Advances in Food Authenticity Testing

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Advances in Food
Authenticity Testing

Edited by

Gerard Downey
Teagasc Food Research Centre
Dublin, Republic of Ireland

AMSTERDAM • BOSTON • CAMBRIDGE • HEIDELBERG

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Contents

List of Contributors xiii

1 Introduction 1
G. Downey
References 3

Part One Advances in Methods for Food Authenticity Testing 5

2 Advances in DNA Fingerprinting for Food Authenticity Testing 7
W.F. Nader, T. Brendel and R. Schubbert
2.1 Introduction 7
2.2 Scientific Background of DNA Fingerprinting and Its Applications 9
2.3 The Methodology of DNA Fingerprinting 11
2.4 DNA Fingerprinting for Authenticity Testing of Rice Varieties 22
2.5 Meat Traceability 27
2.6 Future Trends 28
References 29

3 Advances in Ultraviolet and Visible Light Spectroscopy for Food

Authenticity Testing 35
M.J. Martelo-Vidal and M. V
azquez
3.1 Introduction 35
3.2 Fundamentals of Ultraviolet and Visible Light Spectroscopy
in Food Analysis 37
3.3 Application of UVeVIS Spectroscopy for Authentication
of Foodstuffs 41
3.4 Suitable Foodstuffs for Testing Using Ultraviolet and Visible
Light Spectroscopy Analysis 48
3.5 Case Study: Wine Authentication 59
References 63

4 Advances in Infrared Spectroscopy for Food Authenticity Testing 71

L.E. Rodriguez-Saona, M.M. Giusti and M. Shotts
4.1 Introduction 71
4.2 Vibrational Spectroscopy as a Screening Method 72
vi Contents

4.3 Chemometrics in Vibrational Spectroscopy 78

4.4 Screening Capabilities of Vibrational Spectroscopy to Detect
Adulteration in Foods 84
4.5 The Future of Vibrational Spectroscopy in Food Authentication:
Portable and Handheld Technology 102
Sources of Further Information 107
References 107

5 Advances in Fluorescence Emission Spectroscopy for Food

Authenticity Testing 117
A. Dankowska
5.1 Introduction 117
5.2 What Adulterations Can Fluorescence Emission Spectroscopy
Be Used to Detect? 120
5.3 Applications of Fluorescence Emission Spectroscopy for Food
Authentication e Examples 128
5.4 Future Trends 138
References 140

6 Advances in Nuclear Magnetic Resonance Spectroscopy for Food

Authenticity Testing 147
A.P. Sobolev, S. Circi and L. Mannina
6.1 Introduction 147
6.2 Nuclear Magnetic Resonance Methodologies in Food Analysis 148
6.3 Sample Preparation for Nuclear Magnetic Resonance Analysis 150
6.4 Spectral Assignment and Quantitative Analysis 151
6.5 Adulterations Detected by Nuclear Magnetic Resonance
Spectroscopy 152
6.6 Future Trends 165
References 165

7 Advances in Mass Spectrometry for Food Authenticity Testing:

An Omics Perspective 171
T. Cajka, M.R. Showalter, K. Riddellova and O. Fiehn
7.1 Introduction 171
7.2 Process of Using Mass Spectrometry in the Analysis of Food 172
7.3 Mass Spectrometry-Based Approaches for Food Authenticity
Testing and Adulteration Detection 176
7.4 Future Trends 195
Acknowledgments 195
References 195
Contents vii

8 Advances in Electronic Noses and Tongues for Food

Authenticity Testing 201
M.
Sliwi

nska, P. Wi
sniewska, T. Dymerski, W. Wardencki and
J. Namie
snik
8.1 Introduction 201
8.2 Electronic Nose 202
8.3 Electronic Tongue 205
8.4 Application of Electronic Nose and Tongue in Food Authenticity
Studies 207
8.5 Conclusions 219
References 220

9 Advances in Isotopic Analysis for Food Authenticity Testing 227

K.H. Laursen, L. Bontempo, F. Camin and A. Roßmann
9.1 Introduction 227
9.2 Measurements, Instrumentation, and Applications 231
9.3 Case Studies 239
9.4 Conclusion and Future Trends 245
Sources of Further Information 246
Abbreviations 246
Acknowledgments 246
References 246

10 Advances in Chromatographic Techniques for Food Authenticity

Testing 253
C. Fanali, L. Dugo and L. Mondello
10.1 Introduction 253
10.2 Process of Using Chromatographic Techniques in the Analysis
of Food 254
10.3 Adulterations Which Can Be Detected by Using Chromatographic
Techniques 261
10.4 Foodstuffs Suitable for Testing Using Chromatographic
Techniques 262
10.5 Case Studies 262
10.6 Future Trends 277
Sources of Further Information 277
References 278

11 Advances in Polymerase Chain Reaction Technologies for Food

Authenticity Testing 285
E. Maestri and N. Marmiroli
11.1 Introduction 285
11.2 Process of Using PCR Technologies in the Analysis of Food 288
11.3 Application of PCR Technologies to Detect Adulteration 295
11.4 Case Studies 296
viii Contents

11.5 Future Trends 301

Sources of Further Information and Conclusions 303
Acknowledgments 304
References 304

12 Advances in Differential Scanning Calorimetry for Food

Authenticity Testing 311
T. Nur Azira and I. Amin
12.1 Introduction 311
12.2 Uses of Differential Scanning Calorimetry in the Analysis of Foods 312
12.3 Uses of Differential Scanning Calorimetry in Food Authenticity
Testing 315
12.4 Conclusions and Future Perspectives 329
Sources of Further Information 329
References 330

Part Two Advances in Authenticity Testing 337

13 Advances in Authenticity Testing of Geographical Origin of Food

Products 339
A.M. Pustjens, M. Muilwijk, Y. Weesepoel and S.M. van Ruth
13.1 Introduction 339
13.2 Techniques for Analyzing Isotopes 340
13.3 Techniques for Analyzing Elements 343
13.4 Separation Techniques for Compositional Analysis 344
13.5 (Semi-)Nondestructive Techniques 349
13.6 Other Techniques 357
13.7 Conclusions 358
List of Abbreviations of Analytical Equipment 358
List of Abbreviations of (Multivariate) Statistical Analysis 359
References 360

14 Advances in Authenticity Testing for Meat Speciation 369

J. Amaral, L. Meira, M.B.P.P. Oliveira and I. Mafra
14.1 Introduction 369
14.2 Protein-Based Methods 371
14.3 DNA-Based Methods 376
14.4 Spectroscopic Methods 399
14.5 Final Remarks 402
References 403

15 Advances in Authenticity Testing for Fish Speciation 415

M. Espi~
neira and F. Lago
15.1 Introduction 415
15.2 Methods Used in Fish Speciation 417
Contents ix

15.3 Case Studies 430

15.4 Future Trends 432
Sources of Further Information 434
References 434

16 Authentication of Cereals and Cereal Products 441

D. Cozzolino
16.1 Introduction 441
16.2 Application of NIR and MIR Spectroscopy to Cereal Grain
Authentication 443
16.3 Concluding Remarks 452
References 453

Part Three Advances in Authenticity Testing

for Food Adulteration 459
17 Advances in Testing for Adulteration and Authenticity of Dairy
Products 461
G.A. Abernethy, J.G. Bendall and S.E. Holroyd
17.1 Introduction 461
17.2 Types of Dairy Product Adulteration and Nonauthenticity 463
17.3 Chemical Methods to Combat Nonauthenticity 474
17.4 Spectroscopic Methods to Determine Adulteration 476
17.5 Future Developments 480
Acknowledgment 481
References 481

18 Advances in the Identification of Adulterated Cereals and Cereal

Products 491
S.R. Delwiche
18.1 Introduction 491
18.2 Legislation 493
18.3 Methodology for Phenotyping and Geography 496
18.4 Melamine 500
18.5 Durum 503
18.6 Basmati 505
18.7 Wheat Gluten as an Adulterant 507
18.8 Application of Using NIR Technology for Mixtures of Nonwaxy
(Conventional) and Waxy Wheat 509
18.9 Conclusion 511
Sources of Further Information 512
References 512
x Contents

19 Advances in the Identification of Adulterated Vegetable Oils 519

O. Abbas and V. Baeten
19.1 Introduction 519
19.2 Authenticity Testing of Adulterated Vegetable Oils, Including
Case Studies 523
19.3 Conclusions and Future Trends 534
References 535

20 Advances in the Identification of Genetically Modified Foods 543

M.-A. Fraiture, S. Broeders, P. Herman, I. Taverniers, M. De Loose,
D. Deforce and N.H. Roosens
20.1 Introduction 543
20.2 Processes Used for Identification of Genetically Modified Foods 544
20.3 Case Studies 548
20.4 Future Trends 552
Sources of Further Information 555
Acknowledgments 555
References 556

21 Advances in the Detection of the Adulteration of Alcoholic Beverages

Including Unrecorded Alcohol 565
D.W. Lachenmeier
21.1 Introduction 565
21.2 Processes Used in the Detection of Alcoholic Beverage
Adulteration 566
21.3 Case Studies 575
21.4 Future Trends 577
Sources of Further Information 578
References 578

22 Advances in Adulteration and Authenticity Testing of Herbs

and Spices 585
B. Sasikumar, V.P. Swetha, V.A. Parvathy and T.E. Sheeja
22.1 Introduction 585
22.2 Uses of Spices and Herbs 586
22.3 Adulterants in Spices and Herbs 587
22.4 Techniques for Adulterant Detection 594
22.5 Future Perspectives and Conclusions 612
References 612

23 Tradition Meets High Tech for Authenticity Testing of Fruit Juices 625
P. Rinke
23.1 Introduction 626
23.2 Overview of Methods Applied in Standard Control 628
Contents xi

23.3 Conventional Methods 628

23.4 Overview of Modern Techniques 638
23.5 Case Study Combining Different Methods 653
23.6 Importance of Databases and Documentation 655
23.7 Outlook 656
Acknowledgments 658
References 658

24 Advances in Testing for Adulteration of Food Supplements 667

S.H. El-Ahmady and M.L. Ashour
24.1 Introduction 668
24.2 Adulteration of Food Supplements 673
24.3 Methods of Adulterant Detection 683
24.4 Global Agencies and Authorities Responsible for
Drug and Food Supplement Safety 690
24.5 Future Perspectives 691
References 691

25 Chemometrics for Food Authenticity Applications 701

P. Oliveri and R. Simonetti
25.1 Introduction 702
25.2 Multivariate Data Analysis 703
Acknowledgment 726
References 726

26 Advances in Testing for Adulteration in Honey 729

F. Ulberth
26.1 Introduction 729
26.2 Processes Used in Identification of Adulteration in Honey 732
26.3 Outlook 745
References 745

Index 755
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List of Contributors

O. Abbas Walloon Agricultural Research Centre (CRA-W), Gembloux, Belgium

G.A. Abernethy Fonterra Research & Development Centre, Palmerston North, New
Zealand
J. Amaral University of Porto, Porto, Portugal
I. Amin Universiti Putra Malaysia, Serdang, Selangor, Malaysia
M.L. Ashour Ain Shams University, Cairo, Egypt
V. Baeten Walloon Agricultural Research Centre (CRA-W), Gembloux, Belgium
J.G. Bendall Fonterra Research & Development Centre, Palmerston North, New
Zealand
L. Bontempo Research and Innovation Centre, Trentino, Italy
T. Brendel Eurofins Medigenomix GmbH, Ebersberg, Germany
S. Broeders Scientific Institute of Public Health, Brussels, Belgium
T. Cajka University of California, Davis, Davis, CA, United States
F. Camin Research and Innovation Centre, Trentino, Italy
S. Circi University of Rome, Rome, Italy
D. Cozzolino Central Queensland University, Rockhampton, Queensland, Australia
A. Dankowska Pozna

n University of Economics and Business, Pozna
n, Poland
D. Deforce Ghent University, Ghent, Belgium
M. De Loose Institute for Agricultural and Fisheries Research (ILVO), Merelbeke,
Belgium
S.R. Delwiche USDA, Agricultural Research Service, Beltsville, MD, United States
G. Downey Teagasc Food Research Centre, Dublin, Republic of Ireland
L. Dugo Universita Campus Bio-Medico of Rome, Rome, Italy
T. Dymerski Gdansk University of Technology, Gda
nsk, Poland
xiv List of Contributors

S.H. El-Ahmady Ain Shams University, Cairo, Egypt

M. Espi~
neira ANFACO-CECOPESCA, Vigo, Spain
C. Fanali Universita Campus Bio-Medico of Rome, Rome, Italy
O. Fiehn University of California, Davis, Davis, CA, United States
M.-A. Fraiture Scientific Institute of Public Health, Brussels, Belgium
M.M. Giusti College of Food Agriculture and Environmental Sciences, The Ohio
State University, OH, United States
P. Herman Scientific Institute of Public Health, Brussels, Belgium
S.E. Holroyd Fonterra Research & Development Centre, Palmerston North, New
Zealand
D.W. Lachenmeier Chemisches und Veterin€aruntersuchungsamt (CVUA)
Karlsruhe, Karlsruhe, Germany
F. Lago ANFACO-CECOPESCA, Vigo, Spain
K.H. Laursen University of Copenhagen, Copenhagen, Denmark
E. Maestri University of Parma, Parma, Italy
I. Mafra University of Porto, Porto, Portugal
L. Mannina Sapienza University of Rome, Rome, Italy
N. Marmiroli University of Parma, Parma, Italy
M.J. Martelo-Vidal University of Santiago de Compostela, Lugo, Spain
L. Meira University of Porto, Porto, Portugal
L. Mondello Universita Campus Bio-Medico of Rome, Rome, Italy
M. Muilwijk Wageningen University and Research Centre, Wageningen, The
Netherlands
W.F. Nader Eurofins Global Control GmbH, Hamburg, Germany
J. Namie
snik Gdansk University of Technology, Gda
nsk, Poland
T. Nur Azira International Islamic University Malaysia, Kuala Lumpur, Malaysia
M.B.P.P. Oliveira University of Porto, Porto, Portugal
P. Oliveri University of Genova, Genova, Italy
V.A. Parvathy ICAR-Indian Institute of Spices Research, Kozhikode, Kerala, India
A.M. Pustjens Wageningen University and Research Centre, Wageningen, The
Netherlands
K. Riddellova ALS Czech Republic s.r.o., Prague, Czech Republic
List of Contributors xv

P. Rinke SGF International E.V., Nieder-Olm, Germany

L.E. Rodriguez-Saona College of Food Agriculture and Environmental Sciences,
The Ohio State University, OH, United States
N.H. Roosens Scientific Institute of Public Health, Brussels, Belgium
A. Roßmann Isolab GmbH Laboratory for Stable Isotopes, Schweitenkirchen,
Germany
B. Sasikumar ICAR-Indian Institute of Spices Research, Kozhikode, Kerala, India
R. Schubbert Eurofins Medigenomix GmbH, Ebersberg, Germany
T.E. Sheeja ICAR-Indian Institute of Spices Research, Kozhikode, Kerala, India
M. Shotts College of Food Agriculture and Environmental Sciences, The Ohio State
University, OH, United States
M.R. Showalter University of California, Davis, Davis, CA, United States
R. Simonetti University of Genova, Genova, Italy


M. Sliwi

nska Gdansk University of Technology, Gda
nsk, Poland
A.P. Sobolev National Research Council, Rome, Italy
V.P. Swetha ICAR-Indian Institute of Spices Research, Kozhikode, Kerala, India
I. Taverniers Institute for Agricultural and Fisheries Research (ILVO), Merelbeke,
Belgium
F. Ulberth European Commission, Geel, Belgium
S.M. van Ruth Wageningen University and Research Centre, Wageningen, The
Netherlands
M. V

azquez University of Santiago de Compostela, Lugo, Spain
W. Wardencki Gdansk University of Technology, Gda
nsk, Poland
Y. Weesepoel Wageningen University and Research Centre, Wageningen, The
Netherlands
P. Wi
sniewska Gdansk University of Technology, Gda
nsk, Poland
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Advances in Authenticity Testing
for Meat Speciation 14
J. Amaral, L. Meira, M.B.P.P. Oliveira, I. Mafra
University of Porto, Porto, Portugal

Chapter Outline

14.1 Introduction 369

14.2 Protein-Based Methods 371
14.2.1 Electrophoretic Techniques 371
14.2.2 Immunochemical Techniques 372
14.2.3 Chromatographic and Mass Spectrometry Techniques 374
14.3 DNA-Based Methods 376
14.3.1 PCR-RFLP 379
14.3.2 Species-Specific PCR 383
14.3.3 PCR-Sequencing 383
14.3.4 DNA Barcoding 390
14.3.5 Real-Time PCR 391
14.5.6 Next-Generation Sequencing 392
14.3.7 Biosensors 398
14.4 Spectroscopic Methods 399
14.5 Final Remarks 402
References 403

14.1 Introduction
Nowadays, and particularly after the horse meat scandal in Europe, consumers are
increasingly aware of the problem of food adulteration and consequently demand clear
and reliable information about the composition of foods they are buying and eating.
Meat, a highly appreciated premium source of protein, is among the foods most prone
to suffer adulteration for economic gain. According to EU legislation laying down the
general principles and requirements of food law (European Commission, 2002) and
EU labeling regulations (European Commission, 2001), meat products should be accu-
rately labeled regarding their species content, with food adulteration and misleading
information being considered illegal. However, because of its high demand and value,
frauds in the meat industry and retail markets have become a widespread problem,
especially in ground and comminuted meat products. Adulteration in the meat sector
encompasses many issues, including the substitution of high-quality muscle proteins

Advances in Food Authenticity Testing. http://dx.doi.org/10.1016/B978-0-08-100220-9.00014-X

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370 Advances in Food Authenticity Testing

by other, lower-value meat species or by vegetable proteins, the addition of undeclared

ingredients such as constituents of animal origin (eg, blood plasma, offal), the addition
of nonmeat components such as water and additives, mislabeling regarding the quan-
tity of meat ingredients, the geographical origin of meats and/or the animal feeding
regime, and undeclared processing methods in meat products, such as irradiation
and thawed meat (Ballin, 2010; Montowska and Pospiech, 2011a). All these practices
are of concern, either for economic reasons, as they lead to unfair competition among
producers and deceive consumers, or for health reasons since traceability is compro-
mised. Additionally, they restrict consumers’ freedom of choice, in particular for those
who choose to follow strict diets for religious motives since the consumption of certain
species is not allowed in some religions. For example, pork and its derivatives (among
other species) are strictly forbidden to Muslims and Jews who can only consume halal
or kosher meat, respectively. For those consumers, the presence of certain animal spe-
cies, which are undeclared on the product label, is perhaps of most concern.
Adulteration of meat and meat products because of species substitution is also of
major importance in the case of traditional products and/or game meat (Montowska
and Pospiech, 2012a; Costa et al., 2016a) since both types of products are frequently
more expensive and perceived by consumers as being of superior quality. In the case of
game meat, its consumption has been increasing in recent years, mainly because of its
particular taste and flavor but also because it may be considered a healthier and less-fat
meat, obtained from animals free of hormones and drugs such as antibiotics. However,
game meat supply depends on animal availability and can sometimes be restricted to
hunting seasons. This, together with its generally higher price, makes game meat and
products thereof very prone to adulteration by fraudulent substitution of meat species
(Amaral et al., 2014, 2015). Traditional foods, frequently considered as delicacies, are
another type of product that is highly susceptible to meat species substitution. Among
the regional and traditional products in the European Union labeled with the logos of
protected designation of origin (PDO), protected geographical indication (PGI), or
traditional specialty guaranteed (TSG), there are currently 146 registered products in
the category of “Fresh meat and offal” and 155 in “Meat products (cooked, salted,
smoked, etc.).” From those, several are produced from specific breeds as described
in the specifications accompanying the application for registration. For example, the
Portuguese meat products “Morcela de Estremoz e Borba” (PGI) and “Presunto de
Barrancos” (PDO) are produced from pork meat exclusively from the Alentejana breed
(Sus ibericus), and the lamb meat “Cordeiro Mirandês” refers exclusively to the Churra
Galega Mirandesa breed. Many other examples, similar to these, from other countries
could be mentioned. Since products holding PDO, PGI, or TSG logos generally com-
mand higher prices than other comparable meat products, they are susceptible targets
for economic fraud through undeclared substitution of the specified meat by other meat
from a similar breed.
Considering the relevance of species substitution in the subject of meat adulteration,
this chapter intends to provide an updated overview, attempting to have a global world
coverage on the most recent research studies regarding the development of methodol-
ogies for meat and meat product authenticity testing with respect to speciation and the
applicability to the identification of meat species in commercial products.
Advances in Authenticity Testing for Meat Speciation 371

14.2 Protein-Based Methods

In each species, the genes (genome) predetermine the amino acid composition of the
expressed proteins, their quantity, and rate of synthesis (Montowska and Pospiech,
2011a). Since the proteome of an organism is the result of the expression of its
genome, the analysis of proteins has been frequently suggested for species identifica-
tion purposes. As a result, different protein-based methodologies, including electro-
phoretic, chromatographic, and immunochemical techniques, have been proposed
for meat and meat product authentication studies. However, when evaluating the suit-
ability of these methods, it should be taken into account that protein composition can
vary, not only among species, but also with many other factors, including tissue type,
animal’s age and sex, meat processing treatments, and storage period. Therefore the
application of protein-based methods generally gives better results when applied to
species identification in raw than in processed meat products. In this case the diffi-
culties to overcome are related to the complexity and variability of foods, as well as
the type of processing that can frequently induce protein denaturation or degradation.

14.2.1 Electrophoretic Techniques

Electrophoresis is a powerful technique based on the use of an electric field for the sep-
aration of proteins into distinctive patterns. It includes different electrokinetic separa-
tion techniques according to the principle used for protein separation, such as
differences in electrophoretic mobility, isoelectric point, molecular size, or a combina-
tion of these. Electrophoresis can be conducted either on gel, such as the case of poly-
acrylamide gel electrophoresis (PAGE) and its modification using sodium dodecyl
sulfate as a denaturing agent (SDS-PAGE), isoelectric focusing (IEF), and, more
recently, two-dimensional electrophoresis (2-DE), or in small narrow capillaries,
such as the case of capillary zone electrophoresis and capillary isoelectric focusing
among others. Apart from being routinely used as a tool in different DNA-based
methods and being very useful in proteomic studies performed by mass spectrometry
(MS), electrophoretic methods per se have also been applied for meat species identi-
fication. There are only a few reports on meat authentication by protein gel electropho-
resis, despite being considered a well-known and frequently used technique for species
identification in raw fish and control of marine products (Montowska and Pospiech,
2007, 2012a; Etienne et al., 2000). The first studies used SDS-PAGE and IEF on poly-
acrylamide gel to identify meat cuts of different species (cow, sheep, lamb, goat, red
deer, and rabbit) and obtained somewhat ambiguous results (Hofmann, 1985 referred
in Montowska and Pospiech, 2007). Since several factors, other than the animal spe-
cies, can affect the results achieved by SDS-PAGE, this technique frequently presents
some constraints when applied for species authentication purposes, in particular
regarding meat mixtures and processed meat products, because of its lack of reproduc-
ibility and low discriminating power. IEF has also been used for species identification
in raw meat (Kaiser et al., 1980; Slattery and Sinclair, 1983; Kim and Shelef, 1986;
Skarpeid et al., 1998). Compared to SDS-PAGE, IEF generally presents better band
372 Advances in Food Authenticity Testing

resolution, but gives more complicated and less consistent patterns, which also advises
against its use for the authentication of processed meat products. Better results can be
achieved by means of 2-DE coupled to image analysis software since it allows the sep-
aration of thousands of proteins in one gel and analysis of changes in protein expres-
sion levels, such as isoforms and posttranslational modifications (Montowska and
Pospiech, 2012b). Species-specific differences in the molecular weight and isoelectric
point of skeletal muscle myosin light chain (MLC) isoforms of six species, namely,
cow, pork, chicken, turkey, duck, and goose, were revealed by 2-DE (Montowska
and Pospiech, 2011b). The authors concluded that each species showed a characteristic
pattern on 2-DE gels that can be used for species identification in raw meat mixtures
since even a 2% difference in the sequences of MLC resulted in different electropho-
retic mobilities. Later, the same methodology was applied to investigate if the interspe-
cies differences noticed in the expression of MLC isoforms in raw meat were also
observed in minced meat and meat products (frankfurters and sausages) produced
from the same six species (Montowska et al., 2012b). The results showed that the
MLC suffered only a relatively small degradation after processing, enabling species
identification in all samples when the combination of all the three MLC isoforms
was taken into account, and as long as the content of meat of one species in the mixture
was not lower than 10%. Thus, the authors concluded that MLC isoforms have the po-
tential to be used as markers for the authentication of meat products made from the
analyzed species.
As an alternative to gel electrophoresis, capillary electrophoresis (CE) has also been
applied to meat species authentication studies since it offers high resolving power and
sensitivity in protein analysis. A CE-SDS methodology, developed by Cota-Rivas and
Vallejo-Cordoba (1997) for the analysis of sarcoplasmic proteins, revealed the exis-
tence of qualitative and quantitative differences in raw beef, pork, and turkey meat.
Linear discriminant analysis (LDA) was later used by the same authors for the
interpretation of the quantitative data obtained by CE-SDS (Vallejo-Cordoba and
Cota-Rivas, 1998). The correct classification given by LDA on water-soluble protein
data was 100% for all meat species except pork (94%). The same group also proposed
the use of CE for the discrimination of bovine and ostrich meats (Vallejo-Cordoba
et al., 2010). Although the applicability of CE methodology for meat species differen-
tiation was suggested, in both studies the authors only tested a limited number of meat
samples (n ¼ 42 in total for beef, pork, and turkey samples and n ¼ 20 for beef and
ostrich), thus further work should be performed using samples from different animals
and tissues to confirm these results. Since only fresh meat was used in both studies, the
proposed CE methodologies seem to be applicable only for the authentication of raw
meat. Moreover, they do not seem adequate for species identification or quantification
in meat mixtures since species discrimination was mainly based on quantitative pro-
files of proteins that are common to different species.

14.2.2 Immunochemical Techniques

Immunochemical assays are based on the specific reaction between antibodies and an-
tigens and are frequently used for meat species authentication. The enzyme-linked
Advances in Authenticity Testing for Meat Speciation 373

immunosorbent assay (ELISA) is the most widely used immunochemical technique.

ELISA presents several advantages, such as its simplicity and easy implementation
because it does not require expensive equipment or specialized technicians, it has
high specificity, high sensitivity, and high throughput, and is thus very suitable for
routine application in quality control laboratories (Asensio et al., 2008). On the other
hand, the performance of the assay depends on the availability and specificity of the
chosen antibody to detect a particular species. Both polyclonal and monoclonal anti-
bodies have been used in ELISA for food authentication. The former offers the advan-
tage of recognizing different epitopes of the antigens, being more tolerant to small
changes in the nature of the antigen, but more prone to cross-reactivity with other spe-
cies, in particular with those that are closely related. This explains the differences be-
tween different commercial kits available for the detection of poultry meat, instead of
specifically identifying the presence of chicken or turkey. Another limitation of ELISA
regards the identification of species in processed meat products since proteins can suf-
fer denaturation that leads to specific epitope alterations (Hird et al., 2005) and conse-
quent decreased sensitivity or even false negatives. Moreover, potential target proteins
for ELISA are often irreversibly aggregated, insoluble, and difficult to extract in highly
processed products (Taylor et al., 2009; Kotoura et al., 2012). To overcome this draw-
back, the use of antibodies raised against heat-resistant muscle proteins, such as skel-
etal muscle protein troponin I, has been proposed in different reports (Chen et al.,
1998; Chen and Hsieh, 2000; Djurdjevic et al., 2005; Liu et al., 2006; Zvereva
et al., 2015). Currently, polyclonal antibodies raised against heat-resistant species-
specific muscle-related glycoproteins are included in several commercial kits, which
are being used by regulatory agencies to detect meat species adulteration and to
enforce national and transnational laws and regulations (Asensio et al., 2008). These
kits are, in general, marketed to meet or exceed the United States Department of Agri-
culture Food Safety and Inspection Service protocol standards for the Cooked Meat
Species ELISA. Nonetheless, commercial kits should be used only for qualitative pur-
poses because limits of detection in processed foods can vary with fat content, degree
of thermal processing, tissue, and maturation of meat. Giovannacci et al. (2004)
applied different commercial ELISA kits for the identification of beef, sheep, pork,
and poultry in 40 commercial canned products and found that half of those were in
good agreement with the label, while the others failed because of the presence of un-
declared species. Similarly, L
opez et al. (2011) applied different commercial ELISA
kits for meat species identification in several cooked meat products, including sausages
and hams, either produced in pilot plants or acquired in supermarkets. The methodol-
ogy allowed identification of all animal species described on the labels. The same sam-
ples were also evaluated by SDS-PAGE, which failed to detect some of the species in
some of the samples, possibly because they were only present in low amounts.
A later study by Kotoura et al. (2012) proposed the use of different monoclonal an-
tibodies, raised to detect SDS-denatured proteins, to accurately determine the beef con-
tent in model processed foods and some commercial foods. The proposed method
comprised a sandwich ELISA using two types of monoclonal antibodies, one raised
against denatured beef myoglobin and another against a unique amino acid sequence
of beef myoglobin. This system allowed the determination of beef content in chicken
374 Advances in Food Authenticity Testing

or pork meats, irrespective of the processing conditions; no cross-reactivity with other

food proteins, such as pork, chicken, egg, milk, wheat, buckwheat, peanut, shrimp, and
crab was detected, though some cross-reactivity occurred with lamb.

14.2.3 Chromatographic and Mass Spectrometry Techniques

As alternative protein-based methods, several studies suggest the use of chromato-
graphic techniques, mainly high-performance liquid chromatography (HPLC) coupled
with different detectors and liquid chromatography (LC) hyphenated with different
MS detectors. Giaretta et al. (2013) proposed an anionic exchange ultra-
performance liquid chromatography (UPLC) coupled with diode-array detection for
the separation of myoglobin from raw meat samples of beef, chicken, horse, ostrich,
pork, and water buffalo. Previous to the UPLC analysis, samples were treated with so-
dium nitrite to transform oxymyoglobin and deoxymyoglobin to the more stable met-
myoglobin and desalified on a PD-10 column. This methodology allowed the detection
of 5% (w/w) of pork in raw beef burgers. Although exhibiting low sensitivity and
possibly being only suited for the analysis of raw meat, the authors pointed out the
simplicity of the method and the use of equipment that is commonly present in quality
control laboratories as advantages. HPLC coupled to electrochemical analysis was pro-
posed by Hung et al. (2011) to directly detect peptides and amino acids that exhibit
little or no chromogenic behavior. The authors used a novel copper nanoparticle-
plated screen-printed electrode, which allowed the selective detection of amino acids
and small peptides in protein-rich matrices, while being insensitive to large proteins.
This methodology allowed the differentiation of ostrich from pork, beef, and chicken
meat based on the chromatographic profiles that presented characteristic peaks of
lysine, glutamine, carnosine, and anserine. However, this work only described its
application to differentiate raw meats, each containing single species and without
mentioning the number of samples used for each species and other variability factors
(eg, with different age, sex, breeds) within each species.
More recently, modern proteomics has taken advantage of equipment advances,
namely, the development of soft ionization techniques, such as matrix-assisted laser
desorption ionization (MALDI) and electrospray ionization (ESI) that allow the accu-
rate analysis of peptides by MS methods and, consequently, their use as reliable bio-
markers for meat authentication purposes (Sentandreu and Sentandreu, 2014).
Although other protein-based methods present drawbacks regarding the analysis of
processed meat products, the use of specific peptide markers is related to the primary
structure of proteins, which is known to be relatively resistant to processing (Buckley
et al., 2013; Sentandreu and Sentandreu, 2011). Authentication methods based on pep-
tide identification by MS can also allow differentiation between tissues of the same
species when targeting tissue-specific peptide sequences (Sentandreu and Sentandreu,
2014). Moreover, since high mass accuracy and high sensitivity can be achieved with
modern mass spectrometers, this approach generally allows the detection of low
amounts of undeclared species in meat products (Sentandreu and Sentandreu, 2014).
Nevertheless, the high cost of equipment, the need for specialized technicians, and
Advances in Authenticity Testing for Meat Speciation 375

the duration of the analysis, very important in the case of perishable foods, are seen as
disadvantages.
The use of MS for peptide identification is frequently coupled to a previous sepa-
ration step by LC. Sentandreu et al. (2010) proposed a methodology for the detection
of chicken meat in mixed meats that comprised the extraction of myofibrillar proteins,
the enrichment of target proteins by OFFGEL isoelectric focusing, in-solution trypsin
digestion of MLC3, and the analysis of the generated peptides by LC-MS/MS. Based
on the results obtained by SDS-PAGE and MALDI-TOF MS analysis, MLC3 was
selected because of its heat stability. An enrichment step by OFFGEL fractionation
was included to lower the detection limit of the method. Selection of heat-stable pep-
tides from chicken MLC3 and the use of stable isotope-labeled peptides allowed the
quantitative detection of 0.5% (w/w) of chicken in mixtures with pork meat. The au-
thors considered that, once optimized, the whole protocol has the potential to be car-
ried out within 3e4 days, which is still a very long period when compared to the most
commonly used DNA-based methods.
The use of LC-MS/MS with a multiple reaction monitoring (MRM) method for the
identification of specific peptides was also proposed for the identification of horse and
pork in halal beef (von Bargen et al., 2013) and in highly processed foods (von Bargen
et al., 2014). The method allowed detection of 0.55% (w/w) of horse or pork in beef, or
down to 0.13% pork in beef when MRM3 and a micro-LC system were applied (von
Bargen et al., 2013). Since the selected marker peptides were proven to be sufficiently
stable to heat processing, the same methodology was further used for the identification
of pork and horse meat in highly processed foods (von Bargen et al., 2014). In this
study, the authors also developed a rapid 2-min extraction protocol that allowed an
efficient protein extraction from many types of sample, including highly processed
foods. The method was able to detect pork and horse meat down to 0.24% (w/w) in
a beef meat matrix and was further applied to commercial products, including salami,
sausages, meatballs, canned meat, and Frikandeln. All samples were found to be in
agreement with the labeled information, with the exception of one declaring only
beef in which horse-specific peptides were detected and further confirmed by droplet
digital PCR. The authors suggested that the proposed methodology could be easily
applicable in routine control laboratories since it is fast and does not require any sam-
ple pretreatment or concentration by SDS-PAGE or OFFGEL fractionation.
The use of ambient MS techniques, namely, liquid extraction surface analysis mass
spectrometry (LESA-MS) using tandem electrospray MS, without needing previous
chromatographic separation, was proposed as fast approaches for the discrimination
of five meat species, namely, pork, beef, horse, chicken, and turkey (Montowska
et al., 2014a,b; Fig. 14.1). LESA-MS/MS allowed the identification of species in mix-
tures of cooked meat, namely, the detection of 10% (w/w) of cooked pork, horse, or
turkey meats in a beef matrix and 5% (w/w) of chicken in beef. The method involved
a washing step to remove salts and fats that interfere with efficient ionization, followed
by digestion and direct ionization of dried in-solution tryptic digests from a polymer
surface by LESA-MS, which was significantly faster than other proteomic approaches.
According to the authors, the time of analysis could be shortened to approximately 1 h
by applying microwaves or ultrasonication during the digestion process. On the other
376 Advances in Food Authenticity Testing

Figure 14.1 Distinguishing meat species in cooked beef. (A) Liquid extraction surface analysis
mass spectrometry (LESA-MS) average mass spectra of tryptic digests of cooked meat mix-
tures of beef spiked with 1% (w/w) of horse, pork, chicken, and turkey meat. Orthogonal
partial least squares discriminant analysis (OPLS-DA) score plots of datasets collected from
beef spiked with 10% (B) and 1% (C) of the second meat species, m/z 400e1000, n ¼ 50.
BC, Beef/chicken meat; BH, beef/horse meat; BP, beef/pork; BT, beef/turkey meat.
Reprinted with permission from Montowska, M., Alexander, M.R., Tucker, G.A., Barrett, D.A.,
2014b. Rapid detection of peptide markers for authentication purposes in raw and cooked meat
using ambient liquid extraction surface analysis mass spectrometry. Analytical Chemistry (86),
10257e10265. Copyright © 2014 American Chemical Society.

hand, the sensitivity of this methodology was much lower when compared to LC-MS/
MS (Table 14.1). Applicability of the LESA-MS/MS method was later assessed by the
same group to identify different peptide markers in several types of processed meat
products, including commercial samples and in-house prepared reference sausages.
Results showed that three samples were mislabeled, one pork sausage declaring veal
that presented only peptide markers for pork species and two cocktail sausage samples
that declared turkey that was not detected. Nevertheless, the authors stated that, consid-
ering the low sensitivity of the LESA-MS/MS method, the possibility that the meat
content was below the limit of detection could not be excluded. Additionally, a sample
of horse sausage was also positive for pork meat.

14.3 DNA-Based Methods

Analytical techniques relying on DNA-based methods have rapidly evolved during
recent years as alternatives to overcome the limitations of protein analysis. Compared
to protein-based methods, they present several advantages, specifically the ubiquity of
Table 14.1 Summarized Information About Protein-Based Methods Applied to Meat Species Detection
Method Target Species Target Molecule Type of Product Sensitivity References

Advances in Authenticity Testing for Meat Speciation

2-DE Cattle, pork, Myosin light chain isoforms Raw meat e Montowska and
chicken, Pospiech
turkey, duck, (2011b)
and goose
2-DE Cattle, pork, Myosin light chain isoforms Minced meat Not determined, but content of meat of Montowska and
chicken, mixtures, one species in the mixture should Pospiech
turkey, duck, processed be <10% (w/w) (2012b)
and goose meat products
(frankfurters
and sausages)
CE Cattle and Water-soluble protein and salt soluble Raw meat e Vallejo-Cordoba
ostrich protein fractions et al. (2010)
ELISA with Chicken and Thermostable soluble muscle proteins in Cooked (100 C, 1% (chicken in cooked beef or chicken Djurdjevic et al.
monoclonal turkey chicken (35, 30, 25, and 23.5 kDa) 15 min) in cooked pork) (2005)
antibodies and turkey (29.5, 26, 24.5, and mammalian
22 kDa) meat (pork,
cattle, lamb,
horse, deer)
Sandwich Pork Skeletal muscle troponin I Raw and heat- 0.05% (w/w) (raw, cooked, or Liu et al. (2006)
ELISA with processed autoclaved pork in chicken), 0.05%
monoclonal meat mixtures (w/w) (raw pork in beef), and 0.1%
antibodies (w/w) (cooked or autoclaved pork
in beef mixtures)
ELISA with Cattle SDS-denatured beef myoglobin and Raw and cooked 9.5 ng/mL (detection); 12.8 ng/mL Kotoura et al.
monoclonal peptide with an amino acid sequence meat (quantification) (2012)
antibodies unique to beef myoglobin
Sandwich Mammalian Skeletal muscle protein troponin I Raw meat and 4.8 ng/mL (detection); 8.7e52 ng/mL Zvereva et al.
ELISA (cattle, pork, meat products (quantification range) (2015)

377
lamb, and
horse)
Continued
Table 14.1 Summarized Information About Protein-Based Methods Applied to Meat Species

378
Detectiondcont’d
Method Target Species Target Molecule Type of Product Sensitivity References

OFFGEL Chicken Two peptides (DQGTFEDFVEGLR Raw and cooked 0.5% (w/w) (chicken in pork meat; Sentandreu et al.
isoelectric and ALGQNPTNAEINK) from meat quantification using stable isotope- (2010)
focusing myosin light chain 3 labeled peptides as internal
fractionation standard)
and LC-ESI-
MS/MS
LC-MS/MS Horse and pork Peptides YDIINLR from troponin T, Raw meat 0.55% (w/w) (horse or pork in beef), Von Bargen et al.
and micro TLAFLFAER from myosin-4, and 0.13% (w/w) (pork in beef using (2013)
LC-MS/MS EFEIGNLQSK from myosin-2 micro-LC and MRM3)
using MRM
and MRM3
HPLC-MS/MS Horse and pork Peptides YDIINLR from troponin T, Processed food 0.24% (w/w) (horse or pork in beef) Von Bargen et al.
using MRM TLAFLFAER from myosin-4, matrices (2014)
and MRM3 SALAHAVQSSR from myosin-1
and -4, EFEIGNLQSK from myosin-
2, and LVNDLTGQR from myosin-1

Advances in Food Authenticity Testing

LESA-MS/MS Horse, pork, Several heat-stable peptides from Cooked meat 10% (w/w) (cooked pork, horse, or Montowska et al.
chicken, and MLC1/3f, MLC2f, troponin C, turkey meat), 5% (w/w) (chicken (2014b)
turkey myoglobin, myosin-1, or myosin-4 meat in beef)
LESA-MS/MS Horse, pork, 25 species-specific heat-stable peptides Processed meat e Montowska et al.
chicken, products (2015)
turkey, and
cattle

2-DE, Two-dimensional electrophoresis; CE, capillary electrophoresis; ELISA, enzyme-linked immunosorbent assay; HPLC-MS/MS, high-performance liquid chromatography tandem mass
spectrometry; LC-MS/MS, liquid chromatography tandem mass spectrometry; LC-ESI-MS/MS, liquid chromatography electrospray ionization tandem mass spectrometry; LESA-MS/MS, liquid
extraction surface analysis tandem mass spectrometry; MLC, myosin light chain; MRM, multiple reaction monitoring; SDS, sodium dodecyl sulfate.
Advances in Authenticity Testing for Meat Speciation 379

nucleic acids in every type of cell and their greater stability than proteins. Most DNA-
based methods rely on the polymerase chain reaction (PCR) technique for its speci-
ficity, sensitivity, simplicity, and rapidity, allowing the identification of species of
origin even in complex and processed foods. Specificity of PCR assays is related to
the target sequences based on the appropriate primer choice. For accurate species iden-
tification, the primers should be designed or selected after adequate in silico and
in vitro analysis to confirm their specificity. Both nuclear and mitochondrial genes
can be targeted as species-specific DNA markers. Mitochondrial DNA (mtDNA) is
several-fold more abundant than nuclear, increasing the sensitivity of PCR assays
because of the high number of mtDNA copies per cell. Besides, mtDNA evolves
much faster than nuclear DNA, containing more sequence diversity that enables the
identification of phylogenetically related taxa (Fajardo et al., 2010; Girish et al., 2005).
PCR amplification is based on hybridization of specific primers that flank a target
DNA sequence followed by in vitro synthesis to form millions of copies. DNA
amplification followed by agarose gel electrophoresis to verify fragment size is the
simplest PCR approach to detect the presence of a certain meat species (Fajardo
et al., 2010). Depending on the genetic marker or the aim of the analysis, additional
methods can complement the analysis of PCR products, such as sequencing and re-
striction fragment length polymorphism (RFLP). The vast majority of PCR methods
is based on species-specific primers by means of end-point PCR, multiplex PCR, and
real-time PCR (Ballin et al., 2009; Fajardo et al., 2010). The use of short arbitrary
primers to generate fingerprint patterns of random amplified polymorphic DNA
(RAPD) has been applied to species identification of meats, taking advantage of
the fact that prior knowledge of the target sequence is not required (Arslan et al.,
2005; Calvo et al., 2001; Rastogi et al., 2007). However, difficulties in obtaining
reproducible patterns and the inappropriateness of analyzing admixed meat species
make the applicability of RAPD of limited scope in the case of processed foods
(Fajardo et al., 2010). Other fingerprint DNA-based methods, such as single
sequence repeats (SSRs) or microsatellites, single nucleotide polymorphisms
(SNPs), and PCR with single-strand conformation polymorphisms (SSCPs) have
also been used for meat species differentiation. Analysis of SSR markers facilitated
the discrimination of pork from wild boar, both of which belong to the same species
(Sus scrofa) and therefore are very difficult to differentiate (Conyers et al., 2012).
SNP represents a promising molecular method for meat species traceability, as
described by Yang et al. (2014). PCR-SSCP with capillary electrophoresis targeting
the mitochondrial 12S rRNA gene was successfully developed and applied to differ-
entiate eight avian species based on their distinct patterns (Tisza et al., 2016).

14.3.1 PCR-RFLP
PCR followed by RFLP analysis relies on specific restriction sites recognized by the
restriction enzymes, allowing species identification based on the different patterns ob-
tained (Ballin et al., 2009). This technique has been largely applied to meat and meat
product speciation and has the ability to differentiate closely related species (Fajardo
et al., 2010). The advantages of PCR-RFLP are related to its simplicity, low cost, and
380 Advances in Food Authenticity Testing

suitability for routine analysis (Pfeiffer et al., 2004). On the contrary, the possible ex-
istence of intraspecific mutations at restriction sites (Pereira et al., 2008), the presence
of mixed species, and processing are likely to constrain the application of PCR-RFLP
in meat product authentication (Fajardo et al., 2010).
Table 14.2 summarizes the most relevant information from reported PCR-RFLP
methods, from which it can be inferred that mtDNA markers are mostly targeted to
differentiate domestic and game meat species. Most PCR-RFLP methods use gel

Table 14.2 Summarized Information About Methods of PCR-RFLP

Applied to Meat Species Detection
Target Species Application Target Gene Sensitivity References

Pork, cattle, wild boar, Sausages, Cytochrome b <1% (w/w) Meyer et al.
buffalo, sheep, goat, marinated and (pork) (1995)
horse, chicken, heat-treated
turkey, red deer, roe meats
deer, moose,
antelope, chamois,
mouflon, and
kangaroo
Red deer and sika deer Raw and heat- Cytochrome b e Matsunaga et al.
treated meats (1998)
Buffalo, cattle, sheep, Frozen meat and Cytochrome b e Wolf et al.
goat, hare, red deer, lyophilized (1999)
fallow deer, moose, protein
antelope, gazelle, extracts
wildebeest,
chamois, Pyrenean
ibex, and kangaroo
Pork Meat, D-loop 5% (w/w) Montiel-Sosa
mortadella, (pork) et al. (2000)
pork sausage,
and dry-cured
ham
Ostrich Raw and heat- Cytochrome b e Abdulmawjood
treated meats and Buelte
(2002)
Cattle, goat, sheep, Chicken nuggets, Cytochrome b e Pascoal et al.
pork, quail, wild hamburgers, (2004)
boar, chicken, croquettes,
turkey, red deer, and sausages,
roe deer ham,
tortellini,
moussaka,
ˇ
paté, ravioli,
and
cannelloni
Advances in Authenticity Testing for Meat Speciation 381

Summarized Information About Methods of PCR-RFLP

Table 14.2
Applied to Meat Species Detectiondcont’d
Target Species Application Target Gene Sensitivity References

Cattle, sheep, goat, red Blood and tissue D-loop e Pfeiffer et al.
deer, and roe deer (2004)
Cattle, buffalo, sheep, Raw and heat- 12S rRNA e Girish et al.
and goat treated meats, (2005)
and fried meat
products
Pork Raw meats and Cytochrome b e Aida et al.
fats (halal) (2005)
Cattle, sheep, goat, red Raw and heat- 12S rRNA e Fajardo et al.
deer, fallow deer, treated meats (2006)
and roe deer
Cattle, sheep, goat, Raw meats Cytochrome b 1% (w/w) Maede (2006)
pork, horse, poultry, (beef and
and deer horse)
Chicken, duck, turkey, Raw meats, heat- 12S rRNA e Girish et al.
guinea fowl, and treated meats, (2007)
quail and fried
croquettes
Chamois, Pyrenean Raw and heat- 12S rRNA, e Fajardo et al.
ibex, mouflon, treated meats D-loop (2007a)
cattle, sheep, and
goat
Pork and wild boar Raw meats MC1R e Fajardo et al.
(2008)
Quail, pheasant, red- Raw meats 12S rRNA e Rojas et al.
legged partridge, (2008)
guinea fowl,
capercaillie,
Eurasian woodcock,
woodpigeon, and
song thrush
Red deer, fallow deer, Raw meats 12S rRNA e Fajardo et al.
roe deer, chamois, (2009a,b)
mouflon, Pyrenean
ibex, goat, cattle,
sheep, and swine
Quail, pheasant, red- Raw meats D-loop e Rojas et al.
legged partridge, (2009a)
chukar partridge,
guinea fowl,
capercaillie,
Continued
382 Advances in Food Authenticity Testing

Summarized Information About Methods of PCR-RFLP

Table 14.2
Applied to Meat Species Detectiondcont’d
Target Species Application Target Gene Sensitivity References

Eurasian woodcock,
and woodpigeon
Cattle, pork, buffalo, Raw meats Cytochrome b e Murugaiah et al.
quail, chicken, goat, (2009)
and rabbit
Chicken, turkey, duck, Raw and heat- 12S rRNA, e Stamoulis et al.
goose, pheasant, treated meats Cytochrome b (2010)
partridge,
woodcock, ostrich,
quail, and song
thrush
Cattle, chicken, turkey, Raw meats and Cytochrome c e Haider et al.
sheep, pork, buffalo, blood oxidase (2012)
camel, and donkey subunit 1
Pork Meatballs, Cytochrome b 0.01% (w/w) Ali et al.
streaky (pork) (2012a)
beacon, 0.0001 ng
frankfurters, (pork
and burgers DNA)
Cattle, buffalo, goat, Raw meats Cytochrome b e Kumar et al.
sheep, and pork (2014)
Cattle, sheep, pork, Raw meats, Cytochrome b e Doosti et al.
chicken, donkey, sausages, (2014)
and horse frankfurters,
hamburgers,
and hams
Cat Raw, heat- 18S rRNA 0.01% (w/w) Ali et al.
treated meats (2015a)
and meatballs
Dog Burger Cytochrome b 0.01% (w/w) Rahman et al.
formulations 0.0001 ng (2015)
and (dog
commercial DNA)
burgers

MC1R, Melanocortin receptor 1 gene; PCR-RFLP, polymerase chain reaction restriction fragment length polymorphism.

electrophoresis as a simple and economic strategy to analyze fragment patterns. More

recently, chip-based capillary electrophoresis has emerged as an alternative to conven-
tional electrophoresis for RFLP analysis for meat authentication, producing higher res-
olution band patterns, shorter analysis times, higher versatility, reproducibility, and
suitability for automation (Ali et al., 2012a, 2015a; Fajardo et al., 2009a; Rahman
et al., 2015).
Advances in Authenticity Testing for Meat Speciation 383

14.3.2 Species-Specific PCR

Species-specific PCR has been increasingly used because of its simplicity, high spec-
ificity, and high sensitivity; it is suitable for meat authentication studies even in com-
plex and processed foods. It relies on accurate marker selection (Genbank, http://www.
ncbi.nlm.nih.gov/) and design of specific primers that, after adequate method optimiza-
tion, are able to produce one single PCR product from the target species. Species-
specific PCR is mainly based on conventional PCR analyzed after 30e40 amplification
cycles by agarose gel electrophoresis, known as qualitative or end-point PCR. Since
amplification products are analyzed by the latter phases of amplification (plateau
phase), the results are considered as qualitative, being quantitative or semiquantitative
only in certain cases after cycle number optimization and normalization (Soares et al.,
2010). Table 14.3 summarizes the wide number of species-specific PCR methods
applied to meat species detection, which target mostly mtDNA markers as verified
also in PCR-RFLP methods. A great advantage of species-specific PCR methods is
the ability to provide results on relative and absolute sensitivity, contrary to most
PCR-RFLP methods and other fingerprint techniques.
By the use of two or more specific primer pairs in the same PCR amplification and
after adequate method optimization, we can easily advance to multitarget PCR. Multi-
plex end-point assays are very similar to single target PCR methods, with the great
advantage of allowing simultaneous multitarget detection and becoming therefore
cost-effective and high-throughput methods. Ali et al. (2014b) reported a comprehen-
sive review on the multiplex PCR methods applied to species authentication, relating
the important steps for method development. Table 14.3 also summarizes the reported
multiplex PCR methods for meat species identification in foods.

14.3.3 PCR-Sequencing
Sequencing of PCR fragments allows collection of the full nucleotide information,
which is then compared with a comprehensive reference database to achieve species
identification. Ideally, the target DNA region must consist of a variable sequence
that should be sufficiently informative for species differentiation. Additionally, the
sequence should be flanked by highly conserved regions that are ideal for the design
of universal PCR primers that amplify in a large number of species (Pereira et al.,
2008). As for other PCR-based methods, mtDNA, such as cytochrome b, 12S, and
16S rRNA genes, have been widely used for species discrimination by PCR-
sequencing (Fajardo et al., 2010; Pereira et al., 2008). Regions with considerably
high mutation ratios, such as most animal mtDNA sequences, are recommended to
achieve discrimination of closely related species (Pereira et al., 2008). Currently, the
mitochondrial gene coding for cytochrome c oxidase subunit I (COI) is considered a
reliable DNA barcode for the discrimination of animal species.
PCR-sequencing targeting cytochrome b was used to detect goose species (Anser
anser) in Italian “Mortara” salami (Colombo et al., 2002) and chamois (Rupicapra
rupicapra) meat (Colombo et al., 2004). Horreo et al. (2013) designed universal
primers based on the nucleotide sequences of the 16S rRNA gene of 40 animal species
Table 14.3 Summarized Information About Methods of Species-Specific PCR Applied to Meat Species Detection
Technique Target species Application Target Gene Sensitivity References

Species- Pork Raw meats, heat-treated Porcine growth 0.1% (w/w) Meyer et al. (1994)
specific meats and sausages hormone
PCR
Goat, chicken, cattle, sheep, pig, and Raw and heat-treated Cytochrome b e Matsunaga et al.
horse meats (1999)
Cattle, pork, chicken, and ruminants Ground meats and Short interspersed 0.5 pg (cattle DNA), 0.05 pg Walker et al. (2003)
sausages elements (pork DNA), 5 pg
(chicken DNA)
0.005% (w/w) (cattle),
0.0005% (w/w) (pork),
0.05% (chicken) (w/w)
Chicken, pork, goose, and mule duck Binary liver/foie gras a-Actin 0.1% (w/w) Rodríguez et al.
mixtures (2003a)
Chicken, pork, goose, turkey, and Raw meats, livers, and 12S rRNA 1% (w/w) Rodríguez et al.
mule duck binary liver/foie gras (2003b)
mixtures
Pork, cattle, sheep, and goat Raw and heat-treated 12S rRNA 1% (w/w) Rodríguez et al.
meat mixtures (2004)
Cattle Raw meats, heat-treated Cytochrome b 0.025% (w/w) Pascoal et al.
meats, beef in wheat (2005)
flour, tortellini,
sausages, and ravioli
Cattle, sheep, goat, and deer Raw meats, heat-treated 12S rRNAe 0.05% (w/w) Ha et al. (2006)
meats, and meat tRNAvale16S
meals rRNA
Horse, dog, cat, bovine, sheep, Binary raw meat Cytochrome b 0.1% (w/w) Ilhak and Arslan
porcine, and goat mixtures (2007)
Horse, donkey, and pork Binary raw meat ATPase 8/ATPase 6, 0.1% (w/w) 0.01 ng DNA Kesmen et al.
mixtures ND2 and ND5 (2007)
(dehydrogenase
subunits 2 and 5)

Cattle, sheep, and goat Raw and heat-treated 12S rRNA 0.1% (w/w) Martín et al.
meats (2007a)
Muscovy duck, mallard duck, mule Raw meats, binary raw 12S rRNA 0.1% (w/w) (Muscovy duck Martín et al.
duck, and common teal meat mixtures, and and mule duck in raw and (2007b)
heat-treated meats heat-treated meats)
Red deer, fallow deer, and roe deer Raw and heat-treated 12S rRNA e Fajardo et al.
meats, hams, (2007b)
sausages, and cooked
meats
Chamois, Pyrenean ibex, and mouflon Raw meats, binary raw D-loop 0.1% (w/w) Fajardo et al.
meat mixtures, and (2007c)
heat-treated meats
Rabbit Binary raw and heat- 12S rRNA 0.1% (w/w) (raw and heat- Martín et al. (2009)
treated mixtures of treated)
rabbit meat in oats
Quail, pheasant, partridge, and guinea Binary raw meat
ˇ 12S rRNA 0.1% (w/w) (raw and heat- Rojas et al. (2009b)
fowl mixtures, patés, treated)
braised meats, pickled
meats, and minced
meats
Turkey, chicken, duck, and guinea Liquid and powder eggs Cytochrome b 0.1% (w/w) Nau et al. (2009)
fowl
Chicken, duck, pigeon, and pork Raw and heat-treated Cytochrome b, e Haunshi et al.
meats, blood D-loop (2009)
Continued
Table 14.3 Summarized Information About Methods of Species-Specific PCR Applied to Meat Species Detectiondcont’d
Technique Target species Application Target Gene Sensitivity References

Chicken Raw and heat-treated D-loop 1% (w/w) Mane et al. (2009)

meats, binary meat
mixtures (patty,
kebab, and block)

Quail, pheasant, partridge and guinea Binary raw meat D-loop 0.1% (w/w) (raw and heat- Rojas et al. (2010)
fowl, pigeon, Eurasian woodcock, mixtures treated)
and song thrush
Chicken, turkey, and pork Raw meats, binary raw Cytochrome b 0.1% (w/w) (poultry) Hern
andez-Ch
avez
meat mixtures, and et al. (2011)
heat-treated meats
Cattle Raw and heat-treated D-loop <1% (w/w) Mane et al. (2012a)
meats, binary meat
mixtures (patty,
kebab, and block)
Buffalo Raw and heat-treated D-loop 1% (w/w) Mane et al. (2012b)
meats, binary meat
mixtures (patty,
kebab, and block)
Hare Binary raw meat Cytochrome b 0.01% (w/w) Santos et al. (2012)
mixtures
Chicken Raw meats, blood, and 5-Aminolevulinate 0.1% (w/w) 10 pg (chicken Karabasanavar
binary raw and heat- synthase DNA) et al. (2013)
treated meat mixtures
Pork Raw meats, blood, and D-loop 0.1% (w/w) 10 pg (pork Karabasanavar
binary meats mixtures DNA) et al. (2014)
 Dog Raw and heat-treated Cytochrome b 0.1% (w/w) 0.02 ng (dog Ali et al. (2014a)
meats, spiked DNA)
frankfurters

Cattle, pork, sheep, and chicken Binary meat mixtures, 12S rRNA, 0.09% (w/w) (cattle, pork, Natonek-
animal meals, pork Cytochrome c and sheep), 0.08% (w/w) Wi
sniewska
lard, blood, whey, oxidase subunit I, (chicken) et al. (2013)
casein, and 16S rRNA
hemoglobin
Cattle, hare, rabbit, red deer, and pork Binary meat mixtures, Cytochrome b, 12S 0.01% (w/w) (rabbit, cattle, Amaral et al.
game meat Alheiras rRNA and hare), 0.1% (w/w) (2014)
(red deer and pork)
Duck, partridge, pheasant, quail, Binary meat mixtures, Cytochrome b, 12S 0.01% (w/w) Amaral et al.
chicken, and turkey game meat Alheiras rRNA (2015)
Cattle, sheep, chicken, and donkey Raw meats, ground D-loop, Cytochrome 0.1% (w/w) (chicken and Mousavi et al.
meats, and binary c oxidase subunit donkey) (2015)
meat mixtures II, 12S rRNA, ND2
Multiplex Goat, chicken, cattle, sheep, pig, and Raw and heat-treated Cytochrome b 0.25 ng DNA Matsunaga et al.
PCR horse meats (1999)
Ruminant, poultry, fish, and pork Raw and heat-treated 12S rRNA, tRNA- 0.002% (w/w) (ruminants) Dalmasso et al.
meats, feedstuff, and Val, and 16S (2004)
baby food rRNA
Ruminant, poultry, and pork Raw meats, ground 16S rRNA, 12S e Ghovvati et al.
meats, and sausages rRNA, 12S (2009)
rRNAetRNA-Val
Continued
Table 14.3 Summarized Information About Methods of Species-Specific PCR Applied to Meat Species Detectiondcont’d
Technique Target species Application Target Gene Sensitivity References

Pork and chicken Binary raw meat Cytochrome b, 12S 0.1% (w/w) (pork detection) Soares et al. (2010)
mixtures rRNA 1% (w/w) (pork
quantification)

Cattle, sheep, pork, and poultry Blood and different tRNA-Vale16S 1 ng (pork and sheep DNA), Zha et al. (2010)
tissues rRNA, tRNA-Lys- 5 ng (poultry DNA),
ATPase 8, 12S 0.5 ng (cattle DNA)
rRNAetRNA-Val,
and 12S rRNA
Sika deer, wapiti, red deer, and Blood D-loop, 16S rRNA 0.05 ng (sika deer and wapiti Zha et al. (2011)
reindeer DNA), 0.1 ng (red deer
DNA), 0.5 ng (red deer
DNA), 0.02 ng (reindeer
DNA)
Chicken, beef, pork, and mutton Half-cooked and cooked Cytochrome b 1 ng DNA Zhang (2013)
meats, processed
foods
Soybean, poultry, horse, and pork Reference sausages Lectin, 12S rRNA, 0.01% (w/w) Safdar et al. (2014)
Cytochrome b,
ATPase 6
Cattle, chicken, goat, camel, and Raw meats and sausages Cytochrome b, 12S e Nejad et al. (2014)
donkey rRNA, ND2
 Chicken, duck, and goose Raw and heat-treated 12S rRNA, 1% (w/w) 0.05 ng DNA Hou et al. (2015)
meats, meat mixtures, Cytochrome b,
sausage, kebab, dry D-loop
meat, jerky, and
shredded beef

Donkey, mule, and horse Raw and heat-treated ATPase 8/6, ND2 1% (w/w) Chen et al. (2015)
meats
Cattle, sheep, goat, and fish Raw meats, raw and ATPase 8, tGlu/cytb, 0.1% (w/w) Safdar and Junejo
heat-treated meat 12S rRNA (2015)
mixtures in soybean
meal
Horse, soybean, sheep, poultry, pork, Raw meats, meat Cytochrome b, lectin, 0.01% (w/w) Safdar and Junejo
and cow mixtures, sausages, 12S rRNA, (2016)
meatballs, and cikofte ATPase 6/8
Multiplex Badger, cat, cow, dog, donkey, fox, Tissue, hair, or buccal Cytochrome b 40,000 copies (w1.36 pg) Tobe and Linacre
PCR goat, guinea pig, harvest mouse, cells (2008)
with hedgehog, horse, house mouse,
CGE human, pig, rabbit, rat, red deer, and
sheep
Cat, dog, pork, monkey, and rat Raw meats, binary raw ND5, ATPase 6, 1% (w/w) 0.01e0.02 ng Ali et al. (2015b)
and heat-treated Cytochrome b DNA
meatballs

CGE, Capillary gel electrophoresis; PCR, polymerase chain reaction.

390 Advances in Food Authenticity Testing

that belonged to seven different taxonomic classes (Actinopterygii, Gasteropoda,

Cephalaspidomorphi, Amphibia, Chondrichthyes, Aves, and Mammalia), obtaining
PCR amplicons ranging from 80 to 125 bp. Sequencing analyses confirmed the differ-
ences among species and, although very short, they were considered very informative
and enabled clustering of samples into their respective taxonomic group.
The mitochondrial 12S rRNA gene was first targeted by Girish et al. (2004) for the
differentiation of cattle (Bos indicus), buffalo (Bubalus bubalis), sheep (Ovis aries),
goat (Capra hircus), and mithun (Bos frontalis). The method was successfully applied
to authenticate processed meat products (patties, steam cooked blocks, croquettes, and
autoclaved blocks). Later on, the same authors used the same target gene and
sequencing as a tool for further development of PCR-RFLP methods for meat species
differentiation (Girish et al., 2005, 2007). In fact, sequencing of PCR products has
been frequently used as an intermediate step during the development of several
methods of PCR-RFLP (Fajardo et al., 2006; Rojas et al., 2009a), species-specific
PCR (Haunshi et al., 2009; Santos et al., 2012), and real-time PCR (Laube et al.,
2007; Santos et al., 2012; Kane and Hellberg, 2016).
The major limitation of PCR-sequencing is its unsuitability for identification of spe-
cies in meat mixtures, producing ambiguous sequence results (Girish et al., 2004). The
use of large sequence fragments is not recommended in the case of species identifica-
tion in processed food products that contain low amounts of and degraded DNA
(Pereira et al., 2008).

14.3.4 DNA Barcoding

DNA barcoding relies on sequence variation within a short and standardized region of
the genome, designated as a “barcode,” to provide accurate species identification. This
approach is based on the analysis of the variability within a standard DNA barcode re-
gion, which is useful to establish taxonomic relationships. It involves the PCR ampli-
fication and sequencing of fragments of 400e800 bp by the use of consensus primers
(Hebert et al., 2003). The Barcode of Life Data Systems (BOLD) is designed to sup-
port the generation and application of DNA barcode data, providing a data retrieval
interface that enables searching public records by the use of multiple search criteria.
An ideal DNA barcode should clearly enable a greater interspecies than intraspecies
variability, allowing fast, reliable, automatable, and cost-effective species identifica-
tion (Hebert et al., 2003). The COI barcoding region has been preferred among the
mitochondrial protein-coding genes as the core molecular diagnostic system for animal
species identification (Hebert et al., 2003; Luo et al., 2011).
The introduction of DNA barcoding represents a promising approach for food
authentication, being broadly applied in fish species. DNA barcoding has been suc-
cessful when applied to seafood because the number of species is high in comparison
to other animal sources, such as cattle, sheep, goat, and horse, enhancing the effective-
ness of the approach. Moreover, the classical identification methods are not useful in
many cases, such as processed foods, and in the case of seafood, more than in other
groups, it is possible to go further than the level of species identification (Galimberti
et al., 2013).
Advances in Authenticity Testing for Meat Speciation 391

Although few applications have been published, DNA barcoding has emerged in
recent years as a tool for the authentication of meats and meat products, including
game. Quinto et al. (2016) performed a survey on game meat products sold in the
United States in which the COI gene was successfully used as DNA barcoding to
detect mislabeling. The same research group used the same DNA barcode approach
to verify the labeling of ground meat products sold in the United States (Kane and
Hellberg, 2016). In both studies, BOLD was used to identify sequences at the species
level. In South Africa a DNA barcoding approach was effectively used to confirm that
the reliability of commercial labeling of game meat is very poor (D’Amato et al., 2013)
and for species identification in forensic wildlife cases (Dalton and Kotze, 2011). In
spite of the advantages of DNA barcoding, currently it is not able to identify multiple
species in meat mixtures, requiring the use of other methods, such as real-time PCR or
next-generation sequencing (Hellberg and Morrissey, 2011; Kane and Hellberg,
2016). Another limitation is related to the use of relatively large fragment sizes
(w650 bp), which can disable the barcode amplification when analyzing highly pro-
cessed foods containing degraded DNA (Fajardo et al., 2010).

14.3.5 Real-Time PCR

Real-time PCR is the method of choice in many laboratories for diagnostic tests and
food analysis. It merges PCR chemistry with the use of fluorescent reporter molecules
to monitor the PCR products along amplification cycles. The combination of high
sensitivity and specificity, with reproducibility, low level of cross-contamination,
and reduced time of analysis are advantages that make real-time PCR an attractive
alternative to conventional PCR (Navarro et al., 2015). Probably the most important
benefit of real-time PCR is its capacity to quantify the starting amount of a specific
DNA target, which arises from its ability to measure the target product at early stages
of amplification (exponential). To monitor the progress of amplification, different real-
time PCR chemistries are available and classified into two main groups: double-
stranded DNA intercalating molecules or binding dyes, such as SYBR green I and
EvaGreen; and fluorophore-labeled oligonucleotides, such as the hydrolysis TaqMan
probes (Navarro et al., 2015). The use of DNA binding dyes is the simplest and
most economical approach because it does not need the use of any fluorophore-
labeled oligonucleotides. However, it leads to specific and also to non-specific ampli-
fication products, requiring a melting curve analysis to verify specificity.
The most commonly used real-time PCR universal dye in several research areas,
including meat species identification, is the SYBR green I (Ballin et al., 2009; Navarro
et al., 2015). More recently, the use of third-generation dyes, such as EvaGreen, offers
enhanced fluorescence since it can be used at higher concentrations than SYBR green
I, generating greater signals, increased sensitivity, and excellent stability without
causing PCR inhibition (Wang et al., 2006). The application of real-time PCR with
EvaGreen fluorescent dye has been reported in the identification of Cervidae (Chen
et al., 2009) and hare meat species (Santos et al., 2012). The use of EvaGreen, as a
saturation dye, offers also the advantage of performing high-resolution melting
(HRM) analysis, when using high-resolution instrumentation. HRM analysis involves
392 Advances in Food Authenticity Testing

a more precise temperature control when performing melting curve analysis and an
enhanced data acquisition system as compared to current equipment. HRM analysis
has already demonstrated its potential in food analysis, particularly in differentiating
closely related species and plant cultivars (Druml and Cichna, 2014). In pork meat
traceability, HRM analysis targeting SNP detection was successfully applied as a gen-
otyping method (Yang et al., 2014). The report showed that results of HRM genotyp-
ing were accurate and, in general, consistent with PCR-RFLP and real-time PCR with
TaqMan probes data, but at a much lower cost.
The real-time PCR approach with hydrolysis TaqMan probes has been widely
applied to several areas of research (Navarro et al., 2015), as the most common being
in food authentication, genetically modified organism quantification, and allergen
detection (Ali et al., 2014b; Ballin et al., 2009; Costa et al., 2016b; Mafra et al.,
2008). Hydrolysis probes are designed to bind to a specific region of the target
DNA, with mechanism of action that relies on the 5e30 exonuclease activity of Taq
polymerase, which degrades the bound probe during amplification. TaqMan probes
contain a donor fluorescent moiety at the 50 -end and an acceptor fluorescent moiety
at the 30 -end that quenches the fluorescence emitted from the donor molecule because
of their close proximity. Advantages of using TaqMan probes are related to their rela-
tively simple design and multiplexing capacity, without requiring melting curve anal-
ysis (Navarro et al., 2015). Table 14.4 summarizes the reported real-time PCR methods
for meat species identification in foods, in which can be seen the extensive use of Taq-
Man probes, targeting mostly mtDNA.

14.5.6 Next-Generation Sequencing

In recent years, enormous progress has been made in DNA sequencing technologies.
Next-generation sequencing (NGS) technologies have revolutionized the analysis of
DNA, in terms of speed, read length, and throughput, along with a sharp reduction
in cost per-base. Compared to first-generation sequencing methods, such as Sanger
sequencing, the new methods share three major improvements: (1) they rely on the
preparation of NGS libraries in a cell-free system, without requiring bacterial cloning
of DNA fragments; (2) they produce thousands to several millions of sequencing re-
actions in parallel, instead of hundreds; and (3) the sequencing output is directly
detected without the need for postreaction analysis, such electrophoresis, and the
nucleotide base query is performed cyclically and in parallel (van Dijk et al., 2014;
Fig. 14.2). NGS is becoming a standard approach in a large number of studies in all
species and in many different fields, including resequencing or de novo sequencing
of large and small genomes, metagenomics, and transcriptomics among many other
research and applied areas (Bertolini et al., 2015; Buermans and den Dunnen, 2014;
van Dijk et al., 2014). The huge number of reads generated by NGS has enabled
the sequencing of entire genomes with exceptional speed. This has become possible
in part because of significant advances in data handling by bioinformatic tools.
Despite the high potential of NGS for species identification, only a few studies have
been reported regarding meat species differentiation. Tillmar et al. (2013) described a
DNA-typing method for the determination of mammal species using targeted,
Table 14.4 Summarized Information About Methods of Real-Time PCR Applied to Meat Species Detection

Advances in Authenticity Testing for Meat Speciation

Technique Target Species Application Target Gene Sensitivity References

Real-time PCR Cattle, pork, Binary raw meat mixtures Cytochrome b 0.5% (w/w) Dooley et al.
with TaqMan lamb, (2004)
probes chicken, and
turkey
Horse and Model raw meat mixtures and Cytochrome b 1 pg (donkey DNA) or Chisholm et al.
donkey commercial products (horse 25 pg (horse DNA) (2005)
sausage, horse steak, horse burger,
horse ham, and horse salami)
Duck (mallard Roasted duck, smoked duck, duck
ˇ Cytochrome b Estimated as 0.0001% Hird et al. (2005)
and Muscovy paté, game casserole, and feed (w/w)
duck) samples
Pork Binary mixtures of raw and sterilized 12S rRNA 0.01 ng (pork DNA), 0.5% Rodríguez et al.
pork in beef muscles (w/w) (pork in beef (2005)
mixtures)
Cattle, pork, Home canned food, normal cans, cans Cyclic guanosine 10 genome copies Laube et al.
lamb, goat, for use under tropical conditions, monophosphate (2007)
chicken, and ultra-high heat-treated cans phosphodiesterase,
turkey, and with an amount of at least 0.1% of ryanodine receptor,
duck the respective species and interleukin-2
precursor
Cattle Raw meats, processed meat products, Cytochrome b 35 pg (cattle DNA) Zhang et al.
milk, and cheese (2007)
Cattle, pork, Processed meat products Satellite IV DNA for 0.05% (w/w) (pork, lard, Jonker et al.
horse, cattle and horse, mutton, chicken, (2008)
mutton, cytochrome b for the and turkey), 0.1% (cattle)
chicken, and other species
turkey

393
Continued
 Summarized Information About Methods of Real-Time PCR Applied to Meat Species
Table 14.4
Detectiondcont’d

394
Technique Target Species Application Target Gene Sensitivity References

Chamois and Raw and heat-treated binary mixtures 12S rRNA and D-loop e Fajardo et al.
Pyrenean ibex (2009a,b)

Donkey, pork, Raw and cocked patty samples Mitochondrial ND2, 0.0001% (w/w) 0.0001 ng Kesmen et al.
and horse prepared with binary meat ND5, and ATPase DNA (2009)
mixtures in beef 6/8
Pork Model meatballs spiked with pork Cytochrome b 0.01% (w/w) Ali et al. (2012b)
meat, commercial meatballs of
pork, beef, chicken, mutton, and
chevon/goat
Pork Model chicken nuggets spiked with Cytochrome b 0.01% (w/w) Ali et al. (2012c)
pork meat, commercial nuggets of
pork, beef, chicken, mutton, and
chevon
Rabbit and hare Binary mixtures of processed 12S rRNA 0.1% (w/w) (processed Pegels et al.
muscles [rabbit, hare, and rabbit muscles), 0.01 ng/mL (2013)
plus hare (1:1, w/w)] in oat (each target DNA)
matrix; commercial dog and cat

Advances in Food Authenticity Testing

foods labeled as containing rabbit
Fallow deer, red Meat products labeled as containing Epidermal growth factor 0.5% (w/w) (fallow deer and Druml et al.
deer, and sika deer or game, model game sausage pseudogene red deer), 0.1% (w/w) (2015a)
deer (sika deer)
Roe deer Raw binary meat mixtures with pork, Lactoferrin 0.03% (w/w) Druml et al.
game model sausage (2015c)
Cat and dog Samples of pet food, farm animal Cytochrome oxidase 2 pg (cat DNA), 0.2 pg (dog Espi~neira and
products, and raw materials subunit I DNA) Vieites
(2015)
Horse Heat-processed binary mixtures of 12S rRNA 1 pg horse DNA Pegels et al.
horse in bovine meat, meat-based (2015)
products, and pet foods
 Advances in Authenticity Testing for Meat Speciation
Horse Raw binary mixtures of horse meat in Equine growth hormone 0.1% (w/w) Nixon et al.
beef receptor (2015)

Camel Braised camel meat and charqui Cytochrome b 0.01% (w/w) 5 fg/mL camel Wu et al. (2015)
DNA
Dog Model chicken nuggets with known Cytochrome b 0.01% (w/w) Rahman et al.
amounts of dog meat, halal (2016)
commercial chicken nuggets
Multiplex real- Chicken, duck, Ternary mixtures of pork, duck, and Beta-actin, transforming 1% (w/w) (three target Cheng et al.
time PCR with and pork goose blood growth factor, T-cell species in ternary (2014)
TaqMan probes growth factor mixtures), 0.15 ng for all
species
Roe deer and Raw binary meat mixtures with pork, Lactoferrin, exon 21 of e Druml et al.
deer (the sum several meat species mixtures, epidermal growth (2015b)
of fallow model game sausage factor pseudogene
deer, red deer,
and sika deer)
Cattle and pork Raw binary meat mixtures, minced Cyclic-guanosine 20 genome equivalents Iwobi et al.
meat products, and meat products monophosphate (2015)
-phosphodiesterase,
b-actin
Murine (lab rat, Binary meat mixture of murine meat Cytochrome b 0.1% (w/w), 1 pg of DNA Fang and Zhang
lab mouse, in mutton, meat products spiked per reaction (2016)
and wild with murine meat
mouse)
ˇ
Duck, goose, Model liver paté (containing goose, Cytochrome b, D-loop 2% (w/w) (turkey), 1% K€oppel et al.
chicken, duck, and chicken liver and turkey (w/w) (duck, goose, and (2013)
turkey, and and pork meat), liveremeat chicken), 1% (w/w) (pork)
pork products

395
Continued
Table 14.4 Summarized Information About Methods of Real-Time PCR Applied to Meat Species
Detectiondcont’d

396
Technique Target Species Application Target Gene Sensitivity References

Real-time PCR Cattle and pork Raw and cooked/sterilized meatballs t-Glu-cytb 1% (w/w) (raw and cooked/ L
opez-Andreo
with TaqMan prepared with binary mixtures of sterilized beef), 1% (w/w) et al. (2012)
minor groove beef and pork (raw pork), 5% (w/w)
binding probes (cooked/sterilized pork)
Real-time PCR Pork Raw meat mixtures Cytochrome b 0.1% (w/w) (pork in Yusop et al.
with molecular porkebeef mixtures), (2012)
beacon probe 0.0001 ng pork DNA
Real-time PCR Capercaillie Raw and sterilized binary mixtures of 12S rRNA 0.1% (w/w) Rojas et al.
with TaqMan capercaillie in chicken meat (2011)
probes and real-
Cattle and Milk- and meat-derived food products Cytochrome b, 16S e Drummond et al.
time with
buffalo rRNA (2013)
SYBR green I
dye
Real-time PCR Pork Different mixtures of pork, cattle, Mitochondrial DNA 0.1 ng (pork DNA) Farrokhi and
with SYBR camel, horse, and chicken raw Joozani
green I dye meats, halal-labeled beef, and (2011)
chicken products

Advances in Food Authenticity Testing

Pork Raw binary mixtures in poultry meat, Cytochrome b 0.1% (w/w) 5 pg (pork DNA) Soares et al.
frankfurters, barbecue sausages, (2013)
hamburgers, and nuggets
Real-time PCR Hare Binary raw meat mixtures Cytochrome b 1 pg (hare DNA) Santos et al.
with EvaGreen (2012)
dye
Multiplex real- Cattle and Model sausages and commercial ATPase 8, lectin 0.003% (w/w) (beef), Safdar and
time PCR with soybean sausages 0.001% (w/w) (soybean) Abasıyanık
EvaGreen dye (2013)

PCR, Polymerase chain reaction.

Advances in Authenticity Testing for Meat Speciation 397

Figure 14.2 (A) Simplified representation of a typical next-generation sequencing (NGS)

library preparation workflow. (B) Architecture of a standard Illumina NGS library. Adapters
are indicated in two-tone red and green, the insert is in blue. An index or barcode is indicated in
yellow. Sequencing primers are indicated by arrows. Shown is an Illumina library in which
“P5” and “P7” are the sequences used for flow cell attachment and amplification. In other
technologies the names of these terminal sequences are different, but the principle is the same.
Reprinted with permission from van Dijk, E.L., Auger, H., Jaszczyszyn, Y., Thermes, C., 2014.
Ten years of next-generation sequencing technology. Trends in Genetics (30), 418e426.
Copyright © 2014 Elsevier.

massively parallel sequencing of a short mtDNA sequence by means of NGS. The

method was designed through phylogenetic analyses of DNA reference sequences
of more than 300 mammal species, using binary DNA mixtures. Results obtained
were promising since they allowed discrimination of over 99.9% of mammal species,
with the ability to detect minor components at concentrations as low as 1% (w/w) in
mixed samples (Tillmar et al., 2013). A major advantage of NGS cited by the authors
was the use of short PCR amplicons, which facilitates the analysis of degraded and
poor-quality DNA. The use of universal PCR primers, without requiring prior species
or sequence information and the possibility of detecting minute amounts of DNA, is an
additional advantage of NGS, particularly in the case of forensic and food analysis.
The report of Ripp et al. (2014) showed that NGS of total DNA derived from food-
stuff material can readily identify and quantify species components with high accuracy
by a single experimental assay. Sequence data simulation and real-case Illumina
sequencing of DNA from reference sausages composed of mammalian (pig, cow,
398 Advances in Food Authenticity Testing

horse, sheep) and avian (chicken, turkey) species were able to correctly quantify ma-
terial at the 1% (w/w) level via a read counting approach. The same authors performed
an additional metagenomic step to facilitate identification of traces from animal, plant,
and microbial DNA, including unexpected species, which is prospectively important
for the detection of food allergens and pathogens.
In the report by Bertoloni et al. (2015), the potential of the next-generation
semiconductor-based sequencing technology (Ion Torrent Personal Genome Machine)
was evaluated for the identification of DNA from meat species (pig, horse, cattle,
sheep, rabbit, chicken, turkey, pheasant, duck, goose, and pigeon), as well as from hu-
man and rat, in DNA mixtures, using different universal primers to target the mito-
chondrial 12S and 16S rRNA genes. The approach allowed the detection of low
levels of pork and horse DNA from reads obtained with different universal primer
pairs, which could overcome the potential problems of amplification competition
among species.

14.3.7 Biosensors
Recently, DNA biosensors and DNA biochip technology have been suggested as
high-throughput alternative genetic approaches. Ali et al. (2011) developed a novel
nanobiosensor based on a 3 nm diameter citrateetannate-coated gold nanoparticle func-
tionalized with a fluorophore-labeled probe consisting of a 27-nucleotide AluI-fragment
of pork cytochrome b gene. This biosensor was able to detect 0.5% and 1% (w/w) of
pork in, respectively, raw and autoclaved beef binary meat mixtures, in a single step
without any separation or washing. Considering that the degree of fluorescence depends
on the degree of target binding, the method has the potential for quantitation by the
use of a standard curve generated with known concentrations of probes and targets
(Ali et al., 2011). The same methodology was further applied for the quantitative esti-
mation of spiked pork in burger formulations (Ali et al., 2012d) and in mixed meatball
preparations (Ali et al., 2014c), both with more than 90% accuracy.
DNA biochips rely on the use of microarray analysis and exploit the principle of
hybridization of PCR-amplified fragments on specific probes bound onto the arrays.
The technique presents, in general, the major advantage of allowing for simultaneous
detection of multiple targets, saving time and reducing costs, but it can be limited by
the overall proficiency of the PCR amplification (Iwobi et al., 2011). Iwobi et al.
(2011) tested and compared two commercially available animal DNA biochip detec-
tion systems, the CarnoCheck Test Kit (Greiner Bio-One) and the MEATspecies LCD
Array (Chipron), which allow the simultaneous identification of 8 and 14 animal spe-
cies, respectively (Fig. 14.3). Both test systems are based on the PCR amplification of
consensus DNA regions and species-specific probes that exploit differences within the
mtDNA of the targeted animal species. The two systems showed a good sensitivity,
although this was higher for the LCD array since at least 0.1% of all meat species
in the analyzed samples were detectable using this biochip. The two systems were
applied to the analysis of 70 commercial samples and the results validated against con-
ventional PCR methods, showing a general good performance of both DNA biochips.
A possible disadvantage of the method, in particular for the CarnoCheck Test Kit, is
Advances in Authenticity Testing for Meat Speciation 399

Figure 14.3 LCD Array Meat 1.6 Test System for meat species identification. The figure shows
the spotting pattern of the array while the table lists the capture probes immobilized on each
array (Data Sheet MeatSpecies 1.6, V-I-08, Chipron).
Reprinted with permission from Iwobi, A.N., Huber, I., Hauner, G., Miller, A., Busch, U., 2011.
Biochip technology for the detection of animal species in meat products. Food Analytical
Methods (4), 389e398. Copyright © 2010 Springer Science þ Business Media, LLC.

the need for a special scanner and software to analyze results. As a result, a silicon-
based optical thin-film biosensor DNA chip was developed for the identification of
eight animal species, for which color change results could be interpreted by the naked
eye without the need for expensive instruments (Wang et al., 2015).

14.4 Spectroscopic Methods

In recent years, several studies have focused on the possibility of using spectroscopy
for meat authentication purposes for several reasons including speed and its nonde-
structive character, being able to obtain information about the chemical structure of
molecules without causing sample alteration. Its simplicity, both in regard to sample
preparation and the possibility of being operated by nonexpert technicians, is another
advantage. However, an essential point for the successful application of spectroscopy
relies on the construction of a suitable spectral database, which should include, ideally,
all the possible parameter variations likely to be found in the matrices under evalua-
tion. Based on such databases, useful models can be created, tested, and validated
through the use of appropriate multivariate statistical tools. To date, different
400 Advances in Food Authenticity Testing

techniques have been applied to meat authentication studies, including Raman,

Fourier-transform infrared (FTIR), near-infrared (NIR), and mid-infrared (MIR) spec-
troscopy, among others. However, most studies performed so far only comprise the
evaluation of raw meats and include, generally, a low number of samples to build
up the proposed models. Moreover, several aimed only at differentiating single-
species meats, without evaluating the possibility of meat mixtures.
The use of FTIR spectroscopy and partial least square (PLS) calibration was pro-
posed by Rohman et al. (2011) to detect beef meatball adulteration with pork meat.
Fat extracted from beef meatball adulterated with 1.0e100.0% (w/w) of pork was
analyzed by FTIR spectroscopy in the MIR region (4000e650 cm1) for quantifica-
tion purposes. Statistical results, calculated from the prediction samples set as mean
difference and standard deviation of difference for determination of accuracy (close-
ness of agreement between actual data and the predicted data of FTIR results) and pre-
cision (measure of the random error in a dataset), indicated that the methodology was
appropriate and precise enough to detect adulteration with pork meat. However, the
authors stated that beef and pork samples were randomly obtained from five different
slaughter houses and that the calibration set comprised pork spiked at concentrations of
1%, 3%, 5%, 10%, and 25% (w/w), but the total number of analyzed samples was not
specified. Moreover, instead of using an independent validation dataset, the calibration
model was cross-validated using the “leave-one-out” technique by removing one sam-
ple spectrum at a time. The same technique was later used by the same research group
to detect adulteration of beef meatballs with rat meat (Rahmania et al., 2015). In this
work, besides PLS calibration, principal component analysis (PCA) was used, result-
ing in a clear separation of 100% beef and 100% rat meatballs. For the four commercial
samples analyzed in the study, two presented score plots closer to beef meatballs, be-
ing classified as beef samples, but PCA analysis failed to classify the other two because
they were far away from pure meat samples. Thus, more work is needed to clarify these
results, possibly comprising the use of a larger dataset, including also binary meat
mixtures.
Raman spectroscopy, combined with chemometrics, has been suggested by Boyacı
et al. (2014) for the discrimination of beef and horse meat. Raman spectra were ob-
tained from fat extracted from different parts of horse and beef carcasses and also
from laboratory model mixtures containing 25%, 50%, and 75% (w/w) horse meat.
A calibration dataset including 14 horse and 25 beef samples was used to create a
PCA model based on the preprocessed Raman spectra data. Samples from the valida-
tion dataset included four horse and six beef meat samples, which were all correctly
identified. The performance of the developed model was then tested using the model
meat mixtures adulterated with horse meat, which appeared on the PCA plot separated
from the single meat. Even though the authors concluded that the developed Raman
method was capable of successfully identifying beef containing 25% of horse meat,
they stated that more tests should be performed on a higher number of samples, corrob-
orating the idea that the results from this proof-of-concept study should not be over-
interpreted. Raman spectroscopy was also suggested for the classification of
frankfurters made of chicken, turkey, and mixed meats (Campos et al., 2014) by the
use of training and test sets comprising 38 and 19 samples, respectively. A drawback
Advances in Authenticity Testing for Meat Speciation 401

of this study was the fact that all the samples used to construct the model were
commercially acquired, so there was no assurance that they actually contained the
meat stated on the labels. Additionally, the proportion of correctly classified samples
for the test set was only equal to or higher than 90%, with best results being obtained
for the meat mixture frankfurters. A similar approach was performed by Restaino et al.
ˇ
(2011), who aimed at discriminating meat patés according to the animal species by ˇ
means of NIR spectroscopy and chemometrics. Both beef (n ¼ 5) and pork patés
(n ¼ 7) were correctly classified using PCA followed by stepwise linear discriminant
ˇ
analysis, while pork and beef mixture patés (n ¼ 18) only achieved a 72% correct clas-
sification. Again, the number of samples used in this study should be considered too
low to make any authoritative conclusions. The use of NIR spectroscopy, UVevisible
(UVeVis), and MIR was investigated by Alamprese et al. (2013) for the detection of
minced beef adulteration by turkey meat. By comparing the developed PLS regression
models in terms of prediction errors, best results tested were obtained with NIR
spectra. Nevertheless, considering the root mean square error of prediction values ob-
tained (5.79e9.22), the method was more suited to be used for preliminary sample
screening. Mamani-Linares et al. (2012) proposed to use visible and NIR reflectance
spectroscopy to discriminate meat and meat juices from beef, llama, and horse.
From a total of 79 samples (31 from beef, 21 from llama, and 27 from horse, acquired
in different shops to increase sample diversity) most were correctly classified, although
a few (six) were classified as uncertain. The authors suggested that broadening the cali-
bration set by including more samples of different years, geographic origin, animal
breeds, production systems, and muscles could contribute to develop more robust
models.
Recently, the performance of three different methods based on NIR spectroscopy
was evaluated for detecting adulteration of pure veal sausages with pork meat or fat,
namely, a high-performance Fourier-transform-NIR (FT-NIR) laboratory desktop de-
vice, an FT-NIR equipped with a fiber-optic probe suitable for industrial application
and a handheld FT-NIR spectrometer for on-site use (Schmutzler et al., 2015). After
pretreatment of spectral data, PCA scores were used as input data for support vector
machines classification and validation. Both the laboratory setup and the industrial
fiber-optic setup were able to detect up to the lowest level of adulteration tested
[10% (w/w) pork meat or fat], even when measurements were made through the poly-
mer packaging instead of using a quartz cuvette. Similar results were obtained with the
handheld equipment for sausages adulterated with pork meat, though without enabling
the detection of pork fat at levels lower than 20% (w/w); measurements through the
polymer packaging were possible down to this level.
The use of NIR spectroscopy coupled to image analysis has also been discussed as
a fast, low-cost, and proper alternative since hyperspectral imaging integrates both
spectroscopic and imaging techniques in one system, providing both spectral and
spatial information simultaneously. Kamruzzaman et al. (2013) used NIR hyperspec-
tral imaging for the detection of adulteration in minced lamb meats, as well as to
detect the level of horse meat adulteration in minced beef (Kamruzzaman et al.,
2015). In each case, the authors selected four important wavelengths that were
used to build up a model and an image processing algorithm was developed to apply
402 Advances in Food Authenticity Testing

the model to each pixel in the image. This approach integrates spectroscopy and im-
age analysis, providing both spectral and spatial information simultaneously, thus
allowing to detect a particular type of meat and its distribution in the analyzed sam-
ple. In both studies, results demonstrated that hyperspectral imaging coupled with
multivariate analysis could be successfully applied as a rapid screening technique
for meat adulteration detection. Another technique tested for meat authentication
is 1H NMR spectroscopy. Jakes et al. (2015) proposed a methodology to distinguish
beef from horse meat based on exploiting triglyceride signatures obtained by
60 MHz 1H NMR spectroscopy. The protocol included a simple chloroform extrac-
tion and was tested in two different laboratories for horse and beef meat, including
freeze-thawed samples. The model developed was then applied to 107 extracts pre-
pared from independent samples resulting in all but one being correctly authenti-
cated. The technique proved to be fast, low cost, and simple to use, being a
potential alternative as a high-throughput approach for screening raw meat.

14.5 Final Remarks

Species identification in meats and meat products has been the subject of an increasing
number of reports because of its importance in food authentication and to major ad-
vances in analytical methods. Both proteins and DNA have been the main target mol-
ecules for method development, but the latter have been preferred. Without targeting
particular molecules, the spectroscopic methods have also advanced very recently for
meat species identification based on Raman, FTIR, NIR, and H1 NMR. If, on the one
hand, they can offer advantages, such as simple use, speed, low cost, and minimal or
no sample extraction/preparation, on the other hand they still lack applicability, mainly
to meat mixtures, and mostly rely on chemometrics after constructing suitable spectra
databases. Protein-based methods have demonstrated their effectiveness in meat spe-
cies identification by means of electrophoretic, immunochemical, and chromato-
graphic techniques. Recent advances in MS have prompted the development of
proteomic approaches for meat species differentiation based on the high discriminant
power of peptide biomarkers, which have enhanced specificity when compared to
other protein methods.
DNA-based methods play a crucial role in meat species identification because the
advantages of specificity, sensitivity, simplicity, and low cost can be combined with
their application to complex and processed food matrices. Species-specific PCR
methods targeting mainly mtDNA markers are the most commonly used approaches
for meat species detection in a wide range of meat products, covering all the domestic
and game meat species used as food. For increased specificity and quantitative pur-
poses, real-time PCR with TaqMan probes have also been extensively applied in
meat species identification, being more recently the most reported technique. For
high-throughput species identification, DNA chips are emerging as potential tools
for multiple-species detection, but still need to improve sensitivity and applicability.
Recent technological improvements have revolutionized genomics with the advent
of NGS, which has rapidly become a key technology in basic science and only recently
Advances in Authenticity Testing for Meat Speciation 403

applied in meat species identification. The great potential for meat species identifica-
tion arises from the possibility of identifying more than one species at the same time,
including also unexpected or untargeted species, without any previous information
about their presence, based on the high-throughput reads and bioinformatics. These ad-
vantages are worth exploiting in the near future for forensic food analysis and identi-
fying fraudulent practices in meat and in meat-derived foods.

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