J. Agric. Food Chem.

1997, 45, 627−631 627

Comparison of the Effects of High-Pressure Treatments and Heat

Pasteurization on the Whey Proteins in Goat’s Milk
Xavier Felipe,† Marta Capellas,† and Andrew J. R. Law*,‡
Hannah Research Institute, Ayr KA6 5HL, Scotland, U.K., and Unitat de Tecnologie del Aliments, CeRTA,
Facultat de Veterinària, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain

Goat’s milk was subjected to pressures up to 500 MPa at 25 or 50 °C, and the extent of denaturation
of the individual whey proteins, determined from their loss of solubility at pH 4.6, was examined
by gel permeation FPLC (fast protein liquid chromatography) and SDS-PAGE. On pressure
treatment at 25 °C, β-lactoglobulin readily aggregated, whereas the immunoglobulins and R-lac-
talbumin were more resistant. At 50 °C, the effect was greater, and the immunoglobulins and
R-lactalbumin were also partially denatured. SDS-PAGE showed that after pressure treatment
the proteins in the acid precipitate were disulfide linked. Some small soluble aggregates of
β-lactoglobulin remained in the acid whey from pressurized milk, and these were not present in
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acid whey from heat-treated milk. Thermal and pressure treatments of milk could be distinguished
by their different effects on denaturation of the individual whey proteins. Also, the activity of alkaline
phosphatase in goat’s milk was reduced by pasteurization but not by high-pressure treatment up
Downloaded via UNIV DE LA SERENA on January 20, 2024 at 03:00:58 (UTC).

to 500 MPa for 10 min. The possible use of these differences for monitoring thermal and pressure
treatments in milk, and the importance of high pressure treatment in denaturation of the whey
proteins, are discussed.

Keywords: Whey protein; high pressure; goat’s milk; indices; aggregation; denaturation

INTRODUCTION goat’s milk, the effect of high pressure on denaturation

of the whey proteins has been determined by following
The effect of high pressure on food products has been their loss of solubility at pH 4.6. Differences between
studied since 1899 (Hite, 1899). However, it was only pressure and thermal treatments in the extent of
in the late 1980s that the technology evolved sufficiently denaturation of the whey proteins and inactivation of
for the process to be developed commercially. High- alkaline phosphatase were also examined.
pressure equipment which was previously used for the
production of metals, ceramics, and composites is now
being considered for use in the food industry. Most of MATERIALS AND METHODS
this progress has been made in Japan, where several Sample Preparation. Milk from Murciano-granadina
pressurized foods such as jam and fruit juices have been goats was obtained from the farm of the Universitat Autònoma
available on the market since 1990 (Mertens, 1993). de Barcelona. The fat content was standardized to 4%, and
These treated products have longer shelf lives than the the pH was adjusted to 6.7 with 1 M HCl or NaOH. Each
fresh foods and keep the same flavor and nutritive milk sample for pressure treatment was placed in a tubular
characteristics. Some studies have been carried out on bag made of polyvinylidene chloride (31.8 mm in diameter,
the effect of high pressure on milk. Desobry-Banon et Krehalon, Soplaril Hispania S. A., Spain). The bags were
vacuum packed to exclude air.
al. (1994) showed in their work on reconstituted milk
High-Pressure Treatment. The samples were treated
that this treatment affects the rennet coagulation time,
using discontinuous hydrostatic high-pressure equipment
turbidity, and levels of soluble calcium and nitrogen. (ACB, Nantes, France). The equipment reached 500 MPa in
Johnston et al. (1992) found an increase in hydrophobic about 4 min and took between 90 and 120 s for pressure
groups on the surface of the milk proteins and also reduction. Treatment temperature was taken as the water
noticed a decrease in non-casein nitrogen after treat- temperature inside the cylinder, measured just before applica-
ment, indicating precipitation of the whey protein. tion of pressure. The pressure chamber and the water inside
Johnston et al. (1993) reported that there was an were cooled or heated to the required temperature by a
increase in water-holding capacity, protein hydration constant flow of fluid within the walls of the vessel. Ten
index, gel rigidity, and gel breaking strength of acid- minutes before treatment started, the temperature of each
set gels prepared from pressurized milk. sample was checked. After treatment the samples were
refrigerated for 17 h before analysis or freezing was under-
Pasteurization and microfiltration have been used to taken. The samples were pressurized at 25 or 50 °C, with a
reduce the contaminant microflora in goat’s milk (Gay holding time of 5 or 10 min at the required pressure.
et al., 1993). Pasteurization is the main treatment used Heat Treatment. Skim-milk samples (5.0 mL) were
to reduce the growth of microorganisms in goat’s milk heated in stoppered, thin-walled glass tubes (1 mm wall, 8 mm
but, unfortunately, also brings about undesirable changes internal diameter, 160 mm length) in a waterbath. The milks
in flavor and loss of nutrients. In this study, in order were allowed 2 min 7 s to warm to 83 °C, maintained at this
to determine the suitability of this new treatment for temperature for 1 min, and then rapidly cooled in ice.
Heat Pasteurization. Milk was pasteurized by passing
through a heat-exchanger (Garvia S. A.) at 72 °C at a flow
* Author to whom correspondence should be ad- rate of 500 L/h, with a holding time of 15 s, and an overall
dressed. time of 2 min 30 s for warming, holding, and cooling.
† Universitat Autònoma de Barcelona
Acid Filtrate. Milk samples were warmed to 20 °C and
‡ Hannah Research Institute. adjusted to pH 4.6 with acetic acid (0.83 M) and sodium acetate

S0021-8561(96)00406-2 CCC: $14.00 © 1997 American Chemical Society

628 J. Agric. Food Chem., Vol. 45, No. 3, 1997 Felipe et al.

Figure 1. Elution profiles obtained by gel permeation FPLC

on a Superdex 75 HR 10/30 column in Tris-HCl buffer (pH
7.0, 100 mM Tris, 0.5 M NaCl), showing the decrease in
solubility of the whey proteins at pH 4.6 following heat and
pressure treatments of goat’s milk: (a) acid filtrate from raw Figure 2. Elution profiles obtained by gel permeation FPLC
milk; (b) acid filtrate from milk pressurized at 300 MPa for 5 (conditions as in Figure 1) showing the decrease in solubility
min at 25 °C; (c) acid filtrate from milk heated at 83 °C for 1 of the whey proteins at pH 4.6 with increasing severity of
min. Ig, immunoglobulins; SA/Lf, serum albumin and lacto- pressure treatment of goat’s milk at 25 °C: (a) acid filtrate
ferrin; β-Lg, β-lactoglobulin; R-La, R-lactalbumin. from raw milk. Acid filtrates from milk pressurized for 10 min
at (b) 200 MPa, (c) 300 MPa, (d) 400 MPa, and (e) 500 MPa.
(0.2 M), with a final dilution of 1:1. After allowing the samples Abbreviations as in Figure 1.
to stand for 15 min, the supernatants were passed through
0.22 µm nylon filters (Sartorius, Epson, Surrey, U.K.). aggregation of the protein. However, on pressure treat-
Gel Permeation FPLC. Fast protein liquid chromatog- ment, β-lactoglobulin was most easily denatured, and
raphy (FPLC) was carried out on 50 µL aliquots of acid filtrate the fractions containing immunoglobulins, serum albu-
containing up to 3 mg of protein/mL. The samples were min/lactoferrin, and R-lactalbumin appeared to be much
fractionated by gel permeation FPLC at 20 °C on a Superdex more resistant. As discussed below, there was also a
75 HR 10/30 column (Pharmacia Biotech, St. Albans, U.K.) in small increase in the amount of lower molecular weight
Tris-HCl buffer (pH 7.0, 100 mM Tris, 0.5 M NaCl) at a flow
rate of 0.5 mL/min. The absorbance of the eluate was protein in fraction 2.
monitored at 280 nm, and a total volume of 26 mL was passed The effect of increasing the pressure up to 500 MPa
through the column to ensure complete elution of absorbing at 25 °C on the whey protein fractions in milk is shown
material. Peak areas were corrected for the baseline value, on Figure 2. At a pressure as low as 200 MPa, there
and concentrations of the individual whey proteins calculated was a decrease in the amount of β-lactoglobulin in the
using specific absorbance coefficients at 280 nm: immunoglo- acid filtrate. None of the other fractions was affected,
bulins, 12.1; serum albumin, lactoferrin, 6.9; β-lactoglobulin, although a new peak appeared between the main peak
9.5; R-lactalbumin, 20.1 (Law and Brown, 1994).
SDS-PAGE. Whey proteins were examined by sodium
of the serum albumin/lactoferrin fraction and β-lacto-
dodecyl sulfate (SDS)-PAGE on PhastSystem electrophoresis globulin. On plotting the log of the molecular weight
equipment (Pharmacia Biotech, St. Albans, U.K.) using 20% of the individual whey proteins against elution time
homogeneous gels, in accordance with the manufacturers (Law et al., 1993), the molecular mass of the peak was
instructions. estimated to be about 46 680 Da. The amount of this
Alkaline Phosphatase Activity. Activity was determined protein was greatest at about 300 MPa. Increasing
using a Fluorophos-R fluorometer according to Standard IDF pressure caused a decrease in the amount of this
Method 155 (1992). Each analysis was carried out on three protein. At 300 MPa, the area of the serum albumin/
different milks. lactoferrin fraction increased considerably, and there
was also a slight increase in the region of the main
RESULTS fraction of immunoglobulins (IgG), but not in the minor
The elution profiles obtained by gel permeation FPLC fraction, which corresponded to the higher molecular
of acid filtrates from goat’s milk before and after weight IgM. At higher pressures (400 MPa), most of
thermal and pressure treatments are shown in Figure the β-lactoglobulin was denatured, and there was a
1. The whey protein was separated into four fractions slight decrease in the minor peak of IgM. Finally, at
identified by Law and Brown (1994) as immunoglobulins 500 MPa, there was some denaturation of the immu-
(fraction 1), serum albumin and lactoferrin (fraction 2), noglobulin fraction, but the serum albumin/lactoferrin
β-lactoglobulin (fraction 3), and R-lactalbumin (fraction and R-lactalbumin fractions did not show any ap-
4). Thermal treatment of the milk caused denaturation preciable decrease.
of the whey proteins and a decrease in the amount of The effect of pressure treatment on the whey proteins
whey proteins remaining in solution at pH 4.6. The in milk at 50 °C is shown in Figure 3. At 250 MPa,
fractions containing larger molecular weight proteins, there was marked denaturation of β-lactoglobulin and
such as the immunoglobulins and serum albumin/ the appearance of a small peak immediately before the
lactoferrin, were most easily denatured, whereas β-lac- β-lactoglobulin peak, but little change in the immuno-
toglobulin was more heat-resistant, and R-lactalbumin globulin and R-lactalbumin fractions. At 500 MPa, most
was the most difficult to denature (Law, 1995). Fol- of the β-lactoglobulin was denatured, and compared
lowing pressure treatment of milk at 25 °C, there was with treatments at 25 °C, there were higher levels of
also a decrease in the amount of whey protein remaining denaturation of the immunoglobulin and R-lactalbumin
in solution at pH 4.6 (Figure 1), indicating increased fractions.
Pressure Denaturation of Whey Proteins J. Agric. Food Chem., Vol. 45, No. 3, 1997 629

Figure 3. Elution profiles obtained by gel permeation FPLC

(conditions as in Figure 1), showing the decrease in solubility
of the whey proteins at pH 4.6 with increasing severity of
pressure treatment of goat’s milk at 50 °C: (a) acid filtrate
from raw milk; (b) acid filtrate from milk pressurized at 250
MPa for 10 min; (c) filtrate from milk pressurized at 500 MPa Figure 5. SDS-PAGE pattern on a 20% homogeneous gel
for 10 min. Abbreviations as in Figure 1. for whole whey proteins (WP), whey proteins pressurized at
500 MPa for 10 min at 50 °C (PWP), and the fractions obtained
by gel permeation FPLC as shown in Figure 3 of PWP (500
MPa for 10 min at 50 °C). Lanes 1-4, fractions 1-4, respec-
tively. Abbreviations as in Figure 1.

Figure 4. SDS-PAGE pattern on a 20% homogeneous gel

for whole whey proteins (WP), whey proteins pressurized at
500 MPa for 10 min at 25 °C (PWP), and the fractions obtained
by gel permeation FPLC as shown in Figure 2 of PWP (500
MPa for 10 min at 25 °C). Lanes 1-4, fractions 1-4, respec-
tively. Abbreviations as in Figure 1.
Figure 6. SDS-PAGE pattern on a 20% homogeneous gel
The identities of the whey protein fractions obtained for the proteins precipitated at pH 4.6. Lanes 1r and 5, acid
precipitates from pressurized milk (500 MPa for 10 min at 25
by gel permeation FPLC of acid filtrates from milks °C); lanes 2r and 6, acid precipitates from heat-treated milk
pressure treated at 500 MPa for 10 min at 25 °C were (83 °C for 1 min); lanes 3r and 7, acid precipitates from control
examined in more detail by SDS-PAGE (Figure 4). milk; lane 4, whole whey proteins (WP). Lanes 1r, 2r, 3r, and
Under dissociating conditions, fraction 1 appeared to 4, reduced with 2-mercaptoethanol. Lanes 5, 6, and 7, no
contain mainly immunoglobulins. Fraction 2 contained reduction with 2-mercaptoethanol. R-La, R-lactalbumin; β-Lg,
serum albumin and lactoferrin as major components, β-lactoglobulin, κ-, κ-casein; β-, β-casein; Rs1-, Rs1-casein; Rs2-,
Rs2-casein; Ig, immunoglobulins; SA, serum albumin; Lf,
but a band of β-lactoglobulin was also clearly seen. lactoferrin.
Fraction 3 contained only β-lactoglobulin, and fraction
4 only R-lactalbumin. R-Lactalbumin was not detected little whey protein. However, the acid precipitates from
in any of the other fractions. On similar pressure pressurized milk (lane 1r), and heated milk (lane 2r)
treatment at 50 °C (Figure 5), there was also evidence contain appreciable amounts of whey proteins. Follow-
of the presence of some R-lactalbumin in the serum ing both treatments, most of the β-lactoglobulin pre-
albumin/lactoferrin and β-lactoglobulin fractions. The cipitated at pH 4.6, but most of the R-lactalbumin was
presence of β-lactoglobulin in fraction 2, and of R-lac- resistant to pressure and thermal treatments. How-
talbumin in fractions 2 and 3, was attributed to the ever, there were differences in the behavior of both the
formation of small aggregates which remained soluble immunoglobulins and serum albumin/lactoferrin fol-
at pH 4.6 and which could be disrupted in the presence lowing the two different types of treatment, with less
of 2-mercaptoethanol. of these proteins appearing in the acid precipitate
Several studies have shown that when the whey following pressure treatment. On carrying out SDS-
proteins are aggregated by thermal treatment, they tend PAGE of the acid precipitated proteins from thermal-
to precipitate at pH 4.6 together with the caseins and pressure-treated milks without the reduction step
(Rowland, 1937a,b). As can be seen in Figure 6, the acid with 2-mercaptoethanol (Figure 6), most of the whey
precipitate from nontreated milk (lane 3r) contains very proteins and κ-casein were absent from the running gel,
630 J. Agric. Food Chem., Vol. 45, No. 3, 1997 Felipe et al.

Figure 7. Effect of pressure at 25 °C on the extent of Figure 8. Effect of pressure at 25 °C on the extent of
denaturation of each whey protein fraction, determined by gel denaturation of total whey protein, determined by gel perme-
permeation FPLC of acid filtrates from pressurized milk: (9) ation FPLC of acid filtrates from pressurized milk.
βLg, (b) Ig, (2) RLa.
bulin that were only obtained after a fairly severe heat
and large aggregates appeared in the stacking gel, treatment of 80 °C for 10 min. β-Lactoglobulin, there-
showing that these proteins were disulfide linked. The fore, appeared particularly susceptible to high-pressure
kind of aggregates could not be determined at this stage treatment and, as it is the main constituent of whey
of the work, and they may be a mixture of homo- or protein, almost 50% of total whey protein became
heteropolymers. irreversibly aggregated above 400 MPa (Figure 8).
The denaturation curves for the individual whey In cow’s milk, the activity of alkaline phosphatase is
proteins on pressure-treatment of milk at 25 °C are a useful index of the safety of heat treatment, as its
shown in Figure 7. The serum albumin/lactoferrin thermal inactivation occurs after that of vegetative
fraction has not been plotted, as the presence of a small forms of pathogenic microorganisms. In goat’s milk,
amount of β-lactoglobulin aggregates produced an in- however, the thermal inactivation of this enzyme is not
crease in area following most of the treatments. How- suitable as an index of thermal treatment as it is
ever, previous studies on bovine serum albumin have inactivated at a lower temperature (Williams, 1986). In
indicated that it does not readily aggregate (Hayakawa the present study, following pasteurization of goat’s milk
et al., 1992). The denaturation curve of β-lactoglobulin (72 °C for 15 s), alkaline phosphatase was completely
showed a slow increase up to 150-200 MPa, followed inactivated whereas in raw milk or after pressure
by a rapid increase up to 350 MPa. Above this pressure, treatment (500 MPa for 10 min) the activity was
most of the β-lactoglobulin was aggregated and the level unchanged (data not shown). Wong and Armstrong
of denaturation increased more slowly. Some variability (1992) actually observed increased activity of microbial
in the levels of denaturation between milk samples for alkaline phosphatase at pressures up to 720 MPa.
each treatment was observed, and this was related to
the different initial concentrations of whey proteins. No DISCUSSION
differences could be observed in the levels of denatur-
ation of the immunoglobulins with pressure treatments Thermal and pressure treatments of milk could be
up to 300 MPa, but some aggregation occurred between distinguished by the different susceptibilities of the
300 and 500 MPa. Results are similar to those of whey proteins to denaturation under the two treat-
Howlett et al. (1992), who reported that bovine IgG does ments. On heat-treatment in this and previous studies
not undergo conformational changes below 210 MPa. (Law, 1995), the order of ease of irreversible denatur-
They also found that when the pressure was increased ation of caprine whey proteins, based on loss of solubility
to 820 MPa, some conformational changes and aggrega- at pH 4.6, was immunoglobulins > serum albumin/
tion appeared to occur, the rate of change being faster lactoferrin > β-lactoglobulin > R-lactalbumin. On pres-
between 210 and 460 MPa. This resistance of the sure treatments, however, β-lactoglobulin was more
immunoglobulins may have pharmaceutical-medical readily denatured than the other whey proteins. At a
applications. Only a small amount of aggregation of pressure of 250 MPa at 25 or 50 °C for 10 min,
R-lactalbumin was observed below 500 MPa. This β-lactoglobulin was extensively denatured whereas the
resistance to aggregation could be due to the absence other whey proteins showed little change in their
of free SH groups and the initial difficulty in forming solubility at pH 4.6. At a pressure of 500 MPa for 10
covalent links to other proteins. min at 25 °C, and especially at 50 °C, there were also
Although few studies have been published on the appreciable increases in the levels of denaturation of
inactivation of microorganisms in milk by pressure the immunoglobulins and R-lactalbumin.
treatment (Cheftel, 1995), the available evidence indi- A further difference between thermal and pressure
cates that treatments at 400-500 MPa are suitable for treatment was the appearance in the acid whey, after
reducing the growth of microorganisms. On comparing pressure treatment, of small soluble aggregates of
pressure treatments in the present study with thermal β-lactoglobulin. On heat-treatment, denatured β-lac-
treatment of goat’s milk (Law, 1995; Montilla et al., toglobulin becomes associated with κ-casein on the
1995), it can be seen that pressures of 400-500 MPa micelles and precipitates on subsequent acidification to
caused only a small amount of denaturation of immu- pH 4.6. However, on pressure treatment where only
noglobulins, serum albumin/lactoferrin, and R-lactal- part of the β-lactoglobulin was denatured, it also showed
bumin, similar to that obtained by very mild heat some tendency to form homopolymers or possibly poly-
treatment. However, pressures between 400 and 500 mers with other whey proteins. In studies of isolated
MPa caused high levels of denaturation of β-lactoglo- β-lactoglobulin, Funterberger et al. (1995) similarly
Pressure Denaturation of Whey Proteins J. Agric. Food Chem., Vol. 45, No. 3, 1997 631

found that pressure treatment resulted in formation of Hite, B. H. The effect of pressure in the preservation of milk.
disulfide-linked polymers, and these contained between 1899, Bull. 58, 15-35. West Virginia Univ. Agr. Expt. Sta.,
two and six subunits. In the present study, it was found Morgantown.
that at higher pressures the amount of small polymers Howlett, J. R.; Ismail, A. A.; Armstrong, D. W.; Wong, P. T. T.
was reduced, possibly because of the formation of larger Pressure-induced conformational changes in an antigen and
an antibody and the implications on their use for hyperbaric
polymers that more readily precipitated at pH 4.6. immunoadsorption. Biochim. Biophys. Acta 1992, 1159,
Previous studies on thermal treatment of milk have 227-236.
shown that there is a close correlation between the loss International Dairy Federation. Determination of alkaline
of solubility of denatured whey proteins at pH 4.6 and phosphatase, 1992; Method 155.
the extent to which they are incorporated into the Johnston, D. E.; Austin, B. A.; Murphy, R. J. Effects of high
rennet curd during cheesemaking. If pressure-dena- hydrostatic pressure on milk. Milchwissenschaft 1992, 47
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of the final ripened cheese could be obtained. The set gels prepared from high pressure treated skim milk.
effects of the duration and temperature of the pressure Milchwissenschaft 1993, 48 (4), 206-209.
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Law, A. J. R.; Brown, J. R. Compositional changes in caprine
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