterile distilled water at a concentration of 7.108
cells/ml of water. The cultivation medium was wort from different breweries of
original extract from 10 to 12 % (w/w).

Carrier and yeast immobilisation

Polyvinyl alcohol (PVAL) and polyethylene glycol were liquified using a water bath.
After cooling the solution was mixed with suspension of yeast cells and used to form
droplets. The gelation occurs without any need to add other chemicals due to a
controlled partial drying. After drying, the hydrogels were re-swollen in a stabiliser to
harden the matrix and to remove the polyethylene glycol. The lenticular form (figure
1) is obtained by drying of small droplets (6). Control calcium alginate immobilised
yeast were prepared by a standard dropping method (3).

3
 m
m
-4
3

200 - 400 m

Figure 1: Schematic geometry of LentiKats (6) - modified

Biomass accumulation
Before continuous wort fermentation, a step of aerated batch cultivation of
immobilised yeast was performed in order to accumulate biomass in the carrier
(approx. from 1,3 g to 70 g cell dry mass per kg of the gel) and maintain high yeast
viability. The cultivation medium was saturated with air at oxygen concentration of
approx. 6 mg/l.

Batch fermentation
Batch fermentations were carried out stationary in flasks containing 100 ml of
immobilised PVAL LentiKats or calcium alginate beads and 500 ml of wort, or free
yeast cells of corresponding concentration. The results quoted (figures 3, 4 and 5)
were obtained from parallel experiments.

Continuous fermentation
The primary fermentation was carried out in an up-flow gas lift reactor. The volume
of the reactor was 22.1 l, with an internal diameter of 21 cm. The carrier gas was
recuperated carbon dioxide formed during the wort fermentation, recycled at the flow
rate of 1.3 l/min or carbon dioxide from a gas bottle. The operating temperature was
kept at 15 C. The maturation was provided in a column reactor with packed-beds of
volume 15.8 l. Prior to the maturation a heat treatment of young beer was applied due
to conversion of all -acetolactate to diacetyl (9). Young beer was heated to 60 C,
kept at this temperature for 9 minutes and then cooled to 4 C (figure 2).

Analytical methods
Ethanol and other low molecular weight volatile compounds were determined by gas
chromatography using a flame ionisation detector (FID) and using a column filled
with Porapak QS for ethanol. Analysis of volatile compounds after distillation and
extraction in Likens-Nickerson apparatus (8) employed a column with a free fatty acid
phase (FFAP 8 %). Total nitrogen, free amino nitrogen, total polyphenols, bitterness
and colour were measured according to the current EBC Analytica. Diacetyl
concentration was expressed as the total vicinal diketones (VDK), measured after
thermal conversion of precursors to VDK as described Acker (1). The concentration
of saccharides was determined, after reaction with sulphuric acid,
spectrophotometrically at 488 nm by the phenol method (4). The yeast cell
concentration was expressed as dry mass and determined by drying at 105 C to a
constant weight. The cell concentration of immobilised biomass was determined using
technique of bioluminometry (10). From both reactors, wort and beer tanks
microbiological samples were taken once a week and analysed according to standard

4
methods (13). Characterisation of the beer was determined by a Servo Chem
Automatic Beer Analyser (SCABA 5600, Tecator AB, Hgans, Sweden).

Figure 2: Continuous beer fermentation. 1 - reactor for primary fermentation, 2 -

wort tank, 3 - beer tank, 4 - reactor for maturation, 5 - heater,
6 - control panel, 7 - hold-up coil, 8 - wort pump, 9 - young beer pump,
10 - cooler

RESULTS AND DISCUSSION

Influence of a carrier for yeast immobilisation and fermentation temperature on
beer flavour compounds
As to the date PVAL LentiKats have not been used for beer fermentation, in the first
step we compared flavour compounds formation in young beers produced by batch
fermentation using free, calcium alginate or polyvinyl alcohol immobilised yeast cells
at various fermentation temperatures.

5
 0.20

0.16
Diacetyl (mg/l)

0.12

0.08
4 8 12 16 20
Temperature (C)

Figure 3: Diacetyl concentration in beers produced by batch fermentation of

wort as a function of temperature PVAL LentiKats ; calcium
alginate ; free cells

40

36
Esters (mg/l)

32

28

24

20
4 8 12 16 20
Temperature (C)

Figure 4: Esters concentration in beers produced by batch fermentation of

wort as a function of temperature PVAL LentiKats ; calcium
alginate ; free cells

6
 200

180

Higher alcohols (mg/l)

160

140

120

100
4 8 12 16 20
Temperature (C)

Figure 5: Higher alcohols concentration in beers produced by batch

fermentation of wort as a function of temperature PVAL LentiKats
; calcium alginate ; free cells

Figure 3 shows that yeast entrapped in both, calcium alginate beads or PVAL
LentiKats produced beers with lower diacetyl concentration than the control (free
yeast cells), and the concentration of diacetyl decreased as the temperature was
increased. In contrast, the concentration of diacetyl increased with increasing
temperature with free yeast. We reported similar results using calcium pectate or -
carrageenan (11).
The concentration of total esters increased as the temperature of fermentation was
increased in all studied beers (figure 4). In beer produced by control free yeast the
content of total esters was higher than at the same temperature using calcium alginate
or polyvinyl alcohol entrapped yeast cells. The content of total higher alcohols
(ethanol not included) had similar temperature dependence, it was again higher when
using free yeast cells (figure 5).

Continuous fermentation and character of beer

The proposed continuous system has been already tested and optimised using calcium
alginate and pectate as an immobilisation matrix (3, 12). According to results showed
in figures 3, 4 and 5, the operating temperature of the gas-lift reactor for primary
fermentation was kept at 15 C. To reach optimal content of volatile by products, the
primary fermentation was performed by two different ways. In the first case recycled
carbon dioxide formed during wort fermentation was used as a carrier gas, containing
a part of volatile by products of beer, mainly esters, in the other, commercially
provided carbon dioxide from a gas bottle. The concentrations of flavour compounds
of beer produced in system using recycled carbon dioxide were higher, compared to
the control beer (table 1). The analyses of beer produced in the system using carbon
dioxide from gas bottle show the similar parameters to the control beer.

7
 Parameter Recycled Gas bottle Control
CO2 CO2
Original gravity w/w % 10.1 9.8 9.9
Apparent extract w/w % 2.54 1.99 1.86
Real extract w/w % 3.56 3.48 3.41
Alcohol w/w % 3.41 3.12 3.14
Real attenuation w/w % 64.7 64.4 65.5
Colour EBC 17 15 14
pH 4.36 4.55 4.53
Bitterness EBU 26 21 21
Total nitrogen mg/l 859 847 831
Free amino nitrogen mg/l 218 223 221
Total polyphenol mg/l 127 115 112
Flavour compounds mg/l
Diacetyl 0.11 0.08 0.09
2-Methylpropanol 31.7 29.5 30.1
Butanol 13.4 12.8 11.4
2- and 3-Methylbutanol 57.8 57.2 55.6
2-Phenylethanol 4.1 4.2 4.1
Ethyl formate 0.86 0.38 0.41
Ethyl acetate 10.1 5.4 5.2
Propyl acetate 1.2 0.5 0.2
2-Methylpropyl acetate 0.4 0.09 0.08
3-Methylbutyl acetate 2.09 0.98 1.06
Ethyl hexanoate 0.54 0.13 0.11
Hexyl acetate 3.4 1.2 1.2
Ethyl lactate 1.1 0.4 0.3
2-Methylpropyl hexanoate 0.11 0.03 0.04
Ethyl octanoate 0.04 0.01 0.01
Ethyl decanoate 1.12 0.05 0.03
Ethyl dodecanoate 0.15 0.05 0.04
Ethyl tetradecanoate 0.11 0.02 0.02
Table 1: Steady-state characteristics of beer produced in PVAL LentiKats
continuous system. Carrier gas in gas-lift primary fermentation reactor:
recycled or gas-bottle carbon dioxide. Control - classically produced
beer. Production rate 18 l/day

CONCLUSIONS
The developed immobilised continuous system using PVAL LentiKats could be
applicable for rapid fermentation and maturation of beer. The system was stable for 2
months by the optimal residence time of 24 to 36 hours depending on wort gravity.
The produced beer was characterised by excellent quality with a composition and
flavour profile similar to a control beer. Nine of eleven tasters considered beer
produced using the proposed continuous system to by comparable to a beer produced
by classical fermentation technology.

8
ACKNOWLEDGEMENT
This work was partially supported by VEGA grants No. 1/6252/99, 1/7347/20 and
BCS Engineering Brno, Czech Republic.

REFERENCES
1. Acker, M.E., Journal of American Society of Brewing Chemists, 1985, 43, 168-
170.
2. Andersen, K., Bergin, J., Ranta, B. &, Viljava, T., Proceeding of the 27th
European Brewery Convention Congress, Cannes, 1999, 771-778.
3. Dmny, Z., mogroviov, D., Gemeiner, P., turdk, E., Ptkov, J. &
Malovkov, A., Biotechnology Letters, 1998, 20, 1041-1045.
4. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A. & Smith, F., Analytical
Chemistry, 1956, 28, 350-356.
5. Fouhy, K. & Parkinson, G., Achema Daily, 1997, 3, 20-21.
6. Jekel, M., Buhr, A., Willke, T. & Vorlop, K.D., Chemical Engineering
Technology, 1998, 21, 275-278.
7. Kronlf, J. & Virkajrvi, I., Proceeding of the 27th European Brewery
Convention Congress, Cannes, 1999, 761-770.
8. Likens, S. & Nickerson, G., Proceedings of the American Brewing Chemists,
1964, 5-13.
9. Masschelein, C.A., Ryder, D.S. & Simon, J.P., Critical Review Biotechnology,
1994, 14, 155-177.
10. Navrtil, M., Dmny, Z., Hronsk, V., turdk, E., mogroviov, D. &
Gemeiner, P., Analytical Biochemistry, 2000, 284, 394-400.
11. mogroviov, D. & Dmny, Z., Process Biochemistry, 1999, 34, 785-794.
12. mogroviov, D., Dmny, Z., Gemeiner, P., Malovkov, A. & turdk E.,
Biotechnology Technics, 1997, 11, 261-264.
13. Virkajrvi, I. & Kronlf, J., Journal of American Society of Brewing Chemists,
1998, 56 (2), 70-75.

9
51

A new way for immobilized yeast

systems: secondary fermentation
without heat treatment
Frank Nitzsche1, Gerrit Hhn2, R. Meyer-Pittroff2, Svend
Berger3 & Rainer Pommersheim3
1
Easyproof Laborbedarf GmbH, Laboratoriumstrasse 100, D-46562 Voerde, Germany
2
TU Mnchen, Lehrstuhl fr Energie und Umwelttechnik der Lebensmittelindustrie, D-85350
Freising-Weihenstephan, Germany (e-mail: [email protected])
3
CAVIS Gesellschaft fr Immobilisierungssysteme mbH, Mainz, Germany

Descriptors
Enzymic activity, immobilised yeast, secondary fermentation

SUMMARY
The new plant design includes an additional reactor instead of a heat exchanger. This reactor
is filled with so-called Multi-Layer-Capsules (MLC) containing -acetolactat-
decarboxylase. These in native state immobilized enzymes are not able to pass the membrane.
Due to this special encapsulating technique this enzymes are stable on a high activity level for
a long time. For the maturation process we developed a new carrier material which overcomes
with the financial limitations with at least the same or better properties as known materials.
The duration of the process is not different to the old process layout.

Ein neuer Weg fr Systeme mit immobilisierter Hefe: Nachgrung ohne Wrme-
behandlung

Deskriptoren
Enzymaktivitt, Nachgrung, Trgergebundene Hefe

ZUSAMMENFASSUNG
Die neuartige Anlage beinhaltet einen weiteren Reaktor anstelle eines Wrmetauschers.
Dieser Reaktor ist mit -Acetolactatdecarboxylase, das an so genannte Multi-Layer-
Kapseln (MLC) gebunden ist, gefllt. Die auf natrlichem Wege immobilisierten Enzyme
knnen nicht die Membran passieren. Aus dieser speziellen Einkapselungstechnik resultiert
ein stabiles Enzym mit hohen Umsatzraten und langen Standzeiten. Fr den Reifungsprozess
entwickelten wir ein neues kostengnstigeres Trgermaterial mit letztendlich den selben oder
sogar besseren Eigenschaften als die bekannten Materialien. Die Zeitdauer dieses Prozesses
ist die selbe wie beim alten Prozess.
Une nouvelle voie pour les systmes de levures immobilises : la fermentation secondaire
sans traitement thermique

Descripteurs
Activit enzymatique, fermentation secondaire, levure immobilise

RESUME
Le schma de la nouvelle usine substitue un racteur supplmentaire la place d'un changeur
thermique. Ce racteur est rempli de capsules multicouches (Multi-Layer-Capsules - MLC)
contenant de l'-actolactate dcarboxylase. Ces enzymes immobilises l'tat natif sont
incapables de traverser la membrane. En raison de cette technique d'encapsulation spciale,
ces enzymes sont stables un haut niveau d'activit et pendant longtemps. Pour la maturation,
nous avons dvelopp un nouveau matriau porteur qui surmonte les limitations financires
avec des proprits au moins gales, voire suprieures, celles des matriaux connus. La
dure du processus n'est pas diffrente de celle de lancien procd.

2
INTRODUCTION
The continuous beer maturation is discussed during the last years by means of
lowering production time and production costs. The optimisation of the continuous
maturation process with the aspect of economy plays a particularly large role on that
strongly contested beer market. Many continuous processes are tested for this reason
on their feasibility, and some of them are already applied in industrial scale.
A possibility of improving the economy of such continuous procedures further on is
on one hand the application of lower-priced material for yeast immobilization. Today
used materials have been developed specially for the immobilization of cells or - still
more particularly - for continuous brewing procedures. One major disadvantage of
these products, which are produced in small quantities of few companies are high
costs to due to this reason. If there would be a possibility of using lower-priced
materials, which are produced in sufficient quantity, the costs of continuous maturing
procedures would be reduced. These materials should have similar characteristics as
carrier known materials and it should be possible to use them in usual fermenters.
The most important aspect however is the quality of the final product. Here is a
further aspect. With the usual continuous maturation processes a thermal treatment of
the green beer before maturation (for example 90 C, 10 min) is necessary. During
this thermal treatment 2- acetolactat will be converted to acetoin or diacetyl, which
can be diminished afterwards by immobilized yeast cells. This thermal treatment is
responsible for the appropriate changes, which take place in the beer, e.g. color
modifications and formation of Maillard products and degragation of 2-acetolactate.
Another disadvantage of this thermal treatment is the significant additional energy
consumption. A safe procedure for transformation of preliminary stages of the
vicinale diketons without a thermal treatment would be a further step in the
optimization of the continuous beer maturation.
In the context of a common research project of EasyProof Laborbedarf GmbH,
Voerde, Germany, CAVIS Gesellschaft fr Immobilisierungs-Systeme mbH, Mainz,
Germany and the Chair for Energy and Environmental Technologies in Food Industry,
TU Mnchen-Weihenstephan, Germany, a system for continuous beer maturing was
developed, which works with a new, high-quality and nevertheless economical carrier
material for yeast, and without a thermal treatment of the green beer. Instead of
thermal treatment the system works with immobilized enzyms to convert the
preliminary stages of the vicinale diketons. This sytem is presented here.

PRINCIPAL ITEM
Multi Layer Capsules (MLC)
In extension of a method described by Lim and Sun [3] alginate gel spheres acting as a
matrix for the immobilization of substances or living cells are covered in consecutive
steps with several polymer layers forming a membrane of about 0,8 m thickness
(figure 2).

Each of these layers is optimized with respect to a different property of the capsule's
shell: one layer regulates the permeability, one the mechanical stability and an outer
layer the capsules storage properties. The used materials are natural polymers such as
chitosan and cellulose derivates. The tuning of the membrane for a specific
application is made by the choice of the polymers. The permeability of the membrane

3
can be adjusted in a range between 50 to 200 kDa. Figure 1 shows the schematic
representation of structure of the membrane capsule.

Layer III
Layer II

Layer I

Core Outer Cover

Layer

Figure 1: Schematic representation of structure of the membrane capsule

The core contains the immobilized material either as an aqueous solution (e.g.
enzymes) or as a suspension (e.g. living cells) in alginate. If requested, the alginate
inside the capsule can be gelled or liquified.
Although the capsules can be dried for storage, they have to be used in liquid media.
The membrane resists to an inner pressure of up to 25 N/cm. They should be used in
fluidized bed reactors and should in general not be packed in columns.

Figure 2: Non-encapsulated and encapsulated carrier material

Figure 3: Encapsulated carrier material

4
Figure 2 shows non-encapsulated and figure 3 shows encapsulated carrier material.
The difference of the surface is obvious.

MLC- encapsulated Enzymes

Enzymes are in general very expensive substances, so their recovery in industrial
processes is frequently desired. To lower the costs of the recuperation steps these
substances must be immobilized. Using classical methods for this purpose such as
bonding to surfaces or adsorption on porous materials, up to 70 % of the enzymes
initial activity may get lost in a denaturation process of the molecule.
By the immobilization of the enzymes in multilayer microcapsules (MLC) this loss
of activity is generally below 5 %. The capsules having a liquid core, the activity of
the enzymes is nearly the same as in any other liquid media because they are free
floating and capable to adopt their native conformation.
Some processes like the fermentation of beer require a complete removal of some
additives such as enzymes. So a simple inactivation by heat cant considered, due to
the fact that the protein body of the enzyme would still remain in the beverage. In this
research project CAVIS has developed a product which contains MLC-encapsulated
acetolactate decarboxylase.

PRELIMINARY TESTS
A new carrier material was developed. This new carrier material is called Immopore
and shown in figure 4. In order to avoid the high costs of most conventional
substrates, the surface of open-porous swelling-glass in a spherical shape with a
diameter of 2 up to 4 mm was treated in a special way. Because of that treatment the
yeast cells where enabled to stick to the glass surface. Figure 5 shows a picture taken
with a scanning electron microscopic of the surface of in this way treated porous
glass.

Figure 4: Immopore

5
 200 m

Figure 5: Scanning electron microscopic picture of Immopore

After the surface treatment the next step was the loading of Immopore with yeast.
Therefore bottom-fermented yeast (Saccharomyces carlsbergensis) was suspended in
a malt solution with 10 Plato. The mixture was pumped in cycle through a Immopore
filled reactor, until the malt solution dropped down to 3 Plato. Subsequently, the
reactor was rinsed with fivefold of its own volume of water in order to wash-out free
yeast cells. In the further experiments there where determinations done of cell loading
and fermentation ability.

CELL LOADING
The experiments in a laboratory scale showed that the yeast loading of Immopore is
most comparable (it seems to be higher) with the yeast loading of high-priced
conventional materials. Table 1 shows a comparison between different carrier
materials and their possible cell loading.

6
Table 1: Comparison of some carrier materials and their cell loading (cell number per
100 g substrate or in g dry matter yeast per 100 g carrier material)

Carrier Material Cell Loading Reference

Immopore 1,01012 cn/100 g CM

Schott Siran (Glass) 1,11011-3,91011 cn/100 g CM [1]

spongy glass 7,7109-1,11011 cn/100 g CM [4]
Spongy sinter glass 8-10 g dm/100 g CM [2]

Spezyme GDC (DEAE- 1,31011 cn/100 g CM [1]

Cellulose.)
Spezyme GDC (DEAE-Cellulose) 1,661011 cn/l FBV [5]
Modification of DEAE-Cellulose 51010-11011 cn/100 g CM [1]

Hypermics (Ceramic) 7,81011 cn/100 g CM [1]

Bioceramics (Ceramic) 8,71010 cn/l FBV [5]
cn: cell number; CM: dry matter of carrier material; FBV: fest bed volume

The scanning electron microscopic picture of the loaded Immopore show the
distribution of yeast cells on the surface (figure 6).

10 m

Figure 6: Cells of Saccharomyces carlsbergensis on Immopore

FERMENTATION ABILITY
The results of the test for fermenting ability show that the yeast was successfully
adsorbed. There was no degradation of the fermentation determined, if the carrier
particles were rinsed before, in order to remove freely suspended yeast cells.
Therefore the fermentation primarily takes place via carrier-fixed cells.

Combination of the two processes (main experiments)

Figure 7 shows the schematic experimental setup on a laboratory scale.

Enzym
reactor
filled with
MLC

CO2
Pump Yeast reactor
CO2
filled with
Immopore

Storage vessel 1 Storage vessel 2

Figure 7: Technique system of continuous beer maturing

For the laboratory tests green beer, type Muenchner Hell, was clarified by a candle
filtration, whereby a yeast concentration fewer than 10,000 cells/ml was adjusted. In
an industrial scale the clarification would take place in a separator instead of a candle
filtration. After clarification the green beer was pumped out of the storage vessel 1
through the enzyme reactor. The volume of the enzyme reactor amounts about 0.5 l.
After the enzyme reactor the green beer passes through the yeast reactor, which has a
volume of 5 l. After that the beer is stored in storage vessel 2. The capacity of the
pump allows up to 1.5 l/h, which leads to a retention time of the green beer in the
enzyme reactor of 20 minutes and in the yeast reactor of 200 minutes. In every
laboratory experiment 50 litres were pumped through, which correspondents to a total
duration of the experiment of about 33 hours. The operation of the test range during
one period of 140 hours could also successfully be executed. The operating
temperature of both reactors was set to 18 C.
The matured beer was examined by GC Headspace analytics. From each reactor
samples were analyzed. The content of diacetyl decreased during the continous
maturation from 0,14 mg/l to 0,07 mg/l. So it could be shown that the transformation
of the 2-acetolactats to diacetyl in the enzyme reactor was almost completely effected
and that the reduction of the diacetyls, which took place in the yeast reactor, occured
until far under taste value in beer. The taste of the beer showed no signicicant
differences to conventionally matured beers.

2
RECOVERY
After the experiments the enzyme reactor, after it was rinsed with water, could be
stored at temperatures by 5 C several weeks, without a significantly decrease of
enzyme activity. The yeast reactor was rinsed after the experiments first with water
and cleaned afterwards with sodium hydroxide. After cleaning a steam sterilization
followed.

SUMMARY
The presented system enables a continuous beer maturing in period of fewer than 4
hours. By the application of an immobilized enzyme, the 2-acetolactatdecarboxylase,
a continuous maturation without thermal treatment is possible. Thereby advantages
result in the quality of the later beer, on the other hand there is also a reduction in
energy. By using a new carrier material, called Immopore the capital outlays for the
filling of the yeast reactor can be lowered dramatically. A further advantage of the
new carrier material is that a cleaning with sodium hydroxide is possible, as well as a
sterilization by steam.
For the first time secondary maturation can be done on a cost comparable basis.

REFERENCES
[1] Cashin, M.-M.. (1996): Comparative Studies of Five Porous Supports for Yeast
Immobilisation by Adsorption/Attachment, Journal of the Institute of Brewing,
102 (1), 5-10
[2] Dembrowski, K. (1992): Untersuchungen zur kontinuierlichen Biergrung am
Beispiel des Grfilterverfahrens. Dissertation, Technische Universitt Mnchen,
Fakultt fr Brauwesen, Lebensmitteltechnologie und Milchwissenschaft
[3] Lim, F., A. Sun (1980), Science, 210, 908
[4] Linko, M., Virkajrvi, I., Pohjala, N., Lindborg, K., Kronlf, J., Pajunen, E.
(1997): Hauptgrung mit immobilisierter Hefe - ein Durchbruch? Proceedings
of the 25th EBC Congress, Maastricht, 385-395
[5] Wackerbauer, K., Fitzner, M., Gnther, J. (1996): Technisch-technologische
Mglichkeiten mit immobilisierter Hefe, Brauwelt, 136 (45), 2140-2150

3
52

Application of genome-wide
transcriptional analysis to identify
genetic markers useful in industrial
fermentations
Vincent J. Higgins2, Anthony D. Oliver1, Rachel E. Day2, Ian W.
Dawes2 & Peter J. Rogers1
1
Carlton and United Breweries Ltd, 4-6 Southampton Crescent, Abbotsford, Australia. 3067
(e-mail: [email protected])
2
University of New South Wales, School of Biochemistry and Molecular Genetics, Sydney,
Australia. 2052

Descriptors
Fermentation defect, genetic marker, yeast technology, zinc

SUMMARY
Yeast genome-wide transcriptional analysis technology allows the identification of genes
with significant and specific differential expression. In response to zinc-limiting conditions a
gene was identified that had a much higher differential expression than the genes currently
known to be involved in zinc deficiency. Expression of this gene therefore provides a more
discernible target for rapid quantification using real time PCR, to monitor industrial
fermentations for non-standard conditions. Furthermore, we have successfully shown that
monitoring expression of genes on a genome-wide scale during industrial fermentation may
provide a greater understanding of the causes of defective fermentations and possible
solutions.

Anwendung der genom-bergreifenden Transkriptionsanalyse zur Identifizierung von

genetischen Markern in der industriellen Fermentation

Deskriptoren
Abnormale Grerscheinung, Hefetechnologie, Markierungsgen, Zink

ZUSAMMENFASSUNG
Die genom-bergreifende Transkriptionsanalyse der Hefe ermglicht die Identifikation von
Genen mit bedeutender und spezifischer differenzierter Expression. In Antwort auf Zink-
Limitierung wurde ein Gen identifiziert, das eine sehr viel hhere differenzierte Expression
zeigte als die Gene, die bisher als in der Zink-Defizienz beteiligt bekannt sind. Die
Expression dieses Gens stellt daher ein geeigneteres Ziel fr die schnelle Quantifizierung mit
real time PCR dar, um industrielle Fermentationen unter Standardbedingungen zu
verfolgen. Darber hinaus haben wir erfolgreich zeigen knnen, da die genom-bergreifende
Beobachtung der Expression von Genen whrend der industriellen Fermentation ein besseres
Verstndnis der Grnde fr fehlerhafte Fermentationen und mgliche Lsungen darstellt.

Application de l'analyse transcriptionnelle du gnome pour identifier les marqueurs

gntiques utiles en fermentation industrielle

Descripteurs
Dfaut de fermentation, marqueur gntique, technologie de la levure, zinc

RESUME
La technologie d'analyse transcriptionnelle l'chelle du gnome entier de la levure permet
d'identifier les gnes prsentant une expression diffrentielle significative et spcifique. En
rponse des conditions limitantes en zinc, nous avons identifi un gne ayant une
expression diffrentielle nettement plus leve que les gnes actuellement connus pour
intervenir dans la carence en zinc. L'expression de ce gne apporte donc une cible plus
discernable pour un dosage rapide par PCR en temps rel, pour surveiller les fermentations
industrielles en conditions non standard. Par ailleurs, nous avons russi dmontrer que le
suivi de l'expression gntique l'chelle du gnome entier pendant la fermentation
industrielle pourrait permettre de mieux connatre les causes des fermentations dfectueuses
et de trouver des solutions possibles.

2
INTRODUCTION
The alcoholic fermentation process is a hostile environment, subjecting yeast to
numerous stresses (2). As a result, this can lead to defective or sub-optimal
fermentations in which yeast metabolism is halted or off-flavours are produced (11).
The rapid analysis of gene expression in response to stress using real-time PCR
methods may enable the early detection of conditions that cause defective
fermentations. Genes such as HSP12 (11) and SPI1 (17) showed potential as genetic
markers for stress conditions in alcoholic fermentations. However, the broad range of
conditions that these genes respond would alert the presence of sub-optimal conditions
but not the nature of the stress.
The recent sequencing of the yeast genome, in conjunction with the development of
analytical techniques to probe and quantify the expression levels of a large number of
individual genes (12) has facilitated for a major step forward in the ability to identify
genes that have altered gene expression patterns in response to changing
environmental conditions (10). With zinc deficiency being a major contributor to the
slow down of yeast fermentation in the beer brewing process (3) we confirm the
identification of genes that are highly responsive and specific to zinc limiting
conditions using genome-wide transcriptional analysis. We also demonstrate that this
technique can be used to determine yeast gene expression patterns during industrial
processes.

MATERIALS AND METHODS

Yeast strains, media and culture conditions
The lager brewing yeast (A strain) was obtained from Carlton and United Breweries
(Abbotsford, Victoria, Australia). Yeast were grown in low zinc medium prepared
essentially the same as LZM medium (21) except that maltose replaced glucose as the
carbon source (LZMM). For Northern analysis a yeast colony was inoculated into
LZMM + 4 mg/l Zn medium and grown overnight at 30oC with shaking. Cells were
harvested and washed three times with SDW before inoculation at an optical density
(OD600) of 0.1 into LZMM with and without addition of zinc. Cells were harvested for
RNA isolation at an (OD600) of 0.5. Industrial grade wort at pilot plant level was
inoculated with acid washed strain A cells and total RNA isolated for GeneFilters
microarray analysis at 1 hour and 23 hours into fermentation.

PCR cloning of YOR387c

Forward primer for amplification of YOR387c gene by PCR was YOR387c-F (5
ATAAGAATGCGGCCGCTGTTTTGGCGTATTCTCCAC 3) and the reverse
primer was YOR387c-R (5 CCGCTCGAGAACACGCG TAATTGAAAGGG
3). The PCR used Taq DNA polymerase (Stratagene) and 100 l reactions were
prepared in accordance with the manufacturers instructions. The PCR product was
ligated into the pCR2.1 vector in accordance with the instructions (Invitrogen).

Microarray and Northern analysis

Total RNA for Northern hybridisation and Microarray analysis was isolated from
strain A cells using TRIZOL reagent (Life Technologies Inc., USA) according to the
manufacturer's instructions. Microarray analysis was carried out using GeneFilters
(Research Genetics) containing over 6000 yeast genes. They were hybridised with

3
cDNA produced from total RNA according to the manufacturer's instructions
(Research Genetics). Total RNA was denatured, separated by agarose gel
electrophoresis (10 g), and analysed by Northern blotting. Images indicating gene
expression on the GeneFilters and Northern hybridisations were obtained on a
PhosphorImager (Molecular Dynamics) using ImageQuaNT v4.2a software.

RESULTS
YOR387c and YGL285w are highly homologous genes
In previous microarray GeneFilters results, YOR387c and YGL258w were identified
as having approximately 300-fold higher expression in zinc deficient conditions. This
was about 40-fold higher than the known zinc responsive genes ZAP1 and ZRT1.
Sequence analysis YOR387c and YGL258w showed that they are highly homologous
genes that share 93% identity in protein sequence and 74% similarity with the
Saccharomyces pombe proteins SPAC977.05, SPAC1348.06c and SPBPB2B2.15.
These genes have no known function in their respective cells and have little similarity
to any higher eukaryotic genes.
To establish whether the regulation of YOR387c and YGL258w were suitable for
effective monitoring of zinc deficient conditions in industrial processes, a DNA probe
was constructed to quantify mRNA expression levels using Northern analysis. With
the high homology of the proteins encoded by YOR387c and YGL258w, differing by
only 15 amino acids (figure 1B) a probe that is specific for either transcript was not
possible. The promoters of these genes are virtually identical (figure 1A) and therefore
it is expected that both genes are expressed in a similar manner. The sequences at the
terminal end of the genes diverge rapidly, therefore it was used to generate a reverse
primer for the cloning of the YOR387c gene. The predicted size of the PCR product
using the primers YOR387cF and YOR387cR (Materials and Methods) was 2575 bp
and as expected, a product of approximately 2500 bp was obtained (results not
shown). The 3 A overhang left by the PCR reaction was used to ligate the PCR
product into the pCR2.1 vector creating the plasmid PCRYOR387c (figure 2). The
DNA used as a probe to hybridise to YOR387c and YGL258w mRNA was obtained
by gel purification of a 378 bp fragment created by digesting PCRYOR387c with AccI
and NheI restriction enzymes.

YOR387c/YGL258w expression is specifically responsive to zinc deficiency

To confirm the GeneFilters expression results, YOR387c/YGL285w expression
levels were measured by Northern analysis of total RNA isolated from strain A
growing in zinc-deficient and zinc-replete conditions. The transcript levels of
YOR387c/YGL285w mRNA were very high in zinc deficient conditions whereas in
zinc-replete medium the transcript levels were not detectable (figure 3). The mRNA
levels of ZRT1, a gene known to be induced in response to zinc deficient conditions
was also increased (21). The basal level of ZRT1 expression in medium containing
zinc was relatively high compared to the expression of YOR387c/YGL285w (figure
3). To determine whether the increase in expression of YOR387c/YGL258w was
specific for zinc deficiency and not a general stress response, gene expression was
also tested under oxidative stress and carbon starvation conditions.
YOR387c/YGL285w and ZRT1 expression levels were responsive only to the zinc
depleted conditions. Whereas HSP12 expression was high in all conditions tested

4
except in the control medium that had added zinc (figure 3). As expected, the ACT1
transcript, a commonly used control for standardising RNA concentration, was similar
in all conditions (figure 3). These results indicate that YOR387c/YGL258w are highly
expressed in response to and specific for zinc deficient conditions.

GeneFilters analysis of yeast expression during industrial fermentations

To determine the applicability of whole-genome expression analysis in monitoring
yeast gene expression during industrial processes cells were harvested from a pilot
plant industrial wort fermentation that was pitched with acid washed strain A. Total
RNA was isolated from cells 1 and 23 hours after pitching. The labeled cDNA was
hybridised to GeneFilters and radiation emissions were quantified to provide a
quantitative measure of the relative abundance of mRNA levels (table 1).

Unsaturated fatty acid and sterol synthesis highly active in the first hour of
fermentation
A family of genes involved in lipid, fatty-acid and sterol metabolism were more highly
expressed in the initial phases of fermentation than after 23 hours (table 1). These
included OLE2; the protein that is responsible for conversion of saturated fatty acids
and oxygen to unsaturated fatty acids (9) and expression is induced by hypoxia, early
cell cycle and saturated fatty acids (4, 5). These expression characteristics are similar
to ERG11, which is one of eight genes contained in these results that are required for
the synthesis of ergosterol (6). This step is the rate-limiting enzyme in ergosterol
biosynthesis (20) and apart from having similar activation characteristics as OLE1,
both genes are repressed by the transcription factor ROX1 (14), which is also up-
regulated in the first hour. The identification of genes highly expressed in the first
hour of fermentation that are responsible for synthesis of unsaturated fatty-acids and
ergosterol correlates well with previous observations of very high sterol synthesis
activity in the early stages of fermentation.

Oxidative stress response in first hour of fermentation

OYE2 was up-regulated approximately 19-fold in the first hour of fermentation. Its
upstream region contains a putative Yap1p binding site and its expression is up-
regulated when Yap1p is overexpressed (7). Yap1p is required for activity of the stress
response (STRE) element (CCCCT) in response to oxidative stress but does not bind
the STRE directly. Other genes in these results that contain (STRE) elements in their
upstream activating sequences are SPI1, CTT1, YEF3, TRR1, and CYS3. SPI1
encodes for a cell wall bound protein that has 62% identity to SED1 another gene that
serves a role in protection from oxidative stress (8, 17).
The synthesis of unsaturated fatty-acids and ergosterol requires oxygen, which is
provided by the action of pitching or the blowing of air or oxygen through wort (1).
Over-aeration can be detrimental due to over-production of yeast biomass which
compromises fermentation efficiency and ethanol production and this could be further
compounded as it may potentiate the effect oxidative stress has on these fermentation
characteristics. The metabolic process of fatty acid -oxidation also generates reactive
oxygen species also implicated in the up-regulation of oxidative stress genes.
Unsaturated fatty acids and sterol synthesis also have implications in flavour
formation (19). Studies by Fujiwara et al. (9) showed that the transcription of the
ATF1, a gene encoding a transferase which catalyses the synthesis of acetate esters, it
is co-regulated by the same mechanism as the OLE1 gene and both are regulated in

5
response to cell membrane fluidity. Both acetate esters and ergosterol are synthesised
from acetyl-CoA (15), implicated in this metabolite synthesis is ALD6, a gene up-
regulated 95-fold in these conditions (13). Oxidative stress could also have an effect
on flavour profile since the gene encoding the cysteine generating protein (Cys3p) is
up-regulated by this condition. Cysteine has been shown to have a negative effect on
flavour production and unlike in E. coli, the reaction cannot proceed in the opposite
direction because of the unavailability of O-succinylhomoserine in yeast (16).

CONCLUSIONS
To use real-time quantitative PCR to measure the level of gene expression within
yeast and to detect conditions that are affecting them during fermentation processes,
requires the identification of genes that are effective genetic markers. This
quantification process can take as little as three hours from isolation of mRNA to
finishing the PCR. Genome-wide transcriptional analysis was shown to be effective in
identifying YOR387c and its highly homologous partner, YGL285w, as genes that are
very useful as effective genetic markers for identification of zinc deficient conditions.
These results show that gene expression analysis techniques not only provide an
avenue for identifying genes that can monitor for non-standard fermentation
conditions but can also lead to a greater understanding of their causes and possible
solutions. These insights can then be extrapolated to the wine industry where stuck
fermentation cause greater inefficiencies and higher loss.

ACKNOWLEDGMENTS
Thanks to Carlton and United Breweries and ARC SPIRT grant CO9917629 for
providing funding for this work.

REFERENCES
1. Aries, V., & Kirsop, B.H., Journal of the Institute of Brewing, 1977, 83, 220-223.
2. Attfield, P.V., Nature Biotechnology, 1997, 15, 1351-1357.
3. Bromberg, S.K., Bower, P.A., Duncombe, G.R., Fehring, J., Gerber, L., Lau,
V.K., & Tata M., Journal of the American. Society for Brewing Chemists, 1997,
55,123-128.
4. Cho, R.J., et al., Molecular Cell, 1998, 2, 65-73.
5. Choi, J.Y., Stukey, J., Hwang, S.Y., & Martin, C.E., Journal of Biological
Chemistry, 1996, 271, 3581-3589.
6. Daum, G., Lees, N.D., Bard, M., & Dickson, R., Yeast, 1998, 14, 14711510.
7. DeRisi, J.L., Iyer, V.R., & Brown, P.O., Science, 1997, 278, 680-686.
8. Ezaki, B., Gardner, R.C., Ezaki, Y., Kondo, H., & Matsumoto, H., FEMS
Microbiological Letters, 1998, 159, 99-105.
9. Fujiwara, D., Yoshimoto, H., Sone, H., Harashima S., & Tamai, Y., Yeast, 1998,
14, 711721.
10. Gasch, A.P., Spellman, PT., Kao, C.M., Carmel-Harel, O., Eisen, M.B., Storz, G.,
Botstein, D., & Brown, P.O., Molecular Biology of the Cell, 2000, 11, 4241-
4257.

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11. Ivorra, C., Perez-Ortin, J. E., & del Olmo, M., Biotechnology and Bioengineering,
2000, 64, 698-708.
12. Iyer, V., DeRisi, J., Eisen, M., Ross, D., Spellman, P., Hudson, J., Schuler, G.,
Lashkari, D., Shalon, D., Botstein D., & Brown, P., FASEB J. 1997, 11, 1571,
Suppl. S JUL 31
13. Meaden, P.G., Dickinson, F.M., Mifsud, A., Tessier, W., Westwater, J., Bussey,
H., & Midgley, M., Yeast, 1997, 13, 1319-1327.
14. Nakagawa, Y., Sugioka, S., Kaneko, Y., & Harashima, S., Journal of
Bacteriology, 2001, 183, 745-751.
15. Nordstrom, K., Journal of the Institute of Brewing, 1962, 68, 398407.
16. Ono, B., Tanaka, K., Naito, K., Heike, C., Shinoda, S., Yamamoto, S., Ohmori,
S., Oshima, T., & Toh-e, A., Journal of Bacteriology, 1992, 174, 3339-3347.
17. Puig, S., & Perez-Ortin, J.E., Yeast, 2000, 16, 139-148.
18. Renger, R.S., Van Hateren, S.H., & Luyben, K., Journal of the Institute of
Brewing, 1992, 98, 509-513.
19. Rosi, I. & Bertuccioli, M., Journal of the Institute of Brewing, 1992, 98, 305-314.
20. van den Brink, H.M., van Gorcom, R.F., van den Hondel, C.A., & Punt, P. J.,
Fungal Genetics & Biology, 1998, 23, 1-17.
21. Zhao, H., & Eide, D. J., Molecular & Cellular Biology, 1997, 17, 5044-52.

7
Fold Locus name Gene Characteristics
induction name
Unsaturated Fatty acid synthesis
49 YGL055w OLE1 induced by Cox5Bp, Stearoyl-CoA desaturase

Ergosterol synthesis
10 YMR015c ERG5 cytochrome P450
18 YHR007c ERG11 catalyzes the 14-alpha demethylation of lanosterol
11 YGL001c ERG26 C-3 sterol dehydrogenase, C-4 decarboxylase
7 YML126c ERG13 converts acetoacetyl-CoA to hydroxymethylglutaryl-CoA
27 YPL028w ERG10 acetyl-CoA acetyltransferase
5 YGR175c ERG1 converts squalene & O2 into squalene-2,3-epoxide and H2O
5 YGR060w ERG25 oxidizes 4,4-dimethylzymosterol to carboxylic acid
3 YLR056w ERG3 C5=6 desaturation

Roles in lipid, fatty-acid and sterol metabolism

95 YPL061w ALD6 acetyl-CoA production
21 YPR065w ROX1 represses ERG11, OLE1 and COX5B
6 YKL032c IXR1 binds upstream of COX5B
29 YER141w COX15 cytochrome oxidase assembly
13 YPL117c IDI1 cholesterol biosynthesis pathway
7 YLL013c modulation of the poly(A) status of COX17 mRNA
19 YHR179w OYE2 may be involved in sterol metabolism

Oxidative stress and STRE induced genes

15 YER150w SPI1 has 62% identity to Sed1p
10 YGR088w CTT1 Detoxification of superoxide radicals and H2O2
44 YLR249w YEF3 contains STRE elements
54 YDR353w TRR1 thioredoxin reductase
29 YDR502c SAM2 involved with CYS3
14 YAL012w CYS3 Cystathionine gamma-lyase, generates cysteine
41 YDR077w SED1 serves a role in protection from oxidative stress
10 YGR086c induced by pH and salt
9 YMR002w activated by rapamycin with SED1, SPI1, HSP12

Other
5 YPL089c RLM1 Transcription factor of the MADS
7 YMR043w MCM1 induced by the SWI-SNF complex
6 YBR289w SNF5 part of the SWI-SNF complex
9 YGR160w expressed similar to RHR2
11 YIL053w RHR2 involved in cellular production of glycerol
51 YMR011w HXT2 high-affinity hexose transporter
7 YBR113w Unknown
10 YDR505c PSP1 Unknown
6 YGL122c NAB2 Polyadenylation of pre-mRNA and for mRNA export
13 YOR302w mediates translational regulation
12 YKL054c VID31 vacuole Import and Degradation
29 YBR105c VID24 vacuole Import and Degradation

Table 1: Genes up-regulated in the first hour of fermentation compared to the 23rd
hour.

8
A
Promoter sequence
YOR387c GCTTTTGAATTAGTGTTGCTTCTATTCCTGGAATGGAAGGTAATACTTTC
YGL285w GCTTTTGAATTAGTGTTGCTTCTATTCCTGGAATGGAAGGTAGTAATTTC

YOR387c ATGCATTCTCGCTTTTCGGACTTTTAACAATAAATTAAAAACAATGATAC
YGL285w ATGCATTCTCGCTTTTCGGACTTTTAACAATAAATTAAAAACAATGATAC

YOR387c TTTCATCAACTACCTATACACCCTGCGGGTCAATTATTTTTTTT-CGAAT
YGL285w TTTCATCAACTACCTATACACCCTGCGGGTCAATTATTTTTTTTTCGAAT

YOR387c AACCGCTGAGGTTGGAAATATAGAAACATATTCCAGATCTGTATTTTCAG
YGL285w AACCGCTGAGGTTGGAAATATAGAAACATATTCCAGATCTGTATTTTCAG

YOR387c TTGCTAGAAAAAGGTTAATATAATCATTAAGGTTTTCAGCATATAACAGG
YGL285w TTGCTAGAAAAAGGTTAATATTATCATTAAGGTTTTCAGCATATAACAGG

YOR387c TATAATTGATATATAAGCATCGTAATTTTCATTCAAAATGGAGAGCTACT
YGL285w TATAATTGATATATAAGCATCGTAATTTTCATTCAAAATGGAGAGCTACT

YOR387c GCTTCTGATAGATTGTACAATCTCAAGAAATCAAGAACAACAACCATACC
YGL285w GCTTCTGATAGATTGTACAATCTCAAGAAATCAAGAACAACAACCATACC

Terminal sequence
YOR387c CCAGACGCTGGTTTTGCTTGATTTTTTCCAGCTGCATAATCTGTAGACAT
YGL285w CCAGAAGCTGGTTTTGATTAATCTTTTCTCACATCCTCACTTATAGATAT

YOR387c ATTTTAAGTATATTTTTTAGGCAGTTTTATAAATTAACAGACTATAATTA
YGL285w GTTTTGATTATTTTCGTTATAGACAAAAATCTTTTGAGAAAGCCATGCGG

YOR387c TTTTAGAGAAAAAGACCCAGATTTATACTTCTGACTTTTCTTTTTTATGT
YGL285w AAGTTATTTTTAACTGTAAAGTACAATCGATTTCCTTTAGGCATTTCTAC

B
YOR387c MSFLNIFTFFSVLVSVATAVRFDLTNVTCNNLHGPHCGTYVMEVVGQNGTFLGQSTFAGA
CONS MSFL+IFTFFSVL+SVAT-VRFDLTNVTC--LHGPHCGTYVMEVVGQNGTFLGQSTF-GA
YGL285w MSFLSIFTFFSVLISVATTVRFDLTNVTCKGLHGPHCGTYVMEVVGQNGTFLGQSTFVGA

YOR387c DVLTESAGDAWARYLGQETRFLPKLTTIASNDTKNFSPLIFTTNIYTCNPQSIGDAMVPF
CONS DVLTESAGDAWARYLGQETRFLPKLTTIASN+TKNFSPLIFTTNI-TCNPQSIGDAMVPF
YGL285w DVLTESAGDAWARYLGQETRFLPKLTTIASNETKNFSPLIFTTNINTCNPQSIGDAMVPF

YOR387c ANTVTGEIEYNSWADTADNASFITGLANQLFNSTQYGVQVASCYPNFASVILSTPTVNIF
CONS ANTVTGEIEYNSWADTADNASFITGLANQLFNST-YGVQVASCYPNFASVILSTP-VNIF
YGL285w ANTVTGEIEYNSWADTADNASFITGLANQLFNSTDYGVQVASCYPNFASVILSTPAVNIF

YOR387c AANETLPDYCTAIQLKAVCPPDAGF
CONS --++TLPDYCTAIQLKAVCPP+AGF
YGL285w GKDDTLPDYCTAIQLKAVCPPEAGF

Figure 1A: DNA sequence comparison of YOR387c and YGL258w upstream

activating and terminal regions.
Figure 1B: Protein sequence comparison between YOR387c and YGL258w.

9
 lacZpr
pUC ori
YOR387c
PCRYOR387c
Ampicillin
6.47 kb

Kanamycin
lacZ
F1 ori

Figure 2: Schematic representation of the plasmid PCR2.1-YOR387c.

Zinc No Zinc Stress Starv

ACT1

YOR387c

ZRT1

HSP12

Figure 3: Northern analysis of YOR387c, ZRT1, HSP12 and ACT1 expression in

Zinc (LZMM + 4 mg/l zinc), No Zinc (LZMM), Stress (LZMM + 4 mg/l zinc + 2mM
H2O2) and Starv (Starvation, LZMM + 4 mg/l zinc with no maltose added).

10
53

Metabolic engineering approaches -

opportunities for brewing
Merja Penttil

VTT Biotechnology, P.O.Box 1500, FIN-02044 VTT, Finland (e-mail: [email protected])

Descriptors
Brewers' yeast, genetics, metabolism, wine yeast, yeast technology

SUMMARY
An overview on the current status and future prospects of metabolic engineering of brewers
yeast are given. The knowledge on genetics, physiology and process behaviour and the
complete genome, make S.cerevisiae an attractive organism in this respect. New
developments allow metabolite analyses and measurement of intracellular metabolic fluxes,
and correlation of the data to the proteome and transcription of all genes. This information can
be used to design genetic changes to improve the yeast but also for the molecular monitoring
of yeast behaviour and adjustment of the process conditions accordingly. Specific and generic
examples of metabolic engineering of (brewers) yeast are given, as well as examples on wine
yeasts for comparison and inspiration.

Gentechnische Eingriffe in den Hefemetabolismus Chancen fr die Brauindustrie

Deskriptoren
Bierhefe, Genetik, Hefetechnologie, Stoffwechsel, Weinhefe

ZUSAMMENFASSUNG
Es wird ein berblick ber den momentanen Stand und die Zukunftsaussichten von
gentechnischen Vernderungen am Metabolismus der Bierhefe gegeben. Der Kenntnisstand
des genetischen, physiologischen und Produktionsverhaltens, sowie das komplett
entschlsselte Genom, machen S. cerevisiae zu einem attraktiven Organismus in dieser
Hinsicht. Neue Entwicklungen erlauben Metabolismusuntersuchungen und Messungen der
intrazellulren Stoffwechselwege und die Korrelation dieser Daten mit dem Proteom und
dadurch die Transkription aller Gene. Diese Informationen knnen zur Konstruktion
genetischer Vernderungen der Hefe verwendet werden, aber auch zur molekularen
berwachung der Hefe, um die Prozessbedingungen dementsprechend anzupassen. Spezielle
und allgemeine Beispiele von gentechnischen Vernderungen an der Bierhefe werden
gegeben sowie Beispiele zum Vergleich mit Weinhefe.
Stratgies d'ingnirie mtabolique opportunits pour la brasserie

Descripteurs
Gntique, levure de brasserie, levure de vin, mtabolisme, technologie de la levure

RESUME
Nous prsentons une vue d'ensemble de l'tat actuel et des perspectives futures d'ingnirie
mtabolique de la levure de bire. La connaissance de la gntique, de la physiologie, du
comportement en culture et du gnome complet de S. cerevisiae font de cette levure un
organisme intressant. Les nouveaux dveloppements permettent d'analyser les mtabolites,
de mesurer les flux mtaboliques intracellulaires and de corrler les donnes la transcription
et la traduction de tous les gnes. Ces informations peuvent tre utilises pour effectuer des
modifications gntiques visant amliorer la levure, mais aussi pour une surveillance
molculaire du comportement de la levure et pour l'ajustement en consquence des conditions
mthodologiques. Nous donnons des exemples spcifiques et gnriques d'ingnirie
mtabolique de la levure (de bire), ainsi que des exemples de levures de vin pour
comparaison et inspiration.

2
INTRODUCTION
Biological sciences are currently facing interesting and challenging times. The pros-
pect is that we will be able to understand in the future cellular functions and their in-
teractions at a completely different level than before. This possibility is brought along
with the increasing number of genomes sequenced and the bioanalytical tools, which
are being developed to gather and analyse the information obtained.
The genome data and new bioanalytical methods create new biotechnical opportuni-
ties. In particular metabolic engineering gains significantly from the possibility that
we are able to take into account all cellular reactions and in a quantitative manner.
The concept of metabolic engineering includes more than mere genetic modification
of the production organism (table 1). The aim is to have comprehensive understanding
of the metabolic reactions of the cell and quantification of the faith of the substrate
given to the organism, not only as a product output, but also within the metabolic
network of the cell. This provides means towards modification of the cell so that as
much as possible of the carbon provided to the cell is directed to the metabolite of in-
terest in a particular industrial process.

Metabolic engineering
Table 1: Expertise involved
The various expertise applied cur-
rently in metabolic engineering re- Genetic engineering
Protein engineering
search can also be used to create un- Biochemistry
derstanding of brewer's yeast and its Quantitative physiology
behaviour in the industrial process. Bioprocess engineering
Advanced analytics
Genome-wide approaches
Bioinformatics
Modelling of cell and process
functions

The aim in brewing is obviously not to have the highest possible yield of one com-
pound from the raw materials fed to brewer's yeast. On the other hand, beer is an ex-
cellent example of a product whose type and quality is very dependent on the physiol-
ogy of the process organism, and in particular of the balanced physiology in a rather
complex process. Thus, it would be beneficial for the brewer to be able to understand
the performance of brewer's yeast in its environment and to be able to control it
through process modifications, or if desired through mutagenesis or other types of ge-
netic modifications.

BREWER'S YEAST METABOLIC ENGINEERING

The brewing yeast researches were early active in genetic, and even metabolic engi-
neering. The first examples of successful genetic engineering were done to improve
filtration of beer through expression of a secreted -glucanase in brewer's yeast, to
produce amylolytic yeast strains, and to reduce the production of diacetyl (reviewed in
7). Despite the decrease in genetic engineering attempts in the turn of the 90's due to
hesitations towards GMO research, the progress in the last 15-20 years can be consid-
ered rather significant (see e.g. 3). Most of the targets for genetic engineering of
brewer's yeast listed by Roy Tubb in 1984 (table 2) have been studied and some of

3
them may even be considered to be at least partially fulfilled so that improved strains
suitable for industrial production have been generated. The benefits that the under-
standing of yeast molecular biology and genetics may provide for the brewer have
clearly been demonstrated.
Table 2: Targets for yeast genetics in brewing as listed by R.Tubb 1984 (8).
Raw material choice and cost Improvements in control
of fermentation and beer quality
proteolysis
zymocin/bacteriocin production
amylolytic yeasts
tags for strain identification
lactose utilisation
glucanolytic yeast
cellobiose,cello-
control of flocculation
oligosaccharides utilisation
improved maltotriose utilisation
pentose utilisation
foam and/or cling substances
Efficiency of fermentation
Control of beer flavour
controlled flocculation
adsorption of hop components
maltose utilisation
reduced diacetyl formation
osmotolerance
sulphur compounds
ethanol tolerance
staling compounds
reduced growth rate
reduced synthesis of acetate es-
New products or areas of profitable
ters at high gravity
business
Improvement of value of surplus yeast higher alcohols

The most straight forward and successful examples are those were a single gene, may
it be homologous or heterologous, encoding a secreted protein is expressed in the
yeast such as -glucanase, glucoamylase or protease. In these cases the overall yeast
physiology is not much affected. However, when the changes brought to the yeast
deal more directly with the cellular metabolism the outcome is usually more difficult
to predict and understand, and it may be strain and process dependant. Here the
(allo)polyploid nature of lager brewer's yeast (1) complicates first of all the genetic
modification work and further contributes to the complexity of the physiological re-
sponses. Additional studies and fine-tuning are needed to make the modified strains
acceptable for the process in terms of all brewing characteristics. It is perhaps not su-
prising that, for instance, the attempts to reduce diacetyl formation faced more diffi-
culties when the mitochondrial amino acid pathways of the yeast itself were modified
than when the bacterial -acetolactate decarboxylase gene was introduced into
brewer's yeast (reviewed in 7). In the latter case the new enzyme activity was ex-
pressed in the cytoplasm and dealt with only the excess of the amino acid pathway
intermediate -acetolactate, a precursor for diacetyl. However, the valuable under-
standing we have gained on the lager yeast physiology and flavour formation through
the studies on the yeast amino acid pathways is much more profound than that ob-
tained from the directly successful example on bacterial gene expression.
Considerable success in understanding, modification and control of beer flavour also
in terms of ester and higher alcohol formation, modification of sulphur metabolism,
and on preventing stale flavour formation, has already been made. However, we are
far from full understanding of the contribution of the yeast-derived factors on flavour
nor of all possible metabolic reactions of yeast that contribute in the changing process
conditions. The more complex the issue is and the more genes there are expected to be
involved, the less progress we have yet made. Such is the case for instance with at-
tempts to improve ethanol and osmotolerance, fermentation rate or pentose metabo-

4
lism (from the list in table 2). A more comprehensive picture of the metabolism and
the interrelationships of the individual reactions is needed.

DRIVERS FOR INCREASED UNDERSTANDING OF YEAST PHYSIOLOGY

Reasons for the performance of brewer's yeast even in the conventional batch brewing
process are not well understood. It is apparent that the process conditions and the
physiology of yeast are changing, and in an interrelated manner. But what kind of
physiological phases is the yeast going through and how do the external factors such
as wort composition, oxygen, sugar type and concentration, temperature, pressure, and
finally ethanol accumulation and nutrient starvation affect the yeast metabolism.
Which of these physiological and process phases are required and sufficient for the
production of good quality beer?
New technology, economy and market challenges are bringing up new questions con-
cerning yeast performance and increasing demands for a better understanding of the
physiology of brewing (table 3). Intensified fermentations are expected to pose the
brewer's yeast to new type of stresses brought about for instance by the type and con-
centration of the raw material sugars, or temperature. This may lead to changes in
physiology and problems concerning e.g. flavour formation. In continuous operations,
such as in immobilised systems, the yeast experiences a completely new environment.
Depending on the process, the yeast can be found in more than one physiological con-
dition even though the whole process may approach steady state. How does the biore-
actor design and process operation affect yeast behaviour? Is it feasible to think that
beer could be produced if the yeast does not go through several physiological phases?
How can one keep the yeast in the most productive state in these continuous opera-
tions, so that the process and/or the yeast remain in a steady state for a maximal
amount of time and high quality beer is produced?

Table 3.
Drivers for increased understanding of yeast performance

Intensified fermentations (VHGB, To)

stress factors (adjuncts; osmo-, ethanol and temperature tolerance)
New raw materials/New processes
effect on yeast physiology
Continuous operations
process design (e.g. column design, dilution rate)
effect on the physiological state of yeast
state of optimal yeast performance (fermentation rate, flavour formation)
vitality/ageing of yeast
New products
new processes impact on yeast physiology
flavour control
product stability
Process control and consistency
yeast function markers for process control
strain stability

The new process demands may create new targets for strain improvement, and in any
case new targets demanding understanding. These are the currently fashionable stress

5
responses such as oxygen, osmotic and heat stress. Also factors such as fermentation
rate, nutrient uptake and utilisation, energy metabolism, redox balancing, vitality and
ageing become more important to understand and control with the new process devel-
opments. Along with these general aspects regulating yeast performance, aspects af-
fecting flavour and product stability remain always important. In production of new
types of products these topics obviously have to be re-evaluated.
Process efficiency and consistent product quality demand monitoring and control of
the process operation and a quick reaction to any disturbances in an appropriate way
so that the operation can be continued successfully. Here it is critical to understand the
interplay of the yeast, raw materials and the process, and to have good and early indi-
cators for any unexpected changes in yeast performance.

Bioreactor
- (fed) batch
- immo
- chemostat

IN OUT
Nutrients, "Products""Byproducts"
oxygen, pH IN
? - ethanol - biomass
OUT
- flavours - off-flavours
- etc - etc
?

Extracellular analysis Cellular analysis

- On line/off line -currently off-line
- e.g. HPLC, MS,
- flow cytometry
GC, MIMS
- specific protein/mRNA/
activity assays
Metabolites & fluxes
Figure 1. rapid quencing and
Comprehensive understand- metabolite extraction
ing - and control - of the pro- HPLC, MS, NMR,
cess requires quantitative enzyme assays
analysis of the process pa- labeling and NMR/MS
rameters and the metabolism Genomics
of the yeast in the process. transcriptional profiling
proteomics

BIOANALYTICAL TOOLS WILL PROVIDE UNDERSTANDING ON YEAST

PERFORMANCE IN THE BREWING PROCESS
In order to gain insight into the interplay of the yeast and an industrial process, we
need to be able to measure the process and yeast performance parameters simultane-
ously (figure 1). This would allow us to make correlations, and with increasing infor-

6
mation gain understanding, which should make it possible, for instance, to develop
indicators of yeast performance to be used in process control.
Extracellular analysis from the gas or liquid phase from bioreactors can be done off-
line, but often also on-line. A new development in on-line methods is MIMS (mem-
brane inlet mass spectrometry), which allows measurement of (semi)volatile com-
pounds from the water phase. For instance, ethanol, acetaldehyde, higher alcohols and
esters can be measured during brewing (Ismo Mattila, VTT, unpublished).
The genome information has boosted up the development of a huge amount of various
kind of new bioanalytical tools for the analysis of cellular function and the responses
to environmental changes. The great advantage of the genome-wide methods such as
transcriptional profiling (arrays) (2) and proteomics (figure 2) (5) is, that in principle
(but not always in practise), all cellular reactions can be monitored simultaneously
from a particular condition, for instance from specific stages of fermentation. It would
simply not be possible to carry out the analysis for each cell reaction and gene func-
tion separately. In addition, one is not making any pre-assumptions as to which cell
reactions to measure, and thus - although in an "unintelligent way" - one may reveal
completely new responses of the cell and new yet unknown pathways, which can be
linked to a metabolic consequence such as a change in the flavour profile. A single
change in the yeast metabolic pathway will often have effects even on very distant
metabolic reactions, and these may be impossible to predict. Time course experiments
will provide information on the sequential reactions within the cell caused by a par-
ticular extracellular stimulus, and proteomics combined with analysis of changes in
protein phosphorylation, indications for signalling pathways. The genome-wide ap-
proaches may turn out to be particularly important for brewer's yeast researches be-
cause due to the polyploid nature of the yeast, the analysis of the effect of gene func-
tions is difficult through mutagenesis or classical genetics. Some possible applications
of genome-wide methods (transcriptional profiling, proteomics) are given in table 4.

2D-gel of brewer's yeast proteins For proteome

Laura Salusjrvi, VTT Biotechnology analysis the cellular
proteins are
separated on a 2D-
gel by their apparent
molecular weight
and pI. Each spot
represents a single
protein, which can be
identified by mass
MW spectrometry
100-15 combined with yeast
kDa genome data.
Comparison of spot
signal intensities
from proteomes
obtained from e.g.
different strains or
process conditions
provides information
on the changes that
have occurred. See
Figure 2. pI 3-10 also (5).

7
Table 4: Applications of genome-wide methods.

Analysis of genetic and physiological differences of different yeast strains

Analysis of the causes for strain instability
Analysis of the reasons for performance differences of a strain in different process conditions
Analysis of stress responses and vitality of the strain
Identification of a proteome/transcriptome profile of an optimally performing yeast
Discovery of novel pathways related to yeast performance
Discovery of (unknown) brewer's yeast specific proteins (through proteomics)
Cloning of (unknown) brewer's yeast specific genes (through proteomics)
Discovery of early intracellular triggers (e.g. signalling pathways) leading to a metabolic or per-
formance change of the yeast
Correlation of brewer's yeast performance at genomic and metabolic levels to process parameters
Collecting information for strain improvement approaches
Collecting information for process optimisation
Through accumulating information -
Identification of yeast performance markers for process control

The lager brewer's yeasts are partial species hybrids of still quite unknown nature (1),
suggested to carry in addition to S.cerevisiae genes also genes from closely related
species (S.monacensis, S.bayanus), and transcriptional profiling or proteomics meth-
ods would not give a complete picture. However, through proteomics also the proteins
originating from the non-S.cerevisiae strain can be detected. Since partial amino acid
sequences can be obtained from 2D-gel spots, even cloning of the corresponding gene
can be performed. This way those genes/proteins can be identified, which for instance
differ between particular strains or specifically respond to some process parameters.
Fortunately, new genome sequences are accumulating rapidly, and also sequences of
various yeast species including S.bayanus (4).
Mining the genome information has provided useful suggestions on putative pathway
genes to be tested for their possible relevance for brewing. A good example of the
power of genomics is the finding that there are about 20 putative sugar permeases in
S.cerevisiae. Without genomics, it would have been almost impossible to know this,
nor to proceed towards understanding the substrate specificity and regulation of ex-
pression of these individual permeases, a topic of high importance to the brewer as
well.
Through genome-wide methods we may want to ask questions such as: Does the yeast
genome have candidates, which would carry out a certain metabolic reaction or an
unwanted side reaction? Can we achieve the desired change in the yeast metabolism
by just changing the process conditions? Or can we modify expression of the genes of
the yeast itself rather than use heterologous genes in strain improvement? Is it reason-
able to think that mutagenesis and screening would provide the desired yeast strain.
And can we actually cause more variation in gene expression and metabolism by just
exposing the yeast to new environments than we may ever create by genetic engi-
neering?
What we still know very little about, is how the metabolite levels or metabolic rates
regulate cell function. The methods available for sensitive simultaneous analyses of a
large number of metabolites are still lacking. The metabolic flux analysis methods
such as the fractional enrichment labelling methods (6) combined with MS or NMR
are rather expensive and may currently provide limited information with eukaryotic
microbes due to compartmentalisation of some major metabolic pathways.

8
CONCLUSIONS
Brewers are in a very fortunate situation that (a major part of) the genome of their fa-
vourite organism is sequenced and so much of technique development has been cre-
ated in particular for S.ceverevisiae. There is also a huge amount of data on functional
analyses, protein-protein interactions, signalling pathways, expression of the genes in
various of conditions, etc. The biggest challenge is, however, how to store and handle
the information available, how to manage with data obtained at different cellular in-
formation levels (transcriptome, proteome, metabolome, functional analyses), and
how to correlate that with process parameters. And finally, one needs to make intelli-
gent conclusions, which would allow useful improvements in the process or the or-
ganism. Understanding of the "physionome" (figure 3) will be one of the key topics in
future and a major challenge for the researchers. These activities should also generate
opportunities for the brewers to improve the efficiency of their processes, and the
quality and variety of the products.

Fig. 3. PHYSIONOME
quantification
rates

GENOME
GENOME
GENOME

METABOLOME
RAW
MATERIALS
PROTEOME
PROTEOME
ENVIRONMENTAL
FACTORS
on-line/off-line
analytics
bioinformatics
modelling of the
PRODUCTS process and the
organism
Understanding the yeast physionome will help to keep the cell in the most
productive state, or to modify it for improved biotechnical performance

REFERENCES
1. Andersen, T.H., Hoffmann, L., Grifone, R., Nilsson-Tillgren, T. & Kielland-
Brandt, M.C., European Brewery Convention Symposium, Yeast Physiology - A
new era of opportunity, Nutfield, 1999, Monograph 28, 140-147.
2. DeRisi, J., Vishwanath, R. & Brown, P., Science, 1997, 278, 680-685.
3. European Brewery Convention Symposium, Yeast Physiology - A new era of
opportunity, Nutfield, 1999, Monograph 28.
4. FEBS Lett., 2000, vol. 487 (1).
5. Joubert, R., Brignon, P., Lehmann, C., Monribot, C., Gendre, F. & Boucherie, H.,
Yeast, 2000, 16, 511-522.
6. Maaheimo, H., Fiaux, J., Cakar, Z.P., Bailey, J.E., Sauer, U., Szyperski, T., Eur. J.
Biochem., 2001, 268, 2464-2479.
7. Penttil, M. & Enari, T-M., Biotechnology - Current Progress. Technomic Publish-
ing Co, Lancaster, 1991, 1, 173-202.
8. Tubb, R., Brewer's Guardian, Sept. 1984, 34- 37.

9
54

Influence of the acrospire of malted

barley on the flavour stability and other
quality parameters of beer
Achim Zrcher, M. Krottenthaler & W. Back
TU Mnchen, Lehrstuhl fr Technologie der Brauerei I, D-85350 Freising-
Weihenstephan, Germany (e-mail: [email protected])

Descriptors
Acrospire, grist, lipoxygenase, milling, oxidation, taste stability

SUMMARY
The acrospire of malt is enriched with lipids and lipid degrading enzymes. Further
constituents of the acrospire and the distribution of lipoxygenase (LOX) in malted barley are
presented. In brewing trials the influence of the acrospire on wort composition and beer
quality (e.g. foam, flavour and flavour stability) was shown. Furthermore the influence of
malt storage, grist storage and of malt conditioning before milling on lipid oxidation was
investigated. The impact of grist fineness and the mashing(-in) temperature on the extraction
and inactivation of LOX was investigated. Results indicate how lipid oxidation during wort
and beer production can be reduced.

Einfluss des Keimlings gemlzter Gerste auf die Geschmacksstabilitt und andere
Qualittsparameter von Bier

Deskriptoren
Blattkeim, Geschmacksstabilitt, Lipoxygenase, Oxidation, Schrot, Schroten

ZUSAMMENFASSUNG
Der Blattkeim von Malz ist reich an Lipiden und lipidabbauenden Enzymen. Weitere
Inhaltsstoffe des Blattkeims und die Verteilung der Lipoxygenasen (LOX) im Malzkorn
werden aufgezeigt. In Brauversuchen wurde der Einfluss des Blattkeims auf die Wrze-
zusammensetzung und die Bierqualitt (z. B. Schaum, Geschmack und Geschmacksstabilitt)
untersucht. Des weiteren wurde der Einfluss der Malz- und Schrotlagerung sowie der Malz-
konditionierung auf die Lipidoxidationsvorgnge untersucht. Die Bedeutung der Schrot-
feinheit und der (Ein-)Maischtemperatur auf die Extraktion und Inaktivierung der LOX wurde
untersucht. Die Ergebnisse zeigen, wie die Lipidoxidation bei der Wrze- und Bierbereitung
unterdrckt werden kann.
Influence de l'acrospire de l'orge malte sur la stabilit de l'arme et autres paramtres
de qualit de la bire

Descripteurs
Lipoxygnase, mouture (versement), moture, oxydation, plumule, stabilit organoleptique

RESUME
L'acrospire du malt est enrichi par des lipides et des enzymes de dgradation des lipides. Nous
prsentons d'autres constituants de l'acrospire et la distribution de la lipo-oxygnase (LOX)
dans l'orge malte. Dans les essais de brasserie, l'influence de l'acrospire sur la composition
du mot et la qualit de la bire (p. ex. mousse, arme et stabilit de l'arme) a t dmontre.
Par ailleurs, l'influence du stockage du malt, du stockage des grains et du conditionnement du
malt avant le broyage sur l'oxydation des lipides a t tudi. L'influence de la finesse du
grain et de la temprature du mlange sur l'extraction et l'inactivation de la LOX a galement
t tudie. Les rsultats montrent comment il est possible de rduire l'oxydation des lipides
pendant la production du mot et de la bire.

2
INTRODUCTION
The acrospire is a part of the seedling of germinating barley. It develops from the
embryo during the germination phase of malting and grows below the husk on the
dorsal side of the grain. Low molecular substances resulting from the degradation of
the reserve materials in the endosperm are transported via the ephithelial layer and the
scutellum to the embryo. The new plant tissue is generated for the development of the
acrospire and rootlets. The cells of these tissues remain intact during the whole
operation of malting. The growth of the rootlets and the acrospire is controlled by the
malting conditions. In view of the malting loss and in order to proceed modification
only to a certain extent the growth of the acrospire is limited and usually reaches two-
thirds of the length of the grain.
Lulai et al. (1) have shown that lipoxygenase (LOX) is confined to the germ, acrospire
and rootlets of the germinating barley. Furthermore the acrospire is rich in protein,
lipid material and lipases (2). Several investigations have demonstrated how LOX
activity and lipid oxidation can be influenced by the malting conditions (3, 4, 5).
However the production of pilsner malt always lead to a residual LOX activity in the
kilned malt (6).
Lipid oxidation during wort production occurs by enzymatic oxidation due to LOX
and by auto-oxidation. The oxidation products and their further cleavage to aroma
active compounds have an important influence on the flavour stability of beer (7).
Many efforts were made to minimize lipid oxidation during wort production (e.g.
fractionated milling, low oxygen pick-up in the brewhouse, lowering pH of mashes)
which have been shown to improve the flavour stability of beer.
Separation of the acrospire from malt is not possible without extract loss and
increased capital spending. However, a technological evaluation of the acrospire in
the brewing process gave beneficial information regarding lipid oxidation during wort
production and how beer quality can be affected.

MATERIALS AND METHODS

Acrospire fractionation
Acrospire material was taken from a destoner in a brewery. The material contains
acrospires and other small particles like fractured grains or husks that fall randomly
through the agitated sieves. The acrospire content of this material was increased by
two sieving steps whereby the fraction size 500 m < x < 1250 m was collected. A
further fractionation by form was performed with a trieur resulting in a nearly pure
fraction, except for some splittery husks. Husks were separated after fine milling in a
further sieving step (x = 250 m) as coarse fraction.
For the determination of the distribution of LOX in the kernel the huskless barley
Taiga was malted to give pilsner malt. The procedure used included 48 hours of
steeping at 15C, followed by 5 days germination at 15C and 100% humidity and a
24 hours kilning period. The malted barley was then polished thoroughly to remove
all rootlets and all acrospires. The material removed by polishing was separated into
acrospire and a rootlet fraction by multiple sieving steps.

Mashing and brewing trials

The finely ground malt and acrospires were mashed in laboratory mashings according
to the congress mash procedure. In pilot scale operation (30l) the infusion mashing
started at 50C. Rests for 45 minutes were kept at 63C and at 72C before heating up

3
to 78C for lautertun separation. For hopping, Pellets Typ 45 (110 mg -acid/l) was
used. Conventional fermentation (9.0C) and warm maturation (12.5C) was
performed until diacetyl accept. Hereafter the beers were kept at 0C for 1 week
before filtration.

Analytical methods
LOX activity in malt, grist and mash was determined after purification of the crude
extracts by gel filtration. The separation of hydroperoxide metabolizing enzymes and
inhibitors of lipoxygenase was performed on a Sepharose 12 prep grade column (1.6 x
30 cm). The LOX activity of the partial purified chromatography fraction was assayed
spectrophotometrically according to a modification of the method of Vick and
Zimmermann (8). LOX activity was determined by measuring the increase in
absorbance at 234 nm. In a typical assay 2.4 ml of 0.05 M potassium phosphate buffer
(pH 7.0) and 0.05 ml of freshly prepared enzyme extract were mixed in a 3 ml
cuvette. The reaction was initiated by the addition of 0.05 ml of recently thawed
substrate (8 mM). One unit of LOX activity is equivalent to an increase in absorbance
of one at 234 nm.
The high-pressure liquid chromatography (HPLC) system used for the analyses of
linoleic and linolenic acid hydroperoxides was a Hewlett Packard 1090 with a diode-
array UV detector. After a solid extraction method the hydroperoxides were separated
on a reversed-phase Kromasil column (125 mm x 4.0 mm i.d., M&W) and monitored
by their specific absorption at 234 nm. The eluent system was a mixture of
acetonitrile-water-acetic acid (80:19.95:0.05, v/v/v) at a flow rate of 1 ml/min. For
solid extraction, a C18 column (Chromabond C18ec) was consequently rinsed with 12
ml each of MeOH and H2O. Subsequently, 20 ml sample was eluted through the
column. Then the column was washed with 2 ml of H2O. Linoleic and linolenic acid
hydroperoxides were eluted with 8ml tetrahydrofuran. After being evaporated to
dryness, the residue was dissolved in 2 ml of acetonitrile. This solution was then
subjected to HPLC analysis.

RESULTS AND DISCUSSION

Composition of the acrospire
Table 1. Composition of acrospire and malt
Acrospire Malt
Amount / w/w % 4.8 100
Extract / w/w % dm 68.1 78.7
Moisture / w/w % 6.8 5.1
Protein / % dm 28.4 11.1
Kolbach index / % 37.4 42.3
DMS-P / mg/kg 20.5 6.3
Fat / w/w % 2.0 1.6
Fibre / w/w % 7.2 5.8
Zinc / mg/100g dm 4.84 1.14

The percentage ratio of the amount of the acrospire to the whole kernel of a well
modified malt was 4.8% (table 1). The extract yield of the acrospire fraction was
found to be lowered about 10% compared to malt. In contrast, the content of (soluble)

4
protein and DMS precursor in the acrospire fraction was approximately three fold
higher than in malt. Lipid and fibre material was increased about 20% compared to
malt. Interesting for brewers demand is that zinc content of acrospire material was
increased more than four fold.

Distribution of LOX activity in malted barley

The distribution of LOX in the malted huskless barley variety Taiga is shown in table
2. In a kilned malt the LOX activity was approximately found to be one fourth in the
acrospire, one fourth in the rootlets and one half in the residual kernel. The acrospire
of deculmed malt (free of rootlets) contains more than a third of the LOX activity of
the kernel.
Table 2. Distribution of LOX activity in malted Taiga

%-ratio of LOX activity

Fraction
kilned malt deculmed malt

Rootlets 24.7 -
Acrospire 26.7 35.5

Residual kernel 48.6 64.5

Malt storage
The effect of storage conditions on LOX activity of malt was investigated under
various conditions (figure 1). Malt storage at room temperature was related with a
decline of LOX activity of 25% in the first 2 months after kilning. The decrease of
LOX activity in the first month of storage was higher than in the following month.
When malt was stored under a carbon dioxide atmosphere, the decrease of LOX
activity at room temperature was almost at the same rate. The activity loss of LOX in
malt stored at -18C was negligible. Even though there is a remarkable decrease in
LOX activity during storage of malt, it can not be concluded that long storage periods
are beneficial in regard to flavour stability. To assess the consequences of extended
periods of malt storage further study regarding auto oxidation of lipids and LOX
activity at low aw in malt is necessary.

25

20
LOX-Activity (Units/g)

- 18 C
15
+ 20 C; Air
+ 20 C; CO2
10

0
0 1 2
month

Figure 1: Effect of temperature and atmosphere on LOX activity of stored malt

5
Grist storage
To evaluate the extent of lipid oxidation during prolonged storage periods of grist, the
concentrations of linoleic acid hydroperoxides (HPOD) and linolenic acid hydro-
peroxides (HPOT) were measured by HPLC. Since lipid oxidation occurs to free and
esterified fatty acids (7), grist samples were mashed to hydrolyse a normal quantity of
lipids before extraction. Results (figure 2) indicate that 6 hours of grist storage had
already led to significantly higher concentrations of linoleic and linolenic acid
hydroperoxides. When grist was stored for 60 hours (e.g. if milling is performed
before the weekend), or even longer (240 h), hydroperoxide levels after mashing were
noticeably decreased. This decrease can be ascribed to a further cleavage of
hydroperoxides to secondary lipid oxidation products.

14

12
Concentration (mg/l)

10

2
HPOD
0 HPOT
0 6 60 240
Time (h)

Figure 2: Influence of grist storage time on linoleic acid hydroperoxide (HPOD) and
linolenic acid hydroperoxide (HPOT) concentration in wort

Malt conditioning
Since the acrospire grows directly under the husk, heat treatment was applied with the
objective to inactivate LOX previous to the brewing process. Steam conditioning was
only efficient in reducing LOX activity of malt when water uptake was greater than
2% because of the steam condensation. Due to the high water uptake the
characteristics of these malts for dry milling were completely changed (data not
shown).
Inactivation of LOX by wet conditioning with hot water proved to be more effective.
Figure 3 shows the decrease of LOX activity of malts soaked in hot water. The
conditioning with water at a temperature of 80C reduced LOX activity by 58% of the
initial level within 0.5 minutes. One minute at the same temperature resulted in a
decrease of 75%. When malt was conditioned with water at a temperature of 50C or
65C, rates of inactivation were found to be much lower.

6
 18

LOX activity (Units/ml)

15

12
50 C
9
65 C
6 80 C

0
0 0,5 1
Time (min)

Figure 3: Decrease of LOX activity during wet conditioning of malt

Grist fineness
To investigate the impact of grist fineness on the solubilization of LOX activity three
grists characterised by different degrees of fineness were mashed for 5 minutes at
50C. An increased grist fineness resulted in a significantly higher LOX activity in the
mash extracts (figure 4A). Compared to the Lautertun grist, the rise of activity was
found to be 21% and 30% for the Mashfilter grist and powder grists respectively.

(A) (B)
3
4
Std. + 5 % acrosp.
LOX activity (Units/ml)
LOX activity (Units/ml)

(fine milled)
3 Std. + 5 % acrosp.
2 (intact)
Standard (Std.)
2

1
1

0
Lautertun Mashfilter Powder 0
grist grist grist 0 20 40 60
Time [min]
Figure 4: Impact of grist fineness (A) and acrospire fineness (B) on solubilized LOX
activity in mash

The improved solubilization of LOX by increased grist fineness was found to be

pronounced by the fineness of the acrospire (figure 4B). When acrospire material (5%
of the throw) was added to a standard grist without being milled, only a slight increase
of LOX activity was detectable during 60 minutes of mashing at 45C. In contrast,
when the same amount of acrospire material was fine milled, LOX activity was
already significantly increased shortly after mashing-in and the higher level of LOX
activity was found to persist throughout the entire mashing process.

Mashing temperature and heat stability

Heat stability of LOX was studied under mashing conditions by incubating ground
malt samples in H2O for 60 minutes at varying temperatures (malt:water ratio 1:4).
The LOX activity in the mash was already decreasing at a mashing temperature of
45C (figure 5). At 50C the activity was totally lost in 60 minutes, and at 60C the

7
activity was totally lost in 30 minutes. When mashing was performed at 65C, the
decrease of LOX activity was even more rapid, although 20 minutes after mashing-in
a slightly residual activity was still detectable.
When evaluating the heat stability of LOX under mashing conditions by measuring
the activity in the liquid phase of the mash, simultaneous solubilization and
inactivation makes interpretation of results difficult. Under such experimental
conditions LOX might appear more heat stable than it really is, since gradual
solubilization of activation could mask inactivation in the liquid phase. But even
though LOX is not stable under mashing conditions the experiments confirmed that
LOX activity can be reduced drastically by manipulating the mashing parameters.

2,5
LOX activity (Units/ml)

2,0

1,5 45C
55C
60C
1,0 65C

0,5

0,0
0 10 20 30 40 50 60
Time (min)

Figure 5: Decrease of LOX activity in isotherm mash

Brewing trials
In order to elucidate the influence of the acrospire extract on wort and beer quality,
brews with a commercial pilsner malt and with the addition of 5% acrospire material
to the throw were carried out in pilot scale. Wort analysis of Standard wort and of
Acrospire wort is shown in table 3.

Table 3: Composition of pitching worts. Standard wort: brewed with 100% malt;
Acrospire wort: brewed with 5% acrospire material as substitute for malt

Standard Acrospire
wort wort
Extract / w/w % dm 78.7 77.3
Fermentability / % 82.0 81.7
pH 5.91 5.97
Colour / EBC 5.0 5.5
FAN / mg/l 228 241
total N / mg/l 1061 1178
coag. N / mg/l 15.6 17.1
-Glucan / mg/l 175 170
Polyphenols / mg/) 122 118
Anthocyanogene / mg/l 64 60
Zinc / mg/l 0.13 0.16

8
The addition of 5% acrospire material resulted in some relevant differences in the
constituents of wort. Noticeable increases were found for zinc (+ 23%), total N (+
11%) and FAN (+ 6%) content in the Acrospire wort. In the Acrospire wort, slightly
lowered values for the extract yield, apparent fermentability and the content of
phenolic compounds were observed. A slight increase was detected for the pH of
Acrospire wort. Concentration of DMS and DMS precursor differed only slightly
(data not shown).
Further process control could not point out remarkable differences during
fermentation of worts, and the maturation and filtration of beers. Beer analysis and the
results of sensory evaluation of the beers from the brewing trials are shown in table 4.
Composition of beers showed similar tendencies for extract, pH, colour, and FAN that
were already observed in the corresponding worts. The Acrospire beer differed
mainly in a higher content of total N, and coag. N. There is a possible correlation
between the higher content of total N and coag. N and a better foam stability and a
declined haze stability of the Acrospire beer. To evaluate flavour and flavour stability
the beers were applied to a forced ageing test (1 day shaking, 4 days at 40C)
followed by sensory evaluation of the beers at our expert panel. Acrospire beer was
characterised by a conspicuous spicy and fruity aroma. The bitterness of the beer
came as an aftertaste. Furthermore, flavour stability of the Acrospire beer was judged
to be worse than that of the Standard beer (Alterungsverkostung (9)).
Table 4: Beer analysis and results of sensory evaluation of Standard beer and
Acrospire beer
Standard Acrospire
beer beer
Original Extract / % P 11.2 11.0
pH 4.52 4.56
Colour / EBC 4.0 4.5
FAN / mg/l 119 125
total N / mg/l 791 826
coag. N / mg/l 14.4 16.0
Bitterness / EBC 25 26
Foam / (R&C) 126 130
Foam / s (Nibem) 278 288
Haze / WT 4.0 3.1
Alterungsverkostung 2.0 2.5
DLG-Tasting 4.2 3.9

The concentrations of wort and beer aroma compounds were studied and evaluated
(data not shown). Typical indicators for lipid oxidation, like hexanal, heptanal and
tr,2-cis,6-nonadienal were found to be slightly increased in the Acrospire wort.
Acrospire beer proved to contain a larger quantity of strecker aldehydes, like
methional and 2-me-butanal. Diacetyl and DMS concentrations were well below
flavour threshold.

9
CONCLUSIONS
The acrospire of malt is rich in proteins, lipids and lipoxygenase (LOX). Further
constituents of the acrospire and the distribution of LOX in malted barley are
presented. Grist fineness and acrospire fineness were of notable influence on the
solubilization of LOX in mash. Grist storage periods below 6 hours led to a significant
increase of HPOD and HPOT in worts. When grist storage was longer than 6 hours, a
cleavage of hydroperoxide was detected, indicating secondary lipid oxidation during
grist storage. Furthermore, the potentiality of extraordinary heat treatment during malt
conditioning was tested for inactivation of LOX. The impact of the mashing(-in)
temperature on the solubilization and inactivation of LOX was investigated. Results
indicate that the inactivation of LOX during mashing is most efficient when mashing
starts at temperatures above 60C, even if LOX is already inactivated when mashing
is performed at 45C.
In brewing trials the influence of the acrospire on wort and beer composition as well
as on flavour and flavour stability of beer was investigated. Beer with an added
amount of 5% acrospire material (Acrospire brew) was compared to an allmalt beer
(Standard brew). In the Acrospire wort, noticeable higher concentrations of zinc, total
N, and FAN were found. Furthermore slightly lowered values for the extract yield,
and apparent fermentability were measured. The Acrospire beer differed in a higher
content of total N, and coag. N than Standard beer. This could possibly be correlated
with a better foam stability and a declined haze stability of the Acrospire beer.
Standard beer was chosen by sensory evaluation. The flavour of Acrospire beer was
characterised by a conspicuous spicy and fruity aroma. Moreover bitterness came
through as an aftertaste. Due to the addition of acrospire material flavour stability of
beer was found to decline.

ACKNOWLEDGEMENTS
Work was supported by the Wissenschaftsfrderung der Deutschen Brauwirtschaft
e.V., Bonn, grant B61.

REFERENCES

1 Lulai,E. C.; Baker C.W.: The Altereation and Distribution of Lipoxygenase in Malting Barley and
in Finished Malt. ASBC-Proc. (1975), 33, 154-158.
2 Baxter, D.: Lipoxidases in malting and mashing. J. Inst. Brew. (1982), 88, 390-396.
3 Kaukovirta-Norja, A.; Reinikainen, P.; Laakso, S.; Olkku, J.: Lipoxygenase activity during malting
and storage of malt. EBC Proc. (1995), 193-200.
4 Ketterer, M.: Untersuchungen zum Fettstoffwechsel bei der Keimung und der hieraus resultierenden
Metaboliten. Dissertation TU Mnchen-Weihenstephan. 1994.
5 Yang, G.; Schwarz, P. B.: Activity of Lipoxygenase Isoenzymes During Malting and Mashing.
Journal ASBC 53, 2 (1995), 45-49.
6 van Waesberghe, J.M.W; van Waesberghe W. J.M.: The impact of Lipoxygenase and Microflora
Management on Flavour Stability. EBC Monograph (1994). 44-61.
7 Kobayashi. N.; Kaneda, H.; Kano, Y.; Koshino, S.: Behavior of Lipid Hydroperoxides During
Mashing. Journal ASBC 1994, 141-145.
8 Vick, B. A.; Zimmermann, D.C.: Lipoxygenase and hydroperoxid lyase in germinating water melon
seedlings. Plant Physiol (1976) 57, 780.
9 Narzi, L.; Miedaner H.; Eichhorn, P.: Untersuchungen zur Geschmacksstabilitt des Bieres.
Monatsschrift fr Brauwissenschaft (1999), 3/4, 49-57; 5/6, 80-85.

10
55

Development of novel malt evaluation

method for improving beer flavor
stability
T. Ueda1, K. Sasaki1, K. Inomoto1, K. Kono1, N. Kagami2, K.
Shibata1 & M. Eto1
1
Asahi Breweries Ltd., Brewing Research & Development Laboratory, 1-1-21 Midori, Moriya-
machi, Ibaraki 302-0106, Japan (e-mail: [email protected])
2
Asahi Breweries Ltd., Suita Brewery, 1-45, Nishinoshomachi, Suita, Osaka 564-0071, Japan

Descriptors
Malt constituent, 2-nonenal, staling, taste stability

SUMMARY
An early malt indicator of trans-2-nonenal (T2N) formation in stale beer has been
investigated. For this purpose, pilot brewing trials were carried out, using twenty-three malt
samples. The T2N formation levels in stale beer were highly correlated with the trans-2-
nonenal potential of malt (M-T2N-P). This potential, determined by the nonenal formation
levels of a specially prepared wort, is analyzed by GC-MS in combination with an automated
solid phase extraction system. The M-T2N-P levels varied considerably among more than
300 malt samplestested, suggesting this indicator is useful for evaluating malt on the basis of
beer flavor stability.

Entwicklung einer neuen Methode zur Malzbeurteilung, um die Geschmacksstabilitt

von Bier zu verbessern

Deskriptoren
Altgeschmacksbildung, Geschmacksstabilitt, Malzbestandteil, Nonenal-2

ZUSAMMENFASSUNG
Es wurde ein frher Malzindikator fr die trans-2-Nonenal-Bildung (T2N) in gealtertem Bier
untersucht. Zu diesem Zweck wurden Bierproben einer Pilotanlage aus 23 Malzsorten
getestet. Der T2N-Bildungspegel im gealtertem Bier korrelierte stark mit dem trans-2-
Nonenal-Potenzial (M-T2N-P) des Malzes. Dieses Potenzial, wird durch speziell aufbereitete
Wrze und einem GC-MS in Kombinationen mit einem automatischen Festphasen-
Extraktionssystem bestimmt. Der M-T2N-P-Pegel von mehr als 300 verschiedenen
untersuchten Proben, deutet auf einen hilfreichen Indikator zur Beurteilung von Malz in
Bezug auf die Geschmacksstabilitt in Bier hin.
Dveloppement d'une nouvelle mthode d'valuation du malt pour ameliorer la stabilit
de l'arme de la bire

Descripteurs
Constituant du malt, formation du got dvente, nonnal-2, stabilit organoleptique

RESUME
Nous avons tudi un indicateur prcoce de formation de trans-2-nonnal (T2N) dans la bire
vieillie. A cette fin, des essais pilotes de brassage ont t conduits avec 23 chantillons de
malt. Les niveaux de formation de T2N dans la bire vieillie taient fortement corrls au
potentiel trans-2-nonnal du malt (M-T2N-P). Ce potentiel, dtermin par les niveaux de
formation du nonnal dans un mot spcialement prpar, est analys par CG-SM en
association avec un systme d'extraction en phase solide automatis. Les taux de M-T2N-P
prsentaient de trs importantes variations entre les > 300 chantillons de malt tests, ce qui
suggre que cet indicateur est utile pour valuer le malt sur la base de la stabilit de l'arme
de la bire.

2
INTRODUCTION
Though brewers have made numerous attempts to improve the flavor stability of beer
for the past decades, beer still has relatively poor flavor stability and consumers will
recognize the flavor degradation of beer products after the extended storage period.
While a variety of stale flavor characteristics are noted among pale lager-type beers,
there are many beer brands that are prone to the formation of a cardboard-like flavor.
This flavor can become noticeable even
before the suggested expiry date, if the 0.4
brands are stored under adverse conditions. = 0.80
It is therefore very important to further

Score of Stale
0.3
develop a new brewing technology, which
allows the improvement of beer flavor
stability. In our sensory evaluation for some 0.2
brands, the typical stale character was 0.1 0.2 0.3 0.4 0.5
described as cardboard-like flavor, the T2N (p p b / 3 7 C 1 week )
intensity of which showed a high correlation
with trans-2-nonenal (T2N) formation Fig.1: Relationship between T2N
(figure 1). It was thus considered important and score of stale in aged beer
to minimize the T2N formation in stale beer.
While a variety of technologies have been implemented through the entire brewing
processes to reduce the cardboard flavor (1,2,3), it is the focus of our study to reduce
T2N on the basis of malt quality. The oxidation process of lipid compounds by
oxidases (4,5,6,7), including lipoxygenase, and the antioxidants (8,9) in malt have
been investigated to reduce the T2N levels. In some studies, nonenal potential in wort
has been used to predict the T2N formation in stale beer (9,10,11). It has therefore
been anticipated that the analysis of nonenal potential could be successfully applied to
the malt samples. This led us to investigate the application of nonenal potential to
evaluate malt quality in terms of beer flavor stability.
As a result, M-T2N-P (malt T2N nonenal potential) was found to be significantly
correlated with the T2N formation levels in stale beer brewed in our pilot plant. This
result indicates that M-T2N-P M ashing
P rocess Fresh Stale
analysis is an effective means of M alt W ort B eer
one day B eer
one m onth stored for
selecting malt on the basis of several weeks
N onenal
flavor stability. The main point of w ort analysis T 2N
analy sis
this report is to develop a new preparation
efficient malt evaluation method lab.wort N onenal-
P otential P re d ic tio n T 2N
using the nonenal potential formation
accurately and efficiently (figure Nanalysis
onenal Auto m ated so lid
extraction system
2). In addition we report the
results of brewing trials performed M -T 2N -P
on a commercial scale using low Fig.2: Development of malt evaluation method
M-T2N-P malt samples, and
further discuss several factors affecting M-T2N-P.

MATERIALS AND METHODS

Wort preparation and determination of malt T2N potential (M-T2N-P)
Malt was ground using a Buhler Universal Laboratory Disc Mill (type DLFU) with 0.2

3
mm gap. 60 g of the finely ground malt was mixed with 300 ml water (50C) adjusted
to 10dH with calcium sulphate. The mash was prepared, using a mashing apparatus
with a cover put on the beaker according to the mashing diagram developed in this
study. The mash was then centrifuged (7,500 rpm, 15 min.) and the supernatant was
filtrated through a No.2 filter paper. The nonenal potential of the filtered wort was
determined by the following method.

Determination of nonenal potential (T2N-P)

T2N-P was determined based on the method described by Drost et al (9). Wort
samples were centrifuged, adjusted to pH 4.0 with phosphoric acid, and subsequently
heated in a closed tube for 2 hours at 100C. An internal standard (10-undesenal) was
added for GC-MS analysis, followed by the derivatization with PFBHA
(pentaflulobenzyl-hydroxylamineHCL). T2N was extracted in a solid phase using Sep-
Pak C18 Light and quantified by gas chromatography with a mass spectrophotometer.
To process simultaneously a large number of samples by solid phase extraction, an
automated solid phase extraction system manufactured by Moritex Corporation
(Tokyo Japan) was used.

Determination of T2N in stale beer

T2N was determined by our routine method.

Pilot brewing trials

Twenty-three brewing trials were carried out on a 200 l pilot-plant scale, using a
single malt sample for each trial in order to investigate the effects of M-T2N-P on
T2N formation in stored beer.

Sensory evaluation results

Sensory evaluation by our trained panelists was conducted to examine the stale flavor
intensity of packaged beers stored for one month at 25C and for one week at 37C
respectively.

Commercial brewing trials

Brewing trials were carried out according to our standard production procedures,
using the same brewing conditions other than the ratio of malt blending.

Malt sample
Approximately 300 two-row spring malt samples were selected out of domestic and
imported malts produced in 1999 obtained from malt suppliers worldwide.

Malt analysis
Standard malt analyses were performed, using EBC methods to examine the
relationship with M-T2N-P.
Lipoxygenase activity was determined according to Baxter (12). Malt extracts for the
measurement of Lipoxygenase activity were made after the 5-min incubation of 60 g
malt meal (Buhler-Miag mill 0.2 mm) in 300 ml of water at 50C in a mash bath.
Samples were immediately filtrated through No.2 filter paper. 50 micro-liter of
enzyme extract was incubated in 2.85 ml of buffer at 25C.

4
RESULTS AND DISCUSSION
Optimization of mashing conditions for the determination of M-T2N-P
The preparation of a wort on Temp. (C)
laboratory scale was optimized for
the measurement of M-T2N-P. For 76
this purpose, several conditions of 65
Saccha. Rest
EBC methods were modified,
Protein Rest
including milling, mashing 50
diagram, grist to water ratio and
water hardness. grist / water = 1/5

When the milling conditions were

compared between the fine and Time (min)
60 10 40 16
coarse milling, the fine milling
allowed better solubilization of Fig.3: Mashing Diagram for mash preparation
enzymes and various components, in the measurement of M-T2N-P
including lipoxygenase, and
resulted in the formation of higher
levels of T2N. From these reasons, it was considered that the adoption of fine milling
enables a more reliable analysis of M-T2N-P.
The grist to water ratio was also investigated. In our experiment, T2N potential was
found to rise as the volume of mashing water increased relative to the grist malt.
Therefore we selected a low mash concentration (grist malt : brewing water = 1 : 5) to
improve the reliability of M-T2N-P analysis.
In addition, the mashing diagram described in the EBC method was modified as
shown in figure 3 to approximate the condition (pH, time and temperature) for the
enzymatic reactions, occurring in commercial brewing, and water hardness was also
adjusted with calcium sulphate.

Pilot plant study on relationship

between malt and T2N formation in (ppb)
stale beer 0.3 = 0.87**
i n stale beer

The relationship between malt

T2N

selection and T2N formation in stored 0.2

beer was studied using our pilot plant.
Malt samples were obtained that 0.1
covered a variety of barley growth
0.0
locations, malting companies and
0 10 20
barley varieties. More than 50 samples (ppb)
were chosen and their M-T2N-P was M-T2N-P
analyzed. Twenty-three malts with a Fig.4: M-T2N-P is highly correlated with
wide range of M-T2N-P (5.7 to 21.l T2N formation in pilot brewing trials
ppb) were selected for these trials.
As a result, a high positive correlation (r = 0.87**) was found between M-T2N-P and
T2N formation in stale beer (figure 4). It was also shown the reduction in M-T2N-P
from 20 ppb to 15 ppb caused 33% decrease in T2N formation in the beer stored at
37C for a week.
The high correlation found in this experiment, was presumably attributed to the
following factors: a) wort preparation conditions were optimized by modifying the

5
mashing diagram in the EBC methods to approximate the commercial scale enzymatic
reactions, b) the target compound, T2N, was directly measured, using wort prepared
under the optimized conditions.
It has been known that the oxidation reaction of lipid compounds proceeds via
complex interactions between the reducing compounds (polyphenols, melanoidines,
etc.) and oxidation-related enzymes (lipoxygenase, polyphneol oxidase, peroxidase
etc.). Given the complicated nature of this process, it was difficult to determine
reliably the effects of malt selection on T2N formation in stored beer.
In our view, T2N represents one of the principal final products in oxidative reaction.
Therefore, in developing our method for determining M-T2N-P, nonenal potential
(T2N-P) in wort was targeted in place of the individual indicators used in other
studies, such as precursor of T2N, antioxidants and oxidase activities.

Commercial brewing trials for evaluating the effects of malt selection

The pilot-plant brewing trials showed that the selection of low T2N malt was effective
in reducing T2N. Therefore we conducted brewing trials on a larger commercial
brewery scale in an attempt to further verify the effect of low T2N malt selection. At
the same time, various sensory evaluations were carried out to clarify the effect on
beer flavor.
For brewing trials, several malt samples with different M-T2N-P were mixed to
prepare wort, in contrast to the pilot-plant trials that had used single-malt sample for
each trial. Malts samples were first screened for M-T2N-P and malt blending ratios of
high T2N and low T2N malts were determined.
It was found that the T2N formation levels and score of staling were both lower when
low T2N malt was used (table). In the sensory evaluation, a statistically significant
number of panelists favored the beer produced with low M-T2N-P malt. The similar
tendency was also observed in trials using other commercial brewing plants (data not
shown).
Table : Commercial Brewing trials using malts with different M-T2N levels
High M-T2N-P Low M-T2N-P
malt malt
Harrington Harrington,
Malt Barley Variety of malt used Scarlett, Arapiles, Scarlett, Arapiles,
Samples Asaka-gold, etc Asaka-gold, etc
M-T2N-P(*1) (ppb) 12.4 10.8
T2N-P in cold wort 100 87
Relative T2N T2N in stale beer 37C/ 1W (*3) 100 66
value (*2) T2N in stale beer 25C /1M (*4) 100 68
Number of Panelists 37C1W 5/30 24/30
Sensory (*5) 50/76
in favor 25C1M 11/76
Evalu- 37C1W 0.51 0.36
Score of Staling
ation 25C1M 0.33 0.26
(*1) Weighted average of M-T2N-P for malt used
(*2)T2N values were normalized with high M-T2N-P malt trial being 100.
(*3)Beer stored at 37C for a week
(*4)Beer stored at 25C for a month
(*5)The denominator indicates the total number of panelists participating in the sensory
analysis.

6
Identification of factors having effects on M-T2N-P
Differences of malt quality among malting companies in Europe were investigated in
terms of M-T2N-P (figure 5). The analysis indicated that the average values of M-
T2N-P and its range of distribution varied, depending on malting companies. A
similar trend was observed for the malting companies located in other barley growth
areas (data not shown).
Furthermore we investigated the differences between malting plants. The malting
company H in figure 5 was chosen for this study, because it had shown the widest
range in M-T2N-P values. The data showed that significant differences in M-T2N-P
values were observed for the two malting plants operated by the company H (figure
6). Similarly, the significant differences were also found between two malting plants
operated by another company A.

25 M a x .
(ppb)

A v e .
M- T 2 N - P

20 M i n .

15

10

5
M altin g
A B C D E F G H I J
Com pany
(1 0 ) (7 ) (3 ) (8 ) (8 ) (4 ) (4 ) (1 0 ) (3 ) (5 )

Fig.5: M-T2N-P range of different Malting companies in Europe

Effect of barley varieties and standard

25
malt analysis parameters
(ppb)

Barley varieties, standard malt analysis 20

parameters and lipoxygenase activity were
Max.
examined as possible candidates which 15 +SD
M-T2N-P

Ave.
may affect M-T2N-P, to identify factors -SD
other than the difference between malting 10 Min.
companies. 5
Among the barley varieties studied, one
variety showed some variation in M-T2N- 0
P, but the overall differences observed for plant X plant Y
( n= ) (14) (3)
barley varieties were not so significant as
those found among malting companies Fig.6 : Comparision of M-T2N-P
and plants (figure 6). All the malting between two malting plants
companies were segmented to determine using identical barley variety
the relationship between M-T2N-P, the
standard malt analysis parameters and lipoxygenase activity. We found that there were
a few malting companies where some analysis parameters (protein and water content
etc.) were slightly correlated with M-T2N-P. But, among all the malting companies
examined, no overall significant differences were noted between M-T2N-P and the
standard malt analysis except lipoxygenase activity. There was not a relatively high
correlation(r = 0.49) between M-T2N-P and lipoxygenase activity, which would

7
suggest that residual lipoxygenase activity of malt is not closely linked to M-T2N-P.
This is presumably because many other factors such as antioxidants also influence the
production of nonenal and the absolute amount of lipoxygenase activity varied in
various experiments (9,13).
The lack of correlation between M-T2N-P and barley varieties / standard malt analysis
parameters was presumably due to the fact that all the malt samples surveyed in this
study were within the typical pale-lager malt parameters for the malt, leading to the
narrow range of analyzed values for respective parameters. Our results suggest that M-
T2N-P depends on malting companies and / or plants rather than barley varieties.
These results indicate that there exist certain factors in the malting process and / or in
barley quality that affect the formation of T2N.

CONCLUSION
While the T2N formation in stale beer is influenced by milling, mashing and
fermentation process in addition to the beer type, our study indicates that malt quality
also plays an important role for reducing T2N formation in stale beer.
M-T2N-P identified in our study was highly correlated with the T2N formation levels
in pilot plant trials. The significance of the M-T2N-P was also confirmed in a
comparative commercial-scale studies evaluating beer flavor stability based on T2N
formation in stale beer and sensory tests. In addition, the level of M-T2N-P was found
to depend considerably on the malting process and / or barley quality, while the
standard analysis parameters for all the malt samples were within the standard range
of the specifications for two-row spring malts.
We have therefore concluded that the determination of the M-T2N-P will help us
predict the flavor stability of beer products on the basis of malt quality.

ACKNOWLEGEMENT
The authors wish to thank Asahi breweries, ltd. for allowing the publication of this
paper.

REFERENCE
1. Back, W., Forster, C., Krottenthaler, M., Lehmann, J., Sacher, B., Thum, B.,
Brauwelt Inter., 5, 394-405, 1999
2. Yamaguchi, I., Ueda, T., Ujihara, S., Yamada, M., and Fukushima, S., Proc. 26th
EBC Congress, Maastricht, 1997, 247-255
3. Herrman, H., Master of Brewing Asssociation of America, 36, 1, 49-54, 1999
4. Yabuuchi, S. and Yamashita, H., J. Inst. Brew. 85, 216-218, 1979
5. Schwarz, P.B. and Pyler, R.E., J. Am. Soc. Brew. Chem., 47-53, 1984
6. Kobayashi, N., Kaneda, H., Kano, Y. and Hoshino, S., J. Am. Soc. Brew. Chem.,
52, 141-145, 1994
7. Van Waesberghe, J.W.M., Aural presentation at 2nd International Congress
organized IFBM and ENSAIA in Nancy, 2000
8. Liegeois, C., Lermusieau, G. and Collin, S., Proc. 27th EBC Congress, Cannes,
1999, 461-468

8
9. Drost, B.W., Van den Berg, R., Freijee, F.J.M., Van der Velde, E.G. and
Hollemans, M., J. Am. Soc. Brew. Chem., 48, 124-131, 1990
10. De Buck, A., Aerts, G., Bonte, S., Dupire, S. and Van den Eynde, E., Proc. 26th
EBC Congress, Maastricht, 1997, 333-340
11. Idota, Y., Poster presented at the MBAA, 1999
12. Baxter, E.D., J. Inst. Brew., 88, 390-396, 1982
13. Kaukovirta-Norja, A., Helsinki University of Technology Report, 4, 1998

9
56

Improvement of flavour stability by

reduction of trans-2-nonenal a case
study
Olav Vind Larsen, Sten Aastrup, Henning Nielsen & Anne
Cathrine Lillelund
Alfred Jrgensen Laboratory Ltd., Frydendalsvej 30, DK-1809 Copenhagen-Frederiksberg C,
Denmark (www.ajl.dk, e-mail: [email protected])

Descriptors
2-Nonenal, pH, sparging liquor, taste stability

SUMMARY
The poster relates to a Brewery Group experiencing flavour stability problems in a light lager
from one brewery. Expert Taste Panel evaluation and Trans-2-Nonenal, (T2N - the papery
off-flavour in stale beer) measurement showed that the level of T2N in beer correlated very
well with the flavour stability as perceived by the taste panel following forced ageing tests.
Following an analysis the differences in production processes and parameters at the group
breweries in combination with flavour evaluation and analysis of T2N, improvements were
made, primarily by lowering pH of the sparge water from approx. 6.7 to 5.5.

Verbesserung der Geschmacksstabilitt durch Reduktion von trans-2-Nonenal Eine

Fallstudie

Deskriptoren
Geschmacksstabilitt, Nonenal-2, pH, berschwnzwasser,

ZUSAMMENFASSUNG
Das Poster bezieht sich auf die Erfahrungen einer Brauereigruppe mit dem Problem der
Geschmacksstabilitt eines leichten Lagerbieres in einer Brauerei der Gruppe. Ein Experten-
Panel und die trans-2-Noneal (T2N der Papiergeschmack in gealtertem Bier) Messung
ergaben, dass der Gehalt an trans-2-Nonenal im Bier sehr gut mit der Geschmacksstabilitt
von Proben korrelieren, die einer knstlichen Alterung unterzogen wurden und durch ein
Verkosterpanel untersucht wurden. Daran schloss sich die Analyse der Unterschiede im
Produktionsprozess und der zugehrigen Parameter in der Brauereigruppe an. Dies, die
Evaluierung des Geschmacks der Biere der Gruppe und die Untersuchung auf T2N fhrten zu
Verbesserungen im Brauprozess, vor allem zu einer Absenkung des berschwnzwassers von
ungefhr 6,7 auf 5,5.
Amlioration de la stabilit de l'arme par rduction du taux de trans-2-nonenal tude
de cas

Descripteurs
Eau de lavage des drches, nonnal-2, pH, stabilit organoleptique

RESUME
Ce poster dcrit les problmes de stabilit de l'arme du groupe de brassage dans une bire
lgre d'une brasserie. L'valuation par un panel de goteurs experts et le dosage du trans-2-
nonnal (T2N arme de papier-carton) dans la bire vieillie ont montr que le taux de T2N
dans la bire tait trs bien corrl la stabilit de l'arme peru par le panel d'experts aprs
tests de vieillissement forcs. Aprs avoir analys les diffrences des procds et paramtres
de production dans les brasseries du groupe en association avec l'valuation de l'arme et
l'analyse du T2N, des amliorations ont t apportes, essentiellement en abaissant le pH de
l'eau de lavage de 6,7 env. 5,5.

2
INTRODUCTION
The flavour stability of a beer is a key quality parameter for most breweries, and the
industrys focus on improving flavour stability is growing. A marked change in
flavour during the shelf life of a beer can be perceived as product inconsistency and
can have a damaging effect on the brand perception.
A range of components is known to contribute to the off-flavours in stale beer, e.g.
furfurals, benzaldehyde and trans-2-nonenal.
Trans-2-nonenal (T2N) is the papery off-flavour in stale beer, and can be used as a
useful marker for monitoring changes in flavour stability.
This paper describes the application of a practical method for flavour stability
improvement, based on expert taste panel evaluation and trans-2-nonenal analysis.

METHODS
All beer samples were produced in a series of full scale trial productions and
submitted to the following examinations:

Flavour stability
The beers analysed in this study were submitted to a forced ageing test. Samples
stored at 7 days at 0C were compared to samples stored for 7 days at 38C. This
method is comparable to approximately 2 months storage at 20C.

Flavour evaluation
Fresh and forced aged beers were tasted at random by an expert taste panel using a
flavour profile report with 40 different properties (range: 0-3), and a general
evaluation score (GE range: 1-9).
A difference of more than 0.5-1.0 in GE between fresh and aged beer is considered as
unsatisfactory flavour stability.

T2N analysis
T2N was analysed according to a method developed by Carlsberg Research Center
using a solid phase extraction followed by GC-MS analysis (1). The method has a
detection limit below 0.01 ppb.
The flavour threshold of T2N in beer is in the range 0.05 ppb (Carlsberg (1)) to 0.11
ppb (Meilgaard (2)).

CASE STORIES
Case 1
A regional brewing group experiencing flavour stability problems in one of the
breweries in the group resulting in increased consumer complaints.

Step 1: Can we measure the problem?

Samples of lager beer from 3 breweries in the regional brewing group (BG1-3) were
submitted to the flavour stability tests by taste evaluation and T2N analysis. The
samples were compared with similar analyses of an international lager (Int. Lag.) of
known satisfactory flavour stability. The brewery BG1 had been receiving consumer
complaints about aged character of their beer.

3
The results presented in figures 1 and 2 show that the flavour stability of samples
from brewery BG2 were satisfactory and similar to the international lager.
Furthermore, the results indicated an unsatisfactory flavour stability of the samples
from the other two breweries, BG1 and BG3, compared with the international lager
and the sample from brewery BG2.
In this study the apparently unsatisfactory flavour stability of the lager from brewery
BG1 was further investigated, whereas the 3rd brewery in the group (BG3) was not
further considered in the study.

Fresh Aged Fresh Aged

6,4

General Evaluation (1-9)

0,2 0,19 7,0 6,3
5,7 5,9
6,0 5,6
5,0
0,15
T2N - ppb

0,13 5,0 4,3

0,11 3,9
0,1 4,0
0,07
0,06 3,0
0,05 0,04 0,05
0,04
2,0
0 1,0
1 2 0,0
BG BG 3 .
BG ag BG1 BG2 BG3 Int.
nt.L
I Lag.

Figure 1: T2N analyses of fresh and aged Figure 2: Taste evaluation of fresh
samples of 3 brands (BG1, BG 2, BG 3) and aged samples of 3 brands (BG1,
from the same brewing group compared BG2, BG3) from the same brewing
with an international lager (Int. Lag.). group compared with an international
lager (Int. Lag.).

The taste evaluation scores were correlated with the T2N measurement for the above
samples. The correlation is illustrated in figure 3, demonstrating the good correlation
between T2N and the general taste evaluation score.

7,0
General Evaluation (1-9)

6,0 2
R = 0,93
5,0
4,0
3,0
2,0
1,0
0,0
0 0,02 0,04 0,06 0,08 0,1 0,12 0,14 0,16 0,18 0,2
T2N - ppb

Figure 3: Correlation between T2N and the taste evaluation score (general
evaluation).

4
Step 2: Process examination and trials
From a comparison between the production conditions at BG1 and BG2 (table 1), it
was found that main differences between the 2 breweries were: pH of sparge water
and use of high gravity brewing, both parameters known to affect the flavour stability
of beer (Yasui (3)).

Parameter BG1 BG2

(Selected parameters shown)
Yeast AJL2155 AJL2155
Malt Same supplier and type Same supplier and type
Mash pH 5.6-5.7 5.7-5.8
Wort pH 5.1-5.2 5.2-5.3
Beer pH 4.0 4.15-4.2
Sparge water pH 7.0 6.1
HGB used No Yes

Table 1: Examination and comparison of process data (selected parameters) for the
two breweries BG1 and BG2.

Based on the above process examination, a series of trials were defined and carried
out. The following selected trials gave the most pronounced result:
reduction of brew water pH to 5.5
fermentation of first wort only

The beers from the trials were submitted to flavour stability tests by taste evaluation
and analysis of T2N.
Figures 3 and 4 show that a marked improvement of flavour stability was achieved by
lowering the brew water pH. The T2N level in the fresh as well as the aged samples
was reduced, and a minor drop in general evaluation was noted.

Fresh Aged Fresh Aged

7,0
General Evaluation (1-9)

0,19
0,2 5,5
6,0 5,0 5,2 5,0
T2N - ppb

0,15 5,0
0,11 3,9
0,1 0,07 4,0 3,6
0,07
0,05 3,0
0,02 0,02
0 2,0
f) 1,0
( Re pH
G2 ter ing 0,0
B wa
parg
w s BG2 Low No
Lo No
(Ref) water pH sparging

Figure 4: T2N analyses of fresh and Figure 5: Taste evaluation of fresh

and aged samples of trials, performed and aged samples of trials, performed
at brewery BG1, compared with the at brewery BG1, compared with the
reference (BG2 figure 1). reference (BG2 figure 2).

5
The trials with fermentation of first wort (no sparging), however, gave contradictive
results. The T2N figures in the fresh and aged beers were reduced, but the flavour
score of the aged beer was very low. This should be a subject for further
investigations.
Following a trial. Period the brewery implemented the lowering of the brew water pH
to pH 5.5.

Case 2
A smaller brewery having a flavour stability problem in export products
The brewery received complaints for poor taste from consumers on their export
product. A flavour stability test by taste evaluation and T2N analysis was conducted
on samples from the brewery.
The results of the flavour stability test showed large increases in T2N and a marked
drop in general evaluation score after the forced ageing, indicating unsatisfactory
flavour stability.
The brewery conducted trials with selected changes in process parameters. One of the
trials, mashing-in at 65C, gave a marked improvement compared with the reference,
as seen from figure 5 and 6. The T2N content was reduced in both the fresh and the
aged samples, and only a minor drop in general evaluation after forced ageing was
achieved, resulting in a satisfactory flavour stability of beers from the trial.

Fresh Aged Fresh Aged

0,23
7,0
General Evaluation (1-9)

0,25 5,8 5,8

6,0 5,3
0,2
T2N - ppb

5,0
0,15
4,0 3,6
0,1 0,06
0,06 3,0
0,05
0,02 2,0
0
1,0
ce
en
fer 0,0
Re
Reference 65C mashing

Figure 6: T2N analyses of fresh and Figure 7: Taste evaluation of fresh

and aged samples of a mashing trial and aged samples of a mashing trial
compared with the reference. compared with the reference.

CONCLUSION
The results of this study have demonstrated that good flavour stability of beer can be
achieved by combining process knowledge with a structured analysis of process and
analytical data.
Furthermore, it was shown that a combination of taste evaluation and T2N analyses is
a strong tool for a reliable flavour stability test. T2N is not the only relevant staling
compound to consider, but in many cases the compound can be used as a useful
marker for flavour stability, not least due to the high correlation between T2N and
expert taste panel performance.

6
The structured collection of information about the individual beer in a Flavour
Correlation Data Base allows for an intelligent set up of a few numbers of trials for
possible improvements. In the present examples, lower sparge water pH and increased
mashing-in temperature was applied for successful improvement of flavour stability.

REFERENCES
1. Search and Research, Carlsberg Research Center, Gamle Carlsberg Vej DK-
2500 Copenhagen-Valby, Denmark, 1999.
2. Meilgaard, M., Beer Flavour, doctoral thesis, Technical University of
Copenhagen 1981.
3. Yasui, T., Presentation at WBC2000, Orlando, USA, 2000.

7
57

Linoleic acid hydroperoxides, trans-2-

nonenal and nonenal potential during
the brewing process: evolution and
relationship
Rgis Fournier, Michel Dumoulin & Patrick Boivin
Institut Franais des Boissons de la Brasserie Malterie, Ple Technologique de Brabois, 7, rue
du Bois de la Champelle, B.P.267, F-54512 Vandoeuvre Cedex, France (e-mail:
[email protected])

Descriptors
Flavour formation, linoleic acid, 2-nonenal, peroxide, wort boiling

SUMMARY
Among the carbonyl compounds involved in flavour of beer, trans-2-nonenal is responsible
for the unpleasant cardboard off-flavour. The most credible models link trans-2-nonenal
formation with the degradation of linoleic acid hydroperoxides. The main goal of this study is
to clarify the relationship between theses hydroperoxides and trans-2-nonenal during the
production of beer. Linoleic acid hydroperoxides are produced during the brewing step. With
wort boiling, their degradation leads to trans-2-nonenal. At the end of fermentation, no trans-
2-nonenal (free and potential) can be detected in wort. It seems that trans-2-nonenal in beer
results from formation between fermented wort and beer in bottle.

Linolsurehydroperoxid, trans-2-Nonenal und potenzielles Nonenal whrend des

Brauprozesses: Entwicklung und Beziehungen

Deskriptoren
Aromabildung, Linolsure, Nonenal-2, Peroxid, Wrzekochen

ZUSAMMENFASSUNG
Unter den Carbonylverbindungen, die am Aroma des Bieres beteiligt sind, ist trans-2-Nonenal
fr den unangenehmen Cardboard-Fehlgeschmack verantwortlich. Die glaubwrdigsten
Modelle verbinden die trans-2-Nonenal-Bildung mit der Verminderung von Linolsure-
hydroperoxid. Das Hauptziel dieser Studie ist, die Beziehung zwischen diesen Hydro-
peroxiden und trans-2-Nonenal whrend der Bierproduktion zu erklren. Linolsure-
hydroperoxide werden beim Maischen produziert. Das Wrzekochen fhrt zur Reduzierung
von trans-2-Nonenal. Am Ende der Grung kann kein freies trans-2-Nonenal (freies und
potenzielles) im Jungbier gefunden werden. Es scheint, dass das trans-2-Nonenal, das im Bier
gefunden wird, auf dem Weg vom Jungbier zum Bier in der Flasche entsteht.
Hydroperoxydes de l'acide linolique, trans-2-nonnal et potentiel nonnal pendant le
brassage : volution et relations

Descripteurs
Acide linoligue, cuisson du mot, formation de la flaveur, nonnal-2, peroxyde

RESUME
Parmi les composs carbonyls impliqus dans la flaveur de la bire, le trans-2-nonnal est
responsable du faux got de papier-carton. Les modles les plus vraisemblables attribuent la
formation de trans-2-nonnal la dgradation des hydroperoxydes issus de loxydation de
lacide linolique. Lobjectif principal de cette tude est de clarifier les relations existantes
entre ces hydroperoxydes et le trans-2-nonnal au cours de la fabrication de la bire. Les
hydroperoxydes de lacide linolique sont produits pendant ltape de brassage. Au cours de
lbullition du mot ils se dgradent pour donner le trans-2-nonnal. A lissue de la
fermentation, aucune trace de trans-2-nonnal (libre ou potentiel) ne peut tre dtecte dans le
mot. Il semble que lapparition de trans-2-nonnal dans la bire est le rsultat dune
production entre le mot ferment et la bire en bouteille.

2
INTRODUCTION
As it is now well known, many carbonyl compounds are involved in the flavour
characteristics of the beer. Among them, trans-2-nonenal is thought to be responsible
for the unpleasant cardboard off-flavour. This compound could appear along the
brewing process, as well as the bottling step.

The most credible model links the apparition of trans-2-nonenal with the oxidation of
linoleic acid (1,2). During the brewing process, degradation of certain type of lipid
lead to linoleic acid. Its oxidation allows the formation of two hydroperoxide isomers
(9 and 13 hydroperoxides). The 9-isomer easily leads to trans-2-nonenal.

To understand the formation of trans-2-nonenal in beer, we have to follow the

behaviour of the hydroperoxides obtained from linoleic acid oxidation. For this we
have developped a specific method based on the analysis of alcohols issued from the
reduction of linoleic acid hydroperoxides.

The main goal of this study is to clarify the relationship between hydroperoxides of
linoleic acid and trans-2-nonenal during the brewing process (from malt to beer).

MATERIALS AND METHODS

Malts
Extract variety was used for mashing study. Alexis varity was used for pilot brewing
process study.

Chemicals for analysis of hydroperoxides and trans-2-nonenal

13(S)- and 9(S)-hydroperoxyocta-deca-(9Z,11E)/(10E,12Z)-dienoic acid were
obtained from ICN. Lipoxidase, trans-2-nonenal and 1-chlorononane were obtained
from Sigma.

Brewing processes
The mashing step was specifically studied on a Tepral unity. For this process, 57 g of
malt are mashed with 200 ml following programmed temperature at 50, 63 and 75C.
The brewing and the fermentation steps were studied on a pilot scale unity and the
parameters of the process are those used for the CBMO program. Typically, 300 kg of
malt are mashed with 950 l of water following programmed temperature at 45, 64 and
76C.

Nonenal potential analysis

In wort and beer, trans-2-nonenal can be complexed with proteins and :or sulfides
compounds. To release this nonenal potential, wort/beer are saturated with N2
bubbling. Saturated wort/beer are then heated at 90C during two hours. Then nonenal
released is extract with CS2 and analysed with GC-MS.

Linoleic acid hydroperoxides analysis in wort and beer

This method is based on the wok we have done on the determination of
hydroperoxides potential in malts (4). It is an alternative to the direct determination of
hydroperoxides in wort that is based on the reaction with isoluminol (5).

3
In a first time, wort/beer is centrifuged in presence of ethyl alcohol to stop enzymatic
reaction related to hydroperoxides formation. A fraction of the supernatant is reduced
in presence of NaBH4. This step allows the transformation of hydroperoxides into the
corresponding alcohols. These alcohols are extracted with ethyl oxide/hexane and the
extract thus obtained is analysed with HPLC-Diode Array Detector.
The analytical performances of this method are given below:
recovery : 100-110%
variability : 2-4%
sensibility : 0.1 mol/l (14 g eq. nonenal/l)

Figure 1 illustrates the chromatogram obtained for wort analysis.

Figure 1: Hydroperoxides analysis in wort.

RESULTS AND DISCUSSION

Involvement of hydroperoxides during the mashing step
In figure 2, we have plotted the involvement of 9-hydroperoxide and ratio of 9 to 13-
hydroperoxide during the mashing step.

During the mashing step oxidation of linoleic acid occurs and leads to the formation
of hydroperoxides. The ratio of 9 to 13-hydroperoxide is relatively constant up to
63C with a mean value of 70%. Above 63C, this ratio decreases and reaches a mean
value of 55%. This clearly indicates a change in the oxidation mechanism.

Linoleic acid oxidation can be performed under two distinct mechanisms: enzymatic
oxidation or auto-oxidation.

In malt, lipoxygenase enzyme exists under two isoforms: lox 1 and lox 2. Lox 1 is
predominant in malt and is leading predominantly to the formation of 9-
hydroperoxide (4). Thus, during the first steps of mashing and up to 63C, the
enzymatic oxidation of linoleic could explained the overwhelming formation of 9-
hydroperoxide.

In case of auto-oxidation, 9 and 13-hydroperoxides would be produced in the same

amounts. Above 63C, the decrease of 9 to 13-hydroperoxide near 50% mean value
could be relevant of an auto-oxidation process. Moreover, it would indicated that
lipoxygenase is degradated above 63C.

4
 9-ROOH (Extract) temprature (C) Ratio 9/13 Extract
300,00 80

75
250,00
70

% of 9-hydroperoxide
200,00
65
(mol/l)

150,00 60

55
100,00
50
50,00
45

0,00 40
0 10 20 30 40 50 60 70 80
Time (min)

Figure 2: Involvement of 9-hydroperoxide and ratio of 9 to 13-hydroperoxide

during the mashing step.

Linoleic acid hydroperoxides involvement during pilot brewing and

fermentation
In figure 3 the evolution of hydroperoxides resulting from linoleic oxidation has been
plotted from wort before boiling to beer in bottle.
9-hydroperoxide 13-hydroperoxide

5
(mol/l)

0
Wort bef. Boil. End of boil. After whirl. After Ferm. Middle of End of ferm. Beer
Boil. beginning aeration beginning ferm.

Figure 3: Linoleic acid hydroperoxides involvement from malt to beer.

Until the beginning of wort boiling there is production of 9 and 13-hydroperoxides.

As amounts produced are quite the same, one could say that the oxidation of linoleic
acid is occuring under auto-oxidation pathway.
In general, hydroperoxides are very sensitive to temperature. This could explain the
fast decrease of 9 and 13-hydroperoxides during wort boiling.
Finally, the last part of hydroperoxides, that have not been eliminated during wort
boiling, is degradated during fermentation.

5
Involvement of nonenal potential during brewing and fermentation
In figure 4, we can see the evolution of nonenal potential and 9-hydroperoxide from
the beginning of wort boiling to beer in bottle.
9-hydroperoxide Nonenal potential

1600

1400

1200
(g eq.nonenal/l)

1000

800

600

400

200

0
Boil. beginning End of boil. After whirl. After aeration Ferm. beginning Middle of ferm. End of ferm. Beer

Figure 4: Involvement of nonenal potential and 9-hydroperoxide (X100) from

beginning of wort boiling to beer in bottle.

During wort boiling, the decrease of 9-hydroperoxide is clearly associated with trans-
2-nonenal production. Yeast is added just before the end of aeration step. Thus the
decrease of nonenal potential could certainly be explained by its reduction into
alcohol.

CONCLUSIONS
A new method for hydroperoxides determination in wort and beer has been
developped during this study. Based on hydroperoxides reduction into alcohol, this
method is robust, sensitive and easy to performed in wort and beer.

This new tool was very helpfull to clarify the relationship between hydroperoxides
level and formation of nonenal potential during brewing and fermentation. The mains
conclusions of this work are:
Up to 63C during mashing, hydroperoxides formation is occuring under
enzymatic control. Thus 9-hydroperoxide is produced predominantly over 13-
hydroperoxide. Above 63C, hydroperoxides are mainly obtained from auto-
oxidation of linoleic acid.
All the hydroperoxides produced during mashing disappear with wort boiling and
through fermentation.
The degradation of 9-hydroperoxide during wort boiling seems to associate with
the apparition of trans-2-nonenal in wort.
All the trans-2-nonenal produced during boiling are reduced by the yeast during
fermentation. This last result seems to indicate that nonenal in beer is not directly
related with hydroperoxides of linoleic acid.
On the basis of these results, the trans-2-nonenal that is usually found in beer
should come from pasteurization or bottling steps. Investigations are in progress
to clarify this point.

6
ACKNOWLEDGEMENT
The authors wish to thank the French Malsters and the French Brewers who support
this work.

REFERENCES
(1) Drost, B., Van der Berg, R., Freyee, F.J., Van der Velde, E., Hollemans, M.,
Journal of the American Society of Brewing Chemists, 1993, 397-404.
(2) Lemursieau, G., Nol, S., Ligeois, C., Collin, S., Journal of the American
Society of Brewing Chemists, 1999, 29-33.
(3) Collin, S., Nol, S., Bonte, S., Metais, N., Bodart, E., Peladan, F., Dupire S.,
Proceedings of the European Brewery Convention Congress, Maastricht, 1997,
535-544.
(4) Boivin, P., Malanda, M., Clamagirand, V., Proceedings of the European
Brewery Convention Congress, Cannes, 1999, 397-404.
(5) Kobayashi, N., Kaneda, H., Kano, Y., Koshino, S., Journal of the American
Society of Brewing Chemists, 1994, 141-145.

7
58

Evaluation of the addition of

gallotannins in the brewing liquor for
the improvement of the flavour stability
of beer
Guido Aerts1, Luc De Cooman1, Gert De Rouck1, Zoltan
Pnzes1, Annemie De Buck1, Roger Mussche2 & Joseph van
Waesberghe3
1
KaHo St.-Lieven, Laboratory of Enzyme and Brewing Technology, Gebroeders Desmetstraat
1, B-9000 Ghent, Belgium (e-mail: [email protected])
2
Omnichem, Coopallaan 91, B-9230 Wetteren, Belgium
3
IVEWE, Ginnekenweg 78A, 4818 JC Breda, the Netherlands

Descriptors
Antioxidant, brewing liquor, iso--acid, staling, tannic acid, taste stability

SUMMARY
In most brewing trials, especially in the case of lager beers, the reducing capacity of the malt
and other ingredients seems insufficient to prevent flavour deterioration. This paper deals
with the use of gallotannins, to increase the anti-oxidant power during the mashing process.
Lager beers were prepared, with and without gallotannin addition to the brewing and sparging
liquor, in a pilot brewery on a 200 l scale and evaluated both fresh and after ageing under
forced conditions at 40C. Both the analytical data with regard to Strecker degradation of
amino acids, oxidation of lipids and iso--acids, and the sensory evaluation, demonstrate that
gallotannin addition seems promising to increase the flavour stability of the final beer.

Untersuchung der Verbesserung der Geschmacksstabilitt durch Zugabe von

Gallotannin zum Brauwasser

Deskriptoren
Altgeschmacksbildung, Antioxidantium, Brauwasser, Gerbsure,
Geschmacksstabilitt, Iso--Sure

ZUSAMMENFASSUNG
In den meisten Brauversuchen, besonders im Falle der Lager-Biere, scheint die
reduzierende Kapazitt des Malzes und anderer Bestandteile unzureichend, um
Geschmacks-verschlechterungen zu vermeiden. Dieses Poster beschftigt sich mit der
Verwendung von Gallotannin, um die antioxidative Wirkung whrend des
Maischprozesses zu steigern. Lager-Biere wurden, mit und ohne Gallotanninzugabe
zum Brau- und Anschwnzwasser, in einer 200- l-Versuchsbrauerei hergestellt und
sowohl frisch als auch bei 40C gealtert untersucht. Die Analysendaten hinsichtlich
des Streckerabbaus der Aminosuren, der Oxidation der Lipide und der Iso-a-Suren
als auch die sensorische Auswertung zeigten, dass die Zugabe von Gallotannin eine
viel versprechende Mglichkeit ist, die Geschmacksstabilitt des fertigen Bieres zu
erhhen.

Evaluation de l'effet de l'addition de gallotanins dans leau de brassage pour amliorer

la stabilit de l'arme de la bire

Descripteurs
Acide iso-, acide tannique, antioxydant, eau de brassage, formation du got dvente,
stabilit organoleptique

RESUME
Dans la plupart des essais de brassage, surtout dans le cas des bires fortes (lager), la capacit
rductrice du malt et d'autres ingrdients semble insuffisante pour viter la dgradation de
l'arme. Cet article traite de l'usage des gallotannins pour augmenter le pouvoir antioxydant
pendant le mlange. Des bires fortes ont t prpares avec et sans addition de gallotannins
leau de brassage et de lavage, dans une brasserie pilote l'chelle de 200 l et values
fraches et aprs vieillissement dans des conditions forces 40C. Les donnes analytiques
concernant la dgradation de Strecker des acides amins, l'oxydation des lipides et des iso--
acides et l'valuation sensorielle dmontrent que l'addition de gallotannins semble
prometteuse pour accrotre la stabilit de l'arme de la bire finie.

2
INTRODUCTION
Nowadays, flavour stability is one of the most important quality aspects in the
brewing industry. Huge attention is paid to the development and the implementation
of anti-oxidative beer production systems and to the potential of both endogenous and
exogenous natural anti-oxidants as flavour stabilisers (3). Flavour deterioration
coincides with an increase in and a release of alkanals-alkenals as final products of
auto-oxidation and enzymic degradation of lipids, and with a rise in Strecker
aldehydes (9). Oxidative reactions on iso--acids, especially on the less stable trans
isomers, result in a lower, less fine and harsher bitterness (5). Next to the LOX
content, the anti-oxidant power of the malt, the mashing-in and brewing conditions
are important parameters, affecting the flavour staling in finished beer (4). However,
in most brewing trials, especially in the case of lager beers, the reducing capacity of
the malt and other ingredients seems insufficient to prevent these adverse effects. This
work deals with the use of gallotannins to increase the anti-oxidant power during the
mashing and wort filtration processes. Gallotannins can act as metal-chelating (7),
radical scavenging and reducing agents (8). They are also very effective in binding
aldehydes and in coagulation/flocculation of especially thiol-containing proteins (8).
Some possible negative effects like chelation of oligo-elements (necessary for yeast
growth), inactivation of proteolytic and amylolytic enzymes, coagulation of foam-
active proteins, and an increase in astringency of the beer caused by higher gallate
concentrations, have to be evaluated.

MATERIALS AND METHODS

Preparation of the pilot beers
To examine the potential of gallotannins as natural exogenous anti-oxidants during
mashing and wort filtration, six lager beers were prepared in our pilot brewery on a 2
hl scale. The differences in the mashing-in conditions of the brewing trials are
summarised in table 1. Three pairs of brews, with and without the addition of
gallotannins to the brewing (10 g/hl) and sparging water (5 g/hl), were prepared with
the same batches of raw materials (34 kg pilsner malt and 6 kg maize flakes). To the
brewing water (1.7 hl), obtained by reverse osmosis, Ca2+ was added (40 ppm for the
reference beers, and 80 ppm for the gallotannin beers). For pH correction of the
mash in the brewing trials E and F, lactic acid was used. Wort hopping was performed
by the addition of isomerised hop extracts at the end of wort boiling.

brewing sparging
brewing mashingin conditions water water
experiment addition of gallotannins
Temperature (C) pH 10 g/hl 5 g/hl
A 45 5.8 - -
B 45 5.8 + +
C 62 5.8 - -
D 62 5.8 + +
E 62 5.3 - -
F 62 5.3 + +
Table 1: Summary of the brewing trials on pilot scale (2 hl).

3
Beer analysis
The pilot beers were compared both fresh and after ageing in the dark under forced
conditions at 40C by sensory and analytical evaluation. The staling degree of each
beer sample was scored on a six-point scale, according to Araki et al. (2), by a taste
panel of 12 experienced persons. Standard analyses were carried out according to
Analytica EBC (1) and in house procedures. Cu, Zn and Fe concentrations were
determined by A.A.S. analysis. Aldehydes were extracted from the beers by vacuum
distillation. The extracts were purified by liquid-liquid extraction and subsequently
analysed by CGC. Iso--acids (5) and gallates (unpublished results) were extracted
from the beers by specific liquid-liquid extraction procedures and analysed by proved
HPLC methods.

LOX activity assay

The methods for extraction and assay of potential LOX activity in malt and in the
non-extracted material during mashing have been described earlier (4). One unit of
LOX activity per ml of reaction mixture was defined as an increase in A234nm of
1.0/min.

Aldehyde profiling during brewing

The concentration of some supposed marker aldehydes in worts (+/- gallotannin
addition) was determined during the brewing process as a function of time. The
aldehydes were extracted from the malt and worts by steam distillation and analysed
by CGC.

RESULTS AND DISCUSSION

Evaluation of the pilot beers
The results of the standard analyses for the three reference beers A, C and E and for
the gallotannin beers B, D and F are summarised in table 2.
Beer
A B C D E F
Original extract (P) 11.8 11.4 12.0 11.4 12.3 12.0
Real extract (P) 4.2 4.3 4.3 4.9 4.5 4.7
Alcohol (v / v) 5.0 4.7 5.1 4.3 5.2 4.9
Real attenuation (%) 64.3 62.3 64.2 57.0 63.7 61.2
pH 4.7 4.7 4.5 4.6 4.4 4.5
Soluble protein (g / ml) 296 223 231 316 359 361
Foam stability (s) 397 379 431 450 302 275
Colour (EBC) 6.6 6.9 6.0 6.2 8.0 6.6
Polyphenols (ppm) 123 138 108 152 145 183
Gallates (ppm) 0.68 9.48 0.56 5.80 - -
ITT (s) 252 101 152 115 231 100
Cu (ppm) - - 0.68 0.31 0.43 0.23
Table 2: Standard analyses of the pilot beers.

During brewing, we observed no negative effect of the presence of gallotannins upon

saccharification, indicating that the amylase activity during mashing is not inhibited
or inactivated. The soluble protein content (Bradford assay) of all the beers seems
normal. Also, good foam stabilities (Rudin assay) and comparable colours are
obtained for the different pilot beers. Inhibition of proteolytic activity during mashing

4
was not detected, as the FAN content of the worts was similar ( 160 ppm). The
content of gallates in the gallotannin worts ( 10 ppm) was clearly higher than in the
reference worts ( 0.7 ppm). The presence of these gallates could influence the yeast
flocculation behaviour during fermentation. Gallotannins appear to have a small, but
significant negative effect on yeast performance and attenuation. However, addition
of Zn2+ (0.2 ppm) to the pitching worts resulted, also for the gallotannin beers, in
normal and comparable attenuations ( 64 %) (data not shown). This indicates that the
negative effect of the addition of gallotannins to the brewing and sparging water on
yeast performance is probably only connected with the metal-chelating activity of the
gallotannins during brewing. Next to the worts, also the gallotannin beers contain
higher gallate concentrations than the reference beers. If the clarity of the worts after
lautering had been better for all brewing experiments, this relatively small carry-
through of gallates would have been negligible.

Evaluation of the stability of the pilot beers after forced ageing

The results of the sensory evaluation of the pilot beers are represented in table 3. All
fresh beers were evaluated positively by the tasting panel. The gallotannin beers
were preferred because of their fullness of taste and mouthfeel. After 5 days of storage
at 40C, one can already recognise the difference in stability of the beers. Especially
beer F was largely free of oxidised flavours. After 10 days of storage at 40C, the
reference beers were clearly more aged than the gallotannin beers. A strong
cardboard off-flavour and a sharp bitterness were detectable. Also the colour of the
aged reference beers was darker. In contrast, the aged samples of the gallotannin
beers (especially beer F) still showed a smooth bitterness and the original pale pilsner
colour. A slightly sweeter taste was however perceptible in the aged samples.
Score after ageing at 40C (days)
beer
0 5 10
A 0 - 45
B 0 - 34
C 0 23 3
D 0 12 23
E 0 23 3
F 0 1 2
Evaluation according to Araki et al. (1999)
0: fresh; oxidised flavour not detectable; 1: very weakly aged; 2: weakly aged; 3: moderately
aged; 4: strongly aged; 5: very strongly aged, undrinkable
Table 3: Sensory evaluation of the pilot beers after forced ageing at 40C.

The above results were confirmed by sensory evaluation of the beers CF (data not
shown), stored at 4C in the dark for one year. The cold stored gallotannin beers
were always preferred. Especially the stability of beer F was most striking, suggesting
that the addition of gallotannins to the brewing and sparging water, in combination
with mashing-in at 62C and at pH 5.3, can result in a final product with a
significantly improved organoleptical stability. Figures 13 show (a) the total
bitterness (%) and (b) the relative concentration of trans- and cis- iso--acids (%) as a
function of forced ageing time, for the three pairs of pilot beers, with the same
mashing-in conditions but with or without gallotannin addition.
During the forced ageing period, total bitterness of the gallotannin beers declined by
only a few per cents, as opposed to the situation in the reference beers where a total
bitterness loss of about 10 to 20 % was observed, depending on the beer in question.

5
 (a) (b)

Relative conc. (% )
Total bitterness (% )
100 110
100
90 90
80 80
70
70 60
50
60
0 5 10 15 20
0 2 5 10 15 20
D ays at 40C
D ays at 40C cICH + cIN H (A ) cICH + cIN H (B)
Beer A Beer B tICH + tIN H (A ) tICH + tIN H (B)

Fig.1:Forced ageing of beers A and B at 40C. Total bitterness (%) (Fig. 1a) and
relative concentrations (%) of iso--acids (Fig. 1b) as a function of ageing time.
(a) (b)
100 110
Total bitterness (% )

Relative conc.(% )
100
90
90
80 80
70
70 60
60 50

0 5 10 20 0 5 10 15 20
D ays at 40C
D ays at 40C cICH + cIN H (C) cICH + cIN H (D )
Beer C Beer D tICH + tIN H (C) tICH + tIN H (D )

Fig.2:Forced ageing of beers C and D at 40C. Total bitterness (%) (Fig. 2a) and
relative concentrations (%) of iso--acids (Fig. 2b) as a function of ageing time.
(a) (b)
Relative conc. (% )

110
Total bitterness (% )

100
100
90 90
80
80
70
70 60
50
60
0 5 10 15 20
0 2 5 10 15 20
D ays at 40 C
D ays at 40C cICH +cIN H (E) cICH +cIN H (F)
Beer E Beer F tICH +tIN H (E) tICH +tIN H (F)

Fig.3:Forced ageing of beers E and F at 40C. Total bitterness (%) (Fig. 3a) and
relative concentrations (%) of iso--acids (Fig. 3b) as a function of ageing time.

The stabilising effect of the gallotannin dosage is due to the complete resistance
towards oxidation of cis-iso--acids in all gallotannin beers during the experimental
period. In case of the gallotannin beers D and F also a partial, but significant
stabilisation of the very sensitive trans-iso--acids was noticed. However, cis-iso--
acids always represent the major fraction in the bitter acid profile of beer. Moreover,
cis-iso--acids are about 1.8 times more bitter than their trans counterparts, which
makes the stabilising effect of the gallotannin treatment very interesting from the
sensory point of view. The results in table 2 show a higher reducing power and
smaller Cu2+ concentrations in the gallotannin beers. Also, the polyphenol content of
these beers is higher. Next to higher gallate concentrations, more malt polyphenols in
the reduced state could be present, due to the protective effect of the gallotannins

6
during mashing. As isomerised hopextracts were used for hopping of the wort,
hoppolyphenols cannot be present in the worts and corresponding beers. When
comparing the analytical data obtained from the different beers AF, it appears that
the beers E and F, prepared at mashing-in conditions of 62C and pH 5.3, must be
considered as the most stable beers. In addition, bitterness in the gallotannin beer F
was even more stable than bitterness in the reference beer E. It thus seems very
promising to combine mashing-in at 62C and pH 5.3 with the addition of gallotannin
to the brewing and sparging water. This finding is further indicative of the importance
of LOX activity during mashing, especially at mashing-in. The results of the iso--
acids profiling of the beers as a function of ageing at 40C are only partly confirmed
by the results of the analysis of some supposed marker aldehydes. Whereas formation
of benzaldehyde and -nonalactone was more expressed during ageing of the
reference beer E than in the gallotannin beer F, opposite results were observed for
the C-D pair of pilot beers. Trans-2-nonenal never exceeded a concentration of 0.2
ppb (data not shown).

Effect of gallotannins on protein and lipid oxidation during mashing

Thiol-containing proteins are very easily oxidised. This oxidation will generate
disulphide bridging, resulting in high molecular weight cross-linked proteins. These
protein aggregates do not only reduce the rate of mash filtration, but also can
aggregate or enclose small starch granules thereby inhibiting amylolytic attack.
Muller showed that also H2O2 is produced as a by-product of such oxidation (9). The
presence of gallotannins from the onset of mashing-in could reduce such oxidations
either directly or indirectly which explains the positive effect of gallotannin addition
on wort filtration performance (figure 4). Next to thiol-containing molecules, lipids
composed of unsaturated fatty acids are even more sensitive to oxidative effects. Lipid
oxidation, both enzymic and non-enzymic, generates volatile off-flavours with very
low flavour thresholds. Because of their radical scavenging activity, gallotannins can
act as inhibitors of auto-oxidation processes. However as demonstrated in figure 5,
gallotannins also inhibit the LOX reaction very effectively. Coagulation/precipitation
of the LOX enzymes is not the reason as gallotannins (50 mg/l) reduce the extraction
yield of LOX from malt by only 10 % (data not shown).

180 100
160
140 80
% inhibition
Volume (l)

120
100 60
80
60 40
40 20
20
0 0
0 20 40 60 80 100 120 0 50 100
Time (min)
gallotannins (mg/l)
Beer A Beer B

Fig.4: Effect of the addition of gallotannins Fig.5: Inhibition of the LOX

on wort filtration performance. reaction by gallotannins.
Figure 6 shows the residual LOX activity in the non-extracted material during the
complete mashing process, with and without gallotannin additions. During the
reference brew, the unextracted LOX activity decreases as a function of time, but
increases in the final stage of brewing and is still extractable from the spent grains.

7
Whereas extracted LOX during brewing is very quickly inactivated, there is a
potential role for this enzyme to be active towards lipids and fatty acids throughout
the whole mashing and wort filtration processes, due to its presence and potential
activity in the non-extracted material.

80 80

LOX activity (U/g d m)

Temperatu re (C)

70 60

60 40

50 20

40 0
0 10 20 30 40 50 60 70 80
MALT Time (min ) S PENT
GRAINS

b rewin g s ch eme referen ce ad d itio n o f g allo tan n in s

Fig.6: The residual LOX activity in the non-extracted material during mashing.
The results in figure 6 further point to a decreased potential LOX activity in the non-
extracted material when brewing in the presence of gallotannins, especially during the
first period of mashing.

Effect of gallotannins on aldehyde levels in wort during brewing

Figures 7-10 summarise the changes in aldehyde content from malt throughout the
whole brewing process, including wort filtration, wort boiling and clarification. These
results show that some aldehydes, especially Strecker aldehydes, some Maillard
intermediates but also some fatty acid derived aldehydes are already present in the
malt and most of them are extracted very fast during mashing-in.

referentie
referen ce ad d itio n o f g allo tan n in s
40 40 40 80 40 90 80

30 70 78 80 70
A ld ehy d es (g /100 g )

A ld ehy d es (g /100 g )
A ld ehy d es (g /20 g )

30 30 30
Temp erature (C)

Temp erature (C)

72 72 70

20 63 63 63
60 60
20 20 20 60
10 50 50
50
10 10 10
0 40 40 40

0
0 20 40 60 80
0 30 0 30
0 50 100 0 50 100
Time (min ) Time (min )

h exan al 2-fu rfu ral tran s -2-h exen al

tran s -2-h ep ten al b en zald eh y d e p h en y lacetald eh y d e
tran s -2-n o n en al -n o n alacto n e b rewin g s ch eme

Fig.7: Comparison of some aldehyde concentrations in pilsner malt with those

during mashing as a function of time.

8
The presence of such aldehydes in the malt suggests the presence of their precursors,
which can also be extracted during mashing. The content of the Strecker and Maillard
aldehydes remains fairly constant and increases only at higher temperatures during the
reference brewing trial. The addition of gallotannins results in significantly lower
extracted amounts of phenylacetaldehyde, furfural and benzaldehyde from the malt,
indicating a high binding capacity of gallotannins for such aldehydes. Gallotannins
also appear to inhibit the de novo production of these aldehydes during the whole
brewing process, including wort filtration, wort boiling and clarification. On the
contrary, the effect of gallotannins on the binding of fatty acid derived aldehydes and
on their formation during brewing is more or less negligible. This does not mean that
the gallotannins are not effective inhibitors of initial lipid oxidation. Our results
indicate that most of the fatty acid derived aldehydes are already produced during
malting and kilning, which could explain the relatively small differences between the
reference and gallotannin brewing trials. To evaluate the true effect of gallotannin
on lipid oxidation, determination of fatty acid hydroperoxides, di- and trihydroxy fatty
acids seems required. Further interesting to note is the clear decrease of trans-2-
nonenal in the gallotannin brewing experiment (figure 8).

referentie
referen ce ad d itio n o f g allo tan n in s
1 80 1 80
A ld ehy d es (g /100 g )

A ld ehy d es (g /100 g )

0.8 70 0.8 70
Temp erature (C)

T emp erature (C)

0.6 60 0.6 60

0.440 50 0.4 90 50
30
20 63 63 63 72 72 78 80
70
60

10
0 50
40
0.2 40 0.2 40
0 20 40 60 80
0 30 0 30

0 50 100 0 50 100
Time (min ) T ime (min)

tran s -2-h exen al tran s -2-h ep ten al b en zald eh y d e

tran s -2-n o n en al -n o n alacto n e b rewin g s ch eme

Fig.8: Concentration of some minor aldehydes during mashing in function of time.

referen t ie
referen ce ad d itio n o f g allo tan n in s
A ld ehy d es (g /100 g )

A ld ehy d es (g /100 g )

45 45
45
40 40
40
35
35 35
30 30 30
25
25 25

20

15 20 20
10 15 15
5
10 10
0
5 fi
rst w ort secon d w ort start boi
ng
i
l
5 en d boi
ng
i
l pi
tchi
n g w ort

0 0
t tg g t
or or
t

or
t

t
ng
g
or

or

or

lin lin
lin

ili

i i
w

tw w
w

w
oi

bo b o in g
bo

d
st

nd

s
ng
tb

fir on rt
fir

d
hi
d
co

h
ar

c a n tc
en

t e
se
tc
se

s pi
st

pi

h exan al 2-fu rfu ral tran s -2-h exen al

tran s -2-h ep ten al b en zald eh y d e p h en y lacetald eh y d e
tran s -2-n o n en al n o n alacto n e

Fig.9: Concentration of some aldehydes in wort from filtration to fermentation.

9
 referen t ie

A ld eh y d es (g /100 g )
A ld eh y d es (g /100 g )
referen ce 2.5 ad d itio n o f g allo tan n in s
2.5
2 2.5
2
1.5 2 1.5
1 1.5 1

0.5 1 0.5
0 0.5 0

t
ng
t

g
t

t
ng
0

or

or

or
or

or

or

lin
lin

ili
w
ili

w
w

w
firstwort second wort startboiling end boiling pitching wort

oi
oi

bo
bo

st

nd
st

ng
nd

ng

tb
tb

fir
fir

hi
d
co
hi
d
co

ar
ar

en
en

tc
tc

se

st
se

st

pi
pi
tran s -2-h exen al tran s -2-h ep ten al b en zald eh y d e
tran s -2-n o n en al n o n alacto n e

Fig.10: Concentration of some minor aldehydes in wort from filtration to

fermentation.

CONCLUSIONS
The sensory and analytical results confirm that increasing the reducing or anti-
oxidant power during both mashing-in and wort filtration, results in a remarkable
improvement of the flavour stability of beer. Inhibition of lipid and protein oxidation
can be the main reason for this phenomenon. Addition of gallotannins to the brewing
and sparging water results in a better wort filtration performance, a clear inhibition of
the LOX reaction, a reduction in the concentration of some aldehydes in the wort,
especially some Strecker and Maillard products, and a significant increase in the
reducing power of the beers. Evaluation of the stability of trans- and cis-iso--acids in
beer seems more useful to monitor beer ageing than the determination of small
differences in the level of some supposed marker aldehydes.

REFERENCES
1. Analytica EBC, 5th Edition, 1997, and updates: Brauerei und Getrnke Rundschau, CH-
8047, Zrich, Switzerland.
2. Araki, S., Kimura, T., Shimizu, C., Furusho, S., Takashio, M. & Shinotsuka, K., Journal
of the American Society of Brewing Chemists, 1999, 57, 34-37.
3. Bamforth, C.W., Brauwelt International, 1999, 2, 98-110.
4. De Buck, A., Aerts, G., Bonte, S., Dupire, S., & Van den Eynde, E., Relation Between
Lipoxygenase Extraction During Brewing, Reducing Capacity of the Wort and
Organoleptical Stability of Beer, Proceedings of the 26th Congress of the European
Brewery Convention, Maastricht, The Netherlands, 1997. Oxford University Press, 1997,
333-340.
5. De Cooman, L., Aerts, G., Overmeire, H. & De Keukeleire, D., Journal of the Institute of
Brewing, 2000, 106, 169-178.
6. Muller, R., Journal of the Institute of Brewing, 1997, 103, 307-310.
7. Mussche, R.A. & de Pauw, C., Journal of the Institute of Brewing, 1999, 105, 386-391.
8. Okuda, T., in Polyphenolic Phenomena, ed. A. Scalbert, Paris, INRA Editions, 1993,
pp.221-235.
9. Wackerbauer, K., Hardt, R., Brauwelt International, 1997, 4, 320-327.

10
59

The role of polyphenols and oxidation

processes in brewhouse on beer quality
A. Mikyka, D. Hakov, M. Hrabk, J. rogl & T. Hork
Research Institute of Brewing and Malting PLC, Lpov 15, CZ-120 44 Prague 2, Czech
Republic (e-mail: [email protected])

Descriptors
Carbonyl compound, oxidation, polyphenol, taste stability

SUMMARY
Pilot scale brewing trials have shown that reducing activity of sweet worts, hopped worts and
beers depends on content of malt and hop polyphenols. Aroma hop is the significant source of
polyphenols with reducing activity. Increasing oxidation in brewhouse leads to reduce of
polyphenols content and reducing activity of worts and beers. Protection against oxidation in
brewhouse significantly damaged colloidal stability of beer. Oxidation and protection against
oxidation in brewhouse, both leads to increasing level of carbonyl compounds and damage of
flavor quality of fresh beer. The forming of carbonyl compounds in stored beer increases with
oxidation level in brewhouse.

Die Rolle der Polyphenole und der Oxidationsprozesse im Sudhaus auf die Bierqualitt

Deskriptoren
Carbonylverbindung, Geschmacksstabilitt, Oxidation, Polyphenol

ZUSAMMENFASSUNG
Brauversuche im Pilotmastab haben gezeigt, dass die Reduktionsaktivitt der Vorderwrzen,
der gehopften Wrzen und der Biere vom Inhalt der Malz- und Hopfenpolyphenole abhngt.
Aromahopfen ist die ausschlaggebende Quelle der Polyphenole mit Reduktionsaktivitt.
Strkere Oxidationsprozesse im Sudhaus fhren zu einer Abnahme des Polyphenolgehalts und
der Reduktionsaktivitt der Wrzen und Biere. Der Schutz vor Oxidationsprozessen im
Sudhaus fhrt zu einer erheblichen Abnahme der kolloidalen Stabilitt des Bieres.
Oxidationsprozesse und der Schutz vor diesen im Sudhaus fhren zu einem Anstieg der
Carbonylverbindungen und zu einer Verschlechterung der Geschmacksstabilitt des frischen
Bieres. Die Bildung der Carbonylverbindungen im gelagerten Bier erhht sich mit
zunehmenden Oxidationsprozessen im Sudhaus.
Rle des polyphnols et des procds d'oxydation en brasserie sur la qualite de la biere

Descripteurs
Compos carbonyl, oxydation, polyphnol, stabilit organoleptique

RESUME
Les essais de brassage l'chelle pilote ont montr que l'activit rductrice des mots sucrs,
des mots houblonns et des bires dpend de la teneur en malt et polyphnol de houblon. Le
houblon aromatisant est la source importante de polyphnol activit rductrice.
L'augmentation de l'oxydation dans la brasserie entrane une baisse de la teneur en polyphnol
et de l'activit rductrice des mots et des bires. La protection contre l'oxydation en brasserie
a significativement affaibli la stabilit collodale de la bire. L'oxydation et la protection
contre l'oxydation en brasserie entranent tous deux une augmentation du niveau des
composs carbonyls et de l'altration de la qualit de l'arme de la bire frache. La
formation de composs carbonyls dans la bire stocke augmente avec le niveau d'oxydation
dans la brasserie.

2
INTRODUCTION
A quality of raw materials and technological conditions in a brewhouse can induce
significant changes in colloidal and flavor stability of beer (1,5,6,7,8,12). The role of
polyphenol substances in these processes has been discussed for many years.
Colloidal haze of beer is based on the forming of protein-polyphenol complexes. In
contrary, polyphenols originated from malt and hops are one of the major components
with reducing activity occurring in wort and beer. Beer staling is caused by formation
of unsaturated carbonyl compounds. Antioxidants, such as polyphenols could prevent
formation staling carbonyls. It is well known, that the oxygen uptake in a period of
filtration and bottling deteriorate haze and flavor stability. There is only a technical
problem to minimize aeration of beer. The most important technological step for
achieving good flavor stability of beer is, except malting conditions mashing and wort
boiling.
The aims of this work were:
- to investigate the influence of oxidation conditions in brewhouse on haze stability
of beer.
- to study changes of the taste quality and stability in respect of malt and hop
polyphenols and oxidation conditions in a brewhouse.

MATERIALS AND METHODS

Pilot scale brewing trials
All-malt 12 Plato pale lagers were prepared by the two-mash tune and the classical
two-phase procedure of fermentation (bottom type yeast, strain No 96 of Research
Institute of Brewing and Malting (RIBM) collection) in the pilot scale brewery of
RIBM. The content of malt polyphenols was altered by the treatment of hot sweet
wort by PVPP (100 g/hl). The content of hop polyphenols was modified by the ratio
of dose CO2 - extract and the rest of hop pellets after extraction. Variants of hop
polyphenols dose 0%, 50%, 100% and 200% were tested. 100% represented
equivalent of polyphenols dose by the use of hop pellets. Low, common and high
oxidation level in brewhouse was simulated by air or CO2 - bubbling in the whole
course of mashing and the wort boiling. The beer was filtered, bottled and pasteurized
(28 PU). CO2 was used by the filtration and bottling. Beer was stored in a period of
six months at 20C in a dark place.

Analyses
Sweet wort, hopped wort and beer analyses were carried out according to Analytica
EBC (3). Anthocyanogens were measured using the method of Harris and Ricketts (4).
Determination of oxidized and oxidisable polyphenols was carried out using the
method described by Thompson and Forward (11). The reducing activity was measured
by the two different methods. Determination of DPPH-reducing activity was carried
out according to the method described by Kaneda (5). The method described by
Chapon (2) is based on the formation of a red complex by the reduction of DPFe3.
Carbonyl compounds in the beer were measured using a gas chromatography (9).
Prediction of shelf life was carried out according to the enhancing test described by
avel (10). Temperature profile of this test is 1 day by 0C, 6 days by 50C. The
evaluation of the sensorial quality of the beer and the stored beer was by the panel of
13 trained tasters of RIBM. The overall impression was evaluated in nine-point scale.

3
RESULTS AND DISCUSSION
Wort
About 65% of the malt total polyphenols and 75% of anthocyanogens were removed
by the PVPP treatment of sweet wort. The reducing activity decreased of about 50%
(table 1). It seems to be proved, more then 50% of the reducing activity value by
sweet wort was caused by malt polyphenols. The reducing activity of the sweet wort
showed a strong positive correlation with the content of the total polyphenols as well
as anthocyanogens for both used methods, method by Kaneda and method by Chapon
(table 2). The aroma hops (Saaz hops) is also a significant source of polyphenols with
the reducing activity, comparable with malt. The reducing activity of hop polyphenols
represented about 75% of the malt value (all-malt wort and hop boiled in a buffer)
(table 1).

Wort Wort
Wort Hop
(PVPP- (100% hop
(hop extract) (boiled in a
treated, hop polyphenols)
buffer)
extract)
Total polyphenols
190 79 326 141
(mg/l)
Anthocyanogens (mg/l) 65.2 16.6 116.0 108.1
Oxidisable polyphenols
12.89 0.63 21.83 8.53
(EBU)
Red. activity by Chapon
0.83 0.32 1.06 0.61
(mmol/l)
Red. activity by Kaneda
1.27 0.86 1.40 0.96
(A525)

Table 1: Analysis of the hopped worts and hop (boiled in buffer), oxidation
level control

Sweet wort Beer

Air Control CO2 Air Control CO2
Total polyphenols
Red. activity by Chapon 0.874 0.925 0.976 0.976 0.992 0.993
Red. activity by Kaneda 0.979 0.968 0.960 0.875 0.865 0.894
Anthocyanogens
Red. activity by Chapon 0.866 0.952 0.979 0.977 0.991 0.995
Red. activity by Kaneda 0.980 0.981 0.990 0.879 0.886 0.914

Table 2: Correlation between reducing activity, polyphenols and anthocyanogens -

sweet wort and beer (n = 16, = 0.05, ccrit. = 0.47)

Beer
The content of polyphenols, as well as the reducing activity of beers increased with
the increasing dose of hop polyphenols. A strong positive correlation of the reducing
activity with both polyphenols and anthocyanogenes was detected (table 2). The value
of the reducing activity in the wort and the beer depended on a dose of hop

4
polyphenols and the oxidation level in brewhouse. Partial removing of malt
polyphenols induced significant decrease of reducing activity in beer (figures 1 & 2).

1,40
untreated sweet PVPP - treated
Red. activity by Chapon (mmol/l)

1,20 wort sweet wort

1,00
0,80
0,60
0,40
0,20
0,00
0 50 100 200 0 50 100 200
Dose of hop polyphenols - in% of common dose

Air Control CO2

Figure 1: Reducing activity of beers (method by Chapon)

1,80 untreated sweet PVPP - treated

1,60 wort sweet wort
Reducing activity by Kaneda

1,40
1,20
1,00
(A525)

0,80
0,60
0,40
0,20
0,00
0 50 100 200 0 50 100 200
Dose of hop polyphenols - in % of common dose

Air Control CO2

Figure 2: Reducing activity of beers (method by Kaneda)

The increased oxidation level in a brewhouse induced a decrease of polyphenols

content and reducing activity value of sweet worts, hopped worts and beers. Aeration
in the course of mashing reduced content of polyphenols in about 7% and content of
anthocyanogens in about 25% in sweet wort. Protection against the oxidation in the
brewhouse increased the level of oxidisable polyphenols and significantly damaged

5
colloidal stability of beer. High dose of hop polyphenols negatively affected the shelf
life of the beer, especially by the lack of the malt polyphenols (figure 3).

14
13 untreated sweet
>12 >12
PVPP-treated
Prediction of shelf life (months)

12
wort sweet wort
11
10
9
8
7
6
5
4
3
2
1
0
0 50 100 200 0 50 100 200
Dose of hop polyphenols - in % of common dose

Air Control CO2

Figure. 3: Prediction of shelf life of beers by enhancing test

Several carbonyl markers of the beer stale were determined. Furfural and heptanal are
considered to be heat indicators, 2-me-butanal, 3-me-butanal, benzaldehyd and 2-
phenyletanal are oxygen indicators and 3-me-butan-2-on is general indicator of
beer staling. Oxidation and protection against oxidation in brewhouse, both induced
an increase of total content of carbonyl markers in fresh beer. Concentration of some
carbonyl, stalling markers increased with aeration (i.e. furfural), concentration of
some others increases with protection against oxidation in brewhouse (i.e. 3-me-
butanal, heptanal) (figure 4). Dose of hop polyphenols had positive effect, reduced
total content of carbonyls in fresh beer. Formation of carbonyl compounds increased
with growing oxidation level in brewhouse in the period of beer storage (figure 5).
This is caused especially by the formation large amounts of furfural and 3-me-butanal.
Furfural it seems to be a useful marker of aeration conditions in brewhouse.

6
 500
450
400
350
300
ug/l

250
200
150
100
50
0
Air Control CO2

furfuraldehyd heptanal 2-Me-butanal 3-Me-butanal

benzaldehyd fenylacetaldehyd 3-Me-butan-2-on

Figure 4: Content of carbonyl staling markers in fresh beer

untreated sweet PVPP - treated

3500 wort sweet wort
3000

2500

2000
ug/l

1500

1000

500

0
0 50 100 200 0 50 100 200
Dose of hop polyphenols - in% of common dose

Air Control CO2

Figure 5: Total content of carbonyl staling markers in beer after 6 months storage

Significant damage of the flavor quality of the fresh beer was caused both by
oxidation and the protection against oxidation in brewhouse. Overall impression,
which generally describes the beer popularity with the consumer, was chosen as the
determining criterion. Stalling of beers protected in brewhouse was slow. The
sensorial quality of control beers deteriorated rapidly, continuous in the whole period
of six months storage. After the period of 5 months the beer quality of all three
variants was comparable (figure 6).

7
 8
Overall impression (points)
7

Air
5 Control
CO2
4
0 1 2 3 4 5 6
Storage time (months)

Figure 6: Beer staling effect of oxidation in brewhouse

CONCLUSION
Protection against oxidation in brewhouse increased the content of oxidisable
polyphenols in the beer and significantly damaged the shelf life, the colloidal
stability of beer.
Our results shoved, that both malt and hops are significant source of substances
with reducing activity. The value of reducing activity in wort and beer correlated
with the content of malt and hop polyphenols and anthocyanogens.
It was confirmed, oxidation conditions in brewhouse affects forming of carbonyls.
Content of some stale carbonyl markers in fresh beer increased with oxidation
level, content of other decreased respectively.
Assumed content of carbonyl markers in six months stored beer depended on
oxidation in brewhouse. Protection against aeration had the positive effect, content
of carbonyls significantly decreased.
Furfural is probably a useful indicator of oxidation conditions in brewhouse.
Particular damage of the flavor quality of the fresh beer was caused by oxidation
as well as the protection against oxidation in brewhouse. On the contrary, staling
of these beers proceeded more slow then staling of control beers.

REFERENCES
1. Boivin, P., Malanda, M., Maillard, M.N., Berset, C., Hugues, M., Forget-
Richard, F. & Nicolas, J., Proceedings of the European Brewery Convention
Congress, Brussels, 1995, 159-168
2. Chapon, L., Louis, C. & Chapon, S., Proceedings of the European Brewery
Convention Congress, Estoril, 1971, 307-322
3. European Brewery Convention, Analytica EBC, IV-th edition, Zurich 1997
4. Harris, G. & Ricketts, R.W., Journal of The Institute of Brewing, 1958, 64, 22-
26

8
5. Kaneda H., Kobayashi, N., Furusho, S., Sahara, H. & Koshimo, S., Technical
Quarterly of the Master Brewers Association of the Americas, 1995, 32, 90-94
6. Lermusieau G., Nol, S., Ligerois, C. & Collin, S., Journal of the American
Society of Brewing Chemists, 1999, 57, 29-33
7. Narziss, L., Reicheneder, E. & Lustig, W., Brauwelt International, 1989, 38,
238-
8. Nol, S., Ligeois, C., Lermusieau, G., Bodart, E., Badot, C. & Collin, S.,
Journal of Agriculture and Food Chemistry, 1999, 47, 4323-4326
9. kach,J., ejka, P. & ulk, J., Senzorick stabilita piva (Research report of
RIBM), Prague, 1993
10. avel, J. & Prokopov, M., Kvasn prmysl, 1992, 38, 289-292
11. Thomson, C.C. & Forward, G.P., Journal of The Institute of Brewing, 1969, 75,
37-42
12. Walters, M.T. Ferment, 1997, 10, 111-119

9
60

Comparative study of the stability of

-acids, dihydroiso-
iso- -acids and
tetrahydroiso--acids during beer
ageing
Luc De Cooman1, Guido Aerts1, An Witters1, Marjan De
Ridder1, Annick Boeykens1, Koen Goiris1 & Denis De
Keukeleire2
1
KaHo St.-Lieven, Laboratory of Enzyme and Brewing Technology, Gebroeders
Desmetstraat 1, B-9000 Ghent, Belgium (e-mail: [email protected])
2
University Ghent, Faculty of Pharmaceutical Sciences, Laboratory of Pharmacognosy and
Phytochemistry, Harelbekestraat 72, B-9000 Ghent, Belgium

Descriptors
Beer quality, bitterness, iso--acid, oxidation, staling

SUMMARY
Reduced iso--acids are believed to exhibit a higher resistance to oxidation in the beer matrix,
as compared with iso--acids. This study on the stability of the different bitter acids by HPLC
profiling of selected ageing beers, showed that trans-iso--acids are more prone to oxidation
than their cis counterparts. Tetrahydroiso--acids are extremely resistant to oxidative
deterioration, whilst the "light-stable" dihydroiso--acids are not at all stable on storage. For
"normal" beers, bitterness consistency can be improved by the use of isomerised hop extracts,
or by the partial substitution of iso--acids for tetrahydroiso--acids. For beers packaged in
colourless bottles, dihydroiso--acids are to be used, but oxidative deterioration of these
compounds will occur anyhow. The potential of the hexahydroiso--acids should be re-
evaluated.

Vergleichende Studie zur Stabilitt der Iso--Suren, der Dihydroiso--Suren und der
Tetrahydroiso--Suren whrend der Bieralterung

Deskriptoren
Altgeschmacksbildung, Bierqualitt, Bittere, Iso--Sure, Oxidation

ZUSAMMENFASSUNG
Bei reduzierten Iso--Suren wird vermutet, dass sie im Vergleich zu Iso--Suren eine
hhere Oxidationsresistenz in der Biermatrix gewhrleisten. Die Untersuchung ber die
Stabilitt der unterschiedlichen Bittersuren durch das HPLC-Profil ausgewhlter gealterter
Biere hat gezeigt, dass die trans-Iso--Suren anflliger fr die Oxidation sind, als ihre cis-
Gegenstcke. Tetrahydroiso--Suren sind sehr bestndig gegen einen Zerfall durch
Oxidation, whrend die lichtbestndigen Dihydroiso--Suren nicht immer whrend der
Lagerung stabil sind. Fr normale Biere kann eine gleich bleibende Bittere durch den
Gebrauch von isomerisierten Hopfenextrakten oder durch den teilweisen Ersatz der Iso--
Suren durch Tetrahydroiso--Suren gewhrleistet werden. Fr in Weiglasflaschen
abgefllte Biere sollten Dihydroiso- -Suren verwendet werden, dennoch tritt auch hier eine
Verschlechterung durch Oxidation auf. Das Potenzial der Hexahydroiso--Suren sollte neu
bewertet werden.

-acides, des dihydroiso-

Etude comparative de la stabilit des iso- -acides et des
-acides pendant le vieillissement de la bire
tetrahydroiso-

Descripteurs
Acide iso-, amertume, formation du got dvente, oxydation, qualit de la bire

RESUME
On estime que les iso--acides rduits prsentent une plus grande rsistance l'oxydation
dans la bire que les iso--acides. Cette tude sur la stabilit des diffrents acides amres par
profilage par CLHP de bires vieillissantes slectionnes a montr que les trans-iso--acides
s'oxydent plus que leurs homologues cis. Les ttrahydroiso--acides sont extrmement
rsistants la dgradation oxydative, tandis que les dihydro-iso--acides "photostables" ne
sont pas du tout stables pendant le stockage. Pour les bires "normales" la stabilit de
l'amertume peut tre amliore en employant des extraits de houblon isomriss ou en
procdant une substitution partielle des ttrahydro-iso--acides par des iso--acides. Pour
les bires conditionnes en bouteilles incolores, il est ncessaire d'utiliser des dihydro-iso--
acides mais la dgradation oxydative de ces composs survient de toute faon. Le potentiel
des hxahydro-iso--acides doit tre rvalu.

2
INTRODUCTION
Iso--acids, derived from the hop -acids, are primary flavour constituents of beer
(7). Beer contains six major iso--acids, i.e. the trans- and cis-isomers of
isocohumulone, isohumulone, and isoadhumulone (for the structures, see figure 1).
Together with some minor iso--acids, these compounds are largely responsible for
the characteristic bitter beer taste (15, 18). One of the most striking sensory changes
in ageing beer is a decrease in bitterness intensity and quality, often occurring from
the onset of the ageing process (2). Such undesirable changes are associated with
oxidative transformations on beer storage (1, 12, 19). In this respect, isohumulones
are indeed prone to oxidative decomposition during the brewing process and on
storage of finished beer (3, 18). Iso--acids deterioration in ageing beer has been
reported in several studies and is believed to be caused by free-radical reactions (4,
10, 11, 13, 14, 17, 20). Moreover, iso--acids degradation is often quoted as a
possible pathway to the generation of ageing off-flavours, including staling carbonyls
(2, 4, 5, 18, 19). Recent investigations demonstrated that trans-iso--acids are
significantly less stable in the beer matrix than cis-iso--acids (4, 9, 14, 20). In a
previous study, we emphasised the marked instability of trans-iso--acids in ageing
beer and hence, their adverse impact on bitterness consistency (4). It was clearly
shown that the ratio of the sum of the concentrations of trans-isocohumulone (1a) and
trans-isohumulone (1b) to the sum of the concentrations of cis-isocohumulone (2a)
and cis-isohumulone (2b), defined as the trans-cis ratio, proves to be a reliable
parameter to evaluate bitterness deterioration in ageing beers (4). In contrast to iso--
acids, tetrahydroiso--acids appeared to be remarkably stable on beer storage (4).
These earlier observations prompted us to conduct a more comprehensive study on the
stability of both conventional and reduced iso--acids, including dihydro- and
tetrahydroiso--acids. Nowadays, dihydro- and tetrahydroiso--acids are used in the
preparation of light-stable beers to circumvent the formation of 3-methyl-2-butene-1-
thiol by photosensitised degradation of iso--acids (3, 8) (for the structures of the
reduced iso--acids, see figure 1). Tetrahydroiso--acids are also frequently applied
in normal beers to enhance foam stability and bitterness consistency (3). Besides
their light-proofing properties, it is accepted that reduced iso--acids also exhibit a
higher resistance to oxidative decomposition in the beer matrix in comparison with
conventional iso--acids (6, 14). However, conclusive evidence for this assumption is
not available in literature. We therefore examined the stability of the different bitter
acid types in pilot beers brewed at our institute, and in commercial beers with a
known bitter acid profile.

MATERIALS AND METHODS

Preparation of pilot beers
Three pilsener beers were prepared at our pilot scale brewery (2 hl) from one and the
same brew. By post-fermentation addition of iso--acids, dihydroiso--acids, and
tetrahydroiso--acids, respectively, to green beer derived from the same fermentation
vessel, a similar matrix for these pilot beers is obtained. Beer preparation was carried
out as follows. Grist: pilsener malt (34 kg) and maize flakes (6 kg); brewing water:
reverse osmosis (1.7 hl) with addition of Ca2+ (40 ppm); brewing scheme: 36C (15
min), 49C (30 min), 63C (20 min), 72C (30 min), 78C (120 min, including wort
filtration with lauter tun); wort boiling: 75 min; wort clarification: whirlpool;

3
original gravity: 12.2P; pitching rate: 107 cells/ml; fermentation temp. and duration:
12C, 8 days; hopping: at transfer to cask with either iso--acids (3.13 g/hl; presumed
utilisation: 80%), dihydroiso--acids (4.20 g/hl; utilisation: 85%), or tetrahydroiso--
acids (2.26 g/hl; utilisation: 65%); lagering: in cask (10 days at 2C); beer filtration:
kieselguhr/cellulose sheets (1 m); packaging: manual filling (Servinox) in brown
glass bottles (25 cl).

Beer samples
Two commercial beers, indicated as A (pellet hopping) and E (hopped with
reduced isomerised extracts), and three pilot beers, indicated as B, C, and D,
were studied. Next to fresh beers, the following samples were analysed:
- Beers A, B, C, D, E: samples aged at 60C for 1, 3, 5, 7, and 10 days.
- Beer A: samples kept in the dark at room temperature for 1 to 6, 10 and 12 months.
- Beer E: samples kept in the dark at room temperature for 1 to 6, 9 and 12 months.

-acids
Extraction of bitter acids from beer and HPLC analysis of (reduced) iso-
The bitter acids were extracted from the beers and subsequently analysed by high-
performance liquid chromatography (HPLC) as described earlier (4).

O O O
O
5 5
R R
4 4
HO HO
O OH O OH

Trans-isocohumulone (1a) Cis-isocohumulone (2a)

Trans-isohumulone (1b) Cis-isohumulone (2b)
Trans-isoadhumulone (1c) Cis-isoadhumulone (2c)
O O O O
5 5
R R
4 4
HO HO
O OH O OH

Trans-tetrahydroisocohumulone (3a) Cis-tetrahydroisocohumulone (4a)

Trans-tetrahydroisohumulone (3b) Cis-tetrahydroisohumulone (4b)
Trans-tetrahydroisoadhumulone (3c) Cis-tetrahydroisoadhumulone (4c)
H O O

O O 5 R
HO 4
5
R OH
4 O
HO
HO Ra = CH(CH3)2
OH
Rb = CH2CH(CH3)2
Rc = CH(CH3)CH2CH3
Dihydroiso--acids Trans-iso--acids (1a-c)
Fig. 1: Structures of iso--acids, tetra- and dihydroiso--acids.

4
RESULTS AND DISCUSSION
-acids in a commercial pilsener beer
Evaluation of the stability of iso-
A kettle hopped commercial pilsener, indicated as beer A, was studied as a function
of both spontaneous and forced ageing. Figure 2A shows the iso--acids profile of the
fresh beer. The applied HPLC method allows for the full separation of the 6 major
iso--acids, i.e. trans-isocohumulone (1a), cis-isocohumulone (2a), trans-
isohumulone (1b), cis-isohumulone (2b), trans-isoadhumulone (1c), and cis-
isoadhumulone (2c).

Fig. 2: HPLC analysis of the iso--acids in a commercial pilsener beer A.

Fig. 2A:fresh sample; Fig. 2B: sample after spontaneous ageing for 12 months.

The HPLC analysis of a beer sample that had been stored for one year in the dark at
room temperature, is represented in figure 2B. Comparison between the analyses in
figure 2 points to the differential behaviour of trans- and cis-iso--acids on beer
storage. Trans-isomers are obviously deteriorated at a much faster rate than cis-
isomers. In fact, the small peaks of trans-iso--acids in figure 2B (1a, 1b, 1c) suggest
a high sensitivity of these compounds towards oxidative degradation in the beer
matrix. The contents of iso--acids in samples of beer A that were aged
spontaneously for 1 to 12 months are summarised in table 1.
Ageing time (months)
a b b
Compound 0 1 2 3b 4b 5b 6 b
10 b
12 b

Trans-ich 2.2 1.9 1.7 1.5 1.4 1.2 1.0 0.6 0.5
Cis-ich 5.1 4.9 4.9 4.9 4.8 4.7 4.6 4.6 4.3
Trans-inh 3.5 2.9 2.6 2.4 2.2 1.8 1.5 0.9 0.7
Cis-inh 9.1 8.8 8.8 8.8 8.5 8.2 8.1 7.9 7.4
Trans-iah 1.2 1.1 1.0 1.0 0.8 0.8 0.8 0.6 0.6
Cis-iah 2.4 2.4 2.2 2.3 2.3 2.2 2.1 2.0 2.0
Total (ppm) 23.5 22.0 21.2 20.9 20.0 18.9 18.1 16.6 15.4
Total (%) 100 94 90 89 85 80 77 71 66
T/C ratio (%) 40 35 31 29 27 23 20 16 10
a
= fresh sample
b
= samples stored in the dark at room temperature
Table 1: Iso--acids (ppm) in beer A as a function of spontaneous ageing.

5
After one year, total bitterness is reduced by 34%. The T/C ratio amounts to 40% for
the fresh sample but only 10% for the one year-old beer, reflecting the differential
behaviour of trans- and cis-iso--acids. After one year of spontaneous ageing, about
75% of trans-isomers are lost versus 18% of cis-components. A shelf half-life for
trans-iso--acids of less than one year was also noticed previously in a commercial
top-fermented beer subjected to spontaneous ageing (4). As a consequence, even after
relatively short storage times of only one or two months, distinct decreases in the
levels of trans-iso--acids can be measured (see also table 1). Similar findings as to
the differential behaviour of trans- and cis-iso--acids, were obtained by forced
ageing of beer A at 60C (see table 2). The marked instability of trans-iso--acids
was explained earlier in terms of the stereochemical arrangement of the isohexenoyl
and the prenyl side chains at C(4) and C(5), respectively (4). As shown in figure 1,
only in trans-iso--acids, the proximity of the unsaturated sites provides a pool of
high electron density, allowing ready initiation of radical-type oxidation reactions.
Ageing time (days)
Compound a b b b b b
0 1 3 5 7 10
Trans-ich 2.2 1.7 1.2 1.0 0.7 0.5
Cis-ich 5.1 4.7 4.5 4.5 4.1 4.0
Trans-inh 3.5 2.6 1.7 1.4 0.9 0.7
Cis-inh 9.1 8.3 8.1 8.1 7.2 6.9
Trans-iah 1.2 1.1 0.9 0.9 0.8 0.8
Cis-iah 2.4 2.1 2.1 2.1 1.9 1.7
Total (ppm) 23.5 20.5 18.5 18.0 15.6 14.6
Total (%) 100 87 79 77 66 62
T/C ratio (%) 40 33 23 19 14 11
a
= fresh sample
b
= samples aged in a thermostated room at 60C
Table 2: Iso--acids (ppm) in beer A as a function of forced ageing.
Ageing time (days)
Compound a b b b b b
0 1 3 5 7 10
Trans-ich 2.1 1.9 1.6 1.2 1.2 0.9
Cis-ich 7.9 7.5 7.1 6.7 7.0 6.5
Trans-inh 3.3 2.9 2.3 1.7 1.7 1.2
Cis-inh 11.4 10.4 10.1 9.4 9.9 9.1
Trans-iah 0.8 0.7 0.7 0.6 0.6 0.5
Cis-iah 2.8 2.6 2.6 2.4 2.4 2.3
Total (ppm) 28.3 26.0 24.4 22.0 22.8 20.5
Total (%) 100 92 86 78 81 72
T/C ratio (%) 28 27 23 18 17 13
a
= fresh sample
b
= samples aged in a thermostated room at 60C
Table 3: Iso--acids (ppm) in beer B as a function of forced ageing.

Evaluation of the stability of conventional and reduced iso- -acids in pilot beers
Three pilot beers with a similar beer matrix were prepared as described in Materials
and Methods. After primary fermentation, either iso--acids (beer B), dihydroiso-
-acids (beer C), or tetrahydroiso--acids (beer D), were added. The contents of
iso--acids on forced ageing of Beer B are summarised in table 3. In the case of our
pilot beer, a relatively low T/C ratio (28%) is measured in the fresh sample, which is
typical when using pre-isomerised hop extract (9). Nevertheless, the results obtained
on the pilot beer B (table 3) largely parallel those collected for the commercial beer

6
A (table 2), i.e. clear reductions in total bitterness and especially in the T/C ratio are
observed on forced ageing.
120 120

Relative c onc . (% )
Relative c onc . (% )

100 100
80 80
60 60
40 40
20 20
0 0
0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 9 10
Days at 60C Days at 60C
d-ich(a) d-ich(b)
t-ic h c -ic h t-inh c -inh d-inh(a) d-inh(b)/d-iah(a)
Fig. 3: Iso--acids (%) in beer B as a Fig. 4: Dihydroiso--acids (%) in beer
function of ageing at 60C. C as a function of ageing at 60C.
Figure 3 shows the relative concentrations1 of the major iso--acids, i.e. trans-
isocohumulone (t-ich), cis-isocohumulone (c-ich), trans-isohumulone (t-inh), and cis-
isohumulone (c-inh), as a function of forced ageing time. As opposed to the
differential behaviour of trans- and cis-iso--acids, co- and normal homologues
degrade at a similar rate. When comparing the results in table 3 with those in table 2,
it is further interesting to notice that bitterness was more stable in the pilot beer B
than in the commercial beer A. Closer examination of the quantitative data reveals
that this is mainly due to a greater stability of the trans-iso--acids in the pilot beer.
Obviously, the rate of decline of the T/C ratio varies from one beer to another and
such interesting differences are easily detected by high-resolution HPLC (4).
Ageing time (days)
Compound a b b b b b
0 1 3 5 7 10
Dihydro-ich(a) 6.6 6.1 5.9 5.3 5.1 4.5
Dihydro-ich(b) 10.2 9.4 9.1 8.4 7.8 7.0
Dihydro-inh(a) 5.1 4.6 4.4 4.0 3.8 3.3
Dihydro-inh(b)/ 11.4 10.2 10.0 9.0 8.8 7.7
dihydroiah(a)
Dihydroiah(b) 3.1 2.8 2.8 2.6 2.5 2.3
Total (ppm) 36.4 33.1 32.2 29.3 28.0 24.8
Total (%) 100 91 88 81 77 68
a
= fresh sample
b
= samples aged in a thermostated room at 60C
Table 4: Dihydroiso--acids (ppm) in beer C as a function of forced ageing.

Totally new results were obtained on pilot beer C containing only dihydroiso--
acids. The quantitative data are represented in table 4. Because the identity of the
dihydroiso--acids is not completely solved, the compounds are tentatively indicated
as dihydroisocohumulone (a), dihydroisocohumulone (b), dihydroisohumulone (a),
dihydroisohumulone (b), dihydroisoadhumulone (a)2, and dihydroisoadhumulone (b),
analogous to the identification of iso--acids and tetrahydroiso--acids. At first sight,
the dihydroiso--acids concentration in the fresh sample seems to be rather high
(36.4 ppm), but according to Benitez et al. (3) such a level corresponds to a sensory
1
The content of each iso--acid in the fresh sample was equalised to 100%.
2
Dihydroisoadhumulone (a) is not resolved from dihydroisohumulone (b). Hence, these peaks were
quantified together.

7
bitterness, equivalent to about 25 ppm of iso--acids. Clear reductions in the content
of all dihydroiso--acid components are detected on forced ageing, resulting in a
relative total bitterness of only 68% after 10 days at 60C. The changes in the levels
of the individual components are visualised in figure 4. All dihydroiso--acids show a
similar degradation rate, in contrast to iso--acids. A plausible explanation for this
finding is that the dihydroiso--acids of the applied hop extract would possess the
same configuration, i.e. the cis-configuration as claimed by Ting & Goldstein (16).
According to this view, when comparing the data obtained on the beers B and C
(see tables 3 and 4), dihydroiso--acids would be more sensitive to deterioration in
beer than iso--acids. Anyhow, as apparent from the figures 3 and 4, the currently
available dihydroiso--acids show a faster degradation than cis-iso--acids.
In tetrahydroiso--acids, the double bonds in the side chains at C(4) and C(5) are
reduced (see figure 1). On account of this modification, it has been predicted that
tetrahydroiso--acids should behave very stable towards oxidation in finished beer (3,
18). This view is confirmed by the results obtained on pilot beer D (see table 5 and
figure 5). Only a relatively small reduction in the level of tetra-components (approx.
10%) was detected on forced ageing. In our previous paper, a marked stability of
tetrahydroiso--acids was also noticed in a commercial pilsener, subjected to
prolonged spontaneous ageing (4). By comparing the data collected for the pilot beers,
we can conclude that tetrahydroiso--acids are the most stable bitter acids towards
oxidative deterioration, followed by cis-iso--acids, dihydroiso--acids, and trans-
iso--acids.
Ageing time (days)
Compound a b b b b b
0 1 3 5 7 10
Trans-thich 1.4 1.4 1.4 1.4 1.4 1.3
Cis-thich 4.4 4.4 4.2 4.2 4.1 4.2
Trans-thinh 1.2 1.1 1.0 1.1 1.0 1.1
Cis-thinh 4.2 4.0 4.1 3.6 3.5 3.7
Trans-thiah 0.3 0.3 0.3 0.3 0.2 0.3
Cis-thiah 1.1 1.0 1.0 1.0 0.9 1.0
Total (ppm) 12.6 12.2 12.0 11.6 11.1 11.6
Total (%) 100 97 95 92 88 92
a
= fresh sample
b
= samples aged in a thermostated room at 60C
Table 5: Tetrahydroiso--acids (ppm) in beer D as a function of forced ageing.

120 120
Relative c onc . (% )

Relative c onc .(% )

100 100
80 80
60 60
40 40
20 20
0 0
0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 9 10
Days at 60C Days at 60C
d -ich (a) d -ich (b )
t-thic h c -thic h t-thinh c -thinh d -in h (a) d -in h (b )/d -iah (a)

Fig. 5: Tetrahydroiso--acids (%) in Fig. 6: Dihydroiso--acids (%) in beer

beer D as a function of ageing at 60C. E as a function of ageing at 60C.

8
Evaluation of the stability of reduced iso- -acids in a commercial beer
The study of the pilot beer C revealed instability of dihydroiso--acids on forced
ageing. To determine whether these observations would also hold in other beers, a
commercial light-stable beer E was investigated. As required to light stability, beer
E is completely bittered with reduced iso--acids, the fresh beer containing both
dihydroiso--acids (20 ppm) and tetrahydroiso--acids (3 ppm). In our study on beer
E, samples of the same brew were subjected to forced or spontaneous ageing at
various time intervals. The results are summarised in the figures 6 to 8. In conformity
with our findings obtained on beer C, a relatively strong and similar reduction in all
dihydro-components is observed on forced ageing of beer E (figure 6). When
comparing the results in the figures 6 and 4, it can be noticed that the dihydroiso--
acids behaved more stable in the pilot beer C than in the commercial beer E.
Relative conc. (% )

120 120

Total conc. (% )
100 100
80 80
60 60
40 40
20 20
0 0
0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 9 10 11 12
D ays at 60C Time (months)
t-thich c-thich t-thinh c-thinh Total dihydro Total tetra

Fig. 7: Tetrahydroiso--acids (%) in Fig. 8: Total dihydro- and tetrahydro-

beer E as a function of ageing at 60C. iso--acids (%) in beer E as a function
of spontaneous ageing.

Figure 7 again demonstrates the stability of the tetrahydroiso--acids. As further

shown in figure 8, similar results on the dihydro- and tetrahydroiso--acids were
obtained on prolonged spontaneous ageing of beer E.

CONCLUSIONS
Trans-iso--acids are more prone to oxidation in ageing beer than cis-iso--acids, and
the corresponding trans/cis iso--acids ratio is useful to monitor and evaluate
bitterness deterioration. In contrast with conventional iso--acids, tetrahydroiso--
acids are extremely resistant to oxidative decomposition. Surprisingly, the light-stable
dihydroiso--acids are not at all stable on storage. Thus, the generalised view that
reduced iso--acids exhibit enhanced resistance to oxidative deterioration in the beer
matrix is only true for tetrahydroiso--acids. The decomposition kinetics of the three
bitter acid types in the pilot beers, demonstrated that the most stable bitter principles
towards oxidation in beer are the tetrahydroiso--acids, followed by cis-iso--acids,
dihydroiso--acids, and trans-iso--acids. For beers stored in dark-coloured glass
bottles, bitterness consistency can be substantially improved by the use of pre-
isomerised hop products with a high proportion of cis-iso--acids, and/or by the
partial substitution of iso--acids for tetrahydroiso--acids. Replacement of iso--
acids by dihydroiso--acids in normal beers is not recommended, considering the
susceptibility of the dihydro-components to oxidation. For beers packaged in
colourless glass bottles, dihydroiso--acids should be used as the main bittering

9
principles because of their inherent light-stability. However, special attention must be
paid to the issue of oxidative flavour deterioration in order to slow down, among other
things, dihydroiso--acids decay. It also seems worthwhile to reconsider the potential
benefits of the hexahydroiso--acids.

ACKNOWLEDGEMENT
The authors thank the Flemish Institute for the Promotion of Scientific-Technological
Research in Industry (IWT, Brussels, Belgium) for financial support (HOBU IWT-
Fund, project numbers 970117 and 990070).

REFERENCES
1. Araki, S., Kimura, T., Shimizu, C., Furusho, S., Takashio, M. & Shinotsuka, K., Journal
of the American Society of Brewing Chemists, 1999, 57, 34-37.
2. Bamforth, C.W., Brauwelt International, 1999, 2, 98-110.
3. Benitez, J.L., Forster, A., De Keukeleire, D., Moir, M., Sharpe, F.R., Verhagen, L. C. &
Westwood, K. T., Hops and Hop Products - EBC Manual of Good Practice, Nrnberg:
Fachverlag Hans Carl, 1997.
4. De Cooman, L., Aerts, G., Overmeire, H. & De Keukeleire, D., Journal of the Institute of
Brewing, 2000, 106, 169-178.
5. Hashimoto, N. & Kuroiwa, Y., Proceedings of the American Society of Brewing
Chemists, 1975, 33, 104-111.
6. Held, R., Technical Quarterly of the Master Brewers Association of the Americas, 1998,
35, 133-140.
7. Hough, J.S., Briggs, D.E., Stevens, R. & Young, T.W., Malting and Brewing Science,
Vol. 2, Hopped Wort and Beer, Second Edition, Londen: Chapman and Hall, 1982,
Chapter 23.
8. Hughes, P.S., Cerevisia, 1999, 24 (2), 21-25.
9. Hughes, P.S., Menneer, I.D., Walters, M.T. & Marinova, G., Proceedings of the 26th
Congress of the European Brewery Convention, Maastricht, 1997, 231-238.
10. Kaneda, H., Kano, Y., Koshino, S. & Ohya-Nishiguchi, H., Journal of Agricultural and
Food Chemistry, 1992, 40, 2102-2107.
11. Kaneda, H., Kano, Y., Osawa, T., Kawakishi, S. & Kamada, K., Journal of the American
Society of Brewing Chemists, 1989, 47, 49-53.
12. Kaneda, H., Kobayashi, N., Furusho, S., Sahara, H. & Koshino, S., Technical Quarterly
of the Master Brewers Association of the Americas, 1995, 32, 90-94.
13. Kaneda, H., Takashio, M., Tamaki, T. & Osawa, T., Journal of the Institute of Brewing,
1997, 103, 21-23.
14. King, B.M. & Duineveld, C.A.A., Food Quality and Preference, 1999, 10, 315-324.
15. Moir, M., Journal of the American Society of Brewing Chemists, 2000, 58, 131-146.
16. Ting, P.L. & Goldstein, H., Journal of the American Society of Brewing Chemists, 1996,
54, 103-109.
17. Uchida, M. & Ono, M., Journal of the American Society of Brewing Chemists, 1996, 54,
198-204.
18. Verzele, M. & De Keukeleire, D., Chemistry and Analysis of Hop and Beer Bitter Acids,
Amsterdam, Elsevier, 1991.
19. Wackerbauer, K. & Hardt, R., Brauwelt International, 1997, 4, 320-327.
20. Walters, M.T., Heasman, A.P. & Hughes, P.S., Journal of the American Society of
Brewing Chemists, 1997, 55, 91-98.

10
61

A new technology for beer flavor

stabilization: control of key intermediates
of the Maillard reaction
Rafael Rangel-Aldao1, Adriana Bravo1, Ivn Galindo-Castro1, Beatriz
Snchez1, Lorena Reverol1, Erika Scherer1, Jorge Madrid1, Jos Lus
Ramrez2, Julio Herrera3, Merja Penttil4, Maija-Leena Vehkomki4,
Virve Vidgren4, Hannele Virtanen4 & Silja Home4
1
Empresas Polar, Polar Technology Center, Caracas, Venezuela (e-mail: rafael.rangel@empresas-
polar.com)
2
Institute of Advanced Studies (IDEA)
3
Simn Bolvar University, Department of Chemistry, Caracas, Venezuela
4
VTT Biotechnology, Espoo, Finland

Descriptors
Carbonyl compound, enzymic reaction, non-enzymic browning, stabilisation, staling

SUMMARY
We detected the accumulation of highly reactive -dicarbonyls in aged beers. Six different -
dicarbonyls increased up to 9 fold during storage, and their blockage by specific trapping reagents
retarded flavor deterioration of beer for two months at 28 C. We also purified and cloned a
reductase from brewers yeast (Old Yellow Enzyme, EC1.6.99.1) that was capable of reducing
these intermediates in beer. Overexpression of this enzyme in brewers yeast produced beers in
pilot trials that showed lower concentrations (30-40%) of Strecker aldehydes after primary and
secondary fermentations. This work underlines -dicarbonyls and their own precursors as new
targets to stabilize beer flavor.

Neue Technologie zur Geschmacksstabilisierung von Bier

Deskriptoren
Altgeschmacksbildung, Carbonylverbindung, Enzymreaktion, nichtenzymatische Brunung,
Stabilisierung

ZUSAMMENFASSUNG
Wir stellten die Hufung von hochaktiven -Dicarbonylen in gealtertem Bier fest. Sechs
verschiedene -Dicarbonyle nahmen whrend der Lagerung bis zu 9 Faltungen zu, und ihre
Blockierung durch spezifische abfangende Reagenzien verzgerte die Geschmacks-
erschlechterung von Bier um zwei Monate bei einer Aufbewahrungstemperatur von 28 C. Des
Weiteren reinigten und klonierten wir eine Reduktase aus Bierhefe (Old Yellow Enzyme, EC
1.6.99.1), die in der Lage war, diese Intermediate im Bier zu reduzieren. Die Produktion dieser
Reduktase in Bierhefe fhrte in Pilotversuchen zu Bieren, die eine niedrigere Konzentration (30
40%) an Streckeraldehyden nach der Haupt- und Nachgrung aufwiesen. Diese Arbeit
unterstreicht die Bedeutung von -Dicarbonylen und deren Vorlufer als neues Ziel der
Forschung ber die Geschmacksstabilitt des Bieres.

Nouvelle technologie de stabilisation de l'arme de la bire: contrle des intermdiaires

essentiels de la raction de Maillard

Descripteurs
Brunissement non-enzymatique, compos carbonyle, formation du got d'vent, raction
enzymatique, stabilisation

RESUME
Nous avons dtect l'accumulation d'-dicarbonyles hautement ractifs dans des bires vieillies.
Six -dicarbonyles diffrents ont vu leur concentration multiplie par 9 pendant le stockage, et
leur blocage par des agents pigeants spcifiques a retard la dtrioration de l'arme des bires
pendant deux mois 28 C. Nous avons galement purifi et clon une rductase de la levure de
bire (Enzyme Jaune Vieux, EC1.6.99.1) qui s'est rvle capable de rduire ces intermdiaires
dans la bire. La surexpression de cette enzyme dans la levure de bire a produit, dans des essais
pilotes, des bires prsentant de plus faibles concentrations (30-40 %) d'aldhydes de Strecker
aprs fermentations primaires and secondaires. Ce travail montre que les -dicarbonyles et leurs
prcurseurs sont de nouvelles cibles pour stabiliser l'arme des bires.

2
INTRODUCTION
The flavor stability of beer remains as one of the major challenges of the brewing
industry due to the great complexity and dynamism of the changes involved (1). These,
are caused by a succession of multiple reactions induced mainly by the own chemical
composition of finished beer, the amounts of dissolved oxygen, and by environmental
factors such as exposure to light and the temperature of storage of the packaged product
(2). Given such complexities, we focused our research on the effects of heat treatment on
flavor stability given our tropical temperatures of 28C and higher, and the very low
contents of dissolved oxygen in our packaged beer (<0.08 mg/l).
Chemical indicators of beer flavor deterioration, developed by our group using HPLC
allowed the monitoring of beer aging at a wide range of temperatures during storage, and
correlated very well with flavor deterioration (3). These reporter molecules, came from
early precursors of the Maillard reaction such as 3-deoxy-hexosulose (3-DH) and 1-
deoxy-hexosulose (1-DH). One of the novel indicators, named peak LC18 upon isolation
by HPLC, was very sensitive to the temperature of beer storage even at 0C. It
corresponded to a direct product of the thermal decomposition of 3-DH, to yield 5-
hydroxymethyl furfural (HMF); and its most likely chemical structure was consistent
with that of 3,4-dideoxyhexosulose-3-ene, DDH, an -dicarbonylic product of the
dehydration of 3-DH (3). The role of -dicarbonyl intermediates in beer aging has not
been reported, and there is scanty literature on these type of compounds in the brewing
process (4).
The objective of this work is, therefore, to study the possible role of -dicarbonyls in the
flavor deterioration of beers stored at 28C. Here, we report the formation of these
intermediates in beer and the effect of their blockage on beer flavor stability by either
chemical, or biochemical means using genetically engineered brewers yeast.

MATERIALS AND METHODS

Determination of -dicarbonyls in beer
These reactive compounds were made stable by their derivatization with 1,2-
diaminobenzene to obtain the corresponding hydrophobic and hydrophilic quinoxalines
fractionated by HPLC (5). Chemical structures of the derivatized -dicarbonyls were
determined using High Resolution Gas Chromatography/Mass spectrometry (HRGC-
MS), and High Resolution Nuclear Magnetic Resonance (1H, 13C, DEPT, COSY and
HMQC NMR), as described in (5).

Sensory analysis
A trained tasting panel used a special descriptive form with 37 attributes for the
evaluation. Bitterness was not taken into account in the general score of freshness degree
in the case of the addition of 2 mM aminoguanidine to fresh beer at bottling and
subsequent storage for the indicated weeks at either 0C or 28C.

Enzyme assays
Reductase activity was assayed by measuring the rate of pyrimidine nucleotide oxidation
at 340 nm (6) .

3
Search, isolation, purification, and cloning of a reductase enzyme from brewers
yeast with activity towards -dicarbonyls of beer (Oye2).
In essence, pitching brewers yeast cells were disrupted and submitted to successive
purification steps with a combination of ion and affinity chromatography employing
DEAE-Sepharose, CM-Sephadex, Cibacron Blue, and Red Sepharose columns (6). A
single protein band was obtained after polyacrylamide gel electrophoresis in the presence
of sodium dodecyl sulfate (SDS-PAGE), with a relative molecular mass (Mr) of 44,000.
This protein behaved as a dimer of Mr= 86,000 upon gel filtration chromatography, and
displayed a NADPH-dependent reductase activity towards pure -dicarbonyls (see
Results and Discussion) as well as to Maillard reaction intermediates (6). For partial
amino acid sequencing, the isolated enzyme was further purified with a preparative
reverse-phase ProRPC column, and the amino-terminal sequence determined with the aid
of a ProSequencer unit (6). The terminal amino acid sequence of the purified reductase
showed a 96% identity with Oye1 and Oye2, and 64% with Oye3 yeast reductases (6).
The genes of these enzymes encode for isozymes of the FMN-flavoprotein (7) known as
Old Yellow Enzyme, a NADPH-dependent dehydrogenase (EC 1.6.99.1). The
corresponding DNA sequence of OYE was amplified by PCR with primers designed on
the reported gene sequence of Oye2 (GenBank Accession Number: L06124); and cloned
in plasmid pProEx-HT (Life Technologies, USA) for expression of OYE2 in E. coli strain
JM109. Expression of recombinant OYE2 was induced by addition of 0.6 mM isopropyl-
1--D-galactopyronoside (IPTG) to the culture medium.

Yeast strains
Production yeast strain (Polar) was transformed with either the homologous OYE2 gene
with several modified versions of the ADH1 promoter, or with a truncated (unregulated)
version of the gene of transcriptional activator Yap1 (GenBank Accession Number:
X58693) inserted into plasmid pET13:1 (with CUP1 marker), as described in detail in
(8). For pilot brewing experiments three strains were tested, the untransformed Polar
strain (VTT-A-99168), pETOYE2M (VTT-A-99174) which overexpresses Oye2 protein,
and the strain pCR4 (VTT-A-00178) which overexpresses the incomplete form of Yap1
(8).

Pilot brewing with genetically modified yeast

Brewing trials were carried out in the VTT pilot brewery in 50 l tanks using commercial
wort with an extract content of 17% (m/m). The original gravity was adjusted to 12%
(m/m) during filtration, and pitched with the genetically modified yeast at a pitching rate
of 0.3125 g of yeast/liter to obtain 252 g/50 l. Wort was oxygenated up to a soluble
content of 11-14 mg/l. The main fermentation was carried out at 10C. When the
apparent extract did not change on the successive days, flocculated yeast was removed
and the wort transferred to the secondary fermentation, where it remained until diacetyl
content was reduced below 0.035 mg/l. Temperature was then decreased to -1.5 C, and
secondary fermentation continued for 3 more days at the same temperature. Finally, wort
gravity was adjusted with deoxygenated water to 12 % (m/m) before beer filtration. The
carbon dioxide content in beer was adjusted to 5 g/l.

4
RESULTS AND DISCUSSION
Accumulation of -dicarbonyls in beer upon aging at 28C
Table I shows that six out of seven dicarbonyls detected in beer upon storage at 28C,
increased their concentration from 2 to 9 fold, including 1-DH, an early intermediate of
the Maillard reaction.

-dicarbonyl Concentration (M)

Storage at 0C Storage at 28C
1,4- 0.69 0.41 5.98 0.44
dideoxyhexosulose
1,4- 0.73 0.40 6.63 0.47
dideoxypentosulose
Glyoxal 2.45 0.62 6.58 0.66
Methylglyoxal 1.50 0.61 4.94 0.61
2,3-butanedione 1.22 0.41 6.77 0.45
3-deoxy-2- 231.4 4.6 215.4 4.2
hexosulose
1-deoxy-2,3- 27.1 4.0 47.5 4.4
hexodiulose
Table I: Concentrations of -dicarbonyl compounds in beers stored 4 weeks at
0C and 28C.

To assess the possible involvement of such -dicarbonyls in the generation of flavors

during aging, these intermediates were trapped by the addition of aminoguanidine (AG)
to beer before storage. Aminoguanidine is a well characterized inhibitor of the Maillard
reaction in vivo, being a non-toxic compound used as a possible drug in the treatment of
diabetic complications (9). Aminoguanidine reacts rapidly and irreversibly with -
dicarbonyl compounds to give 3-aminotriazine derivatives as shown below:

Reaction of aminoguanidine with -dicarbonyls to give 3-aminotriazine.

When added to beer during bottling and upon further storage for several weeks at 28C,
2 mM aminoguanidine completely suppressed the formation of HMF in beer as shown in
figure 1. We also observe, however, that in the presence of AG the levels of HMF went
below its initial value as measured by its peak height, indicating that in addition to a
possible blockage of 3-DH decomposition (as precursor of HMF), AG might have reacted
directly with some of the HMF already present in fresh beer.

5
 Control beer
7.00E+05

Beer + 2mM
6.00E+05 aminoguanidine

Area of HMF (V/seg)

5.00E+05

4.00E+05

3.00E+05

2.00E+05

1.00E+05

0.00E+00
0 10 20 30 40 50 60 70

Days of storage at 28C

Figure 1: Addition of aminoguanidine to fresh beer blocks the production

of HMF during storage at 28C

We explored such possibility by adding increasing concentrations of AG to an aged beer

with a high content of HMF and found that at 2mM concentration, AG reduced only 20%
of the HMF present in beer (not shown). Therefore, a direct effect of AG on HMF did not
account for its much larger decrease observed upon storage. So the primary effect of AG
must have been exerted primarily through its irreversible reaction with 3-DH which is
highly reactive and present in fresh beer (5) at a concentration twenty five times (250
M) that of HMF (10 M).
We then tested the sensory effects of addition of 2 mM aminoguanidine to either fresh or
aged beer upon storage at 28C for up to 8 weeks. Figure 2 presents the results of such an
experiment and we see that AG was able to attenuate the loss of the fresh flavor of beer
for the entire period of 8 weeks, and its addition to beer 24 hours before the sensory
analysis did not have an appreciable effect. The latter control would test the effects of a
direct reaction of AG with monocarbonyls that may have accumulated during the
experimental period. We thus come to the conclusion that blockage of the Maillard
reaction in beer, somehow retards deterioration of the fresh flavor for up to 8 weeks at
28C.
We cannot rule out, however, a direct competition of -dicarbonyls and certain
monocarbonyls such as the so called staling aldehydes for AG. These (2), in contrast to
-dicarbonyls such as 3-DH and 1-DH, would react in a reversible fashion, and at a
concentration that is two and a half orders of magnitude (1 M) below that of 3-DH (250
M). Unsaturated aldehydes such as trans-2-nonenal, would react even less with AG
since the former is present at a concentration of 0.5 ppb or 3.6 nM (2), or eight orders of
magnitude below the concentration of a possible competitor such as 3-DH, and six orders
of magnitude less than the baseline levels of the -dicarbonyls (~2 M) detected by us in
fresh beer.

6
 Aminoguanidine + fresh beer
Aminoguanidine added 24h
5
No aminoguanidine added

Degree of freshness
4

1
0 1 2 3 4 5 6 7 8 9

Weeks of storage at 28C

Figure 2: Sensory effects of the addition of 2 mM aminoguanidine to beer

upon storage at 28C for the indicated weeks.

Nobody, however, will use AG as a food additive in beer, and because of this, we
attempted to lower the load of -dicarbonyls in beer by using the enzyme machinery of
brewers yeast. We found, then, an enzyme that reduces -dicarbonyls that accumulate in
beer, Oye2 (6).
This enzyme possesses an NADPH-oxygen oxidoreductase activity, that reduces , -
unsaturated carbonyl compounds at a considerable faster rate than its oxydase activity
due to the reduction of the enzyme prosthetic group (flavin) by NADPH (10). A partial
list for the substrate specificity of Oye2 isolated from our own yeast strain confirms
published results, and can be appreciated in Table II. With the restrictions of partial
substrate solubility tested, the best were -dicarbonyls such as 2,3-hexanodione,
methylglyoxal, and 2,3-butanodione.
The enzyme was also active with oxidative-stress inducing compounds such as
menadione, and to a lesser degree with HMF. Trans-2-nonenal, an ,-unsaturated
aldehyde, was also reduced, but the enzyme did not show activity with saturated
aldehydes such as hexanal and heptanal, or with sugars such as glucose or fructose (not
shown). The specificity of Oye2 for ,-unsaturated carbonyls would then explain its
action on the putative structure of LC18, or DDH (5).
The physiological role of Oye2 is still unknown, but a recent report showed its
involvement in the diauxic shift of yeast (11), and its gene induced by the transcriptional
activator Yap1 that controls the oxidative stress response of yeast (12). Consistent with
such a possible role in brewers yeast, figure 3 shows that all our recombinant strains
transformed with either OYE2 or YAP1 showed resistance to methylglyoxal, a potent
inducer of oxidative stress. Wort can also be a source of oxidative stress to brewers yeast
(13), thus, Oye2 overproducing yeasts might have a considerable advantage during
fermentation; and confer additional flavor stability to the resulting beer by reducing the
initial load of -dicarbonyls and its byproducts of the Maillard reaction downstream.

7
 Enzyme Relative
Substrate Employed activity Activity (%) to
(mmol/min/m methylglyoxal
g) and S.D.
2,3-hexanodione (c) 8.63 0.001 324.80
Methylglyoxal 2.66 0.011 100.00
p-nitrobenzaldehyde (b) 2.34 0.022 88.07
2,3-butanodione (c) 2.31 0.034 86.94
Menadione (b) 1.73 0.001 65.11
Hydroxylmethylfurfural (b) 1.37 0.081 51.56
Benzaldehyde 1.15 0.073 43.28
Glyceraldehyde 1.12 0.028 42.15
3-deoxy-2-hexosulose, 3-DH 1.10 0.011 41.61
(a)
Cyclohexenone (b) 1.04 0.017 39.14
1,3-cyclohexanodione 0.69 0.028 25.96
2-(E)-nonenal (d) 0.45 0.011 16.89
Table II: Substrate specificity of Oye2 with -dicarbonyls and monocarbonyls.
Substrate concentration was 9 mM, except for compounds with partial
solubility in aqueous solution, distinguished with letters in parenthesis.
These concentrations were, 4.0 mM (a), 1.8 mM (b), 4.5 mM (c), and
0.9 mM (d).

[Methylglyoxal]
0 mM 100 mM

Parental

pETOYE2L pETOYE2S

pETOYE2M

Figure 3: Resistance of different transformed yeast strains with OYE2, to

oxidative stress.

Table III shows the sensory evaluation of beers produced at two different pilot trials with
either OYE2 (pETOYE2), or truncated YAP1 (pCR4) with respect to a control lager strain
(8). All fresh beers were very similar and displayed a high score of freshness. Upon
storage at 28C, however, the OYE2 modified strain was capable of retarding the
appearance of the notes of sweet and caramel, and malty and bready, for up to two weeks
when compared to the unmodified yeast strain. Not so with the strain with YAP1
transcriptional regulator (pCR4) which, on its truncated/ irreversibly-active form (8), was

8
supposed to induce a host of reductases, but failed to produce any protective effect on
beer flavor. After four weeks, the attenuation effect on the appearance of the malty and
bready note conferred by the modified strain with OYE2, was lost in one of the trials.
Neither the OYE2, nor the YAP1 modified strains, were capable of preventing the
appearance of papery flavor after four weeks of storage at 28C. This observation is
consistent with the existence of an entirely different route to produce the papery flavor
such as the oxidation of unsaturated fatty acids (2).

28 oC
POLAR pETOYE2M pCR4
trial series III IV III IV III IV
fresh
score 4 4 4 4 4 4
malty + bready - -
papery +
sweet + caramel +
oxidised - - +
positive
2 wk
score 2 2 2 3 2 2
malty + bready + - - +
papery - + +
sweet + caramel + - +
oxidised - - + +
positive +
4 wk
score 2 2 2 2 2 2
malty + bready + - + -
papery + +
sweet + caramel + - -
oxidised + - -
positive -

Table III: Sensory evaluation of beers made with normal and modified strains of
brewers yeast: unmodified (POLAR), overexpressing OYE2 (pETOYE2), and
overexpressing YAP1. Score maximum is 5 toward freshness, and (+) and (-)
signs denote the highest and lowest values, respectively, of the attributes
detected for each trial.

CONCLUSION
These results opened a new approach to the thermal instability of beer flavor by the
blockage of -dicarbonyls from the Maillard reaction that accumulate during beer storage
at 28C. The control of such intermediates could attenuate the appearance of the
deteriorated flavor of beer caused by heat.

9
REFERENCES
1. Evans, D.J., Schmedding, D.J.M., Bruijnje, A., Heideman, T., King, B.M., and
Groesbeek, N.M., J. Inst. Brew., 1999, 105:301-307,.
2. Bamforth, C.W., Technical Quarterly, 2000, 37: 165-171
3. Bravo, A., Snchez, B., Scherer, E. and Rangel-Aldao, R., Proceedings of the
European Brewery Convention Congress, Budapest, 2001, in press.
4. Lieke, Ralf. Bildung von -dicarbonylverbindungen beim Abbau von Amadori-
Umlagerungsprodukten, Dissertation 1999.
5. Bravo, A., Scherer, E., Madrid, J., Herrera, J., & Virtanen, H. Rangel-Aldao, R.,
Proceedings of the European Brewery Convention Congress, Budapest, 2001, in
press
6. Snchez, B., Reverol, L., Galindo-Castro, I., Bravo, A., Ramrez, J., & Rangel-
Aldao, R., Proceedings of the European Brewery Convention Congress, Budapest,
2001, in press.
7. Warburg, O., and Christian, W., Biochem. Z., 1933, 266: 377-411.
8. Vehkomki, M-L., Vidgren, V., Vilpola, A., Virtanen, H., Snchez, B., Galindo-
Castro, I., Penttil, M., and Rangel-Aldao, R., Proceedings of the European
Brewery Convention Congress, Budapest, 2001, in press
9. Feather, M.S., Mossine, V. and Hirsch, J., Amer. Chem. Soc. Symp., 1996. Ser. N
631, 24-32.
10. Karplus, P.A., Fox, K.M. and Massey, V., FASEB J., 1995, 9:1519-1525.
11. DeRisi, J., Vishwanath, R. & Brown, P., 1997, Science, 278, 680-685.
12. Fernandes, L., Rodrigues-Pousada, C., & Struhl, K., Molecular and Cellular
Biology, 1997, 17, 6982-6993.
13. Martin, V., Quain, D.E., Smart, K.A., Proceedings of the European Brewery
Convention Congress, Cannes, 1999, 27: 679-688.

ACKNOWLEDGEMENTS
We dedicate this work to honor Dr. Gerhard Wittl, who as former Technical-Director of
Polar Breweries, originated this project in 1991 and throughout all these years gave his
most enthusiastic support to all researchers and technicians involved. We are also grateful
to Empresas Polar for allowing this work to be published.

10
62

Impact of different parameters on the

absorption integral
Ulrich Peters, Thomas Fgen & Frank-Jrgen Methner
Bitburger Brauerei Th. Simon GmbH, P.O. 1164, D-54621 Bitburg, Germany (e-mail:
[email protected])

Descriptors
Beer analysis result, beer treatment, staling, taste stability

SUMMARY
The objective was to evaluate the impact of storage conditions and some technological
parameters on the absorption integral (AI). The results showed that the AI was influenced
most by temperature, but only above 10 C. Oxygen trials indicate the AI being mainly a
measure for thermal staling rather than oxidative changes. SO2 showed almost no effect on
the AI in fresh beer but a lower increase of AI over time. While shaking of the beer had no
impact on the AI increase beer exposed to light showed a higher increase. Different beers
showed a high correlation of the AI in fresh beer and the increase of AI during time.

Auswirkung verschiedener Parameter auf das Absorptionsintegral

Deskriptoren
Altgeschmacksbildung, Bierbehandlung, Ergebnis von Bieranalysen,
Geschmacksstabilitt

ZUSAMMENFASSUNG
Der Einflu von bestimmten Lagerbedingungen und technologischen Parametern auf das
Absoprtionsintegral (AI) wurde untersucht. Die Ergebnisse zeigen, dass das AI whrend der
Lagerung vorwiegend von der Temperatur beeinflut wird, unterhalb von 10 C jedoch keine
Zunahme zeigte. Sauerstoffversuche untersttzen die These, dass das AI berwiegend die
thermisch verursachte und weniger die sauerstoffinduzierte Alterung beschreibt. SO2 zeigte
im frischen Bier fast keinen Effekt auf das AI, wohl aber auf den Anstieg des AI whrend der
Lagerung. Das Schtteln des Bieres hatte im Gegensatz zum Lichteinflu keine Auswirkung
auf die AI-Zunahme im abgefllten Bier. Bei diversen Bieren aus unterschiedlichen
Brauereien konnte eine Korrelation zwischen dem AI im frischen Bier und dem Anstieg des
AI whrend der Lagerung festgestellt werden.
Impact de diffrents paramtres sur l'intgrale d'absorption

Descripteurs
Formation du got dvente, rsultat danalyse de bire, stabilit organoleptique,
traitement de la bire

RESUME
L'objectif tait d'valuer l'impact des conditions de conservation et de certains paramtres
technologiques sur l'intgrale d'absorption (IA). Les rsultats ont montr que l'IA est surtout
influence par la temprature, mais seulement au-dessus du 10 C. Les dosages d'oxygne
indiquent que l'IA est essentiellement une mesure du vieillissement thermique plutt que des
altrations oxydatives. Le SO2 n'a montr pratiquement aucun effet sur l'IA dans la bire
frache mais l'a lgrement augmente au fil du temps. Alors que le secouement de la bire
n'a aucune consquence sur l'augmentation de l'IA, l'exposition de la bire la lumire a
entran une augmentation plus importante. Diffrentes bires ont montr une forte
corrlation de l'IA dans la bire frache et l'augmentation de l'IA au fil du temps.

2
INTRODUCTION
The absorption integral (AI) is a rather simple and fast method which determines the
sum of some staling compounds and indicators. The objective of this work was to
evaluate the impact of storage conditions like temperature, light and movement as
well as some technological parameters. The impact of temperature was stressed for
evaluating beer complained by consumers. This should lead to an estimate of the
(unknown) storage conditions and therefore of the reason of the complain.

MATERIALS AND METHODS

12 different beers (Pilsener type and non alcoholic beer) from four breweries were
stored under different conditions:
temperature: 5, 10, 23, 32 C,
light and movement: dark and unmoved, dark and shaked, exposed to light
(fluorescent lamp) and unmoved.
One beer was mixed with different levels of oxygen and SO2, resp. Other beers were
brewed to compare decoction and infusion mashing and to give an indication of the
impact of different malts and whirlpool time on the AI.
The AI was determined according to Klein et al. (2) using decarbonated beer for vapor
destillation. The destillates absorbance between 240 and 310 nm was integrated giving
the absorption integral. Other methods like ITT (Indicator Time Test) and oxygen
content were according or similar to MEBAK. SO2 was determined with an HPLC
biosensor system (5).

RESULTS AND DISCUSSION

Time
The absorption integral 20
increased linear over 32 C
time dependent on 15
temperature (figure 1). 23 C
Because a direct
AI [-]

10
correlation of the AI in 10 C
fresh beer and the
change of AI during 5
5 C
storage is not proved
the increase of AI over 0
time 0 5 10 15 20 25
K1 = dAI/dt time [weeks]
was a main topic of
this work. Figure 1: Change of AI over time at different temperatures

Temperature
The impact of temperature on K1 was neither linear nor according to Arrhenius law
but quadratic (figure 2). Below 10 C no change of AI could be determined over time
which is in good accordance to other authors (4). Further experiments (data not
shown) indicate the exact temperature threshold being in the range of 10 to 12 C.

3
 0,7
0,6
0,5
0,4

K1 [1/week]
0,3 Beer A
0,2 Beer B
0,1 Beer C
0,0
-0,1 0 10 20 30 40
temperature [C]

Figure 2: Impact of temperature on increase of AI over time (K1)

According to figure 2 the increase of AI at specific temperature programmes during
storage (alternating 2 weeks temperature 1 and 2 weeks temperature 2) should be the
same. Table 1 shows three trials with varying temperature which prove this indication.
The AI increases stepwise at each temperature and is approx. 6,0 for all trials after 12
weeks. The increase K1 is not influenced by the AI at a specific time but only by the
beer properties itself and the actual temperature.

Temperature programme AI after 1 week [-] AI after 12 weeks [-]

7 / 30 C 4,4 5,9
18,5 / 26,5 C 4,4 5,8
23 C 4,4 6,3

Table 1: Change of AI at different temperature programmes (for details see text)

Oxygen
Klein et al. (2) mentioned an impact of air in the headspace of beer bottles on the AI.
They showed further that furfural is the main component defining the AI, esp. in
staled beer (1). Furfural is rather an heat indicator than an oxygen indicator (3). The
indication of AI being mainly a measure for thermal staling rather than oxidative
changes was proved by the results of different oxygen trials. The injection of air into
bottled beer led to no significant change of AI and K1 (table 2). This was confirmed by
further trials with PET bottles with different oxygen permeation rates (data not
shown), where no differences in AI and K1 were determined.

injected volume of air [ml] AI after 1 week [-] K1 [1/week]

0 5,0 0,26
2 5,1 0,23
4 5,1 0,24
6 5,2 0,22

Table 2: Impact of air in headspace on AI and K1

4
SO2 7
Mixing of SO2 into beer had
almost no effect on the AI in 6
fresh beer but a lower 5
increase of AI over time
4
(figure 3). Contrary to this

AI [-]
5 mg/l SO2
other authors (2) observed a 3
higher AI after adjunction of 10 mg/l SO2
2
SO2.
1 15 mg/l SO2
The ITT was strongly
influenced by the addition of 0
SO2. 0 2 4 6 8 10
time [weeks]

Figure 3: Impact of SO2 on AI

Light and movement
0,8
While shaking of the beer had
no impact on the AI increase 23 C 32 C
0,6
over time the beer exposed to
K1 [1/week]

light showed a higher

0,4
increase of AI (figure 4). At
higher temperatures the
0,2
increase of K1 is higher than
at lower temperatures. This
0,0
means that the additional
dark, not dark, shaked light, not
energy got in by light (and shaked shaked
temperature) is sufficient to
build staling compounds and
indicators determined by the Figure 4: Increase of AI over time at different
AI while the energy brought in storage conditions
by shaking is not.

Correlation of AI in fresh beer and K1

Different Pilsner and light
0,4
beers from different
breweries showed a good
correlation of the AI in 0,3 R2 = 0,94
K1 [1/week]

fresh beer and the increase

of AI during time (figure 0,2
5). A non alcoholic beer
did not fit this correlation. 0,1 Pilsener
Despite this the results of NAB
the trials with SO2 0,0
showed that a good 0 2 4 6 8
correlation cannot be AI after 1 week [-]
expected in every case.
Besides this the standard Figure 5: Correlation of AI after one week and increase
deviation of the AI of AI over time K1 for different Pilsener beers and one
analysis was determined non alcoholic beer

5
as SD = 0,2. This is approximately 10 % of the whole range of values for the shown
Pilsener beers. For a single brewery the values for different batches usually do not
differ so much what means an even higher impact of the standard deviation. For these
reasons it may be advisable to evaluate not only the AI of the fresh beer but also K1.

Production technology
Different trials showed the impact of the production technology (malt quality,
mashing procedure, whirlpool) on the AI and K1 (table 3).

Trial AI after 1 week [-] K1 [1/week]

malt 1 4,3 0,12
malt 2 3,5 0,09

Infusion mashing 4,9 0,17

Decoction mashing 4,8 0,21

10 min whirlpool 4,5 0,61*

60 min whirlpool 5,6 0,68*
* stored at 28 30 C instead of 23 C (other trials)

Table 3: Impact of malt quality, mashing procedure and time of wort in

whirlpool on AI and K1

CONCLUSION
The results proved that AI is a good measure for thermal induced staling compounds
while oxygen does not influence the change of AI over time very much. It is a rather
simple method appropriate for routine applications in breweries. Different variations
of process technology showed small but significant changes of AI. Using AI in
combination with other methods like ITT an estimation of the reason for staling of a
specific beer is possible if the parameters of the fresh beer are known.

REFERENCES
1. Klein, H., Glinsner, T., Natter, M. & Steiner, I., Brauwissenschaft, 2000, 53
(11/12), 217 222
2. Klein, H., Krammer, R. & Natter, M., Proceedings of the European Brewery
Convention Congress, Maastricht, 1997, 553 560
3. Narzi, L, Miedaner, H., Lustig, S., Brauwissenschaft, 1999, 52 (9/10), 164 - 175
4. Wackerbauer, K., Zufall, C. & Legrand, J., Brauwelt, 1999, 139 (40/41), 1796
1803
5. Wessels, D., von der Brelie, A., Gromus, J. & Galensa, R., Brauwissenschaft,
1995, 48 (3/4), 96-101

6
63

Highly sensitive chemical indices of beer

aging
Adriana Bravo, Beatrz Snchez, Erika Scherer & Rafael
Rangel-Aldao
Empresas Polar, Polar Technology Center, Caracas, Venezuela (e-mail:
[email protected])

Descriptors
Beer storage conditions, chemistry, HPLC, 5-hydroxymethylfurfural, non-enzymic browning,
staling

SUMMARY
We monitored beer deterioration upon storage at either 0 C or 28 C by chemical indices
determined by HPLC. A peak named LC18, gradually decreased until being barely detected
after 50 days at 0 C, and asymptotically approached a stationary level after 10 days at 28 C.
Isolation, synthesis, and decomposition of LC18 showed it to be a Maillard reaction
intermediate generated by the decomposition of 3-deoxy-2-hexosulose, and a direct precursor
of 5-hydroxy-methyl-furfural (HMF). Capillary electrophoresis revealed another peak, CE3
which behaved similarly to HMF, and statistical analysis showed that these chemical indices
could be used to assess beer heat damage with high sensitivity.

Hochempfindliche chemische Anzeichen der Bieralterung

Deskriptoren
Altgeschmacksbildung, Chemie, HPLC, 5-Hydroxymethylfurfural, Lagerkellerfhrung,
nichtenzymatische Brunung

ZUSAMMENFASSUNG
Wir beobachteten die Verschlechterung des Bieres nach der Lagerung sowohl bei 0 C als
auch bei 28 C durch chemische Anzeichen, die durch HPLC festgestellt wurden. Ein Peak,
als LC18 bezeichnet, verringerte sich stufenweise, bis er nach 50 Tagen bei 0 C kaum noch
detektiert werden konnte. Nach 10 Tagen, bei 28 C, hatte er sich asymptotisch einem
stationren Zustand angenhert. Die Isolation, Synthese und Aufspaltung von LC18 zeigten,
dass es sich um ein Intermediat einer Maillardreaktion handelt, das durch die Aufspaltung
von 3-Deoxy-2-Hexosulose entsteht und einen direkten Vorlufer von 5-Hydroxy-Methyl-
Furfural (HMF) darstellt. Kapillarelektrophoretische Untersuchungen zeigten einen weiteren
Peak, als CE3 bezeichnet, der sich hnlich wie HMF verhlt. Statistische Analysen zeigten,
dass diese chemischen Anzeichen geeignet sind, um mit groer Empfindlichkeit
Hitzeschdigungen des Bieres nachweisen zu knnen.
Quelques indices chimiques extrmement sensibles du vieillissement de la bire

Descripteurs
Brunissement non-enzymatique, chimie, conditions de garde, formation du got dvente,
HPLC, hydroxymthyl-5 furfural

RESUME
Nous avons suivi la dgradation de la bire pendant le stockage 0 C ou 28 C l'aide
d'indices chimiques dtermins par HPLC. Un pic appel LC18 a progressivement diminu
jusqu' tre peine dtectable aprs 50 jours 0 C et s'est rapproch asymptomatiquement
du niveau stationnaire aprs 10 jours 28 C. L'isolement, la synthse ainsi que la
dcomposition du LC18 a montr qu'il s'agit d'un intermdiaire de la raction de Maillard
gnr par la dcomposition du 3-doxy-2-hxosulose et d'un prcurseur direct du 5-hydroxy-
mthyle-furfural (HMF). L'lectrophorse capillaire a rvl un autre pic, CE3, qui s'est
comport comme HMF et l'analyse statistique a montr que ces indices chimiques peuvent
tre utiliss pour valuer avec une haute sensibilit les dgts thermiques occasionns la
bire.

2
INTRODUCTION
Most of the compounds thought to be involved in the development of stale flavor are
present in both fresh and aged beers (1), thus making it very difficult to establish
chemical correlations with overall flavor balance during beer aging. Hypothetically, a
mathematical model to predict the degree of flavor deterioration would therefore
involve the quantification of many compounds that interact in a complex and dynamic
way.
Instead of such a complex approach, a useful analytical assessment of the degree of
aging has been obtained through the determination of chemical indicators (3,4,5).
These, are compounds whose concentration vary in a continuous form from a fresh to
a deteriorated state and correlate with organoleptic evaluations (2). These compounds
may participate in the reactions involved in beer aging process (intermediates or final
products) but do not necessarily produce the stale flavor. Two types of chemical
indicators have been described so far: those that are very sensitive for exposure at
high temperatures and those that are susceptible to high concentrations of dissolved
oxygen in packaged beer (3).
The concentrations of furfural and 5-hydroxymethyl furfural (HMF) are useful indices
for estimation of heat damage to beer (4,5). Both aldehydes are products of the
degradation of pentoses and hexoses, respectively, in the presence of amino groups
(Maillard reaction, 6). The potential relevance of these aldehydes as chemical
indicators is that they may be reporters of the occurrence of parallel reactions related
to the formation of active flavor compounds.
In this work we report the use of HPLC and Capillary Electrophoresis (CE) to
monitor the progress of beer deterioration at 28C through the determination of new
chemical indicators described here for the first time, that complement the use of HMF
as well. In order to determine whether they could be used to estimate beer heat
damage with high sensitivity, we performed a statistical analysis of these chemical
indicators during the storage of beer produced in four brewing plants. Finally, we
studied the chemical nature of the indices detected by HPLC and found them to be
related to the Maillard reaction as investigated by the use of model system reactions
and the synthesis of the -dicarbonyl intermediate 3-deoxy-2-hexosulose (3-DH).

MATERIALS AND METHODS

Maillard reaction model system
The model system was obtained from a mixture of glucose 1 M and glycine 0.5 M in
distilled water, heated at 90C for 3 hours.
Synthesis of 3-deoxy-D-erythro-2-hexosulose
The synthesis of 3-deoxy-D-erythro-2-hexosulose (3-DH) was performed according to
the method of Madson and Feather (7). Chemical structure was confirmed by mass
spectrometry and [1H]- and [13C]-NMR after derivatization with 1,2-diaminobenzene
to give the 2-(2',3',4'-trihydroxybutyl)quinoxaline (8).
Equipment and reagents
A Waters HPLC System consisting of a 600 pump, Wisp 717 auto sampler,
Millennium 2010 Chromatography Manager v. 2.15, and an Aminex HPX-87H
column (300 x 7.8 i.d.) 9 m held at 55C was utilized. Elution was performed with
0.05 M H2SO4 during 27 min and monitored with a Waters 991 Photodiode Array
Detector. Quantification of HMF, LC18 and LC11 were carried out at 283 nm.

3
Separations were performed using a 270A-HT Applied Biosystems Capillary
Electrophoresis (CE) System with a fused capillary of 50 m i.d. and length 72 cm, in
20 mM sodium citrate buffer, pH 2.5, at a voltage of 15 KV for 20 minutes, and
detection at 200 nm.
Procedure
Beers from 4 brewing plants were stored at 0C and 28C for periods of up to 50 days.
HPLC, CE analysis, and organoleptic evaluations were performed at intervals of three
or four days over the time of analysis. Bottled beers without SO2 and with 10.0 mg/l
of SO2 were stored at 0C and 28C for periods of up to 35 days, and analyzed two
times per week. Beers without SO2 and with increasing amounts of SO2 (5, 10, 15, 20,
30, 40 and 50 ppm) were bottled and analyzed after 72 hours at room temperature.

RESULTS AND DISCUSSION

1. Chemical indices detected by HPLC
Through liquid chromatography analysis it was possible to detect three unknown
peaks (named LC8, LC11, LC18) and HMF, which changed significantly during the
aging process of beer at 28C (figure 1).

LC-8
LC-11
Chrom.B

Chrom.A 5-HMF

LC-18

Figure 1: HPLC chromatogram of a fresh beer (Chrom. A) compared with a beer stored
at 28C for 15 days (Chrom. B). The detected chemical indices of aging are: LC-8, LC-
11, LC18 and HMF.
One of the peaks, LC18, was only present in fresh beers or in those samples stored
shortly at low temperatures. The area of the LC18 peak decreased markedly after beer
pasteurization and further storage at 0C and 28C. The intensity of LC18 correlated
with the beer flavor and aroma deterioration during storage at 0C, as depicted in
figure 2.
The concentration of HMF increased continuously during storage at high temperatures
and thus behaved as an indicator of heat damage. Chemical index LC8 decreased
continuously during the storage at 28C, and also behaved as an indicator or heat
damage. LC11 decreased only after long term storage at 28C.
The combined use of all these chemical indices allowed us to estimate the degree of
heat damage of beers submitted to unknown storage condition.

4
 4.4 LC18 Peak
Flavor
0.012

LC18 peak height (AU x min)

4.2

Beer freshness
4.0

0.009
3.8

3.6
0.006
3.4

3.2
0.003

3.0
0 10 20 30 40 50 60

Days of storage at 0C
Figure 2: Beer freshness evaluation and height of LC18 peak in beers stored at 0C
from 1 to 50 days.
.
2. Statistical analysis of chemical indices
A statistical analysis of the areas of chemical indices and their variations during the
storage at 28C was performed to assess their reliability to follow the thermal aging of
beer. Beers produced in our four brewing plants during a ten month period were used.
Figure 3 shows the box plots of the areas of LC18 of fresh beers, beers after 15 days
of storage at either 0C, or at 28C.

A B C D
Area of LC18

Figure 3: Box plots of the area of LC18 peak of beers produced in four brewing plants:
A,B,C and D during a 10-month period. First box: area of LC18 in fresh beers; second
box: beers stored 15 days at 0C, third box: beers stored 15 days at 28C. Extremes of
the boxes indicate the 25% and 75% percentiles. A line inside the box marks the
median.
Important differences were observed in the area of LC18 peak between beers stored at
0C and 28C, and smaller differences were also obtained from fresh beers and beers
after 15 days at 0C. An analysis of variance of LC18 with respect to the storage
condition, rejected the hypothesis of similitude between groups (p < 10-8), suggesting
that LC18 could be used as a very sensitive indicator of beer freshness. A similar
analysis confirmed that HMF can be used as a marker of beer heat damage.

5
3. Chemical indices detected by HPLC are intermediates of the Maillard reaction
In order to elucidate the chemical structure of LC11 and LC18, we isolated these after
column chromatography and found that were very unstable, yielding many peaks
upon further chromatography. Figure 4 shows the chromatograms of the isolated
LC18 fraction. After lyophilization LC18 had completely disappeared and peaks
corresponding to HMF and 2-furfural were observed instead. Similar results were
obtained when the LC11 peak was isolated. This suggested to us that indices LC11
and LC18 were intermediates of the Maillard reaction.

LC18

Chrom. A
Chrom. B

Figure 4: Chromatograms of the LC18 fraction collected at the end of the HPLC
column and reinjected. Chrom. A, fraction before lyophilization. Chrom. B, fraction
after lyophilization.
Confirmation of this hypothesis was achieved through chromatographic separation of
a Maillard reaction model system under the same conditions of beer analysis,
obtaining peaks of similar retention times to LC8, LC11 and LC18 fractions detected
in beer. Furthermore, we added to a fresh beer an aliquot of this reaction system and
observed the corresponding increase in the areas of LC8, LC11 and LC18 as shown in
figure 5. This result indicated that HPLC is a useful technique to follow Maillard
reaction intermediates in beer and model systems.

Chrom.A
LC8 LC18
LC11

Chrom.B

Figure 5: Increase of analytical indices LC8, LC11, LC18 and HMF by the addition of
Maillard reaction model system to a fresh beer. Chrom. A, beer + model system. Chrom
B, fresh beer.
4. Peak LC18 contains an intermediate between 3-DH and HMF
Given the importance of 3-deoxy-hexosulose (3-DH) as a Maillard reaction
intermediate, we synthesized it and followed its decomposition by HPLC. Figure 6
shows the HPLC chromatograms of the decomposition of synthetic 3-DH at 90C.
The first chromatogram, taken before heating, showed two peaks: one double peak at
Rt =12.0 min., which corresponded to 3-DH, and a peak of HMF (Rt=23.0 min). Upon

6
heating this solution, 3-DH decreased its area while LC18 peak appeared and HMF
area increased continuously, indicating that LC18 may be an intermediate of HMF
formation following the thermal decomposition of 3-DH.

5-HMF
3-DH

LC-18
3 hours
2 hours
1 hour
0 hour

Figure 6: Formation of LC-18 and HMF upon heating a solution of 3-DH. HPLC PDA
chromatograms taken before heating (0 hours), and after 1, 2 and 3 hours of heating a
solution of 3-DH at 90C. Every peak it is plotted at its maximum absorbance.
This result suggested a chemical structure for LC18 that may theoretically correspond
to 3,4-dideoxyhexosulose-3-ene, DDH, a product of the dehydration of 3-DH
(9,10,11). To investigate further into the structure of LC18, a solution of 1,2-
diaminobenze (specific reagent for -dicarbonyls) was added to a Maillard reaction
model system, to wort and to beer, and observed by HPLC that LC18 peak had
completely disappeared (data not shown). This result confirmed the -dicarbonylic
structure of LC18 intermediate.

H
+ DDH

H O C H2 O H O
O O
H H O C H2 C H
H
H C O H H
O H H H
H
O H
3-DH +
H

5-HMF
O O
O O
H O C H2 H O C H2 C H
C H
H

H H

Furylium ion

Figure 7: Proposed mechanism for the conversion of 3DH to HMF (Weenen and Tjan,
1992)
5. Chemical indices LC18 and HMF are sensitive to the addition of SO2 to beer
It is known that SO2 inhibits the non-enzymatic browning produced by Maillard
reaction, therefore, we were interested in studying the effect of SO2 addition to beer
on the behavior of chemical indices LC18 and HMF. We found that the intensity of
LC18 peak decreased as the concentration of SO2 increased, as is depicted in figure 8.
This result suggested that SO2 could have reacted with this intermediate and
decreased its intensity, being also an indirect evidence in favor of the proposed
structure for LC18 as DDH. It has been proposed that the inhibition of browning by
SO2 is a result of the nucleophilic attack by sulphite ion on the ,-unsaturated

7
carbonyl moiety of DDH (12). Such a reaction could, in principle, contribute to the
observed loss of SO2 during beer aging, since intermediates in non-enzymatic
browning reactions are important contributors to irreversible binding of SO2 in foods
(12).

2,0E+05

1,6E+05
Peak area (uVoltxseg)

1,2E+05

8,0E+04

4,0E+04

0,0E+00
0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9
SO2 concentration (mM)

LC18 area HMF area

Figure 8: Area of HMF and LC18 peak as a function of SO2 concentration

Contrary to our expectation, however, the concentration of HMF increased as the
concentration of SO2 added to beer was increased from 0.1 to 0.8 mM, suggesting that
SO2 was probably catalyzing the formation of this furanic aldehyde.

4. New chemical indices detected by Capillary Electrophoresis

Through Capillary Electrophoresis (CE), a novel tool to analyze beer aging, we
identified another peak, called CE3, that increased its area continuously during the
storage at 28C. Figure 9 shows the electrophoregrams of a fresh beer and a beer
stored for 50 days at 28C. It can be seen an important increased of CE3 peak during
this period of storage.
CE3

Beer 50 days at 28C

CE3

Beer 50 days at 0C

Figure 9: Electrophoretic profiles of a fresh beer and the same beer after 50 days of
storage at 28C.
An inverse correlation (r2=0.867) was obtained when CE3 peak height was plotted
against organoleptic evaluation (beer freshness) for beers stored at 28C during 50

8
days (figure 10). This result suggested that CE3 peak could also be used as a sensitive
chemical index of beer heat damage.
.
5.0

4.5

Flavor deterioration 4.0

3.5

3.0

2.5

2.0

1.5

1.0
500 1000 1500 2000 2500 3000

EC3 peak area (microvolts)

Figure 10: CE3 peak area plotted against beer freshness (r2=0.867)
In order to evaluate the use of CE3 peak as chemical index of heat damage, a
statistical analysis was performed of the areas of this peak in beers produced in our
four brewing plants during a ten month period. Figure 11 shows the box plots of the
areas of CE3 of either fresh beers, those stored for 15 days at 0C, or at 28C. In this
figure, we can see significant differences in the area of CE3 peak between beers
stored at 0C and 28C. An analysis of variance of CE3 with respect to the storage
condition, rejected the hypothesis of similitude between groups (p < 4.8 x10-5),
suggesting that CE3 could be used as a very sensitive indicator of beer exposition to
high temperatures.

A B C D
Area of CE3 peak

Figure 11: Box plots of the area of CE3 peak of beers produced in four brewing plants:
A,B,C and D during a 10 month period. First box: area of LC18 in fresh beers; second box:
beers stored 15 days at 0C, third box: beers stored 15 days at 28C. Extremes of the boxes
indicate the 25% and 75% percentiles. A line inside the box marks the median.

9
To assess the chemical structure of CE3 peak, we extracted it from the beer by using a
cation exchange resin and then separated the fractions by preparative RP-HPLC.
However, we could not obtain enough amounts of material to take the NMR spectra.
Experiments are currently under way to determine the chemical structure of CE3, but
its cationic nature and UV absorption spectrum suggest that it could be a nitrogen
aromatic heterocyclic compound.

CONCLUSIONS
Chemical indices detected by HPLC and Capillary Electrophoresis allowed us to
monitor the progress of beer aging at high temperatures. These indices showed to be
very sensitive to the storage at 28C, correlated very well with flavor deterioration
and permitted us to estimate the degree of heat damage of a beer exposed to unknown
conditions.
Chemical indices detected by HPLC, LC11 and LC18, are intermediates of the
Maillard reaction, and the novel indicator contained in peak LC18 is an intermediate
of the thermal decomposition of 3-DH to yield HMF.

REFERENCES
1. Evans, D.J., Schmedding, D.J.M., Bruijnje, A., Heideman, T., King, B.M., and
Groesbeek, N.M., J. Inst. Brew., 105:301-307, 1999.
2. Mathews, S., Singhal, P. and Kulkarni, R., Trends in Food Science and
Technology, 4, 89-91, 1990.
3. Narziss, L; Miedaner, H. and Lustig, S., Monatsschrift fr Brauwissenschaft, N
9/10, 164-175, 1999.
4. Tressl, R, Bahri, D. and Kossa, M., The Analysis and Control of Less Desirable
Flavors in Food and Beverages., Academic Press, 293-318, 1980
5. Madigan, D.J., Am Soc. Brew Chem., 54(4), 146-151, 1998.
6. Ledl, F. and Scheleicher, E., Angewandte Chemie, 29(6), 565-706, 1990.
7. Madson, M.A and Feather, M.S., Carbohydrate Research, 94, 183-191,1981.
8. Morita, N., Mizutani, M, Hayashi, K, Kirihata, M, Ichimono, I., Ueda, H. and
Takagi, M., Bull. Univ. Osaka pref.. Ser. B., 35, 59-70, 1983.
9. McWeeney, D.J., Knowles, M.E. and Hearne, J.F., Journal of the Science of Food
and Agriculture, 25, 735-746, 1974.
10. Weenen, H. and Tjan, S.B., Flavor Precursors. Thermal and Enzymatic
Conversions, ACS Symposium Series N 490. R. Teranishi, Takeoka and Gnter
Editors, Washington, 1992
11. Yaylayan, V., Trends Food Science and Technology, July 20-22, 1990.
12. Wedzicha, B.L. and Vakalis, N., Food Chemistry, 27, 259-271, 1988.

10
64

Identification of -dicarbonylic
compounds in aged beers: their role in
beer aging process
Adriana Bravo1, Erika Scherer1, Jorge Madrid1, Julio
Herrera2, Hannele Virtanen3 & Rafael Rangel-Aldao1
1
Empresas Polar, Polar Technology Center, Caracas, Venezuela (e-mail:
[email protected])
2
Simn Bolvar University, Department of Chemistry, Caracas, Venezuela
3
VTT Biotechnology, P.O. Box 1500, FIN-02044 VTT, Espoo, Finland

Descriptors
Beer storage conditions, carbonyl compound, 5-hydroxymethylfurfural, staling

SUMMARY
We uncovered the accumulation of up to ten -dicarbonyls in beers stored at 28 C,
and determined their structures by 1H and 13C NMR spectroscopy. Four of these -
dicarbonyls have never been reported in beer, and two of them are presumed products
of the Strecker degradation of 1-deoxyosones. Seven -dicarbonyls increased their
concentration up to nine fold during storage at 28 C as compared with control beers
kept at 0 C. Addition of trapping reagents of -dicarbonyls to beer before storage
blocked the formation of 5-HMF, 2-furfural and 2-methylpropanal; and retarded the
deterioration of beer flavor as determined by a descriptive panel of aroma evaluation.

Identifizierung von -Dicarbonyl-Komponenten in gealterten Bieren: Ihre Rolle bei der

Bieralterung

Deskriptoren
Altgeschmacksbildung, Carbonylverbindung, 5-Hydroxymethylfurfural,
Lagerkellerfhrung

ZUSAMMENFASSUNG
Wir wiesen die Aufkonzentrierung von bis zu 10 -Dicarbonylen in Bieren nach, die bei 28
C gelagert wurden und bestimmten ihre Struktur durch 1H und 13C NMR-Spektroskopie.
ber vier dieser -Dicarbonyle im Bier wurde bisher noch nie berichtet, und zwei von ihnen
sind vermutlich Produkte des Streckerabbaus von 1-Deoxyoson. Bei sieben -Dicarbonylen
erhhte sich die Konzentration whrend der Lagerung bei 28 C bis auf das neunfache im
Vergleich zu den bei 0 C gelagerten Bieren. Die Zugabe von Abfangreagenzien fr -
Dicarbonyle zum Bier vor der Lagerung verhinderte die Bildung von 5-HMF, 2-Furfural und
2-Methylpropanol. Des Weiteren wurde eine Verschlechterung der Geschmacksstabilitt
vermieden, was durch ein beschreibendes Verkosterpanel nachgewiesen wurde.

Identification de composs -dicarbonyles dans les bires vieillies et dtermination de

leur rle dans le processus de vieillissement de la bire

Descripteurs
Compos carbonyl, conditions de garde, formation du got dvente, hydroxymthyl-5
furfural

RESUME
Nous avons dcouvert l'accumulation d'une dizaine d'-dicarbonyles dans des bires stockes
28 C et dtermin leurs structures par spectroscopie de RMN du H1 et du C13. Quatre de
ces -dicarbonyles n'ont jamais t dcrits dans la bire et deux d'entre eux sont prsums tre
des produits de la dgradation de Strecker des 1-doxyosones. Sept -dicarbonyles ont vu leur
concentration augmenter pendant le stockage 28 C jusqu' neuf fois par rapport aux bires
tmoins conserves 0 C. L'addition de ractifs de pigeage des -dicarbonyles avant le
stockage de la bire a bloqu la formation de 5-HMF, de 2-furfural et de 2-mthylpropanal et
retard la dgradation de l'arme de la bire dtermin par un panel d'valuation
organoleptique.

2
INTRODUCTION
There appear to be two distinct groups of flavor active aldehydes that contribute to
aged flavors (1), aldehydes that are derived from the oxidative degradation of fatty
acids, and aldehydes derived from the non-oxidative Strecker degradation of amino
acids in the presence of -dicarbonyl compounds (2). The latter are generated by
degradation of carbohydrates catalyzed by amino acids, and by virtue of their
functional groups, they can be very reactive and generate a host of products (2).
The role of -dicarbonyl intermediates in beer aging has not been studied, and there
are few reports in the literature on their detection in the brewing process. Hashimoto
and Koike (3) found an accumulation of 3-deoxy-2-hexosulose (3-DH) during
mashing and kettle boiling which correlated with an increase in color development in
wort. More recently, the formation of 3-DH and 1-deoxy-2,3-hexodiulose (1-DH)
were detected as products of the degradation of Amadori compounds during mashing
and wort boiling in laboratory scale experiments (4).
The purpose of the present investigation was to determine the formation of -
dicarbonyl intermediates during the storage of beer at 28C, and to develop an
analytical method to assess quantitatively the accumulation of -dicarbonyls during
beer aging. In addition, we initiated a long-term research to evaluate the role of -
dicarbonyl intermediates as precursors in the development of flavors related to beer
aging using specific reagents for blocking the -dicarbonyl functionality. The
assessment of -dicarbonyl blockage was performed in two ways: first, by measuring
several compounds related to -dicarbonyl degradation (i.e., HMF and 2-furfural), or
their downstream reaction products (Strecker aldehydes); and, second, by sensory
evaluation of aged beers after the addition of -dicarbonyl trapping reagents at
different points in time.

MATERIALS AND METHODS

Preparative methods for the detection of -dicarbonyl compounds
Alpha-dicarbonyls were converted into stable compounds such as quinoxalines by
their derivatization with 1,2-diaminobenzene (DAB). Dissolved in methanol (460
mM), DAB was added to 13.3 l of beer (60 bottles) under a CO2 stream, and bottles
were recapped and stored at 28C during two months. Beers were extracted with
chloroform, the organic layer dried and concentrated to dryness (hydrophobic
quinoxalines). The aqueous phase contained the hydrophilic quinoxalines.
Isolation of hydrophobic quinoxalines
Were separated by chromatography in a column filled with silica gel (200 g, TLC 60
GF254) in chloroform. The column was eluted with chloroform, acetonitrile and
methanol. Fractions were collected, concentrated in vacuum and analyzed by RP-
HPLC. Quinoxalines from these fractions were purified by semi preparative RP-
HPLC (C18 Prep Nova Pak HR, 6 m, 60 , 19 x 300 mm), using a Waters HPLC
System LC Module I Plus, consisting of a 600 pump, Wisp 717 auto sampler, UV-
visible 486 detector, and Millennium 2010 software, v. 2.15
Isolation of hydrophilic quinoxalines
Hydrophilic quinoxalines from 4 l of aqueous phase were retained in a column of
silica LC-18 (150 g, 4.5 x 9.2 cm) and eluted with acetonitrile (300 ml), concentrated
and lyophilized (520 mg). Quinoxalines were purified by semi preparative RP-HPLC.

3
High Resolution Gas Chromatography/Mass Spectrometry (HRGC-MS)
This was performed with a Hewlett Packard HP6890 GC by using a capillary HP-
5MS column (30 m x 0.25 mm, 0.25 m) and a split-less inlet at 200C. MS analysis
was performed with a Hewlett Packard 5973A mass selective detector. MS in the CI
mode was performed at 170 eV with butane as the reactant gas.
Nuclear Magnetic Resonance (1H,13C, DEPT, COSY and HMQC)
These experiments were performed on a Jeol 400 MHz for 1H and 100 MHz for 13C,
using magnetic susceptibility plugs (Doty Scientific) of zirconia for 5 mm tubes. The
samples were dissolved in dimethylsulfoxide-d6 or chloroform-d3 (Aldrich).
High resolution liquid chromatography
A Waters System consisting of a 600 pump, Wisp 717 auto sampler, Millennium 2010
v. 2.15, and a Nova-Pak C18 column (3.9 x 300, 4 M) was used. Elution was
performed by gradient and monitored with a Waters 996 Photodiode Array Detector.
Quantification of hydrophobic quinoxalines
DAB in methanol was added to bottles of beer under a CO2 stream, bottles were
recapped and stored at 28C and at 0C during one month. An aliquot of beer plus the
internal standard (2-propyl-3-methyl quinoxaline) was extracted with chloroform and
then analyzed by RP-HPLC. External calibration curves were prepared using
quinoxaline (glyoxal), 2-methyl-quinoxaline (methylglyoxal) and 2,3-
dimethylquinoxaline (2,3-butanedione) from Aldrich. To estimate the concentrations
of 1,4-dideoxy-2,3-pentodiulose and 1,4-dideoxy-2,3-hexodiulose, the calibration
curve for methylglyoxal was used.
Quantification of hydrophilic quinoxalines
After extraction with chloroform, beer samples were injected directly to RP-HPLC.
An external calibration curve for quantification of 3-DH and for the estimation of 1-
deoxyhexosulose concentration was prepared from synthetic 2-(2',3',4'-
trihydroxybutyl)quinoxaline.
Quantification of aldehydes
Hydroxy-methyl-furfural was determined by ionic-exclusion HPLC using an Aminex
HPX-87H column (300 x 7.8 i.d.) 9 m. Analysis of 2-furfural, and Strecker
aldehydes was performed by GC with an electron capture detector after derivatization
with O-(2,3,4,5,6-pentafluobezyl)hydroxylamine as described in the literature (5).
Synthetic methods
Synthesis of 3-deoxy-2-hexosulose was made according to the method of Madson and
Feather (6); 2-(2',3',4'-trihydroxybutyl)quinoxaline and 2-(1',2',3'-trihydroxypropyl)
quinoxaline were prepared according to the method of Hofmann (7). The internal
standard (2-propyl-3-methyl-quinoxaline) was prepared by mixing 2,3-hexanedione
and DAB. Spectroscopic data of the synthesized compounds were in agreement with
those reported in the literature.
Sensory analysis
Beers without added SO2 were used to evaluate the effect of aminoguanidine addition.
A descriptive panel of six to eight assessors performed sensory evaluation of beer
flavor. Bitterness was not taken into account in the general score of freshness degree.

RESULTS AND DISCUSSION

1. Detection of -dicarbonyls during beer storage at 28C
To detect and isolate -dicarbonyls generated during beer storage at 28C, these
intermediates were derivatized with 1,2-diaminobenzene to obtain stable compounds,
called quinoxalines (figure 1).

4
 R1 H2N R1 N
O

+ + 2H2O

R2 O H2N R2 N

Figure 1: Derivatization of -dicarbonylic compounds with 1,2-diaminobenzene

Extraction of beer with chloroform gave two distinct groups of quinoxalines, those
that were extracted with the organic phase (hydrophobic quinoxalines), and those that
remained in beer after extraction (hydrophilic quinoxalines). Figure 2 shows a
diagram of quinoxaline isolation from beer; and after several steps of semi preparative
RP-HPLC purification, at least 10 pure compounds and 3 fractions were isolated.
13.3 litres of beer + 1,2-diaminobenzene 4,6 mM

2 month at 28C

Organic phase Extraction with Aqueaous phase

Hydrophobic quinoxalines (3.45 g) 9 l of CHCl3 Hydrophylic quinoxalines (4 l)

Preparative liquid
Preparative liquid chromatography
chromatography on silica
on silica C18

Hydrophylic
quinoxalines 520
mg
I (663 mg) II (197 mg) III (390 mg) G (450 mg)

HPLC HPLC HPLC HPLC

A B C D E F H I J K L M

Figure 2: Diagram of the isolation of quinoxalines from 13.3 l of beer. Quinoxalines

were divided in two groups: hydrophobic and hydrophilic.

2. Chemical elucidation of -dicarbonyl intermediates

Chemical elucidation of the quinoxalines allowed the identification of the
corresponding -dicarbonyls, and it was performed by mass spectrometry and 1H and
13
C NMR, COSY and HSQC experiments. Complete spectroscopic data of the
detected quinoxalines will be published elsewhere.
Quinoxalines A, B, C and G corresponded to the derivatives of glyoxal,
methylglyoxal, 2,3-butanedione and pyruvate by comparison of their spectra with the
pure compounds. Compounds J and K where identified as the quinoxalines of 3-
deoxy-2-hexosulose (3-DH) and 1-deoxy-2,3-hexodiulose (1-DH) by comparison with
the spectroscopic data of pure synthesized compounds. This finding reveals for the
first time the presence of 1-DH in beer or any food system stored at mild conditions
(28C). Compound L was identified as the quinoxaline of 3-deoxy-pentodiulose (3-
DP) by comparison with its mass spectrum reported in the literature.
Compounds D and F where identified as the quinoxalines of 1,4-dideoxy-hexodiulose
(1,4-DDH) and 1,4-dideoxy-2,3-pentodiulose (1,4-DDP) respectively. This is also the
first report on the detection of these compounds in beer or any other food system. So
far, they had only been detected in model systems, and were rationalized as products
of the Strecker degradation of 1-DH and 1-deoxy-pentodiulose (1-DP) as is shown in
figure 3 (8).

5
 CH3
CH3
C O H
C N C COOH
H -H2O
C O + H2N C COOH
C O R
HC OH R
HC OH
HC OH
HC OH
R
CH3 R
CH3 CH3
HC N CH
-CO2 HC NH2 C O
-RCHO -NH3
C O R
C O C O
HC OH + H2O
HC OH CH2
HC OH
HC OH RHC OH
R 1,4-dideoxyosones
R
Figure 3: Strecker degradation of 1-deoxyosones leading to 1,4-dideoxyosones (8)
3. Quantification of -dicarbonyls during the storage of beer at 0C and 28C
The identification of -dicarbonyls formed in beer during the storage at 28C, led us
to develop a quantitative method to determine the concentration of these intermediates
by RP-HPLC during beer storage at either 0C or 28C. Figure 4 shows the overlaid
chromatograms of the hydrophobic fraction of quinoxalines extracted from a beer
stored one month at 0C and the same beer stored one month at 28C.

N CH3
G OH
1,4-dideoxyhexosulose
OH
N

N CH3

1,4-dideoxypentosulose Internal
standard
N OH

glyoxal N
D
N
methylglyoxal
N CH3

N CH3

F
Chrom. B C 2,3-butanodione
B N CH3
Chrom. A A
* * *
*
Unidentified quinoxalines

Figure 4: RP-HPLC chromatograms of the hydrophobic quinoxalines extracted from beer.

Chrom. A, beer stored one month at 0C, Chrom B, beer stored one month at
28C. * Non identified quinoxalines.
As shown in this figure, identified quinoxalines in the hydrophobic fraction were
derivatives of: glyoxal (A), methylglyoxal (B), 2,3-butanedione (C), and also the -
dicarbonyl derivatives of the Strecker degradation of 1-deoxyosones (D and F). All of
these -dicarbonyls increased their area during storage at 28C.
Quinoxalines that remained in the hydrophilic fraction (figure 5) were the derivatives
of 3-DH (K), 1-DH (J), 3-DP (L), an one unidentified -dicarbonyl (H). Of this

6
fraction, only 1-DH and the unidentified -dicarbonyl increased their areas during the
storage at 28C as compared with the same beer stored at 0C.

N
OH
K
3-deoxyhexosulose
N OH

OH
Unknown quinoxaline N CH3
OH
1-deoxyhexosulose
OH
N

OH 3-deoxypentosulose
H pentosulose
N N
OH OH
J OH
OH
N N

I
OH

Figure 5: RP-HPLC chromatograms of the hydrophilic quinoxalines extracted from beer.

Figure 6 shows the calculated concentration of -dicarbonyls accumulated in beer
during one month of storage at 0C and 28C, using an internal standard and external
calibration curves.
60

50
Concentration (M)

40

30

20

10

0
1,4-DDH 1,4-DDP glyoxal methylglyoxal 2,3- 1-DH
Beers at 0C Beers at 28C butanedione

Figure 6: Concentration of -dicarbonyl compounds in beers stored 4 weeks at 0C and

28C.
It can be seen that during storage at 28C most -dicarbonyl compounds detected in
beer increased their concentration from two to nine fold as compared with the same
beer kept at 0C. It is important to emphasize that to detect some of the -dicarbonyls
it was necessary to accumulate them. This was the case for compounds such as 1,4-
dideoxyosones, 1-DG, glyoxal and methylglyoxal. On the other hand, the method of
accumulation offers no insight into the changes of the actual amounts of more stable
intermediates such as 3-DH. This -dicarbonyl is trapped during the first hours of the
storage period, thus blocking it from further reactions, so that its concentration did not
change during the storage at 28C (231 4.6 M).

7
The significant increase of 1,4-DDH and 1,4-DDP as well as 1-DH during the storage
at 28C, are indicatives of the occurrence of the Strecker degradation and Amadori
degradation pathways, reactions observed to take place mainly at higher temperatures
and higher pH. These results also suggest that 1-deoxyosones: 1-DH and 1-
deoxypentodiulose (1-DP) should be very reactive -dicarbonyls -at beer storage
conditions- in the direction of the Strecker degradation. The -dicarbonyl 1-DP was
not detected in beer at 28C, however, and storage at 60C during 24 hours allowed us
to detect two isomeric quinoxalines of 3-DP, and presumably one of them is 1-DP.
Experiments are in progress for their isolation and to obtain 1H and 13C NMR spectra.

4. Accumulation of -dicarbonyls and flavor formation during beer aging

To evaluate the possible role of alpha-dicarbonyls in the generation of flavors during
beer aging, we used two strategies: a) blocking the degradation of these intermediates
before beer storage by the addition of aminoguanidine (AG), and 2) promoting the
alpha-dicarbonyl reaction pathway by addition of methylglyoxal . Aminoguanidine is
a non-toxic compound that reacts rapidly and irreversibly with -dicarbonyl
compounds to give 3-aminotriazine, as is shown in figure 7 (9).
H R1 N
R1 O H2N N N
C + 2H2O
C NH
+
C R2 N NH2
NH2
R2 O
AG 3-aminotriazine
Figure 7: Reaction of aminoguanidine (AG) with -dicarbonyls to give 3-aminotriazine.
We studied the reactivity of DAB and AG, as trapping reagents for -dicarbonyls in
beer. We found that aminoguanidine was less reactive toward -dicarbonyls than 1,2-
diaminobenzene at the pH of beer. At a concentration of 4,6 mM, DAB was able to
react with the total amount of 3-DH (243 M) in beer, while the same concentration
of AG reacted only with 93 M of 3-DH, as shown in figure 8.
300
Concentration of 3-DH quinoxaline ( M)

250

200

150

100

1,2-diaminobenzene
50 Aminoguanidine

0
0 1 2 3 4 5 6 7 8
Reactant concentration (mM)

Figure 8: Detection of 3-DH quinoxaline as a function of increasing concentrations of DAB

and AG. In the case of AG addition, non-reacted 3-DH was determined by
derivatization with DAB. Concentration of 3-DH in beer was 243 M.
Table I shows the effect of different concentrations of AG and DAB on HMF
formation after 2 days of beer storage at 60C. We found that DAB displayed a higher
capacity than AG at the same molar ratio, to inhibit HMF formation, which is in
agreement with their respective reactivities.

8
 Sample Concentration of HMF Inhibition (%) of HMF
(mg/l) 0.02 formation
Control beer at 0C 1.80
Control beer at 60C 4.23
Beer + 2mM AG 2.54 70%
Beer + 1 mM AG 3.75 20%
Beer + 0.5 mM AG 4.14 4%
Beer + 2 mM DAB 1.59 108%
Beer + 1 mM DAB 2.20 84%
Beer + 0.5 mM DAB 2.77 62%
Table I: Effect of the addition of 1,2-diaminobenzene and aminoguanidine (0.5-2 mM) to
beer during bottling in the formation of HMF during 2 days of storage at 60C.
The inhibition of HMF formation was explained as a result of the partial blockage of
3-DH by its reaction with AG or DAB. However, we cannot rule out the contribution
of a Schiff base formation between HMF and AG. Aminoguanidine has been reported
to form stable adducts with ,-unsaturated carbonyls in model reaction systems (12).
This possibility was tested by the addition of increasing concentrations of AG and
DAB to an aged beer. After 24 hours of reaction at room temperature 2 mM of AG
had reacted with only 20% of the initial HMF concentration. We concluded, then, that
even though AG might have reacted with HMF, the primary effect of AG in fresh beer
must have been exerted through its irreversible reaction with 3-DH (and other -
dicarbonyls). It was also shown that AG was able to completely inhibit the formation
of 2-furfural and methyl-propanal during beer storage at 28C (data not shown).
We then tested the sensory effects of the addition of AG to beer on the generation of
flavors after 15 days of storage at 28C. Addition of AG to a fresh beer caused an
alteration of bitterness (some astringency), but had no effects on its aroma and flavor,
as well as on its pH. Figure 9 shows the box plots of beer freshness evaluation of
control beers, after addition of with 1 mM AG or 2 mM AG at the time of bottling
(four experiments, n = 24), and table II shows the descriptive evaluation of the flavor
notes detected by panelists.
5

4
Flavor notes Control 2 mM AG
Beer freshness

Cardboard 20 13
3
Bready 3 0
Sweet 3 0
2
Wine 6 1

1
Table II: Frequency of flavor note detection.
0 Control
1 1 mM2 AG 3 AG
2 mM 4 Total number of evaluations = 24.
Beer treatment

Figure 9: Box plots of beer freshness evaluation for control beers and beer with 1 mM AG
and 2 mM AG added during bottling. Beer freshness: 5 = fresh, 4 = slightly
aged, 3 = aged, still acceptable, 2 = very aged, 1 = extremely aged
An analysis of variance showed a significant difference in the freshness degree
between beers with added aminoguanidine and control beers (p = 0.0001 for 1 mM
AG and p = 0.00044 for 2 mM AG). No significant difference was obtained when
comparing beers with 1 and 2 mM concentration of AG (p = 0.65).

9
Table II shows that aminoguanidine addition during bottling seems to have attenuated
the formation of flavor notes such as bready, sweet and wine, and decreased the
intensity of cardboard flavor note. On the other hand, addition of 20 M of
methylglyoxal to a fresh beer accelerated the appearance of bready, wine and sherry
notes after the storage at 28C (data not shown). We also studied the sensory effect of
the aminoguanidine addition to aged beers (2 to 8 weeks of storage at 28C) to assess
the reaction of AG with accumulated flavor active aldehydes. We found that AG had
a lower effect on the perception of bready and wine notes, and on freshness degree
than the effect of AG added during bottling (data not shown). This result indicates
that addition of aminoguanidine to aged beers does not seem to have a similar
carbonyl-binding effect as the ones of semicarbazide, 2,4-dinitrophenylhydrazine or
hydroxylamine (13,14). Addition of semicarbazide to beer has been reported to
virtually instantaneously eradicate stale aroma from beer (14). In contrast to these
compounds, aminoguanidine seemed to have reacted mainly with precursors
(presumably -dicarbonyl compounds) rather than with the final aldehydes. Taken all
together, these results suggest that the blockage of the -dicarbonyl pathway in beer
inhibited primarily the formation of bready and wine flavor notes during the storage at
28C.

CONCLUSIONS
We detected for the first time the accumulation of -dicarbonyl compounds during
the storage of beer at 28C as compared with beers kept at 0C. Addition of -
dicarbonyl trapping reagents to beer at bottling blocked the formation of Maillard
reaction final products, and inhibited the development of bready and wine flavor
notes during the storage at 28C. This work underlines the relevance of -dicarbonyls
as possible precursors of compounds that deteriorate beer flavor and opens a way for
new strategies to attenuate beer thermal deterioration.

REFERENCES CITED
1. Evans, D.J., Schmedding, D.J.M., Bruijnje, A., Heideman, T., King, B.M., and
Groesbeek, N.M., J. Inst. Brew. 1999, 105, 301-307
2. Ledl, F. and Schleicher, E., Angewandte Chemie, 1990, 29(6), 565-706.
3. Hashimoto, N., Koike, K., Rept. Res. Lab. Kirin Brewery Co., Ltd, 1971, N 14, pp 1-12
4. Liedke, Ralf, Bildung von -dicarbonylverbindungen beim Abbau von Amadori-
Umlagerungsprodukten, Dissertation 1999.
5. Ojala, M., Kotiaho, T., Siiril, J. and Sihvonen, M.L., Talanta, 1994, 41(8), 1297-1309.
6. Madson, M.A and Feather, M.S., Carbohydrate Research, 1981, 94, 183-191.
7. Hofmann, T., Bors, W. and Stettmaier, K., J. Agric. Food Chem., 1999, 4, 379-390.
8. Nedvidek, W, Ledl, F. and Fischer P., Zeitschrift fuer Lebensmittel Untersuchung und
Forschung 1992, 194, 222-228,
9. Hirsch, Baynes, J.W., Blackledge, J.A and Feather, M.S., Carbohydr. Res. 1991, 220, c5-
c6,
10. Al-Abed, Y. and Bucala, R., Chem. Res. Toxicol., 1997, 10, 875-879.
11. Hashimoto, N., Brewing Science, Vol 2. Pollok,R.A. (Editor), Academic Press Inc.,
London, 1981, pp 347-401.
12. Bamforth, Ch.W., MBAA Technical Quarterly, 2000, 37 (2), pp 165-171.

10
65

pH dependence of radical scavenging

activity of polyphenols, phenolic acid
and sulfite
Takashi Nakamura, Oliver Franz & Werner Back

TU Mnchen, Lehrstuhl fr Technologie der Brauerei I, D-85350 Freising-Weihenstephan,

Germany (e-mail: [email protected])

Descriptors
Antioxidant, pH, phenolic acids, polyphenol, sulphites, taste stability

SUMMARY
The hydroxyl radical and superoxide scavenging activities of polyphenols, phenolic acids and
sulfite at various pH levels including beer pH were measured to clarify their roles in flavour
stability of beer. Sulfite, almost all polyphenols and phenolic acids tested were observed to
possess both hydroxyl radical and superoxide scavenging activities at pH 7,0. Although sulfite
showed activities at beer pH of 4,3, the phenolic compounds did not show any or showed
lower activities than sulfite at the same pH. Thus sulfite was suggested to play a more
important role as antioxidant in beer flavour stability than polyphenols and phenolic acids.

pH-Wert-Abhngigkeit der Radikalfnger-Aktivitt von Polyphenolen, Phenolsure

und Sulfit

Deskriptoren
Antioxidantium, Geschmacksstabilitt, pH, Phenolsuren, Polyphenol, Sulfite

ZUSAMMENFASSUNG
Die Hydroxylradikal- und Superoxide-scavenging-Aktivitten von Polyphenolen, Phenol-
suren und Sulfit wurden bei verschiedenen pH-Werten inkl. Bier-pH gemessen, um deren
Einflu auf die Geschmacksstabilitt von Bier zu klren. Es wurde festgestellt, dass sowohl
Sulfit als auch fast alle untersuchten Polyphenole und Phenolsuren bei pH 7,0 eine
Hydroxylradikal- und Superoxide-scavenging-Aktivitt aufzeigen. Fr Sulfit konnte eine
Hydroxylradikal- und Superoxide-scavenging-Aktivitt auch bei pH 4,3 nachgewiesen
werden. Die phenolischen Verbindungen verfgten bei diesem pH-Wert ber keine oder nur
eine geringe Aktivitt. Es ist daher zu vermuten, dass Sulfit als Antioxidans fr die
Geschmacksstabilitt von Bier eine grere Rolle spielt als die Polyphenole und
Phenolsuren.
Variation de l'activit antiradicaux libres des polyphnols, de l'acide polyphnolique et
des sulfites en fonction du pH

Descripteurs
Acides phnoliques, antioxydant, pH, polyphnol, stabilit organoleptique, sulfites

RESUME
Les activits anti-radicaux libres (radicaux OH et superoxydes) des polyphnols, des acides
phnoliques et des sulfites des diffrents niveaux de pH, dont le pH de la bire, ont t
mesures afin d'en clarifier leur rle dans la stabilit aromatique de la bire. Les sulfites,
presque tous les polyphnols et acides phnoliques tests possdaient la fois des activits
anti-radicaux libres OH et superoxydes pH 7,0. Bien que les sulfites aient fait preuve
d'activit au pH de la bire (4,3), les composs phnoliques n'ont prsent aucune activit
pour des activits infrieures celles des sulfites au mme pH. Ainsi, les sulfites auraient un
rle antioxydant plus important que celui des polyphnols et des acides phnoliques dans la
stabilit aromatique de la bire.

2
INTRODUCTION
Free radical reaction has been proposed as the main cause of stale flavor production in
beer7). That is, active oxygen species generated by metal catalysis react with beer
components and produce carbonyl compounds that are responsible for stale flavor.
Polyphenols and phenolic acids are reputed to play an important role in flavor stability
because they may have antioxidant activity. Phenolic compounds in beer are expected
to scavenge active oxygen species and prevent the oxidation of beer components
during storage.
There have been a few reports about the contribution of phenolic compounds to flavor
stability9-11), but the addition of phenolic compounds to beer has not led to an obvious
improvement in flavor stability, although antioxidant activity related to the
compounds was detected by some antioxidant assays10). Opinion on the effect of
proanthocyanidin free barley Caminant has been divided with regards to flavor
stability1, 3). The effect of phenolic compounds on flavor stability of beer has been as
yet unclear.
Our study is aimed to clarify the role of polyphenols and phenolic acids in the flavor
stability of beer. In this paper we report on the pH dependent hydroxyl radical and
superoxide radical scavenging activity of phenolic compounds and sulfite and discuss
their role in the flavor stability of beer.

MATERIALS & METHODS

Reagents
N-t-Butyl--phenylnitrone (PBN), (+)-catechin, ferulic acid, p-hydroxybenzoic acid
and vanillic acid were purchased from Sigma-Aldrich Chem. Co. Gallic acid was
purchased from Fluka. 2-Metyl-6-phenyl-3,7-dihydroimidazo-[1,2-a] pyrazin-3-one
(CLA) was purchased from Tokyo Kasei Kogyo Co., Ltd.

Measurement of hydroxyl radical scavenging activity by the ESR spin-trapping

method
The hydroxyl radical scavenging activity of test compounds was determined using the
Fenton system2) (figure 1). The compound under test was mixed with 0.1 mM ferrous
ion (Fe2+) and 50 mM PBN in an acetate buffer at both pH 4.3 and 7.0, respectively.
Subsequently, 1 mM of hydrogen peroxide (H2O2) was added to the reaction mixture
to generate the hydroxyl radical (OH). The radical was measured as a spin adduct
with PBN by an ESR spectrometer (JEOL Ltd., model JES-FR30, Japan). Both the
reaction and the measurement were carried out at room temperature.
Various concentrations of test compounds were measured to determine their activity.
If a compound under test possesses hydroxyl radical scavenging activity, it scavenges
this radical and quenches the production of PBN spin adduct according to the
concentration in solution. A control blank was evaluated in the same manner by
replacing the test sample with distilled water. Scavenging activities were expressed as
50% inhibition concentration (IC50) calculated from an approximate formula
determined from graphed data.

Measurement of superoxide scavenging activity by the chemiluminescence

method (CL)
The superoxide scavenging activity of the compounds under test was also determined
using the Fenton reaction (figure 1). Superoxide was measured as CLA-dependent CL

3
by a CL-Analyzer (Tohoku Electronic Industrial Co., Ltd., model CLD-110, Japan). A
reaction mixture consisting of the test compound, 0.01 mM ferrous ion and 2.5 nM
CLA in a acetate buffer at pH 4.3 or 7.0 was placed in a sample vessel. First, a base
line was obtained for 30 seconds at 25 C. Subsequently, 1 mM of hydrogen peroxide
was injected into the solution with a syringe and the CL was measured successively
for 30 seconds.

Measurement of chemiluminescence
0.16 mM of catechin (46 mg/l), gallic acid (27 mg/l) and sulfite (10 mg/l) were added
to degassed beer. The CL was measured by a CL-Analyzer according to the method of
Kaneda et al5).

OH + O2- + OH- + 2H
+

Figure 1: Generation of hydroxyl radical and superoxide by the Fenton

reaction and the principle of measurement of the scavenging activity
of both radicals.

RESULTS
Hydroxyl radical scavenging activity
The spectrums of the PBN spin adduct with hydroxyl radical are shown in figure 2. At
pH 7.0, the signal intensity of the spin adduct decreased with the addition of all test
compounds except for ferulic acid. Excessive addition of sulfite was observed to
increase the signal at the same pH. At pH 4.3, only sulfite resulted in the signal being
clearly reduced. p-Hydroxybenzoic acid decreased the signal slightly, but its reactivity
was insufficient to reduce the signal by half even at the maximum concentration
tested. The addition of ferulic acid and vanillic acid had no effect on the signal
production but the addition of catechin and gallic acid increased the signal. As shown
in figure 3, there was a strong exponential correlation between the signal intensity and
concentration of test compounds added.

Superoxide scavenging activity

As shown in figure 4, the CL increased immediately just after the injection of
hydrogen peroxide and began to decrease after several seconds. The CL signal then
approached the base line at the end of measurement period (30 seconds after the

4
injection, figure 4).
p-Hydroxybenzoic acid was the only compound showing no superoxide scavenging
activity at pH 7.0. At pH 4.3 sulfite, ferulic acid and gallic acid quenched the CL
production. Catechin had no effect on the signal while p-hydroxybenzoic acid and
vanillic acid increased it at pH 4.3. The quenching manner was the same as that of
hydroxyl radical (figure 5).

800 100
2

Relative ESR signal intensity (%)

600 R = 0.9733
Signal intensity

400 80
200
0 60
-200
-400 40
-600
20
-800
337 339 341 343 345 347
0
Magnet field (mT)
0.0E+00 5.0E-02 1.0E-01 1.5E-01
no additon 2.1E-02 3.5E-02
6.0E-02 1.0E-01 1.4E-01 Concentration of gallic acid (mM)

Fig. 2: Production of PBN spin adduct Fig. 3: The relationship between the
and its inhibition by the addition relative signal intensity of PBN
of gallic acid at pH 7.0. spin adduct and the
concentration of gallic acid
added

4.0 Test
100
3.5 compound 2
R = 0.9676
Relative CL intensity (%)
(x 10 counts/sec.)

2+
3.0 Fe 80
CL intensity

2.5 CLA
2.0 60
pH 4.3
1.5
4

1.0 H2 O2 40
0.5
0.0 20
0 10 20 30 40 50 60
0
Time (sec.)
0.0E+00 2.0E-02 4.0E-02 6.0E-02
no addition 1.7E-02 2.6E-02
3.5E-02 5.2E-02 Concentration of sulfite (mM)

Fig. 4: Production of CLA-dependent Fig. 5: The relationship between the

chemiluminescence and its relative CL intensity and the
inhibition by the addition of concentration of sulfite added.
sulfite at pH 4.3.

5
Suppression of CL production in beer by addition of catechin, gallic acid and
sulfite
The CL production in beer was examined by the addition of 0.16mM catechin, gallic
acid and sodium sulfite. The beer pH was not changed by the addition of test
compounds. As shown in figure 6, sulfite and gallic acid quenched CL production by
25% and 5%, respectively. The addition of catechin had no influence on the CL
production in beer.

3.0
Chemiluminescence intensity

2.5
no addition
(x 10 counts/ min)

2.0
catechin
1.5
gallic acid
1.0 sulfite
4

0.5
0.0
0 10 20 30 40 50 60
Time (min.)

Figure 6: Effect of the addition of 0.16 mM catechin, gallic acid and sulfite to
beer on the production of chemiluminescence.

DISCUSSION
The methods described here to measure hydroxyl radical and superoxide scavenging
activity were newly developed to evaluate the role of polyphenols and phenolic acids
as antioxidants in the flavor stability of beer. In this study, the radical scavenging
activity was measured not only at a beer pH of 4.3 but also at a neutral pH of 7.0 to
determine the pH-dependence of the radical scavenging activity.
Hydroxyl radical and superoxide were selected for the methods used here because
these radicals possess strong reactivity and have been well studied by measurements
of Lag-Time4) (EA-Value8)), and CLA-dependent CL intensity6), respectively. As it is
clear that these two radicals have a great effect on flavor stability, the antioxidant
properties of the phenolic compounds were expected to be clearly evaluated by this
experiment.
The radical scavenging activity of the phenolic compounds showed a variety of
activities depending on the test compounds and pH (table 1). Compared to pH 4.3, the
radical scavenging activities of compounds tested were always higher at pH 7.0. All
tested phenolic compounds showed no hydroxyl radical scavenging activity at pH 4.3
while it was revealed that sulfite had this scavenging activity at the pH 4.3. The
maximum concentration of the test compounds indicated in table 1 was determined by
their solubility in water.
The relative activities of polyphenols and phenolic acids versus sulfite are shown in
table 2. As it is well known that sulfite displays antioxidant activity in beer, it is
interesting to compare the scavenging activities of phenolic compounds to those of

6
sulfite. This comparison was possible because sulfite showed scavenging activities for
both radicals at both values of pH.
Although their activities were lower than that of sulfite, all of the phenolic compounds
tested showed either hydroxyl radical or superoxide scavenging activities at pH 7.0.
On the other hand the activities of the phenolic compounds were faint or at a non
detectable level at the beer pH of 4.3, although the activity of sulfite remained at a
significant level.
The effects of excessive addition of phenolic compounds and sulfite to beer were
studied by CL measurements to confirm the results obtained in this study. It has been
reported that the CL intensity decreases when antioxidant is added to beer8, 11).
Catechin and gallic acid were chosen because catechin has been well studied for its
antioxidant activity and gallic acid was found to scavenge superoxide slightly at pH
4.3. The results obtained in this study (figure 6) suggest that only sulfite suppresses
oxidative reactions in beer. They support the data shown in table 1.
Thus, it is suggested that sulfite is a more important antioxidant than polyphenols and
phenolic acids in beer. As sulfite is also expected to mask some carbonyl compounds
(cardboard flavor), it can prevent the flavor deterioration of aged beer more
effectively than phenolic compounds.
It is clear that the scavenging activity of the phenolic compounds is greatly affected
by pH and is at the same time radical-species dependent (table 2). Ferulic acid was
found to react only with superoxide but not with the hydroxyl radical at both values of
pH. These findings indicate that the antioxidant activity of beer compounds must be
tested at the beer pH and the measuring method should be chosen carefully.

Table 1: Hydroxyl radical and superoxide scavenging activity (IC50) of the phenolic
compounds and sulfite at pH 4.3 and pH 7.0.

pH pH 4.3 pH 7.0
Test compound Hydroxyl Superoxide Hydroxyl Superoxide
\Active oxygen radical (mM) (mM) radical (mM) (mM)
1) 2) -1
Catechin N.A. (6.0) N.A. (5.9) 2.9 x 10 2.0 x 10-2
Ferulic acid N.A. (2.4) 1.4 x 100 N.A. (2.4) 5.0 x 10-2
-2
p-Hydroxybenzoic N.A. (8.5) N.A. (8.5) 8.3 x 10 N.A. (8.5)
acid
Gallic acid N.A. (8.3) 4.9 x 100 8.0 x 10-2 1.1 x 10-2
Vanillic acid N.A. (4.8) N.A. (4.7) 3.8 x 10-1 6.2 x 10-2
Sulfite 1.4 x 100 2.8 x 10-2 4.3 x 10-2 5.9 x 10-4

1) N.A. indicates No Activity

2) A number in parenthesis indicates the maximum concentration of test compounds.

7
Table 2: Relative scavenging activity of the phenolic compounds versus sulfite at pH
4.3 and pH 7.0.

PH pH 4.3 pH 7.0
Test compound Hydroxyl Superoxide Hydroxyl Superoxide
\Active oxygen radical (%) (%) radical (%) (%)
Catechin - - 15 3
Ferulic acid - 2 - 1
p-Hydroxybenzoic - - 52 -
acid
Gallic acid - 1 54 5
Vanillic acid - - 11 1
Sulfite 100 100 100 100

CONCLUSIONS
The hydroxyl radical and superoxide scavenging activity of polyphenols, phenolic
acids and sulfite at pH 4.3 and pH 7.0 were measured. Sulfite showed clear evidence
of scavenging activity for both radicals at both values of pH. Although all of the
phenolic compounds tested were found to possess scavenging activity for either
radical at pH 7.0, they showed faint or non detectable levels of scavenging activity at
pH 4.3 as compared with sulfite. Only sulfite clearly quenched CL production, but not
catechin and gallic acid when they were added to beer.
Thus, sulfite is suggested to play a more important role as antioxidant in the flavor
stability of beer than polyphenols and phenolic acids. The antioxidant activity of beer
compounds must be tested at the beer pH and the measuring method should be chosen
carefully.

FUTURE WORK
These methods have been modified to measure the radical scavenging activity of wort
at pH 5.5. The role of phenolic compounds in the mashing process will be evaluated
by using these methods.

ACKNOWLEDGMENT
I would like to thank Mr. E.Tawada of Kirin Brewery Co., Ltd. for giving the author
the opportunity to study at the Technical University of Munich, Weihenstephan.
Professor W. Back is thanked for his support and permission for this presentation at
the EBC congress.

REFERENCES
1. Andersen, M.L., Outtrup, H., Riis, P & Skibsted, L.H., Proceedings of the
European Brewery Convention Congress, Cannes, 1999, 133-140.
2. Arima, Y., Hatanaka, A., Tsukihara, S., Fujimoto, K., Fukuda, K. & Sakurai, H.,

8
 Chem. Pharm. Bull., 1997, 45, 1881-1886.
3. Back, W., Forster, C., Krottenthaler, M., Lehmann, J., Sacher, B. & Thum. B.,
Brauwelt, 1997, 137, 1667-1692.
4. Forster, C., Schwieger, J., Narzi, L., Back, W., Uchida, M., Ono, M. & Yanagi,
K., Monatsschrift fr Brauwissenschaft, 1999, 52, 86-93.
5. Kaneda, H., Kano, Y., Kamimura, M., Kawasaki, S. & Osawa, T., J. Inst. Brew.,
1991, 97, 105-109.
6. Kaneda, H., Kano, Y., Osawa, T., Kawakishi, S. & Koshino, S, Proceedings of
the European Brewery Convention Congress, Lisbon, 1991, 433-440.
7. Kaneda, H., Kobayashi, N., Takashio, M., Tamaki, T. & Shinotsuka, K, Technical
Quarterly, 1999, 36,41-47.
8. Uchida, M., Suga, S. & Ono, M., J. Am. Soc. Brew. Chem., 1996, 54, 205-211.
9. Walters, M.T., Heasman, A.P, & Hughes, P.S., J. Am. Soc. Brew. Chem., 1997,
55, 83-89.
10. Walters, M.T., Heasman, A.P, & Hughes, P.S., J. Am. Soc. Brew. Chem., 1997,
55, 91-98.
11. Walters, M.T., Ferment, 1997, 10,111-119.

9
66

DPPH-scavenging activity of beer and

polyphenols measured by ESR
Oliver Franz & W. Back
TU Mnchen, Lehrstuhl fr Technologie der Brauerei I, D-85350 Freising-Weihenstephan,
Germany (e-mail: [email protected])

Descriptors
Antioxidant, beer treatment, phenolic acids, polyphenol, spectroscopy

SUMMARY
A specific analysis was evaluated to determine the qualitative effect of polyphenols on the
antioxidative activity of beer. The antiradical potential of phenolic substances was measured
against DPPH with the Electronspinresonanz-Spectroscopy. As there are precise differences
in the results, you have the possibility to estimate the radical-scavenging activity of each
substance. Furthermore you can evaluate the effect of phenolic substances on the
antioxidative activity of beer. It is shown, that the addition of phenolic acids can increase the
antiradical potential of beer, which can be measured with this method.

Mittels ESR gemessene DPPH-Scavenging-Aktivitt von Bier und Polyphenolen

Deskriptoren
Antioxidantium, Bierbehandlung, Phenolsuren, Polyphenol, Spektroskopie

ZUSAMMENFASSUNG
Um den qualitativen Einfluss der Polyphenole auf die antioxidative Aktivitt von Bier
beurteilen zu knnen, wurde eine spezifische Analyse erarbeitet. Als Parameter wurde
das antiradikalische Potenzial der einzelnen phenolischen Substanzen gegenber dem
stabilen Radikal DPPH mittels Elektronenspinresonanz-Spektroskopie gemessen. Die
Ergebnisse zeigen deutliche Unterschiede zwischen den Substanzen und ermglichen
es, ihre Radikal-fnger-Aktivitt beurteilen zu knnen. Zudem kann ihr Beitrag zur
antioxidativen Aktivitt des Bieres gewichtet werden. Die Methode zeigt, dass mit
dem Zusatz von einzelnen phenolischen Suren das antiradikalische Potenzial von
Bier gesteigert werden kann.
Mesure par ESR de l'activit antiradicaux libres DPPH de la bire et des polyphnols

Descripteurs
Acides phnoliques, antioxydant, polyphnol, spectroscopie, traitement de la bire

RESUME
Une analyse spcifique a t conduite afin de dterminer l'effet qualitatif des polyphnols sur
l'activit antioxydante de la bire. Le potentiel anti-radicaux des substances phnoliques a t
mesur contre le DPPH par rsonance lectronique de Spin (ESR). Comme il existe des
diffrences prcises entre les rsultats, on a la possibilit d'estimer l'activit anti-radicaux
libres de chaque substance. Par ailleurs, on peut valuer l'effet des substances phnoliques sur
l'activit antioxydante de la bire. Il est dmontr que l'addition d'acides phnoliques peut
augmenter le potentiel anti-radicaux de la bire, qui peut tre mesur par cette mthode.

2
INTRODUCTION
There is an increasing relevancy of flavor stability as a quality criteria of beer. The
fact that beer is exposed to the process of natural aging, we have to talk about flavor
instability [2]. The ingredients of beer are subject to oxidation, which is also caused
by free radicals [3]. These are mainly reactive species of oxygen [6]. The endogenous
antioxidative activity (EAA) of beer expresses the ability to prevent the reactions and
correlates significantly with the flavor stability. In this context, sulphur dioxide plays
an important role in preventing the generation of the hydroxyl radical, which is said to
be the most reactive radical. To determine the EAA, the Lag-Time of beer can be
measured [5].
Other important antioxidative compounds are radical scavengers like phenolic
substances. Kaneda used a spectralphotometric method to evaluate the Reducing
Activity of beer against 1,1-Diphenyl-2-picryl-hydrazyl (DPPH) [4,1]. Kaneda
evaluated only the amount of reduced DPPH, but not the reaction rate. As DPPH is a
stable free radical it can be measured directly by Electronspin-resonance-
Spectroscopy (ESR). A method was developed to determine the antiradical potential
(ARP) of beer and phenolic substances by ESR. This analysis complements other
measurements of antioxidative activity like the Lag-Time.

MATERIALS
Reagents
1,1-Diphenyl-2-picryl-hydrazyl (DPPH), Caffeic Acid, (+)-Catechin, Chlorogenic
Acid, (-)-Epicatechin, (-)-Epigallocatechin, (-)-Epigallocatechin Gallate, Gentisic
Acid, Ferulic Acid, p-Hydroxybenzoic Acid, Syringic Acid and Vanillic Acid were
purchased from Sigma, Germany. (+)-Epicatechin Gallate and Protocatechic Acid
were purchased from Roth, Germany. Gallic Acid was purchased from Fluka,
Germany.

Preparation of the DPPH-solution

The solution was prepared in accordance to the publication of Kaneda.
- 0.1 M acetate buffer was set to pH 4.3 by addition of hydrochloric acid.
- The buffer was mixed with ethanol in a volumetric ratio of 1 : 2.
- The concentration of DPPH was 1.86 x 10-4 mol/l.

Electronspinresonance-spectroscopy
The mesurements of DPPH were performed with an Electronspinresonance-
Spectrometer (ESR) from JEOL Ltd. (Model JES-FR30), Japan.
As a stable free radical, DPPH can be measured directly by ESR (figure 1). For
evaluation, the peak height of the middle wave was calculated. Mn2+ was used as a
standard ESR marker. The relative response was calculated as the quotient of both
peak heights.

Peak height of DPPH

Relative response = Peak height of Mn2+

3
 1500
DPPH
1000

500
2+

Response
Mn
0

-500

-1000

-1500
337 339 341 343 345 347
Magnetic Field [mT]

Figure 1: ESR-spectrum of DPPH

The following ESR-settings were used:

Power 4 mW
C.Field 342.000 mT
SwWid 5 mT
SwTime 0.5 min
ModWid 0.1 mT
Amp 79
TimeC 0.03 sec

METHOD
The general idea of this method is to determine the decrease of the radical DPPH after
addition of a beer sample or any substance with reducing activity.
- To determine the zero point, which represents 100 % of DPPH, the DPPH-
solution was measured without any addition.
- To measure the antiradical potential (ARP) of beer and phenolic substances, the
following mixtures were prepared:

BEER
- The volumetric mixing ratio of the DPPH solution to degased beer is 14 : 1.

PHENOLIC SUBSTANCES
- To prepare the polyphenol solutions, aqua bidest was used. The concentration of
the phenolic substances was the same as DPPH (1.86 x 10-4 mol/l).
- The mixing ratio with the DPPH-solution was 1 : 1.

After determination of the zero point, the mixtures were prepared and shaken for a
few seconds. The measurement started one minute after mixing. The cavity with the
mixed solution could be left in the ESR and every further minute a measurement was
done. After 10 minutes the collection of data was stopped.
The resulting graph represented the scavenging activity of the sample solution against
DPPH (figure 2). The unit of the amount of DPPH was expressed as a percentage.

4
 100
90
80
70

DPPH in %
60
50
40
30
20
10
0
0 1 2 3 4 5 6 7 8 9 10
Time [min]

Figure 2: DPPH scavenging activity

To evaluate the graph, a trendcurve (6th grade polynomial) was calculated, followed
by integration of the area beyond the curve over a time period of ten minutes (fig. 3).

100

90

80

70
DPPH in %

60

50

40

30

20

10

0
0 1 2 3 4 5 6 7 8 9 10
Zeit in min

Figure 3: Integration of the area beyond the trendcurve

If there is no DPPH scavenging activity, it will result in an area of 1000. With an

increase of the antiradical activity, the area will decrease. To evaluate the antiradical
potential (ARP), the area above the curve is calculated (figure 4). The ARP provides
information about the amount of reduced DPPH and the reaction rate.

ARP = 1000 area beyond the trendcurve

100

90

80

70
DPPH in %

60

50

40

30

20

10

0
0 1 2 3 4 5 6 7 8 9 10
Zeit in min

Figure 4: Graph of the ARP

5
RESULTS & DISCUSSION
Phenolic substances
Several phenolic acids, which are found in beer, were measured. In figure 5 and table
1 you can see the scavenging activity and the ARP of derivates of the cinnamic acid:

100

90

80

70

60 Ferulic Acid
DPPH [%]

Caffeic Acid
50
Chlorogenic Acid
40 Cumaric Acid

30

20

10

0
0 1 2 3 4 5 6 7 8 9 10
Time [min]

Figure 5: DPPH scavenging activity of hydroxycinnamic acids

Hydroxycinnamic Acids ARP

Caffeic Acid 684
Chlorogenic Acid 632
Ferulic Acid 288
p-Cumaric Acid 261

Table 1: ARP of hydroxycinnamic acids

Caffeic acid and chlorogenic acid (5-caffeoyl-quinicacid) show a high ARP, which
can be explained by the ortho-dihydroxy structure at the benzene ring.
Figure 6 and table 2 display the scavenging activity and ARP of derivates of the
bencoic acid:

100

90

80

70
Gallic Acid
60 p-Hydroxybenzoic Acid
DPPH [%]

Syringic Acid
50
Vanillic Acid
40 Protocatechic Acid
30 Gentisic Acid

20

10

0
0 1 2 3 4 5 6 7 8 9 10
Time [min]

Figure 6: DPPH scavenging activity of hydroxybencoic acids

6
 Hydroxybencoic Acids ARP
Gallic Acid 816
Gentisic Acid 580
Protocatechic Acid 293
Syringic Acid 272
p-Hydroxybencoic Acid 157
Vanillic Acid 109

Table 2: ARP of hydroxybencoic acids

Gallic acid possesses the highest ARP. It has a trihydroxy structure, which seems to
have a strong antiradical activity.
In figure 7 and table 3, the scavenging activity and ARP of several flavonoids are
shown:

100

90

80

70

60 Epicatechin
DPPH [%]

Epicatechin Gallate
50
Epigallocatechin
40 Epigallocatechin Gallate
30

20

10

0
0 1 2 3 4 5 6 7 8 9 10
Time [min]

Figure 7: DPPH scavenging activity of flavonoids

Flavonoids ARP
Epigallocatechin Gallate 667
Epigallocatechin 555
Epicatechin 532
Epicatechin Gallate 230

Table 3: ARP of flavonoids

Epigallocatechin gallate shows a high ARP. An explanation is the trihydroxy structure

at the epicatechin. In addition it is esterified with a gallic acid.

7
The results make it possible to create a ranking of the phenolic substances by the
order of their antiradical potential (table 4).

Substance ARP
1 Gallic Acid 815
2 Caffeic Acid 684
3 Epigallocatechin Gallate 667
4 Chlorogenic Acid 632
5 Gentisic Acid 580
6 Epigallocatechin 555
7 Epicatechin 532
8 Protocatechic Acid 293
9 Ferulic Acid 288
10 Syringic Acid 272
11 p-Cumaric Acid 261
12 Epicatechin Gallate 230
13 p-Hydroxybenzoic Acid 157
14 Vanillic Acid 109

Table 4: Ranking of phenolic substances by their ARP

To evaluate the positive antiradical effect of phenolic substances in beer, several acids
were added to beer (figure 8).

1000 931
900 870
816 811
773 776
800

700

600
ARP

500

400

300

200

100

0
Beer + Gallic Acid + Gentisic Acid + Protocatechic + Syringic Acid + p-
Acid Hydroxybencoic
Acid

Figure 8: Change of the ARP of beer by addition of phenolic substances

Figure 8 shows an increase of the ARP of beer by the addition of phenolic substances.
Gallic acid, which has the highest ARP of the studied substances, led to the strongest
increase of the ARP in beer. To evaluate the influence of phenolic substances
contained in hop, a hopped and an unhopped beer were measured. Table 5 shows the
positive effect of hopping on the ARP of beer.

ARP
Unhopped Beer 795
Hopped Beer 887

Table 5: ARP of hopped and unhopped beer

8
Even an unhopped beer has a good ARP, which is also caused by the phenolic acids
coming from the malt. But the addition of hop can increase the ARP of beer
significantly.

Beer
To determine the ARP of beers, the volumetric ratio of the beer and DPPH-solution is
important. The reaction rate can vary most intensive. In figure 9 the DPPH-
scavenging activity of several lager beers is diagrammed. The beers show significant
differences in their ARP (table 6).

100

90

80

70
A
60
DPPH [%]

B
50 C
D
40 E
30 F

20

10

0
0 1 2 3 4 5 6 7 8 9 10
Time [min]

Figure 9: DPPH scavenging activity of lager beers

Lager Beers ARP

A 480
B 631
C 567
D 665
E 631
F 850

Table 6: ARP of lager beers

The method works just as well with top-fermented beer, like wheatbeer, dark beer and
nonalcoholic beer.

CONCLUSION
In contrast to other methods of measurement, the antiradical potential (ARP)
considers the amount of reduced DPPH and the reaction rate.
Phenolic substances possess a very good antiradical activity aginst DPPH. With the
ARP it is possible to create a ranking of phenolic substances. The addition of phenolic
acids to beer lead to an increase of the ARP.
Beers show significant differences in their ARP. Therefore, the ARP is a new method
to evaluate the antioxidative activity in beer and can be used as a complementary
parameter to other analysis like the Lag-Time.

9
ACKNOWLEDGEMENT
This project is sponsored by Wissenschaftsfrderung der Deutschen Brauwirtschaft
e.V. and AIF Otto von Guericke e.V. (AIF 12605).

REFERENCES
1) Brand-Williams, W., Cuvelier, M. E. and Berset, C.: Use of free radical method to
evaluate antioxidant activity, Food Science and Technology, 28, 1995.
2) Dalgliesh, C. E.: Flavour Stability, Proc. Congr. Eur. Brew. Conv., 1977.
3) Kaneda, H. et al.: Detection of Free Radicals in Beer Oxidation, J. Food Sci., 53,
1988.
4) Kaneda, H., Kobayashi, N., Furusho, S., Sahara, H. and Koshino, S.: Reducing
Activity and Flavor Stability of Beer, MBAA Tech. Quart., 32, 1995.
5) Uchida, M. and Ono, M.: Improvement for Oxidative Flavor Stability of Beer
Rapid Prediction Method for Beer Flavor Stability by Electron Spin Resonance
Spectroscopy, J. Am. Soc. Brew. Chem., 54, 1996.
6) Uchida, M. and Ono, M.: Improvement for Oxidative Flavor Stability of Beer -
Role of OH-Radical in Beer Oxidation, J. Am. Soc. Brew. Chem., 54, 1996.

10
67

Generation of foaming proteins along

the malting and brewing processes
Didier Marion1, Sandrine Jgou1,2, Jean-Paul Douliez1, Thrse
Gaborit1 & Patrick Boivin2
1
Institut National de la Recherche Agronomique, Unit de Biochimie et Technologie des
Protines, B.P. 71627, F-44316 Nantes Cedex 03, France (e-mail : [email protected])
2
Institut Franais des Boissons de la Brasserie Malterie, Ple Technologique de Brabois, 7 rue
du Bois de la Champelle, B.P. 267, F-54512 Vandoeuvre Cedex, France

Descriptors
Albumin, chemical composition, foam stability, malt modification

SUMMARY
The lipid transfer protein 1 (LTP1), an abundant albumin of barley seeds, accounts for about
50% of the proteins of beer foam. From seeds to beer, the LTP1 undergoes structural
modifications including unfolding and glycation through Maillard reactions. These
modifications improve the foaming properties of the protein (Jgou et al., J. Agric. Food
Chem., 2000, 48, 5023-5029). To identify the key stages of processes that lead to these
physico-chemical modifications, a detailed study was carried out on the structure of LTP1
forms isolated throughout malting and brewing.

Die Bildung von Schaumproteinen in Laufe des Vermlzungs- und Brauprozesses

Deskriptoren
Albumin, chemische Zusammensetzung, Malzlsung, Schaumbestndigkeit

ZUSAMMENFASSUNG
Das Lipid-Transfer-Protein 1 (LPT1), ein Eiwei, das in Gerstenkrnern reichlich vorhanden
ist, ist fr etwa 50% der Proteine im Bier verantwortlich. Whrend der Bierbereitung ndert
sich das LPT1 strukturell, einschlielich der Entfaltung und Glykation infolge von Maillard-
Reaktionen. Infolge dieser Modifizierungen verbessert sich das Schaumverhalten der Proteine
(Jgou et al., J. Agric. Food Chem., 2000, 48, 5023-5029). Zur Identifikation der wichtigsten
Phasen der Prozesse, die diese physio-chemischen Modifizierung bewirken, wurde ein
detailliertes Studium der whrend des gesamten Vermlzungs- und Brauprozesses isolierten
LPT1-Formen durchgefhrt.
Formation de protines moussantes au cours du maltage et du brassage

Descripteurs
Albumine, compos chimique, dsagrgation du malt, stabilit de la mousse

RESUME
La protine de transfert de lipides (LTP1), une albumine majeure de lorge, reprsente
environ 50% des protines de la mousse de la bire. Du grain la bire, la LTP1 subit des
modifications structurales comprenant dnaturation et glycosylation par raction de Maillard.
Ces modifications amliorent les proprits moussantes de la protine (Jgou et al., J. Agric.
Food Chem., 2000, 48, 5023-5029). Afin didentifier les tapes cls des procds qui
conduisent ces modifications, une tude dtaille a t conduite sur la structure des
diffrentes formes de LTP1 isoles aux cours des diffrentes tapes du maltage et du
brassage.

2
INTRODUCTION
The beer foam quality depends mainly on malt-derived polypeptides, especially
protein Z (Mr 43 kDa) and 9 kDa lipid transfer protein (LTP1)(1,2). These barley
albumins are the major proteins that contribute to formation and stability of beer foam
(2,3). In a first attempt, we have focused our work on LTP1 in regard to the numerous
data available on the structure and physico-chemical properties of this protein family
(see 4 for a recent review). Furthermore LTP1 is an interesting model proteins in
regard to its known allergenic properties in most plant-derived foods (5). The
structure of barley and other plant LTP1s is characterised by a four-helix bundle and
a C-terminal arm that are stabilised by four disulphide bonds. Moreover the presence
of a hydrophobic cavity is the binding site for hydrophobic and amphiphilic lipids (4).
Although LTP1s are surface active proteins (6), the native barley seed LTP1 displays
poor foaming properties (2). This protein becomes a foam promoting form only after
unfolding during wort boiling (3). So we have studied the structural and chemical
modifications of LTP1 along the malting and brewing processes in relation with their
foaming potential in beer.

MATERIAL AND METHODS

Preparation of malt, wort and beer samples
Four kg of a spring barley cultivar (Hordeum vulgare, cv. Scarlett) were micromalted
and microbrewed at the IFBM using standard procedures. Green malts were prepared
from barley by three alternating steeping and air rest periods. Then, germination of
grains was conducted for five days at an average of 16C and green malts were kilned
with the following successive air temperatures: 50C, 64C and 80C. The mashing
profile consisted of a 45C stand for 20 minutes followed by ramping to 64C and
then to 74C. After lautering, worts were boiled for 90 minutes, then fermented and
stored several days prior to filtration and bottling.

Extraction and purification of lipid transfer proteins

Soluble proteins were extracted from 1 kg solid material (barley, malt) by a gentle
stirring with 4 l of distilled water for 4 hours at room temperature. After
centrifugation, the soluble material was lyophilised for purification. 800 ml of beer,
previously degassed, were dialysed (dialysis tubing cut-off 3.5 kDa) against
desionised water in order to remove low molecular weight compounds. LTP1 from the
different samples was purified as previously described (7) with successive
chromatographies on a cation exchange column (Pharmacia, France), Sephadex G-50
size exclusion gel (Pharmacia, France), and semi-preparative reversed phase HPLC
(CIL, Bordeaux, France). The purity of fractions was controlled by SDS-PAGE in
presence of 2-mercaptoethanol (8).

Mass spectrometry
Protein molecular masses were measured using a Perkin-Elmer API III+ (Sciex,
Thornill, Canada) triple quadrupole mass spectrometer equipped with an atmospheric
pressure ionisation source (Electro-spray mass spectrometer, ES-MS). The sample
analysis (1 mg/ml) was achieved either by infusion at 5 l/min or by an on-line
coupling between MS and RP-HPLC (LC-MS). Elution was carried out on a RP-
HPLC column (Symmetry C18 Waters, Milford, MA) at a flow rate of 0,25 ml/min
(40C) with a split to the MS ionisation source which was set at a flow rate of 30
l/min. Ion detection was performed in positive mode and molecular masses were
determined from charge m/z using Biomultiview 1.2 (Software package Sciex).

Circular dichroism spectroscopy

The secondary structure of proteins was determined by circular dichroism (CD) in the
far UV (from 190 nm to 250 nm). The measurements were performed at 25C on a
CD6 Jobin-Yvon dichrograph. Proteins were solubilized in desionised water at a final
concentration of 0.5 mg/ml. A quartz cell of 0.2 mm path length was used. Data were

3
expressed as mean-residue ellipticity. Secondary structures were determined by using
the CONTIN software.

Production of polyclonal and monoclonal antibodies

LTP1+LTP1b purified from barley seeds (cv. Plaisant) and emulsified with Freunds
adjuvant (complete for the initial injection and incomplete adjuvant for boosters) were
subcutaneously administered to three rabbits (around 360 g/injection). Sera were
collected after the seventh injection and their specificity was tested by ELISA.
Immunoglobulins against total LTP1 (LTP1 + LTP1b) were purified by two steps of
ammonium precipitation and desalted against PBS(8mM Na2HPO4, 1.5 mM KH2PO4,
2.7 mM KCl, 140 mM NaCl).
The monoclonal antibody (Mab) specific for LTP1b were prepared according to the
method described by Klher and Milstein (9). The Mabs were obtained from ascite
liquid and stored at 20C.

Enzyme linked immunosorbent assay

LTP1 content was determined by ELISA according the method described by Engvall
and Perlmann (10). Each well was coated with 100 l of LTP1 standard or assay
solution. The plate, left overnight at room temperature, was rinsed with PBS
containing Tween 20 at 0.05% (v/v) thereafter called Tween-PBS. PBS (250 l)
containing 1% (v/w) bovine serum albumin (BSA) were added to each well. After 1
hour at 37C, the plate was washed with Tween-PBS and 100 l of diluted specific
immunoglobulins was added. The antigen-antibody complex was revealed by goat
anti-mouse IgG antibodies conjugated with radish peroxidase and diluted to 1/3000 in
Tween-PBS. After 1 hour of incubation at 37C, the plate was rinsed and 100 l of
substrate (0.04% (w/v) o-phenylenediamine and 0.02% H2O2 in 0.05M citrate buffer
pH 5.5 were added. The plate was incubated during 30 min at 37C and the enzyme
activity was inhibited by addition of 25 l of HCl 2N. Absorbance measurements
were performed at 490 nm and 630 nm.

Foaming measurements
To analyze the foaming properties of LTP1 fractions, the apparatus previously
described by Loisel et al. (11) was used. Eight ml of protein solution were poured into
a reservoir ending by a column (2 cm x 20 cm) and foams were produced by sparging
solutions with air at a constant flow rate (15 ml.min-1) through a porous disk (porosity
= 2 ; radius = 2 cm). Conductivity was determined with a pair of platine electrodes
located at the bottom of the column. Gas sparging continued until 35 ml of foam was
reached. This fixed foam volume was detected by a camera. When bubbling stopped,
the drainage kinetic was recorded by conductivity measurements. The conductivity
was related to the volume of liquid sustained by foam within the lamellae (12).

Analysis for free sulphydryl groups in protein fractions

Colorimetric reactions were conducted under the conditions described by Ellman (13).
2 mg of protein were suspended in 3 ml of Tris-HCl pH 8 containing 6M urea and 3
mM EDTA. 100 l of DTNB solution (40 mg DTNB solubilized in 10 ml of buffer)
was added into the solution. The reaction mixture was incubated at room temperature
for 15 min. The absorbance was recorded at 420 nm against a protein-free blank
containing the DTNB assay solution.

RESULTS AND DISCUSSION

Variation of LTP1 et LTP1b content from barley to malt
It was previously shown that LTP1 is present in barley as two forms, LTP1 (9,689 Da)
and LTP1b (9,983 Da)(7). It was recently shown that LTP1b corresponds to LTP1
with a covalently linked adduct of 294 Da. The structure of this adduct is still
unknown (14). The variation of LTP1b and total LTP1 (LTP1+LTP1b) contents was

4
followed along the malting process by ELISA. The amount of LTP1b was almost
constant during steeping and throughout germination (figure 1) as previously
observed (15). The content of LTP1b increased significantly from the end of
germination to the end of the kilning step 50C. Then, the content remained constant
until finished malt was obtained. The amount of total LTP1 evolved similarly to that
of LTP1b. The raise of total LTP1 content on kilning reflected the increase of the
LTP1b level in malt.
LTP1b LTP1+LTP1b Water content

200 50

180
40
160

% (water content)
140 30
mg/100g

120
20
100

80 10

60
0
barley

End 50C

Begin. 64C

End 64C

End 80C

Malt
5 Day germin.

Figure 1: Total LTP1 (LTP1+LTP1b) and LTP1b contents from barley to malt as
determined by ELISA

Glycation of LTP1b on kilning

9984
Intensity, cps

10146

10294
9821
10308 10618
-W 10456
-W 10785
-W 10920
11099 11317

9600 9800 10000 10200 10400 10600 10800 11000 11200

Mass, Da

Figure 2: Mass spectrum of LTP1b recovered from finished malt showing the
Maillard reaction adducts (+162Da) and dehydration rearrangement (-W).
Peak at 9,821 Da corresponded to LTP1b without the C-terminal Tyr91.

5
The determination of masses of LTP1 during the kilning process, from the beginning
of step 64C to the beginning of step 80C, showed the appearance of Maillard
reaction products (figure 2). This reaction include the initial condensation reaction
between proteins (amine groups from N-terminal and lysine residues) and reducing
sugars to form Amadori intermediate rearrangement products (16). Only LTP1b was
observed along the kilning process, a result that confirmed the data obtained by
ELISA. Then, the glycation level of LTP1b, increased until 80C. The glycation can
be explained to the abundance of glucose and maltose in barley malt (17).

Denaturation of LTP1 on brewing

In contrast with malt less LTP1b was recovered from beer suggesting that hydrolysis
of the 294 Da adduct was achieved during brewing (figure 3). Finally the three major
LTP1 fractions recovered by HPLC showed that some disulphide bonds were broken.
Full denaturation was only observed, by circular dichroism, for the form in which all
4 disulphide bonds were reduced (table 1). Furthermore, no denaturation of LTP1 was
observed in finished malt. Finally, the glycation level of LTP1 did not change along
the brewing process (figure 3).

9689
Intensity, cps

9851

10013
9526

9983 10175
10145 10309
9412 9820 1033710471

9400 9600 9800 10000 10200 10400

Mass, Da
Figure 3: Mass spectrum of LTP1 from beer showing the 162 Da Maillard reaction
adducts. Beer LTP1b (F3) provided similar spectrum. Peak at 9,526 Da
corresponded to LTP1 without the C-terminal Tyr91.

Secondary Free-SH
structure (CD)
LTP1 Barley -helix 0
LTP1b -helix 0

LTP1b Malt -helix 0

LTP1 F1 Random coil 8

LTP1 F2 Beer -helix 4
LTP1b F3 -helix 2

Table 1: Secondary structure and free-SH content of LTP1 and LTP1b fractions from
barley, malt and beer. Malt and beer protein fractions were glycated.

6
Foaming properties of beer LTP1
The foaming properties of the different LTP1 fractions were studied. Interestingly
LTP1b displayed the best foam capacity but poor stability. In beer, reduction of one
disulphide bond were obviously sufficient to improve the stability of foam provided
by glycated LTP1b (table 2). LTP1 was unable to form a foam and only the
denaturated LTP1 with all disulfide bonds reduced, led to the formation of stable
foams. So, at this stage of our investigation it appeared that full denaturation of LTP1
was required to obtain a good foam while this conformational change was not
necessary for glycated LTP1b. Finally, controlling the balance between glycated
LTP1b and denaturated LTP1 levels along the brewing process should be essential to
optimise the foaming properties of beer.

FC T1/2
LTP1 Unstable Unstable
Barley foam foam
LTP1b 1.64 34
Barley
LTP1 F1 1.00 83
LTP1 F2 0.57 22
LTP1b F3 1.00 71

Table 2: Foaming properties of barley and beer LTP1 polypeptides. FC : foam

capacity (ml foam liquid /ml injected gas) ; T1/2 : time (sec.) for a 50%
reduction of foam liquid.

CONCLUSIONS
Many complex biological and physico-chemical events occur on malting and brewing
processes that improve the foaming properties of LTP1. On kilning up to 50C,
synthesis of LTP1b occurs, probably in relation to a response to heating stress. It is
known for many plants that the synthesis of LTP1 is induced in response to many
biotic and abiotic stress (4). Subsequent heating up to 80C induces glycation of
LTP1b through Maillard reaction. This reaction is improved by both increasing
temperature and decreasing water content of seeds on kilning. On brewing partial
denaturation of LTP1b and its conversion in LTP1 by hydrolysis of the 294 Da adduct
is probably achieved on wort boiling. The different glycated LTP1 forms of beer
display specific foam capacity and/or stability. Therefore the control of heating
diagrams on malting and brewing processes is essential to improve the foaming
properties of beer. These structural and chemical modifications should also decrease
and even suppress the allergenic properties of LTP1. Therefore heating parameters are
essential to provide a safe beer with good foaming characteristics.

ACKNOWLEDGEMENT
We gratefully thank M. Malanda and M. Dumoulin from IFBM for preparing malt and
beer samples. This work was granted by Malteurs de France and Brasseurs de France.
S.J. received financial support from the French Ministry of Education and Research
and the French Ministry of Agriculture (AQS and CIFRE contracts).

7
REFERENCES
1. Kaersgaard, P. & Hejgaard, J., The Journal of the Institute of Brewing, 1979,
85, 103-111.
2. Srensen, S.B., Bech, L.M., Muldbjerg, T.B. & Breddam, K., Master Brewers
of the American Technical Quarterly,1993, 30, 136-145.
3. Bech, L.M., Vaag, P., Heinemann, B. & Breddam, K., Proceedings of the
European Brewery Convention Congress, Brussels, 1995, 561-568.
4. Douliez, J-P., Michon, T., Elmorjani, K. & Marion, D., Journal of Cereal
Science, 2000, 32, 1-20.
5. Asero, R., Mistrello, G., Roncarolo, D., de Vries, S.C, Gautier, M.F., Ciurana,
C.L., Verbeek, E., Mohammadi, T., Knul-Brettlova, V., Akkerdaas, J.H.,
Bulder, I., Aalberse, R.C. &, van Ree, R., International Archives of Allergy and
Immunology, 2000, 122, 20-32.
6. Subirade, M., Salesse, C., Marion, D. & Pzolet, M., Biophysical Journal, 1995,
69, 974-988.
7. Jgou, S., Douliez, J.P., Moll, D., Boivin, P. & Marion, D., Journal of
Agricultural and Food Chemistry, 2000, 48, 5023-5029.
8. Laemmli, U.R., Nature (London) 1970, 227, 680-687.
9. Klher, G. & Milstein, C., Nature, 1975, 256, 495-497.
10. Engvall, E. & Perlmann, P., Journal of Immunology, 1972, 109, 129-135.
11. Loisel, W., Gueguen, J. & Popineau, Y., Food Proteins, Schwenke K. D. &
Mothes R., eds, 1993, 320-323.
13. Ellman, G.L., Archives of Biochemistry and Biophysics, 1958, 74, 443-450.
12. Guillerme, C., Loisel, W., Bertrant, D. & Popineau, Y., Journal of Texture
Studies, 1993, 24, 287-302.
14. Douliez, J.P., Jgou, S., Pato, C., Larr, C., Moll, D. & Marion, D., Journal of
Agricultural and Food Chemistry, 2000, 49, 1805-1808.
15. Evans, D.E. & Hejgaard, J., Journal of the Institute of Brewing, 105, 1999, 159-
169.
16. Maillard, L.C., Comptes Rendus de lAcadmie des Sciences, Paris, 1912, 154,
743-752.
17. Allosio, N., Quemener, B., Bertrand, D. & Boivin, P., Journal of the Institute of
Brewing, 2000, 106, 45-52.

8
68

Interfacial mechanisms underlying lipid

damage of beer foam
Peter J. Wilde, Fiona A. Husband, Daniel Cooper, Alan R.
Mackie, A. Patrick Gunning, Victor J. Morris, Nicola
Woodward & Clare Mills

Institute of Food Research, Norwich Research Park, Norwich NR4 7UA, United Kingdom (e-
mail: [email protected])

Descriptors
Beer quality, foam stability, lipid, polypeptide, reaction mechanism, surface activity

SUMMARY
Beer foam is highly susceptible to lipid damage because it is protein stabilised. The
destabilisation mechanism has been studied using surface visco-elasticity and atomic force
microscopy imaging of mixed protein:lipid interfaces. The lipids formed domains that
weakened the interface and destabilised the foam. Longer chain fatty acids were most
damaging. Hydrophobic beer polypeptides interacted strongly with the long chain fatty acids,
and prevented lipid damage of beer foam. Understanding the destabilisation mechanism, the
critical levels of lipids required, and the source of the protective components, should allow the
development of a beer with greater resistance to lipid damage.

Durch Lipide hervorgerufene Schdigungen des Bierschaums und der Grenz-

flchenmechanismen

Deskriptoren
Bierqualitt, Lipid, Oberflchenaktivitt, Polypeptid, Reaktionsmechanismus,
Schaumbestndigkeit

ZUSAMMENFASSUNG
Bierschaum ist gegen Lipidbeschdigung in hohem Grade empfindlilch, weil er durch Protein
stabilisiert wird. Der Dentabilisierungsmechanismus wurde mit Hilfe der Oberflchen-
rheologie und mittels der Darstellung der Protein-Lipid-Schnittstellen durch Atom-
mikroskopie untersucht. Die Lipide bildeten Gebiete, die die Schnittstelle schwchten und
den Schaum denstabilisierten. Lngere Fettsurenketten wirkten am zerstrendsten.
Hydrophobe Polypeptide im Bier wirkten stark auf die langkettigen Fettsuren ein und
verhinderten die durch die Lipide hervorgerufene Zerstrung des Schaums. Durch das
Verstndnis des Destabilisierungsmechanismuses, durch Kenntnis der kritischen
Konzentrationen an Lipiden und durch Kenntniss der schtzenden Bestandteile sollte es
mglich sein, Biere zu entwickeln, die gegen eine Schaumzerstrung durch Lipide nicht so
anfllig sind.

Mcanisme d'interface la base de laffaissement de la mousse de bire par les lipides

Descripteurs
Lipide, mcanisme de raction, polypeptide, qualit de la bire, stabilit de la mousse, tensio-
activit

RESUME
Une altration des lipides dans la mousse de la bire est hautement probable car ils sont
stabiliss par les protines. Le mcanisme de dstabilisation a t tudi en utilisant la visco-
lasticit de surface et la microscopie de force atomique d'interfaces mlanges
protines/lipides. Les lipides ont form des domaines qui affaiblissaient l'interface et ont
dstabilis la mousse. Les acides gras possdant les chanes les plus longues taient les plus
endommags. Les polypeptides hydrophobes contenus dans la bire ont interfr fortement
avec les acides gras chane longue, et ont vit une altration des lipides contenus dans la
mousse de la bire. La comprhension du mcanisme de dstabilisation, les taux critiques de
lipides ncessaires, et l'origine des composants protecteurs devraient permettre de mettre au
point une bire prsentant une meilleure rsistance la destruction des lipides.

2
INTRODUCTION
Beer foam is one of the first quality traits observed by the consumer; hence the
research into beer foam stability has received much attention over recent years. Beer
foam is stabilised by proteins and polypeptides, principally from the malt, which have
survived the brewing process. Much work has concentrated on some of the proteins in
beer, which contribute to foam stability such as hydrophobic peptides [22, 24] a 40kD
protein [15, 26] and barley LTP1 [23]. There are several factors that can improve
(foam positive) or decrease (foam negative) foam stability [2]. Foam positives include
hop acids [1, 4], metal ions [3] and PGA [20]. Foam negatives include lipids, fats and
surfactants [7, 13, 19]. Proteins stabilise foams by forming a strong elastic film on the
foam bubble surface [18]. The factors listed above act on the mechanical strength of
the protein adsorbed layer and increase (foam positives) or decrease (foam negatives)
the surface elasticity. Interfacial research at the Institute of Food Research has
focussed on the competition between proteins and lipids in food foams and emulsions.
We have demonstrated how lipids breakdown the surface elasticity of an adsorbed
protein layer and hence induce coalescence in protein stabilised foams [8, 25] and
emulsions [9]. Recent work, by the authors, using atomic force microscopy, revealed
for the first time, the true mechanism of lipid induced surface destabilisation of
proteins [16, 17]. This was found to be a purely physical mechanism where lipids
adsorbed into the interfacial protein layer and formed domains. The domains
expanded as more lipid adsorbed, compressing the adsorbed protein into smaller but
thicker regions, ultimately displacing them from the interface altogether [16]. The
mechanism was termed orogenic displacement, after the geological process by which
mountain ranges are formed. During the early stages of displacement, the presence of
lipid domains in the adsorbed protein film, weakens the film, reducing its stabilising
ability and resulting in increased levels of coalescence. This paper investigates the
applicability of this mechanism to beer foam, considering that beer foam is
susceptible to lipid damage.

MATERIALS AND METHODS

An all malt, additive free beer was provided by Brewing Research International
(Nutfield, Surrey, UK). Fatty acids were used to destabilise the beer foam, these were
oleic acid (C18:1) and linoleic acid (C18:2) both 99% pure, purchased from Sigma
Aldrich (Gillingham, UK) and used without further purification.

Foam stability was measured using the micro-conductivity technique [25]. Here, 2 ml
of beer was sparged with nitrogen at a back pressure of 20 psi. through a single orifice
(20 m diameter) in a glass jet. This produces a very narrow size distribution of
bubbles (700 70 m diameter), also the mean bubble diameter is not influenced by
surface tension effects [25]. Therefore it is possible to add lipids or other surface
active components without affecting the physical structure of the initial foam. 2.6 ml
of foam was formed and then sparging stopped, and the foam was allowed to collapse.
The foam density was monitored using conductivity, which followed the dynamics of
foam drainage and collapse. The foam density is expressed as a percent liquid content
of the foam and is calculated according to Lemlich [10 ] thus:

Foam density = 3K - 2.5K 4/3 + 0.5K2

3
Where K is the conductivity of the foam relative to the bulk conductivity of the
sample. The stability of the beer was measured in the presence of a range of
concentrations of the fatty acids. The foam stability was calculated thus:

Foam stability (%) = 100. C300 / C0

Where C0 and C300 are the foam conductivities at time = 0 and 300 s drainage
respectively.

Surface rheology was measured using a CIR-100 interfacial rheometer (Camtel

Instruments, Royston, UK). This instrument uses a Du Nouy ring oscillating in
normalised resonance mode [21]. The ring is oscillated in air at its resonant frequency
of approximately 3 Hz. When the ring is placed at an interface, the resonant frequency
and amplitude will change depending on the elasticity and viscosity of the interface.
Feedback currents restore the frequency and amplitude of the oscillations back to
resonance. The values of the feedback signal required to restore the frequency are
used to calculate the surface shear elastic modulus (G), and the signal required to
restore the amplitude enables calculation of the viscous component (G). The radial
amplitude of the ring during these measurements was 5x10-3 rad. The surface
elasticity was determined for beer adulterated with between 0 and 28 M oleic acid.

Imaging of the adsorbed layer was performed using atomic force microscopy (AFM).
The air-water interface is too deformable to be able to image in-situ, therefore the
adsorbed layer was transferred onto a 1 cm2 sheet of mica, hydrophobised with a
mixture of 5% dichloromethylsilane, 10%1,1,1-trichloroethane, 5%
tetrakis(ethoxy)silane in toluene. The beer sample, with or without 5 M linoleic acid,
was carefully poured into a small dish and allowed to stand for a few minutes. The
adsorbed layer was transferred onto the mica using the Langmuir-Schaeffer method.
This entailed mounting the mica horizontally, and slowly lowering it until the face
made contact with the surface of the sample, the surface layer would now be adsorbed
to the mica. The mica was carefully removed and rinsed in water to remove excess
sample and prevent secondary adsorption. The mica was mounted in the stage of the
AFM and the transferred interface was imaged, using the mica as a solid, atomically
flat substrate. The sample was imaged under butanol to improve resolution, which has
already been shown not to influence the structure of the film [16].

RESULTS
Figure 1 shows the foam drainage curves for beer adulterated with different
concentrations of linoleic acid (C18:2). This fatty acid was chosen as it is a common
lipid, often found at relatively high concentrations in beer, and thought to be
responsible for some lipid damage. The drainage behaviour of the beer in the absence
of any lipid is typical for beers of a similar character using this method. In the
presence of linoleic acid the initial density (at time = 0) of the beer foam is largely
unaffected. However, as foam drainage continues, beer in the presence of the fatty
acid drained more rapidly. The extent of drainage increased with more added lipid.
Therefore the stability of the foam was steadily reduced with increasing levels of
linoleic acid. The increased drainage of liquid from the foam is due either to increased
levels of liquid flow through the foam, or more bubble coalescence events.

4
In separate experiments (data not shown) hydrophobic polypeptides were added back
to the beer in the presence of lipid and the foam stability was restored.
14

Foam liquid content (%)

12 Linoleic Acid
concentration
10 (M)
8
0
6
2.5
4 5
2 10
0
0 100 200 300 400
Time (sec)

Figure 1: Foam drainage curves using foam micrconductivity method for

beer adulterated with different concentrations of linoleic acid

Binding experiments showed that this protein fraction strongly interacted with linoleic
acid. Figure 2 shows the calculated foam stability values for beer as a function of
linoleic acid and oleic acid concentrations. Both fatty acids are effective at
destabilising the beer foam. The amount of either fatty acid required to reach
minimum foam stability was less than 10 M (3 ppm). No recovery in foam stability
was observed over these concentration ranges as has been observed in previous
studies on surfactants [8, 14, 25].

60

50 Linoleic acid
Foam Stability (%)

40 Oleic acid

30

20

10

0
0 5 10 15 20 25 30
Fatty Acid Conc. (M)
Figure 2: Foam stability of beer as a function of added fatty acids.

It is widely accepted that the destabilising effect of surfactants and lipids is due to
disruption of the surface elasticity of the protein adsorbed layer. Therefore the surface

5
rheology of the mixed beer fatty acid samples was studied. Figure 3 shows the
surface elastic modulus of beer alone and one adulterated with 7.1 M oleic acid as a
function of adsorption time.
6000
Surface elastic modulus ( N/m)
5000
Beer
4000

3000

2000

1000
Beer + 7.1M Oleic Acid
0
7.1M Oleic acid
-1000
0 200 400 600 800 1000
Time (s)
Figure 3: The surface elasticity of beer as a function of adsorption time
in the presence and absence of 7.1 M oleic acid.

The surface elasticity of the oleic acid alone is also shown. Similar to the foam
stability results, the beer alone shows typical values of the surface elasticity found for
beers of a similar character. As the proteins and polypeptides adsorb, they unfold and
aggregate at the surface resulting in a time dependent increase in the surface elasticity
as the interface aged.

6000
Surface Elastic Modulus ( N/m)

5000

4000

3000

2000

1000

0
0 5 10 15 20 25 30
Fatty Acid Concentration (M)
Figure 4: Effect of oleic acid concentration on the surface shear
elasticity of beer.

6
 In the presence of the fatty acid, the surface elasticity is much reduced, developing to
only 75 N/m, 70 times less than the beer alone. Figure 4 shows the surface elastic
modulus after 14 minutes adsorption as a function of oleic acid concentration. The
decrease in elastic modulus is remarkably similar to the observed decrease in foam
stability (figure 2).

a b
Figure 5: AFM images of surfaces formed by the adsorption of (a) beer and
(b) beer +5 mM linoleic acid. Image sizes are (a) 5x5 m; (b) 7.5x7.5 m.

The interfacial rheology gives averaged information over the whole interface. To
reveal the true nature of the destabilisation process, it is possible to image the
interface using atomic force microscopy (AFM). Figure 5 shows images of two
interfaces. Figure 5a is beer alone and figure 5b is beer adulterated with 5 M linoleic
acid. AFM is a probe microscope, which gives topographical information, therefore in
the images, the lighter regions are thicker and the dark regions are the thinnest. The
beer alone shows a fairly uniform primary adsorbed protein layer in the background.
The image also shows a great deal of aggregation, with the largest aggregates being
around 20 nm thick. In the presence of fatty acid (figure 5b) the number of aggregates
is dramatically decreased, and the appearance of holes is observed. Sampling the
interface 2 hours later it was found that the holes increased in size and number.

DISCUSSION
It is well known that lipids and other small molecular weight surfactants damage
protein-stabilised foams [8, 19]. In many foods, it is difficult to exclude lipids, and the
competition with proteins for interfacial area often results in less than ideal foaming
behaviour. In beer the levels of intrinsic lipids are usually quite low, however
extrinsic lipids can be a real problem [2]. Extrinsic lipids are those that are introduced
during dispense or consumption, their sources include dirty glassware or dispensing
equipment and consumption of fatty foods. Therefore to maintain a stable foam, the
beer must be resistant to the foam damaging effects of lipids. To devise a robust
strategy to tackle this issue, we must acquire a thorough understanding of the precise
mechanism underlying lipid damage of beer foam. The first stage in this was to
develop controlled destabilised beer systems which. In this case we added linoleic or
oleic acids to an all malt, additive free beer and found that they significantly reduced

7
the stability of the beer (figure 1). The small difference in the amount of lipid required
to destabilise the beer foam (figure 2) is not surprising since both fatty acids are C18
and unsaturated. Oleic acid has one double bond and linoleic has two. In the presence
of a hydrophobic protein fraction from beer, the damaging effect of the lipid was
negated. It is likely that this protein fraction, through its strong interactions with the
lipids, restabilises the foam by chelating the lipids. This prevents lipid adsorption and
hence destabilisation, in a manner similar to that found for puroindoline [7] a lipid
binding protein from wheat.

The foaming technique used here has been shown to produce a constant bubble size
distribution, independent of surface tension effects [25], therefore any changes in
drainage behaviour are due to changes in interfacial properties of the bubbles and
coalescence effects. Disproportionation was minimised as the foam was sparged with
nitrogen and the bubbles were all a very similar size. The foam drainage rates
increased as the fatty acid concentration increased. This was probably due to
increased levels of bubble coalescence within the foam, as visual inspection of the
foams indicated a greater rate of coarsening in the presence of the fatty acids.

Previous work using surfactants showed that at higher surfactant concentrations, foam
stability recovered [8, 14, 25]. This was due to the inherent foam stabilising properties
of the surfactant itself. In the present case of fatty acids, no foam recovery was
observed over the concentration range studied. Therefore the fatty acids were acting in
a purely destabilising manner. The previous work with surfactants found that Tween
20 was particularly effective at destabilising foams, however, destabilisation wasnt
observed until concentrations of around 20 to 30 M Tween 20. Therefore, the fatty
acids studied here are highly effective at destabilising beer foam, being effective at
concentrations below 5 M. It is possible that the presence of ethanol in the beer aids
solubility and hence surface activity of the fatty acids.

Previous work on model protein and surfactant systems has shown that the lipid
induced destabilisation is a result of a reduction in the interfacial rheology of the
adsorbed protein layer [5]. When proteins adsorb at an interface, they unfold and
aggregate and thus develop an elastic interface. This was observed to happen for the
beer in this study (figure 3). The elasticity continued to develop over the experimental
time as the proteins and polypeptides partially unfolded and aggregated at the surface.
The mechanical properties of the protein stabilised interface are key to the foam
stability, that is, highly elastic interfaces can withstand more perturbations and
deformation, and thus will remain stable for longer. This premise was put forward by
Rehbinder over 60 years ago [11], and there has been much work linking the elastic
properties of protein stabilised interfaces with stability [12, 15, 18]. Since the stability
of protein foams are dependent on the mechanical properties of the interface, factors
which reduce the surface elasticity are likely to damage foam stability. It can be seen
from figures 3 and 4 that the addition of fatty acids drastically reduces the surface
elasticity of the beer. The fatty acid concentrations required to reduce the surface
elasticity and the foam stability of the beer are remarkably similar. Therefore it is
likely that the stability of the beer foam in this case is highly dependent on the
elasticity of the layer of protein adsorbed onto the bubble surface.

It is widely accepted that the cause of instability in mixed protein-lipid foams is the
reduction in surface elasticity. However this is only the effect, to reveal the underlying

8
cause, it is necessary to probe the interfacial structure using an approach with much
greater spatial resolution. Atomic force microscopy (AFM) was used to investigate
the interfacial structure of the beer samples with nm resolution. Figure 5a shows an
image of the interface formed by the beer alone, displaying a quite uniform
background containing the primary adsorbed layer of proteins and polypeptides. In
addition, there appeared to be a great number of aggregates associated with the
interface. Whether or not the aggregates contribute to the foam stability of the beer is
not known and may require further investigation. In the presence of linoleic acid
(figure 5b), most of the aggregates disappear, and much thinner regions become
apparent. From our previous work on mixed protein surfactant systems, the thinner
darker regions are in fact surfactant or lipid domains. Once the lipid begins to adsorb
into the protein network, other lipids preferentially adsorb into regions containing
lipid rather than the protein. This causes an increase in the surface pressure of the
lipid domain and so it begins to expand (Subsequent samples of the beer + linoleic
acid sample, showed that the holes did indeed increase in size). As the lipid domains
expand, the protein network is compressed until, if there is sufficient lipid, it
collapses, and is finally displaced into solution. This displacement process is generic
and has been observed for a number of model protein-surfactant systems. The critical
stage in this process is the expansion of the lipid domains. In order to expand and
reduce the elasticity of the interface, the surface pressure applied by the domain has to
overcome the elastic resistance of the protein network. In previous work it was found
that proteins with poor surface elastic properties were more easily disrupted than a
protein, which formed a more elastic surface. [6, 16]. Therefore the surface elasticity
of the adsorbed protein layer is extremely important for resisting lipid damage of
foam. Other components which are known to improve beer foam stability such as hop
acids and PGA are known to increase the surface elasticity, and hence they could be
increasing the resistance of the beer foam to lipid damage.

CONCLUSIONS
In summary, the combination of surface rheology and AFM has revealed the
underlying mechanism of lipid-induced damage of beer foam. Which is that lipids
adsorb into the surface protein layer, forming lipid rich domains, these domains
expand, reducing the elasticity of the surface network. The weakened adsorbed layer
is more prone to coalescence; hence the foam stability is decreased. The lipid binding
properties of proteins in the beer are able to reduce the effect of the lipid damage.
The elasticity of the adsorbed protein network is a key attribute, which resists lipid
destabilisation. Future work will focus on the effects of enhanced lipid binding
proteins in beer and for using surface rheology as a predictive indicator of
functionality.

ACKNOWLEDGEMENT
The authors would like to acknowledge the BBSRC for funding through LINK
(AFM100) and through the core strategic grant to the Institute, and to Bass Brewers
Ltd, Scottish Courage Brewing Ltd and BRI for permission to publish this work.

9
REFERENCES
1. Asano, K. & Hashimoto, N., Report of the Research Lab of Kirin Brewery, 1976
19, 9-16.
2. Bamforth, C.W. & Cope, R., American Society of Brewing Chemists Journal,
1987, 45, 27-32.
3. Bishop, L.R., Whitear, A.L.& Inman,W.R., The Journal of the Institute of
Brewing, 1974 80, 68-81.
4. Clark, D.C., Wilde, P.J. & Wilson, D.R., The Journal of the Institute of Brewing,
1991 97, 169-172.
5. Clark, D.C., Wilde, P.J., Bergink-Martens, D., Kokelaar, A. & Prins, A., Food
Colloids and Polymers: Structure and Dynamics,(Eds. E. Dickinson and P.
Walstra) RSC, London, 1993, 354-364.
6. Clark, D.C., Mackie, A.R., Wilde, P.J. & Wilson, D.R., Royal Society of
Chemistry: Faraday Discussion, 1994, 98, 253-262.
7. Clark, D.C., Wilde, P.J. & Marion, D., The Journal of the Institute of Brewing,
1994, 100, 23-25.
8. Coke, M., Wilde, P.J., Russell, E.J. & Clark, D.C., Journal of Colloid and
Interface Science, 1990, 138, 489-504.
9. Cornec, M., Wilde, P.J., Gunning, P.A., Mackie, A.R., Husband, F.A., Parker,
M.L. & Clark, D.C., Journal of Food Science, 1998, 63, 39-43.
10. Datye, A.K. & Lemlich, R., International Journal of Multiphase Flow, 1983, 9,
627-636.
11. Izmailova, V.N., Yampolskaya, G.P. & Tulovskaya, Z.D., Colloids and Surfaces
A-Physicochemical and Engineering Aspects, 1999, 160, 89-106.
12. Kim, S.H. & Kinsella, J.E., Journal of Food Science, 1985, 50, 1526-1530.
13. Klopper, W.J., Proceedings of the European Brewery Convention Congress,
Salzburg, 1973, 14, 363-371.
14. Lee, J.C.& Tynan, K.J., Proceedings of the 2nd International Conference On
Bioreactor Fluid Dynamics, 1988, 353-377.
15. Maeda, K., Yokoi, S., Kamada, K.& Kamimura, M., American Society of
Brewing Chemists Journal, 1991, 49,14-18.
16. Mackie, A.R., Gunning, A.P., Wilde, P.J. & Morris, V.J., Journal of Colloid and
Interface Science, 1999, 210, 157-166.
17. Mackie, A.R., Gunning, A.P., Wilde, P.J. & Morris, V.J., Langmuir, 2000, 16,
8176-8181.
18. Mitchell, J.R. in Developments in Food Proteins - 4 (Ed. Hudson B.J.F.)
Elsevier, London, 1986, 291-338.
19. Roberts, R.T., Keeney, P.J. & Wainwright, T., The Journal of the Institute of
Brewing, 1978, 84, 9-12.
20. Sarker, D.K. & Wilde, P.J., Colloids and Surfaces B-Biointerfaces, 1999 15,
203-213.
21. Sherrif, M. & Warburton, B., Polymer, 1974, 15, 253-254.
22. Slack, P.T.& Bamforth, C.W., The Journal of the Institute of Brewing, 1983, 89,
395-401.
23. Sorenson, S.B., Bech, L.M., Muldbjerg, M., Beenfeldt, T. & Breddam, K.,
MBAA Technical Quarterly, 1993, 30, 136-145.
24. Wenn, R.V., The Journal of the Institute of Brewing, 1972, 78, 404-406.
25. Wilde, P. J., Journal of Colloid and Interface Science, 1996, 178, 733-739.
26. Yokoi, S., Maeda, K., Xiao, R., Kamada K.& Kamimura M., Proceedings of the
European Brewery Convention Congress, Zurich, 1989, 593-600.

10
69

The background of the adherence of

beer foam to the glass
Helene C. M. Mocking-Bode1, Piet S. Peereboom1, Emile
T.M.J. Martynowicz2 & Anneke C. Douma1
1
TNO Nutrition and Food Research Institute, Utrechtseweg 48, P.O. Box 360, 3700 AJ Zeist,
the Netherlands (e-mail: [email protected])
2
Haffmans B.V., P.O. Box 3150, 5902 RD Venlo, the Netherlands

Descriptors
Biochemistry, cling, laboratory equipment, reaction mechanism

SUMMARY
For breweries the adherence of beer foam to the glass (cling) is a quality characteristic of beer
that is of great importance, since it is easily observed by the consumer. After drinking of the
beer, sufficient foam should cover the glass. In the present paper, the effect of several
biochemical and physicochemical factors on beer cling behaviour has been studied.
Equipment for analyzing cling behaviour in a simple and fast way has been optimized.

Der Hintergrund der Haftfhigkeit des Bierschaums am Glas

Deskriptoren
Biochemie, Laboratoriumausstattung, Reaktionsmechanismus, Schaumhaftung

ZUSAMMENFASSUNG
Fr Brauereien ist die Haftfhigkeit des Bierschaums am Glas ein bedeutendes
Qualittskriterium, da es auch vom Verbraucher leicht zu erkennen ist. Nachdem das Bier
getrunken wurde, sollte ausreichend Schaum am Glas haften. In dieser Studie wurden
unterschiedliche biochemische und physikalisch-chemische Faktoren, die das Anhaftverhalten
von Bier beeinflussen, untersucht. Die fr die Analyse des Haftverhaltens notwendigen
Apparaturen wurden auf einfache und schnelle Art optimiert.
Base de l'adhrence de mousse de bire au verre

Descripteurs
Adhrance de mousse, biochimie, matriel de laboratoire, mcanisme de raction

RESUME
Pour les brasseries, l'adhrence de la mousse de la bire sur le verre (adhsivit) constitue un
critre de qualit extrmement important de la bire, car ce phnomne est facilement
perceptible par le consommateur. Une quantit suffisante de bire devrait recouvrir le verre
une fois que la bire a t bue. Ce poster tudie l'effet de plusieurs facteurs biochimiques et
physico-chimiques sur la raction d'adhsivit de la bire. Les quipements utiliss pour
analyser facilement et rapidement la raction d'adhsivit de la bire ont t optimiss.

2
INTRODUCTION
The visual appearance of a beer is an important factor in determining the perceived
quality. Both the stability of the foam head and the adherence of beer foam to the
glass (cling, lacing) have a significant visual impact.
Several methods of quantifying beer foam stability have been developed and are used
in quality control at the brewery. Quantitative studies of beer foam stability indicate
that foaming behaviour is dependent on the interaction of various beer components,
among them proteins. Several groups of proteins and some specific proteins are
claimed to affect foam stability. These include high molecular weight proteins (> 5
kD), hydrophobic proteins (Bamforth et al., 1993), the 40 kD protein or protein Z
(Yokoi et al, 1989) and LTP which is 10-12 kD in size (Srensen et al., 1993; Lusk et
al., 1995). Interactions between proteins may also be relevant (Douma et al., 1997).
Several methods for determining the adherence of beer foam to the glass have been
described. Ziliotto et al. (1964) used a turbidity meter to determine the quantity of
foam lacing. Klopper (1973) described an apparatus which measures the light
reflected by the foam adhering to the glass. Jackson and Bamforth (1982) collected
the foam adhering to the glass and determined the light absorption of this material at
230 nm and compared it with the absorption of the whole beer. Van Strien and
Wassenaar (1990) presented equipment with which cling is quantified by measuring
the infrared transmission of the foam adhering to the glass. Their system mimicked
the drinking behaviour of the consumer.
None of these methods are in routine use. The analysis takes too long or the
equipment seems to be too expensive for routine quality-control. Consequently,
relatively little information is available on foam lacing.
In this work the effect of several parameters on beer cling behaviour has been studied,
with a series of beers strongly differing in foam lacing, using Kloppers method and
equipment (Klopper, 1973). Some improvements to Kloppers method are proposed.

MATERIALS AND METHODS

Foam was produced with the Haffmans Inpack 2000 sampling device/foam flasher.
Foam stability values were determined with the NIBEM-T Foam Stability Tester from
the same firm. Immediately after the foam stability measurement, the quantity of cling
is measured essentially as described by Klopper (1973). Black screens were used to
minimise interference by ambient light. The amount of cling is assayed by quantifying
the amount of foam which is left behind on the glass after the foam head has
collapsed. This is performed by measuring the reflection caused by the foam on the
glass surface. Calibration for 100 % cling was performed using a glass covered with
white paper. Measurements of foam stability and cling were performed in
quadruplicate at room temperature. The standard deviation for the cling measurement
was 5.6.
Hydrophobic proteins were quantified as described by Bamforth et al. (1993), high
molecular weight proteins by the method of Bradford (1976) and specific beer
proteins as described by Douma et al. (1997). Dynamic surface rheological
measurements were carried out as described by Kokelaar et al. (1991).

3
RESULTS AND DISCUSSION
Foam stability and cling of the set of beers
Our study included both lager beers and special beers. The alcohol content of the
special beers ranged from 0 11 %, the CO2-content from 5.2 9.5 %. The series of
beers covered a broad range of foam stability and cling values. The variation in beer
foam stability was larger within the special beers than within the lager beers. The
special beers on average showed higher cling values (table 1).

Table 1: Foam stability and cling values of the beers

Studied

Range of values Range of values

for foam stability for foam cling
(sec) (%)

Lager beers 240 321 16 67

(14 brands)

Special beers 205 367 35 84

(18 brands)

The relation between foam stability and cling

It was investigated whether foam cling could be predicted by the NIBEM foam
stability measurement. Across all the beers tested, both lager and special beers, no
clear relationship could be seen between foam stability and cling. This may be due to
the fact, that the group of beers differs in a number of respects, such as alcohol
content, bitterness and CO2-content. When the analysis was restricted to the lager type
beers, which are more similar in composition, the beers with the higher foam stability
values also tended to have a higher foam cling (figure 1). However, it was not
possible to predict the foam cling values directly from the foam stability values.
Therefore foam stability and foam cling are separate foam quality characteristics, that
should be judged separately.

The influence of temperature on foam stability and cling

No significant effect of temperature on foam cling was observed. This is in contrast to
foam stability, which increases at lower temperatures. In figure 2 an example for one
of the brands is shown. Similar results were obtained for other brands. The effect of
temperature on beer foam stability has also been reported by van Akkeren and
Ansems (1999).

4
 100
90
80
foam adhesion (%)

70
60 la g e r
50
40 s p e c ia l
30
20
10
0
150 200 250 300 350 400
N ib e m fo a m (s e c )

Figure 1: The relation between cling and foam stability

500

400

300 foam
200 cling

100

0
4 9 15 20
beer temperature (C)

Figure 2: The influence of temperature on foam stability and

cling. Foam stability values in sec., cling values in %.

The effect of protein composition on cling

Protein components play an important role in beer foam stability. The total quantity of
high molecular weight protein and hydrophobic protein, the 40 kD protein (protein Z)
and a 10-12 kD protein (LTP) have been described to have a foam positive effect.
Therefore it might be expected, that an increase in the content of these specific
proteins or protein groups also results in an improvement of the extent of foam lacing.
It was investigated whether this was indeed the case. The measures of 40 kD protein
and 10 kD protein are not directly comparable with the measures of total and
hydrophobic protein, due to the differences in the measurement technique.

5
The results show that within the set of beers a substantial range of variation in the
content of certain specific proteins and protein classes occurs. This variation is clearly
greater in the special beers than in the lager beers (table 2).

Table 2: Content of specific proteins and protein classes of the beers studied

High molecular Hydrophobic 40 kD protein 10 kD protein

weight proteins protein (mg/l) (mg/l)
(mg/l) (mg/l)

Lager beers 141 316 46 79 84 - 234 1.6 5.8

Special beers 81 575 26 91 5 383 0 2.6

In order to test for a relationship between protein content and cling behaviour within
the lager beers, these beers were divided into a group of beers with a relatively high
foam lacing (values > 30) and a group of beers with a relatively low cling (values <
30).
The quantity of high molecular weight proteins appeared to be somewhat higher in the
group of beers with the higher cling values. Between the two groups of beers no clear
relation with the quantity of hydrophobic protein or the 10 and 40 kD protein could be
observed (table 3). Principal component analysis (PCA) could not detect any
combinations of protein factors that would explain the differences in observed cling
behaviour (data not shown).

Table 3: Average quantity of specific proteins and protein classes in lager beers
with cling values < 30 and > 30

High molecular Hydrophobic 40 kD protein 10 kD protein

weight proteins protein (mg/l) (mg/l)
(mg/l) (mg/l)

Cling 246 59 141 4.1

values
< 30

Cling 293 64 139 3.7

values
> 30

6
The results suggest that more factors than only proteins are of importance in the cling
behaviour of beer. The quantities of hop components were not determined in this
study. Bamforth and Jackson (1983) indicated that although hop substances play a
role in lacing, they do not account for the differences in lacing observed between
brands.
Factors negative for lacing may be important. The results may also have been
influenced by the possible presence of foam stabilizing agents.
Preliminary results indicate that physico-chemical parameters, such as surface tension,
the surface dilational modulus and the ratio between elastic and viscous behaviour
also do not appear suitable to predict foam lacing on their own (data not shown).

Suggestions for improvement of cling measurement

The advantage of the cling analysis method used in this study is that it allows rapid
measurement of cling behaviour, immediately after the NIBEM foam analysis.
Measuring cling in this way takes very little longer than a foam stability test.
The equipment described by Klopper (1973) is vulnerable to interference by ambient
light. In this study, this was minimised by enclosing the scanning unit with black
screens. With this kind of modification the instrument would provide a good basis for
a commercial cling meter. Special attention should be paid to:
- the stability of the light source with regard to the light intensity
- the centering of the glass during measurement
- standardization and simplification of the calibration procedure
- inclusion of a data processing mode

CONCLUSIONS
The amount of cling varies strongly from beer to beer. For lager beer, there is a
tendency for higher NIBEM foam stability values to result in higher cling values. For
special beers this could not be determined. Foam stability is strongly influenced by
temperature, whereas cling behaviour is hardly affected. Therefore, foam stability and
foam cling are independent quality parameters.
Cling behaviour cannot be attributed solely to specific proteins or classes of proteins.
Other beer components, either alone or in combination with proteins, may be
responsible for differences in foam lacing between beers.
The equipment used in this study for the analysis of foam lacing allows rapid
measurement of cling behaviour connected to foam stability analysis by the NIBEM
method. Suggestions for optimizing this apparatus are presented, which provide a
basis for commercialisation.

REFERENCES
1. Akkeren, F.J.J. van & Ansems, W.J.H., In: EBC Monograph 27, European
Brewery Convention Symposium Beer Foam Quality, October 1998, Amsterdam,
The Netherlands, ISBN: 3-418-00769-4, 1999, 48-61
2. Bamforth, C.W. & Jackson, G., Proceedings of the European Brewery
Convention Congress, London, 1983, 331-338

7
3. Bamforth, C.W., Canterranne, E., Chandley, P. & Ohnishi, A., Proceedings of the
European Brewery Convention Congress, Oslo, 1993, 331-3401
4. Bradford, M.M., Analytical biochemistry, 1976, 72, 248-254
5. Douma, A.C., Mocking-Bode, H.C.M., Kooijman, M., Stolzenbach, E., Orsel, R.,
Bekkers A.C.A.P.A. & Angelino, S.A.G.F., Proceedings of the European
Brewery Convention Congress, Maastricht, 1997, 671-679
6. Jackson, G. & Bamforth, C.W., Journal of the Institute of Brewing, 1982, 88,
378-381
7. Klopper, W.J., Proceedings of the European Brewery Convention Congress,
Salzburg, 1973, 363-371
8. Kokelaar, J.J., Prins, A. & de Gee, M., Journal of Colloid and Interface Science,
1991, 146, 507-511
9. Lusk, L.T., Goldstein, H. & Ryder, D., Independent role of beer proteins,
melanoidins and polysaccharides in foam formation. Journal of the American
Society of Brewing Chemists, 1995, 53, 93-103
10. Srensen, S.V., Bech, L.M., Muldbjerg, M., Beenfeldt, T. & Breddam, K.,
MBAA Technical Quarterly, 1993, 30, 136-145
11. Strien, J. van & Wassenaar, F.R., Monatsschrift fr Brauwissenschaft, 1990, 3,
106-111
12. Yokoi, S., Maeda, K., Xiao, R., Kamada, K. & Kamimura, M., Proceedings of the
European Brewery Convention Congress, Zurich, 1989, 593-600
13. Ziliotto, H.L., Bockelmann, J.B. & Tirado, W., Journal of the American Society
of Brewing Chemists, 1962, 77-80

8
70

Trihydroxyoctadecenoic acids having

negative effects on beer foam are
produced by enzymatic factors present
in malt
Hisao Kuroda, Naoyuki Kobayashi, Hirotaka Kaneda,
Masachika Takashio & Ken Shinotsuka
Sapporo Breweries Ltd., Brewing Research Laboratories, 10 Okatome, Yaizu, Shizuoka 425-
0013, Japan (e-mail: [email protected])

Descriptors
Enzymic activity, foam stability, lipoxygenase, malt constituent, oleic acid, peroxidase

SUMMARY
We have confirmed that trihydroxyoctadecenoic acids (THOD), oxidized products of linoleic
acid (LH) having a beer foam collapse activitiy, are produced by enzymatic factors present in
malt. Partially purified lipoxygenase1 transformed LH to THOD. Partially purified peroxidase
did not have this activity, however incubating both enzymes produced more THOD than
lipoxygenase 1 alone did. We speculate accumulation of THOD in the mashing is due to these
enzymes. It suggests importance of controlling not only lipoxygenase but peroxidase to
reduce THOD in beer. This finding would partly explain the practical experience that high-
temperature mashing-in results in improved beer foam.

Trihydroxyoctadecensuren mit negativem Einfluss auf den Bierschaum werden durch

enzymatische Faktoren im Malz gebildet

Deskriptoren
Enzymaktivitt, Lipoxygenase, Malzbestandteil, Oleinsure, Peroxidase,
Schaumbestndigkeit

ZUSAMMENFASSUNG
Wir haben besttigt, dass Trihydroxyoctadecensuren (THOD) und oxidierte Linolsure (LH)
einen negativen Einfluss auf die Schaumstabilitt haben. Sie werden durch enzymatische
Faktoren aus dem Malz erzeugt. Teilweise gereinigte Lipoxigenase 1 setzt LH zu THOD um.
Teilweise gereinigte Peroxidase zeigt eine nicht so starke Aktivitt. Dennoch produzieren
beide Enzyme zusammen mehr THOD als Lipoxigenase 1 alleine. Wir vermuten, dass sich
THOD whrend des Maischprozesses anreichert. Das deutet darauf hin, dass nicht nur auf die
Lipoxigenase, sondern auch auf Peroxidase im Hinblick auf die THOD-Reduzierung im Bier
zu achten ist. Diese Entdeckung wrde teilweise die praktische Erfahrung erklren, dass hohe
Einmaischtemperaturen den Bierschaum verbessern.
Des facteurs enzymatiques prsents dans le malt produisent des acides trihydroxy-
octadcnoques ayant des effets ngatifs sur la mousse de bire

Descripteurs
Acide olique, activit enzymatique, constituant du malt, lipoxygnase, peroxydase, stabilit
de la mousse

RESUME
Nous avons confirm que certains facteurs enzymatiques prsents dans le malt produisent des
acides trihydroxyoctadcnoques (THOD), produits d'oxydation de l'acide linolique (AL)
provoquant l'effondrement de la mousse de bire. La lipo-oxygnase 1 partiellement purifie a
transform l'AL en THOD. La peroxydase partiellement purifie n'a pas montr cette activit;
toutefois, l'incubation de ces deux enzymes a produit plus de THOD que la lipo-oxygnase 1
seule. Il est permis de supposer que l'accumulation de THOD dans le brassin est due ces
enzymes. Cela montre l'importance de contrler non seulement la lipo-oxygnase, mais
galement la peroxydase pour rduire les THOD dans la bire. Cette observation expliquerait
en partie ce fait d'exprience que le brassage haute temprature donne une meilleure mousse
la bire.

2
INTRODUCTION
Trihydroxyoctadecenoic acids (THOD) are the oxidized products of linoleic acid (LH)
which reduce the head retention of beer (9). Although they were already found in
beers in the early 1970s (3), where and how they are produced was not investigated
precisely. Recently, we have found that THOD were produced during mashing and
they survive in the final beer (5). Since the production of THOD decreased by raising
the mashing-in temperature, it was suggested that the production was due to some
enzymatic activities exist in the malt (6).
Malt has lipoxygenase (LOX) activity and it produces lipid hydroperoxides during
mashing (4). THOD are probably transformed from linoleic acid hydroperoxide
(LOOH), but the biochemical analysis of this reaction was insufficient. The fact that
the amount of THOD produced during mashing did not correlate to the LOX activity
of the malt implies the existence of other factors (6). Baur found protein fraction from
soybean containing LOX and peroxidase (POD) could transform LH into THOD (1).
Moll reported that the transformation of LOOH to THOD could be achieved using a
commercial product of horse radish POD (7). It is well known that malt has at least 10
active isozymes of POD, whose activities survive through kilning (2).
The aim of this study is to examine whether LOX and POD in malt could transform
LH to THOD. If they can catalyze this reaction, we might be able to improve beer
quality by repressing the activities of these enzymes during mashing or selecting malt
with less activities.

MATERIALS AND METHODS

Analysis of THOD producing activities of mash extract and gel-filtered extract
Seventy grams of malt meal was added to 200 ml of water prewarmed at 50 C and
stirred for 2 minutes. After chilling on ice, it was centrifuged at 10,000 g. The
supernatant was filtered through Miracloth (Calbiochem) and used as the mash
extract. The mash extract (5 ml) was subjected to a Hiprep Desalting column
(Amersham Pharmacia biotech) using 10 mM Tris-HCl (pH 7.2) as the eluent. The
mash extract (4.75 ml) or the void fraction of the mash extract (4.25 ml) mixed with
0.5 ml of 1 M sodium acetate buffer (pH 5.5) was incubated with 0.25 ml of 40 mM
LH dissolved in ethanol and incubated at 50 C for 20 minutes. The extraction and
determination of THOD was performed as described (5).

Enzyme assays
The LOX and POD activities were determined by the method of Yang (10) and
Clarkson (2), respectively.

Analysis of THOD producing activities of partially purified LOX and POD

One gram of malt meal was extracted with 10 ml of 0.1 M sodium acetate (pH 5.5),
0.1% Tween 20, 5 mM DTT, CompleteTM protease inhibitor (Roche) and cetrifuged at
15000 g for one hour. The supernatant was desalted as described above and subjected
to a column of Resource Q (Pharmacia) equilibrated with 10 mM Tris-HCl (pH 7.2),
then it was eluted with an NaCl gradient (0-1 M). LOX1 (fractions 40-44) and POD
(fractions 1-20) were added to 4.75 ml of 0.1 M sodium acetate buffer (pH 5.5) at a
final concentration of 0.8 U/ml and 11 U/ml, respectively. Incubation with LH and the
determination of THOD were performed as already described.

3
RESULTS
Mash extract transforms LH into THOD
The incubation of LH and the mash extract resulted in the accumulation of THOD; the
concentration of THOD in the reaction mixture increased from 9.5 ppm to 33 ppm.
Because the accumulation was not observed with the heat denatured mash extract
(boiled for 5 minutes), or without the incubation of LH, it is suggested that THOD
were produced by some enzymatic activities and they were derived from LH.
The mash extract was subjected to gel-filtration and a void fraction (M.W. > 5000)
was incubated with LH. This fraction was also able to produce THOD isomers in
proportions similar to those found in mashing (figure 1).

100 peak2
80 peak1
Figure 1. GC-MS analysis of products
Signal

60 transformed by gel-filtrated malt extract

40 (>M.W.5000). Peak 1 and 2 were identified
native
20
as 9,12,13- and 9,10,13- THOD,
heat denatured respectively.
23.8 23.9 24 24.1 24.2 24.3 24.4 24.5 min

Analysis of the THOD transforming activities of LOX and POD from malt
LOX and POD were partially purified in order to examine the activity to transform
LH into THOD. By subjecting the crude malt extract to Resource Q anion exchange
chromatography, these enzymes were successfully separated (figure 2).

Abs 280nm NaCl (%) POD LOX

1.4 2.5 45 1
40 0.9
1.2
2 35 0.8
LOX activity (U/mL)

POD activity (U/mL)

1 0.7
30
Abs 280nm

NaCl (M)

0.8 1.5 0.6

25
0.5
0.6 20
1 0.4
15 0.3
0.4
0.5 10 0.2
0.2 5 0.1
0 0 0 0
0 10 20 30 40 50 60 70
Fraction
Figure 2. Partial purification of LOX and POD

The major LOX activity in malt was LOX1, but some trace activity of LOX2 was also
observed. The trace amount of POD activity was observed for the fractions 37-39 and
40-42, however, more than 99.9% of the activity was localized in fractions 2-14.
Aliquots of LOX (fractions 40-44) and POD (fractions 2-14) were added to a buffer
solution at the concentration of residual activity observed during mashing and
incubated with LH. We expected that THOD would be obtained in the reaction
mixture incubated with both LOX and POD, however, THOD was produced by only

4
partially purified LOX1. The production of THOD was stimulated by incubation of
POD, while POD alone could not produce THOD. Although partially purified LOX
and POD could transform LH to THOD, the amount of THOD produced was only
10% compared to that observed in the reaction mixture with the mash extract and LH
(table 1).

Table 1. Production of THOD by partially purified LOX and POD

F40-44 (LOX) F1-20 (POD) Substrate THOD (ppm)
- - EtOH N.D.*
- - Linoleic acid N.D.*
- + EtOH N.D.*
- + Linoleic acid N.D.*
+ - EtOH N.D.*
+ - Linoleic acid 1.6
+ + EtOH N.D.*
+ + Linoleic acid 2.4
*Not detected.

Possible role of POD to produce THOD during saccharification step

Although we need careful examinations, it is suggested that POD has a stimulative
effect on the production of THOD. POD is a heat stable enzyme and its residual
activity is still found at the end of the saccharification step (figure 3). After heat
inactivation of LOX in the protein rest, LOOH still exists at the end of mashing and
THOD accumulates at a decreased rate (6). We speculate that the production of
THOD at the end of mashing is due to the transformation of LOOH by POD.

LOX POD Temp

Lionleic acid hydroperoxide (Ref. 4)
THOD (Ref. 6)
100 0.9 16

0.8 14
80 0.7 12
LOX (U/mL)

POD (U/mL)
Temp (C)

0.6
60 10
0.5
8
0.4
40 6
0.3
4
20 0.2

0.1 2

0 0 0
0 10 20 30 40 50 60 70
Time (min)
Figure 3. Residual acitivity of LOX and POD in mashing.
Concentrations of linoleic acid hydroperoxide and THOD were
presented arbitrarily according to the references indicated.

5
CONCLUSIONS
Present study shows the existence of enzymatic factors which transform LH to THOD
in malt. Because the transformation by LOX and POD was not as efficient as that of
the mash extract, some other enzymatic factors besides these enzymes may exist. It is
possible that fractions separated by Resource Q might contain this activity, which
could be the key enzymes for the production of THOD. We are now searching for
these fractions which can transform LOOH into THOD.
It was also shown that POD stimulated the accumulation of THOD. POD is
implicated in the color and haze development of the beer, however, their substrates
and products in mashing have not been identified. POD might produce THOD by
transforming LOOH during mashing, and it suggests the importance of controlling not
only LOX but POD to reduce the production of THOD. In fact, Narzi reported that
the foam quality of beer could be improved by raising the temperature of mashing-in
from 65 C to 70 C (8); LOX is usually inactivated almost completely at the both
temperatures. Further study on the transforming activity of purified malt peroxidases
will clarify this issue.
We hope our studies will contribute to the fundamentals of lipid oxidation and the
lipid-oxidative enzymes during mashing. In addition, utilizing the molecular breeding
techniques, it would be possible to develop novel barley cultivars with modified lipid-
oxidative enzymes, which could not be achieved in traditional breeding; the malt
produced from these varieties are expected to improve the foam quality or flavor
stability of the beer.

REFERENCES
1. Baur, C., Grosch, W., Wieser, H. & Jugel, H., Zeitschrift fr Lebensmittel-
Untersuchung und-Forschung, 1977, 164, 171-176.
2. Clarkson, S.P., Large, P.J. & Bamforth, C.W., Phytochemistry, 1992, 31, 743-
749.
3. Drost, B.W., van Eerde, P., Hoeksta, S.F. & Strating, J., Proceedings of the
European Brewery Convention Congress, Estoril, 1971, 451-457.
4. Kobayashi, N., Kaneda, H., Kano, Y. & Koshino, S., Journal of Fermentation
and Bioengineering, 1993, 76, 371-375.
5. Kobayashi, N., Kaneda, H., Kuroda, H., Kobayashi, M., Kurihara, T., Watari, J.
& Shinotsuka, K., Jounal of the Institue of Brewing, 2000, 106, 107-110.
6. Kobayashi, N., Kaneda, H., Kuroda, H., Watari, J., Kurihara, T. & Shinotsuka,
K., Jounal of Bioscience and Bioengineering, 2000, 90, 69-73.
7. Moll, C., Biermann, U. & Grosch, W., Journal of Agicultural and Food
Chemistry, 1979, 27, 239-243.
8. Narzi, L., Reicheneder E., Jogasuria, P., Eichhorn, P., Mayer, W. & Gommelt
T., Brauwelt international, 1990, II, 126-134.
9. Yabuuchi, S and Yamashita, H., Jounal of the Institue of Brewing, 1979, 85,
216-218.
10. Yang, G., Shwarz, P. B. & Vick, B. A., Cereal Chemistry, 1993, 70, 589-95.

6
71

Monitoring barley lipid transfer protein

levels in barley, malting and brewing
Lance T. Lusk, Henry Goldstein, Kelly Watts, Alfonso Navarro
& David Ryder
Miller Brewing Company, 3939 W. Highland Boulevard, Milwaukee, WI. 53208, U.S.A. (e-
mail: [email protected])

Descriptors
Barley constituent, foam stability, immunology, malting, protein

SUMMARY
Barley lipid transfer protein (LTP1) is an important foam stabilizing protein. Monoclonal
antibodies to native and denatured, foam-stabilizing LTP1 were prepared, epitope mapping
was performed, and enzyme-linked immunoassays were developed. The fate of LTP1 from
barley through malting and the brewhouse to the packaged beer was followed. Transformation
of LTP1 from its native structure to its denatured foam stabilizing form begins earlier in the
process than previously reported. The ability to quantify foam stabilizing components is a
prerequisite for developing malting and brewing programs in order to have a consistent,
pleasing head of foam.

Die berwachung des Gehaltes an Gerstenlipidtransferprotein in Gerste, beim Mlzen

und beim Brauen

Deskriptoren
Gerstenbestandteil, Immunologie, Malzherstellung, Protein, Schaumbestndigkeit

ZUSAMMENFASSUNG
Das Gerstenlipidtransferprotein (LTP1) ist ein fr die Schaumstabilitt wichtiges Protein. Es
wurden monoklonale Antikrper zu nativen und denaturierten, schaumstabilisierenden LTP1
hergestellt, eine Epitopmarkierung wurde eingerichtet und ein ELISA-Test wurde aufgebaut.
Die Entwicklung des LTP1 von der Gerste ber die Vermlzung und das Sudhaus bis hin zum
fertig verpackten Bier konnte so verfolgt werden. Die Vernderung des LTP1 von seiner
nativen Form in seine denaturierte, schaumstabilisierende Form erfolgt im Herstellungs-
prozess frher als bisher angenommen. Die Mglichkeit der Quantifizierung der schaum-
stabilisierenden Komponenten ist eine Voraussetzung fr die Entwicklung von Mlzungs- und
Brauprogrammen, die eine angenehme stabile Blume auf das sptere Bier bringen.
Surveillance des taux de LTP d'orge pendant le maltage et le brassage

Descripteurs
Constituant de l'orge, immunologie, maltage, protine, stabilit de la mousse

RESUME
La protine de transfert des lipides (LTP1) d'orge est une importante protine de stabilisation
de la mousse. Nous avons prpar des anticorps monoclonaux contre la LTP1 native et
dnature, effectu une cartographie des pitopes et mis au point des tests ELISA. Le devenir
de la LTP1 d'orge lors du maltage et du brassage jusqu' la bire emballe a t suivi. Le
passage de la LTP1 de sa forme native sa forme dnature (qui est celle qui stabilise la
mousse) commence plus tt qu'on ne le pensait jusqu'ici. La capacit de quantifier les
composants stabilisateurs de mousse est une condition pralable au dveloppement de
programmes de maltage et de brassage pour avoir une tte de mousse homogne et agrable.

2
INTRODUCTION
Barley lipid transfer protein (LTP1) is an important foam protein. However, no single
component is responsible for foam stability, and interactions of components are
important and poorly understood. Other macromolecules associated with beer foam
formation and stability are protein Z (9), a 17,000 Dalton -1 hordein (14),
melanoidins, and polysaccharides (11).

Sorensen et al. (13) and Bech et al. (2) showed that LTP1 contributes to foam
formation. Using polyclonal antibodies, Bech et al. showed that throughout the
brewing process lipid transfer protein was transformed from the native barley form to
the denatured foam promoting form (2). Lusk et al. showed that the denatured LTP1
(foam-LTP) is a foam-stabilizing beer protein especially in the presence of
isohumulone (11,12). Jgou discovered that there are two forms of barley LTP1 (10).
Modifications of both forms occur during the brewing process. Evans and co-
investigators used polyclonal antibodies against LTP1, barley proteins Z4, and Z7 to
examine these proteins in germination, kilning and brewing (3,4,5,6). Protein Z4 was
positively correlated with foam stability but Z7 and LTP1 were not (5). Contrarily,
Evans et al. (6) showed that the LTP antibodies reacted with drained beer foam, but
protein Z antibodies did not react with drained beer foam.

The objectives of the current research were to develop immunological methodologies

based on monoclonal antibodies for analyzing lipid transfer protein and use the
methods to probe the brewing process. Furthermore, these procedures will help with
barley breeding, brewing raw material selection, and optimization of malting and
brewing regimes to unravel some of the interactions mentioned above.

MATERIALS AND METHODS

Lipid Transfer Protein isolation
LTP1 was isolated from barley following the procedure of Sorensen et al. (13) except
that a final preparative reverse-phase HPLC step was added due to an inability to
achieve the degree of purification previously described. RP-HPLC was performed
using a Zorbax (Wilmington, DE) PRO-10/300 Protein Plus C3 column. The sample
was eluted using a gradient of 10-50% acetonitrile with a background of 0.1%
trifluoroacetic acid. Foam-LTP was isolated using the foam fractionation and column
chromatography procedures described by Lusk et al. (11). Protein Z was obtained
from this same separation. Concentrations of purified LTP1 and foam-LTP were
determined by amino acid analysis.

Monoclonal antibody production, characterization and ELISA development

Monoclonal antibodies were prepared by ProtoPROBE (Milwaukee, WI). LTP1
elicited an antigenic response in balb/c mice without conjugation. Foam-LTP
conjugated with ovalbumin elicited an antigenic response, but no antigenic response
was elicited with unconjugated foam-LTP or with foam-LTP conjugated to itself (1).
Incomplete Freund's adjuvant was used to prime mice for ascites production. Four
weeks after the initial immunization spleen cells were harvested and fused with a
myeloma cell line (ATCC CRL 1581 Sp2/0-Ag14). Successful fusion products were
screened for immunoreactivity and cross-reactivity with the appropriate proteins. Cell
lines with high affinity binding were subcloned and screened for the production of

3
monoclonal antibodies. Plate wells (Costar, Corning, NY) were coated with antigen
and incubated overnight at 4C. Subsequent steps were performed at room
temperature. Plates were blocked with 10% nonfat powdered milk (Carnation, Nestle
USA, Solon, OH) in Dulbeccos phosphate buffered saline (DPBS, Sigma, St.
Louis, MO). Antibody-containing cell culture media was added and the plates were
incubated for 1 hr. Plates were then sequentially developed with alkaline phosphatase
conjugated goat antibody to mouse IgG (Sigma, A5153) and Sigma 104 reagent.

Epitope mapping was performed on a select population of antibodies using

immobilized peptides (Chiron Technologies, MitoKor Inc., San Diego, CA) (7).
Peptide segments of 17 amino acids, each overlapping in the LTP1 sequence by five
residues, were obtained for the complete sequence of barley lipid transfer protein
(SWISS-PROT accession number P07597). Peptides recognized by the antibodies
were developed using peroxidase-conjugated goat anti-mouse IgG (H+L chain)
(Kirkegaard & Perry Laboratories, Gaithersburg MD) and 2,2-azinobis(3-
ethylbenzthiazoline-sulfonic acid) reagent (Sigma).

Sandwich enzyme linked immunosorbant assays (ELISA) were developed for the
detection and quantification of LTP1 and foam-LTP. Plate wells were coated with
12.5 g monoclonal capture antibody in 50 l carbonate buffer (100 mM, pH 8.3) and
incubated overnight at 4C. Subsequent steps were performed at room temperature.
Plates were blocked with 100 l 10% nonfat powdered milk in DPBS. Antigen (50 l)
was added and the plates were incubated for 2 hr. Conjugated alkaline phosphatase
detection antibody diluted in DPBS (50 l) was added and the plates were incubated
for 2 hr. Detection substrate, 100 l of BluePhos (Kirkegaard & Perry Laboratories),
was added and the plates were incubated in the dark for 1 hr. Absorbance at 650 nm
was measured with a SPECTRAmax PLUS plate reader, and calculations were
performed using SOFTmax PRO version 3.0 software (Molecular Devices,
Sunnyvale, CA).

Sample extraction and preparation

Robust barley was malted in a micro-malting unit (Joe White Malting Systems,
Victoria, Australia). Samples were collected throughout the malting process: barley;
end of steeping; germination at 24 hr, 48 hr, 72 hr, 96 hr, 120 hr, and 144 hr; and end
of kilning. Barley and green malt samples were lyophilized for 48 hr and then ground
in a cyclone mill (UDY Corp., Ft. Collins, CO) equipped with a 0.5 mm screen.
Extracts were prepared from 50 mg samples in 1 ml of 0.05 M sodium phosphate
buffer, pH 6.0. Samples were mixed intermittently for 2 hr with the aid of a vortex
mixer, followed by centrifugation at 15000 x g for 10 min. Grain samples for LTP1
were diluted 1:50 in DPBS; grain samples for foam-LTP were not diluted. Beer
samples for foam-LTP were mixed with 1M Tris-HCL, pH 9, to achieve a pH of 7-8.

RESULTS
LTP1 and foam-LTP analysis
N-terminus amino acid sequencing (16 residues) and SDS-polyacrylamide gel
electrophoresis showed that barley LTP1 had been purified. Characterization of
purified foam-LTP was previously described (11). Circular dichroism results
demonstrated that LTP1 had the expected alpha helical structure with minima at 208
nm and 222 nm (10). The circular dichroism spectrum of foam-LTP was typical of a

4
denatured protein (figure 1). Both proteins were analyzed at 0.1 mg/ml in water in a
0.1 cm cell using a spectrapolarimeter at room temperature (Jasco J-720, Tokyo,
Japan). The molecular weight determined by matrix-assisted laser desorption mass
spectrometry (9,992 Daltons) suggests that the isolated LTP1 was the LTP1b isoform.
LTP1b may be an LTP1 isoform or post-translationally modified LTP1(10).

Figure 1: Circular dichroism spectra of LTP1 (solid line) and foam-LTP (dashed
line).

Specificity of LTP1 and foam-LTP monoclonal antibodies

Foam-LTP and LTP1 reacted quite specifically with the monoclonal antibodies (tables
1 & 2). The monoclonal antibodies did not react with protein Z isolated from beer or
with an irrelevant negative control protein.

Foam-LTP clones LTP1 Foam- Protein Z Negative control

LTP
1H2.1 0.841 1.414 0.396 0.245
2C1.1 0.702 1.684 0.233 0.096
2E3.1 0.130 1.560 0.450 0.073
3D1.1 0.495 1.543 0.162 0.099
3D11.1 0.186 0.429 0.176 0.146
Table 1: Monoclonal antibodies toward foam-LTP. ELISA 405 nm absorbance values.

LTP1 clones LTP1 foam-LTP Protein Z Negative control

1A3.1 1.116 0.261 0.242 0.169
1D4.1 1.068 0.144 0.237 0.219
1F3.1 1.698 0.195 0.208 0.140
2C12.1 1.432 0.192 0.173 0.201
3A11.1 1.476 0.142 0.140 0.111
3F7.1 1.544 0.162 0.187 0.203
3G1.1 1.018 0.245 0.274 0.165
Table 2: Monoclonal antibodies toward LTP1. ELISA 405 nm absorbance values.

5
Epitope maps
Monoclonal antibodies recognize specific peptide segments of proteins or specific
three-dimensional structures. Epitope mapping was performed for the purpose of
finding two monoclonal antibodies to each protein that reacted to different parts of the
protein molecule. Monoclonal antibodies were identified that contained epitopes to
either the N- or the C- terminus of foam-LTP. Monoclonal antibody, 3D1.1,
recognized the N-terminus of foam-LTP and monoclonal antibody, 2E3.1, recognized
the C-terminus (figure 2). Antibody 3D1.1 was chosen as the capture antibody for
ELISA development.

Figure 2: Epitope maps of monoclonal antibodies exhibiting specificity toward the

N-terminus (3D1.1; white bars) or the C-terminus (2E3.1; black bars)
peptide segments of foam-LTP.

Only one monoclonal antibody that exhibited specificity toward LTP1 yielded a well-
defined linear epitope map. The epitope map for monoclonal antibody 3G1.1 (figure
3) was well defined as a linear sequence of peptides encompassing the B helix of
the LTP1 three-dimensional structure (8). Antibody 3G1.1 was chosen as the capture
antibody for ELISA development. The other six monoclonal antibodies to LTP1 had
profiles with low absorbance values. Immobilized peptides are linear peptides and
these monoclonal antibodies to LTP1 may be specific to a three-dimensional structure
that includes more than one peptide segment. Mapping techniques for non-linear
epitopes are not well developed, and this type of mapping was not attempted.

Alkaline phosphatase conjugated monoclonal antibodies

Alkaline phosphatase conjugated antibodies to foam-LTP and LTP1 were challenged
with both foam-LTP and LTP1. The monoclonal antibodies are specific to the form of
lipid transfer protein to which they were raised (table 3). Alkaline phosphatase
conjugated antibody 2E3.1 became the detection antibody for the foam-LTP ELISA,
and alkaline phosphatase conjugated antibody 2C12.1 became the detection antibody
for the LTP1 ELISA.

6
ELISA standard curves
The standard curves for foam-LTP and LTP1 had sigmoidal shapes when protein
concentration was plotted vs. absorbance on semi-log axes. Log-logit fits were used
for quantification. The useful range for LTP1 quantification was from 2 g/ml to 8
g/ml. The useful range for foam-LTP quantification was from 6 g/ml to 60 g/ml.

Figure 3: Epitope maps of two monoclonal antibodies exhibiting specificity toward

LTP1. Monoclonal antibody 3G1.1 (white bars) yielded a well-defined
linear epitope toward LTP1. 2C12.1 (black bars) typifies antibodies
exhibiting nonspecific or nonlinear epitope maps.

Alkaline phosphatase (AP) conjugated foam- LTP1 Negative

antibody LTP Control
2E3.1 AP-foam-LTP >2 0.103 0.095
3D1.1 AP-foam-LTP 0.574 0.062 0.057
2C12.1 AP-LTP1 0.069 1.359 0.059
3G1.1 AP-LTP1 0.144 1.635 0.105
Goat Anti-rabbit IgG AP conjugated 0.092 0.085 0.101
antibody

Table 3: Alkaline phosphatase conjugated antibodies showed selective reactivity to

either LTP1 or foam-LTP. ELISA 405 nm absorbance values.

Barley and malt analysis

LTP1 concentrations were examined in malt prepared from thirteen barley varieties.
Malted barley LTP1 levels ranged from 2.3 mg/g to 4.1 mg/g (table 4). Evans and
Hejgaard (4) reported values of 0.53 mg/g to 0.97 mg/g for 42 samples from different
malting barley varieties. Evans and Hejgaard analyzed barley rather than malt, and
they did not report reducing water content by lyophilization as performed in the
current work. Both factors could influence LTP1 levels.

The concentration of LTP was monitored throughout the malting process (figure 4).
The malting cycle was extended for an extra 48 hours to study the effects of extended

7
germination. There was little increase in LTP1 concentration during malting, in
agreement with Evans and Hejgaard (4) and Bech et al. (2). A slight increase in LTP1
concentration was noted at the end of kilning. Contrarily, Evans and Hejgaard
reported a decrease in LTP1 level due to kilning (4). A trace of foam-LTP was
detected in barley, which may represent lipid transfer protein that the cell has not
completely processed or that is improperly folded. An increase in foam-LTP
concentration was observed at 144 hr of germination. This increase was followed by
an additional increase at the end of kilning. The increase in foam-LTP at the end of
kilning is most likely due to the heat during kilning. LTP1 denaturation during kilning
has not been previously reported.

Malt mg/g Malt mg/g

A 2.3 H 3.0
B 2.4 I 3.0
C 2.6 J 3.3
D 2.8 K 3.5
E 2.8 L 3.7
F 2.8 M 4.1
G 2.9

Table 4: LTP1 levels in lyophilized malted barley.

Figure 4: LTP levels during an extended germination cycle. LTP1 levels (bars) are
quantified as mg/g and foam-LTP levels (diamonds) are quantified as g/g.

Beer analysis
LTP levels were examined during commercial-scale brewing of a Pilsner-style beer
(figure 5). The brewhouse vessel size was 590 hl and the fermentation vessel size was
5900 hl (filled with 10 brews). The LTP1 concentration at kettle full was less than in
the mash, which is a result of dilution from lautering. More than 50% of the LTP1
that was soluble in the wort was converted to foam-LTP. A low level of foam-LTP
was observed at mash-in. This is in agreement with the observation that foam-LTP is

8
detected in malt. Most of the foam-LTP was generated during the kettle boil, in
agreement with Bech et al. (2).

The foam-LTP concentration in the fermentation vessel was lower than that of wort
(figure 6). As we only followed one of the ten brews used to fill the 5900 hl
fermentation vessel, it could happen that the other brews contained lower
concentrations of foam-LTP. Also additional losses may occur during the settling and
cooling of the boiled wort, and some dilution takes place with the addition of the
liquid adjunct and the yeast. The foam-LTP level on the first day of fermentation was
higher than on subsequent days, which may indicate that foam-LTP is lost in the foam
head that forms during fermentation. The final decrease in foam-LTP level was at
finishing which is the result of diluting the high gravity brew to package release
strength.

Figure 5: LTP1 (open bars) and foam-LTP (black bars) in the brewhouse.

Figure 6: Foam-LTP levels during fermentation and finishing.

9
CONCLUSIONS
Monoclonal antibodies were prepared that are specific to the native structure of barley
lipid transfer protein (LTP1) and to the denatured conformation of barley lipid
transfer protein as found in beer foam (foam-LTP). Unique monoclonal antibodies to
foam-LTP were identified that recognize epitopes to either the N-terminus or C-
terminus of the protein. Most monoclonal antibodies to LTP1 apparently recognize
noncontiguous regions of the LTP1 three-dimensional structure.

LTP1 levels were stable during malting. Low levels of foam-LTP were detected in
barley and during malting, and there was an increase in the level of foam-LTP during
kilning. The largest conversion of LTP1 to foam-LTP occurred in the kettle. Foam-
LTP levels decreased during fermentation. Foam-LTP levels may become predictable
based upon malt LTP1 analysis once the transformation and losses in brewing are
better understood.

REFERENCES
1. Avrameas, S., Immunochemistry, 1969, 6(1), 43-52.
2. Bech, L., Vaag, P., Heinemann, B. and Breddam, K., Proceedings of the
European Brewery Convention Congress, Brussels, 1995, 561-568.
3. Evans, D.E, MacLeod, L.C. and Lance, R.C.M., Proceedings of the European
Brewery Convention Congress, Brussels, 1995, 225-232.
4. Evans, D.E. and Hejgaard, J., The Journal of the Institute of Brewing, 1999,
105(3), 159-169.
5. Evans, D.E., Sheehan, M.C. and Stewart, D.C., The Journal of the Institute of
Brewing, 1999, 105(2), 171-177.
6. Evans, D.E., Sheehan, M. and Logue, S.J., Australian. Barley Technical
Symposium, 1997, 5 pages.
7. Geysen, H.M., Rodda, S.J., Mason, T.J., Tribbick, G. and Schoofs, P.G., Journal
of Immunology Methods, 1987, 102, 259-274.
8. Heinemann, B., Andersen, K.V., Nielsen, P.R., Bech, L.M and Poulsen, F.M.,
Protein Science, 1996, 5(1), 13-23.
9. Hejgaard, J. and Kaersgaard, P., The Journal of the Institute of Brewing, 1983,
89, 402-410.
10. Jegou S., Douliez J.P., Molle D., Boivin P. and Marion D., Journal of
Agriculture and Food Chemistry, 2000, 48(10), 5023-5029.
11. Lusk, L. T., Goldstein, H. and Ryder, D., Journal of the American Society of
Brewing Chemists, 1995, 53(3), 93-103.
12. Lusk, L., Ting, P., Goldstein, H., Ryder, D. and Navarro, A., European Brewery
Convention Beer Foam Quality Symposium, Amsterdam, 1998, 166-187.
13. Sorensen, S.B., Bech, L.M., Muldbjerg, M., Beenfeldt, T. and Breddam, K.
Technical Quarterly of the Master Brewers Association of the Americas, 1993,
30,136-145.
14. Vaag, P., Bech, L.M., Cameron-Mills, V., and Svendsen, I., Proceedings of the
European Brewery Convention Congress, Cannes, 1999, 157-166.

10
72

Better analytical techniques for beer

and brewing raw materials analysis
F. Richard Sharpe
Brewing Research International, Lyttel Hall, Nutfield, Surrey RH1 4HY, United Kingdom (e-
mail: [email protected])

Descriptors
Analysis method, antioxidant, extract yield, extraction, homogeneity, malt modification,
sulphur compound

SUMMARY
Through studies on raw material and beer quality BRi have developed numerous new
analytical techniques. Four methodologies are now described. Beer Fingerprinting uses novel
extraction methods followed by gas chromatography coupled olfactometry. Malt Assessment
measures homogeneity in 10 minutes, and is being evaluated for its ability to predict degree of
modification and extract yield. Sulphur Volatile Fingerprinting looks at some 20 medium to
non-volatile sulphur components ranging from dimethyl sulphide to thiazole and the
Antioxidant Tests assess individual as well as collective antioxidants in malt,wort and beer.
Robustness, accuracy, repeatability and cost of analysis are reviewed in each case.

Bessere Analysentechniken fr die Bier- und Rohstoffanalyse

Deskriptoren
Analysemethode, Antioxidantium, Extraktausbeute, Extraktion, Homogenitt, Malzlsung,
Schwefelverbindung

ZUSAMMENFASSUNG
Durch die Untersuchung von Rohstoffen und Bier hat BRi zahlreiche neue Analysetechniken
entwickelt. Vier Methoden werden beschrieben. Fr einen Bier-Fingerabdruck wird eine
ungewhnliche Extraktionsmethode, gefolgt von einer olfaktometrischen Gas-
chromatographie, verwendet. Eine neuartige Malzbestimmung misst die Homogenitt in 10
Minuten und ermglicht eine Vorhersage des Lsungsgrades und des Extraktgehalts. Mit
einem Fingerabdruck der flchtigen Schwefelverbindungen werden zirka 20 mittel- bis
nichtflchtige Schwefelkomponenten charakterisiert, die von Dimethylsulfid bis zu Thiazolen
reichen. Ein Antioxidantien-Test ermittelt sowohl einzelne Antioxidantien, als auch die
Gesamtmenge der Antioxidantien im Malz, der Wrze und im Bier. Die Widerstands-
fhigkeit, Genauigkeit, Wiederholbarkeit und die Kosten der Analysen werden bei jedem
Verfahren diskutiert.
De meilleures techniques analytiques pour l'analyse des bires et matires premires de
brassage

Descripteurs
Antioxydant, compos soufr, dsagrgation du malt, extraction, homognit, mthode
d'analyse, rendement en extrait

RESUME
Au moyen d'tudes sur la qualit des matires premires et de la bire, BRi a dvelopp
plusieurs techniques analytiques. Quatre mthodes sont maintenant dcrites. La technique des
empreintes de bire (beer fingerprinting) met en oeuvre de nouvelles mthodes d'extraction
suivie d'une olfactomtrie couple la chromatographie gazeuse. L'valuation du malt mesure
l'homognit en 10 minutes et fait l'objet d'tudes cherchant valuer la capacit prvoir le
degr de modification et le rendement d'extraction. L'empreinte du soufre volatil tudie
quelque 20 milieux de composs soufrs non volatiles, allant du sulfure de dimthyle aux
thiazoles. Le test des antioxydants value les antioxydants individuels et collectifs dans le
malt, le mot et la bire. La robustesse, l'exactitude, la rptabilit et le cot des analyses sont
valus dans chaque cas.

2
INTRODUCTION
New analytical techniques are being developed all the time and at the Brewing
Research International (BRi) there are now four new techniques available. All these
techniques focus on product quality assessment and they are described here with
detail on the equipment required, the time taken for the test, its robustness and
repeatability.

BEER AROMA FINGERPRINTING

This technique can fingerprint the aroma of a beer brand and so characterize the
changes in aroma as a result of the use of different hops, malt or brewing processes.
The technique can also be used to investigate contaminated samples or to study
ageing. This powerful methodology utilizes the traditional separation technique of gas
chromatography (GC) and the unique human sniff recognition of aromas, which
combine to produce the GC-olfactometry technique.2 At BRi this has been linked with
a fingerspan (rheostat) tool which allows quantification of the aromas. The three
techniques are combined to produce an aroma fingerprint.

The aroma fingerprint could then be further characterized, if required, by GC and

mass spectrometry to investigate individual components or groups of associated
aromas e.g. hop-derived aromas or off aromas associated with ageing.

In this method aroma is first concentrated by an extraction technique, which has no

apparent effect on the product composition this technique can be applied to beer,
wort, hops, malt or other beverages or raw materials. The raw materials are extracted
following an infusion.

Extracts are evaluated using a GC-FID system, fitted with an olfactory port.
Chromatography is usually on 30 m DB wax columns of 0.32 mm diameter and
having 0.5 m film thickness. These are temperature programmed from 40C to
240C at 5C/min. Injection size is 1 l and run time is ca. 30 minutes.

Trained sniffers record their impressions of aroma by employing aroma descriptors

against time. At the same time the intensities of the aromas are recorded by using a
finger-span rheostat.

If further investigation is required the GC eluant can be split and channelled to a mass
spectrometer for specific component identification.

The GC-olfactometry technique has already been applied in five ways:

Brand Fingerprint here the brands gas chromatographic aroma profile is
produced with an overlay of the rheostat olfactory assessment. This is shown in
figure 1.
Brand Identity Fingerprint this builds on the brand fingerprint described above
by using mass spectrometry to identify the GC and olfactory observations.
Brand Benchmark Fingerprint again building on the brand fingerprint. This
compares the fingerprints from a number of different brands, which can then be
used to identify the characteristic differences in brands.

3
 1000 600000
Intensity
900
FID
500000
800

700
400000

600
Intensity/mV

FID/mV
500 300000

400

200000
300

200
100000

100

0 0
0.00 5.00 10.00 15.00 20.00 25.00 30.00
Retention time/minutes

Figure 1: Comparison of FID GC aroma print and rheostat olfactory assessment

of the aroma of a beer brand. The olfactory assessments are shown as
spikes falling to the x-axis.

Brand Contaminant Assessment this variation is ideal for troubleshooting an odd

aroma in a product. Very often an aroma does not show up on a standard GC-FID
assessment because it is present in too small a quantity. The human nose is
considerably more sensitive however; therefore a characteristic aroma can be
searched for in the olfactory analysis and then identified by mass spectrometry.
Brand Ageing Profile not all brands age at the same rate or in the same way and
the technique is ideally suited to identify some of the key changes in aroma
occurring in the first few weeks of ageing. These aromas are first detected in
traditional organoleptic assessment and can then be picked out using GC-
olfactometry and identified by mass spectrometry.

The methodology has also been applied to:

the setting of a brand aroma specification

assisting with new product development and product matching

assessing the effect of raw materials and/or packaging

optimising aroma from process changes

imparting particular flavour notes to a brand e.g. hop character

The robustness of the technique is as good as any GC investigation. The

reproducibility is taken into consideration by utilizing five assessors who are each
highly trained and have extensive experience of product flavour and aroma.
Repeatability does vary since this is a subjective assessment, however, the training of
the assessment team reduces variability to a minimum. The time taken to conduct the
assessment using a single assessor is 30 minutes. The technique is often repeated to
confirm the result.

4
ENDOSPERM QUALITY ASSESSMENT
Assessing the mealy and steely character of barley is a long established practice for
choosing barleys suitable for producing quality malt. Assessing malt for its
homogeneous modification is particularly helpful in predicting performance in
process.3
Both assessments rely on an endosperm evaluation, which has traditionally been
determined by a Farinator, a simple tool used to cut the grain prior to evaluation of the
endosperm quality by eye. Typical Farinated sections of grain are shown in figure 2.
Mealy endosperm

Mealy and steely

STEELY
Steely

Figure 2: Farinated sections of grain showing mealy and steely endosperm.

Farination relies on sectioning the grain in one place and making a personal visual
assessment. It is open to error especially if the grain is cut in a single plane when the
true character of the grain can be misinterpreted. This is more clearly shown in figure
3.

Mealy
area

Steely grain ?

Mealy
area

Mealy grain ?

Figure 3: Assessment of grain by Farination.

5
BRi have found that it is important to assess the whole grain and have devised a
technique which results in light being passed through a grain sample whereby the
mealy grains absorb the light and are seen as dark and the steely grains transflect the
light and are seen as opaque.

BRi have used this principle to develop a simple instrument, the light transflectance
meter (LTm), to assess barley and malt and other cereals for mealiness and steeliness
and degree of sample homogeneity.

Cereal grains are placed in a rotating carousel and light is passed through the whole
grain. A measure of the light transflected is recorded for 97 grains and compared to
reference standards.

The result is an assessment of grain transflectance, which enables evaluation of the

percentage mealiness and degree of homogeneity of a sample to be calculated with
remarkable accuracy. An example of the tests printout is shown in figure 4.

1.0
Chariot Epic Fanfare
Light transflectance value

0.8

0.6

0.4

0.2

0.0
1 21 41 61 81

Number of grains

Figure 4: LTm assessment of barley samples.

The test requires no sample preparation, takes approximately 12 minutes and is semi-
automatic. Data handling is easy through a Microsoft Excel programme and no
subjective interpretation is required. The test is non-invasive and gives robust and
reproducible results. It is essentially a measure of % mealiness/steeliness in barley
and malt and correlates with the Carlsberg Calcofluor modification test. Homogeneity
of the barley and malt sample can be assessed and it is also possible to detect
contamination by chit malt above the 5% level. The test is being developed to assist in
the prediction of brewhouse performance.
The instrument design is robust and repeatability is less than 5% LTm value for both
barley and malt.

6
The test is available for evaluation through BRi and at present it is undergoing
commercialisation. A few machines are available for hire and sale.

SULPHUR FINGERPRINTING
Sulphur compounds are an integral part of beer flavour. Some of them are essential to
create a balanced product; others are often regarded as detrimental. BRi has
developed inert and sensitive extraction processes which, combined with selective
separation and detection, can monitor most of the highly volatile compounds
(hydrogen sulphide, DMS, methanethiol etc.) as well as the less volatile compounds
such as the thiazoles and thiophenes.

The assessment can help with setting of brand specifications, product matching, new
product development, assessing the effect of raw materials, monitoring process
changes, tracking product ageing, selecting hop aroma products and the processes
used to impart specific aromas, assessing the effect of some packaging materials,
troubleshooting odd sulphury flavours, and tracking the creation and disappearance of
sulphur volatiles during beer production.

Beer is extracted using an inert halocarbon solvent which qualitatively and

quantitatively dissolves a majority of the sulphur species present in the beer. The
extract, including an internal standard, is then concentrated by evaporation before
being taken up into hexane ready for analysis. The extract so produced is evaluated
using GC coupled to a Sievers detector. The basic analysis evaluates eleven
compounds and the full spectrum analysis offers 18 assessments. Many other sulphur
species can be observed in the fingerprint and, while these cannot yet be identified
with certainty, it is possible to analyse a total sulphur screen in order to plot the
overall flavour or change in flavour that occurs with time. It is interesting to note that
during ageing some sulphur volatiles decline quite rapidly while others are relatively
constant.

As a further example of the use of the technique, the malt-derived and hop-derived
organosulphur components in a beer can be monitored through the process. Figure 5
illustrates how the technique has been used to identify where the majority of the
sulphury character is derived and what influences processing has on the sulphur
content.

This technique is looking at very small traces of component composition and like
most GC techniques has a high degree of variability. Repeatability of analysis is of
the order of +/- 15% concentration. The extraction and analysis takes 3 hours to
perform.

7
 3

Hop-derived (16.2)
2.5
Malt-derived (11.4)
Peak area ratio

1.5

0.5

er
2

9
g

er
l

ol

er
oi

oi
or

or
n

po

ay

ilt
lt

be
-b

-b
hi

da

da

fi

-f
d
l
as

st
re

e-
ir

al
g

st
n

on

on
po

in
m

pr

n
o

po
-w

fi
ch

ti

ti

ti
ta

ta

ta
st

it

en

en

en
p
po

rm

rm

rm
fe

fe

fe

Figure 5: Origin and fate a malt and hop derived organosulphur compound
with retention times, in minutes, shown in brackets.

ANTIOXIDANTS
Antioxidants protect against free radical mediated oxidative damage and can
potentially reduce the adverse effects of oxygen, which include the development of
stale flavours during beer storage. Many antioxidants are inherent in brewing raw
materials (e.g. reduced proteins and polyphenols from the hops and malt), whereas
others are formed during processing (e.g. melanoidins produced during kilning of
malt). These compounds provide protection from oxidation during the brewing
process, and also during storage of the beer. Knowledge of the type and level of
antioxidants present can influence the choice of raw materials used. Similarly, the fate
of these antioxidants during malting, hop processing and brewing can give useful
insight into the optimisation of antioxidant yield.

BRi uses two antioxidant tests. Total activity is quantified using the ABTS radical
cation decolourisation assay.1 Specific antioxidant species can be identified and their
relative importance as antioxidants estimated using the post-column
chemiluminescence HPLC assay.4 In both techniques raw materials, beer and other
drinks, e.g. wine, can be assayed. 150 grams of raw materials and/or a 100 ml of
liquid are required (to allow for repeatability of data).

The beers, or other drinks, are used directly, though dilution may be necessary for
samples containing high concentrations of antioxidants. Raw materials are extracted
into an acetate buffer solution. The ABTS method measures the ability of a sample to
quench the in-situ production of a radical cation chromophore using UV
spectrophotometry. This gives an insight into the total concentration of antioxidant
species within the sample.

8
The HPLC method separates antioxidants on a C18 reverse phase column. These
compounds then enter a reaction chamber where their effectiveness in quenching a
radical-mediated post-column chemiluminescent reaction can be evaluated. The
separate species can be identified by comparison with standards.

The tests can be used to evaluate total antioxidants in malt samples. Figure 6 shows
how malt colour correlates with the total antioxidants in malt, and how the level
increases to a maximum around 400 EBC colour.

12
Total Antioxidant Activity (mM)

10

0
400 800 1200 1600
ColourEBC

Figure 6: Total antioxidants versus malt colour.

Total antioxidants in beer can also be evaluated and the contributions from malt can
be identified. In this work at BRi, the malt analysed was then used to produce the
lager, which was subsequently analysed for antioxidant species. Many malt-derived
antioxidants were found in the lager. Additional antioxidants were either hop- or
process-derived. These antioxidants can then be followed throughout the beer ageing
process and their decrease in a reference beer over 52 weeks storage at 25C is shown
in figure 7 where the chemiluminescence HPLC assay has been used to make specific
identification.
The antioxidant assays are accurate in characterization of individual components
which can be determined within a coefficient of variation of less than 5%. The ABTS
assay takes ca 0.5 day to conduct and the chemiluminescent assay ca 1.5 days.

9
 30

0 w e e ks
C o n c e n tra tio n ( m g / l)

25
2 6 w e e ks

4 0 w e e ks
20

5 2 w e e ks
15

10

Total Ascorb ic acid Poly p he nolic Ca tech in Ep icatechin

an tioxid an t + V an illic acid m ateria l
con te nt

Figure 7: Change in specific beer antioxidants with age.

CONCLUSION
Four analyses have been presented all of which assist in evaluating the quality of beer
and brewing raw materials. All four tests are in routine use at BRi. The endosperm
evaluation is particularly useful for assessing barley samples prior to malting and the
homogeneity of the malt produced. The other three tests are exceptionally useful in
new product development, product and process analysis, flavour troubleshooting and
stability assessment.

ACKNOWLEDGMENT
Thanks go to all the BRi staff, and in particular Sachin Chandra, for their help in
developing these tests.

REFERENCES
1. Araki, S., Kimura, T., Shimizu, C., Furusho, S., Takashio, M. & Shinotsuka, K.,
The American Society of Brewing Chemists, 1999, 57 (1), 34-37.
2. Etievant, P.K., Journal of Agriculture Food and Chemistry, 1999, 47, 1673-1680.
3. Palmer, G., Brewing and Distilling International, 1997, 28 (3), 18-20 & 23.
4. Walters, M.T., Hughes, P.S. & Bamforth, C.W., Proceedings of 24th Convention
of the Institute of Brewing Asia Pacific Section, 1996, 103-107.

10
73

A new test for measuring endosperm

structure and quality
L.K. Wheaton, R.E. Muller, C.D. Booer, & S. Chandra
Brewing Research International, Lyttel Hall, Nutfield RH1 4HY, United Kingdom (e-mail:
[email protected])

Descriptors
Barley, homogeneity, malt modification, refractometry, quality analysis

SUMMARY
A new instrument, based on light transflectance, has been designed to measure quickly the
mealy/steely parameter of barley endosperm structure. Ninety-seven whole husked grains are
analysed in each test giving an indication of the homogeneity of the entire sample. Malt
samples have been assessed and values correlate with modification scores obtained from the
calcofluor test. Malt values also correlate with another new test developed at BRI which
measures ease of wort separation. The light transflectance method is quick, simple, non-
destructive and robust and could be used in a malting laboratory as an assessment of
endosperm quality and sample homogeneity.

Ein neuer Test zur Messung der Endospermstruktur und -qualitt

Deskriptoren
Gerste, Homogenitt, Malzlsung, Refraktometrie, Qualittskontrolle

ZUSAMMENFASSUNG
Ein neues Instrument, basierend auf Lichttransflektion, wurde darauf abgestimmt, den
Paramter mehlig/fest in der Gersten-Endospermstruktur schnell zu messen. In jedem Test
werden 97 vollstndig geschlte Krner analysiert und geben Aufschluss ber die
Homogenitt der untersuchten Proben. Malzproben wurden hergestellt und die Werte
korrelierten mit denen, die durch einen Calcofluortest erhalten wurden. Die Malzwerte
korrelierten ebenfalls mit denen eines anderen neuen am BRI entwickelten Tests, der die
Luterfhigkeit misst. Die Lichttransflektionsmethode ist schnell, robust, einfach,
zerstrungsfrei und knnte in einem Malzlabor zur Einschtzung der Endospermqualitt und
der Probenhomogenitt verwendet werden.
Nouveau test de mesure de la structure et de la qualit de l'endosperme

Descripteurs
Analyse de qualit, dsagrgation du malt, homognit, orge, rfractomtrie

RESUME
Un nouvel appareil, fond sur la transflectance de la lumire, a t conu pour mesurer
rapidement le paramtre farineux/vitreux de la structure de l'endosperme de l'orge. Quatre-
vingt-dix-sept grains d'orge entiers (avec balle) sont analyss dans chaque test, donnant une
indication de l'homognit de la totalit de l'chantillon. Des chantillons de malt ont t
valus et les valeurs sont corrles aux scores de modification obtenus par le test au
calcofluor. Les valeurs du malt sont galement corrles un autre nouveau test dvelopp
chez BRI qui mesure la facilit de sparation du mot. La mthode de transflectance de la
lumire est rapide, simple, non destructive et robuste, et elle peut tre utilise dans un
laboratoire de maltage comme mthode d'valuation de la qualit de l'endosperme et de
l'homognit de l'chantillon.

2
INTRODUCTION
Non-homogenous barley samples can result in problems during malting leading to
poor malt modification. This will also be reflected in the performance of the malt in
the brewhouse, resulting in poor extracts and high wort viscosities, as well as filtration
difficulties and hazes in the final product. Although there are many ways to assess
embryo quality there is no simple, robust, rapid and quantitative method which can
identify endosperm structure quality.

This paper introduces a new method (The LTm meter) which assesses both barley and
malt endosperm for mealiness and modification respectively and gives an indication
of the homogeneity of the sample. Also the relationship between the LTm meter
reading and another new method which measures wort separation is given.

The Transflectance principle

There are two different types of endosperm structures: mealy and steely. These
differences occur due to the packing of the endosperm material into the grain during
grain filling. The mealy endosperm (figure 1) is an open structure where starch
granules are loosely packed into a dense protein matrix. The steely endosperm (figure
2) consists of a tightly packed matrix of starch, protein and cell wall material. More
commonly there are mixtures of the two structures (2, 3).

Figure 1: SEM micrograph of a mealy Figure 2: SEM micrograph of a steely

endosperm endosperm

When light is passed through these types

of structures different effects are seen.
Figure 3 shows dehusked grains on a
light box where it is possible to see the
internal structure of the grains. Some of
the grains appear dark, others appear
light and translucent and some even
seem to possess the properties of both.
By careful selection of grains it is
possible to show that the lighter grains
are steely and the darker ones are mealy.

Figure 3: Dehusked grains on a light box

3
MATERIALS AND METHODS
The Light Transflectance meter (LTm)
This method relies on the quantitative measurement of the transmission of a laser light
through a barley grain. Dehusking is avoided as the laser is able to penetrate the husk.
Ninety seven husked grains are placed into the holes in a carousel (figure 4) and then
this is placed into the LTm meter (figure 5). The first position is automatically located
by the machine. This is an empty slot giving maximum light throughput (high LTm
value). Next is a neutral density filter giving a standard reading and lastly a blanked
cell resulting in no light being detected (low LTm value). These three readings supply
check values each time a sample is run. Each run takes six minutes.

Figure 4: Sample holder Figure 5: Light Transflectance meter

The amount of light passing through the endosperm is measured and this value
recorded to an Excel spreadsheet using EasyLog for Windows. The values are
then ordered by magnitude.

Malt modification
The Carlsberg Calcofluor stain method was used to assess malt modification (1).

Malting schedules
When comparing the LTm values with Calcofluor values, the BRI micro-malting
plant (4) was used to prepare malt samples. A sample of 350 grams of barley was
screened over a 2.2 mm sieve. A three steep schedule was used with times, 7, 17, 7,
17, 1 hrs alternating wet/dry. Kilning was for 8 hrs at 45C and 16 hrs at 65C.

When measuring LTm values of malts and comparing with Vmax values (see
Filtration cell) the malts were prepared as followed. Standard malt and chit malt,
prepared by germinating the standard variety for just one day, were produced using
the BRI 50 kg maltings. These were then mixed together in the proportions 5%, 10%
and 20% chit malt.

Filtration cell
The filtration cell was also developed at Brewing Research International. This
equipment measures the ease of wort separation by measuring flow rate and extract
volume of a thick laboratory mash in a standard time.

4
 Vmax (g) is the theoretical maximum weight of
extract obtainable and is calculated by measuring wort
run-off rate of a thick laboratory mash (liquor:grist
ratio of 3:1) in a pressurised cell.

Mashing conditions are isothermal at 64C for 1 hour.

Mash temperature is then raised to 78C and run-off
performed at this temperature. The Vmax value
produced is then compared to that of a malt of known
performance.

In general, high Vmax values indicate a good

performance and low Vmax indicates poor run-off
performance.
Figure 6: BRI Filtration Cell

RESULTS AND DISCUSSION

Relationship between visual assessment of mealiness and LTm value of barley
After initial studies the limits of the LTm values for different endosperm structures
were determined. In general an LTm value below 200 mV indicates a mealy
endosperm structure, above 400 mV a steely endosperm and between these two values
are endosperms with both mealy and steely characteristics. Individual LTm scores are
measured for all the ninety-seven grains which can be illustrated in a line graph, as
shown in figure 7. This graph indicates how the results from the LTm meter can
highlight differences between four different barley samples with varying degrees of
mealiness, the bottom two lines showing mealy samples and the top two lines
showing steely samples. An indication of the homogeneity of the sample is also given
by the slope of the line. The more homogeneous the sample the flatter the slope. For
example, sample B is more homogeneous than sample A.
1000

800
LTm value (mV)

600

steely
400

200
mealy

0
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97
number of grains

mealy A mealy B steely A steely B

Figure 7: Samples showing different levels of mealiness and homogeneity

5
Prior to the development of the LTm meter the only method to measure barley
endosperm was visual assessment of a transverse section. This method was not
quantitative, only one plane of the endosperm was available and results relied on
operator judgement.

A variety of barley samples were of varying malting quality were used to compare
visual assessment with LTm values. After measuring the endosperm structure using
the LTm meter, each grain was visually assessed by cutting it transversely and scoring
the structure for mealiness. Using datalogging techniques we were able to establish
the LTm value that corresponded to a particular grain and so could compare the two
values. A very good correlation between visual assessment and the barley LTm value
was found, as shown in figure 8.
steely
1.6

1.2
Barley LTm value (ln mean)

0.8

0.4
R2 = 0.9887

0.0
0 10 20 30 40 50 60 70 80 90

-0.4
mealy Visual assessment (% mealy)

Figure 8: Correlation between methods for measuring endosperm quality

Relationship between Malt LTm value and calcofluor modification

The principle of LTm meter is the measurement of light transmitted through a
structure. Variations in the density of the packing of the endosperm causes differences
in the amount of light that is scattered within the grain and therefore the amount of
light being detected. Barley grain is more densely packed than the corresponding malt
and so scatters less light. During malting, the grain becomes apparently more mealy
so the LTm meter can be used to assess the endosperm structure of malt as well as
barley (results not shown).

In addition to measuring cell wall modification as with the calcofluor method, LTm is
also sensitive to modification of small starch granules which are inherent in the steely
endosperm (figure 2).

A correlation between the malt LTm values and the corresponding calcofluor
modification value for that malt is shown in figure 9. LTm provides similar analysis to
Calcofluor but is far more rapid, requiring minimal sample preparation, and is cheaper
and non-destructive.

6
 100

90

Calcofluor modification (%)

80

70

60

2
R = 0.9586 50

40
-2.2 -2.0 -1.8 -1.6 -1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0
Malt LTm value (ln mean)
mealy steely

Figure 9: Relationship between Malt LTm value and Calcofluor modification

Relationship between admixture, filtration performance and malt LTm value

When a batch of well modified malt was mixed with varying proportions of a poorly
modified sample of the same variety the LTm meter was able to detect a difference
between the samples. A decrease in the %mealy value for the given variety was
noticed, as illustrated in figure 10. This suggests that for a given variety the LTm
meter is able to detect an admixture resulting in an increase in heterogeneity.

98

97
Malt LTm value (% mealy)

96

95

94

93 2
R =1

92
0 5 10 15 20
% chit malt

Figure 10: Detection of an admixture for a single variety

7
Heterogeneity can cause brewhouse problems. Another new instrument, the Filtration
cell, has also been developed at BRI which measures wort separation. The flow rate
and extract volume of a thick laboratory mash are measured which produces a Vmax
value. The higher the Vmax value the better the run-off.

Samples of varying modification were assessed by the LTm meter and using the
Filtration cell. A strong correlation was found between the % mealy values obtained
from the LTm meter and the Vmax values, as shown in figure 11.

98
2
R = 0.9693
97
Malt LTm value (% mealy)

96

95

94

93

92
10 12 14 16 18 20 22
Vmax (g)

Figure 11: Correlation between % mealy and Vmax value

CONCLUSIONS
Malting barley homogeneity is important for the production of high quality malt.
Commonly used methods for assessing endosperm structure and homogeneity rely on
operator judgement and some are time consuming. The LTm meter measures grain for
mealiness in barley and degree of modification in malt quickly and quantitatively. The
equipment can detect variations in homogeneity for a single variety and can also be
used in conjunction with other new method, the filtration cell, to assess filtration
performance.

The measurement of homogeneity and its relationship with filtration performance can
provide practical information for both maltsters and brewers.

Although all of the work described here is related to barley and malt quality, the
method is also able to reveal internal structure in wheat (results not shown).

8
REFERENCES
1. Aastrup, S., Gibbons, G.C. and Munck, L., Carlsberg Research Communications,
1981, 46 (1), 77-86.
2. Chandra, G.S., Proudlove, M.O. and Baxter, E.D., Journal of the Science of Food
and Agriculture, 1999, 79, 37-46.
3. Chandra, G.S., Wheaton, L.K., Schumacher, K. and Muller, R.E., Journal of the
Institute of Brewing, 2001, 107, 39-47.
4. Kelly, G.R., Brewing and Distilling International, 1987, 17 (12) 40-41.

ACKNOWLEDGEMENT
The authors would like to thank Mr G. Gasson and Mr S. Garland (BRI Specialist
Workshops) for the design and construction of both the LTm meter and the filtration
cell. The authors would also like to thank the Chief Executive Officer of BRI for
permission to publish. This project was part supported by the Home Grown Cereals
Authority, U.K.

9
74

Development of a biosensor allowing

reliable and fast dectection of
mycotoxins
Bram van der Gaag, Edwin Stigter, Sabine Spath, Sjaak van
Veen & Maarten Schans

TNO Nutrition and Food Research, Utrechtseweg 48, P.O. Box 360, 3700 AJ Zeist, the
Netherlands (e-mail: [email protected])

Descriptors
Barley quality, biological analysis method, malt quality, mycotoxin, quality analysis, sensor

SUMMARY
Product integrity is an important issue in the barley-beer chain. Therefore the quality of the
raw materials is carefully monitored. The occurrence of mycotoxins in barley and malt is a
point of increasing attention. Current methods to analyze these components are labour-
intensive, expensive and require specialist expertise. In this paper the development of a new
assay for mycotoxins is described, namely a biosensor-based inhibition assay, which has the
potential to detect multiple mycotoxins in a single measurement. The device, which is easy to
use and requires no highly qualified personnel, will result in a significant reduction in analysis
costs.

Entwicklung eines Biosensors fr den zuverlssigen und schnellen Nachweis von

Mykotoxinen

Deskriptoren
Gerstenqualitt, Malzqualitt, Methode zur biologischen Analyse, Mykotoxin,
Qualittskontrolle, Sensor

ZUSAMMENFASSUNG
Produktreinheit ist ein wichtiger Faktor in der Gerste-Bier-Kette. Folglich wird die Qualitt
der Rohstoffe sorgfltig berwacht. Das Auftreten der Mykotoxine in Gerste und Malz ist ein
Punkt zunehmender Aufmerksamkeit. Die aktuellen Methoden zum Analysieren dieser
Bestandteile sind arbeitsintensiv, kostspielig und bentigen Fachkenntnis. In diesem Poster
wird die Entwicklung einer neuen Analyse fr Mykotoxine, und zwar einer auf Biosensoren
basierenden Hemmung, beschrieben, die das Potenzial hat, mehrere Mykotoxine in einer
einzigen Messung zu ermitteln. Die Analyse, die einfach zu verwenden ist und kein hoch
qualifiziertes Personal bentigt, fhrt zu einer deutlichen Reduzierung der Analysekosten.
Dveloppement d'un biocapteur permettant la dtection fiable et rapide des mycotoxines

Descripteurs
Analyse de qualit, capteur, mthode danalyse biologique, mycotoxine, qualit de lorge,
qualit du malt

RESUME
L'intgrit du produit est une question importante dans la chane orge-bire. Par consquent,
la qualit des matires premires est surveille avec soin. La prsence de mycotoxines dans
l'orge et le malt est un point important de nos proccupations. Les mthodes actuelles
d'analyse de ces composants sont lourdes, coteuses et ncessitent des connaissances
spcialises. Dans ce poster, nous dcrivons le dveloppement d'un nouveau dosage des
mycotoxines, savoir un dosage par inhibition avec biocapteurs, capables de dtecter
plusieurs mycotoxines en une seule mesure. Le systme, facile utiliser et ne ncessitant pas
de personnel hautement qualifi, entranera une rduction significative des cots d'analyse.

2
INTRODUCTION
Mycotoxins are toxic fungal metabolites that can occur in primary food products such
as nuts, cereals and fruits as a result of mould growth. Some mycotoxins have been
proved strong carcinogenic agents like aflatoxin B1; others are under suspicion to
have carcinogenic effects. Currently a few hundred mycotoxins are known which are
often produced by the genera Aspergillus, Penicillium and Fusarium. The most
prominent toxins are aflatoxins, deoxynivalenol, zearalenone, ochratoxin, fumonisin
and patulin.
The occurrence of mycotoxins in both unmalted and malted cereals is a point of
increasing attention. Legislation regarding the allowed levels of mycotoxins present in
food and feed products and in raw materials is expected soon in several countries.
Apart from the issue of food safety and legislation, a number of problems in the
barley-beer chain have been associated with the use of fungal infected barley and/or
the derived malt. A prerequisite for monitoring and controlling the levels of
mycotoxins in the barley-beer chain is the availability of a reliable, economical and
easy to use assay to determine mycotoxin levels. Current methods for mycotoxin
determination include TLC, ELISA and HPLC. Several methods using one of these
detection techniques have AOAC approval. Especially the HPLC method is a very
sensitive method, which however is also the most costly and time consuming due to
extraction and cleanup procedures necessary. The above mentioned techniques do not
provide the means to the industry to control mycotoxin levels in the chain.
The technique described here aims at providing the chain with an assay that allows
reliable, speedy and easy analysis of samples at intake and/or in the field. Multiple
mycotoxins can be detected in one single measurement thus allowing the chain to
efficiently monitor and control the levels of mycotoxins in the barley-beer chain.

THE ASSAY
Materials
Aflatoxin B1, zearalenone, ochratoxin A, deoxynivalenol (DON) and fumunosin B1
were purchased at Sigma. The aflatoxin B1 and zearalenone antibodies were from
Sigma. The anti ochratoxin A antibody was from TNO and the anti fumonisin B1 and
DON antibodies were a gift from Dr. Chris Maragos, USDA. All antibodies were
monoclonals of murine origin.
Carboxymethoxylamine, N-hydroxysuccinimide (NHS), hexanediamine, 2-[N-
morpholino]ethanesulfonic acid (MES) and guanidine chloride were from Sigma.
Dimethyl sulphoxide (DMSO), pyridine, ethanolamine and acetonitrile were from
Baker. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was purchased at
Fluka. Boric acid and glycine were from Merck. MycoSep 224 cleanup columns were
purchased at Romer Laboratories.
The CM5 sensor surfaces, HEPES buffered saline (HBS) and the BIACORE 2000
used for these analysis were provided by Biacore AB.

Principle of the technique

The assay that is presented here is designed as an inhibition assay, in which the
principle of detection is based on surface plasmon resonance (SPR). In principle SPR
is a measurement of mass concentration changes that occur at the sensor surface due
to binding of molecules. At a specific wave length and angle of incidence light will be
absorbed by free electrons in the thin metal film of the sensor surface and the intensity

3
of the reflected light will decrease. The angle where this phenomenon takes place
changes as the mass concentration on the sensor surface changes. The changes in
mass concentration thus measured by the biosensor are those due to binding and
dissociation of interacting molecules, in this case between the immobilised antigen
(mycotoxin) and the specific antibody in the sample. For a more detailed description
of the phenomenon and technique used, see Johnsson et al (2). An example of an
aflatoxin B1 assay based on this technique has been described by Daly et al. (1).
With the SPR equipment used, the BIACORE 2000, the measurements are limited to
molecules larger than 10-15 kD. To overcome this limitation, the assay presented here
has been designed as an inhibition assay in which a fixed concentration of mycotoxin
specific antibodies is mixed with a sample containing an unknown concentration of
mycotoxin. The mixture is then passed over a sensor surface on which a mycotoxin is
immobilised. Non-complexed antibodies are then measured as they bind to the
mycotoxin on the sensor surface. The sensor surface contains four serial connected
flowcells, making it possible to detect multiple mycotoxins in a single measurement.

Methods
Immobilisation of mycotoxins to the sensor surface
In order to be able to immobilise the aflatoxin and zearalenone on the sensor chip
surface the mycotoxins were modified as described by Thouvenot and Morfin (4). The
aflatoxin B1 and zearalenone were carboxylated using carboxymethoxylamine (10 mg
per 5 mg of mycotoxin) in pyridine (7.5 ml). This mixture was shaken overnight at
200 rpm protected from light. The pyridine was evaporated to dryness and the
mycotoxins were redissolved in DMSO. Immobilisation on the CM5 sensor surface
took place using a modification of the standard EDC-NHS reaction as described by
Jnsson (3). Every flowcell of a docked sensor chip was immobilised separately. A
diamine spacer was coupled to a EDC-NHS activated CM5-sensorchip after which
EDC-NHS activated aflatoxin or zearalenone were injected. For blocking of non-
reacted groups an ethanolamine injection was used.
For immobilisation of fumonisin, aminocaproic acid was used as a spacer. For the
immobilization of DON, the method described by Xiao et al. (5) was used using BSA
as a carrier protein. Before use each flow cell is regenerated 20 times with a 6 M
Guanidine Chloride solution in 50 mM glycine pH 2.0

Sample preparation
Samples were extracted with acetonitrile-water. 10 g sample is mixed with 40 ml 90%
acetonitrile and blended at high speed for two minutes. The resulting mixture is
allowed to precipitate and a 5 ml aliquot was removed for further clean up on a
Mycosep 224 MFC column. The resulting filtrate was diluted ten times with HBS and
used for analysis. Standards for calibration were prepared in 8.4% acetonitrile-water.

The concentrations of mycotoxins in the samples were determined by HPLC and GC.
Calibration and sample concentration determinations on the BIACORE 2000
equipment were done according to a standard method, using a buffer flow of 20 l per
minute and an injection of 70 l the appropriately diluted sample, mixed 1:1 with an
antibody solution-mix consisting of antibodies against the four mycotoxins that were
determined. This injection was followed by a regeneration, which consisted of an
injection of 6.0 M guanidine chloride solution in 50 mM glycine pH 2.0 during one
minute.

4
Results
The figures 1 a-d show the calibration curves for four mycotoxins that were
determined simultaneously. These calibration curves are the result of multiple
separate measurements.

140 70
A B

response (RU)
120 60
response (RU)

100 50
80 40
60 30
40 20
20 10
0 0
0.001 0.01 0.1 1 10 100 1000 0.1 1 10 100 1000

concentration zearalenone (ng/ml) concentration DON (ng/ml)

150 250
response (RU)

C D
response (RU)

120 200
90 150

60 100

30 50

0 0
1 10 100 1000 10000 0.01 0.1 1 10 100
concentration fumonisin B1 (ng/ml) concentration aflatoxin B1 (ng/ml)

Figure 1 a-d: Calibration curves as obtained for the four mycotoxins in a

simultaneous analysis (N = 6) . A = zearalenone, B = DON, C =
fumonisin B1 and D = aflatoxin B1.

Figure 2 shows the correlation between results as obtained with the multi-analyte SPR
sensor and results obtained via GC-HPLC for DON, zearalenone, fumonisin B1 and
aflatoxin B1.

5
 1500

sensor (ng mycotoxin/g) DON

1000
fumonisin B1

zearalenone

500 aflatoxin B1

0
0 500 1000 1500

HPLC/GC (ng mycotoxin/g)

Figure 2: Correlation between data obtained via the biosensor and data obtained
via HPLC/GC.

CONCLUSIONS
A biosensor for the simultaneous detection of four mycotoxins has been developed.
Extraction and clean-up of the sample require approximately 15 minutes.
Additionally, the measurement takes 10 minutes, including regeneration of the sensor
chip surface, making a total of approximately 25 minutes for the simultaneous
determination of four mycotoxins in a single sample.
The Fusarium and Aspergillus toxins tested can all be detected in the relevant
concentrations (see table 1).

Table 1: Overview of detection limits and reproducibility as currently obtained with

the multi analyte biosensor based assay.

mycotoxin limits (ng/g) Detect. limit(ng/g) RSD (%)

aflatoxin B1 2 0.2 2 - 10
zearalenone 200 0.01 16 - 19
ochratoxin A 3 0.1 4-9
fumonisin B1 1000 50 2-8
deoxynivalenol 500 0.5 2-5

Applying commercially available SPR devices allows the development of low-cost

equipment which can be used in the field and/or at intake facilities to monitor and

6
control the levels of mycotoxins in the barley beer chain. Other applications for the
technology (detection of pesticide residues etc.) are currently being looked into.

REFERENCES
1. Daly, S.J., Keating, G.J., Dillon, P.P., Manning, B.M., OKennedy, R, Lee, H.A.
& Morgan, R.A., J. Agri. Food Chem., 2000, 48, 5097-5104
2. Johnsson, B, Lfs, S & Lindquist, G., Anal. Biochem., 1991, 198, 268-277
3. Jnsson, U., BioTechniques, 1991, 11, 620-627
4. Thouvenot, D & Morfin, RF., Appl. Environ. Microb., 1983, 45, 16-23
5. Xiao, H, Clarke, J.R., Marquardt, R.R. & Frohlich, A.A., J. Agri. Food Chem.,
1995, 49, 2092-2097

ACKNOWLEDGEMENT
Dr Chris Maragos (USDA) is acknowledged for the supply of the fumonisin and DON
antibodies. BIAcore AB (Uppsala, Sweden) is acknowledged for the use of the
BIACORE 2000 to perform the multi analyte analysis.

7
75

Development of a process control system

for the optimisation of the cytolytic,
proteolytic and amylolytic degradation
during mashing
G. Fischer1, M. Mitzscherling1, T. Becker1, A. Delgado1, T.
Dickel2, M. Krottenthaler2 & W. Back2
1
TU Mnchen, Lehrstuhl fr Fluidmechanik und Prozeautomation, D-85350 Freising-
Weihenstephan, Germany (e-mail: [email protected])
2
TU Mnchen, Lehrstuhl fr Technologie der Brauerei I, D-85350 Freising-Weihenstephan,
Germany

Descriptors
Enzymic activity, laboratory equipment, mashing conditions, process control

SUMMARY
Process accompanying measurement of the enzymatic degradation procedures within the
mashing tun enables an instant response to the specific and alternating characteristics of malt.
Therefore, a pilot measurement equipment is designed to achieve online monitoring of the
significant quantities. These physical parameters were specified to extract the command
quantity by means of chemometry and artificial neural networks (ANN). Furthermore, the
developed photometric and chemical screening tests, necessary to show the correlation
between the enzymatic conversion and the measured course of the significant quantities, give
practicable tools for brewing laboratories to shorten analysis effort and time.

Entwicklung eines Prozesskontrollsystems fr die Optimierung der cytolytischen,

proteolytischen und amylolytischen Lsung whrend des Maischens

Deskriptoren
Enzymaktivitt, Laboratoriumausstattung, Maischefhrung, Prozesteuerung

ZUSAMMENFASSUNG
Prozessbegleitende Messungen der enzymatischen Lsung innerhalb des Maischvorgangs
ermglichen es, sofort auf die spezifischen und wechselnden Eigenschaften des Malzes zu
reagieren. Deshalb wurde ein Versuchsmessaufbau zusammengestellt, um die signifikanten
Parameter online zu berwachen. Diese physikalischen Parameter wurden spezifiziert, um die
Befehlsanzahl durch Chemometrie und knstliche neuronale Netze (ANN) zu bestimmen.
Auerdem ermglichen die entwickelten photometrischen und chemischen Screeningtests, die
notwendig sind, um die Korrelation zwischen enzymatischen Umwandlungen und
gemessenen Verlufen der signifikanten Parameter zu zeigen, Brauereilaboren die
Analysenkosten und -aufwand zu senken.

Dveloppement d'un systme de contle des procds pour l'optimisation de la

dgradation cytolytique, protolytique et amylolytique pendant le brassage

Descripteurs
Activit enzymatique, commande de procd, conditions de brassage, matriel de laboratoire

RESUME
Le processus accompagnant les procdures de mesure de la dgradation enzymatique dans la
cuve de brassage permet une rponse instantane aux caractristiques spcifiques et
changeantes du malt. Pour cette raison, nous avons mis au point un appareillage de mesure
pilote permettant d'exercer une surveillance en ligne des quantits significatives. Ces
paramtres physiques ont t spcifis pour extraire la quantit de commandes au moyen de la
chimiomtrie et de rseaux neuronaux artificiels (RNA). Par ailleurs, les tests de dtection
chimique et photomtrique, ncessaires pour montrer la corrlation entre la conversion
enzymatique et l'volution mesure des quantits significatives, donne des outils utilisables
par les laboratoires de brasserie pour raccourcir les travaux et la dure d'analyse.

2
INTRODUCTION
Up to now recipes and management of the mashing process are performed by a time
controller, primarily based on empirical experiences. Hence, there are still
economisation potentials by means of process controlled mashing. Beyond this, mere
process accompanying measurement of the enzymatic degradation procedures within
the mashing tun enables an instant response to specific and alternating characteristics
of the raw material malt. Therefore, a pilot measurement equipment is developed to
achieve online monitoring of the significant quantities. These are physical parameters
which correlate with the solid degradation and the progress of the solution of the malt
constituents, as there are only laboratory analyses to observe the enzymatic activities
and conversions so far.

CONCEPTION OF THE SYSTEM

Online measurement and management of the mashing process enables the
determination of the state of the enzymatic degradation. Hence, impacts of varying
malt characteristics can be detected during the process procedure and the
interpretation of the significant quantities permits preventive process management
instead of compensating measures.
Process monitoring in order to control the mashing procedure demands a permanent
and quick overview of the actual state of the degradations. However, the duration of
the existing analytical methods sharply reduces the practicability of measuring the
significant quantities like the portions of -glucan, free amino acid or the
saccharification of the mash in a direct way. Therefore, it is necessary to gain an
image of the process by measuring apace ascertainable sensitive quantities as there are
physical parameters. For this reason the measured quantities are the conductivity, the
density, the pH-value, the saccharification, the temperature and the viscosity of the
matrix fluid.

DIR VIR QIRT

Sacc.

QIRT
pH

QIRT
Cond.

Figure 1: Piping and Installation Diagram (P&ID) of the pilot measurement system

3
In order to separate the solid particles from the matrix fluid a hydrocyclone is used.
This allows the continuous clarification of the mash and beyond this the re-entry of
the solids into the mash tun. Within the hydrocylone there are two vortices. The
primary vortex moves from the inlet near the top downwards the cyclone wall
carrying the coarse particles into the underflow outlet, whereas the fine solids within
the interior secondary vortex flow upwards towards the overflow outlet. The cut size
depends on the cyclone design and decreases with reduced cyclone diameter, reduced
inlet diameter, reduced outlet diameter, reduced cone angle, reduced vortex finder
length or increased body length [7].

Conductivity
The conductivity is mainly influenced by phosphate, chloride, sulfate, potassium,
magnesium and calcium, which are introduced on the one hand by the brewing liquors
hardness and on the other hand by the grist. Whereby the portion of the water remains
constant as far as there is an accurate water softening. The total conductivity consists
of the aggregate of this basis and the portion caused by the malt constituents such as
the named ions and solved amino acids or smaller peptides. As the degradation of
phosphate runs parallel to those of the proteins both increase the conductivity at the
same time and vice versa the course of the conductivity gives a sensitive state
description of the proteolysis. Figure 2 shows the measured correlation between the
conductivity and the solution of nitrogen during an isothermal 45C-mashing. Herein
the proteolysis was measured as soluble nitrogen by the Kjeldahl-Method.

550 1,1

1,05 Conductivity [S/cm]

Soluble N [mg/l]

500 1

0,95

450 0,9

0,85

400 0,8
0 10 20 30 40
Time [min]

Figure 2: Course of the soluble nitrogen (Soluble N) and of the

Conductivity during an isothermal 45C-Mashing

Density
Due to the continuous depolymerisation and degradation of the grist particles
increasing fractions of the malt constituents become soluble. Hence, a steady density
enhancement of the matrix fluid results. At this, measurements of the wort density
during an Eyben-Mashing-Procedure show the particular relevance of the heating-up
periods. Therein are the combined activities of different enzymes available, as for
example proteolytic and amylolytic ones in the temperature range around 55C, a
circumstance that leads to a steeper increase in density than it could be detected

4
during the rests. Figure 3 shows the mentioned course of the density during the
mashing.

6,05 1,035

6 1,03

Density [g/cm^3]
pH-Value

5,95 1,025

5,9 1,02

5,85 1,015

5,8 1,01
10 30 50 70 90 110
Time [min]

Figure 3: Course of the pH-Value and the Density during an Eyben-

Mashing-Procedure

pH-Value
The pH-value changes as well as the buffering capacity increases due to the solved
phosphates with the preceding activity of the malt phosphatases and the degradation
of the malt phosphates. This leads to a release of primary phosphates, which in the
case of deionisised water decrease the pH-value. This effect is likely to refer to the
hindered reaction with the cations out of the brewing liquor. Further there are single
acids and amino acids as well which influence the content of the hydronium
concentration within the mash. In figure 3 the course of the pH-value during the above
mentioned Eyben-Mashing-Procedure is pictured.

Saccharification
Solved starch is detectable due to its iodine-complex spectra. At this, there appear
different peaks for complexes with amylose in comparison to those formed with
amylopectin. The latter show maximum absorption at wave lengths between 530 and
570 nm whereas the extinction of amyloseiodinecomplexes is at maximum in the
range of 620 to 640 nm. Linear molecules as -amylolysis-products of amylose have
an achromatic limit of 18 to 20 glucose-units whereas chained oligosaccharides
exceed this limit by a multiple. Several authors determined the correlation between
wavelength of the maximum absorbance and the degree of polymerisation for non-
ramified starch components to be logarithmic [3, 4]. On the basis of this knowledge it
is possible to estimate whether the mash saccharification is finished or further
amylolysis is necessary. Furthermore there is no absolute starch or even
polycarbohydrate concentration measurement required as the main information for the
truncation of the mashing process is the saccharification of the mash. The qualitative
character of this analysis compared to quantitative requirements simplifies its
fulfilment.

5
Viscosity
On account of the gelatinisation grist solids increase their volumes whereas the matrix
fluid is subjected to changes in rheology, for example increase in the viscosity whilst
the gelatinisation. However, there appear not only changes in viscosity, changes in
flow behaviour are likely as well [5, 8]. In doing so, the flow behaviour shifts from
newtonian to non-newtonian. Within the next step of amylolysis the degradation of
carbohydrate polymers leads to a decrease of viscosity. The total course of the
rheological mutations allows to determine the degree of raw polymers which still have
to be processed by the hydrolases. One major aspect herein is the content of -glucan
in the matrix fluid. Figure 4 shows the shift in flow behaviour from newtonian to non-
newtonian and vice versa during an Eyben-Mashing-Procedure. The figured quantity
power law index is a measure whether the flow behaviour is newtonian or not. The
value One characterises newtonian flow behaviour and an index smaller than One
shear thinning behaviour.

1,2

1,1
Power Law Index

0,9

0,8

0,7

0,6
0 20 40 60 80 100
Time [min]

Figure 4: Course of the Flow Behaviour Index during an Eyben-Mashing-

Procedure

CHEMICAL DETECTION OF THE PROGRESS OF THE ENZYMATIC

BREAKDOWN DURING MASHING
The object for the Institute of Brewing Technology I within this project is to detect the
progress of enzymatic breakdown during mashing in order to obtain the points of
optimal solution. Therefore, the three main enzymatic processes need to be examined.
This is done by the determination of -glucan for cytolysis, soluble nitrogen as well as
free amino nitrogen (FAN) for proteolysis and the photometric iodine test for
amylolysis. The points of sufficient solution of the macromolecules are regarded to be
350 mg/l -glucan, 1000 mg/l soluble N and 20 mg/100 ml FAN, and photometric
iodine test below 0.3 [6].
Continuous monitoring of the mashing process produces a large number of samples.
Therefore, analysis methods are needed, which will be easy to handle and will lead to
fast results. These requirements are fulfilled by photometric analysis. -glucans can
be easily detected by calcofluor flow injection analysis [2], soluble nitrogen can
quickly be measured by forming the difference of the extinction at 215 and 225 nm

6
[1]. The photometric iodine test can be simplified in order to obtain relative changes
to an initially determined absolute value. Further conveniences of photometry are the
low costs and low environmental pollution.
Figure 5 shows an example of the proteolytic and amylolytic test results of a congress
mashing procedure which have been obtained during a process of the above-
mentioned parameters. The figure shows that the proteolytic breakdown is completed
within 5 min after mashing in, which is indicated by reaching the values of 1000 mg/l
for soluble nitrogen and 20 mg/100 ml FAN. This result recommends heating up the
mash to the next enzymatic temperature optimum at this point of time. The amylolytic
breakdown is already completed within 15 min after reaching the temperature
optimum for -amylase (70 C). This is indicated by reaching a sufficient gravity
value of 16 % or more and a value for the iodine test below 0.3. These results suggest
an earlier termination of the mashing process.

50 Point of sufficient 90
Proteolysis
Gravity [WW %] - Iodine Test

40 78

Temperature [C]
FAN [mg/100ml]

Nitrogen [mg/l]
30 66

20 54

10 Point of sufficient 42
Amylolysis
0 30
0 10 20 30 40 50 60 70 80 90 100 110
Time [min]

Figure 5: Enzymatic Breakdown of Proteins and Starch during a Congress Mashing

Procedure characterised by Gravity, Iodine Test *60, FAN,
Temperature and Soluble N

DATA PROCESSING
Herein the correlation of the array of the measured physical parameters with the
technological quantities mainly -glucan content, soluble nitrogen content, free
amino nitrogen and saccharification has to be specified. Even though every single
measured value gives some basic information on the progress, the actual state of the
mashing process is not congruent with the single processes. As described above
cytolysis could be detected by viscosity measurement, proteolysis by conductivity and
pH-value and amylolysis by viscosity and spectrometric measurements, but all the
analyses for their own are not sensitive enough to sufficiently specify the course of
the degradations. Therefore, it is indispensable to use the total array of quantities to
characterise every single process. At this, in order to gain the command quantity the
single quantities have to be processed by statistical methods as for example principle
components regression. Further processing will include the use of an artificial neural
network, if the accuracy of the results gained with the other evaluation methods
requires this measure.

7
CONCLUSIONS
The data of the measured quantities demonstrate the possibility of monitoring the
degradation progresses of the enzymatic processes within the mashing tun by
observing apace ascertainable sensitive quantities. Further progress has to be made in
monitoring each of the three main processes. This requires to find an appropriate
analysis and evaluation method of the measured values to reproduce the course of
cytolysis, proteolysis and amylolysis as it is represented by the chemical detections.
Therefore, it is necessary to observe mashing processes by chemical reference
analyses and by measurement of the quantities array. In the end, process control
strategies could be developed on the basis of the gained knowledge.

REFERENCES
[1] Analytica-EBC, Nrnberg, 1998, Method 4.9.2.
[2] Analytica-EBC, Nrnberg, 1998, Method 8.13.2.
[3] Bailey, J.M. & Whelan, W.J., The Journal of Biological Chemistry, 1961, 236,
969 973.
[4] Bendetzkii, K.M., Jarowenko, W.L. & Lukjanowa, L., Starch, 1972, 24, 136.
[5] Hoog, D., Annemller, G. & Senge, B, Brauwelt, 1997, 137, 1606 1610.
[6] Pfenninger, H., Brautechnische Analysenmethoden der MEBAK Band 2,
Freising: Selbstverlag der MEBAK, 1993, Chapter 2.
[7] Svarovsky, L., Hydrocyclones, Eastbourne: Holt, Rinehart and Winston Ltd.,
Chapter 7, 1984.
[8] Werner, F. & Mersmann, A., Chemie Ingenieur Technik, 1998, 70, 890-894.

8
76

From seeds to beer: development of a

quantitative detection method of
transgenic maize
Pierre Brignon, Marc Vaitilingom, Luc Didierjean, Estelle
Carniel, Aurlie Brelin & Christophe Bastien
TEPRAL-Kronenbourg, 68, Route dOberhausbergen, F-67037 Strasbourg Cedex, France (e-
mail : [email protected])

Descriptors
Analysis method for cereals, deoxyribonucleic acid, genetics

SUMMARY
With the recent release of EU food regulation on genetically modified organisms (GMO)
labelling, ingredients and beers made with maize need to be checked. The method we
developed consists in two steps, extraction of very pure DNA from these matrices, then
amplification of the DNA released by real-time quantitative PCR. The procedure is easy to
handle, accurate, sensitive (0,001% transgenic maize detected in grits for instance) and fast
(whole procedure routinely performed on a 24-sample set in under 12 h.). It is currently used
for the needs of Kronenbourg breweries. It also listed as a reference method in normalisation
groups at the French (AFNOR) and EU (CEN) level.

Von der Saat zum Bier: Entwicklung einer quantitativen Nachweismethode von
transgenem Mais

Deskriptoren
Analysenmethode fr Getreide, Genetik, Mais, mengenmssige Analyse, Recht

ZUSAMMENFASSUNG
Nach der neuen EU-Lebensmittelregelung kann auf die Kennzeichnung von GMO verzichtet
werden. Deshalb ist es notwendig, Zutaten und Bier, die aus Mais hergestellt wurden, zu
kontrollieren. Die von uns entwickelte Methode besteht aus zwei Schritten. Zunchst wird aus
der Matrix sehr reine DANN extrahiert. Anschlieend erfolgt eine Vermehrung durch eine
quantitative Echt-Zeit-PCR. Die Analyse ist leicht durchfhrbar, sehr genau, empfindlich
(Beispielsweise kann eine 0,001% Zugabe von transgenem Mais ermittelt werden) und
schnell (Die normalerweise auf 24 h ausgelegte Analyse kann in 12 h durchgefhrt werden).
Derzeit wird die Analyse in den Kronenbourg Brauereien eingesetzt. Sie wird aber auch als
Referenzmethode der Normungs-Komitees von Frankreich (AFNOR) und der EU (CEN)
aufgefhrt.
De la semence la bire: dveloppement d'une mthode quantitative de dtection du
mas transgnique

Descripteurs
Analyse quantitative, gntique, lgislation, mais, mthode danalyse de crales

RESUME
Avec la publication rcente de la rglementation alimentaire de lUE sur l'tiquetage des
OGM, les ingrdients et bires fabriqus avec le mas doivent tre vrifis. La mthode que
nous avons dvelopp compte deux tapes: extraction d'ADN trs pure partir des matrices,
puis amplification de l'ADN libre par PCR quantitative en tant que telle. Cette technique
est facile utiliser, exacte, sensible (0,001 % de mas transgnique dtect sur les grilles, par
exemple) et rapide (analyse complte effectue en routine sur un jeu de 24 chantillons en
moins de 12 heures). Elle est actuellement utilise pour les besoins des brasseries
Kronenbourg. Elle est aussi recense comme mthode de rfrence dans les groupes de
normalisation au niveau franais (AFNOR) et europen (CEN).

2
INTRODUCTION
With the growing consumer concern over GMOs, many countries and governments
have decided to label their derived foodstuffs. In Europe, Novel Food Regulation
(278/97/EC) has been implemented by a new legislation requiring the labelling of
food containing more than 1% GMO (49/2000/EC). Additives and flavourings are
covered by separate modified regulation (20/2000/EC) and requires labelling if there
is no substantial equivalence as indicated by the presence of recombinant protein or
DNA. Therefore beers likely to contain GMOs from raw materials or ingredients need
to be checked.
Here we describe the quantitative detection method of transgenic maize we developed
with specific adaptations for the brewing industry.

MATERIALS AND METHODS

Materials
Standard curves were calibrated with commercial transgenic maize reference
standards (Fluka).

DNA isolation
DNA was isolated from reference standards and samples as described previously (2).
Depending on starting material, various amounts and volumes of lysis buffer [10 mM
tris (pH 8), 150 mM NaCl, 2 mM ethylenediaminetetraacetic acid (EDTA), 1% (w/v)
sodium dodecylsulfate and 0.8 M guanidine hydrochloride] were used (table 1).

Material Quantity Lysis buffer (ml)

Reference standards 1g 30
Grits 2g 30
Starch 2g 40
Beer 40 ml 10

Table 1: Starting material quantities and lysis buffer volumes used in the DNA
isolation procedure.

200 l (20 mg/ml) of protease from Streptomyces griseus (Sigma) was added then the
sample was incubated at 50 C for 2 h. The extracted DNA was purified using the
Wizard protocol (Promega). An additional step using Qiaquick spin colums (Qiagen)
was performed. DNA was then eluted in EB buffer (Qiagen) and then quantified
spectrophotometrically with a Lambda Bio spectrophotometer (Applied Biosystems).

Primers and probes

The technology we applied for our quantitative detection method is called real-time
quantitative polymerase chain reaction (PCR) using the ABI Prism 7700 sequence
detection system (Applied Biosystems). Briefly this technique is based on TaqMan
chemistry (1) in our procedure: a fluorogenic probe is hybridising within the target
sequence bound by usual PCR primers. The probe is labelled with a fluorescent
reporter dye on the 5 end and with a fluorescent quencher dye on the 3 end. Due to
the closeness of the quencher to the reporter, the reporter fluorescence is suppressed.
During PCR, the 5 to 3 exonuclease activity of Taq DNA polymerase degrades the

3
hybridised probe and separates the two dyes. The resulting increase of fluorescence is
proportional to the amount of specific PCR products.
For the transgenic Maximizer maize event 176 (Novartis) quantification, two
PCR systems were designed (see table 2 and our previous work, 2), one for the total
detection of maize (amplification of a part of 10-kDa zein gene) and the other for the
specific detection of transgenic maize (amplification of a part of cryIA(b) gene).
Endogenous probe was labelled with the fluorescent reporter VIC (molecule non
revealed by the supplier) on the 5end and transgenic probe with FAM (6-
carboxyfluorescein) on the 5end. The fluorescent quencher dye, TAMRA (6-
carboxytetramethylrhodamine) was located on the 3end of the probes.

PCR System Name Sequence

Endogenous Primer Zetm1 5'-TGTTAGGCGTCATCATCTGTGG-3'
(69 bp Primer Zetm3 5'-TGCAGCAACTGTTGGCCTTAC-3'
product) Probe Zetmp 5'-ATCATCACTGGCATCGTCTGAAGCGG-3'
Transgenic Primer Crytm1 5'-GTGGACAGCCTGGACGAGAT-3'
(106 bp Primer Crytm2 5'-TGCTGAAGCCACTGCGGAAC-3'
product) Probe Crtmp 5'-AACAACAACGTGCCACCTCGACAGG-3'

Table 2: Primers and probes used in this study.

During PCR cycling, the sequence detector software (Applied Biosystems) calculates
the Rn values which reflect the quantity of fluorescent probes degraded and fit an
exponential function generating a real-time amplification plot for each well. The point
at which the amplification plot crosses the threshold is defined as Ct. Ct is reported as
the fractional cycle number reflecting a positive result. The amount of DNA in an
unknown sample is measured by interpolation from a standard curve of Ct values
generated from known starting DNA concentrations. Total DNA quantity was
determined by reporting the Ct VIC value on the endogenous standard curve, and
transgenic DNA quantity was fixed by reporting the Ct FAM value on the transgenic
standard curve. Because both endogenous and transgenic PCR systems were
performed in the same tube, a direct comparison between total and transgenic DNA
was possible. The percentage of transgenic material was then determined as being the
ratio of transgenic to total DNA quantities.

PCR conditions
Amplification reactions (50 l) were performed with the TaqMan PCR Core
Reagents (Applied Biosystems Roche Molecular Systems). They contained 25 nM
of endogenous primers, 100 nM of transgenic primers, 200 nM of endogenous and
transgenic probes, dATP, dCTP and dGTP, each at a concentration of 400 M, 800
M of dUTP, 2.5 U of AmpliTaqGold DNA Polymerase, 0.5 U of AmpErase uracil
N-glycosylase (UNG), 6.5 mM MgCl2 and 1 x TaqMan buffer A. For the generation
of standard curves, stock DNA from the 2 % transgenic maize event 176 (Fluka)
was diluted to the following concentrations: 10 ng/l, 5 ng/l, 1 ng/l, 0.5 ng/l, 0.25
ng/l, 0.1 ng/l and 0.05 ng/l. 10 l were included in the amplification reaction
giving a range from 100 to 0.5 ng and 2 to 0.01 ng endogenous and transgenic DNA
quantities respectively.

4
PCR reactions were run with the ABI Prism 7700 Sequence Detection System
(Applied Biosystems) with the following program: 2 min at 50 C, 10 min at 95 C
and 50 cycles 15 sec at 95 C and 1 min at 60 C.

RESULTS AND DISCUSSIONS

DNA extraction from ingredients and beer
Before PCR amplification, DNA must first be extracted from sample to analyse. Five
samples were analysed, one starch, two grits (grits 1 and grits 2) and two beers (beer 1
and beer 2 being produced with starch and grits 1 respectively). For routine analysis,
we wanted to develop a fast and easy to handle procedure with an efficient DNA
extraction yield and without residual inhibitors of the enzymatic amplification
reaction. In the case of starch for instance, maize DNA content is very low but on the
other side polysaccharide concentration is high and is known to inhibit Taq
polymerase. We clearly show that both objective, sufficient DNA quantity for
amplification and elimination of inhibitors has been reached (table 3 and figure 1).

Material OD260 OD280 OD260/OD280 DNA (ng/l)

Starch 0.025 0.016 1.56 25
Grits 1 0.179 0.127 1.41 179
Grits 2 0.224 0.15 1.49 224
Beer 1 0.005 0.003 1.67 5
Beer 2 0.007 0.004 1.75 7

Table 3: Estimated quantities of purified DNA from samples analysed by optical

density measurements.

M 1 2 3 4 5

Figure 1: Gel electrophoresis (2% agarose in Tris-acetate-EDTA) PCR products of 10

kDa zein gene of genomic DNA extracted from starch, grits 1, grits 2, beer
1 and beer 2 (lanes 1 to 5 respectively). Lane M: 100 kb ladder (Gibco
BRL).

5
Quantitative detection of transgenic maize
The transgenic content of grits, starch and beer was quantified as follows: total and
transgenic DNA quantities were estimated using standard curves generated with a
scale of 2% Maximiser maize diluted DNA (figures 2 and 3), then percentage of
transgenic DNA was calculated from the ratio of transgenic to total DNA quantities
(table 4).

Endogenous Standard Curve R2 = 0,9981

4
Log (endogenous
DNA amount)

2
0
-2
-4
20 25 30 Ct 35 40

Figure 2: 10-kDa zein gene PCR product detection in real time: Amplification plot
generated by known amounts of DNA (lanes A7 to H7: 100 ng, 50 ng, 10
ng, 5 ng, 2.5 ng, 1 ng, 0.5 ng and 0.1 ng of 2% Maximiser maize; lane G1:
negative control) with endogenous PCR system (Zetm1, Zetm3 and Zetmp
oligonucleotides). The upper part is the standard curve from the data given
in the amplification plot.

6
Transgenic Standard Curve 2
Log (transgenic DNA R = 0,9929
2
1
0
amount)

-1
-2
-3
-4
-5
20 25 30 Ct 35 40

Figure 3: cryIA(b) gene PCR product detection in real time: Amplification plot
generated by known amounts of DNA (lanes A7 to H7: 100 ng, 50 ng, 10
ng, 5 ng, 2.5 ng, 1 ng, 0.5 ng and 0.1 ng of 2% Maximiser maize; lane G1:
negative control) with transgenic PCR system (Crytm1, Crytm3 and Crtmp
oligonucleotides). The upper part is the standard curve from the data given
in the amplification plot.

Material Ct DNA (ng) % transgenic

Starch VIC: 24.22 Total: 56.01 0.4
FAM: 30.13 Transgenic: 0.23
Grits 1 VIC: 22.07 Total: 211.29 0.04
FAM: 31.66 Transgenic: 0.09
Grits 2 VIC: 20.36 Total: 607.68 0.01
FAM: 32.81 Transgenic: 0.05
Beer 1 VIC: 38.39 Total: 0.01 -
FAM: - Transgenic: -
Beer 2 VIC: 37.74 Total: 0.01 -
FAM: Transgenic: -

Table 4: Determination of transgenic content in samples analysed.

These results demonstrate that our procedure is adapted to beer ingredients and final
products. Concerning beers 1 and 2, due to the very low amount of total DNA, no
transgenic signals were detected.

CONCLUSION
We developed a fast, easy to handle and accurate method to quantify transgenic maize
in foodstuffs. Depending on the sample, we reached a level of sensitivity of 0.001%

7
transgenic maize with a coefficient of variation of 5% (grits in that case). Moreover,
we routinely perform the whole procedure (DNA extraction and quantification) on a
24-sample set under 12 h. The results presented in this study were obtained from grits
starch and beer but our procedure was successfully transposed to syrups and
flavourings.
We are currently working on the detection of other transgenic maize commercially
available in the USA and on the event specific transgene detection.

REFERENCES
1. Holland, P.M., Abramson, R.D., Watson, R. and Gelfand, D.H., Proc. Natl. Acad.
Sci. USA., 1991, 88, 7276-7280.
2. Vatilingom, M., Pijnenburg, H., Gendre, F. and Brignon, P.J., Agric. Food
Chem., 1999, 47, 5261-5266.

8
77

Development and validation of a rapid

wort and beer filterability test
Chris Ford1, Doug Stewart2, Martin Barski1 & Evan Evans1
1
Adelaide University, Barley Biochemistry and Brewing Research Laboratory, Department of
Plant Science, Waite Campus, PMB1, Glen Osmond, SA 5064 South Australia (e-mail:
[email protected])
2
Adelaide Malting Company, Cavan, SA 5094 South Australia

Descriptors
Accelerated method, beer, determination of filterability, malt quality, wort

SUMMARY
A small-scale syringe-based filtration test (SWIFT) was developed to provide rapid, simple
and cheap measurements of wort filterability. Initial trials showed SWIFT to be highly
correlated with existing methods of determining beer filterability (Vmax, DE filtration). The
results of trials using over 40 Australian malts show that beer filterabilities measured by
SWIFT correlate well with SWIFT data for both hot water extracts and worts derived from
the malts. Further work indicates that good discrimination of malts is possible, and that the
test may form a useful aid to barley breeders in addition to brewers and maltsters.

Entwicklung und Validierung eines schnellen Tests zur berprfung der Filtrierbarkeit
von Wrze und Bier

Deskriptoren
Bestimmung der Filtrierbarkeit, Bier, Malzqualitt, Schnellverfahren, Wrze

ZUSAMMENFASSUNG
Es wurde ein kleiner, spritzenbasierender Filtrationstest (SWIFT) entwickelt, um schnell,
einfach und preiswert die Filtrierbarkeit von Wrze bestimmen zu knnen. Erste Versuche
zeigten die sehr gute Korrelation des Swift mit existierenden Methoden zur Bestimmung der
Filtrierbarkeit von Bier (Vmax, Kieselgurfiltration). Die Resultate der Versuche, in denen ber
40 australische Malze verwendet wurden, zeigen, dass die durch SWIFT gemessene
Filtrierbarkeit von Bier gut mit den SWIFT-Ergebnissen sowohl aus Heiwasserextrakten als
auch von Wrzen aus Malz korrelierten. Weitere Arbeiten zeigten, dass eine gute
Unterscheidung des Malzes mglich ist, und dass der Test nicht nur ein ntzliches Hilfsmittel
fr Brauer und Mlzer, sondern auch fr Braugerstenzchter ist.
Dveloppement et validation d'un test rapide de filtrabilit du mot et de la bire

Descripteurs
Bire, mthode de dtermination de la filtrabilit, procd acclr, mot, qualit du malt

RESUME
Un test de filtration sur seringue petite chelle (SWIFT) a t dvelopp pour permettre des
mesures rapides, simples et peu coteuses de la filtrabilit du mot. Les premiers essais ont
montr que le SWIFT est hautement corrl aux mthodes existantes de mesure de la
filtrabilit de la bire (Vmax, filtration sur DE). Les rsultats des essais sur plus de 40 malts
australiens montrent que la filtrabilit des bires mesure par le SWIFT est bien corrle aux
donnes SWIFT pour les extraits l'eau chaude et les mots drivs des malts. D'autres
travaux montrent qu'une bonne discrimination des malts est ainsi possible et que le test peut
s'avrer une aide utile aux producteurs d'orge, outre les brasseurs et les malteurs.

2
INTRODUCTION
Filtration plays a critical part in the production of bright beer. Consumer perception of
product quality is based predominantly on visual cues, and invariably clarity is a
major factor for beer acceptability. Some form of filtration is commonly used
immediately prior to bottling or kegging. Breakdowns in filtration cellars due to
blockages can be expensive and have consequences in downstream operations
including packaging and dispatch. Tests to predict filterability can provide early
indications of potential problems and are widely used to provide assurance of
successful beer filtration prior to large-scale packaging of the product.
We previously developed a small-scale test that predicts beer filterability from
measurements made on sweet wort [1,2]. The Small-scale Wort rapId Filtration Test
(SWIFT) uses negative pressure generated by a 10 ml syringe to draw clarified wort
through a 0.45 micron filter, and has the advantages of being simple to perform, quick
and cheap compared to traditional methods of filterability determination. SWIFT was
shown to correlate well with existing methods for assessing beer filtration efficiency,
including membrane and diatomaceous earth-based protocols [2]. These early results
suggested that SWIFT might provide an alternative to more involved tests of
filterability commonly used in breweries and maltings.
Our recent work, presented here, has extended the original scope of SWIFT,
examining the correlation between the filterability of hot-water extracts, worts and
beers derived from a large number of Australian malts using SWIFT and Vmax
measurements.
We have also examined the sensitivity of SWIFT with regard to the discrimination of
malt samples, as an aid particularly to barley breeders in early multi-site evaluation of
potential new malting varieties. It is hoped to increase the scope of SWIFT to include
barley breeders, maltsters and brewers in the list of potential users.

METHODS
The SWIFT membrane filterability test
The original SWIFT test was described in our earlier work [1]. Clarified extract, wort
or degassed beer is drawn through a nylon (Polyamide) membrane filter (13 mm
diameter, 0.45 micron pore size), using negative pressure generated from a syringe
with its plunger withdrawn to a fixed volume. A two-way tap beneath the filter unit is
opened to start the filtration period, and closed after a pre-determined interval (usually
60 seconds). The use of a 10 place rack allows concurrent measurement of multiple
samples with a five-second gap between the start of each measurement; this is
illustrated in figure 1.

3
 Figure 1: SWIFT apparatus showing concurrent analysis of 9 samples

In a significant modification to the published procedure, disposable syringe filters

were tested as replacements for the refillable units used previously. The reliability of
these filters was improved dramatically by pre-flushing with sterile-filtered water.
This removed air-bubbles that otherwise blocked the passage of liquid during
filtration, causing inaccuracies between replicate samples.

Other analyses
Hot water extracts were produced from 5 g malt samples using a method based on the
Analytica EBC protocol. Boiled worts (diluted to 10Plato following removal of cold
break material) and beers were made using the Small Scale Brewing procedure
described earlier [3]. Hot water extract analyses were performed according to
protocols based on those described in the Analytica EBC. Vmax measurements were
made using the method described in [3]. All SWIFT determinations were made in
triplicate, and the results analysed using the statistical package Genstat.

RESULTS
A comprehensive survey of Australian malts confirms the durability of SWIFT
24 malts from barleys grown across Australia were analysed. Hot water extracts
prepared from 5.00 g samples of grist (0.2 mm grind) were tested for SWIFT
filterability and a range of quality parameters (viscosity, extract, soluble protein, wort
and malt -glucan, and Kolbach index). Boiled worts and the fermented beers
produced from them were tested for filterability using SWIFT and Vmax (beers only).
The results are presented in table 1.

4
Table 1. Correlation data of malt filterability and malt quality data, 24 malts

HWE SWIFT

Wort SWIFT

Beer SWIFT

Beer VMAX

Viscosity

Kolbach
Soluble

Wort -
Protein
Extract

Glucan

Glucan
Malt -

Index
HWE
*
SWIFT
WORT
0.613* *
SWIFT
Beer
0.631* 0.864* *
SWIFT
Beer
0.695* 0.768* 0.8* *
VMAX
Extract 0.146 0.153 0.103 0.075 *
Viscosity -0.73* -0.727* -0.691* -0.616* -0.039 *
Soluble
0.319 0.38 0.346 0.457 -0.255 -0.327 *
Protein
Wort -
-0.301 -0.242 -0.348 -0.509 0.09 0.408* -0.43 *
Glucan
Malt -
0.25 0.151 0.073 0.157 -0.561 -0.162 0.391 -0.061 *
Glucan
Kolbach
0.372 0.229 0.275 0.3 0.235 -0.179 0.612* -0.357 -0.486* *
Index

* P<0.05

SWIFT filterability of hot water extracts (HWEs) and beer Vmax data were
significantly correlated (r = 0.7, n = 24, P <0.05)
SWIFT filterability data for HWEs and boiled worts had a statistically significant
correlation of 0.61, while the correlation HWEs and beer SWIFT values was 0.63.
Boiled wort and beer SWIFT data showed a correlation of 0.86, suggesting that
boiling had a beneficial effect on the ability of SWIFT to predict beer filterability
As seen in earlier work [1], beer SWIFT correlated well with the Vmax test for
filterability (r = 0.8; n = 24, P <0.05). This provides further evidence that SWIFT
is a reliable measure of potential filterability
SWIFT filterability and viscosity of HWE were highly correlated (r = -0.73; n =
24, P <0.05); there were no other significant malt parameters that correlated with
SWIFT filterability.

In a separate series of trials, SWIFT filterabilities for HWEs were compared with
those assayed using extracts boiled for 60 minutes prior to equilibration to 24C.
Correlations of SWIFT data between the two treatments ranged from 0.6 (n = 16) to
0.85 (n = 12, in both cases P <0.05), suggesting that changes in extract composition
during the boil were reflected in altered filterability data. Further work is underway to
determine the nature of these changes, but the removal of proteinaceous material by
the hot and cold breaks is a likely candidate.

SWIFT discrimination of closely specified malts

We were concerned that although SWIFT provides a good determinant of potential
beer filterability from HWE or wort samples, the ability of the test to discriminate
samples with similar specifications may be limited. For it to be of value in breeding
programs, in which incremental improvements in malt quality (including filterability)

5
are the normal outcome, the SWIFT test must be capable of robust and reliable
detection of very small differences in filterability. As a preliminary evaluation of the
sensitivity of SWIFT, eight malts representing Australian barleys grown for malting
(n = 5) or feed purposes (n = 3) were used to prepare HWEs as described above.
SWIFT determinations of filterability were made for each HWE (6 replicates); the
trial was designed and analysed as a Randomised Complete Block Design. Malt
specifications (density, wort -glucan and viscosity) were determined as described
above. Results are displayed in figure 2 and table 2

A statistically significant difference (Variance ratio = 39.48, n = 48, P <0.001)

was seen in the mean SWIFT filterability data for the 8 malts tested
A clear discrimination in mean SWIFT filterability occurred between the feed
varieties and 3 of the malting varieties
SWIFT data for 2 malting varieties did not allow their discrimination from feed
varieties
Wort viscosity was correlated with SWIFT value (r = -0.71, P <0.05); but wort -
glucan and density showed no significant correlation with SWIFT. 91% of the
variation in viscosity between the 8 varieties was explained by wort -glucan
levels.

These data suggested that the SWIFT test was indeed capable of discriminating malts
that were otherwise closely specified. Additional trials are underway to develop the
discriminative power of SWIFT still further, by comparing filterability data over
varying time periods and filter sizes. We are also testing the filterability of over 40
barleys grown over seven sites in South Australia to provide a full evaluation of the
SWIFT test in our barley breeding program.

6
Table 2: Correlation table of SWIFT filterability and malt quality data

WORT -
SWIFT DENSITY VISCOSITY
GLUCAN
SWIFT *
DENSITY -0.051 *
VISCOSITY -0.705* -0.443 *
WORT -
-0.527 -0.538 0.956* *
GLUCAN

CONCLUSIONS
The SWIFT test has been extensively evaluated and shown to be a reliable predictor of
beer filterability.
Further development of SWIFT is underway to improve the discrimination of
similarly specified malts.
We are currently examining SWIFT determinations of HWE filterability from barleys
in advanced breeding trials at several sites across South Australia; these results will be
critical in determining the future of SWIFT as an aid to our breeding programs.

ACKNOWLEDGMENT
This work is supported by the Grains Research and Development Corporation of Australia.
Philippa Tansing and Rebecca Fox of the Barley Quality Evaluation Laboratory, Department
of Plant Science, Adelaide University, are gratefully acknowledged for their assistance with
preparation and analysis of hot water extracts.

REFERENCES
[1] Stewart, D., Freeman, G. and Evans, E. (2000). Development and assessment of a small-
scale wort filtration test for the prediction of beer filtration efficiency. J. Inst. Brew.,
106, 361-366.
[2] Stewart, D., Freeman, G. and Evans, E. (2000). Better prediction of the Beer Filtration
Efficiency of malt, Proceedings 26th Convention of the Institute of Brewing-Asia Pacific
Section, Singapore. 47-51.
[3] Stewart, D.C., Hawthorne, D. and Evans, D.E. (1998). Cold Sterile Filtration: A Small
Scale Filtration Test and Investigation of Membrane Plugging, J. Inst. Brew., 104, 321-
326.
78

A novel and rapid method for the

automatic and simultaneous
determination of total and viable cell
concentration in pitching yeast slurries
Chris A. Boulton1, Wendy G. Box1, John Carvell2 & Kirsty
Turner2
1
Bass Brewers Limited, Technical Centre, P.O. Box 12, Cross Street, Burton on Trent DE14
1XH, United Kingdom (e-mail: [email protected])
2
Aber Instruments Ltd, 5, Science Park, Aberystwyth SY23 3AH, United Kingdom

Descriptors
Accelerated method, automatic analysis equipment, pitching yeast, viability

SUMMARY
Precise measurement of total and viable cell concentration in yeast slurries is required for
accurate control of pitching rate and assessment of yeast quality. Apparatus is described in
which these analyses can be made rapidly and automatically using a small sample of pitching
yeast slurry. Radio-frequency impedance is used to measure viable yeast concentration.
Simultaneously, a bulk fluorescence measurement, in the presence of a membrane potential
dye, allows the determination of the dead cell count. From these two outputs, the viability is
computed, automatically. Results are presented which compare analyses made with this
apparatus and those obtained using conventional methods.

Eine neue und schnelle Methode fr die automatische und simultane Bestimmung der
Gesamt- und vermehrungsfhigen Zell-Konzentration in der Anstellhefe

Deskriptoren
Anstellhefe, Ausrstung zur automatischen Analyse, Lebensfhigkeit, Schnellverfahren

ZUSAMMENFASSUNG
Ein exaktes Messen der Gesamt- und vermehrungsfhigen Zell-Konzentration in der
Anstellhefe ist notwendig, um die Vermehrungsrate zu kontrollieren und eine Aussage ber
die Hefequalitt treffen zu knnen. Hier wird die Vorrichtung beschrieben, in der diese
Analysen einer Anstellhefeprobe schnell und automatisiert durchgefhrt werden kann. Um die
vermehrungsfhige Hefekonzentration zu messen, wird ein Hochfrequenzwiderstand
verwendet. Gleichzeitig erlaubt eine Massenfluoreszenzmessung, in Anwesenheit eines
Membran-Potenzialfarbstoffes die Ermittlung der Zahl der toten Zellen. Diese zwei Werte
erlauben es, die Vermehrungsfhigkeit automatisch zu berechnen. Die hier vorgestellten
Ergebnisse werden im Vergleich zu Ergebnissen dargestellt, die mit herkmmlichen
Methoden ermittelt wurden.

Mthode nouvelle et rapide pour la dtermination automatique et simultane des

concentrations de cellules totales et de cellules viables dans des mlanges de levure
densemencement

Descripteurs
Levain, matriel danalyse automatique, procd acclr, viabilit

RESUME
Une mesure prcise de la concentration en cellules totales et en cellules viables dans les
mlanges de levure est indispensable pour un contrle exact du taux densemencement et
l'valuation de la qualit des levures. Nous dcrivons un appareil permettant d'effectuer
rapidement et automatiquement ces analyses sur un petit chantillon de levure
densemencement. Les mesures sont effectues par impdance de radiofrquences pour
dterminer la concentration en levure viable. Simultanment, une mesure par fluorescence en
prsence d'un colorant de potentiel de membrane permet de dterminer le nombre de cellules
mortes. Avec ces deux chiffres, la viabilit est calcule automatiquement. Nous prsentons
des rsultats comparant les analyses effectues avec cet appareil et ceux obtenus par des
mthodes conventionnelles.

2
INTRODUCTION
Precise control of yeast pitching rate is essential for good fermentation control. A
necessary pre-requisite is the determination of the total and viable yeast concentration
of pitching slurries. The results of this analysis are used to calculate the appropriate
quantity of slurry required to achieve the desired pitching rate. In addition, the relative
proportions of live and dead cells in the slurry can be used as an indication of yeast
quality. The traditional method of measuring total and viable yeast cell concentration,
using a haemocytometer and the vital stain, methylene blue, is slow, labour-intensive
and requires skilled laboratory personnel. Furthermore, the accuracy and
reproducibility of the procedure is poor. This is a consequence of errors introduced by
the necessity for dilution of slurries prior to counting and operator fatigue when
performing microscopic analyses. In addition, it is generally accepted that methylene
blue staining is inaccurate when used with yeast of low viability [7].
The dye, DiBAC4 (Bis-[1,3-dibutylbarbituric acid] trimethine oxonol) is excluded
from viable cells which possess a plasma membrane transmembrane potential.
Conversely, dead cells take up the dye and become fluorescent when excited with
light of a suitable wavelength [5]. It has been suggested that this dye can be used in
conjunction with flow cytometry for the automatic determination of viability of
brewery pitching yeast [1]. This method largely eliminates operator errors and the
fluorophore reportedly provides accurate results with yeast of low viabilit [8].
It has been demonstrated previously that radio-frequency impedance measurments can
be used to provide an instant and precise measure of viable yeast concentration, either
on-line or in the laboratory. The measurement is unaffected by viability and can be
applied directly to pitching yeast slurries without dilution [2,3,4]. The device
overcomes many of the disadvantages of the traditional method, however, since the
sensor is responsive only to the viable fraction of yeast slurries ipso facto it does not
provide information regarding viability.
Here is described a modified laboratory yeast analyser in which a combination of
radio-frequency impedance and bulk fluorescence staining are used to quantify both
the viable and dead fractions of pitching yeast slurries.

MATERIALS AND METHODS

The fluorophore, DiBAC4 was obtained from Molecular Probes Inc., Eugene, Oregon,
USA.
Samples of yeast slurry were removed from brewery storage vessels and if not used
immediately stored at 4oC prior to use. In some experiments mixtures of viable and
dead yeast cells were prepared. The dead fraction was obtained by heating a portion of
yeast slurry for 15 min at 65oC. Mixtures of pre-determined total cell count and
viability were prepared based upon analyses using a haemocytometer and methylene
blue staining [6]. Trub was obtained from a sample taken from the base of a brewery
whirlpool immediately after copper cast. Pure trub was recovered from residual wort
by filtration. The effect of acid washing on yeast viability was simulated by adding
phosphoric acid to chilled yeast slurry to achieve pH 2.2. The slurry was then held for
2 h at 4oC.

The apparatus used in this study (figure 1a) was a prototype which consisted of a
modified standard RF impedance yeast laboratory analyser (Aber Instruments
Limited, Aberystwyth, UK). The device is fitted with a stirred 10 ml sample chamber

3
(figure 1b). A 4-pin electrode system provided the means for measuring total viable
cell counts via radio frequency impedometry. Bulk fluorescent measurements were
made following the addition of DiBAC4. The system excited the slurry at a
wavelength of 470 nm and detected the resultant fluorescence of dead cells at 520 nm.

Figure 1a: An impedometric laboratory yeast analyser

Sample chamber
Fluorimeter

Electrodes

Excitation at
470nm
Radio frequency
field

Detection at
520nm

Magnetic stirrer

Figure 1b: Diagrammatic representation of sample chamber with combined RF

impedance and fluorimetric detectors for viable and non-viable yeast cell detection.

4
RESULTS AND DISCUSSION
In order to make valid viability determinations the measured fluorescence intensity
must be independent of dye concentration, total yeast cell concentration and yeast
strain identity. The effect of varying DiBAC4 concentration on fluorescent intensity of
yeast slurries of a fixed total cell concentration but varying viability is shown in figure
2. The total yeast cell concentration was 8 x 108 cells/ml, a typical value for pitching
yeast slurries cropped from cylindroconical fermenters.

30

25
96.6% viable
Fluorescence intensity

90% viable
20
85% viable
80% viable
15 70% viable
0% viable
10

0
0 2.5 5 7.5 10 12.5 15 17.5
g/ml)
DiBAC4 concentration (

Figure 2: Effect of dye concentration on fluorescent intensity (arbitrary units) for

yeast of varying viability. Slurries of an ale yeast, at the viabilities indicated, were
prepared by mixing live and heat-killed cells, as described in the Methods. The total
yeast cell concentration was 8 x 108 cells/ml. Yeast was suspended in beer.

Predictably, total fluorescence intensity was dependent on viability. For all viabilities
in the range that would be encountered in production brewing, fluorescence intensity
was independent of dye concentration providing that the latter was in excess of a
threshold value of approximately 10 g/ml. In all subsequent determinations, a
DiBAC4 concentration of 12.5 g/ml was used.
Plots of fluorescence intensity against viability for individual yeast strains were
shown to be linear but with differing gradients and overall magnitude (figure 3). The
total yeast concentrations of the slurries used in this experiment ranged from 6 x
108/ml to 1 x 109/ml. Therefore, some of the observed differences in fluorescence
intensity were clearly yeast cell concentration related. In addition, strain-specific
differences in cell size and the number of intracellular dye-binding sites may be
implicated. From a practical standpoint, the instrument will require calibration for
each yeast strain.

5
 10
Lager Strain1
9 Ale Strain 1
Lager Strain 2
8
Fluorescence Intensity

Ale Strain 2
7

0
50 55 60 65 70 75 80 85 90 95 100
Viability (%)

Figure 3: The relationship between fluorescent intensity and viability for four
different yeast strains. Yeast slurries were removed from brewery storage vessels and
mixtures of live and heat-killed cells prepared, as described in the Methods, to give a
range of viabilities between 70 and 95%. Total yeast counts ranged from 6 x 108/ml to
1 x 109/ml.

The effect on fluorescence intensity of using yeast mixtures of varying total cell
count but constant viability is shown in figure 4.

90% viability
8 75% viability

7
Fluoresence Intensity

0
300 400 500 600 700 800
cells/ml ( millions)

Figure 4: Effect of total yeast count on fluorescent intensity for two lager yeast
slurries.

6
Here measurements were made using dilutions of two slurries of the same yeast strain
with viabilities of 90% and 75%, respectively. The results indicated that the total yeast
cell concentration had a small but significant effect on measured fluorescence
intensity. This deviation from ideal behaviour was observed within the range of total
cell concentrations that would be encountered in pitching yeast slurries. To correct for
this potential source of error a software algorithm was developed and applied in all
subsequent determinations.
Using the software algorithm, the prototype instrument was used to provide estimates
of viability for a number of samples of slurry of a lager yeast strain (figure 5). Yeast
slurries with varying viability were prepared by mixing viable and heat-killed cells.
The total cell count varied between 5 x 108/ml and 1 x 109/ml. It was evident that
there was a good correlation between viability as measured using methylene blue
staining and the viability predicted from the measured fluorescence intensity.

100
95
Predicted viability (%)

90
85
80
75
70
65
60
55
50
50 60 70 80 90 100
% viability (methylene blue)

Figure 5: Comparison of viability measured by methylene blue staining compared

with viability predicted from bulk fluorescence measurements.

The results described so far were based on trials using mixtures of viable and heat-
killed cells. This approach was necessary to obtain yeast slurries with a range of
viabilities sufficiently wide to test the efficacy of the method. However, it may be
criticised in that such artificial mixtures are not necessarily reflective of yeast slurries
actually encountered in production brewing.
A possible problem is the presence of variable levels of trub in yeast slurries.
Previous work has demonstrated that this material does not interfere in the RF
impedance measurement of viable yeast concentration [2]. However, Boyd et al.,
2000 [1] reported that trub particles have the ability to bind DiBAC4 and, therefore,
potentially interfere in the viability measurement. The effect on viability
measurements of supplementing yeast slurries with varying concentrations of trub is
shown in figure 6.

7
 100

95
Predicted viability (%)

90

85

80

75

70
0 1 2 3 4 5 6
Trub (% wet wt. per g slurry)

Figure 6: Effect of varying trub concentration on viability of a lager yeast slurry (total
cell count ca 5 x 108 cells/ml) determined by bulk fluorescence measurement. Trub
was isolated from wort as described in the Methods and added to the yeast slurry at
the concentrations indicated.

Within the range tested (1 to 5% g wet weight trub per g slurry) there was no
significant effect on the derived viability measurement.
In many breweries, pitching yeast is subjected to transient exposure to low pH
conditions to reduce bacterial loading. Where yeast is already stressed, this treatment
can cause a significant reduction in viability. The effects on viability of this were
assessed by acid washing a lager pitching yeast slurry, as described in the Methods.
Prior to treatment, the slurry was stored for 2 weeks at 4oC. The intention was that the
stress induced by this prolonged period of starvation would exaggerate the deleterious
effects of acid washing and thereby, magnify observed changes in viability. Viability
before and after acid washing was assessed by methylene blue staining and from bulk
fluorescence intensity. In addition, the change in viable cell concentration, as
determined by radio frequency impedance was also monitored. The latter value was
expressed as a viability by calculation using the RF impedance output and with a
knowledge of the total cell count as measured by haemocytometery. The results
(figure 7) indicated that during acid washing, the viability declined from an initial
value of 90% to approximately 60% at the end. All three methods of viability
assessment showed reasonable agreement.

8
 100

80
Methylene blue
Viability %

60 RF Impedance
Fluorimetry

40

20

0
Before acid wash After acid wash

Figure 7: Effect of acid washing on viability assessed by methylene blue, bulk

fluorimetry and radio frequency impedance. The yeast was a lager pitching slurry,
suspended in beer (total count 3.5 x 108 cells/ml). Further details are provided in the
text.

The results of trials described so far have indicated that bulk fluorescence intensity
measurements can be used to assess the viability of pitching yeast slurries. The
proposed mode of operation for automatic and simultaneous analysis of yeast slurries
for viable cell count and viability using the modified laboratory analyser is shown in
figure 8.

Select appropriate yeast strain

Put 10ml of slurry into sample

Check mixing is ok

Press measure button

Fluorophore is automatically
added to sample

Wait 10 seconds Display of viable yeast count

Wait 20 seconds Display of % viability

Press wash button

Figure 8: Schematic representation of proposed measurement procedure

CONCLUSIONS
It has been demonstrated that bulk fluorescence measurements can be used to measure
the viability of pitching yeast slurries. In combination with radio frequency

9
impedance measurements, the viability and viable cell concentrations of yeast slurries
can be assessed using a single laboratory instrument. Results are obtained in less than
a minute and the analyses require minimal laboratory skills. The results of the
viability test, using yeast populations with viability and physiological condition that
would be encountered in production brewing showed a good correlation with those
obtained by methylene blue staining and manual counting. In the absence of another
independent reference method, it is not possible to compare the relative precision of
the two techniques. Clearly, the failure to demonstrate a perfect correlation may have
been due to shortcomings in either or both methods. For the same reasons, it is not
possible to claim that the fluorescence viability measurement affords greater accuracy
than methylene blue staining, at low yeast viability. However, since fluorescence
staining can be applied directly to undiluted yeast slurry, any errors associated with
sample preparation will be eliminated. Similarly, the automatic detection of bulk
fluorescence removes inconsistencies associated with manual counting. Therefore, it
would be predicted that the repeatability of the fluorescence approach would be
superior to the methylene blue staining technique. Perhaps, repeatability is of greater
importance than absolute precision for routine analysis of pitching yeast.
Based on these trials it is concluded that the instrument could be used in a brewery
production laboratory for the assessment of yeast condition and calculation of pitching
rates. Refinement of the existing prototype instrument is in progress and the launch of
a commercial version is anticipated. There remains a requirement for methods for
assessing the physiological status of pitching yeast. A further application of the device
described here is the use of alternative fluorophores capable of probing aspects of
yeast physiology relevant to fermentation performance.

ACKNOWLEDGEMENT
The authors thank the Directors of Bass Brewers Limited for permission to publish
this paper.

REFERENCES
1. Boyd, A., Attfield, P. and Veal, D., Journal of the Institute of Brewing, 2000, 106, 319-
324.
2. Boulton, C.A., Maryan, P.S., Loveridge, D. and Kell, D.B., Proceedings of the European
Brewery Convention Congress, Zurich, 1989, 653-661.
3. Carvell, J.P., Austin, G., Matthee, A., Van de Spiegle, K. and Cunningham, S., Journal of
the American Society of Brewing Chemists, 2000, 58, 57-62.
4. Cunningham, S., Brewing Yeast Performance, Blackwell Science, Oxford, UK., 2000, 62-
73.
5. Dinsdale, M.G., Lloyd, D. and Jarvis, B., Journal of the Institute of Brewing, 1995, 453-
458.
6. IOB Recommended Methods of Analysis, Institute of Brewing, London, UK, 1997, 21.33
and 21.35.
7. Jones, R.P., Process Biochemistry, 1987, 22, 118-133.
8. Vincent, S., Boyd, A., Attfield, P., Rogers, P. and Veal, D., Proceedings of the 28th
Convention of the Institute of Brewing Asia Pacific Section, 2000, 79-81.

10
79

Real time monitoring of viable yeast

concentration in production fermenters
using novel RF impedance probes
John Carvell1, Robert Todd1, Stephen Cunningham1 & Takeshi
Yonezawa2,3
1
Aber Instruments Ltd, 5, Science Park, Aberystwyth SY23 3AH, United Kingdom (e-mail:
[email protected])
2
Suntory Ltd., Japan
3
Heriot-Watt University, ICBD, Edinburgh, Uinited Kingdom

Descriptors
Cylindro-conical tank, equipment, flocculation, real-time operation, viability, yeast growth

SUMMARY
Reliable instrumentation for monitoring the progress of yeast growth and flocculation is
required for production fermenters. In this paper we describe a novel method using RF
impedance measurements with a new flush electrode for measuring the viable yeast
concentration in real time in a range of brewing vessels. When the probe was installed either
in the side of a fermenter or a re-circulation loop, the critical phases of yeast growth and
flocculation during fermentation were identified. The probe could also be adapted to become
a submergible in situ version for measuring the distribution of live yeast within a
production fermenter.

Die Echtzeitberwachung der vermehrungsfhigen Hefekonzentration in Grtanks

durch RF-Impedanz-Sonden

Deskriptoren
Anlage, cylindrokonischer Tank, Flockung, Hefewachstum, Lebensfhigkeit, Rechtzeitbetrieb

ZUSAMMENFASSUNG
Fr die berwachung des Hefewachstums und der Hefeflockung in Grtanks sind
zuverlssige Messinstrumente erforderlich. In diesem Poster beschreiben wir eine RF-
Impedanz-Messung mit einer neuen glatten Elektrode zur Echt-Zeit-Messung der
vermehrungsfhigen Hefekonzentration. Nachdem die Sonde entweder an der Seite eines
Grtanks oder in einer Umlaufleitung installiert wurde, konnten die kritischen Phasen des
Hefewachstums und der Hefeflockung whrend der Grung identifiziert werden. Die Sonde
konnte auch als eingetauchte in situ Version zum Messen der Verteilung der lebenden Hefe
innerhalb eines Grtanks verwendet werden.
Surveillance en temps rel des concentrations de levures viables dans des fermenteurs de
production au moyen de nouvelles sondes impdance de radiofrquence

Descripteurs
Croissance de la levure, floculation, matriel, tank cylindroconique, traitement en temps rel,
viabilit

RESUME
Les fermenteurs de production exigent une instrumentation fiable pour surveiller l'volution
de la croissance et de la floculation des levures. Dans ce poster, nous dcrivons une nouvelle
mthode de mesure par impdance de radiofrquence (RF) utilisant une nouvelle lectrode
pour mesurer la concentration en levure viable en temps rel dans une srie de cuves de
brassage. Lorsque la sonde est installe sur le ct d'un fermenteur ou dans une boucle de
recirculation, les phases critiques de croissance et de flocculation de la levure pendant la
fermentation ont t identifies. La sonde peut galement tre adapte pour donner une
version immergeable "in situ" permettant de mesurer la distribution des levures vivantes dans
un fermenteur de production.

2
INTRODUCTION
The use of Radio-Frequency (RF) Impedance or cellular capacitance for the on-line
measurement of viable yeast biomass in brewery applications such as pitching and
cropping is common (Boulton and Clutterbuck, 1989; Boulton et al, 1993). These
applications involve the measurement of concentrated samples typically 20 to 60%
yeast solids. Recently, a number of brewers have become interested in monitoring
brewery fermentations on-line and have recognised the advantages reliable
information on the progress of yeast growth and flocculation can have on controlling
the process. Traditional methods of measuring yeast during fermentation require a
representative sample to be removed from the vessel and the information is often
limited due to the limited number of samples that are analysed. The methods usually
used for assessing yeast growth are also labour intensive and may be unreliable as
they can be prone to human error. The limitations with using methylene blue to
determine yeast viability are well known (Pierce, 1970) and it has been shown that the
RF impedance measurements are much more accurate over a significantly wider
viability range (Carvell et al, 2000).
Although the RF Impedance technique is widely used in stirred fermenters in
biotechnology for yeast, bacteria, animal and plant cells mixed results had been
obtained from brewery fermenters. Clearly, the spatial distribution of yeast within the
fermenter is likely to be uneven and time-varying, so measuring at a single point will
not give such clear information as in the fully mixed fermenters. Also, the extremely
slow fluid velocities in the brewing fermenter might allow yeast and/or trub to settle
on the measuring electrodes, creating random noise in the measurements.
The signal obtained from the RF impedance method is cell size dependent and at low
biomass concentrations, this becomes critical in determining the minimum
concentration that can be detected from the background noise. Large yeast (10
microns in diameter) can be detected at much lower cell concentrations than small
yeast that are 5 microns in diameter. The errors involved when measuring at typical
fermenter cell concentrations are also significantly affected by yeast size (Carvell et
al, 2000). The purpose of this work was to develop a reliable method for monitoring
the growth and flocculation of yeast during fermentation.

METHODS
The Yeast Monitor 320 (Aber Instruments, UK) has been specifically designed for
measuring yeast in high density samples such as pitching slurries and during yeast
cropping, therefore various modifications to the existing instrument had to take place
resulting in the development of the Yeast Monitor 330LC (Aber Instruments, UK).
The electronics have been adapted to improve the accuracy and minimise instrument
drift over the course of the fermentation. A new probe with 4 flush platinum electrode
configuration has also been introduced (figure 1a). This prevents yeast and/or trub
settling on the electrodes and giving erroneous data. Part of the results presented have
been obtained with a novel submersible head amplifier and probe that can be lowered
to varying depths within the fermenter. These various modifications have optimised
the measurement in low yeast concentration conditions.
The equipment was initially tested in laboratory scale fermentations conducted in a 30
litre vessel. The vessel had a 25 mm probe port fitted approximately a third of the way
up. The growth media was produced from a commercially available brewing kit and
had Original Gravity 1.044 (11Plato). The yeast employed was samples obtained

3
freshly from brewery yeast storage vessels or dried yeast re-hydrated in wort prior to
pitching. The pitching rate was 11 x 106 cells ml-1 and the fermentation temperature
ranged from 20 to 25C as temperature control was not available on the vessel.

Fig 1a The flush 4-pin electrode Fig 1b Novel Sumersible probe for
for monitoring viable yeast monitoring the spatial distribution of
concentrations in brewery yeast in ferementers in real time.
fermentations on-line

The instrument was also fitted in the pilot brewery facility at the International Centre
for Brewing and Distilling in a 2 hl fermentation vessel. The wort was all lager malt
with OG 1.044 (11Plato), the yeast was a brewing lager strain of Saccharomyces
cerevisiae. The fermentation temperature was maintained at 13C throughout the
fermentation. The main purpose of this experiment was to test the equipment in a
brewery environment where quantities of non-yeast particulate matter such as trub
would be present.
Trials were also conducted at production scale in conjunction with a major
international brewer, therefore the site and the fermentation conditions are
confidential.
Preliminary data is also presented using a novel submersible probe (figure 1b) that
could be lowered to varying depths within a production fermenter.

RESULTS
Laboratory fermentations
The purpose of this set of experiments was to optimise the instrumentation "in-house"
to ensure that reliable result would be obtained in larger scale fermenters. The data
contained in figure 2a was obtained using fresh brewer's yeast that was classified as
small being approximately 6m in diameter and non-flocculant. The instrument was
zeroed in cell free wort prior to pitching and on addition of the yeast gave a reading of
0.6 units. The readings increased to a maximum of 1.8 units after 14 hours of
fermentation and was maintained until the end of fermentation. The maximum signal
was equivalent to approximately 40 x 106 cell ml-1, which was low and most probably
due to limited dissolved oxygen within the wort limiting cell replication. Some noise
was observed during the fermentation, but was equivalent to +/- 1 x 106 cell ml-1
which was within 5% of the total signal. Dried brewer's yeast was also tested and was
rehydrated prior to use. The viable cell numbers for pitching were determined by
manual cell counts adjusted for viability with methylene blue. The gain of the

4
instrument was also increased to improve the capacitance signal and improve the
sensitivity of the instrument. There was also a decrease in the background noise and a
good fermentation profile was observed (figure 2b) as compared to the previous data
(figure 2a).

2.5 7
YM Units, Conductivity (mS/cm)

YM units, Conductivity (mS/cm)

6
2
5
1.5 4
3
1
2
0.5 1

0 0
0 12 24 36 48 60 72 0 5 10 15 20
Fermentation time (hours) Fermentation time (hours)

YM Conductivity (mS/cm) YM (Units) Conductivity (mS/cm)

Figure 2a: Laboratory scale fermentations Figure 2b: Laboratory scale

using fresh brewer's yeast fermentations using fresh brewer's
yeast

Pilot plant
The pilot plant results showed that the trub did not interfere with the yeast
measurement during the growth phase (figure 3). The time interval between readings
was also varied during the fermentation and this accounts for the elevated signal noise
at the start of fermentation. Data was missed between 72 to 144 hours due to problems
with the data logger and accounts for the plateau in the trace.

1.6 18
1.4 16
YM Units, Conductiv ity

14
Temperature (oC)

1.2
12
1
(mS/cm)

10
0.8
8
0.6
6
0.4 4
0.2 2
0 0
0 48 96 144 192 240 288 336 384 432
Fermentation time (hours)

YM Conductivity Temperature

Figure 3: On-line monitoring of a pilot plant beer fermentation

During the first 24 hours of fermentation (lag phase) the RF Impedance signal started
to increase whereas the cell numbers as determined by manual cell counting, remained
constant. As the YM is affected by cell size changes this increased signal may have
been due to an increase in the cell size and bud formation, rather than an increase in
cell number. This information may be useful to the brewer as it is an indication that
the yeast is metabolically active.
The Yeast Monitor signal began to decrease after 144 hours of fermentation as the
yeast had begun to flocculate and sediment to the bottom of the vessel. When the

5
vessel was cooled after 168 hours there was a corresponding drop in conductivity and
also a more rapid decrease in the capacitance signal. After the initial drop, the
decrease in the capacitance signal became more gradual due to the yeast cells falling
to the bottom of the fermenter. During lagering when the temperature was further
reduced the capacitance reading returned to zero indicating that the number of yeast in
suspension is less than the detection limit of the instrument.

Production fermenters
Initial data was obtained from the probe being fitted in a recirculation loop on a
production fermenter at the Guinness St James Gate brewery and some data were
previously reported (Carvell et al 1999). The challenge was to repeat these data when
the probe was fitted in the side of a production fermenter as the fluid velocity past the
probe would be a lot lower.
Results from initial trials of the instrument fitted to the side of a production scale
fermenter have shown that the fermentation can be monitored satisfactorily in this
way and that the results correlated well with manual yeast counts (figure 4). The yeast
was pitched into the first brew and the dilution effect of the yeast concentration to
approximately 20 x 106 cells ml-1 as the fermenter continued to be filled can be clearly
seen. The on-line data correlated very well with manual cell counts during late log and
stationary phase (i.e. 71 to 109 hours of fermentation), but differences were observed
at the end of fermentation and were due to the yeast cells flocculating. These
differences may have been due to problems associated with counting clumped cells or
changes in the electrical properties of flocculated and non-flocculated yeast.

140000000 1.6

1.4
120000000
Conductivity,mS/cm Temp,deg C/10
1.2
100000000

1
80000000
cells/ml

0.8

60000000
Aber-CC 0.6
lab
40000000 Conductivity
0.4
Temp/10

20000000
0.2

0 0
1
21
41
61
81
101
121
141
161
181
201
221
241
261
281
301
321
341
361
381
401
421
441
461
481
501

time(15mins)

Figure 4: Production scale fermentation monitored using RF Impedance

Submersible electrode
The submersible probe was tested in a production scale whisky fermenter (data
courtesy of T. Yonezawa of ICBD Heriot Watt University and Suntory, Japan). The
probe was placed placed two thirds the way down the vessel. Such a probe was ideal
in this situation as the production vessels were oak fermenters that could not be
adapted to take an additional probe. The data presented in figure 5 clearly show that
the growth and decline in live yeast cell concentration with peak biomass
concentration being reached 20 hours after pitching. It was also noted that the death
phase after 20 hours coincided with a drop in measured capacitance, but an increase in

6
wort conductivity was also observed which was due to yeast autolysis. The yeast
death phase is of interest as it is during this period when important flavour compounds
are produced.

55 5
53 4.5
C
Capacitance
GConductivity
51

Conductance (S/cm)
4

Capacitance (pF)
49
47 3.5
45 3
43 2.5
41
2
39
37 1.5
35 1
0 10 20 30 40 50 60 70 80 90 100
Fermentation Periods (hrs)

Figure 5: A Scotch whisky fermentation monitored using a submersible electrode

CONCLUSIONS
The data presented highlights that RF Impedance can successfully measure viable
yeast concentration on-line in real-time during brewery fermentations. There was
minimal interference from trub and the instrument noise was acceptable (within 1
million cells ml-1 when measuring down to 20 million cells ml-1). The development of
a probe with 4 flat electrodes that are flush with the end of the probe has significantly
improved the performance of the instrument in non-stirred fermentation systems.
Using the RF Impedance method to follow yeast growth during brewery fermentations
on-line can provide useful information to the brewer. The initial pitching rate can be
checked and the lag phase prior to the exponential growth phase can be monitored to
ensure that the fermentation is proceeding correctly. The instrument can also measure
the peak cell number to ensure sufficient yeast growth has occurred. The decrease in
cell numbers at the end of fermentation can also be monitored to determine that the
yeast sedimenting at the correct time. The rate of yeast growth can have a critical
effect on the time taken to complete fermentation and/or reduce impurity levels. The
real-time measurement of the viable yeast concentration gives an early indication of
growth profiles out-with the desired specification may allow corrective action to be
taken.
Due to the improved instrument accuracy at low yeast concentrations it is now ideal
for a number of other applications such as the Kraussen process or monitoring
propagation vessels, ensuring that the correct amount of yeast is transferred and
leading to improved plant productivity. It is hoped that developments to the
instrumentation will further improve its accuracy especially with small yeast cells at
the low cell concentrations such as those encountered after pitching.
Measurements at yeast concentrations typically found during brewery fermentations
are difficult as the measured capacitance may be very small. Care must therefore be
taken to minimise the effects of varying temperature and conductivity which can
cause errors in the capacitance measurement. As the capacitance measured depends
on yeast cell diameter as well as on the concentration, low concentrations of small
yeast cells present the biggest challenge to measurement accuracy. The data presented
in figure 6 identifies the typical measurement errors when using the Yeast Monitor

7
330LC. The errors may be significantly smaller than shown when interfering variables
such as conductivity and temperature are optimally controlled. Further developments
are aimed at reducing these errors even further.

25
Figure 6: Estimated
6 errors expected with
20 the Yeast Monitor
330LC with varying
cell size.
15
+/- % Error

Cell Diameter
7 (microns)
10

5
10

0
0 50 100 150 200 250 300
Yeast Concentration (million cells/ml)

Initial data from the submersible probe show that fermentations can be accurately
monitored using this device. Further research with this equipment is planned to
investigate the movements of yeast within a fermenter during fermentation. As the
instrument can be multiplexed, up to 8 different points within the fermenter can be
monitored simultaneously. The information gained from such a system will show how
yeast concentration varies over the depth or other areas of the vessel during
fermentation. It may also be an important tool in determining how different fermenter
designs will impact on the yeast mixing patterns over fermentation.

ACKNOWLEDGEMENT
The authors wish to express their gratitude to Robert Todd and Zoe Philips (Aber
Instruments), Chris Boulton (Bass Brewers, UK), Takeshi Yonezowa (Suntory,
Japan), International Centre for Brewing and Distilling, (Scotland) and Gearoid Cahill
(Guinness Ireland) and the other groups that took part in these trials.

REFERENCES
1. Boulton, C.A., Maryan, P.S. and Loveridge, D., Proceedings of the European
Brewery Convention Congress 1989, 653-661.
2. Boulton, C.A. and Clutterbuck, V.J., Proceedings of the European Brewery
Convention Congress, 1993, 509-516.
3. Carvell, J.P., Austin, G., Matthee A., Van de Spiegle, K and Cunningham, S.,
Journal of the American Society of Brewing Chemists, 2000, 58, 57-62.
4. Carvell, J.P., Cahill, G. and Todd R., Poster at ASBC Annual Meeting, 1999.
5. Pierce, J.S,. The Journal of the Institute of Brewing, 1970, 76, 442-443.

8
80

The use of GC-olfactometry to assess

hop aromatic quality
Guillaume Lermusieau & Sonia Collin
Universit Catholique de Louvain, Unit de Brasserie et des Industries Alimentaires, Croix du
Sud, 2 Bte 7, B-1348 Louvain-la-Neuve, Belgium (e-mail: [email protected])

Descriptors
Aromatic compound, beer aroma, hop constituent, sensory analysis result

SUMMARY
For many years, research on hop aromatic compounds has focused on the qualitative analysis
of markers allowing discrimination between hopped and unhopped beers. Unfortunately, the
usual techniques such as GC-FID or GC-MS yield poor information. Our strategy was
therefore to use GC-olfactometry to pinpoint hop derived compounds that really influence
beer flavour. This technique alllowed us to identify new compounds of interest. Comparisons
with hop dichloromethane extracts enabled us to evidence very active compounds occurring at
diverse concentrations according to the cultivar. On the basis of our results, we propose a new
explanation for the quality of the Saaz variety.

Die Verwendung der GC-Olfaktometrie zur Beurteilung der Qualitt des Hopfenaromas

Deskriptoren
Aromatische Verbindung, Bieraroma, Ergebnis von sensorischen Analysen, Hopfen-
bestandteil

ZUSAMMENFASSUNG
Fr viele Jahre hat die Forschung auf dem Gebiet der Hopfenaromen ihr Augenmerk auf die
qualitative Analyse von Markern gerichtet, mit denen man den Unterschied zwischen
gehopftem und ungehopftem Bier untersuchen konnte. Leider liefern die blichen Methoden
wie GC-FID oder GC-MS nur wenige Informationen. Unsere Strategie war es, mit Hilfe der
GC-Olfaktometrie genau zu bestimmen, welche Hopfenbestandteile wirklich Einfluss auf das
Bieraroma haben. Vergleiche mit Dichloromethanextrakten ermglichten uns, ber den
Nachweis der Konzentration sehr aktiver Substanzen die einzelnen Hopfensorten zu
unterscheiden. Aufgrund unserer Ergebnisse schlagen wir eine neue Qualittsbestimmung der
Saazer Sorten vor.
Lutilisation de la GC-olfactomtrie pour estimer la qualit aromatique des houblons

Descripteurs
Arme de la bire, compos aromatique, constituant du houblon, rsultat d'analyse
sensorielle

RESUME
Depuis plusieurs annes, les recherches sur les composs aromatiques du houblon se sont
concentres sur lanalyse qualitative des molcules permettant de diffrencier les bires
houblonnes et non-houblonnes. Malheureusement, les techniques habituelles telles que la
GC-FID et la GC-MS apportent peu dinformations. Notre stratgie ft ds lors dutiliser
lanalyse olfactomtrique en sortie de colonne chromatographique pour rechercher les
composs qui interviennent rellement dans larme de la bire. Cette technique nous a
permis de mettre en vidence de nouveau composs importants. Des comparaisons avec des
extraits de houblon au dichloromthane ont galement permis disoler certains composs trs
actifs organoleptiquement et prsents diverses concentrations selon les varits. Sur la base
de ces rsultats, nous sommes en mesure de proposer une nouvelle explication pour les
qualits aromatiques de la varit Saaz.

2
INTRODUCTION
Aromatic hops are usually defined only according to their low -acid content (< 5%),
as opposed to bitter varieties. The brewers experience is therefore crucial to selecting
the best cultivars taste-wise. To give brewers indicators of a more quantitative nature,
we have attempted to determine what, exactly, an aromatic hop is4.

Many compounds found in hops are known to survive up to the final beer. This is the
case of -terpineol, caryophyllene oxide, geraniol, humuladienone, humulene
epoxides, humulenol II, humulol, linalool, linalool oxide, citronellol, geranyl acetate
and terpinen-4-ol 6,9. Yet who could say which of these are both organolepically
active and desirable? The list, furthermore is probably incomplete, as extremely low
concentrations of certain compounds, undetectable by standard chromatographic
detectors, are often sufficient to modify beers aromatic features.

Most published work to date has focused on the complete identification of volatiles by
gas chromatography-coupled with FID or mass spectrometry. Unfortunately, the
human detector, i.e. the nose, is often much more sensitive than the most advanced
physico-chemical techniques. For this reason, we have preferred the opposite
approach: instead of determining the chemical composition of hopped beer, we have
used AEDA-GC-Olfactometry10 to construct their aroma profiles and have evidenced
which hop compounds are the most important for beer character in terms of odour and
intensity. The next step was obviously to determine the structures hidden behind these
odours on the basis of their retention times on two chromatography columns, the SCD
detector responses, and their mass spectra.

BREWING, EXTRACTION AND ANALYTICAL PROCEDURE

Three lagers were brewed, one without hop and two with hop pellets, either Saaz or
Challenger, at a hopping rate of 1.8 g/l. 15 Litres of a 12P industrial wort were boiled
1h15 and the hop was added 7 min before the end. Fermentations and maturations
were conducted in 3-l EBC-tubes at 12C for 5 days, 13C for one day, 14C for one
day, 15C for three days, 7C for 3 days and 0C for 24 hours.

Triangular tests were conducted with the three beers described above. The panel was
asked to smell only the glass headspace in order to avoid any interaction of aroma
with the bitter taste imparted to beer by isohumulones. The tests were carried out in
three sessions with a panel of 12 assessors, each session comparing 2 beers. For each
session, the panel was also asked to describe the aroma of the product.

An optimised XAD-2 extraction procedure2,3,5 was used to recover beer aromatic

compounds.

GC-Olfactometry analytical conditions

For the sniffing analyses, we used a Chrompack CP9001 gas chromatograph equipped
with a splitless injector maintained at 250C and opened after 0.5 min. Compounds
were separated using a 50 m x 0.32 mm, wall coated open tubular (WCOT) apolar CP
SIL5 CB capillary column (1.2 m film thickness) connected to an FID detector. The
oven temperature was set to rise from 36 to 120C at 20C.min-1, to 250C at
2C.min-1, and then to remain constant at 250C for 30 min. To assess the olfactory

3
potential of the compounds, a T-junction was used at the end of the capillary column.
50% of the eluent was sent to the FID detector maintained at 280C and connected to
a Shimazu CR6-A integrator, while the other part was directed to a GC-odour port at
250C. In the latter case, the eluent was diluted with a large volume of air (20
ml.min-1) previously humidified using an aqueous copper (II) sulphate solution. 2 l
of the beer extract were injected.

GC-Sievers analytical conditions

The column was directly connected to a Sievers 355 SCD (sulphur
chemiluminescence detector). In the 800C combustion room of the detector, the air
and hydrogen flows were maintained at 40 and 100 ml.min-1, respectively. A 6-psi
airflow was applied in the ozone generator under a vacuum (150-275 Torr) obtained
by an Edwards oil-sealed RV5 pump.

SENSORIAL ANALYSES
Three beers were compared, one brewed without hop, one with Saaz, and one with
Challenger. In the latter two cases, only late hopping was investigated.

As depicted in table 1, the three beers were recognized to a significant extent in

triangular tests, although panellists found likeness between the unhopped and the Saaz
beer.

Comparison Detection rate

Beer without hop >< Beer + Saaz 67%*
Beer without hop >< Beer + Challenger 92%*
Beer + Saaz >< Beer + Challenger 82%*

Table 1: Rate (%) of correct answers in triangular tests (* significant with a 5%

threshold7).

The beer brewed without hop had a strong fruity flavour quite similar to that of cider.
The Saaz-hopped beer was characterized by a "beer-like" flavour with fresh and
pleasant spicy notes, while the Challenger-hopped beer exhibited cheese-like heavy
flavours (table 2).

Beer brewed without hop Beer + Saaz Beer + Challenger

Fruity (banana) fruity (citrus) hop
Flowery Flowery sulfur
Fresh Fresh heavy
Sweet Spicy cheesy
Cider-like Beer-like aggressive
Pleasant Pleasant unpleasant

Table 2: Odour description of the three beers.

Figure 1 shows the aromagrams drawn for our three beers. More than forty odours
were perceived in the beer brewed without hop (figure 1a). The horizontal line

4
represents the threshold above which all the odours should be perceived even without
synergy (calculated on the basis of isoamyl acetate concentration compared to its
flavour threshold5). The other aromas may also interact but in synergy rather than
alone. When hop was added at the end of boiling (figures 1b & c), the number of
peaks almost doubled. A few odours appeared as determinants of beer flavour even
though most were characterized by an n value below 5 (n = number of GC-O
detections through the dilutions).

In all three beers we found, as expected, esters and fusel alcohols such as isoamyl
alcohol, -phenylethanol, isoamyl acetate, ethyl caproate, and ethyl caprylate (table
3). 4-Vinyl guaiacol and 2-aminoacetophenone appeared as two important additional
flavouring compounds. Among the odours also present in the unhopped beer, two
appeared more intense when hop was added. The former was a fruity/sweet aroma
identified as -nonalactone while the latter revealed to be -damascenone (table 3).

N
Odours (R.I.) Beer Beer + Beer + Compounds
without hop Challenger Saaz
Solvent (711) 8 7 7 isoamyl alcohol
Sweet (849) 7 7 7 isoamyl acetate
fruity/rum (974) 5 6 6 ethyl caproate
Lily (1092) 7 7 8 -phenylethanol
fruity/flowery (1178) 4 4 4 ethyl caprylate
Honey (1274) 6 6 5 2-aminoacetophenone
Dentist (1289) 6 6 7 4-vinyl guaiacol
Fruity (1325) 4 6 6 -nonalactone
apple/peach (1372) 4 7 7 -damascenone

Table 3: Odours common to extracts of all three beers (n = number of times the odours were
perceived through the dilutions). R.I. = Retention Index calculated on the basis of
alcanes on a CP-Sil 5 CB chromatography column.

Some new flavours were found in hopped beers only (table 4): a typical spicy/hoppy
odour at R.I. = 810 with an n value of 8-9. With such an intensity, it clearly
influences the beers organoleptic quality. Until now this key compound has never
been mentioned in the literature because no peak is detected by usual detectors.
Dimethyltrisulfide was another important aroma in fresh hopped beers, with its well-
known "onion" odour usually associated with staling1.

Table 4 shows six other flavours found only in both hopped beers. Among them, only
linalool had been mentioned previously as contributing to the aroma of hopped beer.

5
A
n 9

0
600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700

retention index = Ik
(CP-Sil 5 CB)

B
n 9

0
600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700

retention index = I k
(CP-Sil 5 CB)

C
n 9

0
600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700

reten tio n index = I k

(CP-Sil 5 CB)

Figure 1: Aromagrams of the unhopped (a), Challenger (b) and Saaz (c) beers.

6
 N
Odours (R.I.) Beer Beer + Compounds
Beer + Saaz
without hop Challenger
spicy/hop/leek (810) - 9 8 unknown
sweat/fruity (938) - 6 6 unknown
sweat/grapefruit (953) - 6 6 unknown
onion soup (961) - 7 7 dimethyltrisulfide
Coriander (1083) - 5 6 linalool
Plastic (1126) - 5 5 unknown
Phenol (1391) - 6 5 unknown
Strawberry (1441) - 5 6 ethyl cinnamate

Table 4: Odours common to extracts of both hopped beer but absent from the beer brewed
without hop.

As described in table 5, many unpleasant odours were perceptible only when the beer
was hopped with Challenger. This probably explains the heavier flavour of this beer.
On the other hand, few typical Saaz beer aromas were detected, most of them being,
however, fresh and pleasant. Such is humuladienone previously mentioned in the
literature as a determining hop compound8.

n
Odours (R.I.) Beer Beer + Compounds
Beer + Saaz
without hop Challenger
Cheesy (734) - 6 - dimethyldisulfide
sweat/rubber (913) - 5 - diethyldisulfide
woody/flowery (1050) - 5 - unknown
Geranium (1070) - 5 - unknown
burned plastic (1165) - 6 - unknown
Unpleasant (1357) - 5 - unknown
Perfume/pine (1360) - x 5 geranyl acetate
Cheesy (1393) - 5 - unknown
Flowery (1421) - - 5 unknown
Pine (1537) - - 6 unknown
Terpene (1569) - - 6 unknown
flowery/fresh (1580) - x 5 humuladienone

Table 5: Odours found only in one type of hopped beer.

Both fresh hops were further analysed by GC-olfactometry. 45 GC-O peaks, mainly
described as corresponding with cooked vegetable, cabbage, cheesy, catty, and sweaty
odours, were detected in the Challenger variety. On the other hand, only 19 odours
were found in the Saaz variety, all of which were attributed much more pleasant
descriptors such as fruity, greenery, or pine. Most of the hop flavours found in fresh
beers resulted from transformations occurring during boiling and fermentation. Only 2
intense odours found in beer proved to have derived directly from hop: the famous
spicy/hoppy odour at R.I. = 810 and the coriander aroma from linalool.

Since the main aromatic differences between the two cultivars appeared to be due to
sulfur-like flavours, we analysed the two samples by GC-SCD (Sulphur
Chemiluminescence Detection) (figure 2). As foreseeable, the Challenger variety was
characterized by huge amounts of sulfur compounds (82), while the Saaz variety

7
contained almost none. Among the six hop varieties tested, the Saaz cultivar yielded
the lowest number of SCD peaks (table 6). On this basis, a non-quality
classification of hops, taking into account the increasing number of sulfur peaks, has
been proposed; it seems to correspond quite well with the brewers' experience.

(a)

(b)

Figure 2: SCD-chromatograms of (a) Saaz and (b) Challenger hops.

8
 Number of SCD peaks
Variety
(area > 5000 V.sec)
Saaz 10
Tettnang 19
Nugget 19
Hallertau Hersbrcker 20
Styrian Goldings 37
Challenger 82

Table 6: Comparison of the amount of sulfur compounds in 6 hop varieties.

CONCLUSION
The Challenger variety is characterized by large amounts of sulfur compounds leading
to a beer with heavy unpleasant flavours. On the other hand, Saaz hop exhibits only a
few peaks with the SC-detector while some delicate specific odours are detected by
GC-olfactometry in the resulting beer.

Among the hop compounds previously mentioned in the literature, only linalool and
humuladienone seem to have a determining influence on the hoppy character of beer.
On the other hand, new compounds were evidenced, among which a typical
spicy/hoppy flavour, found as one of the most intense peaks in both hop and beer
aromagrams. Yet a large number of hop-related compounds found in beer seem to be
derived from hop modifications occurring during boiling and/or fermentation.

As a general conclusion we advice the growers, sellers and brewers to perform SCD
analyses of hops to assess the real aromatic potential of each cultivar.

ACKNOWLEDGMENT
Guillaume Lermusieau is grateful to the Interbrew-Baillet Latour Foundation
(Leuven, Belgium) for financial support.

REFERENCES
1. Gijs, L., Perpte, P., Timmermans, A. & Collin, S., Journal of Agricultural and
Food Chemistry, 2000, 48, 6196-6199.
2. Guyot, C., Chevance, F., Lermusieau, G. & Collin, S., Journal of Agricultural
and Food Chemistry, 2000, 48, 5850-5855.
3. Hawthorne, D.B., Kavanagh, T.E. & Clarke, B.J., Journal of the American
Society of Brewing Chemists, 1987, 45, 23-27.
4. Lermusieau, G. & Collin, S., Journal of the American Society of Brewing
Chemists, 2001, 59, 39-43.
5. Lermusieau, G., Bulens, M. & Collin, S., Journal of Agricultural and Food
Chemistry, 2001, in press.
6. Moir, M., J. De Clerck Chair V, 1992, Leuven, Belgium.
7. Sauvageot, F., Techniques danalyses et de contrle dans les industries
agroalimentaires: principes et techniques danalyses ; Linden G., Ed. ;
Lavoisier : Paris, France, 1991 ; Vol. 2, 381-448.

9
8. Shimazu, T., Hashimoto, N. & Eshina, T., Journal of the American Society of
Brewing Chemists, 1975, 33, 7-12.
9 Tresll, R., Friese, L., Fendesack, F. & Kppler, H., Journal of Agricultural and
Food Chemistry, 1978, 26, 1422-1426.
10. Ullrich, F. & Grosch, W., Journal of the American Oil Chemist Society, 1988,
65, 1313-1317.

10
81

Analytical device for measuring the

ethanol concentration in beer based on
NIR absorption
Gerald Zanker1 & Roman Bene2
1
Brau Union sterreich AG, Triesterstrasse 357, A-8055 Graz, Austria (e-mail:
[email protected])
2
Anton PAAR GmbH, Krtnerstrasse 322, A-8054 Graz, Austria

Descriptors
Alcohol percentage, analysis method for beer, infrared radiation, laboratory equipment

SUMMARY
Beer analysis is important for improving product quality and production control. It is pre-
dominantly based on wet-chemical analytical techniques. Here an alternative is presented: a
near infrared analyser - the Alcolyzer - for the rapid measurement of the ethanol concentration
in beer. The Alcolyzer differs from other similar commercial devices because it only requires
a two point calibration with water and an arbitrary aqueous ethanol solution. The accuracy of
the Alcolyzer was validated by measuring more than 150 beers of various types and origins.
This yielded the following specifications:
range: 0 15 % v/v repeatability: 0,01 % v/v
accuracy: 0,04 % v/v resolution: 0,01 % v/v
As a result, the Alcolyzer can be considered as a new standard instrumentation/ method for
the ethanol determination in beer.

Analytische Einheit, basierend auf NIR-Absorption zur Messung der thanol-

konzentration im Bier

Deskriptoren
Alkoholgehalt, Analysenmethode fr Bier, infrarote Strahlung, Laboratoriumausstattung

ZUSAMMENFASSUNG
Die Bieranalyse ist fr eine Verbesserung der Produktqualitt und zur Steuerung des
Herstellungsprozesses wichtig. berwiegend basiert sie auf nasschemischen analytischen
Techniken. Alternativ dazu wird hier ein Nah-Infrarot-Detektor der Alcolyzer fr eine
schnelle Messung der thanolkonzentration im Bier vorgestellt. Der Alcolyzer
unterscheidet sich von anderen hnlichen kommerziellen Detektoren durch eine Zwei-Punkt-
Kalibrierung mit Wasser und einer willkrlichen wssrigen thanollsung. Die Genauigkeit
des Alcolyzer wurde durch Messung von mehr als 150 Bieren verschiedener Art und
Ursprungs validiert. Die folgenden Spezifikationen haben sich dabei ergeben:
Bereich: 0 15% v/v Wiederholbarkeit: 0,01% v/v
Genauigkeit: 0,04% v/v Auflsung: 0,01% v/v.
Daraus folgt, dass der Alcolyzer als neue Standardmethode/-instrument fr die thanol-
ermittlung im Bier angesehen werden kann.

Systme analytique de mesure de la concentration d'thanol dans la bire par

absorption NIR

Descripteurs
Matriel de laboratoire, mthode danalyse de la bire, radiation infrarouge, teneur en alcool

RESUME
L'analyse de la bire est importante afin d'amliorer la qualit du produit et le contrle de
production. Elle repose essentiellement sur des techniques analytiques en milieu humide.
Comme alternative, nous prsentons un analyseur dans le proche infrarouge - l'Alcolyzer -
pour une mesure rapide de la concentration d'thanol dans la bire. L'Alcolyzer diffre
d'autres appareils dj sur le march par un talonnage en deux points avec de l'eau et une
solution aqueuse arbitraire d'thanol. L'exactitude de l'Alcolyzer a t valide en mesurant
plus de 150 bires de diffrents types et origines, donnant les spcifications suivantes:
limites: 0 15 % v/v rptabilit: 0,01 % v/v
exactitude: 0,04 % v/v rsolution: 0,01 % v/v
En consquence, l'Alcolyzer peut tre considr comme un nouvel appareil standard pour le
dosage de l'thanol et de la bire.

2
INTRODUCTION
The simple, fast and accurate determination of alcohol content in beers is very
important for beer fermentation, quality assessment, trading and even consumption.
Traditional analysis methods such as distillation or GC are time consuming and
require experienced operators. Simpler methods such as the combined density and
refractive index method or boiling point determination (Ebulliometer) tend to be
inaccurate, because the underlying measuring properties are non-specific to alcohol.
Near infrared (NIR) spectroscopy-based methods can be both fast and accurate, but
currently suffer from the need for elaborate calibrations and the limited applicability
of these calibrations.
Anton Paars new, patented method [1] of using NIR spectroscopy to determine beer
alcohol content avoids the shortcomings of present NIR-based systems. Its application
led to a NIR-based dedicated alcohol analyzer offering simple calibration, simple
operation and outstanding performance at a very attractive price.

METHOD
NIR absorption spectra are caused by the combinations and overtones of the
fundamental vibrations of the various molecules contained in the samples. These
spectra are considered to be analytically poorly specific because NIR absorption
bands tend to be broad and overlapped. This apparent disadvantage of using NIR
spectra for quantitative analysis is counter-balanced by powerful, inexpensive opto-
electronic components, by the sophisticated evaluation algorithms which are available
and by practical advantages such as easy sample handling due to reasonable sample
cell volume etc. However, major disadvantages have remained until now when using
NIR spectroscopy for precise quantitative analysis: the time-consuming procedure of
acquiring representative calibration sample sets, the skilled personnel required for
modeling the calibrations and the limited applicability of such calibrations, resulting
in large errors if sample constituents were not properly represented by the calibration
sample sets.

0.62

0.61 A
2

0.60
A
3
0.59

0.58
A
absorption

0.57
A
1
0.56

1 2 3
0.55

0.54

0.53

0.52
1165 1170 1175 1180 1185 1190 1195 1200
wavelength

Figure 1: Evaluation method

A new approach by Anton Paar of using NIR spectroscopy for a dedicated alcohol
analyzer overcomes the above disadvantages. The main idea is to use a small, highly
alcohol-specific spectral range only, combined with a simple but powerful evaluation
algorithm. This strategy virtually eliminates the influence of other known sample
components. Extremely high repeatability and resolution of the measured sample
absorbance is required to arrive at the requested small error (< 0.05 % v/v) and high
repeatability (0.01 % v/v) of the determined alcohol content.
A highly alcohol-specific range of the spectrum was identified between 1150 and
1200 nm [1]. The evaluation method uses the significant alcohol peak in this area and
two spectral points very close to it for defining the baseline (see figure 1). Extensive
investigations showed that the alcohol results based on this type of evaluation are
virtually free of influences from other known beer constituents [2]. This allows
calibrations to be done simply with water for the zero point and a binary ethanol/water
mixture for slope adjustment. This therefore eliminates the problems of traditional
NIR analysis requiring large numbers of different calibration samples to achieve
merely narrow ranging calibrations and all related pitfalls.
In order to achieve the required accuracy and repeatability of the measurement result,
a spectral resolution of 2 nm and a high wavelength stability ( 0.01 nm) as well as an
extremely high resolution and stability of the measured absorbance (< 0.01 mAU) are
necessary. This requires a stable and compact optical set-up and a low noise, highly
linear electronic circuitry.

APPARATUS
Based on this new approach, a bench-top instrument for beer analysis called Alcolyzer
was developed. It utilizes a straight optical set-up without any moving parts (see
figure 2). The instrument consists of an electronically modulated LED as a near
infrared light source, a condenser lens to generate a parallel beam, a 1 ml stainless
steel sample cell with glass windows and 7 mm optical path length, a collimator lens
to focus the parallel beam onto the spectrometer entry slit and a miniature grating
spectrometer with a detector array (InGaAs). The sample cell is temperature
controlled by a thermoelectric solid state (Peltier) thermostat with a stability of
0.01 C. Almost all the optical components are custom designed. Analogue signal
electronic circuitry is minimized to preamplifiers and analogue-to-digital converters.
Synchronous demodulation with a one second integration time and signal processing
of all spectral channels is done in the digital domain.
A measurement is carried out in approximately one minute, whereby most of the time
is required for sample filling and attemperating. The Alcolyzer can be filled with
samples either by syringe, peristaltic pump or automatic sample changer. The basic
configuration measures the alcohol content of beer in % volume per volume. By
optionally connecting a density meter, the Alcolyzer can also display alcohol %
weight per weight, density, real extract, original gravity, degree of fermentation, and
energy value. Results are displayed, stored in an internal data memory and transferred
to a printer or PC. A barcode reader and a keyboard can be connected for easy sample
identification.
Under normal circumstances, the Alcolyzer requires a re-adjustment with pure water
once per day to once per week. About four times per year, a re-adjustment with an
ethanol/water mixture of known concentration is necessary. This is sufficient for the
alcohol content determination of beers with an alcohol range of 0 to 15 %v/v with an
error of less than 0.05% v/v at the 95% confidence level. Sample preparation other
than the degassing of samples is not necessary.

4
 Sample out

Dispersive Element

Detector
Light source
Lens Array
Lens

Glass window
Sample in
Cuvette

Thermostatting of
the cuvette

Figure 2: Optical set-up

Figure 3: Alcolyzer

5
RESULTS
The tests have been carried out at the Puntigam brewery (Graz, Austria). During the
test trial the ethanol concentration of about 200 various beers (mostly from Austria)
were measured with the Alcolyzer and distillation as the reference method [3].
Different types of beers manufactured and bottled by the Puntigam brewery were
investigated. These include lager beers, pale beers, alcohol-free beers, turbid beers,
beer/lemonade mixtures (Radler), strong beers (about 7 % v/v alcohol and more than
14 P original gravity) and beers directly from the fermentation tanks. Additionally,
some beers from Germany, the U.S.A., England, the Czech Republic, Slovakia,
Ireland, Denmark, Poland and Romania were measured. The measurement set-up
consisted of an Alcolyzer, a density meter DMA 4500 and an automatic sampler SP-
1m (all from Anton Paar, Austria).

Repeatability
To evaluate the repeatability, the following procedure was chosen: The automatic
samplers vessels were filled with one type of beer (Puntigamer Mrzen, Austria) and
distillate water, so that 5 vessels of beer were followed by 2 water vessels. In total, 18
beer vessels and 6 water vessels were placed in the carousel of the automatic sampler
SP-1m. The measurement took about 1 hour. The repeatability corresponds to the
standard deviation of the measurement series with one type of beer, since neither
sample, operator, measurement device, nor environmental conditions were changed
within the short time of the measurement. A repeatability of 0.010 % v/v was
determined.

Accuracy
The accuracy was evaluated by comparing the ethanol concentration determined by
the Alcolyzer and distillation. According to the error propagation law, the standard
deviation of the differences between the Alcolyzer distillation (dif ) includes the
accuracy (acc ), repeatability of the Alcolyzer (Alc ), and repeatability of distillation
(dist ). The following is valid:

dif
2
= acc + Alc + dist
2 2 2
equation 1

The repeatability of the distillation is 0.02 % v/v [4]. The accuracy within a
confidence interval of 95% can be simply computed by multiplying of the standard
deviation acc by 1.96.

Table 1 compares the Alcolyzer to the reference method for all the beer types
measured. It can be seen that the mean deviation between the Alcolyzer and
distillation is around the zero for all the beer types. The Alcolyzer therefore shows no
systematic error. The overall accuracy for the 95% confidence interval is about
0.04 % v/v.
It has to be stressed that the same calibration model was used for the all the beers
measured. Hence, the model works for the naturally turbid beers and beers from the
fermentation where the yeast was not filtrated before measuring with the Alcolyzer.

6
Beer type # samples Min. alcohol Max. alcohol Mean Accuracy
[% v/v] [% v/v] deviation (95% conf)
[% v/v] [% v/v]
From 39 1.20 6.15 0.01 0.05
fermentation
Lemonade/beer 12 2.50 3.30 0.01 0.04
(Radler)
Alcohol-free 21 0.00 0.46 0.03 0.04
Strong 14 5.40 10.10 -0.02 0.09
OG > 14P
Other, e.g. 117 3.00 6.00 0.00 0.04
pale, pilsner,
light, dark, etc.
Total 203 0.00 10.10 0.00 0.04

Table 1: Accuracy of the Alcolyzer for various beer types

CONCLUSION
The new Alcolyzer by Anton Paar represents a new approach in using NIR
spectroscopy for the quantitative analysis of alcohol in beer. It avoids the
shortcomings of present NIR-based analyzers, while maintaining their advantages.
Only de-gassing of the sample is necessary prior to the measurement. The Alcolyzer
offers simple calibration, simple operation, high accuracy and fast results at a very
attractive price.

REFERENCES
1. Bene, R., et al., Verfahren zur spektroskopischen Bestimmung der
Konzentration von Alkoholen mit 1 bis 5 Kohlenstoffatomen, in Austrian patent
A323/99, 1999, Joanneum Research: Austria.
2. Bene, R., et al. Analytical Device for Measuring of Ethanol Concentration in
Beverages Based on NIR Absorption. in NIR'99, 1999,Verona.
3. Pfenninger, H., ed. Brautechnische Analysenmethoden, 1997, Selbstverlag der
MEBAK: Freising - Weihenstephan.
4. Bene, R. and J. Pleschiutschnig, Infrarot Alkomat fr Wein, 1996, Joanneum
Research: Graz.

7
82

Solid Phase Microextraction the new

alternative for the determination of the
vicinal diketones in beer
T. Hork, V. Kellner, J. ulk, M. Jurkov & P. ejka

Research Institute of Brewing and Malting PLC, Brewing Institute Prague, Lpov 15, CZ-
120 44 Prague 2, Czech Republic (e-mail: [email protected])

Descriptors
Analysis method for beer, diacetyl, extraction, vicinal diketones

SUMMARY
The measurement of the content of the vicinal diketones in beer is important because these
compounds, especially diacetyl give beer a buttery off-flavour in concentrations above the
flavour threshold. This poster examines the new possibility of the selective determination of
diacetyl and pentandione by gas chromatography and Solid Phase Microextraction (SPME).
The evaluation of SPME conditions is shown. Validated parameters of an optimalized
method were obtained. This new method is a suitable alternative to conventional static
headspace technique, not only for the economical attraction but also due to the excellent pre-
concentration of analytes, which determines free vicinal diketones without having to heat
samples.

Fest-Phasen-Mikroextraktion Die neue Alternative zur die Ermittlung der vicinalen

Diketone im Bier

Deskriptoren
Analysenmethode fr Bier, Diacetyl, Extraktion, vicinale Diketone

ZUSAMMENFASSUNG
Die Messung des Gehalts an vicinalen Diketonen ist wichtig, insbesondere da Diacetyl dem
Bier einen butterartigen Geschmack verleiht, sobald seine Konzentration ber dem
Geschmacksschwellenwert liegt. Dieses Poster zeigt die neue Mglichkeit der Bestimmung
von Diacetyl und Pentandion durch Gaschromatographie und Fest-Phasen-Mikroextraktion
(SPME). Es wird die Auswertung der SPME Bedingungen gezeigt. Validierte Parameter einer
optimierten Methode wurden erreicht. Diese Methode ist eine Alternative zur herkmmlichen
statischen Head-Space-Technik und dies nicht nur aufgrund der konomischen Vorteile,
sondern auch wegen der ausgezeichneten Vorkonzentratierung der Proben, die nicht mehr
erhitzt werden mssen, um die freien vicinalen Diketone bestimmen zu knnen.
La micro-extraction en phase solide nouvelle alternative pour la dtermination des
dictones vicinales dans la bire

Descripteurs
Diactyle, dictones vicinales, extraction, mthode danalyse de la bire

RESUME
Les mesures de la teneur en dictones vicinales dans la bire sont importantes car ces
composs, surtout le driv diactyle, donne la bire un arme de beurre aux concentrations
suprieures au seuil. Ce poster analyse la nouvelle possibilit d'une dtermination slective du
diactyle et de la pentanedione par chromatographie gazeuse et micro extraction en phase
solide (SPME). L'valuation des conditions de SPME est prsente. Des paramtres valids de
la mthode optimise ont t obtenus. Cette nouvelle technique constitue une alternative
satisfaisante la technique conventionnelle d'espace de tte statique, non seulement par son
intrt conomique mais galement en raison de l'excellente concentration des substances
analyser, qui dtermine les dictones vicinales libres sans avoir chauffer les chantillons.

2
INTRODUCTION
One of the most important groups of flavour compounds in beer are the vicinal
diketones (VDK): diacetyl (2,3-butanedione) and 2,3-pentanedione. These compounds
are formed amongst many other compounds during the fermentation step of the
brewing process. The most important is especially diacetyl, giving beer a buttery off-
flavour in concentrations above its flavour threshold (10).
Several different methods have been devised to determinate the concentrations of
VDK in beer. Spectrophotometric methods have been widely used; the most
frequently used method is the one according to Gjertsen (5). These methods involve
steam distillation. However, distillation has the disadvantage of an incomplete
fractionation of diacetyl from the closely related compounds, -acetolactate, a
precursor to diacetyl, and acetoin, which will result in an overestimation of the
diacetyl concentration in beer.
The chromatographic methods enable selective determination both diacetyl and 2,3-
pentanedione. A few HPLC methods have been published (2, 8, 11). Another methods
are based on the gas chromatographic headspace analysis with electron capture
detection and 2,3-hexanedione as the internal standard (7, 9, 12). Instead of the
classical static headspace can be used Solid Phase Microextraction (SPME).
SPME is a fast, simple and solventless alternative sampling technique (1, 14) and has
been applied to a number of flavour and taint analyses of beverages (4, 13, 6, 3). In
particular, headspace SPME would be the favoured choice for the analysis of VDK in
beer because of the high volatility of the analytes, and also the headspace SPME
method reduces some interference from other beer constituents in beer.
This study presents the evaluation of SPME conditions included the choice of the best
type of the SPME fiber coating, effects of addition of salt (sodium chloride), time of
sampling and influence of ethanol concentration on the analysis of the VDK.
Validated parameters of optimalized method were obtained.

MATERIAL AND METHODS

Diacetyl (DA), 2,3-pentanedione (PE) and 2,3-hexanedione (IS) were purchased from
Merck (Darmstadt, F. R. G.). The SPME holder for manual sampling and the fibers
coated with a 100 m polydimethylsiloxane (PDMS), 65 m Carbowax-
divinylbenzene (CD) and 75 m Carboxen-polydimethylsiloxane were obtained from
Supelco (Bellefonte, USA).

Sample preparation and headspace SPME procedures

Bottled commercial beers containing 4,0 5,5 % V/V of alcohol were kept cooled
(4 C) until they were analysed. 5 % V/V ethanol was used for the evaluation of the
method and for the calibration curves. Headspace SPME was applied for all analyses.
The SPME fiber was exposed to the headspace above 3 ml of the sample with the
addition of 12 g of 2,3-hexanedione as internal standard (final concentration:
40 g/l) in a 10 ml glass vial with an aluminium-coated septum. The vial was
vigorously shaken for 10 s prior to the commencement of headspace SPME.

GC analysis
The GC analysis was carried out using a Carlo Erba 5300 Mega Series gas
chromatograph. For the conventional headspace technique the Dani 3950 sampling

3
unit was used. Analytes were thermally desorbed from the coated fiber of the SPME
in the hot injector of the GC and were separated on 60 m x 0,32 mm i. d. fused silica
capillary column of J&W Scientific DB 624 with 1,8 m film thickness. The GC
column was maintained at 75 C for 10 min, ramped at a rate of 5 C/min to 130 C
and held at this temperature for 4 min. The fiber remained in the injector for 5 min in
order to condition it for the next analysis. The split-splitless injector was used, and the
split vent was opened after 0,5 min. Temperatures of the injector and the electron
capture detector were 200 and 150 C, respectively. The carrier gas was helium 5,0
with a column head pressure of 100 kPa at 75 C.

RESULTS AND DISCUSSION

Development of the method
Table 1 shows the comparison of the three different SPME fibers for relative
efficiency in extracting the analytes. The best sensitivity was founded for Carboxen,
however the ECD was saturated above at about 200 ppb (data not shown). So 65 m
Carbowax-divinylbenzene coating fiber was used in all subsequent experiments.

Table 1: Comparison of relative efficiency of Polydimethylsiloxane-, Carbowax-

Divinylbenzene-, and Carboxen-coated fibers in the extraction of the VDK.

Compound PDMS CW/DVB CARBOXEN

Diacetyl 1,0 4,5 150
2,3-pentanedione 1,0 4,9 53
2,3-hexanedione 1,0 2,8 6,6

The effect of the addition of salt (sodium chloride) on the extraction of the VDK was
examined by the addition of varying concentrations of salt (0,5 2 g) to 3 ml of the
sample. Figure 1 demonstrates that the salting-out effect obviously took place. The
salting-out effect reached saturation about 1,5 g of salt addition for DA and about
1,0 g of salt addition for PE. The values of the response ratio of DA to IS and PE to IS
are shown in figure 1 and are relatively consistent, with an average of 0,118 and
0,864, respectively and a coefficient of variation of 4,3 % and 2,8, respectively. This
observation demonstrates that the values of the response ratio are constant regardless
of the significant salting-out effect.

Figure 1: Effect of the addition of salt (NaCl) on the extraction of the VDK.
Diacetyl 2,3-pe ntane dione
1200000 10000000 1,2 00

1000000 8000000
0,120
0,900
Response ratio of PE/IS
Response ratio of DA/IS
Response of diacetyl

Response of PE

800000
6000000
0,080 0,600
600000
4000000
400000
0,040 0,300
2 000000
200000

0 0,000 0 0,000
0 0,5 1 1,5 2 0 0,5 1 1,5 2

A mount of NaCl added (g) A mo u n t o f NaCl ad d ed (g )

Response of DA Response ratio DA /IS Resp o n se o f PE Resp o n se ratio o f PE/IS

4
The effect of the lenght of the sampling time on the extraction of the VDK is shown in
figure 2. The responses increased lineary over a range of 0,5 2 min and then only
slightly up to 40 min, while the values of the response ratio of DA to IS and PE to IS
remained constant throughout all extraction times with an average of 0,130 and 0,898,
respectively and a coefficient of variation of 3,8 % and 2,6 %, respectively.

Figure 2: Effect of the length of the sampling time on the extraction of the VDK.
D iace tyl 2,3-pentandione

9000000 1,20
0,15
1,00
1200000

Response ratio PE/IS

0,12

Response ratio DA/IS

Response of PE
6000000 0,80
Response of DA

800000 0,09 0,60

0,06 3000000 0,40

400000
0,03 0,20

0 0,00 0 0,00
0,5 1 2 5 10 20 30 40 0,5 1 2 5 10 20 30 40
Extraction time (minutes) Extraction time (min utes)

Resp onse of DA Resp onse ratio of DA /IS Response of PE Response ratio of PE/IS

The influence of varying concentrations of ethanol on the extraction of the VDK is

shown in figure 3. With the increasing concentration of ethanol the responses of the
VDK decreased, however the response ratio of DA to IS and PE to IS remained
constant with an average of 0,121 and 0,911, respectively and a coefficient of
variation of 3,9 % and 3,0 %, respectively.

Figure 3: The influence of varying concentrations of ethanol on the extraction

of the VDK.

Diacetyl 2,3-pentanedione
0,18 1,20
1600000 12000000
0,14
Response ratio of
Response ratio of

Response of PE
Response of DA

1200000 0,80
0,10 8000000
PE/IS
DA/IS

800000
0,06
4000000 0,40
400000 0,02

0 -0,02 0 0,00
4 4,5 5 6 4 4,5 5 6
Concentration of ethanol (% V/V) Concentration of ethanol (% V/V)

Response of DA Response ratio of DA/IS Response of PE Response ratio of PE/IS

Comparison of SPME and static headspace (SHS) analysis was performed by

determination of the VDK in 7 different beer samples. Measured values of DA and PE
using SPME were within -6 to 11 % and -3 to 7 %, respectively of the values obtained
by SHS.
Figure 4 shows the chromatogram of a beer sample. The peaks of DA, PE and IS were
clearly free from interference by other GC eluents.

5
 Figure 4: Chromatogram of beer. 1 diacetyl, 2 2,3-pentanedione, 3 2,3-
hexanedione (internal standard).

Method validation
A calibration curve throughout a range of the VDK concentration from 10 g/l to
1000 g/l in the 5 % V/V ethanol solution showed a better fit to a quadratic curve
than to a linear curve. For DA and PE the correlation coefficient to a straight line was
0,9940 and 0,9936, respectively, for a quadratic curve fit was 0,9995 and 0,9997,
respectively.
Table 2 shows validated parameters. Detection limit of quantitation was estimated as
signal-to-noise ratio of 7:1. The accuracy of the method was investigated by
conducting the VDK recovery test. The test was performed by measuring naturally
occurring the VDK in 7 different beers followed by measuring the same beer spiked
with a known concentration of each diketone (100 g/l or 200 g/l). The repeatability
of the method was investigated by repeating the headspace SPME analysis (10 times
on the same day) of the same beer sample.
The results indicate that the method has very good repeatability and that the
concentration of the VDK is accurately determined.

Table 2: Detection limit, Recovery and Repeatability of the VDK Obtained by the
Headspace SPME Method. (CV coefficient of variation)

Detection Spike 100 g/l Spike 200 g/l Repeatability

limit
(g/l) Recovery (%) CV (%) Recovery (%) CV (%) CV (%)
Diacetyl 0,8 103 6,6 104 5,2 3,7
2,3-pentanedione 0,2 104 5,7 101 7,2 4,1

6
CONCLUSION
We have demonstrated that the responses of the VDK are related in the addition of
salt, length of sampling and ethanol concentration, while the response ratio of DA/IS
and PE/IS remains constant regardless of the changes in any of the above conditions.
The procedure devised for the practical extraction of the VDK from beer by
headspace SPME utilizes 3 ml of beer with the addition of 2,3-hexanedione as internal
standard (final concentration: 40 g/l) and 1,5 g of sodium chloride, shaken manually
for 10 s followed by headspace SPME using CD coating fiber for 30 min at ambient
temperature. This method is rapid, low cost, robust, sensitive, selective and provides
quantitative results that are comparable to those of existing static headspace methods.

REFERENCES
1. Arthur, C. L. & Pawliszin, J., Anal. Chem., 1990, 62, 2145-2148.
2. Baumann, R.A., Gooijer, C., Velthorst, N.H., Frei, R.W., Strating, J., Verhagen,
L.C. & Velthuijzen-Doorduin, R.C., Inter. J. Environ. Anal. Chem., 1986, 25,
195-207.
3. Fisher, C. & Fisher, U., J. Agric. Food Chem., 1997, 45, 1995-1997.
4. Garcia, D.D., Magnaghi, S., Reichenbcher, M. & Danzer, K., J. High Resolut.
Chromatogr., 1996, 9 (19), 257-262.
5. Gjertsen, P., Undstrup, S. & Trolle, B., Monatsschr. Brauwiss., 1964, 17, 232-
234.
6. Hawthorne, S.B., Miller, D.J., Pawliszyn, J. & Arthur, C.L., J. Chromatogr.,
1992, 603, 185-191.
7. Liebel, M. & Seeleitner, G., Mitt. Versuchsstn. Grungsgewerbe Wien, 1982,
7/8, 81-85.
8. Moree-Testa, P. & Saint-Jalm, Y., J. Chromatogr., 1981, 217, 197-208.
9. Postel, W. & Meier, B., Z. Lebensm. Unters. Forsch., 1981, 173, 85-89.
10. Shimwell, J.L. & Kirkpatrick, W.F., J. Inst. Brew., 1939, 45, 137-139.
11. Verhagen, L.C., de Jong, R.L. & Strating, J., Proceedings of the EBC Congress,
Madrid, 1987, 615-622.
12. White, F.H. & Wainwright, T., J. Inst. Brew., 1975, 81, 37-45.
13. Yang, X. & Peppard, T., J. Agric. Food Chem., 1994, 42, 1925-1930.
14. Zhang, Z. & Pawliszyn, J., Anal. Chem., 1993, 65, 1843-1852.

7
83

Recovery of volatiles from gasses of

fermenting wort
M. Arnould1, M. Bes2, F. Harmegnies3, F. Delvaux1,
J.-L. Escudier2 & G. Derdelinckx1
1
Katholieke Universiteit Leuven, Centre for Malting and Brewing Science, Faculty of
Agricultural and Applied Biological Sciences, Kardinaal Mercierlaan 92, B-3001 Heverlee,
Belgium (e-mail: [email protected])
2
Institut National de la Recherche Agronomique, Unit Exprimentale dOenologie de Pech
Rouge, Institut des Produits de la Vigne, F-11430 Gruissan, France
3
Meura Technologies, Voie Minckelers 1, B-1348 Louvain-la-Neuve, Belgium

Descriptors
Alcohol free beer, aromatic compound, carbon dioxide, fermentation by-product, flavouring
agent, volatile compound

SUMMARY
CO2 gasses released during wort fermentation contain a large spectrum of flavour active
molecules mainly originating from yeast biosynthetic activity and hop. After modelisation,
three food grade resins were industrially tested and trapped up to 90 g aromas per kilo of
resin; mainly esters were recovered. This resin treatment can be a powerful source of natural
valuable bioflavouring molecules to improve the quality of alcoholic free beer and for the
food industry. It will also be a new and useful solution in the next future to reduce the charge
(considered as waste) of the CO2 gasses liberated during fermentation.

Rckgewinnung flchtiger Substanzen aus Gasen grender Wrze

Deskriptoren
Alkoholfreies Bier, aromatische Verbindung, flchtige Substanz, Grungsnebenprodukt,
Geschmacksstoff, Kohlendioxide

ZUSAMMENFASSUNG
Das Kohlendioxid, das whrend der Grung der Wrze frei wird, enthlt ein breites Spektrum
aromaaktiver Substanzen, die hauptschlich aus dem Hopfen und aus der Biosynthese der
Hefe stammen. Nach Erstellung eines Modells wurden drei Absorberharze aus dem
Lebensmittelbereich industriell getestet. Sie waren in der Lage bis zu 90 g Aromen pro
Kilogramm Harz aufzufangen; vor allem Ester wurde absorbiert. Die Behandlung mit
aromabeladenen Harzen knnte eine interessante Mglichkeit sein, alkoholfreie Biere oder
andere Lebensmittel qualitativ zu verbessern. Es ist auerdem eine neue und ntzliche
Lsungsmglichkeit zur Reduktion des (als Abfall eingestuften) bei der Grung frei
werdenden CO2.
Recupration des composs volatils partir des gaz du mot en fermentation

Descripteurs
Aromatisant, bire sans alcool, compos aromatique, compos volatil, dioxyde de carbone,
sous-produit de fermentation

RESUME
Le gaz carbonique libr durant la fermentation du mot contient un large spectre de
molcules aromatiques provenant principalement du houblon et de lactivit biosynthtique de
la levure. Aprs modlisation, trois rsines ont t testes industriellement et nous avons t
en mesure de piger jusqu 90 g darmes par kilo de rsine; en majorit, nous rcuprons
des esters. Ce traitement au moyen de rsines peut tre une importante source de molcules
aromatiques naturelles permettant damliorer la qualit des bires sans alcool et pour
lindustrie alimentaire en gnral. Ce sera galement une solution dans le futur pour rduire la
charge (considre comme un dchet) du gaz carbonique libr durant la fermentation.

2
INTRODUCTION
Fermentation gasses released during wort fermentation contain a large spectrum of
biosynthesised flavour molecules. These originate mainly from biosynthetic activity
of yeast and hop. They are commonly waste but can also be considered as an
interesting and valuable source of natural aromas. Three hydrophobic food grade
resins (crossed linked polystyrene porous beads) were tested to trap those aromas
from the fermentation gasses.
The physical phenomenon occurring on the surface of the resin is Van der Waals
adsorption. In this case the adsorbed aromas are held onto the resin by molecular
cohesion and dipole-dipole interaction which enable adsorption of low polar
substances. The general principle is that hydrophobic or non polar materials will be
attracted to hydrophobic surfaces from relatively hydrophilic media.
Interactions between aromatic molecules and the resin as a function of different
parameters were modelled in liquid solution.
Applying different conditions (type of resin, temperature, alcoholic concentration),
resins saturated at industrial scale were eluted at lab scale. This way, we found out the
optimal conditions to recover all the bulk of these aromatic molecules or to select,
regarding their chemical characteristics, some of them.
Finally industrial tests were performed to validate the previous observations and to
optimise the adsorption and desorption step of this process providing us finally an
aromatic extract with an interesting economic added value and partially cleaned
fermenting gasses.

SAMPLE ANALYSIS
All the samples were analysed by direct injection in a gas chromatograph VARIAN
3300 equipped with a FID detector. The column used was a CP-WAX 52 CB 50 m x
0.32 mm, 1.2 m film from Chrompack. The temperature program increased from 50
C to 250 C at 5 C/min, then held at 250 C for 5 minutes.

Gas samples
Gas samples from the fermenting wort before and after resin treatment were collected
in 1 l sample bags. These were equipped with a septum and purchased at SKC
Company. 6 l of external standard (8.09 g 1-pentanol per litre of ethanol) were
injected in through the septum. After 30 minutes equilibration time, 1.2 cm of the
gas were injected in the GC and the quantity of gas was measured by pumping the
content of the bag in a graduated collector.

Liquid samples
Liquid samples with a low ethanol content were extracted with ethyl acetate (2) in the
presence of an internal standard (1-pentanol). A volume of 1.2 l of the extracted
liquid was injected in the GC.
Liquid with a high ethanol content were analysed in the presence of an external
standard (1-pentanol). A volume of 1.2 l of the extracted liquid was injected in the
gas chromatograph.

3
RESULTS AND DISCUSSION
Adsorption
First experiments were performed on liquid model solutions to study the capacity of
the three resins for some aromas. One cm of resin was putted in contact with 100 mg
of the selected molecule dissolved in 1 litre of water. The bottle was capped and
stirred overnight to reach thermodynamic equilibrium. The concentration of aromas
remaining in the liquid was measured and from this result, the capacity of the resin
was deduced (table 1).

Resin type B K C C
Ethanol percentage of the model solution 0% 0% 0% 10 %

Molecules with a LESS

Hydrophobic character i-Butanol 8.5 13.9 17 <2
Isoamyl alcohol 14.3 19.7 23.5 <2
Isoamyl acetate 34 59 68 8
Molecules with a MORE Ethyl caproate 41 78 83 45
Hydrophobic character

Table 1: Adsorption capacities (g/l resin) of three food grade resins for different
aromatic molecules and influence of 10 % ethanol in the model solution on the
adsorption capacity of resin C.

Regarding the flavouring molecules fixed, molecules with the highest hydrophobicity
show the highest affinity for the resin. Resin C is characterised by the highest
adsorption capacities. Ethanol was shown to decrease the adsorption capacities of
aromatic molecules. This last observation is still more significant in the case of less
hydrophobic molecules.

At industrial scale, enrichment in esters against higher alcohols was observed

comparing the composition of the fermenting wort to the composition of the extract
adsorbed on the resin. This clearly appeared looking at the relative concentration of
isoamyl alcohol compared to isoamyl acetate (table 2). The first enrichment in esters
is observed in the escaping fermentation gasses. Then a second enrichment is
observed when looking at the composition of the extract obtained from elution of a
saturated resin.

Fermenting CO2 composition Composition of the

wort aromas adsorbed on the
composition resin

Isoamyl alcohol 98-99 50-60 15-25

Isoamyl acetate 1-2 40-50 75-85

Table 2: Relative composition (%) of the couple isoamyl alcohol-isoamyl acetate in

the fermenting wort, the escaping fermentation gasses and the extract obtained after
desorption of a saturated resin.

4
Quantification of the fermentation gasses composition in aromas before and after resin
treatment was allowing us to explain this second enrichment (figure 1). At the
beginning of adsorption phase both molecules are totally adsorbed on the resin. Then
when less adsorption sites are available, isoamyl alcohol is no more totally adsorbed
while, the acetate ester is still totally adsorbed. Later on, isoamyl alcohol is even
washed out because of competition taking place with molecules being more
hydrophobic and having higher adsorption capacities than isoamyl alcohol.

Adsorption > Desorption

100
Aromatic molecule adsorbed (%)

80
60
40
20
Isoamyl acetate
0
Isoamyl alcohol
-20 0 10 20 30 40 50
-40
-60
-80 Desorption < Adsorption
-100
Time (days)

Figure 1: Comparison of the fermenting wort gas composition before and after
flowing over the adsorption unit. Results are expressed in percentage of the molecule
adsorbed by the resin (Delta in/out).

BREWERY A BREWERY B
FERMENTATION
CONDITIONS
Type of yeast strain Bottom fermenting Top fermenting
Wort original gravity 11 P 17 P
Fermentation temperature 12 C 24 C
GAS COMPOSITION (g/l)
Higher alcohols
n-Propanol <1 6-15
i-Butanol <1 10-30
Isoamyl alcohol 5.3 40-100
Esters
Ethyl acetate 13.6 100-320
Isoamyl acetate 3.7 30-80
Ethyl caproate <1 3-11
Ethyl caprylate <1 7-25
Ethyl caprate <1 3-7

Table 3: Fermentation conditions and gas composition of the fermenting wort from
breweries A and B.

5
Industrial scale trials were carried out at two breweries (A: bottom fermentation ; B:
top fermentation). Noticeable differences between the gasses released were stated
(table 3). The logic consequence is that at brewery B where flavour content of the
escaping gasses was richer, the adsorption phase period was reduced by a factor close
to 10. So, 10 times more aromas were collected (adsorbed) for the same volume of
beer produced.

Desorption
First, experiments were performed on liquid model solutions to study kinetic of
desorption according to different parameters. To achieve this experiment, 1 cm of
resin was putted in contact with 100 mg of the selected molecule dissolved in 1 litre
of water. The bottle was capped and stirred overnight to reach thermodynamic
equilibrium. The resin was collected and putted into contact with 400 ml of eluting
solvent under stirring. Aliquots of the eluting solvent were taken up at different
determined moments and analysed.
In figure 2, influence of the type of molecule, ethanol concentration and temperature
of the eluting solvent are illustrated. Molecules with a less hydrophobic character
have a higher desorption rate. It also appeared that higher ethanol concentrations of
the eluent or warming it up improve the desorption performances.

100
90
80
Total desorbed amount (%)

70
60
50 Isoamyl alcohol (66 % Ethanol - 20C)
40
Isoamyl acetate (66 % Ethanol - 20C)
30
Isoamyl acetate (35 % Ethanol - 20C)
20
10
Isoamyl acetate (35 % Ethanol - 56C)

0
0 20 40 60 80 100 120 140
Time (min.)

Figure 2: Relative influence of the type of molecule, temperature and ethanol

concentration on the kinetic of desorption expressed in percent.

Aware of these informations on model solutions, lab scale tests were carried out to
find out the best eluting conditions. These tests were performed using a 70 ml glass
column (2 cm internal diameter) in which 50 ml of resin was poured; it was
industrially saturated in aromas escaped from the fermentation gasses. The eluting
solvent was then flown over the column at a rate of 100 ml/h which is equivalent to
2BV/h (two times the resin bed volume per hour). After 5 BV (250 ml) have been
collected, elution was stopped.
Figure 3 is illustrating at laboratory scale the impact of lowering the ethanol
concentration of the eluent on the recovery of aromas in 5 BV (250 ml). The decrease
of ethanol concentration in the eluent provokes a reduction of the quantity of aromas

6
that can be recovered in 5 BV. One can notes as well that molecules with a more
hydrophobic character as ethyl caproate will be much more affected by a lower
ethanol concentration than molecules with a less hydrophobic character as isoamyl
alcohol.

As a consequence, this feature enables (based on the hydrophobic characteristics of

the molecules) to separate aromas adsorbed on the resin by eluting the resin using
different ethanol concentrations.

Total
120

Amount eluted (%) compared to the amount

Isoamyl alcohol
Isoamyl acetate

eluted for a 100 % ethanol eluent

100
Ethyl caproate
80

60

40

20

0
100 90 80 70 60 50 40 30 20 10 0
Ethanol percentage in the eluent

Figure 3: Relative amount (%) of aromas eluted with 5 Bed Volume (1 BV = volume
equal to the volume of the resin bed) compared to the amount desorbed using an 100
% ethanol eluent.

2500

2000
Concentration (mg/L)

1500

1000
100 % Ethanol - 20C
66 % Ethanol - 20C
500 66 % Ethanol - 56C

0
0 1 2 3 4 5
Eluted BV

Figure 4: Influence of the combination of ethanol and temperature on the

concentration of ethyl caprylate in the different elution fractions.

7
The influence of warming up the solvent to improve elution was also studied at
laboratory scale. By washing the resin with 66 % ethanol at 56 C, more aromas were
recovered than by using the same eluent at 20 C. Nevertheless, the amount is still
weak compared to what can be recovered using 100 % ethanol.

At industrial scale, resin B, C and K were tested in the two breweries and in the same
conditions (gas debit of 9 m/h/l resin). After saturation, the resins were eluted using
95 % ethanol at a velocity of 0.5 BV/h. Elution results presented (figure 5) show that
resin C is having the highest adsorption capacities in both breweries (60 g of aromas
per litre of resin recovered in brewery B). Moreover, more concentrated fermentation
gasses (table 3 brewery B) improve the adsorption capacity of the resin. It was also
established that within 2 BV, between 90 and 95 % of the aromas trapped by the resin
can be recovered. The extract is mainly composed by esters (75 %), some isoamyl
alcohol and hop oils (table 4).

70

60

50
g aromas / L resin

40 Brewery A
30 Brewery B

20

10

0
Resin B Resin C Resin K

Figure 5: Amount of aromatic molecules (g aromas / l resin) that can be recovered by

the three resins at the two breweries.

Esters Higher alcohols & hop oils

Isoamyl acetate 23.8 Isoamyl alcohol 7.6

Ethyl caproate 11.4 2-Phenylethanol 0.072
Ethyl caprylate 8.4 Myrcene 2.6
Ethyl caprate 2.8 beta-Caryophyllene 0.8
alpha-Humulene 1.7

TOTAL : 59.2

Table 4: Example of an extract composition (g aromas / l resin) after elution of resin

C saturated at brewery B.

8
CONCLUSIONS
Resin beds can be used to treat up to at least 15 m of fermenting gas per litre of
resin and per hour. Flowing the gas over the resin unit can be performed without
extra-compressor because of the weak pressure losses generated by resins (1).
The obtained results show that the treatment of fermentation gasses by flowing
them over a resin bed can be:
o a powerful source of valuable bioflavouring molecules
o a new and useful solution in the next future to reduce the flavour / odour
charge of the CO2 gasses liberated during the fermentation.
An amount 60 g of aromas per litre of resin can be recovered (75 % being esters).
Depending on their origin (characteristics of the fermenting wort, top-bottom
fermenting yeast, original gravity), the recovered compounds were showed to be
powerful flavouring agents to improve the quality of beverages as alcoholic free
beer with an addition of maximum 0.01 to 0.02 % of ethanol (using this solvent as
eluent of the resin) to the beverage and a natural flavour source for the food
industry in general.

REFERENCES
1. Arnould M. & Harmegnies F., Final Report of the European CRAFT Project of
Brite Euram BE S2 5328, 2000.
2. Williamson S.A. & Iverson W.G., Journal of the American Society of Brewing
Chemists, 1993, 51(3), 114-118.

ACKNOWLEDGEMENT
This work was part of the CRAFT project of Brite Euram (BE S2 5328). M. Arnould
and G. Derdelinckx are grateful to Mr. X. Jacquet (BOCCARD S.A.), project leader.

9
84

Application of voltametry in beer

analysis
A. Barros1, J. Rodrigues1, P. Almeida1, L. Guido1,
P. Rodrigues1, J. Santos1, J.M.Machado Cruz2 & A.A. Ferreira2
1
Centro de Investigao em Qumica da Universidade do Porto, Departamento de Qumica da
Facudade de Cincias da Universidade do Porto, Rua do Campo Alegre, 687, P-4169-007
Porto, Portugal (e-mail: [email protected])
2
Unicer-Bebidas de Portugal S.A., Apartado 1044, P-4466-955 S. Mamede de Infesta,
Portugal

Descriptors
Aldehyde, analysis method for beer, diacetyl, staling, sulphur dioxide, voltametry

SUMMARY
The purpose of this work is to show that voltametry is an easily applicable methodology that
can be used to solve many analytical brewing problems. Examples of application in beer
analysis are presented. A first example is a voltametric method for determination of diacetyl
during beer fermentation; the determination takes about 15 minutes, and its application on-
line is under investigation. In a second example voltametry is used to determine aldehydes
relevant to beer ageing; at the same time this method allows the determination of free and
total SO2, which also play a key role in the understanding of beer ageing.

Anwendung der Voltametrie bei der Analyse von Bier

Deskriptoren
Aldehyd, Altgeschmacksbildung, Analysenmethode fr Bier, Diacetyl, Schwefeldioxid,
Voltametrie

ZUSAMMENFASSUNG
Das Ziel dieser Arbeit ist es, zu zeigen, dass Voltametrie eine einfach anzuwendende
Methode ist, die die Lsung mehrerer analytischer Probleme in der Brauerei erlaubt. Einige
Beispiele seien hier erwhnt. Der erste Fall betrifft die Bestimmung des Diacetyls whrend
der Grung. Die Bestimmung dauert etwa 15 Minuten und an der Einbindung als Online-
Messung wird derzeit gearbeitet. Eine andere Mglichkeit betrifft die Analyse der Aldehyde
und gleichzeitig die Bestimmung des freien und des gesamten SO2, die ebenfalls alle eine
wichtige Rolle beim Verstndnis der Bieralterung spielen.
Application de la voltamtrie l'analyse de la bire

Descripteurs
Aldhyde, diactyle, dioxyde de soufre, formation du got dvente, mthode danalyse de la
bire, voltamtrie

RESUME
Le but du prsent travail est de dmontrer que la voltamtrie est une mthodologie
d'application facile, qui permet la rsolution de plusieurs problmes analytiques en brasserie.
Voici quelques exemples. Un premier cas concerne la dtermination du diactyle pendant la
fermentation de la bire: lanalyse seffectue seulement en quelques minutes (environ 15) et
sa dtermination en ligne est dj en recherche. Une autre possibilit concerne l'analyse des
aldhydes, et, simultanment, la dtermination du SO2, libre et total, composs trs importants
pour la comprhension du vieillissement de la bire.

2
INTRODUCTION
Voltammetry is an important instrumental method of analysis that played a
remarkable role in many laboratories since its invention in the middle twenties. The
basis of voltammetric analysis is the establishment of a relationship, generally linear,
between the concentration of the analyte and the intensity of the current that flows
through an electrode dipped into a solution of the analyte when the potential of the
electrode is varied in a convenient way. This current is due to the oxidation (anodic)
or to the reduction (cathodic) of the chemical species, which means that only
electroactive species can be determined by voltammetry.
In the old days the method was known by the name polarography and its inventor,
the Czech Heyrovsky, won the Nobel Prize in Chemistry in 1959. Polarographic
techniques were the first, other than time consuming volumetric and gravimetric
techniques, that were available for the determination of metals in solution. For the
first time, solutions below milimolar level could be analysed without using extremely
elaborate techniques [5]. After 1965, with the advent of other methods of analysis,
like atomic absorption spectrometry and gas chromatography, polarography almost
disappeared from the laboratories specially because academic electrochemists turned
decisively away from analysis and toward the study and control of interfacial
processes [11]. Polarography reached its nadir around 1967 and was at that time
almost defunct. The environmental crisis had only just awakened the public
conscience, and atomic absorption spectrophotometry was at its peak of vigour [3].
The new interest in voltammetric analysis was undoubtedly associated with the
application of anodic stripping voltammetry (ASV) in the determination of trace metal
ions. Stripping analysis has received special interest because it was the most sensitive
electroanalytical technique available during the eighties, combining the advantages of
very low detection limits, multielement capabilities, and low cost [16]. A
representation of the detection limits obtained with different voltammetric techniques
is shown in figure 1. A typical example of the use of anodic stripping voltammetry
(ASV) is the direct analysis of metal ions in tap water illustrated in figure 2. The
potentials of the voltammetric peaks obtained allow a preliminary identification of the
metal ions present. Afterwards, these metal ions can be quantified using the method of
standard additions.
100 10 1 0,1 0,01 0,001 Cu Pb Cd Zn

ppm
classic polarography

normal pulse polarography

2 nA
differential pulse polarography

square wave voltammetry

stripping methods (previous accummulation step )

Analysis of Pb2+ (1 ppm) and

Cd2+ (1 ppm) by polarography: +100 mV (30 s accummulation at 1200mV) -1200 mV
C
A classic
B normal pulse B Figure 2. Determination of metal ions in tap water,
C differential pulse A using Anodic Stripping Voltammetry (ASV)
25.0 mL tap water plus 0.1 mL conc. HClO 4;
------ addition of 200 L lead (II) ion 1x10-5 M;
........ addition of 200 L cadmium (II) ion 3x10-6 M
Figure 1. Detection limits of voltammetric techniques
[result: 13 ppb lead (II) and 3 ppb cadmium (II)]

3
Another important stripping technique, specially when flowing systems are
considered, is adsorptive stripping voltammetry (AdSV), as it simplifies the analytical
procedure, improves the selectivity and sensitivity of the determination and increases
the sample throughput. A higher selectivity is achieved automatically in flowing
systems as other electroactive components whose faradaic response could interfere in
the signal recording are removed from the vicinity of the electrode by the carrier
stream during the reduction step [9]. A schematic representation of the main types of
voltammetric stripping methods of analysis can be seen in figure 3.
In this work examples are presented showing that several beer compounds can be
determined using voltammetric techniques. A first case is the determination by
adsorptive stripping voltammetry of diacetyl during beer fermentation using a flow
system. The results are similar to those obtained by gas chromatography and the
determination takes less than 15 minutes; the application of the method on-line is
under development. In a second example it is shown that some compounds related
with beer ageing can be assessed by voltammetry. In fact, in some preliminary work,
two voltammetric peaks were obtained in the analysis of beer distillates, showing
opposite tendencies with beer storage (figure 4). Complementary studies proved that
these two peaks were due to SO2 (left peak) and acetaldehyde (right peak), two
compounds that play a key role in the staling reaction mechanisms leading to the
flavour changes that occur in beer [1].

double effect of
deposition step {
lower determination limits (concentration)
elimination of interferences (extraction)
ip (nA)

50
acetaldehyde,

TECHNIQUE METHODOLOGY 40
deposition 30 SO2
ASV Mn+(aq) + Hg + ne M (Hg)
stripping 20
deposition
species (solution) species (adsorbed) 10
AdSV 0
species
adsorbed
stripping
{ Reduction (AdCSV)
Oxidation (AdASV)
Tensammetry
-500 -700 -900 -1100 E (mV)

Figure 3. Main stripping voltammetric techniques Figure 4. Analysis of beer distillates.

fresh beer; - - - - forced ageing (7 days at
37 C; .... natural ageing (6 months of shelf).

EXPERIMENTAL
Voltammetric work was performed using an Autolab PGSTAT10 voltammetric
system (Eco Chimie), controlled with a PC equipped with GPES for Windows -
version 4.4 software. Sampling time is an important experimental parameter to be
controlled in this work; with this software, current is sampled during the last 20 ms of
the pulse period for pulse times longer than 40 ms, and during the last half of the
pulse for pulse times shorter than 40 ms. A Metrohm 663VA voltammetric stand was
used in the HMDE mode. The three-electrode potentiostatic system was completed
with a glassy carbon auxiliary electrode and a AgCl/Ag (3M KCl) reference electrode.
Measurements were made at 25 C.
Before analysis the solution contained in the voltammetric cell was deoxygenated for
10 minutes by passing nitrogen (except in flow analysis in which oxygen removal was
not necessary). Stirring was switched on during the deoxygenation period and
switched off 10 seconds before initiating the scan.

4
All the solutions were prepared with reagents of analytical grade and with distilled
water further purified in a Millipore system. Hydrochloric acid was used in the
preparation of buffer solutions with pH less than 2; solutions with other values of pH
were prepared from 0.1 M solutions of di-sodium hydrogenophosphate or sodium
acetate, adjusting pH with 1M HCl or 1M NaOH. Other experimental conditions that
were specifically used in some applications will be indicated during the presentation
of the applications.

VOLTAMMETRIC DETERMINATION OF DIACETYL IN BEER USING A

FLOW SYSTEM
Introduction
The work presented here appears in the sequence of the development of a batch
method for the voltammetric determination of oxalic acid published some years ago
[12], a method that can be applied to other alfa-dicarbonyl compounds including
diacetyl. The voltammetric determination of diacetyl in batch conditions was studied
afterwards, both in beer [2,13] and in other food products [14]. The voltammetric
determination of diacetyl (and other alfa-dicarbonyl compounds) is preceded by a
derivatisation reaction with o-phenylenediamine (OPDA) to form 2,3-
dimethylquinoxaline (DMQ), the same reaction that is proposed by EBC for the
spectrophotometric determination of diacetyl [4]; the voltammetric signal is due to the
reduction of DMQ:
CH 3 H
NH 2 O=
=CC N CH 3
N CH 3 N CH 3
+ + 2H 2 O
=
O C + 2H + + 2e -
NH 2 CH 3 N CH 3 N CH 3 N CH 3
OPDA DIACETYL DM Q DM Q H

Experimental
Stock solutions of diacetyl (1x10-3 M) were prepared weekly from a more
concentrated diacetyl solution. Diacetyl was obtained from Aldrich GMBH. Stock
solutions of 2,3-dimethylquinoxaline (5x10-4 M) were prepared by dissolution of the
solid compound (Aldrich GMBH) in water. The o-phenylenediamine (OPDA)
derivatisation solution was prepared by dissolution of the solid compound (Merck) in
water and was prepared fresh every day, owing to its instability.
Figure 5 represents the flow system developed for the determination of diacetyl and
that was used for the analysis of diacetyl in beer that follows. The determination starts
with the continuous pumping of the beer sample into the lower cavity of the
pervaporation cell (a cell that was specially constructed and that is similar to the one
presented in reference [8]), which is plunged in a bath at 90 C. Due to the warming,
diacetyl is distilled and forced to pass to the upper cavity of the pervaporation cell
through a hydrophopbic membrane that separates both cavities (non-hydrophobic
compounds are retained in the lower cavity). The upper cavity contains a solution of
the derivatising reagent, OPDA, which converts diacetyl in DMQ. The upper solution
is then forced to pass through the voltammetric flow cell (that was specially
constructed, and represents an evolution of the one presented in reference [15]),
equipped with a hanging mercury drop electrode (HMDE), where DMQ is
determined. The voltammetric conditions applied to the hanging mercury drop
electrode (HMDE) for the determination of diacetyl in flow using the system depicted
in figure 5 are the following: square wave mode with a frequency of 500 Hz,
accumulation potential of -0.600 V, initial and final potentials of 0.600 V and 1.00

5
V respectively, step potential of 1 mV and potential amplitude of 5 mV; the
adsorption time was 5 seconds.

peristaltic injection
pump valve HMDE

hydrophobic waste
derivatisation membrane
solution

phosphate buffer 1. analysis sub-system

!!!
!!
!! pervaporation
!!! module
peristaltic
pump
waste
water bath
(90o C)

beer 2. distillation sub-system

Figure 5. Flow system for the determination of diacetyl in beer. 1. Distillation sub-
system beer is heated at about 90 oC, releasing gaseous diacetyl, which crosses the hydrophobic
membrane. 2. Analysis sub-system diacetyl reacts with OPDA with formation of DMQ that is
determined by voltammetry at the HMDE.

Results and discussion

The results for the analysis of diacetyl in beer during fermentation are presented in
figure 6 and are similar to those obtained using gas chromatography. The first curve is
for beer alone; on the other three curves an amount of 21.5 ppb of diacetyl was
successively added to the beer sample.
i (nA) d
c a - beer ( diacetyl not added)
150
b - 21.5 ppb diacetyl added
c - 43.0 ppb diacetyl added
b d - 64.5 ppb diacetyl added
100
a

50

|diacetyl| obtained 27 ppb

0
-700 -800 -900 E (mV)

Figure 6. Voltammetric flow analysis of diacetyl in beer using the method of standard
additions. Flow rate = 0.3 mL/min; phosphate buffer 0.1 M, pH 7 as eluent; derivatising solution:
OPDA 0.05 %; analysis time = 15 minutes (distillation 10 min.; determination and cleaning 5 min.)

Although the figure represents a situation where the method of standard additions was
used, the method of the calibration curve is also applicable. So, if a calibration curve
is obtained previously, diacetyl can be determined in 15 minutes, as this is the time for
each analysis, including the washing of the system. An investigation is now being
conducted in order to reduce the time of analysis to 5 minutes only. The idea is to
have in the system three pervaporation cells working out of phase and a unique
voltammetric cell (the really expensive part); with this modification three samples can
be determined during the same period of 15 minutes, because the time needed for the
determination at the voltammetric cell (including washing) is less than five minutes
and then it is not the limiting step.

6
VOLTAMMETRIC DETERMINATION OF ALDEHYDES AND SO2
Introduction
Aldehydes can be determined voltammetrically in either acidic or basic media. In
basic electrolytes aldehydes are determined directly, while in acidic electrolytes
carbonyl compounds condense with a wide variety of amines to produce the
azomethine group, - CH = N - which is also electrochemically active. The derivatives
of carbonyl compounds are more easily determined than the free carbonyl compounds
themselves, where complicating side reactions such as hydration, acetal formation or
enolisation tend to lower the concentration of the free carbonyl group [6].
By reacting with hydrazine under acidic conditions, acetaldehyde can be
quantitatively converted into an hydrazone (figure 7). Once formed, this electroactive
adduct is stable. No deterioration of the voltammetric signal was observed after one
hour of standing. The reaction of hydrazine with the aldehyde is not immediate. The
samples are mixed with the supporting electrolyte (0.1 M acetate buffer pH 4.0) and
allowed to stand at least five minutes before running the voltammogram.
The reduction of the hydrazone at the DME (dropping mercury electrode) is a two-
electron process (figure 8) and the current is proportional to acetaldehyde
concentration.
NH2
O N NH2 N NH2 HN
N2H4 + R C R C + H2O RC + 2H + + 2e - R C H
H H H H
Figure 7. Derivatization of carbonyls Figure 8. Reduction reaction of hydrazones
with hydrazine to form hydrazones at the DME (dropping mercury electrode)

In the case of sulphur dioxide the voltammetric determination has to be carried out in
slightly acidic solution. The reduction signal at DME is associated to the reduction of
sulphurous acid, H2SO3. Differential pulse polarography has been previously
developed for the determination of sulphites in food [7] and this work presents an
adaptation of the method to the specific analysis of sulphur dioxide in beer. The
method is highly specific and sensitive and is applicable to a wide variety of beers.

Experimental
A purge-trap apparatus (adapted from reference 7) was developed for the extraction of
acetaldehyde and sulphur dioxide from beer (figure 9). In the analysis, 1 ml of non-
degassed beer was added to 20 ml of 0.01 N NaHO, previously saturated with
nitrogen, in a gas collect tube dipped in a water-bath at 45 C directly connected to the
voltammetric cell. Acetaldehyde was purged with nitrogen for 20 minutes at 0.5 l/min,
collected in an 10 C thermostatized voltammetric cell containing 20 ml of 0.1 M
acetate buffer (pH 4.0) with 0.02 M hydrazine dihydrochloride and analysed. After the
removal of acetaldehyde, the sample of beer was acidified with 1 ml of 12 M HCl and
SO2 was purged with nitrogen for 15 minutes at 0.4 ml/min into the 15 C
thermostatized voltammetric cell containing 20 ml of 0.1 M acetate buffer (pH 5.0)
and determined. The voltammograms were obtained under the conditions given below
and the calculations were based on a calibration curve. The square wave voltammetric
determinations were performed in deoxygenated solutions (by bubbling nitrogen for 5
minutes) using the following voltammetric conditions for the potential scan:
frequency = 25 Hz; pulse amplitude = -25 mV; step potential = -2 mV.

7
RESULTS AND DISCUSSION
Voltammetric analysis of acetaldehyde
Acetaldehyde is removed by bubbling nitrogen through the beer sample in alkaline
conditions (pH > 9.0) at 45 C. At this temperature it is expected that acetaldehyde
represents almost all the released aldehydes. At pH>9 all forms of SO2 are converted
into SO32-, which does not form adducts with carbonyl groups (figure 10). Thus total
acetaldehyde (including that released from the adducts with HSO3-) is purged with
nitrogen from the basic sample, collected in electrolyte-trapping solution and then
determined by voltammetry.

pKa11.7 SO2 + H2O " HSO3- + H+

N2
N2 pKa27.2 HSO3- + H2O " SO32- + H+
flow meter N2 +
aldehydes
(or SO2)
SO2 mixture HSO3-

beer (1) 0 1 2 3 4 pH

HSO3- mixture SO32-

voltammetric cell (2) 5 6 7 8 9 pH

Figure 9. (1) Purge-trap apparatus to transfer Figure 10. Effect of pH on the equilibrium of SO2
acetaldehyde (at pH>9) and SO2 (at pH<1) from beer species in aqueous solution.
to the voltammetric cell. (2) Voltammetric analysis of
acetaldehyde hydrazone (pH 5.0) and SO2 (pH 4.0).

The acetaldehyde content of a pilsener type beer was determined from a calibration
curve in acetate buffer pH 5.0 / 0.02 M hydrazine (figure 11-A). The peak potential
observed for derivatised acetaldehyde is -1.1 V (vs Ag/AgCl) but it can be shifted by
small changes in pH. The voltammetric cell is maintained at 10 C in order to increase
acetaldehyde solubility, thus avoiding escape of the gas. Studies on the influence of
the purging time (figure 11-B) lead to the conclusion that 20 minutes are sufficient to
completely transfer acetaldehyde from beer samples. In these conditions recoveries of
more than 90% acetaldehyde added to beer can be achieved (data not shown). The
acetaldehyde content obtained for the analysed beer is 8.5 ppm.

Voltammetric analysis of sulphur dioxide

Total sulphur dioxide (free of carbonyls) is extracted from beer by the same procedure
described for acetaldehyde. After removal of total acetaldehyde the beer sample is
acidified to pH<1.0. At this pH all the bisulphite (HSO3-) was converted into SO2
(figure 10) and can be easily removed by purging the tube with nitrogen for 15
minutes (figure 12-B). And as carbonyl-HSO3- adducts tend to dissociate at low pH
this enables the rapid transfer and quantification of total sulphur dioxide present in
beer. A typical square wave voltammogram for SO2 exhibits a well-defined peak at
0.60 V versus Ag/AgCl reference electrode (figure 12). Beer samples were analysed
and the total SO2 concentration was calculated by interpolation of a calibration curve
in acetate buffer pH 4.0. After a purging time of 15 minutes the average recovery was
found to be 95% (data not shown).

8
 3 ppm 15 min
N2
10 min
2 ppm bubbling
5 min

I/A
I/A

1ppm

E/V (vs. AgCl/Ag) E/V ( vs. AgCl/Ag)

Figure 11. Voltammetric determination of acetaldehyde in beer. A calibration

curve. B effect of purging time on the transfer of acetaldehyde from beer.

800 ppb

600 ppb 10 min N2 bubbling

I/A

5 min N2 bubbling
I/A

400 ppb

200 ppb

E / V (vs. Ag/AgCl) E / V (vs. Ag/AgCl)

Figure 12. Voltammetric determination of SO2 in beer. A calibration curve. B

effect of purging time on the transfer of SO2 from beer.
The proposed voltammetric method is highly specific for sulphur dioxide, with no
need for derivatisation or colour development reactions. In order to assess the quality
of the proposed method, determination of total SO2 for a set of 10 beer samples was
carried out by voltammetry and by the AOAC recommended method using p-
rosaniline [10]. The results obtained with this method were 25% higher than the
voltammetric ones, but higher SO2 levels by the p-rosaniline method were already
reported when compared to Monier-Williams and coulometric methods [17].
Problems of turbidity of some samples, especially those of high gravity beers and dark
beers, affecting colorimetric methods, can also be overcome using voltammetry.
Free sulphur dioxide is difficult to determine with accuracy due to changes in
equilibrium with carbonyls by altering the original composition of the beer matrix.
Even a small change introduced by diluting the beer sample gives rise to a shift in the
equilibrium between sulphite complexes and free carbonyls. Preliminary results have
shown that 80-95% of acetaldehyde is combined with sulphur dioxide at typical
concentrations found in beer. Work is in progress on the determination of free sulphur
dioxide assuming that all the acetaldehyde is in the adduct form. It is believed that the
error introduced by this approximation is smaller than the error involved in the
dilution of beer sample for the analysis.

9
CONCLUSIONS
The aim of this work is to try to prove that the application of voltammetry in beer
analysis can be an interesting option in the future. Two applications are reported to
justify this opinion.
In one of the applications a flow method is described for the determination of diacetyl
by adsorptive stripping voltammetry. The method can be applied directly to beer and
investigations proceed in order to reduce the analysis time from 15 to 5 minutes.
In the other application a voltammetric method was developed which combines the
measurement of acetaldehyde and sulphur dioxide in beer. In addition to its simplicity
and rapidity, this new approach seems to be very attractive for the analytical
determination of beer staling, since acetaldehyde and SO2 play a key role in the beer
ageing process.
In conclusion, it is the opinion of the authors that voltammetry, an easily applicable
methodology not involving a considerable investment (no more than 15 000 euros),
can be used in the future to solve many other analytical brewing problems.

ACKNOWLEDGEMENTS
A. Barros, J. Rodrigues, P. Almeida, L. Guido, P. Rodrigues and J. Santos thank
Fundao para a Cincia e a Tecnologia (F.C.T.) and Unicer Bebidas de Portugal S.
A. for financial support. This work was patronized and financed by Program Praxis
XXI and by FEDER, as part of the Project 2/2.1/TPAR/463/95.

REFERENCES
[1] Bamforth, C.W., MBAA Technical Quarterly, 2000, 37, 165-171.
[2] Barros, A.A., Rodrigues, J.A., Almeida, P.J., Rodrigues, P.G., Fogg, A.G., Analytica
Chimica Acta, 1999, 385, 315-323.
[3] Bond, A.M., Modern Polarographic Methods in Analytical Chemistry, New York:
Marcell Dekker, Inc., 1980, Chapter 1.
[4] European Brewery Convention. Analytica EBC, 4th Edition, Brauerei und
Getrnke-Rundschau, Zurich, 1987.
[5] Flato, J.B., Analytical Chemistry, 1972, 44, 75A-87 A.
[6] Fleet, B. and Keliher, P., Analyst, 1969, 94, 659-663.
[7] Holak, W. and Patel, B., Journal of the Association of Official Analytical Chemists,
1987, 70, 572-578.
[8] Isquierdo-Ferrero, J.M., Fernndez-Romero, J.M., Luque de Castro, M.D., Analyst,
1997, 122, 119-122.
[9] Kalvoda, R., Kopanica, M., Pure & Applied Chemistry, 1989, 61, 97-112.
[10] Methods of Analysis of ASBC, American Society of Brewing Chemists, 8th Ed.,1992,
Method Beer-21.
11] Osteryoung, J., Accounts of Chemical Research, 1993, 26, 77-83.
[12] Rodrigues, J.A., Barros, A.A., Analytica Chimica Acta, 1993, 273, 531-537.
[13] Rodrigues, J.A., Barros, A.A., Cruz, J.M.M., Ferreira, A.A., The Journal of the Institute
of Brewing, 1997, 103, 311-314.
[14] Rodrigues, J.A., Barros, A.A., Rodrigues, P.G., Journal of Agricultural and Food
Chemistry, 1999, 47, 3219-3222.
[15] Taylor, L.R.M., Chemtec, 1994, 38-44.
[16] Wang, J., Stripping Analysis, Florida: VCH Publishers, Inc., 1985, Preface.
[17] Zeller, S., Wills, K. and Huffman, E., Brewers Digest, 1988, 63, 24-27.

10
85

Electrochemical determination of heavy

metals in beer
Pavel Dostlek1, Jaroslav epika1, Jan Enge1, Richard Koplk2
& Eva urdov3

Institute of Chemical Technology, Technick 5, CZ-166 28 Praha 6, Czech Republic

1
Department of Fermentation Chemistry and Bioengineering (e-mail:
[email protected])
2
Department of Food Chemistry and Analyses
3
Department of Analytical Chemistry

Descriptors
Analysis method for beer, contamination, electrode, heavy metal, purity

SUMMARY
Determination of trace metals is based on deposition on the surface of electrode using a
constant potential and metal deposit is stripped by application of constant current and of time
dependent potential is measured (galvanostatic stripping chronopotentiometry-GSCP).
Brewing raw materials and intermediate products were analysed in brewing process. Only
metal traces are transported from raw materials to final beer. Soft-drink and wine samples
were analysed for lead content. Beer represents the purest beverage from point of view lead
concentration. Brewing process is a self-cleaning process from heavy metal content point of
view.

Elektrochemische Bestimmung von Schwermetallen im Bier

Deskriptoren
Analysenmethode fr Bier, Elektrode, Kontamination, Reinheit, Schwermetall

ZUSAMMENFASSUNG
Die Bestimmung von Spurenmetallen basiert auf der Ablagerung dieser auf der Oberflche der
Elektrode, die ber ein konstantes Potenzial verfgt. Das abgelagerte Metall wird regelmig
wieder abgelst und der Potenzial-Unterschied zwischen diesen Zeiten wird gemessen
(GSCP). Brauereirohstoffe und -zwischenprodukte wurden whrend des Brau-prozesses
untersucht. Lediglich Spurenmetalle werden von den Rohstoffen bis in das fertige Bier
getragen. Soft-Drinks und Weinproben wurden auf ihren Bleigehalt hin untersucht.
Brauereirohstoffe und Zwischenprodukte im Brauprozess wurden analysiert. In Bezug auf die
Bleikonzentration stellt Bier das reinste Getrnk dar. Vom Gesichtspunkt des Schwermetall-
gehaltes stellt das Brauen einen Selbstreinigungsprozess dar.
Dosage lectrochimique des mtaux lourds dans la bire

Descripteurs
Contamination, lectrode, mtal lourd, mthode danalyse de la bire, puret

RESUME
La dtermination des mtaux lourds s'appuie sur leur dposition la surface d'une lectrode
avec un potentiel constant, le dpt mtallique tant limin par application d'un courant
constant et en mesurant un potentiel en fonction du temps (chronopotentiomtrie
galvanostatique GSCP). Les matires premires de brassage et des produits intermdiaires
ont t analyss dans le processus de brassage. Seuls les mtaux lourds passent des matires
premires la bire finie. La teneur en plomb d'chantillons de boissons sucres et de vin a t
ainsi analyse. La bire reprsente la boisson la plus pure du point de vue de la teneur en
plomb. Le brassage est un processus hautement nettoyant du point de vue de la teneur en
mtaux lourds.

2
INTRODUCTION
There are two groups of analytical methods used for determination of trace amounts of
heavy metals in samples, electrochemical and spectrometric. Last years with
development of electronics became again an expansion of electrochemical methods,
which were almost forced out by spectrometric methods. Dominant position in
spectrometric methods takes atomic absorption spectrometry (AAS) with different
ways of atomisation and introduction of sample into the atomisator (F AAS, GF AAS).
Multielemental analysis is represented namely by inductively coupled plasma (ICP-
AES, ICP-MS) emission and mass spectrometry.
Stripping determination of trace amounts of metals is based on deposition on the
surface of electrode (so called preconcentration) using a constant potential. In next
stage the deposit is stripped by application of constant current and measurement of
time dependent potential (galvanostatic stripping chronopotentiometry-GSCP). This
method is highly selective, the cost per one determination is low and it is possible to
determine several metals simultaneously. An electrochemical flow-through cell with a
porous working electrode made of vitreous carbon particles and plated with mercury
was used for the determination of Zn, Cd, Pb and Cu using GSCP in a flow system.

MATERIAL AND METHODS

Decomposition technique
Wet ashing under elevated pressure with focussed microwave heating in 3 ml of 65%
HNO3 was used. (Plazmatronika BM-1S, PL). Samples (solid samples malt and hop -
approximately 0.5 g, liquid samples water, sweet wort, wort, degassed beer
approximately 10 g) were weighted accurately into polytetrafluoroethylene digestion
bombs. Digestion was then completed using a microwave oven for 3 minutes at low
power followed by 7 minutes at high power. After cooling for 10 minutes, the digests
were transferred quantitatively into volumetric flasks (50 ml) and diluted to volume
with deionised water.

Measurements
Determination of zinc, copper, cadmium and lead by GSCP
Electrochemical analyser Istran EcaFlow GP 130 produced by Istran (Bratislava, SK)
which is suitable for laboratory research and also for monitoring of metals in aqueous
solutions e.g. in beverage industry was used for electrochemical measurements.
Scheme of electrochemical analyser is shown in figure 1.
The analysis is carried out in full automatic mode. First, a given volume of sample
solution is pumped through the cell with porous working electrode set to the
deposition potential where the trace metals are collected. The heart of the system is the
patented compact electrochemical cell. This cell is shown in figure 2. In the next step
constant current to the optimum electrolyte solution strips the deposit whereas the
change of potential of the working electrode is monitored and evaluated. The
evaluation (transformation) of sample chronopotentiometric curve is shown in figure
3. After the measurement of the sample, background signal is measured automatically
in a similar way just by treating the electrolyte solution instead of the sample solution.
The background signal is then substracted from the sample signal giving the
background-corrected net signal. By integrating the stripping peaks, the concentration
of the corresponding trace elements is calculated by making use by automatic standard
addition. Example for simultaneous measurement of zinc, copper, cadmium and lead is

3
shown in figure 4. Operating conditions for this measurement are summarised in table
1.

Table 1: GSCP operating conditions

Electrode Istran E 56-LMF

Electrolyte sodium acetate 0.1 mol.l-1
Accumulation current -5000 A
Initial potential -1600 mV
Final potential 350 mV
Stripping current 200 A
Dwell time 10 s
Measurement time 30 s
Regeneration time 10 s
Regeneration potential 200 mV
Sample volume 1 ml
Standard addition volume 0.1 ml

Determination of zinc, copper, cadmium and lead by ICP-MS

Elan 6000 (Perkin Elmer Sciex) ICP mass spectrometer, with peristaltic pump Gilson
212, was used for determination of Cd, Cu, Pb and Zn in sample digests. The solutions
were aspirated into nebulizer in 3 5 % v/v HNO3. Operating conditions for this
measurement are summarised in table 2. The following isotopes were used for signal
evaluation: Cd 112, Zn 66, Cu 65, Pb 208. In 115 and Bi 209 were applied as internal
standards.

Table 2: ICP MS operating conditions

Sweeps/Reading 5
Dwell time / ms 200
Gas flow rates/ l.min-1
Coolant 17.0
Auxiliary 1.2
Nebulizer 0.8
Forward power /W 1000
Sample uptake rate/ ml.min-1 1.0

4
 Optional membrane filter
Flow- electrolyte
through Peristaltic
cell pump vent 1
Waste
standard
vent 2

Analyser is fully controlled by

an IBM compatible PC sample

Figure 1: Scheme of electrochemical analyser Istran EcaFlow GP 130

Outlet tubing
PTFE block
with electrodes Auxiliary
platinum
Reference electrode
Ag/AgCl
Porous working
electrode
electrode made
Cover of from vitreous
working carbon and plated
electrode with mercury

Inlet tubing

Figure 2: Scheme of electrochemical flow-through cell

5
 Zn

Pb
Cd Cu

Figure 3: Chronopotentiometric curve for sample and standard solution

Heavy metal concentrations in beers

120
1 0 8 .8

100 9 2 .9

80

median
ppb

60 5 5 .4
maximum
40 3 2 .6

20
6 .5 8 .5
0 .5
0 .2
0
Zn Cd Cu Pb

Figure 4: Heavy metal concentrations in beers

6
 Lead concentration in beverages
ppb
45
40
35
30
25 4 1 .8

20
15
10
5 6 .5 7 .5

0
beer soft drink wine

Figure 5: Median lead concentration in beverages

M etal b alance in b rew ing p rocess

120

100
metal content %

80
ra w m a teria ls
sw eet w o rt
60
w o rt
b eer
40

20 18
20
7 9
5 2 6
1 2 1 3 2
0
Zn Cd Cu Pb

Figure 6: Metal balance in brewing process raw material - 100% metal content

7
RESULTS
A total of 200 bottles of different beers were analysed by GSCP for determination of
zinc, copper, cadmium and lead. Resultant medians and maximum values are shown in
figure 4. GSCP method was validated by ICP-MS method. As maximum and mean
lead concentration was relatively high, soft-drink (50 samples) and wine (50 samples)
samples were analysed for lead content. Median lead concentrations are shown in
figure 5. Beer represents the purest beverage from point of view lead concentration.
Metal balance for zinc, copper, cadmium and lead is shown in figure 6. Only metal
traces are transported from raw materials to final beer. Brewing process is a self-
cleaning process from heavy metal content point of view.

CONCLUSION
GSCP is suitable method for simultaneous metal determination in beers.
Beer is very clean product from metal concentration point of view (namely lead).
Brewing process is self-cleaning process. This process reduce amount of heavy
metal in beer.

LITERATURE
1. Ostapczuk, P.: Anal. Chim. Acta, 273, 1993, 35-40.
2. Beinrohr, E., Csmi, P., Manov, A., Dzurov, J.: Fres. J. Anal. Chem., 349, 1994,
625-632.
3. Bond, A.M.: Anal. Chim. Acta, 40, 1999, 333-379.
4. Dostlek, P., Koplk, R., Patzak, M.: Czech J. Food Sci., 17, 1999, 73-76.
5. Matsushige, I., Oliviera, E.: Food Chem., 47, 1993, 205-207.

8
86

The effect of pH on protein-polyphenol

particle size
Karl J. Siebert & P.Y. Lynn
Cornell University, Dept. of Food Science & Technology, Geneva, NY 14456, USA
(www.nysaes.cornell.edu, e-mail: [email protected])

Descriptors
Gliadin, haze measurement, particle size, pH, tannic acid

SUMMARY
It is known that variations in haze particle size affect human and instrumental perceptions of
turbidity, as well as filtration and sedimentation behavior. Haze intensity was known to be
influenced by pH, but this had not been studied in detail. The haze-active (HA) protein
gliadin and the HA polyphenol tannic acid were combined in various proportions in buffer
model systems at several different pHs. Haze intensity and particle size distribution were
observed under each condition. The greatest haze intensity and the largest particle size were
observed near pH 5, which is thought to be close to the isoelectric point of gliadin.

Der Auswirkung des pH-Wertes auf die Protein-Polyphenolteilchengre

Deskriptoren
Gerbsure, Gliadin, Korngre, pH, Trbungsmessung

ZUSAMMENFASSUNG
Es ist bekannt, dass Schwankungen der Trbungsteilchengre sowohl die visuelle, als auch
die instrumentell beobachtbare Trbung verndern knnen, aber auch das Filtrations- und
Sedimentationsverhalten beeinflussen. Die Trbungsintensitt wird vom pH-Wert beeinflusst,
jedoch wurde dies noch nie im Detail untersucht. Das trbungsbildende Protein Gliadin und
das trbungsbildende Polyphenol Tannin wurden in unterschiedlicher Konzentration bei
verschiedenen pH-Werten in einem Modell-Puffer-System kombiniert. Bei jeder Variation
wurden die Trbungsintensitt und die Partikelgrenverteilung beobachtet. Die grte
Trbungsintensitt und die grten Partikel beobachtete man bei einem pH-Wert von
ungefhr 5, was dem isoelektrischen Punkt von Gliadin sehr nahe kommt.
Effet du pH sur la taille des particules de protines-polyphnols

Descripteurs
Acide tannique, dimension des particules, gliadine, mesure du trouble, pH

RESUME
Il est connu que les variations de la taille des particules de trouble affectent les perceptions
humaines et instrumentales de la turbidit, ainsi que le comportement en filtration et
sdimentation. On sait que l'intensit du trouble est influence par le pH, mais cela n'a pas t
tudi en dtails. La gliadine, protine du trouble (PT) et l'acide tannique, polyphnol PT, ont
t associs diverses proportions dans des systmes tampons modles diffrents pH.
L'intensit du trouble et la granulomtrie des particules ont t observs dans chaque
condition. Le trouble le plus intense et la granulomtrie la plus forte ont t observes aux
environs de pH 5, qui serait proche du pI de la gliadine.

2
INTRODUCTION
Protein-polyphenol interaction is the most frequent cause of haze formation in beer
and a number of other beverages. It was previously shown in model systems that not
only the concentrations of haze-active (HA) protein and HA polyphenol, but also their
ratios in a sample influence the intensity of the turbidity (7, 11). If HA protein
concentration is held constant and HA polyphenol is increased, the haze at first
increases, but then reaches a peak and later declines. Similarly, if HA polyphenol
concentration is held constant and HA protein is increased, the haze at first increases,
but reaches a maximum and then declines. A hypothetical model that explained this
behavior was proposed (4, 7, 11). This was based on the concept that HA proteins
have a limited number of sites to which polyphenols can bind and HA polyphenols
have at least two ends that can attach to HA proteins. When the number of HA
polyphenol ends is roughly equal to the number of protein binding sites, a large
network, corresponding to large particle size and maximum light scattering, would
result. With an excess of protein to polyphenol, essentially all of the polyphenols
would be able to join proteins together, but there would be insufficient polyphenols to
connect many of these protein dimers to one another. This would result in smaller
particles and less light scattering. With a polyphenol excess, most of the attachment
sites in the HA proteins would be occupied by HA polyphenols, but in most cases the
other end of the polyphenol would be unable to find a vacant site on a protein
molecule to attach to. This would also result in smaller particles and less light
scattering.

The hypothesized mechanism was consistent with a number of observed behaviors. In

particular, it accounted for the mechanisms of adsorbent action in beverages (8-10)
and explained how silica gel could remove haze-active but not foam-active protein
from beer (9). It also explained why there are large differences in the effectiveness of
silica gel and polyvinylpolypyrrolidone in apple juice, which is polyphenol-rich, and
in beer, which is protein-rich (7).
In a recent study (5) both haze intensity measurements and particle size analyses were
carried out in model systems at pH 4.5 (at the high end of the beer pH range); this
showed that indeed the particle size does vary with the protein/polyphenol ratio and
the largest haze intensities coincided with the largest particle sizes. Further, some fine
structure in the pattern revealed quantized behavior. Ridges of haze intensity occurred
at gliadin/TA ratios of 2:1 and 5:1 and particles of only a few discrete sizes were
seen. Most changes in the patterns were shifts in the proportion of the haze particles
of these sizes.
In one model system study of protein-polyphenol interaction, the effects of pH and
alcohol were investigated (7). Alcohol had little influence on haze intensity near pH 3
and a modest effect at beer pH. On the other hand, pH had a dramatic effect on haze.
The same amounts of protein and polyphenol produced very little haze at pH 3 but
about seven times more haze in the beer pH range. The haze declined at higher pH.
Since the size of haze particles has a major impact on their sedimentation and
filtration behavior (2, 3, 12), it was of considerable interest to study the effect of pH
on particle size.

3
EXPERIMENTAL
Chemicals Gliadin was obtained from Sigma (St. Louis, MO). Tannic acid (TA)
was obtained from Mallinckrodt Chemical. The buffer was 0.02 M sodium phosphate
adjusted to the indicated pH (3.0, 4.0, 4.5, 5.0, 5.5, 6.0 or 6.5). Gliadin quantities were
weighed as the dry powder. Tannic acid stock solutions were prepared fresh daily at
10 times the target concentration, and 10 ml of the stock solution was made to a final
sample volume of 100 ml.

Gliadin (100, 200, 300, 400, 500 mg/l) and TA (40, 60, 80, 100, 120, 140 mg/l) were
combined in all possible combinations in buffer in 150 ml beakers. In each case the
beaker contents were stirred at 300 rpm for 1 min, then incubated at 25C in a water
bath for 30 min, and finally stirred again for 1 min. Haze intensity measurements and
particle size analysis were then carried out.

Haze intensity measurements were made with a Model 2100AN ratio turbidimeter
(Hach Co., Loveland, CO) in the ratio mode using 13 mm diameter cuvettes. Results
were expressed in nephelos turbidity units (NTU).

Particle size distribution patterns were estimated with a laser diffraction particle size
analyzer, Model LS120 (Coulter Instruments, Hialeah, FL) using the Fraunhofer
optical model. This instrument presents results for a sample over the particle diameter
range of 0.1 900 m as either the number % or the volume % of the total particles in
each of 100 channels of logarithmically increasing diameter.

The volume % data were converted into area % and then the individual channel
contributions to the total haze intensity were estimated as described previously (5, 6).

RESULTS AND DISCUSSION

Gliadin is, like barley hordein, a grain prolamin (proline-rich, alcohol soluble protein)
that has been shown to be quite haze-active (4). Tannic acid differs from most of the
beer polyphenols involved in haze formation (proanthocyanidin dimers) in that it has
five groups that provide strong attachment to proline-rich proteins (4). However,
gliadin and tannic acid are reasonably well-defined substances that are commercially
available and suitable for model system studies.

Gliadin and tannic acid were combined in various proportions in buffers of different
pH. In each case haze developed and the haze intensity and particle size distribution
pattern were observed. The haze intensity patterns at each pH are presented in figures
1-4.

In a previously reported study at pH 4.5, there appeared to be some fine structure in

the plot, with ridges corresponding approximately to gliadin: tannic acid weight ratios
of 2:1 and 5:1 (5), see figure 3. It can be seen that similar patterns also occurred at a
number of other pHs. Overall, the highest haze intensities were observed at pH 5 and
pH 5.5 (data not shown), with lower hazes at both lower and higher pHs. In the beer
pH range, the same amounts of gliadin and tannic acid produced much more haze at
pH 4.5 than at pH 4.0.

4
 500 10 12 500
7 9
8
6
400 6 400 20

6 7
300 300
5
6
200 4 200
20 4080
3
100 2 100
40 60 80 100 120 140 40 60 80 100 120 140
Tannic Acid (m g/L) Tannic Acid (m g/L)

Figure 1: Contour plot of haze Figure 2: Contour plot of haze

intensity (NTU) at various protein and intensity (NTU) at various protein and
polyphenol concentrations at pH 3.0. polyphenol concentrations at pH 4.0.

500 500
150 250
50 150 300
400 100 400
100 200
200
150
300 300
200 250
100
200 150 200
50 100
50 50
100 100
40 60 80 100 120 140 40 60 80 100 120 140
Tannic Acid (m g/L) Tannic Acid (m g/L)

Figure 3: Contour plot of haze Figure 4: Contour plot of haze

intensity (NTU) at various protein and intensity (NTU) at various protein and
polyphenol concentrations at pH 4.5. polyphenol concentrations at pH 5.0.

Human sensory perception of haze intensity was successfully modeled as a function

of particle concentration and diameter (1). It was shown from this equation that
turbidity is proportional to the particle diameter squared (6), and this made it possible
to estimate the haze intensity contribution from each channel determined by the
particle size analyzer. There is insufficient space to display all the individual results.
Of those in the beer pH range, results for pH 4.5 were published previously (5) and
those for pH 4.0 are shown in figure 5, over the particle diameter range 0.1 900 m.
It can be seen that particles of only a relatively small number of sizes were observed
in each individual plot. As the protein: polyphenol ratio varied, shifts between
particles of different sizes occurred. The larger haze intensities and to some extent
larger particles tended to correspond to intermediate gliadin: TA ratios.

5
a4
G
Q
12.5 17 8.33 18 6.25 24 5.0 33 4.17 38 3.57 38

0 0 0 0 0 0

4 0.1 0.1 0.1 0.1 0.1 0.1

10 12 6.67 15 5.0 38 4.0 25 3.33 24 2.86 28

0 0 0 0 0 0

4 0.1 0.1 0.1 0.1 0.1 0.1

7.5 10 5.0 13 3.75 14 3.0 15 2.5 22 2.14 31

0 0 0 0 0 0

4 0.1 0.1 0.1 0.1 0.1 0.1

5.0 8 3.33 10 2.5 10 2.0 17 1.67 21 1.42 116

0 0 0 0 0 0

4 0.1 0.1 0.1 0.1 0.1 0.1

2.5 5 1.67 6 1.25 12 1.0 34 0.83 29 0.71 40

0 0 0 0 0 0

0.1 0.1 0.1 0.1 0.1 0.1

40 60 80 100 120 140

Fig. 5. Estimated haze intensity (NTU) contributions of 0.1 - 900 m particles at

various protein and polyphenol concentrations at pH 4.0. The numbers in the upper
right corner are the total haze intensity in NTU of the sample, while the upper left
shows the gliadin:TA ratio.

100

80

60

40
0.54 2.0 7.9
20 191

Particle diameter (um)

Figure 6: Summed estimated haze intensity (NTU) contributions of particles of each

size at pH 3.0.

The estimated NTU contributions for all 30 conditions for each pH were summed for
each size channel. The results are shown in figures 6-9. Very different haze intensities
were observed with the same concentrations of protein and polyphenol at different
pHs. It is apparent that in each case particles of particular sizes predominate. While
the haze intensity was very low at pH 3 (figure 6), the pattern (particle diameters and
relative sizes of peaks) somewhat resembled that seen at pH 4 (figure 7). At pH 4.5

4
there was a definite shift to greater amounts of smaller particles (figure 8); the second
highest peak had similar particle diameter to a peak seen at lower pHs. The pH 5.0
results showed the peak at small diameter to be even more dominating (figure 9). It
was sharper, and the other peaks were relatively less important. At pH 5.5 (data not
shown above pH 5.0) there was a distinct shift to larger particle size and a nearly
bimodal distribution. The pattern at pH 6.0 was quite similar to that of pH 5.5,
although of lower amplitude. The pH 6.5 pattern shifted toward smaller diameter
particles.

100

80

60

40 6.6
0.35
190
20

Particle diam eter (um )

Figure 7: Summed estimated haze intensity (NTU) contributions of particles of each

size at pH 4.0.

100

80 0.27
4.6 9.5
60

40
45 191
20

Particle diam eter (um )

Figure 8: Summed estimated haze intensity (NTU) contributions of particles of each

size at pH 4.5.

5
 0.45

100

80
5.0 9.5 41
60

40

20

Particle diam eter (um )

Figure 9: Summed estimated haze intensity (NTU) contributions of particles of each

size at pH 5.0.

Most evidence indicates that protein-polyphenol interaction is not ionic, but rather
involves a combination of hydrophobic and hydrogen bonding (4). Since the
formation of haze particles involves bridging together of protein molecules by
polyphenols, the effect of pH in that case might be inhibition of haze development
through repulsion when the protein has either a high net negative or positive charge.
In such a case the greatest interaction would likely occur near the isoelectric point,
where the protein has net neutral charge. However, charge profile plots estimated for
gliadin and hordein (figure 10) are similar and indicate pIs well above any pH used
in this study (near 8). It is interesting to note that both prolamins have a net charge of
approximately +8 at beer pH (3.9-4.6).

20
15 hordein
10 gliadin
5
0
-5
-10
-15
-20
-25
0 2 4 6 8 10 12 14
pH
Figure 10: Molecular charges of gliadin and hordein at various pHs estimated from
amino acid sequences at web site: http://bioweb.pasteur.fr/seqanal/tmp/iep/.

6
Based on the results of this study, variations in pH in the normal beer range would be
expected to have significant effects on haze particle size as well as haze intensity.

Beers are rich in HA protein and low in HA polyphenol (7). Lowering HA protein
levels at constant HA polyphenol could lead to increased particle size and have an
impact on haze intensity. Chillproofing treatments that reduce the level of HA protein
in beer as a result could lead to formation of larger colloidal particles. However,
adsorbents typically remove both some protein and some polyphenol in complexes,
rather than only one or the other component (8,9), so the situation is complicated.

CONCLUSIONS
Haze intensity in a protein-polyphenol model system was definitely influenced by pH
as well as protein and polyphenol levels. At pH 3 haze intensity was very low. Haze
intensity increased markedly as pH was increased through pH 4 and 4.5 and reached a
maximum at pH 5 to 5.5. Further increases in pH resulted in declines in haze
intensity. This indicates that the pH of a beer in the normal range is likely to have an
influence on the formation of particles. This in turn should have an effect on apparent
visual turbidity, on sedimentation of the beer during storage or centrifugation, and on
removal of particles by filtration.

Overall, the greatest haze intensity at each pH tended to occur when both gliadin and
TA concentrations were high and in balance. The greatest haze intensity sometimes
coincided with large particle size, but sometimes was associated with large numbers
of small particles. Most samples contained particles mainly concentrated in two or
three size ranges. Shifts in the proportion of these occurred as protein/polyphenol
ratio was varied.

REFERENCES
1. Carrasco, A. and Siebert, K.J., Human visual perception of haze and relationships
with instrumental measurements of turbidity. Thresholds, magnitude estimation
and sensory descriptive analysis of haze in model systems, Food Quality &
Preference, 10: 421-436, 1999.

2. Freeman, G.J., Particle removal by powder filters, Journal of the Institute of

Brewing, 101: 439-445, 1995.

3. Reed, R.J.R. and Freeman, G.J., Beer filtration - fitting the filter aid to the beer,
Ferment 6: 64-67, 1993.

4. Siebert, K.J., Effects of protein-polyphenol interactions on beverage haze,

stabilization, and analysis, Journal of Agricultural and Food Chemistry, 47: 353-
362, 1999.

5. Siebert, K.J., Effect of protein/polyphenol ratio on the size of haze particles,

Journal of the American Society of Brewing Chemists, 58: 117-123, 2000.

7
6. Siebert, K.J., Relationship of particle size to light scattering, Journal of the
American Society of Brewing Chemists, 58: 97-100, 2000.

7. Siebert, K.J., Carrasco, A. and Lynn, P.Y., Formation of protein-polyphenol haze

in beverages, Journal of Agricultural and Food Chemistry, 44: 1997-2005, 1996.

8. Siebert, K.J. and Lynn, P.Y., Mechanisms of adsorbent action in beverage

stabilization, Journal of Agricultural and Food Chemistry, 45: 4275-4280, 1997.

9. Siebert, K.J. and Lynn, P.Y., Mechanisms of beer colloidal stabilization, Journal
of the American Society of Brewing Chemists, 55: 73-78, 1997.

10. Siebert, K.J. and Lynn, P.Y., Comparison of polyphenol interactions with PVPP
and haze-active protein, Journal of the American Society of Brewing Chemists,
56: 24-31, 1998.

11. Siebert, K.J., Troukhanova, N.V. and Lynn, P.Y., Nature of polyphenol-protein
interactions, Journal of Agricultural and Food Chemistry, 44: 80-85, 1996.

12. Yor Hang, L., Pecar, M., Sudarmana, D., Peel, R., Freeman, M. and Hawthorne,
D., Effect of storage conditions on the filterability of beer, Technical Quarterly,
Master Brewers Association of the Americas, 29: 37-41, 1992.

8
87

Applications of chemometrics in
brewing
Karl J. Siebert
Cornell University, Dept. of Food Science & Technology, Geneva, NY 14456, USA
(www.nysaes.cornell.edu, e-mail: [email protected])

Descriptors
Analysis method for brewing, chemical and physical analysis method, data processing,
process control

SUMMARY
Multivariate analysis procedures have occasionally been applied to brewing problems and
appear particularly appropriate and useful. Exploratory Data Analysis methods can reveal
how many fundamental properties are represented in a data set and could be used to
collectively examine a brewerys analytical procedures to find and reduce redundancy.
Pattern Recognition procedures can identify beer brands or raw material cultivars and detect
adulteration. They also have use in multivariate QA/QC. Empirical modeling has many
applications in process control and in discerning relationships between product composition
and properties. Multivariate calibration can measure either multiple analytes or single
analytes in difficult matrices.

Anwendungen der Chemometrie beim Brauen

Deskriptoren
Analysenmethode zur Bierherstellung, Datenverarbeitung, Methode zur chemischen und
physikalischen Analyse, Prozesteuerung

ZUSAMMENFASSUNG
Multivariante Analyseverfahren sind gelegentlich bei Problemen im Brauprozess angewendet
worden und schienen besonders sinnvoll und ntzlich. Untersuchende Datenanalysemethoden
knnen aufdecken, wie viele grundlegende Eigenschaften in einem Datensatz dargestellt
werden und konnten dazu verwendet werden, im brauanalytischen Verfahren Redundanzen zu
finden und zu verringern. Verfahren zur Strukturaufklrung knnen Biermarken oder
Rohstoffvarietten kennzeichnen und Verflschung aufdecken. Sie finden auch Anwendung
in multivarianten QA/QC-Systemen. Das empirische Modell hat viele Anwendungen in der
Prozesssteuerung und im Erkennen der Beziehungen zwischen Produktaufbau und
Produkteigenschaften. Multivariante Kalibrierung kann entweder mehrfache Parameter oder
einzelne Parameter in schwierigen Matrizen messen.
Applications de la chimiomtrie en brasserie industrielle

Descripteurs
Commande de procd, mthode danalyse pour la fabrication de la bire, mthode physico-
chimique danalyse, traitement des donnes

RESUME
Les techniques d'analyse multifonctionnelles ont t parfois appliques aux problmes de
brasserie, o elles s'avrent particulirement appropries et utiles. Les mthodes d'analyse
exploratoire des donnes peuvent rvler combien de proprits fondamentales sont
reprsentes dans un ensemble de donnes et peuvent tre utilises pour examiner
collectivement les mthodes analytiques d'une brasserie pour reprer et rduire la redondance.
Les techniques de reconnaissance de modles (Pattern Recognition) peuvent identifier les
marques de bire ou de matires premires ou dtecter les fraudes. Elles ont galement un
intrt dans le CQ/AQ multifonctionnel. Un modelage empirique a de nombreuses
applications dans le contrle des mthodes et pour discerner les relations entre la composition
et les proprits du produit. L'talonnage multifonctionnel peut mesurer plusieurs substances
analyser ou des substances uniques dans des matrices difficiles.

2
INTRODUCTION
The pattern of measurements carried out in brewing today is largely multivariate in
nature. A number of different measurements are typically made on each raw material
or on each beer produced on either the production or pilot plant scale. In addition, a
number of commonly used analytical methods produce multiple results from a single
sample (most notably multiple peaks in chromatography and absorbances at multiple
wavelengths in spectroscopy). The large quantity of results produced in modern
laboratories often makes gaining overall understanding of the information they
contain difficult.

Most human perceptions are a result of phenomena where multiple compounds

together produce a physical (haze, foam or color) or direct sensory phenomenon
(olfaction or taste). As a result multivariate methods are needed to successfully study
and develop understanding of these phenomena.

The application of good measurement principles and multivariate mathematics and

statistics to chemical data is the province of Chemometrics. There are problems in
using some of the classical multivariate statistical methods in that their assumptions
(e.g. minimal collinearity between measurements, high sample to measurement ratio)
are often violated by the nature of typical chemical or sensory analysis results.
Modern chemometric methods are robust to these situations (2, 6).

Chemometric methods have been used in brewing for about the last 20 years with
many reported applications to sensory and chemical analysis data; a review of this
topic was recently prepared (3).

EXPLORATORY DATA ANALYSIS (EDA)

Exploratory Data Analysis (EDA) is designed to produce useful information from
large and complicated data sets. In most cases this involves reducing the
dimensionality by minimizing redundancy and noise while retaining the meaningful
information. Two main approaches are used here, Cluster Analysis (CA) and
Principal Components Analysis (PCA)/Factor Analysis. An EDA data set is typically
represented as a matrix with samples as rows and measurements as columns.

Simple Distances Dendrogram from Hierarchical

x2 3 Cluster Analysis
1
d13
2
3
d23

1 1 Similarity 0
d12 2

x1

Figure 1: Diagram of hierarchical clustering based on distance.

3
Cluster Analysis uses a measure of similarity between samples (most often distance in
multidimensional space, or occasionally correlation) to represent similarity. The
results of hierarchical clustering are typically presented as a dendrogram (tree
diagram), see figure 1. Non-hierarchical clustering procedures also are used and these
perform well in some situations (6).

120 120

100 100

80 E 80 E
E E

x 2 60 E x 2 60 E
E E E E
E E E E
40 E 40 E
E E
20 E 20 E

0 0
0 20 40 60 80 100 120 0 20 40 60 80 100 120
x1 x1

120 120

100 100
E
80 E 80 E
E

x E E
60 x 2 60 J
2 E J EEJJJ
J
E E E JJJ E
40 E 40 JJ E
E E
20 E 20 E

0 0
0 20 40 60 80 100 120 0 20 40 60 80 100 120
x x1
1

Figure 2: Two-dimensional example of Principal Components Analysis. Samples are

plotted on two measurement axes (upper left) and a line accounting for maximum
variance is constructed (upper right). Points are projected onto the line and their
location can be expressed as a single number, the PC score (lower left). Additional
PCs are constructed orthogonal to the previous (lower right).

PCA focuses on the variance in a data set, see figure 2. Each original measurement is
considered to be a different dimension and the results for a sample represent its
coordinates in the multidimensional space. Often the information contributed by some
of the measurements is somewhat redundant. The first principal component (PC) is
extracted by projecting a line through the data along the axis that explains maximum
variance (this often resembles a regression line). Each sample is projected
perpendicularly onto the line and its location along this single dimension is called its

4
score on this PC axis. The PC score is a linear function of all the original
measurement values for a sample, each multiplied by a different coefficient.
Subsequent PCs are extracted in turn, in each case constrained to be orthogonal (at
right angles) in multidimensional space to all the previous PCs. This makes the PCs
totally uncorrelated and thus non-redundant in information content. While as many
PCs as original measurements can be extracted, typically only a small number (often
2-4) are needed to represent most of the information. This results in a considerable
reduction in dimensionality and facilitates plotting and understanding the data. For
example, in a study of human visual perception of turbidity, PCA was applied to
descriptive analysis results where a set of samples was rated on each of six attributes
(1). The first two PCs accounted for over 98% of the variance in the data set,
indicating that although the panel rated six attributes, fundamentally panelists
perceived only two phenomena. PCA revealed this and also showed which of the
attributes were redundant. PCA has frequently been applied to sensory and chemical
analysis data in brewing (3).

In Factor Analysis the PC structure is rotated in multidimensional space to improve

the alignment between the original measurements and the PCs. This often helps in
developing conceptual understanding of the results.

Future possibility
In most brewing companies the analytical methods that are employed have been
individually determined to be useful for monitoring or controlling the raw materials or
process. Rarely have the methods been looked at collectively. Since it is quite likely
that there is considerable redundancy between at least some of the measurements, it is
almost certain that more work than necessary is being carried out. It would be very
interesting to determine the optimum combination (the minimum number of methods
that would contain all the useful information). This could be approached by carrying
out PCA on results of each analysis from a range of samples at a particular process
point.

PATTERN RECOGNITION (PARC)

Pattern recognition is used to determine the classifications of samples from their
patterns of measurements. For this, each sample must have a class identification (e.g.
raw material cultivar or place of growth, beer brand or brewery, etc.). The class
identifications of a training set of samples are used to develop rules that estimate the
class identity of new samples.

In k-Nearest Neighbor Analysis (KNN) the distance of a sample from others in

multidimensional space is used to make a classification, see figure 3. For example,
using 3-NN, if, of three samples closest to one being classified, two belong to class B
and one to class C, the sample would be identified as class B (majority wins).

In Linear Discriminant Analysis (LDA), see figure 4, a boundary between each pair
of classes is constructed. In two dimensions this is a line, in three a plane and in more
a hyperplane. Samples are identified by the side of the boundary on which they are
located.

5
 x2 J
x2 H 3-NN
H H
H H
H H
H B H
H J H B
H
H B B
B H B B B
B B
B B

x1 x1

Figure 3: Example of KNN using the Figure 4: Example of linear

three nearest neighbors to assign class discriminant analysis. The solid line
identity to the sample represented by separates the triangle and square
the solid circle. classes. The circle would be judged a
member of the triangle class.

With SIMCA, a model for each class that completely bounds it is constructed by
making a PC model of the samples belonging to the class and using the dispersion of
the samples to establish boundaries in all dimensions (see figure 5). The number of
significant PCs is estimated for each class. This may be 0, which indicates the
samples are not homogeneous, 1, 2 or some higher number. The number of significant
PCs determines the shape of the class envelope (6). If there are no significant PCs, the
shape is a sphere. For one significant PC it is a cylinder, for two a box and for three or
more a hyperbox (a multidimensional flat sided envelope).

X2 J
J J J
J J J
J J
J J J J
J
J J
K

Figure 5: SIMCA constructs a PC model of X1

each class and establishes boundaries in all H
directions based on sample dispersion. Class H
L is bounded by a one-component model H
H
while class K is modeled by a two- H L
X3
component model.

In a study of capillary GC patterns of hop essential oils, a number of varieties grown

in Europe and North America were compared (5). LDA was fairly successful in
classifying samples according to their cultivar, while SIMCA was completely

6
successful. However, SIMCA found no significant PCs for the Hallertau samples,
indicating that this group was not homogeneous.

Unlike LDA, SIMCA bounds classes on all sides, which reveals samples that do not
belong to any known class. It is also useful when a sample may be an outlier in any
direction in a multivariate sense (see figure 6). This has application in detecting
adulteration and in multivariate QA/QC.

X3 J J
J
J
H J
H HH
J
J J
HH H J J
HHH X2
J J J
J

Figure 6: SIMCA model when one class is

surrounded by other samples, e.g. in detecting
adulteration or performing multivariate
X1 QA/QC.

Possible future application

Since a beer brand must simultaneously satisfy multiple criteria, multivariate QA/QC
should take into account the entire pattern of results rather than make a series of
univariate tests. A SIMCA model of the class of good or acceptable samples could
be used to assess the quality of new samples (i.e. to determine if they are well within
the envelope of good quality, close to the edge, or outside it).

EMPIRICAL MODELING
Empirical modeling makes no assumptions about the fundamental form of
mathematical relationships. Rather it employs good experimental design to efficiently
collect data suitable for construction of mathematical (response surface) models that
explain behavior within a certain region. This is useful for gaining an understanding
of and optimizing system behavior, where the system may be an on-line or laboratory
analytical method, a process or unit operation, or a product composition to property
relationship.

One variable at a time experiment designs have frequently been used but are unable to
detect interactions between variables and can fail to find true optima in a number of
situations. Statistical experiment design employs multiple combinations of variable
levels to thoroughly explore a region of interest and is well suited to gentle curvature
and interaction, phenomena that are frequently seen. A number of statistical
experiment designs exist and these provide great efficiency in collecting useful data
for the purpose at hand. Screening designs (often fractional factorials) establish which
of a list of factors actually have a significant influence on a response variable. Modest
numbers of variables can be explored with full factorials or composite designs.
Balanced reductions of some combinations are practical with large numbers of

7
factors, and can conserve resources, including time. Once the data are collected
(response or responses at each experimental condition), modeling is used to construct
an equation describing the behavior.

In multivariate modeling a row in a matrix represents one combination of conditions

(the independent variables or xs) and one or more corresponding response
observations (dependent variable(s) or ys). A regression procedure such as multiple
linear (or ordinary least squares) regression (MLR), principal components regression
(PCR) or partial least squares (PLS) regression is used to develop an equation in
which the behavior of a dependent variable is a function of some combination of
independent variables:
y = b0 + b1x1 + b2x2 + + bnxn

Such a formula can be used to predict behavior and to select optima. MLR (figure 7)
develops the equation through a least squares model of all the independent variables.
It assumes that the variables are not highly correlated and that there are significantly
more samples than x variables. This is usually the case in a statistically designed
experiment, but often not so with other data.

p y

X y = f(X
n n
Figure 7: In Multiple Linear Regression (MLR) the y variable is modeled from all the
x variables.

PCR first computes principal components, t, of the x variables and then relates some
of the t variables to the response (figure 8). Due to the nature of PCA, this removes
any correlation between the variables used for modeling and also increases the sample
to variable ratio (because fewer t variables are needed to represent the information in
the x variables).

p m y
T = f(X)
X T y = f(T)
n n
Figure 8: In Principal Components Regression (PCR) the y variable is modeled from
principal components (t) of the x variables.

PCA seeks to explain variance rather than to predict a response, and can be improved
upon for modeling by focusing on the PCs with best ability to predict a response
rather than to explain variance. That is how PLS operates (see figure 9).

8
 p m y
T = f(X)
X T
y = f(T)
n n

Figure 9: In Partial Least Squares Regression (PLS) the y variable is modeled from
principal components (t) of the x variables that predict y.

In a model system study of the effects of conditions on haze intensity, various

concentrations of the haze-active (HA) protein gliadin were combined with different
amounts of the HA polyphenol tannic acid at various pHs and alcohol contents
according to a statistical design (4). The results were modeled, resulting in a five
dimensional equation (the four independent variables plus the response). This can be
viewed by holding two of the variables constant and representing the predicted effect
of the other two on the response (see figure 10).

160
140
120

Haze (NTU)
100
80
60
40
20
100 500
80 400
60 300
Tannic acid 40 200
Gliadin
(mg/L) 20 100 (mg/L)

Figure 10: Example of a composition to property model: Response surface model

showing the effects of protein (gliadin) and polyphenol (tannic acid) concentrations
on haze intensity in a buffer model system; predicted for 6%(v/v) alcohol and pH 3.7.
Reproduced with permission from J. Agric. Food Chem., 1996, 44, 1997-2005 (4).
Copyright 1996 Am. Chem. Soc.

Numerous applications of empirical modeling have been reported in brewing (3).

Laboratory applications include ruggedness testing of methods (an ASBC and EBC
official method), response surface modeling and optimization of method conditions,
and development of calibration models (e.g. for NIR measurements). Many models of
unit operations in malting and brewing have been constructed, particularly of mashing
and fermentation. Models of filtration, packaging and hop utilization in the kettle
have also been described. Models describing product properties as a function of beer

9
composition have been developed to describe foam, haze, flavor and flavor stability.
One model of compound properties (flavor thresholds of acids in beer) as a function
of molecular parameters has been reported.

Future possibility
While multivariate calibration has been used to simultaneously determine several
analytes in hops, barley, malt and beer by NIR, an application not reported yet in
brewing is the use of multivariate calibration for determination of a single analyte in a
difficult matrix. This could substitute relatively simple measurements and
complicated mathematics for complete separation of a compound before detection.

CONCLUSIONS
Chemometrics has considerable potential to improve extraction of information from
measurements for several purposes. EDA can greatly reduce dimensionality and aid in
improving understanding of data sets. PARC can be used to monitor raw materials
and products to detect adulteration and to improve evaluation of quality. Multivariate
modeling can be used to gain better knowledge and control of measurements,
processes and product properties, as well as to promote understanding of human
perceptions of them.

REFERENCES
1. Carrasco, A. and Siebert, K.J., Human visual perception of haze and relationships
with instrumental measurements of turbidity. Thresholds, magnitude estimation
and sensory descriptive analysis of haze in model systems, Food Quality &
Preference, 10: 421-436, 1999.

2. Massart, D.L., Vandeginste, B.G.M., Buydens, L.M.C., De Jong, S., Lewi, P.J.
and Smeyers-Verbeke, J., Handbook of Chemometrics and Qualimetrics: Part A,
Elsevier Science, Amsterdam (Vandeginste B.G.M., Rutan S.C., eds. Data
Handling in Science and Technology); Vol. 20A, 1997.

3. Siebert, K.J., Chemometrics in brewing - a review, Journal of the American

Society of Brewing Chemists, in press.

4. Siebert, K.J., Carrasco, A. and Lynn, P.Y., Formation of protein-polyphenol haze

in beverages, Journal of Agricultural and Food Chemistry, 44: 1997-2005, 1996.

5. Stenroos, L.E. and Siebert, K.J., Application of pattern-recognition techniques to

the essential oil of hops, Journal of the American Society of Brewing Chemists,
42: 54-61, 1984.

6. Vandeginste, B.G.M., Massart, D.L., Buydens, L.M.C., De Jong, S., Lewi, P.J.
and Smeyers-Verbeke, J., Handbook of Chemometrics and Qualimetrics: Part B,
Elsevier Science, Amsterdam (Vandeginste B.G.M., Rutan S.C., eds. Data
Handling in Science and Technology); Vol. 20B, 1998.

10
88

Development of image analysis

technology for breweries
Gearid Cahill1, Padraig K. Walsh2 & Dan Donnelly1
1
Guinness Ltd., St. Jamess Gate, Dublin, Ireland (e-mail: [email protected])
2
Dublin City University, School of Biotechnology, Dublin 9, Ireland

Descriptors
Glycogen, image analysis, staining, vitality, yeast propagation, yeast technology

SUMMARY
Near real time information regarding the quality of brewing yeast is important in a
fermentation management system. In our brewery, image analysis techniques have been
developed which provide near real time information on brewing yeast quality and physiology.
Imaging techniques have been developed to determine changes in yeast cell size during
propagation, fermentation and storage. A novel rapid technique has been developed using a
combination of image analysis and cellular staining to determine the glycogen content of
yeast on an individual cell basis. These techniques are described and their application in a
brewery yeast management process are explored.

Entwicklung einer Bildanalysentechnik fr Brauereien

Deskriptoren
Bildanalyse, Frbung (Biologie), Glykogen, Hefetechnologie, Hefevermehrung, Vitalitt

ZUSAMMENFASSUNG
Die Annherung an Echtzeit-Informationen in Bezug auf die Qualitt der Bierhefe ist ein
bedeutender Punkt im Grungsmanagement. In unserer Brauerei wurde eine Bildanalyse
entwickelt, die nahezu Echtzeit-Informationen in Bezug auf die Bierhefequalitt und
Hefephysiologie liefert. Die Bildanalyse wurde ent-wickelt, um nderungen der Hefezell-
gre whrend der Propagation, der Grung und der Lagerung zu detektieren. Eine neue,
schnellere Technik, die eine Kombination aus Bildanalyse und Zelllinien verwendet, um den
Glycogengehalt der Hefe zu bestimmen, wurde entwickelt. Diese Techniken werden hier
beschrieben und deren Anwendungen im Hefemanagement einer Brauerei werden untersucht.
Dveloppement de la technologie d'analyse d'image pour les brasseries

Descripteurs
Analyse d'image, coloration (biologie), glycogne, propagation de la levure, technologie de
la levure, vitalit

RESUME
Il est important, dans un systme de gestion des fermentations, de disposer d'informations
presque en temps rel sur la qualit de la levure de bire. Dans notre brasserie, nous avons
dvelopp des techniques d'analyse d'image apportant des informations presqu en temps rel
sur la qualit et la physiologie de la levure de bire. Des techniques d'imagerie ont t mises
au point pour dterminer les variations de taille des cellules de levure pendant la propagation,
la fermentation et le stockage. Nous avons notamment dvelopp une nouvelle technique
rapide combinant l'analyse d'image et la coloration cellulaire pour dterminer la teneur
individuelle en glycogne des cellules de levure. Ces techniques sont dcrites et leur
application dans un procd de traitement de la levure de bire est discute.

2
INTRODUCTION
Many reported biotechnological applications of image analysis concern the
morphological and physiological characterisation of industrially-important micro-
organisms, including filamentous micro-organisms (13), dimorphic yeast (7) and yeast
(3).With increasing pressure on brewery production capacity and the prevalence of
high-gravity brewing, there are increased demands placed on brewery yeast. Increased
automation and improved fermentation plant go some of the way towards meeting the
demands placed on breweries. However, yeast management cannot remain unaltered if
the goals of maintaining yeast quality and fermentation performance are to be met in
an ever-changing brewery environment. New technologies for the rapid assessment of
yeast quality are of great importance. Image analysis is such a technology.
The morphology of yeast cells can be related to its physiological status, for example, it
is known that some yeast strains elongate when exposed to stress (11). Image analysis
of yeast during fermentation provides near-real-time information about the
distribution of yeast cell size and the proportion of the population which exist as
single cells, budding cells and clusters (16).
Techniques have been developed to determine the actual physiological state of yeast
(as opposed to its morphology) using a combination of staining techniques and image
analysis. A non-destructive method involving modern fluorescent staining techniques
combined with image analysis have been used to measure the intracellular pH of intact
yeast cells. This technique illustrates the evolutionary direction of image analysis.
Early methods involved the processing of monochrome images to yield physical
measurements of cells (9). Later methods combined staining techniques to identify
cells or regions of interest in images based on staining or non-staining (15). Currently,
imaging techniques have become more sophisticated, and are capable of estimating
physiological parameters such as intracellular pH (6) and glycogen content (5).
Image processing times depend on the complexity of the analysis and on computer
processing speeds. Processing times for micro-organisms are continuously decreasing,
for example; 83 hr (1), 17 hr (1), 4 hr (8), 89 min. (7), 8 minutes (5) and 80 sec (2).
Other applications for image analysis include determination of brewing materials
quality, for example; thousand corn weights can be determined rapidly and
measurement of cross contamination of cereals is also possible (14). Image analysis
technology has been successfully used in packaging plants, for example the keg plant
in our brewery in St. Jamess Gate uses imaging technology to identify keg faults
(dirty keg, beer leakage, incorrect or missing keg cap) and divert kegs from production
conveyors. Commercially available inspection systems are available to inspect bottles
after filling (e.g. Akitek, Montreal, Canada). Inspection includes checks for foreign
particles; bottle neck damage, incorrect, missing or damaged crowns, and fill level
(underfill & overfill). Inspection systems can operate at high speed (72,000 bottles per
hour).

FUNDAMENTALS OF IMAGE ANALYSIS

Vision is an essential part of mankinds understanding and interaction with his
environment. The invention of the microscope in the 17th century by Antonie van
Leeuwenhoek has played a significant part in the advancement of our understanding of
the principles of microbiology. Whereas photography has been in existence for over

3
100 years, development of image analysis has been totally dependent on the evolution
of other technologies e.g. camera, video and computer technology.
Image analysis refers to the digitisation of an image into a grid of pixels which enables
the measurement of the light intensity at each pixel. An eight-bit black and white
image is recorded as an array of pixels with a range of 256 (28) light intensity values
ranging from black (0) to white (255), which surpasses the sensitivity of the human
eye (10). Colour images consist of three separate colour bands (red, green and blue),
each with an intensity range from 0 to 255. Colour images require much greater
resolution to compare to the sensitivity of the human eye, e.g. 12-bit (212 colours).
An image analysis system consists of five main components, a personal computer
(PC), a framegrabber board, a camera, appropriate magnification lenses and image
analysis software (see figure 1). Depending on the application, the video camera can
be attached to a microscope, a macro lens or even a a telescope. The framegrabber
board installed in the PC converts the signal from the video camera into a digitised
format consisting of 100,000s of pixels. Once digitised, the images can be processed
using the image analysis software.

The main elements of image analysis

Image acquisition - images are relayed from a video camera to a computer via a
framegrabber board. Once stored in the memory of the computer as an image file, the
appropriate image analysis software can be used to process the image.

B
A

D
C

Figure 1: A schematic of a typical image analysis system incorporating a video

camera (A), PC monitor (B), PC processor and framegrabber board (C) and
microscope (D).

4
Image enhancement filtering techniques have been developed to enhance image
quality by eliminating or reducing picture defects e.g. blurring, poor illumination,
electrical interference and dirt on the camera lens.

Feature identification - features of interest are identified by distinguishing them from

the image background and from other objects which may be present. The most widely
used method of feature detection is thresholding. This method is based on the
assumption that the features of interest consist of, or are bounded by, pixels which are
significantly different from the image background by being darker or brighter. By
setting an appropriate threshold value, the operator selects all pixels in the image with
a specific range of intensity values. Features are then identified as a connected group
of pixels.

Feature classification - it is likely that other objects in the image have been detected
which are not of interest. Classification based on size and shape of all detected objects
allows the filtering out of all unwanted objects.

Feature measurement - the features of interest can then be measured and the data
output sent to a spreadsheet for analysis. Basic measurement of features include
length, breadth, cross-sectional area, perimeter and circularity.

EXPERIMENTAL
Yeast strains and fermentation conditions
Brewery strains of ale and lager yeast were used and are identified as strain 1164 and
strain 7012 respectively. Standard ale and lager brewery wort was used in all
experiments as indicated. 100 ml of lager wort was dispensed into 250 ml
Erylenmeyer flasks and sterilised for propagation trials. These flasks were inoculated
with an overnight culture of yeast and incubated with shaking at 24C. Fermentations
were conducted in standard brewery lager or ale wort with an original gravity as
indicated in the text. All fermentations were conducted in EBC fermentation tubes.
The fermentation temperatures for lager and ale fermentations were 15 and 24C
respectively and the pitching rate was 15 and 10 million cells per ml respectively. The
wort was saturated with air prior to pitching. Viability was measured using Methylene
Blue.

Image Analysis methods

Yeast cell size was determined at 400 x magnification using a JVC KY-F55B colour
video camera (Victor Company of Japan Ltd., Japan) attached to a Nikon Optiphot
microscope (Nikon Corp., Tokyo). The images were processed with Optimas 6.1
image analysis software (Media Cybernetics, Washington, USA). The algorithm for
determination of cell size has been described previously (2).
Glycogen content of yeast was determined using a novel method based on the
combination of image analysis and glycogen staining of whole yeast cells using I2:KI
solution (5). The intensity of staining is directly proportion to the glycogen content of
the yeast high-glycogen yeast stain dark brown while low-glycogen yeast stain pale
yellow. Two important advantages of this method are 1) rapid analysis and 2) the

5
measurement of the glycogen distribution within the yeast population and the mean
glycogen content.

RESULTS
The impact of wort gravity on the cell size of lager yeast
Lager yeast were propagated in worts with original gravities (OG) of 7.5, 10 and
10.5P. Whereas the cell counts at the end of propagation were similar (figure 2), the
biomass yield (data not shown) and mean cell size of the yeast increased with
increasing wort OG (figure 3). Similar findings have been observed for ale yeast (2).
Osmotic effects alone, from either the increased solids content in the wort at the start
or from increased ethanol concentration at the end of propagation should result in a
decrease in cell size. This is clearly not the case. The change in cell size is most likely
a stress response by the yeast to its environment. To evaluate this phenomenon, the
propagated yeast were used to pitch lager fermentations at OG 12.5P. Fermentation
rates were similar in all cases. However, the yeast viability decreased towards the end
of fermentation for yeast which were propagated at higher gravities (figure 4). Greater
losses in viability were observed for high gravity ale yeast fermentations using yeast
propagated at different gravity. Viability figures of 93, 90 and 85% were measured at
the end of high gravity fermentation for yeast propagated in wort gravities of 7.5, 12.5
and 17.5P respectively (2).

Glycogen measurement
Coupling of image analysis technology with yeast staining techniques results in a
more sophisticated method which gives more information regarding yeast physiology.

160

140

120
Cell Counts (million / ml)

100

80

60

40
OG 7.5P
20 OG 10P
OG 12.5P

0
0 10 20 30 40 50 60 70 80
Time (h)

Figure 2: The impact of wort gravity on lager yeast cell numbers

6
 225

220

215
Mean Cell volume ( m )
3

210

205

200

195

OG 7.5P
190
OG 10P
OG 12.5P
185
0 10 20 30 40 50 60 70 80
Time (h)

Figure 3: The impact of wort gravity on lager yeast cell size during propagation.

99
OG 7.5P
OG 10P
OG 12.5P
98

97
% Viability

96

95

94
0 50 100 150 200
Time (h)

Figure 4: The impact of propagation wort gravity on lager yeast viability during
fermentation.

7
Glycogen content is an indicator of yeast vitality. However, using glycogen content
has not universally resulted in optimisation of yeast pitching rates (12). To date,
glycogen analysis has been focussed on determination of a mean glycogen content for
a yeast population. Mean glycogen measurement is only of use if the glycogen content
of individual yeast cells in a population is distributed uniformly about the mean value.
This is not always the case. Using image analysis, a novel technique has been
developed which determines the mean glycogen content of a yeast population and in
addition can determine the distribution of cell glycogen content throughout the
population. This method has uncovered some interesting findings.
The range of cellular glycogen content in a brewing yeast population can be very
broad. It has been reported previously that the range of cellular glycogen content is
greater in cropped ale yeast compared to propagated yeast and that a broad range of
cellular glycogen content is indicative of poor yeast quality (5). Further work has
indicated that the glycogen distribution can differ during fermentation. Figure 5
indicates the different cellular glycogen distribution of ale yeast in fermentation versus
in the yeast crop (or plug) towards the end of fermentation. It should be noted that the
glycogen distribution of both samples is not uniformly distributed around the mean.
Secondly, there is a significant difference between the glycogen content and range of
glycogen content of the yeast in suspension and in the yeast which has sedimented into
the FV crop.
Yeast can persist in many different local environments during the fermentation before
ending up in the yeast crop; i.e. in the yeast head (ale and stout only), in suspension
and in the yeast crop for different periods of time. The yeast crop in the base of a

60

50

40
Frequency (%)

30

20

10
Ferm
Plug

0
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
Optical Density

Figure 5: The distribution of glycogen concentration in two ale yeast populations

towards the end of fermentation.

8
fermenter contains regions with different local temperatures which can impact on
yeast quality (4). Glycogen dissimilation occurs during the latter stages of
fermentation and these findings indicate that dissimilation is greater in the yeast plug
where there is severe nutrient limitation and increased local temperatures. Therefore,
it is not surprising that there is a wide range of yeast vitality in a cropped yeast
population (as determined by glycogen content). Methods which determine mean
glycogen are therefore of limited use.

CONCLUSIONS
Image analysis has been demonstrated to be a powerful analytical tool in brewery
yeast management. The systems described augment the information available to a
skilled microbiologist. Simple cell size determinations are therefore very useful for
determining subtle difference in yeast morphology which are a result of physiological
changes in the yeast. Combining the technology with standard staining techniques
provides even greater information regarding the entire population of yeast cells as
opposed to the population mean. This work indicates that the glycogen distribution in
yeast populations is not always uniform and therefore measure of mean glycogen
content is of limited use.
As the technology advances it is likely that the distribution of vitality indicators within
a population will be routinely reported alongside the mean value. Such an approach, if
applied to the image analysis method for intracellular pH (6) could greatly increase the
information available regarding yeast quality.

REFERENCES
1. Adams, H.L. and Thomas, C.R., The use of image analysis for morphological
measurements on filamentous microorganisms, Biotechnol. Bioeng., 1988,
32:707-712.
2. Cahill, G., Murray, D.M., Walsh P.K. and Donnelly, D.,The effect of the
concentration of propagation wort on yeast cell volume and fermentation
performance, J. Am. Soc. Brew. Chem., 2000, 58:14-20.
3. Cahill, G., Walsh P.K. and Donnelly, D., Improved control of brewery yeast
pitching using image analysis, J. Am. Soc. Brew. Chem., 1999, 57:72-78.
4. Cahill, G., Walsh, P.K. and Donnelly, D., A study of thermal gradient
development in yeast crops, Proc. 27th Congr. Eur. Brew. Conv., Cannes, 1999,
695-702.
5. Cahill, G., Walsh P.K. and Donnelly, D., Determination of yeast glycogen
content by Individual Cell Spectroscopy using image analysis, Biotechnol.
Bioeng., 2000, 69:312-322.
6. Imai, T. and Ohno, T., Measurement of yeast intracellular pH by image
processing and the change it undergoes during growth phase, J. Biotechnol.,
1995, 38:165-172.
7. OShea, D.G. and Walsh, P.K., Morphological characterisation of the dimorphic
yeast Kluyveromyces marxianus var. marxianus NRRLy2415 by semi-
automated image analysis, Biotechnol. Bioeng., 1996, 51:679-690.

9
8. Packer, H.L., Keshavarz-Moore, E., Lilly, M.D. and Thomas, C.R., Estimation
of cell volume and biomass of Penecillium chrysogenum using image analysis,
Biotechnol. Bioeng., 1992, 39:384-391.
9. Pons, M.N., Vivier, H., Remy, J.F. and Dodds, J.A., Morphological
characterization of yeast by image analysis, Biotechnol. Bioeng., 1993, 42:1352-
1359.
10. Russ, J.C., The Image Processing Handbook, 2nd Ed., CRC Press, Boca Raton,
1995, 481-487.
11. Shimozaka, M., Masui, S., Togawa, Y. and Okazaki, M., Cell aggregation and
elongated cell morphology caused by a mutation conferring increased sensitivity
to indole-3-acetic acid in the yeast Saccharomyces cerevisiae, J. Ferment.
Bioeng., 1991, 72:485-487.
12. Slaughter, J.C. and Nomura, T., Intracellular glycogen and trehalose contents as
predictors of yeast viability, Enzyme Microb. Technol., 1992, 14:64-67.
13. Treskatis, S.K., Orgeldinger, V., Wolf, H and Gilles, E.D., Morphological
characterisation of filamentous microorganisms in submerged cultures by on-
line digital image analysis and pattern recognition, Biotechnol. Bioeng., 1997,
53:191-201.
14. Van Laarhoven, H.P.M., Duijnhouwer, I.D.C., Angelino, S.G.A.F., Image
analysis as a practical tool in barley-malt-beer chains, Proc. 26th Congr. Eur.
Brew. Conv., Maastricht, 1999, 183-189.
15. Vanhoutte, B., Pons, M.N, Thomas, C.R., Louvel, L. and Vivier H.,
Characterisation of Penecillium chrysogenum physiology in submerged cultures
by colour and monochrome image analysis, Biotechnol. Bioeng., 1995, 48:1-11.
16. Zalewski, K. and Buchholz, R., Morphological analysis of yeast cells using an
automated image processing system, J. Biotechnol., 1996, 48:43-49.

10
89

Sensory analysis - a bridge with the

consumer. The use of an external expert
sensory panel
Paul Hegarty, Janice Chilver & Lee Threapleton

Bass Brewers Ltd, Bass Technical Centre, P.O. Box 12, Cross Street, Burton on Trent DE14
1XH, United Kingdom (e-mail: [email protected])

Descriptors
Beer quality, quality control, sensory analysis, taste panel

SUMMARY
Reliable flavour data is essential for controlling and optimising beer quality. Historically
flavour data has been seen as subjective and results have lacked credibility. A panel of
external expert tasters has been recruited that produces highly repeatable and robust sensory
data. Procedures for recruiting, training and operating the panel are described. Extensive
training, performance monitoring and coaching dramatically improves the repeatability of
sensory results. The panel is used for quality control of existing products and the assessment
of trial beers. Results are also used, in conjunction with external consumer research, to
optimise existing products and identify the optimum new product formulations.

Sensorische Analyse Eine Brcke zum Verbraucher. Der Einsatz eines externen
Experten-Sensorikpanels

Deskriptoren
Bierqualitt, Gteregelung, sensorische Analyse, Verkostergremium

ZUSAMMENFASSUNG
Verlssliche Geschmacksdaten sind unerlsslich fr die Kontrolle und Optimierung der
Bierqualitt. Historische Geschmacksdaten haben sich als subjektiv herausgestellt, und diese
Ergebnisse erweisen sich nicht als vertrauenswrdig. Sehr gut reproduzierbare und gefestigte
sensorische Daten wurden durch das Zusammenstellen eines externen Panels, bestehend aus
extern geschulten Testern, erhalten. Die Vorgehensweise der Auswahl, der Ausbildung und
der Arbeitsweise des Panels werden in dieser Arbeit beschrieben. Umfassendes Training, die
Beobachtung der Umgebung und Unterweisung der Panelteilnehmer lie die Reproduzier-
barkeit der sensorischen Ergebnisse deutlich ansteigen. Das Panel wird fr die Qualitts-
kontrolle von bereits bestehenden Produkten genutzt, hilft aber auch bei der Beurteilung von
Versuchsbieren. In Zusammenarbeit mit der externen Verbraucherforschung werden die
Ergebnisse auch zur stetigen Verbesserung bereits bestehender Produkte sowie zur Erstellung
optimaler neuer Produktrezepturen verwendet.
L'analyse sensorielle un pont vers le consommateur. Recours un panel externe
d'experts sensoriels

Descripteurs
Analyse sensorielle, contrle de qualit, jury de dgustation, qualit de la bire

RESUME
Il est essentiel de disposer de donnes fiables sur l'arme pour contrler et optimiser la qualit
de la bire. Les donnes historiques sur l'arme ont t juges subjectives et les rsultats
manquaient de crdibilit. Un panel d'experts externes goteurs a t recrut, aboutissant
des donnes sensorielles extrmement reproductibles et solides. Les procdures de
recrutement, de formation et de fonctionnement du panel sont dcrites. Une formation
approfondie, le suivi et l'encadrement des sances de gotage amliorent de faon
spectaculaire la rptabilit des rsultats sensoriels. Le panel est employ pour le contrle de
qualit des produits existants et l'valuation des bires l'essai. Les rsultats sont galement
utiliss en association avec les tudes de consommation externe pour optimiser les
produits existants et laborer la formule optimale des nouveaux produits.

2
INTRODUCTION
Whilst not all good beers are successful brands, all successful brands are good
intrinsic products. Successful brands perform well in unbranded consumer tests. The
challenge for marketeers is to develop successful brand images; the challenge for
technical brewers is to develop and produce consistently good beers. The key to this is
to identify what drives drinker preference. In consumer research, drinkers can say
what they like but find it almost impossible to say why they like products. Most
qualitative consumer research comments are simply repetitions of liking or disliking
(nice, nasty, refreshing etc). It is of no value to go back to brewers and ask them to put
more "nice" into the beer or less "nasty"! We need to be able to identify the specific
product parameters that drive preference.
There are a number of sources of product quality information that can be used to
optimise beers (eg chemical analysis, sensory analysis and consumer research). The
goal is to understand how changing product specifications affects drinker preference.
It is possible with statistical techniques to inter-relate different sets of data (figure 1).
However this is only possible if the underlying data is robust. If you put rubbish into a
statistical model, you will get rubbish out.
The brewing industry has been very successful at improving the reliability of its
analytical methods. We have internationally agreed methods with greatly improved
precision, international proficiency testing schemes and many new techniques such as
HPLC (1) and GC/MS that allow far more effective analysis of beers.
Whilst most companies do not share the details of their methodologies, consumer
research techniques have also improved greatly. This is often driven by close co-
operation between technical and marketing staff to ensure that consumer research
methods are robust and they satisfy both technical and marketing needs.
Chemical analysis and consumer research data can now be regarded as very reliable,
but flavour data has often been perceived to be a weak link because sensory results
often show poor repeatability and limited accuracy.
It is possible that one could try to model drinker preference with analytical results
alone. Beer has been shown to contain over a thousand different compounds and
many of these are flavour active at minute levels (parts per billion and even parts per
trillion) and compounds interact both positively and negatively (3,4). It is not possible
to assess beer quality accurately using analysis alone. In order to accurately assess
beer quality, you must assess flavour.
This leaves a conundrum; sensory information is essential for ensuring that our
products meet the needs of drinkers but sensory information is perceived to be
unreliable. Fortunately sensory information need not be unreliable provided
appropriate sensory disciplines are adopted (2,3). Whilst improvements in chemical
analysis have been largely driven by improvements in methods, the only way to
enhance sensory results is by improvements in the people who make the assessments.

ENHANCING SENSORY RESULTS

There are four steps to improving the reliability of sensory performance:
1. recruiting tasters with the correct sensory skills
2. rigorous training
3. monitoring of the performance of tasters
4. on going coaching to maintain and improve performance.

3
 Sensory Analysis
Chemical Analysis

Computer Statistics
Packages

Consumer Research

Figure 1: Relating internal quality data to external consumer research results.

Most brewing companies use their own employees as tasters. This leaves little or no
scope for selecting people with good sensory ability; one has to use the pool of people
who are available. Employees are busy and the time available for sensory training is
limited.
Disciplines for monitoring taster performance are well established (5). These rely on
assessing replicate samples and carrying out statistical analysis to compare the
repeatability of results. This approach allows you to establish whether observed
differences in sensory score are statistically significant relative to differences in scores
between replicates and between individual tasters. This provides valuable insight into
areas of panel weakness such as inconsistency of scoring between tasters and poor
replication between samples. Such information is used to target coaching to enhance
the performance of individual tasters and the panel as a whole. The success of the
coaching in enhancing performance is monitored by on going statistical monitoring of
the panel performance. Thus there is an on-going cycle of tasting, performance
monitoring and coaching (figure 2).
Adoption of sensory disciplines can be used to produce highly reliable data; there is
consistency from day to day; the precision of results is quantified in the same way as
the results of a chemical test and the cycle of performance monitoring and coaching
can deliver enormous improvements in both the accuracy and precision of results.
The disadvantage of such an approach is that it is very time consuming - both for the
sensory analysts who run the sessions and for the tasters. In particular the tasters need
to be available for several hours a week to carry out replicate tasting and to receive
training. In is essential that every taster is available for each of the replicate
determinations in order to quantify the repeatability of their results.
The traditional in house panel of company employees will never be available for this
amount of time. They are employed to carry out a "day job" and cannot be released
for several hours a week for tasting. The demands of their jobs mean that they often
have to miss tasting. The panellists on our in house sensory panel typically attended
fewer than 50% of tastings. This was not a reflection of a lack of commitment but
rather a reflection of the pressure of their work commitments.

4
 TRAIN RECRUIT

MONITOR

TASTE

Figure 2: The cycle of training of a sensory panel.

RECRUITING AN EXTERNAL SENSORY PANEL

In 1999, it was decided to recruit a panel of external expert tasters to ensure that a
pool of committed tasters was available to produce robust sensory data. An
advertisement was placed in a local newspaper and there were 70 applications. A
three stage selection process was carried out involving an initial screen of CVs; an
interview and a practical tasting session. Selection was based on number of criteria:

Availability. The key to a successful panellist is availability. It would be easy to

see the role of beer taster as a "fun" job so great care was taken to stress that the
role is very serious and the importance of attending all sessions. Panellists were
asked about commitments that could impinge on attending sessions such as other
employment or family commitments.
Long term commitment. A considerable amount of time must be invested in
training each panellist and it takes many months for panellists to achieve peak
performance. We were looking for people who saw the role as a job for several
years and not a few months. Thus we excluded people such as students looking for
employment in "gap years" and people who saw the role as a stepping stone to
other roles within Bass.
Verbal Skills. It is important for panellists not only to be good tasters but also to
be able to describe the flavour of products. We asked people to describe their
favourite drinks (not necessarily beer) and why they like them.
Basic scientific, computing and mathematical skills. There is a considerable
amount of "scientific jargon" used in brewing. We ensured that people would be
happy tasting materials with "chemical" names (e.g. iso amyl acetate, dimethyl
sulphide). Panellists would also need to be able to use a computer terminal during
tasting sessions and to have the mathematical knowledge to understand feed back
on their performance (averages, percentages, graphs, scoring scales etc).
Sensory interest. We asked applicants about their hobbies; we were looking for
people who showed an interest in the flavour of food and drink (cooking, growing
herbs, wine tasting, home brewing etc).

5
 Plate 1: The Bass Brewers Expert Panel.

Interpersonal skills and team working. It is essential that the panel operates
together as team. Great care was taken to ensure that the balance of the team was
right (men versus women; younger versus older). Every member of the panel must
be able to contribute and we avoided applicants who were either very timid or
very domineering.

Despite the very wide range of skills and abilities that were needed , it was possible to
identify a pool of people who performed well on all criteria.

PANEL TRAINING AND OPERATION

The recruitment process identified a panel of 10 people aged between mid twenties
and early 50s (Plate 1). There were seven women and three men. The panel is
employed for three 3 hour sessions per week (Tuesday, Wednesday and Thursday
mornings).
Panellists underwent an initial two month training period. The philosophy was to train
them to be tasters and not just "beer" tasters. In addition to receiving training in the
well established beer flavour characters they were exposed to a wide range of foods
and drinks (cheese, chocolate, mineral water, colas etc) and asked to identify flavour
descriptors. This not only broadened their sensory experience but also taught them the
process of developing sensory descriptors from first principles. This ability would be
needed for profiling non beer drinks for which there is no established flavour
terminology.
Sensory data is captured using a bespoke computerised sensory data capture system.
This allows an enormous range of sensory methods to be used (difference tests,
flavour profiling, ranking tests etc). It also allows total flexibility in the sensory

6
descriptors that are used and allows the panel to profile any product type. One of the
greatest benefits of the computerised system is that each panellist immediately sees
their own sensory score relative to the mean score for the rest of the panel. This
provides the opportunity to turn the end of each tasting session into a training exercise
using the samples under evaluation. This is a very powerful tool in improving
consensus between the different members of the panel.
Another significant benefit is that the real time availability of data enables the sensory
analyst to carry out rapid evaluation of results from simple spider plots to more
complex multivariate manipulations such as principle component analysis. These can
be displayed on the screen in a matter of minutes.

PANEL PERFORMANCE
Repeatability of results
The repeatability of results from the new panel is shown in comparison to results
obtained with the old internal panel (figure 3). Statistical 95% confidence limits for
the old panel were typically over a range of 20 -30 %. The 95% confidence range for
the expert panel is now less 5% on the same scale. This improvement was achieved by
routinely assessing beers in triplicate and providing immediate feedback to panellists
on their scores relative to the rest of the panel. Panel performance monitoring and
coaching is very effective in increasing the performance of panellists.

BEFORE AFTER
7 7
6
6
5
5
4
Sensory Mean

Upper 95% CL
4 3
Sensory Mean

Upper 95% CL Mean

3 2 Lower 95% CL
Mean
2 Lower 95% CL 1
1 0
-1
0
-2
-1
Rum
Sweetness

Floral

Warming

Bitterness

Aftertaste

Refreshing
Carbonation

Lemon Cooked
Astringent

Lemon Zest

Alcoholic
Complexity

Lime Spicy

Yellow Colour

Green Colour
Pulp/Cloudyness
Thickness/Viscosity

Fermented Character

Lime Fresh/Sherbet

Lemon Fresh/Sherbet
Acid/Sourness/Sharpness

Overall Fruit Intensity

Non-Citrus Fruit Character

-2
Dms

Dms
Sweet

Toffee

Thick
Herbal

Herbal
Floral

Floral
Hoppy

Hoppy

Warming
Rubber

Bitter

Rubber
Malty

Malty
Grainy

Grainy
Acid/Sour
Alcoholic

Alcoholic
Sulphidic

Sulphidic

Astringent
Stale/Cardboard

Stale/Cardboard
Estery/Fruity

Estery/Fruity

Attribute Attribute

Figure 3: The performance of the external panel (right hand picture) relative to the
old internal panel (left hand picture). The upper and lower lines show the 95%
confidence limits for the scores for each sensory attribute.

Holistic assessment of beer sensory quality

Internal sensory data can only be used to explain external consumer research results if
the internal data fully describes every aspect of the beer quality that the consumer
uses to judge beers. Historically our sensory data was limited to solely flavour data.
Overlooking appearance data ignores some of the major drivers of product preference
- colour, clarity and foam quality. In addition to flavour data, the expert panel use a
range of appearance descriptors to capture every aspect of beer quality (figure 4). For
instance, these flavour results suggest that the two beer samples are very similar. It is

7
only when the appearance results are included that it can be seen that the stability and
texture of the foam on the two beers is dramatically different.

foam colour
yeasty 90 foam creamyness
metallic foam lacing
rancid 80 beer colour
acid/sour 70 temperature
diacetyl 60 carbonation

phenolic 50 nucleation
40
herbal alcoholic
30

meaty 20 estery/fruity
10
musty/mouldy/earthy hoppy
0

stale/sherry/honey floral

stale/cardboard grainy

catty/ribes malty

aldehydic toffee

astringent burnt/roast
Beer 1
thick sulphidic - H2S
warming sulphidic - onion/garlic
bittersweet DMS sulphidic-cooked veg
Beer 2

Figure 4: A spider diagram of the sensory profile of two beers showing appearance
and flavour results.

Correlation with consumer research data

The two improvements in the results from the Expert Panel that allow externally
generated consumer data to be explained are that the noise in the data is dramatically
reduced and that the data captures all aspects of product quality, not just flavour.
Provided that the appropriate consumer research techniques are used, it is possible to
correlate the internal sensory data and external consumer data to identify which
product parameters are driving preference and which parameters have a negative
effect on liking. Such techniques will show that consumer preference can be increased
by subtle changes in sensory profile - increasing the levels of some characters and
reducing the levels of others.

Assessment of drinks other than beer

Bass Brewers have been successful over the past decade in developing a range of new
products which are not beer based. These Flavoured Alcoholic Beverages (FABs) are
fruit based drinks whose flavour cannot be described using standard beer flavour
profile terminology. The expert panel has been used to generate new flavour
terminology to describe the different fruit flavours found in FABs. This illustrates the
importance of training panellists to be "tasters" and not solely "beer tasters". For
example a sensory map for lemon and lime based products is shown in figure 5
describing the appearance and flavour of various FABS - both Bass and competitor
products.

8
 r u m b a s e c it r u s p r o d u c t

W a r m in g f e r m e n t e d b a s e le m o n 2
Rum rum b a s e le m o n
v o d k a b a s e le m o nle m o n f r e s h / s h e r b e t
f e r m e n t e d b a s e le m o n 1 ferm ente d ch ara cter
- 2 .0 2 c o m p le x it y 2 .0 2
a c id / s o u r n e s s / s h a r p n e s s
sw eetne ss
lim e s p ic y le m o n z e s t
lim e f r e s h / s h e r b e t s p ir it b a s e le m o n
G r e e n c o lo u r
s p ir it b a s e lim e Y e llo w c o lo u r O v e r a ll f r u it in t e n s it y

v o d k a b a s e lim e
- 2 .0 2

Figure 5: A PCA plot of sensory results for Lemon and Lime Flavoured Alcoholic
Beverages (FABs).

Publicity
From the start it was appreciated that a brewery recruiting beer tasters would be a
wonderful media story! The Bass Brewers Communications Department were
involved in the recruitment process from the outset to ensure that there was no
negative publicity and capitalise on any positive PR. Stories appeared in newspapers,
radio and television, at both local and national level. These portrayed Bass and the
brewing industry in a very positive light. Messages included "beer flavour is very
diverse and attractive", "women are good beer tasters and like drinking beer";
"brewers care so much about quality that they employ people just to taste beer".

Financial justification
Operating an external panel results in increased sensory costs because there is an on
cost of salaries for 10 people working 9 hours per day. Balanced against this, there are
savings that can be identified by carrying out high quality sensory analysis internally.
Previously sensory analysis had to be contracted out because the internal panel did not
have the capability of carrying out the work required (e.g. non beer products). Fewer
external consumer tests are needed when a reliable internal panel is available.
Products can be optimised more effectively before going to external tests. Correlating
internal and external data produces very accurate diagnostic information that renders
extensive "follow up" consumer testing unnecessary. Annually such savings are
approximately three times greater than the cost of employing the panel. Thus it is
possible to identify a very sound commercial justification for running an external
panel.

CONCLUSIONS
A panel of external part time tasters who are employed solely to taste beer can be used
to produce very high quality sensory data. The precision and accuracy of the external

9
panel is very much greater than a panel of internal tasters because it is possible to
spend far more time monitoring and coaching tasters to improve performance. This
panel has the capability of profiling all aspects of beer quality (flavour and
appearance) and also assessing other types of alcoholic drinks. The improved
accuracy and precision of the external panel makes it possible to correlate internal
sensory results with externally generated consumer preference data. Such studies can
identify the factors that drive consumer preference. This makes it possible to develop
products that far better match the needs of drinkers. Whilst employing people to taste
beer has an additional cost, savings on both contract sensory assessment and external
consumer research are about three times greater than the employment costs of the
panel.

ACKNOWLEDGEMENT
We would like to thank the Directors of Bass Brewers for permission to publish this
paper. We would also like to thank Janice Chilver for her enormous enthusiasm in the
day to day running of the panel and also to thank all the panellists for their
commitment and dedication to achieving sensory excellence.

REFERENCES
1. R. Cope, P.K. Hegarty, F.H. White (1997) Proceedings of the EBC Congress,
Maastricht, 599-606.
2. P.K. Hegarty (1994) Brewers Guardian, Oct. 23-30.
3. P.K. Hegarty (1997) Proceedings of the EBC Congress, Maastricht, 569-578.
4. P.K. Hegarty, R. Parsons, C.W. Bamforth, S.W. Molzahn (1995) Proceedings of
the EBC Congress, Brussels, 515-522
5. P.K. Hegarty, F.H. White (1993) Proceedings of the EBC Congress, Oslo, 429-
436.

10
90

Consumer perception of risk

Joachim Scholderer

Centre for Market Surveillance, Research and Strategy for the Food Sector (MAPP), The
Aarhus School of Business, Haslegaardsvej 10, DK-8210 Aarhus V, Denmark
(www.mapp.asb.dk, e-mail: [email protected])

Descriptors
Brewing industry, consumer research, genetics, health hazard, psychology

Deskriptoren
Brauindustrie, Genetik, Gesundheitsrisiko, Psychologie, Verbraucherforschung

Descripteurs
Etude du march(consommation), gntique, industrie brassicole, psychologie, risque pour
la sant
INTRODUCTION
Scientists and regulators are regularly baffled by public responses to risk, especially
when the issue at stake seemed unproblematic or at least technocratically solvable as
long as it was only discussed within the expert community. In terms of such
polarizations, the 1970s were the age of dissent over nuclear power, while the 1990s
saw the emergence of gene technology as an issue of public debate. The first decade of
the new millennium aspires to become the age of food safety, and once again, a major
research effort is made to find out how consumers confidence can be restored.
Brewing, as a particular branch of food manufacturing, has in the past been able to
dodge implication in major risk debates. The latest crisis in a related industry was the
temporary banning of several brands of the Coca-Cola Co. in 1999 in Belgium
following symptoms of nausea and vomiting amongst people who had consumed
them. Applications of gene technology in all industries a topic which is likely to
result in confrontations are still in the pre-market phase, and a high-profile debate is
to be expected as soon as the first products are launched.
In the following, I will briefly review the state of the art in risk perception research,
covering structure, process, and the social dynamics of risk debates. After that I will
present results from a recently completed research project. In this project, we
specifically looked into consumers perceptions of gene technology applied to
brewing, and how these perceptions related to consumers attitudes and choice
behavior.

Structure
Seminal research on the driving forces behind public opposition to new technologies
focused on the structure of consumers risk judgments, unsettled by the observation
that consumers systematically overrated the riskiness of some technologies, but
systematically underrated the riskiness of others. Slovic, Fischhoff and Lichtenstein
(1980) laid the foundations to what nowadays is called the psychometric approach
to risk perception. They asked their subjects to evaluate altogether 90 hazards on 18
dimensions. The hazards included technologies (such as nuclear power, gene
technology, aviation), activities (skiing, smoking), chemical substances (alcohol,
pesticides, dynamite), and societal hazards (crime, terrorism).
The judgment dimensions included voluntariness, immediacy of effect, knowledge
about the risk by those exposed, scientific knowledge, control over the risk, newness,
chronic vs. catastrophic, common vs. dread, severity of the consequences, and so on.
The authors found that, when aggregated over subjects, the dimensions could be
reliably represented by two underlying factors: (a) a dread factor, and (b) a familiarity
factor. Notably, only the dread factor was a reliable predictor of subjects overall risk
judgments.
The basic structure turned out to be remarkably stable across replications. Despite its
intuitive appeal, however, the model has recently been subjected to harsh critique. The
first problem is that the model is based on between-hazards comparisons. Strictly
speaking, there is no evidence whatsoever that people engage in such comparisons
when encountering a given hazard in their daily lives. The second problem is the pre-
selection of judgmental dimensions. Research using open-ended methods has found
that many consumers spontaneously use dimensions that are not included in the
standard set (e.g., tampering with nature), whilst other dimensions that figure

2
prominently in the standard set (e.g., risk to future generations) are hardly ever used
spontaneously.

Process
These problems have led risk perception researchers to put more emphasis on the
psychological processes that lead to specific risk judgments. The classic approach here
is the heuristics and biases tradition, originally devised to explain how people form
probability judgments when their factual knowledge is incomplete. Tversky and
Kahneman (1973) showed that people judge the frequeny of an event category by the
ease with which instances of this category can be retrieved from memory. This
phenomenon (the availability heuristic) was also called upon by Slovic et al. (1980)
to explain why dreaded, catastrophic risks are systematically overrated as compared to
common, dispersed risks.
A second approach has focused on the way people conceptualize hazardous processes.
Such mental models can be faulty, and emprirical research has in fact suggested that
laypeople may lack certain scientific concepts altogether (e.g., dose-response
relationships; Slovic et al., 1995), apply them incorrectly (e.g., ordinary tomatos do
not contain genes; Biotechnology and the European Public Concerted Action, 1997),
or may even engage in peculiar forms of dualist thinking (e.g., non-equivalence of
natural and man-made chemicals; Kraus, Malmfors & Slovic, 1992). Faulty mental
models lead to faulty judgments and may thus explain some of the more bewildering
instances of public outrage.
A third approach has looked into the relation between attitudes and risk perception.
Katz (1960) was the first to propose that attitudes may have a knowledge function,
providing us with a basis to judge social objects even if we do not know enough to
evaluate them objectively. Siegrist and Cvetkovich (2000) found that the degree to
which consumers risk perceptions depend on their general socio-political attitudes is
determined by the amount of knowledge consumers possess about the technology in
question: the less consumers knew about a particular technology, the more they tended
to infer its riskiness from their general attitudes toward the regulatory system.

Social dynamics
The social dynamics of risk debates are surely the most fascinating issue here, but
unfortunately also the hardest to assess: what is it that makes some risks a public
outrage, while others appear not to bother consumers at all? Kasperson et al. (1988)
have suggested that a hazard as such is only the initial input to a social amplification
process. A hazard has to make its way through the individual risk perception of
consumers, their social networks, may be highlighted by certain pressure groups who
put it on their agenda, evolve into a public debate, cause ripple effects in other
industries and technological areas, and may thus result in long-term consequences for
considerably larger social systems than seemed initially affected.
Viewed in a social dynamics perspective, the question about the processes that
transform a mere hazard into public outrage becomes at least heuristically tractable. In
the following, I will present results from a project where we tried to pinpoint the
reasons for the ongoing failure of introducing genetically modified foods in European
consumer markets. The project has special relevance for the European brewing
industry as we conducted all studies with the same set of product examples, including
a lager produced from genetically modified yeast.

3
CONSUMER PERCEPTION OF NOVEL BREWING TECHNOLOGIES:
THE CADE-GENTECH PROJECT
Expert perceptions versus consumer perceptions
It is a truism in risk perception research that the risk conceptualizations of experts
differ considerably from those of consumers (Sjberg, 1999). Previous research has
identified serious incongruities with regard to, for example, microbiological hazards
(Miles, Braxton & Frewer, 1998) and chemical hazards (Slovic et al., 1995). In a
series of qualitative studies, we investigated which risks and benefits experts associate
with GM foods, and compared them to those that consumers associate with GM foods
(Bredahl, 1999; Scholderer, Balderjahn, Bredahl & Grunert, 2000; Scholderer,
Balderjahn & Will, 1998).
The studies were simultaneously conducted in Denmark, Germany, Italy, and the UK.
As example applications, we used beer produced from GM yeast and yoghurt
produced from GM starter cultures. Relating the results of both groups yielded two
major points where expert perceptions contradicted consumer perceptions. A first
point was the experts expectation that GM foods would gain a dominant position in
the functional foods market. A defining characteristic of functional foods is that they
claim healthiness. However, the attribute genetically modified was in all countries
and product categories associated with perceptions of unwholesomeness and
unhealthiness.
The second point was the experts plan to communicate the potential for more
sustainable production as added value to consumer markets. The GM beer we used as
an example claimed that reduced energy expenditure during production would provide
an environmentally sound product. However, only British and Italian consumers were
inclined to accept the claim. Danish consumers were ambivalent. German consumers,
constituting the largest beer market in Europe, completely ignored the environmental
soundness claim and considered genetically modified beer an unnatural product.
The results of these studies suggested serious incompatibilities between expert and
consumer perceptions. The same attributes that were considered key benefits by
experts were considered risks by consumers. It was not difficult to conclude that these
incongruities would cause substantial problems for communication activities.
However, the incongruencies went even further. In addition to the risks and benefits
perceived, we also investigated experts beliefs about the processes that led consumers
to form attitudes and risk perceptions and how these could be affected (Scholderer &
Balderjahn, 1999).
A majority of the experts believed that negative consumer attitudes resulted from a
lack of information. Lack of information was thought to cause uncertainty about the
risks and benefits of GM foods and, subsequently, negative evaluation of the entire
technology on terms of the precautionary principle. Providing the public with
objective information, the experts thought, would enable consumers to rationally
weigh risks against benefits, proceed to a positive attitude, and act upon this through
an informed purchase decision.

Attitude structure: bottom-up or top-down?

The attitude formation model underlying these assumptions is a bottom-up one:
perceptions of specific risks and benefits are seen as antecedents of consumers
overall risk and benefit evaluations, not as concequences. As shown by Siegrist and
Cvetkovich (2000; see above), however, such unprejudiced evaluation can only be

4
expected when consumers are already familiar with a certain product or technology.
At the time of writing, though, not a single product had been approved yet for
marketing in the European Union according to the Novel Food Directive (Regulation
EC 258/97), so that direct experience with GM products is something consumers
cannot actually be believed to have.
In a survey involving respresentative samples of Danish, German, Italian and UK
consumers, we investigated the degree of interrelatedness among consumers
perceptions of 15 specific risks and benefits associated with GM foods (Bredahl,
2000; also see Scholderer, Bredahl & Frewer, 1999). If consumers judged these risks
and benefits independently and only then formed an overall evaluation, the covariation
among these perceptions should be neglectable. If, on the other hand, consumers
judged the risks and benefits on the basis of their prior attitudes towards gene
technology, their covariation should be substantial. And in fact, the 15 risks and
benefits could reliably be represented by a common perceived-risk factor and a
common perceived-benefit factor.
The common factors could in turn be reasonably well predicted by general socio-
political attitudes. Among these were, in line with previous research, attitudes towards
technological progress (cf. Frewer, Shepherd & Sparks, 1996; Hamstra, 1991; Sparks,
Shepherd & Frewer, 1994), attitudes towards environment and nature (Frewer,
Howard & Shepherd, 1997; Frewer, Howard, Hedderley & Shepherd, 1997; Hamstra,
1995; Siegrist, 1998), and trust in the institutions that regulate emerging technologies
and manage their risks (Hoban, Woodrum & Czaja, 1992; Siegrist, 1999, 2000).
The existing evidence suggests that, at the time being, bottom-up processes play only
a minor part in the formation of consumers attitudes. When asked to evaluate GM
foods they have no experience with, consumers seem to spontaneously construct
product-related attitudes as instances of higher-order attitudes and values, chiefly
those towards environment and nature, modern technologies, and the institutions
involved in their development and regulation.

Information strategies, attitude change and product choice

In a large experimental study involving consumer samples from Denmark, Germany,
Italy and the UK, Frewer, Scholderer, Downs and Bredahl (2000) tested the effects of
different information strategies on consumers attitudes to GM foods as well as on
actual product choice. None of the strategies could change consumers attitudes.
Instead, they had a strikingly uniform effect: all information strategies independent
of their design and evaluative tendency were more likely to activate existing
attitudes than the no-information condition in the control group, where consumers
only saw product examples with a genetically modified information on the label.
The activation of pre-existing attitudes did not result in attitude change, but in
attitude-consistent product choice. Consumers in the information groups chose GM
products significanly less often than consumers in the control group.
The pattern is consistent with Fazios (1986) theory of automatic attitude activation.
In this theory, attitudes are defined as object-evaluation links that are stored in
memory. A strong attitude is one that consists of many object-evaluation links. When
external cues (e.g., information about gm foods) are present in a given situation, and
these cues are directly or indirectly associated with the attitude object, then the
attitude is accessed from memory. Once activated, subsequent information processing
and behavior become more consistent with the attitude (Fazio, Powell & Williams,
1989). Hence, it seems that the voluntary provision of information materials prior to

5
the actual market introduction is not only an ineffective strategy to change consumers
attitudes to GM foods, but may even be dangerous when consumers product choices
are considered.

Is direct experience the key?

Reviewing the existing evidence, Scholderer, Bredahl & Frewer (1999) have
concluded that direct experience with GM products may be the only way in which the
detrimental attitude activation processes outlined above may be circumvented. In a
recent experiment (Scholderer, Bech-Larsen & Grunert, in preparation), we have put
this hypothesis to test. A total of 750 consumers from Denmark, Norway, Sweden and
Finland was invited to taste cheese samples. One group was told that the cheese they
had liked best was genetically modified. A second group was told that the cheese they
had liked best was genetically modified and, in addition, beneficial to their health (low
calories). Finally, a group of control subjects was told that the cheese they had liked
best was conventionally produced.
The results showed that participants who believed they had tasted a GM product had
significantly more positive attitudes to GM after the experiments as compared to
participants who thought they had tasted a conventional cheese. When they also
believed that the GM cheese had an additional health benefit, the attitudes were even
more positive. Most interestingly, a structural analysis indicated that in this particular
group, attitudes to GM were significantly less dependent on general socio-political
attitudes than in the other two groups. It can be concluded that direct product
experience in conjunction with tangible consumer benefit is in fact a strategy that can
improve public acceptance of food biotechnology.

CONCLUSIONS
Risk perception is a highly complex phenomenon. This is less due to a large number
of determinants (in fact, these are often very few) than to a multitude of processes that
can underlie consumers' responses to risk. The case of gene technology in brewing and
food production may serve as a reminder how important it is to know how consumers
arrive at the judgments we commonly refer to as risk perception. In this particular
case, the psychological processes we believe to have uncovered have much more in
common with social phenomena like stereotyping and prejudice than with the rational
risk-benefit trade-off many stakeholders expect consumers to engage in when
evaluating novel technologies.
In such a situation, ill-founded attempts to communicate with consumers may do more
harm than good. We saw in the information experiments described above that
consumers' negative attitudes to gene technology were not only resistant to external
information, but were even bolstered because the messages seemed to act as a cue that
activated pre-existing attitudes and thereby increased their detrimental influence on
product choice. Experience-based interventions, on the other hand, appeared to be the
only way to bypass such effects, as demonstrated in the cheese-tasting experiments
also decribed above.
The implications of this are almost ironic. In the end of the "hot phase" of the GM
debate that preceded the quasi-moratorium on GM foods in the European Union, all
major stakeholder groups reached a silent consensus about the information policy they
would adopt. This policy was primarily eductional and based on the notion of

6
complete transparency. In light of the empirical studies discussed above, it appears
that this was a wrong decision, at least as far as the aim of improving consumer
acceptance is concerned. In the meantime, however, the then implicit consensus has
become declared consumer policy, and it would be politically almost impossible to
revert to an approach that is less based on information and transparency, and more on
direct, experience-oriented promotional activities.

REFERENCES
1. Biotechnology and the European Public Concerted Action, Nature, 1997, 387,
845-847.
2. Bredahl, L., Appetite, 1999, 33, 343-359.
3. Bredahl, L., Determinants of Consumer Attitudes and Purchase Intentions with
Regard to Genetically Modified Foods, Aarhus: MAPP, 1999.
4. Fazio, R.H., in R.M. Sorrentino & E.T. Higgins (Eds.), The Handbook of Moti-
vation and Cognition. Foundations of Social Behavior, New York: Guildford
Press, 1986.
5. Fazio, R.H., Powell, M.C. & Williams, C.J., Journal of Consumer Research,
1989, 16, 280-288.
6. Frewer, L.J., Howard, C., Hedderley, D. & Shepherd, R., Public Understanding
of Science, 1999, 8, 35-50.
7. Frewer, L.J., Howard, C. & Shepherd, R., Science, Technology & Human Val-
ues, 1997, 22, 98-124.
8. Frewer, L.J., Scholderer, J., Downs, C., Bredahl, L., Communicating about the
Risks and Benefits of Genetically Modifed Foods: Effects of Different
Information Strategies, Aarhus: MAPP, 2000.
9. Frewer, L.J., Shepherd, R. & Sparks, P., British Food Journal, 1994, 96, 26-32.
10. Hamstra, A.M., Biotechnology in Foodstuffs: Towards a Model of Consumer
Acceptance, The Hague: SWOKA, 1991.
11. Hamstra, A.M., Consumer Acceptance Model for Food Biotechnology: Final
Report, The Hague: SWOKA, 1995.
12. Hoban, T., Woodrum, E. & Czaja, R., Rural Sociology, 1992, 57, 476-493.
13. Kasperson, R.E., Renn, O., Slovic, P., Brown, H.S., Emel, J., Goble, R.,
Kasperson, J.X., & Ratick, S., Risk Analysis, 1988, 8, 177-187.
14. Katz, D., Public Opinion Quarterly, 1960, 24, 163-204.
15. Kraus, N.N., Malmfors, T. & Slovic, P., Risk Analysis, 1992, 12, 215-232.
16. Miles, S., Braxton, D.S. & Frewer, L.J., British Food Journal, 1999, 101, 744-
762.
17. Scholderer, J. & Balderjahn, I., in L. Hildebrandt, D. Annacker & D. Klapper
(Eds.), Marketing and Competition in the Information Age, Brussels: European
Marketing Academy, 1999.
18. Scholderer, J., Balderjahn, I., Bredahl, L. & Grunert, K.G., European Advances
in Consumer Research, 2000, 4, 123-129.
19. Scholderer, J., Balderjahn, I., Will, S., Communicating the Risks and Benefits of
Genetically Engineered Food Products to the Public: The View of Experts from
Four European Countries, Aarhus: MAPP, 1998.
20. Scholderer, J., Bredahl. L., & Frewer, L.J., ESF Workshop on Risk and Deci-
sion-Making, Universiteit van Amsterdam, 09-11 December 1999.

7
21. Siegrist, M., Personality and Individual Differences, 1998, 24, 861-866.
22. Siegrist, M., Journal of Applied Social Psychology, 1999, 2093-2106.
23. Siegrist, M., Risk Analysis, 2000, 20, 195-204.
24. Siegrist, M. & Cvetkovich, G., Risk Analysis, 2000, 20, 713-720.
25. Sjberg, L., Human Ecology Review, 1999, 6, 1-9.
26. Slovic, P., Fischhoff, B. & Lichtenstein, S., in R. Schwing & W.A. Albers
(Eds.), Societal risk assessment: How safe is safe enough, New York: Plenum
Press, 1980.
27. Slovic, P., Malmfors, T., Krewski, D., Mertz, C.K., Neil, N. & Bartlett, S., Risk
Analysis, 1995, 15, 661-675.
28. Sparks, P., Shepherd, R. and Frewer, L.J., Agriculture and Human Values, 1994,
11, 19-28.
29. Tversky, A. & Kahneman, D., Cognitive Psychology, 1973, 5, 207-232.

8
91

Mycotoxins in the total chain from

barley to beer
Liisa Vanne & Auli Haikara

VTT Biotechnology, P.O. Box 1500, FIN-02044 VTT, Finland (e-mail: [email protected])

Descriptors
Barley, contamination, legislation, moisture content, mycotoxin

SUMMARY
Mycotoxins can be formed during barley cultivation, storage and processing. The most
important toxin producers in the field are Fusarium moulds. To prevent growth of storage
fungi, the grain should be dried to moisture content of 13-14 % immediately after harvest. In
malting and brewing, a part of mycotoxins is destroyed, but the formation and carry-over of
toxins through the process into beer are possible. In the European legislation, limits for
mycotoxins are under preparation. In the research of mycotoxins, growth of fungi and
interactions with other microbes are studied. New detection methods for mycotoxins and the
fungi is an important part of the research.

Mycotoxine in der gesamten Kette von der Gerste bis zum Bier

Deskriptoren

Gerste, Kontamination, Mykotoxin, Recht, Wassergehalt

ZUSAMMENFASSUNG
Mycotoxine knnen whrend des Gerstenwachstums, der Gerstenlagerung oder der Malz-
produktion entstehen. Die wichtigsten Toxinbildner in diesem Bereich sind Fusariumpilze.
Um das Wachstum von Lagerungsschimmel zu vermeiden, sollte das Korn nach der Ernte
schnellstmglich auf einen Wassergehalt von 13 bis 14% getrocknet werden. Durch das
Mlzen und Brauen wird ein Teil des Mycotoxins zerstrt, aber die Entstehung und das
Verschleppen des Toxins durch den gesamten Prozess hinweg in das fertige Bier ist mglich.
In der Mycotoxin-Forschung wurde das Wachstum der Pilze und deren Zusammenspiel mit
anderen Mikroorganismen studiert. Neue Detektionsmethoden fr Mycotoxine und die Pilze
sind ein wichtiger Bereich der Forschung auf diesem Gebiet.
Rle des mycotoxines dans la chane totale allant de l'orge la bire

Descripteurs
Contamination, lgislation, mycotoxine, orge, teneur en eau

RESUME
Les mycotoxines peuvent se former pendant la culture, le stockage et le traitement de l'orge.
Dans ce domaine, les principaux producteurs de toxines sont les moisissures du genre
Fusarium. Pour prvenir la croissance des champignons pendant le stockage, l'orge doit tre
sche jusqu' une humidit de 13-14 % immdiatement aprs la rcolte. Pendant le maltage
et le brassage, une partie des mycotoxines est dtruite, mais la formation et le passage des
toxines d'une tape l'autre pendant le processus de fabrication de la bire sont possibles.
Dans la lgislation europenne, des taux-limites de toxines sont en prparation. Dans la
recherche des mycotoxines, la croissance des champignons et leurs interactions avec d'autres
microbes sont tudies. Les recherches visent en grande partie mettre au point de nouvelles
mthodes de dtection des mycotoxines et des champignons.

2
INTRODUCTION
The growing barley plant in the field belongs to a complex ecosystem affected by
numerous biotic and abiotic factors. The microbial load of the barley has a significant
impact on the total production chain of malt and beer. Harmful metabolites of fungi
can be transmitted to products and cause processing problems and adverse health
effects. Studies of individual mycotoxins and their occurrence in different parts of the
malt and beer production chain have been carried out, but more systematic
information is needed for the evaluation of the risk of mycotoxins in malting and
brewing.

BARLEY IN THE FIELD

In the field, the microbial flora of barley is predominated by gram-negative bacteria,
but varying numbers of yeasts, actinomycetes and filamentous fungi are also present
(Haikara et al., 1977, Flannigan, 1996). The most important fungal contaminants in
the field belong to the genera Fusarium, Alternaria, Cladosporium,
Helminthosporium and Epicoccum (Lacey, 1989). The fungal contamination starts at
the blooming and ear emergence stage and it is affected both by factors determined by
the barley cultivar (Axberg et al., 1997, Prom et al., 1999), the fungal strain and the
environmental conditions (Noots et al., 1998). The fungal contaminants can be host-
specific parasites, saprophytes or epiphytes of plants (Schmidt, 1991). Due to their
need of a high water activity (aw >0.85) for growth, these fungi are often referred to as
field fungi (Chelkowski, 1991). However, the spores of these organisms can remain
viable for prolonged periods in storage and be activated if the conditions are
favourable for their growth.

Several species of Fusarium are important plant pathogens causing head blight in
cereals. In cool climates, Fusarium culmorum and F. poae are the most common
causative agents of this disease, whereas F. graminearum is the predominant species
in USA, Canada (Vandemeulebroucke, 2000) and southern parts of Europe
(Schildbach, 1995).

Harvesting
Ecologically, harvesting causes a major change in the microbiological balance of
barley. The availability of water decreases considerably and a new microbial
inoculum is spread by the harvesting machinery, field dust and crop residues (Magan
& Lacey, 1988, Noots et al., 1998). Crop maturity, weather conditions at harvesting
and agricultural practices also have an impact on the microbial load of barley entering
storage (Mills, 1989). Immediate drying of the crop below aw 0.7 restricts efficiently
the growth of most fungi (Jecfa, 2001). Due to climatic conditions, efficient drying is
a common practice in northern Europe. However, in case of extensive rainfall during
harvest, delayed drying or improper storage conditions, there is a risk of continuous
microbial activity in barley (Bennett & Richard, 1996). Because of the changed
conditions, the microbial flora starts to undergo a slow modification. The specific
plant-fungus associations are destroyed and the predominant fungal flora in the
harvested grain consists of saprophytic organisms mainly belonging to the genera
Aspergillus and Penicillium (Noots et al., 1998). They are present in small numbers in
the field and the lower water activity and higher temperature in the grain storage can
favour their growth.

3
In table 1, the prerequisities for growth of common field and storage fungi are
presented. The optimal growth temperatures are similar in both groups, but the
minimum water activity needed for the growth of Penicillium and Aspergillus is
clearly lower than is needed for the growth of field fungi.

Fungal species Temperature C Water activity aw

Field fungi Range Optimum Minimum Optimum
Alternaria alternata -2 - 32 20-25 0.88 1.00
Cladosporium cladosporioides -5 - 32 24-25 0.88 1.00
Cladosporium herbarum -10 - 32 25 0.85 0.95
Fusarium avenaceum -3 - 37 25 0.89 1.00
Fusarium culmorum <0 - 37 25 0.89 0.99
Fusarium graminearum >5 24-26 0.89 0.98-0.99
Fusarium poae 2 - 39 22-28 0.89 1.00
Fusarium sporotrichioides -2 - 35 22 27 0.88 1.00
Storage fungi
Penicillium brevicompactum 12 - 30 23 0.78 - 0.82 1.00
Penicillium aurantiogriseum -2 - 32 23 0.81 - 0.83 >0.98
Penicillium verrucosum 0 - 31 20 0.81 - 0.83 nd
Aspergillus fumigatus 10 - 55 40-42 0.85 0.98- 0.99
Aspergillus ochraceus >10 28-32 0.76 - 0.83 0.95
Aspergillus restrictus 9 - 40 30 0.71 - 0.75 0.96

Table 1: Growth conditions of field and storage fungi. (Sweeney and Dobson, 1998,
Bottalico & Logrieco, 1998, Axberg et al., 1997, Reiss, 1998, Jecfa, 2001, Bilgrami &
Choudhary, 1998)

BARLEY IN STORAGE
Stored barley is a man-made ecosystem in which living, respiring grain interacts with
both microorganisms and the environment (Magan & Lacey, 1988). Mould growth
and spoilage are determined predominantly by the availability of water and
temperature. If the grain has been dried before storage, the risk of enhancing
microbial activity during storage is low. However, slight changes in the determinative
parameters can cause a rapid deterioration of the crop. The fungal inoculum is
distributed unevenly in the grain bulk. If the conditions at one spot allow the
proliferation of xerophilic microbes, their metabolic activity changes the local
environment more favourable for other types of organisms (Noots, 1998). As a result,
a variety of adverse effects can be caused to the stored barley. Discoloration of
kernels and changes in the carbohydrate, fat and protein content of the kernels may
lead to the rejection of the lot, whereas the production of secondary metabolites like
gushing factors, allergens and mycotoxins can cause problems at a later stage if
transmitted into malting and brewing processes (Hernndez et al., 2000).

Secondary metabolism
By definition, mycotoxins are fungal secondary metabolites that occur as natural
contaminants in agricultural products and are toxic when ingested via a natural route
of administration, mainly orally (Abramson, 1998). Secondary metabolism is a stage
in the fungal life cycle after a phase of rapid cell growth. When an essential nutrient
starts to restrict cell proliferation, the accumulating products of primary metabolism
induce the synthesis of new enzymes for the production of compounds like enzymes,
organic acids, antibiotics and mycotoxins. The ability to produce given metabolites is

4
species or strain specific and usually a mycotoxigenic strain produces several toxins
simultaneously. The biological purpose of mycotoxins has not been fully elucidated,
but the production is assumed to be a part of the defensive mechanisms of the fungus
against other fungi or animals (Smith & Moss, 1985). In laboratory experiments with
a toxigenic Penicillium verrucosum growing alone or in paired cultures with other
fungi in irradiated barley, varying degrees of restriction in both seed infection and
ochratoxin production have been observed It is obvious that the amount of
mycotoxins is constantly fluctuating. There can be biosynthesis, degradation and
stimulation of the metabolite simultaneously in different parts of the grain bulk and
the total amount of the compound is an integrate of them. (Ramakrishna et al., 1996).

IMPORTANT MYCOTOXINS IN BARLEY

The production of mycotoxins in the field is dependent on the presence of a barley
variety sensitive to the fungus, presence of a toxigenic fungal strain and other factors
including weather conditions, use of fungicides and numerous biological interactions.
It has been shown by molecular biological methods in field experiments that some
fungicides recommended for the control of ear diseases are effective only against
pathogenic but non-toxigenic fungi. (Edwards et al., 2001).

Trichothecenes are a group of mycotoxins produced by a variety of Fusarium species,

although a number of other genera are also known to produce these compounds, e.g.
Cephalosporium, Trichoderma, Stachybotrys and Myrothecium. Chemically,
trichothecenes are tricyclic sesquiterpenes and they can be designated into four
subclasses: Type A has a functional group other than ketone at position C-8, whereas
type B has a ketone at C-8. Type C trichothecenes have a second epoxy group and
type D contains a macrocyclic ring between C-4 and C-5 (Sweeney & Dobson, 1998).
Trichothecenes are usually formed already in the field, but they can also be produced
in inappropriate storing conditions and during processing of barley.
Deoxynivalenol (DON) is a type B trichothecene that occurs predominantly in grains
like wheat, barley and maize and less often in oats rice, rye, sorghum and triticale. In
a data analysis of 1662 barley samples from 13 countries, 59 % were found to contain
DON, The production of this toxin is associated primarily with Fusarium
graminearum and F. culmorum (Jecfa, 2001). It has been shown that DON can be
formed continuously during head maturation and development of Fusarium head
blight (Prom et al., 1999). The toxic effects caused by DON are mainly acute
toxicoses, whereas genotoxicity has not been confirmed. The Joint FAO/WHO
Committee on Food Additives (Jecfa) has established a provisional maximum daily
intake (PMTDI) of 1 ug/kg of body weight (Jecfa, 2001). In USA, FDA has set an
advisory maximum level of 500 ug/kg in food and feed (Giese, 2000). It has been
estimated that if the concentration of DON is equivalent to or less than 100 ug/kg
grain, the health risk is low or zero. In regions with a low incidence of Fusarium head
blight, DON concentrations from 10 to 50 ug/kg are found (Chelkowski, 1998). The
level of preformed DON does not decrease significantly during barley storage (Beattie
et al., 1998).

T-2 and HT-2 toxins are type A trichothecenes that are produced by the saprophytic
fungus Fusarium sporotrichioides. They are normally not found in grain at harvest,
but can result from water damage to the grain, especially in cold weather. T-2
concentrations from 0.1-21 ug and HT-2 from 0.4-15 ug/kg barley have been found

5
(Jecfa, 2001). T-2 toxin is a potent inhibitor of protein synthesis and it is considered to
be considerably more toxic than DON. However, when ingested with food, the
toxicity of T-2 with DON is roughly similar. In the animal gut, HT-2 is a primary
metabolite of T-2 and there is little information about the toxicity of HT-2 alone. A
group PMTDI of 60 ng/kg for T-2 and HT-2 together has been set by Jecfa (Jecfa,
2001).

Nivalenol (NIV) is produced by Fusarium cerealis (F. crookwellense), F. poae and to

a lesser extent by F. culmorum and F. graminearum. It causes immunotoxic and
haematotoxic effects in mammals. Information about the genotoxicity of NIV is
inadequate. A temporary TDI of 0-0.7 ug/kg bw has been set by the EU Scientific
Committee of Food (SCF) (SCF, 2000b).

Trichothecenes are usually produced in mixtures even under pure culture conditions
and it is often difficult to identify the causative toxins in a toxicosis, even though the
fungal species is known (Sweeney & Dobson, 1998). Because these toxins have a
common basic chemical structure, they may also share common mechanisms of toxic
action. It remains to be considered, whether a group TDI level should be assigned for
trichothecenes. (SCF, 2000b).

Ochratoxin A (OTA) is a derivative of isocoumarin linked to L-phenylalanine and it is

the most toxic of the isocoumarin compounds. It is produced predominantly by
Penicillium verrucosum in cool and temperate climates and Aspergillus ochraceus in
warmer regions of the world (Sweeney & Dobson, 1998). OTA is a potent
nephrotoxin, teratogen and carcinogen (Jecfa, 2001). The optimum aw for OTA
production is around 0.95-0.99, but it can be formed even at an aw of 0.80. The
optimum temperature for OTA production is 31 C (Sweeney & Dobson, 1998.). A
TDI of maximum 14 ng/kg bw has been established by Jecfa (Jecfa, 2001).
It has been thought that OTA is not produced in field conditions. However, low levels
of this toxin have been found in freshly harvested barley (Gareis, 1999, Thellmann &
Weber, 1997).

Zearalenone (ZEA) is a nonsteroidal estrogenic mycotoxin produced by several

Fusarium species. It can be found worldwide in cereal crops and it has been
implicated in numerous mycotoxicoses in farm animals, especially in pigs (SCF,
2000a). It binds competitively to estrogen receptors in vitro. ZEA has a low acute
toxicity and the evidence concerning its carcinogenicity in humans and experimental
animals is yet inadequate for the classification of the compound as a carcinogen. ZEA
causes alterations in the reproductive tract of laboratory animals and it has been
suspected as a causative agent of precocius pubertal changes in young children. In
grain, zearalenone is usually present together with other mycotoxins. In USA, no
regulatory level has been set (Giese, 2000), whereas a temporary TDI of 0.2 ug/kg
body weight has been set by the SCF (SCF, 2000a).

Combinations of mycotoxins have been tested in vitro against T-lymphocyte cells of

pig. There are indications that in some cases the synergistic effect can be more potent
than the effects of single toxins. (Bernhoft et al., 2001).
In table 2., recent results of mycotoxin surveys in barley are presented.

6
 Toxin Country n Positive Maximum Reference
% g/kg
DON Great Britain 106 71 370 Prickett & al., 2000
DON Malting barley, 30 76 311 Prickett & al., 2000
Great Britain
T-2, Poland 24 50 2.40 Perkowski & al., 1997
HT-2 24 20 0.37
Tricho- Finland 10 50 96 Laitila & al., 2000
thecenes
OTA Germany 22 19 0.495 Wolff, 2000
OTA USA 103 11 17 Trucksess, 1999
OTA Malting barley, 30 13 13.8 Prickett & al., 2000
Great Britain
OTA Great Britain 131 26.7 17.8 Scudamore & al., 1999
OTA Malting barley, 89 10.1 0.15 Gareis, 1999
Germany

Table 2: Mycotoxins in stored barley

MYCOTOXINS IN MALTING
The microbiological status of a barley lot entering the malthouse is a result of the
contamination and growth or inactivation of the population during the preceding steps
(Noots et al., 1998). Susceptible lots are usually rejected already based on other
quality parameters than the presence of mycotoxins, but in some situations it is
possible that a grain lot containing a toxigenic fungal inoculum or mycotoxins is
accepted for malting (Hernndez et al., 2000).
Systematic studies of the fate of mycotoxins during malting are few. It is known that
the processing conditions in steeping and germination favour the growth of most
microbes (Haikara et al., 1977, Petters et al., 1988). It has been found that most of the
preformed DON is washed out during steeping, but new toxin formation can take
place during germination and in the beginning of the kilning stage (Schwarz et al.,
1995, Munar & Sebree, 1997). The elevated temperature of malt kilning is not high
enough to destroy mycotoxins. Gareis (1999) studied OTA in freshly harvested barley
from commercial maltings in Germany. The samples (ca. 500 g) were analysed again
after five months in storage, after which they were malted and brewed. Low amounts
of OTA were found at every stage. It could be concluded that there was neither
formation of new toxin nor degradation of the toxin already present during malting.
Schwarz et al. (1995) studied the effect of malting on mycotoxins by using barley
containing unacceptable levels of DON. Almost 90 % of the DON present was
washed out during steeping, but the mould growth during germination resulted in
production of new DON and, additionally, small amounts of ZEA.

MYCOTOXINS IN BREWING AND BEER

Surveys about mycotoxins in beer are continuously being carried out in different
countries. The products studied include usually both domestic and imported beers. In
addition to the mycotoxins originating from barley, aflatoxins and fumonisins can also
be found in the final products. These toxins are transmitted to beer via maize-based
raw materials or adjuncts. Aflatoxins are the most important mycotoxins worldwide,

7
produced by Aspergillus flavus and A. parasiticus. They are considered to be the most
toxic natural compounds and classified as proven human carcinogens
(Vandemeulebroucke, 2000). In a study by Scott & Lawrence (1997) the high
incidence of aflatoxins could be localised to beers imported from Mexico and Brasil
(see table 3).
Fumonisins (FB) are 1,2,3-tricarboxylic acids produced by Fusarium verticillioides
and F. proliferatum. They have a low acute oral toxicity, but a part of them can
accumulate in the liver and kidneys. Fumonisins were found in 1988 and there is no
systematic collection of human exposure data yet. There is a possible correlation
between fumonisins and high frequency of oesophageal cancer in some parts of the
world (SCF, 2000c.).

Toxin Country of n positive Maximum Reference

study % g/l
DON Argentine 50 44 221 Molto & al., 2000
DON / NIV Korea 54 26/43 23 / 38 Shim & al., 1997
DON / NIV Canada 50 18/6 50 / 0.84 Scott & al., 1993
Ochratoxin A Germany 161 63 0.33 Meyer & Neugebauer, 2000
Germany 318 63 0.293 Bresch & al., 2000
Italy 61 50 0.135 Visconti & al., 2000
Denmark 9 55 0.026 Guldborg, 1997
Fumonisin USA 29 86 0.013 Hlywka & Bullerman, 1999
Canada 41 24 0.059 Scott & Lawrence, 1995
Aflatoxin USA 16 19 0.006 Scott & Lawrence, 1997
USA/Mexican 8 63 0.076 Scott & Lawrence, 1997
beers

Table 3: Mycotoxins in beer

Hernndez et al. (2000) have carried out an extensive study concerning Fusarium
toxins in wheat, wheat malt and beer manufactured from infected raw material. In
brewing, most of the ZEA present in malt was removed to the spent grains and it
could not be detected in beer. Instead, a three-fold increase in the amount of DON was
found during wort boiling and the DON level in the final beer was approximately on
the same level as in malt. The increase of DON during brewing was explained by the
activation of toxin precursors not detectable in malt. It has also been found out that
trichothecenes can inhibit the growth of brewing yeasts in laboratory conditions
(Boeira et al., 1999a, 1999b).

PREVENTION OF MYCOTOXIN FORMATION

The reduction of sources of toxigenic fungal inoculum is an important tool in the
prevention of mycotoxin production. Before sowing, old seed heads and other
substrate material have to be removed. Mechanical damage should be minimised
during crop cultivation and good sanitation has to be practised at all stages of
harvesting (Codex, 2000).
The main postharvest strategy involves efficient drying and keeping the stored grain
in well-cleaned storage silos below 0.7 water activity.

8
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(4/5), 89-94.
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drying and storage, Elsevier, Amsterdam, 53-66.
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agriculture and food safety. Marcel Dekker, Inc., New York, 45-64.
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session, Beijing, China, 20-24 March 2000.
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Environ. Microbiol., 67 (4), 1575-1580.
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16. Gareis, M., 1999, Archiv fr Lebensmittelhygiene, 50 (4/5), 83-87.
17. Giese, J., 2000, Food Technology, 54 (10), 90-91.
18. Guldborg, M., 1997, Brygmesteren, 54 (4), 16-17.
19. Haikara, A., Mkinen, V. & Hakulinen, R., 1977, Eur. Brew. Conv., Amsterdam
1977, 36-46.
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21. Hlywka, J.J. & Bullerman, L.B., 1999, Food Additives and Contaminants, 16
(8):319-321.
22. Jecfa 2001. www.fao.org/es/ESN/Jecfa/Jecfa.htm
23. Lacey, J., 1989, J. Appl. Bacteriol. Symp. Suppl. 11S-25S
24. Laitila, A. & Haikara, A., 2000, World Brewing Congress, Orlando, FL, 29th
July - 2nd August 2000, 109.
25. Magan, N. & Lacey, J., 1988, Int. J. Food Microbiol., 7, 245-256.
26. Meyer, R.A. & Neugebauer, S., 2000, Nahrung, 44 (1), 58-59.
27. Mills, J., 1989, J. Food Prot., 52 (10), 737-742.

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28. Molto, G., Samar, M.M., Resnik, S., Martinez, E.J. & Pacin, A., 2000, Food
Additives and Contaminants, 17(9):809-813.
29. Munar, M.J. & Sebree, B., 1997, Gushing - a maltster`s view, J. Am. Soc. Brew.
Chem., 55 (3), 119-122.
30. Noots, I., Delcour, J.A. and Michiels, C.W., 1998, Critical Reviews in
Microbiology, 25, 2, 121-153.
31. Perkowski, J., Jelen, H., Kiecana, I. & Golinski, P., 1997, Food Additives and
Contaminants, 14 (4), 321-325.
32. Petters,. H.I., Flannigan, B. & Austin, B., 1988, J. Appl. Bact., 65, 279-297.
33. Prickett, A.J., Macdonald, S. & Wildey, K.B., 2000, HGCA Project report No.
230. Central Science Laboratory, Sand Hutton, York, Great Britain, 28 p.
34. Prom, L.K., Horsley, R.D., Steffenson, B.J. & Schwarz, P.B., 1999, J. Am. Soc.
Brew. Chem., 57 (2), 60-63.
35. Ramakrishna, N., Lacey, J. & Smith, J.E., 1996, J. Food Prot., 59 (12), 1311-
1317.
36. Reiss, J., 1998, Schimmelpilze. Springer, 308 s.
37. SCF 2000a. http://europa.eu.int/comm/food/fs/sc/scf/out65_en.pdf
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39. SCF 2000c. http://europa.eu.int/comm/food/fs/sc/scf/out73_en.pdf
40. Schmidt, H. 1991. In: Chelkowski, J., 1991, (ed.) Cereal grain: Mycotoxins,
fungi and quality in drying and storage, Elsevier, Amsterdam, 53-66.
41. Schwarz, P.B., Casper, H.H. & Beattie, S., 1995, J. Am. Soc. Brew. Chem., 53
(3), 121-127.
42. Scott, P.M., Kanhere, S.R. & Weber, D., 1993, Food Additives and
Contaminants, 10 (4), 381-389.
43. Scott, P.M. & Lawrence, G.A., 1995, J. Food Prot., 58(12), 1379-1382.
44. Scott, P.M. & Lawrence, G.A., 1997, J. AOAC Int., 80 (6), 1229-1234.
45. Scudamore, K.A., Patel, S. & Breeze, V., 1999, Food Additives and
Contaminants, 16 (7), 281-290.
46. Schildbach, R., 1995, Einflu natrlicher Umweltfaktoren auf die
Schimmelpilz-Kontamination an Braugerste, Brauwelt, 135 (5/6), 211-212, 214,
216-218.
47. Shim, W-B., Kim, J-C., Seo, J-A & Lee, Y-W., 1997, Food Additives and
Contaminants, 14 (1), 1-5.
48. Smith, J.E. & Moss., M.O., 1985, Mycotoxins formation, analysis and
significance. John Wiley & Sons, New York, 31-33.
49. Sweeney, M.J. & Dobson, A.D.W., 1998, Int. J. Food Microbiol., 43, 141-158.
50. Thellmann, A. & Weber, W., 1997, Deutsche Lebensmittel-Rundschau, 93 (1),
1-3
51. Trucksess. M.W., Giler, J., Young, K., White, K.D. & Page, S.W., 1999, J.
AOAC International, 82 (1), 85-89.
52. Vandemeulebroucke, C., 2000, Cerevisia Belg. J. Brew. Biotechnol., 25 (4), 19-
26
53. Visconti, A., Pascale, M. & Centonze, G., 2000, J. Chromatography A, 888
(1/2), 321-326
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10
92

Using HACCP to prevent engineering

related product safety and quality risks
Alan I. Kennedy
Scottish Courage Brewing Ltd, Technical Centre, 160 Canongate, Edinburgh EH8 8DD,
Scotland, United Kingdom (e-mail: [email protected])

Descriptors
Contamination, engineering, glycol, HACCP, heat exchanger, product safety

SUMMARY
Hazard Analysis Critical Control Point (HACCP) studies have been in place in breweries for a
number of years, using a risk assessment approach to identify and prevent consumer safety
issues (and, in some cases, quality issues) in the final product. The paper will describe a
number of important engineering and hygiene related areas, including tank linings,
replacement frequencies for valve and flow plate rubbers and return to service of equipment.
Plate heat exchangers will be used as an example to show how HACCP principles can be used
to reduce a known beer contamination risk to an acceptable level.

Der Einsatz von HACCP zur Vermeidung von technisch bedingten Risiken bei der
Produktsicherheit und Produktqualitt

Deskriptoren
Glykol, HACCP, Kontamination, Produktsicherung, Technik, Wrmeaustauscher

ZUSAMMENFASSUNG
Die Risikoanalyse kritischer Kontrollpunkte (HACCP) ist seit einigen Jahren Bestandteil in
Brauereien und verwendet Risikoabschtzungen, um Aspekte der Verbrauchersicherheit im
fertigen Produkt zu identifizieren und sicherzustellen (in manchen Fllen auch Qualitts-
belange im fertigen Produkt). Die vorgestellte Arbeit beschreibt einige wichtige technische
und hygienische Gesichtspunkte, einschlielich Tankauskleidungen, Austauschintervalle fr
Ventil- und Durchflusstellergummis sowie der Instandsetzung der sonstigen Ausstattung. Am
Beispiel eines Plattenwrmetauschers wird gezeigt, wie HACCP-Regeln dazu verwendet
werden knnen, um ein bekanntes Bierkontaminationsrisiko auf ein akzeptables Niveau zu
reduzieren.
Utilisation de l'HACCP pour la prvention des risques sur la scurit et la qualit du
produit lis la fabrication

Descripteurs
Contamination, changeur de chaleur plateaux, glycol, HACCP, ingnirie, scurit des
produits

RESUME
Depuis un certain nombre d'annes, l'industrie brassicole met en oeuvre des tudes par
HACCP (Hazard Analysis Critical Control Point), mthode d'valuation des risques visant
identifier et prvenir les problmes de scurit (et, dans certains cas, de qualit) du produit
fini pour le consommateur. Cette confrence dcrira un certain nombre de questions
importantes d'ingnirie et d'hygine, dont le revtement des cuves, la frquence de
remplacement des soupapes et des caoutchoucs des plateaux et le retour en service du
matriel. Les changeurs thermiques plateaux seront pris comme exemple pour illustrer la
manire dont on peut utiliser les principes de l'analyse HACCP pour rduire un risque connu
de contamination de la bire un niveau acceptable.

2
INTRODUCTION
Product risk assessment to assure consumer safety has been a legal requirement in the
food industry in Europe since the early 1990s. The most commonly used technique
for carrying out risk assessments is HACCP (Hazard Analysis Critical Control Point).
A HACCP study satisfies the requirements of the Food Safety (General Food
Hygiene) Regulations of 1995 and can be used as a due diligence defence in a case
of product contamination. Critical hazards in a food business operations, the Critical
Control Points, are identified and effective monitoring and control measures are put in
place at each of these points. Regular review and audit of the HACCP studies are also
introduced to verify the procedures in place. Within Scottish Courage Brewing it was
decided that HACCP studies should not only consider the more obvious aspects of
consumer safety related to product contamination (to comply with legislation) but also
include within the scope of the studies other potential hazards, mainly
microbiological. It was intended that this would result in a document that, once
produced, would be used by the breweries on a day-to-day basis, rather than some
previous studies which were produced solely to satisfy the legal requirement and
seldom consulted again. It was also hoped that by considering the hazards in
categories B and C (below) the company would be well prepared for future
developments in the area of consumer safety. To achieve this, identified hazards were
divided into three categories:

Category A - Harm to the Consumer

Any foreign body which may choke, cut or damage the teeth of the consumer
(e.g. glass, metal fragments); chemical contaminants (e.g. nitrosamines, lead,
caustic) and pathogenic microbes (e.g. Cryptosporidium, Salmonella, E.coli).

Category B - Perceived 'Harm' to the Consumer

Foreign bodies (e.g. insects, mould or rat droppings) in final product;
chemicals (e.g. oil, propylene glycol) and microbiological (e.g. spoilage of
final product).

Category C - Consumer Perception 'Won't Buy Again'

Foreign bodies (e.g.. insects in brewhouse); chemicals (e.g. taints in sugar
syrup causing butyric acid off-notes in beer) and microbiological
contamination (e.g. spoilage in process causing taints in beer, but microbes
killed by pasteurisation).

The HACCP studies produced at the Scottish Courage breweries have previously been
described at the EBC Symposium Quality Issues and HACCP in Stockholm in
19971.

HACCP AND ENGINEERING ISSUES

A number of engineering related issues have been identified that should come under
the scope of brewery HACCP studies to assure product safety and quality. These areas
include:

tank linings where scheduled regular visual inspection of vessels should be

introduced to identify problems with vessel surface deterioration;

3
 the frequency of change of valve, door, flowplate and other rubber seals and
gaskets, where a schedule for replacement should be devised to ensure that these
items are replaced before failure occurs. The HACCP system is based on
preventative measures and does not allow for reaction to equipment failure;
procedures for the return to service of valves and pumps that have been removed
from the plant for repair, where it must be assured that these pieces of equipment
are safe for use prior to being replaced. Also, spare valves and pumps held in store
must be fully assessed prior to use to ensure that they are not a source of
microbiological contamination, rubber fragments, metal fragments or oil and
grease;
plate heat exchangers (PHEs), which will be used as an example of how the
principles of HACCP can be used to assess and reduce a known product risk in the
brewing industry. Glycol contamination from a plate heat exchanger was the cause
of a major product recall incident in the UK in 1998;
in addition, HACCP studies should be cross-linked with Engineering ISO standard
operating procedures to provide an audit trail.

PLATE HEAT EXCHANGERS

Plate heat exchangers are used widely in the beverage industry. Efficient heat
exchange depends on the product and coolant streams being brought into close
contact, separated by thin-walled metal plates. There are three proprietary types of
secondary / tertiary coolants used widely in the brewing industry; monopropylene
glycol (commonly known as propylene glycol), industrial methylated spirits (IMS)
and brine. It is essential that plate integrity is maintained in order to avoid the mixing
of coolant and product streams. Within the brewing industry, plate heat exchangers
are known contamination risk factors. Reliance cannot be placed solely on the use of
food grade coolants, as contamination is likely to arise over time through contact with
non food grade materials (pipe surfaces, anti-corrosion chemicals, etc). It is not
possible to design and maintain a risk-free system; the objective must be to use the
principles of HACCP to minimise risk and reduce it to an acceptable level.

HACCP RISK ASSESSMENT OF PLATE HEAT EXCHANGERS

A HACCP risk assessment of the range of plate heat exchangers in use in the brewery
showed that they could be split into three categories of risk; low, medium and high.

Low risk category

Example: Wort coolers
The process of wort cooling is usually continuous and any stoppages should be
infrequent and of a short duration. Some minor risk of contamination will remain
during line stoppages where residual glycol pressures may theoretically exceed the
wort pressure.

Medium risk category

Example: Filter chillers
Filter chillers do not operate continuously and operating experience indicates that
stoppages during transfer do occur regularly especially during quality changes.

4
Ensuring that product pressures are greater than glycol during normal operation does
not deal with the risk of contamination during stoppages, which can be frequent.

High risk category

Example: Smallpack chillers
Two different types of heat exchanger processes exist within smallpackaging areas,
chillers upstream of Bright Beer Tanks (BBT) and chillers downstream of BBT.
Chillers upstream of BBT trim chill product as it is transferred into bulk storage tanks
i.e. BBT and as a result these chillers can be treated in a similar way to filter chillers.
Chillers downstream of BBT normally feed directly onto fillers via small buffer tanks.
The operation is known to be stop/start and no large buffer such as a BBT exists to
prevent slugs of glycol passing direct to the filler should catastrophic failure take
place.

RISK REDUCTION METHODS

A number of risk reduction measures can be considered, with a view to the selection
of the most appropriate method of protection in each PHE application. All PHEs
should be regularly tested.

Regular testing of PHEs

Regular testing of all PHEs using a non-intrusive method should take place on a 6
monthly basis to ensure that any cracks are identified and plates replaced.
Replacement plates should be sourced from approved suppliers who provide a
guarantee on plate integrity. Freeze-up of PHEs should be avoided wherever possible,
and inspection of the PHE may require to be increased in frequency if a PHE is liable
to freeze-up.

Modification of pressure differentials

Modification of pressure differentials in PHEs can be carried out to ensure that
product pressures are greater than that of the refrigerant during normal operation. The
target is a differential of 1 bar, with an absolute minimum of 0.5 bar differential. This
form of pressure balance need not be implemented where double skin PHEs are
installed.

Flushing
Flushing alone remains a viable solution for heat exchangers which should operate
continuously with minimal stoppages. On completion of a transfer or prior to start up
the line should be flushed either to the manifold or to a suitable drain. This solution
applies to such processes as chillers that cool liquor used in the brewing process. On
start up, a timed liquor purge should be sent out to drain. Flushing is also a practical
solution to such processes as tanker bay chillers, where a tanker should be emptied or
filled as one continuous operation.

Recirculation loops
Including a heat exchanger in a recirculation loop ensures that risks of contamination
are reduced further than pressure modifications alone, since product pressures during
upsets in operations are maintained above that of coolant. This solution is preferred
for applications where regular stoppages of forward flow occur since it minimises the
need for flushing and hence product losses. It should be noted that including an

5
exchanger in a recirculation loop is not always practical to implement since the
recirculation loop must be routed back to the suction of the main feed pump. In some
circumstances this pump may be a significant distance from the chiller, or numerous
feed pumps may exist making routing to pump suction difficult to achieve.

Double skin heat exchangers

Double skin exchangers, as their name suggests, provide two separate plates between
product and coolant. The advantage of such a solution is that double protection is
provided against contamination and if one plate cracks any leak should be detectable
by regular inspection via a leak path to the base of the affected plates. Double skin
exchangers offer significant advantages in that the existing pipework layouts and
processes remain largely unaffected. In addition, pressure modifications will not be
required. Fewer plant items requiring maintenance will result from this solution and
running costs of recirculation (e.g. booster pumps) will be avoided.

However, there are significant areas of concern regarding double skin exchangers. If a
plate cracks there is a potential microbiological risk. Any micro-contamination of
product would be detected quickly by routine sampling, but the risk to plant hygiene
would have to be eliminated by the introduction of regular visual inspection and
pressure testing of the heat exchanger. Catastrophic plate failure is still theoretically
possible but highly unlikely. This risk could be minimised further by installing filters
on glycol feed lines to the exchanger. Double skin exchangers take up more space
than conventional exchangers due to poorer thermal efficiencies requiring additional
plates. The double skin solution is sometimes more costly than recirculation loops, at
an average cost of 30,000 (approximately 50,000 Euros) per unit installed.

In this solution, daily inspections of the double skin PHEs should be carried out to
identify any plate failure, as seen by liquid dripping from the area between the double
skin. This check can be carried out during the line check on route start-up.

Tertiary systems
Tertiary systems, where an intermediate heat exchanger is installed which cools a
closed low pressure glycol system, which in turns cools the product stream, provides a
high level of protection against product contamination by glycol. Furthermore, by
fitting alarmed level measurement on the systems header tank, any leak in a plate will
be detected almost immediately. Tertiary systems, however, require an increased
flowrate of glycol, typically 30%, and may have a significant impact on utilities.
Tertiary systems are costly at up to 50,000 (approximately 80,000 Euros) per unit
depending on process conditions and give more items of plant to maintain and
operate.

HEAT EXCHANGER APPLICATION SOLUTIONS

A selection of the risk reduction methods described can be applied at each heat
exchanger application to reduce the risk of glycol contamination to an acceptable
level.

Low risk heat exchangers

Maintaining product pressures higher than that of the glycol coolant and flushing
should be all that is needed in low risk areas.

6
Medium risk heat exchangers
In these areas, ensuring that product pressures are greater than glycol, and
recirculating product through the exchanger during line stoppages should be adequate
control measures. In some cases, this may not be possible if the heat exchangers are
fed from multiple pumps located a significant distance from the chiller itself. The
recommendation here would be to install double skin exchangers.

High risk heat exchangers

Tertiary systems should be installed in these areas, where any plate failure would
immediately be identified by the local glycol header tank level alarm.

TESTING AND MAINTENANCE OF PLATE HEAT EXCHANGERS

A review of existing testing and maintenance practices within Scottish Courage
Brewing sites was carried out and historical data on plate pack failures was collated.
The BLRA Guide for Best Practice for Coolants and Cooling Systems2 was also
consulted. The ultimate aim was to produce Group standards for the testing and
maintenance of plate heat exchangers which would ensure optimal plant performance,
but also further reduce the risk of product contamination (so satisfying HACCP
requirements). Examples of the regimes in place are given below.

Double skin heat exchangers

Pressure testing is on a 6 monthly basis, on both sides of the heat exchanger. Visual
inspection at 4 weekly interval to examine for external leakage, mechanical damage,
corrosion, etc is also carried out, as is inspection and cleaning of glycol strainer.
Service of plate packs is at a minimum frequency of 5 years. Sampling of product for
contamination with glycol from each chiller is carried out on a weekly basis.

Single plate heat exchangers tertiary systems

System testing is at a defined frequency, combined with bench testing of pressure
gauges and inspection and cleaning of glycol strainers. Inspection of glycol header
tank and confirmation of alarm signal operation is also carried out. Visual inspection
at 4 weekly interval to examine for external leakage, mechanical damage, corrosion,
etc is also in place. In addition, a record of operational pressure and temperature
parameters and confirmation of correct product / glycol differential pressure is
required. Service of plate packs is at a minimum frequency of 5 years. Sampling of
product for contamination with glycol from each chiller is carried out on a weekly
basis.

SYSTEM TESTING
The method of choice for testing plate packs is a non-intrusive conductivity method
(Testex), where a saline solution is placed on one side of the plate, with water on the
other. Leaks in the plates are detected using conductivity measurements. Frequency of
testing can be from every 6 to 9 months, depending on heat exchanger type,
application and historical data.

7
Stripping down plate packs for inspection on a regular basis, for example every two
years can introduce more risk than it prevents. It is preferable to have a planned
replacement schedule in place for each heat exchanger based on age, type, application
and risk assessment. However, certain heat exchanger are opened up for special cleans
or following freeze-up and any findings made during such an activity should be
recorded and available for HACCP purposes.

PROPYLENE GLYCOL ANALYSIS

Analysis of beer and in-process samples for glycol can be carried out by a gas
chromatography method. Butanol is used as the extractant, and ethylene glycol is run
as an internal standard. The lower limit of detection achieved with this method is in
the area of 1 3 ppm. Beer samples can routinely be found to contain glycol at levels
of up to 30 ppm, glycol being produced naturally by the brewing yeast present. The
viability and vitality of the yeast can affect this level. Any sample found to contain
glycol above this level would give cause for concern, prompting an investigation with
beer put on hold or ultimately destroyed.

ACKNOWLEDGEMENT
The author would like to thank the Directors of Scottish Courage Brewing Ltd for
permission to publish this paper.

REFERENCES
1. Kennedy, A.I. and Hargreaves, L.L. (1997). Is there improved quality in brewing
through HACCP ? In: European Brewery Convention Monograph XXVI
Quality Issues and HACCP, Stockholm, Sweden, pp 56-62, Verlag Hans Carl,
Germany.
2. Brewers and Licensed Retailers Association (2000). Coolants and Cooling
Systems: a Guide for Developing Best Practice.

8
93

Disinfectant testing against brewery-

related biofilms
E. Storgrds, M. Nrhi & G. Wirtanen
VTT Biotechnology, P.O. Box 1500, FIN-02044 VTT, Espoo, Finland (e-mail:
[email protected])

Descriptors
Beer spoilage organism, comparison, disinfectant, efficiency, lethality

SUMMARY
Disinfectants were tested against true biofilms grown in liquid media, against biofilm models
grown on filter paper on agar media and against biofilm constructs formed in poloxamer-
hydrogels. Brewery isolates of Dekkera anomala, Lactobacillus brevis, L. lindneri, Pectinatus
frisingensis and Enterobacter sp. were used as test organisms. Ethanol- and isopropyl alcohol-
based disinfectants were effective against brewery-related biofilms. The effects of peracetic
acid based-formulations were variable. Epifluorescence microscopy in situ showed that living
cells could still be detected after disinfection treatments, although the degree of reduction of
the living population was high. Disinfectant testing methods had a major effect on results
obtained.

Tests von Desinfektionsmitteln gegen brauereispezifische Biofilme

Deskriptoren
Bierschdliche Organismen, Desinfektionsmittel, Letalitt, Vergleich, Wirkungsgrad

ZUSAMMENFASSUNG
Es wurden Desinfektionsmittel an Biofilmen auf unterschiedlichen Nhrmedien getestet. Es
handelte sich um Biofilme auf flssigen Nhrmedien, an Filterpapieren auf Agar-Nhrbden
und um Biofilmbruchstcke, die in Poloxamerhydrogelen gebildet wurden. Isolate aus der
Brauerei, wie Dekkera anomala, L. lindneri, Pectinatus frisingensis und Enterobacter sp.,
wurden als Testorganismen verwendet. Auf Ethanol- und Isopropyl-Alkohol-Basis
hergestellte Desinfektionsmittel waren am Effektivsten gegen die brauereispezifischen
Biofilme. Die Ergebnisse bei den auf Peressigsure basierenden Mitteln waren unter-
schiedlich. Die in situ Epifluoreszenzmikroskopie zeigte, dass nach der Desinfektions-
mittelanwendung immer noch lebende Zellen nachgewiesen werden konnten, obwohl der
Abttungseffekt auf die lebenden Zellen sehr gro war. Desinfizierende Testmethoden
bildeten den Haupteffekt bei den beobachteten Ergebnissen.
Essais de dsinfectants contre les biofilms de brasserie

Descripteurs
Comparaison, dsinfectant, efficacit, ltalit, microorganisme contaminant

RESUME
Des dsinfectants ont t tests contre de vritables biofilms pousss en milieu liquide, des
biofilms modles pousss sur papier filtre avec milieu glos et des biofilms artificiels forms
dans des poloxamres-hydrogels. Des isolats de brasserie de Dekkera anomala, Lactobacillus
brevis, L. lindneri, Pectinatus frisingensis et Enterobacter ont t utiliss comme micro-
organismes d'essai. Des dsinfectants base d'thanol et d'isopropanol ont t efficaces contre
les biofilms de brasserie. L'effet de formule base d'acide practique tait inconstant. La
microscopie d'pifluorescence in situ a montr qu'il tait encore possible de dtecter des
cellules vivantes aprs l'application des dsinfectants, mme si le degr de rduction de la
population vivante tait lev. Les mthodes d'essai des dsinfectants ont eu un effet majeur
sur les rsultats obtenus.

2
INTRODUCTION
The aim of disinfection is to reduce the surface population of viable microorganisms
after cleaning and to prevent microbial growth during the interproduction time. The
purpose of disinfectant testing is to establish whether products meet the requirement
of destroying harmful organisms to a defined level. In the guidelines of the European
Committee for Standardisation, the assessment of the efficacy of disinfectants is
established in 3 phases. In phase 1, the activity of the agent is tested in suspension
using standardised hard water. In phase 2 step 1, the activity is tested in suspensions
including interfering substances such as bovine albumin. In phase 2 step 2, the activity
is tested against microorganisms dried onto surfaces. Phase 3 refers to field tests
under practical conditions.
Standardised tests are needed for fair comparison of various formulations in the same
conditions. However, standardised conditions are often not the same as realistic
conditions. Approved standard tests are, understandably, not up-to-date with the latest
findings of biofilm research. Microorganisms that are exposed to disinfection on food
or beverage processing surfaces are those that remain after the cleaning stage and are
thus likely to be surface-attached (6). Adherent cells have been shown to be more
resistant to disinfectants than freely living (planktonic) cells (4). Microbial biofilms
exhibit a broad spectrum of resistance to antimicrobials. The concentrations of some
disinfectants must be increased ten to one hundred fold (7), or even to one thousand
fold (1) in order to obtain the same degree of inactivation for biofilm bacteria as for
cells in suspension.
The resistance to antimicrobial components observed in biofilms is caused by a
variety of mechanisms. The exopolymer matrix on its own does not represent a
significant barrier to diffusion, due to its high water content. However, it can restrict
access of antimicrobials to the inner parts of the biofilm by quenching chemically
reactive agents or by binding highly charged compounds. Furthermore, extracellular
enzymes bound within the matrix increase diffusion limitation through destruction of
susceptible compounds. The shortage of nutrients and oxygen for deeper lying cells
results in spatial gradients of growth rate within the biofilm community. The slower-
growing cells at the core of the biofilm generally survive longer than the more rapidly
metabolising cells at the periphery of the biofilm. Slow growing cells express
dormant, starvation phenotypes which often overexpress non-specific defences such
as shock proteins, multi-drug efflux pumps and further exopolymer synthesis. Finally,
it is believed that attachment of microorganisms to surfaces causes the expression of
biofilm-specific phenotypes, possibly regulated by quorum sensing mechanisms,
which may also contribute to biofilm resistance. Collectively these mechanisms
probably result in the resistance observed in biofilms (2).
Living microorganisms on production surfaces are a source of contamination, which
in turn may cause economic losses. Therefore reliable means of assessing disinfectant
efficacy are needed. However, passing standardised tests does not necessarily mean
that the disinfectant will perform well in process conditions. When disinfectants are
assessed for their use in food hygiene, they are usually tested against microbes in
suspension. These tests are referred to as suspension tests. Although suspension
tests can be used to assess the activity of disinfectants under a range of conditions,
they provide no information about how products actually perform on contaminated
surfaces. To overcome this shortcoming, different kinds of "surface tests" are being
developed. The aim of this work was to describe and discuss alternative disinfectant
testing methods.

3
ANALYTICAL METHODS
Disinfectants were tested against true biofilms grown in wort sucrose broth (12),
against biofilm models grown on filter paper on agar media (3) and against biofilm
constructs formed in Poloxamer F127 hydrogels (5). All biofilms or biofilm models
were formed on stainless steel coupons (AISI 304, 2B). The incubation time for true
biofilms was 4 days, for biofilm models 48 hours and for hydrogel-enclosed
microorganisms 6 hours. The test coupons were immersed in the disinfectant solution
for 5 minutes at room temperature followed by immersion in the inactivation solution
(14) to stop the action of the agent. All experiments were performed with three
replicates.
Ten disinfectants were tested at their recommended use concentrations, among them
ethanol-based, isopropyl alcohol-based and peracetic acid-based formulations (table
1). The ethanol- and isopropyl alcohol-based disinfectants are designed to be used
locally at critical points by spraying or wiping onto surfaces. The formulation low in
ethanol concentration contained quaternary compounds and synthetic tensides. The
formulations based on peracetic acid contained hydrogen peroxide and they are
frequently used for post-cleaning disinfection in CIP operations.

Table 1: Disinfectants tested.

Disinfectant Main active agent Product concentration

tested
A, B, C, D Ethanol 2 % or 100 %
(20 70 %)
E, F Isopropyl alcohol 100 %
(15- 60 %)
G, H, I, J Peracetic acid (PAA) 0.25 1.0 %
(4 15 %)

Brewery isolates of product spoiling wild yeasts and bacteria were used as test
organisms in pure and in two-species cultures together with Enterobacter sp. (table 2).
The plate count method was used for detection of viable microorganisms. The
microbicidic effect (ME) was calculated according to the formula

ME = log Nc log Nd, where

Nc = cfu (colony forming unit) in the control

Nd = cfu in the treated sample.

In addition, surfaces covered by biofilm were stained with 5 mM CTC (5-cyano-2,3-

ditolyl tetrazolium chloride) for 3 hours at 30C. The stain was fixed with 5%
formaldehyde for 5 minutes, and counterstained with DAPI (4',6-diamidino-2-
phenylindole, 1 g/ml) for 3 minutes at room temperature. The stained surfaces were
examined by epifluorescence microscopy (Olympus BX-60, Japan) and image
analysis (analySIS). 50 fields were analysed from each triplicate sample.

4
Table 2: Test strains from VTT Culture Collection (13).

Species VTT Cultivation conditions

code
Dekkera anomala C-91183 YM (Difco 0712) 26C aerobic
Saccharomyces C-00387 YM (Difco 0712) 26C aerobic
cerevisiae C-00388
C-00389
C-00390
Lactobacillus brevis E-88388 MRS (Oxoid CM361) 30C anaerobic
L. lindneri E-91454 NBB (Dhler 4709) 30C anaerobic
Pectinatus frisingensis E-79100 PYF (Suihko, 1999) 30C anaerobic
Enterobacter sp. E-86263 TSA (Oxoid CM131) 26/30C aerobic/
anaerobic

RESULTS AND DISCUSSION

True biofilms
A disinfectant is regarded as effective against microbes growing on surfaces, when its
ME is 3 log units (10). In the proposed CEN test (prEN 13697), the requirement for
bactericidal activity is 4 log units and for fungicidal activity 3 log units. However,
in this test pure cultures of microbes are only dried onto the surface, not grown to
form biofilm.
The effects of the disinfectants based on ethanol or isopropyl alcohol against true
biofilms were generally very good (ME = 3.6 9.0). The most resistant strain against
the alcohol-based disinfectants was L. brevis, but still the ME was 3 log units for all
the formulations (figure 1). The alcohol-based disinfectants were also effective
against the two-species biofilms regardless of the higher cell numbers compared to the
pure culture biofilms.

ME (log cfu)
9
8
7
6
5
4
3
2
1
0
A B C D E F
L. brevis L. brevis + Enterobacter sp. D. anomala + Enterobacter sp.
Figure 1: Effects of ethanol-based (A-D) and isopropyl alcohol-based (E-F)
disinfectants against biofilms of microbes isolated from the brewery environment.

5
The effects of the PAA-based disinfectants against true biofilms were variable (ME =
1.5 8.9). A trend towards higher activity with higher concentration was observed.
The most resistant biofilms against PAA-based formulations were those containing L.
lindneri or P. frisingensis. The PAA-based agents were not sufficiently active against
these biofilms at concentrations of 0.25 - 0.5 % (ME < 3), whereas the disinfectant
recommended to be used at a 1 % concentration proved to be effective (figure 2).

ME (log cfu)
9
8
7
6
5
4
3
2
1
0
G (0.25%) H (0.3%) I (0.5%) J (1%)

L. lindneri L. lindneri + Enterobacter sp. P. frisingensis + Enterobacter sp.

Figure 2: Effects of PAA-based (G-J) disinfectants against biofilms of microbes

isolated from the brewery environment. Concentration used in brackets.

Because working with true biofilms is laborious and difficult to standardise,

alternative methods are being developed in which microorganisms are in the biofilm-
state. Such methods are the biofilm model described by Charaf and co-workers (3)
and the poloxamer-hydrogel method described by Gilbert and co-workers (5).

The biofilm model

The PAA-based disinfectants were tested against newly isolated S. cerevisiae wild
yeast strains using the biofilm model (3). The disinfectants were not sufficiently
effective against most of the wild yeasts at a concentration of 0.25% (ME < 3), and in
some cases practically no effect was observed (figure 3). By increasing the
concentration to 0.5%, all disinfectants performed satisfactorily against the wild
yeasts (ME > 4.8).

Compared to working with true biofilms, much less work and consumables were
needed with the biofilm model. Furthermore, the method was also suitable for
anaerobic Pectinatus-bacteria. The resistance of microorganisms to disinfectants was
slightly lower in the biofilm model than in true biofilms, but the resistance could
probably be increased by extending the incubation time from 2 days to 3 or 4 days.

6
The simplicity and reproducibility of the method makes it an attractive alternative in
biocide testing.

ME (log cfu)
9
8
7
6
5
4
3
2
1
0
G H I J
Yeast isolates: C-00387 C-00388 C-00389 C-00390

Figure 3: Effects of PAA-based (G-J) disinfectants at a 0.25% concentration against

biofilm models of S. cerevisiae strains VTT C-00387, C-00388, C-00389, C-00390
isolated from the brewery environment.

Biofilm constructs in poloxamer-hydrogels

Poloxamer F127 is a non-toxic, di-block copolymer of polyoxyethylene and
polyoxypropylene. Aqueous solutions show thermoreversible gelation, being liquid at
temperatures below 15C and solid gels at temperatures above 15C (5). Chilled
poloxamer in appropriate liquid medium can be inoculated with a test organism and
pipetted onto a surface. When transferred to a normal incubation temperature, the gel
solidifies. The use of this technique in biocide testing is based on the fact that gel-
enclosed populations mimic localised high cell densities of biofilms. Furthermore,
SDS-PAGE of cell envelope preparations has shown that the poloxamer-grown cells
exhibit a biofilm rather than a planktonic phenotype (5).
When tested against biofilm constructs in poloxamer-hydrogels, the microbicidic
effects of the disinfectants were much lower than those obtained against true biofilms
or biofilm models (table 3). These low effects have been claimed to mimic the effects
obtained in real disinfection situations (5). Other authors have reported effects in the
range of 0.4 - 3 logarithmic units for various disinfectants (8, 14), which are close to
those obtained in this study. However, no recommendations for acceptable thresholds
have hitherto been given for this method.

A weak point of the method is that the cells are entrapped in an artificial matrix and
not in their own extracellular polymeric substances. This may lead to results not
applicable to real situations. Furthermore, the obligate anaerobe Pectinatus could not
survive in the poloxamer-gel despite the use of strict anaerobic techniques throughout
the test protocol. Thus the method may not be suitable for testing of disinfectant

7
activity against anaerobic bacteria, which nevertheless are important in brewery
microbiology.

Table 3. Microbicidic effects (ME) of disinfectants against poloxamer-grown cells.

Disinfectant Range Average

A, B, C, D 0.02 - 0.53 0.19

(ethanol)
E, F 0 - 2.57 0.62
(isopropyl alcohol)
G, H, I, J 0 -1.24 0.50
(PAA)

Epifluorescence microscopy of CTC-DAPI stained cells

Surviving cells after disinfectant treatment can be detected in situ by epifluorescence
microscopy. Actively respiring microorganisms are visualised by staining with CTC,
which is reduced via electron transport activity to fluorescent CTC-formazane. This
compound accumulates intracellularly, producing a red dot in living cells.
Counterstaining of CTC-treated samples with the DNA-specific fluorochrome DAPI
allows estimation of active and total populations within the same preparation (11).
This method usually indicates higher levels of residual microbial activity compared
with the plate count method, due to the presence of viable but non-culturable cells (9).
On the basis of the microscopy results (table 4), the viable microbial populations in
the true biofilms were in most cases reduced by >90% (median 92 - 99%) by the
disinfectants. Thus, the reduction was generally lower than the recommended 3 log
units or 99.9%, and viable cells were present after the disinfectant treatments. The
method could not be applied to pure culture bacterial biofilms due to low cell density,
or to obligately anaerobic Pectinatus -bacteria due to unspecific staining. Moreover,
the cells stressed by the disinfectant treatment may stain dimly, which further
decreases the reliability of this method. However, the technique is useful for
visualising viable but non-culturable cells.

Table 4: Microbicidic effects of disinfectants according to microscopy results.

Reduction (%) in viable cells as estimated by image analysis of the area stained red by
CTC.

Disinfectant D. anomala D. anomala + L. brevis + L. lindneri +

Enterobacter sp. Enterobacter sp. Enterobacter sp.
A 96.0 0.4 50.0 3.4 99.3 0.2 95.5 0.2
B 68.0 4.1 79.1 1.5 99.0 0.2 97.2 0.2
C 94.8 2.4 90.4 0.4 99.1 0.8 92.7 0.4
D 97.7 0.6 92.5 0.9 99.3 0.2 97.9 0.8
E 94.2 0.4 66.0 0.4 96.5 1.0 97.4 1.0
F 95.7 2.6 91.9 0.9 98.8 0.5 99.2 0.2
G 79.4 9.9 99.8 0.3 100 0 100 0
H 89.2 0.5 92.1 1.0 99.7 0 92.3 2.6
I 91.5 0.5 94.3 0.4 99.6 0.1 80.3 5.3
J 98.6 0.1 97.5 0.5 99.5 0.2 98.5 0.2

8
CONCLUSIONS

Ethanol- and isopropyl alcohol-based disinfectants were effective against
brewery-related biofilms.

The microbicidic effects of PAA-based disinfectants were highly dependent on the
concentration used. At sufficient concentrations they were effective against
brewery-related biofilms.

Results obtained by epifluorescence microscopy in situ showed that viable cells
could still be detected after disinfection treatments. Thus, careful removal of
biofilms prior to disinfection is essential.

The testing procedure had a major effect on the test results obtained. Cultivation
of true biofilms on surfaces produced the most resistant populations but was
laborious and difficult to standardise. Biofilm models grown on filter paper were
simple to prepare and gave reproducible results in disinfectant testing. Biofilm
constructs in poloxamer-hydrogels may not give results relevant to real conditions,
and the method was not applicable for anaerobic Pectinatus -bacteria.
Epifluorescence microscopy was superior to the methods based on cultivation as it
also detected non-culturable cells, but image analysis needs further automation to
be applicable for disinfectant testing.

ACKNOWLEDGEMENT
The authors thank Ms. Merja Salmijrvi and Mr. Kari Lepist for skilful technical
assistance. Financial support received by the Finnish malting and brewing industry, by
the National Technology Agency (Tekes) and by VTT is gratefully acknowledged.

REFERENCES
1. Allison, D.G. and Gilbert, P., 1995, Modification by surface association of
antimicrobial susceptibility of bacterial populations, J. Ind. Microbiol., vol. 15:
311-317.
2. Allison, D.G., McBain, A.J and Gilbert, P., 2000, Biofilms: problems of control.
In: (eds.) Allison, D.G., Gilbert, P. Lappin-Scott, H.M. and Wilson, M.
Community structure and co-operation in biofilms, Soc. Gen. Microbiol.
Symposium series, NO. 59, 309-327.
3. Charaf, U.K., Bakich, S.L. and Falbo, D.M., 1999, A model for efficacy
assessment of antimicrobials versus biofilm bacteria. In: Wimpenny, J., Gilbert,
P., Walker, J., Brading, M. ja Bayston, R. (eds.), Biofilms. The Good, The Bad
and The Ugly. Bioline, Cardiff, 171-177.
4. Frank, J.K. and Koffi, R.A., 1990, Surface-adherent growth of Listeria
monocytogenes is associated with increased resistance to sanitizers and heat, J.
Food Prot., vol. 53: 550-554.
5. Gilbert, P., Jones, M.V., Allison, D.G., Heys, S., Maira, T. and Wood, P., 1998,
The use of poloxamer hydrogels for the assessment of biofilm susceptibility
towards biocide treatments, J. Appl. Microbiol., vol. 85: 985-990.
6. Holah, J.T., 1992, Industrial monitoring: hygiene in food processing. In: Melo,
L.F., Bott, T.R., Fletcher, M. and Capdeville, B. (eds), Biofilms Science and
Technology, Kluwer Academic Publications, Dordrecht, 645-659.

9
7. Holah, J.T., Higgs, C., Robinson, S., Worthington, D. and Spenceley, H., 1990, A
conductance-based surface disinfection test for food hygiene, Lett. Appl.
Microbiol., vol. 11: 255-259.
8. Hrknen, P., Salo, S., Mattila-Sandholm, T., Wirtanen, G., Allison, D.G. and
Gilbert, P., 1999, Development of a simple in-vitro test system for the
disinfection of bacterial biofilms, J. Water Sci. Technol., vol. 39: 219-225.
9. McFeters, G.A., Yu, F.P., Pyle, B.H. and Stewart, P.S., 1995, Physiological
methods to study biofilm disinfection, J. Ind. Microbiol., vol. 15: 333-338.
10. Mosteller, T.M. and Bishop, J.R., 1993, Sanitizer efficacy against attached
bacteria in a milk biofilm, J. Food Prot. 56: 34-41.
11. Rodriguez, G.G., Phipps, D., Ishiguro, K. and Ridgway, H.F., 1992, Use of a
fluorescent redox probe for direct visualization of actively respiring bacteria,
Appl. Environ. Microbiol., vol. 58: 1801-1808.
12. Storgrds, E., Simola, H., Sjberg, A-M. and Wirtanen, G., 1999, Hygiene in
gasket materials used in food processing equipment. Part 1: new materials.
TransIChemE, Part C, Food Bioprod. Proc., vol. 77: 137-145.
13. Suihko, M.-L., 1999, VTT culture collection. Catalogue of strains. 4th ed. VTT
research notes 1980, Espoo. 298 p.
14. Wirtanen, G., Salo, S., Allison, D.G., Mattila-Sandholm, T. and Gilbert, P., 1998,
Performance evaluation of disinfectant formulations using poloxamer-hydrogel
biofilm constructs, J. Appl. Microbiol., vol. 85: 965-971.

10
94

Designing packaging for brand growth

A. Ray Morgan
5 Points Solutions, LLC, 2441 Oldfield Road, NW, P.O. Box 20305, Atlanta, Georgia 30325,
U.S.A. (e-mail: [email protected])

Descriptors
Beverage industry, consumer research, marketing, packaging material

SUMMARY
This presentation will discuss the strategic importance of beverage packaging in delivering
tangible brand value. Marketplace examples are presented to demonstrate how beverage
companies design packaging to generate interest, recognition and awareness, loyalty, and
proprietary equity. These illustrations capture a method for understanding packaging
attributes as a means for creating unique differentiation and meaningful specialness for
targeted users. Ultimately how packaging satisfies consumer needs sets the expectation for
future purchase intent and delivers the potential for brand growth.

Verpackungsdesign fr Markenwachstum

Deskriptoren
Getrnkeindustrie, Marketing, Verbraucherforschung, Verpackungsmaterial

ZUSAMMENFASSUNG
Die Prsentation beschftigt sich mit der strategischen Bedeutung von Getrnkeverpackungen
als lieferbaren und greifbaren Markenwert. Exemplarische Marktpltze werden gezeigt, um
zu demonstrieren, wie Getrnkehersteller ber ihr Verpackungsdesign Interesse wecken,
Wiedererkennungswert, Markenbewusstsein und Loyalitt schaffen. Diese Beispiele zeigen
eine Methode, um Verpackungsmerkmale dafr zu benutzen, einmalige Unterscheidung und
nachhaltige Spezialisierung auf die Zielgruppe zu ermglichen. Letztlich entscheidet die
Zufriedenheit der Verbraucher mit der Verpackung ber die Aufmerksamkeit bei zuknftigen
Kufen und liefert so das Potenzial fr ein Markenwachstum.
Conception de lemballage pour la croissance des marques

Descripteurs
Etude du march (consommation), industrie des boissons, marketing, matriau d'emballage

RESUME
Cette prsentation discutera de l'importance stratgique de lemballage des boissons pour une
valorisation tangible des marques. Des exemples tirs du march sont prsents pour
dmontrer comment les fabricants de boissons conoivent leurs emballages pour susciter
l'intrt, la reconnaissance, la sensibilit la marque (awareness) et la loyaut du
consommateur. Ces illustrations permettent de mieux comprendre les attributs de lemballage
comme moyen de crer une diffrenciation exclusive et une particularit signifiante pour la
clientle vise. En fin de compte, la faon dont lemballage satisfait aux besoins du
consommateur permet d'esprer de futures intentions d'achat et constitue le potentiel de
croissance de la marque.

2
 Designing Packaging
for
Brand Growth
Presented By:
Ray Morgan, Principal
5 POINTS SOLUTIONS, LLC
Atlanta, GA USA
May 15, 2001

Thank you Mr. Chairman, I am delighted to be here. As a veteran beverage industry

executive, my remarks today are based on experience from over thirty years in the
soft drink business. Being responsible for global packaging and trademark
management at Colca-Cola was indeed challenging and rewarding.

1999 package mix comparison

US soft drink and European beer
G las s
1 .0 %

C ans
2 5 .0 %
Plas tic
C an s
4 7 .0 %
5 2 .0 % G la s s
7 5 .0 %

US soft drinks Europe - beer

Source: Beverage Marketing Corp, share of packaged gallonage 2

I would like to begin by referencing these charts comparing the package mix of US
soft drinks and European beer. You will see the higher penetration of cans and also
that of plastic in the US.

3
 1999 package mix comparison
US beer and European beer

C ans
2 5 .0 %
G las s
4 4 .0 % C an s
5 6 .0 % G la s s
7 5 .0 %

US -beer Europe - beer

Source: Beverage Marketing Corp, share of packaged gallonage 3

When you compare beer packaging in the US to European beer packaging the
similarities of the bottle/can relationship are nearly the same as soft drinks, the
exception being plastic for soft drinks and glass for US beer. With these similarities
and the opportunities ahead with packaging materials especially plastics, soft drink
packaging experience can be invaluable to the beer industry.

growth of US soft drink packaging

100
90
80
billions of units

70 cans
60
plastic
50
40 glass
30 total
20
10
0
75

84

86

88

90

92

94

96

98
19

19

19

19

19

19

19

19

19

Source: Beverage Marketing Corp, share of units

4

This chart shows the immense growth of soft drinks in the US over the last 25 years
as indicated by the black line. It also shows the conversion of glass packaging to
plastic and the incredible growth of cans.

4
 US soft drink growth packages

large multi-serve plastic bottles in the

early 80s
can multi-packs in the mid 80s
larger size single drink in the late 80s
proprietary plastic packaging in the 90s

Growth of soft drinks in the US can be attributed to several packaging initiatives.

First, in the early 80s, the introduction of 2 liter PET allowed soft drinks to increase
in home penetration adding significantly to the potential for consumption occasions.
In the mid 80s, can multipacks were also used to increase home inventory. As with
big bottles, it was quickly learned that once in the home, soft drinks were consumed at
a faster rate. In the late 80s, as consumers were conditioned to drink more soft drinks,
the conversion from small size individual containers mostly cans to half liter and 20
ounce containers for out of home consumption created another growth wave. In the
early 90s, proprietary packaging was reintroduced using plastic bottles and even an
attempted to create a proprietary shaped can.

the changing role of packaging in the US

YESTERDAY TODAY
contain, identify, describe contain, identify, describe,
promote, market
low cost value-added

all channels channel-specific

single packaging multiple packaging

type/size types/sizes

all occasions occasion-specific

Source: Beverage Marketing Corp 6

This chart illustrates the changing role of packaging in the US.

5
 an opportunity for beer in Europe
because a successful launch of plastic
packaging for beer has the potential to grow
the category:
marketing experiences in US soft drink package
conversion to plastic can create an advantage for
beer implementation
consumer perception and attitude
reaction from the retail trade
environmental concerns
cannibalization of glass
changes in distribution

And this is why I believe that there is indeed a great opportunity for the beer industry
to grow by simply learning from soft drink mistakes.

key factors affecting

beverage growth
PITA Model
population number of people
incidence* how often one drinks
transaction size* how much one buys
amount* how much one drinks

* factors beverage companies can influence

There are four factors which affect beverage consumption and are represented in what
is termed the PITA model. Marketing initiatives target three of these factors:
incidence, transaction size and amount. Of these, transaction size has the greatest
correlation to growth.

6
 building a beverage brand
requires many tools
great product
believable advertising promise
consistent quality
affordable price
recognizable name
embedded brand values
uniqueness/authenticity
heritage
great packaging

And of course great packaging!

great packaging, because it

creates more brand impressions than

advertising
is the tangible connection the product
has to the consumer
creates consumer dem and

10

7
 developing great packaging
requires
thorough understanding of product
positioning, category relevance, and the
competition
clearly defined brand objectives and
strategies
development of consumer based principles to
guide design
agreed upon measures of success
imagination
11

packaging must also tangibly

deliver the advertising promise
communicate
graphics
inform consumers about product attributes
form
create brand recognition
function
container
hold and protect the liquid
fulfill a positive drinking experience
12

8
 consumers have choices
will it taste good?
will it be good for me?
what will my friends think if I buy it?
will it be fun?
is it a good value for the money?

13

two factors influence

purchase intent most

psychological needs
functional needs
30%

70%
14

9
 and, the package must deliver
functional needs
operational performance
material characteristics

psychological needs
recognizable imagery
uniqueness

15

when it comes to choice

psychological needs
purchase 1/3
decision
functional needs
2/3
16

Functional consideration far outweighs branding when it comes to why consumers

make choices. If the product works and meets expectation, the branding becomes
important and the psychological needs are met. If the product does not meet
expectations, then the opposite happens and the psychological needs are negatively
influenced. Imagine if you buy a can of Guinness and the widget does not produce the
foam expected, the consumer becomes suspicious of the package and the product and
may be less likely to buy it again.

10
 packaging attributes enhance
purchase intent
marketing tools purchase factors package attributes
functional attributes
product easy to open
easy to hold/use
name easy to carry
functionality sturdy
packaging safe
recyclable
advertising
communication
psychological
visual appearance
values quality
appealing
uniqueness/Authenticity easy to recognize
heritage

17

Considering the marketing tools available and the need to meet functional and
psychological needs, packaging has an important role because it is the tangible
component to deliver product attributes. Functionally, the package must be easy to
open, hold, and use. It must be easy to carry, sturdy and safe and above all recyclable.
Psychlogically, the package must present quality and be appealing throughtout the use

ideal package

functionality appearance

18

cycle.

An ideal package is a balance of functional and psychological values. When designed

with the consumer in mind, the package is more likely to hit the target of optimal
balance.

11
 package positioning example

functionality appearance

19

Here is an example of a food product called Jelly from Hell. The package conveys
great imagery. Shoppers can find it easily on the shelf and know that whatever it is
inside, it is hot. I bought this product purely out of novelty interest. It has great
imagery and appearance on shelf. When I got it home, I found that the wax covered
lid was virtually impossible to open. After several minutes of chipping away, this
Jelly from Hell went to the bin.

package positioning example

functionality appearance

20

Here is an interesting example of near total functionally. Without reading the text,
first time consumers would not know it was potato chips.

12
 package positioning example

functionality appearance

21

This syrup package demonstrates a great balance of functionality and appearance.

Molded in the shape of a pitcher, the container can be micro waved to a perfect
temperature by using the built in indicator. The easy open top can be resealed for
storage.

examples of good packaging

22

There are many great packages that satisfy consumer needs. These are a few.

13
 what is common to all these
packages?

different
better
special

23

for beverages
In the 1920s Coke
introduced its
proprietary shaped
bottle and today it is
an icon known to
anyone, even in the
dark

24

14
 beer has its icons too

25

limitations of generic packaging

All too often, the
only thing different is
the label
What that says to
the consumer is that
everything else is
the same.

26

15
Generic packaging is just that generic. There is nothing different, better, or special
about it. What is in it might as well be a commodity and the package discounts the
specialness of the brand.

highly intrusive packaging

often goes too far!

27

Packaging that goes overboard on imagery like the Jelly from Hell may create strong
shelf impact but like these beverage bottles from Japan and Switzerland, they are all
far from delivering functional benefits and most likely lived short market lives.

generic soft drink packaging

Negatively impacts
imagery
Detrimental to brand
values
Limits marketing
initiatives

28

16
I can attest to the negative impact of generic soft drink packaging on imagery, brand
values and marketing initiatives. For you, beer is not just beer. And the packaging you
use is very important to the quality, taste and uniqueness of every product.

new packaging opportunities

bottle shaped can plastic beer bottles

29

There are many new package alternatives available to you. The Japanese are
producing beer packed in an aluminium bottle. And, in America, Miller has launched
products packed in plastic bottles.

ATTENTION!
dont fall into the trap of
selling only what you
makeinstead, make and
sell what consumers want to
buy

30

17
 good packaging always satisfies
consumer needs

Psychological needs
30%
30% Functional Psychological
Functional needs

70%

70%
Purchase Decision

31

good packaging is always

different
better
special

32

18
 Ray Morgan

A Marketing Consultancy
Atlanta, GA USA

33

Thank you for your invitation. I am honored to present to the European Brewery
Convention.

19
95

Beer in plastic bottles highlights EBC

Symposium November 2000
David R. Wiggins
Bass Brewers Ltd, Bass Technical Centre, Cross Street, P.O. Box 12, Burton on Trent DE14
1XH, United Kingdom (e-mail: [email protected])

Descriptors
Barrier technology, brewing industry, consumer research, plastic bottle, returnable bottle,
shelf life

SUMMARY
This paper serves to summarise the information that was shared by the technical community
of the EBC at its meeting last year. It will review the range of barrier technologies employed
by brewers and packaging suppliers to produce a container that ensures the delivery of our
product to consumers with the optimum freshness and shelf life characteristics. Issues
surrounding recyclability of PET and the development of returnable bottle systems will be
discussed. To balance the technical information comment will be made about some initial
consumer acceptance issues concerning beer in plastic packaging.

Bier in Kunststoffflaschen Highlights des EBC Symposiums vom November 2000

Deskriptoren
Barriertechnologie, Brauindustrie, Haltbarkeitsdauer, Kunststoffflasche, Mehrwegflasche,
Verbraucherforschung

ZUSAMMENFASSUNG
In diesem Vortrag sollen Informationen vom EBC-Symposium PET-Verpackung, das in Oslo
im November 2000 stattfand, zusammengefasst werden. Es wird eine bersicht ber die
heutigen Barrieretechniken fr Plastikflaschen vorgestellt, die garantieren sollen, dass unser
Produkt Bier mit optimaler Frische und Lagerfhigkeit dem Kunden geliefert werden kann.
Aspekte der Wiederverwertung von PET und die Entwicklung von wiederbefllbaren
Flaschensystemen werden diskutiert. Als Ausgleich zu den technischen Informationen werden
auch Ergebnisse ber die Akzeptanz von Bier in Kunststoffverpackungen beim Verbraucher
prsentiert.
La bire en bouteille plastique temps forts du Symposium EBC, novembre 2000

Descripteurs
Bouteille consigne, bouteille en plastique, dure de conservation, tude du march
(consommation), industrie brassicole, technologie d'tanchit

RESUME
Cet expos rsume les informations changes par la communaut technique de l'EBC lors de
son meeting de l'anne dernire. Il passe en revue l'ventail des technologies d'tanchit
employes par les brasseurs et les fournisseurs d'emballages pour produire un conteneur
assurant la fourniture de notre produit aux consommateurs avec une fracheur et une dure de
vie optimales. Les questions concernant les possibilits de recyclage du PET et la mise en
place d'un systme de consigne seront discutes. Pour quilibrer les informations techniques,
nous commenterons certains aspects initiaux de l'acceptabilit, par les consommateurs, d'une
bire en bouteille plastique.

2
OBJECTIVE
The purpose of this paper is to summarise the highlights of the EBC Symposium held
from 27 28 October 2000 in Oslo.

The meeting presented an insight into the work going on around the world on the
development of plastic as a packaging form for beers. The symposium covered
consumer issues, development of the bottle and closure, filling technology, labelling
and recycling. In order to keep within the time constraints of the Budapest meeting I
will only cover consumer issues and package development highlights. The full update
covering the filling and recycling questions is available from the EBC.

The work presented is not mine, it is that carried out by the presenters and their
colleagues at the Oslo meeting. I would therefore like to acknowledge their part in this
presentation and thank the following people in particular:

Ludovic Auvray Heineken

Frank-Jrgen Methner Bitburger Brauerei
Johan Robbrecht Interbrew
Peter Heyes Crown Cork and Seal
Stephen Ott Alcoa

INTRODUCTION
Why plastic as a packaging format for beer?

We discovered in Oslo that there are many hurdles to be overcome when developing a
new package format for our products. The most significant ones are oxygen ingress,
carbon dioxide loss and flavour stability. In the course of my presentation I will
demonstrate how these have been addressed and what result this has on shelf life.

I believe that as an industry we should recognise the work done by our technical
colleagues and applaud them for the science that they have brought to the
development of this exciting new package.

However some of the work done by our commercial departments leads me to question
why. Plastic does not afford the same levels of protection to our products that more
recognised packaging formats do, nor is it readily accepted by consumers as an
alternative to glass or can that brings them benefits when consuming our products.

The possible exception being that as it is lighter it attracts less environmental

penalties, in some parts of Europe it is therefore seen as an eco friendly package (in
others the opposite applies!).

Our challenge then as we go away from Budapest is to use the foundations laid in
packaging technology to develop a truly innovative pack format that bring real
consumer benefit while enhancing the growth of all our businesses.

To give a full understanding of the work done to date I will now take us through the
following:

3
 bottle structure and supply
barrier performance
closure performance
current developments.

As a conclusion I will return to the consumer issues.

BOTTLES
Brewers around the world have developed their own version of plastic beer bottles;
the vast majority have selected PET as the material for these packs. However each has
then applied modification to the basic material to establish what they believe to be an
acceptable shelf life.

The barriers in most common use are:

Multilayer PET/EVOH developed by Pechiney/ANC
Multilayer PET/PA developed by Schmallbach Lubeca
Multilayer PET/Oxygen Scavenger developed by Continental PET
Interior Coated PET developed by TetraPak and Sidel
Exterior Coated PET developed by CC&S (Constar), Sipa, Krones

As an alternative barrier to be used for multi trip bottles:

PEN developed by PLM Rexam

Whichever technology a brewer has selected to work with the objectives are constant,
that is to protect the beer from oxygen ingress, carbon dioxide loss and light
degradation, all of which contribute to poor consumer acceptance of the product. The
key indicators therefore of acceptability are measurements of taste and appearance.

Each brewer has his own interpretation of taste acceptability based on the nature of
his product; I do not intend to dwell on this here. The factors that we are able to reach
a degree of agreement over are:

Oxygen ingress
depends on beer sensitivity to oxidation
1 ppm of oxygen for very sensitive products
probably 2 to 3 ppm for typical products
1 ppm equates to 0.0015 ml/bottle/day in a 33cl bottle.

Carbon dioxide loss

influences taste
10% loss regarded as maximum that is tolerable over required shelf life.

Having established performance criteria a huge amount of excellent work has been
carried out by all the brewers comparing their selected barrier technology with current
package formats. One of the best summaries is that presented by Frank-Jurgen
Methner and his team.

4
This work also begins to introduce the difference between closure technologies.

BARRIER PERFORMANCE

3
Monolayer PET + screw cap
Multilayer PET/PA + crown
Interior Coating + screw cap
Multilayer PET/Scavenger + crown
2
total O 2 [mg/l]

Glass bottle + crown

0
0 4 8 12
time [weeks]

Figure 1: Oxygen Ingress into PET Bottles

(Carbonated, Degassed Water)

0,4

Monolayer PET + screw cap

Multilayer PET/PA + crown
0,3
Interior Coating + screw cap
total O 2 [mg/l]

Multilayer PET/Scavenger + crown

0,2
Glass bottle + crown

0,1

0,0
0 4 8 12
time [weeks]
Figure 2: Oxygen Content of Beer in PET
Bottles

5
 oxygen ingress
5 [mg/l/week]
PET bottle without scavenger screw cap
0,19
PET bottle with scavenger screw cap
4
glass bottle
total O2 [mg/l]

1
0,016
0 -0,006
0 6 12 18 24
time [weeks]
Figure 3: Oxygen Ingress - Impact of Scavenger/Barrier
Screw Cap (Carbonated, Degassed Water)

0,8

PET bottle without scavenger screw cap

0,6 PET bottle with scavenger screw cap

Glass bottle
total O 2 [mg/l]

0,4

0,2

0,0
0 6 12 18 24
time [weeks]

Figure 4: Oxygen Ingress Impact of

Scavenger/Barrier screw Cap (Beer)

0,6

Time for
10 % loss
[months]
CO 2 content [%]

0,5 9

46
4
0,4 2
Interior Coating + screw cap
Glass bottle + crown
Multilayer PET/PA + crown
Multilayer PET/Scavenger + crown
0,3
1 4 8 11
time [weeks]

Figure 5: Loss of CO2

6
 30

25
after 3 months
CO 2 loss [%]

20 after 6 months

15

10

0
glass bottle

II, Std. screw cap

Exterior Coating,
Interior coating I,

Interior coating I,
scavenger/barrier

scavenger crown
PET/Scavenger,
Interior coating
Std. screw cap

Std. crown

Multilayer
screw cap

Figure 6: Loss of CO2 after 3 and 6

months

change of
CO2
0,50 [%/week]
Glass bottle
PET bottle without scavenger screw cap
0,48
PET bottle with scavenger screw cap
CO 2 content [%]

0,46 0,0001
- 0,0003
0,44

0,42 - 0,0013

0,40
0 6 12 18 24
time [weeks]

Figure 7: Loss of CO2 Impact of

Scavenger/Barrier Screw Cap

CLOSURES
The initial developments at Bass Brewers retained the crown as the preferred closure
in the belief that this would meet less consumer resistance.

It is now common practice to include an oxygen scavenger in the liner to remove gas
that is retained in the package or enters with time.

7
Subsequent developments around the world have seen the use of twist of crowns,
aluminium roll on closures and plastic screw closures.

The technical merits of each are shown in the following table courtesy of Stephen Ott
at Alcoa.
5

4
Turbidity [EBC]

0
glass bottle

Interior coating I,

Interior coating II,

Interior coating I,

Exterior Coating,

scavenger crown
PET/Scavenger,
scavenger/barrier

Std. screw cap

Std. screw cap

Std. crown

Multilayer
screw cap

Figure 7: Analytical Changes III: Turbidity after 6

months

Developments continue on closures, I believe that the choice of closure will ultimately
be a major factor in the consumer acceptance of plastic as a beer package. The closure
will enable us to provide benefits in terms of ease of use, recloseability and sharing
which cannot be achieved with current packages.

TECHNICAL SUMMARY
The testing work carried out has shown the following performance characteristics of
plastic beer packages:

Permeation Rates of oxygen

Multilayer PET with scavenger < Coating < Multilayer PET/PA < Monolayer PET

Permeation rates of carbon dioxide

Coating < Multilayer PET/PA < Multilayer PET with scavenger

Beer Quality
Coating < Multilayer PET/PA, PET with scavenger, PET/EVOH, PEN <
Monolayer PET

Scavenger/barrier closure perform better than standard closures.

CONSUMER
I opened this paper with the question Why?

8
The technical community have developed a first class package, and have
demonstrated to us that our products are protected satisfactorily, albeit with reduced
shelf life when compared with glass.

I will now summarise the work done by us at Bass Brewers and the work shown in
Oslo by Ludovic Auvray from Heineken.

We are seeing that consumers initial reaction is that plastic is wrong for beer, it taints
products (we can prove this is incorrect), it is cheap and downgrades the product, it is
for soft drinks. However some do see a positive modern picture and welcome the
opportunity to see packs in more contemporary shapes.

When confronted with the plastic pack for the first time some of the misconceptions
start to break down. The light weight, the strength and reseal ability are soon
recognised. As suppliers of packaged products we need to address and demonstrate
that we can enhance these functional benefits by addressing the emotional concerns.
The concerns need to be addressed and turned into benefits.

Brands that chose to adopt plastic as a packaging medium must build on its benefits,
create structures and identify usage occasions that will encourage new consumers to
try their product because of it attractiveness.

So to close I would ask that the commercial sides of our business now rise to the
challenge of plastic as a new packaging medium rather than a glass or metal
replacement and pick up the work done by their technical colleagues.

9
96

Validation of PET-bottles for the filling

of beer
Christian Drr & Horst Weisser
TU Mnchen, Lehrstuhl fr Brauereianlagen und Lebensmittel-Verpackungstechnik,
Weihenstephaner Steig 22, D-85350 Freising-Weihenstephan, Germany (e-mail:
[email protected])

Descriptors
Beer quality, permeability, plastic bottle, polyethylene terephthalate, shelf life

SUMMARY
Due to different advantages of plastic bottles compared with glass bottles the brewing
industry intend to fill beer into PET bottles. The correlated problems were nvestigated in this
work. Enhancing the barrier properties leads to a fractional amount of oxygen permeation.
Light protection can be obtained by additives to the PET. The real oxygen permeation and the
measured permeation (using a gas-in-gas analyser) bring up differences and hence a
distinction between the accounted shelf-life and the real shelf-life. Additives to the beer are
limited able to enhance shelf-life. The impact of oxygen permeation through closure rises
with the enhancement of bottle barrier properties. A restricted shelf-life prediction is possible.

Bewertung von PET-Flaschen fr Bier

Deskriptoren
Bierqualitt, Durchlssigkeit, Haltbarkeitsdauer, Kunststoffflasche, Polythylenterephthalat

ZUSAMMENFASSUNG
Auf Grund verschiedener Vorteile von Kunststoffflaschen gegenber Glasflaschen mchten
Brauereien Bier in Kunststoffgebinde abfllen. Die damit verbundenen Probleme wurden in
dieser Arbeit untersucht. Durch Verbessern der Gasbarriereeigenschaften der Flaschen kann
der O2-Durchgang durch die Flaschenwand auf einen Bruchteil verkleinert werden. Der
Lichtschutz kann durch Zustze zum Packstoff PET gewhrleistet werden. Tatschlicher und
gemessener Sauerstoffeintritt (Gas-in-Gas-Messung) stimmen nicht miteinander berein.
Dadurch unterscheiden sich die rechnerischen von den tatschlichen Haltbarkeiten. Zusatz-
stoffe knnen die Haltbarkeit des Bieres nur bedingt verlngern. Durch die barriere-
verbessernden Manahmen steigt der Einfluss der O2-Durchlssigkeit des Verschlusses auf
die Haltbarkeit. Die Vorhersage der Produktlebensdauer ist nur bedingt mglich.
Validation des bouteilles en PET pour le conditionnement de la bire

Descripteurs
Bouteille en plastique, dure de conservation, permabilit, polyethylne terephthalate,
qualit de la bire

RESUME
En raison des avantages des bouteilles en plastique par rapport aux bouteilles en verre, la
brasserie industrielle a intrt tester la bire en bouteilles PET. Les problmes affrents ont
t tudis dans ce poster. Le renforcement des proprits de barrire conduit un niveau
fractionnel de permabilit l'oxygne. La protection contre la lumire peut tre obtenue par
des additifs au PET. La permabilit relle l'oxygne et la permabilit mesure ( l'aide
d'un analyseur gaz-gaz) montrent des diffrences et donc une distinction entre la dure de vie
thorique et la dure de vie relle. Les additifs la bire ont peu de capacits augmenter la
dure de vie. L'impact de la permabilit l'oxygne par le systme de fermeture augmente
avec le renforcement des proprits de barrire de la bouteille. Une prvision restreinte sur la
dure de stabilit est possible.

2
INTRODUCTION
Various advantages of PET-bottles are well-known in the bottling industry. Compared
to glass bottles the low density allows to reduce the packaging weight remarkably.
Furthermore the unbreakability of plastic bottles is of interest since this is a major
issue of product liability.
The remaining problem of plastic bottles arises from their gas permeation properties.
In regard to the beer production the loss of carbon dioxide and the ingress of oxygen
[5, 6] have to be named (figure 1). Filling of plastic bottles is more difficult than
filling beer into glass bottles. Due to the thin bottle walls and the geometric affinity to
glass bottles there is more headspace which causes oxygen ingress in great quantity.
Another problem, especially for filling beer, is the light permeability. Thus it is a
challenge to find a way of filling beer into plastic bottles and guaranteeing shelf-life
of more than three months.
For these investigations the newest inventions of plastic bottles were tested. In this
project various solutions of enhancing the gas barrier properties of plastic bottles were
compared. We worked with different test methods to measure CO2-loss and to
measure the oxygen permeation into the bottles and through the closures. The
measurement of the light transmission rates through the bottle walls was also studied.

partial pressure
[bar]

2 CO2

1
O2

N2

outside bottle wall inside

Figure 1: Partial pressure conditions in- and out-side the filled bottle [4]

BOTTLE TESTING
Measuring the oxygen ingress into the bottles was carried out with a Mocon
equipment. Therefore the bottles were moulded into a form of brass (figure 2) with a
wax mixture (80 % paraffine, 20 % poly-iso-buthylene). Depending on the bottle type
the bottles were purged with carrier gas for 24120 hours. Sub-sequent the gas was
led over the electro-chemical sensor to determine the content of oxygen.
Measuring the CO2-loss was carried out with different test methods. First the testing
was performed with a Permatran C4/40 by Mocon. The bottles were put into a special
fixture and then the gas-in-gas permeation was detected. The second used method was
the measurement of the CO2-content of bottled beer (Blom and Lund, EBC-method)

3
for a period of two months.
The light transmission was tested with an UVIKON Spectral Photometer.

Tube

Gastubes
Gastight connexions
Carrier gas
To the sensor

Figure 2: Bottle fixture for Mocon OxTran

CLOSURE TESTING
Measuring the oxygen ingress through the closures was also done with Mocon
equipment. The closures were mounted on a special fixture of brass. In difference to
the analysis regulation [2] the relative humidity of the purging gas was set to 95
% r. h. Thus the O2-permeation of closures with oxygen scavenging function could be
measured.

TESTING ADDITIVES FOR BEER

Some additives to protect beer from oxidation are well known for beer filled into glass
bottles. The influence of a large quantity of oxygen, as it is given when beer is filled
into plastic bottles, to the additives and their influences on flavour stability and aging
of beer is not known yet. Storing trials were performed by analysing ageing
components and making tasting trials. PEN-bottles (weight 36 g) and closures with
barrier liner and oxygen scavenging function were used for these trials.

Sulphite
Dipotassium disulphite is given in crystal form. As a solution in water sulphurous acid
arises (eq. 1).
K2S2O5 + H2O
2 SO32- + 2 K+ + 2 H+ (1)

Sulphite reacts with oxygen to sulphate (eq. 2). This is the reason for the reducing
character of dipotassium disulphite.
SO32- + 1/2 O2
SO42- (2)

1 mol of dipotassium disulphite is equivalent to 2 mol sulphite. 2 mol sulphite bind 1

mol of elementary oxygen. Due to this reducing ability the use of dipotassium
disulphite can improve the taste stability. For these trials a dosage of 15 mg SO2 per

4
litre beer was used. The content of SO2 in beer was about 1,5-2,5 times larger than the
natural content.

Ferulic acid
Ferulic acid represents one of the most powerful antioxidants [7, 8]. It is well known
as a precursor of 4-vinyl-guaiacol. The R1 in the structure equation (figure 3) means
hydrogen (H), the R2 stands for the methyl ether (OCH3) [1]. The reducing parts of the
molecule are the enol group and the carboxyl group. For these trials a concentration of
10 mg ferulic acid per litre beer was used.

Figure 3: Structural formula of ferulic acid

(+)-Catechin
Catechins are so-called flavan-3-ols. Catechins can be used as antioxidants. The used
(+)-catechin is the simplest representative of the group of the flavanols. In the
structural formula given in figure 4 the R and the R1 stand for hydrogen (H). The
reduction potential of (+)-catechin is given by the dienol group [1]. For the trials a
dosage of 10 mg (+)-catechin per litre beer was used [7, 8].

Figure 4: Structural formula of (+)-catechine

SHELF-LIFE TESTS
Shelf-life tests were conducted with different types of beer and different bottle types.
A part of these trials were storage tests under varying environmental conditions such
as temperature and partial oxygen pressure. For these trials PEN-bottles with a weight
of 45 g were used. They were closed with standard plastic closures.

5
RESULTS
Bottle testing
The results of the different tested bottles are shown in figure 5. The permeation rates
of PET-bottles, PEN-bottles, multilayer-bottles, coated bottles and bottles with a
integrated oxygen scavenging function are presented. The permeation rates were
measured with Mocon OxTran.

Figure 5: Permeation rates of different bottle types (volume 0,5 l)

Figure 6: Permeation rates of different closures

6
Testing of closures
Figure 6 shows the results of the measurements of different closures. We tested
standard plastic closures, plastic closures with barrier liner (EVA) and closures with
an oxygen scavenging function as well.

Loss of CO2
In figure 7 the content of carbon dioxide in beer stored in PEN-bottles is shown versus
a storage time of 8 weeks. The blue line shows the results of bottles which were stored
at 23 C and the black line shows the results of bottles which were stored at only 6 C.

Figure 7: Contents of CO2 in at different temperatures stored beer (blue 23 C, black

6C)

Sulphite
Figures 8-9 show the findings of the taste trials (ageing taste trials according to
Eichhorn [3]) and the analyses of ageing components.
Ferulic acid and (+)-catechin
Figures 8-9 show also the findings of the taste trials (ageing taste trials according to
Eichhorn [3]) and the analyses of ageing components.

Ferulic acid and (+)-catechin

Figures 8-9 show also the findings of the taste trials (ageing taste trials according to
Eichhorn [3]) and the analyses of ageing components.

7
 Figure 8: Ratings of taste trials according to Eichhorn [3]

Figure 9: Sum of ageing components in beer with different antioxidants

Shelf-life tests
Figures 10-11 show the results of the shelf- life tests under varying conditions. A part
of the probes was stored in an atmosphere of pure oxygen to force the oxygen ingress
into the bottle. The other samples were stored under standard conditions (normal
atmosphere). Aim of this investigation was to find a short term test for predicting
shelf life of beer bottled in plastic.

8
 Figure 10: Ratings of taste trials according to Eichhorn [3]

Figure 11: Sum of ageing components in beer



9
CONCLUSIONS
By enhancing the barrier properties plastic bottles are approximately as gas tight as
glass-bottles. These solutions are only available for disposable bottles. On the other
hand filling beer in reusable bottles basically results in a larger amount of oxygen.
A possibility to enhance shelf-life of beer could be the use of antioxidants.
Using sulphite as antioxidant led to a larger amount of ageing components.
After a storing time of 12 weeks the tasting trials show nearly the same
ratings as for beer stored in glass bottles.
Using (+)-catechin and ferulic acid led to higher amounts of ageing
components, too.
The rating of the ageing taste trials showed significantly better values than
for glass bottles.
There seems to be a masquerade effect on the age off flavour by the used
antioxidants.
The loss of CO2 does not restrict shelf-life even in returnable bottles.
There is only a loss of 9 % CO2 in bottles stored at 6 C and a loss of 15 %
CO2 in bottles stored at 23 C within half a year.
The loss of CO2 can be compensated with a more intensive carbonisation
before filling.
The comparison of beer stored in oxygen atmosphere and in normal atmosphere
does not show any correlation between oxygen concentration and ageing of the
beer.
Installing a simple test method to predict shelf-life of beer stored in plastic
bottles is not possible with enhancing oxygen concentration and temperature.

REFERENCES
[1] Belitz, H.-D., Grosch, W., Lehrbuch der Lebensmittelchemie, 4. Aufl., Berlin,
Springer, 1992
[2] DIN 53 380 Teil 3 07.89 (Entwurf): Bestimmung der Gasdurchlssigkeit
Sauerstoffspezifisches Trgergasverfahren zur Messung an Kunststofffolien und
Kunststoffformteilen
[3] Eichhorn, P., Untersuchungen zur Geschmacksstabilitt des Bieres, Mnchen,
T.U., Fak. fr Brauwesen, Lebensmitteltechnologie und Milchwissenschaft,
Diss., 1991
[4] Hertlein, J., Bornarova, K., Weisser, H., Eignung von Kunststoffflaschen fr die
Bierabfllung, Brauwelt, 137 (1997), Nr. 21-22, 860-866 and Brauwelt
International (1997), No. III, 204216
[5] Heyse, K.-U. (Red.): Stabilitt des Bieres vom Malz bis in die Flasche,
Brauwelt, 137 (1997), Nr. 38, 16701672
[6] Narzi, L., Abri der Bierbrauerei. 6. Aufl., Stuttgart, Enke, 1995
[7] Walters, M.T. et al, Comparison of (+)-Catechin and Ferulic Acid as Natural
Antioxidants and their Impact on Beer Flavor Stability, Part 1: Forced-Aging, J.
Am. Soc. Brew. Chem., 55 (1997), No. 2, 8389
[8] Walters, M.T. et al, Comparison of (+)-Catechin and Ferulic Acid as Natural
Antioxidants and their Impact on Beer Flavor Stability, Part 2: Extended Storage
Trials, J. Am. Soc. Brew. Chem., 55 (1997), No. 3, 9198

10
97

Development of a standardised stained

beer bottle for testing bottle washers
Karl Wackerbauer, Hartmut Evers & Knut Soltau
VLB Berlin, Seestrasse 13, D-13353 Berlin, Germany (e-mail: [email protected])

Descriptors
Bottle washing, efficiency, evaluation, standardisation

SUMMARY
The poster treats the development of a standardised procedure of coating bottles with different
substances and the search for a suitable method to evaluate a substance for the use as standard
stain. The applied method is simple and inexpensive. No special equipment is needed and the
fixed substances which are used exist in every brewery. Standardised stained bottles provide
an objective evaluation for bottle washers without interrupting the running process. This can
help to decrease the consumption of cleaning agents and water and to save energy.

Ausarbeitung eines Verschmutzungsstandards fr Bierflaschen zur berprfung von

Reinigungsmaschinen

Deskriptoren
Beurteilung, Flaschenreinigung, Standardisierung, Wirkungsgrad

ZUSAMMENFASSUNG
In dem Poster wird eine standardisierte Methode zur knstlichen Verschmutzung von
Bierflaschen vorgestellt, mit welcher die Effektivitt von Flaschenreinigungsmaschinen
berprft werden kann. Die angewandte Methode ist einfach durchzufhren und preiswert.
Das zum "Coating" notwendige Equipment ist ebenso in jedem normalen Brauereilabor
vorhanden wie auch die verwendeten fixierten Substanzen. Der Einsatz standard-
verschmutzter Flaschen erlaubt eine objektive Beurteilung der Flaschenreinigungsmaschine
bei laufendem Betrieb. Die Auswertung ist einfach und schnell durchzufhren. Damit ist der
Betriebskontrolle ein Instrument an die Hand gegeben, den Verbrauch an Wasser,
Reinigungsmitteln wie auch an Energie zu reduzieren.
Dveloppement d'une bouteille de bire teinte standardise pour tester les lavages

Descripteurs
Efficacit, valuation, lavage de bouteilles, standardisation

RESUME
Le poster traite du dveloppement d'un procd standardis de revtement des flacons par
diffrentes substances et de la recherche d'une mthode approprie pour valuer une substance
utilisable comme teinte standard. La mthode utilise est simple et peu coteuse. Elle ne
requiert aucun matriel particulier et les substances fixes qui sont utilises existent dans
toute brasserie. Les bouteilles teintes standardises apportent une valuation objective des
lavages sans interrompre le processus en cours. Cela peut aider diminuer la consommation
d'agents nettoyants et d'eau et conomiser de l'nergie.

2
INTRODUCTION
Yet there is no objective means of evaluating the cleaning effect of bottle washers.
Standardised stained bottles provide an objective evaluation for bottle washers. The
results of evaluation can help to decrease the consumption of cleaning agents and
water and to save energy.
The poster treats the development of a standardised procedure of coating bottles with
different substances, the search for suitable methods to estimate a substance for the
use as standard stain and a visual evaluation method for industrially cleaned bottles.

Aims of this work

To develop a test bottle with a standard stain showing a correlation between the
cleaning effect on the bottle and the efficiency of the bottle washer.
To find a simple and inexpensive method to produce standardised stained bottles.
To detect malfunctions in bottle washers without interrupting the running process.

MATERIALS AND METHODS

Kieselguhr
Starch

Preparation of bottles
- Mix 10 % kieselguhr, 18 % starch with 72 % water.
- Stirr for 5 minutes.
- Fill 10 g in each bottle.
- Coat the inner bottle surface by rolling on level ground.
- Dry for 3 hours at 90 C.
- Cool down to room temperature.
The bottles are stable for at least four weeks.

RESULTS
To find suitable substances for the test bottles a cleaning simulation in laboratory
scale was developed. The cleaning process consists of two caustic soaks and a rinsing
with deionised water after every steep. The estimation of the cleaning effect is done
by scaling before and after cleaning, haze measurement, pH-control, titration of the
NaOH- and soda-content in the caustic and by visual evaluation of the residues in the
bottles. This method was applied to kieselguhr
starch, yeast, spent grains, glass colour and hop extracts. A mixture of 10 %
kieselguhr, 18 % starch and 72 % water gave the best results.

3
 Figure 1: Different stains of kieselguhr, starch
and water after cleaning

Evaluation method
An evaluation grid is applied to the cleaned bottle (figure 2), dividing its surface into
segments. The bottom is also treated as segmentated, giving 30 segments per bottle.

Figure 2: A: Evaluation grid; B: Naming of segments for evaluation

Each segment is evaluated in opposite light concerning residue spreading and residue
thickness using the following scheme:

Spreading: Thickness:
0 = none 0 = no residues
1 = 1 - 20 % 1 = less than before
2 = 21 - 40 % 2 = like before cleaning
3 = 41 - 60 % 3 = thicker than before
4 = 61 - 80 %
5 = 81 -100 %

4
 Flchenbelegung 5
A B C D E
4
Mndung 0 0 0 0 0 0
3
Hals 0 0 1 0 0 1
2
Schulter 0 0 2 1 0 3
Zylinder 1 0 0 4 2 0 6 1
Zylinder 2 0 0 1 0 0 1 0
Boden 0 0 0 0 0 0 E

ng
D

Sc Hals
du
C

r
lte
n

r1
0 0 8 3 0 11 B

hu
M

de

r2
A

lin

de

n
de
Zy

lin

Bo
Zy
Flchenbelegung

Schichtdicke 3
A B C D E
Mndung 0 0 0 0 0 0 2
Hals 0 0 2 0 0 2
Schulter 0 0 2 2 0 4 1
Zylinder 1 0 0 3 3 0 6
Zylinder 2 0 0 0 0 0 0 0
Boden 0 0 0 0 0 0 E

ng
D

s
du

S Hal
C

er
n

t
r1
B

ul
0 0 7 5 0 12

ch

de

r2
A

lin

de

n
de
Zy

lin

Bo
Zy
Schichtdicke

Figure 3: Example for a visual evaluation of a cleaned bottle

The graphics (figure 3) give a 3-dimensional picture of the residues in the bottle.

CONCLUSIONS
It is possible to produce standardised stained bottles in a simple and inexpensive way.
The cleaning behaviour of the stain correlates with the cleaning efficiency of bottle
washers. For the future it is necessary to collect data with this means of testing, so that
conclusions from specific residue spreadings to certain specifications of bottle
washers can be drawn.
98

Seeing the light using innovative

instrumental analysis to assess new
packaging
Peter G. Hill1, Stefan Lustig1 & Hans-Jrgen Barklage2
1
Brauerei Beck & Co, Am Deich 18/19, D-28199 Bremen, Germany (e-mail: [email protected])
2
Nienburger Glas, Gr. Drakenburger Strasse 132, D-31582 Nienburg, Germany

Descriptors
Bottle colour, gas chromatography, lightstruck flavour, ultra-violet radiation

SUMMARY
A clear glass bottle was considered by marketing to be the packaging of choice for a new light
beer. Clear glass, however, provides no protection against UV-light, resulting in the formation
of the lightstruck off-flavour when the beer is subjected to light. This is particularly a problem
in beers brewed according to the German Reinheitsgebot, as no modified hop products may
be used. Consequently, a clear glass bottle with built-in UV-protection was developed and its
protective properties assessed using a highly sensitive novel method of sulphur analysis,
SPME-GC-PFPD. The light beer in its innovative new packaging was subsequently
successfully launched.

Ein Glasklarer Fall die Beurteilung eine neue Verpackung mittels innovativer
instrumenteller Analytik

Deskriptoren
Flaschenfarbe, Gaschromatographie, Lichtgeschmack, ultraviolette Strahlung

ZUSAMMENFASSUNG
Ein Weiglasflasche wurde aus Marketingsicht als die beste Verpackung fr ein neues
Lightbier-Produkt empfunden. Klarglas schtzt das Bier allerdings nicht vor UV-Licht, mit
der Folge der Bildung eines Lichtgeschmacks wenn Bier Lichteinflssen ausgesetzt wird.
Dieses Problem ist besonders bei Reinheitsgebot-Bieren akut, da keine modifizierten Hopfen-
produkte eingesetzt werden drfen. Deshalb wurde eine Klarglasflasche mit eingebautem UV-
Schutz entwickelt und mittels einer neuartigen Methode der empfindlichen Schwefelanalytik,
SPME-GC-PFPD, auf ihre Schutzqualitten untersucht. Das Lightbier in dieser innovativen
Verpackung wurde erfolgreich im Markt eingefhrt.
Voir la lumiere utilisation de mthodes innovantes d'analyse instrumentale pour
valuer un nouveau conditionnement

Descripteurs
Chromatographie en phase gaseuze, couleur de bouteille, got de lumire, radiation
ultraviolette

RESUME
Une bouteille en verre transparent a t envisage par le service marketing comme
l'emballage de choix pour une nouvelle bire lgre Toutefois, le verre transparent ne protge
pas contre le rayonnement UV, ce qui entrane la formation d'arme photognr lorsque la
bire est soumise la lumire. C'est notamment un problme pour les bires brasses selon la
rgle de puret allemande (Reinheitsgebot), car elle interdit toute utilisation de produits de
houblon modifis. En consquence, nous avons mis au point une bouteille en verre
transparent incorporant une protection anti-UV; ces proprits protectrices ont t values
l'aide d'une nouvelle mthode extrmement sensible d'analyse du souffre, la SPME-GC-
PFPD. La bire lgre dans son nouvel emballage innovant a donc t lance avec succs.

2
INTRODUCTION
Brauerei Beck & Co recently developed a new product - a light beer - for the
American market. For marketing reasons a clear glass bottle was considered to be the
packaging of choice. Clear glass, however, provides no protection against UV-light.
Consequently, light in the spectral range between 350500 nm (1) can, in the presence
of a suitable photosensitiser such as riboflavin, cause the photo-degradation of iso--
acids. As a result the 3-methyl-2-butenyl radical intermediate is formed, which can
then react further with sulphur donors such as hydrogen sulphide or the amino acid
cysteine to form 3-methyl-2-butene-1-thiol (3-MBT). 3-MBT is the compound held
primarily responsible for the objectionable skunky flavour of beer which has been
exposed to light.

It is possible to make beers light stable by using modified hop products such as rho or
tetra iso--acids. These reduced iso--acids, however, may not be used in beers
brewed in accordance with the German Reinheitsgebot, as is the case with all Beck &
Co products. Therefore an alternative method of protection against light influences
was investigated: the development of a clear glass bottle with a built-in UV-barrier.

The new clear glass bottle with a built-in UV-barrier was developed by Nienburger
Glas, Germany, a subsidiary of Beck & Co. The UV-protection was achieved by
adding a small quantity of vanadium to the glass. However, it is very difficult to keep
the vanadium stable in the required oxidation state V, as this is dependent on the
carbon content of all raw materials. Nienburger Glas accomplished this by employing
a patented system which keeps the carbon levels low and stable and the vanadium in
oxidation state V.

The main problem was the evaluation of the amount of UV-protection afforded by the
new clear glass bottle. Sensory analysis of the lightstruck flavour is notoriously
difficult: the nose quickly becomes saturated by the pungent skunky aroma, making
objective tasting unfeasible. A recently-developed sensitive method of sulphur
analysis using a combination of solid phase microextraction and gas chromatography
with pulsed flame photometric detection (SPME-GC-PFPD) (2) was therefore
employed to determine the concentration of 3-MBT in illuminated samples.

MATERIALS AND METHODS

Illumination
Samples (without labels) were exposed to light under defined conditions using a
purpose-built illumination system: the bottles were rotated slowly and light was shone
from a set distance using special day-light lamps.
Analysis
75 m Carboxen/PDMS SPME fibres were used, a Varian 8200 CX Autosampler
with SPME III agitation modifications and a heated carousel being used for SPME
sampling and injection. The GC analyses were carried out using a Varian 3800 GC
equipped with a 1079 split/splitless injector and a pulsed flame photometric detector.
A specially-designed 0.8 mm SPME injector liner was used to prevent peak-
broadening during injection. The chromatographic separation was carried out using a
combined polar/non-polar capillary column: a non-polar VA-1 column (100% DMPS)
(60 m x 0.25 mm x 0.5 m) was preceded by a short piece of DB-Wax column (PEG)
(10 m x 0.25 mm x 0.5 m).

3
9 ml of beer, cooled to 0C to prevent the loss of volatile compounds, was pipetted
into a 15 ml glass vial and internal standards were added. The vials were warmed to
45C in the autosampler and the SPME fibre subsequently exposed to the SPME fibre
for 32 minutes. Following extraction the fibre was desorbed in the GC injector for 3
minutes at 250C in the splitless mode. The split was turned on after 0.8 minutes. A
hydrogen carrier gas flow of 2.7 ml/min was maintained throughout the run. The
column oven was held at 32C for 7 minutes, increased to 110C at 7C/min,
increased to 190C at 11/min, increased to 235C at 22C/min and held for 6
minutes.

RESULTS
Transmission
A measurement of the transmission properties of the new clear glass bottle with a
built-in UV-barrier showed that the transmission of UV-light in the wavelength range
300 400 nm was significantly reduced in comparison to a standard green glass
bottle. In comparison to a standard clear glass bottle, transmission was reduced across
the whole measured spectrum from 300 800 nm (figure 1).

normal clear glass

UV-barrier clear
glass

green glass

Figure 1: Transmission curves for normal clear glass, clear glass with a built-in UV-
barrier and green glass bottles
Analyses
The beer samples were removed from the illumination apparatus at regular intervals
and analysed. The extreme sensitivity of the SPME-GC-PFPD system allowed 3-MBT
to be detected after only one hour of illumination. Even at this early point the
increased UV-protection of the clear glass bottle with a built-in UV-barrier was
clearly evident.

4
Excerpts of SPME-GC-PFPD chromatograms of beer bottled in normal clear glass,
green glass and UV-barrier clear glass and illuminated for 8 hours can be seen in
Figures 2, 3 and 4 respectively.

Figure 2: Excerpt of an SPME-GC-PFPD chromatogram of beer bottled in normal

clear glass and illuminated for 8 hours

Figure 3: Excerpt of an SPME-GC-PFPD chromatogram of beer bottled in green

glass and illuminated for 8 hours

Figure 4: Excerpt of an SPME-GC-PFPD chromatogram of beer bottled in UV-

barrier clear glass and illuminated for 8 hours.

5
From the three chromatograms it can be seen that smallest 3-MBT peak is seen in the
UV-barrier clear glass bottle sample, the largest 3-MBT peak being seen in the clear
glass bottle sample. No 3-MBT was determined in any of the non-illuminated control
samples.

The results of the SPME-GC-PFPD analyses are summarised in table 1 below.

Illumination Time
1 hour 4 hours 9 hours
Standard green glass 0.048 0.124 0.171
UV-Barrier clear glass 0.020 0.052 0.076

Table 1: Concentrations of 3-MBT (g/l) in illuminated beer samples in different

glass bottles

CONCLUSIONS
The results of the transmission measurements and the SPME-GC-PFPD analyses
allow the following conclusions to be drawn:

The UV-barrier clear glass bottle from Nienburger Glas provided greater UV-
protection than either a normal clear glass bottle or a green bottle. The desired UV-
protection was achieved through technological measures to stabilise vanadium in the
oxidation state V during production of the glass. This technology provides a viable
alternative to the use of modified hop products to prevent lightstruck formation, of
particular interest to breweries brewing in accordance with the Reinheitsgebot.

The recently-developed SPME-GC-PFPD method allows the simple, inexpensive and

extremely sensitive analysis of sulphur compounds, including 3-MBT. This method
can be routinely employed as an alternative to the difficult and unreliable sensory
analysis of the lightstruck flavour in beer.

On the basis of the results presented, the range of wavelengths responsible for the
formation of the lightstruck flavour in beer can be narrowed down considerably. The
SPME-GC-PFPD results show that the UV-barrier clear glass bottle provides more
UV-protection than the green glass bottle, which in turn provides better protection
than a standard clear glass bottle. There is, however, only one part of the transmission
spectrum for the three glass samples where transmission for the UV-barrier glass is
lower than that for the green glass whilst at the same time the normal clear glass
transmission is highest: this is the wavelength range 380 - 410 nm. The inference is
that light in this wavelength range is, in the presence of a photosensitiser such as
riboflavin, responsible for the photo-degradation of iso--acids and the subsequent
formation of the lightstruck flavour.

REFERENCES
1. J. Templar, K. Arrigan, W.J. Simpson, Brewers Digest, 70 (1995) 18
2. P.G. Hill, R.M. Smith, J. Chromatography A, 872 (2000) 203

6
99

Standard interface between the field

and production level of filling and
packaging lines
Tobias Voigt, Thomas Rdler, & Horst Weisser

TU Mnchen, Lehrstuhl fr Brauereianlagen und Lebensmittel-Verpackungstechnik,

Weihenstephaner Steig 22, D-85350 Freising-Weihenstepan, Germany (e-mail:
[email protected])

Descriptors
Data processing, efficiency, filler, optimization

SUMMARY
The major task of the presented project in order to increase the acceptance of data acquisition
systems is to reduce the costs for the implementation and to formulate general methods for
analysing and optimising a filling-line. For this reason an interface specification between the
field and production level of filling-lines in breweries was developed and resumed in a
manual of best practice for the breweries and machine suppliers. This contains techniques for
the signal processing and coding, uniform definitions for equipment status and key figures,
new effective methods for the buffer and failure analysis and filling-reports worked out in
examples.

Standard-Schnittstelle fr die BDE-Anbindung bei Getrnkeabfllanlagen

Deskriptoren
Datenverarbeitung, Fller, Optimierung, Wirkungsgrad

ZUSAMMENFASSUNG
Das vorgestellte Projekt hat zum Ziel die Akzeptanz von Datenerfassungs-Systemen im
Abfllbereich zu erhhen, indem die Kosten fr deren Installation gesenkt und einheitliche
Methoden fr die Datenauswertung und Anlagenoptimierung festgelegt werden. Hierzu wurde
eine Standardschnittstelle fr die Verbindung der Leit- und Feldebene einer Abfllanlage
spezifiziert und in einem sowohl von Brauereien als auch Maschinenherstellern zu nutzendes
Standardpflichtenheft zusammengefasst. Dieses beschreibt die Techniken zur Signal-
kodierung und -verarbeitung. Es liefert einheitliche Definitionen fr Maschinen- und
Anlagenzustnde sowie Kennzahlen. Darber hinaus sind neue Methoden zur effektiven
Pufferbeurteilung und Schwachstellenanalyse sowie Empfehlungen fr die Gestaltung des
technische Abfllberichtswesens mit Beispielberichten enthalten.
Interface standard entre le niveau de la production et celui du terrain pour les lignes
dembouteillage et demballage

Descripteurs
Efficacit, optimisation, soutireuse, traitement des donnes

RESUME
La tche majeure du projet prsent est d'augmenter l'acceptation des systmes d'acquisition
de donnes et de rduire les cots de mise en uvre, ainsi que de formuler des mthodes
gnrales pour analyser et optimiser une ligne de conditionnement. Pour cette raison, une
spcification d'interface entre le terrain et la production de chanes de conditionnement en
brasseries a t dveloppe et rsume dans un manuel de meilleures pratiques pour les
brasseries et quipementiers. Cet ouvrage contient des techniques de traitement et de codage
du signal, des dfinitions uniformes pour le statut du matriel ainsi que des chiffres cls, de
nouvelles mthodes efficaces pour l'analyse des checs et des rapports de conditionnement
prsents comme exemples.

2
INTRODUCTION
Data acquisition systems for filling lines have been in operation in breweries over a
decade and the possibilities and the benefits of such systems have also been treated in
many publications, e.g. [3, 6]. Even so, in practice the acquisition systems have not
achieved the general objectives of breweries such as increasing productivity, assuring
product and process quality and dito improving bottling operations [2, 10].
One will only find a few individual and special systems realized in breweries which
have been developed at a high expense of work and money. These individual systems
are opposed to the result of a survey in the German brewing industry that in 90 % of
all breweries 90 % of the reports required about the bottling area are similar.

OBJECTIVES AND APPROACH

The initial position for standardization were several problems which occurred during
the realization of data acquisition systems, such as heterogeneous information
structures, insufficiently prepared machine controllers and an overhead of acquired
data.
The major task of the project is to reduce the cost for the implementation and to
formulate general methods for analyzing and optimizing a filling line in order to
increase the acceptance of data acquisition systems for filling lines.
Therefore templates have been worked out to support breweries in matters of data
acquisition at their filling lines. These have been summarized in a manual of good
practice that includes specifications for machine suppliers and system vendors.
Contents of the manual of good practice are:
- recommendations for the system architecture,
- specification of the standard interface for the preparation of data in the machine
controllers,
- documentation forms for the provided data sets,
- basic functions for data analysis and
- technical reports for the main requirements.
The arising data bases are necessary among the daily work in the breweries for a
variety of scientific studies in the future within the bottling area like simulation, data
mining, control strategy, maintenance activities, and benchmarking [8]. The basic
approach of our project was to consider both the basic data requirements of breweries
about their filling lines and the IT of the machine controller. Thus the project,
sponsored by the Deutscher Brauerbund, was treated by the authors in coordination
with the industrial based working party of Deutscher Brauerbund and the machine
suppliers KRONES and KHS. The main attention was focused on developing the
interface specification between the field and production level of filling lines in
breweries.

RESULTS
The IT in filling lines is shown in figure 1. At present PLCs are operating within the
machines, but the open information structure also considers the PC based technology.
Next to direct connection of machine controllers with the data acquisition server
master PLCs can be useful with larger facilities. They work as data concentrator or

3
data spooler. For the process bus Profibus FMS or FDL are cost-efficient systems
[7, 9].
The future belongs, however, to industrial Ethernet, that gets more and more popular
in production.

Connection by a Data Connection by a

Acquisition Server Master PLC
Office Network
(Ethernet, TCP/IP) Data Acquisition
Server/Data Base
Operating,
Monitoring,
Reporting DB

System Bus
(industrial Ethernet)

Data Aquisition Master PLC

DB Server/Data
Base
Process Bus
(Profibus FDL, FMS or
industrial Ethernet)

PLC
Standard
Industrial-PC Interface

Figure 1: System architecture of data acquisition systems for bottling facilities

A common difficulty in setting up a data acquisition system is retrieving information

from the machine controllers. As shown in figure 2, a lot of data is handled in a PLC-
program. The interesting data has to be transfered to a reserved data-array and has to
be updated regularly.
Subsequent data transfer leads to:
- complex program work
- unnecessary expense
- longer PLC-cycle times

Data-array
FB SB for the
PB ... ...
Standard
... DW 55 DW 45 Interface
... ... ...
... DB
OB 1
... FB DB DW 55
... ... ... DW 23
PB ... DW 23 ...
...
...
... ...
DW 67
DB
... DW 45
FB DB
... ... DW 67
... ... ...
... ...

Figure 2: Data transfer to the machine controller

Techniques for signal processing are binary coding (bit by bit) or integer coding
(coding by an integer-number), which are used for the state of the machine, the

4
program and the operating status. Double words are suitable for parameters and
measured data and long double words are required for counters which have to be
rotary counters.
Table 1: Data structure for the standard interface
Bit
category DBB ... ... ... ... ... ... ... ...
machine
semi-
state, Manual automatic
... off automatic
operating operation operation
operation
mode1
pro- Pro- Change main-
... Start up run down clean break
gram1,2,3 duction over tenance
operator Starving/
operating Operatin equipment Externa block
1,3 ... Ready inter- starving blocking
state g failure l failure -ing
vention branch line
machine
specific ...
messages4
notice of ...
5 ...
16-bit integer-word for notices of failure
failure
...
pointer6 ...
16-bit integer-word for pointer messages
program ...
...
16-bit integer-word for program steps
step
...
...
16-bit integer-word for parameter 1
para- ...
...
16-bit integer-word for parameter 2
meters
...
...

...
...
16- or 32-bit integer-word for measured value 1
measured ...
...
16- or 32-bit integer-word for measured value 2
values
...
...

...
...
16-bit low-word for counter 1
...
...
16-bit high-word for counter 1
...
counters7 ...
16-bit low-word for counter 2
...
...
16-bit high-word for counter 2
...
...
...
1 Instead of coding bit by bit also a 16-bit integer-word is applicable to code this information
2 If there is no program for cleaning, changes and maintenance service, an information for the time
account auxiliary production time (see figure 2) has to be set up. Additional programs have to be
specific for each machine.
3 In conjunction with the respective programs the operation states are necessary for the calculation of
key figures and have to be applied XOR or for every machine.
4 This position is also suitable to apply all messages (notice of failure and pointer), that are provided
by the controller Messages have to be declared with a message-type in general (notice of failure or
pointer).
5 Notices of failure should be coded as a 16-bit with priorities ascending by time of occurence. If this
cannot be realized by the machine supplier, bit by bit coding is necessary at the position for machine
specific messages.
6 If all pointers are coded bit by bit, the 16-bit integer-word can be resigned.
7 For the calculation of operating figures the counters of the produced goods are indispensable.

5
Uniform definitions of the above described states and data were summarized in a
standardized data structure for each machine (table 1).
A detailed documentation of the provided data is necessary for the parameterization of
an acquisition server. Digital tables, as appended to the manual of good practice, are
recommended for this.

Table 2: Getting operating times from measured data [4]

effective runtime Equipment failure period external failure period auxiliary production time
production + Production + equipment production + ready or clean, change over,
operating failure external failure or maintenance, break
generall runtime starving or blocking
operation time
production
working time

The well known key times of filling lines [4] are defined by the measured data in
table 2 (italics illustrate the corresponding data of table 1.). The calculation of the
automatically calculable operating figures is specified as followed:

- efficiency of a machine E (according to DIN 8782):

ratio of effective runtime and overall runtime
effektive runtime Q eff E effective runtime
E = = =
general runtime Q estE effective runtime + equipment failur period

- supply rate of a filling-line A (according to DIN 8782):

ratio of effective output and nominal output
filled quantity
effective output Q eff A general runtime
A = = =
no min al output Q n A no min al output

- automatically calculable supply rate of a filling-line (according to the manual of

good practice):
ratio of effective output# and nominal output
filled quantity
# #
effective output Qeff A operation time
#A = = =
no min al output Qn A no min al output

New effective methods for the buffer and failure analysis of filling lines including the
failure tracking were completed in the manual to give the IT-Industry an outline of
requirements for practical and well designed analysis tools.
Recommendable features for the data analysis at filling lines are [1, 5]:
- line visualization
- histograms and trend charts
- tools that support the analysis of critical points
- data acquisition with reference stipend to charges and shifts
- operating figures and times
- preventive maintenance.

6
The data requirements of breweries can be resumed in different reports like batch-,
shift-, article-, filling-, machine-, and QM-report, which were worked out in examples
and appended to the manual of good practice.

CONCLUSION
With this specification a standardized interface for the bottling area is available for the
first time, which drastically reduces the engineering cost for the implementation of
data acquisition systems. This interface are the bridges for the gap between the field
level and the information and monitoring level and also existing ERP- or IT-Systems.
Now the breweries are given the chance to choose the optimal bottling line from the
technical point of view and an optimal data acquisition tool from the IT point of view.
From now on the machine suppliers KHS and KRONES will configure their machine
PLCs according to the developed standard. Others will follow.

REFERENCES
[1] Bechmann, F., Kehl, H., Rdler, Th., Weisser, H., Computer Aided
Techniques in Breweries, Zoeterwoude: European Brewery Convention
Technology & Engineering Forum, Report, 1998.
[2] Bechmann, F., Kehl, H., Weisser, H., Betriebsdatenerfassung in der Brau- und
Getrnkeindustrie, Brauwelt, 136 (1996), Nr. 3, 76-78.
[3] Cavalli, R., Monitoring and Information System Applied to the Bottling Area.
In: EBC (Ed.): Proceedings of the 24th Congress in Oslo (1993), Oxford:
Oxford University Press, 577-585.
[4] DIN 8782: Getrnke-Abflltechnik Begriffe fr Abfllanlagen und einzelne
Aggregate, Berlin: Beuth, 1984.
[5] Hrte, F.L., Efficiency analysis of packaging lines, Delft: University of
Technology Delft, Department of Mathematics and Computer Science,
Statistics, Stochastics and Operation Research Unit, MSc-thesis, 1997.
[6] Knudsen, J.W., High Level Information Technology in Bottling. In: EBC
(Ed.): Proceedings of the 25th Congress in Bruxelles (1995), Oxford: Oxford
University Press, 457-466.
[7] Polke, M. (Hrsg.): Prozessleittechnik. 2. Auflage, Mnchen und Wien:
Oldenburg, 1994.
[8] Rdler, T., Modellierung und Simulation von Abfllinien. Freising-
Weihenstephan: TU Mnchen, Lehrstuhl fr Brauereianlagen und
Lebensmittel-Verpackungstechnik, Dissertation, 1999.
[9] Schnell, G. (Hrsg.), Bussysteme in der Automatisierungstechnik.
Braunschweig und Wiesbaden, 1993.
[10] Weisser, H., Betriebsdatenerfassung in der Getrnkeindustrie, Flssiges Obst,
64 (1997), Nr. 9, 502-506.

7
100

Heineken Filled Bottle Inspector (FBI) -

from pilot to operation
B.C.J. LANDMAN
Heineken Technical Services B.V., P.O. Box 510, 2380 BB Zoeterwoude, the Netherlands (e-
mail: [email protected])

Descriptors
Efficiency, filled bottle inspector, high-capacity equipment

SUMMARY
Heineken has been developing an inspection device to detect foreign particles including glass
fragments with very high detection efficiencies at filling line speed of more than 40.000 BPH.
A pilot version of the Heineken FBI has been tested at the brewery in Zoeterwoude and
refinements have been made to various parts of the system. Because of the succesful results of
the Pilot, two FBIs are on order now to implement into a new packaging line of Heineken.
The Poster will give an overview of objectives, requirements and operating principle of the
FBI; results/ experiences based on the on line testing in the Zoeterwoude brewery; special
features in the industrialized FBIs in the new line and economic aspects.

Heineken Filled Bottle Inspector (FBI) von der Pilotanlage bis zum Betrieb

Deskriptoren
Hochleistungsanlage, Vollgutausleuchter, Wirkungsgrad

ZUSAMMENFASSUNG
Heineken entwickelte eine Inspektionseinheit zur Detektion von Fremdpartikeln,
einschlielich Glasfragmenten, mit sehr hoher Effizienz, bei einer Leistung von mehr als 40
000 Flaschen pro Stunde. Eine Pilotversion des Heineken FBI wurde in der Brauerei in
Zoeterwoude getestet und dort in vielen Bereichen verfeinert. Nach den erfolgreichen
Ergebnissen der Pilotanlage wurden zwei dieser Anlagen bestellt, um sie in eine
Verpackungslinie von Heineken einzubauen. Dieses Poster gibt einen berblick ber die
Ziele, Anforderungen und Wirkungsweise des FBI; Ergebnisse/Erfahrungen des Online-Tests
in Zoeterwoude; spezielle Merkmale des industriellen Einsatzes und konomische Aspekte.
Contrle du remplissage des bouteilles (FBI) de Heineken "du pilote l'oprationnel"

Descripteurs
Efficacit, installation haute capacit, mireuse de bouteilles pleines

RESUME
Heineken a mis au point un systme de contrle permettant de dtecter les particules
trangres, y compris les fragments de verre, avec une trs haut pouvoir de dtection sur une
ligne de conditionnement de plus de 40.000 bouteilles/heure. Une version pilote du FBI de
Heineken a t teste la brasserie de Zoeterwoude et des amliorations ont t apportes aux
diffrentes parties du systme. En raison des excellents rsultats du pilote, deux systmes FBI
vont bientt tre mis en uvre dans une nouvelle ligne de conditionnement de Heineken. Le
poster donne une vue d'ensemble des objectifs, exigences et principes opratoires du FBI; des
expriences/rsultats bass sur les tests en ligne de l'usine de Zoeterwoude; des
caractristiques spciales des FBI industrialiss de la nouvelle ligne et ses aspects
conomiques.

2
101

Flexible packaging strategies

Knud-Erik Pedersen
Danbrew ltd. A/S, Rahbeks All 21, DK-1801 Copenhagen-Frederiksberg C, Denmark (e-
mail: [email protected])

Descriptors
Bottling line, engineering, finance, multipack, packaging material

SUMMARY
The prevailing trends towards multipacks influence all links of the supply chain, to buy, to
make, to store, to move and to sell. The presentation starts with an overview of the different
pack types that are currently available in to-days competitive market.
Hereafter, moving upstream from sales, one of the most capital-binding parts of the supply
chain emerges: The packaging department. The major technical and economical issues with
dedicated lines for returnable and non-returnable bottles versus one multi-purpose line will be
addressed. Key parameters as layout, capacity, investment, floorage and operating cost
including manning will be compared.

Flexible Verpackungsstrategien

Deskriptoren
Finanzierung, Flaschenflllinie, "Multipack, Technik, Verpackungsmaterial

ZUSAMMENFASSUNG
Der andauernde Trend hin zu Multipack-Verpackungen beeinflusst alle Schnittstellen in der
Versorgungskette, vom Einkauf ber Herstellung, Lagerung, Transport bis zum Verkauf. Die
Prsentation wird anfangs eine bersicht geben ber die heute auf dem Markt befindlichen
Umverpackungen. Vom Verkauf zurck zur Herstellung wird einer der grten kapital-
indenden Bereiche aufgedeckt: die Verpackungsabteilung. Die Hauptfaktoren hinsichtlich
technischer und wirtschaftlicher Aspekte mit spezifischen Linien fr Mehrweg- und
Einwegflaschen gegenber Vielzwecklinien wird angesprochen. Schlsselparameter wie
Auslegung, Kapazitt, Investition, Grundflche und Betriebskosten inklusive Personalkosten
werden verglichen.
Les stratgies d'emballage flexible

Descripteurs
Gestion financire, groupe d'embouteillage, ingnirie, matriau d'emballage, multipack

RESUME
La tendance actuelle aux multipacks influence tous les maillons de la chane de distribution:
achat, fabrication, stockage, transport et vente. Notre expos commence par une revue
gnrale des diffrents types de packs actuellement proposs aux acteurs du march.
Ensuite, en amont de la vente, apparat l'une des pices les plus immobilisatrices de capital de
la chane de distribution: le dpartement Conditionnement. Nous aborderons les principales
questions techniques et conomiques, en considrant particulirement les bouteilles
consignes et non consignes par opposition aux chanes multi-usages. Nous comparerons les
paramtres essentiels, tels que la prsentation, la capacit, l'investissement, la surface au sol
ainsi que les cots opratoires incorporant le personnel.

2
INTRODUCTION
If you study the European brewery map you will find that nearly 90 percent of all
breweries have annual sales less than one million hl per year. Question number 1 for
many of those breweries is: What would be the optimal solution in connection with
investment in new packaging facilities or upgrading of existing systems? One medium
speed, multi purpose combi line or a number of dedicated, but in turn changeable
packaging lines.
In order to answer this question, capital and operating expenditures for a medium
scale one million hl European brewery have been analysed.
Two different scenarios with real values from practice have therefore been
established.
Scenario 1:
One dedicated line at low speed for returnable bottles, glass and PEN in plastic
crates
One dedicated line at low speed for non returnable bottles
One repack line for crates and display trays with or without toppack
Scenario 2:
One multipurpose combi line at medium speed for returnable and non-returnable
bottles in all kinds of secondary and tertiary packaging

Packaging cost factors

The cost factors as illustrated in figure 1 have all been identified and analysed. Only
the cost of packaging materials is assumed to be equal for both scenarios.

Packaging Losses in
Materials Production
Nominal Batch
Output sizes

Line
Packaging Manning
Efficiency
Costs
Annual Utility
Production Consumption

Building
Plant and Maintenance
Area
Equipment

Figure 1: Main cost factors in packaging.

ASSUMPTIONS
A medium scale brewery will typically make bottles, cans and kegs on three or four
packaging lines. Well knowing that there are big differences among European
breweries - in the UK for instance, more than 60% is sold in kegs and casks which is
about the same percentage for cans in Sweden and non-returnable bottles in Italy - the
split in table 1 represents a good average.

3
 Primary Packaging % Share Annual sales, hl/a
Bottles 65 650,000
Can 27 270,000
Kegs 8 80,000
Total 100 1,000,000
Table 1: Assumed split of primary packaging into bottles, cans and kegs.

The 650,000 hl bottled beer represents 15 different beers in 3 different bottles with 5
bottle sizes and 9 bottle shapes and colours. The split of bottles and secondary /
tertiary packaging is illustrated in figure 2 and 3.

38 cl 50 cl
P EN RG B
8% 28%

25 cl
NRGB
15%
33 cl
RG B 33 cl
34% NRGB
15%

Bottle mix for 650,000 hl/a

Figure 2: Split of returnable glass and PEN and non-returnable bottles.

250,000

200,000 Wraps,
54 Display Tray Fully Enclosed,
150,000
Film Only
hl/a

4s, 6s,
4s, 6s, 8s, 10s, 12s
100,000 Toppacks,
Multipacks
Loose Bottles
50,000
24 Crate 20 Crate 24 Wraparound
0
33 cl 38 cl 50 cl 25 cl 33 cl
Returnable Returnable Returnable Non- Non-
Glass Bottles PEN Bottles Glass Bottles Returnable Returnable
Glass Bottles Glass Bottles

Figure 3: The total packaging mix with 80 SKUs.

Returnable bottles are packed in crates and display trays. Multipacks are collated in
trays and shrinkwrapped or collated on flat board (pad) and shrinkwrapped, or
collated and wrapped in film only.

4
The supply chain
The prevailing trend towards multipacks for non-returnable bottles and display trays
for returnables will certainly influence all five links of the supply chain, i.e. to buy, to
make, to store, to move, to sell; and the packaging department in particular.

Beer Ware- Depot

Raw Wort Fer- Pack-
Pro- house/ Struc-
Mate- Pro- men- aging
cess- Dis- ture
rials duction tation
ing patch Primary Secondary

Supplier Brewing Packaging Distribution

Buy Make Store Move Sell
Figure 4: Schematic illustration of the supply chain for a brewery.

In todays competitive market the brewer is forced to optimise each link in a way that
ensures overall chain performance. The chain is not stronger than the weakest link
is true, especially when each function activity traditionally tends to focus on own
performance.
For example, longer production runs on one product certainly gives fewer
changeovers, but inventory levels and storage costs will increase. This causes
inflexibility to meet customers demands that in the worst case could affect sales.
Another example is purchase of cheap but inferior packaging materials as for example
crown corks, labels, glue or corrugated board. The buyer on his part of the supply
chain can boast of savings that may be lost tenfold later in the packaging lines. Lower
efficiencies because of perpetual machine stops owing to some packaging materials
being out of spec.s give higher production costs and impedes production plans.
Hence, margins are reduced and sales might as well be affected.

LAYOUT AND TECHNICAL SOLUTIONS

To cater for realistic variations in forecasts and assumptions, the calculation shows
that the dedicated lines together must be rated more than 30% higher than the combi
line. Utilisation of the dedicated lines is not as high as one should expect. With the
wide range of products, bottles, secondary and tertiary packaging, even dedicated
lines, will loose parts of the working time for changeover. The basic parameters are
listed in table 2.

Nominal Output / Efficiency RGB/PEN NRGB Repack Combi

Nominal output, Glass 36,000 36,000 42,000 54,000
Nominal output, PEN 36,000 42,000 42,000
Utilisation, DIN 8782 5.5 64% 63% 56% 56%
Table 2: Nominal output and Utilisation of bottling lines.

Dedicated bottling line for returnable glass and PEN

The layout is shown in figure 5. It is made with open and spacious operating areas for
lowest possible manning. The heart of the line is the filler area with discharge of
bottle washer, EBI filler/crowner/capper and labeller. The second area is crate and

5
bottle washing including bottle and crate sorting. The third area is the dry part arena
with conventional packing and palletising machines.
There is no tunnel pasteuriser as most of the returnable bottles are plate pasteurised.

Filler Area Bottle Washer/Sorting Area Dry Part Area

MANUAL SORTING
CRATES + EMPTY

CRATE WASHER
BOTTLE WASHER BOTTLES ON
DE- EURO PALLETS
CRATER DESTRAPPER

CRATE MAGAZINE
SORTER
BOTTLE
PRODUCTION
DEPALLE- PALLETS EMPTY
EBI TISER EURO PALLETS
IN STACKS
36,000 bph CIP-SET PALLET
PASTEURISER

FILLER INSPECTION
RGB / PEN DEFECTIVE
PLATE

FCI EURO PALLETS

PALLE- IN STACKS
CROWNER ECI TISER
CRATER DEFECTIVE
CC BUFFER REST PALLETS
DISPENSER TANK BEER
TANK
LABELLER
BINDER CRATES + FULL
FBI BOTTLES ON
EURO PALLETS

LABELLER

Figure 5: 36,000 bph Returnable Bottling Line for Glass and PEN.

Obviously, it would have been nice to have a dedicated bottling line for PEN. This
can, however, not be justified because of the relatively small part of the sales. Some
aspects of the time consuming task of changing from glass to plastic are listed below:
The caustic solution in the bottle washer must be dumped. Thus not even water
and detergents but also heat energy will be lost.
The interior parts of the bottle washer must be thoroughly cleaned before it can be
filled with fresh water and caustic again.
The whole line and the conveyors in particular will have to be cleaned for all glass
debris and dirt that can scuff the PEN bottles or cause them to turn over.
The filler must be adjusted for a lower vacuum.
All centring tulips in the filler must be shifted in order to avoid the risk of glass
particles damaging the mouth of the PEN bottles.
The crowner must be by-passed with a feed worm for feeding PEN bottles into the
special capper.
Protein glue must be used instead of casein glue because the labelling pressure.
The above procedure takes about a full shift to carry out.

Dedicated bottling line for non-returnable bottles

As for the returnable line the layout is u-shaped with 3 operating areas:
1. The rinser / filler / crowner area with EBI and labeller. The layout is made for by-
pass of plate pasteurised products.
2. The packer area with two independent packaging legs, i.e. multipacker followed
by a tray shrink packer and a wraparound packer for corrugated cardboard.
3. The palletising area with bulk glass depalletiser and palletiser robot in close
vicinity.

6
 Multi Packer Packer Area
SHRINK
MULTIPACKER TRAY PACKER TUNNEL
Individual or FREE
HEIGHT
parallel run Dual
WRAP AROUND PACKER
palle-
tising
LABELLER
FBI 1x100%
LABELLER 2x50%
ROBOT
STRETCH
Filler PALLETISER
WRAPPER
FILLER PALLET FULLS ON EURO
Area36,000 bph MAGAZINE PALLETS
EMPTY EURO
NRGB BUFFER REST PALLETS IN STACKS
TANK BEER
TANK
CC RINSER TUNNEL PASTEURISER BULK GLASS BULK GLASS
DISPENSER DEPALLETISER PALLETS
DESTRAPPER
EBI
PALLET PAD
MAGAZINE MAGAZINE

Bulk Glass infeed

Figure 6: 36,000 bph line for non-returnable bottles.

The multipacker leg features a triple pressureless laning system. Hence, all kinds of
twin lane wraps and triple lane fully enclosed formats can be produced. A by-pass
conveyor for the multipacker enables loose bottles in tray + shrinkwrap or film only
multipacks to be produced.
Obviously, the idea of having dedicated packers is to maintain full flexibility with a
minimum of changeover. In addition, different formats and bottle sizes can be set up
on one packer while the other one is still running. Another advantage is that the same
product can be packed in two different formats at the same time. The ratio between
settings would typically be 50/50 or 40/60 as required, depending on the batch size.
The line efficiency is also less affected by downstream troubles compared with a
single combi packer solution. If one of the legs should break down the parallel packer
can ramp up from its set ratio speed to 100%, thus keeping the filler running.
The palletising system with an arm type robot is designed for simultaneous handling
of two different products and can cater for any variation in the packing area.

Dedicated repacking line for crates and display trays

The repack line can handle empty and filled goods at the same time. There are three
different modes of operation:
1. Repacking of full bottles from crates into trays. The balance is maintained by
simultaneous repacking of returned empty bottles in trays into crates.
2. Repacking of full bottles from crates into trays without incoming empties. The
surplus of crates is palletised and discharged whereas empty trays are supplied.
3. Re-packing of returned empty bottles in trays into crates without simultaneous
repacking of full bottles. The surplus empty trays and the required crates are
handled in the opposite order as mentioned under mode No. 2.
The finished pallet contains 5 54 4 = 1080 bottles which is up to 20% more than a
conventional pallet. Each quarter pallet is stretchwrapped with a special film. Hereby
the finished goods are effectively protected from light and dust during storage and
transportation. Wrapping for stability reasons is not necessary. Empties for example,
are returned without straps.

7
Multipurpose combi line for returnable and non-returnable bottles
There are three individual infeed systems for new glass in bulk and returned empty
bottles in crates and display trays as illustrated in figure 7. The bottle and crate sorting
area in front of the bottle washer is designed for by-pass of new bottles. However, in
order to eliminate surplus crates because of the foreign bottle sorting among other
things, new bottles can also be mixed with the returned empties.
The heart of the line is the area from discharge of bottle washer to empty bottle
inspection, rinsing, filling and infeed of the tunnel pasteuriser. A replaceable feed
worm ensures that returnable bottles can by-pass the carousel rinser and go straight
into the filler. As for the dedicated line there is also a by-pass of the capper / crowner
depending on whether glass or PEN is produced. The layout after the filler enables by-
pass of the tunnel pasteuriser for all plate pasteurised products.

Sorting/Washing Display Tray Handling STRETCH

WRAPPER LABELLER
BULK GLASS
DEPALLETISER Palletising
MANUAL SORTING
MIDDLE TRAY BULK GLASS
CRATE WASHER

BOTTLE WASHER BOTTOM TRAY PALLETS

CRATE MAGAZINE

TOP PACK LABELLER MIDDLE TRAY

APPLICATORS STRETCH
TOP TRAY WRAPPER BOTTOM TRAY

BOTTLE TRAYS + EMPTY

SORTER BOTTLES ON
DEPALLETISER EURO PALLETS

TRAY WASHER
BOTTLE PRODUCTION

INSPECTOR
TRAY DRYER
TURNERS PALLET STACKS

PALLET
BY-PASS D2 TRAYS + FULL
EBI ECI D1 D3 PALLET BOTTLES ON
LOADER EURO PALLETS
54,000 bph R / NR BOTTOM LABELLER DEFECTIVE EURO
Glass / PEN RINSER CIP-SET TRAY
MIDDLE TRAY STRETCH WRAPPER
TOP TRAY
PALLET STACKS
PASTEURISER

CROWNER RINSER
TRAY SORTER CRATES + EMPTY
DESTRAPPER BOTTLES ON
PLATE

CAPPER
EURO PALLETS
DEPALLE-
BUFFER REST TISER PRODUCTION
TANK BEER DECRATER PALLET PALLET STACKS
TANK INSPECTOR PALLET MAGAZINES
FILLER DEFECTIVE EURO
BY-PASS PALLET STACKS
CRATER PALLE-
TISER STRETCH WRAPPER FINISHED PRODUCT
ON EURO PALLETS
Filling BINDER LABELLER
BY-PASS SHRINK
BY-PASS TUNNEL

TUNNEL PASTEURISER
MULTIPACKER COMBI PACKER

LABELLERS Labelling Packing

Figure 7: 54,000 bph combi line for returnable and non-returnable bottles.

The labelling area is furnished with two medium size labellers with by-pass for bottles
with pressure sensitive labels. They can run comfortably at moderate 33,000 bph and
ramp up to around filler speed if one of them goes down. In addition, changeover can
be made on one labeller during the last hour of a batch run while the other one at
maximum speed keeps pace with the filler.
There are three independent packaging legs after the labellers: One leg for returnable
bottles in display trays. The other one is dedicated for crates while the third leg is for
non-returnable bottles. In order to ensure full flexibility for the returnable part in
crates and display trays, two independent palletising plants are installed. They can run
together at any ratio between the two formats. The non-returnable bottles are
palletised over the crate robot palletiser, stretch wrapped and finally barcode labelled.

COMPARISON OF MAIN COST DRIVERS

The maximum available working time is based on three-shift operation 6 days per
week 50 weeks per year. The selected 10% three months average sales peak is
satisfied, partly by surplus line capacity partly by stock building for the months above
the peak. The manning schedule and the calculated gross working time is stated in
table 3 below.

8
 Manning Level Dedicated Lines Combi Line Reduction
Operators + supervisor 13 6
Relief persons 3 1
Craftsmen 4 2
FLT drivers 4 2
Manning per shift 24 11 -54%
Gross working time, h/year 9,394 6,217 -34%
Total, full time equivalent 53 45 -16%
Table 3: Manning level based on 1540 working hours per year.

The gross working time is calculated on basis of the necessary operating time and the
non-productive ancillary time which is the scheduled (realistic) downtimes for
cleaning, maintenance and changeover. The relatively low manning is due to
operators being educated as process technicians and craftsmen being trained as multi-
skilled mechanics and electricians. The hourly rates for operators and craftsmen /
supervisors are 30 / 36 Euro respectively.
The total building area for the packaging lines including warehousing and the
associated capital costs are verified in table 4 and 5. The bottling halls are complete
with all access areas, auxiliary and amenity functions as well as offices and stores.

Building Area Dedicated Lines Combi Line Reduction

Bottling hall 5,900 4,000 -32%
Auxiliary functions / stores 1,800 1,400 -22%
Buffer store / expedition 6,500 6,000 -8%
Total, m2 14,200 11,400 -20%
Table 4: Building area for bottling lines and buffer store.

Capital Expenditures Dedicated Lines Combi Line Savings

Plant and Equipment 26.4 19.8 -25%
Buildings 11.9 9.3 -22%
Total, mill. EUR 38.3 29.1 -24%
Table 5: Capital expenditures for plant and buildings.

The cost of plant and equipment is based on a mix of estimates and actual contracts.
All commissioning costs, training and spare parts are included. The average prices for
bottling halls with installations and noise abatement and for the storage function are
1020 / 645 EUR/m2 respectively. Plant and buildings are depreciated over 10 / 20
years.

CONCLUSION
The result of the analysis and comparison of the cost factors reveals a difference of
2.2 Euro/hl or a saving of 12% in favour of the combi line. These savings alone,
however, are not big enough to justify the combi line. Therefore, some less tangible
advantages and disadvantages must be addressed.

9
 0.91 Depreciation, Buildings
19.1
EUR/hl 4.06 Depreciation, Plant 0.71
16.9
3.05
EUR/hl
1.95 Warehouse
1.84
1.10 Other Costs
0.98 Utilities 1.00
0.89
2.22 Maintenance 1.96
2.48 Manning Costs 2.01

Three One
Dedicated 5.40 5.40 Combi
Packaging Materials
Lines Line

Figure 8: Comparison of production costs for the two scenarios.

Advantages of the combi line versus dedicated lines

The biggest advantage of the combi line option - besides the savings - is the flexibility
to cater for future unknown changes in the market.
The core parts of the combi line can always be used regardless of changes in the mix
of primary and secondary packaging.

Disadvantages of the combi line versus dedicated lines

The disadvantages of the combi line are summarised below:
One big line only must have a higher nominal speed than a number of dedicated
lines.
A flexible combi line is technically more complicated to control and maintain.
Efficiencies of complex lines (measured over the operating time) are usually lower
compared with dedicated lines at low speed.
Production runs per batch are shorter resulting in more frequent changeovers.
Line utilisation is lower, not only because of more frequent changing over, but
also due to lower efficiencies during fine tuning and start-up of line after a
changeover.

For many existing breweries, however, one decisive point could easily disqualify the
combi line solution: It may not be possible to install a spacious multipurpose line in
old buildings with many columns and fixed walls.

10
102

Supply chain management information

system development in a just in time
environment with driver sales
operations
Eduardo Bezares

Compaia Cervecera de Canarias S.A., Avda. Angel Romero 18, E-38009 Santa Cruz de
Tenerife, Spain (e-mail: [email protected])

Descriptors
Brewing industry, information management, logistics, planning, supply

SUMMARY
The information pipeline system needs models that use linear programming logic to identify
optimal solutions to complex problems. With a sophisticated set of decision support tools, the
system reinforces the whole supply chain and their linkages, rising the efficiency through
improving delivery methods, reducing lead times, getting better planning systems and total
logistics costs. Attainment the right balance among technology, processes and people,
produce a top customer service level, showing that the blending of New IS technologies and
traditional brewery operations, it is old wine in new bottles, the latest phase in the ongoing
evolution of beer business.

Entwicklung eines Informationssystems fr das Supply Chain Management unter Just-

In-Time Bedingungen

Deskriptoren
Beschaffung, Brauindustrie, Informationswesen, Logistik, Planung

ZUSAMMENFASSUNG
Das Informationsverteilungssystem braucht Modelle, die lineare Programmierlogik
verwendet, um optimale Lsungen fr komplexe Probleme zu ermglichen. Mit einem
ausgefeilten Werkzeug zur Untersttzung von Entscheidungen verstrkt das System die
gesamte Versorgungskette und ihre Schnittstellen. Durch verbesserte Liefersysteme und
verkrzte Abwicklungszeitrume lassen sich die Planungssysteme verbessern und die Kosten
der gesamten Logistik senken. Durch Erreichung des richtigen Gleichgewichts zwischen
Technologie, Prozessen und Menschen wird ein sehr hoher Grad an Kundenservice erreicht.
Dveloppement d'un systme d'information pour la gestion des Chaines
d'Approvisionnement dans un environnement de "Juste Temps"

Descripteurs
Acquisition, fonction logistique, gestion de l'information, industrie brassicole, planification

RESUME
Le systme d'information central ncessite des modles faisant appel la logique de
programmation linaire pour identifier les solutions optimales des problmes complexes.
Avec un ensemble sophistiqu d'outils d'aide la dcision, le systme renforce l'ensemble de
la chane d'approvisionnement, augmentant l'efficacit par l'amlioration des mthodes de
fourniture, la rduction des temps d'acheminement, l'obtention de meilleurs systmes de
planification et la baisse des cots logistiques totaux. Lorsque le bon quilibre entre la
technologie, les procds et le personnel est atteint, cela se traduit par un service de haute
qualit au client, ce qui montre que le mlange des nouvelles technologies IS et des
oprations traditionnelles de brassage n'est que la dernire en date des phases de l'volution de
l'industrie brassicole. C'est du vin vieux dans des outres neuves.

2
The main purpose of this lecture is to present a view about the main logistics
problems we have in our Brewery located in the Canary Islands, an ultraperipherical
and fragmented region, how we have tried to solve them and the results we have
achieved.

First of all, I would like to make a short introduction of the company:

TODAY

ISO 9002
B
C T
1997 O EFQM U
Q
N S
1995 M
S SAP R/3 S I
Y PROJECT O N E
T
S L S E X
1 Q I
9 TQM T Y S C
1996
9 M TQM D S
6 PROJECT E - Training S E
INICIATIVES A T
M L
T E - Self-
E M Assessment L
1997
D E
- Model
1999 Application N
C
- Award E
PAST FUTURE

If I could only show you one figure on this, it would be this one, made by Alfonso our
Corporate Development Manager, which speaks of the effort and the constant
commitment of over 600 people in the quest for excellence. CCC was founded in
1939 and since then has been producing to the highest quality standards on the
market, reaching one million hectolitres on sales last year.

Highlighting the main facts, we can see that over only the last five years we have
achieved ISO-9002 certification and introduced a total quality management
programme supported by an implemented integrated SAP R-3 4.6 computer system, a
really powerful tool, well known as an Enterprise Resource Planning System. Since
last year we have joined the European Foundation on Quality Management; with its
model of excellence we are trying to continue, along the route of total quality in a
continuing quest for competitive advantage.

I have several times put emphasis on our search for excellence. Why? Because it is
the reason which led us to develop our Supply Chain Management Information
System form now called logistics information system (LIS) the LIS is the only way to
manage the complex network of facilities with excellence and the flow of materials
and finished products among those facilities, and from/to suppliers/consumers in our
customer-driven company, responding always to market requirements.

Why is this case so special?

We have some factors, the first one is related to our geographical location and that is
why I would not like to omit to tell you where we are. I would say that we are a long
way away, although the expression they use now is that of an Outlying Region. At a
distance of over a thousand miles from the continent of Europe.
The distance is not the only constraint. We have a very rough orography in most of
the islands which does not help us in our logistics tasks. This orography implies a
complex logistics network.
Added to the distance and the orography problem we have to consider that the
territory of the Canary Islands is fragmentary, in that it consists of seven islands,
which are grouped relatively closely together but are separated by the waters of the

3
Atlantic Ocean. And although it may seem that the islands are close together, the fact
is that there are no bridges between them, Therefore for the movement of 70% of our
supplies and 50% of the finished product we depend on the frequency of shipping
services, and for example in the case of the islands of Tenerife and Lanzarote, there
are only two direct connections by sea per week. This significant dependence on sea
transport and hence on ports causes considerable logistical costs, which have been on
a gradually decreasing trend over the last few years.
Although the islands may seem very close, appearances are deceptive. The equivalent
distance overland taking into account the average time to be calculated for the journey
of a trailer, with beer kegs, going from Tenerife to Lanzarote would be equivalent, in
the context of a continental land mass, to nearly 7000 kilometres, that is to say a
similar distance to that which unites (or divides) the eastern and western seaboards of
North America. The islands are not so close together as they seem.

The way I use to calculate that estimated time is part of our logistical model, this is
one of our computer-based analytical tools that facilitate the solution to problems.
Going deeper into this we appreciate that with the two above mentioned islands, the
frequency of services between them, and the journey time, we establish a model to
determine the average delivery time as a function of the day on which the demand
arises on the other island. We study the variability in delivery times as a function of
the standard deviation from the average for that variable to establish a level of service
as a constant deduced from the normal function or Poison distribution.

Once the delivery time has been calculated, we can establish the coverage in the
warehouse at the destination, according to variability and the level of service
determined.

This geographical scenario is one of the key problems we face every day for making
possible the Optimum Supply Chain Management at CCC; with that primary problem
we have some others unusual in our business, such as our driver sales operation
system (without pre-orders from customers), the market mix (we are in the market
with a multi-product portfolio and is difficult to foresee the customer behaviour, in the
Canary Islands like a tourist area your consumer pattern changes every year, which
makes long and medium term forecasting quite difficult), the distribution philosophy,
reaching sixteen thousand customers directly without wholesalers or intermediaries,
and finally the JIT environment: At CCC we are trying to match production to
customer demand, bottling and producing only enough product to replenish what the
customer has sold to compensate the absence of economies of scale.

Managing this logistical situation optimally requires clear logistical objectives, and a
good model, based on a physical network, the best possible organisation of human
resources by processes and good information tools for decision making. This model
will underlie the Logistics Information System and will show us that the whole
would be greater than the sum of its parts.

4
 Place/
Customer Service
Levels

Inventory Transportation
Carrying Costs Costs
LOGISTIC
COST
TRADE OFF Lot Quantity Warehousing
Costs Costs

Order Processing
and
Information Costs

Logistics Objetive:
Minimize total cost, given the customer service objetive where:
Total costs = Transportation cost + Warehousing costs + Order processing & information cost + Lot
quantity cost + Inventory carrying cost

The optimisation of the process as a whole requires the reduction of costs to a

minimum, while at the same time guaranteeing an outstanding level of service to the
customer and a clear market orientation inspired by the values of the Company.
With a logistical network which reaches the sixteen thousand customers directly by
means of nine regulatory warehouses, twenty tractor units, 80 trailers and two
hundred trucks.

Warehouses of Factory (Finished

Supplier Raw Materials Product Transport Depot Client
Warehouses)

DORADA

Supply Production Transport Storage Distribution

SIG

Management Physical Materials & Products Flow

of Materials Distribution
Information Flow

This great investment in storage and transport is very considerable although much less
than it seems because at CCC people are the most important element. That is why we
are an organisation without internal barriers. Used to working in teams with TQM
culture. The Rolling Forecast group is the interdepartmental team who co-ordinate the
Supply Chain Management with formal monthly meetings and weekly data
interchange and informal daily communications.

Sales Strategical
Director
Logistics
Organization Tactical
Logistics
LogisticsSubdirector
Subdirector

Administration
Administration
Expedition
Expedition Operative
Plant
PlantWarehouses
Warehouses
Vehicles
VehiclesWorkshop
Workshop

5
Accustomed to working by processes you see on the figure both co-ordination and
operation activities and Logistics influence all the decision making throughout the
Chain. From Purchasing Planning to delivery to customers.

Coordinatin Bottling &

Estratgic
Capacity Plan Logstics ProductionPlan Purch. Plan
plan
Activities Plan

Gestin de
Stocks

Operational
Production Warehousing Transport &
Purchasing Deliveries
&Distribution Shippment
Activities

To manage well by process is why we have developed the logistical model, based on
equipment, people and systems and techniques such as mathematical calculations and
quantitative methods for taking decisions. In the figure we can see the way to
calculate the coverage as explained before.

S HIP P ING LINE : TRAS M EDITERRANEA

DEP ARTURE : TUES DAY & THURS DAY FROM TENERIFE

c DEM AND ARRIV AL LEAD TIM E

o FRIDAY M ONDAY 4
M ONDAY W EDNESDAY 3
b TUESDAY W EDNESDAY 2
3
e
W EDNESDAY FRIDAY
THURSDAY FRIDAY 2

r
Ave ra ge Le a d Tim e 2.8 S t. De v 0.83666003
t S stock ca lcula tion
u S stock= D x L.T. + D x S .L.F..L.T.V . X S .D.L.T + S .L.F.D.V . X S .D.D. X S QR( S .L.F.T.V . X S .D.L.T.)
S .D.: DEM AND S TANDARD DEV I.

r V a ria bility is e va lua te d w ith sta nda rd de v,

S .L.F.= S ERV ICE LEV EL FACTOR
D: AV ERAGE DEM AND

e L.T.: LEAD TIM E

L.T.V .: LEAD TIM E V ARIABLITY
D.V .= DEM AND V ARIABILITY
S .L.T.: LEAD TIM E S TANDARD DEV IATION

Even all this is little enough when it comes to satisfying all of the variability of the
market and the environment. With this driver calculation plan which sets the cover
to be maintained at each branch on sales days.

We have developed a medium and short term forecasting system, being realistic a
forecast serves no real purpose. It is merely a part of a larger process; demand
forecasting has no intrinsic value. It has meaning only when combined with the
scheduling and procurement processes through the MRP; and that is what we do.

This may sound a little strange but the forecast is always wrong. We all know how
accurate the local TV weather forecast is. So why would we expect an estimate of the
demand for a product to be any more accurate, particularly with uncontrollable factors
such as the economy and the competition?, Our key to success is how we manage the
forecast error.

6
To foresee demand we apply methods of medium and short term forecasting which
analyse trends and seasonalities such as the one on the slide, based on the exponential
smoothing forecasting tool. In order to determine the forecast value, all that is
required is the previous forecast value, the most recent past value and the alpha
smoothing factor. The speed with which the forecast reacts to a modification in the
pattern depends on the smoothing factor.
As a difference among other companies, we use long term forecast (made by
marketing and sales and medium and short term (monthly and daily forecast) to feed
the SOP and the DRP.
Forecasts include marketing, sales and maintenance inputs, for that we have within
SOP a decentralised tool to capture data from the different areas.

Forecast
Model

To validate the theoretical model based on the physical network and the key data
inputs such as coverage or smoothing factors we apply also simulation models which
enable one to get it right first time, to check that the values calculated on paper are
going to deliver the desired results. Based on the Excel Solver or with SAP utilities.

At this moment it is time to introduce our digital system.

On this central nervous system we have had the biggest challenge, the difference
between what companies spend and what they receive in return, in Systems
investments comes from an incomplete understanding of the possibilities combined
with a lack of vision, as to the potential offered by technology to get the right
information to anybody in the company at the right time. With our efforts we have
avoided this common problem.

I like to compare the nervous system of the human body with the Information System:
the first sets off reflexes, which enable us to react promptly to danger, and other
needs. It also provides us with the necessary information to evaluate the options
available and to select a course of action. We remain alert to what is important and the
nervous system excludes all of the information that is not necessary at that time.
Companies must have a similar kind of nervous system: capable of functioning in a
fluid and efficient manner, in order to react promptly to emergencies and
opportunities, to rapidly communicate valuable information to those members of the
organisation that require it, so as to take decisions at once and make contact with
clients.
I agree with the founder of Microsoft that this is the best way to make improvements .

7
The star of this system is the ERP (Enterprise resource Planning) we are using SAP
and really with an exceptional level of performance and almost unlimited potential.
Linked with SAP all the new stars from the new economy in the slide (made by Pedro
our IS Subdirector).

PURCH Productin Bottling Bottling Wareh Distri wareho Distrib CUSTO

Planning ouse b. use . MER
1 1 2 2

PP/
MM MM/PP PP / SOP MM MM SD EDI
DRP
With an exceptional level of performance and almost unlimited potential.

TRANSPT.
MGMT
PM
KRONES-SAP INTERPH.
Note: ready for VMI & Routing
project
FI/CO
HR

For us Internet does not represent a break from the past, will let us to reach the total
integration, The merge between the SCM and the CRM with the e-business will take
us to the last stage in the value chain optimisation, where real time information from
consumers will assist even the development of new products and strategies.

Logstics
Marke- Product Producti Customer
Sales Orders Purch. & Distri-
ting Developm. on butin
Service

Procurement
Procurement Electronic
Electronic
Internet
Internet Internet
Internet Call
Call e-Procure-
e-Procure- Warehouse
Warehouse
&&supplier
supplier supply
supply Call
Callcenter
center
marketing
marketing sales
sales center
center ment
ment &&logistics
logistics
managment
managment chain
chain
Technology
Technologyenabled
enabled Customer
Customer
EDI
EDI Shipping
Shipping
selling
selling service
service
Sales
Sales Order
Order
configuration
configuration
system
mgmt.
mgmt.
system
ERP
ERP

The system is on the alert, ready twenty four hours a day to detect needs. Generating
product movements within the logistical network, in close collaboration with our
suppliers and customers and always at their service, the system therefore activates
packaging, production and supplies.

8
 Planificatin Mat 01
System
Deliveries to
Batch size Depots
Del 01
calculation
Mat N
Redondeo Deliveries to
Depots

Supply class
DRP
Mat 01 Deliveries
Lead time intradepot

SStock
Del N

Mat N Deliveries
Sales by days intradepot

Historical sales Planificatin

S705 S605 Profile LO2
vrs 000 Forecas t table Fo reca st
S801 S801 => S076
vrs. 000 (i nput) vrs. 002
vrs. 001 (output)
Events Usuers

Stocks

SStocks Bottli ng Planni ng

S076
Forecast vrs 002

Last year sal es

And if there are unforeseen eventualities we have linked the LIS with the Outlook
through the Logistics Early Warning System, and in emergency cases it even sends us
an e-mail giving us a margin to continue to improve our ratios, all of them
successfully benchmarked.

ratios selection
Stocks

Exceptions
Anlisis Outlook
Sales Requisitions comunication
(SAP) by e-mail

Safety Stocks Condicitions

9
As a conclusion I like to highlight the importance of a good SCM information System
to assist the processes continuous improvement, reducing costs and increasing
customer service level, We have not completed the job, we will keep developing our
information system to adapt it to the market change.
Because in this way if there is one thing we are sure of it is that this race for
excellence in which we always like to be has no finish.

- 60% Intradepot deliveries to 0,3% sales (*)

- 35% Stock reduction to 5 sales days (*)
R
- 20% Customer Service Level to 99,8% (*)
E
S - 40% Planning Administration costs to 0,6 f.t.e.
U
L - 30% Replenishment lead time reduction
T
S - 20% Primary distribution fleet reduction
All this levels are excellents (Benchmark with Danbrew, Carlsberg & Guinness during

the SCM Seminar. Denmark 05/00).

1) Crandall, R.E., Production Planning in a variable demand environment, P&IC

Journal, Vol. 34, n4, 1998
2) Drucker, Peter, Managing in a time of great change, 1996, Edhasa
3) Gable, John, White, A., Logistics Technology in the Twenty-first Century, The
Free Press, 1994.
4) Martinez Budria, E., Actas Congreso Panamericano. Santander. Servicio de
Publicaciones M de Fomento, 1998.
5) Maslanka, Bryan.American Production & Inventory Control Society Journal,
March 2001
6) Porter, E., Strategy and the Internet, H.B.R., March 2001.

10
103

Implementing environmental
management systems
Tom Woollard

Environmental Resources Management Ltd, 8 Cavendish Square, London W1M 0ER, United
Kingdom (e-mail: [email protected])

Descriptors
Brewing industry, environmental protection, management

SUMMARY
How do you make sure that the most significant environmental risks and opportunities are
managed efficiently and effectively in a brewery?
Over the past five years many European companies have implemented superficial, self-
serving, bolt-on and bureaucratic environmental management systems which are largely
ineffective - this presentation aims to describe ways in which companies can avoid this
situation and, instead; implement streamlined, integrated and flexible environmental
management systems that identify and manage the real issues.

Die Implementation von Umweltmanagementsystemen

Deskriptoren
Brauindustrie, Management, Umweltschutz

ZUSAMMENFASSUNG
In welcher Weise knnen wir sicherstellen, da Brauereien effizient und effektiv mit den
wichtigsten Umweltrisiken umgehen? In den vergangenen fnf Jahren haben viele
europische Unternehmen oberflchliche, selbstndig arbeitende und brokratische Umwelt-
Managementsysteme sowie sogenannte bolt-on-Systeme implementiert, die grtenteils als
ineffektiv zu bezeichnen sind. Die Absicht dieser Prsentation ist, Methoden zu beschreiben,
mit denen die Unternehmen dieser Situation vorbeugen knnen und stattdessen reibungslos
funktionierende, integrierte und flexible Umweltmanagementsysteme implementieren knnen,
mit denen sich die wirklichen Themen identifizieren und verwalten lassen.
Mise en oeuvre des systmes de gestion de lenvironnement

Descripteurs
Gestion de l'entreprise, industrie brassicole, protection de l'environnement

RESUME
Comment sassurer que les risques et les opportunits les plus importants pour
lenvironnement sont grs de manire efficace et rentable dans une brasserie? Depuis cinq
ans, beaucoup de grandes entreprises europennes ont mis en place des systmes de gestion
de lenvironnement lourds et bureaucratiques, au seul service de leurs intrts. Ils sont pour la
plupart inefficaces. Cette prsentation indique comment les entreprises peuvent viter cette
situation et mettre en oeuvre des systmes de gestion de lenvironnement rationnels, intgrs
et flexibles, permettant didentifier et de grer les vrais problmes.

2
An effective management system need not be certified but it focuses on delivery of
real performance improvements. It should be streamlined, non-bureaucratic and
concentrate on a companys key risk issues. It should also be integrated into core
business activities and other support systems to allow it to compliment rather than
complicate operations. The system should be clear, articulate and consistent with
environmental policies, standards and codes of practice which are easy to understand,
by employees, at all levels within the company. Details of the processes and
responsibilities required by an EMS should also be made readily available to staff
through internal channels such as intranet sites.

There are therefore four basic implementation principles for a successful EMS:
Build on existing systems which are internally accepted and already work well
Embed environmental management into all core operations, rather than as a stand-
alone system
Evaluate the need for every single document produced for the system, avoid
bureaucracy
Use ISO 14001 as a checklist, not an absolute.

An EMS needs to contain the right balance of physical (emissions abatement,

emergency response equipment and bunding) and management (inspections, auditing,
monitoring) controls. Too much emphasis on either of these individual elements can
reduce the capability of the system to deliver optimal outputs.

A system which is excessively bureaucratic can be expensive and stifle initiative,

while environmental management initiatives implemented without sufficient
expertise, and no EMS, are likely to cause chaos and have significant cost
implications. For example, a company is more vulnerable to spillages, explosions,
casualties, fatalities, fines, compensation claims and reputation damage under these
circumstances. Some internal expertise can assist a company to improve its
performance without a formal management system but often this leads to sporadic and
localised initiatives, which are not effective in contributing to large-scale change.
Continuous progress is most easily delivered by combining a structured EMS with the
right level of internal expertise.

Streamlining existing systems and training employees, at all levels, contributes

directly to improving the levels of management and expertise within a company.
Global Structure of Environmental Management

Bu sin ess Pr in cip les

En viron men tal Po licy

Performance and In cid ent
A ssu rance

Globa l Requir em en ts
Reporting

Training

Regional/ Op erating comp an y sp ecific g uid a nce

Site System s

Environmental issues and impacts occur at a number of different levels: site, regional
and corporate. At the site level more specific process management systems are
required, while at the corporate level strategic policy making and definition of
overriding business principles are more important. However, staff at each of these
levels will require training and education and will be involved in performance
reporting for common issues.

Environmental management issues need to be addressed at both the operational and

the strategic level. Relevant operational issues include transport, waste, energy,
contaminated land and visual impacts. The focus for management of these issues will
usually be at site and regional levels. Strategic issues include stakeholder relations
(investors, shareholders, pressure groups etc.) and products and services. These issues
are more appropriately dealt with at the corporate level. However, there are issues that
straddle the two levels, such as product life cycle and supply chain. Very few
environmental issues can be tackled in isolation and require co-ordination throughout
a companys structures to be effectively managed.

Having decided to develop an EMS companies next have to address the question of
where to direct the resources for their environmental management. Should the
emphasis be on corporate strategy or on site-level operational issues?

2
 Environm ental exposure is not constant along the value

Fuel consum ption

Hig h
GHG em issions
Traffic congestion
En
vir
on
me
nt
al
Fo Freshwater supplies, soil
fertility, pesticides,biodiversity
ot Packaging waste
Climate change & crop
Health im pacts
pri yields/pest outbreaks
Reputation risk
nt Local com munities
Reputation risk Regulatory compliance
Eco-eficiency
Clean-up liabilities
Reputation risk
Low

N atural Transportation M anufacturing Product Consum ers

Resources Sites D istribution

Value Chain

As the diagram above illustrates, environmental exposure is not constant along the
value chain. Many of the more obvious environmental impacts of manufacturing, such
as spillages and emissions occur at the site level. However, the environmental
footprint at an individual site is usually much lower than that of the extensive chain of
impacts which occur before the basic natural resources arrive at a site. These wider
impacts include natural resource use, product use and consumer health implications
and create a much greater overall footprint. The most effective EMS will ensure that it
captures the management of environmental issues throughout the value chain,
prioritising those issues that present the most immediate, as well as significant, risks.

Most management systems, however, tend to focus on operational compliance,

allocating the most significant amount of their management resources to the site level.
At manufacturing sites compliance is obviously a significant issue, associated with
retention of operational licences and permits. EHS manuals, training and working
groups and internal reports are usually the foci of site level management processes.
At this level performance data and key indicators are more easily collected and
defined so companies are keen to arrange auditing and certification to demonstrate the
more tangible areas of their performance.

The wider issues of natural resource consumption, transportation, distribution and

consumption are therefore often neglected. This results in an imbalance between
environmental risks and management effort, with a concentration of overall
management efforts on the site level compliance issues, despite the fact that this often
proportionally represents only a small amount of the overall risk.

The strategic issues of policy, performance and reputation opportunities and risks
exist outside the boundaries of the individual but should not become consigned to ad-
hoc and sporadic management initiatives which cannot effectively control them.

3
Third party interest is another important factor in determining where to focus
management resources. The scope of interest is clearly different depending upon the
parties involved, as illustrated below:

I RRC KLD

OEKOM OEKOM

KLD OECD
OECD

CEP CEP CEP

Organisation SI GMA SI GMA SI GMA

/
I nitiative
UN Global
UN Global Compact UN Global Compact
Compact

SAM-DJSGI SAM-DJSGI SAM-DJSGI SAM-DJSGI

GRI GRI GRI GRI GRI

Raw Material Transportation Manufact uring Product Product Use

Suppliers Sites * Distribution

Value Chain
* Three schemes also demonstrate an interest in shared manufacturing facilities (JVs)

These differences may also influence the priority given to management of each aspect
within the value chain, depending upon the relative importance/relevance of these
external parties to a company.

Implementation of a streamlined and integrated EMS is approached through a process

of risk identification. At the site level the physical impacts could include issues such
as:
Electricity consumption and production
Natural resource consumption
Chemicals and water treatment processes
Waste production
Discharges
Consumption of volatile substances
Run-off
Effluent generation and treatment
Air emissions.

While off site/peripheral activities to be assessed could include:

Sewer integrity
Landfill
Waste disposal (landfill, agriculture, reclamation)
Integrity of discharge pipes
Breaches of consents/permits

4
 Transportation impacts (nuisance, noise, emissions, spillages, odour, fuel
consumption)

Wider stakeholder issues, are illustrated in the diagram below:

Water Utilities
Environmental Policy/Strategy
Widnes Environmental/Community Policy
Code of Practice on Sludge in Agricultur4
WwTW
Mission Statement
Relevant instructions & procedures:
avoidance of pollution of watercourse from
installations (CO/91/13 - CO/95/3)
Sewage sludge tankers - Vacuum type (CO/91/16)
Relevant sections of: Preparation of post-incident review reports
QA manual (CO/91/5)
Local Local procedures Communication about pollution incidents
Community Emergency Plan (CO/91/6)
Pollution incidents - cost recovery (CO/96/38)
Various expectations Visits by groups & school parties to operational
(e.g. noise, odours, sites (TEMP 10)
traffic)
Checking on-site flow recording devices (TEMP 15)
Regulatory reporting (WMP/OP6)
Input of OMS data (WMP 03/05)
Monitoring asset performance (WMP 05/01)
Local Authority
Emergency Plan Environment
Planning Perimission Agency
IPC Authorisations (no AM2867
Schedule A of
Agreement of 2 April 1998) WWTW
Operational Information in Tranche 1 & 2
Management of and supplementary
Sewage Network documentation Filtrate quality
Flowrate

An effective EMS takes each of the elements described above (risks, opportunities,
scale, operations, strategy and stakeholder concerns) into consideration and prioritises
them, then integrates their management into existing management processes and
structures. An EMS should combine certain recognised elements common to all
organisations but should also reflect the specific operational, management and
strategic issues specific to the company it is designed for.

5
104

Production-integrated environmental
protection in the brewing industry
M. Kaschek1, H. Chmiel1, V. Mavrov2 & H. Janke2
1
Institute for Environmentally Compatible Process Technology, Im Stadtwald 47, D-66123
Saarbrcken, Germany (e-mail: [email protected])
2
University of the Saarland, Department of Process Technology, Im Stadtwald 47, D-66123
Saarbrcken, Germany

Descriptors
Anaerobic effluent treatment, brewing industry, environmental protection, lubricant,
membrane filtration, sodium hydroxide, waste water

SUMMARY
The objective of a research project (BMBF 01 ZF9501/3) was to determine the optimum
processes for the treatment and reuse of brewery effluents and the suitability of the separated
pollution load for anaerobic fermentation. The research areas were:
treatment of alkaline solutions
treatment of washing water
treatment of vapour condensate
treatment of conveyor belt lubricants
anaerobic fermentation of the concentrated pollution load.
All processes were developed on a laboratory scale and tested in breweries. Results showed
that these partial streams could be successfully treated by membrane processes, combined
partly with pre-filtration and downstream disinfection.

Produktionsintegrierter Umweltschutz in der Brauindustrie

Deskriptoren
Anaerobe Abwasserklrung, Brauindustrie, Membranfiltration, Natriumhydroxid, Schmier-
mittel, Schmutzwasser, Umweltschmutz

ZUSAMMENFASSUNG
Im Rahmen eines BMBF-Forschungsvorhabens (01 ZF9501/3) wurde untersucht, mit welchen
Verfahren unterschiedliche Fluidstrme zur Wiederverwendung aufbereitet werden knnen.
Es wurde untersucht, in wie weit sich die separierten Schmutzfrachten fr eine anaerobe
Vergrung eignen. Die Themen gliedern sich in:
Laugenaufbereitung
Aufbereitung von Waschwasser
Aufbereitung von Brdenkondensat
 Aufbereitung von Kettengleitmitteln
Anaerobe Vergrung der Schmutzfrachtkonzentrate
Alle Verfahren wurden im Labormastab entwickelt und erfuhren jeweils eine gro-
technische Erprobung in verschiedenen Brauereien. Es konnte gezeigt werden, dass die
Aufbereitung aller untersuchter Teilstrme mittels Membranverfahren, zum Teil in
Kombination mit speziellen Vorfiltrationsverfahren und nachgeschalteten Desinfektions-
verfahren, mglich ist.

La protection de lenvironnement intgre dans la production dans lindustrie brassicole

Descripteurs
Fermentation anarobie, gestion des eaux, procds membranes, rcupration des lessives
alcalines, recyclage des vapeurs

RESUME
Le but dun projet de recherches (BMBF 01 ZF9501/3) tait de dterminer les procds idals
pour le traitement des effluents manent des brasseries et laptitude de la charge polluante
spare pour la fermentation anarobie. Les domaines de recherches taient:
traitement des lessives alcalines
traitement des liquides laveurs
traitement du condensat vaporeux
traitement des lubrifiants
fermentation anarobie de la charge polluante concentre.
Tous les procds ont t dvelopps lchelle laboratoire et mis en marche dans des
brasseries. Les rsultats montrent que le traitement de ces effluents est possible par des
procds membranes, combins partiellement avec la filtration prparatoire et la
dsinfection subsquente.

2
INTRODUCTION
Within the framework of a research project supported by the German Federal Ministry
for Education and Research, various liquid material streams and the related
production processes were investigated to determine how environmental pollution can
be prevented while reducing costs and/or increasing quality. This publication will deal
with the most important results of the work.

INVENTORY ANALYSIS AND RESEARCH PROGRAMME

Extensive preliminary tests showed the different weak points in the liquid material
streams. Detailed tests were conducted with CIP caustic solutions from the brewing
house, fermentation and storage cellars and regeneration of PVPP as well as the
caustic cleaning solution from the bottle washing machine. For water recycling the
main focus centred on the rinsing water from the bottle washing machine, vapour
condensate and conveyor belt lubricants.

Vapour Lubri- Wash

condensate
Production line cants water

Process
water Fermentation Bottle Product
Wort Filtration
and washing
boiling
storage

CIP- Washing
PVPP- machine Mixing and
base base base equalising
tank

Retentates and wastewater with high COD

Indirect discharge or
Anaerobic
on-site wastewater treatment
biological
treatment

Figure 1: Brewery fluid streams and their treatment

Compact anaerobic treatment of partial streams was chosen to eliminate pollution load
involving tests to determine the extent to which anaerobic partial stream treatment is a
viable option and if the concentrated caustic solutions can be used as a means of pH
adjustment.
A detailed inventory analysis, a technical concept, laboratory-scale and industrial-
scale pilot tests and an analysis of economic efficiency formed the project structure.

TREATMENT OF CAUSTIC SOLUTIONS

The concept for the CIP caustic solutions from the brewing house, fermentation,
storage and filtration cellars involved holding and extensively treating the acids or
caustic solutions via a process combination of pre-treatment and membrane filtration.
In line with the overall concept, the concentrates were routed to anaerobic
degradation.

3
 Energy

Base 50 % Storage tank for Pipes

cleaning-in-place and
Water
base Alkaline cleaning solution 2-4 %, 80 C container

Regenerated
base,
Pretreatment
Membrane
70 C
Filtration

Particulate matter

Concentrates

Energy
Anaerobic degradation

Low-contaminated
wastewater

Figure 2: Flow diagram for the treatment of CIP bases

Treatment of CIP caustic solutions

The main focus of the tests was centred on developing a suitable membrane process.
In an initial membrane screening process, temperature and pH stability as well as the
various retention values for COD at different cut-offs were observed. Centrifugation
of cleaning solutions, currently applied in some production sectors, was used as a
comparison.
As additives such as surfactants, complexing agents and disinfectants etc. are added to
the cleaning solutions, conflicting objectives are encountered in fluid treatment: on
the one hand, the waste load should be totally removed and on the other hand the
cleaning substances should not be separated. The degree of retention of the cleaning
substances was determined for the different membranes. The best compromise proved
to be a cut-off of roughly 1kD.

high

Economic feasibility Economically

should be assessed feasible
Concentation

No economic
feasibility

low

multi-use cleaning solutions single-use cleaning solution

low Use of cleaning solutions high
Figure 3: Economic efficiency of fluid treatment in CIP processes using
membrane processes

4
The economic efficiency of this process has only a limited dependence on the price of
caustic solutions and acids as well as on individual costs for water and wastewater.
Generally speaking, treatment is not profitable for multi-use cleaning solutions with
relatively low concentrations of acids and caustic solutions. This applies particularly
in cases where the plant to be cleaned has not been optimised for a CIP process, i.e.
where there are large dead volumes causing 10-30% of the washing solutions to be
lost in each washing process. These quantities must then be replaced thus obviating
the need to dispose of the washing solution.
Membrane separation processes are only a viable option in cases where a single-use
cleaning process has to be applied and the cleaning intervals are lengthy, such as in
PVPP regeneration or when concentrations are high.
The sector where economic efficiency should be assessed depends on individual cost
structure. Secondary effects such as pH reduction in wastewater, prevention of shock
loading and temperature problems can also be significant.

Treatment of PVPP regeneration solutions

In general, a single-use caustic cleaning solution without additives and without
noteworthy concentration of particulate substances is applied in PVPP regeneration.
The pilot plant for the treatment of the caustic solution and its recirculation consists of
an intermediate buffer for the spent caustic solution (9m3) and a nanofiltration unit
with a membrane cut-off of 200 D. As part of the post-rinsing water is also collected,
recovery of the fluid quantity is 100% and roughly 80% for the caustic solution. The
mean flow rate of the plant was approximately 500 l/h.
Assuming that a membrane with long-term stability (at least one year) is used, that the
price of caustic solution is roughly 100 Euro/to (50% NaOH) and that the cost of fresh
water and wastewater is approx. 3 Euro/m3, pay-back time would be roughly three
years. The quantity of recycled caustic solution would be roughly 4000 m3/a. Pay-
back times would be shorter for larger plants. However it is crucial that each case is
considered individually.

TREATMENT OF FLUIDS FROM BOTTLE WASHING MACHINES

The concept for the treatment of effluents from bottle washing machines can be
divided into the sub-sections of caustic solution treatment and water recycling. The
goal was to produce water of drinking quality from the treatment of post-rinsing water
which usually exits the washing machine via the pre-soaking stage. This water is to
replace a certain fresh water quantity. In bottle washing machines in mineral water
production the water from the pre-soaking stage can be used instead of post-rinsing
water.

Treatment of caustic solutions

As the level of impurities in the washing water is proportional to the concentration of
impurities in the caustic solution and its transfer into the post-rinsing stage, the waste
load in the caustic solution should be reduced by a membrane process.
The waste load mainly depends on the extent to which the bottles contain beer residue
and labels. Optimisation potential lies in thoroughly emptying residue and in keeping
the level of impurities entering the cleaning process from the pre-soaking stage as low
as possible. Good resistance to caustic solutions should be taken into account when
choosing bottle labels. The inflowing COD load caused by surfactants is proportional

5
to the quantity of caustic solution used per bottle. Loads are negligible if 10 to 50 ml
of caustic solution are used per bottle.
returned water
additives fresh water
caustic soda
additives

residue pollutant
bottle labels transfer

pre-soak Z1 Z2 Z3
WW1 WW2 WW3
alkaline cleaning warm water rinsing
bottle bottles bottles
bottle
inlet
outlet
highly loaded
waste water

MS MS

purified water

anaerobic
treatment
MS: membrane separation

Figure 4: Concept for treating water and alkaline cleaning solutions in bottle
washing machines

Similar to the treatment of CIP caustic solutions, the choice of membranes was based
on resistance to caustic solutions and temperature. In this case, there was also a
conflict of objectives between the retention of impurities and additives. A cut-off of
1 kD was accepted as a compromise. Alternatively, nanofiltration with a cut-off of
200 D can be selected and the relevant additives added later. The choice of process to
be used mainly depends on the choice of additive components, on the water quality
and the concentration of impurities. The type and cost of washing components are
also of significance.

100
CSB
90 LK 1
LK3
80 LK2
LK4
70
Rckhalt [%]

60

50

40

30

20

10

0
UF 50 kD UF 1 kD NF3 NF4
Membran

Figure 5: Retention of additive mixtures and their individual components

6
The molecular weights of the washing additives are in the range of 150 to 300 D. It
can be assumed that a membrane with a cut-off of 1kD would allow these molecules
to permeate whereas retention of this size of molecule would be up to roughly 90%
using a membrane with a cut-off of 200 D (NF3).
The test with the additive mixture and the COD as a measured quantity seemed to
confirm this relation (figure 5 - black bar). It was only the tests involving the
individual substances which showed that retention by all membranes of the non-ionic
surfactant LK4 was very extensive. Further tests showed that polyether sulfone (PES),
which is a polymer used to produce most of the membranes resistant to temperature
and caustic solutions, adsorbed or absorbed the surfactant while partly losing its
separation properties and permeability.

Treatment of washing water

The demonstration plant for the treatment of post-rinsing water consisted of a multi-
layer sand filter, two-stage bag filtration, a reverse osmosis stage as well as UV
disinfection. Flow rate was roughly 4 m3/h.
Two-stage, two-strand Reverse osmosis
bag filtration stage

Coarse filter Fine filter

Feed- Pre- Permeate UV dis-

Tank treatment Tank infection

Product water
of drinking quality

Concentrate
Figure 6: Flow diagram of the demonstration plant for the treatment of
post-rinsing water

Stable permeability rates of approximately 0.2 l/m2hbar were achieved at yields of

between 80 and 60%. However this value must be judged as being relatively low and
did not meet with expectations. The resultant total costs are roughly 4 Euro/m3,
depreciation being 10 years.
The quality criteria as stipulated in the drinking water regulations could be complied
with and even exceeded for the ionogenic components.
Table 1: Water quality during the treatment of post-rinsing water by reverse osmosis
Parameter Unit WW1 Treated water Drinking water
regulations
El.conductivity mS/cm 3300 50 <2000
Turbidity FNU 50 0.1 1.5
PH - 6.5-9.8 5.0-10.4 6.5-9.5
COD mg/l 500 8 -
Sodium mg/l 500 5 150
Calcium mg/l 30 0.5 400
Magnesium mg/l 7 0.2 50
Aluminium mg/l 5 <0.05 0.2
Hydrogen carbonate mg/l 1000 10 -
Cleaning additives mg/l n. d. n. d. -
Colony count 1/ml 300,000 70 100
Coliform bacteria 1/100 ml - n. d. n. d.

7
A likely use for this recycled water would be production points where partially
desalinated water of high temperature (up to 60C) must be used, such as in a low-
pressure steam generator. Pay-back times for this type of plant are considerably
shorter than plants with direct recirculation due to the additional energy recovery and
the costs saved for partial desalination. During the test period, the microbiological
standards for drinking water were also fulfilled.

TREATMENT OF VAPOUR CONDENSATE

Vapour condensate constitutes up to 3% of the total wastewater stream of a brewery.
The level of impurities is very low and salt content is equivalent to that of partially
desalinated water. Temperature is usually between 30 and 40C but depends greatly
on the efficiency of heat recovery.
The process combination consisted of two-stage membrane filtration with pre-
treatment and downstream UV disinfection. The objective was to produce water of
drinking quality, in which case the very low-molecular components in the vapour
condensate such as DMS (dimethyl sulfide) had to be separated.

Water of
drinking
quality

1st membrane 2nd membrane

Pretreatment stage stage Disinfection

Base Acid Biocide

CIP plant
Figure 7: Demonstration plant for the treatment of low-contaminated process water

The process combination was tested for roughly one month. After an initial start-up
period, stable permeability rates of roughly 1.4 l/m2hbar were achieved, these being
seven times higher than those reached in the treatment of water from the bottle
washing machine.
Total costs for such water treatment with the low-pressure reverse osmosis membrane
are between 1 and 1.5 Euro/m3 depending on the size of the plant. Costs are
considerably higher if UV oxidation is chosen for the second stage instead of a second
membrane stage.
To summarise: for the treatment of water in breweries and water reuse, vapour
condensate is the more economical option for process water treatment. The best
possible use for such water is boiler make-up water. The quality is adequate for low-
pressure steam generators and the ion exchanger units operate only as a safety
measure. Decisively less strain is put on the ion exchanger unit in high-pressure
systems.

8
RECYCLING OF CONVEYOR BELT LUBRICANTS
The collection and treatment of conveyor belt lubricants was incorporated into the
research project for various reasons: on the one hand, the cost of lubricants is very
high. On the other hand, the biocide effect of these lubricants, although considerably
less intensive, is still present in the mixing and equalising tank or in the wastewater
treatment plant. A further common motive was to prevent the formation of aerosols
and to ensure filling areas remain as dry as possible so that micro-organisms do not
form and spread.
A concept was elaborated which involved lubricant collection, pretreatment, treatment
by microfiltration and UV disinfection. The best possible membrane was a ceramic
membrane with a pore size of 0.2 m and stable permeability rates of 50 l/m2hbar
were achieved. Yields amounted to almost 100% without impairment to permeate
quality. Daily cleaning and disinfection of the plant is recommended for hygienic
reasons.
Water
Concentrated
lubricants

Spent conveyor Treated

belt lubricants lubricants
Pre- Membrane UV
treatment filtration disinfection

Sludge
Figure 8: Flow diagram of the treatment of conveyor belt lubricants

In an on-site test lasting several months, analysis of the permeate quality (roughly 50 l
permeate per hour) showed that turbid matter was reduced to below detection limit
and colony count was reduced to well below the limit value for drinking water. These
results were all the more remarkable as the membrane plant was not cleaned nor
disinfected during the entire test period.
A cautious estimation of economic efficiency is based on the assumption, that the
concentration of conveyor belt lubricants is 0.2%. Taking a price of 2.5 Euro/m3 for
fresh water and wastewater and 1.65 Euro/kg for lubricant concentrate, total costs for
conveyor belt lubricants would be 4.80 Euro/m3.
Considering all the costs involved, total costs for recycled conveyor belt lubricants
would be roughly 3.5 Euro/m3.

ANAEROBIC TREATMENT OF PARTIAL STREAMS

Currently, standard wastewater treatment concepts in breweries usually involve
mixing all wastewater partial streams in a mixing and equalising tank with subsequent
neutralisation if necessary. The wastewater is then routed to an on-site or a municipal
treatment plant. The costs for discharge to municipal wastewater treatment depend on
the concentration of impurities, thus making partial degradation of the waste load a
viable option.
Within the framework of the overall concept, tests were conducted to determine the
extent to which the concentrates together with the highly contaminated wastewater
partial streams could be degraded by anaerobic fermentation and if the spent caustic
solutions from the production sectors under study could be used as a means of pH
adjustment.

9
 Raw Production line Product (beer)
materials Brewing Storage Filtration Filling
85-90 % von Q total
10-15 % von Qtotal
100 %
Highly contaminated Low-contaminated
80 % partial streams partial streams
(COD > 1 500 mg/l) (COD < 1 500 mg/l)
Membrane filtration
Spent bases from:
20 %
Cleaning-in-place two-stage
PVPP regeneration Use as anaerobic
Bottle washing 100 % pH adjustment pretreatment
( app. 80%)

Municipal sewage treatment

Figure 9: Management concept for wastewater and bases

In addition to the tests in a laboratory reactor, all tests were performed on a pilot scale
in a container unit in a brewery over a period of several months. The plant consisted
of a two-stage unit with separate acidification reactor and a methane production stage.
Flow rates were up to 0.6 m3/d at a mean COD value of 15,000 mg/l in the feed.
Within realistic limits (800 to 20 000 mg/l) COD in the feed did not affect cleaning
efficiency. Degradation greater than 80% can be expected for standard brewery
wastewater and shock loadings higher than 6 times the original loading could be
accommodated without an adverse effect on efficiency. Consumption of caustic
solutions amounted to between 0.2 and 0.37 g NaOH/g COD at a pH value of 6.2
Spent cleaning solutions from CIP processes, bottle washing machines and PVPP
regeneration are basically suitable as a means of pH adjustment. With the exception of
the polyphenols, the impurities are suitable for anaerobic degradation. The
consumption of caustic solutions in breweries is almost equivalent to the requirements
for caustic soda solution needed for the treatment of partial streams.
In assessing the economic efficiency of the anaerobic treatment of the partial streams
of highly contaminated wastewater streams compared to the anaerobic treatment of
entire streams, it can be stated that the treatment of partial streams results in a cost
advantage of roughly 30% for larger breweries. Treatment of total streams is the more
economical solution for smaller breweries.
Profitability strongly depends on individual circumstances such as cost structure,
possibilities for uncoupling wastewater streams etc.

10
105

Environmentally sustainable alternative

uses for brewery by-products
P.J.M. Bruijn1, T.R. Noordman1, P.C. Deurinck1 & S. Grass2
1
Heineken Technical Services B.V., P.O. Box 510, 2380 BB Zoeterwoude, the Netherlands
(e-mail: [email protected])
2
2B Biorefineries, Neugutstrasse 66, CH-8600 Duebendorf, Switzerland (www.2bio.ch)

Descriptors
Brewing industry, by-product disposal, combustion, energy policy, feed, spent grains

SUMMARY
The preferred use for spent grains is as feed for ruminants. If this outlet cannot be used, a
sustainable alternative outlet is necessary. Spent grains can be separated in two fractions. A
fraction high in proteins and fats suitable as an animal feed for pigs and poultry. Other by-
products with nutritional value like malt dust, trub and yeast can be added to this fraction.
The second fraction mainly consists of fibrous matter and can be used for energy generation.
The project combines efficient and environmentally sound use of the nutritional value of by-
products with the generation of energy from biomass.

Umweltvertrgliche Alternativen fr die Verwertung von Brauereinebenprodukten

Deskriptoren
Brauindustrie, Energiewirtschaft, Futtermittel, Nebenproduktbeseitigung, Treber,
Verbrennung

ZUSAMMENFASSUNG
Die bevorzugte Verwertung von Biertrebern liegt in der Verftterung an Wiederkuer. Wenn
dieser Verwertungsweg ausfllt, wird eine Alternative bentigt. Biertreber knnen in zwei
Fraktionen getrennt werden. Die eine Fraktion, bestehend aus Proteinen und Fett, kann als
Futter fr Schweine und Puten verwendet werden. Zu dieser Fraktion knnen andere
Brauereinebenprodukte, wie Malzstaub, Trub oder Hefe, zugegeben werden. Die zweite
Fraktion, die hauptschlich aus dem Faseranteil der Biertreber besteht, kann zur
Energieerzeugung verwendet werden. Das Projekt vereint die effiziente und sinnvolle
Verwendung des Nhrstoffgehalts von Nebenprodukten mit der Erzeugung von Energie aus
Biomasse.
Usages alternatifs cologiquement acceptables des sous-produits de l'industrie
brassicole

Descripteurs
Aliment pour animaux, combustion, drche, gestion de l'nergie, industrie brassicole, rejet
de sous-produits

RESUME
L'usage prfr des drches usages est l'alimentation pour les ruminants. Si ce dbouch
n'est pas utilisable, il faut trouver un autre dbouch acceptable. Les drches usages peuvent
tre spares en deux fractions. Une fraction riche en protides et en matires grasses,
convenant comme nourriture pour les porcs et la volaille. Cette fraction peut tre complte
par d'autres sous-produits ayant une certaine valeur nutritionnelle, comme la poussire de
malt, le trouble (trub) et la levure. La seconde fraction se compose essentiellement de
matires fibreuses et peut constituer une source d'nergie. Le projet combine l'emploi efficace
et cologiquement sain de la valeur nutritionnelle des sous-produits la gnration d'nergie
partir de la biomasse.

2
INTRODUCTION
Spent grains are produced in high quantities as a by-product of brewing (18 kg wet
weight/hl beer). Other by-products that are marketable include surplus yeast (2.5 kg
wet weight/hl), whirlpool trub (approximately 0.8 kg wet weight/hl) and malt dust.
The fibre content of spent grains is 160 g/kg dry matter making them unsuitable as a
feed for non- ruminant animals e.g. pigs. At present, in Europe and many other
countries, Heineken sells spent grains as cattle feed. This outlet is very attractive for
the breweries as no investment is required and the marketing and sales can be
contracted out to specialist firms. However there are drawbacks to the use of spent
grains as cattle feed. Spent grains are bulky, due to the high water content (70-80 %
w/w), making handling and transport inefficient. They are vulnerable to
microbiological decay; therefore quality control is an important aspect in handling
spent grains. In addition, production of spent grains is high in summer when demand
for cattle feed is low. The sale of spent grains, as cattle food is also not a global
solution, as this market does not exist in some African countries and in Singapore.
Finally, there is growing competition from by-products of other industries. These
factors led Heineken to investigate other markets for these brewery by-products.
Three criteria were set for these alternative markets: - they should be economically
viable (profitable with minimal investment in capital and manpower), environmentally
sound and sustainable and also provide a global solution.
This paper reports on a development project between Heineken Technical Services
and 2B Biorefineries, a Swiss firm specialising in separation technology, which began
in 1998. 2B Biorefineries adapted a grass separation method for use with spent grains.
The process separates spent grains into two useful fractions, a protein concentrate
and a fibre concentrate. In addition, there is a waste water stream.
The protein concentrate has a low fibre content and can be fed to non-ruminant
animals e.g. pigs and poultry. This product has a similar market to soybean meal.
Surplus yeast, whirlpool trub and malt dust can all be added to this product. The fibre
concentrate is high in fibre and low in protein and can be burnt to produce steam.
This fuel is a renewable energy source and does not contribute to global warming.

PRINCIPLE ITEM
Materials and methods
Joint research with 2B Biorefineries began in 1999 and initial trials used
approximately 1300 kg spent grains (wet weight) per batch trial. The process has been
significantly adapted and recently (2001), trials started on a continuous plant which is
able to process up to 4500 kg wet spent grains/h. This plant, located at 2B
Biorefineries, can process spent grains from a brewery producing 2 million hl beer per
year. Spent grains used for these trials were full malt, roller milled and separated
from the wort in a lauter tun. They were provided by Heineken Switzerland.
The separation process is a relatively simple mechanical technique, uses equipment
that is available and allows some flexibility in the products.

3
 Waste water
Spent grains Protein
concentrate

Mixing Decanter
tank

Fibre
concentrate
Shearing Fibre
device separation

Figure 1: Schematic diagram of the spent grain processing plant

The spent grains are diluted with recirculated liquid in a mixing tank and are then fed
into a shearing device (figure 1). Here the shear forces separate the protein particles
from the husk. The fibres are then pressed from the liquid, which is mainly fed back to
the mixing tank to dilute spent grains. The fibre concentrate is a wet product. An
external consultancy firm has investigated the possibility of converting this fraction
into energy. Part of the recirculated liquid, is treated in a decanter to obtain the
protein concentrate (figure 1) and the liquid is partly discharged as waste water. This
protein concentrate is also a wet product. An institute for animal nutrition has
evaluated the nutritional and economic aspects of this product. Analytical data
obtained in these studies has been used in least cost calculations. Diets were set
according to Dutch CLO requirements for fattening pigs. Simple acceptance trials
were carried out on pigs. The environmental sustainability of this process is the
subject of a comparative Life Cycle Assessment by the Dutch Centre for Agriculture
and the Environment. The results of this research are not yet available. This project
was subsidised by the Dutch Government. This process is the subject of a patent
application submitted by 2B Biorefineries.

Results
The process is continuous and separates grains into a protein concentrate and a fibre
concentrate and waste water. The nitrogen content of the fibre concentrate was
selected as the key quality parameter because it is important to keep this value as low
as possible to minimise emission of NOx gases, when this fraction is burnt to produce
energy, and to maximise the nitrogen content of the protein fraction. The fibre
concentrate, produced using the standard process settings, typically had a nitrogen
content of 2.38 % on a dry weight basis.

Protein concentrate
The flexibility of the process allows the protein concentrate to be produced with a
dry matter content of 20 - 30 %. A 20 % slurry can be pumped and is suitable for

4
delivery to pig farms equipped to use wet feed whereas, the drier 30 % product is a
paste suitable for further drying for use as a poultry feed.
For pig feed the protein concentrate must have a crude fibre content of 100 g /kg
dry matter. The crude fibre content of spent grains (160 g/kg dry matter) can be
reduced more than 30-fold in the separation process to produce a protein concentrate
of 5 g/ kg dry matter. At this highest quality the concentrate can be used as a feed for
piglets and poultry. Nutritional analysis showed that this protein concentrate was
suitable for fattening pigs of between 45 and 110 kg body weight and can replace up
to 20 % of their daily diet. Crucially, pigs accepted this protein concentrate as part of
their diet. However, in the future feeding trials may be necessary. Processing the spent
grains from a 2 million hl brewery would produce approximately 2000 tons of this
protein concentrate (dry weight) per year.
There is an inverse relationship between the crude fibre content of the protein
concentrate and its economic value. The value of this product is related to the price of
soybean meal. The current market price (November 2000) for soybean meal (88 % dry
matter and 45 % protein) is 230 /ton. Typically the highest quality protein
concentrate has a protein content of 65 % and a market value equivalent to 110 %
that for soybean meal when sold as a wet pig feed (all values calculated back to 88 %
dry matter). This market value increases to 175 % that of soybean meal when sold as
dried poultry feed. In addition to generating higher net revenue, drying the protein
concentrate offers more flexibility as the dried poultry feed has a longer shelf life and
transportation costs are lower.
Surplus yeast, whirlpool trub and malt dust may also be added to the protein
concentrate. Addition of these by-products would increase the quantity of protein
concentrate by approximately 30 % but have only a small influence on both the
protein and crude fibre content. This mixed product would have the same estimated
market price.
The protein concentrate can also be mixed with untreated spent grains to produce a
product that is still of acceptable quality for pig feed purposes (crude fibre content
100 g/kg dry matter) but has a lower protein content and hence a lower market value.
The quantity of pig feed produced by a 2 million hl brewery would be 4000 tons in
this case. Obviously, intermediate qualities can be achieved by altering the mix ratio
of high quality protein concentrate and spent grains. This flexibility allows
production of pig feed to be adjusted to both the quality and quantity demanded by
local markets.

Fibre concentrate
The fibre fraction has a dry matter content of 40 %, although further optimisation of
the process should increase this to 45 %. The quantity produced is directly related to
the quality of the protein concentrate. For a 2 million hl brewery, production of the
highest quality protein concentrate (crude fibre content 5 g/kg dry matter) would
result in 5500 ton dry matter of fibre concentrate. This is reduced to 3500 ton dry
matter if maximum production of pig feed (crude fibre content 100 g/kg dry matter) is
achieved, by blending the protein concentrate with spent grains.
Analysis of the fibre concentrate found the product to be similar to wet sawdust. It is
suitable as a renewable energy source. The fuel is a clean biomass which does not
contribute to global warming. The levels of nitrogen (2.38 % dry weight) and sulphur
in the fibre concentrate are low enough to comply with Dutch regulations for flue
gas emissions (1).

5
The fibre concentrate can be burnt or gasified to produce energy either on-site or
by a third party e.g. an electricity generating station using biomass. On-site direct
combustion would be optimal and, due to the similarity of this product to wet sawdust,
available equipment would only require minor modifications. A moving grate
combustion plant accepts products with a dry matter content of 40 %. The water in
the fibre concentrate evaporates during combustion, thus reducing the net energy
yield. The NOx emission is estimated to be below 400 mg NOx / Nm3, meeting the
current Dutch legislation (1). Cyclones, and possibly an electrostatic filter, are
necessary to separate dust from exhaust gases. However, combustion tests are
necessary to validate the expectations made above and to check aspects like
transportation of fibres to and the possible formation of deposits in the combustion
chamber of the steam boiler.
Analysis of the energy yield of the fibre concentrate found the lower heating value to
be 19.27 MJ/kg at 93 % dry matter which may be calculated back to 7.2 MJ/kg at 45
% dry matter. For a 2 million hl brewery the capacity of the combustion installation
would be 4 MWth (6000 h operation time). At an estimated efficiency of 70 %
approximately 60,000 GJ can be generated. This is about 30 % of the fossil fuel
requirements of a modern brewery (specific energy consumption 100 MJ/hl).

Waste water
Waste water, produced as a result of treatment of 36,000 tons of wet spent grains in a
separation plant, would have a Chemical Oxygen Demand (COD) of 240 tons. This is
equivalent to an increase of 12 % in the COD of the waste water produced by a
modern brewery. The volume of waste water would be 12,000 m3, which is equivalent
to an increase of only 1.2 %.

Economic aspects
Basic cost calculations have been made for processing spent grains from a 2 million hl
brewery producing 36,000 ton/year wet spent grains i.e. the capacity of our current
separation plant (see table 1 for specifications).

Specification Value
Running time (h/yr) 8000
Capacity (kg wet spent grain/h) 4500
Power requirement (kW) 135
Surface requirement (m2) 150
Table 1: Specifications for the Pilot Separation Plant.

Investment cost are estimated to be 1.7 million (accuracy 30 %) for the fully
installed plant. Study estimates for running costs have been calculated for our pilot
separation plant (see table 2 for specifications).

6
 Cost Specification Cost / year ()
Energy ( 135 kW @ 0.096 /kWh) 104,000
Materials (conservation and cleaning) 56,000
Maintenance 40,000
Waste water ( dependent on local 20,000
situation)
Total running cost 220,000
Table 2: Estimated running costs for separation plant (accuracy 30 %).

At present, there is no combustion plant for energy production from the fibre
concentrate or drying equipment to produce poultry feed from the protein
concentrate, so calculations of net present value (NPV) are based on the sale of both
these concentrates to third parties at the brewery gate. The protein concentrate was
assumed to be 170 /ton (calculated as 88 % dry matter), which is equivalent to 75 %
of the price of soybean meal, leaving a 30 % incentive. The fibre concentrate was
assumed to be 20 /ton (at 45 % dry matter), equivalent to 60 % of the price of fossil
fuels. It was assumed that the interest rate was 7.5 % and depreciation was 15 years.
No account has been taken of transportation costs for either these products or spent
grains. Obviously, as a result of the reduction in volume there will be considerable
transport savings with these new products. The cost calculations show that the NPV is
highly sensitive to the value of the two products and spent grains. Where there are
currently positive revenues from spent grains e.g. the Netherlands, installation of a
separation plant would give no added economic advantage at present. However, in
countries where there is no revenue (some African countries) or negative revenue (in
Singapore the spent grains are transported to Malaysia), installation of separation
plants would be advantageous (figure 2).

NPV (106 )
12
10
Singapore
8
6
Africa
4
2 The Netherlands

0
-2 -10 0 10 20

-4
Spent grains price (/ton)

Figure 2: Economic viability of installation of a separation plant. The price used for
spent grains is for the wet product at the brewery gate.

7
On-site combustion of the fibre concentrate increases the economic viability of this
process by reducing both the energy costs for the brewery and transportation costs. It
is our objective to decrease both investment and running costs for the separation plant
by further optimisation of the process.

Environmental aspects
The fibre concentrate is a renewable energy source. In addition, use of this clean
biomass reduces the emission of fossil carbon dioxide. Approximately 30 % of the
thermal energy requirements of a modern brewery can be provided by use of this
fraction to produce energy on-site.
Sale of the protein concentrate to farmers for pigs instead of spent grains for cattle
reduces the number of customers by 80 % depending on local conditions. This is
achieved by both a reduction in the quantity of product and the fact that, in general,
pig farms have more animals than cattle farms. In the Netherlands the pig farms have
1000 pigs compared with 65 cows per cattle farm.
This considerable reduction in the quantity of protein concentrate to be transported,
compared with that of spent grains, leads to important savings on transportation which
has environmental as well as economic benefits.
Today, security and traceability of animal feed are becoming increasingly important.
Here another benefit of the separation process for spent grains becomes apparent;
reduction in the number of customers allows the products to be traced back to the
producer more easily.

CONCLUSIONS
The separation process is a novel process producing two marketable products, a
protein concentrate and a fibre concentrate. The protein concentrate is suitable as
a feed for non-ruminant animals including pigs and poultry. The process is flexible
and allows the quality of this product to be adjusted to the demands of the local
market. In addition, other by-products including surplus yeast can be added to this
product. The fibre concentrate is similar to wet sawdust and it is likely that proven
combustion techniques will enable steam to be produced from this biofuel.
The separation process is economically viable and in many countries economically
advantageous. Use of the fibre concentrate as a fuel is environmentally sustainable
and savings on fossil fuel have been calculated to be 30 MJ/hl equivalent to 30 % of
the thermal energy requirements of a modern brewery. Heineken is committed to
environmentally friendly solutions. This alternative use of spent grains is
environmentally friendly, offers a long-term solution and is applicable globally.

REFERENCES
1. Nederlandse emissierichtlijn lucht (NeR). Infomil, Informatiecentrum
Milieuvergunningen (September 2000).

ACKNOWLEDGEMENTS
P. Bruijn, P. Deurinck and R. Noordman would like to thank 2B Biorefineries for their
collaboration on this project and permission to publish this work.

8
106

Enhancing the value of spent yeast and

brewers spent grain
Peter J. Rogers, Michael Pecar, Aldo Lentini, Adrian Gardner &
Joseph Kulandai
Carlton and United Breweries Ltd, 4-6 Southampton Crescent, Abbotsford, Australia 3067 (e-
mail: [email protected])

Descriptors
Agriculture, by-product disposal, fungicide, spent grains, waste yeast

SUMMARY
Spent brewers grain derived products exhibit strong inhibition towards plant fungal
pathogens, including Rhizoctonia, Microdochium and Fusarium sp in laboratory and
agricultural field trials. The product from spent grain enhances visual appearance, plant
growth and resistance to disease. The results are due partially to nutrient availability, and
significantly to a series of changes which promote the suppressive character of soil - the
antagonistic character of the microflora to pathogen domination - which is desirable for
cereal crop and turf production. Concentrated yeast (20% w/w) has been developed for food
applications, eg sandwich spreads, with acceptable sensory properties, to replace
conventional more dilute brewers yeast.

Alternative Verwertung von Resthefe und Trebern aus dem Brauprozess

Deskriptoren
Abfallhefe, Fungizid, Landwirtschaft, Nebenproduktbeseitigung, Treber

ZUSAMMENFASSUNG
Produkte aus Biertrebern weisen starke Hemmung gegenber Erregern von Pilzkrankheiten
an Pflanzen auf, u.a. von Rhizoctonia, Microdochium und Fusarium sp. Dies zeigten sowohl
Laborversuche als auch landwirtschaftliche Feldversuche. Das Produkt aus Biertrebern
verbessert die uere Erscheinung der Pflanze, das Wachstum und den Widerstand gegenber
Krankheiten. Die Ergebnisse zeigen Einflsse durch die Verfgbarkeit von Nhrstoffen und
besonders durch eine Reihe von unterdrckenden Einflssen des Bodens antagonistischer
Einfluss der Mikroflora gegenber der Dominierung von Pathogenen die fr die
Getreideernte, aber auch fr die Bodenkrume wichtig sind. Konzentrierte Hefe wurde fr
Nahrungsmittelanwendungen, z.B. als Brotaufstriche, mit annehmbaren sensorischen
Eigenschaften entwickelt. Solches Konzentrat knnte herkmmliche, eher dnnbreiige
Brauerhefe ersetzen.
Comment augmenter la valeur des levures et drches usages

Descripteurs
Agriculture, drche, fongicide, levure rsiduelle, rejet des sous-produits

RESUME
Les produits drivs des drches usages prsentent un puissant effet inhibiteur sur les
champignons phytopathognes, dont Rhizoctonia, Microdochium et Fusarium, en laboratoire
et dans les terres agricoles. Le produit des drches usages amliore l'aspect visuel, la
croissance des plantes et leur rsistance aux maladies. Les rsultats sont partiellement ds
la disponibilit des nutriments et significativement une srie de changements qui favorisent
le caractre suppressif du sol - l'antagonisme de la microflore la domination des pathognes
- ce qui est souhaitable pour la rcolte des crales et la production de tourbe. Des formes
concentres de levure (20 % p/p) ont t dveloppes pour les applications alimentaires, p.
ex. en sandwichs, avec des proprits sensorielles satisfaisantes, pour remplacer la levure de
bire classique, plus dilue.

2
INTRODUCTION
Brewers spent grain and spent yeast are sources of a wide range of chemicals and
macromolecules. Typically however these resources have been disposed of at low cost
for cattle feed (1) and as resource material for the food industry (eg 2). Greater value
adding although highly desirable, still depends on access to markets and the
development of processes that are aligned to core business capabilities (3). We have
sought to couple the development of novel products to existing coproduct activity.
Our yeasts are already used for the manufacture of food spreads (4). Yeast ghosts
from the yeast autolysis process can be used for the manufacture of glucan products.
Glucans are considered to be immunostimulants and more recently their cholesterol
lowering activity has been recognised (5,6). In 1997 our company launched Shrimp
Activa a micron sized yeast glucan preparation for use as an immuno-stimulant
additive for incorporation into shrimp feed (7-9). Trials in Thailand, South America
and Australia showed that Shrimp Activa increased shrimp yields significantly.
The introduction of cross flow filtration for beer recovery from yeast, has also
provided highly concentrated yeast streams. We have consequently sort to enhance the
value of the yeast through a greater understanding of our customers requirements,
and the physiology, and rheological and organoleptic behaviour of yeasts recovered
after different operations in the plant.
More recently spent grain has been fractionated to make a product that is suited to turf
management applications as well as other cropping activities.

CONCENTRATED YEAST
Cross flow filtration
Spent yeast was concentrated using a 95m2 ceramic cartridge cross flow filtration unit.
This enabled beer recovery and potentially provided savings for our customers
through compression of the yeast extract process. Table 1 compares the traditional
spent yeast with the crossflow derived yeast, with and without water diafiltration (2
vol water). The two yeasts had comparable viability. There was a noticeable loss of
protein after cross flow filtration. Hot water extracts were prepared and concentrated
and dried. The cross flow yeast extract was similar overall to the control spent yeast,
apart form a small reduction in the protein content. The dia-filtrated yeast produced an
extract with a lower protein content and a higher carbohydrate content (Table 1).

Sensory issues
Analysis of cross flow yeast extract (no dia-filtration) identified acetoin, dimethyl
disulfide (DMDS), 2methylbutenol, methylethyl ketone, and methanthiol, which were
not detected in the control yeast extract.
DMDS was considered offensive even in ppb levels, when incorporated into yeast-
based products. Pilot plant trials showed these volatiles were due to yeast metabolism.
Adding back beer, or beer proteins, or reducing the pH of the concentrated yeast slurry
to <4 prior to extraction could repress the formation of the meaty DMDS flavour.
Concentrated yeast prepared using discharge centrifuges in series, was also capable of
producing a very similar meaty volatile profile in yeast extracts. Similarly, these
flavour characteristics could be suppressed by the steps described above.

3
Taken together these data showed that brewing yeast are able to produce a range of
flavours, which may have serious consequences or alternatively provide opportunities
in food processing.
Sample Protein Vitality DW Extrac Soli Protei CH As Fat
t yield ds n O% h

Spent yeast 45 90.5 28 39 63 41,5 12 9 0.4

Cross flow yeast 42 90 23 38 60 40 11.5 8.5 .3
Cross flow 42.5 90 22.5 42 59 34 17.5 7.5 0.3
diafiltered
Table 1: Comparison of typical spent yeast and cross flow yeast diafiltration. Italics data
refers to yeast extract made from these yeasts.

GLUCANS
Glucans have been implicated in stimulation of the immune system in many
organisms and as causative agents for the reduction of plasma cholesterol levels in
animals and man (10,22). In invertebrate animals, there is a non-self recognition
system for recognizing microorganisms - the prophenoloxidase activating system
(proPO-system) (8). When activated it produces melanin deposits on the foreign
intruder. The proPO-system is turned into its active form by miniscule amounts
(picograms/litre) of fungal beta-1,3-glucans, bacterial lipopolysaccharides or peptido-
glycans. As a consequence, a series of serine proteinases are induced to their active
form. The final result is the specific proteolytic cleavage of prophenoloxidase and the
active enzyme phenoloxidase can then produce melanin from phenols, which is toxic to
fungi and trigger other defense mediators (12). These elicitors can gain access through
wounds and through the gut. Particles > ~1 micron are excluded from the midgut, the
hepatopancreas, where synthesis and secretion of digestive enzymes, and all
absorption of dietary products occurs (13). So it is important to gain access to the
midgut if the immune response is targeted through the feed chain. We previously
reported (9) a process for milling yeast envelopes sometimes referred to as ghosts that
are recovered after hot water extraction. The milling reduces particle size to ~0.1
micron in diameter. The particles have high glucan content (60%) and a high glucan
recognition characteristic according to the Fungitec G Test Kit glucan assay, similar to
the LAL endotoxin test (not shown) (11, 14).
There is evidence that exogenous glucans can stimulate the immune system of shrimp
(prawns) (8,15). This was confirmed when we conducted prawn farm trials in northern
Australia, Indonesia, Thailand and Ecuador which all showed a significant
improvement in the survival rate of the prawns - by as much as 50% compared to the
controls. Improvements in yield and resistance to bacterial challenge were also
observed (Figure 1 & Figure 2) in trials carried out in the three regions.
Challenge trials were carried out with Litopenneas vannamei by monitoring mortality
after a LD50 dose of Vibrio parahaemolyticus (Figure 1). Mortality for populations
fed with Shrimp Activa supplements (1%) was 20% as opposed to 50% for the control
(minus Shrimp Activa). Some commercial glucan preparations, and also whole yeast,
and yeast envelopes that were not were milled were less effective. This may be related
to the small mean particle size of Shrimp Activa and hence better access to the
absorptive mid gut of the shrimp. This has not been unambiguously established. The
milling process appears to shear off some of the external mannan from the yeast cell

4
wall and expose the underlying glucan matrix, and also reduce the particle size (9).
Linkage analysis showed that glucan content is greater than 60% on a dry weight basis
(data not shown). White spot virus remains the greatest threat to commercial shrimp
farming. Although glucans provide vigour, they do not immunise against the virus.

80
W i t h V ib r i o
c h a l le n g e
60
% survival

40

20

c o n tro l p lu s c o n tro l p lu s
fe e d A c tiv a fe e d A c tiv a

Figure 1: Survival of Shrimp (Litopennaeus vannamei) fed with a control formulation and
1% Shrimp Activa incorporated into the diet and challenged with Vibrio
parahaemolyticus. These trials were carried out at the AquaLab hatchery and pond
farm in Ecuador.
B lack tiger shrim p B la ck tige r shrim p
(P . m onodon) (P . m onodon)
N orthern A ustralia SouthernT hailand
4000 4000

30
Average pond survival %

30

2000 2000 20
20
Kg / ha

10
10

plus control plus control

Shrim p fee d S hrim p feed
A ctiva A ctiva

Figure 2: The effect of BioTurf supplementation on survival and yield of black tiger shrimp
in culture.

BIOTURF
BioTurf is a product that originated from a desire to value add, and minimise trade
effluent discharge from the breweries. Spent grain is a source of complex
carbohydrates that may have biological activity (16). The Kirin Brewery Co. for
instance has developed a food made from spend brewer's grain for patients afflicted
with ulcerative colitis In agriculture, yeast extracts have been used to fortify plants and
to stave off disease with various reported rates of success (17). These extracts contain
high levels of complex carbohydrates and at first we considered spent grain would be
able to supply similar molecules that may be biologically active in plant systems.
There are at least 50 published references describing the effects of various yeast
extracts against plant diseases, both in vitro and in vivo (eg ref 17). There are also

5
reports of composts that promote suppressive quality in soils, especially those that
are used regularly for monoculture farming (18 - 21). Suppressiveness refers to
pathogen resistance, and usually in the context of broad acre, cereal crop production.
Suppressiveness in soils has been attributed to antagonistic effects of existing
microflora, faster propagation of bacteria in suppressive than in susceptible soils and
reduced germination of inocula in the former and latter kinds of soil. The complex
microflora as well as the composition of the flora is believed to keep pathogens in
check. There is evidence for the control of take-all disease (Gaeumannomyces
graminis) in wheat by such a mechanism (18). Our spent grain has a typical microbial
profile, and the well being promoted in dairy cattle supplied with spent grain has
anecdotally been attributed to the microflora - at least in part.
Taken together we believed there was a possibility that spent grain fractions could
stimulate plant well being by providing nutrients (N,P and K, and organics), by
stimulating defence mechanisms, and by promoting soil suppressive quality.
We restricted our preliminary investigations to studying the effects of formulations
based on yeast fractions, spent grain fractions and hop extracts, on commercial turf,
growth and health. Turf management in Australia and in North America centres on
recreational turf such as golf courses and horse racing tracks. In Australia there are
over 450 first class golf courses that have large curatorial budgets. Turf is a
monoculture and there are many varieties of grass in commercial use. Nevertheless it
is likely that the results on turf may be extended to include other monoculture grass
related crops such as rye and barley and wheat.

BioTurf composition
Spent grain was fractionated as shown in Figure 2. The compositions of the soluble
and particulate fractions from spent grain are shown in Table 2. These fractions in turn
were combined with yeast extract and glucan to make BioTurf (Table 3). Both liquid
and dry, powder product was prepared.
BioTurf also contains significant amounts of the amino acids glutamine and
glutamate, aspartate and asparagine, proline, leucine and lysine. It also retains a
mixture of microrganisms.

Biological Activity of BioTurf

The core spent grain fractions were trialled on commercial turf at the Huntingdale
Golf Course in Melbourne, and at turf farms in central Victoria (Table 4). Both the
particulate and the soluble fractions stimulated growth compared to control turf that
received no additions or turf treated with inorganic fertilisers. Trials were carried out
on sandy and heavy soils and loam. In all cases BioTurf improved appearance, the rate
of growth (45% reduction in time to maturity after sowing), reduced fungal scar
appearance such as the typical Rhizoctonia horse shoe, and enhanced the cation
exchange capacity of the underlying soil (up to 3 fold increase for sandy soil).

Product Solids Feed for: Activity

Spent Grain 20% dsb Filter press
Spent Grain Liquor 8% dsb Decanter Biologically Active*
Wet Particulate Paste 25% dsb Drier / stabilised Biologically Active
Dry particulate Fraction >92% w/w% Biologically Active And Stable
Soluble Fraction 4% dsb Evaporator / Drier Biologically active
Dry Soluble Fraction >92% w/w Some Biological Activity
Table 2: Summary of Brewers Spent Grain Fractionation Process

6
 Dry Particulate Fraction (w/w%)
Total phosphorus 0.05
Total potassium 0.1
Total nitrogen 6.4 7
Total protein (approx. TN x 6.25) ~ 40-
Total carbohydrate (fermentable) 1
Dry Soluble Fraction (w/v%)
Total phosphorus 0.01
Total potassium 0.03
Total nitrogen 0.7
Total protein (approx. TN x 6.25) 4.4
Total carbohydrate (fermentable) 4
Table 3: Composition of the particulate and soluble fractions isolated from Brewers Spent
Grain

Carbohydrates and Lipids BioTurf Liquid (%w/v) BioTurf Powder

(%w/w)
Fructose 0.06 0.4
Glucose 0.22 1.6
Maltose 0.24 2.5
Maltotriose 0.44 4
Total fermentable sugars 0.96 9.5
Non-Fermentable Sugars 4.3 54
Lipids (%w/w) 1 15
Table 4: Composition of BioTurf

SPENT GRAIN FRACTIONS APPLICATION APPEARANCE SCORE

RATE
Control Converging grass 2
Spent Grain soluble Liquid 60 L / 100 m 2 Strong, thick, green grass 5
& particulate structure
Spent Grain soluble Dried 4 kg / 100 m2 Strong, thick, green grass 5
& particulate >92% w/w structure
Spent Grain 20%dsb 6 kg / 100 m Strong, thick, green grass
2 5
particulate paste structure
Spent Grain Dried 3.5 kg / 100 m Thick, green, apparent
2 5
particulate dried >92% w/w stimulation growth
Spent Grain soluble Liquid 10 L / 100 m2 Slight improvement 3
Spent Grain soluble Dried 3.5 kg / 100 m2 Slight improvement 3
>92% w/w
No treatment i.e. no 1
additions but water
Commercial As per suppliers 2
inorganic fertilizer instructions
Table 5: Effect of Spent Grain Fractions on turf appearance following trials on bent grass,
couch grass, Fescue grass, and rye grass at a turf farm in central Victoria, and golf
courses in the sand belt area of Melbourne.

The positive effects of BioTurf on turf quality that we achieved in these trials were
better than the results achieved with inorganic fertilisers, even when these treatments
were supplemented with fungicides.

7
Pathogen control
BioTurf can provide basic nutrition in the form of N, P and K. The apparent reduction
in the incidence of plant disease in BioTurf treated areas also suggested that BioTurf
may enhance resistance to pathogens and foster suppressive soil character.
Suppressiveness is typically characterised by high populations of the following soil
microrganisms- 1) fluorescent pseudomonads, 2) actinomycetes sp, 3) spore forming
bacteria and 4) saprophytic fungi. BioTurf treatment increased the levels of some of
these organisms (Figure 3).
Pathogens were isolated from samples collected from the farms and the golf course.
Two significant pathogens were isolated. Microdochium bolleyi (De Hoog &
Hermanides Nijhof) was isolated from couch and bent grass (golf course) and
Rhizoctonia solani (Kuhn) from bent grass subsoil. Other pathogens included
Fusarium species.
Poison well trials evaluated the effect of BioTurf on the spread of pathogenic mycelia
across the surface of agar plates (Figure 4). In other tests, BioTurf was added to the
agar medium to test the effect on the spread of inoculated pathogens across the
surface.
1 00

50

A B C D + L ow + H igh
C ontrol B ioT urf B ioT urf

Figure 3: Effect of BioTurf addition to couch grass in open field trials, on the population of
the actinomycetes, endospore forming bacteria, saphrophytic fungi, and
fluorescent pseudomonads. Cell numbers on the y axis: A) fluorescent
pseudomonads cfu / g soil; B) actinomycetes x 107 cfu / g soil; C) endospore
forming bacteria x 103 / g soil, D) fungi cfu / g soil.

Figure 4: In vitro well experiment - growth of Microdochium bolleyi in the presence of; left
photograph, a). water wells (control); b) sterilised Bioturf wells; c) unsterilised
Bioturf wells. Right photograph composite, M. Bolleyi mycelium diameter after

8
 seven days growth on PDA poisoned with BioTurf - a) 0% b) 0.25% c) 0.5%
d) 0.75% e) 1% f) 10% w/v.

Studies such as those shown in Figure 4 showed that Bioturf significantly reduces the
mycelium diameter of M.bolleyi and has a smaller effect on R.solani. Distributing
BioTurf throughout the agar poisons the growth medium and inhibits both pathogens.
Heat-treated BioTurf and combinations of BioTurf and antibiotics also elicited an
inhibitory response in well and poisoned agar tests. Thus chemical components of
Bioturf appear to inhibit the growth of M.bolleyi, and to a lesser extent R.solani.
Mycelium diameter after 7days mm

50

PD A PDA PDA
+ 0 .1 % + 1%
B io T u rf B io T u rf

Figure 5: M.Bolleyi mycelium diameter after seven days growth on potato dextrose agar
(PDA) Poisoned With BioTurf - a) 0%, b) 0.1% and, c) 1% w/v

The biological components are particularly active in restricting the growth of both
pathogens. BioTurf appears to result in increased growth in commercial bent grass
varieties used for recreational turf. While an increase in growth may be desirable
because it allows plants to outgrow pathogens, or to reach maturation more quickly, it
is on the other hand likely that this effect may be better suited to other commercial
crops where increased production is important. Thus BioTurf has the potential to be
used on cereals such as wheat or rice, which are closely related to turf grasses and also
to cotton which is particularly susceptible to infection by Fusarium oxysporum..

ACKNOWLEDGEMENT
We thank Professor David Guest and Christine Hall, Botany Department University of
Melbourne, for their skilful assistance and advice.

REFERENCES
1. Davis, C.L., Grenawalt, D.D. and McCoy, G.C., Journal of Dairy Science, 1983,
66, 73 79.
2. Chen, E., Alli, I., Ervin, V., Crowe, N.E., Baker, B.E., 1993, Canadian patent,
no 1317413.

9
3. Anheuser Busch Company Product Stewardship Program web site, 2001,
www.abehsreport.com/data/
4. Vegemite web site, 2001, www.vegemite.com.au
5. Stone, B.A. and Clarke, A.E., Chemistry and biology of (1 3) beta glucans.
LaTrobe University Press, Melbourne, 1992, 525 pp.
6. Nicolosi, R., Bell, S.J., Bistrain, B.R., Greenberg, I., Forse, R.A. and Blackburn,
G.L., American Journal Clinical Nutrition, 1999, 70, 208 212.
7. Shrimp Activa web site, 2001, www.shrimpactiva.com.au
8. Soderhall, K., Cerenius, L., Current Opinion in Immunology, 1998, 10, 23-28.
9. Wheatcroft , R., Langereis, W., Kulandai, J., Gilbert, R.W., Sime, K.J. and
Smith, C.G., 1996, Patent JP 701745.
10. Bohn, J.A.Q. and BeMiller, J.N., Carbohydrate Polymers, 1995, 28, 3 14.
11. Ohno, N., Uchiyama, M., Tsuzuki, A., Tokunaka, K., Miura, N.N., Adachi, Y.,
Aizawa, M.W., Tamura, H., Tanaka, S., Yadomae, T., Carbohydrate Research,
1999, 316, 162-172.
12. Graham, D.G., Tiffany, S.M. and Vogel, F.S., Dermatology, 1978, 70, 113-116.
13. Thornqvist, P-O and Soderhall, K., Diseases in Asian Acquaculture III, Asian
Fisheries Society, Philipines, Diseases in Asian Acquaculture III, 1997, Asian Fisheries
Society, Philipines, 1997 pp 56-63.
14. Aketagawa, J., Tanaka, S., Tamura, H., Shibuta, Y., Saito, H., Jourmal
Biochemistry, 1993, 113, 683-686.
15. Cerenius, L., Liang, Z., Duvic, B., Keyser. P., Hellman, U., Palva, T., Iwanaga,
S. and Soderhall, K., Jouirnal of Biological Chemistry, 1994, 269, 29462
29467.
16. Raston, C.L., Chemistry. Australia, 2000, 67, 21 23.
17. Wilson, C.L. and Plusey, P.L., Plant Disease, 1985, 69, 375 389.
18. Hornby, D., Annual review of Phytopathology, 1983, 21, 65 85.
19. Weller, D.M., Annual review Phytopathology, 1996, 26, 379 407.
20. Birch, R.G., Australian Plant Pathology 1986, 15, 51-56.
21. Gutterson, N., Critical Reviews BioTechnology, 1990, 10, 69 91.
22. Nicolosii, R., Bell, S.J., Bistriam, B.R., Greenberg, I., Forse, R.A. and
Blackburn, G.L., American Journal of Clinical Nutrition, 1999, 70, 208 212.

10
107

Use of spent grains

Werner L. Kepplinger1 & Gerald Zanker2
1
Institut fr Verfahrenstechnik des industriellen Umweltschutzes, Peter Tunner Strasse 15,
A-8700 Leoben, Austria (e-mail: [email protected])
2
Brauunion sterreich, Poschacherstrasse 35, Postfach 281, A- 4010 Linz , Austria

Descriptors
Air pollution, by-product disposal, combustion, spent grains

SUMMARY
In the brewing process spent grains are obtained as a by-product. In future some problems
will occur:
decreasing use of malt returns in agriculture
restricted possibilities and/or strict legal limitations for waste disposal and land fill
intermediate storage only possible after expensive treatment.
The basic idea of the process development of Brau Union Austria and the Institute of Process
Engineering of the University of Leoben was the combination of mechanical predrying and
combustion in a bio-mass vessel. The main problems like low efficiency of mechanical
drying, NOx and SO2-emissions in the vessel as well as the use of the press-water could be
solved in a 3-years research programm which was sponsered by the Austrian
"Forschungsfrderungsfonds". Basic results of lab-scale tests could be confirmed in two
campaigns on industrial plants (continous press filter and 1000 and 3000 kW biomass vessel).

Die Verwendung von Biertrebern

Deskriptoren
Luftverschmutzung, Nebenproduktbeseitigung, Treber, Verbrennung

ZUSAMMENFASSUNG
Im Brauprozess fallen Trebern als Nebenprodukt an. In der Zukunft wird dies zu einigen
Problemen fhren:
abnehmende Verwendung in der Landwirtschaft;
eingeschrnkte Mglichkeiten oder strikte gesetzliche Rahmenbedingungen fr die
Abfall-entsorgung;
Zwischenlagerung nur nach aufwndiger Behandlung.
Die Grundidee der beteiligten Partner war die Kombination von mechanischer Vortrocknung
mit der Verbrennung in einem Biomasse-Kessel. Die Hauptprobleme wie die geringe
Effizienz der Vortrocknung, die NOx- und SO2-Emissionen im Kessel wie auch die
Verwendung des Presswassers konnten in einem dreijhrigen Forschungsprogramm gelst
werden. Dieses Forschungsvorhaben wurde vom sterreichischen Forschungsfrderungsfonds
untersttzt. Grundlegende Ergebnisse im Labormastab konnten in zwei Kampagnen an
industriellen Anlagen besttigt werden (kontinuierliche Filterpresse und 1000- and 3000-kW-
Biomasse-Kessel).

Utilisation des drches usages

Descripteurs
Combustion, drche, pollution de l'air, rejet de sous-produits

RESUME
Les drches usages sont un sous-produit du maltage. A l'avenir, certains problmes se
poseront:
moindre usage du malt retourn dans l'agriculture
restriction des possibilits et/ou limitations lgales strictes pour l'vacuation et l'pandage
des dchets
le stockage intermdiaire n'est possible qu'aprs un traitement coteux.
L'ide de base du dveloppement du procd de Brau Union Autriche et de l'Institut
d'Ingnirie des Mthodes de l'Universit de Leoben tait d'associer un prschage mcanique
la combustion dans un fermenteur. Les principaux problmes, comme la faible efficience du
schage mcanique, les missions de NOx et de SO2 dans le fermenteur ainsi que l'emploi de
l'eau de pressage, pourraient tre rsolus par un programme de recherche de 3 ans sous l'gide
du "Forschungsfrderungsfonds" autrichien. Les rsultats fondamentaux d'essais l'chelle du
laboratoire pourraient tre confirms dans deux campagnes en installations industrielles
(filtre-presse en continu et fermenteurs de 1000 et 3000 kW).

2
INTRODUCTION
Spent grains are a by-product in the brewing process. They have a moisture content of
appr. 80 %. Approximately 20 kg are produced per hectolitre of beer. In this paper the
integration of an utilisation plant for 20 000 tpy of wet spent grains will be discussed.
This equals the yearly production of a brewery with a capacity of 1 million hectolitres
of beer per year.
The common use of spent grains is food for cattle. In wet form the spent grains are not
stable and have to be consumed within 2 or 3 days otherwise biological degradation
starts. To make a stable and storable product drying is necessary. This was commonly
done in contact dryers because of minimised emmissions. This methode, however, is
relatively expensive and is a high burden in the sales price. Generally can be said that
the costs for thermical drying compared with the mechanical removing of water is
considerably higher. Only at the end of the drying the removal of the final amounts of
water the thermic treatment becomes cheaper.
Another serious problem in the future is the development of the population of cattle.
Figure 1 shows this development in the federal country of Styria in Austria wich
reflects the general trend in Central Europe. The problem could become still more
serious because of different animal deseases. Together with this problem and the
restricted or expensive deposing off requires a economical as well as ecological
feasible solution for the use of spent grains. Legal limitations for landfill materials
such as the limitation of the organic carbon content with maximal 5 % will be
introduced in nearly all member states of the EC.

2 50
No. Of cattle (thousands)

2 00

1 50

1 00

50

0
197 0 1 98 0 1 990 1 993 199 5 200 0

Development of cattle in Styria

Figure 1: Development of the population of cattle in Styria

A number of alternative solutions had been proposed to find applications for spent
grains:
- Additive in making of bricks with low density
- Production of pressed panels for insolation in the building industry (here a problem
might be the attack of insects or bacterias)
- Additive in human or animal food
- Co-combustion in powerstations, here the problem is the very low calorific value
(see figure 10).

3
Under these preconditions a new process for the energetic utilisation of spent grains
was developed in cooperation of the Brau-Union sterreich and the Institute of
Process Engineering of the University of Leoben, Austria.

The Austrian fund for the promotion of research (Forschungsfrderungsfonds der

gewerblichen Wirtschaft) supported the project.
Figure 2 summarizes the problems and requirements for the process development:
- high water content/low calorific value
- mechanical drying is necessary
- press water has high organic load
- separate thermal drying has to be avoided
- heat recovery by combustion
- disposal is expensive
- full recycling is necessary

Problems with thermal

utilisation of the spent grains

High water content of the grains, approx. 80%

Mechanical drying necessary
Press water - High organic content
Thermal drying - high energy requirement and emissions
Combustion - NOx content exceeds 20 x limit
Disposal of approx. 150 t Ash

Figure 2: Problems and requirements for the process development.

Of great importance are the emissions to the air. In Europe the TA-Luft is a
generally accepted guide for gaseous emissions. In figure 5 the relative values for the
following gas components are illustrated: NOx, CmHn, SO2, CO and dust.

Silo
big bags
Clarifier 1 Clarifier 2 Press

Existing silo
Buffer 420 m3 Collection
Existing plant

Wet Steam for

spent Stack
Mech. brewery
grains dried
spent
Continuous grains Intermediate Bio mass Waste gas
pressure bunker vessel treatment
filter

Anaerobic Ash for

treatment further
use

Treated
water

Figure 3 depicts the basic flow diagram of the process with its connection to the
brewery.

4
The wet spent grains from the production with an water content of appr. 80 % are fed
to the continuous pressure filter via an intermediate storage bin to compensate
deviations in the production. From here they are continuously charged to the pressure
filter. The water content is now reduced to a level below 58 %. The dried material is
now much more stable and can be stored in an intermediate bin to compensate the
different requirement of the subsequent bio-vessel. They can be stored now for several
days without danger or unwanted organic reactions. This is important for the weekly
start-up of the plant to have sufficiant fuel for the bio-vessel. The press water can be
either processed in ananaerobic water treatment or utilized internally in the brewery.
In the case of an anaerobic waste water treatment Methan-containing gas is produced
which is an additional fuel in the bio-mass vessel. The ash obtained after the
combustion can be utilized as fertilizer additive. Wet cleaning of the flue gas is
normally not necessary. In the bio-mass vessel approx. 60% of the steam requirement
for the brewery can be produced.
Figure 4 shows in a Sankey diagram the main mass flows of the process. The values
are related to a 1 million hectolitre per year brewery. Depicted are the streams of
water, solids and steam, not considered is the mass flow of the combustion air.

Figure 4: Sankey diagram of the main mass flows of the process

1 00

90

80
% TA-Luft limits

70

60

50

40

30

20

10

0
NOx SO2 Cm Hn CO D ust

Relative emissions of Bio Mass vessel

Figure 5: Relative values of NOx, CmHn, SO2, CO and Dust

5
It can be seen that all limits for bio-mass vessels of this size are met and this without
any special waste gas treatment. These measurements were made during a series of
pilot tests in commercial scale. It has to be mentioned that this tests were made on
bio-mass vessels which were not optimized for the combustion of spent grains.

Cross section of continuous pressure filter

Figure 6: The mechanical removal of a high amount of water is possible with this
equipment.

In the speach is a short video sequence available which shows a section of a

continuous pressure filter during an industrial scale test. The throughput during this
test periode was appr. 5 t/h of wet spent grains. This dried material was collected and
used for a subsequent combustion test on a commercial bio-mass vessel, to proof the
suitability for that purpose.

Grain Press

Figure 7: The arrangementmof the optional press water cleaning unit behind the
continuous pressure filter.

With this equipment a very clean press water can be obtained with very low content of
suspended matter. The filtercake can be recycled to the wet spent grains. So a fully
closed recycling loop is possible.

6
 Bio mass vessel

Figure 8: The cross section of a typical bio-mass vessel suited for the combustion of
pre-dried spent grains.

The thermal capacity of these vessels ranges between 2 000 to over 10 000 kWh. The
possible deviation from the nominal capacity of this type of vessels is +/- 25 % e.g.
nominal capacity 4 000 kWh, minimum capacity 3 000 kWh and maximum capacity
of 5 000 kWh. This flexibility allows to cope with the variation of the energy
requirement during the brewing cycle. The refractory lining in the vessel partly serves
as reflector for the radiation heat to maintain a further preheating and drying of the
spent grains. By this means a very well controlled combustion can be reached. Also
variations in the moisture content of the spent grains can be compensated.

Combustion

Figure 9 shows a video sequence respectively in a still photo the the very good
combustion behaviour of the spent grains in the vessel, even the test equipment was
designed for saw dust and bark and not for spent grains.

7
 2100 0

1600 0

C alo rific Valu e kJ/kg

1100 0

600 0

100 0

-400 0

Dry substance 100 0

Moisture 0 100

Net calorific value Vs Moisture content

Figure 10 shows that the calorific value of the dry substance is similar to lignite coal
or dry wood with appr. 21 MJ/kg. The effective calorific value depends on the water
content. To obtain the highest possible energy recovery a water content below 60 % is
desired. The wet spent grains with an water content of 80 % have a very low calorific
value due to the evaporation of the water content. The calorific value can be
calculated according the equation

H = (1-w ).21000 w.2250 H.....calorific value, w = water content

Above 40 % dry substance (or 60 % water) the combustion properties improve

considerably.

1 8 .0 0
R e q u i re m e n t N m 3
1 6 .0 0 B io G a s N m 3

1 4 .0 0

1 2 .0 0

1 0 .0 0

8 .00

6 .00

4 .00

2 .00

0 .00
22 : 3 0 0:10 1:50 3: 3 0

Natural gas and Bio gas utilisation

Figure 11 depicts the energy requirement in a brewery during three brewing batches.

It can been seen that periodes of high energy requirement change with periodes with
low energy requirement. The area in the lower part of the picture show the energy
produced from thr bio-mass vessel,the area above this line show the amount of
imported primary energy. In this case appr. two thirds of the total energy are supplied
by the bio-mass vessel.

8
 100%
C aO
MgO
80% K 20
P 2O5

60%

40%

20%

0%
Sp ent g rains Saw du st B ark

Comparison of ash

Figure 12: Here is compared the chemical composition of ashes of different origin.

We see here ash from spent grains,saw dust and bark. The ash from the spent grains
show a very different picture. The high P2O5-content is a very important issue because
it is an effective fertilizer component. Therefore the ash can be added to standard
fertilizers like the well established NPK-fertilizers (N stands for nitrogen, P for
phosphorus and K for kalium = potassium). Depending on the ash content yearly appr.
150 tons of ash could be utilized as fertilizer additive.

3 .5

2 .5
Value in ATS

1 .5

0 .5

0
as h s aw d u st b a rk

Value of fertiliser components

Figure 13 shows the value of the ash components

From the economical point of view the P2O5-content has a certain value. This is also
a credit to the production costs.

9
 R even u es fo r 1 Mill. h L /y
8
0,53
0,49 0,49
7
AT S [Mill.]

million ATS/year
6
P ayb ack
5 p erio d (years)

2
0,09
1

0
s a le o f g r a in s [1 .] T h e r m a l re c y c lin g [1 .] + P r e s s W a te r [1 .] + a d d itio n a l
+ e x is tin g a n a e r o b ic R e c y c lin g a n a e ro b ic tr e a tm e n t
tr e a tm e n t

Revenue

Figure 14 compares 4 different cases:

- Sale of spent grains as cattle food
- The utilisation of spent grains as fuel and the treatment of the press water in an
existing anaerobic water treatment plant.
- As before- but including of the installation of an anaerobic treatment, the same
revenue, longer pay-back periode
- As case 2 but with internal recycling of the press water to the brewery, same
revenue, shortest pay back periode. Not considered in these cases is a future
scenario that spent grains have to be deposed off. The expected cost will be in the
range of 150-300 Euro/ton as it is today for special or toxic wastes.

Commercial application proven by industrial scale tests

All environmental requirements can be fulfilled

(no additional CO2 production by using Bio gas)

Full integration into brewing process

Economical feasibility

Independence of cattle feed

Figure 15: Summary.

Figure 15 summarizes at a glanze:

Commercial application proven by industrial tests
All environmental issues fulfilled
Full integration into a brewery
Economical feasible
Independent of sales as cattle food..

10
108

The role of environmental biotechnology

for the brewing industry
T.L.F.M. Vereijken & W.J.B.M. Driessen
Paques B.V., P.O. Box 52, 8560 AB Balk, the Netherlands (www.paques.nl, e-mail:
[email protected])

Descriptors
Biotechnology, brewing industry, ecology, efficiency, environmental protection

SUMMARY
The role of environmental biotechnology for industry has changed considerably over the past two
years. The brewing industry has been at the front end of these changes, for instance by
implementing ISO 14001 certification, setting industry standards, and combining economy and
ecology with investments in new environmental biotechnologies. The European Committee of
Environmental Technology Suppliers Associations (Eucetsa) is using their experiences in the
field (for instance in the brewing industry), to serve as examples of so-called Best Available
Technologies, which form the basis for content of future environmental permits. This closes the
loop. The paper is using examples to argue that ecological efficiencies from "being green" are
probably larger than the initial gains from passive or conventional end-of-pipe approaches.

Die Rolle der Umweltbiotechnologie fr die Brauindustrie

Deskriptoren
Biotechnologie, Brauindustrie, kologie, Umweltschutz, Wirkungsgrad

ZUSAMMENFASSUNG
Die Rolle der Umweltbiotechnologie fr die Industrie hat sich in den letzten zwei Jahren
erheblich gendert. Die Brauindustrie befindet sich erst am Anfang vom Ende dieser
Entwicklung, beispielsweise durch Einfhrung der ISO 14001, Aufstellen von Industrie-standards
oder durch Verbindung konomischer und kologischer Interessen bei der Investition in neue
Umweltbiotechnologien. Der Europische Verband der Zulieferindustrie fr Umwelt-
biotechnolgie (Eucetsa) nutzt seine Erfahrungen auf diesem Gebiet, um (beispielsweise in der
Brauindustrie) die bestverfgbaren Technologien bereitzustellen. Dies ermglicht es, eine Basis
fr eine zufriedenstellende zuknftige Umwelt zu schaffen. Damit schliet sich der Kreis. Das
Poster verwendet Beispiele, die von den kologischen Vorteilen berzeugen und damit
klarstellen, dass dies weit mehr Vorteile bringe, als die ursprnglichen Absichten von passiven
oder End-of-Pipe-Lsungen.
Rle de la biotechnologie environnementale dans l'industrie brassicole

Descripteurs
Biotechnologie, cologie, efficacit, industrie brassicole, protection de l'environnement

RESUME
Le rle de la biotechnologie environnementale pour l'industrie a considrablement chang au
cours des deux dernires annes. L'industrie brassicole a t la proue de ces changements, par
exemple en mettant en oeuvre la certification ISO 14001, en tablissant des normes industrielles
et en associant l'conomie et l'cologie par des investissements dans de nouvelles biotechnologies
prservant l'environnement. Le Comit Europen des Associations de Fournisseurs de
Technologies Environnementales (Eucetsa) utilise son exprience en la matire (par exemple dans
l'industrie brassicole) pour servir d'exemple des "Meilleures Technologies Disponibles", formant
la base du contenu des futurs permis environnementaux. Ainsi la boucle est boucle. En
s'appuyant sur des exemples, cet article dfend l'ide que les efficiences cologiques d'une
"attitude verte" sont probablement plus importantes que les gains initiaux d'approches de fin de
chane (end-of-pipe) passives ou conventionnelles.

2
INTRODUCTION
The role of environmental technology for industry has changed considerably over the past
few years. In the seventies, the general notion developed that pollution, caused by
industrial processes should not be left to nature, but should be cleaned up. The principle
approach of: the polluter pays developed and is still the basis of regulations, sometimes
under construction, in some countries. Today, many industries like the brewing industry
go one step further and accept that only by a responsible use of raw materials and natural
resources we can assure that sufficient remains for future generations. The European
framework for sustainable development and related aspects are described, and illustrated
with three examples from practice in which the brewing industry played or can play a
leading role.

THE EUROPEAN AND BUSINESS PERSPECTIVE

In the past decades a range of regulations and tax systems have been developed to prevent
pollution and to tax companies for their environmental impact. Over the past 20 years
some 200 pieces of Environmental legislation have emanated from the European
Commission in the form of various Directives and Regulations. This Euro legislation in
turn has to be incorporated by European Governments into National Environmental
Legislation. This process is taking too much time in view of the European Commission,
and an increasing number of (legal) enforcement actions are undertaken to direct
countries into compliance with EU-Directives. Almost 20% of these so-called reasoned
opinions refer to implementation of environmental Directives. With respect to
enforcement liabilities a Framework Directive is possibly launched in 2001.
One Directive with important environmental legislation is the European IPPC Directive1
(96/61/EC). The abbreviation IPPC stands for Integrated Pollution Prevention and
Control. It is a set of common rules on permitting for industrial installations, specified in
a listing (on which breweries are currently not yet mentioned). The installations listed are
not allowed to operate unless they have a permit. Integrated means that the permits must
take into account the whole environmental performance of the plant, that is emissions to
air, water and land, energy-efficiency, risk management, etc. These permits must be
based on the concept of Best Available Techniques (BAT). It is evident that the licensing
authorities need some assistance to find out which techniques are BAT. The European
Commission organizes an exchange of information between experts from EU member
states, industry and environmental organizations.
One particular organization, the European Committee of Environmental Technology
Suppliers Associations (EUCETSA) currently represents over 800 environmental
technology (ET) companies organized in 9 (nine) national associations in Europe.
EUCETSA is therefore in a unique position to transfer practical know-how, since the
market mechanism has selected economical efficiency and applicability of their solutions.
One of EUCETSAs goals2 is to initiate the transfer of suitable environmental
technologies into the BAT-reference documents (BREF), the guidelines for permitting.
For each sector it takes about two years to complete the work and produce a BREF. The
fifteen EU member states had until the end of October 1999 to adjust their national
legislation in line with the IPPC Directive. However, at the time of writing (May 2001)

3
several Member States (Ireland, Belgium, Luxemburg, Germany, Italy Spain and Greece
experienced delays in transposing to the Directive.
The brewing industry has been at the basis of one BAT in particular, being anaerobic
technology. Since 1984, the number of anaerobic installations for brewery effluent has
rapidly increased to about 300, and is growing at the same pattern. Reactor configurations
have improved considerably throughout the years3. The spin-off of these projects within
the brewing industry has lead to a widespread application of anaerobic technology in
other industries, some of which already have to comply within the framework of the IPPC
directive.
Multiple initiatives for sustainable development are also embedded in the Fifth
Environmental Action Program (19982002), in which 2.7 billion Euro (20%) is
budgeted for the segment energy, environment and sustainable development. The new
Sixth Environmental Action Program is just launched.
In response to regulatory pressures, public scrutiny, liability threat and competitive issues
standard bodies, regulators and industry associations began working together to produce a
standard for Environmental Management, leading to the publication of the ISO 14001
Standard in September 1996. Companies are adopting these standards to demonstrate to
their customers, suppliers and the general public that they have a management system in
place capable of dealing with all their environmental impacts4.
Self-regulation and pro-active measures by companies themselves can be very effective.
They often work favorable in two directions: for the companies themselves as well as
towards the environment. Quantitative and qualitative knowledge about environmental
effects, and understanding the relationship with in-plant activities will be a very useful
tool to:
Improving overall cost control
Overall environmental control and improvement.
Enhanced image and market share.
Meeting customers environmental expectations.
Conservation of materials and energy.
Maintaining good public and community relations.
Improved relations with local authority and Governments Regulators.
Obtaining insurance premiums at reasonable cost.
The results create incentives to make more use of available data and to take the necessary
steps to complement the available knowledge with new research and developments. This
is leading towards increased awareness amongst industry and researchers that (new)
technologies are required at all levels to effectively deal with pollution caused by
industrial processes and to minimize the impact of increased economic activities.
The development of environmental technologies for this purpose is a complicated
process. The fundamental research work, its funding and the practical applicability of
achieved results are some of the issues at stake. Between initial research and full
availability of the technology often many years go by, and in some areas the technology
supply can not keep track of the demand necessary to achieve the goals of sustainable
environment. Emerging technologies initially may not be that well affordable too, until
application on a larger (international) scale has been accomplished, which will bring
down investment levels and operation cost.

4
An important other factor is the availability of technology and (more important) the
experienced results in full-scale applications. An in-depth exchange of information
regarding learning curves is crucial. In the brewing industry however, exchange of
technical information in this respect is more common, and well stimulated by the
European Brewery Convention4 and other organizations.
Some of these factors may have caused a general intuition that ecology and technology
are opposite forces. It is understandable that (smaller) companies intuitively move away
from measures to prevent environmental discharges in their production processes, instead
of considering opportunities to produce in a more effective as well as environmental
friendly way.
Partnerships between stakeholders like companies, suppliers and universities will lead
towards faster results, and are essential for further exploration of the potentials of
environmental biotechnology for industry.

THE PRACTICAL PERSPECTIVE

Better overall efficiencies and the tighter environmental restrictions result in a new
framework for environmental technology development, in which sustainability and
economy are the keywords. Below, current sustainable developments are described in the
field of: 1. Brewery effluent technologies; 2. Biogas cleaning technologies and 3.
Biological side-stream filtration in (cooling-) water systems, to illustrate this point.

Example 1: The area of brewery effluent technologies

The characteristics of brewery effluent are changing to smaller flows, higher organic
loads, and increased adverse effects of inhibiting compounds are more likely to occur.
This in turn will lead to different requirements for biological treatment plants, some of
which have been acknowledged, demonstrated on full scale and described here. Others
are currently in development.
Biological treatment systems can be divided in anaerobic and aerobic processes. In
comparison with conventional aerobic systems advantages of anaerobic systems are
mostly related to: no energy required for aeration, energy production in the form of
biogas, smaller footprint and low surplus sludge production. To meet more stringent
effluent discharge limits anaerobic pre-treatment is mostly combined with aerobic post-
treatment. Brewery effluent is not really complex to treat biologically and the aerobic and
anaerobic processes in use and related performance aspects are generally well understood
and described5,6.
Anaerobic conversions of organic compounds work favourable in two ways: The energy
derived from the produced biogas is so-called Green Energy, since no fossilized carbon
was used to generate it, and secondly, the emission of carbon dioxide is far less than the
levels of alternative energy-intensive aerobic treatment methods. The major benefit does
not come from the use of the produced biogas, but from the choice of not using the
aerobic alternative. The facts that new national laws will force industry into use of green
energy, and upcoming carbon dioxide emission taxes illustrate the relevance of this
argument, which is currently somewhat undervalued. Calculations show that using biogas
from anaerobic effluent treatment for energy purposes is an economically feasible option
to deal with these developments.

5
The brewing industry began to apply Upflow Anaerobic Granular Sludge Bed systems
(UASB) for the anaerobic treatment of their effluent in 19845. In fact, it was the first
application of anaerobic technology for cold and dilute industrial effluent treatment.
These experiences lead to improved reactor concepts, and again the brewing industry was
amongst the first to apply. The first six anaerobic Internal Circulation (IC) reactors at a
brewery have been in operation since 1989 and the capacity was expanded in 1998. The
plant was built after a two-year research program with a semi-technical IC-reactor of 20
meter in height, which provided the necessary design information. Currently over 100 IC-
plants have been built, many of them in the brewing industry, replacing the initially used
UASB concept.
Industry is in need of compact wastewater treatment systems, capable of producing high
quality effluents and handling nutrient removal. The following parameters determining
volumetric conversion capacities of biological reactors for industrial effluent treatment
are important7:
1. Biomass conversion capacity; determined by the bacterial kinetic parameters like,
growth rate, maintenance and yield as well as by process conditions like pH,
temperature and water activity.
2. Mass transport; in which hydrodynamics, reactor geometry and physical structure of
the biomass are the determining factors.
3. Biomass concentration; governed by retention of biomass, for which aggregation of
bacteria, type of settler system and viscosity of the fluid are the crucial parameters.
With the introduction of granular technology in the seventies and with the application of
well-mixed systems like IC along the nineties a big progress has been made in industrial
wastewater treatment. Coming from conversion rates of 1 kg COD/(m3.day) towards rates
of 30 kg COD/(m3.day), the volumetric conversion potential for anaerobic processes
seems to be optimally exploited.
The latest development is anaerobic treatment in combination with extended aerobic
treatment in a Circox reactor, with integrated denitrification8. The aerobic post treatment
technology that suits the compact anaerobic technology very well operates with sludge on
carrier. The granular biomass is suspended in an airlift reactor. Also in this technology,
use is made of granular biomass, allowing for biomass concentrations of about 30 gr
VSS/l. Due to these high concentrations and the good mixing properties relatively high
aerobic volumetric conversion properties of 5 kg COD/(m3.day) are obtained, which is a
magnitude higher when compared to low loaded activated sludge systems.
The combination of IC and Circox technologies has already been successfully applied for
treatment of several industrial effluents, and (of course) also in the brewing industry. The
integrated denitrifying capacity was developed to satisfy the increased demands with
respect to nitrogen effluent concentrations. The first denitrifying Circox was installed at a
potato processing company in the Netherlands in June 1998. The complete closed
installation requires 300 m2 of surface area (when compared to the area required if
conventional activated sludge had been applied, the surface area would be about 1.500
m2). Clearly the technology provides an answer for industrial sites where wastewater
treatment is imposed, but where space is lacking.
Design concepts have been modified to allow anaerobic treatment in urban areas where
breweries are typically located. A compact IC-Circox combination in the brewing
industry was built at the Grolsch brewery in Enschede. At the World Brewing Congress

6
in Orlando (Florida, USA) in July 2000 this compact concept was reviewed. About 40
breweries have applied similar concepts since the introduction in 1996. The installation at
the Hasserder Brewery in Germany now includes the new denitrifying Circox
technology to accommodate nitrogen demands. Other applications match different
effluent demands.

Example 2: The area of biogas cleaning technologies

Since 1990, the Dutch company Paques has been engaged in the development and
installation of treatment systems based on biotechnological processes to remove sulfur
compounds from water, air and gaseous streams. The biological desulfurization processes
developed by Paques are known under the product name Thiopaq.

One of the Thiopaq processes removes

hydrogen sulfide (H2S) from gas streams.
A significant number of Thiopaq-
scrubbers desulfurizing biogas or sour gas
are operated. In conjunction with Shell
Global Solutions this process was also
demonstrated for the removal of H2S from
natural gas and syn-gases. In the process
H2S is absorbed and oxidized into
elemental sulfur. The brewing industry is
currently using this technology as well.
H2S has to be removed because sulfides
are:
Corrosive.
Malodorous: a smell like rotten eggs.
Toxic: even at very low levels it can
cause headache and nausea.
Sulfurdioxide emissions when the gas
is used as a fuel gas.

T
Thiopaq scrubber at
Grolsch Bierbrouwerij Nederland B.V.

The Thiopaq process can be considered as a caustic scrubber combined with a

bioreactor, in which the spent caustic solution is regenerated. In the scrubber, hydrogen
sulfide is absorbed under alkaline conditions, i.e. at pH 8 - 9. The absorption of H2S
proceeds according to the following equation:

H 2 S + NaOH NaHS + H 2 O (1)

From this equation it follows that alkalinity is consumed. High H2S removal efficiency is
feasible, because the H2S concentration in the washing liquid entering the scrubber will

7
be virtually zero. The alkalinity consumption due to the absorption of H2S is
compensated in a bioreactor through the oxidation of hydrogen sulfide to elemental sulfur
which proceeds under oxygen controlled conditions:
NaHS + O S o + NaOH
2 (2)

The Thiopaq process uses bacteria of the genera Thiobacillus to oxidize the hydrogen
sulfide. These bacteria grow very fast and are known to be highly resistant to varying
process conditions. A small part of the dissolved sulfide will be oxidized to sulfate
according to:

2 NaHS + 4O2 2 NaHSO 4 Na 2 SO4 + H 2 SO4 (3)

As a result of this side reaction, some caustic soda is required to neutralize the formed
sulfuric acid. A small bleed stream is withdrawn from the system to ensure that the build-
up of sodium sulfate and other salts is prevented. The bleed stream (containing sodium
salts and some sulfur particles) is harmless and can generally be discharged.

GAS out
NAOH

NUTRIENTS WATER

GAS IN
PURGE

AIR
SULFUR

ABSORPTION REACTOR SULFUR RECOVERY

SECTION SECTION SECTION

Figure 3: Thiopaq process for gas desulfurization.

Some of the characteristics found for the Thiopaq process are:

High removal efficiencies for hydrogen sulfide from process gases.
High biological activity, which absorb peak loads.
Most odor causing components can be removed.
Operation at ambient or elevated pressures, as well as at various temperatures.
Low operational costs due to recovery of caustic.
No need for discharge of sulfide containing waste stream.

8
 No need for use of chelating compounds (chemical RedOx processes) and no
subsequent production of hazardous bleed streams.
Production of elemental sulfur as re-usable byproduct.

Example 3: Biological side-stream filtration in (cooling-) water systems

Biological activity in recirculating cooling water systems may lead to Microbial Induced
Corrosion (MIC) and lower heat exchange efficiencies. The use of oxidizing biocides as a
tool to control excessive biofilm formation in a (cooling) water system can be
characterised as fighting the symptoms, because it does not take away the main cause of
biofilm growth: the availability of nutrients. Due to frequent use of oxidizing biocides the
microbial growth rate in a cooling water system studied9 was even higher (1 - 2 d-1) than
the dilution rate (0.33 d-1).
By means of an Astrasand continuous sand filter used as a side stream biofilter it is
possible to achieve considerable reductions in nutrients and in suspended biomass
contents. Due to the first order kinetics of nutrient removal and due to the improved
filtration efficiencies at increasing influent biomass contents, it is concluded that a side
stream biofilter introduces a self-regulating process element in the (cooling) water
system, leading to a faster recovery of the water quality after upset conditions.
Considerable savings in the use of oxidizing agents can be established.
Particularly the biological removal of nutrients leads to reductions in growth rates in the
cooling water system, leading to potential reductions in oxidizing biocide usage of at
least 70 to 80 %. Besides obtaining a cleaner and more stable water system, the
reductions made in hypochlorite dosages lead to lower chloride contents and thus to a
lower conductivity and corrosiveness of the (cooling) water. This positive side effect may
lead to further minimisation of the blow-down flow and the use of blow-down related
chemicals.

CONCLUSIONS
Environmental biotechnology can improve economy and ecology when applied in
industrial processes. The European legislative framework that will be implemented is also
set up along this principle. Three areas of potential applications were elaborated, and
show that the brewing industry played an important role, and can continue to set
examples in this field for the benefits of their economy, the industry and the environment.
Strategic partnerships will help to accelerate these developments to occur.

REFERENCES
1. The European Commission. Council Directive 96/61/EC, Brussels, 24 September
1996.
2. EUCETSA Policy Manifest. Brussels, March 2001 (www.eucetsa.org);
3. Driessen, W., Habets, L. and Vereijken, T. (1997) Novel anaerobic anaerobic process to
meet strict effluent plant design requirements, Ferment, Vol 10, No. 4, August 1997,
U.K., 243-250.

9
4. European Brewery Convention (2001) EBC manual of good practice: Brewery Effluent
(to be published).
5. Vereijken, T.F.L.M., Swinkels, K.T.M and Hack, P.J.F.M. (1986) Experience with
the UASB-system on brewery wastewater, Proceedings of the NVA-EWPCA Water
Treatment 1986, 15 - 19 September, Amsterdam, 283-296.
6. Vereijken, T., Driessen, W., Yspeert, Y., (1999) Aspects of managing brewery
effluent composition, IOB Africa Convention 1999, Nairobi, Kenya.
7. Mulder, R., Vereijken, T.L.F.M. and Vellinga, S.H.J. (2001) Future perspectives in
reactor development, (to be published);
8. Frijters C.T.M.J., Vellinga S., Jorna T. and Mulder R. (2000). Extensive nitrogen
removal in a new type of airlift reactor, Wat. Sci. Tech., 41(4-5), 469-476.
9. Daamen* E.J., Wouters*J.W. and Savelkoul**J.T.G. (2000) Side stream biofiltration
for improved biofouling control in cooling water systems, Wat. Sci.Tech., 41 (4-5),
445-451.

10
109

State detection and control of overloads

in the anaerobic treatment of brewery
waste water using fuzzy logic
E. Murnleitner, T. Becker & A. Delgado
TU Mnchen, Lehrstuhl fr Fluidmechanik und Prozeautomation, D-85350 Freising-
Weihenstephan, Germany (e-mail: [email protected])

Descriptors
Anaerobic effluent treatment, bioreactor, model simulation, process control

SUMMARY
Anaerobic waste water treatment is still difficult to handle, particularly overloads can shut
down an entire plant. Therefore a two stage anaerobic waste water treatment was modelled
and controlled, so that the biological state of the reactors could be predicted. Based upon this,
proper control actions were enacted automatically. Even very strong fluctuations in the
loading of both concentration and volumetric flow rate, could thereby be handled
successfully. Moreover, effluent concentrations could be kept low without using TOC, COD
or equivalent measurements. Applying such a control system, waste water treatment can be
made more effective, more stable and less supervision will be necessary.

Bestimmung des Stadiums und Kontrolle der berladung von anaeroben Brauerei-
abwasserbehandlungsanlagen mit Fuzzy-Logic

Deskriptoren
Anaerobe Abwasserklrung, Bioreaktor, Modellbildung, Prozesteuerung

ZUSAMMENFASSUNG
Die anaerobe Abwasserbehandlung ist nach wie vor schwer zu handhaben. Besonders eine
berladung kann eine funktionierende Anlage zum Absturz bringen. Aus diesem Grund
wurde eine zweistufige anaerobe Abwasserbehandlung modelliert und kontrolliert, um den
biologischen Status der Reaktoren vorhersagen zu knnen. Darauf basierend ergaben sich
automatisch befriedigende Kontrollmechanismen. Sogar sehr starke Fluktuationen beim
Beladen beider Stufen und hohe volumetrische Durchflussraten konnten auf diese Weise
erfolgreich gehandhabt werden. Des Weiteren konnte die Ausflusskonzentration auch ohne
Messsysteme, wie TOC oder COD, niedrig gehalten werden. Die Anwendung eines solchen
Kontrollsystems fhrt zu einer effektiveren und stabileren Abwasserbehandlungsanlage, die
zudem mit weniger Personaleinsatz auskommt.
Dtection et contrle des tats de surcharge lors du traitement anarobie des eaux uses
de brasserie au moyen de la logique floue

Descripteurs
Bioracteur, commande de procd, modlisation, traitement anarobie des effluents

RESUME
Les dchets anarobies aprs traitement restent difficiles manipuler et les surcharges, en
particulier, peuvent mettre en panne toute une usine. Par consquent, nous avons modlis et
contrl un systme de dtection des dchets anarobies en deux temps, de manire pouvoir
prdire l'tat biologique des fermenteurs. Bases sur ce modle, des mesures de contrle
appropries ont t dclenches automatiquement. Mme les importantes fluctuations dans le
chargement de la concentration et du dbit volumtrique ont pu tre traites avec succs. Par
ailleurs, la concentration des effluents a pu tre garde un niveau faible sans recourir au
TOC, au COD ou des mesures quivalentes. En appliquant un tel systme de contrle, on
peut rendre le traitement des dchets plus efficace et plus rgulier, avec moins de surveillance.

2
INTRODUCTION
Fuzzy Logic and other cognitive Tools, like Artificial Neural Nets, have already been
used at the Chair for Fluid Mechanics and Process Automation for applications in the
food and beverage industry for years [1, 2], e.g. the control of the lautering process or
the phase detection of the fermentation process. As such processes are characterised
by their complexity and inlinearity, cognitive methods are predestined for that usage.

Even in economical breweries the wastewater produced is two to six times the volume
of beer produced. This wastewater contains at least 2 g COD (chemical oxygen
demand) per litre of wastewater. Anaerobic treatment offers the advantage of the
elimination of the main part of the pollutants, which saves disposal charges, as well as
production of energy (biogas) and fewer sludge compared to the aerobic technology
(figure 1). However, anaerobic treatment has also some drawbacks. These are the
more difficult handling and the extreme long characteristical times, whereby
particularly an overload can lead to a complete break down of the plant. A restart
takes weeks to months which has restricted the user acceptance compared to
conventional aerobic plants. But this problem can be circumvented by using
intelligent control systems. Up to now comparatively few control engineering is
applied in the wastewater
treatment. The controlled
variables are mostly
regarded isolated, e.g.
temperature and pH are
held constant and
controlled by a local
controller. Connecting the
particular measurands and
manipulated variables, one
can increase the efficiency
of wastewater treatment
systems as well as the
stability essentially. Even
though such controllers
would be possible by
using classical control Figure 1: Bacterias and chemical compounds of the anaerobic
engineering, the effort digestion process.
would be considerable due
to its complexity. Therefore we applied a fuzzy logic system for the state detection
and the control of the anaerobic wastewater treatment, as much qualitative knowledge
was available in literature and moreover, the fuzzy logic handles the concept of the
partial truth, whereby statements like pH is high or tank is full can be handled
easily.

The anaerobic digestion is a complex process in which organic matter is degraded into
methane and carbon dioxide (figure 1). It usually is described in four steps, (1)
hydrolysis, (2) acidogenesis, (3) acetogenesis, and (4) methanogenesis.
Since hydrolysis (1) and acidification (2) occur mostly much faster than the
acetogenic (3) and methanogenic (4) processes, and taking into account that the two
latter bacteria live in symbiosis, the anaerobic digestion often is separated into an
acidification reactor (1, 2) and a methane reactor (3, 4). In the hydrolytic step (1),

3
complex substances, like carbohydrates, fat and proteins are converted into the
monomers. These compounds are fermented and reduced components are formed (2).
As in the acidification step not only acetate which is a substrate for the methanogenic
bacteria but also propionate and other highly reduced substances like butyrate, ethanol
and lactic acid are formed, a further step (3) is required which converts the latter
products into acetate which than can be consumed by methanogenic bacteria (4). For
(3) and (4) hydrogen and propionate play a key role because accumulation leads to a
doom loop [1, 2]. In such a case, hydrogen, which is produced in step (2) is not
consumed fast enough in step (4). As hydrogen inhibits step (3), propionate is
accumulated, which in turn inhibits step (4). As propionate is accumulated, pH drops
and methanogenesis stops at pH lower than 6.

FUZZY LOGIC EXPERT SYSTEM

The two stage anaerobic wastewater
(a) low normal high treatment was modelled and
1 controlled. The biological state of
truth value

the reactors (acidification buffer

0.5 tank and an anaerobic filter reactor)
could be predicted using a fuzzy-
0 logic system and based upon this,
0 500 1000 proper control actions were taken
hydrogen [ppm] automatically in order to avoid an
overload. The system was designed
to handle very strong fluctuations in
(b) shortage normal overload toxic the concentration of the substrate
1
and the volumetric loading rate.
truth value

0.5

Input values
0
0 1 2 3 4 5
state ID
state
(c) no feed keep more
1
measures
truth value

0.5

0 output values
-1 0 1 2 3
COG = 0.67 feed change
Figure 3: The structure of the
Figure 2: Fuzzy variables (methane reactor): (a): Fuzzy logic expert system
hydrogen(2). (b): state(2). (c): feed setpoint (2).
Exemplary truth values are shown. COG = centre of
gravity (a defuzzification method).

4
Hydrogen concentration together with methane concentration, gas production rate, pH
and the fill level of the acidification buffer tank were used as input variables for the
fuzzy logic system. The manipulated variables were the flow rate from the
acidification buffer tank into the methane reactor, the temperature and pH of both
reactors, the circulation rate of the methane reactor, back flow form the methane
reactor into the acidification, and the control of the feed into the acidification buffer
tank. However, the inputs are not directly mapped to the output variables. Instead,
intermediate variables for the states and global measures were introduced in order to
make the Fuzzy system more structured and to gain more insight into the process
(Figure 3).

Selected rules of the Fuzzy logic expert system (c2 = methane reactor):

BEGIN RULEBASE state

...
IF hydrogen_c2 = high THEN state_c2 = overload
IF pH_c2 = low THEN state_c2 = overload WITH 0.5
...
END RULEBASE state

BEGIN RULEBASE measures

...
IF state_c2 = overload THEN feed_c2 = no_feed
IF state_c2 = overload AND (feed_c2_avg = no_feed OR fill_c1 = full)
THEN condition_c2 = better
...
END RULEBASE state

BEGIN RULEBASE output

...
IF conditions_c2 = better AND pH_c2 = low
THEN pH_c2 = optimal
...
END RULEBASE output

The complete rulebase and the shape of all fuzzy logic variables as well as the
Materials and Methods used, are described more in detail in [3].

RESULTS
The developed control system was successfully tested (A) on a fully automated lab
scale two stage anaerobic digester and (B) on a dynamic simulation model. Different
types of wastewater from brewery and food processing industry were successfully
applied. Even a restart of feeding with very high COD loading (> 100 000 mg/l) after
several days of stand by was handled successfully. Moreover, effluent concentrations
could be kept low without using TOC, COD or equivalent measurements. Finally, the
control system was validated using a complex dynamic simulation model which was
connected with the fuzzy logic software. The control system could also detect and
avoid overloads using the simulation model with different types of wastewater and
loadings.

5
 8 120
(a)
7
100
6
filling level

filling level [%]

feed rate [d-1]
80
5

4 60
3
40
feed rate
2
20
1

0 0
0 5 10
time [h]

2000 80
(b)
70
methane
1500 60
hydrogen [ppm]

methane [%]
50
1000 40
30
500 20
hydrogen
10
0 0
0 5 10
time [h]

1
(c) overload
normal
truth value

shortage

0
0 5 10
time [h]

Figure 4: Experiment without control. (a) shows the feed (volumetric flow into the acidification buffer tank
per unit of volume of the methane reactor (d-1)) and the filling level of the acidification buffer tank (%).
Switching of wastewater (from 3.3 to 28 g COD l-1) is indicated with a dotted line. (b) concentrations of
hydrogen (ppm) and methane (%) in the off-gas of the methane reactor. (c) truth values (degree of
membership) of the fuzzy sets of the state variable for the methane reactor. Overload is indicated with a thick
line, normal with a smaller line and shortage with a dotted line.

6
 8
(a) 120
7 filling level
6 100

filling level [%]

feed rate [d-1] 5 80
4
60
3
feed acidif. 40
2

1 feed methane r. 20

0 0
0 5 10 15 20 25 30 35 40
time [h]

1000 70
(b)
methane 60
750
50
hydrogen [ppm]

methane [%]
40
500
30
hydrogen
20
250
10

0 0
0 5 10 15 20 25 30 35 40
time [h]

1
(c)
overload
normal
truth value

shortage

0
0 5 10 15 20 25 30 35 40
time [h]

Figure 5: CONTROL of the experiment with the modified fuzzy variable hydrogen. (a) shows the feed rate
into the acidification buffer tank normalised with the volume of the methane reactor per hour and the fill level
of the acidification buffer tank. Begin of dosage of the higher concentrated wastewater (28 g/l COD) is
indicated with a dotted line. (b) shows the concentrations of hydrogen (ppm) and methane (%) in the off-gas of
the methane reactor. (c) shows truth values (degree of membership) of the fuzzy sets of the state variable for
the methane reactor. Overload is indicated with a thick line, normal with a smaller line and shortage with a
Figure 4 shows the feed, gas concentrations and detected states for an experiment,
dotted line.
7
 4
where only the state detection
(a) of the Fuzzy system was
used. The suggested control
3 methane reactor actions of the Fuzzy system
gas flow [d-1]

were not used. Here it can

2 clearly be seen that after
change of the wastewater
1 acidification
concentration from 3.3 to 28
g /l lead to an increase of the
hydrogen concentration and
0
0 5 10 15 20 25 30 35 40 detection of an overload. The
time [h] effluent COD in this
uncontrolled experiment
30 increased steadily and the
(b) experiment was stopped at a
25 influent
COD of approx. 3 g/l.
20
COD [kg m-3]

Figure 5 shows, how the

15
control system can avoid the
acidification
10 overload. Here the output
variables of the Fuzzy expert
5
methane reactor
system were applied to the
0 system (i.e. used as
0 5 10 15 20 25 30 35 40 manipulated variables for the
time [h] sub controllers). When a
slight overload was detected
after 7.5 hours (Figure 5a),
Figure 6: Gas production rate and COD of the controlled feed into the methane
experiment. reactor was decreased.
(Figure 5c). Influent and
effluent COD concentrations are shown in Figure 6. The effluent was relative low
compared to the influent during the whole experiment.

CONCLUSIONS
Using an anaerobic wastewater pre-treatment system in general
- costs for disposal of wastewater can be saved or at least reduced,
- energy can be produced by utilisation of the produced biogas and
- only few sludge is produced

Applying a control system as presented, additionally

- the treatment can be made more effective, therefore smaller reactors can be
constructed
- the stability can be enhanced (i.e. very strong variations in the loading can be
handled)
- specialised staff, which often is not available, can be saved.
- the effluent can be kept nearly constant and low without the need of COD or TOC
measurement.

Transparently, all these enumerations can help to save resources and costs.

8
Although this control concept was successfully tested on laboratory scale reactors and
using a simulation model, transfer of the results for an industrial scale application
appears possible and is planned for the next future.

REFERENCES
1. Kurz T., Becker T., Fellner M., Schmitz M., Delgado A., Murnleitner E.,
Cognitive Computing in Brewing Technology. - In: 7th European Congress on
Intelligent Techniques & Soft Computing (EUFIT), Aachen, Germany, 13 -16
September 1999, 235 f
2. Kurz, T., Fellner, M., Becker, T, Delgado, A., Proceedings of the European
Brewery Convention Congress, Cannes, 1999, 743 - 750.
3. Murnleitner, E., Becker T., Delgado, A, State detection and control of overloads
in the anaerobic wastewater treatment process using fuzzy logic, Water Research,
2001, Accepted.

9
Development and demonstration of
polymerase chain reaction based
methods for process control in
breweries

Chris Powell, Katherine Smart, Auli Haikara (coordinator), Riikka Juvonen, Erna
Storgrds, Teija Koivula, Irmeli Koskinen, Jutta Suksi, Leila Peltoniemi-Laajanen,
Eberhard Geiger, Thomas Schuhbeck, Reiner Springer, John Hammond, Samantha
Walker, Gemma Marsden, Marie Maugueret, Gudrun Vogeser, Andreas Scherer, Jeff
Hodgson, Benham Taidi, Xanthe Green, Alan Kennedy, Tore Hage, Stefan Loch-
Ahring, Elke Neumann, Stefan Lustig, Andreas Eidtmann, Sven Schppner, Martina
Specketer, Thomas Sommer, Terry Dando, Torbjrn Ingemansson (scientific officer).

INTRODUCTION
Although beer is considered to be an unfavourable habitat for microorganisms,
contamination of the process by certain bacteria or wild yeasts can lead to production
inconsistencies and quality defects in the final product (figure 1). Traditional methods
for detecting contaminants in breweries are based on cultivation and therefore only
confirm the presence of microorganisms after a delay of several days or weeks. As
such, they do not allow proactive process control or confirmation of product quality.
Furthermore, identification in this manner does not present information about the
spoilage potential of the detected organisms, hence there is a need for more effective
quality monitoring tools. Polymerase Chain Reaction (PCR) is an in vitro nucleic acid
amplification technique that allows rapid and specific detection of low levels of
microbes. Using molecular techniques (figure 2) such as PCR technology (figure 3) it
is possible to provide insight into both the origin of the contaminant and the potential
damage that it may cause to final product quality. By detecting brewery contaminants
at an early stage, the risk of spoilage may be reduced considerably. In recent years,
various applications have been described for specific beer spoilage bacteria and there
is a considerable interest in the brewing industry for the implementation of the
technology. However, further technical developments and thorough evaluation of the
technology are required before it can be introduced to routine quality control in
breweries. The primary objective of the project is to develop novel, practical PCR
based methods for the detection of spoilage bacteria and wild yeasts. It is proposed
that this technology could be incorporated into routine brewery quality control.
Figure 1a: Scanning Electron Micrograph of a biofilm, demonstrating contamination
of beer with bacteria and wild yeast. Provided by Erna Storgrds, VTT
Biotechnology.

Figure 1b: Gram stained Pediococcus and Lactobacillus. Provided by John

Hammond, BRI.

2
Figure 1c: Light Micrograph demonstrating brewing yeast contaminated with
Lactobacillus. Provided by John Hammond, BRI.

1 2 3 4 5 6 7 8 9 10 11

Figure 2: Restriction Fragment Length Polymorphism (RFLP) illustrating genetic

variation between L. brevis (1), L. brevisimilis (2), L. buchneri (3), L. casei (4), L.
coryniformis (5), L. curvatus (6), L. frigidus (7), L. lindneri (8), L. plantarum (9), L.
delbrueckii (10), Size Marker II (11). Provided by Dr. Reiner Springer, Lehrstuhl fr
Technologie der Brauerei II.

3
DNA AMPLIFICATION USING PCR

Figure 3a: The principles of PCR technology. Adapted from

http://allserv.rug.ac.be/~avierstr/principles/pcr.html

4
 Enzymatically Enzymatically
Chemically
extracted DNA extracted DNA
extracted DNA
(Batch 1) (Batch 2)

R1 R2 R3 R4 R1 R2 R3 R4 R1 R2 R3 R4 M U

900bp

R1 R2 R3 R4 R1 R2 R3 R4 R1 R2 R3 R4 M U

900bp

Figure 3b: PCR technology

R1 Lactobacillus paracasei paracasei, R2 L. paracasei paracasei (Isolate 2), R3
L. paracasei paracasei (Isolate 3), R4 Pediococcus pentosaceus.
M Molecular weight ladder (800bp band brightest).
U Chemically extracted DNA from an artificially contaminated yeast slurry.

Reproduced with kind permission (Taidi, B. and Hodgson, G. (2000). Detection of

Lactic acid bacteria in pitching slurries using PCR. In: Brewing Yeast Fermentation
Performance. p136-139. Ed K. Smart. Blackwell Science).

5
OBJECTIVES OF THE PROGRAMME
The BREWPROC (European Commision Project Number QLK1-CT-2000-01251)
programme will comprise two distinct phases, including a research and development
phase (figure 4) and a demonstration phase (figure 5). The project is a 36 month
assignment (initiated on the 1/1/01) involving the cooperation and close collaboration
between ten partners and two subcontractors (figure 6). Groups from Finland,
Germany, Norway and the United Kingdom representing Brewing Companies,
Research Institutes and Universities will collaborate to achieve seven key objectives:

1. Production of robust PCR based methods for spoilage organisms in brewery

process samples and final beer;
2. Demonstration of the applications of PCR technology to routine food control;
3. Evaluation of the technical performance and practicality of the new PCR
applications;
4. Demonstration of the real benefits of PCR in brewery QC in comparison to
established methods;
5. Production of standard PCR protocols for brewery samples in reference
manuals;
6. Exploitation of developed PCR kit prototypes;
7. Transfer of PCR technology to the brewing industry.

APPLICATIONS
The PCR protocols and kit prototypes resulting from the project will be used to detect
the most common beer spoilage organisms and trace their origins in the production
line. It is anticipated that the developed methods can be used as a basis for the
application of PCR technology in other fields of food production.

6
 R & D PHASE

The R & D phase involves

1. The development of novel, practical PCR based methods for the
detection of spoilage bacteria and wild yeasts
In bright and unfiltered beer samples
Pitching yeast slurry
Fermenting wort

2. A variety of methodological approaches including

DNA-PCR and Reverse Transcriptase (RT)-PCR combined to
gel electrophoresis
PCR-ELISA
Fluorogenic on-line PCR techniques

PCR for spoilage PCR for spoilage Improved detection

organisms in bright organisms in methods for PCR
beer. P1-4, P10 unfiltered process amplicons. P2-4,
samples. P1-4 P7 and P8

Figure 4: The research and development phase

DEMONSTRATION PHASE

The demonstration phase involves

1. Evaluation of the new methodology through independent testing in
research and brewery QC laboratories for
Performance and ease of use
Cost benefits
Work safety issues
Sensitivity to DNA contaminations
2. Dissemination of results to ensure full awareness of the new
methodology via
The World Wide Web
EBC activity
Training workshops and symposia
Publications

Dissemination of Industry evaluation. Method

knowledge. P1-5 P1-10 and SC1 comparison. P10,
and P10 SC1 and SC10

Figure 5: The demonstration phase

7
 Figure 6: Participants in the European collaboration

ACKNOWLEDGEMENT
The authors would like to thank the European Commission Quality of Life and
Management of Living Resources Programme for financial support.

8
The New International Calibration Standards (ICS)
for the HPLC Analysis of Isomerized and Reduced Isomerized -Acids

Presented on behalf of The International Subcommittee for Isomerized Hop -

Acids Standards by Richard J.H. Wilson (Chairman)

This poster is dedicated to the memory of the late Dr Heinrich (Heini)

B. Pfenninger, formerly a member of the above Subcommittee

Introduction
Until now, the calibration of HPLC methods for the analysis of iso--acids and
reduced iso--acids to an internationally agreed standard has only been possible in
the case of the iso--acids, for which purpose the use of a commercially prepared
standard comprising the dicyclohexylamine (DCHA) salts of purified iso--acids
has formerly been recommended.

In 1998, at the Annual Meeting of the ASBC, a paper was delivered by Dr John Paul
Maye1 in which he described not only an improved method for the preparation of
DCHA iso--acids, but also methods for obtaining pure calibration standards of the
three, commercially important, reduced iso--acids. Subsequently, it was proposed
that an International Committee be formed, bringing together representatives from
ASBC, EBC, IoB (now IGB) and BCOJ for the purpose of first evaluating Dr Mayes
methods, then moving on to the preparation of each standard type in quantity
sufficient for issue as an officially approved, HPLC standard.

Since its formation, the International Subcommittee for Isomerized Hop -Acids
Standards has worked hard to progress its mission and the new standards have now
been produced and approved. They are available from ASBC and Labor Veritas
(acting for EBC).

1
Maye et al, J. Am. Soc. Brew. Chem. 57(2), 55-59, (1999).
Description of Standards
The four, new International Calibration Standards are:

-acids)
DCHA-Iso, ICS-I1 (DCHA salts of trans- form, iso-
-acids)
DCHA-Rho, ICS-R1 (DCHA salts of cis- form, rho-iso-
-acids, as mixed cis- and trans- forms)
Tetra, ICS-T1 (Tetrahydroiso-
-acids)
DCHA-Hexa, ICS-H1 (DCHA salts of cis- form, hexahydroiso-

Each standard was evaluated for purity and composition by HPLC, elemental analysis,
melting point and UV absorption. Cohumulone ratios were also established and the
long-term stability of such preparations assessed. (All four standard types are
considered stable in cold (freezer) storage, though DCHA-Iso may have slight
instability at ambient storage temperature).

An isocratic method for HPLC analysis of unknown samples (based on EBC Method
7.8) was used to finally establish purity values (shown below) for each standard, and
is recommended for general use. This method uses a mobile phase based on acidic
methanol, a reverse phase, C-18 ODS column and requires UV detection at 270 nm.

DCHA-Iso DCHA-Rho Tetra DCHA-Hexa

Parameter (ICS-I1) (ICS-R1) (ICS-T1) (ICS-H1)
% Purity by HPLC * 97.5 98.4 97.9 98.7
(major homolog peaks only)

Cohumulone Ratio ** 0.53 0.20 0.41 0.53

cis:trans Ratio < 0.01 > 50 ~ 1.0 > 50

* As assessed by modified EBC Method 7.8 ** Area of cohumulone peaks / Total peak area of major homologs

Preparation of DCHA-Iso
In each case, the new standards were made from commercial preparations of the
appropriate iso--acids or reduced iso--acids. Somewhat similar processes were
used for the production of all three standards prepared as DCHA salts, illustrated here
for DCHA-Iso, ICS-I1:
 Commercial Iso--acids Hexane + 20% H 2SO 4 Iso--acids
(as 30% solution of K + salts) (acid form in hexane)

Dry over anhydrous MgSO 4

Remove drying agent
Rotary evaporate
Add 1 mole equiv. of DCHA Dissolve in dry acetone
Stir 18 h. Place in freezer at - 4 oC
DCHA-Iso--acids After 24 h, filter and collect crystals Iso--acids
(crude preparation) (acid form in acetone)

Recrystallize 2 x from cold ethanol

Pure, white crystals

DCHA-Iso, ICS-I1 Yield ~ 7%

When used according to the specified instructions for storage, handling and use,
especially including chromatography by the recommended HPLC method, this
-acids = 64.5%
standard is deemed to have the following composition: Total Iso-

Structures of DCHA - Iso-alpha-acids The above figure takes into account

O
O only these major forms of the total iso-
-acids content:
H DCHA - trans-Isocohumulone

HO
O
-
H2N
+
trans-isocohumulone,
(mw: 529.8)
O
trans-isohumulone &
trans-isoadhumulone.
O
O Conventionally produced worts and
beers will contain both trans and cis
H DCHA - trans-Isohumulone
-
O
HO H2N
+

(mw: 543.8)
forms of iso--acids, as too will
O
samples of non-reduced, isomerized
hop products and the worts or beers
O
O made from them. However, at 270 nm
the extinction coefficients for all forms
H DCHA - trans-Isoadhumulone
- +
O
HO H2N
(mw: 543.8)
of the iso--acids are known to be all
O
quite similar in acidic, methanolic
solutions such as the mobile phase of
2
the recommended method , hence ICS-I1 is considered suitable as a standard for all
mixtures of isomers.

2
Biendl & Hartl, Monatsschr. Brauwiss. 3/4 102-107, (1995)
Purity of DCHA-Iso by HPLC Analysis
The following chromatograms (of a single analysis) illustrate (1) the three major
peaks upon which the calibration is based and (2) the minor peaks that are also present
in the standard.

DCHA-Iso Column: 250 x 4.6 mm, 5 C18 Nucleosil

ICS-I1 Mobile Phase: 750 ml MeOH /
340 ml H2O / 10ml H3PO4 /

Isohumulone
Isocohumulone
1 ml 0.1M Na2EDTA
Temperature: 35 oC

Isoadhumulone
Wavelength: 270 nm
trans-Isoposthumulone?

Unknown 5
Unknown 3
Unknown 2
Unknown 1

Unknown 4

Peak spectra, as obtained from a photo-diode array (PDA) detector scanning at the
peak maxima, are shown below:

Isocohumulone Unknown 3
Unknown 1

Isohumulone Unknown 4
Unknown 2

Isoadhumulone Unknown 5
Isoposthumulone ? -acid ?)
(Iso-

Some of the minor peaks are believed to be trans forms of minor iso--acids,
including trans-isoposthumulone. Cis forms (of even the major iso--acids homologs)
are essentially absent.
DCHA-Rho and DCHA-Hexa
Commercial preparations of rho-iso--acids and hexahydroiso--acids are known to
be comprised almost entirely of the cis forms of the various homologs. DCHA salts
are nevertheless easily formed, contrary to the case of the iso--acids, which only
form crystalline DCHA salts of the trans isomers. ICS-R1 and ICS-H1 were
recrystallized from diethyl ether, and in each case have a balance of isomers and
homologs that is considered appropriate for the analysis of both the post-fermentation
bittering agents from which they were prepared and the beers made from them.

Tetra
Tetrahydroiso--acids do not readily form DCHA salts. However, it is possible to
crystallize the free acids from organic solvent solution so as to produce an essentially
pure preparation. ICS-T1 was prepared by repeated blending and recrystallization
from hexane of material obtained from commercial batches of tetrahydroiso--acids
that had been made from either - or -acids. By this means, a product (in ~ 5%
yield) was deliberately formulated to contain a balanced blend of the various
homologs and isomers that occur in commercial products.
O O
O O

R R

H H
Tetra ICS-T1
OH OH
HO (contains both cis- & trans- forms HO
of tetrahydroiso-alpha-acids)
O O
cis- trans-

Even so, the question arises as to whether the balance of components would seriously
distort analytical data where an unknown sample is substantially dissimilar to the
standard and the calibration is based (as is instructed for all of the new standards) on
applying an averaged response factor to all of the major homologs and isomers peaks.
Prior to the preparation of ICS-T1, six test preparations of widely differing
composition were made, ranging from essentially all cis- form to all trans- form, and
having peak area based cohumulone ratios from 0.21 to 0.83. The HPLC response
factors at 270 nm of four of these preparations in the mobile phase of the
recommended method were later compared against that of the new standard:
 % Peak Cohumulone Cis:trans Response
Standard (CoH homologues)
Purity Ratio Factor*
C2 98.5 0.21 25 : 1 13.65
ICS-T1 97.5 0.41 0.98 :1 13.68
C1 99.1 0.61 0.69 : 1 13.78
D1 99.0 0.67 1.3 : 1 13.89
D2 99.0 0.60 220 : 1 14.03
-8
* Response factor = (Area of major tetrahydroiso--acids peaks x 10 ) / (mg x % peak purity)

Clearly, the decision to recommend the use of a single response factor for ICS-T1 is
justified.

Further Work
The Subcommittee plans to continue its work, making checks on stability and also
trying to establish in more detail the exact identity, where not already known, of each
of the component compounds in these complex preparations.

Acknowledgements
The Chairman wishes to thank all Subcommittee members for their valuable
contributions, but especially to Dr John Paul Maye, Dr James (Gus) Guzinski and Dr
Paul Hughes who were responsible for the actual preparation of the standards. Also, to
the Technical Committees of ASBC, EBC, IGB and BCOJ for their support of this
work.
Curriculum vitae of (co)authors - A

Aastrup, Sten (contribution nr. 56)

Born in 1950.
Studies: MSc in Biology at the University of Copenhagen, Denmark
(1979).
Appointments: 1979-1990: Scientist and Senior Scientist at the
Carlsberg Research Center; 1990-1994: Head of the Carlsberg
Maltings; since 1994: Senior Consultant at Alfred Jrgensen
Laboratory.

Abe, Mayumi (contribution nr. 39)

Born in 1972.
Studies: Food Chemistry at the Kanagawa Nutrition University,
Japan (1993).
Appointments: since 1993: Researcher in Research Laboratory for
Brewing at Kirin Brewery Co. Ltd.

Abraham, Norbert (contribution nr. 28)

Appointments: 1985: Beck & Co, Germany: Consultant for
Logistics and Production (PROLAB, BDE and SAP); since 2000:
Nord IT: Consultant for SAP R/3 PM/MM/PP

Aerts, Guido (contribution nr. 58, 60)

Born in 1951.
Studies: M. Sc. degree in Chemistry at the University of Gent,
Belgium (1973); PhD in Biochemistry at the University of Gent
(1980).
Appointments: 1973-1980: assistant professor in Biochemistry,
Laboratory of Biochemistry, University of Gent; since 1980:
professor in Biochemistry and General Chemistry at the KaHo St.-
Lieven, Gent; since 1991: Head of the Biochemistry Department at the KaHo St.-
Lieven, Gent. Research topics: flavour stability, enzyme technology.

Allosio-Ouarnier, Nathalie (contribution nr. 19)

Born in 1972.
Studies: Engineer in Food Technology (ENITIAA, Nantes, France),
1996. Honours in Process Technology, University of Nantes, 1996.
PhD in Biotechnology and Food Industry, Institut National
Polytechnique de Lorraine, Nancy, 1999.
Appointment: since 1999: Institut Franais des Boissons de la
Brasserie Malterie (Vandoeuvre les Nancy): Raw Material Manager:
in charge of new barley variety tests; Process and Biochemical
Research Manager: in charge of different research projects; Quality Assurance
Manager: in charge of three quality systems (ISO9002, COFRAC accreditation, Good
Laboratory Practices).

Almeida, Paulo Joaquim Ferreira (contribution nr. 84)

Born in 1967.
Studies: Degree in Chemistry (Scientific option) at the Faculty of Science and
Technology, University of Coimbra (1989); PhD in Analytical Chemistry at the
Faculty of Science, University of Porto, Portugal.
Appointments: 1989: Monitor at the Chemistry Department, Faculty of Science and
Technology, University of Coimbra; 1990: First Assistant at the Chemistry
Department, Faculty of Science and Technology, University of Coimbra; 1991-1992:
First Assistant at the Chemistry Department, Faculty of Science, University of Porto;
1992-2000: Second Assistant at the Chemistry Department, Faculty of Science,
University of Porto; since 2001: Lecturer at the Chemistry Department, Faculty of
Science, University of Porto.

Alt, Axel (contribution nr. 5, 6)

Born in 1969.
Education: Study of Chemistry (1990-1997) and PhD (2001) at the
University of the Saarland, Saarbrcken, Germany: Investigation of
beer and its constituents on potential cancer preventive activity;
since 03/2001: Post-doc at the Institute for Pharmacognosy and
Analytical Phytochemistry, University of the Saarland, Saarbrcken;
Member of GDCh.

Andersen, Helge A. (contribution nr. 44)

Born in 1939.
Studies: PhD, Dr.Sc. University of Copenhagen, Denmark.
Appointments: 1965-1968: Research Ass. in Biochemistry at the
Royal Veterinary and Agricultural University, Copenhagen; 1968-
1972: Research Ass. at the Biological Institute of the Carlsberg
Foundation, Copenhagen; 1973-1982: Lecturer at the Biological
Institute of the Carlsberg Foundation, Copenhagen; 1978-1982: Head
of the Biological Institute of the Carlsberg Foundation, Copenhagen; 1983-1991:
Visiting scientist at the University of Copenhagen; 1991-1992: Visiting scholar at the
University of California, Los Angeles, U.S.A.; since 1992: Senior Researcher at the
Carlsberg Laboratory, Copenhagen.

Arimura, H (contribution nr. 39)

Born in 1971.
Studies: Agricultural Chemistry at the Kagoshima University, Japan
(1996).
Appointments: 1996-1998: staff in Fukuoka Brewery, Kirin; since
1998: Researcher in the Research Laboratory for Brewing.

2
Arnould, Michel (contribution nr. 83)
Born in 1973.
Studies: Bachelor's degree Candidat en Sciences Agronomiques,
Universit Catholique de Louvain, Belgium, 1991-1994; Master's
degree Ingnieur Chimiste et des Bio-Industries, Universit
Catholique de Louvain, 1994-1997; Master's degree Master's in
Malting and Brewing Sciences, Katholieke Universiteit Leuven,
1997-1998.
Appointments: 1998-2000: Research Engineer at Katholieke Universiteit Leuven;
2000-2001: Research Engineer at Brewery Orval. Main research activities: aromas,
treatment of by-product for valorisation, hop

Asakura, Takashi (contribution nr. 21)

Born in 1947.
Studies: Faculty of Agriculture and Veterinary at the Nihon
University, Japan (1970).
Appointments: 1970: Maltster in Nihon Nousan Kako Co.Ltd.; 1970-
1990: Maltster in Nihon Nousan Kako Co.Ltd.; 1991-2000: Malt
Quality Analysis in Plant Breeding at the Plant Bioengineering
Research Laboratories (PBRL), Sapporo Breweries Ltd.; since 2000:
Group Manager in Cereal Chemistry at PBRL.

3
Curriculum vitae of (co)authors - B

Back, Werner (contribution nr. 15, 17, 22, 23, 39, 54, 65, 66, 75)
Born in 1942.
Technical graduation as Brewer and Malster (1965); Diploma
Engineer at the Faculty of Brewing and Food Technology of the
University Munich-Weihenstephan, Germany (1969); Scientific
Assistent at the Institute of Technical Microbiology and Technology
of Brewing (1970-1976); Doctorate, theme: "Taxonomy of Beer
Spoiling Cocci" (1974).
Head of Microbiology and Quality Control at Dhler GmbH, Darmstadt (1976);
University Lecturing Qualification (Habilitation), theme: "Taxonomic Examinations
on Beer Spoiling Bacteria" at the Faculty of Brewing, Food Technology and Dairy
Science of the University Munich-Weihenstephan (1980); Member of Management of
Dhler GmbH, Darmstadt (1983-1988).
Since 1985 publicly ordered and sworn expert for Food Microbiology (Chamber of
Industry and Commerce Darmstadt); University Professor at the Chair of Brewing
Technology I at the University Munich-Weihenstephan (1988); since 1992: Head of
the Chair of Brewing Technology I and Beverage Technology at the Weihenstephan
Center of Life and Food Sciences, University Munich.

Barklage, Hansjrgen (contribution nr. 98)

Born in 1950.
Studies: Physics at the Technical University of Clausthal (TUC),
Germany (1969-1970); Thesis in Glass Technology (1978), also at
the TUC.
Appointments:1978-1991: Senior Scientist in Glass Processing at the
German Society for Glass Technology; since 1984 lectures in glass
technology at the TUC; secretary of different committees for glass
technological problems; research, development and consulting in co-operation with
many different glass manufacturers and suppliers of the glass industry. 1992: joined
Nienburger Glas GmbH.: 1992-1996: Manager for Glass Technology. 1994: Honorary
Professor for glass technology at TUC; 1996: Senior Manager for Technology and
Production; 1998: Chairman of the Comittee for Glass Furnaces and Energy of the
DGG; 2000: Technical Director.

Barros, Aquiles A. (contribution nr. 84)

Born in 1950.
Studies: Degree in Chemical Engineering, 1973, Faculty of Engineering - University
of Porto, Portugal; PhD in Analytical Chemistry, 1986, Faculty of Science -
University of Porto.
Aggregation in Chemistry, 1999, Faculty of Science - University of Porto.
Appointments: all at the Faculty of Science of the University of Porto: Second
Assistant, 1973-1976; First Assistant, 1976-1986; Lecturer, 1986-1995; Associated
Professor, 1995-2001. President of the Chemistry Department, 1996-1997.
Bartsch, Helmut (contribution nr. 5, 6)
Born in 1940.
Studies: BSc, MSc in Organic Chemistry, University of Heidelberg,
Germany (1965); PhD in Chemistry at the University of Heidelberg
(1968). Topic: Elucidation of the structure of phorbol. Habilitation in
Experimental Oncology, Medical School in Hannover, Germany
(1979); docent and external faculty member.
Appointments: 1968-1970: Research Associate at the Institute of
Biochemistry, German Cancer Research Center, Heidelberg; 1971-1973: Postdoc at
McArdle Laboratory for Cancer Research, Madison, Wisconsin, USA (with E.C. and
J.A. Miller); 1974-1980: Scientist at WHO, Unit of Carcinogenesis, IRAC, Lyon,
France; 1981-1993: Unit Chief at WHO, Unit of Environmental Carcinogenesis and
Host Factors, IRAC, Lyon; since 1993: Full Professor (Chair) at the University of
Heidelberg, Head of the Division of 'Toxicology and Cancer Risk Factors', German
Cancer Research Center, Heidelberg. Scientific interest: (i) genetic cancer
susceptibility markers, (ii) genetic alterations in human tumorigenesis, (iii)
biomarkers for cancer etiology and prevention, (iv) cancer chemoprevention.

Bastin, Patrick (contribution nr. 46)

Born in 1975.
Studies: Industrial Engineering Degree at the Institut Meurice,
Brussels, Belgium (1999).
Research training period: from March to June 1999 in Molecular
Biochemistry at the Jean-Marie Wiame's Institut, Brussels.
Industrial training period in July 1999 in Fermentation Technology at
the Brewery of Scourmont, Chimay, Belgium.
Appointment: since January 2000 employed by Meurice Research
and Development at the Department of Brewing Sciences and Fermentation
Technology of the Institut Meurice; for the period from January 2001 till June 2001:
Research training period at IFBM, Nancy, France.
Participation in scientific meetings: Lecture entitled Potentialits de production et
dutilisation dantioxydants naturels extraits de levure at the ARFB workshop Bire
et sant: les bienfaits de la nature dans un verre.

Baxter, E. Denise (contribution nr. 9)

Brewing Research International (BRi), UK, represents many brewing
and malting companies across the world. She joined BRi in 1972
from Edinburgh University. Her research at BRi has covered several
aspects of malting biochemistry and physiology, in particular protein
degradation during malting. She became Head of the Raw Materials
Team in 1987 and continued in that position until 1990. She is
currently Director of Membership Services and Head of Food Safety
at BRi and a member of its Executive Board.
She assumed responsibility for Food Safety matters in 1986. This post involves
initiating and managing a programme of research relating to food safety for the
Brewing Industry as well as potential healthy ingredients in beer. This includes
maintaining monitoring services for potential contaminants in beer and brewing raw
materials, co-ordinating malting and brewing trials to evaluate both contaminants and
micronutrients, and providing an information service on food safety and health for
Member Companies. As Head of Food Safety she is a Member of several brewing

2
industry committees. Where necessary she liaises with the relevant government and
Food Industry bodies. She also lectures widely at seminars and conferences on food
safety in brewing and health benefits of moderate beer consumption.

Bechmann, Frank (contribution nr. 28)

Born in 1965.
Studies: 1989: Degree in Brewing Science at Technical University of Munich-
Weihenstephan, Germany; 1995: PhD in Engineering at Technical University of
Munich-Weihenstephan.
Appointments: 1996: GIW-Geschftsfhrer Gesellschaft fr Informations-technologie
Weihenstephan.

Becker, Hans (contribution nr. 5, 6)

Born in 1940.
Education: study of pharmacy at the University of Mainz, Germany
1961-1964; PhD at the University of Karlsruhe, 1970, "Essential oil
production in intact plants and in in vitro-cultures"; Professor for
Pharmaceutical Biology at the University of Heidelberg. 1973-1987.
Present professional position: Full Professor (Universittsprofessor
C4) for Pharmacognosy and Analytical Phytochemistry (Succession of late Professor
Dr. Egon Stahl) at the University of the Saarland; 1994-1996 Dean of Faculty of
Mathematics and Natural Science.
Present research: secondary compounds (terpenes and phenols) from bryophytes,
biosynthesis of terpenes, cancer preventing agents from food and medicinal plants.
Member of GDCh, Gesellschaft fr Arzneipflanzenforschung, American Society of
Pharmacognosy, DPhG, Groupe Polyphenole.
Since 1997 Board Member Ausschuss Analytik der Deutschen Homoeopathischen
Arzneibuchkommission; Board Member of "Biozentrum Uni Wrzburg"; Editorial
Board of Chromatographia.

Becker, Thomas (contribution nr. 29, 75, 109)

Born in 1965.
Studies: Food and Biotechnology at the Technical University Munich
-Weihenstephan, Germany (1991); Doctoral Thesis (1995):
Entwicklung eines rechnergesttzten enzymintegrierten Flie-
injektionssystems und seine Verwendung in der biotechnologischen
Prozeleittechnik und Qualittskontrolle.
Appointments: Engineer Geo-Konzept GmbH (1992-1993);
Scientific Assistant at the Chair of General Chemistry and Biochemistry (1994-1996);
Head of the department of Process Automation at the Chair of Fluid Mechanics and
Process Automation at the Weihenstephan Center of Life and Food Sciences,
Technical University Munich (since 1996).

3
Bene, Roman (contribution nr. 81)
Born in 1958.
Studied Electrical Engineering at the Czech Technical University in
Prague where he received his master's degree in 1983. In the same
year he joined the Charles University at Prague, Faculty of
Mathematics and Physics as a research assistant. He worked in the
field of photoacoustics, investigating its use as a tool for non-
destructive testing of layered samples and for studying the laser
ablation processes in the UV range. He received his PhD degree from Charles
University in 1988. In 1989, he joined the Institute of Physical Electronics of the
Slovak Academy of Sciences. He worked on the design, development and testing of
IR focal plane arrays based on pyrodetectors and PtSi Schottky diodes. In 1991, he
joined the Institute of Chemical and Optical Sensors at Joanneum Research. He was
active in the development and characterization of optical sensors, qualitative
spectroscopy in VIS and NIR spectral ranges, and in measurement techniques. In the
field of NIR spectroscopy, Joanneum Research has co-operated with an industrial
company Anton PAAR GmbH since 1994. The NIR spectroscopy became a strategic
direction for this company and it was decided to involve it directly among their
research activities. Since 1999, he is working at Anton PAAR GmbH. He is
responsible for managing of applied research, primary for NIR spectroscopy.

Berg, Harry (contribution nr. 49)

Born in 1965.
Studies: Chemical Engineering at Kokkola Institute of Technology,
Finland, 1991.
Appointments: 1986-1987: Trainee at Sinebrychoff Helsinki brewery;
1991-1993: Shift Supervisor at Sinebrychoff Helsinki brewery; !993-
1999: Restaurant Brewer at Kappeli brewery; since 2000:
Development Engineer at Sinebrychoff Kerava brewery.

Bergin, Joe (contribution nr. 49)

Has a Science degree in Microbiology and has spent his entire
working life (22 years) in brewing Research and Development with
Guinness, Ireland. Over the years, he has worked on various aspects
of the brewing process. He has worked for a number of years on new
product development (both brewed and formulated) before
concentrating on the process side. The last 10 years has been spent on
process improvement and optimisation while evaluating and assessing
new technologies for possible applications within the brewing industry. The bulk of
his recent work in the last 2-3 years has centred around immobilised yeast bioreactors
and their potential application in the brewing industry, ranging from non-alcoholic
beer production to both primary and secondary fermentation and also wort
acidification. He has also worked extensively on membrane applications for alcohol
removal from beer, recovery of beer from waste yeast, and gas exchange applications.
He currently holds the position of Process Innovation Manager with the Process
Support & Innovation Group. This groups work ranges from support function to our
own and third party operations right through to step change innovative process
solutions.

4
Bertrand, Dominique (contribution nr. 19)
Is a Senior Researcher in the French public organisation INRA
(National institute of agricultural research). Doctor thesis
Application of multidimensional analyses in Near Infrared
spectroscopy" in 1988. His research has been devoted to the
development of Near and Mid Infrared spectroscopy in food science,
and to the application of artificial vision for the quality control of
agricultural products. In this field his main interests are about
chemometrics applied to physical measurements such as spectra or digitised images.
He has contributed to more than 150 scientific publications or communications in
international congresses. He recently edited a book on the analytical applications of
Near and Mid Infrared spectroscopy in agriculture and food science.

Bes, Magali (contribution nr. 83)

Born in 1971.
Studies: Engineer from "Institut des Sciences de l'Ingnieur de l'Universit de
Montpellier" (ISIM), 1994. Specialties: Science and technology of food industries.
Engineer of experimentation, IMECA, BIP, 1994.
Appointments: 1995: Engineer of development, Skalli Fortant de France; 1996-1999:
Engineer of research, 12 distilleries and TRIAL; since 1999: Research engineer,
manager of the team "process, new technology and new product", INRA Pech Rouge.
Main research activities: aromas, technology of fractionation, extraction, processing
of vegetable, treatment of by-product for valorisation.

Bezares, Eduardo (contribution nr. 102)

Born in 1963.
Studies: Msc Industrial Engineer at the Basque Country University
(1982-1988); MBA La Laguna University (1994-1995); PhD
Econom. & Business Management, La Laguna University Spain
(1995-); Dip. Industrial Organization. London School of Economics.
U.K. (1994); Dip. Logistics, British Institute of Logistics, (1990-
1994).
Appointments: 1987-1988: Sales Dept. Sist. Telemticos. Madrid, Spain; 1988-1989:
Tr. Project Engineer Royal Dutch Shell, The Netherlands; since 1989 employed by
Ca. Cervecera de Canarias, S.A. (S.A.B.I. Group): 1989-1995: Logistics Manager;
since 1995: Logistics Subdirector. Since 1994: Vice-President Spanish Shippers
Council. Since 1994: Member Board of the Directors Tenerife Port Authority. Since
2001: Vice-President Canary Islands Industrial Association. Since 1994: Logistics,
Transport and SCM Professor University of Preston USA. Since 1996: Logistics
Professor University of La Laguna Spain. Author of numerous articles and speaker at
seminar and conference programs concerning Logistics, SCM, Quality and R&D. In
the public sector he has served as an advisor to the Canary Islands Dept. of Transport
and Dept. of Economy & Commerce, Spanish Deputy Congress and EU DGVII.
Founder of the world wide ONG AIESEC in the Canary Islands. MILDM, MCEL,
AIRTE, MBTAC, MAPICS.

5
Boeykens, Annick (contribution nr. 60)
Born in 1975.
Studies: Academic degree in Industrial Engineering - Biochemistry
at the KaHo St. - Lieven, Gent, Belgium (1998).
Appointments: September 1998 - July 1999: Research and
Development laboratory assistant at Omnichem n.v., Wetteren; July
1999 - October 2000: Assistant Scientist in Malting and Brewing
Science at the Laboratory of Enzyme and Brewing Technology of
KaHo St. - Lieven. Current position: responsible for practical courses in Malting and
Brewing Science and assistant scientist in Malting and Brewing science. Research
topics: High Tech Hopping, Flavour Stability.

Bohak, Ingrid (contribution nr. 17)

Born in 1964.
Graduated from Technical University of Munich-Weihenstephan,
Germany, in Brewing Science and Beverage Technology in 1989.
After receiving her diploma from university, she started in 1990 to
work as Scientific Assistant at the Chair for Brewery Technology I
under Prof. Dr. Werner Back. Her area as leader of the department
Beverage Technology and Beverage Microbiology encloses development of new soft
drink products as well as research on microbiological questions appearing in soft
drink industry and brewery and taxonomic studies on beverage relevant bacteria and
yeasts.

Boivin, Patrick (contribution nr. 13, 19, 20, 46, 57, 67)
Born in 1957.
He is Scientific and Quality Director at the French Malting and
Brewing Institute. He has been lecturer in malting technology at the
Louvain-la-Neuve University since 1996 and Visiting Professor at
Nancy Food Engineer School. He received BSc in Biology and
Biochemistry from Rouen University (1983), MSc in Biotechnology
from Compigne Technology University (1984) and PhD in Microbiology,
Enzymology and Bioconversion from Compigne University (1987). He was Post-
Doctoral Fellow at Baylor University, Texas, U.S.A., 1987-1989. He received Master
in Business and Administration in 1998 from French Institute of Management. Since
1989 he has been working at the French Malting and Brewing Institute as Scientific
Director. He is a member of the EBC Brewing Science Group, Chairman of the
Flavour Sub-group and a member of the French Barley-Malt-Beer Committee. He is
co-ordinator of the French procedure for assessing pesticide used in malting barley.
He is a member of ACCC and polyphenol group. He has published several papers,
reviews and patents.

Bolshaw, Louise (contribution nr. 8)

Is a graduate research scientist in the Beverage Product Quality
department at Brewing Research International, UK. She gained her
degree in Applied Biology through part-time studies from the
University of Greenwich, after first completing a Higher National
Certificate at the North East Surrey College of Technology. Since
joining BRI in 1992, she has worked on a number of different
research areas within the Product Quality department, including investigations into

6
Colloidal Stability and Cider polyphenols and Mouthfeel. She is currently working in
the area of Flavour Stability and is responsible for the HPLC analyses of Hops,
polyphenols and antioxidants.

Bonacchelli, Bruno (contribution nr. 24)

Born in 1971.
Studies: Chemical Engineering at the University of Lige, Belgium (1994).
Appointments: 1995-1998: Research Engineer at the Centre Wallon de Biologie
Industrielle (University of Lige) in collaboration with Meura (project on beer
filtration); since 1999: Research Engineer at Meura Technologies (R&D department
of Meura s.a.), in charge of different projects on brew house and fermentation.

Booer, Chris D. (contribution nr. 73)

Born 1951.
Studies: Joint Honours Degree in Botany and Microbiology at the
University College of Swansea, UK (1973).
Appointments: Since 1973 employed by Brewing Research
International: initially as a Research Scientist and latterly as Senior
Maltster. Since 2000, Member of the Institute of Brewing English Working Party and
Chairman of the English Micromalting Group.

Boulton, Chris A. (contribution nr. 78)

Born 1948.
Education: First degree and PhD in Biochemistry, University of Hull,
UK.
Appointments: University of Hull, Department of Biochemistry,
Research Technician (1970-1977); Research Assistant (1977-1980);
Post-Doctoral Research Fellow (1980-1984); Bass Brewers, R&D
Department: Fermentation Microbiologist (1984-1989); Project Manager
Microbiology (1989-1993); Senior Research Scientist (1993-1995); Development
Scientist (1995-present).

Box, Wendy G. (contribution nr. 41, 78)

Born 1953.
Studies: HNC Applied Biology (specialising in Microbiology) at
Trent Polytechnic, Nottingham, UK (1974).
Appointments: 1972-1973: Research Technician (Analytical) at
Bovril Foods Ltd; since 1973: employed by Bass Brewers as
Scientific Assistant - Microbiology (1973-1976); Senior Technician -
Genetics (1977-1984); Senior Technician - Analytical (1985-1988);
Senior Technician - Microbiology (1989-1997) and Research
Technologist (1997-).

7
Bravo Melet, Adriana (contribution nr. 48, 61, 63, 64)
Born in 1961.
Studies: BSc in Chemistry at the Central University of Venezuela
(1988, with honors). PhD in Analytical Chemistry at the Simon
Bolivar University (2001). Co-author in 2 utility patents.
Appointments: 1988-1993: Associate Scientist at the Laboratory of
Biotechnology, Polar Technology Center; 1993-1999 Chief
Scientist at the Laboratory of Biotechnology, Polar Technology
Center; since 1999: Corporate Manager of Chemistry, R&D Unit of
Empresas Polar, Caracas-Venezuela.

Brignon, Pierre (contribution nr. 76)

Born in 1965.
Studies: PhD in Plant Molecular Biology at the Universit Louis Pasteur of
Strasbourg, France (1992).
Appointments: 1993-1994: post-doc training in the laboratory of Pr Pere
Puigdomenech, departemento de genetica molecular, CID-CSIC Barcelona; 1994-
2000: Head of Molecular Biology department of Tepral, Kronenbourg breweries;
since 2000: Head of Tepral.

Bruijn, Paul J.M. (contribution nr. 105)

Born in 1961.
Studies: Mechanical Engineering at Hogere Technische School,
degree in 1983; Biology at the University of Leiden (the
Netherlands), degree in 1988.
Employed by Heineken Technical Services (HTS) since 1989:
Scientist at HTS Research and Development 1989-1996; topics by-
products, waste water treatment, cleaning and disinfection.
Environmental Specialist at HTS Project Services since 1996; main
topics include water and waste water management (development of improvement
programs), handling of by-products (research and operational support).

Buck, Annemie, de (contribution nr. 58)

Born in 1955.
Studies: A two year study in Chemistry at the University of Gent,
Belgium (1975); academic degree in Industrial Engineering -
Biochemistry at KaHo St.- Lieven, Gent (1979).
Appointments: 1979-1990: Assistant Scientist at the Faculty of
Agricultural Sciences, University of Gent; since 1990: Professor in
Biochemistry at the KaHo St.-Lieven, Gent. Responsible for the
practical courses in Biochemistry and Food Analysis, theoretical courses in Enzyme
Technology. Research topic: Enzyme Technology.

8
Curriculum vitae of (co)authors - C

Cahill, Gearid (contribution nr. 88)

Studies: BSc Biotechnology at Dublin City University, Ireland
(1987); MSc Immobilised Yeast Studies at Dublin City University
(1990); PhD Brewery Yeast Management at Guinness & Dublin City
University (1999).
Appointments: 1990-1991: Fermentation Operator at Interbio Ltd.,
Dublin; 1991- 1996: Environmental Consultant at Bord na Mona,
Kildare; 1996-2000: Development Technologist at Guinness Dublin;
currently Innovation Manager at Guinness Dublin. Member of IOB and MBAA.
Member of MBAA Editorial Committee.

Cantrell, I (contribution nr. 42)

Carvell, John (contribution nr. 78, 79)

Born in 1955.
Studies: Biochemistry at Bangor University (1976) and PhD at
University of Newcastle, UK (1980).
Appointments: 1980-1985: Production Manager at British
Fermentation Products; 1985-1987: Senior Product Development
Engineer at APV, Crawley; 1987-1989: Biotechnology Sales
Manager, APV Baker; 1989-1993: Senior Product Manager, Alfa
Laval (UK); 1993-2001: Sales and Marketing Director Aber Instruments Ltd (UK).
Presented with Queens Award for Export in 1998. Board member of Export
Association (UK). Since 1998 member of ASBC with poster presentations in 1998
and 1999. Member of IOB and MBAA with poster presentations at IOB Asia Pacific
Meetings in 1998 (Perth) and 2000 (Singapore).

ejka, Pavel (contribution nr. 82)

Born 1951.
Studies: Institue of Chemical Technology - Prague, Faculty of
Fermentation Chemistry and Bioengineering, MSc degree 1974;
Institute of Microbiology in Academy of Sciences of the Czech
Republic, PhD degree 1987.
Appointments: since 1975: Research Institute of Brewing and
Malting Prague; 1975-1990: Head of the Laboratory of Checking;
1990-2000: research worker; since 2000: Quality Manager of Accredited Analytical
Testing Laboratory.
epika, Jaroslav (contribution nr. 85)
Born in 1940.
Studies: Chemistry at the Institute of Chemical Technology in Prague,
Czech Republic, Faculty of Food Technology, graduated with
Dipl.Ing. in Chemistry (1962). Specialised in Hop Chemistry and
Utilisation and obtained PhD (1972) from the same Institute.
Appointments: 1963: postgraduate study at ICT Prague; 1965:
Assistant Professor for Brewing Science; 1969: Technical Assistant at
Staropramen Brewery in Prague; 1972: Assistant Professor for Brewing Science;
1988: Associated Professor for Food Technology; 1989: Vice-dean of the faculty of
Food and Biochemical Technology; 1996: Scientific Secretary of the Research
Institute of Brewing and Malting; 1997: Head of Department of Fermentation
Chemistry and Bioengineering; 1996: Member of the EBC Brewing Science Group.

Chandra, Sachin (contribution nr. 8, 73)

Born 1964.
Studies: B.Sc (Hons) Chemistry from Osmanda University, India;
M.Sc (Eng) Process Biotechnology (1986) and PhD in Chemical
Engineering at the University of Birmingham U.K. (1990).
Appointments: 1986-1990: Research Associate in Chemical
Engineering at the National Engineering Laboratory in U.K.; 1990-
1992: Post-Doctoral Research Associate at the University of Bath, U.K.; since 1992:
employed by Brewing Research International: 1992-1999: Senior Scientist; since
1999: Head of Product Quality.

Chmiel, Horst (contribution nr. 5, 104)

Born in 1940.
Education: 1963: BSc in Chemical Engineering, Frankfurt, Germany;
1967: DIC in Chem. Eng., Technical University Aachen; 1971: PhD
in Chem. Eng., Technical University Aachen; 1973: Habilitation in
Biochemical Engineering in Medicine, Technical University Aachen.
Professional experience: 19671971: Post-graduate Research,
Institute for Chem. Eng.,Technical University Aachen; 19711975:
Post-doctoral appointment, Heimholtz-Institute Aachen; 19761992: Director of the
Fraunhofer-Institut fr Grenzflchen- und Bioverfahrenstechnik (Interfacial
Phenomena and Biochemical Engineering) Stuttgart; 19791986: apl. Professor at the
University of Stuttgart; 19861995: full Professor at the University of Stuttgart; since
1992: full Professor at the University of Saarland and Director of the Gesellschaft fr
umweltkompatible Prozetechnik (Institute for Environmental Compatible Process
Technology), Saarbrcken, Germany.

Collin, Sonia (contribution nr. 80)

PhD in Chemical Sciences from the University of Namur, Belgium
(1988). Professor of Malting and Brewing Sciences at the University
Catholique de Louvain, Louvain-la-Neuve, since 1993. She is
currently Head of the Department of Brewery and Food Industries
(INBR). She is a member of the American Society of Brewing
Chemists and of the EBC Brewing Science Group. She has published
more than 75 papers in scientific journals, mainly on flavour stability,

2
sulfur aroma, parazines and hops. She was recently chairman of the IXth J. De Clerck
Chair The oxygen paradox in the brewing process (2000).

Cooman, Luc, de (contributions nr. 7, 58, 60)

Born in 1961.
Studies: MSc (Plant Physiology and Biochemistry), University of
Gent, Belgium (1984); PhD in Biochemistry, University of Gent
(1991).
Appointments: 1984-1991: Assistant Professor in Plant Biochemistry,
Laboratory of Plant Biochemistry, University of Gent; 1992-1994:
post-doc position (same affiliation), funded by the Flemish Institute
for the Promotion of Scientific-Technological Research in Industry (IWT, Brussels);
1994-1997: Senior Research Associate at the Laboratory of Pharmacognosy and
Phytochemistry, University of Gent; 1997-2000: Senior Research Associate at the
Laboratory of Enzyme and Brewing Technology, KaHo St.- Lieven, Gent. Current
position: Professor in Malting and Brewing Science at the KaHo St.-Lieven,
Laboratory of Enzyme and Brewing Technology. Research topics: Hops, Beer
Flavour Stability.

Cooper, Daniel J. (contribution nr. 68)

Studies: BSc in Brewing and Distilling (1st class hon., 1995, Heriot
Watt University, UK), PhD High gravity brewing and its negative
effect on head retention (1999, Heriot Watt University).
Appointments: 1999-to present, Institute of Food Research,
postdoctoral researcher. Associate member of The Institute and
Guild of Brewing.

ulk, Ji (contribution nr. 82)

Born 1952.
Studies: Institute of Chemical Technology - Prague, Czech Republic,
Faculty of Food Preservation and Meat technology; MSc degree
1975; Institute of Chemical Technology - Prague, Faculty of
Fermentation Chemistry and Bioengineering; PhD degree 1987.
Appointments: 1975-1977: Koliprojekt-Prague: Food Engineer; 1978-
1980: Koliprojekt-Prague: Development worker; 1980-now:
Research Institute of Brewing and Malting Prague (RIBM): Research worker; since
2001: Technical Head of Accredited Analytical Testing Laboratory. Since 1997:
member of the MEBAK Committee.

Cunningham, Stephen (contribution nr. 79)

Studies: Graduated in 1994 from the University of Dundee, UK, with
an honours degree in Microbiology. In 1995 commenced a PhD at
the International Centre for Brewing and Distilling at Heriot Watt
University investigating the effects of acid washing and high gravity
brewing on the fermentation performance of brewers yeast.
Appointments: 1994-1995: Research technician at the University of
Dundee. Since 1998: Technical Sales Engineer with Aber
Instruments Ltd responsible for sales of brewery instrumentation

3
within the UK and Ireland.

urdov, Eva (contribution nr. 85)

Born in 1959.
Studies: Faculty of Science, Charles University Prague, Czech
Republic, specialization Analytical Chemistry MSc (1978), PhD
degree in Analytical Chemistry (1993).
Appointment: Assistant Professor at Department of Analytical
Chemistry, Institute of Chemical Technology Prague.

4
Curriculum vitae of (co)authors - D

Davis, Daniel (contribution nr. 33)

Studies: University of Reading, UK, BSc in Biotechnology (1992-
1996), University of Reading, PhD in Biotechnology (1996-1999).
Appointments: Fermentation Scientist, Brewing Research
International (2000-2001); Fermentation Plant Scientist, Lonza
Biologics (2001-date).

Dawes, Ian W. (contribution nr. 52)

Born in 1945.
Studies: BSc in Food Technology at University of New South Wales,
Australia; DPhil in Biochemistry from University of Oxford, UK
(1969).
Appointments: 1969-1970: Junior Research Fellow Linacre College
& Guinness Research Fellow in Microbiology Oxford University;
1970-1972: Postdoctoral Fellow of the Arthritis Foundation in
Molecular Biology, University of Wisconsin, Madison, USA and Rosenstiel Basic
Medical Sciences Research Center, Brandeis University, Massachusetts. 1972-1988:
Lecturer then Reader in Microbiology, Edinburgh University; since 1989: Professor of
Genetics, School of Biochemistry and Molecular Genetics, University of New South
Wales; 1991-1996: Head, Biochemistry & Molecular Genetics, University of New
South Wales; 1998 Visiting Professor, Institute for Genetics, University of Salzburg,
Austria; since 1998: Associate Dean Research, Faculty of Life Sciences, UNSW;
1984-1988: Editor: Microbiological Sciences; Member of Editorial Boards: Journal of
General Microbiology (1980-86); Yeast (1985- ); Molecular Microbiology (1989-
1996); FEMS Yeast Research (2000- ).

Day, Rachel E. (contribution nr. 52)

Born in 1973.
Studies: BSc Hons 1 in Biochemistry and Molecular Genetics at the
University of New South Wales, Australia. Currently completing a
PhD in the School of Biochemistry & Molecular Genetics, UNSW
and Carlton & United Breweries, Victoria. Recipient of Australian
Postgraduate Industry Award. Thesis title: Maltotriose utilisation in
Saccharomyces cerevisiae.
Appointments: November 1995- February 1998: Research Assistant in Molecular
Parasitology group, UNSW, working with Crithidia luciliae and Tritrichomonas
foetus investigating the purine transport. Publications: Rachel E. Day and Annette M.
Gero (1997) Stimulated transport of hypoxanthine in Crithidia luciliae: relationship
to purine stress. Parasitology, 114, 19-27. Annette M.Gero, Rachel E. Day and
Simone T. Hall (1997) Stimulated transport of adenosine, guanosine and
hypoxanthine in C. luciliae: Metabolic machinery has a distinct advantage over the
host, International Journal of Parasitology, 27, 241-249.
Debourg, Alain (contribution nr. 45, 46)
Is Lecturer in brewing sciences and fermentation technology at the
Institut Meurice and at the Free University of Brussels, Belgium, and
since academic year 1993-1994, Head of the department of Brewing
Sciences and Fermentation Technology.
From 1987 to 1991, he was Research Assistant of the National
Belgian Fund for Scientific Research and had studied regulatory
mechanisms involved in yeast amino acid metabolism and presented
his Doctorate at the University of Brussels.
He is a member of the Belgian Biochemistry Society and the American Society of
Brewing Chemists. In June 1996 he also became Vice-Chairman of the EBC Brewing
Science Group and from 1 October 2000 he is Chairman of this Group.
He is actually coordinator of different research and education programmes as well at
national as at European level, collaborating with many universities and industries
worldwide.
The department of Brewing Sciences and Fermentation Technology is involved in
consultancy activities around the world for technical support and development of new
activities.

Delgado, Antonio (contribution nr. 29, 75, 109)

Born in 1956.
Studies: Energy and Process Engineering at the University GH
Essen, Germany (1981); Doctoral Thesis (1986): Untersuchung der
turbulenten Strmung von Polymerlsungen in einem zwei-
dimensionalen Kanal mittels Laser-Doppler-Anemometrie;
Professoral thesis (1993): Gravitationskompensierte Strmungen
rotierender newtonscher und nichtnewtonscher Fluide.
Appointments: Scientific Assistant at the subject area Fluid
Mechanics of the University GH Essen (1981-1986); Head of the department of Fluid
Mechanics and Microgravity Utilisation at the Center of Applied Aerospace
Technology and Microgravity at the University Bremen (1987-1992); Head of the
department of Advance Development and Research at the Vorwerk Elektrowerke
Stiftung & Co KG (1992-1996); Lecturer at the University Bremen (1993-1995);
Head of the Chair of Fluid Mechanics and Process Automation at the Weihenstephan
Center of Life and Food Sciences, Technical University Munich (since 1995); Studies
Dean of the Faculty Brewing and Food Technology (since 1999); First Pro-Dean of
the Weihenstephan Center of Life and Food Sciences, Technical University Munich
(since 2000).

Delvaux, Freddy (contribution nr. 83)

Born in 1945.
Studies: 1963-1965: Bachelor's degree: Kandidaat Landbouwkundig
Ingenieur, Katholieke Universiteit Leuven, Belgium; 1965-1968:
Master's degree: Ingenieur voor de Scheikunde en de Landbouw-
industrien, Katholieke Universiteit Leuven; 1972: PhD degree:
Doctor in de Landbouwwetenschappen, Katholieke Universiteit
Leuven.
Appointments: 1973-1990: Research and Quality Director at the Artois (Interbrew)
brewery; since 1991: Professor and brewery technology consultant at the Katholieke
Universiteit Leuven. Main research activities: Malting and Brewing Science.

2
Derdelinckx, Guy (contribution nr. 83)
Born in 1954.
Studies: 1972-1974: Bachelor's degree: Kandidaat Landbouwkundig
Ingenieur, Universit Catholique de Louvain, Belgium; 1974-1978:
Master's degree: Landbouwkundig ingenieur voor tropische en
subtropische gebieden, Universit Catholique de Louvain; 1978-
1979: Master's degree: Speciaal diploma in de brouwerij, Universit
Catholique de Louvain; 1986: PhD degree: Doctor in de Landbouw-
wetenschappen, Universit Catholique de Louvain.
Appointments: 1980-1987: Laboratory assistant, Laboratoire de brasserie, Universit
Catholique de Louvain; 1987-1993: Senior Lecturer, Laboratoire de brasserie,
Universit Catholique de Louvain; 1982-1993: Manager of the analysis station,
Laboratoire de brasserie, Universit Catholique de Louvain; since 1993: Assistant
Professor, Centre for Malting and Brewing Science, Katholieke Universiteit Leuven;
since 1993: Invited Assistant Professor, Universit Catholique de Louvain; since
1999: Invited Assistant Professor, University Dunarea de Jos, Galati Romania; since
1995: Titular of the P.F. Martens Chair for Brewing Research, Katholieke Universiteit
Leuven; since 1984: Scientific adviser of Brewery Martens, Bocholt, Belgium; since
1982: Technical consultant of several breweries of special beers and top fermented
beers; Manager of brewing yeast bank,Centre for Malting and Brewing Science,
Katholieke Universiteit Leuven. Member of the Institute of Brewing, Member of the
American Society of Brewing Chemists.
Main research activities: Belgian beer types/special beer types, top fermentation,
bottle fermentation, aboriginal fermented beverages (banana wine and palm wine),
natural flavour production Brettanomyces sp/Dekkera sp, phenolics.

Deurinck, Patricia C. (contribution nr. 105)

Born in 1969.
Studies: 1988-1993: Chemical Engineering, K.U.Leuven, Belgium; 1993-2000: PhD
(modelling of catalysts), K.U.Leuven.
Appointment: since 2000: Brewing Operations Scientist at Heineken Technical
Services, the Netherlands.

Dickel, Torsten (contribution nr. 75)

Born in 1974.
Studies: Brewing and Beverage Technology at the Technical
University Munich-Weihenstephan, Germany (1999).
Appointments: Scientist Process Technology in Brewing at the Chair
of Brewing Technology I and Beverage Technology at the
Weihenstephan Center of Life and Food Sciences, Technical
University Munich (since 1999).

Dillemans, Monique (contribution nr. 45, 46)

She is Research Assistant in the department of Brewing Science at
Institut Meurice, Brussels, Belgium. She got her Industrial Engineer
Degree from the Institut Meurice in 1978, a Master Degree in
Biological Sciences from the University of Louvain in 1980 and the
European Certificate in Cosmetology from the University of
Brussels in 1990. She is responsible of different research programs
concerning extraction, purification and characterization of yeast

3
growth factors active on yeast and mammalian cells. Many of her results have been
communicated in international meetings and publications.

Donnelly, Dan (contribution nr. 88)

Studies: BSc, MSc at National University of Ireland, Cork; PhD at
Dublin City University (1990).
Appointments: 1990-1993: Post Doctoral Research at National
Biotechnology Centre and the National Microelectronic Research
Centre in Cork; 1993-2000: Manager of Process Technology Section
at Guinness, Dublin; 2000 to date: Process Support Manager at
Guinness, Dublin. Member of the Royal Society of Chemistry,
Member of the EBC Brewing Science Group.

Drr, Christian (contribution nr. 96)

Born in 1969.
Studies: Graduate engineer at the Faculty of Brewing and Food
Technology of the Technische Universitt Mnchen in Freising-
Weihenstephan, Germany (1998).
Appointments: Since 1998 research associate at the Chair of Brewery
Installations and Food Packaging Technology of the Technische
Universitt Mnchen in Freising Weihenstephan.

Dostlek, Pavel (contribution nr. 85)

Born in 1963.
Studies: Chemistry and Chemical Engineering at the Institute of
Chemical Technology in Prague, Czech Republic, Faculty of Food
Technology (1985).
Appointments: 1986: postgraduate study at ICT Prague; 1987:
Assistant Scientist in Food Technology; 1990: Assistant Professor for
Brewing Science; 1993: Biotechnology Pilot Plant, Dublin City
University; 1996: Food Technology, Hebrew University, Agricultural Faculty,
Rehovot, Israel; 1997: Lecturer, Department of Fermentation Chemistry and
Bioengineering, ICT Prague.

Douma, Anneke C. (contribution nr. 69)

Born in 1958.
Studies: 1976-1983 MSc in Biology at the State University of
Groningen, the Netherlands, specializing in Microbiology, Molecular
Biology, Electron Microscopy and Teaching (1983). PhD in Yeast
Physiology from the same University in 1989.
Appointments: 1989-1995: project leader at the Department of Plant
Biotechnology of the TNO Nutrition and Food Research Institute.
1995present: Agro-NIBEM, Product Manager Beverages of the same TNO institute.
Member of the EBC Brewing Science Group and Chairman of the Foam Subgroup.
Member of the EBC Barley and Malt Committee and the EBC Analysis Committee.

4
Driessen, Willie J.B.M. (contribution nr. 108)
Studies: MSc Environmental Technology at Agricultural University
of Wageningen, the Netherlands (1989).
Appointments: Research and Process Engineer, Ahlstrom Aquaflow
Oy, Finland (1989-1990); Senior Process Specialist, Paques Water
Systems B.V., the Netherlands (1990-1996); Area Manager, Paques
B.V. (1996 to present); External teacher anaerobic treatment courses,
Stichting Wateropleidingen B.V., Nieuwegein, the Netherlands.

Dumoulin, Michel (contribution nr. 20, 57)

Born in 1968.
Studies: Doctor of Philosophy in Chemistry at Claude Bernard
University Lyon, France (1997). Chemical Engineer with a
specialization in analytical chemistry at the Ecole Suprieure de
Chimie Industrielle de Lyon (1994).
Appointments: since 1998: Analytical Manager at the Institut
Franais des Boissons, de la Brasserie et de la Malterie.

5
Curriculum vitae of (co)authors - E

Eger, Carsten (contribution nr. 28)

Born in 1964.
Studies: 1989: Degree in Brewing Science at Technical University of
Munich-Weihenstephan, Germany; 1990: PhD in Engineering at
Technical University of Munich-Weihenstephan; Chair of
Technology of Brewery II, thesis: Rapid detection of beer-spoiling
bacteria with immunochemical methods.
Appointments: 1994: Beck & Co, Trainee; 1996: Beck & Co, Special
Tasks; 1999: Beck & Co, Head of Research and Development; 2000: Beck & Co,
Head of Research and Development and Quality Management.

Enge, Jan (contribution nr. 85)

Born in 1976.
Studies: Chemistry and Chemical Engineering at the Institute of
Chemical Technology in Prague, Czech Republic, Faculty of Food
Technology (1994).
Appointments: since 1999: postgraduate study at ICT Prague -
Department of Fermentation Chemistry and Bioengineering.

Escudier, Jean-Louis (contribution nr. 83)

Born in 1951.
Engineer from national high school in agro-foods industries: ENSIA, 1976, Massy
Paris, France. Specialities: cane and beet sugars-alcohol distillery-milk beer.
1976-1981: Technical manager, Rum factory; 1981-1983: IAA research engineer, St
KTI Paris; 1983-1997: Research engineer, INRA Pech Rouge; since 1998: Director,
of the Oenologic experimental unit of INRA (Pech Rouge). Main research activities:
separative techniques (tangential micro-filtration, electrodialysis) and vinification
technology.

Evers, Hartmut (contribution nr. 97)

Born in 1952.
Studies: Brewing Technology and Food Technology at the
Technical University of Berlin, Germany (1976-1978 Diploma
Brew master; 1983-1989 Qualified Engineer).
Appointments: 1966-1969: Apprenticeship in Brewing at the
Dortmunder Actien Brewery; 1969-1976: Practical work in several
Breweries; 1978-1983: Assistant Scientist in Distillation and
Solvent Extraction at the Research and Teaching Institute of Distillation and
Fermentation in Berlin; 1989-1997: Senior Assistant Scientist at the Research and
Development Institute in Brewing Science and Technology (Prof. Wackerbauer) at
the Technical University of Berlin; Thesis for the PhD: Kieselguhrfree Filtration;
1996: PhD in Brewing Science. Since 1997: Leader of the Technical Department at
the Research and Teaching Institute of Brewing in Berlin (VLB); 2000: teaching
profession and assistant lecturer at the Technical University of Berlin in Equipment
and Machinery of the Brewery and Malt house
Curriculum vitae of (co)authors - F

Farkas, Gabriella (contribution nr. 43)

Born in 1972.
Studies: BSc in Food Technology at the College of Food Industry,
Szeged, Hungary (1996); MSc in Food Engineering (spec. in food
biotechnology) at the University of Horticulture and Food Industry,
Budapest, Hungary (1999). Currently: PhD student at the Szent Istvan
University, Budapest, Hungary (formerly: the University of
Horticulture and Food Industry).
Professional experiences: participation and preparation of several papers in the
National Scientific Congress for Higher Education Students (1994-1999); one month
practice-research at the Heriot-Watt University, ICBD, Edinburgh, Scotland (1998);
as a PhD student holds Brewing technology laboratory practices for undergraduate
students; currently a spending six-month Erasmus Scholarship at Technische
Universitt Mnchen, Freising-Weihenstephan, Germany.
Research activities: effect of micro- and macro elements on the fermentation
performance of yeast (thesis); yeast cell immobilization, continuous fermentation,
non-alcoholic and low-alcoholic beer, genetic improvement of brewers yeast.

Ferreira, Antnio A. (contribution nr. 84)

Born in 1955.
Studies: Licentiateship in Pharmacy at the University of Oporto,
Portugal (1981); post-graduation Course in Brewing Science at the
Catholic University of Oporto (1991); General Management Course at
the High Institute of Management Studies of Oporto (1994).
Appointments: Pharmaceutical Manager at the Portuguese Air Forces
(1981-1983); Responsible for the Biochemical Analysis at LABMAT Clinical
Analysis Laboratory (1983-1984); Responsible for the Instrumental Analysis at the
Central Laboratory of Unicer (1984-1989); Responsible for the Instrumental and
Chemical Analysis at the Central Laboratory of Unicer (1989-1990); Quality Control
Manager at Unicer (1990-1991); Director of Quality and Development Dept. at
Unicer (since 1991); Member of the EBC Analysis Committee (since 1992).

Fick, Michel (contribution nr. 13)

Born in 1960.
Studies: Biochemical Engineering and Food Technology at the
Institut National des Sciences Appliques (1982), Toulouse,
France; PhD in Bioprocess Enginering at the Institut National
Polytechnique de Lorraine (INPL) (1986), Nancy.
Appointments: 1987-1994: Lecturer in Biochemical Engineering at
the Agronomic and Food Industry School (ENSAIAINPL), Nancy;
Scientist at the Laboratory of Chemical Engineering Sciences (LSGCCNRS), Nancy;
since 1994: Professor in Biochemical Engineering at the Agronomic and Food
Industry School (ENSAIAINPL), Nancy; Scientist at the Laboratory of Chemical
Engineering Sciences (LSGCCNRS), Nancy.
Fillaudeau, Luc (contribution nr. 27)
Born in 1970.
Studies: Chemical Engineering and Chemistry at H.E.I. (Hautes
Etudes Industrielles, France, 1994) and Food Technology at U.T.C.
(Universit de Technologie de Compigne, 1998).
Appointments: 1995-1996: Inside Sales Assistant in C.M.Inc.
(Philadelphia, USA); 1996-1998: Scientist Assistant (membrane
processes) in INRA-LGPTA (National Research Institute of
Agronomy - Laboratory for Food Process Engineering and Technology in Villeneuve
d'Ascq, France); since 1998: Scientist (food process: heat treatment, new technology)
at INRA-LGPTA.

Finglas, Paul (contribution nr. 4)

Is Senior Research Scientist in the Nutrition Health and Consumer
Science Division, Institute of Food Research. He graduated from
Aston University in Birmingham, UK, in 1981 with a BSc Honours
degree in Chemistry. His research interests have included food
composition including HPLC and immunochemical methods for the
determination of vitamins in foods, quality assurance, and certified
reference materials; relationships between nutrient intake and
biochemical markers of status; and the bioavailability of organic
microcomponents. His current interests include folate absorption and metabolism
using stable isotopes, and interactions with B-vitamins and homocysetine. He is
author of over 100 research publications in these areas. He is a member of several
committees and expert groups including FSA Optimal Nutrition Programme,
European Standardisation Committee (CEN) on Vitamins, and the Nutrition Society.
He is editor of Food Chemistry and Trends in Food Science and Technology and has
also served as adviser to FAO's Food and Nutrition Division; Food, Nutrition and
Health Programme (EU FP5), and IAEA in Vienna. He is the Scientific Coordinator
for the current EU project Folate: from Food to Functionality and Optimal Health.

Fischer, Gunther (contribution nr. 75)

Born in 1972.
Studies: Technical Graduation as Brewer and Maltster (1995);
Brewing and Beverage Technology at the Technical University
Munich-Weihenstephan, Germany (2000).
Appointments: Scientist Process Automation in Biotechnology at the
Chair of Fluid Mechanics and Process Automation at the
Weihenstephan Center of Life and Food Sciences, Technical
University Munich (since 2000).

Ford, Christopher M. (contribution nr. 10, 77)

Born 1962.
Studies: BSc (Hons), Applied Biology (Hatfield Polytechnic, 1984);
DPhil (University of Sussex, UK, 1988).
Research Career: 1988-1990: Postdoctoral Research Associate,
Division of Virology, National Institute for Medical Research, Mill
Hill London; 1990-1995: Research Fellow, Australian Research
Council Special Research Centre for Basic and Applied Plant
Molecular Biology, Department of Plant Science, Adelaide University; 1995-1999:

2
Research Fellow, Cooperative Research Centre for Viticulture, Department of
Horticulture, Viticulture and Oenology, Adelaide University; 2000-present: Barley
Biochemistry and Brewing Research Laboratory, Department of Plant Science,
Adelaide University.

Forster, Adrian (contribution 16, 17)

Born in 1942.
Studies: Technical University of Munich-Weihenstephan, Germany,
Dipl.-Ing. (1966); Dr.-Ing. (1972).
Appointments: Head of Laboratory for Hop Extraction, Barth, & Co.
(1973-1976); Plant Manager (1976-81); Managing Technical
Director Hop Extraction and Pelleting HVG Barth, Raiser GmbH
& Co. KG (1981- ). Published extensively on hop related topics.

Forster, Clemens (contribution nr. 15)

Born in 1968.
Studies: Brewing Science and Beverage Technology at the Technical
University of Munich-Weihenstephan, Germany (1992); 1993-1996:
Doctoral thesis about Flavour formation during kilning of malt and
the impact of kilning technology on beer flavour and flavour
stability at the Chair for Brewery Technology in Weihenstephan.
Appointments: 1997-2000: Assistant professor at the Institute for
Brewery Technology and Beverage Microbiology in Weihenstephan. Areas of
research: impact of raw materials and brewing technology on beer flavor and flavor
stability, analytical methods to determine flavor stability and antioxidant activity of
beer and its optimization during the brewing process. Since 2000: Brewing
Development Leader in Technology Development Department of Labatt Brewing
Company Ltd., Canada.

Fournier, Regis (contribution nr. 57)

Born in 1967.
Studies: Degree and Honour in Plant Life Sciences and Molecular
Biology, University Louis Pasteur (Strasbourg, France), 1991. PhD in
Molecular Biology, University Henri Poincare, Nancy, 1997.
Appointments: 1998-2000: Post doctoral position in the Molecular
Immunology Division of the Medical Research Foundation (Royal
Perth Hospital, Perth, Australia). Study of the regulation of the human
Interleukin-5 transcription. Since mid 2000: Molecular Biology
Division Manager, in charge of the development of the Molecular Biology Division at
the Institut Franais des Boissons, de la Brasserie et de la Malterie, Vandoeuvre ls
Nancy.

Frank, Norbert (contribution nr. 5, 6)

Born in 1945.
Studies: Chemistry at the University of Basel, Switzerland; PhD in Chemistry at the
University of Freiburg, Germany (1974). Topic: Structure elucidation of
polysaccharide derivatives using NMR-spectroscopy.
Appointments: 1974-1975: Postdoctoral fellow at the Institute of Organic Chemistry,
University of Heidelberg, Germany; 1975-1986: Scientist in the Division of

3
Metabolism of N-nitroso compounds, German Cancer Research Center, Heidelberg;
1986-1987: Visiting scientist at the National Cancer Research Center, Tokyo, Japan;
1987-1994: Senior scientist in the Division of Molecular Toxicology at the German
Cancer Research Center; since 1995: Senior scientist in the Division of Toxicology
and Cancer Risk Factors, German Cancer Research Center; since 1996 Member of the
Workgroup Cancer Chemoprevention, Division of Toxicology and Cancer Risk
Factors, German Cancer Research Center. Special interest in (i) the analysis of DNA
adduct formation as biomarkers in carcinogenesis, (ii) principle mechanisms of cancer
chemoprevention, (iii) in vivo evaluation of chemopreventive agents.

Franz, Oliver (contribution nr. 65, 66)

Born in 1970.
Study of Brewing and Beverage Technology at the Technical
University of Munich-Weihenstephan, Germany (1992-1998):
Graduate engineer 9/1998 Theme of diploma thesis: Investigation
on the endogenous antioxidative activity (Lag-Time) during the
brewing process of lager in practice.
Since 2/1999: Doctoral thesis at the chair for Brewing Technology I
(Prof. Back) in Weihenstephan, Theme: Systematic investigations on the endogenous
antioxidative activity of beer in consideration of technological features; since
5/2000: Head of the laboratory for GC / HPLC - Analytics at the chair for Brewing
Technology I (Prof. Back) in Weihenstephan.

4
Curriculum vitae of (co)authors - G

Gaag, Bram, van der (contribution nr. 74)

Born in 1962.
Studies: BSc in Medical Microbiology.
Appointments: 1988-1992: Technician at the monoclonal facility of
TNO Nutrition and Food Research Institute in Zeist, the Netherlands;
1992-2000: Sensor Technology Department of the same institute,
first as a Technician, later as Study Director and from 1997-2000 as
Product Manager Biosensors. 2001-present: Study Director within the Biomolecular
Screening Group of the Department of Analytical and Molecular Pharmacology of
TNO Pharma in Zeist.

Gaber, Richard F. (contribution 44)

Born in 1952.
Studies: Yeast Genetics and Molecular Biology at the University of
Wisconsin, Madison, U.S.A. (1982).
Appointments: 1983-1986: Postdoctoral Fellow, Massachusetts
Institute of Technology/Whitehead Institute for Biomedical Research;
1986-1992: Assistant Professor, Northwestern University; 1992-1999:
Associate Professor, Northwestern University; since 1999: Professor,
Northwestern University; 2000-2001: Visiting Professor at the Carlsberg Laboratory,
Copenhagen.

Gahr, Andreas (contribution 17)

Born in 1965.
Studies: 1986-1988: Apprenticeship in the Augustiner Brewery,
Munich, Germany; 1988-1994: Studies at the Technical University
Munich-Weihenstephan; Diploma Brewmaster.
Appointments: 1994-1998: Employee at the Chair for Brewery
Technology I, Technical University Munich-Weihenstephan; since
1998: Head of the Research Brewery, Hopfenveredlung HVG Barth,
Raiser GmbH & Co. KG, St. Johann.

Galindo-Castro, Ivn (contribution 47, 48, 61)

Born in 1958.
Studies: BSc in Molecular Genetics at the Central University of
Venezuela (1988); PhD in Molecular Biology at the Simon Bolivar
University (1996, with honours). Appointments: 1988-1990:
Assistant Scientist at the Laboratory of Molecular Genetics, Central
University of Venezuela; 1990-1993: Associate Scientist at the
Laboratory of Biotechnology, Polar Technology Centre; 1993-1999:
Chief Scientist at the Laboratory of Biotechnology, Polar Technology Centre; since
1999: Corporate Manager of Biotechnology, R&D Direction of Empresas Polar,
Caracas.
Gamal-Eldeen, Amira (contribution nr. 5, 6)
Born in 1969.
Studies: BSc in Zoology at Ain Shams University, Egypt: MSc in Immunology at
Cairo University.
Appointments: Scientific work at the Clinical Immunochemistry Laboratory, National
Research Center, Cairo; since 1999: PhD student in the Workgroup Cancer
Chemoprevention at the Division of Toxicology and Cancer Risk Factors at the
German Cancer Research Center Heidelberg, Germany focussing on mechanisms of
anti-oxidative activity as a common principle in chemoprevention.

Gardner, Adrian (contribution nr. 106)

Born in 1946.
Studies: educated in Brisbane, Australia in Industrial Chemistry,
Industrial Engineering, Marine Biology and Business Administration.
Appointments: work history within Castlemaine Perkins Limited
(Brewers) from 1964 to 1969. The Coca Cola Company in Australia,
the United Kingdom and Canada in Production Services (1969-1973).
University of Queensland Department of Zoology tutoring (1974-
1976). Carlton and United Breweries Australia (1977 to present) in various
operational roles. Present position involved international technical liaison for the
Fosters Brewing Group, identification and development of new technologies and
commercialisation of intellectual property. IOB member since 1982. Chairman of
Strategic Planning Committee (Asia Pacific) from 1988 to 2000, President of the
Institute and Guild of Brewing since July 2000, Member of RACI and MBAA.

Gerhuser, Clarissa (contribution nr. 5, 6)

Born in 1964.
Studies: Pharmacy at the University of Wrzburg, Germany (1989), PhD in
Pharmaceutical Biology, University of Munich (1993). Topic: Investigation of the
antiviral activity of Thuja occidentalis L., Cucurbita pepo L. and Ecballium elaterium
[L.] A. Rich. - Isolation of podophyllotoxin lignans and new ribosome-inactivating
proteins. Since 1998: Docent and external faculty member at the University of
Heidelberg, Germany.
Appointments: 1993-1995: Postdoctoral Research Associate at the University of Illinois
at Chicago, USA; 1995-1996: Visiting Research Assistant Professor, University of
Illinois at Chicago, USA; since May 1996: Group leader of the Workgroup Cancer
Chemoprevention in the Division of Toxicology and Cancer Risk Factors, German
Cancer Research Center, Heidelberg. Main research interest: (i) identification of plant-
derived compounds and synthetic analogs with potential chemopreventive activity, (ii)
investigation of molecular mechanisms of chemopreventive activity. Member of the
American Association for Cancer Research, American Society of Pharmacognosy,
American Society for Biochemistry and Molecular Biology, International Society of
Cancer Chemoprevention.

2
Goiris, Koen (contribution nr. 60)
Born in 1976.
Studies: Academic degree in Industrial Engineering - Biochemistry
at the KaHo St.- Lieven, Gent, Belgium (2000).
Appointments: 2000-present: Assistant Scientist in Malting and
Brewing Science at the Laboratory of Enzyme and Brewing
Technology of the KaHo St.- Lieven. Research topic: High Tech
Hopping.

Goldstein, Henry (contribution nr. 71)

Studies: Chemistry at New York University, Tulane University and
the University of Connecticut, U.S.A., where he received the AB, MS
and PhD degrees, respectively. After his doctoral work was
completed, he was employed at the American Sugar Company, later
know as AMSTAR Corporation in New York City. For the past
twenty-five years he has been in the Research Department of the
Miller Brewing Company. His research interests include hop
chemistry, down-stream hop compounds, beer flavor, beer foam and the physical
stability of beer. He was a member of the American Society of Brewing Chemists and
that organizations Program Committee. In 1996 he was presented the American
Society of Brewing Chemistrys Eric Kneen Memorial Award for excellence in
scientific publication. In 2000 he was awarded the Miller Achievement Award for his
research and development efforts in the production of down-stream hop products and
Kraft Foods ABCD Award in recognition of extraordinary efforts and achievement.
He is the author or co-author of numerous articles and presentations dealing with
chemistry, food science and brewing science and technology as well as an inventor or
co-inventor in two dozen U. S. and foreign patents. He has been a member of the
American Chemical Society for over thirty years.

Gourdon, Antoine (contribution nr. 27)

Born in 1963.
Studies: Biology and Neurophysiology at Ecole Normale Suprieure
of Saint-Cloud (France).
Appointments: 1987-1990: Scientist Assistant at Laboratory of
Neuro-physiology in collge de France; 1991-1999: employed by
Vivendi; 1991-1993: Scientific Director of Environment Research
Centre (Creed) in Limay; 1993-1998: Manager of hazardous waste incineration plant
Sotrenor; 1998-1999: Development Director of Sarp Industries; since 1999: Manager
of ProFiltra.

Grass, Stefan (contribution nr. 105)

Born in 1960.
Studies: 1980-1986: Dipl. Ing. Agr. ETH, Zurich, Switzerland.
Appointments: 1986-1989: Plss Staufer AG, Oftringen, R+D
Engineer; 1990-1993: Carbotech AG, Zrich, Environmental
Consulting; 1993-1994: University of California, Davis, Guest
Scientist
Energy utilization of biomass; 1995-1996: Feasibility Study, Ethanol from Biomass,
work at ETH laboratory; 1996-present: Foundation of 2B AG, 2B Biorefineries,

3
Dbendorf, Switzerland; partner responsible for Research and Development and spent
grain technology.

Guido, Luis (contribution nr. 84)

Born in 1972.
Studies: Degree in Biochemistry (specialisation in Applied Biochemistry) at the
Faculty of Science of Porto University, Portugal (1995). Since 1998: Doctorate
Student at the Faculty of Science of Porto University.
Appointments: 1995-1996: Scientist at the Institute for Molecular and Cell Biology
(IBMC); 1996-1998: First Assistant at the Wood Engineering Department,
Polytechnic Institute of Viseu; since 1999: First Assistant at the Chemistry
Department of Faculty of Science of Porto University in Analytical Chemistry.
Member of the Portuguese Society for Biochemistry (Biotechnology), Portuguese
Electrochemical Society.

Gunkel, Jrg (contribution nr. 11)

Born 1969.
Studies: Food and Fermentation Technology (main emphasis on
Brewing Technology) at the Technische Universitt Berlin, Germany
(TUB); Master of Engineering / M.Eng. (1997). Currently PhD
student at the TUB (Prof. Schildbach), thesis deals mainly with the
influence of the malting barley genotype on different final (apparent)
attenuation levels.
Appointment: 1997-2000: Research Scientist at the Versuchs- und Lehranstalt fr
Brauerei in Berlin (VLB), Abteilung Forschungsinstitut fr Rohstoffe (FIR) (Research
and Teaching Institute for Brewing in Berlin, Dept.: Research Institute for Raw
Materials).

Gunning, Patrick A. (contribution nr. 68)

Studied Physics at Cambridge, UK.
Appointments: Worked at the Institute of Food Research since 1985
on topics ranging from polysaccharide chemistry, rheology and varied
biophysical techniques. Since 1990 has specialised in the new
technique of scanning probe microscopy (SPM). This began with
scanning tunnelling microscopy and progressed to atomic force
microscopy. The work has led to over 35 publications in scientific
journals on the application of SPM to biology in general and food related topics in
particular. Co-author of the current best-selling book on atomic force microscopy
Atomic Force Microscopy for Biologists published by Imperial College Press in
December 1999. Most recently was co-author of a series of papers, which have
demonstrated unequivocally the mechanism by which low molecular weight
surfactants induce displacement of interfacial protein films.

4
Curriculum vitae of (co)authors - H

Haikara, Auli (contribution nr. 12, 91)

Studies: 1965, MSc in Nutrition Chemistry, 1981 LicPhil and 1984
PhD in Microbiology at the University of Helsinki, Finland.
Appointments: 1965-1973: Research Scientist at the Research
Institute of the Finnish Brewing Industry; 1973-1978: Senior
Research Scientist at the Biotechnical Laboratory of the Technical
Research Center of Finland (VTT); 1979-1986, 1988-1993: Manager
of Microbiology Section at the Biotechnical Laboratory of VTT;
1987: Research Professor in Fermentation Technology at the
Biotechnical Laboratory of VTT; since 1993: Docent in Industrial Microbiology in
Department of Applied Chemistry and Microbiology of the University of Helsinki;
1994-1999: Manager of Process Microbiology Group at VTT Biotechnology; since
2000: Manager of Detection and Control of Contaminants Group at VTT
Biotechnology; Member of the EBC Brewing Science Group, Detection of
Contaminants and Gushing sub-groups; Member of the American Society of Brewing
Chemists and editorial Board of ASBC.
Research interests: microflora of cereals, gushing of beer, beer spoilage organisms,
brewery hygiene, rapid detection methods and antimicrobials produced by lactic acid
bacteria.

Hamilton, Alisdair (contribution nr. 41)

Born 1973.
Studies: BSc in Brewing and Distilling at Heriot Watt University,
UK (1994), Hons in Microbiology at Heriot Watt University (1995).
Appointments: 1997-1998: Quality Assurance Laboratory Assistant
within Bass Brewers; 1998-2000: Senior Research Technologist Bass
Brewers; 2000-2001: Account Manager within Sales Function of
Bass Brewers and presently National Account Executive in that
function (2001-). Associate Member of the Institute of Brewing.

Hammond, John R.M. (contribution nr. 33, 37)

Studies: Biochemistry at University of Cambridge, UK (1965-1968);
PhD in Chemical Microbiology at University of Cambridge (1968-
1972).
Appointments: Group Leader (Microbiology) and Senior
Microbiologist, Guinness, Park Royal (1971-1985); Head of
Fermentation, Brewing Research Foundation (1985-1995); Head of
Quality Services, BRF International (1995-1996); Head of
Information and Communications, Brewing Research International (1996-1998);
Head of Membership Services, Brewing Research International (1998-1999);
currently Laboratory Director, Brewing Research International. Chairman EBC
Microbiology Group (1988-1995); Chairman of EBC Brewing Science Group - Yeast
Genetics and Physiology Sub-Group (1999-date); Member of EBC Technology and
Engineering Forum; Fellow of the Institute of Brewing and of the Royal Society of
Chemistry, Member of ASBC.
Hariri, Ahmed (contribution nr. 13)
Born in 1972.
Studies: Study of steeping at Chemical Engineering Sciences Lab -
ENSAIA, INPL, France.
Appointments: 1991: Mathematical baccalaureat (Algeria); 1991-
1996: Engineer of food industry (Algeria).
1998-1999: Master of science in Biotechnology and Food industry
(ENSAIA, INPL); 1999-2001: PhD in Biotechnology and Food
industry at ENSAIA, INPL.

Harmegnies, Frdrique (contribution nr. 24, 83)

Born in 1971.
Studies: Biochemical Engineering at Institut Suprieur Industriel Catholique (ISIC
Mons, Belgium), graduated in 1993.
Appointements: 1993-1995: Research Engineer at ISIC (project on beer filtration in
collaboration with Meura); 1995-1998: Research and Development Engineer at Meura
S.A ; since 1998: Research and Development Engineer at Meura Technologies

Hakov, Danua (contribution nr. 59)

Born in 1955.
Studies: Institute of Chemical Technology in Prague, Faculty Food
and Biochemical Technology, Specialization: Fermentation
Chemistry and Bioengineering (1979).
Appointments: 1979-1982: PhD student in the Institute of
Microbiology, Academy of Sciences of the Czech Republic, Prague;
1982-1984: home, child care; 1985-1989: Technologist in
Hydroprojekt, Prague; since 1989: Researcher in the Department of Technology,
Research Institute of Brewing and Malting, PLC, Prague.

Hegarty, Paul (contribution nr. 89)

Born in 1960.
Graduated from Sheffield University, UK, with a degree in
Biochemistry and completed a PhD in Plant Cell Couture at the
Wolfson Institute of Biotechnology in Sheffield. Following
postdoctoral research at Kings College London into monoterpene
metabolism, he joined Bass Brewers in 1986 and has held a number
of technical roles. He is currently Analytical Services Manager
working in the Bass Technical Centre in Burton on Trent. In this
position he is responsible for running an analytical, sensory, consumer testing and
trouble shooting service for the company. The author of 30 technical publications, he
is a former chairman of the Institute of Brewing Research Committee and Brewers
Guild Training Committee and is currently the Chairman of the IGB Sensory Sub-
group and a member of the IGB Analysis Committee.

Heiss, Elke (contribution nr. 6)

Born in 1973.
Studies: Biochemistry at the University of Regensburg, Germany, and the University
of Kent at Canterbury, UK (1992-1997).
Appointments: 1997-1998: Diploma student at the Department of Cell Biology and
Plant Physiology, University of Regensburg, Germany (Characterization of

2
glycosylation-defect mutants of S. cerevisiae); since 1998: PhD in the Workgroup
Cancer Chemoprevention student at the Division of Toxicology and Cancer Risk
Factors, German Cancer Research Center Heidelberg, with focus on the elucidation of
mechanistic aspects of chemopreventive compounds and on the establishment of the
MMOC model.

Heyerick, Arne (contribution nr. 7)

Born in 1974.
Studies: BSc in Biochemistry, Ghent University, Belgium (1996) -
thesis on The Phyto-oestrogenic Activity of Hops (Humulus
lupulus L.); PhD in Biochemistry, Ghent University (2001) thesis
on Unraveling the Mechanism of the Lightstruck Flavor of Beer.
Appointments: 1997-2001: PhD fellowship from the Interbrew-
Baillet Latour Foundation with a position at the Ghent University,
Faculty of Pharmaceutical Sciences, Laboratory of Pharmacognosy and
Phytochemistry; since 2001: Scientific Collaborator for an industrial research project
on the valorization of estrogens from hops and investigations towards an innovative
phytotherapeuticum with potent estrogenic activity, Ghent University, Faculty of
Pharmaceutical Sciences, Laboratory of Pharmacognosy and Phytochemistry.

Higgins, Vincent J. (contribution nr. 52)

Born in 1962.
Studies: BSc in Biological Sciences at University of Western Sydney;
PhD in Biochemistry and Molecular Genetics from University of
New South Wales, Australia.
Appointments: 1989-1994: Research Officer in the Strain
Development Group at the Burns Philp Yeast Research and
Development Laboratories in Australia. 1994-1998: Evaluation of
IP/patent and research feasibility as a Senior Research Scientist at the Burns Philp
Laboratories exploring the use of recombinant and classical methods of gene
manipulation in the production of novel and useful strains of industrial yeast. Since
1998: Senior Research Officer investigating gene regulation at the School of
Biochemistry and Molecular Genetics, University of New South Wales. Research
liaisons were closely linked with the Cooperative Research Centre for Food Industry
Innovation and collaborative work in yeast gene expression technologies with Carlton
and United Breweries.

Hill, Peter G. (contribution nr. 98)

Born in 1968.
Studies: 1990, Degree in Chemistry and Analytical Chemistry at
Loughborough University of Technology, UK; 1999, PhD in
Analytical Chemistry at Loughborough University of Technology, in
co-operation with Brauerei Beck & Co, Bremen, Germany; thesis:
The Analysis of Sulphur Compounds in Beer.
Appointments: 1988-1989: 1 year practical session in the research
department of Horseracing Forensic Laboratories, Newmarket, UK; 1993: Research
chemist at Brauerei Beck & Co, Bremen, Germany; since 2000: Head of
Instrumental Analysis at Brauerei Beck & Co.

3
Hodgson, Jeff A. (contribution nr. 31, 40)
Received a BSc and PhD in Biochemistry from the Universities of
Glasgow (1975) and Cambridge (1979), UK, respectively. This was
followed by postdoctoral work on yeast genetics and metabolism at
Glasgow and Strathclyde Universities. Joined Scottish & Newcastle
in 1988 as Fermentation Scientist. Then held posts of Fermentation
Manager, Sensory Manager and Group Microbiologist during
subsequent years. Currently Innovation & Development Manager
responsible for product, packaging, process and plant developments.
Member of EBC Brewing Science Group and Detection of Sub-contaminants
Subgroup.

Hhn, Gerrit (contribution nr. 51)

Born in 1969.
Studies: Technology of Brewing and Beverage at the TU Mnchen,
Freising-Weihenstephan, Germany (1996).
Appointments: since 1996: Assistant Scientist at the Chair for
Energy and Environmental Technologies of the Food Industry, TU
Mnchen, Freising-Weihenstephan

Home, Silja (contribution nr. 61)

Studies: Helsinki University of Technology, Chemical Engineering
and Biotechnology department, MSc 1970, D.Tech. 1993.
Appointments: VTT (Technical Research Centre of Finland)
Biotechnology: 1970-1979: Research Scientist; 1979-1999: Head of
the Malting and Brewing Research Group; since 2000: Manager,
Malting and Brewing Research.
Author/co-author of many publications.
Member of the EBC Analysis Committee and the EBC Barley & Malt Committee.
Member of ASBC and IGB.

Hork, Tom (contribution nr. 59, 82)

Born 1967.
Studies: Natural Science Faculty at the Charles University in Prague,
Czech Republic, MSc degree 1991.
Appointments: 1991-1994: Hygienic Station of the Capital Prague,
analyst; 1994-1997: Ecochem Prague, analyst; 1997-now: Research
Institute of Brewing and Malting, PLC-Prague, research worker.

Hori, Toshihiko (contribution nr. 39)

Born in 1968.
Studies: Agricultural Chemistry at the Kyoto University, Japan
(1993).
Appointments: 1993-1997: staff in Kyoto Brewery, Kirin; since 1997:
Researcher in the Research Laboratory for Brewing.

4
Hoschke, goston (contribution nr. 43)
Born in 1938.
Studies: Chemical Engineering at the Technical University Budapest,
Hungary (1961): Dr.Ing. Technical University Budapest (1966); PhD,
Hungarian Academy of Sciences (1979).
Appointments: 1962-1983: staff member (1980-1983: Associate
Professor) in Department of Agricultural Chemical Technology at the
Technical University Budapest; 1983-1991: Head of Food
Biotechnology Division at Central Food Research Institute, Budapest; 1991-1999:
Professor at University of Horticulture and Food Industry; 1991-1996: Dean of
Faculty of Food Industry; 1996-1998: Vice Rector; since 1991: Head of Department
of Brewing and Distilling, since 1999: Professor at Szent Istvn University; since
1999: Dean of Faculty of Food Science; since 1993: Member of the Working Party on
Downstream Processing and Recovery of Bioproduct at European Federation or
Biotechnology, since 1998: Member of EBC Brewing Science Group.

Hrabk, Milo (contribution nr. 59)

Born 1969.
Studies: Biotechnology and brewery technology in the Institute of
Chemical Technology (ICT) in Prague, Czech Republic (1993),
external PhD student in ICT.
Appointments: 1993-1996: technologist in Experimental and
development Center of the Research Institute of brewing and malting
Prague (RIBM); 1996-1998: deputy manager of Experimental and
development Center; since 1998: Head of Experimental and development Center of
RIBM.

Husband, Fiona A. (contribution nr. 68)

Studies: BSc in Biochemistry (University of Glasgow, UK, 1989).
Appointments: 1988: Shell Research Centre, Kent; 1990-1992:
Ocean Protein Associates Limited, UK; 1992-Present, Institute of
Food Research.

Hutter, Karl-Joseph (contribution nr. 38)

Born in 1943.
Studies: 1965-1970: Brewery technology TU-Berlin (VLB),
Germany; graduation: March 1974 TU-Berlin; vocational training:
1960-1962, Brauerei Frankenheim, Dsseldorf.
Occupation: 1970-1979: Scientist, Fraunhofer-Gesellschaft; since
1979: Scientist, German Cancer Research Center, Heidelberg.
Habilitation: May 2000, TU-Dresden. Lectureships: 1985-1992:
University of Heidelberg; 1994-1999: University of Hohenheim; since 1995:
Fachhochschule Mannheim; since 2000: TU-Dresden. Memberships: Deutsche
Gesellschaft fr Zytometrie, EBC Brewing Science Group.

5
Curriculum vitae of (co)authors I / J

Imai, Takeo (contribution nr. 39)

Born in 1959.
Studies: Agricultural Chemistry at the Tokyo University, Japan
(1985).
Appointments: 1985-1989: Researcher in Brewing Science
Laboratory, Kirin Brewery Co.Ltd.; 1989-1994: Researcher in
Central Laboratory for Key Technology; 1994-1996: Staff in
Production Division; 1995: PhD, Theme: The Physiology of the
Brewing Yeast.; 1996-1998: Guest Researcher in the Laboratory of Brewing
Technology I at the University Munich-Weihenstephan; 1998-: Manager in Research
Laboratory for Brewing, Kirin. Member of Japan Society for Bioscience,
Biotechnology and Agrochemistry.

Inomoto, Kumiko (contribution nr. 55)

Studies: Food Chemistry at the Kagawa Nutrition University, Japan
(1986).
Appointments: since 1986 employed by Asahi Brewery Ltd.: 1986-
1993: Center R&D Lab as Analyst in Quality Assurance Section;
1993-1995: Brewing R&D Lab Process Engineering Section; 1995-
1999: Process Engineering R&D Lab as Assistant Chief in System
Technology Section; 1999-present: Brewing R&D Lab as Chief in Brewing Science
Section.

Isenberg, Torsten (contribution nr. 28)

Studies of Technical Computer Sciences (1986-1990) at the
Fachhochschule Hamburg, Germany; University degrees: Dipl. Ing.,
EUR Ing.
Appointments: since 1988: at Werum Software & Systems: focus on
ERP/MES (logistics, batch production processes): Project
management in several MES projects in pharmaceutical industry e.g.
MSD England - Implementation of MES in factory for production of
solids with FDA validation; Fette - Product development and
customer projects for standardized interface to tablet presses; Krka Slovenia -
Implementation of MES in factory for production of solids with focus on automated
material flow control.
Project management in several MES projects in beverages industry e.g. Deinhard AG
- Implementation of MES for filling lines with focus on line control in a sparkling
wine factory; Flensburger brewery - Implementation of MES for a production control
system for the area brewery (Sudhaus); Beck & Co. - Implementation of MES in the
whole brewery with focus on material flow control, line control, data acquisition and
evaluation.
Ito, Kazutoshi (contribution nr. 21)
Born in 1949.
Studies: Faculty of Agriculture (postgraduate course) at the Tohoku
University, Japan, PhD (1952).
Appointmants: 1977: Research Associate in Faculty of Agriculture,
Tohoku University; 1980-1982: Post-Doctoral Program at University
of California, Davis, USA; 1982-1989 Research Associate in Faculty
of Agriculture, Tohoku University; 1989-2000: Research Scientist in
Plant Biotechnology the Plant Bioengineering Research Laboratories
(PBRL), Sapporo Breweries Ltd.; since 2000: Director of PBRL.

Janke, Hans-Dieter (contribution nr. 104)

Born in 1951.
Studies: 19691974: Degree in Biology at the State University of
Kharkov; 19741979: Scientist at the Institute for Microbiology at
the Martin Luther University in Halle-Wittenberg. PhD thesis under
the guidance of Professor W. Fritsche.
Appointments: 19791991: Scientist at the Central Institute for
Microbiology at the Academy of Science in Jena. Head of the
working group Microbial degradation of pollutants; 19911994: Scientist in the
Department of Chemical Microbiology (Head of Department: Professor H.-J.
Knackmuss) at the Fraunhofer Institute for Interfacial Phenomena and Biochemical
Engineering in Stuttgart, Germany; 1992: Habilitation for the subject Microbiology
at the University of Stuttgart; 19941995: Project Manager at Jena Umwelttechnik
GmbH, Germany; since 1995: Head of the working group Bioengineering at the
Institute for Environmentally Compatible Process Technology (upt), Saarbrcken.

Jenkins, Cheryl (contribution nr. 40)

Born in 1976.
Studies: Nutrition and Food Science BSc Hons at Oxford Brookes
University, UK (1999); Scottish Courage Brewing Scholar, studying
the effect of serial repitching on yeast physiological state and
brewing yeast fermentation performance at Oxford Brookes
University (PhD) (1999-2002).
Appointments: Vacation assistant with the Ministry of Agriculture
Fisheries and Food (MAFF), Food Safety Branch, Risk Assessment Division, July
1997- September 1997. Undergraduate work placement at Hook Norton Brewery,
September 1997-July 1998. Society of General Microbiology vacation scholarship to
study yeast viability and vitality, July-September 1998.

Jitariouk, Nikola (contribution nr. 27)

Born in 1956.
Studies: Chemical Engineering, Radiation Chemistry and Membrane
Technology at the Mendeleev institute of chemical technology
(Moscow, Russia).
Appointments: 1979-1994: employed by Joint Institute of Nuclear
Research; 1979-1982: Engineer; 1982-1987: Chief of Laboratory;
1988-1991: Senior Scientist; 1991-1994: Chief of Department; 1994-
1995: Senior Scientist at laboratory of irradiated polymers at Nuclear Research Centre
of Saclay; 1995-1997: Senior Scientist at Environment Research Centre (Creed) of

2
Vivendi in Limay; since 1997: Research and Development Director of ProFiltra; since
1999: Chairman of ProFiltra.

Jurkov, Marie (contribuiton nr. 82)

Born in 1956.
Studies: Natural Science Faculty at the Charles University in Prague;
MSc degree in 1980; Institute of Microbiology, Academy of Sciences
of the Czech Republic, Prague, PhD degree in 1991.
Appointments: 1980-1992: Institute of Microbiology, Academy of
Sciences of the Czech Republic, Prague, research worker; 1993-now:
Research Institute of Brewing and Malting Prague (RIBM), research
worker; since 2001: Metrology Manager of the Accredited Analytical Testing
Laboratory.

3
Curriculum vitae of (co)authors - K

Kalita, Jogen C. (contribution nr. 7)

Born in 1961.
Studies: BSc Honours in Zoology (1981), secured Ist Class, Ist position and MSc in
Zoology (major in Animal Physiology) (1986) secured Ist Class, Ist position with
academic gold medal, Gauhati University, India; awarded with Commonwealth Staff
Scholarship for PhD study at Kings College, London, UK (1995); PhD in
Physiology, Kings College, London (1998).
Appoinments: 1986-1988: Junior Research Fellow, Department of Reproductive
Biology and Medicine, All India Institute of Medical Sciences, New Delhi, India;
1988-1995: Lecturer in Zoology, Gauhati University, India; 1995-1998:
Commonwealth Scholar, Kings College, London; 1998-1999: Postdoctoral Research
Associate in Physiology, Kings College, London; since 1999: Reader in Zoology
(Animal Physiology and Biochemistry), Gauhati University, India, and actively
engaged in screening of plants of North-East India for estrogenicity and for evaluation
of their effects on reproductive functions.

Kaltner, Dietmar (contribution nr. 16, 17)

Born in 1969.
Graduated from the Technical University of Munich-Weihenstephan,
Germany, in Brewing Science and Beverage Technology (1991-
1997). From 1986 to 1989 he had several practical apprenticeships in
domestic breweries. After receiving his diploma from university, he
started to work on his doctorate at the Chair for Brewery Technology
I in Weihenstephan (1997-2000). His area of research was impact of
technology on hoppy flavor in beer with special regard to new analytical methods and
technological measures for its optimization in the brewing process. Since 2000:
Manager of Research & Development department at the Hopfenveredlung HVG
Barth, Raiser GmbH & Co. KG in St. Johann.

Kaneda, Hirotaka (contribution nr. 70)

Born in 1958.
He graduated from Kyushu University, Japan, in 1984 with an MS
degree in Food Hygienic Chemistry and then joined Sapporo
Breweries, Ltd. He received PhD degree in Food Science at Nagoya
University in 1994. He had studied as a guest researcher in National
Institute of Bioscience and Human-Technology from 1996 to 1998.
He is Senior Biochemist in Brewing Research Laboratories of
Sapporo Breweries, Ltd.
He received the Eric Kneen Memorial Award from the American Society of Brewing
Chemists in 1995 and the Technical Award from the Agricultural Chemical Society of
Japan in 2000.
Kaneko, Takafumi (contribution nr. 21)
Born in 1962.
Studies: Faculty of Agriculture at the Kyoto University, Japan
(1985).
Appointments: 1985: Research Scientist in Plant Cell Research at the
Plant Bioengineering Research Laboratories (PBRL), Sapporo
Breweries Ltd.; 1985-2000: Research Scientist in Plant
Biotechnology at PBRL; since 2000: Group Manager in Plant
Biotechnology at PBRL. Since 2001: Doctor (Title of doctor thesis: Studies on
polymorphism of beta-amylase in barley grain).

Kaschek, Martin (contribution nr. 104)

Born in 1962.
Education and professional experience: 1978-1981: Training period
for information electronics technician at the Siemens company in
Heidenheim, Germany; 1981-1984: Technical High School in Aalen.
1984-1989: Degree in Technical Cybernetics at Stuttgart University;
1989-1991: additional studies at the Technical University and the
University of Vienna. Main subject: Biomedical Technology.
Professional experience: 1992: Scientist at the Fraunhofer Institute
for Interfacial Phenomena and Biochemical Engineering in Stuttgart; since 1993:
Scientist at the Institute for Environmentally Compatible Process Technology (upt) in
Saarbrcken.

Kellner, Vladimr (contribution nr. 82)

Born in 1949.
Studies: Institute of Chemical Technology - Prague, MSc degree,
1972; The Academy of Sciences of the Czech Republic, Institute of
Macromolecular Chemistry - Prague, PhD degree, 1976.
Appointments: 1972-1976: PhD student (Institute of Macromolecular
Chemistry - Prague); 1977-1978: research worker (Institute of
Macromolecular Chemistry - Prague); since 1979: Research Institute
of Brewing and Malting Prague (RIBM): 1979-1980: research worker (RIBM); 1980-
1985: Head of the Department of Special Analyses (RIBM); since 1985: Section
Manager - Analytical Services and Research (RIBM); and since 2001: Manager of
Accredited Analytical Testing Laboratory. Since 1991 coopted member of the EBC
Analysis Committee; since 1994 member of the EBC Analysis Committee; since 1996
chairman of the Beer Sub-committee of the EBC Analysis Committee; since 1995
chairman of the editorial board of the Czech beverage journal Kvasny Prumysl.

Kennedy, Alan I. (contribution nr. 40, 92)

Born 1965.
Education: BSc (Hons) Applied Microbiology, University of
Strathclyde, Glasgow, UK (1987); PhD Department of
Phytochemistry, University of Strathclyde, 1991 (thesis entitled
Biologically Active Secondary Metabolites from Medicinal and
Aromatic Plants).
Career: 1991-1993: Lecturer / Researcher in the Plant Science
Department at the Scottish Agricultural College; 1993 to date: Scottish Courage
Brewing Ltd, various positions including Fermentation Research Scientist and Senior

2
Microbiologist at the Technical Centre, Microbiology Team Leader at Fountain
Brewery, Quality Assurance Advisor in the Group Microbiology Department.
Currently, Process Development Manager in the Innovation & Development
Department with responsibility for the Central Microbiology laboratory, HACCP,
hygiene, laboratory and process auditing and new product development. Member of
the IGB, ASBC and Institute of Food Science and Technology (IFST) and co-
chairman of the EBC Microbiology Sub-Committee. Industrial supervisor for three
PhD students in the School of Biological and Molecular Sciences, Oxford Brookes
University.

Kepplinger, Werner (contribution nr. 107)

Born in 1942.
Studies: Hhere Technische Lehranstalt Linz/Austria (The Chemical
Engineering) Montanuniversitt Leoben (Dept. Metallurgical Science
D iplom Ingenieur); Montanuniversitt Leoben (Thesis on COREX-
Process - Dr. mont.).
Appointments: 1962-1967: Vereinigte sterreichische Eisen- und
Stahlwerke/Linz, Department of Coal Chemistry; 1968-1970: Voest-
Alpine Linz, Chemical Engineering Department; 1970-1978: Chemie
Linz, Technical manager of Licensing and Engineering department; 1978-1993: VA-
Industrieanlagenbau GmbH Linz; 1979: Member of COREX development team;
1985: Head of department for reduction metallurgy; 1992: Director of R&D-
department of VA-Industrieanlagenbau GmbH; 1993: Montanuniversitt Leoben:
Professor of Process Engineering (Environmental and Industrial Processes).
(Co-)author of 3 books. Appr. 70 publications. Appr. 300 patents.

Keukeleire, Denis, de (contribution nr. 7, 60)

Born in 1943.
Studies: BSc in Chemistry, Ghent University, Belgium (1966); PhD
in Chemistry, Ghent University (1971) - thesis on The Absolute
Configuration of Hop Bitter Acids; Aggregation for Higher
Education in Organic Chemistry, Ghent University (1982) - thesis on
The Chemistry of Hop Bitter Acids; postdoctoral studies at
California Institute of Technology, Pasadena, California, USA,
University of Geneva, and University of Bonn; awards for research
on the chemistry of hops and beer; author or co-author of more than 300 scientific
publications and 4 books; ca. 400 lectures in 37 countries.
Appointments: 1966-1972: PhD fellowship from the Belgian National Fund for
Scientific Research; 1972-1991: Senior Research Associate of the Belgian Fund for
Scientific Research with a position at the Laboratory of Organic Chemistry, Ghent
University; 1991-1992: Associate Professor at the Laboratory of Plant Biochemistry,
Ghent University; since 1992: Full Professor at the Faculty of Pharmaceutical
Sciences, Ghent University, and director of the Laboratory of Pharmacognosy and
Phytochemistry; since 1998: Member of the Research Council of the Ghent
University; since 1999: Member of the Advisory Commission for Plant Preparations
to the Belgian Minister of Social Affairs, Public Health, and Environment.
Research interests: hops (bitter acids, essential oils, polyphenols, advanced hop
products, light-stability, oxidative transformations, bioactivities), estrogenic plants.

3
Kielland-Brandt, Morten C. (contribution nr. 44)
Born in 1944.
Studies: MSc in Biochemistry and Genetics, University of
Copenhagen, Denmark (1970); PhD in Genetics, University of
Copenhagen (1974). Postdoctoral trainee at Department of Genetics,
University of Washington, Seattle, U.S.A. (1974 and 1975).
Appointments: from 1976: Research scientist at the Carlsberg
Laboratory, Copenhagen; 1984-1985: Visiting scientist at MIT and the
Whitehead Institute, Cambridge, Massachusetts; from 1986: Professor at the Carlsberg
Laboratory; 1987-2001: Head of the Department of Yeast Genetics, Carlsberg
Laboratory; 1994: Member of the Royal Danish Academy of Sciences and Letters; 2000:
Adjunct Professor at the Institute of Molecular Biology, University of Copenhagen;
2001: Head of the Department of Physiology, Carlsberg Laboratory.

Kihara, Makoto (contribution nr. 21)

Born in 1966.
Studies: Faculty of Agriculture at the Hokkaido University (1988),
Faculty of Agriculture (master course) at the Hirosaki University,
Japan (1990).
Appointments: 1990: Research Scientist in Plant Cell Research at the
Plant Bioengineering Research Laboratories (PBRL), Sapporo
Breweries Ltd.; 1990-1994: Research Scientist in Cell Technology at
PBRL; 1994-1997: Research Scientist in Plant Biotechnology at
PBRL; 1997-2000: Deputy Manager in Plant Biotechnology at PBRL; since 2000:
Cereal Chemist at PBRL. Since 1999: Doctor (Title of doctor thesis: Establishment of
technique for the production of transgenic plants using protoplast culture system in
barley).

Kleemola, Tuija (contribution nr. 12)

Born in 1971.
Studies: 1995 MSc (Tech.) and since 1998 PhD student in Chemical
Engineering at the Helsinki University of Technology, Finland.
Appointments: 1995: Masters Thesis: Brewery by-products and
wastes, VTT Biotechnology; since 1995: Research scientist at VTT
Biotechnology. Research grants: 1997: Niilo N. Santasalo and Lauri
N. Santasalo Foundation for the study of the hygiene and recycling of
steeping water; 1998: Food Research Foundation for the scholarship
to USA. Research interest: Gushing of beer, microflora of cereals.

Klimo, Karin (contribution nr. 6)

Born in 1955.
Education: Biological research technician at the German Cancer Research Center,
Heidelberg, Germany (1970-1973).
Appointments: since 1997: scientific work at the German Cancer Research Center,
Heidelberg. 1973-1981: Division of 'Cellular Immunology'; 1981-1996: Division of
'Toxicology and Cancer Risk Factors; since 1997: member of the work group 'Cancer
Chemoprevention' in the Division of 'Toxicology and Cancer Risk Factors'. Main
research focussing on inhibition of phase 1 enzyme activity, anti-inflammatory effects
and estrogen (ant)agonism of chemopreventive compounds.

4
Knauft, Jutta (contribution nr. 6)
Born in 1945.
Education: Chemical research technician at the Fachschule fr Chemotechniker,
Augsburg, Germany (1963-65)
Appointments: 1966-68: Technical assistant at Boehringer Mannheim in Tutzing;
since 1972: scientific research assistant at the German Cancer Research Center,
Heidelberg: 1972-95: Divisions of 'Molecular Toxicology'; since 1995: Division of
'Toxicology and Cancer Risk Factors'; since 1996: member of the Workgroup 'Cancer
Chemoprevention' in the Division of 'Toxicology and Cancer Risk Factors', German
Cancer Research Center, Heidelberg; Main research: (i) metabolism of N-nitroso
compounds, (ii) mechanisms of cancer chemopreventive agents with focus on anti-
inflammatory mechanisms (inhibition of cyclooxygenase), (iii) testing of
chemopreventive compounds in animal models.

Kobayashi, Naoyuki (contribution nr. 70)

Born in 1965.
Studies: Agricultural Biological Chemistry at the University of
Tokyo, Japan (1989); 1989-1991: Graduate student at Department of
Applied Biological Chemistry, the University of Tokyo; 1998: PhD
from Department of Applied Biological Chemistry, the University of
Tokyo.
Appointments: 1991-present; Research Scientist in Brewing
Research Laboratories, Sapporo Breweries Ltd.

Koplk, Richard (contribution nr. 85)

Born in 1962.
Studies: Food Chemistry at Institute of Chemical Technology,
Prague, Czech Republic, MSc (1986), PhD (1992).
Appointment: since 1993: Assistant Professor at the Department of
Food Chemisty and Analysis, Institute of Chemical Technology,
Prague.
Research area: determination and speciation of trace elements in food.

Kotaviita, Erja (contribution nr. 12)

Born in 1956.
Studies : MSc in Analytical Chemistry at bo Akademi University,
Turku, Finland (1980); Brewmaster at the Scandinavian Brewing
School, Copenhagen, Denmark (1988).
Appointments : 1981-1986: Laboratory Manager, Raisio Wheat
Starch Factory, Raisio Factories; since 1986: Development Manager,
Raisio Malt, Raisio Group plc; since 1994: Member of Oy
Panimolaboratorio (Research Institute of the Finnish Brewing Industry).

Kreisz, Stefan (contribution nr. 23)

Born in 1970.
Studies: November 1991 to September 1997: Study at the University of Munich-
Weihenstephan., Germany, in Brewing and Beverage Technology; 9-97 Graduation.
Doctoral thesis: November 1997 to present: Research work concerning the filterability
of wort and beer at the Institute for Brewing Technology I, University of Munich-
Weihenstephan, under the direction of Professor Back.

5
Work Experience: November 1997 to present: Research Associate at the Institute for
Brewing Technology. Consultation of breweries, supervision of thesis. March 1992 to
April 1992: Versuchs- und Lehrbrauerei Weihenstephan, Germany. Internship in
malting. May 1991 to August 1991 Calwer-Eck Brau and Stuttgarter Hofbrau AG,
Germany. Internship in all relevant departments of the brewery.

Krottenthaler, Martin (contribution nr. 22, 54, 75)

Born in 1963.
Studies: Brewing and Beverage Technology at the Technical
University Munich-Weihenstephan, Germany (1989); Doctoral thesis:
October 1989-April 1992: Development of a method which allows to
predict beer taste and other beer quality parameters of new barley
varieties in an early stadium of breeding.
Work Experience: May 1992 to July 1995, Assistant of Management
at the Spaten-Lwenbru Brewery, Munich. This includes the
creation of a new alcohol free beer and the temporal direction of malting and brewing
facilities. Malting and test brews in big scale with scientific evaluation of the malt-
and beer-quality were done; since July 1995: Assistant Professor at the Chair of
Brewing Technology I and Beverage Technology at the Weihenstephan Center of Life
and Food Sciences, University Munich-Weihenstephan. Responsible for research and
lessons concerning malt- and wort production.

Kuroda, Hisao (contribution nr. 70)

Born in 1960.
Studies: Biology (Molecular biology of plant cells) at the graduate
school of Nagoya University, Japan (1989).
Appointments: 1989-1999: Research Scientist at Plant
Bioengineering Research Laboratories, Sapporo Breweries Ltd.;
1999-present: Research Scientist at Brewing Research Laboratories,
Sapporo Breweries Ltd.

Kurz, Tomas (contribution nr. 29)

Born in 1970.
Studies: Brewing and Beverage Technology at the Technical
University Munich-Weihenstephan, Germany (1997).
Appointments: Scientist Process Automation in Biotechnology at the
Chair of Fluid Mechanics and Process Automation at the
Weihenstephan Center of Life and Food Sciences, Technical
University Munich (since 1997).

6
Curriculum vitae of (co)authors - L

Landman, Berco C.J. (contribution nr. 100)

Born in 1955.
Studied Mechanical Engineering at the University of Delft, the
Netherlands, gaining an MSc degree in measurement and control
in1983.
From 1983 to 1986 he was at Procter & Gambles European
Technical Centre in Brussels as Packaging Development Manager.
He joined Heineken in 1986 as a specialist in the Packaging
Engineering department of Heineken Technisch Beheer BV. In 1991
he became Section Manager of the Bottling and Packaging Engineering department of
Heineken Technical Services. He is currently Senior Manager Packaging Technology
of the Research and Development department of Heineken Technical Services.

Larsen, Olav V. (contribution nr. 56)

Born in 1968.
Studies: MSc. in Chemical Engineering and Food Technology at the
Danish Technical University (1995). Master brewer from the
Scandinavian School of Brewing (1998).
Appointments: Director and Head of Process Consultancy
Department of Alfred Jrgensen Laboratory.

Lee, Mark (contribution nr. 33, 37)

Studies: BSc in Molecular Biology, University of Manchester, UK
(1990-1994).
Appointments: QC Scientist, Dista Products (1991-1993);
Fermentation Scientist, Brewing Research International (1995-1999);
Head of Fermentation, Brewing Research International (1999-2000).

Lentini, Aldo (contribution nr. 106)

Born in 1959.
Graduated from La Trobe University (Australia) with a BSc (Hons)
and a PhD in Organic Chemistry / Biochemistry in 1987. After a 18
months of Post-doctoral studies he joined the Group Technical
Services Department of Carlton and United Breweries (CUB) in
1988, as a Research Officer.
In 1993 he was promoted to Senior Research Scientist within the
Research and Development Department of CUB. In 2000, he became the Principal
Fermentation Biotechnologist within CUBs National Operations Support Analytical
Team.
His current research interests are in the areas of yeast physiology, yeast management
systems, yeast propagation, yeast nutrient requirements, yeast strain optimisation,
fermentation process systems and flavour development. Fermentation R&D studies
have been involved in the brewing, wine and distillation areas. Other areas of interest
are in malt (varieties and specifications) and wort production.
Over the past 13 years, he has presented his R&D work on yeast physiology to
numerous National and International, Brewing and Scientific meetings/conferences.
He is a member of the Institute and Guild of Brewing, The American Society of
Brewing Chemists, The Royal Australian Institute of Chemistry, The Australian
Biotechnology Association and The Australian Society of Biochemistry and
Molecular Biology. He is currently the State (Victorian) Representative for the
Institute and Guild of Brewing.

Lermusieau, Guillaume (contribution nr. 80)

Born in 1975.
Studies: 1993-1998: Chemistry and Biotechnology Engineering with
a specialisation in Brewing Technologies, Universit Catholique de
Louvain, Louvain-la-Neuve (Belgium). Since October 1998: PhD
student in the field of beer ageing and hop flavours in the Brewing
Sciences Laboratory of Louvain-la-Neuve. Since October 2000:
MBA student in the field of International Management supervised by
Mercer University (Atlanta & Macon, USA), in United Business Institutes
(Brussels). Trainings: 1994 and 1995: Hall & Woodhouse Ltd., Blandford (England).
1996: Unicer, Porto (Portugal).
Publications & books: J.Am.Soc.Brew.Chem. (1999, 2001), Cerevisia (1999, 2001),
EBC Congress (1999), Food Science and Technology COST 96 (1999), J. Agric.Food
Chem. (1999, 2000), J. Food Chem. (2001), Molecular Methods of Plant Analysis
vol. 1: Taste and Aroma (book in press).

Lillelund, Anne C. (contribution nr. 56)

Born in 1970.
Studies: BSc in Chemical Engineering at the Engineering Academy
of Denmark (1994). Master Brewer from the Scandinavian School of
Brewing (1996).
Appointments: 1996-1999: Brewmaster at the Albany brewery,
Denmark; since 1999: Brewing Consultant at Alfred Jrgensen
Laboratory.

Linder, Markus B. (contribution nr. 12)

Studies: MSc (tech.), Helsinki University of Technology, 1993; PhD,
Helsinki University of Technology, Finland, 1997.
Appointments: 1992-1993: Finnish Red Cross - Blood transfusion
service, Project Researcher; 1993-1994: Uppsala University,
Sweden, Department of Biochemistry, Research Scientist; 1994-
present: VTT Biotechnology, Research Scientist.
Research interests: Protein engineering, protein production and
purification, biophysical chemistry.

2
Lommi, Heikki (contribution nr. 49)
Born 1949.
Studies: MSc, Organic Chemistry, Helsinki University, Finland,
1974.
Appointments: Research Scientist and R&D Project Manager in
Finnsugar Ltd (1975-1981); Department Manager in R&D Center in
Finnsugar Ltd (1981-1984); Corporate R&D manager in Finnsugar
Ltd (1984-1987); Business Manager, Protein Adsorbents in SPEC
(Starch Processing Enzyme Company) (1987-1992); Business Manager, Technology
Licensing in Cultor Ltd, Technology Center (1992-1997); Division and Sales
Manager, Immocon in GEA Tuchenhagen Scandinavia A/S (1997-05/2000); since
May 2000: R&D Manager Tuchenhagen Brewery Systems GmbH .

Londesborough, John (contribution nr. 37)

Born 1942.
Studies: B.A. (Biochemistry) Oxford, U.K., 1965; D.Phil. ("Studies of isocitrate
dehydrogenase") Oxford, 1969; Postdoctoral studies at Case-Western, Cleveland,
USA and Northwestern, Chicago, USA.
Appointments: 1972-1994: Senior biochemist and project leader in the Research
Laboratories of Alko Ltd., Helsinki, Finland; 1995-1996: Biochemist in the
Biotechnology Unit of Primalco Ltd, Rajamki, Finland; 1996 present: Senior
biochemist and group leader at VTT Biotechnology, Espoo, Finland.
Research interests: metabolic engineering and stress physiology.

Lusk, Lance T. (contribution nr. 71)

Joined the Miller Brewing Company in 1980 and is currently a Principal
Research Scientist. His research interests include foam properties, beer
physical stability, and beer flavor stability. He is a member of the
American Society of Brewing Chemistry, American Chemical Society,
American Association for the Advancement of Science, and The Protein
Society. In 1996 he was presented the American Society of Brewing
Chemistrys Eric Kneen Memorial Award for excellence in scientific
publication. He studied Biology at the University of Chicago and
Biochemistry at Roosevelt University (Chicago, U.S.A.) where he received AB and MS
degrees respectively. From 1972-1980 he was employed at the University of Chicago
Specialized Center of Research in Atherosclerosis.

Lustig, Stefan (contribution nr. 98)

Born in 1964.
Studies: 1988: Engineering degree in Brewing Technology at the TU
Mnchen-Weihenstephan, Germany; 1994: PhD in Brewing
Technology at the TU Mnchen-Weihenstephan; 1994 Thesis on
Flavour Stability: Staling Relevant Volatiles Analytical Methods
and Technological Influences
Appointments: 1989: Trainee position at Brauerei Beck & Co,
Bremen; 1990-1995: Research Assistant at Lehrstuhl fr Technologie der Brauerei I,
TU Mnchen-Weihenstephan; 1991: Head of GC Department at the same Institute;
1995: Head of the department Development & Technology at Brauerei Beck & Co,
Bremen; 1999: Head of Quality Department at Brauerei Beck & Co. Since 2000
Member of MEBAK (Mitteleuropische Brautechnische Analysenkommission).

3
Lynn, P.Y. (contribution nr. 86)

4
Curriculum vitae of (co)authors - M

Machado Cruz, Jos M. (contribution nr. 84)

Born in 1938.
Studies: Chemical Engineering at Porto University, Portugal (1965);
Cryptogamy, Fermentation and Bromatology at Faculty of Pharmacy-
Porto University; Biotechnology at Faculty of Pharmacy-Porto
University (1984).
Specialized training: General Management, Brewing Science,
Microbiology, Molecular Genetics, Biochemical Engineering, Soft Drinks
Technology, Quality Assurance, Packaging of Foods-Chemical Problems, Gas-Liquid
Chromatography, Flavour and Flavour Stability, Risk Assessment (HACCP), Hygiene
Practice for the Brewing and Soft Drinks Industries.
Appointments: 1965-1980: Laboratory Quality Control Manager at Companhia Unio
Fabril Portuense, S.A.R.L.; 1980-1991: Director of Industrial Quality; 1991-1996:
Director of Research and Development; 1996-2000: Adviser on Technical and
Regulatory Affairs to the Executive Board at Unicer-Unio Cervejeira, S.A. Since
2001: Adviser on Technical and Regulatory Affairs to the Executive Board at Unicer-
Bebidas de Portugal, S.A.
Since 1974: member of the EBC Council; 1971-1996: member of EBC Biochemistry
Group; 1992-1996: member of EBC Microbiology Group; since 1996: member of
EBC Brewing Science Group. Since 1988: member of the Technical Committee of
the Union of EU Soft Drinks Association (Unesda); since 1992: member of the
Committee on Market and Technology of "The Brewers of Europe" (CBMC),
Chairman of the Technical Committee of "Associao Nacional dos Industriais de
Refrigerantes e Sumos de Frutos" (ANIRSF); since 1996: member of "Comisso do
Mercado Interno" of the Portuguese Food Federation. 1992-2000: President of the
Portuguese Brewers and Maltsters Association. Member of "Ordem dos Engenheiros
Portugueses".

Mackie, Alan R. (contribution nr. 68)

Studies: 1979-1982: Physics with electronics at the University of East
Anglia, UK.
Appointments: since 1983 employed by the Institute of Food
Research: 1983-1990: Professional & Technical officer, Process
Physics Dept.; 1990-1993: Scientific Officer; 1993-: Band 6
Research Scientist, Food Material Science Dept.

Madrid, Jorge D. (contribution nr. 61, 64)

Born in 1973.
Studies: BSc student in Chemistry at the Universidad Simn Bolvar,
Venezuela (2001). Appointments: 1997: Lab assistant at the Central
Laboratory of Polar Brewery in Barcelona, Venezuela; 1998-2001:
Lab Assistant and Assistant Scientist of the Polar Technology Center
in Caracas. Has experience in Sensory analysis and aroma research,
analytical and food chemistry, GC and GC-MS. Member of the
Venezuelan Chemical Society.
Madsen, Peter L. (contribution nr. 44)
Born in 1966.
Studies: Chemical Engineering at the Technical University of
Denmark (1992).
Appointments: 1992-1996: Graduate student at University of
Copenhagen; 1996: Assistant Research Professor at the Technical
University of Denmark; 1997-1998: Visiting Research Scientist at
the National Institutes of Health, Bethesda, USA; 1999-2000:
Postdoctoral Fellow at the Carlsberg Laboratory, Copenhagen; since
2001: Genetic Toxicologist at H. Lundbeck A/S, Copenhagen.

Marz, Anna (contribution nr. 43)

Born in 1949.
Studies: Dipl.biologist: Attila Jozsef University, Faculty of Sciences,
Szeged (Hungary), 1973; DSc, A. Jzsef University, 1977; PhD,
1989; Doctor Habil: Microbiology-biotechnology-molecular biology,
University of Horticulture and Food, 1996.
Appointments: 1976-1979: Assistant Lecturer; 1979-1982: Lecturer
in Department of Microbiology, A. Jzsef University, Szeged; 1982-
1985: Senior scientist, Head of Laboratory of Biotechnology, Central Food Research
Institute, Budapest; 1985-1989: Lecturer; 1990-1998: Associate Professor; since
1998: Professor; since 1997: Head of department in the Department of Microbiology
and Biotechnology, St Istvan University, Faculty of Food Science, Budapest; since
2000: vice Dean in Faculty of Food Science.
1995-1997: Representative at the Hungarian Academy of Sciences, Academy Board;
since 1994: Secretary of the Mycological Section, Hungarian Microbiological
Society; since 1995: National representative at the Working Party of Microbial
Physiology, European Federation of Biotechnology; since 1997: National
representative of the International Commission for Yeasts.

Martynowicz, Emile T.M.J. (contribution nr. 69)

Born in 1967.
Studies: PhD Degree in Chemical Engineering (1994) at Delft University of
Technology, the Netherlands.
Appointments: since 1999: Manager Research & Development at Haffmans B.V. His
department is responsible for the development of the Nibem-T foam stability tester
and at present developing a clingmeter.

Mavrov, Valko (contribution nr.104)

Born in 1945.
1970: Master of Science in Chemical Engineering at the Technical
University of Prague, Czech Republic; 1974: PhD in Chemical
Engineering at the Technical University of Prague. 1974-1990:
Lecturer in Water Technology at the University of Bourgas,
Bulgaria; 1981: Habilitation and Associate Professor at the
University of Bourgas; 1990-1992: Scientist at the Max-Planck
Institute for Biophysics, Frankfurt on Main, Germany; since 1992: Lecturer in
Separation Processes at the University of the Saarland, Saarbrcken; 1998:
Habilitation and Associate Professor at the University of the Saarland, Saarbrcken.

2
Research field: separation processes particularly membrane processes Water and
waste water treatment.

Methner, Frank-Jrgen (contribution nr. 62)

Born in 1953.
Studies: 1975-1980: Brewing Technology at the Technical
University of Berlin, Germany, final examination: master degree in
engineering science (Dipl.-Ing.). 1980-1981: Quality control at the
Schlsser Brewery, Dsseldorf; 1982-1986: Scientific collaborator at
the Research Institute of Brewing and Malting Science at the
Versuchs- und Lehranstalt fr Brauerei (VLB), Berlin; 1982-1986:
PhD (Dr.-Ing.) in Brewing Sciences at the Technical University of
Berlin; since 1987: Director for Brewing Technology and Quality Management at the
Bitburg Brewery, Bitburg.

Meyer-Pittroff, Roland (contribution nr. 51)

Born 1942.
After studying Mechanical Engineering at the Technical Universities
(TU) of Brunswick and Munich/Germany, brief engineering work in
gas turbine manufacturing.
1967-1972: research work at the Institute for Thermodynamics at the
TU Munich. Thesis for the doctorate about thermodynamic equation
of state.
1972-1984: Engineer and Manager at the Kraftwerk Union AG (subsidiary of
Siemens AG) in the field of thermal power plants. Since 1984 full Professor and Head
of the chair for Energy and Environmental Technologies of the TU Munich in
Freising-Weihenstephan; 19921994: Decan of the Faculty of Brewing.
Arbitrator at the International Court of Buisiness Chamber of Austria. Member of the
board of Society for Energy Technology within the Society of German Engineer
(VDI) and the Bavarian State Brewery Weihenstephan. Honorary professor of the
Polytechnical University Timisoara/Romania. Author and co-author of more than 260
publications.

Mikyka, Alexander (contribution nr. 59)

Born in 1956.
Studies: Fermentation Chemistry and Bioengineering in the
Institute of Chemical Technology in Prague (ICT), Czech
Republic (1980).
Appointments: 1980-1988: Research worker in Biochemical
department of the Research Institute of Brewing and Malting in
Prague (RIBM); 1989: Senior Assistant in Department of
Fermentation Chemistry and Bioengineering ICT; 1990-1994:
Head of Biochemical Department of RIBM; 1995-1996: Head of
Technological Department of RIBM; since 1997: Head of Research Department in
Prague of RIBM.

3
Milligan, Stuart R. (contribution nr. 7)
Born in 1950.
Studies: BSc in Zoology, Oxford University, UK (1971); PhD,
Oxford University, (1974).
Appointments: 1974-1975: Browne Junior Research Fellow in
Zoology, The Queen's College, Oxford; 1975-1991: Lecturer in
Physiology, King's College, London; since 1991: Senior Lecturer in
Physiology, King's College, London; since 1999: Director of the
Endocrinology and Reproduction Research Group, King's College
London. 1988-1993: Editor of Oxford Reviews of Reproductive Biology; 1987-1991,
since 1997: Council of Management of Journals of Reproductions and Fertility; since
1994: Editorial Board of Reviews of Reproduction. 1989-1994: Business Secretary of
the Society for the Study of Fertility; since 1997: Treasurer of the Society for the
Study of Fertility.

Mills, E.N. Clare (contribution nr. 68)

Studies: BSc in Biochemistry (Bristol, UK), 1979; PhD Biochemistry (University of
Kent at Canterbury), 1984.
Appointments: 1979-1980, Glaxo Group Research, Ware; 1983-1984, UK
Environment and Toxicology Division, Department of Health, Medical; 1985-present,
Institute of Food Research. 2000-present, Secretary of Industrial Biochemistry and
Biotechnology Group of Biochemical Society; 2001: Member of Royal Society
Expert Group on Genetically Modified Organisms for Food Use.

Mitchell, Mack C. (contribution nr. 2)

Born in 1952.
Education: 1970-1972: Davidson College, Davidson, NC; 1972-1974: Johns Hopkins
University, Baltimore, MD, B.A. 1974; 1973-1977: Johns Hopkins School of
Medicine, Baltimore, M.D., 1977; 1977-1979: Internship and Residency in Internal
Medicine, Johns Hopkins Hospital, Baltimore, Maryland; 1979-1980: Fellow in
gastroenterology, Johns Hopkins School of Medicine, Preceptor: Dr. Willis C.
Maddrey; 1980-1982: Fellow in gastroenterology and clinical pharmacology,
Vanderbilt University School of Medicine, Preceptor: Dr. Steven Schenker.
Current position: since 1996: Chairman, Department of Medicine, Carolinas Medical
Center, Charlotte, North Carolina; Clinical Professor, Department of Medicine,
School of Medicine, University of North Carolina at Chapel Hill. Previous positions:
1991-1996: Chairman, Department of Medicine, Greater Baltimore Medical Center
Baltimore, Maryland; 1989-1996: Associate Professor of Medicine, The Johns
Hopkins University School of Medicine, Baltimore, Maryland; 1982-1989: Assistant
Professor of Medicine, The Johns Hopkins University School of Medicine.
Honors: 1971: Phi Eta Sigma Freshman Honorary Fraternity, Davidson College;
1971: Freshman Chemistry Award, Davidson College; 1977: Phi Beta Kappa, Johns
Hopkins University; 1977: Alpha Omega Alpha, Johns Hopkins School of Medicine;
1981: American Liver Foundation Research Fellowship; 1981: National Research
Service Award; 1983: American Gastroenterology Association Interdisciplinary
Award; 1985: New Investigator Award, NIAAA (AA06865); 1991: Fellowship
American College of Physicians; 1993: Fellowship American College of
Gastroenterology; 1993: Teaching Award, Greater Baltimore Medical Center; 1991-
1995: Voted one of Baltimore's Best physicians, annually; 1995-2000: Voted one of
Best Doctors in America, Southeast region.

4
Membership in professional societies: American Association for Study of Liver
Diseases; American College of Physicians, Fellow; Johns Hopkins Medical and
Surgical Association; American Federation for Clinical Research (Eastern Section
Councilor, 1984-1989); Research Society on Alcoholism.
Other professional activities: Governor's Council (Maryland), American College of
Physicians 1992-1996; Chairman, NIAAA Alcohol Research Centers Grant Review
Committee 1995; NIAAA study section Biomedical-1 1989-93; VA Merit Review
Board on Alcohol & Clinical Pharmacology 1988-1991; NIAAA ad hoc study section
for alcohol research centers 1988; VA ad hoc review board for Alcohol Research
Centers 1990; AASLD Research Committee, 1991-94; Alcoholic Beverage Medical
Research Foundation, President 1989-, Member of biomedical research council 1984-
1987; Reviewer for Hepatology, Gastroenterology, Annals of Internal Medicine,
Digestive Diseases and Sciences, Medicine, American Journal of Physiology,
American Journal Clinical Nutrition, Alcoholism: Clinical & Experimental Research,
Journal of Clinical Investigation.
Research interests: effects of ethanol on glutathione metabolism; mechanisms of drug-
induced and alcoholic liver injury; role of oxidant stress in hepatic transplant
rejection; treatment of chronic hepatitis.

Mitzscherling, Martin (contribution nr. 75)

Born in 1973.
Studies: Brewing and Beverage Technology at the Technical
University Munich-Weihenstephan, Germany (1999).
Appointments: Scientist Process Automation in Biotechnology at the
Chair of Fluid Mechanics and Process Automation at the
Weihenstephan Center of Life and Food Sciences, Technical
University Munich (since 1999).

Mochaba, Franciska (contribution nr. 42)

Born in 1957.
Studies: Bsc, (1979) National Univerisity of Lesotho, South Africa;
Post-graduate diploma Industrial Microbiology, (1981) Msc (1982) at
Heriot-Watt University, UK; PhD Plant Pathology (1989) at the
University of the Witwatersrand, South Africa.
Appointments: 1979-1982: Teaching assistant University of Lesotho;
1982-1991: Lecturer University of Lesotho; 1991-1996: Research
Scientist - Brewing Research Department, South African Breweries;
since 1996: Senior Research Scientist - South African Breweries, Sandton, South
Africa.
Research interests: yeast glycogen metabolism and measurement using Near Infrared
Spectorscopy, yeast quality measurements e.g. protease release, magnesium release
and pH as indicators of yeast quality, flocculation mechanism and malt factors that
influence flocculation. Member of the Institute of Brewing, Africa Section since
1992.

5
Mocking-Bode, Helene C.M. (contribution nr. 69)
Born in 1955.
Studies: BSc Analytical Chemistry and Biochemistry.
Appointments: 1972-1981: Research Technician at the National
Institute for Barley, Malt and Beer (NIBEM); 1981-present: Research
Technician at Agro-NIBEM, product group Beverages, the TNO
Nutrition and Food Research Institute Zeist, the Netherlands.

Morgan, A. Ray (contribution nr. 94)

Born 1947.
Studies: Bachelor of Science degree, Chemistry and Physics at North
Georgia College (1965-1969), Bachelor of Science degree,
Mechanical Engineering at Southern Technical Institute in association
with Georgia Institute of Technology (1975-1979).
Appointments: 1969-1979: Quality Assurance, Flavor Base and New Product
Development Chemist at Coca-Cola USA; 1979-1984: Innovation Engineer at The
Coca-Cola Company, awarded 27 international design and utility patents; 1984-1998:
Director, Packaging and Innovation at Coca-Cola USA, Vice President, Packaging
and Marketing Services Worldwide at The Coca-Cola Company; Since 1999:
President at 5 Points Solutions, LLC, a strategic marketing consultancy for innovation
in new products, packaging, branding and market development.

Morris, Victor J. (contribution nr. 68)

Studies: BSc [Physics & Chemistry 1st class hon., 1966], MSc Solid
state physics [1967], PhD Solid state physics [1972], DSc Molecuar
biophysics [1991].
Appointments: Ministry of Defence fellowship 1971-1974; Medical
research fellow 1974-1976; ICI fellow 1976-1979; Senior scientific
officer IFR, 1979-1983; Principal scientific officer IFR, 1983-87;
Senior principal scientific officer IFR, 1987-1999; Principal senior
scientist IFR, 1999-. Editorial boards: Carbohydrate Polymers, International J.
Biological Macromolecules, Trends in Food Science & Technology, Current Opinions
in Colloid & Interface Science. Chartered Physicist, Chartered Chemist, Fellow
Institute Physics, Fellow Royal Society Chemistry, Member: Society Chemical
Industry, British Society of Rheology, British Biophysical Society. 266 papers, 2
books [Atoms in Contact & Atomic Force Microscopy for Biologists].

Muller, Robert E. (contribution nr. 73)

Born in 1957.
Studies: Joint Honours Degree in Microbiology and Biochemistry at
the University of Leeds (1979), PhD in Biochemistry at the
University of Glasgow, UK (1982), Post-Doctoral Research in
Molecular Biology, University of Osaka, Japan (1983-1985).
Appointments: since 1985 employed by Brewing Research
International, initially as a Research Scientist, then as Senior Scientist and latterly as
Head of Cereal Science. Member of the Institute of Brewing Technical Committee.

6
Murnleitner, Ernst (contribution nr. 109)
Born in 1967.
Studies: Food Science and Biotechnology at the University of
Agriculture in Vienna, Austria and at the Delft University of
Technology, the Netherlands (1996).
Appointments: Scientist Process Automation in Biotechnology at the
Chair of Fluid Mechanics and Process Automation at the
Weihenstephan Center of Life and Food Sciences, Technical
University Munich, Germany (since 1997).

Mussche, Roger A. (contribution nr. 58)

Studies: Academic degree in Industrial Engineering - Chemistry at
the KaHo St. Lieven, Gent, Belgium; Academic degree in
Industrial Engineering - Biochemistry at the CTL, Gent; Ir. Degree
in Microbiology-Brewery Sciences at the University of Leuven; PhD
at the Ale University of Seattle, USA.
Appointments: Member of the lecturers board at the University of
Leuven for the Post University Degree in Brewing Sciences; was a
member of the Plant Biochemistry Laboratory at the University of Gent for two years;
from 1971: Quality Control Manager at Omnichem; from 1980: Development
Manager at Omnichem; from 2001: Scientific Advisor Manager.
He published a lot of articles about hops, beer bitterness analysis, tannins and
gallotannins, special beers production and total shelf-life program of beers. He is
Lecturer at the University of Leuven in beer shelf-life and tannins; at the Ale
University of Seattle in special beers production. He is visiting professor at the
Hardware University CEOR, India.

7
Curriculum vitae of (co)authors - N
Nakamura, Takashi (contribution nr. 65)
Born in 1966.
Studies: Enzymology and Protein Engineering, M.S. degree at the
Tohoku University, Japan (1992).
Appointments: since 1992 employed by Kirin Brewery Co., Ltd.:
1992-1999: Scientist at the Research Laboratory for Brewing of
Technology Development Department. Since 1999: Guest scientist at
the Institute for Brewing Technology I (Prof. Back) Technical
University of Munich Weihenstephan, Germany.

Nakari-Setl, Tiina (contribution nr. 12)

Studies: 1989 MSc and 1995 PhD in biochemistry at the University

of Helsinki, Finland.
Appointments: 1988-1989: Research Assistant, Teaching Assistant
and Research Scientist at the Department of Medical Chemistry at the
University of Helsinki; 1990-1992: Research grant of the Foundation
for Biotechnical and Industrial Fermentation Research; since 1993
employed by VTT Biotechnology.
Research interests: Biotechnical and molecular aspects of protein production in fungi.
Molecular biology, biochemistry and biotechnical applications of fungal
hydrophobins.

Nrhi, Marko (contribution nr. 93)

Born 1968.
Studies: Applied Microbiology, Applied Biochemistry and Polymer
Technology at the Helsinki University of Technology, Finland
(2000).
Appointments: 1998: Research Assistant at the Laboratory of
Polymer Techology at the Helsinki University of Technology; 1999-
2000: Research Trainee at the Biotechnical Laboratory of the
Technical Research Centre of Finland; since 2000: Research Scientist at the
Laboratory of Biochemistry and Microbiology at the Helsinki University of
Technology.

Navarro, Alfonso (contribution nr. 71)

Studied Chemistry at the University of Valencia, Spain, where he
also obtained his PhD degree in Biochemistry and Molecular
Biology. He also holds a Research Diploma in Molecular Cytology
by the Cytological Research Institute of Valencia and the University
of Kansas, College of Health Sciences (USA). He performed his post-
doctoral training at the Pasteur Institute in Paris, France. He was
Director of Research for the Spanish Association of Research in
Brewing and Malting (INVESCEMA) for 13 years. In 1997, he joined Miller Brewing
Company as Director of Research and Development. He was a member of the EBC
Biochemistry and Microbiology Committees. Currently, he is a member of the EBC
Brewing Science Group and of the Editorial Board of the Journal of the American
Society of Brewing Chemists.

Nedervelde, Laurence, van (contribution nr. 45, 46)

Researcher at the department of Brewing Sciences and Fermentation
Technology at the Institut Meurice in Brussels, Belgium, since
academic year 1994-1995 where she is in charge of different research
projects as well at academic level as in collaborative projects with
industry.
To obtain her graduation as Engineer in Food and Chemical
Industries at the University of Brussels she presented a research work
on the regulatory mechanisms involved in yeast amino acid
metabolism.

Neske, Walter (contribution nr. 28)

Born in 1949.
Appointments: 1971: Beck & Co Maintenance for electrical
engineering; 1991: Beck & Co Head of the department for electrical
maintenance and plant development.

Neumann, Isabell (contribution nr. 5, 6)

Born in 1975.
Education: Biological Research Technician in Berlin, Germany (1996-1998).
Appointments: 1998-99: Technical Assistant in the Division of Tumor Progression
and Immune Defense at the German Cancer Research Center, Heidelberg, since 1999:
member of the Workgroup 'Cancer Chemoprevention' in the Division of 'Toxicology
and Cancer Risk Factors', German Cancer Research Center, Heidelberg. Main interest
in anti-oxidative, radical-scavenging and anti-tumor promoting effects of
chemopreventive agents and in the establishment of the MMOC model.

Nielsen, Henning (contribution nr. 56)

Born 1939.
Studies: MSc. in Chemical Engineering at the Danish Technical University (1962).
Master brewer from the Scandinavian School of Brewing (1965).
Appointments: 1965-1974: Process Consultant at Alfred Jrgensen Laboratory; 1974-
1988: Technical Director at Faxe Brewery, Denmark; 1990-2000: Director and Head
of Process Consultancy Department of Alfred Jrgensen Laboratory; since 2000:
Senior Consultant at Alfred Jrgensen Laboratory.

Nielsen, Peter S. (contribution nr. 44)

Born in 1961.
Studies: Master degree in Biology at Department of Molecular
Biology and Plant Physiology, University of Aarhus (1987); PhD in
Natural Sciences, University of Aarhus, Denmark (1991).

2
Appointments: 1991-1994: Postdoctoral Fellow at Plant Molecular Biology
Laboratory, Agricultural University of Norway; 1995-1996: Postdoctoral Fellow at
Environmental Science and Technology Department, Ris National Laboratory; 1996-
1999: Assistant Research Professor at Center for Process Biotechnology, Technical
University of Denmark; 1999-2001: Postdoctoral Fellow at the Carlsberg Laboratory,
Copenhagen.
Nitzsche, Frank (contribution nr. 51)
Born in 1960.
Studies: Brewing Technologie at Technische Universitt Mnchen-
Weihenstephan, Germany (1983-1989), PhD at Chair for Technology
of Malt- and Wortpreparation (Prof. Dr. Narziss) (1989-1991).
Appointments: since 1991 employed by Knig Brauerei GmbH & Co
KG, Duisburg: 1991-1993: Head R&D; 1993-1997: Head QA; since
1997: Head Production and QA; since 1998: CEO EasyProof
Laborbedarf GmbH, Voerde.

Noordman, T.Reinoud (contribution nr. 105)

Graduated as a Chemical Engineer at the University of Groningen, the Netherlands, in
1991. He has been working at the University of Groningen from 1992 until 1998 and
has been involved in various projects concerning membrane filtration (desalination,
waste water treatment, modelling) and product development (improvement of the
shelf life of sweet products). In April 2000 he obtained a PhD degree in Chemical
Engineering on his work in the field of membrane filtration.
Since January 1999 he is working as Senior Scientist at the department R&D Process
Development & Optimisation of Heineken Technical Services, Zoeterwoude. He is
involved in new developments in separation processes (membrane filtration for bright
beer, treatment of spent grains).

3
Curriculum vitae of (co)authors - O

Ohtake, Yasuyuki (contribution nr. 14)

Born in 1959.
Studies: BS in Agriculture (1982) and PhD at the Faculty of Agriculture, University
of Tokyo, Japan (1990).
Appointments: since 1982: employed by Asahi Breweries, Ltd.; 1982-1983: Research
Scientist at Central research Laboratories, Asahi Breweries, Ltd.; 1983-1984:
Research student at Fermentation Research Institute, Agency of Industrial Science
and Technology; 1984-1985: Research Scientist at Central Research Laboratories,
Asahi Breweries, Ltd.; 1985-1986: Research student at Research Institute for Food
Science, Kyoto University; 1986-1992: Research Scientist at Central Research
Laboratories, Asahi Breweries, Ltd.; 1992-1994: Postdoctoral Fellow at Section on
Genetics of Simple Eukaryotes, Laboratory of Biochemical Pharmacology, National
Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of
Health, USA; 1994-1995: Deputy Manager at Central Research Laboratories, Asahi
Breweries, Ltd.; 1995-1999: Deputy Manager at Bioscience Research & Development
Laboratory; 1999-2001: Manager at Foods & Pharmaceuticals Research &
Development Laboratory; 2001-: Executive Researcher at Key Technology Research
Laboratory.

Oliver, Tony (contribution nr. 52)

Born 1950.
Studies: Chemical Engineering at the University of Queensland.
Associate Members Examination.
Appointments: 1976-1994: employed by Uncle Bens of Australia:
1976-1980: Reseaarch Scientist; 1980-1986: Project Engineer/Senior
Project Engineer; 1987-1988: Business Planning Manger; 1988-1994:
Project Manager; since 1995: employed by Carlton and United
Breweries: 1995-2000: Senior Process Engineer; 2001-current: facilitator Beer and
Beverage Capability. Membership: Australia Institute of Chemical Engineers (1976-
current), Institute and Guild of Brewing (1995-current).

Olkku, Juhani (contribution nr. 12)

Born 1944.
Studies: MSc in Chemical Engineering and Food Technology at the
Helsinki University of Technology, Finland (1969); PhD in Food and
Agricultural Engineering at the Univeristy of Massachusetts, USA
(1976).
Appointments: 1969-1971: Technical Manager in Makeis- ja Mehu
Oy; 1971-1974: Research Assistant at the Univeristy of
Massachusetts; 1975: Research Assistant of the Finnish Academy of Sciences at the
Queen Elisabeth College, University of London; 1976-1982: Research Scientist in
Food Physics and Food Process Engineering in Food Research Laboratory of the
Technical Research Center of Finland (VTT); 1982-1985: Genetic Engineering
Project Manager in Orion Corporation; since 1985: Research and Development
Director in Polttimo Companies Ltd and since 1999: Managing Director of LP
Research Centre Ltd. Member of several professional societies, associations and
companies and their boards.
Ottow, Kim E. (contribution nr. 44)
Born in 1973.
Studies: 1995-1996: Enrolled at the University of Copenhagen,
Denmark (Biochemistry). 1996-1997: Military service. 1997-2000:
continued studies at the University of Copenhagen. 2000: BSc in
Biochemistry. 2000: started laboratory work on a Master Thesis at the
Carlsberg Laboratory, Copenhagen.

Ozaki, Kazutaka (contribution nr. 14)

Born in 1966.
Studies: Chemistry at Chiba University, Japan (1990).
Appointments: since 1990: employed by Asahi Breweries, Ltd. Since 1995: member
of the BCOJ Analysis Committee. Since 2000: Vice-Chairperson of the BCOJ
Analysis Committee. Since 2001: Chief Researcher at Key Technology Research
Laboratory. Member of BCOJ and ASBC.

2
Curriculum vitae of (co)authors P / Q

Pajunen, Esko (contribution nr. 49)

Born in 1945.
Studies: Chemical Engineering and Food Technology at the Helsinki
University of Technology, Finland (1970).
Appointments: 1970-1973: Senior Assistant Scientist in
Biochemistry at the Helsinki University of Technology; 1973-1978:
Scientist Process Technology in Brewing at the Biotechnical
Laboratory of the Technical Research Center in Finland; 1978-1979:
Director of Research and Development at the Finnish Food Industries Federation;
since 1979: employed by Sinebrychoff Brewery: 1979-1987: Research and
Development Manager; 1987-1992: Production Director; since 1993: Senior Vice-
President Development, Research, Technology. Since 1987: Chairman and Director
of Oy Panimolaboratorio (Research Institute of the Finnish Brewing Industry). Since
1997: Board Member of the Brewing Research International (BRi). 1998-1999: Vice
President of the European Brewery Convention; since 1999: President of EBC.
Member of the EBC Council, EBC Brewing Science Group, EBC Technology &
Engineering Forum (Chairman), IOB, MBAA and ASBC.

Pauls, Sebastian (contribution nr. 28)

Appointments: Nord IT, Consultant for PROLAB.

Pecar, Michael (contribution nr. 106)

Studies: obtained a Bachelor of Engineering in Chemical
Engineering at RMIT in 1987. He also has a Master of Engineering
at RMIT in 1988.
Appointments: joined Carlton and United Breweries in 1988 in the
R&D Department, where his major role was filter optimisation and
sterile filtration development. In 1990, he had a twelve month stint
at Abbotsford Brewing Department and in 1993 he joined the
Process Engineering Department where his primary role was in the sterile filtration
aspects of Carlton Cold Filtered Bitter production. He has been part of the process
engineering area of the profitability group (1995-2000) looking at by-product
utilisation, automatic DE dosing, yeast concentration processes and general process
engineering. In late 2000 he joined the operations support engineering team where he
will continue to provide general processing engineering support.

Peereboom, Pieter S. (contribution nr. 69)

Born in 1950.
Studies: BSc Biochemistry and Microbiology.
Appointments: 1972-1981: Technician at the National Institute for
Barley, Malt and Beer (NIBEM); 1981-present Technician at Agro-
NIBEM, product group Beverages, the TNO Nutrition and Food
Research Institute, Zeist, the Netherlands.
Penttil, Merja (contribution nr. 12, 47, 53, 61)
Studies: 1981 MSc and 1987 PhD in Genetics at the University of
Helsinki, Finland.
Major appointments: 1978-1979: Research Assistant at the Institute
of Occupational Health (Helsinki); 1981-1984: The Neste
Foundation scholar; 1985-1989: Research Scientist at VTT
Biotechnical laboratory; 1988 (3 mo): Visiting Scientist at Kirin
Brewery (Tsukuba, Japan); 1989-1991: Senior Research Scientist;
1991-1996: Group Leader "Molecular biology of yeast and
filamentous fungi"; 1996-1999: Chief Scientist and Group Leader "Production
microbes"; since1999: Research Professor in Biotechnology at VTT Biotechnology.
Research interests: Molecular biotechnology, metabolic engineering, protein
production.

Pnzes, Zoltn (contribution nr. 58)

Born in 1978.
Studies: Chemical Engineering and Food Technology at the
University of Debrecen, Hungary (2001) with thesis in Malting and
Brewing Science at the KaHo St.-Lieven (September 2000-March
2001).

Peters, Ulrich (contribution nr. 62)

Born in 1966.
Studies: Brewing Technology at Technical University of
Munich/Weihenstephan, Germany (1993).
Appointments: 1993-1998: Assistant Scientist in Brewing
Technology/ Process Automation at Technical University of
Munich/Weihenstephan; 1999 PhD in Brewing Technology at
Technical University of Munich/Weihenstephan; since 1998:
employed by Bitburg Brewery, Bitburg; 1998-2001: Head of Pilot Brewery; since
2001: Head of production.

Pocock, Victoria (contribution nr. 7)

Born in 1954.
Studies: BSc in Reproductive Physiology, Endocrinology, and
Animal Behaviour, Kings College, London, UK (1999).
Appointments: since 1999: Research Associate, Kings College,
London for a project on reproductive effects of phytoestrogens; since
1999: Lecturer in Endocrinology and Vertebrate Biology, Kings
College, London.

Powell, Chris D. (contribution nr. 41)

Born 1974.
Studies: BSc in Biology and Environmental Biology at Oxford
Brookes University, UK (1996); PhD Rainbow scholarship supported
by the BBSRC from Bass Brewers and Oxford Brookes University
(2001) entitled Yeast Cell Age and Brewing Fermentation
Performance.

2
Appointments: 1996: Research post at Oxford Brookes University investigating
aspects of heavy metal toxicity in fission yeast; 1996-1997: Research position
researching oxidative stress in brewing yeast (Oxford Brookes); 1997-1998: Research
Scientist in R & D at Bass Brewers. Since 2001 employed as a Research Scientist
(European collaboration) to develop and evaluate rapid methods for bacterial
identification.

Preu, Thomas (contribution nr. 15)

Born in 1969.
Studies: Brewing Science and Beverage Technology at the
Technical University of MunichWeihenstephan, Germany (1997).
1997-2001: Doctoral thesis about Technological measures to gain
malty dark beers of high flavour stability after characterization of
the key odorants of selected dark beers at the Chair for Brewery
Technology in Weihenstephan.
Since 2001: Assistant professor at the Institute for Brewery
Technology and Beverage Microbiology in Weihenstephan. Areas of research: impact
of raw materials and brewing technology on beer flavour and flavour stability.

Quain, David E. (contribution nr. 41)

Born 1952.
Studies: BSc in Brewing and Biochemistry at Heriot Watt University,
UK (1976), PhD in Yeast Biochemistry from the University of
Liverpool (1979).
Appointments: 1970-1972: Laboratory Assistant at Brewing
Research International (BRi); 1979-1982: Fermentation Scientist at
BRi; since 1982 employed by Bass Brewers as Research Project
Manager (Microbiology) (1982-1987); QA Projects Manager (Burton Brewery)
(1988-1991); Company Microbiologist (1991-1994); Logistics Research Project
Manager (1994-1995) and Head of Research (1995-).
Fellow of the Institute of Brewing, member of the EBC Brewing Science Group, the
Research Committee at the International Centre for Brewing and Distilling at Heriot
Watt University and the Process Panel at BRi.

3
Curriculum vitae of (co)authors - R

Rdler, Thomas (contribution nr. 99)

Born in 1965.
Vocational Training and University Education: Aprenticeship in the
dairy industry with Milchwerke Westfalen eG in Herford,
Germany (1985-1987); Study of Technology and Biotechnology of
Food, Technical University of Munich (1987-1992); PhD (Dr.-Ing.)
examination, PhD-thesis: Modelling and Simulation of Filling
Lines, Technical University of Munich (1999).
Appointment: Associate Research at the Chair of Brewing Plants and Food Packaging
Technology, Center of Life and Food Sciences, Technical University of Munich
(1993-2000).

Rangel-Aldao, Rafael (contribution nr. 47, 48, 61, 63, 64)

Born in 1946.
Studies: Medicine at University Central of Venezuela, Caracas
(1969); Master of Sciences (1976), and Doctor of Philosophy (PhD),
Department of Molecular Biology, Albert Einstein College of
Medicine, New York (1977).
Appointments: 1969-72: Instructor of Biochemistry at the Faculty of
Medicine, University Central of Venezuela; 1977-78: Research
Associate, Department of Molecular Pharmacology, Albert Einstein College of
Medicine; since 1978 at University of Carabobo, Maracay, Venezuela: Associate
Professor, Department of Biochemistry (1979-1983), Professor of Biochemistry
(1983-1987). Since 1987 employed by Empresas Polar, Caracas: National Manager of
Biotechnology (1987-1999), Director of Research and Development (1999 to
present). Professor of Biotechnology at University Simon Bolivar (1988-1999) and at
Institute of Advanced Studies, Caracas (1999-2001). Member of the American
Society of Biochemistry and Molecular Biology, American Chemical Society,
American Society of Brewing Chemists. Member of the editorial boards of Biological
Research, and Electronic Journal of Biotechnology.

Rath, Frank (contribution nr. 11)

Born in 1957.
Studies: 1980-1986: Agricultural Science at the Rheinische Friedrich-
Wilhelms-University of Bonn, Germany; 1993: PhD.
Appointments: 1986: Scientific Collaborator at the Research
Department/Plant Production and Physiology, Weissheimer
Malzfabrik, Andernach; 1986-1990: Scientific Collaborator at the
Research Institute of Raw-Materials within the Research an Teaching
Institute of Brewing in Berlin (VLB); 1990-1998: Head of the Research
Department/Plant Production and Physiology, Weissheimer Malzfabrik, Andernach;
since 1999: Head of the Research Institute of Raw-Materials within the Research an
Teaching Institute of Brewing in Berlin (VLB).
Reverol-Viloria, Lorena (contribution nr. 48, 61)
Born in 1969.
Studies: BSc in Biology at the Central University of Venezuela,
Caracas, Venezuela (1994). Since 2000: undergraduate student at
the Dept. of Food Science, Simon Bolivar University.
Appointments: 1994-1995: Research Assistant in the Laboratory of
Biochemistry, Simon Bolivar University; 1995-present: Assistant
Scientist at the Laboratory of Biotechnology, Polar Technology
Center. Areas of expertise included protein purification, protein
sequencing, bacterial and yeast culture, cell transformation, DNA sequencing, cloning
and a variety of laboratory techniques such as PAGE, PCR, FPLC, HPLC, etc.
Experience and special training on microorganism isolation and culture, microbial
identification and food preservation.

Rezessy-Szab, Judit (contribution nr. 43)

Born in 1943.
Studies: Dipl. Chemical Engineer at the Technical University
Budapest, Hungary (1968); Economical Engineer at the Technical
University Budapest (1975); Dipl. Ing. in Biotechnology at the
Technical University Budapest (1987); Dr.univ. (1984): Study of the
decay of Candida utilis yeast by interference microscope.
Appointments: Assistant (1968-1971), Head of Fermentation pilot
plant (1971-1979) at the Factory of Pharmaceutical and Chemical
Products Chinoin; 1979-1983: Research Fellow at the Microbiological Research
Group of Hungarian Academy of Sciences; Research Fellow (1983-1989) then Head
of Bioengineering Section (1989-1991) at the Central Food Research Institute,
Budapest. Since 1991: Lecturer of the Department of Brewing and Distilling at the
Szent Istvan University (formerly University of Horticulture and Food Industry).
Research activities: physiology of micro-organisms, improvement programs for
breeding of microbes and optimisation of media composition, control of fermentation
processes and study of different fermentation systems and development of
fermentation technology.

Ridder, Marjan, de (contribution nr. 60)

Born in 1976.
Studies: Academic degree in Industrial Engineering - Biochemistry at
the KaHo St. - Lieven, Gent, Belgium (1998).
Appointments: 1998-present: Assistant Scientist in Malting and
Brewing Science at the Laboratory of Enzyme and Brewing
Technology of the KaHo St.-Lieven
Research topics: foam stability, high tech hopping.

Righelato, Renton (contribution nr. 3)

Born in 1943.
His training as a Microbiologist at Bristol and London was followed
by a research career spanning the food, drink and pharmaceutical
industries. After a post-doctoral fellowship at the Dutch Institute of
Public Health, he moved to Glaxo, where he led their fermentation
research teams in Cumbria in the UK. He then joined Tate & Lyle,
the multinational food and agribusiness group, as Manager of

2
Fermentation R&D and was appointed their corporate Director of Research and
Technology for from 1980 to 1989. From 1989-1995 he had his own company at the
University of Reading, focusing on research strategy and technology transfer in the
food chain. Much of their work was on nutrition and functional foods for
multinational companies, SMEs, European and UK government agencies.
In 1995 he became Director General of Brewing Research International, and led its
restructuring before he retired from that post in the summer of 2000. Amongst other
things, he championed nutrition research at BRI and he his colleagues worked with
the EBCs Technology and Engineering Forum to ensure the completion of the series
of EBC Manuals of Best Practice.
He has had many public appointments in the food arena in the UK and the EU. He is a
visiting professor at the University of Reading and is currently a consultant, working
on food chain and conservation issues working with the UKs Ministry of Agriculture
Fisheries and Food and food and drink companies.

Robert, Paul (contribution nr. 19)

Doctor in Chemistry.
Engineer in research at I.N.R.A. (Nantes, France). Specialist in vibrational
spectroscopy and physico-chemistry of macromolecules.

Rodrigues, Jos A. (contribution nr. 84)

Born in 1964.
Studies: Degree in Chemistry (1986) and PhD in Analytical Chemistry at the Faculty
of Science of Porto University, Portugal (1998).
Appointments: 1987: Assistant at the Chemistry Department of Faculty of Science of
Porto University; 1998: Lecturer at the Chemistry Department of Faculty of Science
of Porto University. Member of the Portuguese Society of Chemistry and Portuguese
Electrochemical Society.

Rodrigues, Pedro M. Gonalves (contribution nr. 84)

Born in 1973.
Studies: 1998: Degree in Biochemistry, Specialisation in Food Industries, at the
Faculty of Science of Porto University, Portugal. Since October 1999: doctoral
student at the Faculty of Science of Porto University.
Appointments: 1996 to 1997: Specialisation training in Food Industries, at
Electroanalysis Laboratory- Chemistry Investigation Centre of Porto University; since
1997: Scientist associated to the project A contribution to the improvement of the
quality control on the process of brewing (Grant for Young Scientists- Initiation to
Scientific Investigation) at the Faculty of Science of Porto University and UNICER-
Bebidas de Portugal, S.A.; since 2001: Scientist associated to the project
Determination of -ketoacids and -diketones as products of cellular metabolism
at the Faculty of Science of Porto University and UNICER- Bebidas de Portugal,
S.A.; since 1999: studies are being conducted as invited scientist at UNICER-
Bebidas de Portugal, S.A., as part of doctorate investigation; since 1998: Junior
Consulter for Food Quality/Safety Assessments (HACCP strategy, food processing,
food technology) at ERGOMOL, Lda.

3
Roelens, Frederik (contribution nr. 7)
Born in 1977.
Studies: Pharmacist, Ghent University, Belgium (2000).
Appointments: since 2000: Assistant at the Laboratory of
Pharmacognosy and Phytochemistry, Faculty of Pharmaceutical
Sciences, Ghent University; PhD project on Investigation of the
Bioactivity Spectrum of the Phytoestrogen from Hops, 8-
Prenylnaringenin, and Semi-synthetic Analogs.

Rogers, Peter J. (contribution nr. 52, 106)

Is the Manager of New Technology within Operations at Carlton and
United Breweries, Australia. He has been with the company since
1997. Prior to joining the Fosters Brewing Group, he was a member
of the Faculty of Science at Griffith University. He worked closely
with the Queensland meat industry for several years to launch a local
manufacturing industry for the production of high valued commodity
proteins such as purified bovine blood proteins. He is an Adjunct Professor at Griffith
University and is a member of the Industry Advisory Board of the University of
Melbourne. He completed his postgraduate studies at the Australian National
University in Canberra, in developmental biology specialising in yeast differentiation
and has worked at the Max Planck Institute for Experimental Medicine, UCSF, at the
BHP Melbourne Research Laboratory, at various times Melbourne University,
Monash University, the Australian National University, and at various major meat
works including South Burnett Pastoral, and Tancred Brothers. He is on the Board of
the Malting Barley Quality Improvement Program, and the Advisory Process Panel of
Brewing Research International. He is a member of the Australian Industry Research
Group, the IGB, the Master Brewers Association of America, the American Society of
Brewing Chemists, the American Institute of Food technologists, and Australian
Society of Biochemistry and Molecular Biology and the Australian Biotechnology
Association. He is the immediate Past President of the Australian Biotechnology
Association.
He has a particular interest in value adding process development in the food and
beverage industry. He has a continuing interest in technical education. He is keen to
see greater communication and collaboration between companies like CUB which
operate in technically complex commodity markets and researchers-so as to develop
mutually beneficial strategic collaborations. He has initiated strategic relationships
with researchers at the University of New South Wales. Macquarie University, VUT,
Melbourne University and Griffith University.

Rong, Haojing (contribution nr. 7)

Born in 1972.
Studies: BSc in Pharmacy, Shanghai Medical University, P.R. China
(1994); MSc in Pharmaceutical Sciences, Ghent University, Belgium
(1997); PhD in Pharmaceutical Sciences, Ghent University (2000)
thesis on the phytoestrogenic properties of hops (humulus lupulus
l.).
Appointments: 1994-1996: Research Associate in Quality Control,
Bristol-Myers-Squibb, Shanghai, P.R. China; 1996-2000: Scientific Collaborator,
Ghent University, Faculty of Pharmaceutical Sciences, Laboratory of Pharmacognosy

4
and Phytochemistry; since 2001: Research Scientist in Pharmacokinetics and Drug
Metabolism, Amgen Inc., Thousand Oaks, California, USA.

Rouck, Gert, de (contribution nr. 58)

Born in 1974.
Studies: Academic degree in Industrial Engineering - Biochemistry at
the KaHo St. - Lieven, Gent, Belgium (1996).
Appointments: 1997-present: responsible for the pilot brewing plant
(2-4 hl) of the KaHo St. - Lieven; practical courses in malting and
brewing science; Assistant Scientist in Malting and Brewing Science.
Research topics: flavour stability, mixed fermentation, foam stability,
high tech hopping.

Ruddlesden, J. David (contribution nr. 41)

Born 1947.
Studies: BSc in Chemical Engineering, University of Birmingham,
UK (1968), MSc in Malting and Brewing Science, University of
Birmingham (1974). Diploma Institute of Brewing (1981).
Appointments: Process Plant Design Engineer, Hick Hargreaves
Bolton (1968-1970), Brewer, Bass Ireland (1970-1973). Various
production and project brewing posts, Bass, Runcorn, (1974-1990),
Process Design Engineer Bass (1990-1995). Product Support Manager, Bass Burton
(1995-).

Ryder, David (contribution nr. 71)

David Ryder is vice president brewing, research and quality assurance
for the Miller Brewing Company. He has served that position since
August 1995.
Ryder began his brewing career in England at Associated British
Maltsters. He then joined the South African Breweries Beer Division and
was later named director of research and development for that groups
brewing and malting concerns at the Delta Corporation, Ltd. Ryder was
subsequently international technical consultant with Artois Breweries in
Belgium, during which period he was also responsible for operations in parts of Africa and
China. He joined Miller Brewing Company as Director of Research in June 1992 and was
later named director of research and quality in February 1995.
Prior to joining the Miller Brewing Company, he was vice president, technical services at
J.E. Siebel Sons Co., Inc. in Chicago IL. He was also director of education for the Siebel
Institute of Technology.

5
Curriculum vitae of (co)authors - S

Sacy, Hubert (contrubtion nr. 1)

Has been Director General of ducalcool - a Quebec/Canada-based
not-for-profit organization devoted to alcohol education - since it
was founded eleven years ago. As such, he supervises all prevention,
information, education and communication campaigns, programs
and operations for both the general public and specific target groups.
He holds degrees in law, political science and French literature, as
well as a diploma in social marketing from the cole des hautes
tudes commerciales de Montral.
He is a Member of the Board of the International Council on Alcohol and Addictions
and the Chair of its Alcohol Education Section.
He also chairs the Social Responsibility Committee of the Canadian Association of
Liquor Jurisdictions.
He is guest lecturer on communication, public relations and social marketing at cole
nationale dadministration publique (the Quebec school of public administration), the
University of Montreal, the University of Sherbrooke, the University of Laval, and the
University of Quebec at Montreal (UQAM).

Sakamoto, Kanta (contribution nr. 14)

Born in 1968.
Studies: Agricultural Chemistry at the Graduate School of the
University of Tokyo, Japan (1994).
Appointments: since 1994 employed by Asahi Breweries, Ltd.: 1994-
1995: Member of the BCOJ Analysis Committee. Since 1998: Chief.
1998-1999: Guest Researcher at the University of Groningen in the
Netherlands. Since 2001: Scientist at Key Technology Research Laboratory.
Member of JSBBA, JSLAB.

Snchez, Beatriz (contribution nr. 47, 48, 61, 63)

Born in 1969.
Studies: BSc in Biochemistry (Cum Laude) at the Simon Bolivar
University, Venezuela (1992). PhD in Cell Biology at the Simon
Bolivar University (2001). Appointments: 1992-1996: Assistant
Scientist at the Laboratory of Biotechnology, Polar Technology
Centre; 1996-1998: Intern at the Dept. of Neurobiology, Stanford
University; Intern at Roche Bioscience, California, U.S.A.; since
1998: Associate Scientist at the Laboratory of Biotechnology, Polar Technology
Centre, Caracas.

Santos, Joo R.S. (contribution nr. 84)

Born in 1978.
Studies: Degree in Chemistry at the Faculty of Sciences of Porto University, Portugal
(1999). Since 1999: Masters student of Chemistry at the Faculty of Sciences of Porto
University.
Appointments: since November 1999: Scientist in Laboratory Electroanalisys IV -
Chemistry Investigation Centre of Porto University - in the ambit of Food Industries;
since February 2001, scholarship-holder BIC Bolsa Investigao Cientfica,
associated to the project Determinao de cetocidos e dicetonas como
produtos do metabolismo celular, at the Faculty of Sciences of Porto University.
Since April 2001: member of Sociedade Portuguesa de Qumica (SPQ).

Sasaki, Katsuya (contribution nr. 55)

Studies: Environmental Chemistry and Engineering at Graduate
School of the Tokyo Institute of Technology, Japan (1998).
Appointments: since 1998 employed by Asahi Brewery Ltd.: 1998-
1999: Foods & Pharmaceuticals R&D Laboratory as Assistant Chief;
1999-present: Brewing R&D Laboratory as Assistant Chief in
Brewing Science Section.

Schans, Maarten J. (contribution nr. 74)

Born in 1965.
Studies: MSc Biology at the University of Groningen, the
Netherlands, specialising in molecular biology and plant cell
biotechnology (1989).
Appointments: 1989-1999: Scientist Process Technology - Raw
Materials at Heineken Technical Services. Since 1999: Project leader
at Agro-NIBEM, Product Group Beverages at the TNO Nutrition and
Food Research Institute.

Scherer Holmblad, Erika (contribution nr. 61, 63, 64)

Born in 1972.
Studies: BSc in Chemistry at the Universidad Simn Bolvar, Caracas, Venezuela,
1996.
From April 1996 to the present she has worked as Assistant Scientist at the
Laboratory of Biotechnology, Polar Technology Center. Areas of expertise include
analytical chemistry, HPLC, GC, GC-MS and Capillary Electrophoresis. Has some
experience and training in sensory analysis of beer by tasting and by GC-
olfactometry, and has also worked in food and beverage chemical analysis.

Scherf, Hans-Rudolf (contribution nr. 6)

Born in 1937.
Studies: Biology, Chemistry and Physics at the University of Bonn, Germany; PhD
(1967).
Appointments: 1967-1993: Scientist in the Division of Toxicology and
Chemotherapy, German Cancer Research Center, Heidelberg; since 1993: Senior
scientist in the Division of Toxicology and Cancer Risk Factors, German Cancer
Research Center; since 1997: Member of the Workgroup Cancer Chemoprevention,
Division of Toxicology and Cancer Risk Factors, German Cancer Research Center.
Main research topics: induction of terminal differentiation in cultured human cells by
selected chemopreventive compounds.

2
Schmidt, Roland (contribution nr. 16)
Born in 1957.
1978-1984: Studies at the Technical University Munich-
Weihenstephan, Germany.
Since 1984: Head of Laboratory and Manager of Quality
Assurance at NATECO2 GmbH & Co. KG, Wolnzach.

Schneider, Jan (contribution nr. 22)

Born in 1970.
Studies: Graduate Engineer at the Faculty of Brewing and Food
Technology of the Technische Universitt Mnchen, Germany
(1991-1996).
Internships and Practical experience in breweries and malt factories:
Euler in Wetzlar, Ihring-Melchior in Lich, Bayerische Staatsbrauerei
in Weihenstephan and Gordon Biersch in San Jose / San Francisco
and at the in Mlzerei Sulzbach.
University: Doctorate (since January 1997), Assistant professor (since November
1997) at the chair of brewery installations and food packaging technology.

Scholderer, Joachim (contribution nr. 90)

Born in 1969.
Studies: Psychology (Free University of Berlin, Germany, 1996).
Appointments: 1997-1998: Research Assistant, Department of
Marketing, University of Potsdam, Germany; 1998-2000: Lecturer,
Department of Marketing, University of Potsdam; since 2000:
Assistant Professor, Centre for Market Surveillance, Research and
Strategy for the Food Sector (MAPP), The Aarhus School of
Business, Denmark. Member of Society for Risk Analysis (SRA),
Association of Consumer Research (ACR), European Marketing Academy (EMAC).

Sharpe, F. Richard (contribution nr. 72)

Obtained his first degree in Chemistry. He then studied for his PhD at
the Brewing Research Foundation where he investigated the
chemistry of beer flavour, hop oil and the extraction of hops with
liquid carbon dioxide. He joined Whitbread plc in 1979 and after a
twenty year career in science and technology left his position as
Director of Beverage Research and Development to join Brewing
Research International as their Technical Director.
He is the author of 61 publications and two patents. He is a Fellow of the Royal
Society of Chemistry, a Fellow of the Institute of Food Science and Technology and a
Fellow of the Institute of Brewing. He is Chairman of the Institute of Brewings
Analysis Committee, a member of the EBC Analysis Committee and a member of the
Heriot Watt Research Committee. He is a visiting Professor of Food and Beverage
Safety at the University of Luton and his science interests are flavour, hop chemistry,
biotechnology and beer foam.

3
Siddique, Rukhsana (contribution nr. 35)
Studies: BSc in Microbiology at Canterbury University, Kent, UK
(1996). MSc in Food Science & Biotechnology at Strathclyde
University, Glasgow (1997). She is currently undertaking an MPhil at
Oxford Brookes University investigating the acidification power test
and brewing yeast membrane integrity.
Research specialisation: 1996 at Canterbury University, Kent,
investigated the aspect of growth inhibition of Geotricum candidum against different
CO2 concentration; 1996-1997 at Strathclyde University, Scotland, investigated the
optimisation of incubation time and temperature used in immuno magnetic separation
methods to isolate E.coli:O157:H7 from water.

Siebert, Karl J. (contribution nr. 86, 87)

Born in 1945.
Studies: Biochemistry at Penn State University, U.S.A., B.S. (1967),
M.S. (1968), PhD (1970).
Appointments: from 1971-1989 employed by the Stroh Brewery
Company, Detroit: 1971: Research Associate, 1971-1973: Head,
Research & Development Section, 1973-1982: Manager, Research &
Development Laboratory, 1982-1989: Director of Research. Since
1970 employed by Cornell University, Geneva, NY: 1970-present: Professor of
Biochemistry, 1970-1975: Chairman of the Food Science & Technology Department
and Associate Director, Cornell Institute of Food Science. 1970-1976: Board
Member, Cornell Research Foundation. Member of ASBC (Technical Committee
member 1983-1989 & Chairman, 1986-1988, Journal Editorial Board 1980-1992,
1996-), MBAA, American Chemical Society, Institute of Food Technologists, and
International Chemometrics Society. Awards & Honors: National Science Foundation
Fellowship, 1967-1970. MBAA Presidential Award 1986 & 1990. Honorary
Professor, Moscow (Russia) State Academy of Food Production, 1996. ASBC Eric
Kneen Memorial Award, 1998 & 1999; ASBC Award of Distinction, 1999.

Simon, Karin (contribution nr. 16)

Born in 1972.
1988-1991: Apprenticeship laboratory assistant, Munich.
1994-1999: Studies at the Technical University Munich-
Weihenstephan, Germany; Dipl.-Ing. Foodtechnology (FH)
Since 2000: Research & Development, NATECO2 GmbH & Co. KG,
Wolnzach.

Smart, Katherine A. (contribution nr. 32, 35, 40)

Completed a BSc (Hons) in Biological Sciences at Nottingham
University, UK, and was awarded the Rainbow Research Scholarship
to complete a PhD in Brewing Yeast Physiology at Bass Brewers,
Burton-on-Trent. She then moved to Cambridge University to take up
an appointment as Research Fellow in the Department of Plant
Sciences where she worked on bioactive surfaces, biofouling and
bacterial contamination of beverages. In 1992 at the age of 25, she
became a Lecturer in Microbiology and Fermentation at Oxford Brookes University.
Now the Scottish Courage Reader in Brewing Science and a Royal Society Industrial

4
Fellow, she has a Research group comprising two postdoctoral researchers, five PhD
students and one MPhil student. She has published extensively in the brewing press
was awarded the Institute of Brewing Cambridge Prize in 1999. She serves on the
IGB Technical and International Committees. She is a member of the Editorial board
for the Journal of the ASBC, and is a member of the ASBC programme committee.
Her main research interests include brewing yeast handling, stress responses, ageing
in Saccharomyces cerevisiae and brewing microbiology.

Spillmer, Frank (contribution nr. 28)

Born in 1966.
Appointments: 1999: Beck & Co, Germany, Research and
Development, Consultant for automatic Data Collection.

rogl, Ji (contribution nr. 59)

Born in 1937.
Studies: Institute of Chemical Technology (ICT), Prague, Czech
Republic (1960).
Appointments: 1960-1963: Head of Control Laboratory of Nord
Moravian Breweries; 1963-1967: Assistant at Department of
Fermentation chemistry and Technology ICT; 1967-1997: Head of
Research Laboratory of Pilsner Urquell Brewery, Pilsen; since 1997:
External Adviser of Pilsner Urquell Brewery, Pilsen, Research
Department in Prague of Research Institute of Brewing and malting, Prague.

Stewart, Graham G. (contribution nr. 34, 36)

Is the Director and Professor of the International Centre for Brewing
and Distilling, Heriot-Watt University, Edinburgh, Scotland. He
received his BSc Hons, Microbiology and BSc Hons Biochemistry
from the University of Wales at Cardiff, and PhD and DSc degrees
from Bath University. He was a lecturer in Biochemistry in the
School of Pharmacy at Portsmouth College of Technology (now
Portsmouth University) from 1967 until 1969. From 1969 to 1994 he
held a number of technical positions with Labatts in Canada and from 1986 to 1994
was Director of Brewing Technical Affairs for John Labatt Ltd. He became a Member
of the Institute of Brewing in 1969, was elected a Fellow in 1987, and was the
Institutes President 1999-2000. He is also a Member of the MBAA, the ASBC and
the Institute of Brewing Studies. He has recently been elected the International
Director of the ASBC. He holds Fellowships in the Institute of Biology and the
American Academy of Microbiology. He is a 1983 and 1998 recipient of the Master
Brewers Association of the Americas Presidential Award. In addition to co-authoring
and editing a number of books, he has published over 200 original papers, patents and
reviews.

5
Stigter, Edwin (contribution nr. 74)
Born in 1968.
Studies: BSc Biotechnology at the Hogeschool Rotterdam en
Omstreken, the Netherlands (1994).
Appointments: 1994-1996: Technician in Biochemistry at the TNO
Nutrition and Food Research Institute; since 1996: Senior Technician
in Biosensor research at the same Institute.

Storey, Kim (contribution nr. 33)

Studies: BSc Microbiology, Kings College, University of London, UK
(1990-1993).
Appointments: Microbiology Technician (Environmental Water),
Robens Institute (1993-1995); Fermentation Scientist, Brewing
Research International (1995-1999); Fermentation/Instrumentation
Technician, University of Westminster (1999-date).

Storgrds, Erna (contribution nr. 93)

Studies: Microbiology at the Helsinki University, Finland, MSc 1985,
PhD 2000.
Appointments: 1983-1984: Production Hygienist at the
Pharmaceutical plant of Orion Yhtym Oy; 1987-1988: R&D Scientist
at the Laboratory of Genetechnology, Orion Yhtym Oy; 1988-2000:
Research Scientist at VTT Biotechnical Laboratory; since 2000:
Senior Research Scientist at VTT Biotechnical Laboratory. Member
of EBC Microbiology Group, later the EBC Brewing Science Group
since 1992; Chairman of the Detection of Contaminants Subgroup since 1993; Vice
chairman of the Finnish Brewmasters Association since 1997; Member of the
Microbiology Subcommittee of the EBC Analysis Committee since 1998.

6
Curriculum vitae of (co)authors T

Taidi, Behnam (contribution nr. 31)

Is currently in charge of promoting and progressing the strategic
research programme at Scottish Courage Brewing trough appropriate
use of internal and external resources. Behnam, a biochemist, has
been involved in brewing research and innovation since 1993 when,
having obtained his PhD in the area of Microbial Physiology and
Biochemistry joined Brewing Research International. Later on, as a
Fermentation Team Leader, he was in charge of yeast and
fermentation research at BRi.
He joined SCBL in 1996 and has since been involved and responsible for various
yeast, fermentation and microbiology projects, laboratory and site audits and a
number of non-core responsibilities. As a Process Project Team Leader he has also
been in charge the Pilot Brewing facility at SCBL. He has been a lecturer to the IOB
Brewster and International courses and is currently a lecturer to the extensive Scottish
Courage internal training courses for the IOB Associateship Examinations. He
regularly attends international scientific and brewing meetings where he normally
presents aspects of his work.

Takashio, Masachika (contribution nr. 70)

Born in 1946.
Studies: Agricultural Chemistry (Fermentation) at the University of
Tokyo, Japan (1971).
Appointments: 1971-1976: Research Scientist at Brewing Research
Laboratories of Sapporo Breweries Ltd.; 1976-1981: Research
Scientist at the Institute of Microbial Chemistry (Tokyo); 1980-1988:
Senior Research Scientist at R&D Laboratories of Sapporo Breweries
Ltd.; 1989-1993: Chief Representative of Sapporo Breweries Ltd. to
New York, USA; 1994-1997: General Manager of Brewing Research Laboratories.
1998-2000: Deputy-director; since 2001: Director. Since 2001: Board Member of
BCOJ. Member of Japan Society of Bioscience, Biotechnology and Agrochemistry,
Brewing Society of Japan, ASBC, New York Academy of Sciences, and others.

Takeuchi, Toshihiko (contribution nr. 39)

Born in 1953.
Studies: Agricultural Chemistry at the Nagoya University, Japan
(1975).
Appointments: 1993-1997: Brew master of the Shiga plant, Kirin;
1997-2000: Manager of the Brewing Group at the Research
Laboratory for Brewing; since 2000: Deputy Plant Manager of the
Tochigi plant, Kirin.
Tapani, Kaisa-Maija (contribution nr. 49)
Born in 1968.
1991-1998: MSc in Microbiology at the University of Helsinki,
Finland; 1995-1996: Pro Gradu thesis, VTT Biotechnology and
Food Research: Influence of lactic acid bacteria on the toxigenic
Fusarium fungi in malting; 1996-1997: Research Assistant at VTT
Biotechnology and Food Research; since 1998: Process Researcher
at Oy Sinebrychoff Ab.

Threapleton, Lee (contribution nr. 89)

Born in 1957.
Joined Bass Brewers as QA technician in 1977. Operated the Tower
Brewery tasting facility in Tadcaster from 1979-1982 and recruited
and trained the first internal brewery Flavour Profile panel. Gained a
First in chemistry with the Open University in 1990. He has held a
number of technical and production roles in the company, analytical
chemist, QA Manager and Brewery Production Manager at Cannon
Brewery in Sheffield. In 1998 recruited as Sensory Manager at the
Bass Technical Centre in Burton on Trent. In this role he is responsible for the
development of the Bass Expert Panel and Company sensory training and provides
sensory information for Production, Marketing and New Product Development.

Thum, Bernhard (contribution nr. 15)

Born in 1961.
Studies: Food Chemistry at the Friedrich-Alexander-University of
Erlangen-Nrnberg, Germany (1990).
1990-1991: Trainee at the Regional Office for Health Investigation of
North Bavaria; 1991-1992: Analytical Research at the Institute for
industrial and social medicine of the Friedrich-Alexander-University
of Erlangen-Nrnberg; 1992-1996: Doctoral thesis about Model
investigations about formation of carbonyl compounds during storage of beer at the
Chair for Brewery Technology and Beverage Microbiology of the Technical
University of Munich-Weihenstephan; 1997-2000: Assistant professor at the Chair for
Brewery Technology and Beverage Microbiology of the Technical University of
Munich-Weihenstephan. Areas of Research: Analytical methods for the evaluation of
beer flavour; Flavour thresholds of beer odorants and influence of additive or
competitive effects to beer aroma; Impact of raw materials and brewing technology on
beer flavour; Changes in the flavour profile during storage of beer.
Since 2001: Lecturer in Chemistry and Analytics at the Municipal School for dental
techniques, chemistry, biology and drugstore professionals; development and
introduction of new curriculum for laboratory technicians with main focus in
instrumental analytics.

Thurston, Patrick (contribution nr. 40)

Has worked in the Brewing Industry for over 20 years. He currently works for
Scottish Courage Ltd. as Regional Production Support Manager (South). In this role
he is responsible for all Quality Assurance, Quality Systems, Operational
Improvement and some capital project activities at the Berkshire Brewery.

2
Previously he has worked for both Scottish Courage Ltd. and Courage Ltd. as
Franchise & Process Control Manager in which he set up a number of overseas
licence brewing/ packaging operations. Prior to this he held a number of posts with
Grand Metropolitan Brewing (GMB) including QA Manager Stag Brewery and Group
Microbiologist.
Before working for GMB, he obtained his PhD in yeast lipid metabolism whilst
studying at BRi and Bath University. He is the author of several scientific
publications.

Tigel Gil, Rafael (contribution nr. 24)

Born in 1951.
Studies: Engineer in Agronomy at the Universit Catholique de Louvain (UCL),
Belgium (1979); Special Diploma on Brewing at the Universit Catholique de
Louvain (1980).
Appointments: 1980-1983: Research Engineer at the Brewing Department of UCL;
1983-1986: Research Engineer / University Assistant in the Process Department of
UCL; since 1986: Research and Development manager of Meura s.a. (Director of
Meura Technologies).

Todd, Robert W. (contribution nr. 79)

Born 1951.
Studies: Electronic Engineering at University of Southampton (UK)
and PhD on Adaptive Control Techniques in Prosthetics.
Appointments: Joseph Lucas Lecturer at University of Southampton
for 5 years; Technical Director of the Centre Of Alternative
Technology in Machynlleth Mid Wales; Chairman and Technical
Director of Dulas Engineering (UK); 1988 to present Research and
Development Director of Aber Instruments Ltd (UK).
Received Ambrose Flemming Award of the Institute of Electrical Engineers in 1974.
Published over 40 papers and has been named as inventor on six instrumentation-
related patents.

Turner, Kirsty (contribution nr. 78)

Studies: Gained a BSc (Hons) Microbiology degree from the
University of Leeds, UK (1993). In 1994 undertook a PhD at the
University of Liverpool investigating the starvation stress responses
of Salmonella typhimurium.
Appointments: 1993-1994: Research Scientist at CAMR, Porton
Down; 1999-2000: Research Scientist with the National Institute for
Biological Standards and Control; 2000-present: Microbiologist with
Aber Instruments Ltd.

3
Curriculum vitae of (co)authors U / V

Ueda, Tsutomu (contribution nr. 55)

Studies: Biotechnology at the Osaka University, Japan (1992).
Appointments: since 1992 employed by Asahi Brewery Ltd.: 1992-
1995: Fukusima brewery as mashing section staff; 1995-1997: Asahi
Beer Malt Ltd. as Malting Supervisor; 1997-1999: Brewing R&D
Lab as Chief in Quality Assurance Section; 1999: Brewing Research
International (BRi) as a visiting researcher; 1999-present: Brewing
R&D Lab as Chief in Brewing Science Section; since 1998: Malt
specialist in Asahi Research and Development Center.

Veen, Sjaak J.F., van (contribution nr. 74)

Born in 1955.
Studies: 1983 MSc Physical Chemistry, University of Amsterdam,
the Netherlands.
Appointments: 1983-1986: Lecturer Pharmaceutical Analysis,
University of Utrecht; 1986-1997: Group leader of the Sensor Group
at the TNO Nutrition and Food Research Institute; 1997-present:
Product Manager Sensor Technology Group.

Vehkomki, Maija-Leena (contribution nr. 47, 61)

Born in 1959.
Studies: MSc (Genetics) at the University of Oulu, Finland (1987).
Appointments: since 1989 Research Scientist at the VTT
Biotechnology. Presently working on molecular biology of brewer's
yeast.

Vereijken, Tom L.F.M. (contribution nr. 108)

Studies: BA in Food Technology, Friesland College of Food
Technology, (1981), MBA from Universities of Nyenrode (NL) and
Rochester (USA) (1999). Honorary Award for Technological
achievement by University of Valencia (1992).
Appointments: Bavaria N.V. (1981); Process Specialist Paques B.V.
(1987); Head of Technology Department Paques B.V. (1993);
Branche Manager (1996). Active in implementation of environmental
biotechnologies in industrial applications. Elected Chairman of the
European Committee of Environmental Technology Suppliers Associations
(EUCETSA) in October 2000.
Vidgren, Virve (contribution nr. 47, 61)
Born in 1970)
Studies: MSc (Genetics) at the University of Helsinki, Finland.
(1998).
Appointments: since 1998 Research Scientist in the metabolic
engineering group at the VTT Biotechnology.

Viljava, Tapio (contribution nr. 49)

Born in 1955.
Studies: Chemical Engineering in Helsinki University of Technology,
Finland, MSc 1980.
Appointments: Acting teaching assistant (Industrial Chemistry) in
HUT 1980; Research engineer, Teknos Paint Ltd. 19801984; since
1984 different research positions in the Research Center of Finnish
Sugar Co., later Cultor Ltd. and further Danisco Corp., presently
Project Manager in the Danisco Sugar & Sweeteners Development Center, Kantvik,
Finland.

Vilpola, Arvi (contribution nr. 47)

Born in 1955.
Studies: Process Engineer at the Technical High School of Tampere,
Finland (1979).
Appointments: since 1980: Research Engineer (malting and
brewing) at the VTT Biotechnology.

Virant, Majda (contribution nr. 18)

Born in 1949.
Studies: Chemical Technology at the Univestity of Ljubljana, Faculty
of Natural Science and Technology (1971).
Appointments: 1972-1990: Head of the Laboratory at the Institute of
Hop Research and Brewing alec; since 1990: Head of the
Department for Brewing at the Institute of Hop Research and
Brewing alec; 1985-1990: Member of the Editorial board of the
professional journal Beer Brewing Edited by Beer and malt industry of Yugoslavia -
Business association); since 1992: member of Technical Association of the Standards
and metrology institute at Ministry of Science and Technology; since 1993: Chair
person of technological Commission and member of Editorial board of Beer Brewing
Bulletin of the Association of Slovene Brewers; since 1993: member of professional
group preparing Regulations on beer at the Ministry of Agriculture, Forestry and
Food, Republic of Slovenia; since 1996: member of the Commission for preparing
criteria for laboratories for official testing at the Ministry of Agriculture, Forestry and
Food, Republic of Slovenia; since 1997: Member of the EBC Analysis Committee
and EBC Barley & Malt Committee.

2
Virtanen, Hannele (contribution nr. 47, 61, 64)
Born in 1945.
Studies: MSc (organic chemistry and biology) at Helsinki University,
Finland (1974).
Joined VTT Biotechnology in 1986. Presently working in the Flavour
Group, main field GC analyses.

Voetz , Michael (contribution nr. 11)

Born in 1964.
Studies: Biology at the Johannes Gutenberg University, Mainz and
Molecular Biology and Genetics at the University of Cologne,
Germany (1985-1990).
1990-1995: Max-Planck-Institute for Plant Breeding Research, Dept
Genetic Principles of Plant Breeding (Prof. Jeff Schell), PhD 1995.
1994: DAAD grant for scientific work at the Oregon State
University, U.S.A., Dept of Crop & Soil Sciences.
1995-2000: scientific collaborator at the Research Department/Biotechnology,
Weissheimer Malz, Andernach; since 2000: head of the biotechnology/PCR
laboratory at the Research Institute for Raw-Materials within Research and Teaching
Institute of Brewing in Berlin (VLB).

Voigt, Tobias (contribution nr. 99)

Born in 1973.
Studies: Diploma Engineer at the Faculty of Brewing and Food
Technology of the University Munich-Weihenstephan, Germany
(1993-1999). Internships and Practical experience: Gentner Brewery,
Wolframseschenbach/Mittelfr; Spaten-Franziskaner Lwenbru
Brewery, Munich; Gordon Biersch Brewing Company, San Jose,
(CA) USA.
University: PhD-Student, Research Associate (since April 1999) and Assistant
professor (since March 2001) at the chair of brewery installations and food packaging
technology, Technische Universitt Mnchen/Weihenstephan.

Vundla, W., (contribution nr. 42)

3
Curriculum vitae of (co)authors W

Waesberghe, J.W.M., van (contribution nr. 58)

Born in 1937.
Studies: MSc degree in Tropical Agriculture, Food Technology and
Economics at Wageningen University, the Netherlands (1960).
Appointments: served several multinationals as Research Manager or
Technical Director. Brewing, Winemaking, Malting, Distilling, Tea
and herb processing, Soy processing, Sorghum processing, Solid-
state fermentation, Fruit products, juices and soft drinks.
Since 1975: self-employed as an independent consultant to the food and beverage
industries. Specialized in upstream processing to control flavour-stability. In
brewing involved in the conceptual engineering of maltings (to ensure micro flora-
management and LOX-management) and brew houses (new approaches to milling,
mash-separation-techniques, de-intensified wort boiling and forced volatile-stripping).

Wagner, Florian (contribution nr. 23)

Born in 1972.
Professional Training as a Brewer at Brewery "Hochstiftliches Brauhaus Motten-
Bayern," Germany (1992-1994).
Studies: Brewing and Beverage Technology at the Munich-Weihenstephan University
of Technology (1995-2001). Engineer of Brewing and Beverage Technology at
Brewery "Hochstiftliches Brauhaus Fulda", Germany (2001).

Walker, Caroline J. (contribution nr. 4, 8, 9)

Is a specialist in the area of Biochemistry and Cereal development
with wide international research experience. Over the last 15 years
she has conducted research at Bristol University (U.K.), U.C. Davis
and Clemson University, SC. She was also a fellow at the Carlsberg
Laboratories in Copenhagen where she worked on characterizing
mutant barley varieties. Currently she is a Principal Scientist at BRi
and leads research in the area of wholesomeness. Her interests are in investigating the
healthful aspects of beer and maximizing the potential of raw materials to contribute
to its nutritional value.

Walsh, Padraig K. (contribution nr. 88)

Studies: B.E. National University of Ireland, Dublin; M.S. University
of Missouri-Rolla, USA; Ph.D. National University of Ireland,
Dublin.
Appointments: 1984-present: Lecturer, School of Biotechnology,
Dublin City University; 1999-2000: Sabbatical year at Heriot-Watt
University, Edinburgh. Associate Member of the Institution of
Chemical Engineers.
Watts, Kelly (contribution nr. 71)
Obtained a BS degree in Microbiology from The University of Florida,
USA. She also holds a BS degree in Medical Technology from Florida
Atlantic University, USA. She continued her secondary studies at The
University of Florida, USA, where she obtained her doctorate in
Molecular Biology and Microbiology. In 1998, she joined Miller
Brewing Company as a Research Biochemist working in The Cereal
Chemistry Department.

Wehrenberg, Horst (contribution nr. 28)

Appointments:
Nord IT - Management of Nord IT.

Weisser, Horst (contribution nr. 22, 96, 99)

Born in 1938.
Studies: Chemical Engineering BSc 1960, Food Engineering MSc
1962 at Universitt Karlsruhe, Germany (Uni-KA). PhD (Dr.-Ing.)
1972 in Food Process Engineering at Uni-KA.
Appointments: Universitt Karlsruhe: Scientific Assistant, Institute
of Food Process Technology, Uni-KA 1962-1972. Lecturer, 1973-
1974; Assistant Professor 1974-1987 Institute for Food Process
Engineering, Uni-KA. Technische Universitt Mnchen in Freising-Weihenstephan:
Professor and Head, Chair for Brewery Installations and Food Packaging Technology
1987-present.
Research: 1. In the field of brewery plant: operational data acquisition in beverage
factories, especially breweries, design of filling plant, storage technology, membrane
filtration of beer mash. 2. In the field of packaging technology: processes of aseptic
packaging, diffusion processes (water vapour, oxygen) in food and packages, fracture
mechanics of glass and glass packages, closing and sealing technology of beverages
containing CO2, ecological effects of various packaging systems, freezing and
thawing behaviour of food.
Membership: EBC Working Group on Process Technology; Technology Group
(Technischer Ausschu) of Deutscher Brauer-Bund. Author and co-author of 160
publications on food engineering, food analysis, food packaging and computer aided
processing.

Wheaton, Lynne K. (contribution nr. 73)

Born 1967.
Studies: Degree in Chemistry at Kingston Polytechnic.
Appointments: since 1987: employed by Brewing Research
International as a Scientist.

2
Wiggins, David R. (contribution nr. 95)
He is a graduate in Packaging Technology he has spent the past 22 years working in
Packaging Development in a wide range of industries. He has practical experience in
Food, Toiletries, Cosmetic and Pharmaceutical packaging and is currently the Head of
Packaging Development at Bass Brewers where he is responsible for the development
and implementation of packaging and graphics projects as well as packaging
innovation.

Wilde, Peter J. (contribution nr. 68)

Studies: BSc in Biophysics at the University of East Anglia, Norwich
UK (1985), PhD Interfacial mechanisms underlying the stability of
protein stabilised foams and emulsions, Institute of Food Research
and University of East Anglia (2000).
Appointments: 1985-1999: Research Scientist, Institute of Food
Research, Norwich, UK; 1999-present: Senior Research Scientist,
Institute of Food Research. Since 1996: Member of EBC Foam sub-
group.

Wirtanen, Gun (contribution nr. 93)

Born in 1961.
Studies: Chemical Engineering at the Helsinki University of
Technology, Finland (MScTech, 1988; DTech, 1996).
Appointments: 1986-1987: Research Trainee at VTT Food Research
Laboratory; 1988-1993: Research Scientist at VTT Food Research
Laboratory; 1994-1997: Research Scientist at VTT Biotechnology and
Food Research; 1997-1999: Senior Research Scientist at VTT
Biotechnology and Food Research; Senior Research Scientist since 2000 and Group
Manager since 2001 at VTT Biotechnology.
Member of the board of the R3-Nordic Association on Contamination Control since
1999 and of the board of the Food Hygiene Group of the Society of Food Researchers
since 2000.

Witters, An (contribution nr. 60)

Born in 1974.
Studies: Academic degree in Industrial Engineering - Biochemistry
at the KaHo St. - Lieven, Gent, Belgium (2000).
Appointments:
2000-present: Quality Manager and Technical Responsible in the
Laboratory of Food Science and Technology at the University of
Gent.

Wolfe, Caroline (contribution nr. 4)

Is a post-doctoral Research Scientist in the Division of Nutrition,
Health and Consumer Sciences at Institute of Food Research, UK.
She gained her degree and PhD in Biochemistry at the University of
Essex, specialising in the biophysics of cell membranes. She joined
the IFR in 1995 to work on the effect of diet on the functionality and
fertility of spermatozoa, using fluorescence techniques. She then
conducted research in the field of microbiology, looking into the

3
growth behaviour of Listeria monocytogenes. For the past two years her current
research has been investigating the absorption and metabolism of synthetic and
naturally occurring folates in humans using stable isotope techniques.

Woollard, Tom (contribution nr. 103)

Born 1964.
Education: BSc Ecology, St Andrews University, PhD Ecology,
Bristol University, UK.
Professional Affiliations: Registered Principal Auditor (IEMA), Lead
Assessor for ISO 14001
Synopsis: Is Director of Corporate Advisory Services (CAS) - a team
of 15 consultants specialising in Strategy and Communications, EHS
Management Systems and Corporate Social Responsibility. His team
advises FTSE and Fortune 100 companies on a variety of strategy and implementation
projects globally.
His core expertise lies in auditing, EHS Management Systems, Reporting and Report
Verification and strategic advice. He has conducted over 400 EHS due
diligence/compliance audits of manufacturing facilities in Europe, Eastern Europe,
Russia, China and the Former Soviet Republics, Australia and Central America. He
has directed and managed over 50 EHS Management Systems assignments and was
responsible for developing a new service for ERM called EHS Systems
Streamlining which reduces EHS risk whilst at the same time improving the
operational efficiency of major manufacturing sites and companies. EHS systems
streamlining delivers systems which are lean, integrated with core activities and
focused on reducing risk and maximising performance.
In 1997 he helped to establish ERMs ISO 14001 Certification Body (ERM CVS) by
training more than 100 ERM consultants worldwide. He has certified sites for Bristol
Myers Squibb and Baxter Healthcare in the UK, Italy and Russia.
He has designed and presented over 50 training courses on EHS and CSR issues. His
clients include, P&O, TRW, ABB, IUCN, EBRD, Unilever and ITT.
He as verified Corporate Environmental and Sustainability reports for Glaxo
Wellcome, Body Shop, Bass, BP, General Utilities, Tetra Pak, Orange and has helped
numerous companies gather data and generate public reports. More recently he has
provided strategic advice covering Reputation Risk, Socially Responsible Investment
(SRI) and internal assurance to a variety of major FTSE 100 companies. Strategy
clients have included, Anglo American, Rio Tinto, M&S, Bass Brewers, Prudential
and Railtrack.
He has provided expert witness on public reporting to the House of Commons Audit
Committee. He contributes to ACBE Committees on sustainable development and
SRI and regularly gives presentations to conferences in the UK and Europe.
He has written a variety of scientific papers on ecological issues and has contributed
to numerous management papers and trade and broadsheet articles on Auditing,
Management Systems, Corporate Reporting, Reputation Risk, SRI, Rating Agencies
and Green Funds.

4
Wright, Anthony J.A. (contribution nr. 4)
Has accumulated diverse biochemical experience over the 34 years
that he has worked for the Institute of Food Research (Norwich, UK).
This has included, in chronological order, the isolation and
characterization of membrane structures from plant tissue, the
analysis of total vitamin folate in foods by microbiological assay,
investigation of factors affecting the regulation of non-haem iron
absorption by mucosal epithelial cells, relationships between nutrient
intake and biochemical markers of status in humans, and the bio-
availability of organic micro-components of fruit and vegetables. Currently employed
as a Senior Research Scientist, his present interests includes red cell folate analysis &
folate 1-carbon metabolism.

5
Curriculum vitae of (co)authors Y / Z

Yasuda, Y. (contribution nr. 39)

Born in 1972.
Studies: Food Chemistry at the Ohtsuma University, Japan (1993).
Appointments: since 1993: Researcher in the Research Laboratory
for Brewing, Kirin.

Zandycke, Sylvie M., van (contribution nr. 35)

Born 1973.
Studies: studied Biochemical Engineering and Fermentation at the
Institut Meurice (Brussels, Belgium), she completed her degree in
September 1996. During that time, she obtained an Erasmus
studentship for a 6-month project on brewing yeast cell ageing at
Oxford Brookes University, UK. She obtained her PhD on The Role
of Catalase and Glutathione on Replicative Lifespan in Saccharomyces cerevisiae in
July 2000 at Oxford Brookes University.
Appointments: since March 2000 she is employed as Project Manager for Smart
Brewing Services at Oxford Brookes University. She is involved in contract research,
microbiological analysis, development of methods and kits for the brewing industry.
Her main research interests include ageing in Saccharomyces cerevisiae, brewing
microbiology and yeast quality.

Zangrando, T. (contribution nr. 26)

Born in 1933.
Studies: Brewing Technology in Weihenstephan, Germany, 1959.
Appointments: 1960-1998: Brew master and Manager of various Italian breweries.
1962-1980: Member of the EBC Analysis Committee. Since his retirement he works
as Brewing Consultant and Editor of the Italian brew technical magazine Birra e
Malto.

Zanker, Mag. Gerald (contribution nr. 81, 107)

Born in 1955.
Studies: 1980 -1982: College for Chemistry at the Fachhochschule
fr Chemotechnik in Graz, Austria; 1986-1993: Study of Chemistry
at the Grazer Karl Franzens-University.
Appointments: Senior Assistant in the central laboratory of the
Steirerbrau AG (1982-1988), Head of the Steirerbrau laboratories
(1988-1990), research work until 1998, Head of the laboratory
Puntigam, since 1998 in the Brauunion sterreich. Since 1984: Member of NAK
(Nationales Analysenkomitee sterreich); since 1996: Member of MEBAK.
Zrcher, Achim (contribution nr. 54)
Born in 1969.
Apprenticeship to a Brewer and Maltster (1990-1992) at the Htt-
Brauerei, Baunatal and at the Darmstdter Privatbrauerei, Darmstadt,
Germany. From 1992 to 1998: study of Brewing and Beverage
Technology at the TU Munich-Weihenstephan. Graduate Engineer
since 1998. Practical training at the Binding Brauerei, Frankfurt
(1992 and 1995) and at the Gabriel Sedlmayr Spaten-Franziskaner
Bru KGaA, Munich (1998). From beginning of year 1999 doctoral thesis at the
Lehrstuhl fr Technologie der Brauerei 1, Weihenstephan. Since 2000 head of the
GC- / HPLC-Laboratory of the Lehrstuhl fr Technologie der Brauerei 1,
Weihenstephan.

2
PARTICIPANTS EXHIBITION DENMARK
SCIENTIFIC INSTITUTES &
The Scandinavian School of Brewing
BREWING SCHOOLS
Gamle Carlsberg Vej 16
28 th EBC Congress, Budapest 2001
DK-2500 COPENHAGEN-VALBY
______________________________
tel. +45 33 272 400
AUSTRIA fax +45 33 272 401
e-mail [email protected]
sterreichisches Getrnke Institut contact person: Carsten Bryde Andersen
Berufsschule fr Brauer, Mlzer und
Destillateure FINLAND
Michaelerstrasse 25
VTT Biotechnology
A-1180 WIEN
P.O. Box 1500
tel. +43 1 479 69 24 12
FIN-02044 VTT
fax +43 1 479 69 24 11
tel. +358 9 456 5115
e-mail [email protected]
fax +358 9 455 2103
contact person: Dr. H. Schwarz
e-mail [email protected]
BELGIUM contact person: Silja Home

Institut Meurice FRANCE

Dept. of Brewing Science & Fermentation
ENSAIA
Technology
Institut National Polytechnique de Lorraine
Avenue E. Gryson 1
Ecole Suprieure dAgronomie et des Industries
B-1070 BRUSSELS
Alimentaires
tel. +32 2 526 7351
2 avenue de la Fort de Haye
fax +32 2 526 7301
B.P. 172
e-mail [email protected]
F-54505 VANDOEUVRE LES NANCY CEDEX
contact person: Alain Debourg
tel. +33 3 83 59 58 01
fax +33 3 83 59 58 04
Universit Catholique de Louvain
e-mail [email protected]
Unit de Brasserie et des Industries
contact person: Dr. Michel Fick
Alimentaires
Croix du Sud 2, bte 7
B-1348 LOUVAIN-LA-NEUVE IFBM / Qualtech
tel. +32 10 472 913 Ple Technologique de Nancy-Brabois
fax +32 10 472 178 7, rue du Bois de la Champelle
e-mail [email protected] B.P. 267
contact person: Guillaume Lermusieau F-54512 VANDOEUVRE CEDEX
tel. +33 3 8344 8800
CZECH REPUBLIC fax +33 3 8344 1290
e-mail [email protected]
Institute of Chemical Technology Prague contact persons: Jean Lavie & Franoise Lacour
Dept. of Fermentation Chemistry and
Bioengineering GERMANY
Technick 5
CZ-166 28 PRAHA 6 Doemens Academy
tel. +420 2 311 3278 Stefanusstrasse 8
fax +420 2 2435 5051 D-82166 GRFELFING/MNCHEN
e-mail [email protected] tel. +49 89 858 050
contact person: Jaromir Ftala fax +49 89 858 0526
e-mail [email protected]
Research Institute of Brewing and Malting contact persons: Dr. Fritz Schur & Dr. Fritz Briem
in Prague
Lpov 15 Technische Universitt Berlin
CZ-120 44 PRAHA 2 Fakultt III, Fakultt fr Prozesswissenschaften
tel. +420 2 2491 5384 Fachgebiet Brauwesen
fax +420 2 2492 0618 Seestrasse 13-15
e-mail [email protected] D-13353 BERLIN
contact person: Tomas Horak tel. +49 30 4508 0290
fax +49 30 453 3052
e-mail [email protected]
contact person: Dipl.-Ing. Andreas Ludwig
Technische Universitt Mnchen Heriot-Watt University
Lehrstuhl fr Technologie der Brauerei I International Centre for Brewing and
D-85350 FREISING-WEIHENSTEPHAN Distilling
tel. +49 8161 713 269 Riccarton
fax +49 8161 713 883 EDINBURGH EH14 4AS
e-mail [email protected] Scotland
contact person: Prof.Dr. W. Back tel. +44 131 451 3453
fax +44 131 449 7459
Versuchs- und Lehranstalt fr Brauerei e-mail [email protected]
in Berlin contact person: Dr. J.H. Bryce
Seestrasse 13-15
D-13353 BERLIN Institute and Guild of Brewing
tel. +49 30 4508 0291 33 Clarges Street
fax +49 30 453 6069 LONDON W1J 7EE
e-mail [email protected] tel. +44 20 7499 8144
contact person: Dipl.-Ing. Olaf Hendel fax +44 20 7499 1156
e-mail [email protected]
SLOVENIA contact person: B. Pegnall
Institute of Hop Research and Brewing alec
Oxford Brookes University
Cesta alskega tabora 2
School of Biological and Molecular Sciences
SI-3310 ALEC
Gipsy Lane Campus
tel. +386 3 712 1600
Headington
fax +386 3 712 1620
OXFORD OX3 0BP
e-mail [email protected]
tel. +44 1865 483 248
[email protected]
fax +44 1865 483 242
contact person: Iztok Koir
e-mail [email protected]
SPAIN contact person: Dr. Katherine Smart

Escuela Superior de Cerveza y Malta U.S.A.

Jos Gultirrez Abascal, 2
Alltech Biotechnology Center
E-28006 MADRID
3031 Catnip Hill Pike
tel. +34 91 336 3190
NICHOLASVILLE, Kentucky 40356
fax +34 91 336 3009
tel. +1 859 885 9613
e-mail [email protected]
fax +1 859 885 6736
contact person: Elena Roche
e-mail [email protected]
THE NETHERLANDS contact person: Dr. Pearse Lyons

TNO Nutrition and Food Research Siebel Institute of Technology

Utrechtseweg 48 4055 West Peterson Avenue
P.O. Box 360 CHICAGO, IL 60646-6001
3700 AJ Zeist tel. +1 773 279 0966
tel. +31 30 694 4486 fax +1 773 463 7688
fax +31 30 694 4295 e-mail [email protected]
e-mail [email protected] contact person: Keith Lemcke
contact person: Maarten J. Schans

UNITED KINGDOM
Brewing Research International
Coopers Hill Road
NUTFIELD, Surrey RH1 4HY
tel. +44 1737 822 272
fax +44 1737 822 747
e-mail [email protected]
contact person: Prof.Dr. F.R. Sharpe
European Brewery Convention

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List of Participants

ARGENTINA

Oliemans, Jos
Cerveceria y Malteria Quilmes S.A.
Avenida 12 de Octubre y Gran Canaria
1878 Quilmes Buenos Aires
ARGENTINA
+54 11 4349 1730
+54 11 4349 1724
[email protected]
Technical Director

AUSTRALIA

Ford, Christopher
Adelaide University
Department of Plant Science
Waite Campus
PMB1 Glen Osmond
SA 5064
AUSTRALIA
+61 8 8303 6738
+61 8 8303 7109
[email protected]
Research Fellow

Gardner, Adrian
Carlton & United Breweries
GPO Box 753 F
Melbourne 3001
AUSTRALIA
+61 3 9633 2629
+61 3 9633 2878
[email protected]
President: Institute & Guild of Brewing

Miles, Mel
Foster's Brewing Group Ltd.
77 Southbank Boulevard
Southbank Victoria 3006
AUSTRALIA
+61 3 9633 22 40
+61 3 9633 24 44
[email protected]

Rogers, Peter
Carlton & United Breweries
4-6 Southampton Crescent
Abbotsford, 3016
AUSTRALIA

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European Brewery Convention

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+61 3 9420 6522

+61 3 9420 6915
[email protected]
Manager of New Technology

AUSTRIA

Benes, Roman
Anton Paar GmbH
Krntner Strasse 322
8054 Graz
AUSTRIA
+43 316 257 480
+43 316 257 257
[email protected]
R&D

Huber, Christian
Brau Union sterreich AG
Dr. Beurle-Strasse
3250 Wieselburg
AUSTRIA
+43 74 16 502 200
+43 74 16 502 248
[email protected]
Braumeister

Kepplinger, Werner
Montanuniversitt Leoben
Franz-Josef-Strasse 18
8700 Leoben
AUSTRIA
+43 3842 4020
+43 3842 402 308
Ordentl. Univ.Prof

Liebl, Markus
Brau Union sterreich AG
Poschacherstrasse 35
4021 Linz
AUSTRIA
+43 732 6979 21 03
+43 732 6979 23 66
[email protected]
Vorstandsmitglied

Majan, Gerd
VULCASCOT
Getrnkeindustriebedarf Handelsges.m.b.H.
Muthgasse 25
1190 Wien

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AUSTRIA
+43 1 369 44 77 24
+43 1 369 44 77 924
Technical Engineer

Markovic, Romeo
VULCASCOT
Getrnkeindustriebedarf Handelsges.m.b.H.
Muthgasse 25
1190 Wien
AUSTRIA
+43 1 369 44 77 24
+43 1 369 44 77 924
Sales Manager

May, Alexander
M. May Industrievertretungen GmbH
Waldmhlgasse 5
2380 Perchtoldsdorf
AUSTRIA
+43 1 869 70 95
+43 1 869 709 54
[email protected]
Geschftsfuhrer

Natter, Michael
Brau Union sterreich AG
Poschacherstrasse 35
4020 Linz
AUSTRIA
+43 732 6979 21 10
+43 732 6979 21 12
[email protected]
Leiter Qualittssicherung u. Entwicklung

Pelz, Dieter
Brau Union sterreich AG
Triesterstrasse 357-359
8055 Graz
AUSTRIA
+43 316 502 3613
+43 316 502 3770
[email protected]
Managing Director

Pozsgay, Martin
Brau Union sterreich AG
Alanovaplatz 5
2320 Schwechat
AUSTRIA
+43 1 701 40 41 29

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European Brewery Convention

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+43 1 701 40 41 50
[email protected]
Braufuhrer

Schwarz, Helmuth
sterreichisches Getrnke Institut (GI)
Michaelerstrasse 25
1130 WIEN
AUSTRIA
+43 1 479 69 24 12
+43 1 479 69 24 11
[email protected]
General Manager

Uebersberger, Hartwig
Stamag Stadlanermalzfabrik Ges.
Smolagasse 1
1220 Wien
AUSTRIA
+43 1 28808 215
+43 1 28808 18
[email protected]
Director

Walzl, Manfred
State Mental Clinic
AUSTRIA
+43 664 20 99 490

Zanker, Gerald
Brau Union sterreich AG
Triesterstrasse 357-359
8055 Graz
AUSTRIA
+43 316 502 32 63
+43 316 502 32 03
[email protected]
Leiter Qualittssicherung

Zeilinger, Franz
Brau Union sterreich AG
Brgerbru Innsbruck
Ing. Etzelstrasse 11
6020 Innsbruck
AUSTRIA
+43 512 59 738 65 36
+43-512 59 738 65 26
[email protected]
Braumeister

BELGIUM

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European Brewery Convention

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List of Participants

Adam, Pierre
Interbrew S.A.
Vaartstraat 94
3000 Leuven
BELGIUM
+32 16 24 5124
+32 16 24 7889
Engineer Brewing Process

Aerts, Guido
KaHo St.-Lieven
Department KIHO-C.B.O.K.
Gebr. Desmetstraat 1
9000 Gent
BELGIUM
+32 9 265 86 13
+32 9 225 62 69
[email protected]
Prof

Arnould, Michel
C.M.B.S K.U. Leuven
92 Kardinaal Mercierlaan
3001 Heverlee
BELGIUM
+32 16 32 14 60
+32 16 32 15 76
[email protected]

Bastin, Patrick
Institut Meurice
Avenue E. Gryson, 1
1070 Brussels
BELGIUM
+32 2 526 73 50
+32 2 526 73 01
[email protected]
Researcher

Bauduin, Claude
Meura S.A.
Chausse d'Antoing 55
7500 Tournai
BELGIUM
+32 69 88 42 42
+32 69 88 42 49
[email protected]
Area Sales Manager

Bonachelli, Bruno

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European Brewery Convention

Date: 2001-06-01
Page: 1
List of Participants

Meura S.A.
Chausse d'Antoing 55
7500 Tournai
BELGIUM
+32 69 88 42 42
+32 69 88 42 49
[email protected]
R & D Engineer

Bonte, Sabine
Interbrew
Vuurkruisenlaan cd/co
3000 Leuven
BELGIUM
+32 16 24 7647
+32 16 20 6594
[email protected]
Plant Quality Manager

Braekeleirs, Robert
Meura S.A.
Chausse d'Antoing 55
7500 Tournai
BELGIUM
+32 69 88 42 42
+32 69 88 42 49
[email protected]
Deputy Sales Manager

Catteeuw, Marnix
Interbrew
Vaartstraat 94
3000 Leuven
BELGIUM
+32 16 24 7542
+32 16 24 7889
[email protected]
Resource Manager Knowledge Management

Coesens, Marc
Brewery Duvel Moortgat
Breendonkdorp 58
2870 Puurs
BELGIUM
+32 3 860 94 00
+32 3 886 46 22
mcoesens@duvel,be
Lab Assistent

Coppens, Theo
Cargill Malt

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European Brewery Convention

Date: 2001-06-01
Page: 1
List of Participants

Zijpstraat,155
B 3020 Herent
BELGIUM
+32 16 23 99 52
+32 16 53 61 20
[email protected]
General Superintendent Malt

Daems, Veerle
Interbrew
Vaartstraat 94
3000 Leuven
BELGIUM
+32 16 31 520 2
+32 16 24 78 89
[email protected]
Reource Manager Sensory

Daeyaert, Glenn
Sopura S.A.
Patrijzenweg 67
9160 Loveren
BELGIUM
+32 71 46 80 10
+32 71 45 25 90
[email protected]
Regional Sales Director

Daniels, Jacques
Belgomalt SA
Chausee de Charleroi 40
5030 Gembloux
BELGIUM
+32 81 625 840
+32 81 61 25 34
[email protected]
Quality Assurance Manager

De Brackeleire, Christian
Meura S.A.
Chausse d'Antoing 55
7500 Tournai
BELGIUM
+32 69 88 42 42
+32 69 88 42 49
[email protected]
Technical Director

De Bruyn, Jan
Albert Maltings N.V.
Kanaaldijk 2

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Page: 1
List of Participants

2870 Ruisbroek
BELGIUM
+32 3 860 04 11
+32 3 886 83 99
General Manager

De Cooman, Luc
KaHo St.-Lieven
Department KIHO-C.B.O.K.
Gebr. Desmetstraat 1
9000 Gent
BELGIUM
+32 9 265 86 13
+32 9 225 62 69
[email protected]

De Keukeleire, Denis
University of Ghent
Faculty of Pharmaceutical Sciences
Harelbekestraat 72
9000 Ghent
BELGIUM
+32 9 264 80 55
+32 9 264 81 95
[email protected]
University Professor

De Rouck, Gert
KaHo St.-Lieven
Gebr. Desmetstraat 1
9000 Gent
BELGIUM
+32 9 265 86 13
+32 9 225 62 69
[email protected]
Asisstent

De Schouwer, Peter
Boortmalt
Dorpsstraat 11
3190 Boortmeerbeek
BELGIUM
+32 15 50 11 11
+32 15 50 11 99
[email protected]
Adj.Sales Director

Debourg, Alain
Institut Meurice
Department of Brewing Science
1, Avenue E. Gryson

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European Brewery Convention

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List of Participants

1070 Brussels
BELGIUM
+32 2 526 73 51
+32 2 526 73 01
[email protected]
Professor

Delvaux, Freddy
Katholieke Universiteit Leuven
Center for Malting and Brewing Science
Kasteelpark Arenberg 22
3001 Leuven
BELGIUM
+32 16 32 17 37
+32 16 32 19 97
[email protected]
Professor

Demunter, Damien
Institut Meurice
Avenue E. Gryson 1
1070 Brussels
BELGIUM
+32 2 526 73 30
+32 2 526 13 01
Researcher

Derdelinckx, Guy
Kuleuven - CMBS
Kasteelpark 22
3001 Heverlee
BELGIUM
+ 32 16 32 16 34
+ 32 16 32 15 76
[email protected]
Prof. Dr.

Dufait, Alain
Cargill
Zijpstraat 155
B 3020 Herent
BELGIUM
+32 16 23 99 52
+32 16 20 12 77
[email protected]
R&D Manager

Dumortier, Tom
Interbrew
Vaartstraat 94
3000 Leuven

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List of Participants

BELGIUM
+32 16 24 7331
+32 16 24 7889
[email protected]
Engineer Brewing Microbiology

Dupire, Stphane
Interbrew
Vaartstraat 94
3000 Leuven
BELGIUM
+32 16 24 7396
+32 16 24 7889
[email protected]

Elo, Alexandre
Omnichem SA
Industrial Research Park - Fleming
1348 Louvain-La-Neuve
BELGIUM
+32 10 48 32 73
+32 10 45 25 53
[email protected]
Sales Manager

Franzi-Gauger, Susan
E-malt nv
Louis Schmidtlaan 114, bus 3
1040 Etterbeek
BELGIUM
+33 476 92 09 81
+33 476 92 09 80
[email protected]
Manager

Gauger, Holger M.
H.M. Gauger bvba
Hoogstraat 64
1930 Zaventem
BELGIUM
+32 2 720 95 05
+32 2 720 58 68
[email protected]
Director

Geerts, Jan
Interbrew NV
Interbrew - DWC Aalst
Gentsestraat 80
9300 Aalst
BELGIUM

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European Brewery Convention

Date: 2001-06-01
Page: 1
List of Participants

+32 53 73 97 17
+32 53 73 97 68
[email protected]
Plant Manager DWC Maltings

Hermia, Jacques
Universit Catholique de Louvain
Unit des Procds
Voie Minckelers, 1
1348 Louvain-La-Neuve
BELGIUM
+32 10 47 23 24
+32 10 47 23 21
[email protected]
Professor

Janssens, Philippe
Interbrew
Vaartstraat 94
3000 Leuven
BELGIUM
+32 16 31 56 49
+32 16 24 56 24
[email protected]
Head Pilot brewery

Keersmaecker, Jacques
Interbrew
Vaartstraat 135
3000 Leuven
BELGIUM
+32 16 31 58 66
+32 16 31 58 71
[email protected]
Procurement Raw Materials Europe Manager

Lermusieau, Guillaume
Universit Catholique de Louvain la Neuve
Place Croix du Sud 2 BTE 7
1348 Louvain-La-Neuve
BELGIUM
+32 10 47 35 94
+32 10 47 21 78
[email protected]
Engineer

Lestiboudois, Andrew
Interbrew Belgium NV
Vaartstraat 94
3000 Leuven
BELGIUM

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European Brewery Convention

Date: 2001-06-01
Page: 1
List of Participants

+32 16 24 77 74
+32 16 20 65 94
[email protected]
Plant Manager

Lietar, Claudine
CERIA, Revue des Fermentations
Institut Meurice
Avenue E. Gryzon 1
1070 Brussels
BELGIUM
+32 2 526 73 50
+32 9 228 46 68

Lietar, Jacques - Yves

Biocon
145 Scheldestraat
9040 St. Amandsberg
BELGIUM
+32 9 228 49 71
+32 9 228 46 68
[email protected]
Director

Luyssaert, Philippe
Meura S.A.
Chausse d'Antoing 55
7500 Tournai
BELGIUM
+32 69 88 42 42
+32 69 88 42 49
[email protected]
Area Sales Manager

Malcorps, Philippe
Interbrew
Vaartstraat 94
3000 Leuven
BELGIUM
+32 16 24 7787
+32 16 24 7889
[email protected]
Resource manager Fermentation & Microbiol.

Meulemans, Stephane
Yakima Chief
19, Otto De Mentockplein
1853 Strombeek-Bever
BELGIUM
+32 2 263 06 10
+32 2 263 06 13

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List of Participants

[email protected]
Vice President of Sales

Mussche, Roger
Omnichem N.V.
Cooppallaan 91
9230 Wetteren
BELGIUM
+32 9 365 33 93
+32 9 369 82 64
[email protected]
Scientific Advisor

Neven, Hedwig
Duvel Moortgat NV
Dorp 58
2870 Puurs
BELGIUM
+32 3 886 71 21
+32 3 886 46 22
[email protected]
Adj. Production Director

Nickmans, Marco
Boortmalt
Dorpsstraat 11
3190 Boortmeerbeek
BELGIUM
+32 15 50 11 11
+32 15 50 11 99
[email protected]
Adj.Dir.Gen

Pellaud, Jerome
Interbrew S.A.
Vaartstraat 94
3000 Leuven
BELGIUM
+32 16 31 5782
+32 16 24 7889
[email protected]
Engineer Chemistry

Rahier, Georges
Universit Catholique de Louvain
Unit des Procds
Voie Minckelers, 1
1348 Louvain-La-Neuve
BELGIUM
+32 10 47 23 32
+32 10 47 23 21

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European Brewery Convention

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[email protected]
Senior Researcher

Rock, Jean-Marie
Orval Brewery
Brasserie d' Orval
6823 Villers-devant Orval
BELGIUM
+32 61 311 261
+32 61 312 927
[email protected]

Roggemans, Patrick
Alfa Laval
Bazellaan, 5
1140 Brussels
BELGIUM
+32 2 728 39 12
+32 2 728 39 85
Application Development Manager

S Almeida, Alfredo
Meura S.A.
Chausse d'Antoing 55
7500 Tournai
BELGIUM
+32 69 88 42 42
+32 69 88 42 49
[email protected]
Commercial Director

Schoorens, Jose
Sopura S.A.
Rue de Trazegnies 199
6180 Courcelles
BELGIUM
+32 71 46 80 10
+32 71 45 25 90
[email protected]
Regional Sales Director

Staelens, Dimitri
Interbrew S.A.
Vaartstraat 94
3000 Leuven
BELGIUM
+32 16 24 7624
+32 16 24 7889
[email protected]
Engineer Brewing Process

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European Brewery Convention

Date: 2001-06-01
Page: 1
List of Participants

Thirionet, Hubert
Boortmalt
Dorpsstraat 11
3190 Boortmeerbeek
BELGIUM
+32 15 50 11 11
+32 15 50 11 99
[email protected]
General Director

Tigel Gil, Rafael

Meura S.A.
Chausse d'Antoing 55
7500 Tournai
BELGIUM
+32 69 88 42 42
+32 69 88 42 49
[email protected]
R & D Manager

Timmermans, Paul
Interbrew NV
Vaartstraat 94
3000 Leuven
BELGIUM
+32 16 24 74 00
+32 16 31 58 20
[email protected]

Toye, Jan
Palm Brouwerij NV
Steenhuffeldorp 3
1840 Londerzeel
BELGIUM
+32 52 31 74 34
+32 52 30 37 33
[email protected]
Administrator

Tys, Jean-Pierre
Boortmalt
Dorpsstraat 11
3190 Boortmeerbeek
BELGIUM
+32 15 50 11 11
+32 15 50 11 99
[email protected]
Export Dir.

Van Den Berg, Steven

Hogeschool Gent - CTO

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European Brewery Convention

Date: 2001-06-01
Page: 1
List of Participants

Voskenslaan 270
9000 Gent
BELGIUM
+32 9 242 42 36
+32 9 243 87 78
[email protected]
Wertenchappelyk medewerker

Van Den Bogaert, Bernard

Brewery De Koninck N.V.
Mechelsesteenweg 281
2018 Antwerpen
BELGIUM
+32 3 218 40 68
+32 3 218 44 67

Van den Bussche, Jeroen

Meura S.A.
Chausse d'Antoing 55
7500 Tournai
BELGIUM
+32 69 88 42 42
+32 69 88 42 49
[email protected]
Area Sales Manager

Van Den Eynde, Erik

Interbrew S.A.
Vaartstraat 94
3000 Leuven
BELGIUM
+32 16 24 7698
+32 16 24 7889
Director Technology Planning 6 Process

Van der Heggen, Eddy

Interbrew Hoegaarden
Stoofkenstraat 46
3320 Hoegaarden
BELGIUM
+32 16 76 98 45
+32 16 76 76 78
[email protected]
Head of Production

Van Herreweghen, Willem

Brouwery Palm N.V.
Steen Huffeldorp 3
1840 Londerzeel
BELGIUM
+32 52 31 74 61

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European Brewery Convention

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+32 52 30 92 80
[email protected]
Director Quality and Logistics

Van Regenmortel, Alfons

Brewery Duvel Moortgat
Breendonk-Dorp 58
2870 Puurs
BELGIUM
+32 3 886 71 21
+32 3 886 46 22
[email protected]
Prod & Tech. Manager

Vandemeulebroucke, Claudine
Federation of the Belgian Maltsters
Nerumstraat 7
9340 Lede
BELGIUM
+32 53 810 990
+32 53 809 230
[email protected]
Secretary General

Vanhamel, Sonja
Alken-Maes Breweries
Waarloosveld 10
2550 Waarloos
BELGIUM
+32 11 59 03 10
+32 11 59 03 09
[email protected]
Head of Production Department

Vanthournhout, Cedric
Yakima Chief
19 Otto de Mentockplein
1853 Strombeek-Bever
BELGIUM
+32 2 263 06 10
+32 2 263 03 13
[email protected]
Sales Manager

Velings, Maurice
Sopura S.A
Rue de Trazegnies 199
6180 Courcelles
BELGIUM
+32 71 46 80 10
+ 32 71 45 25 90

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[email protected]

Verschaeve, Stefaan
Alken-Maes Breweries
Waarloosveld 10
2550 Waarloos
BELGIUM
+32 15 30 90 19
+32 15 30 94 60
[email protected]
Director

Willaert, Ronnie
Hogeschool Gent
Lab. Brouwerij- en Fermentatietechnol.
Voskenslaan 270
9000 Gent
BELGIUM
+32 9 242 42 36
+32 9 243 8778
[email protected]
Prof.

BRAZIL

Corte, Odair Otavio

Corn Products Brasil
Av. do Caf, 277
torre B - 2 andar
04311-000 Sao Paulo - SP
BRAZIL
+55 11 507 077 08
+55 11 507 078 31
[email protected]
Sales Manager

Guercia, Homero Francisco

Ambev
Av. Maria Coelho Agviar, 215
Bloco F-7 Andar - CEP: 05804-900
BRAZIL
+55 11 37 48 90 99
+55 11 37 41 14 57
[email protected]
Corporate Manager Quality Assurance

Mauhin da Cruz, Maria Helena

Cervejarias Kaiser
Av.Pres. Humberto d Alencar Castelo Branco
2911-12321-150- Bairro Rio Abaixo
Jacarei-SP

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Page: 1
List of Participants

BRAZIL
+55 12 355 16 17
+55 12 352 16 16
[email protected]
Quality Control Central Manager

Oliveira, Sidney
Primo Schincariol Ind. Cerv.e Refrig.
Av. Primo Schincariol, 2300 Bairro Itaim
Itu/ Sao Paulo CEP:13.312-250
BRAZIL
+55 11 4022 95 00
+55 11 4022 95 55
[email protected]
Brew Master

Reichert, S.
Ambev
Av. Maria Caelho Agviar,215
Bloco F-6 Andar-05804-900
BRAZIL
+55 11 3741 3805
+55 11 3741 1457
R&D Manager

BULGARIA

Batchvarov, Valentin
Institute of Cryobiology and Food Industries
Brewing Technology Department
Samokovsko shose 2a
Sofia 1138, Gorubljane
BULGARIA
+359 2 955 53 61
+359 2 955 53 61
[email protected]
Head of Brewing Technology Departement

CANADA

Hood, John
Molson Breweries Canada
33 Carlingview Drive
Etobicoke, Ontario M9W 5E4
CANADA
+1 416 679 75 01
+1 416 798 83 91
[email protected]
Vice President, Brewing Quality

Joy, Richard

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List of Participants

Westcan Malting Ltd.

P.O. 113
Alix, Alberta
CANADA
+1 403 747 2777
+1 403 747 2068
[email protected]
QC & Technical Services Manager

Mcgarrity, Michael
Labatt Brewing Company Ltd.
197 Richmond St
London, Ontario N6A-4H3
CANADA
+1 519 667 73 43
+1 519 667 73 50
[email protected]
Principal Scientist

Sacy, Hubert
duc'alccol
606, Carthcart Suite 700
Montral, Qubec H3 B1 K9
CANADA
+1 514 875 74 54
+1 514 875 59 90
[email protected]
Director General duc'alcool

Steer, James T.
Molson Canada
33 Carlingview Dr.
Etobicoke, Ont. M9W 5E4
CANADA
+1 416 679 75 00
+1 416 798 83 91
[email protected]
Sr. Technical Consultant

CHINA

Eyben, Donald
Beijing Boortmalt Malting Company (BBMC)
Jiliamiao South Fengtai District
100070 Beijing
CHINA
+86 10 6374 55 93
+86 10 63 74 55 93
[email protected]
Managing Director

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European Brewery Convention

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COLOMBIA

Bonnells, Jorge
Cervecera Union S.A.
Cra. 50 A No.38-39
Medelln
COLOMBIA
+574 375 68 66
+574 372 34 88
[email protected]

Castao, Dario
Cervecera Union S.A.
Cra. 50 A No.38-39
Medelln
COLOMBIA
+574 375 68 66
+574 372 34 88

Marin Montealegre, Javier

Bavaria S.A.
Avda Boyscs 9-02
COLOMBIA

Salazar, Vicente
Cervecera Union S.A.
Cra. 50 A No.38-39
Medelln
COLOMBIA
+574 375 68 66
+574 372 34 88

COSTA RICA

Feoli, Fernando
Florida Ice and Farm Co
Apdo. 795
1000 San Jose
COSTA RICA
+506 443 22 22
+506 441 78 66
[email protected]

CROATIA

Geuns, Guy
Slavonija Slad - Boortmalt
Urije bb
35400 Nova Gradiska
CROATIA
+385 35 361 576

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+385 35 362 030

[email protected]
President, General Manager

Maric, Vladimir
Karlovac Brewery
Faculty of Food Technology and Biotechnology
Pierottieur 6
Zagreb
CROATIA
+385 1 4605 290
+385 1 4836 424
[email protected]

CYPRUS

Georgiades, Nicos
Keo Ltd.
P.O. Box 50209
3602 Limassol
CYPRUS
+357 5 85 31 04
+357 5 57 88 68
[email protected]
Brewery Manager

CZECH REPUBLIC

Celeda, Libor
Yakima Chief, Inc
Ceske Druziny 35
16000 Praha 6
CZECH REPUBLIC
+420 2 3122 346
+420 2 2431 50 96
[email protected]
Sales Director, Eastern Europe

Cepicka, Jaroslav
Institute of Chemical Technology Prague
Technick 5
16628 Praha 6
CZECH REPUBLIC
+420 2311 32 78
+420 2243 550 51
[email protected]
Head of Department

Crous, Steve
S.A. Breweries
c/o Pilsner Urquell a.s.

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List of Participants

U Prazdroje 7
304 97 Plzen
CZECH REPUBLIC
+420 19 706 2937
+420 19 706 2738
[email protected]
Technical Development Director

Denny, Steve
Trazsk Pivovary A.S.
Ndrazni 84
150 54 Praha 5
CZECH REPUBLIC
+420 2 571 91 465
+420 2 573 16 740
[email protected]
Supply Director

Dostlek, Pavel
Institute of Chemical Technology Prague
Technick 5
166 28 Praha 6
CZECH REPUBLIC
+420 224 354 037
+420 224 355 051
[email protected]
Assistant Professor

Famera, Jiri
Pilsner Urquell a.s.
U. Prazdroje 7
30497 Plzen
CZECH REPUBLIC
+420 19 706 20 59
+420 19 706 23 41
Quality Ass. Manager

Fiala, Jaromir
Institute of Chemical Technology Prague
Technick 5
166 28 Prague 6
CZECH REPUBLIC
+420 2 2435 4037
+420 2 2435 5051
[email protected]
Assistant Professor

Fridrich, Stanislav
Radegast Brewery J.S.C.
73951 Nosovice
CZECH REPUBLIC

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Date: 2001-06-01
Page: 1
List of Participants

+420 658 64 1454

+420 658 64 1414
Technical Director

Halaska, Eduard
Haffmans - Regom Instruments
Marie Cibulkov 34
140 21 Praha 4
CZECH REPUBLIC
+420 2 41 40 22 06
+420 2 41 40 02 90
[email protected]
Manager

Hlavcek, Jan
Pilsner Urquell a.s.
U. Prazdroje 7
30497 Plzen
CZECH REPUBLIC
+420 19 706 2160
+420 19 706 2704
Plant Manager

Hork, Thoms
Research Institute of Brewing and Malting
Lpov 15
12044 Praha 2
CZECH REPUBLIC
+420 2 2492 21 11
+420 2 2491 53 91
[email protected]
Research Worker

Hnigova, Vera
Prazske Pivovary a.s.
Ndrazn 84
15054 Praha
CZECH REPUBLIC
+420 2 571 91 372
+420 2 571 91 365
[email protected]
Quality Assurance Director

Kanak, Ivo
Radegast Brewery J.S.C.
739 51 Nosovice
CZECH REPUBLIC
+42 658 602 225
+42 658 602 550
Plant Manager

Congrex CX System: Copyright 1996-1998 by CONGREX Sweden AB

European Brewery Convention

Date: 2001-06-01
Page: 1
List of Participants

Kellner, Vladimr
Section Manger Analytial Services & Researh
Lpov 15
12044 Praha 2
CZECH REPUBLIC
+420 2 24 92 21 11
+420 2 2491 53 91
[email protected]

Kosar, Karel
Research Institute of Brewing and Malting
Lpov 15
12044 Praha 2
CZECH REPUBLIC
+420 5 452 101 03
+420 5 453 212 25
[email protected]
Director

Mares, Borivoj
Budweiser Budvar Brewery
K.Svetle 4
37021 Ceske Budejovice
CZECH REPUBLIC
+420 38 770 54 02
+420 38 731 11 36
[email protected]
Dipl. Ing

Mikyska, Alexander
Research Institute of Brewing and Malting
Lpov 15
12044 Praha 2
CZECH REPUBLIC
+420 2 24 91 55 75
+420 2 24 92 06 18
[email protected]
Head of resarch dept. Praha

Prucha, Pavel
Pilsner Urquell a.s.
U.Prazdroje 7
30497 Plzen
CZECH REPUBLIC
+420 19 706 28 12
+420 19 706 23 41
[email protected]
Quality Manager

Psota, Vratislav
Research Institute of Brewing and Malting

Congrex CX System: Copyright 1996-1998 by CONGREX Sweden AB

European Brewery Convention

Date: 2001-06-01
Page: 1
List of Participants

Lpov 15
12044 Prague
CZECH REPUBLIC
+420 2 2491 55 75
+420 2 4532 12 25
[email protected]
Ing.

Savel, Jan
Budweiser Budvar Brewery
K. Svetl 4
37021 Ceske Budejovice
CZECH REPUBLIC
+420 38 7705 118
+420 38 7705 120
[email protected]
Head of Research Dept.

Steenberg, Johann
S.A. Brewers
c/o Pilsner Urquell a.s.
U Prazdroje 7
304 97 Plzen
CZECH REPUBLIC
+420 19 706 2938
+420 19 706 2738
[email protected]

Vesely, Jan
Pilsner Urquell a.s.
Czech Beer & Malt Association
Lpov 15
120 44 Prague 2
CZECH REPUBLIC
+420 2 249 145 42
[email protected]
Chairman of the Association

DENMARK

Aastrup, Sten
Alfred Joergensen Laboratory Ltd.
30 Frydendalsvej
1809 Fredericksberg C
DENMARK
+45 33 55 88 00
+45 33 55 88 01
[email protected]
Senior Consultant

Andersen, Carsten Bryde

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List of Participants

The Scandinavian School of Brewing

Gamle Carlsberg Vej 16
2500 Valby
DENMARK
+45 33 27 24 00
+45 33 27 24 01
[email protected]

Breddam, Klaus
Carlsberg Research Laboratory
Gamle Carlsberg Vej 10
2500 Copenhagen Valby
DENMARK
+45 33 27 52 40
+45 33 27 47 64
[email protected]

Due, Jens
Danbrew Ltd. A/S
Rahbeks Alle 21
1801 Frederiksberg C
DENMARK
+45 33 85 55 00
+45 33 85 56 72
[email protected]
President- Chief Executive

Eiken, Jens
Carlsberg Corporate Operations Control
Ny Carlsberg Vej 142
1799 Copenhagen
DENMARK
+45 3327 27 40
+45 3327 48 04
[email protected]

Erdal, Kenneth
Carlsberg Breweries A/S
100 Ny Carlsberg Vej
1760 Copenhagen V
DENMARK
+45 33 27 44 70
+45 33 27 47 25
[email protected]
Group Quality Manager

Funch Jensen, Birgitte

Carlsberg Corporate Operations Control
Ny Carlsberg Vej 142
1799 Copenhagen V
DENMARK

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List of Participants

+45 3327 27 09
+45 3327 48 04
[email protected]

Gaarde, Klaus
Damas A/S
Industrivej 2
Vester Aaby
5600 Faaborg
DENMARK
+45 63 61 82 00
+45 63 61 68 51
[email protected]
Man.Director

Gubbi Jorgensen, Kim

Danish Malting Group A/S
Spirevej 5
4760 Vordingborg
DENMARK
+45 55 97 50 00
+45 55 97 50 50
[email protected]
Chief Executive Officer

Guldborg, Merethe
Carlsberg Danmark A/S
Ny Carlsberg Vej 142
1760 Copenhagen V
DENMARK
+45 33 27 44 80
+45 33 27 47 54
[email protected]

Henriksen, Jens
Damas A/S
Industrivej 2
Vester Aaby
5600 Faaborg
DENMARK
+45 63 61 82 00
+45 63 61 68 51
[email protected]
Sales Manager

Johansson, Bo
Danfoss A/S
A801
6430 Nordborg
DENMARK
+45 7488 2797

Congrex CX System: Copyright 1996-1998 by CONGREX Sweden AB

European Brewery Convention

Date: 2001-06-01
Page: 1
List of Participants

+45 7488 48 45
[email protected]
Regional Manager

Jorgensen, Jens Alro

Danbrew Ltd. A/S
Rahbeks Alle 21
1801 Frederiksberg C
DENMARK
+45 33 85 55 50
+45 33 21 15 18
[email protected]
Key Account Manager

Kielland-Brandt, Morten C.
Carlsberg Research Laboratory
Gamle Carlsberg Vej 10
2500 Copenhagen Valby
DENMARK
+45 33 27 53 31
+45 33 27 47 65
[email protected]
Professor

Kristiansen, Axel
Carlsberg Corporate Operations Control
Ny Carlsbergvej 142
1799 Copenhagen V
DENMARK
+45 3327 27 05
+45 3327 48 04
[email protected]

Larsen, Olav Vind

Alfred Joergensen Laboratory Ltd.
30 Frydendalsvej
1809 Fredericksberg C
DENMARK
+ 45 33 55 88 00
+ 45 33 55 88 01
[email protected]
Director, Head of process Consultancy Dept.

Lisbjerg, Soren
Carlsberg Breweries A/S
100 Ny Carlsberg Vej
1760 Copenhagen
DENMARK
+45 33 27 33 00
+45 33 27 48 48
[email protected]

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List of Participants

Technical Director, Western Europe

Munksgaard, Jannik
Danbrew Ltd. A/S
Rahbeks Alle 21
1801 Frederiksberg C
DENMARK
+45 33 85 55 07
+45 33 21 15 18
[email protected]
Technical Manager

Nielson, Olav
Alfa Laval Scandi Brew
Rosenkaeret 18
2860 Soeborg
DENMARK
+45 39 67 00 44
+45 39 67 00 33
[email protected]
Sales & Technology Manager

Nohr Bak, Soren

Danbrew Ltd. A/S
Rahbeks Alle 21
1801 Frederiksberg C
DENMARK
+45 33 85 55 53
+45 33 21 15 18
[email protected]
Head of Function

Pedersen, Knud-Erik
Danbrew Ltd. A/S
Rahbeks Alle 21
1801 Frederiksberg C
DENMARK
+45 33 85 55 70
+45 33 21 15 18
[email protected]
Manager, Packaging Department

Petersen, Mogens Toftegaard

The Danish Brewery Group A/S
Faxe Bryggeri
Torvegade 35
4640 Faxe
DENMARK
+45 56 77 19 20
+45 56 77 19 29
[email protected]

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European Brewery Convention

Date: 2001-06-01
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List of Participants

Quality Assurance Manager

Piper, Jens-Ulrik
Danbrew Ltd. A/S
Rahbeks Alle 21
1801 Frederiksberg C
DENMARK
+45 33 85 55 06
+45 33 21 15 18
[email protected]
Vice President

Scholderer, Joachim
The MAPP Centre
The Aarhus School of Business
Haslegaardsvej 10
8210 Aarhus V
DENMARK
+45 89 48 64 87
+45 86 15 01 77
[email protected]
Associated professor

Steenholt, Torsten
Carlsberg Breweries A/S
Ny Carlsberg Vej 100
DK-1760 Copenhagen V.
DENMARK
+45 33 273 327
+45 33 274 741
[email protected]
N.P.D. Manager

ESTONIA

Josua, Lembit
Saku Brewery ltd
Harju County
Tallinna Str. 2
75 501 Saku
ESTONIA
+372 6508 345
+372 6508 401
[email protected]
Laboratory Manager

Krblane, Enn
Saku Brewery Ltd
Harju County
Tallinna str. 2
75 501, Saku

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Date: 2001-06-01
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List of Participants

ESTONIA
+372 6508 351
+372 6508 401
[email protected]
Head Brewer

FINLAND

Ahvenainen, Juha
VTT Biotechnology
P.O Box 1500
02044 VTT
FINLAND
+358 9 456 51 60
+358 9 455 21 03
[email protected]

Elonheimo, Juhani
Raisio Malt
P.O. Box 101
21201 Raisio
FINLAND
+358 2 44 32 111
+358 2 44 32 904
[email protected]
Marketing Manager

Grnqvist, Anders
Oy Sinebrychoff AB
P.O. Box 87
04201 Kerava
FINLAND
+358 9 294 99 224
+358 9 294 994 08
[email protected]
Development Manager

Haikara, Auli
VTT Biotechnology
Tietotie 2
P.O. Box 1500
02044 VTT
FINLAND
+358 9 456 51 30
+358 9 455 21 03
[email protected]

Hartwall, Peter
Oy Hartwall AB
Ristipellontie 2-4
P.O Box 31

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European Brewery Convention

Date: 2001-06-01
Page: 1
List of Participants

00391 Helsinki
FINLAND
+358 9 540 21
+358 9 540 24 34
[email protected]
Brewmaster

Home, Silja
VTT Biotechnology
Tietotie 2, Espoo
P.O. Box 1500
02044 VTT
FINLAND
+358 9 456 5115
+358 9 455 21 03
[email protected]
Manager Malting & Brewing

Jskelinen, Kimmo
Oy Sinebrychoff AB
P.O. Box 87
04201 Kerava
FINLAND
+358 9 294 992 34
+358 9 294 994 81
[email protected]
Production Director

Kangas-Heiska, Tapio
Oy Sinebrychoff AB
P.O. Box 87
04201 Kerava
FINLAND
+358 9 294 99 281
+358 9 294 99 481
[email protected]
Brewmaster

Kleemola, Tuija
VTT Biotechnology
Tietotie 2, Espoo
P.O. Box 1500
02044 VTT
FINLAND
+358 9 456 7141
+358 9 455 2103
[email protected]
Research Scientist

Kotaviita, Erja
Raisio Malt

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Date: 2001-06-01
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List of Participants

P.O. Box 101

21201 Raisio
FINLAND
+358 2 443 26 77
+358 2 443 29 04
[email protected]
Development Manager

Kronlf, Jukka
Hartwall Plc
P.O. Box 44
19101 Lahti
FINLAND
+358 3 862 280
+358 3 734 22 21
[email protected]
Research Manager

Lommi, Heikki
Tuchenhagen Brewery Systems GmbH
Koivuhaankuja 2
01510 Vantaa
FINLAND
+358 9 8700 3155
+358 9 8700 3152
[email protected]
R & D Manager

Londesborough, John
VTT Biotechnology
P.O. Box 1500
02044 VTT
FINLAND
+358 9 456 59 96
+358 9 455 20 28
[email protected]
Senior Biochemist

Lhdesmki, Matti
Primalco Ltd.
Grain Processing
61330 Koskenkorva
FINLAND
+358 6 425 92 12
+358 6 425 93 52
[email protected]
Production Manager

Maunula, Hannu
Raiso Malt
P.O. Box 101

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European Brewery Convention

Date: 2001-06-01
Page: 1
List of Participants

21201 Raisio
FINLAND
+358 2 443 26 40
+358 2 443 29 04
[email protected]
Vice President

Maunula, Mikko
Polttimo Companies Ltd.
P.O Box 22
15141 Lahti
FINLAND
+358 3 864 11
+358 3 781 08 23
CEO

Multanen, Lauri
Oy Hartwall AB
Box 33
95401 Tornio
FINLAND
+358 40 558 56 30
+358 16 433 601
[email protected]
Factory Manager

Olkku, Juhani
LP Research Centre Ltd.
P.O. Box 22
15141 Lahti
FINLAND
+358 3 864 11
+358 3 864 288
[email protected]
R&D Director

Pajunen, Esko
Oy Sinebrychoff AB
P.O Box 87
04201 Kerava
FINLAND
+358 9 294 99 208
+358 9 294 99 490
[email protected]
President

Pelttari, Pentti
Brewery Olvi Oyj
Box 16
74101 Ilsalmi
FINLAND

Congrex CX System: Copyright 1996-1998 by CONGREX Sweden AB

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List of Participants

+358 17 83 85 301
+358 17 83 85 215
[email protected]
Head Brewer

Penttil, Merja
VTT Biotechnology
P.O.Box 1500
02044 VTT
FINLAND
+358 9 456 45 04
+358 9 455 21 03
[email protected]
Research Professor

Pietil, Kirsti
VTT Biotechnology
P.O Box 1500
02044 VTT
FINLAND
+ 358 9 456 51 11
+ 358 9 455 21 03
[email protected]
Research Scientist

Pohjolainen, Riina
Raisio Malt
P.O. Box 101
21201 Raisio
FINLAND
+358 400 88 55 47
+358 2 443 29 04
[email protected]
Head Technologist

Pyri, Sami
Suomen Unilever Oy Diverseylever
Hitsaajankatu 4 A
00810 Helsinki
FINLAND
+358 9 759 06 50
+358 9 759 78 12 38
[email protected]
Technical sales Represnt.

Ranta, Bo
Oy Sinebrychoff AB
P.O. Box 87
04201 Kerava
FINLAND
+358 9 294 991

Congrex CX System: Copyright 1996-1998 by CONGREX Sweden AB

European Brewery Convention

Date: 2001-06-01
Page: 1
List of Participants

+358 9 294 994 81

[email protected]
Head Brewer

Rasi, Jorma
Hartwall Plc.
P.O. Box 44
15101 Lahti
FINLAND
+358 3 862 11
+358 3 734 22 21
[email protected]
R & D Manager

Salmi, Pentti
National Product Control Agency
P.O. Box 210
00531 Helsinki
FINLAND
+358 9 3967 2750
+358 9 3967 2798
[email protected]
Head of Dep.

Segersven, Bo
Oy Sinebrychoff AB
P.O. Box 87
04201 Kerava
FINLAND
+358 9 294 992 25
+358 9 294 994 98
[email protected]

Stenholm, Katharina
Polttimo Companies Ltd.
P.O. Box 22
15141 Lahti
FINLAND
+358 3 864 11
+358 3 864 296
[email protected]
Project Manager

Stenius, Vilma
Oy Sinebrychoff AB
P.O. Box 87
04201 Kerava
FINLAND
+358 929 49 91
+358 9 294 994 08
[email protected]

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European Brewery Convention

Date: 2001-06-01
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List of Participants

Laboratory Manager

Storgrds, Erna
VTT Biotechnology
P.O. Box 1500
02044 VTT
FINLAND
+358 9 456 4526
+358 9 455 2103
[email protected]

Therman, Rolf
Hartwall Plc.
P.O. Box 31
00391 Helsinki
FINLAND
+358 9 540 21
+358 9 540 23 99
[email protected]
Technical Director

Toivola, Ansa
Oy Sinebrychoff AB, Pori Plant
P.O.Box 49
28101 Pori
FINLAND
+358 2 622 67 81
+358 2 622 66 02
[email protected]
Lab Manager

Vanhatalo, Ilkka
Oy Sinebrychoff AB
PL 49
28101 Pori
FINLAND
+358 2 622 67 79
+358 2 622 66 02
[email protected]
Production Manager

Vanne, Liisa
VTT Biotechnology
P.O. Box 1500
02044 VTT
FINLAND
+358 9 456 4460
+358 9 455 2103
[email protected]
Research Scientist

Congrex CX System: Copyright 1996-1998 by CONGREX Sweden AB

European Brewery Convention

Date: 2001-06-01
Page: 1
List of Participants

Vehkomki, Maija-Leena
VTT Biotechnology
P.O.Box 1500
02044 VTT
FINLAND
+358 9 456 78625
+358 9 455 2103
[email protected]
Research Scientist

Vehvilinen, Anna-Kaisa
Oy Hartwall AB Lapin Kulta
Box 33
95401 Tornio
FINLAND
+358 16 43 37 11
+358 16 43 36 01
[email protected]
Manager of Quality Insurance

Vehvilinen, Heikki
Hartwall Plc.
PO Box 44
15101 Lahti
FINLAND
+358 3 862 331
+358 3 734 22 21
[email protected]
Head brewer

Vesala, Erkki
Hartwall Plc.
PB 31
00391 Helsinki
FINLAND
+358 9 5402 392
+358 9 5402 513
[email protected]
Purchasing Manager

Virtanen, Hannele
VTT Biotechnology
P.O.Box 1500
02044 VTT
FINLAND
+358 9 456 5116
+358 9 455 2013
[email protected]
Research Scientist

Yl-Anttila, Juha

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European Brewery Convention

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List of Participants

Polttimo Companies Ltd.

P.O Box 22
15141 Lahti
FINLAND
+358 3 864 11
+358 3 864 296
[email protected]
Director, Malt Division

FRANCE

Aime, Francois
IFBM
7 rue du Bois de la Champelle
B.P. 267
54512 Vandoeuvre Cedex
FRANCE
+33 3 83 44 88 00
+33 3 83 44 12 90
[email protected]
Technical Director

Arbogast, Marc
Brasserie Fischer S.A.
7 Route de Bischwiller
67300 Schiltigheim
FRANCE
+33 3 88 33 82 03
+33 3 88 33 82 87
[email protected]
Directuer Shaligie et Developement

Auroux, Claude
Malteurop
6 Alle Ren Fonck - BP 1041
51688 Reims Cedex 2
FRANCE
+33 326 78 61 00
+33 326 78 61 48
[email protected]
Commercial Director

Bauer, Gilbert
Brasseries Kronenbourg
B.P. 13
67037 Strasbourg Cedex 2
FRANCE
+33 3 88 27 44 00
+33 3 88 27 48 98
[email protected]
Quality and Development Director

Congrex CX System: Copyright 1996-1998 by CONGREX Sweden AB

European Brewery Convention

Date: 2001-06-01
Page: 1
List of Participants

Bazard, Daniel
Brasseries Kronenbourg
68 rte d'Oberhausbergen
B.P. 13
67037 Strasbourg Cedex 2
FRANCE
+33 3 88 274 605
+33 3 88 274 622
[email protected]
Chef de fabrication

Bendler, Erik
DSM Food Specialities
20, Rue du Ballon
59041 Lille Cedex
FRANCE
+33 3 28 36 74 08
+33 3 28 36 74 25
[email protected]
Technical Service Manager

Boivin, Patrick
IFBM
Ple Technologique de Nancy - Brabois
7 rue du Bois de la Champelle
P.O. Box 267
54512 Vandoeuvre Cedex
FRANCE
+33 3 83 44 88 00
+33-3-83 44 12 90
[email protected]
Scientific and Quality Director

Bonnenfant, Christine
Malteurop
6 Alle Ren Fonck - BP 1041
51688 Reims Cedex 2
FRANCE
+33 326 78 61 00
+33 326 78 61 48
Export Sales Manager

Brignon, Pierre
Brasseries Kronenbourg
68 rte d'Oberhausbergen
B.P. 13
67037 Strasbourg Cedex 2
FRANCE
+33 3 88 274 060
+33 3 88 274 846

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List of Participants

[email protected]
Directeur Industrielle

Brunelle, Rgis
Malteurop
6 Alle Ren Fonck - BP 1041
51688 Reims Cedex 2
FRANCE
+33 326 78 61 00
+33 326 78 61 48
[email protected]
Director of Malteurop Deutschland

Chaudron, Philippe
Malteries Franco Suisses
74 rue de Alouettes
36100 Issoudun
FRANCE
+33 2 54 21 13 43
+33 2 54 03 17 78
[email protected]
Generla Manager

Cothenet, Bruno
Malteries Soufflet
Quai Sarrail
10400 Nogent / Seine
FRANCE
+33 3 25 39 41 70
+33 3 25 39 44 80
[email protected]
Eastern European Malting Director

Crequer, Jean-Paul
Malteries Soufflet
Quai Sarrail
10400 Nogent / Seine
FRANCE
+33 3 25 39 41 70
+33 3 25 39 44 80
[email protected]
Area Manager

Darrigo, Fabrice
Brasseries Kronenbourg
68 rte d'Oberhausbergen
B.P. 13
67037 Strasbourg Cedex 2
FRANCE
+33 3 88 274 640
+33 3 88 274 776

Congrex CX System: Copyright 1996-1998 by CONGREX Sweden AB

European Brewery Convention

Date: 2001-06-01
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[email protected]
Quality Manager

De Bry, Luc
INRA
B.P. 71627
44316 Nantes Cedex 3
FRANCE
+33 2 40 67 52 15
+33 2 40 67 50 25
[email protected]
E.U. Marie Curie Fellow

De Keersmaeker, Joseph
Cargill Malt
18/20 rue des Gaudines
B.P. 215
78108 Saint Germain-en-Laye Cedex
FRANCE
+33 327 41 88 99
[email protected]
Consultant

Delatte, Jean-Louis
Malteries Soufflet
Quai Sarrail
10400 Nogent / Seine
FRANCE
+33 3 25 39 41 70
+33 3 25 39 44 80
[email protected]
Vice Technical Manager

Deloison, Marc
Malteries Soufflet
Quai Sarrail
10400 Nogent / Seine
FRANCE
+33 3 25 39 41 70
+33 3 25 39 44 80
[email protected]
Eastern Europe Vice Director

Dessy, Alain
Les Brasseurs de Gayant
B.P. 89
59502 Douai Cedex
FRANCE
+33 3 27 93 26 22
+33 3 27 93 26 24
[email protected]

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Technical Manager

Didierjean, Luc
Brasseries Kronenbourg
68 rte d'Oberhausbergen
B.P. 13
67037 Strasbourg Cedex 2
FRANCE
+33 3 88 274 080
+33 3 88 274 053
[email protected]
Direction Industrielle Brasseries Kronenbourg

Dumoulin, Michel
IFBM
Ple Technologique de Nancy - Brabois
7 rue du Bois de la Champelle
B.P. 267
54512 Vandoeuvre Cedex
FRANCE
+33 3 83 44 88 00
+33 3 83 44 12 90
[email protected]
Analytical Manager

Entzmann, Francoise
Malteries Soufflet
Quai Sarrail
10400 Nogent / Seine
FRANCE
+33 3 25 39 41 70
+33 3 25 39 44 80
[email protected]
Research & Development Engineer

Fick, Michel
ENSAIA
2, avenue de la Fort de haye
BP 172
54505 Vandoeuvre cedex
FRANCE
+33 383 595 801
+33 383 595 804
[email protected]
Professor

Fillaudeau, Luc
INRA-LGPTA
369, Rue Jules Guesde, B.P. 39
59651 Villeneuve d'Ascq
FRANCE

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Date: 2001-06-01
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+33 3 20 43 54 36
+33 3 20 43 54 26
[email protected]
Charg de Recherches

Fliss, Myriam
Malteries Soufflet
Quai Sarrail
10400 Nogent / Seine
FRANCE
+33 3 25 39 41 70
+33 3 25 39 44 80
[email protected]
Research & Development engineer

Fournier, Rgis
IFBM
7 rue du Bois de la Champelle
54500 Vandoeuvre Cedex
FRANCE
+33 3 83 44 88 00
+33 3 83 44 12 90
[email protected]
Molecular Biology Division, Manager

Frelon, Pierre Grard

Malteries Franco Suisses
74 rue des alouettes
36100 Issoudun
FRANCE
+33 2 54 21 13 43
+33 2 54 03 17 78
[email protected]
Commercial Manager

Gay, Pascal
Malteries Soufflet
Quai Sarrail
10400 Nogent / Seine
FRANCE
+33 3 25 39 41 70
+33 3 25 39 44 80

Gosselin, Yves
Lesaffre Developpement
147 Rue Gabriel Peri
B.P. 6027
59706 Marcy-en-Baroeul Cedex
FRANCE
+33 3 20 81 62 21
+33 3 20 89 20 33

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[email protected]
Project Manager

Gros, Christophe
IFBM
7 rue du Bois de la Champelle
B.P. 267
54512 Vandoeuvre Cedex
FRANCE
+33 3 83 44 88 00
+33 3 83 44 12 90
[email protected]

Gutfreund-Jaquet, Laurence
Brasseries Kronenbourg
68 rte d'Oberhausbergen
B.P.13
67037 Strasbourg Cedex 2
FRANCE
+33 3 88 274 809
+33 3 88 274 053
[email protected]
Diretion Brass

Hariri, Ahmed
ENSAIA
2, avenue de la Fort de Haye
BP 172
54505 Vandoeuvre Cedex
FRANCE
+33 383 595 959
+33 383 595 804
[email protected]
phD student

Huvet, Daniel
Malteurop
6 Alle Ren Fonck - BP 1041
51688 Reims Cedex 2
FRANCE
+33 326 78 61 00
+33 326 78 61 48
[email protected]
Managing Director

Kohl, Jean-Marie
Brasserie Heineken S.A.
19 Rue des Deux Gares
92500 Rueil Malmaison
FRANCE
+33 1 47 14 36 75

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+33 1 47 14 28 20
[email protected]
Development and Quality Manager

Lacour, Francoise
IFBM
7 rue du Bois de la Champelle
B.P. 267
54512 Vandoeuvre Cedex
FRANCE
+33 3 83 44 88 00
+33 3 83 44 12 90
[email protected]
Sales Manager

Lavie, Jean
IFBM
7 rue du Bois de la Champelle
B.P. 267
54512 Vandoeuvre Cedex
FRANCE
+33 3 83 44 88 00
+33 3 83 44 12 90
[email protected]
Director

Lefort, Stphane
Cargill Malt
18 Rue des Gaudines
78100 Saint-Germain-en-Laye
FRANCE
+33 1 30 61 35 30
+33 1 30 61 36 50
[email protected]
Malt Sales Manager

Lehrmann, Philippe
Malteries Franco-Suisses
74 Rue des alouettes
30100 Issoudun
FRANCE
+33 2 54 21 13 43
+33 2 54 03 17 78
[email protected]
General Manager

Lemaire, Michel
DSM Food Specialties
20 rue de Ballon
59041 Lille Cedex
FRANCE

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European Brewery Convention

Date: 2001-06-01
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List of Participants

+33 3 28 36 74 00
+33 3 28 36 74 25
[email protected]
Technical Manager

Lesaffre, Patrick
International Malting Company ( I.M.C )
90 rue de Lille
59520 Marquette
FRANCE
+33 320 21 82 30
+33 320 55 64 01
[email protected]
President

Levinson, Jrome
Malteurop
6 Alle Ren Fonck - B.P. 1041
51688 Reims Cedex 2
FRANCE
+33 326 78 61 00
+33 3 26 78 61 48
Export Manager

Lopez, Michel
DSM Food Specialties
15 rue des comtesses
59472 Seclin
FRANCE
+33 3 20 96 43 96
+33 3 20 96 55 70
[email protected]
Scientist

Malpote, Jean-Yves
Brasseries Kronenbourg
B.P. 13
67037 Strasbourg Cedex 2
FRANCE
+33 3 88 27 43 80
+33 3 88 27 43 13
[email protected]
Process and neur products Manager

Marie, Dominique
Malteurop
6 Alle Ren Fonck - BP 1041
51688 Reims Cedex 2
FRANCE
+33 326 78 61 00
+33 326 78 61 48

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European Brewery Convention

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[email protected]
Quality Assurance Manager

Marion, Didier
INRA
B.P. 71627
44316 Nantes Cedex 3
FRANCE
+33 2 40 67 50 56
+33 2 40 67 50 25
[email protected]

Mattiato, Bruno
Malteurop
6 Alle Ren Fonck - BP 1041
51688 Reims Cedex 2
FRANCE
+33 326 78 61 00
+33 326 78 61 48
[email protected]
Agent

Mayance, Jacques
Brasseries Kronenbourg
B.P. 13
67037 Strasbourg Cedex 2
FRANCE
+33 3 88 27 43 72
+33 388 27 43 13
[email protected]
International Assistance Manager

Meens, Hubert
Malteurop
6, Alle Ren Fonck - BP 1041
51688 Reims Cedex 2
FRANCE
+33 326 78 61 00
+33 326 78 61 48
[email protected]
Director of B. M.B.M., subsidiary in China

Mengoli, Luigi
Brasseries Heineken
19 Rue des Deux Gares
92500 Rueil Malmaison
FRANCE
+33 1 47 14 36 55
+33 1 47 14 29 25
[email protected]
General Technical Manager

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Date: 2001-06-01
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Mercier, Christiane
IFBM
7 rue du Bois de la Champelle
B.P. 267
54512 Vandoeuvre Cedex
FRANCE
+33 3 83 44 88 00
+33 3 83 44 12 90
Scientific Adviser

Merle, Rene
Brasserie Fischer
7, Route de Bischwiller
67300 Schiltigheim
FRANCE
+33 3 88 33 82 00
+33 3 88 33 82 01
Directeur Technique

Morand, Robert
Malteurop
6 Alle Ren Fonck - BP 1041
51688 Reims Cedex 2
FRANCE
+33 326 78 61 00
+33 3 26 78 61 48
[email protected]
Industrial Director

Notardonato, Jacky
Brasseries Kronenbourg
68 rte d'Oberhausbergen
B.P. 13
67037 Strasbourg Cedex 2
FRANCE
+33 3 88 27 44 88
+33 3 88 27 42 06
[email protected]
Directeur Industriel et

Proth, Jacques
Brasseries Kronenbourg
68 rte d'Oberhausbergen
B.P. 13
67037 Strasbourg Cedex 2
FRANCE
+33 3 88 274 029
+33 3 88 274 053
[email protected]
Direction Industrielle brasseries Kronenbourg

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Date: 2001-06-01
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List of Participants

Reux, Frdric
176 rue du Temple
75003 Paris
FRANCE

Riedl, Fritz
Brasserie Fischer
7, Route de Bischwiller
67300 Schiltigheim
FRANCE
+33 3 88 33 82 00
+33 3 88 33 82 01
Maitre -Brasseur

Tourrette, Pierre
Association des Brasseurs de France
25 Boulevard Malesherbes
75008 Paris
FRANCE
+33 1 42 66 29 79
+33 1 42 66 07 66
[email protected]
President - Delegue general

Wallart, Christian
Wallart S.A.
B.P. 36
59004 Lille Cedex
FRANCE
+33 3 20 93 66 71
+33 3 20 92 80 74
[email protected]
Director

Vincon, Bernard
Malteurop
6 Alle Ren Fonck - BP 1041
51688 Reims Cedex 2
FRANCE
+33 326 78 61 00
+33 326 78 61 48
[email protected]
Director of Intermalta

Yvonneau, Gilles
Brasseries Heineken S.A.
19 Rue des Deux Gares
92500 Rueil Malmaison
FRANCE
+33 1 47 14 36 68

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European Brewery Convention

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+33 1 47 14 28 20
[email protected]
Head of Quality Insurance in Production

Zimmermann, Didier
Brasseries Kronenbourg
68 rte d'Oberhausbergen
B.P. 13
67037 Strasbourg Cedex 2
FRANCE
+33 3 88 321 620
+33 3 88 314 435
[email protected]
Engineer

FYROM, MACEDONIA

Blazeska, Pande
Prilepska Pivarnica - AD
"Cane Kuzmanoski"-1
Prilep 7500
FYROM, MACEDONIA
+389 98 21 451
+389 98 34 843
[email protected]
Main Director for Technical Issues

Ckatroski, Vladimir
Prilepska Pivarnica - AD
"Cane Kuzmanoski"-1
Prilep 7500
FYROM, MACEDONIA
+389 98 21 451
+389 98 34 843
[email protected]
Chief of Bewhouse

Cosic, Danijela
Pivara Skopje
Str. "808" No. 12
P.O. Box 1184
1000 Skopje
FYROM, MACEDONIA
+389 2 611 341
+389 2 611 482
[email protected]
Engineer in quality control

Janevski, Svetozar
Pivara Skopje AD
Str. " 808" No.12

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List of Participants

P.O. Box 1184

1000 Skopje
FYROM, MACEDONIA
+389 91 612 372
+389 91 613 180
[email protected]
General Manager

Petreski, Tihomir
Pivara Skopje
Str. "808" No. 12
P.O. Box 1184
1000 Skopje
FYROM, MACEDONIA
+389 2 611 248
+389 2 611 482
[email protected]
Marketing Manager

Popovski, Aleksandar
Pivara Skopje
Str. "808" No.12
P.O. Box 1184
1000 Skopje
FYROM, MACEDONIA
+389 2 612 190
+389 2 611 482
[email protected]
Sales Coordinator

Slavkova, Snezana
Pivara Skopje
St. 808 No.12 Skopje
P.O. Box 1184
FYROM, MACEDONIA
+389 2 61 35 35
+389 2 31 14 82
[email protected]
Production Manager

Subotic, Obrad
Pivara Skopje
St. "808" No.12
P.O. Box 1184
1000 Skopje
FYROM, MACEDONIA
+389 2 611 314
+389 2 611 482
[email protected]
Engineer in beer and malt production

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GERMANY

Adler, Karl-Werner
Lwenbru AG
Nymphenburger Strasse 4
80335 Mnchen
GERMANY
+49 89 52 00 32 22
+49 89 52 00 36 89
[email protected]
Director International Brewing

Balk, Georg
Spaten-Lwenbru-Group
Marsstrasse 46-48
80335 Mnchen
GERMANY
+49 89 5122 24 19
+49 89 5122 25 54

Becker, Heinrich
Privatbrauerei Gaffel
Eigelstein 41
50668 Kln
GERMANY
+49 221 160 061 43
+49 221 160 061 37
[email protected]
Geschftsfhrender Gesellschafter

Bellmer, Horst-Gevert
Brauerei Beck & Co.
Am Deich 18/19
28199 Bremen
GERMANY
+49 421 5094 43 15
+49-421-509 43 03
[email protected]
Managing Director/Vice Pres. of EBC

Beyer, Klaus
Versuchs-und Lehranstalt fr Brauerei ( VLB )
Seestrasse 13
13353 Berlin
GERMANY
+49 30 45 080 291
+49 30 45 36 069
[email protected]
Geschftsfhrer

Bosch, Hansjrg

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Date: 2001-06-01
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List of Participants

Deutscher Brauer-Bund e.V.

Postfach 20 04 52
53134 Bonn
GERMANY
+49 228 959 06 30
+49 228 959 06 18
[email protected]
Geschftsfuhrer

Burgess, David
Holsten-Brauerei AG
Holstenstrasse 224
22765 Hamburg
GERMANY
+49 40 38 101 916
+49 40 38 101 211
[email protected]
Technical Consultant

Bhler, Thomas
Fachverlag Hans Carl
Redaktion Brauwelt
Andernacher Strasse 33 A
90411 Nrnberg
GERMANY
+49 911 952 85 37
+49 911 952 85 60
[email protected]
Editor

Delgado, Antonio
TU Mnchen
Lehrstuhl fr Fluidmechanik und Prozessautomation
Weihenstephaner Steig 23
85354 Freising-Weihenstephan
GERMANY
+49 8161 71 3272
+49 8161 71 4510
[email protected]
Professor

Dickhoff, Rolf
KHS Till GmbH
Kapellenstrasse 47 - 49
65830 Kriftel
GERMANY
+ 49 6192 491 123
+ 49 6192 241 97
[email protected]

Doetsch, Michael

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Date: 2001-06-01
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List of Participants

Holsten-Brauerei AG
Holstenstrase 224
22765 Hamburg
GERMANY
+49 40 38 101 740
+49 40 38 101 211
[email protected]
Director Quality Assurance

Drohmann, Christian
BASF AG
ZKS/B - B1
67056 Ludwigshafen
GERMANY
+49 621 60 22 308
+49 621 60 20 313
[email protected]
Research Scientist

Dziondziak, Klaus
Holsten Brauerei AG
Holstenstrasse 224
22765 Hamburg
GERMANY
+49 40 38 101 722
+49 40 38 101 389
[email protected]
Head R & D

Drr, Christian
TU Mnchen
Lst. fr Brauereianlagen
und Lebensmittel-Verpackungstechnik
Weihenstephaner Steig 22
85350 Freising
GERMANY
+49 8161 71 50 58
+49 8161 71 50 58
[email protected]
Scientist

Eger, Carsten
Brauerei Beck & Co.
Am Deich 18/19
28199 Bremen
GERMANY
+49 421 50 94 43 91
+49 421 50 94 81 43 91
[email protected]
Head of Research and Develop.

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European Brewery Convention

Date: 2001-06-01
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List of Participants

Eggers, Guido
Knig-Brauerei GmbH & Co. KG
Friedrich-Ebert-Strasse 255-263
47139 Duisburg
GERMANY
+49 203 455 24 61
+49 203 455 25 63
Leiter Qualittssicherung

Eisemann, Rudolf, A.W.

Fa.Rudolf Eisemann GmbH & Co. KG
Hopfen & Malz
Im Hopfengarten 1-3
74937 Spechbach
GERMANY
+49 6226 43 53
+49 6226 422 33
[email protected]

Eisenbeiss, Christian R.
Holsten-Brauerei AG
Holstenstrasse 224,
22765 Hamburg
GERMANY
+49 40 38 101 386
+49 40 38 101 853
[email protected]
Chairman of the Supervisory Board

Esslinger, Michael
Freiberger Brauhaus AG
Am Frstenwald
09599 Freiberg
GERMANY
+49 3731 363 100
+49 3731 363 291
[email protected]
Sprecher des Vorstands

Evers, Hartmut
Versuchs-und Lehranstalt fr Brauerei ( VLB )
Seestrasse 13
13353 Berlin
GERMANY
+49 30 45 080 225
+49 30 45 36 069
[email protected]
Head of The Technical Dep.

Fischer, Gunther
TU Mnchen

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Lehrstuhl fr Fluidmechanik und Prozeautomation

Weihenstephaner Steig 23
85350 Freising
GERMANY
+49 8161 71 5172
+49 8161 - 71 4510
[email protected]
Engineer

Forster, Adrian
Hopfenveredlung St. Johann
Mainburgerstr.15
93358 St.Johann/Hallertau
GERMANY
+49 9444 878 10
+49 9444 878 78
[email protected]
Technical Managing Director

Franz, Oliver
Technical University of Munich
Lehrstuhl fr Technologie der Brauerei I
Weihenstephaner Steig 20
85354 Freising-Weihenstephan
GERMANY
+49 8161 713 662
+49 8161 713 883
[email protected]
Scientific employee (Dipl.-Ing.)

Fritzsche, Erhard
Krombacher Brauerei GmbH & Co.
Hagener Str. 261
57223 Kreuztal-Krombach
GERMANY
+49 2732 880 400
+49 2732 880 11 401
[email protected]
Geschftsfhrer Technik

Furukubo, Susumu
Suntory Ltd.
Biberstr 25
85354 Freising
GERMANY
+49 81 61 691 79
+49 81 61 691 81
[email protected]
Production Division

Gamal-Eldeen, Amira

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Deutsches Krebsforschungszentrum
Im Neuenheimer Feld 280
69120 Heidelberg
GERMANY
+49 6221 423 331
+49 6221 423 359
[email protected]
Biochemist

Gaub, Reiner
Pall Corporation
Phillip-Reis-Strasse 6
63303 Dreieich
GERMANY
+49 61 03 307 310
+49 61 03 307 339
[email protected]
Business Line Manager

Gauger, Frank
Cargill Malz
Postfach 100247
38202 Salzgitter
GERMANY
+49 5341 224 601
+49 5341 224889
[email protected]
Commercial Manager

Geiger, Eberhard
Lehrstuhl fr Technologie der Brauerei II
Alte Akademie 3
85350 Freising
GERMANY
+49 81 61 713 329
+49 81 61 713 101
[email protected]
Univ.-Prof.Dr.

Geils, Herbert Eilert

Durst Malz
Postfach 1330
76603 Bruchsal 1
GERMANY
+ 49 7251 506 0
+49 7251 506 54
[email protected]
Managing Director

Gerhuser, Clarissa
German Cancer Research Center

Congrex CX System: Copyright 1996-1998 by CONGREX Sweden AB

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Date: 2001-06-01
Page: 1
List of Participants

Div. C0200
Im Neuenheimer Feld 280
69120 Heidelberg
GERMANY
+49 6221 42 3306
+49 6221 42 3359
[email protected]
Group leader Chemoprevention

Gerlach, Eberhard
A. Ziemann GmbH
Schwieberdinger Strasse 86
71636 Ludwigsburg
GERMANY
+49 7141 40 82 29
+49 7141 40 82 22
[email protected]
General Manager

Goebel, Lucas
Brauindustrie
Schloss Mindelburg
87714 Mindelheim
GERMANY
+49 8261 9990
+49 8261 999 395
goebel@sachon
Editor in chief

Gomez, Marcos
BASF AG
ZKS/B - B1
67056 Ludwigshafen
GERMANY
+49 621 60 212 76
+49 621 60 20 313
[email protected]
Research Scientist

Grimpe, Gnter
Pure Malt Products Ltd.
Eibenstrae 55
90513 Zirndorf
GERMANY
+49 911 601 645
+49 911 605 388
[email protected]
Sales Director

Guggeis, Helmut
Schlossbrauerei Kaltenberg

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List of Participants

Augsbruger Str. 41
822 56 Frstenfeldbruck
GERMANY
+49 8141 2 43 151
+49 8141 2 43 181
Technischer Betriebsleiter

Hayler, Dieter
Schlossbrauerei Kaltenberg
Augsburger Str. 41
822 56 Frstenfeldbruck
GERMANY
+49 8141 243 106
+49 8141 243 181

Hendel, Olaf
Brauerei-Forum / VLB Berlin
Seestrasse 13
13353 Berlin
GERMANY
+ 49 30 45 080 255
+ 49 30 45 080 210
[email protected]
Editor

Heyse, Karl-Ullrich
Fachverlag Hans Carl GmbH&Co
Andernacher Strasse 33 A
90411 Nrnberg
GERMANY
+49 911 952 85 22
+49 911 952 85 60
[email protected]
Editor-in-chief

Hiby-Durst, Andreas
Durst Malz
Postfach 1330
76603 Bruchsal 1
GERMANY
+ 49 7251 506 0
+ 49 7251 506 54
[email protected]
Managing Director

Hill, Peter
Brauerei Beck & Co.
Am Deich 18/19
28199 Bremen
GERMANY
+49 421 50 94 40 31

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European Brewery Convention

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+49 421 50 94 44 37
[email protected]
Head of Instrumential Analysis

Homann, Frank
Warsteiner Brauerei
Am Waldpark
59581 Warstein
GERMANY
+49 2902 881 798
+49 2902 882 798
[email protected]
Dipl. Braumaister

Hutter, Karl-Josef
Eichbaum-Brauereien AG
Postfach 120562
Kfertalerstrasse 170
68056 Mannheim
GERMANY

Hyland, Tim
Cargill Malt
Rdenkenstrae 51
38239 Salzgitter-Beddingen
GERMANY
+49 5341 224 804
+49 5341 224 888
[email protected]
Plant Manager

Hhn, Gerrit
Weihenstephaner Steig 22
85350 Freising-Weihenstephan
GERMANY
+49 8161 715 151
+49 8161 714 415
[email protected]
Wissenschaftlicher Assistent

Irie, Ryoichi
Asahi Breweries Ltd.
Adalbart Str. 58
10179 Berlin
GERMANY
+49 30 2759 05 61
+49 30 2759 05 61
[email protected]
Assistant Section Manager

Ishida, Fumito

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Sapporo Breweries Ltd.

Sophienstr. 3
80333 Mnchen
GERMANY
+ 49 89 550 295 67
+49 89 550 295 68
[email protected]
Chief Representative

Junkersfeld, Lydia
Deutscher Brauer-Bund e.v.
Postfach 20 04 52
53134 Bonn
GERMANY
+49 228 959 06 33
+49 228 959 06 18
[email protected]
Referentin des Geschftsfurung

Kaltner, Dietmar
Hopfenveredlung St.Johann
Mainburgerstr. 15
93358 St.Johann/Hallertau
GERMANY
+49 9444 878 26
+49 9444 878 78
[email protected]
R&D

Kaschek, Martin
Im Stadtwald 47
66123 Saarbrcken
GERMANY
+49 681 9345 373
49 681 9345 380
[email protected]
Dipl. -Ing./Engineer

Kimura, Takashi
Asahi Breweries Ltd.
Mainburger Str.19
85356 Freising
GERMANY
+49 8161 938 792
+49 8161 938 791
[email protected]
Assistant Section Manager

Klein, Fritz Michael

Bavaria-St.Pauli-Brauerei GmbH
Hopfenstrasse 15

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20359 Hamburg
GERMANY
+49 40 31 809 200
+49 40 31 809 560
[email protected]
Geschftsfhrer

Kogin, Akira
Suntory Ltd.
Biberstr 25
85354 Freising
GERMANY
+49 81 61 691 79
+49 81 61 691 81
[email protected]
Assitant Brewmaster

Kraus-Weyermann, Thomas
Weyermann Malting Company
Brennerstrasse 17-19
96052 Bamberg
GERMANY
+49 951 932 20 18
+49 951 356 04
[email protected]

Kreisz, Stefan
Lehrstuhl fr Technologie der Brauerei I
Weihenstephaner Steig 20
85354 Freising
GERMANY
+49 8161 71 32 66
+49 8161 71 38 83
[email protected]
Research Associate

Kurz, Tomas
TU Mnchen
Lehrstuhl fr Fluidmechanik und Prozessautomation
Weihenstephaner Steig 23
85354 Freising
GERMANY
+49 8161 71 32 47
+49 8161 71 45 10
[email protected]
Engineer

Ludwig, Andreas
VLB Berlin/ TU Berlin
Seestrasse 13
13353 Berlin

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GERMANY
+49 30 450 80 296
+49 30 453 30 52
[email protected]
University Lecturer

Ludwig, Gebhard
Tivoli Malz GmbH
Reichsbahnstrasse 99
22525 Hamburg
GERMANY
+49 40 540 02 228
+49 40 540 02 372
[email protected]

Lustig, Stefan
Brauerei Beck & Co
Am Deich 18/19
28199 Bremen
GERMANY
+49 421 50 94 43 04
+49 421 50 94 44 37
[email protected]
Head of Quality Department

Manke, Winfried
Weissheimer Malz
Schaarstrasse 1
56626 Andernach
GERMANY
+49 2632 400 236
+49 2632 400 267
[email protected]
Quality Manager

Menu, Sjef
C.Thywissen GmbH
Ursula Strasse 49
50354 Hrth
GERMANY
+49 2233 974 730
+49 2233 974 73 72
[email protected]

Methner, Frank-Jrgen
Bitburger Brauerei Th. Simon GmbH
Rmermauer 3
54634 Bitburg
GERMANY
+49 6561 14 24 44
+49 6561 14 27 65

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[email protected]
Manager Quality-Control & Technology

Meyer-Pittroff, Roland
Bayer. Staatsbrauerei Weihenstephan
Weihenstephaner Steig 22
85350 Freising-Weihenstephan
GERMANY
+49 8161 713 436
+49 8161 714 415
[email protected]
Board member

Michel, Rudolf
HRCH. Huppmann GmbH
Heinrich-Huppmann Strasse 1
97318 Kitzingen
GERMANY
+49 9321 303 276
+49 9321 303 222
[email protected]
Director Brewing & Technology

Mitter, Willi
Simon H. Steiner, Hopfen, GmbH
Auhofstrasse 18
84048 Mainburg
GERMANY
+49 8751 860 50
+49 8751 860 580
[email protected]
Technical Director

Modrok, Alexander
Sartorius AG
Weender Landstr. 94 - 108
37070 Gttingen
GERMANY
+49 551 308 3700
+49 551 308 3754
[email protected]
Marketing Manager Brewing Industry

Mbius, Jrgen
Bhler GmbH
Ernst-Amme-Str 19
38114 Braunschweig
GERMANY
+49 531 594 29 14
+49 531 594 21 90
[email protected]

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Director Brewing and Malting

Mller-Hergt, Gustavo
Warsteiner Brauerei
Am Waldpark
59581 Warstein
GERMANY
+49 2902 88 13 11
+49 2902 88 23 11
[email protected]
Technischer Geschftsfhrer

Nakamura, Takashi
Technical University of Munich
Institute for Brewing Technology I
Weihenstephaner Steig 20
85354 Freising -Weihenstephan
GERMANY
+49 8161 715 271
+49 8161 713 883
[email protected]
Researcher

Niemsch, Klaus
Stabifix Brauerei-Technik KG
Lochhamerschlag 17
82166 Graefelfing
GERMANY
+49 89 854 19 63
+49 89 85 59 33
[email protected]
Managing Director

Oechsle, Dietmar
Seitzchenk Filtersystems
Bettringer Strasse 42
73550 Waldstetten
GERMANY
+49 7171 401 268
+49 7171 401 114
[email protected]
Executive Vice Director

Otomo, Ken
Sapporo Breweries Ltd.
Sophienstr. 3
80333 Mnchen
GERMANY
+ 49 89 550 295 69
+49 89 550 295 68
[email protected]

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Assistant Manager

Papp, Andreas
BASF AG
Domstr. 17
20095 Hamburg
GERMANY
+49 40 32905 140
+49 40 32905 500
[email protected]
Segment Manager - Beverages

Peters, Ulrich
Bitburger Brauerei Th. Simon GmbH
Rmermauer 3
54634 Bitburg
GERMANY
+49 6561 14 23 62
+49 6561 14 25 44
[email protected]
Head of Pilot-plant

Pichlmaier, Johann
HVG e.G.
Preysingstr. 10
85283 Wolnzach
GERMANY
+49 8442 925 80
+49 8442 30 60
[email protected]
Managing Director

Preuss, Thomas
Lehrstuhl fr Technologie der Brauerei I
Weihenstephaner Steig 20
85350 Freising
GERMANY
+49 8161 715 271
+49 8161 713 883
[email protected]
Wissenschaftlicher Angestellter

Rasmussen, Erik Juul

Hannen Brauerei GmbH
Senefelderstrasse 25
41066 Mnchengladbach
GERMANY
+49 2161 667 760
+49 2161 667 491
[email protected]
Gechfts Fuhrer

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Rath, Frank
Versuchs-und Lehranstalt fr Brauerei ( VLB )
Seestrasse 13
13353 Berlin
GERMANY
+49 30 450 801 54
+49 30 450 801 512
[email protected]

Riese, Jens
Aspera Brauerei Riese GmbH & Co.
Rheinstrasse 146-152
45478 Mlheim an der Ruhr
GERMANY
+49 208 588 980
+49 208 592 641
[email protected]
Managing Psrtner

Rost, Andreas
Holsten Brauerei AG
Holstenstrasse 224
22765 Hamburg
GERMANY
+49 40 38 101 719
+ 49 40 38 101 500
[email protected]
Chairman of the Board

Ruiz, Carlos
HVG e.G.
Preysingstr. 10
85283 Wolnzach
GERMANY
+49 8442 925 80
+49 8442 30 60
[email protected]
International Sales Manager

Rusitzka, Peter
Karlsberg Brauerei KG Weber
Postfach 1351
66404 Homburg
GERMANY
+49 6841 105 212
+49 6841 105 498
[email protected]
Managing Director

Sarx, Hans-Georg

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Weissheimer Malz
Schaarstrasse 1
56626 Andernach
GERMANY
+49 2632 400 215
+49 2632 400 210
weissheimerat-online.de
Partner

Schiessl, Franz
Privatbrauerei Diebels GmbH & Co.KG
Brauerei-Diebels-Strasse 1
47661 Issum
GERMANY
+49 2835 304 01
+46 2835 303 05
[email protected]
Geschhftsfhrer Technik

Schill, Carl Otto

Schill-Malz GmbH & Co KG
Ludwig-Schwamb Strasse 9-11
67574 Osthofen
GERMANY
+49 6242 910 20
+49 6242 9102 11
[email protected]
Managing Director

Schill, Peter
Schill-Malz GmbH & Co KG
Ludwig-Schwamb Strasse 9-11
67574 Osthofen
GERMANY
+49 6242 910 20
+49 6242 9102 11
[email protected]
Managing Director

Schmitt, Traudel
Fachverlag Hans Carl
Andernacher Str.33 A
90411 Nrnberg
GERMANY
+49 911 952 85 58
+49 911 952 85 47
[email protected]
Executive Publisher

Schmitz, Karl Georg

Holsten Brauerei AG

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Holstenstrasse 224
22765 Hamburg
GERMANY
+49 40 38 101 693
+49 40 38 101 560
[email protected]
Director Brewery Operations

Schmucker, Fritz Ludwig

Deutsche Gesellschaft fr Hopfenforschung
Postfach 1148
8529 Wolnzach-Hll
GERMANY
+49 84 42 35 97
+49 84 42 28 71
Geschftsfuhrer

Schneider, Jan
TU Mnchen
Weihenstephaner Steig 22
85354 Freising
GERMANY
+49 8161 71 5058
+49 8161 71 4515
[email protected]
Assistant Professor

Schorn, Rudolf
Warsteiner Brauerei
Am Waldpark
595 81 Warstein
GERMANY
+49 2902 88 12 87
+49 2902 88 22 87
[email protected]
Leiter Forschung und Entwicklung

Schuch, Christopher
KHS Maschinen -und Anlagenbau AG
Planinger Str. 139-147
55543 Bad Kreuznach
GERMANY
+49 671 852 22 01
+49 671 852 26 90
Manager beverage Technology

Schur, Fritz
Doemens Academy
Stefanusstrasse 8
Graefelfing / Munich
GERMANY

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+49 170 224 39 43

+49 89 89 86 09 11
[email protected]
Managing Director

Simon, Axel Th.

Bitburger Brauerei Th. Simon GmbH
Rmerstrasse 3
Postfach 1164
54621 Bitburg
GERMANY
+49 6561 14 22 04
+49 6561 14 8 22 04
[email protected]
Dr. - Engineer

Stempfl, Wolfgang
Doemens e.V.
P.O. Box 1325
82155 Grfeling
GERMANY
+49 89 858 05 20
+49 89 858 05 26
[email protected]
Head of wba - Technikum

Stippler, Kurt
Anton Steinecker Maschinenf. GmbH
Raiffeisenstrasse 30
85356 Freising
GERMANY
+49 81 61 953 170
+49 81 61 953 157
[email protected]
Manager Technology Dept.

Strauss, David
Krones AG
Bhmerwaldstr. 5
93068 Neutraubling
GERMANY
+49 170 630 5918
+49 9401 703 056
[email protected]

Strohofer, Michael
Flottweg GmbH
Industriestr. 6-8
841 37 Vilsbiburg
GERMANY
+49 8741 301 355

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+49 8741 301 303

[email protected]

Temme, Wilmar
Knzel Maschinenbau GmbH
Industriestrasse 7
95336 Mainleus
GERMANY
+49 92 29 880
+49 92 29 881 88
[email protected]
Betriebswirt Geschftsfuhrer

Treptau, Bernd
Frstlich Frstenbergische Brauerei KG
Postplatz 1-4
78166 Donaueschingen
GERMANY
+49 771 86 350
+49 771 86 396
[email protected]
Gedchftsfhrer

Wackerbauer, Karl
TU/ VLB
Artuswall 13
13465 Berlin
GERMANY
+49 30 401 12 88
+49 30 401 38 79
[email protected]
Prof.

Weihe, Klaus
Simon H. Steiner, Hopfen, GmbH
Postfach 1373
84044 Mainburg
GERMANY
+49 8751 86 05 0
+49 8751 86 05 80
[email protected]
Vice President

Weisser, Horst
TU/ Mnchen
Lehrstuhl fr Brauereianlagen und
Lebensmittel-Verpackungstechnik
Weihenstephaner Steig 22
85350 Freising
GERMANY
+49 8161 713 126

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+49 8161 714 515

[email protected]
Professor

Weissheimer, Helmut
Friedrich Weissheimer Malzfabrik
Schaarstrasse 1
56626 Andernach
GERMANY
+49 2632 400 216
+49 2632 400 210
[email protected]
Managing Partner

Wiese, Michael
Tivoli Werke AG
P.O.Box 540209
22502 Hamburg
GERMANY
+49 40 540 020
+49 40 540 02 312

Voetz, Michael
Versuchs-und Lehranstalt fr Brauerei ( VLB )
Seestrasse 13
13353 Berlin
GERMANY
+49 30 450 802 34
+49 30 453 13 90
[email protected]

Voigt, Tobias
TU-Mnchen
Lehrstuhl fr Brauereianlagen und
Lebensmittel-Verpackungstechnik
Weihenstephaner Steig 22
85350 Freising
GERMANY
+49 8161 71 4377
+49 8161 71 4515
[email protected]
Wissenschaftlicher Mitarbeiter

Volz, Detlef
C.Thywissen
Industriestrasse 34
41460 Neuss
GERMANY
+49 2131 26041
+49 2131 2604 220
[email protected]

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Manager

von Bayern, Prinz, Luitpold

Schlossbrauerei Kaltenberg
Augsburger Str. 41
822 56 Frstenfeldbruck
GERMANY
+49 8141 2 43 106
+49 8141 2 43 181
[email protected]
Managing Director

Vongeheur, Hermann-Otto
A. Ziemann GmbH
Schwieberdinger Str. 86
716 36 Ludwigsburg
GERMANY
+49 71 41 40 82 34
+49 71 41 40 82 22
[email protected]
General Manager

Zarnkow, Martin
TU Mnchen
Lehstuhl fr Technologie der Brauerei I
Weihenstephaner Steig 20
85354 Freising
GERMANY
+49 8161 71 32 63
+49 8161 71 38 83
[email protected]
Research Associate

Ziehl, Jrgen
BASF AG
67056 Ludwigshafen
GERMANY
+49 621 60 947 87
+49 621 60 946 11
[email protected]
Product Manager

Zimmermann, Thomas
Joh. Barth & Sohn
Freiligrathstr. 7/9
90482 Nuernberg
GERMANY
+49 911 5489 375
+49 911 5489 340
[email protected]
Technical Services Manager, QM

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Zrcher, Achim
TU Mnchen
Lehrstuhl frTechnologie der Brauerei 1
Weihenstephaner Steig 20
85354 Freising
GERMANY
+49 8161 713 662
+49 8161 713 883
[email protected]
Scientist

GREECE

Hollemans, Marja
Athenian Brewery S.A.- Heineken Group
102 Kifissos Avenue
12241 Egaleo - Athens
GREECE
+30 1 5384 320
+30 1 5384 220
[email protected]
Control Manager

Moraitis, Dimitris
Athenian Brewery S.A.- Heineken Group
102 Kifissos Avenue
12241 Egaleo - Athens
GREECE
+30 1 5384 310
+30 1 5385 361
[email protected]
Technical Director

HUNGARY

Bakone Horvath, Ibolya

Brau Union Hungria Srgyrak RT
Quality Control Department
Vndor Sndor u.1.
9400 Sopron
HUNGARY
+36 99 516 195
+36 99 516 251
[email protected]
Microbiologist

Balazs, Ferenc
Dreher Breweries
7-11 Street Jaszberenyi
1106 Budapest

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HUNGARY
+36 1 432 9760
+36 1 432 9762
[email protected]
Process Manager

Baranyai, Zoltn
Amstel Brewery Hungary
Tzolt t 2.
2922 Komrom
HUNGARY
+36 34 340 884
+36 34 345 312
[email protected]
Quality Assurance Manager

Bndek, Gyrgy
Assoc. of the Hungarian Brewers
Fogarasi u. 18
1148 Budapest
HUNGARY
+36 1 221 2806
+36 1 221 2806
[email protected]

Cosser, Keith
South African Breweries ( SABIM Kft )
Maglodi Ut 17
1106 Budapest
HUNGARY
+36 1 432 99 03
+36 1 432 99 04
[email protected]
Packaging Consultant

Farkas, Gabriella
Szent Istvn University
Dep. of Brewing and Distilling
Menesi Ut 45
1118 Budapest
HUNGARY
+36 1 372 6214
+36 1 372 6214
[email protected]
Ph. D. Student

Ficsor, Attila
Dreher Breweries
7-11 Street Jaszberenyi
1106 Budapest
HUNGARY

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+36 1 432 9711

+36 1 432 9759
[email protected]
Technical Director

Gati, Levente
Alltech Hungary KFT
Gyr u.2
2040 Budars
HUNGARY
+36 23 418 939
+36 23 418 930
[email protected]
General Manager

Helembai, Jeno
Alltech Hungary KFT
Gyr u.2
2040 Budars
HUNGARY
+36 23 418 939
+36 23 418 930
[email protected]
Director of Sales

Hoschke, Agoston
Szent Istvn University
Department of Brewery and Distilling
Mnesi t. 45
1118 Budapest
HUNGARY
+36 1 372 62 15
+36 1 372 62 14
[email protected]
Head of Department

Jozsa, Zoltn
Amstel Brewery Hungary
Tzolt t 2.
2922 Komrom
HUNGARY
+36 34 340 884
+36 34 345 312
[email protected]
Eng. and Pack. Manager

Kindl, Gbor
Albadomu Malta Bt.
2400 Dunajvros
Szalki-sziget
HUNGARY

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+36 25 513 100

+36 25 513 108
[email protected]
Managing Director

Kovacs, Karoly
HUNGARY

Mester, Raimund
Brau Union Hungria Srgyrak RT
Vndor Sndor u.1.
9400 Sopron
HUNGARY
+36 99 516 194
+36 99 516 243
[email protected]
Technical Support Mgr

Nemeth, Andras
Dreher Breweries
7-11 Street Jaszberenyi
1106 Budapest
HUNGARY
+36 1 432 9524
+36 1 432 9713
[email protected]
Brewhouse Manager

Nemeth, Arpd
Brau Union Hungria Srgyrak RT
Vndor Sndor u.1.
9400 Sopron
HUNGARY
+36 99 516 145
+36 99 516 226
[email protected]

Patard, Guy
Malterie Soufflet Magyarorszag
Csengery u. 111
8800 Nagykanizsa
HUNGARY
+36 93 326 593
+36 93 310 168
Maltings Manager

Spi, Rbert
Borsodi Sorgyr Rt.
POB 6
Bcs 3574
HUNGARY

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+36 46 318 400

+36 46 318 388
[email protected]
Purchasing and Malting Director

Scheller, Ludwig
Albadomu Malta Bt.
H-2400 Dunajvros
Szalki-sziget
HUNGARY
+36 25 513 100
+36 25 513 108
[email protected]
Managing Director

Simon, Eva
Dreher Breweries
7-11 Street Jaszberenyi
1106 Budapest
HUNGARY
+36 1 432 9775
+36 1 432 9838
[email protected]
Quality Manager

Tokaji, Zsolt
Amstel Brewery Hungary
Szabadsg Ut 117
2040 Budars
HUNGARY
+36 23 507 333
+36 23 507 330
[email protected]

Tmr, Lszl
Association of Hungarian Brewers
Magldi Ut. 17
1106 Budapest
HUNGARY
+36 1 431 71 08
+36 1 261 64 60
[email protected]
President

Varj, Peter
Brau Union Hungria Srgyrak RT
Quality Control Department
Vndor Sndor u.1.
9400 Sopron
HUNGARY
+36 99 516 128

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+36 99 516 251

[email protected]
Quality Assurance Manager

IRELAND

Cahill, Gearid
Guinness Ltd
Technology House
St. James's Gate
Dublin 8
IRELAND
+353 1 453 67 00
+353 1 408 48 16
[email protected]

Campbell-Crawford, Nigel
Irish Brewers Association
13 Stamer St.
Dublin 8
IRELAND
+353 1 475 54 51
+353 1 475 54 51
[email protected]
Dr.

D'arcy, James
Beamish Crawford Ltd.
South Main Street
Cork
IRELAND
+353 21 49 111 89
+353 21 49 111 11
jim.darcy@beamish
Production Director

Horan, Hugho
Greencore Malting Group
The Maltings
Athy Co Kildare
IRELAND
+353 507 40 300
+353 507 310 46
[email protected]
Tech. Dir.

Jones, Phil
Malting Company of Ireland
The Maltings
South Link, Togher
Cork

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IRELAND
+353 21 432 33 75
+353 21 431 32 53

Jordan, Patrick
Irish Brewers Association
Confederation House
84 Lower Baggot Street
Dublin 2
IRELAND
+353 1 605 15 58
+353 1 638 15 58
[email protected]
Director

Walsh, Padraig
Dublin City University
School of Biotechnology
Dublin 9
IRELAND
+353 1 700 53 93
+353 1 700 54 12
[email protected]
Lecturer i Bioprocess Engineering

ISRAEL

Slootweg, Gysbert
Tempo Beer /Heineken International
P.O. Box 127
Netanya 42101
ISRAEL
+972 9 863 06 12
972 9 865 0720
[email protected]

ITALY

Breitenstein, Frank
Carlsberg Italy
Via Olona, 103
21056 Inovno Olona ( VA )
ITALY
+39 0332 208 111
+39 0332 200 801
[email protected]
Direttore Tecnico Centrale

Cason, Michele
Saplo Spa Soc. Prod. Lavorazione Orzo
Via Naro 39

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00040 Pomezia (Roma)

ITALY
+39 6 9120 194
+39 6 9120 116
[email protected]

Cavalli, Roberto
Birra Peroni Industriale SpA
Direzione Generale
Via Renato Birolli 8
00155 Roma
ITALY
+39 6 225 442 92
+39 6 225 443 13
[email protected]
Director of Operations

Mammi, Giuseppe
Heineken Italia SpA
Via Spirano 26
24040 Comun Nuovo (BG)
ITALY
+39 035 409 306
+39 035 409 222
[email protected]
Technologist

Marturano, Gennaro
SpA Birra Peroni Industriale
Via R.Birolli 8
00155 Roma
ITALY
+39 062 254 44 01
+39 062 228 34 45
[email protected]
Brewmaster

Sbuelz, Raffaele
SpA Birra Peroni Industriale
Via R.Birolli 8
00155 Roma
ITALY
+39 062 254 44 91
+39 062 254 43 13
[email protected]
Responsabile Laboratorio Centrale

Scopel, Adelchi
SpA Birra Peroni Industriale
Via R.Birolli 8
00155 Roma

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ITALY
+39 062 254 43 07
+39 062 254 43 13
[email protected]
Brewing Director

Zangrando, Tullio
Birra E Malto
Via Trento 79
32034 Pedavena
ITALY
+39 0439 30 37 37
+39 0439 31 72 73
[email protected]
Editor

Zasio, Giorgio
SpA Birra Peroni Industriale
Via R.Birolli 8
00155 Roma
ITALY
+39 062 254 43 09
+39 062 254 43 13
[email protected]
Dipl. Brewmaster

JAPAN

Eto, Masakazu
Asahi Breweries, Ltd
1-21, Midori 1-Chome,
Moriya-Machi
Kitasoma-Gun
Ibaraki 302-0106
JAPAN
+81 297 46 15 10
+81 297 46 12 06
[email protected]
General Manager Brewing R&D Laboratory

Fukui, Nobuyuki
Suntory
1-1-1 Wakayamadai
Shimamoto-Cho, Mishima-Gun
Osaka 618-8503
JAPAN
+81 75 962 88 02
+81 75 962 82 62
[email protected]
Institute for fundamental research

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Furusho, Shigeki
Sapporo Breweries Ltd. / BCOJ
10 - Okatohme,
Yaizu,
Shizuoka 425-0013
JAPAN
+81 54 629 79 83
+81 54 629 31 44
[email protected]
Manager

Harayama, Koichi
Asahi Breweries Ltd
1-21, Midori 1 Chome,
Moriya-Machi
Kitasuma-Gun
Ibaraki 301-0106
JAPAN
+81 297 46 15 04
+81 297 46 15 05
[email protected]
Deputy Manager

Hirota, Toru
Kirin Brewery Company Limited
1-17-1, Namamugi 1
Tsurumi-ku
Yokohama 230-8628
JAPAN
+81 45 521 20 75
+81 45 521 50 53
[email protected]

Hosoda, Hiroshi
Miyake Industries Co., Ltd
Taito-Ku, Kotobuki 3-17-7
Tokyo, 111-0042
JAPAN
+81 3 5828 26 66
+81 3 5828 26 67
[email protected]
Design section

Imai, Takeo
Kirin Brewery Co., LTD.
1-17-1, Namamugi, Tsurumi-ku,
Yokohama, 230-8628
JAPAN
+81 45 503 90 93
+81 45 503 82 85
[email protected]

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Manager

Kawasaki, Yasutsugu
Suntory Ltd.
Yakayamadai 1-1-1,
Shimamotocho, Mishima-Gun
Osaka
JAPAN
+81 75 962 7314
+81 75 962 8915
[email protected]
New Production

Kihara, Makoto
Sapporo Breweries Ltd.
Kizaki, Nitta, Gunma
370-0393
JAPAN
+81 276 56 1455
+81 276 56 1605
[email protected]
Cereal chemist

Kinoshita, Muneshige
Asahi Breweries,Ltd.
1-23-1,Azumabashi,Sumida-Ku
Tokyo
JAPAN
+81 3 5608 5216
+81 3 5608 5124
[email protected]
Senior General Manager

Koyanagi, Masahiro
Asahi Breweries Ltd
1-21, Midori 1 Chome,
Moriya-Machi
Kitasuma-Gun
Ibaraki 301-0106
JAPAN
+81 297 46 18 26
+81 297 46 18 29
[email protected]

Kuroda, Hisao
Sapporo Breweries
10 Okatome, Yaizu
Shizuoka 425-0013
JAPAN
+81 54 629 7983
+81 54 629 3144

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[email protected]
Biochemist

Miyake, Hidekazu
Miyake Industries Co. Ltd
Taito-ku, Kotobuki 3-17-7
Tokyo 111-0042
JAPAN
+81 3 5828 26 66
+81 3 5828 26 67
[email protected]
President

Nagatani, Kazuya
Alcoa CSI Japan
Sk Building 2-7-4 Nishi-Shibashi
Minato-ku Tokyo
JAPAN
+81 355 11 00 36
+81 355 11 21 61
[email protected]

Nakatani, Kazuo
Suntory Tonegawa Brewery
2712 Akaiwa Kurakake
Chiyoda Machi Gunmaken
JAPAN
+81 276 86 52 11
+81 276 86 54 82
[email protected]
General Manager

Nakatsu, Hirotaka
Kirin Brewery Co Ltd
1-17-1 '
Yokohama 230-8625
JAPAN
+81 45 521 49 19
+81 45 509 39 25
[email protected]

Ohkochi, Motoo
Kirin Brewery
1-17-1,Namamugi,Tsurumi-ku
Yokohama, 230-8628
JAPAN
+81 45 503 9001
+81 45 503 8285
[email protected]
Brewmaster

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Ohno, Toshihiko
Kirin Brewry Co., Ltd
Central Laboratories for Key Technology
1-13-5, Fukuura, Kanazawa-ku
Yokohama 236-0004
JAPAN
+81 45 788 7200
+81 45 788 4041
[email protected]
General Manager

Ozaki, Kazutaka
Asahi Breweries Ltd
1-21, Midori 1-Chome
Moriya-Machi
Kitasoma-Gun
Ibaraki 302-0106
JAPAN
+81 297 46 15 04
+81 297 46 15 05
[email protected]
Deputy Manager

Sakamoto, Kanta
Asahi Breweries Ltd
1-21, Midori 1-Chome
Moriya-Machi
Kitasoma-Gun
Ibaraki 302-0106
JAPAN
+81 297 46 15 04
+81 297 46 15 05
[email protected]
Chief

Shibata, Kazunori
Asahi Breweries Ltd.
1-21, Midori 1-Chome
Moriya-machi
Kitasoma-Gun
Ibaraki 302-0106
JAPAN
+81 297 46 15 13
+81 297 46 15 14
[email protected]
Manager

Sone, Hidetaka
Kirin Brewery Co., Ltd.
Central Laboratories for Key Technology
1-13-5, Fukuura, Kanazawa-ku

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Yokohama 236-0004
JAPAN
+81 45 788 7220
+81 45 788 4047
[email protected]
Senior Research Scientist

Takahashi, Koichiro
Asahi Breweries Ltd.
1-21, Midori 1-Chome
Moriya-Machi
Kitasoma-Gun
Ibaraki 302-0106
JAPAN
+81 297 46 15 12
+81 297 46 12 06
[email protected]

Takahashi, Susumu
Sapporo Breweries Ltd.
37-1 Kizaki, Nitta, Gumma
370-0321
JAPAN
+81 276 56 1455
+81 276 56 1605
[email protected]
Senior Breeder

Takashio, Masachika
Sapporo Breweries Ltd.
10 Okatohome
Yaizu
Shizuoka 425-0013
JAPAN
+81 54 629 7980
+81 54 629 3144
[email protected]
Director of Brewing Research Laboratories

Tanikawa, Mitsuru
Kirin Brewery Company Limited
Shinkawa 2-10-1
Chuo-ku
Tokyo 104-8288
JAPAN
+81 3 55 40 35 12
+81 3 55 40 35 36
[email protected]

Ueda, Tsutomu
Asahi Breweries Ltd.

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1-21, Midori 1-Chome

Moriya-machi
Kitasoma-Gun
Ibaraki 302-0106
JAPAN
+81 297 46 15 13
+81 297 46 15 14
[email protected]
Chief

Uemura, Kazuhiko
Asahi Breweries
1-1 Midori 1-Chome, MoiIya
Ibaraki, 302-0106
JAPAN
+81 297 45 7111
+81 297 45 7131
[email protected]
Brewer

Watari, Junji
Sapporo Breweries Ltd
Brewing Research Laboratories
10 Okatome, Yaizu
Shizuoka 425-0013
JAPAN
+81 54 629 7980
+81 54 629 3144
[email protected]
General Manager

Yamashita, Akitsugu
Sapporo Breweries Ltd.
Brewing Material & Purchasing Dep.
4-20-1 Yebisu, Shibuya-ku
Tokyo 150-8686
JAPAN
+813 5423 7244
+81 3 5423 7265
[email protected]
Manager

Yuji, Nishida
Suntory Ltd
Research Institute for New Product Development
1-1-1, Wakaymadai, Shimamoto-Cho, Mishima-Gun
618-8503 Osaka
JAPAN
+81 75 962 73 14
+81 75 962 89 15
[email protected]

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Research Institute for New Product Development

LATVIA

Gavrilovs, Vitalijs
Aldaris Brewery
44 Tvaika St
Riga 1005
LATVIA
+371 702 32 01
+371 702 32 24
[email protected]
President, Chairman of Board

Sure, Inara
A/S Aldaris
44 Tvaika Str.
Riga 1005
LATVIA
+371 7 023 231, 023241
+371 7 023 240
[email protected]
Production and Technical Development Dep. Director

LUXEMBOURG

Thix, Andre
Micro Matic S.A.
18, Rue de Drinklange
P.O. Box 33
9911 Troisvierges
LUXEMBOURG
+352 97 90 30
+352 97 90 60
[email protected]
Group Sales Director

Wagner, Peter
Brasserie Bofferding
2, Boulevard J.F. Kennedy
4930 Bascharage
LUXEMBOURG
+352 509 011 236
+352 509 011 282
Techn.Director, Dipl-Brewmaster

MEXICO

Dominguez, Agustin
Cerv. Cuauhtemoc Moctezuma Toluca
Carretera Mexico-Toluca Km 59.5

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Toluca Edo. de Mexico C.P. 50071

MEXICO
+1 5272 79 81 44
+1 5272 89 81 45
adommac.ccm.femsa.com.mx
Brewing Manager

Santana, Felipe
J.Jess Ponce #625
Col.Lomas de Circunvalacin
28010 Colima, Colima
MEXICO
consultant

Sierra, J. Antolin
Cerveceria Cuauhtemoc Moctezuma Toluca
Av. Alfonso Reyes 2202 Nte. Col. Bellavista
Monterrey, N.L.
MEXICO
+1 528 328 51 20
+1 528 328 51 27
[email protected]
Director of Process Development

MOROCCO

Bagoulla, Lahsen
SBM Brasseries du Maroc
Bd. Ahl Loghlam, B.P 2660- Ain Sebaa
Casablanca
MOROCCO
+21 22 75 46 46 / 75 49 20
+21 22 75 49 64
Directeur Industriel

Khrouz, Hssain
SBM Brasseries du Maroc
Bd. Ahl Loghlam. B.P 2660-An Sebaa
Casablanca
MOROCCO
+21 22 75 46 46, 22 75 49 20
+21 22 75 49 64
Chef Production

Lahlou, Lamfadel
SBM Brasseries du Maroc
Bd. Ahl Loghlam. B.P 2660-Ain Sebaa
Casablanca
MOROCCO
+212 2 75 46 46, 22 75 49 20
+212 2 75 49 64

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Directeur Dexploitation

NETHERLANDS

Bouwmeester, Dini
Heineken Technical Services B.V.
P.O. Box 510
2380 BB Zoeterwoude
NETHERLANDS
+31 71 545 62 10
+31 71 545 70 34
[email protected]
Production Manager Brewing

Bruijn, Paulus
Heineken Technical Services
P.O. Box 510
2380 BB Zoeterwoude
NETHERLANDS
+31 71 545 6859
+31 71 545 7204
[email protected]
Environmental Specialist

Coucke, Andre
European Patent Office (EPO)
Patentlaan 2
PO BOX 5818
2280 HV Rijswijk
NETHERLANDS
+31 703 402 444
+31 703 403 988
[email protected]
Examiner

de Groen, Andries
Grolsche Bierbrouwerij Nederland B.V.
P.O. Box 55
7500 AB Enschede
NETHERLANDS
+31 53 483 31 66
+31 53 483 31 15
[email protected]
Logistics Director

De Man, Thomas
Heineken Internationaal Beheer B.V.
P.O. Box 530
2380 BD Zoeterwoude
NETHERLANDS
+31 71 545 6055

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+31 71 545 7450

[email protected]
Corp. Prod. Policy & Control Director

Doderer, Albert
Heineken Technical Services B.V.
Burgemeester Smeetsweg 1
2382 PH Zoeterwoude
NETHERLANDS
+31 71 545 72 70
+31 71 545 72 08
[email protected]

Douma, Anneke
TNO Nutrition and Food Research Institute
P.O. Box 360
3700 AJ Zeist
NETHERLANDS
+31 30 69 44 381
+31 30 69 44 295
[email protected]
Product Manager Beverages

Driessen, Willie
Paques B.V.
P.O. Box 52
8560 AB Balk
NETHERLANDS
+31 514 60 85 00
+31 514 60 33 42
[email protected]
AREA MANAGER

Govaert, Arthur
Heineken International B.V
P.O. Box 530
2380 BD Zoeterwoude
NETHERLANDS
+31 71 545 6920
+31 71 545 7580
[email protected]
Engineering Policy Manager

Gross, Marc
Heineken International
P.O Box 530
2380 BD Zoeterwoude
NETHERLANDS
+31 71 545 74 22
+31 71 545 73 70
[email protected]

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Regional Technical Manager

Haeck, Jos
Mouterij Kloosterzande
P.O. Box 32
4587 ZG Kloosterzande
NETHERLANDS
+31 114 681 930
+31 114 681 202
[email protected]
Manager

Haitink, Maarten
Heineken International
P.O. Box 530
2380 BD Zoeterwoude
NETHERLANDS
+31 71 545 60 84
+31 71 545 74 50
[email protected]
Brewing Policy Manager

Heemskerk, Piet
Heineken
Burg Smeetsweg 1
2382 PH Zoeterwoude
NETHERLANDS
+31 71 545 64 31
+31 71 545 74 50
[email protected]
European Production Coordinator

Heinen, Nico
Royal Grolsch N.V.
P.O. Box 55
7500 AB Enschede
NETHERLANDS
+31 53 4833 200
+31 53 483 31 82
[email protected]
Technical Director

Holterman, Menno
Haffmans B.V
Marinus Dammeweg 30
5928 PW Venlo
NETHERLANDS
+31 77 323 23 00
+31 77 323 23 23
[email protected]
Managing Director

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Hughes, Paul
Heineken Technical Services
Burgemeester Smeetsweg 1
2382 PH Zoeterwoude
NETHERLANDS
+31 71 545 7941
+31 71 545 7208
[email protected]

Kakebeeke, Marien
Heineken Nederland B.V.
P.O. Box 530
2380 BD Zoeterwoude
NETHERLANDS
+31 71 545 61 41
+31 71 545 78 81
[email protected]
Director Suply cHAIN Mgmt

Kool, Willem
Heineken International
P.O. Box 510
2380 BB Zoeterwoude
NETHERLANDS
+31 71 545 71 22
+31 71 545 74 50
[email protected]
Operations Strategy Manager

Landman, Berco
Heineken Technical Services B.V
Burgemeester Smeetsweg 1
P.O Box 510
2380 BB Zoeterwoude
NETHERLANDS
+31 71 545 71 88
+31 71 545 67 78
[email protected]
Senior Manager Packaging

Meersman, E.
Bavaria NV
P.O. Box 1
5737 ZG Lieshout
NETHERLANDS
+31 499 428 111
+31 499 428 269
[email protected]

Muller Kobold, Wouter

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Grolsche Bierbrouwerij Nederland B.V.

P.O. Box 55
7500 AB Enschede
NETHERLANDS
+31 53 483 33 33
+31 53 483 35 85
[email protected]
Manager Grolsch Innov. & Techn.

Peppink, Jan Willem

Grolsche Bierbrouwerij Nederland B.V.
P.O. Box 55
7500 AB Enschede
NETHERLANDS
+31 53 483 32 80
+31 53 483 31 59
[email protected]
Director Production

Reuchlin, Henri
Centraal Brouwerij Kantoor
Herengracht 282
1016 BX Amsterdam
NETHERLANDS
+31 20 625 22 51
+31 20 622 60 74
[email protected]

Schans, Maarten J.
TNO Nutrition and Food Research Institute
Utrechtseweg 48
P.O. Box 360
3700 AJ Zeist
NETHERLANDS
+31 30 694 44 86
+31 30 694 42 95
[email protected]
Projectleader Malting and Brewing Techn.

Schippers, Hennie
European Brewery Convention
P.O. Box 510
2380 BB Zoeterwoude
NETHERLANDS
+31 71 545 66 14
+31 71 541 00 13
[email protected]
Assistant Secretary EBC

Schuurman, Rik
Norit Process Technology B.V.

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List of Participants

Opaalstraat 22
7554 TS Hengelo
NETHERLANDS
+31 74 256 00 00
+31 74 256 00 01
[email protected]
Account Manager Drinks

Seldeslacht, Dirk
Interbrew Nederland N.V
Ceresstraat 13
Postbus 3212
4800 MA Breda
NETHERLANDS
+31 1761 525 21 92
+31 1761 525 24 18
[email protected]

Sloesen, Jos
Haffmans B.V.
Marinus Dammeweg 30
5928 PW Venlo
NETHERLANDS
+31 77 323 23 00
+31 77 323 23 23
[email protected]
Senior Product Manager

Swinkels, G.J.C.M
Bavaria Maltings
P.O. Box 1
5737 ZG Lieshout
NETHERLANDS
+31 499 428 111
+31 499 428 234
[email protected]

Swinkels, L.G.F.M
Bavaria N.V
P.O. Box 1
5737 ZG Lieshout
NETHERLANDS
+31 499 428 111
+31 499 428 269
[email protected]

Tiktak, K.
Heineken Technical Services
Burgemeester Smeetsweg 1
2382 PH Zoeterwoude
NETHERLANDS

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+31 71 545 7046

+31 71 545 7800
[email protected]

Ubbink, Herman
Heineken
Rietveldenweg 37
5222 AP 's-Hertogenbosch
NETHERLANDS
+31 73 620 93 00
+31 73 620 93 04
[email protected]
Brewery Manager

Van den Berg, Rob

Heineken Technical Services B.V
P.O Box 510
2380 BB Zoeterwoude
NETHERLANDS
+31 71 545 70 20
+31 71 547 77 30
[email protected]
Departement Manager

van der Stappen, Leo

Royal Grolsch N.V.
P.O. Box 55
7500 AB Enschede
NETHERLANDS
+31 53 483 32 00
+31 53 483 31 82
[email protected]
Director Technological control and Services

Van Dieren, B.
Bavaria NV
P.O. Box 1
5737 ZG Lieshout
NETHERLANDS
+31 499 428 111
+31 499 428 269
[email protected]

van Wijngaarden, Marjolein

European Brewery Convention
P.O. Box 510
2380 BB Zoeterwoude
NETHERLANDS
+31 71 545 00 47
+31 71 541 00 13
[email protected]

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Secretary General EBC

Vereijken, Tom
Paques B.V.
P.O.Box 52
8560 AB Balk
NETHERLANDS
+31 514 608 500
+ 31 514 603 342
[email protected]
Marketing Manager

Verkoelen, Frank
Haffmans B.V.
Marinus Dammeweg 30
5928 PW Venlo
NETHERLANDS
+31 77 323 23 00
+31 77 323 23 23
[email protected]
Senior Product Manager

Voss, Hans-Peter
Heineken Technical Services BV
Burgemeester Smeetsweg 1
2382 PH Zoeterwoude
NETHERLANDS
+31 71 545 7696
+31 71 545 7208
[email protected]

NEW ZEALAND

Inglis, Tom
New Zealand Hop Marketing Board
P.O.Box 3205
Richmond, Nelson
NEW ZEALAND
+64 3 544 11 51
+64 3 544 60 07
[email protected]
Board Chairman

NORWAY

Aasprong, Anne
Ringnes AS
Thv. Meyersgt. 9-11
P.O. Box 7152 Maj.
0307 Oslo
NORWAY

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+47 22 06 95 00
+47 22 06 97 30
[email protected]
Development Manager

Hage, Tore
Ringnes AS
P.O. Box 7152 Maj.
0307 Oslo
NORWAY
+47 933 25 164
+47 22 06 99 02
[email protected]
Process Development Manager

Haukeli, Alf Dagfin

Ringnes AS
P.O. Box 7152 Maj.
0307 Oslo
NORWAY
+47 2206 99 60
+47 2206 96 85
[email protected]
Division Quality Assurance Manager

Helland, Ole
Hansa Borg Bryggerier ASA
P.O. Box 24 Kokstad
5863 Bergen
NORWAY
+47 55 96 44 02
+47 55 96 44 09
[email protected]
Brewmaster

Kioenig, Jahn
Hansa Borg Bryggerier
P.O Box 7
1701 Sarpsborg
NORWAY
+47 69 12 63 32
+47 69 12 63 44
[email protected]
Techn.Director

Kjekshus, Svenn Erik

Ringnes AS
P.O Box 7152 Maj.
0370 Oslo
NORWAY
+47 220 698 50

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+47 220 696 85

[email protected]
Technical Director

Linde, Kari Erland

Hansa Borg Bryggerier ASA
P.O. Box 24 Kokstad
5863 Bergen
NORWAY
+47 55 96 44 21
+47 55 96 44 29
[email protected]
Laboratory Manager

Olsen, ge Willy
Hansa Borg Bryggerier
P.O. Box 7
1701 Sarpsborg
NORWAY
+47 69 12 62 80
+47 69 12 63 44
[email protected]
Brewmaster

POLAND

Baranowski, Krysztof
Institute of Biotechnology
Przem Rol -Spoz
Ul Rakowiecka 36
02-532 Warszawa
POLAND
+48 22 606 36 07
+48 22 849 04 26
Dr.

Panek, Krysztof
Browar Belgia
Ul. Sciegiennego 499
25 116 Kielce
POLAND
+48 41 362 0431
+48 41 362 0432
Quality Manager

Sierens, Piet
Browar Belgia
Ul. P Sciegiennego 499
25-116 Kielce
POLAND
+48 41 362 0431

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+48 41 362 0432

[email protected]
Production Manager

PORTUGAL

Brandt Petersen, Gert

UNICER - Bebidas de Portugal, S.A
Apartado 1044
4466-955 S.Mamede de Infesta
PORTUGAL
+351 22 905 22 06
+351 22 905 23 00
[email protected]
Member of the Board

Devreux, Andr
Cereuro-Cervejeira Europeia, SA
Estrada de Portela, 8
2795 Carnaxide
PORTUGAL
+351 21 424 34 66
+351 21 424 34 41
[email protected]
Teacher

Estevens Pombeiro, Maria Jose

Central de Cervejas S.A.
Estrada da Alfarrobeira
Apartado 15
2626 - 851 Vialonga
PORTUGAL
+351 21 952 86 48
+351 21 952 22 58
[email protected]
Development and Quality Control Director

Ferreira de Oliveira, Manuel

Unicer-Bebidas de Portugal S.A.
Apartado 1044
4466-955 S. Mamede de Infesta
PORTUGAL
+351 22 905 25 00
+351 22 905 22 13
[email protected]
Chairman and C.E.O.

Girio, Francisco
INETI
Dept. of Biotechnology
Estrada Do Pago Do Lumiar, 22

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1649-038 Lisboa
PORTUGAL
+351 21 716 51 41
+351 21 716 36 36
[email protected]
Head of Research

Guido, Lus
University of Porto
Faculty of Sciences
Rua do Campo Alegre, 687
4169-007 Porto
PORTUGAL
[email protected]
Assistant

Machado Cruz, Jos Miguel

UNICER - Bebidas de Portugal, S.A
Apartado 1044
4465-955
S. Mamede de Infesta
PORTUGAL
+351 22 905 2208
+351 22 905 2213
[email protected]
Technical Adviser/Ing

Mendes, Joao Paulo

Central de Cervejas S.A.
Estrada da Alfarrobeira
Apartado 15
2626 - 851 Vialonga
PORTUGAL
+351 21 952 86 48
+351 21 952 22 58
[email protected]
Brewmaster

Mendonca Fonseca, Joao Diogo

UNICER - Bebidas de Portugal, S.A
Apartado 1044
4466-955
S.Mamede de Infesta Codex
PORTUGAL
+351 22 905 23 71
+351 22 905 24 90
[email protected]
Bottling Hall Manager

Monteiro Ferreira, Antnio Augusto

UNICER - Bebidas de Portugal, S.A

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Apartado 1044
4466-955
S.Mamede de Infesta Codex
PORTUGAL
+351 22 905 23 04
+351 22 905 24 59
[email protected]
Quality Director

Moreira Da Silva, Pedro Manuel

UNICER - Bebidas de Portugal, S.A
Apartado 1044
4466-955
S.Mamede de Infesta Codex
PORTUGAL
+351 22 905 23 71
+351 22 905 24 90
[email protected]
Director of the Brewery of L. Balid

Paisana Joaquim, Jose

UNICER - Bebidas de Portugal, S.A
Apartado 1044
4466-955
S.Mamede de Infesta Codex
PORTUGAL
+351 22 905 23 71
+351 22 905 23 00
[email protected]
Laboratory Manager

Ribeiro-Telles, Maria Da Graca

Central de Cervejas S.A.
Estrada da Alfarrobeira
Apartado 15
2626 - 851 Vialonga
PORTUGAL
+351 21 952 86 48
+351 21 952 22 58
[email protected]
Quality Director

Ricca Goncalves, Cristina

UNICER - Bebidas de Portugal, S.A
Apartado 1044
4466-955
S.Mamede de Infesta Codex
PORTUGAL
+351 22 905 23 78
+351 22 905 24 59
[email protected]

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Research and Development Director

Teixeira, Joao
Empresa de Cervejas da Madeira Lda
Pizo-Parque Industrial da Zona Oeste
9304-003 Camara de Lobos
Madeira
PORTUGAL
+351 291 911 183
+351 291 911 192
[email protected]

Vasconcelos, Laura
Central de Cervejas S.A.
Estrada da Alfarrobeira
Apartado 15
2626 - 851 Vialonga
PORTUGAL
+351 21 952 86 48
+351 21 952 22 58
[email protected]
ID & D Manager

REPUBLIC OF KOREA

Song, Tae Young

Oriental Brewery Co., LTD.
Doosan Tower 16th Floor
18-12, Eulchiro 6-Ga, Chung-Gu,
Seoul, Korea
REPUBLIC OF KOREA
+82 2 3398 5100
+82 31 633 7648
[email protected]
Vice President

Yang, Myung Guk

Oriental Brewery Co.,Ltd.
27, Shinha-Ri, Bubal-Eup, Icheon-Shi
Kyunggi-Do
REPUBLIC OF KOREA
+82 31 630 8575
+82 31 633 7648
[email protected]
Deputy Manager

ROMANIA

Berkesy, Corina-Michaela
S.Cberemalt S.A
ROMANIA

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Magyari, Zoltan
Brauunion Romania
Hargita Str. 100 Miercurea-Ciuc
Jud Hargita
ROMANIA
+40 66 171 425

RUSSIAN FEDERATION

Anisimov, Sergey
ZAO NPO Elevar
Klara Zetkin Str., 4/6
125299 Moscow
RUSSIAN FEDERATION
+ 7 95 150 90 84
+ 7 95 159 66 60
[email protected]
General Director

Kizilov, Denis
Brau-El-Info Ltd
Klara Zetkin Str. 4/6
129299 Moscow
RUSSIAN FEDERATION
+7 95 156 04 50
+7 095 159 66 60
[email protected]
Deputy General Director

Lashova, Maria
ZAO NPO Elevar
Klara Zetkin Str., 4/6
125299 Moscow
RUSSIAN FEDERATION
+7 95 159 95 59
+7 95 159 66 60
[email protected]
Assistant General Director

Mamkaeva, Lioubov
ZAO NPO Elevar
Klara Zetkin Str., 4/6
125299 Moscow
RUSSIAN FEDERATION
+ 7 95 150 90 84
+7 95 159 66 60
[email protected]

SINGAPORE

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List of Participants

Ronteltap, Alexander Daniel

Asia Pacific Breweries Pte Ltd
459 Jalan Ahmad Ibrahim
Singapore 639934
SINGAPORE
+65 860 31 01
+65 860 30 10
[email protected]
Technical Manager

SLOVAKIA

Markus, Stanislav
Pivovar a Sladovna Gemer a.s.
Cukrovarsk 67/1,
97901 Rimavsk Sobota
SLOVAKIA
+421 866 563 18 28
+421 866 563 13 89
[email protected]
Manager

Pramuk, Michal
Slovenske zdruzenie vyrobcov piva a sladu
Blumentlska c.19
81613 Bratislava
SLOVAKIA
+421 7 50 22 52 40
+421 7 55 42 15 25
[email protected]
Honorary Chairman

Rutgers, Adriaan B
Heineken Slovensko A.S.
Stefanikova 79
94901 Nitra
SLOVAKIA
+421 818 760 21 07
+421 818 760 21 07
adriaan.rutgers/heineken/hslovensko/[email protected]
Technical Director

Smogrovicova, Daniela
Slovac Technical University
Faculty of Chemical Technology
Department of Biochemical Technology
Radlinskeho 9
81237 Bratislava
SLOVAKIA
+421 7 367 085
+421 7 367 085

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[email protected]
Assoc. Prof.

SLOVENIA

Bajd, Ivanka
Brewery Union
Pivovarniska 2
1000 Ljubljana
SLOVENIA
+386 1 47 17 410
+386 1 47 17 405
[email protected]
Head of Development

Kirn-Godec, Regina
Pivovarna Lasko
Trubarjeva 28
3270 Lasko
SLOVENIA
+386 3 734 84 13
+386 3 573 17 95
[email protected]
Technology Head

Kocar, Bogoslav
Brewery Union
Pivovarniska 2
1000 Ljubljana
SLOVENIA
+386 1 47 17 404
+386 1 47 17 405
[email protected]
Production Manager

Lavric, Dimitrije
Brewery Union
Pivovarniska 2
1000 Ljubljana
SLOVENIA
+386 1 47 17 203
386 1 47 17 207
mitja.lavric@pivo-union-si
President of Board

Oset, Matej
Pivovarna Lasko
Trubarjeva 28
3270 Lasko
SLOVENIA
+386 3 734 84 30

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+386 3 573 17 95
[email protected]
Beer Production Head

Rape, Andrej
Brewery Union
Pivovarniska 2
1000 Ljubljana
SLOVENIA
+386 1 47 17 401
+386 1 47 17 373
[email protected]
Technical Director

Virant, Majda
Institute of Hop Research and Brewing Zalec
Ul. Zalskega tabora 2
3310 Zalec
SLOVENIA
+386 371 216 10
+386 371 216 20
[email protected]
Head of the Department for Brewing

Vojvodic, Alenka
Pivovarna Lasko
Trubarjeva 28
3270 Lasko
SLOVENIA
+386 3 734 84 14
+386 3 573 17 95
[email protected]
Technologist

SOUTH AFRICA

Mochaba, Franciska
South African Breweries
P.O Box 782 178
Sandton 2146
SOUTH AFRICA
+27 11 881 85 62
+27 11 881 80 72
[email protected]

Stafford, Bob
South African Breweries
65 Park Lane, Sandown
P.O. Box 782 178
Sandton 2146
SOUTH AFRICA

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+27 11 881 8057

+27 11 881 8379
[email protected]
Engineering Development Consultant

SPAIN

Alvarez-Scholl, Rafael
Heineken Espaa, S.A.
Avda. de Andaluca, 1
41007 Sevilla
SPAIN
+34 954 97 99 73
+34 954 97 99 16
[email protected]
Quality Assurance Manager

Bezares Carretero, Eduardo

Ca. Cervecera de Canarias
Avda. Angel Romero 18
38008 Santa Cruz de Tenerife
SPAIN
+34 639 78 20 80
+34 922 20 50 84
[email protected]
Subdirector Logistica

Fumanal, Antonio J.
La Zaragozana S.A.
c/ Ramon Berenguer IV No 1
50007 Zaragoza
SPAIN
+34 976 27 28 46
+34 976 37 33 85
[email protected]
Technical Manager

Gerstner, Jean Claude

Heineken Espana S.A.
Retama 3
28045 Madrid
SPAIN
+34 91 506 97 52
+34 91 467 06 84
[email protected]
Technical General Manager

Inaraja, Carlos
Heineken Espaa S.A.
Carretera Nacional I, Km 23.5
San Sebastin de Los Reyes

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28709 Madrid
SPAIN
+34 91 652 58 11
+34 91 652 64 79
[email protected]
Quality Manager

Landaluce, Rufino
Landaluce, S.A.
P.O.Box 43
39300 Torrelavega
SPAIN
+34 942 82 4411
+34 942 84 5151
[email protected]
Engineer

Martnez Fras, Javier

Heineken Espaa, S.A.
Avda. de Andaluca 1
41007 Sevilla
SPAIN
+34 95 497 99 99
34 95 497 99 11
[email protected]

Munguia, Juan Luis

Cerveceros de Espaa
c/ Karmele San Martin 31
2019 San Sebastian
SPAIN
+34 943 213 451
+34 94 33 10 111
Brewmaster

Navarro Marzal, Alfonso

Cirilo Amors 24
46004 Valencia
SPAIN
+34 963 51 33 99
+34 963 51 33 99
[email protected]

Perero, Pedro
A.E.T.C.M
Ramirez de Prado, no 8, 1 F
28045 Madrid
SPAIN
+34 91 527 7255
+34 91 528 5507
[email protected]

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Secretario

Posada, Javier
Spanish University Brewing School
Ferrocarril, 40 - 1 DCHA
28045 Madrid
SPAIN
+34 91 539 95 41
[email protected]
Director of Spanish University

Roche, Elena
Spanish University Brewing School
Lerida, 1
28020 Madrid
SPAIN
+34 606 671 407
+34 91 336 30 09
[email protected]

Rouco, Carlos
A.E.T.C.M
Ramirez de Prado, no 8, 1 F
28045 Madrid
SPAIN
+34 91 527 7255
+34 91 528 5507
President

Rumeu, Andres
Compaa Cervecera Canarias, S.A
Avenida Angel Romero, 19
38009 Santa Cruz de Tenerife(Canary Islands)
SPAIN
+34 922 229 340
+34 922 204 600
[email protected]
Technical Director

Sangrador, Carlos F.
Mahou S.A.
Ctra. Nal. 2, Km 47
19208 Alovera ( Guadalajara )
SPAIN
+34 949 26 82 66
+34 949 26 82 28
[email protected]
Laboratory Manager

Torrent, Josep
San Miguel, Fcas. de Cerveza y Malta , S.A.

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Avenida de la Industria Pol. Ind El Segre

Apdo. Correos 67
25080 Lleida
SPAIN
+34 973 70 54 00
+34 973 70 54 28
[email protected]
Research I+D

Vidal, Fernando
San Miguel, Fcas. de Cerveza y Malta, S.A.
c/ Urgell, 240
08036 Barcelona
SPAIN
+34 93 227 24 06
+34 93 227 24 16
[email protected]
Director of Quality & Development

SWEDEN

Aas, Tnu
Baltic Beverages Holding HB
Box 20182, Masugnsvgen 28
161 02 Bromma
SWEDEN
+46 8 799 84 17
+46 8 29 13 03
[email protected]
Project Manager

Cardelli, John
Spindal Europe Nord AB
Brodalsvgen 1
43338 Partille
SWEDEN
+46 31 44 33 40
+46 31 44 33 42
[email protected]
Manager

Johansson, Claes-Gran
Baltic Beverages Holding AB
P.O. Box 20182
161 02 Bromma
SWEDEN
+46 70 661 72 49
+46 8 29 13 03
[email protected]
Quality Manager

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Krantz, Mats
Baltic Beverages Holding AB
P.O Box 20182
161 02 Bromma
SWEDEN
+46 8 799 84 00
+46 8 29 13 03
[email protected]
Director

Larsson, Tommy
Carlsberg Sverige AB
161 86 Stockholm
SWEDEN
+46 8 757 74 66
+46 8 98 10 79
[email protected]
Product Development

Lindberg, Anders
Carlsberg Sverige AB
161 86 Stockholm
SWEDEN
+46 8 75 773 59
+46 8 988 540
[email protected]
Head Brewer

Malmberg, Christian
Carlsberg Sverige AB
161 86 Stockholm
SWEDEN
+46 8 757 76 70
+46 8 98 10 79
[email protected]
Product Development Manager

Olsson, Bengt
Svenska Malt AB
Exportgatan 1
302 45 Halmstad
SWEDEN
+45 650 704 02
+46 650 706 55
[email protected]
Managing Director

Romander, Sven
Swedish Brewers Association
P.O. Box 8104
10420 Stockholm

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SWEDEN
+46 8 566 213 40
+46 8 566 213 10
[email protected]
Technical Manager

Svensson, Eivor
Perten Instruments AB
Ideon
223 70 Lund
SWEDEN
+46 46 286 87 30
+46 46 12 98 79
[email protected]
Product Manager

Welz, Klaus
Spendrups Bryggeri AB
Vrby All 72, Box 9
143 01 Vrby
SWEDEN
+46 8 736 66 00
+46 8 740 04 30
[email protected]
Braumeister

Wendler, Anders
Carlsberg Sverige AB
161 86 Stockholm
SWEDEN
+46 8 75 770 00
+46 8 988 540
[email protected]
Head Brewer

Wiesgickl, Albert
Abro Brewery
P.O. Box 23
598 21 Vimmerby
SWEDEN
+46 492 165 75
+46 492 136 90
Head Brewer

Wirack Nilsson, Monica

Baltic Beverages Holding AB; BBH
Box 20 182
3161 02 Bromma
SWEDEN
+46 706 94 96 70
+46 31 91 13 15

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[email protected]
Brewmaster

SWITZERLAND

Drost, Martijn
Feldschlsschen-Getrnkegruppe
Theophil-Roniger-Strasse
4310 Rheinfelden
SWITZERLAND
+41 61 833 47 41
+41 61 833 47 00
[email protected]
Head of Engineering & Process Mgmt.

Hannemann, Wolfgang
Novozymes Switzerland AG
Neumatt
4243 Dittingen
SWITZERLAND
+41 61 765 61 11
+41 61 765 64 44
[email protected]
Tech. Marketing Manager

Janser, Elmar
Novozymes AG
Neumalt
4243 Dittingen
SWITZERLAND
+41 61 765 61 11
+41 61 765 63 33
[email protected]
Marketing Manager Brewing & Alcohol

Schrder, Harald
Filtrox AG
SWITZERLAND

Zuber, Jrg
Filtrox AG
Moo
SWITZERLAND

UNITED KINGDOM

Andrews, John
Briggs of Burton PLC
Briggs House, Derby Street,
Burton-on-Trent DE14 2LH
UNITED KINGDOM

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+44 1283 566 661

+44 1283 545 978
[email protected]
Chairman

Baker, David
Young & Co' s Brewery PLC
The Ram Brewery
Wandsworth, London SW18 4JD
UNITED KINGDOM
+44 20 8875 7125
+44 20 8875 7100
Brewer

Boughton, Richard
FlavorActiv Limited
Sanderum House
Oakley Road
Chinnor, Oxon OX9 4TW
UNITED KINGDOM
+44 1844 354 154
+44 1844 354 508
[email protected]
Managing Director

Boulton, Christopher
Bass Brewers
Technical Centre,
PO Box 12, Cross Street
Burton upon Trent DE14 1XH
UNITED KINGDOM
+44 01283 513 936
+44 01283 513 944
[email protected]

Bryce, James Hutchison

Heriot-Watt University
Centre For Brewing and Distilling
Riccarton
Edinburgh EH14 4AS
Scotland
UNITED KINGDOM
+44 131 451 34 53
+44 131 451 30 09
[email protected]
Senior Lecturer

Butterworth, Michael
Institute & Guild of Brewing
33, Clarges Street
London W1J 7EE

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UNITED KINGDOM
+44 20 7499 81 44
+44 20 7499 11 56
[email protected]
Technical Manager

Canterranne, Evelyne
Flavoractiv Limited
Sanderum House
Oakley Road, Chinnor
Oxon OX9 4TW
UNITED KINGDOM
Development Manager

Carvell, John
Aber Instruments Ltd.
5 Science Park
Aberystwyth, SY23 3AH
UNITED KINGDOM
+44 1970 636 300
+44 1970 615 455
[email protected]
Director

Cooper, Daniel
Institute of Food Research
Norwich Research Park
Colney
Norwich, Norfolk NR4 7UA
UNITED KINGDOM
+44 16 03 255 200
+44 16 03 507 723
[email protected]
Postdoctoral Researcher

Cox, Simon
Carlsberg-Tetley Brewing Ltd
P.O Box 142
The Brewery
Leeds LS1 1QG
UNITED KINGDOM
+44 113 259 47 69
+44 113 259 47 69
[email protected]
Brewery Director

Craig, Harry
DSM Food Specialties
7 Malvern Road
Knutsford, Chshire
UNITED KINGDOM

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+44 1565 63 47 57
+44 1565 65 37 37
[email protected]

Don, Ken
Young & Co' s Brewery PLC
The Ram Brewery
Wandsworth, London SW18 4JD
UNITED KINGDOM
+44 20 8875 7000
+44 8875 7100
Head Brewer

Dyter, Adrian
Pauls Malt Ltd
P.O.Box 54 Kentford
Newmarket, Suffolk CB8 7QU
UNITED KINGDOM
+44 1638 55 55 30
+44 1638 55 55 00
[email protected]
Export Manager

Elderfield, Peter
Scottish Courage Brewing Ltd.
Fountain Brewery, Fountainbridge
Edinburgh, EN3 9YY
UNITED KINGDOM
+44 131 229 93 77 ext 3282
+44 131 229 12 82
[email protected]
Operations Improvement Manager

Fels, Stephane
DCL Yeast
Salatin House, 19 Cedar Road
Sutton, Surrey SM2 5JG
UNITED KINGDOM
+44 2086 43 18 18
+44 2086 43 64 54
[email protected]
Breing Projects Manager

Gimbel, L.S Tom

Steiner Hops Ltd
UNITED KINGDOM

Hammond, John
Brewing Research International
Lyttel Hall
Nutfield

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Surrey RH1 4HY

UNITED KINGDOM
+44 1737 82 22 72
+44 1737 82 27 47
[email protected]
Laboratory Director

Hegarty, Paul
Bass Brewers Ltd
Bass Technical Centre
PO Box 12, Cross Street
Burton on Trent DE14 1XH
UNITED KINGDOM
+44 1283 513930
+44 1283 513944
[email protected]
Analytical Services Manager

Hodgson, Jeff
Scottish Courage Brewing Ltd
Technical Centre
160 Canongate
Edinburgh EH8 8DD
UNITED KINGDOM
+44 131 248 11 40
+44 131 248 11 01
[email protected]
Innovation & Development MGR

Jaques, David
Interbrew UK Ltd
The Technical Centre
Park Street
Luton, Beds. LU1 3ET
UNITED KINGDOM
+44 1582 39 65 32
+44 1582 39 66 30
[email protected]
Director of Technical Services

Jenkins, Cheryl
Oxford Brookes University
School of Biological and Molecular Sciences
Gipsy Lane
Headington
Oxford OX3 0BP
UNITED KINGDOM
+44 1865 483 845
+441865 48 44 10
[email protected]
Research Student

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Date: 2001-06-01
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List of Participants

Jones, Hilary
Scottish Courage Brewing Ltd
John Courage House
1 Broadway Park
Edinburgh EH12 9JQ
UNITED KINGDOM
+44 131 316 62 05
+44 131 316 62 83
[email protected]
Technical Support Director

Kennedy, Alan I.
Scottish Courage Brewing Ltd.
Sugarhouse Close
160 Canongate
Edinburgh, EH8 8DD
UNITED KINGDOM
+44 131 248 1145
+44 131 248 1101
[email protected]

Kent, Robert
Bass Brewers Ltd
Burton Brewery
P.O. Box 217
Station Street
Burton-on-Trent DE14 IBG
UNITED KINGDOM
+44 128 351 38 33
+44 128 351 35 78
[email protected]
Quality Assurance Manager

Keogh, Ian
Bass Brewers Ltd
P.O. Box 12
Cross Street, Burton on Trent
Staffordshire
UNITED KINGDOM
+44 1283 513 619
+44 1283 513 944
NPD Manager

Kierstan, Marek
Brewing Research International
Lyttel Mall
Nutfield, Surrey
RH1 4HY
UNITED KINGDOM
+44 1737 822 272

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+44 1737 822 747

[email protected]
Chief Executive

Knox, Robert
Charles Wells Ltd
Havelock St
Bedford MK40 4LU
UNITED KINGDOM
+44 1234 272 766
+44 1234 279 001
[email protected]
Technical Director

Lees-Jones, Michael
J. W. Lees & Co ( Brewers ) Ltd.
Greengate Brewery
Middleton Junction
Manchester M24 2AX
UNITED KINGDOM
+44 1 61 643 24 87
+44 1 655 37 31
[email protected]
Operations Brewer

Lees-Jones, Richard
J. W. Lees & Co ( Brewers ) Ltd.
Greengate Brewery
Middleton Junction
Manchester M24 2AX
UNITED KINGDOM
+44 1 61 643 24 87
+44 1 345 39 64
[email protected]
Chairman

Madsen, Kasper
Carlsberg-Tetley Brewing Ltd.
Northampton Brewery
140 Bridge Street
Northampton
UNITED KINGDOM
+44 1604 668 200
+44 1604 668 384
[email protected]
Production Director

Marchbanks, Christopher John

BDI
18, Brizlincote Lane
Burton Upon Trent DE15 0PR

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UNITED KINGDOM
+44 1283 56 1626
+44 1283 56 1120
[email protected]
Freelance technical writer

Middleton, John
Briggs of Burton PLC
Briggs House, Derby Street,
Burton-on-Trent DE14 2LH
UNITED KINGDOM
+44 1283 566 661
+44 1283 545 978
[email protected]
Sales Director

Molzahn, Stuart
Bass Brewers Limited/Technical Centre
P.O. Box 12, Cross Street
Burton on Trent
Staffs DE14 1XH
UNITED KINGDOM
+44 1283 513 948
+44 1283 513 944
[email protected]
Director of Technical Innovation & Services

Morton, Nick
Scottish Courage Brewing Ltd.
John Smiths Brewery
The Brewery
Tadiaster LS24 95A
UNITED KINGDOM
+44 1937 832 091
+44 1937 837 340
[email protected]
Draught Packaging & Warehouse MGR

Mostert, Fred
Micro Matic AIS
Sanderum House
Oakley Road,
Chinnor, OX9 4TW
UNITED KINGDOM
+44 1 844 353 993
+44 1 844 354 144
[email protected]

Murray, James
Brewing Research International
Lyttel Hall, Nutfield

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Surrey RH1 4HY

UNITED KINGDOM
+44 1737 82 42 32
+44 1737 82 27 47
[email protected]
Associate Director

Negus, Emma
Institute & Guild of Brewing
33, Clarges Street
London W1J 7EE
UNITED KINGDOM
+44 20 7499 8144
+44 20 7499 11 56
[email protected]
Office Manager

Nelson, Larry
Brewers' Guardian
18-20 Hill Rise
Richmond
Surrey TW10 6UA
UNITED KINGDOM
+44 20 8410 2843
+44 20 8332 8996
[email protected]
Editor

Noble, C. Stuart
Bass Brewers Limited
Bass Technical Centre
P.O. Box 12, Cross Street
Burton-On-Trent DE14 1XH
UNITED KINGDOM
+44 1283 511 000
+44 1283 513 944
[email protected]
Production Standards Brewer

Pegnall, Brian
Institute & Guild of Brewing
33, Clarges Street
London W1J 7EE
UNITED KINGDOM
+44 20 7499 8144
+44 20 7499 11 56
[email protected]
Chief Executive

Peters, Ann
Bass / Interbrew

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List of Participants

Bass Brewers
Manor Park, Turk Street
Alton Hampshire
UNITED KINGDOM
+44 14 20 541 177
+44 14 20 541 366
[email protected]
Quality Support Manager

Powell, Chris
Oxford Brookes University
Gipsy Lane
Headington
Oxford OX3 0BP
UNITED KINGDOM
+44 1865 48 44 13
+44 1865 48 44 10
[email protected]
Research Scientist

Quain, David
Bass Brewers
Technical Centre,
P.O Box 12, Cross Street,
Burton-on-Trent DE14 1XH
UNITED KINGDOM
+44 1283 513896
+44 1283513944
[email protected]
Head of Research

Reckelbus, Benoit
DCL Yeast
Salatin House, 19 Cedar Road
Sutton, Surrey SM2 5JG
UNITED KINGDOM
+44 2086 43 18 18
+44 2086 43 64 54
[email protected]
Brewing Technologist

Redman, John
Greene King plc
Abbot House, Westgate Brewery
Bury St. Edmunds
Suffolk IP29
UNITED KINGDOM
+44 128 471 42 02
+44 128 471 44 56
[email protected]
Brewing & Distribution Director

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List of Participants

Righelato, Renton
Ashbourne Biosciences
63 Hamilton Road
Reading, RG1 5RA
UNITED KINGDOM
+44 118 926 4513
+44 118 926 8170
[email protected]
Professor

Shardlow, John
Interbrew U.K.
Porter Tun House
500 Capability Green
Luton LU1 3LS
UNITED KINGDOM
+44 1582 397 471
+44 1582 397 563
[email protected]
Brewing Development Engineer

Sharpe, Richard F.
Brewing Research International
Lyttel Hall
Coopers Hill Road
Nutfield Surrey RH1 4HY
UNITED KINGDOM
+44 1737 824 237
[email protected]
Technical Director

Siddique, Rukhsana
Oxford Brookes University
B.M.S Gipsy Lane Campus
Headington
Oxford OX3 0BP
UNITED KINGDOM
+44 1 865 48 32 87
+44 1 865 48 44 10
[email protected]
Research Student M.Phil

Simpson, Peter
Simpsons Malt Limited
Tweed Valley Maltings
Berwick Upon Tweed
Northumberland, TD15 2UZ
UNITED KINGDOM
+44 1 289 330 033
+44 1 289 306 602

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[email protected]
Director

Simpson, Richard
Simpsons Malt Limited
Tweed Valley Maltings
Berwick Upon Tweed
Northumberland, TD15 2UZ
UNITED KINGDOM
+44 1 289 330 033
+44 1 289 306 602
[email protected]
Director

Simpson, Simon
Simpsons Malt Limited
Tweed Valley Maltings
Berwick Upon Tweed
Northumberland, TD15 2UZ
UNITED KINGDOM
+44 1 289 330 033
+44 1 289 306 602
[email protected]

Smart, Katherine
Oxford Brookes University
School of Biological and Molecular Sciences
Headington
Oxford OX3 0BP
UNITED KINGDOM
+44 1865 483 248
+44 1865 483 242
[email protected]
Reader in Brewing Science

Sole, Stan
Crisp Malting Group Ltd
Great Ryburgh, Fakenham
Norfolk NR21 7AS
UNITED KINGDOM
+44 1328 829 391
+44 1328 829 776
[email protected]
Group Technical Manager

Spika, Gero
Ineos Silicas Ltd
P.O. Box 26
Warrington, Cheshire
WA5 1AB
UNITED KINGDOM

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+44 1925 416 471

+44 1925 416 113
[email protected]

Stewart, Graham G.
Heriot-Watt University
International Centre For Brewing & Distilling
Riccarton
Edinburgh EH14 4AS
Scotland
UNITED KINGDOM
+44 131 451 31 84
+44 131 449 74 59
[email protected]
Professor and Director

Taruoka, Makoto
Asahi Beer Europe Ltd.
17 Connaught Place
London W2 2EL
UNITED KINGDOM
+44 20 7706 8330
+44 20 7706 4220
[email protected]
Brewing Supervisor

Thomas, Martin
Bass Brewers
137 High Street
Burton-on-Trent DE14 1JZ
UNITED KINGDOM
+44 1283 513234
+44 1283 513214
[email protected]
Production Director

Thorburn, William
PureMalt Products Ltd.
Victoria Bridge
Haddington
East Lothian EH41 4BD
UNITED KINGDOM
+44 1620 824 696
+44 1620 822 018
[email protected]
Sales Director

Thurston, Pat
Scottish Covrage Brewing Ltd.
Berkshire Brewery, Imperial Way
Reading, Berks, RG2 0PN

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UNITED KINGDOM
+44 118 922 29 88 ext 4725
+44 118 922 23 83
[email protected]
Regional Production Support Manager

Turner, Bruce
PureMalt Products Ltd.
Victoria Bridge
Haddington
East Lothian EH41 4BD
UNITED KINGDOM
+44 1620 824 696
+44 1620 822 018
[email protected]
Chairman

Walker, Caroline
Brewing Research International
Lyttel Hall
Nutfield
Surrey RH1 4HY
UNITED KINGDOM
+44 1737 82 22 72
+44 1737 82 27 47
[email protected]

Walker, Grahame
Pall Europe Ltd
Europa House
Havant Street, Portsmouth PO1 3PO
UNITED KINGDOM
+44 239 230 22 69
+44 161 790 38 73
[email protected]
Senior Sales Mgr. Brewing Europe

Van Zandycke, Sylvie

Smart Brewing Services
Oxford Brookes University
School of BMS
Gipsy Lane Campus
Oxford OX3 0BP
UNITED KINGDOM
+44 1865 48 44 13
+44 1865 48 44 10
[email protected]
Project Manager

Ward, Peter
Thomas Hardy Holdings Limited

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3, The Old Brewery, Highstreet

Theale, Berkshire RG7 5AH
UNITED KINGDOM
+44 1189 304 176
+44 1189 304 191
[email protected]
Director

White, Harry
Bass Brewers
Bass Technical Centre
P O Box 12
Burton-on-Trent DE14 1XH
UNITED KINGDOM
+44 1283 513 986
+44 1283 513 944
[email protected]

White, Philip
Oxford Brookes University
Gipsy Lane Campus
Headington
Oxford OX3 0BP
UNITED KINGDOM
+44 1865 483 287
+44 1865 484 410
[email protected]
PhD Student

Wiggins, David R.
Bass Technical Centre
P.O. Box 12, Cross Street
Burton-on-Trent DE14 1XH
UNITED KINGDOM
+44 1283 511 000
+44 1283 513 944
[email protected]
Head of Packging Development

Wilkes, David
Pauls Malt Ltd
P.O.Box 54 Kentford
Newmarket, Suffolk CB8 7QU
UNITED KINGDOM
+44 1638 55 55 20
+44 1638 55 55 00
[email protected]
Managing Director

Wilson, Richard
Steiner Hops Ltd

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List of Participants

319A High Street,

Epping
Essex, CM16 4DA
UNITED KINGDOM
+44 1992 572 331
+44 1992 573 780
[email protected]
Group Technical Director

Woollard, Tom
Environmental Resources
Management Ltd.
8 Cavendish Square
London W1M 0ER
UNITED KINGDOM
+44 20 7465 7200
+44 20 7465 7272
[email protected]

UNITED STATES

Annand, Kirk
Siebel Institute of Technology
4055 W. Peterson Ave.
Chicago, IL 60646
UNITED STATES
+1 773 279 0966
+1 733 463 7688
Director of education

Benstein, Phillip
New Belgium Brewing Company
500 Linden
Fort Collins, CO 80524
UNITED STATES
+1 970 221 0524
+1 970 221 0535
[email protected]
Brewer

Davis, Michael
American Malting Barley Association
740N Plankinton Avenue
Milwaukee WI 53203
UNITED STATES
+1 414 272 46 40
+1 414 272 46 31
[email protected]
President

Doerge, Harald T.

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List of Participants

Lupofresh Inc.
214 Observation Drive
Yakima, WA 98901
UNITED STATES
+1 509 877 21 94
+1 509 877 40 32
[email protected]
President

Gimbel, Louis
S.S. Steiner, Inc.
UNITED STATES

Heissinger, Heinrich
Anheuser-Busch INC
One Busch Place, OSC-6
St. Louis
MO 63118-1852
UNITED STATES
+1 314 577 23 65
+1 314 577 45 74
[email protected]
Vice President, Brewing Research & Technol

Held, Rudolph W.
Kalsec, INC
3713 West Main Street
P.O. Box 50511
Kalamazoo, MI 49005-0511
UNITED STATES
+1 616 349 9711
+1 616 349 9055
[email protected]
Marketing Managar - Hops

Hertrich, Joseph
Anheuser-Busch, Inc.
One Busch Place 0SC-6
St. Louis
MO 63118 -1852
UNITED STATES
+1 314 577 9176
+1 314 577 4574
[email protected]
Director, Malting

Hysert, David
John I. Haas, Inc.
P.O. Box 1441
Yakima
Washington, 98907

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UNITED STATES
+1 509 469 4011
+1 509 469 4080
[email protected]
Vice President Technical Director

Klimovitz, Raymond
Master Brewers Assoc. of the Americ.
9094 165th Street
Chippewa Falls,
Wisconsin 54729
UNITED STATES
+1 715 720 6884
+1 715 720 7217
[email protected]
Technical Director

Kraemer, Gerhardt
Anheuser Busch Co.
One Busch Pl.
St. Louis
MO 69118
UNITED STATES
+1 314 577 27 47
+1 314 577 91 70
[email protected]
Sr Vice President World Brewing Technology

Lusk, Lance T.
Miller Brewing Company
3939 W. Highland Blvd
Milwaukee
WI 53208
UNITED STATES
+1 414 931 27 32
+1 414 931 25 06
[email protected]
Principal research scientist

Maye, John Paul

Haas Hop Products
5185 MacArthur Blvd
Washington, DC 20016
UNITED STATES
+1 202 777 4827
+1 202 777 4895
[email protected]
Technical Director

Mitchell, Mack C.
Alcoholic Beverage Medical Research Foundation

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List of Participants

Department of Internal Medicine

Carolinas Medical Center
P.O. Box 32861
Charlotte, NC 28211
UNITED STATES
+1 704 355 3165
+1 704 355 7626
[email protected]
President

Morgan, Ray
5 Points Solutions, LLC
PO Box 20305
Atlanta, GA 30325 USA
2441 Oldfield Rd, NW
Atlanta, GA 30327
UNITED STATES
+1404 351 86 66
+1 404 351 86 66
[email protected]
Principal

Munroe, James
Anheuser-Busch, Inc.
One Busch Place, 36-5
St. Louis MO 63118-1852
UNITED STATES
+1 314 577 9968
+1 314 865 8942
[email protected]
Director, Brew. Tech. Services

O'Conell, Brendan
Yakima Chief
P.O. Box 209
Sunnyside, WA 98944
UNITED STATES
+1 509 839 9022
+1 509 839 5570
[email protected]
VP of operations

Otteson, William
Rahr Malting Co.
301 4th Ave 8 Str 567
Minneapolis, MN 55415
UNITED STATES
+1 612 332 51 61
+1 612 332 68 41
[email protected]
Vice President Sales

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Papazian, Charlie
Association of Brewers - USA
736 Pearl Street
Boulder, Colorado 80302
UNITED STATES
+1 303 447 0816 ext. 111
+1 303 447 2825
[email protected]
President

Pringle, Alastair
Anheuser Busch Inc
Brewing Technical Service 156-2
St. Louis MO 63118
UNITED STATES
+1 314 577 45 40
+1 314 577 07 82
[email protected]
Research Director

Rahr, William
Rahr - Westcan Malting
800 west 1st Ave
Shakopee, MN 55379
UNITED STATES
+1 952 496 7008
+1 952 496 7055
[email protected]
International Sales

Rahr, Jr, Guido R.

Rahr Malting Co.
567 Grain Exchange
P.O Box 15186
Minneapolis, Minnesota 55415
UNITED STATES
+1 612 332 51 61
+1 612 332 68 41
Chairman of the Board

Ryder, David S.
Miller Brewing Company
3939 West Highland Boulevard
Milwaukee, WI 53201
UNITED STATES
+1 414 931 30 62
+1 414 931 24 52
[email protected]
Vice President Brewing, Research & QA

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List of Participants

Salazar, Gerardo
John I. Haas, Inc.
5185 MacArthur Blvd.
Washington, DC 20016
UNITED STATES
+1 202 777 4819
+1 202 777 4895
[email protected]
Director of International Sales Executive

Schwarz, Harald
S.S. Steiner, Inc
655 Madison Avenue
New York, NY 10021-8078
UNITED STATES
+1 212 838 89 00
+1 212 593 42 38
[email protected]
Director Business Development

Seabrooks, John
Miller Brewing Company
3939 West Highland Blvd.,
P.O Box 482
Milwaukee, Wisconsin 53201-0482
UNITED STATES
+1 414 931 37 43
+1 414 931 25 06
[email protected]

Sfat, Michael
Bio-Technical Resources
1035 S. 7th Street
Manitowoc, WI 54220
UNITED STATES
+1 920 684 55 18
+1 920 684 55 19

Siebert, Karl
Cornell University
Food Sci. & Tech. Dept.
Geneva, NY 14456-0462
UNITED STATES
+1 315 787 2299
+1 315 787 2284
[email protected]
Professor of Biochem

Teass, H.A.
McNab Incorporated
20 North MacQuesten Parkway

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Mount Vernon, New York

10550
UNITED STATES
+1 914 699 16 16
+1 914 699 16 71
[email protected]
Director of Development

Thoet, Bryan
Conagra Malt
1220 Main Street, Suite 440
Vancouver, WA 98660
UNITED STATES
+1 360 759 43 25
+1 360 759 4331
[email protected]
V. P. Sales

Villa, Keith
Coors Brewing Company
B.C. 600
Golden, Colorado 80401
UNITED STATES
+1 303 277 6393
+1 303 277 6834
[email protected]
Research Associate

Zastrow, Klaus
2700 Bopp Road
St. Louis, MO 63131-3221
UNITED STATES
+1 314 432 5862
+1 314 432 1339
[email protected]
Consultant

VENEZUELA

Bonfanti, Adelmo
C.A.Cervecera Regional
Estrado Aragua
Zona Industrial Santa Rosalia
Avenida 2 Norte, Cagua
VENEZUELA
[email protected]
Vice President

Bravo, Adriana
Empresas Polar
Av. Principal los Cortijos de Lourdes

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Caracas 1071
VENEZUELA
+58 212 202 3062
+58 212 202 3065
[email protected]
Corporate Manager of Chemistry

Galindo-Castro, Ivn
Empresas polar
Av. Principal Los Cortijos de Lourdes
Caracas 1071
VENEZUELA
+58 212 202 30 62
+58 212 202 30 65
[email protected]
Corporate Manager of Biotechnology

Rangel-Aldao, Rafael
Empresas Polar
Av. Principal Los Cortijos de Lourdes
Caracas 1071
VENEZUELA
+58 212 202 30 62
+58 212 202 30 65
[email protected]
R & D Director

Tresselt, Joaquin
Cervecera Polar C.A.
4ta. Transversal Los Cortijos
Centro Empresarial Polar, p2
Caracas 1070
VENEZUELA
+58 220 234 24
+58 220 234 79
[email protected]
Technical Director

Zufall, Carsten
Cervecera Polar C.A.
4ta. Transversal Los Cortijos
Centro Empresarial Polar, piso 2
Caracas 1070
VENEZUELA
+58 220 234 95
+58 220 234 79
[email protected]
Master Brewer
Number of Participants 701

Congrex CX System: Copyright 1996-1998 by CONGREX Sweden AB

 European Brewery Convention
Secretariat General
c/o Burgemeester Smeetsweg 1, P.O. Box 510, NL-2380 BB Zoeterwoude, The Netherlands
tel. +31 71 5456047/5456614 fax +31 71 5410013
e-mail: [email protected] web: www.ebc-nl.com

Founded in 1947

Mission Statement
EBC is the European expert organisation for all areas of brewing and malting
science, technology and technical matters, whilst promoting co-operation
between all parties involved for the benefit of the European Brewing Industry.

Objectives
to promote, in the interest of the brewing industry and taking account of
the interests of consumers and the community at large

development of brewing and malting science and technology

application of best practices in brewing and malting technology
transfer of knowledge from other industries into brewing and malting

to identify new scientific and technical opportunities

to act as the European advisory expert body of science and technology

in the brewing & malting industry

to encourage all actions in support of pre-competitive aspects of

product quality

to encourage all actions in support of wholesomeness, safety & health

and environmental issues

Members
National Brewery Associations of 22 subscribing European countries:
Austria Finland Ireland Portugal Sweden
Belgium France Italy Romania Switzerland
Bulgaria Germany Latvia Slovakia
Czech Republic Great Britain The Netherlands Slovenia
Denmark Hungary Norway Spain
Council
The Council consists of representatives of the members and is
responsible for controlling all actions. It elects a President & 4 Vice
Presidents from amongst Council Members for a 4-year period; they can
be re-elected once.

Board
The Board consists of the President, the Vice Presidents and the
Secretary General. It is responsible for the continuous work and prepares
action to be taken by the Council. Current composition:

President Esko PAJUNEN Sinebrychoff, Finland

Vice Presidents Horst-Gevert BELLMER Beck & Co., Germany
Stuart W. MOLZAHN Bass, Great Britain
Leo A. VAN DER STAPPEN Grolsch, the Netherlands
Jan VESELY Pilsner Urquell, Czech Rep.
Secretary General Marjolein VAN WIJNGAARDEN

Activities
to form standing Committees & Groups
to organise biennial Congresses
to organise Symposia
to publish material resulting from the above activities in the form of
publications and press releases
to establish working relationships with representative bodies of the
brewing and malting industries
to establish an expert/expertise database of brewing and malting
scientists

Co-operation with
CBMC (the Brewers of Europe a trade organisation)
ASBC (American Society of Brewing Chemists)
MBAA (Master Brewers Association of the Americas)
BCOJ (Brewery Convention of Japan)
EUROMALT (cooperation to be formalized)

Committees & Groups

Analysis Committee
Barley & Malt Committee
Brewing Science Group
Technology & Engineering Forum
Congresses
Held every second year since 1947. Dissemination of up-to-date
knowledge on different aspects of brewing science and technology
presented in high quality lectures, posters and poster debates. Invited
lectures cover topics from within and outside the brewing and malting
industry. All contributions are published in the Congress Proceedings.

28th Congress: Budapest 2001

! 42 lectures, 6 of which are Invited Lectures

! 66 posters

! 22 countries represented in scientific programme

! number of delegates: 760

! number of partners: 178

! number of countries: 45

29th Congress: Dublin, Ireland, 18-22 May 2003

30th Congress: Prague, Czech Republic, 14-19 May 2005

Symposia
Two-days meeting where up-to-date knowledge of a well-defined
subject of scientific or technological nature is discussed by specialists.
The contributions are published in the series of EBC MONOGRAPHS.

Publications
See List EBC Publications, published elsewhere on this cd-rom. All
EBC publications can be ordered from:

Fachverlag Hans Carl tel. +49 911 95285-0

Postfach 990153 fax +49 911 95285-61
D-90268 Nrnberg e-mail [email protected]
Germany web www.hanscarl.com
Committees and Groups appointed by the Council of the
European Brewery Convention

Committee / Group Chairman

Analysis Committee Dr. C.-G. Johansson

Baltic Beverages Holding AB
Masugnsvgen 28
P.O. Box 20182
S-16102 Stockholm
Sweden
E-mail: [email protected]

Barley and Malt Committee Mr. R. van den Berg

Heineken Technical Services B.V.
P.O. Box 510
2380 BB Zoeterwoude
The Netherlands
E-mail: [email protected]

Brewing Science Group Dr.Ir. A. Debourg

Haute Ecole Lucia de Brouckre
Institut Meurice
Department of Brewing Science
Avenue Emile Gryzon 1
B-1070 Brussels
Belgium
E-mail: [email protected]

Technology and Engineering Forum Mr. E. Pajunen

Oy Sinebrychoff Ab
Alikeravantie 40
P.O. Box 87
FIN-04201 Kerava
Finland
E-mail: [email protected]
Twenty fifth report on the activities of the
Analysis Committee
Claes-Gran Johansson, Chairman
Baltic Beverages Holding AB, P.O. Box 20182, S-161 82 Bromma, Sweden (e-mail: [email protected])

During the last two years the Analysis Committee held four meetings: in Cannes (France) 30
May 1999, in Copenhagen (Denmark) 22 October 1999, in Helsinki (Finland) 12 May 2000,
and in Freising-Weihenstephan (Germany) 15 November 2000.

CHANGES IN MEMBERSHIP
Currently there are members from 28 members from 18 countries. Membership changes
during the last two years:

Resigned: Mr. M. Bnard (France)

Mr. L.C. Verhagen (Netherlands)
Dr. F.H. White (U.K.)
Dr. P. Chandley (U.K.)

In April 2001, the oldest member of the Analysis Committee, Dr. Heinrich Pfenninger from
Switzerland, passed away.

New members: Dr. S. Bech Srensen (Denmark)

Dr. V. Clamagirand (France)
Dr. G. Mller-Hergt (Germany)
Mrs. M. Brugmans (Netherlands)
Mrs. H. Harrison (U.K.)
Dr. R. Sharpe (U.K.)

ACTIVITIES
In 1999 a questionnaire was carried through in the member countries to identify missing as
well as obsolete methods.

The content of each section of Analytica-EBC is taken care of by a Section Sub-committee,

whilst testing of a specific method is performed in ad hoc Sub-committees formed for each
method. The work in relation to the different sections is summarised below:

Barley and Cereal Adjuncts (Chairman: Silja Home, FIN). The method Sieving Test for
Barley has been revised regarding the definition of damaged barley kernels. The calculation in
the method Extract in Maize has been corrected. Husk quality is a parameter for which there
presently is no method, but where a demand exists according to the questionnaire referred to
above. A new -amylase preparation (from Megazyme) for use in the -amylase method has
been approved as replacement for the old one (from Boehringer), which is no longer
available.

Malt and Coloured Malts (Chairman: Kenneth Erdal, DK). The Boiled Wort Colour method
has been tested and is now an accepted EBC method. The flow injection method for -glucan
is re-evaluated and has been tested collaboratively. More strict specifications of standards and
reagents, as well as a more precise description of critical steps in the method are given in
order to improve the precision of this method. The Fine/coarse Extract Difference in the
Congress Mashing has been archived due to the low precision of the method. The
Identification of Barley Varieties by Electrophoresis can also be applied for malt, and the
method is accepted for this application.

The 15th EBC Standard Malt has been analysed in a ring analysis and approved. In addition as
serving as control sample, this malt shall be used as the reference material for the calibration
of friabilimeters. The friability value of the 15th standard malt has been obtained from the
Friability Calibration Network (FCN), which is also analysing friability in the standard malt at
regular intervals as a stability check. A separate ring analysis with malt of different levels of
friability has also been conducted, showing similar precision data over the range of friability
tested.

Hops (Chairman: Martin Biendl, D). An International Sub-committee with participation also
from ASBC and BCOJ has worked with the production and calibration of standard materials
for reduced iso--acids. These standards, iso--, tetra-, hexa-, and rho-, are now
commercially available. With these standards available, the standardisation of HPLC methods
for these compounds has started. A simplified method for the Lead Conductance Value in
Hop Pellets and Hop Extracts has been tested and approved. Instead of preparation of the
stock solution, the simplified version allows the direct titration of the diethyl ether phase.
Precision data as well as mean values are similar to the original method. The determination of
Hop Oils by Steam Distillation has been collaboratively tested and approved for inclusion.

Wort (Chairman: Walter Hagen, D). The method for the measurement of Viscosity in Wort is
also applicable with other viscosimeters than capillary ones. A ring analysis of Calcium in
Wort has been conducted. The determination of Iso--acids in Wort and Beer by HPLC is
under collaborative testing.

Beer (Chairman: Vladimir Kellner, CZ). An interlaboratory test of NDMA analyis was
conducted to elucidate what precision data can be obtained by the methods that are in use at
different laboratories. Considering that all participating laboratories analysed by their own
method, and the very low concentrations, the results were satisfactory. The Foam
measurement by the NIBEM method has been collaboratively tested and the results are under
evaluation. Recovery of iso-octane for bitterness is included. A method for the determination
of Aluminium in Beer by Graphite Furnace Atomic Absorption Spectrometry is going to be
tested.

Packaging (Chairman: Josep Torrent, E). This Sub-committee is dealing with the collation of
methods for use in packaging - primarily methods for packaging operations control but also
for the control of packaging materials.
Microbiology (Chairman: Tore Hage, N). This Sub-committee has worked with a complete
up-date and revision of Analytica Microbiologica, a work that now has come to an end.
During autumn 2001 this will be published on CD-ROM. A demo version was on display at
the EBC Congress in Budapest.

Statistics (Chairman: Peter Varj, H). Collaborative trials for the determination of precision
data of the following methods in the Beer section have been conducted and the values
included into Analytica: Colour, Final Attenuation, Total Nitrogen, Free Amino Nitrogen, and
VDK by Spectrophotometry. New methods have been accepted for inclusion in Analytica:
Collaborative Trials, Trueness, Sampling Design, and Specification and Tolerance Bands.

Sensory Analysis (Chairman: Roland Walter, F). The Sub-committee is working with a
sensory analysis method to be used in quality control.

PUBLICATIONS
The second update of Analytica-EBC was published. It includes the following new methods:
- Boiled Wort Colour
- Dimethyl Sulphide and Other Lower Boiling Point Volatile Compounds in Beer by Gas
Chromatography.

The following press releases have been published on behalf of the EBC Analysis Committee
during the last two years:
- Calibration (Reference) value for Partly Unmodified Grains and Glassy Corns (submitted
by Dr. F.H. White)
- Determination of DMS and other volatile compounds in beer by headspace capillary gas
chromatography and flame ionisation detection (submitted by Dr. S. Dupire)
- ICE-2 is stable (submitted on behalf of the EBC/ASBC Joint Hop Standard
Subcommittee)
- Determination of the total soluble nitrogen content of malt and beer by the Dumas
combustion method: collaborative trial (submitted by Dr. B. Johnson, IOB, and Dr. C.G.
Johansson, EBC)
- Determination of the N-Nitrosodimethylamine content of beer and malt. (submitted by Dr.
V. Kellner)
- Determintaion of boiled wort colour (submitted by Dr. S.A.G.F. Angelino)
- Analytica-EBC - Precision values of wort analyses (submitted by Dr. W. Hagen and Dr.
H. Schwarz)
- Determination of the repeatability and reproducibility of EBC accepted methods: V
Beer. (submitted by Mr. M. Bnard)
- Reports from the meetings of the International Subcommittee for Isomerised Hop -Acids
Standards (five reports submitted by Dr R.J.H. Wilson)
- Determination of the LCV (Lead Conductance Value) of Hop Pellets by a simplified
version of method EBC 7.5 (submitted by Dr. M. Biendl)
- Determination of the LCV (Lead Conductance Value) of Hop Extracts by a simplified
method EBC 7.6 (submitted by Dr. M. Biendl).
 Analysis Committee

Claes-Gran Johansson (Chairman)

Xavier Casta-Sitjas (Vice Chairman)

Objectives
Standardise methods of analysis
Review and select methods
Identify absences and deficiencies among present methods
Collate and publish methods in Analytica-EBC
Promote international acceptance of standard methods through cooperation
with international organisations
Collaborate with other groups within EBC
Provide analytical expertise on behalf of the European Brewing Industry

Members
The Analysis Committee has 28 members from 18 countries. The national
member organisations nominate one or two members each.

Collaboration with other organisations

ASBC (US) and BCOJ (Japan).
MEBAK and the analysis committee of IOB
Co-opted members of the Hops SC from the hops industry.

Organisation
Main Committee Section Trial
Decides which Subcommittees Subcommittees
methods should be Permanent Formed for the testing
tested, and included subcommittees. Each of a specific method.
into Analytica-EBC. one covers a section in Organises and
The different issues are Analytica-EBC. They evaluates the
however prepared in review and update the collaborative trials.
the subcommittees. content of the different
sections.
 Analysis Committee

Validation Process
Input for testing of new methods is e.g.:
Industry requirements
New methodology or instrumentation
Legislation
EBC groups demands

The Section Subcommittees The Trial Subcommittee The Main

evaluate the proposals. What is plans, conducts, and Committee decides
the relevance of the method? evaluates the collaborative if the tested method
How are the specificity, trial of the method. should be accepted,
accuracy, and precision of the Calculates precision values: and the method is
method in question? For what repeatability, r95, and then published in
purpose will the method be reproducibility R95, and Analytica-EBC.
used? For trading or for gives recommendations to The result of the
process ntrol? Is it related to the main committee. trial is published in
health and safety? the brewing press.

Activities
Section SC Method under evaluation Methods under testing in
collaborative trials
Barley -Total starch
- Husk quality
Malt - NDMA - -glucans by FIA (re-evaluation)
- Friability
Hops - HPLC method for - Hop oils by distillation
reduced iso--acids
Wort - Oxalate - Calcuim in wort
Beer - Sensitive proteins - SO2 in beer
- CO2 methods - Iso--acids in beer by HPLC
- Aluminium in beer by GF-AAS
- Foam by NIBEM-method
Sensory Analysis - QC method
Packaging - Packaging materials
- Packaging control
Microbiology Total revision of Analytica
Microbiologica
Statistics - Collaborative trials
- Accuracy
- Sampling design
- Tolerance bands
 Analysis Committee

Standard materials
EBC Standard Malt. This malt has reference values for all major malt analytes,
and is the reference material for the calibration of friabilimeters.
ICE-2. Calibration extract for - and - acids. Joint EBC/ASBC.
Isomerised Hop -acid Standards (iso--acids and reduced iso--acids).
Joint EBC/ASBC/BCOJ.

These materials are available from Weihenstephan (Malt) and

Labor Veritas (ICE-2 and Isomerised Hop -acid Standards).

Publications
Analytica-EBC Analytica Microbiologica
The accepted methods are published in The third, completely revised
Analytica-EBC. The fifth edition was version will be published on CD-
published in 1998 and contains more than ROM during this year. A pre-view
175 methods covering the entire process version is on display in the booth of
from raw materials to final products. It is Fachverlag Hans Carl.
updated annually.

Reports
During the last two years the following reports have been published on behalf of the
EBC Analysis Committee:

Calibration (Reference) value for Partly Unmodified Grains and Glassy Corns (F.H.
White)
Determination of DMS and other volatile compounds in beer by headspace capillary
gas chromatography and flame ionisation detection (S. Dupire)
ICE-2 is stable (Submitted on behalf of the EBC/ASBC Joint Hop Standard
Subcommittee)
Determination of the total soluble nitrogen content of malt and beer by the Dumas
combustion method: collaborative trial (B. Johnson, IOB, and C.G. Johansson, EBC)
Determination of the N-Nitrosodimethylamine content of beer and malt. (V. Kellner)
Determintaion of boiled wort colour (S.A.G.F. Angelino)
Analytica-EBC - Precision values of wort analyses (W. Hagen and H. Schwarz)
Determination of the repeatability and reproducibility of EBC accepted methods: V
Beer. (M. Bnard)
Reports from the meetings of the International Subcommittee for Isomerised Hop -
Acids Standards (five reports submitted by Dr R.J.H. Wilson)
Determination of the LCV (Lead Conductance Value) of Hop Pellets by a simplified
version of method EBC 7.5 (M. Biendl)
 Determination of the LCV (Lead Conductance Value) of Hop Extracts by a simplified
method EBC 7.6 (M. Biendl)

MEMBERS OF THE ANALYSIS COMMITTEE

Country Name e-mail
Austria Dipl.Brmst. Siegfried Hoffman [email protected]
Dr. Helmuth Schwarz [email protected]
Belgium Mr. Jef Dionys [email protected]
Mr. Laurent Mlotte [email protected]
Bulgaria Dr. Valentin Batchvarov [email protected]
Switzerland Vacant
Czech Republic Dr. Vladimir Kellner [email protected]
Germany Dr. Walter Hagen [email protected]
Dr. Gustavo Mller-Hergt [email protected]
Denmark Dr. Steen Bech Srensen [email protected]
Mr. Kenneth Erdal [email protected]
Spain Mr. Xavier Casta Sitjas [email protected]
Mr. Josep Torrent Xifr [email protected]
France Mr. Vincent Clamagirand [email protected]
Mr. Roland Walter [email protected]
Finland Dr. Silja Home [email protected]
Mrs. Ansa Toivola [email protected]
Great Britain Prof. Richard Sharpe [email protected]
Mrs. Helen Harrison [email protected]
Hungary Dr. Peter Varj [email protected]
Italy Dr. Raffaele Sbuelz [email protected]
Norway Mr. Tore Hage [email protected]
Netherlands Dr. Anneke Douma [email protected]
Mrs. Monique Brugmans [email protected]
Portugal Dr. Antnio Ferreira [email protected]
Mrs. Maria Jos Pombeiro [email protected]
Sweden Dr. Claes-Gran Johansson [email protected]
Slovenia Mrs. Majda Virant [email protected]

Co-opted in Dr. Martin Biendl [email protected]

Hops Dr. Klaus Kammhuber [email protected]
Co-opted in Dr. Alan Kennedy [email protected]
Microbiology SC Dr. Tomoo Ogata
Mrs Marta Orive i Camprubi [email protected]
Dr. Gudrun Vogeser [email protected]
Dr. Erna Storgrds [email protected]
Report on the activities of the Barley &
Malt Committee
Rob van den Berg, Chairman
Heineken Technical Services B.V., P.O. Box 510, 2380 BB Zoeterwoude, the Netherlands (E-
mail: [email protected])

Since the previous EBC Congress 12 new winter barley varieties and 25 new spring
barley varieties have been tested.
The trial results have been published in much detail in the annual report meant to be
studied by the experts. A summary which is to be considered as a judged opinion has
been published in the brewing press.
The Committee uses critical success factors to judge its own performance since one
year now.

OBJECTIVES
Whereas national breeding and selection programmes result in the national
registration of malting barley varieties, it is the task of the EBC Barley & Malt
Committee to make available the information on the performance of upcoming
malting barley varieties in the various growing areas all over Europe.
This information is of interest to breeders, maltsters and brewers in North, West and
Central Europe in order to know in time how barley varieties, bred in any country,
perform in any barley growing area in Europe. This information is also of interest to
breeders, maltsters and brewers in Eastern and Southern Europe, where only limited
breeding activities take place; so they heavily rely on this Europe-wide information.
Because of the growing interest of companies in malting and brewing operations in
East and South Europe this information organised by the EBC Barley & Malt
Committee is to their benefit as well.
Since EBC has the experience of organising the trials for many decades already and
since it probably will offer the widest and best comparable results, the Barley & Malt
Committee does continue its work in an effective and efficient way. Every year a ring
analysis is organised to make sure that all laboratories and pilot maltings involved do
perform in an appropriate way.

Besides the organisation of the Europe-wide information on the performance of

upcoming barley varieties, the communication of these results is a key factor. Both the
overall information and the results per country are of great value for the relevant
parties in the chain; the Committee considers it a critical success factor to offer the
information in an appropriate way and also to discuss the outcome with the
chainpartners.
The Committee will also establish and apply improved methods for testing barley
varieties and formulate requirements for malting barley and malt. It will not make use
of separate working groups for this, but will rely on the existing EBC Analysis
Committee and EBC Brewing Science Group. Through firm relationships with these
bodies and of course with the malting and brewing industry, the Committee expects to
achieve this goal in an efficient way.

MODE OF OPERATION
The EBC Barley & Malt Committee consists of representatives from the EBC
countries where the National Organisations have decided to appoint a delegate to the
Committee. From amongst its members, the Committee elects a Chairman and up to 3
Vice Chairmen who together form the Steering Committee which is reponsible for:
! supervising the trials
! liaison with other EBC Committees and Groups
! communication and reporting
! reviewing the activities considering critical success factors.

The EBC Barley & Malt Committee will:

! select the national barley varieties to be tested according to strict, transparent rules
! manage the barley seed
! organise the national trials
! organise the quality tests
! produce the national report in a standard format in time
! communicate the output in the member country with all relevant members at the
appropriate time
! provide a summary of the trial results in the brewing press.

The Barley & Malt Committee meets twice a year. Mid January the progress of the
various activities is discussed and the selection of spring barley varieties to be tested
in the regions North, West and Central is made. Three times 6 new spring barley
varieties are selected.
In the meeting held in late spring/early summer, the progress of various activities is
again reviewed also considering the critical success factors, while at the same time 6
spring barley varieties to be tested in region South and 6 winter barley varieties to be
tested in all four regions are selected.

FACTORS OF SUCCESS
Critical success factors have been formulated. These factors have to do with
1a. the quality of the selection process of the barley varieties
1b. the exchange of barley varieties from one country to another country
2. the reliability of the analytical data in the annual report
3. the quality of communicating the results
4. the costs involved.

2
EBC Barley & Malt Committee
Chairman: Rob van den Berg

Objectives
It is the aim of the Committee to support the adequate supply of malting barley
in Europe, considering the changing conditions:
For the rural production;
For the technology of breeding, malting and brewing;
Of consumers behaviour
Caused by the introduction of HACCP in 1995;

The committee seeks to establish, where appropriate, in association with the

EBC Analysis committee, the EBC Brewing Science Group and the malting
and brewing industry, improved methods for testing malting barley and
formulate requirements for malting barley and malt.

The Committee seeks to communicate the results of its activities to all parties
in the supply chain such that they are aware of promising new malting barley
varieties.

Success Criteria
The Committee is at present quantifying the relative success of its work via a
number of parameters. One of which is to ensure that 100% of successful
malting barley varieties in participating countries have been trialled by EBC.

EBC Malting Barley Trials

EBC Barley & Malt Committee organize malting barley field trials in 18
member countries. The countries are divided into 4 regions : West, Central,
North, South.
Each year a maximum of 6 new Winter and 6 new Spring varieties are grown
in each region and the resultant agronomic and micro-malting results are
compared with previously agreed standard varieties. Varieties normally are
trialled for 2 years. The results of the trials are published annually. An
Executive Summary is also produced and circulated widely within the industry.
Results Field Trials Harvest 2000

Region Central North South West

Winter Esterel Esterel Esterel Esterel

Standard Angora Angora Angora Angora
Varieties Clarine Clarine Clarine Clarine
Regina Regina Regina Regina
Year 1 Vanessa Vanessa Vanessa
Year 2 Opal Anita Opal

Region Central North South West

Spring Barke Barke Barke Barke

Standard Scarlett Scarlett Scarlett Scarlett
Varieties
Year 1 Alliot Cellar
Prestige Brise
Year 2 Annabell Pasadena Cecilia Hanka
Viskosa Saloon Luberon Jersey
Astoria
Sabel

Varieties highlighted are those that after completion of first year trials (Year
1) or second year of trials (Year 2) are considered by the relevant Regional
Trials Chairmen to be at least equal to and in some cases an improvement
on the control varieties in terms of agronomic and /or micromalting
performance. These varieties may be of interest to the Malting and Brewing
Industry in the near future. More specific information is available from the
Executive summary.
Report on the activities of the Brewing
Science Group
Alain Debourg, Chairman
Institut Meurice, Avenue E. Gryson, 1 , B-1070 Brussels, Belgium (e-mail :
[email protected])

3rd TECHNICAL MEETING, PILSEN URQUELL BREWERY, 26-29

SEPTEMBER 2000

At the kind invitation of Pilsen Urquell Brewery, Budejovicky Budvar Brewery and
Cesky Svaz Pivovaru a Sladoven, the EBC Brewing Science Group held its 3rd
Technical Meeting in Pilsen. It was attended by 56 participants from 22 different
countries. The following 23 scientific papers were presented and discussed during 3
days:

Day 1, Morning session, Chairman: James Bryce

* Erna Storgrds, Marko Nahri and Gun Wirtanen: Disinfectant testing against
brewery related biofilms.
* Pavel Dostalek, Jaroslav Cepicka, J. Enge, R. Koplik and E. Curdova:
Electrochemical determination of heavy metals in beers.
* Jan Savel: Thermal sugar degradation during beer ageing.
* Louis F. Guido, J.M. Machado Cruz, Helena Cunha, Antonio A. Ferreira and
Aquiles A. Barros: Analytical evaluation of compounds involved in beer ageing.
* Jrme Pellaud, Philippe Malcorps and Stphane Dupire: Matrix effect on calcium
oxalate precipitation in beer.

Day 1, Afternoon session, Chairman: Erna Storgrds

* F. Cardoso, L. Vivero, A. Custodio, A. Clemente, Laura Vasconcelos: Prototype
test kit for dsRNA virus detection.
* P. Perpete, P. Van Cutsem, A.M. Corbisier Colson and S. Collin: AFLP, a new
method for the analysis of brewersyeast DNA polymorphism.
* Jeff Hodgson, Xanthe Green and Behnam Taidi: Use of PCR to monitor mixed
yeast ale fermentation.
* Alastair Pringle, John Jackson and Joe Maurer: Brewers yeast characterization.
* John Hammond, Mark Lee and Gareth Dunne: Yeast stress gene expression and its
relevance to brewing.

Day 2, First morning session, Chairman: John Hammond

* Jukka Kronlf: Combined fermentation and maturation with immobilized yeast.
* Daniela Smogrovicova, J. Patkova and Z. Domeny: Stress tolerance of immobilised
brewers yeast.
Day 2, Second morning session, Chairman: Alain Debourg
* Dan Donnelly and Gearoid Cahill: Near real time analysis of yeast glycogen levels
using image analysis.
* David Quain: Early warm cropping of yeast.

Day 2, Afternoon session, Chairman: Alain Debourg

* Laurence Van Nedervelde, M. Dillemans, P. Bastin, Alain Debourg and Patrick
Boivin: Evaluation of antioxidant properties of Saccharomyces cerevisiae.
* Karen Van den Bremt, F.R. Delvaux, H. Verachtert and G. Derdelinckx:
Methylotrophic Candida mutant strains as bioflavouring microorganisms in beer.

Business Meeting

Day 3, Morning session, Chairman: Patrick Boivin

* K. Wackerbauer and M. Rauschmann: How to avoid beta-glucan-gel formation
during ultrafiltration of beer particulary with the use of special malts.
* Joseph Skach, P. Havlova, J. Susta, A. Gavendova and M. Pajurek: Influence of
malt lipoxygenase activity on beer flavour stability.
* J. Rautio and John Londesborough: Maltopermease activity through wort
fermentations.
* Calum Mc Cafferty, James M. Brosnan and James Bryce: Limit dextrinase - does
its malt activity relate to its activity in brewing?
* Auli Haikara, A. Laitila and T. Kleemola: Effect of different Fusarium species and
climatic conditions on the quality of barley and malt.
* Alfonso Navarro: Use of differential scanning calorimetry in malt quality
evaluation.

REPORT OF SUBGROUP ACTIVITIES

Foam, Chairman: Anneke Douma
The actual size of the Foam Subgroup is 21 people coming from 6 EBC countries,
Japan and the USA.
The next meeting of the Subgroup will be held on 14-15 November 2001 at Brewing
Research International (BRI).

Emerging Fermentation Systems, Chairman: David Ryder

At the kind invitation of Professor Renton Righelato, a meeting of the Emerging
Fermentation Systems Subgroup with the theme Fermentation Systems to enter the
Next Millennium was organized at Brewing Research International (BRI) on
November 24 & 25, 1999, immediately following the EBC Symposium on Yeast
Physiology A New Era of Opportunity held in the same U.K. venue. This extra
special meeting of the sub-group was dedicated to the memory of Professor Charles
A. Masschelein (1928-1999) whose inspiration led to the formation of the EBC
Subgroup for Immobilized Enzymes and Cell Systems the former name of this
subgroup. A total of 16 papers were presented by scientists from 10 countries, and
covered fermentation intensification, primary fermentation systems, yeast systems for
fermentation of the future, future fermentation systems & control of by-products, and
the focus for fermentation systems of the future.

2
The next meeting of the Subgroup will probable be organized for September 2001, in
Portugal, in combination with meeting of the Yeast Genetics and Physiology
Subgroup.

Yeast Genetics and Physiology, Chairman: John Hammond

The former Subgroup Chairman Dirk Iserentant informed the Chairman of the
Brewing Science Group in June 1999 that, for professional reasons, he was not able to
continue his work as chairman of this Subgroup. As all members of the Group were
convinced that Yeast Genetics and Physiology was an essential research field, the
decision was taken to nominate a the new Chairman. John Hammond kindly agreed to
act as new Chairman of this Subgroup.
No meeting was organized this year as the EBC Symposium Yeast Physiology - A
New Era of Opportunity was held on November 22 & 23, 1999 at BRI. 56 Scientists
and brewers from 14 different countries were present and 16 papers were presented.
All contributions are published as EBC Monograph 28.
The next meeting of the Subgroup is scheduled for September 2001 in Portugal.

Detection of Contaminants, Chairman: Erna Storgrds

A meeting of the Subgroup was planned to be arranged in Lisbon by kind invitation of
AICP - Associaao da Industria Cervejeira Portuguesa on May 29th 2000. The
meeting was announced on March 1st 2000 to all the members of the Subgroup and of
the Brewing Science Group. However, only two offers to present a paper were
received. Under these circumstances, the meeting had to be cancelled.
Under the 5th Framework Programme of the European Community, a combined R &
D and Demonstration project related to the Subgroup activities has started on January
the 1st, 2001. The name of the project is Development and Demonstration of
Polymerase Chain Reaction based Methods for Process Control in the Brewing
Industry, and the objective is to improve the microbiological quality and safety of
European beer through implementation of PCR technology in brewery QC. The
project is going to occupy five of the Subgroup members in the near future, and
meetings of both the Subgroup and the Brewing Science Group will be used as
platforms for dissemination of the project results.

Malting Barley Genetics and Physiology, Chairman: James H. Bryce

The Subgroup held its first meeting during the 16th Long Ashton International
Symposium on Biotechnology of Cereals: Tools, Targets and Triumphs in
September 1999. At this first meeting the following discussions and conclusions were
reached.
(a) Breeders, farmers, maltsters and brewers need to interact effectively. Overall the
aim is a higher yield of barley and malt at a cheaper cost.
(b) Problems faced by maltsters with regard to extract, enzymes, viscosity, and
nitrogen were discussed. The meeting considered ways that would enable effective
screening for high quality malting barley to be carried out. It was envisaged that this
would require micro-malting and simple tests. QLTS were mentioned. There is clearly
a need to explore aspects important in terms of quality.
(c) At this meeting, different groups present outlined their skills in transformation,
micro-malting, various analytical techniques, and mutagenesis.
(d) The overal conclusion was that improved means of establishing malt quality were
required.

3
The second meeting of the Subgroup was held in June 2000 during the EU-COST
meeting at Heriot-Watt University. It was agreed that the objectives of the EBC
Brewing Science Group would best be achieved by:
(i) seeking research funding
(ii) holding technical meetings
The delegates discussed the 5th Framework programme 1998-2002 (Quality of life and
Management of Living Resources) with a view to obtaining funding from the avenue
entitled cell factory . Following detailed discussions it was agreed that
homogeneity of malting barley should be a major focus as this is a key factor in
producing high quality malt. The Subgroup will aim to produce a submission for
funding that provides the basis for new analyses and closes specific gaps in our
knowledge about malting barley. Funds will also be sought to hold a technical
meeting.

Gushing, Chairman: Poul Sigsgaard

A ring test of the rapid physical gushing test was carried out in 1999 and the results
were reported to the EBC Analysis Committee. The Committee concluded:The
precision of the collaborative trial were unacceptable. The procedure will be
reconsidered. It is planned to perform a new ring test among the more than ten major
laboratories, which are alredy using the technique in their routine control.

Flavour and Flavour Stability, Chairman: Patrick Boivin

A Flavour and Flavour Sability Subgroup meeting was organised at IFBM (Nancy, F)
the 25-26 October 1999. 32 people attended this meeting representing 10 breweries.
This meeting was very interesting and active during the question time. A booklet with
all 14 presentations was sent to all participants and EBC Brewing Science Group
members.
The future actions of this Subgroup are the following:
(a) Organisation of an EBC Flavour and Flavour Stability Symposium with
presentations (international speakers) and posters. This Symposium will be held in
Nancy in October 2001.
(b) Organisation of target meeting with limited number of people: 5 to 8 experts on
the specific subject that we want to discuss deeply. The objective of the target group
will be:
- to propose recognised methods to measure for example antioxidant activity on
malt, hop, wort and beer; trans-2-nonenal in beer; DMSO; maize oxidation...
- to propose a scheme of specific product with the impact of raw material and process
- to propose the need of future work on a specific project.

Contaminants, Chairman: Stphane Dupire

This subgroup having as main objective the detection of chemical contaminants has
been created during last Technical Meeting of the Brewing Science Group. A first
membership list includes 11 scientists from 8 countries.
A first meeting will be organised during EBC Congress in Budapest in order to define
the objectives of this new subgroup.

4
Brewing Science Group
Alain Debourg, Chairman
Institut Meurice
Avenue E. Gryson 1, B-1070 Brussels, Belgium
Tel: + 32 2 526 73 51 fax: + 32 2 526 73 01
E-mail: [email protected]

Objectives
To promote better understanding and technical advancement of science and
innovation in the brewing industry.
To provide a forum for discussion of scientific research in brewing, typically in
relation to:
- biochemistry
- chemistry
- microbiology
- physics
- genetics and physiology.
To have clear missions executed in Sub-groups of limited duration.

Activities
Biennial Technical Meeting held in years alternate to EBC Congresses.
Papers presented at the Technical Meetings are published in a (confidential)
bulletin and circulated to the members of the Group.
Sub-group Meetings.

Foam Sub-group
Chairman: Anneke Douma
TNO Nutrition and Food Research Institute, Food Science and Technology Dept.
Utrechtseweg 48, P.O.Box 360, NL-3700 AJ Zeist, the Netherlands
Tel: + 31 30 6944381 fax: + 31 30 6944295
E-mail: [email protected]

Objectives
Formation, stability and appearance of beer foam and cling:
- methods of analysis
- biochemistry of foam
- physical chemistry of foam
- the effect of the raw materials on foam
- the effect of processing on foam
- foam stabilizing and destabilizing ingredients
- consumer perception of foam
Activities

EBC Symposium Beer Foam Quality, Amsterdam, 26-27 October 1998, 14
lectures, EBC Monograph 27.

Next Sub-group Technical Meeting: Brewing Research International (BRI),
Nutfield, 14-15 November 2001.

Flavour and Flavour Stability Sub-group

Chairman: Patrick Boivin
IFBM, Ple Technologique de Nancy-Brabois,
7, rue du Bois de la Champelle, P.O. Box 267, F-54500 Vandoeuvre Cedex, France
Tel: + 33 3 83448803 fax: + 33 3 83441290
e-mail: [email protected]

Objectives
Better knowledge of raw material, process and storage on organoleptic stability of
beer.
Exchange of expert knowledge during scientific meeting of the Sub-group.

Activities

Sub-group Technical Meeting at IFBM, Nancy, 25-26 October 1999 attended by
32 people, 14 lectures on malt, hop, yeast, fermentation, nonenal formation, beer
storage, new analytical methods and sensory analysis.

31st EBC Symposium on Flavour and Flavour Stability, Nancy, France, 28-30
October 2001.

Detection of Contaminants Sub-group

Chairman: Erna Storgrds
VTT Biotechnology
Tietotie 2, Espoo, P.O. Box 1500, FIN-02044 VVT, Finland
Tel: + 358 9 4564526 fax: + 358 9 4552103
E-mail: [email protected]

Objectives
The objective of the Detection of Contaminants Sub-group is to arrange a forum for
the exchange of ideas and experiences related to the detection of microbial
contaminants in the malting and brewing process. An important task is to distribute
information about new rapid detection methods as early as possibly to persons
working in this field.

Activities

Sub-group Technical Meeting, VTT, Espoo, 18 May 1998, 9 lectures on PCR,
ATP bioluminescence, riboprinting, ELISA.

5th Framework Programme of the European Community: combined R & D and
Demonstration project: Development and Demonstration of Polymerase Chain
Reaction based Methods for Process Control in the Brewing Industry.

This is also presented in a separate poster on this topic.

2
Contaminants Sub-group
Chairman: Stphane Dupire
Interbrew S.A.
Vaartstraat 94, P.O. Box 87, B-3000 Leuven, Belgium
Tel: + 32 16 247396 fax: + 32 16 247889
E-mail: [email protected]

This Sub-group has been created during the last Technical Meeting of the Brewing
Science Group. A first meeting will be organised during the Congress, Meeting
Room Mozart IIIIII, Monday May 14 from 17.45-18.15 hrs.

Malting Barley Genetics and Physiology Sub-group

Chairman: James H. Bryce
Heriot-Watt University, International Centre for Brewing and Distilling
Riccarton, Edinburgh EH14 4AS, Scotland, United Kingdom
Tel: + 44 131 4513453 fax: + 44 131 4513009
E-mail: [email protected]

Objectives
The aims of this Sub-group are two-fold:
1. To improve techniques for establishing malt quality;
2. To facilitate research and dissemination of knowledge that leads to the
improvement of malt quality.

This is a new Sub-group and is still in the process of formulating the areas of research
which it wishes to take forward. The areas so far identified are:
1. A better understanding of water uptake into grains could lead to substantial
savings in water and energy costs;
2. The implications of proanthocyanidins and other components in barley with
respect to health;
3. Malting barley research has generally been done on single batches of barley
whereas, on an industrial scale, barley may be blended prior to malting. The
implications of this lack of homogeneity need to be assessed.

Activities

First Technical Meeting during 16th Long Ashton International Symposium in
September 1999.

Second Meeting during EU-COST meeting at Heriot-Watt University, June 2000.

At these meetings, members outlined their areas of expertise and discussed their
knowledge of research projects funded by the EU, and from national and industrial
sources.
It has been agreed that clusters of members from within the Sub-group should be
formed to put together appropriate research proposals to EU programmes.

3
Gushing Sub-group
Chairman: Poul Sigsgaard
The Scandinavian School of Brewing
Gamle Vartov Vej 30, DK-2900 Hellerup, Denmark
Tel: + 45 39 202555 fax: + 45 39 200102
E-mail: [email protected]

Objectives
To discuss and evaluate tests for gushing potential in barley, malt and beer.

Activities
A ring test of a rapid physical gushing test was carried out in 1999 and the results
were reported to the EBC Analysis Committee. The Committee concluded: The
precision of the collaborative trial were unacceptable. The procedure will be
reconsidered. However, more than 10 major laboratories within the malting and
brewing industry are already using the method in routine quality control. It is planned
to carry out a new ring test in 2001.

Emerging Fermentation System Sub-group

Chairman: David Ryder
Miller Brewing Company
3939 West Highland Boulevard, Milwaukee WI 53201, U.S.A.
Tel: + 1 414 9313062 fax: + 1 414 9312452
E-mail: [email protected]

Objectives
Current interests are concerned with all aspects related to fermentation intensification,
primary fermentation systems and yeast fermentation systems for the future.

Activities

Sub-group Technical Meeting, Brewing Research International (BRI), Nutfield,
24-25 November 1999, Fermentation Systems to enter the Next Millenium, 16
lectures.

Next Sub-group Technical Meeting: Portugal, September 2001.

Yeast Genetics and Physiology Sub-group

Chairman: John Hammond
Brewing Research International
Lyttle Hall, Nutfield, Surrey RH1 4HY, United Kingdom
Tel: + 44 1737 824202 fax: + 44 1737 822747
E-mail: [email protected]

Objectives
Current interests are chiefly concerned with the application of molecular biology to
understanding yeast physiology, understanding the genome structure of brewing

4
yeasts, maintenance of yeast quality, the effects of stress on yeast and improving the
efficiency of fermentation.

Activities

EBC Symposium Yeast Physiology - a New Era of Opportunity, BRI, Nutfield,
22-23 November 1999, 16 lectures, EBC Monograph 28.

Next Sub-group Technical Meeting: Portugal, 26-28 September 2001.

5
List of EBC Publications
EBC publications can be ordered from:
Fachverlag Hans Carl
Postfach 990153, D-90268 Nrnberg, Germany
tel. : +49 (911) 95285-0 e-mail : [email protected]
fax : +49 (911) 95285-61 internet : http://www.hanscarl.com

MONOGRAPHS OF EBC SYMPOSIA

The EBC Monographs contain papers presented at the EBC Symposia, incl. discussion.
The following titles are available:
2 Barley and Malting, 1975, 304 pp 37,84
3 Computerized Process Control, 1976, 432 pp 42,95
4 Noise Abatement, 1977, 252 pp 32,72
8 Brewing Economics & Technical Management, 1982, 280 pp 39,88
9 Biotechnology, 1983, 294 pp 42,95
13 Hops, 1987, 288 pp 41,93
14 Water in the Brewing Industry, 1988, 214 pp 36,81
15 Plant Biotechnology, 1989, 168 pp 33,75
16 Separation Processes, 1990, 276 pp 42,95
17 Packaging, 1990, 220 pp 37,84
18 Wort Boiling and Clarification, 1991, 234 pp 39,88
19 Waste Reduction in Brewery Operations, 1992, 216 pp 39,88
20 Instrumentation and Measurement, 1992, 214 pp 39,88
21 Process Hygiene, 1994, 183 pp 34,77
22 Hops, 1994, 302 pp 43,97
23 Malting Technology, 1994, 216 pp 39,88
24 Immobilized Yeast Applications in the Brewing Industry,
1995, 259 pp 44,99
25 Draught Beer, Packaging & Dispense, 1996, 198 pp 39,88
26 Quality Issues and HACCP, 1997, 180 pp 36,81
27 Beer Foam Quality, 1998, 220 pp 44,99
28 Yeast Physiology A new era of opportunity, 1999, 228 pp 44,99
29 Assuring Product Safety in the Brewing Industry, 2000,
124 pp 35,28

in preparation:
30 Bottling Beer in Plastic Bottles, 2000
31 Flavour and Flavour Stability, 2001

PROCEEDINGS OF EBC CONGRESSES

Proceedings of previous EBC Congresses (from the 16th Congress held in 1977 up to and
incl. the 27th Congress held in 1999) are still available; prices upon request.

28th Congress, Budapest 2001 (available on CD-ROM only) 70,00

 EBC MANUALS OF GOOD PRACTICE
In 1990, the European Brewery Convention established the Technology & Engineering
Forum with the responsibility of developing technical Manuals of Good Practice for
malting and brewing. The project has been supported by the AIR Programme of the
European Union.
The objectives of these Manuals are the characterisation of new (more efficient
production) systems with reduced environmental impact; the assessment of existing
processes and machinery in order to disseminate best performance, practice and
standards; the evaluation of existing systems for maintaining the quality and safety of
products and the identification of the underpinning science and technology.
The intention of the Manuals is also to work out weaknesses and shortcomings of current
systems thereby identifying the needs and the directions for future research and
development. This will lead to improved systems based on either current or new
concepts. The Manuals do not contain recent research findings or concepts that have not
had an opportunity to prove themselves in day-to-day operations.
The Manuals provide factual information for those working at the interface between
brewing science and the brewing process. Newcomers will find a whole range of
practical information. Experienced practitioners will find the manuals valuable as a
source of information of factual and practical issues. The data and processes put together
in these Manuals can be used to derive professional economic decisions from.
The mechanism applied to produce the Manuals is through Working Groups of
experienced persons interacting in order to establish a statement that is more substantive
than the knowledge and experience of any individual. This good practice statement is
enhanced by exposure to readers who are experienced and knowledgeable in the area
and whose suggestions and comments are incorporated in the publication. Final editing of
the Manuals is carried out by Quality Managers at Brewing Research International (BRI)
on behalf of the EBC Technology & Engineering Forum.

Beer Pasteurisation, 136 pp sold out

Brewery Utilities, 200 pp 48,57
Hops and Hop Products, 186 pp 48,57
Beer Filtration, Stabilisation and Sterilisation, 200 pp 115,04
Milling, 102 pp 94,59
Malting Technology, 226 pp 132,94
Fermentation and Maturation, 208 pp 120,15
Water in Brewing, 144 pp 99,70
titles in preparation:
Process Quality Assurance
Brewery Waste Water Treatment
Wort Boiling
Utility Distribution Systems
Mashing Technology
Mash Separation

2
 ANALYTICA-EBC
The book containing the standardised analytical methods written in a style according to
the international standard ISO 78/2; precision values for the methods have been
determined according to the protocols given in ISO 5725. More than 175 methods cover
the whole process flow from raw materials to analyses of finished products. 5th edition
published in 1998; updated annually.

5th Edition, format: DIN A4, in ring-binder

approx. 500 pages, numerous illustrations and diagrams
English language edition exclusively 250,53
First update, May 1999 42,95
Second update, May 2001 58,80

ANALYTICA-MICROBIOLOGICA-EBC
Recommended methods presented in four sections: microbiology, microbiological
techniques, yeast analysis and detection of contaminants; hygiene legislations are added
as annex.
Will be published on CD-ROM in autumn 2001. 99,-

THESAURUS
EBC Thesaurus, 2nd edition (2 Vol. Set) 45,-

from:

Elsevier Scientific Publishing Company

P.O. Box 211, 1000 AE Amsterdam, The Netherlands
tel. : +31 (20) 4853642
fax : +31 (20) 4853598
e-mail : [email protected]

the following publication can be ordered:

ELSEVIERS DICTIONARY OF BREWING

January 1983, 264 pp 144,90
(Edition in four languages:
English, French, German and Dutch
and based on the Thesaurus compiled by
the European Brewery Convention.
It includes approx. 4000 terms.
Basic table in English.)
Also available on CD-ROM 176,50

Postage is not included in the above prices.

3
 European Brewery Convention

List of Member Organisations

Liste der Mitgliedsorganisationen
Liste des Associations Membres
Country Member Organisation Council Member
Land Mitgliedsorganisation Ratsmitglieder
Pays Association Membre Membre du Conseil

A sterreichisches Getrnkeinstitut (GI) M.A. Liebl

Michaelerstrasse 25
A-1182 Wien
E-mail: [email protected]

B Centre Technique et Scientifique A. Debourg

de la Brasserie de la Malterie et E. van den Eynde
des Industries Connexes (CBM)
Maison des Brasseurs; GrandPlace 10
B-1000 Bruxelles
E-mail: [email protected]

BG Union of Brewers in Bulgaria A. Nenov

18 Serdika Str. P. Paunkov
BG-1000 Sofia
E-mail: [email protected]

CH Schweizerischer Bierbrauerverein R. Eisenring

Bahnhofplatz 9
Postfach 6325
CH-8023 Zrich
E-mail: [email protected]

CZ Cesky Svaz Pivovaru a Sladoven K. Kosar

Lpov 15 J. Vesely
CZ-12044 Praha 2
E-mail: [email protected]

D Deutscher Brauer-Bund e.V. H.-G. Bellmer

Annabergerstrasse 28 A.Th. Simon
Postfach 200452 + 200453
D-53134 Bonn
E-mail: [email protected]

DK Bryggeriforeningen K. Erdal
Frederiksberggade 11 N. Hald
DK-1459 Copenhagen K
E-mail: [email protected]
E Cerveceros de Espaa J.F. Vidal
c/ Almagro 24-2 izda.
E-28010 Madrid
E-mail: [email protected]

F Association des Brasseurs de France M. Arbogast

25 Boulevard Malesherbes
F-75008 Paris
E-mail: [email protected]

FIN Oy Panimolaboratorio P.J. Hartwall

P.O. Box 16 E. Pajunen
FIN-02151 Espoo
E-mail: [email protected]

GB The Institute and Guild of Brewing D. Jacques

33 Clarges Street S.W. Molzahn
London W1J 7EE
E-mail: [email protected]

H Association of the Hungarian Brewers R. Spi

Magldi t 17
P.O. Box 126
H-1475 Budapest 10
E-mail: [email protected]

I Associazione degli Industriali R. Cavalli

della Birra e del Malto
Viale di Val Fiorita 90
I-00144 Roma
E-mail: [email protected]

IRL Irish Brewers Association N. Campbell-Crawford

Confederation House J. DArcy
84/86 Lower Baggot Street
Dublin 2
E-mail: [email protected]

LV Latvijas Alus Daritaju Savieniba Mrs. I. Shure

(Brewers Association of Latvia)
Brivibas iela 68 - Apt. 2
LV-1011 Riga
E-mail: [email protected]

N Norsk Bryggeri- og S.E. Kjekshus

Mineralvannindustris Forening
Essendropsgate 6 - 2nd floor
P.O. Box 7087 Majorstua
N-0306 Oslo
E-mail: [email protected]

2
NL Centraal Brouwerij Kantoor M.G. Kakebeeke
Herengracht 282 L.A. van der Stappen
Postbus 3462
NL-1001 AG Amsterdam
E-mail: [email protected]

P Associaao da Indstria F.M. Ferreira do Amaral

Cervejeira Portuguesa (AICP) J.M. Machado Cruz
Avenida Almirante Reis 115-3
P-1050-014 Lisboa
E-mail: [email protected]

RO Romanian Brewers Association V. Versescu

George Enescu Street 27-29
Etaj 2
Bucuresti 1
E-mail: [email protected]

S Svenska Bryggarefreningen M. Krantz

Polhemsgatan 29
P.O. Box 8104
S-10420 Stockholm
E-mail: [email protected]

SK Slovensk Zdruzenie Vyrobcov S. Markus

Piva a Sladu
Blumentlska 19
SK-81613 Bratislava
E-mail: [email protected]

SI Association of Slovene Brewers M. Oset

Pivovarna Union A. Rape
Pivovarniska 2
SI-1000 Ljubljana
E-mail: [email protected]

BOARD E. Pajunen President (FIN)

H.-G. Bellmer Vice President (D)
S.W. Molzahn Vice President (GB)
L.A. van der Stappen Vice President (NL)
J. Vesely Vice President (CZ)
Mrs. M. van Wijngaarden Secretary General

3
SECRETARIAT: Mrs. M. van Wijngaarden (Secretary General)
Mrs. H. Schippers (Assistant Secretary)

P.O. Box 510

2380 BB Zoeterwoude
The Netherlands

tel. : +31 (71) 545 6047 / 545 6614

telefax : +31 (71) 541 0013
e-mail : [email protected]
internet : http://www.ebc-nl.com

4
List of EBC Congresses
The European Brewery Convention aims at co-ordinating the scientific work carried
out in different countries in the technical fields of Brewing and Malting. One of the
means by which it seeks to attain this object is the organising of international
congresses once in every two years, with the particular aim of providing the
opportunity to exchange views on definite subjects.

Congresses held up to date are:

1st Scheveningen, The Netherlands, 1947

2nd Lucerne, Switzerland, 1949
3rd Brighton, Great Britain, 1951
4th Nice, France, 1953
5th Baden-Baden, Germany, 1955
6th Copenhagen, Denmark, 1957
7th Rome, Italy, 1959
8th Vienna, Austria, 1961
9th Brussels, Belgium, 1963
10th Stockholm, Sweden, 1965
11th Madrid, Spain, 1967
12th Interlaken, Switzerland, 1969
13th Estoril, Portugal, 1971
14th Salzburg, Austria, 1973
15th Nice, France, 1975
16th Amsterdam, The Netherlands, 1977
17th Berlin, Germany, 1979
18th Copenhagen, Denmark, 1981
19th London, Great Britain, 1983
20th Helsinki, Finland, 1985
21st Madrid, Spain, 1987
22nd Zurich, Switzerland, 1989
23rd Lisbon, Portugal, 1991
24th Oslo, Norway, 1993
25th Brussels, Belgium, 1995
26th Maastricht, The Netherlands, 1997
27th Cannes, France, 1999
28th Budapest, Hungary, 2001

Proceedings of a number of congresses are still available. See list of EBC

publications.