Handbook of Seafood and Seafood Products Analysis PDF

HANDBOOK OF Seafood and Seafood Products Analysis HANDBOOK OF Seafood and Seafood Products Analysis Edited by LEO M.L. NOLLET FIDEL TOLDRÁ Boca Raton London New York CRC Press is an imprint of the Taylor & Francis Group, an informa business CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2010 by Taylor and Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S. Government works Printed in the United States of America on acid-free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number: 978-1-4200-4633-5 (Hardback) This book contains information obtained from authentic and highly regarded sources. Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume responsibility for the valid- ity of all materials or the consequences of their use. 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TX385.H36 2010 641.3’92--dc22 2009034833 Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com Contents Preface..................................................................................................................................ix Editors..................................................................................................................................xi Contributors...................................................................................................................... xiii PART I: CHEMISTRY AND BIOCHEMISTRY 1 Introduction—Importance of Analysis in Seafood and Seafood Products, Variability and Basic Concepts.....................................................................................3 JÖRG OEHLENSCHLÄGER 2 Peptides and Proteins.................................................................................................11 TURID RUSTAD 3 Proteomics..................................................................................................................21 HÓLMFRÍÐUR SVEINSDÓTTIR, ÁGÚSTA GUÐMUNDSDÓTTIR, AND ODDUR VILHELMSSON 4 Seafood Genomics......................................................................................................43 ASTRID BÖHNE, DELPHINE GALIANA-ARNOUX, CHRISTINA SCHULTHEIS, FRÉDÉRIC BRUNET, AND JEAN-NICOLAS VOLFF 5 Nucleotides and Nucleosides......................................................................................57 M. CONCEPCIÓN ARISTOY, LETICIA MORA, ALEIDA S. HERNÁNDEZ-CÁZARES, AND FIDEL TOLDRÁ 6 Lipid Compounds.......................................................................................................69 SANTIAGO P. AUBOURG 7 Lipid Oxidation..........................................................................................................87 TURID RUSTAD 8 Volatile Aroma Compounds in Fish...........................................................................97 GUÐRÚN ÓLAFSDÓTTIR AND RÓSA JÓNSDÓTTIR v vi ◾ Contents PART II: PROCESSING CONTROL 9 Basic Composition: Rapid Methodologies...............................................................121 HEIDI NILSEN, KARSTEN HEIA, AND MARGRETHE ESAIASSEN 10 Microstructure.........................................................................................................139 ISABEL HERNANDO, EMPAR LLORCA, ANA PUIG, AND MARÍA-ANGELES LLUCH 11 Chemical Sensors.....................................................................................................153 CORRADO DI NATALE 12 Physical Sensors and Techniques.............................................................................169 RUTH DE LOS REYES CÁNOVAS, PEDRO JOSÉ FITO SUÑER, ANA ANDRÉS GRAU, AND PEDRO FITO-MAUPOEY 13 Methods for Freshness Quality and Deterioration...................................................189 YESIM OZOGUL 14 Analytical Methods to Differentiate Farmed from Wild Seafood............................215 ICIAR MARTÍNEZ, INGER BEATE STANDAL, MARIT AURSAND, YUMIKO YAMASHITA, AND MICHIAKI YAMASHITA 15 Smoke Flavoring Technology in Seafood.................................................................233 VINCENT VARLET, THIERRY SEROT, AND CAROLE PROST PART III: NUTRITIONAL QUALITY 16 Composition and Calories........................................................................................257 EVA FALCH, INGRID OVERREIN, CHRISTEL SOLBERG, AND RASA SLIZYTE 17 Essential Amino Acids..............................................................................................287 M. CONCEPCIÓN ARISTOY AND FIDEL TOLDRÁ 18 Antioxidants.............................................................................................................309 NICK KALOGEROPOULOS AND ANTONIA CHIOU 19 Vitamins...................................................................................................................327 YOUNG-NAM KIM 20 Minerals and Trace Elements...................................................................................351 JÖRG OEHLENSCHLÄGER 21 Analysis of n-3 and n-6 Fatty Acids..........................................................................377 VITTORIO M. MORETTI AND FABIO CAPRINO PART IV: SENSORY QUALITY 22 Quality Assessment of Fish and Fishery Products by Color Measurement..............395 REINHARD SCHUBRING 23 Instrumental Texture...............................................................................................425 ISABEL SÁNCHEZ-ALONSO, MARTA BARROSO, AND MERCEDES CARECHE Contents ◾ vii 24 Aroma.......................................................................................................................439 JOHN STEPHEN ELMORE 25 Quality Index Methods............................................................................................463 GRETHE HYLDIG, EMILÍA MARTINSDÓTTIR, KOLBRÚN SVEINSDÓTTIR, RIAN SCHELVIS, AND ALLAN BREMNER 26 Sensory Descriptors..................................................................................................481 GRETHE HYLDIG 27 Sensory Aspects of Heat-Treated Seafood.................................................................499 GRETHE HYLDIG PART V: SAFETY 28 Assessment of Seafood Spoilage and the Microorganisms Involved.........................515 ROBERT E. LEVIN 29 Detection of Fish Spoilage........................................................................................537 GEORGE-JOHN E. NYCHAS AND E.H. DROSINOS 30 Detection of the Principal Foodborne Pathogens in Seafoods and Seafood-Related Environments................................................................................557 DAVID RODRÍGUEZ-LÁZARO AND MARTA HERNANDEZ 31 Parasites....................................................................................................................579 JUAN ANTONIO BALBUENA AND JUAN ANTONIO RAGA 32 Techniques of Diagnosis of Fish and Shellfish Virus and Viral Diseases.................603 CARLOS PEREIRA DOPAZO AND ISABEL BANDÍN 33 Marine Toxins..........................................................................................................649 CARA EMPEY CAMPORA AND YOSHITSUGI HOKAMA 34 Detection of Adulterations: Addition of Foreign Proteins.......................................675 VÉRONIQUE VERREZ-BAGNIS 35 Detection of Adulterations: Identification of Seafood Species.................................687 ANTONIO PUYET AND JOSÉ M. BAUTISTA 36 Veterinary Drugs......................................................................................................713 ANTON KAUFMANN 37 Differentiation of Fresh and Frozen–Thawed Fish...................................................735 MUSLEH UDDIN 38 Spectrochemical Methods for the Determination of Metals in Seafood.....................................................................................................................751 JOSEPH SNEDDON AND CHAD A. THIBODEAUX 39 Food Irradiation and Its Detection..........................................................................773 YIU CHUNG WONG, DELLA WAI MEI SIN, AND WAI YIN YAO viii ◾ Contents 40 Analysis of Dioxins in Seafood and Seafood Products.............................................797 LUISA RAMOS BORDAJANDI, BELÉN GÓMARA, AND MARÍA JOSÉ GONZÁLEZ 41 Environmental Contaminants: Persistent Organic Pollutants.................................817 MONIA PERUGINI 42 Biogenic Amines in Seafood Products......................................................................833 CLAUDIA RUIZ-CAPILLAS AND FRANCISCO JIMÉNEZ-COLMENERO 43 Residues of Food Contact Materials.........................................................................851 EMMA L. BRADLEY AND LAURENCE CASTLE 44 Detection of GM Ingredients in Fish Feed...............................................................871 KATHY MESSENS, NICOLAS GRYSON, KRIS AUDENAERT, AND MIA EECKHOUT Index.................................................................................................................................889 Preface There are several seafood and seafood products, which represent some of the most important foods in almost all types of societies, including those in developed and developing countries. The intensive production of fish and shellfish has raised some concerns related to the nutri- tional and sensory qualities of cultured fish in comparison to their wild-catch counterparts. In addition, there are several processing and preservation technologies, from traditional drying or curing to high-pressure processing, and different methods of storage. This increase of variability in products attending the consumers’ demands necessitates the use of adequate analytical methodol- ogies as presented in this book. These analyses will be focused on the chemistry and biochemistry of postmortem seafood; the technological, nutritional, and sensory qualities; as well as the safety aspects related to processing and preservation. This book contains 44 chapters. Part I—Chemistry and Biochemistry (Chapters 1 through 8)—focuses on the analysis of the main chemical and biochemical compounds of seafood. Chapter 1 provides a general introduction to the topics covered in this book. Part II—Processing Control (Chapters 9 through 15)—describes the analysis of technological quality and the use of some nondestructive techniques. Various methods to differentiate between farmed and wild seafood, to check freshness, and to evaluate smoke flavoring are discussed in these chapters. Part III—Nutritional Quality (Chapters 16 through 21)—deals with the analysis of nutrients in muscle foods such as essential amino acids, omega fatty acids, antioxidants, vitamins, minerals, and trace elements. Part IV—Sensory Quality (Chapters 22 through 27)—covers the sensory quality and the main analytical tools to determine the color texture, the flavor and off-flavor, etc. Sensory descrip- tors and sensory aspects of heat-treated seafood are also discussed. Finally, Part V—Safety (Chapters 28 through 44)—is concerned with safety, especially related to analytical tools, for the detection of pathogens, parasites, viruses, marine toxins, antibiotics, adulterations, and chemical toxic compounds from the environment generated during processing, or intentionally added, that can be found in either cultured or wild-catch seafood. The last chapter also deals with the analysis of genetically modified ingredients in fish feed. This book provides an overview of the analytical tools available for the analysis of seafood, either cultured fish or their wild-catch counterparts, and its derived products. It also provides an extensive description of techniques and methodologies for quality assurance, and describes ana- lytical methodologies for safety control. In summary, this handbook deals with the main types of analytical techniques available worldwide, and the methodologies for the analysis of seafood and seafood products. ix x ◾ Preface We would like to thank all the contributors for their excellent work. Their hard work and dedication have resulted in this comprehensive and prized handbook. We wish them all the very best in their academic and/or scientific careers. Leo M.L. Nollet Fidel Toldrá Editors Dr. Leo M.L. Nollet is the editor and associate editor of several books. He edited for Marcel Dekker, New York—now CRC Press of Taylor & Francis Group—the first and second editions of Food Analysis by HPLC and the Handbook of Food Analysis. The Handbook of Food Analysis is a three-volume book. He also edited the third edition of the Handbook of Water Analysis, Chromatographic Analysis of the Environment (CRC Press) and the second edition of the Handbook of Water Analysis (CRC Press) in 2007. He coedited two books with F. Toldrá that were published in 2006: Advanced Technologies for Meat Processing (CRC Press) and Advances in Food Diagnostics (Blackwell Publishing). He also coedited Radionuclide Concentrations in Foods and the Environment with M. Pöschl in 2006 (CRC Press). Nollet has coedited several books with Y.H. Hui and other colleagues: the Handbook of Food Product Manufacturing (Wiley, 2007); the Handbook of Food Science, Technology and Engineering (CRC Press, 2005); and Food Biochemistry and Food Processing (Blackwell Publishing, 2005). Finally, he also edited the Handbook of Meat, Poultry and Seafood Quality (Blackwell Publishing, 2007). He has worked on the following five books on analysis methodologies with F. Toldrá for foods of animal origin, all to be published by CRC Press: Handbook of Muscle Foods Analysis Handbook of Processed Meats and Poultry Analysis Handbook of Seafood and Seafood Products Analysis Handbook of Dairy Foods Analysis Handbook of Analysis of Edible Animal By-Products Handbook of Analysis of Active Compounds in Functional Foods He has worked with Professor H. Rathore on the Handbook of Pesticides: Methods of Pesticides Residues Analysis, which was published by CRC Press in 2009. Dr. Fidel Toldrá is a research professor in the Department of Food Science at the Instituto de Agroquímica y Tecnología de Alimentos (CSIC) and serves as the European editor of Trends in Food Science & Technology, the editor-in-chief of Current Nutrition & Food Science, and as a member of the Flavorings and Enzymes Panel at the European Food Safety Authority. In recent years, he has served as an editor or associate editor of several books. He was the editor of Research Advances in the Quality of Meat and Meat Products (Research Signpost, 2002) and the associate editor of the Handbook of Food and Beverage Fermentation Technology and the Handbook of Food Science, xi xii ◾ Editors Technology and Engineering published in 2004 and 2006, respectively, by CRC Press. He coedited two books with L. Nollet that were published in 2006: Advanced Technologies for Meat Processing (CRC Press) and Advances in Food Diagnostics (Blackwell Publishing). Both he and Nollet are also associate editors of the Handbook of Food Product Manufacturing published by John Wiley & Sons in 2007. Professor Toldrá has edited Safety of Meat and Processed Meat (Springer, 2009) and has also authored Dry-Cured Meat Products (Food & Nutrition Press—now Wiley-Blackwell, 2002). He has worked on the following five books on analysis methodologies with L. Nollet for foods of animal origin, all to be published by CRC Press: Handbook of Muscle Foods Analysis Handbook of Processed Meats and Poultry Analysis Handbook of Seafood and Seafood Products Analysis Handbook of Dairy Foods Analysis Handbook of Analysis of Edible Animal By-Products Handbook of Analysis of Active Compounds in Functional Foods Toldrá was awarded the 2002 International Prize for Meat Science and Technology by the International Meat Secretariat. He was elected as a fellow of the International Academy of Food Science & Technology in 2008 and as a fellow of the Institute of Food Technologists in 2009. Contributors M. Concepción Aristoy Isabel Bandín Instituto de Agroquímica y Tecnología de Departamento de Microbiología y Alimentos Parasitología Consejo Superior de Investigaciones Instituto de Acuicultura Científicas Universidad de Santiago de Compostela Burjassot, Valencia, Spain Santiago de Compostela, Spain Marta Barroso Santiago P. Aubourg Instituto del Frío Instituto de Investigaciones Marinas Consejo Superior de Investigaciones Consejo Superior de Investigaciones Científicas Científicas Madrid, Spain Vigo, Spain José M. Bautista Faculty of Veterinary Sciences Department of Biochemistry and Molecular Kris Audenaert Biology IV Department of Plant Production Universidad Complutense de Madrid Faculty of Biosciences and Landscape Ciudad Universitaria Architecture Madrid, Spain University College Ghent Ghent, Belgium Astrid Böhne Institut de Génomique Fonctionnelle de Lyon Ecole Normale Supérieure de Lyon Marit Aursand University of Lyon SINTEF Fisheries and Aquaculture Lyon, France Trondheim, Norway Luisa Ramos Bordajandi Instrumental Analysis and Environmental Juan Antonio Balbuena Chemistry Department Cavanilles Institute of Biodiversity and General Organic Chemistry Institute Evolutionary Biology Consejo Superior de Investigaciones University of Valencia Científicas Valencia, Spain Madrid, Spain xiii xiv ◾ Contributors Emma L. Bradley Corrado Di Natale Food and Environment Research Agency Department of Electronic Engineering York, United Kingdom University of Rome Tor Vergata Rome, Italy Allan Bremner Allan Bremner and Associates Carlos Pereira Dopazo Mount Coolum, Queensland, Australia Departamento de Microbiología y Parasitología Frédéric Brunet Instituto de Acuicultura Institut de Génomique Fonctionnelle de Lyon Universidad de Santiago de Compostela Ecole Normale Supérieure de Lyon Santiago de Compostela, Spain University of Lyon Lyon, France E.H. Drosinos Laboratory of Food Quality Control and Cara Empey Campora Hygiene Department of Pathology Department of Food Science & Technology John A. Burns School of Medicine Agricultural University of Athens University of Hawaii Athens, Greece Honolulu, Hawaii Fabio Caprino Mia Eeckhout Dipartimento de Scienze e Technologie Department of Food Science and Technology Veterinari per la Sicurezza Alimentare Faculty of Biosciences and Landscape Università degli Studi di Milano Architecture Milan, Italy University College Ghent Ghent University Association Ghent, Belgium Mercedes Careche Instituto del Frío Consejo Superior de Investigaciones John Stephen Elmore Científicas Department of Food Biosciences Madrid, Spain University of Reading Reading, United Kingdom Laurence Castle Food and Environment Research Agency Margrethe Esaiassen York, United Kingdom Nofima Marked Tromsø, Norway Antonia Chiou Department of Science of Dietetics-Nutrition Eva Falch Harokopio University Mills DA Athens, Greece Trondheim, Norway Ruth De los Reyes Cánovas Pedro Fito-Maupoey Institute of Food Engineering for Institute of Food Engineering for Development Development Polytechnic University of Valencia Polytechnic University of Valencia Valencia, Spain Valencia, Spain Contributors ◾ xv Delphine Galiana-Arnoux Marta Hernandez Institut de Génomique Fonctionnelle de Lyon Molecular Biology and Microbiology Ecole Normale Supérieure de Lyon Laboratory University of Lyon Instituto Tecnologico Agrario de Castilla y Lyon, France León Valladolid, Spain Belén Gómara Aleida S. Hernández-Cázares Instrumental Analysis and Environmental Instituto de Agroquímica y Tecnología de Chemistry Department Alimentos General Organic Chemistry Institute Consejo Superior de Investigaciones Consejo Superior de Investigaciones Científicas Científicas Burjassot, Valencia, Spain Madrid, Spain Isabel Hernando María José González Department of Food Technology Instrumental Analysis and Environmental Universidad Polite′cnica de Valencia Chemistry Department Valencia, Spain General Organic Chemistry Institute Yoshitsugi Hokama Consejo Superior de Investigaciones Department of Pathology Científicas John A. Burns School of Medicine Madrid, Spain University of Hawaii Honolulu, Hawaii Ana Andrés Grau Institute of Food Engineering for Grethe Hyldig Development Aquatic Process and Product Technology Polytechnic University of Valencia National Institute of Aquatic Resources Valencia, Spain (DTU Aqua) Technical University of Denmark Nicolas Gryson Kongens Lyngby, Denmark Department of Food Science and Technology Francisco Jiménez-Colmenero Faculty of Biosciences and Landscape Department of Meat and Fish Science and Architecture Technology University College Ghent Instituto del Frío Ghent University Association Consejo Superior de Investigaciones Ghent, Belgium Científicas Ciudad Universitaria Ágústa Guðmundsdóttir Madrid, Spain Department of Food Science and Nutrition School of Health Sciences Rósa Jónsdóttir Science Institute Matís University of Iceland Icelandic Food Research Reykjavik, Iceland Reykjavik, Iceland Nick Kalogeropoulos Karsten Heia Department of Science of Dietetics-Nutrition Nofima Marine Harokopio University Tromsø, Norway Athens, Greece xvi ◾ Contributors Anton Kaufmann Vittorio M. Moretti Kantonales Labor Zurich Dipartimento de Scienze e Technologie Zurich, Switzerland Veterinari per la Sicurezza Alimentare Università degli Studi di Milano Young-Nam Kim Milan, Italy Department of Nutrition and Health Sciences Duksung Women’s University Heidi Nilsen Seoul, South Korea Nofima Marine Tromsø, Norway Robert E. Levin Department of Food Science George-John E. Nychas University of Massachusetts Laboratory of Microbiology and Amherst, Massachusetts Biotechnology of Foods Department of Food Science and Technology Empar Llorca Agricultural University of Athens Departamento de Tecnología de Alimentos Athens, Greece Universidad Politécnica de Valencia Valencia, Spain Jörg Oehlenschläger Max Rubner-Institute María-Angeles Lluch Federal Research Centre for Nutrition and Department of Food Technology Food Universidad Politécnica de Valencia Hamburg, Germany Valencia, Spain Guðrún Ólafsdóttir Iciar Martínez Syni Laboratory Services Instituto de Investigaciones Marinas (CSIC) and Consejo Superior de Investigaciones University of Iceland Científicas Reykjavik, Iceland Vigo, Spain Emilía Martinsdóttir Ingrid Overrein Matís SINTEF Fisheries and Aquaculture Iceland Food Research and Reykjavík, Iceland Department of Biotechnology Norwegian University of Science and Kathy Messens Technology Department of Food Science and Technology Trondheim, Norway Faculty of Biosciences and Landscape Architecture Yesim Ozogul University College Ghent Department of Seafood Processing Ghent University Association Technology Ghent, Belgium Faculty of Fisheries Cukurova University Leticia Mora Adana, Turkey Instituto de Agroquímica y Tecnología de Alimentos Monia Perugini Consejo Superior de Investigaciones Department of Food Science Científicas University of Teramo Burjassot, Valencia, Spain Teramo, Italy Contributors ◾ xvii Carole Prost Isabel Sánchez-Alonso Food Aroma Quality Group Instituto del Frío LBAI—ENITIAA Consejo Superior de Investigaciones Rue de la Géraudière Científicas Nantes, France Madrid, Spain Ana Puig Rian Schelvis Department of Food Technology Wageningen IMARES Universidad Politécnica de Valencia Institute for Marine Resources & Ecosytem Valencia, Spain Studies IJmuiden, the Netherlands Antonio Puyet Faculty of Veterinary Sciences Reinhard Schubring Department of Biochemistry and Molecular Department of Safety and Quality of Milk Biology IV and Fish Products Universidad Complutense de Madrid Federal Research Institute for Nutrition and Ciudad Universitaria Food Madrid, Spain Max Rubner-Institut Hamburg, Germany Juan Antonio Raga Cavanilles Institute of Biodiversity and Christina Schultheis Evolutionary Biology Institut de Génomique Fonctionnelle de Lyon University of Valencia Ecole Normale Supérieure de Lyon Valencia, Spain University of Lyon Lyon, France David Rodríguez-Lázaro Food Safety and Technology Thierry Serot Research Group Food Aroma Quality Group Instituto Tecnologico Agrario de Castilla y LBAI—ENITIAA León Rue de la Géraudière Valladolid, Spain Nantes, France Claudia Ruiz-Capillas Della Wai Mei Sin Department of Meat and Fish Science and Analytical and Advisory Services Division Technology Government Laboratory Instituto del Frío Hong Kong, People’s Republic of China Consejo Superior de Investigaciones Científicas Ciudad Universitaria Rasa Slizyte Madrid, Spain SINTEF Fisheries and Aquaculture Trondheim, Norway Turid Rustad Department of Biotechnology Joseph Sneddon Norwegian University of Science and Department of Chemistry Technology McNeese State University Trondheim, Norway Lake Charles, Louisiana xviii ◾ Contributors Christel Solberg Vincent Varlet Faculty of Biosciences and Aquaculture Food Aroma Quality Group Bodø University College LBAI—ENITIAA Bodø, Norway Rue de la Géraudière Nantes, France Inger Beate Standal SINTEF Fisheries and Aquaculture Véronique Verrez-Bagnis Trondheim, Norway Ifremer Nantes, France Pedro José Fito Suñer Institute of Food Engineering for Oddur Vilhelmsson Development Department of Science Polytechnic University of Valencia University of Akureyri Valencia, Spain Akureyri, Iceland Hólmfríður Sveinsdóttir Jean-Nicolas Volff Division of Biotechnology and Biomolecules Institut de Génomique Fonctionnelle de Lyon Matís Ecole Normale Supérieure de Lyon Iceland Food Research University of Lyon SauđárkrÓkur, Iceland Lyon, France Kolbrún Sveinsdóttir Yiu Chung Wong Matís Analytical and Advisory Services Division Iceland Food Research Government Laboratory Reykjavik, Iceland Hong Kong, People’s Republic of China Chad A. Thibodeaux Michiaki Yamashita Department of Chemistry Food Biotechnology Section McNeese State University National Research Institute of Fisheries Lake Charles, Louisiana Science Yokohama, Japan Fidel Toldrá Instituto de Agroquímica y Tecnología de Yumiko Yamashita Alimentos Food Biotechnology Section Consejo Superior de Investigaciones National Research Institute of Fisheries Científicas Science Burjassot, Valencia, Spain Yokohama, Japan Musleh Uddin Wai Yin Yao Corporate Quality Assurance Analytical and Advisory Services Division Albion Fisheries Ltd. Government Laboratory Vancouver, British Columbia, Canada Hong Kong, People’s Republic of China CHEMISTRY AND I BIOCHEMISTRY Chapter 1 Introduction—Importance of Analysis in Seafood and Seafood Products, Variability and Basic Concepts Jörg Oehlenschläger Contents 1.1 World Catch and Harvest.................................................................................................. 3 1.2 Variability of Aquatic Animals........................................................................................... 5 1.3 Special Problems with Aquatic Animals............................................................................. 5 1.4 Benefits and Risks.............................................................................................................. 6 1.5 Sampling............................................................................................................................ 6 1.6 Analytical Methodologies................................................................................................... 7 1.7 Analytical Problems........................................................................................................... 8 1.8 Trends and Outlook........................................................................................................... 9 References..................................................................................................................................10 1.1 World Catch and Harvest Seafood has by far the greatest variety of all animal-based foods. Whereas the species consumed as warm-blooded mammals (beef, pork, lamb, goat, and donkey) or poultry (hen, turkey, geese, and duck) are represented by very few species, fishes and other aquatic animals show an abundant 3 4 ◾ Handbook of Seafood and Seafood Products Analysis number of species and variability. The fish group alone is represented by 25,000–35,000 species. However, only a little proportion of this large number of about 5% is present in the world’s oceans in amounts huge enough to allow an economical use (catch and following processing). Further, only some of these 5% have the desired sensory properties and give a good or satisfying fillet yield that catching and processing them can be justified. Another difference compared with land-living animals is the fact that the quality (size, state of maturity, nutritional status, infestation with parasites, burden of pollutants, etc.) of aquatic animals when captured by fishing techniques is—with few exceptions—completely unknown. Although land-based animals are today tailor made according to industry’s and consumer’s wishes in weight, body composition, appearance, and sensory properties, in the case of captured seafood we have to accept what we find in the trawl despite modern advanced technology of sonar and echo sounders. Further, fish and other seafood are highly perishable products when stored without chilling. They deteriorate at ambient temperature in a few days, and only correct storage of wet fish in melting ice or of certain products at chilled temperatures can prolong the shelf life up to weeks or months. The total world seafood supply for 2007 amounted to 143 million tons. The world’s aquacul- ture provided 52 million tons (36%), and the captured fish, 91 million tons (64%) of the total supply. Although the amount of captured fish is almost constant at a level around 90 million tons/ year since 1990 after a continuous growth for more than 40 years, aquaculture is dramatically growing (1960: 2 million tons, 1970: 4 million tons, 1980: 7 million tons, 1990: 16 million tons, 2000: 40 million tons, including plants). The stagnation of captured fish is mainly due to fully exploited or partially overfished stocks. The most important primary product producing countries of marine and inland (freshwater) fisheries in 2005 were China (17.1 million tons), Peru (9.4 million tons), the United States (4.9 mil- lion tons), Indonesia (4.4 million tons), Chile (4.3 million tons), Japan (4.1 million tons), India (3.5 million tons), Russia (3.2 million tons), Thailand (2.6 million tons), and Norway (2.4 million tons). The top 10 species being caught in huge amounts in 2005 were Anchoveta (10.2 million tons), Alaska Pollock (2.8 million tons), Atlantic herring (2.3 million tons), Skipjack tuna (2.3 million tons), Blue whiting (2.1 million tons), Chub mackerel (2.0 million tons), Chilean jack mackerel (1.7 million tons), Japanese anchovy (1.6 million tons), Largehead hairtail (1.4 million tons), and Yellowfin tuna (1.3 million tons). Most fish was caught in the Pacific Ocean (Northeast and Southeast) followed by Northeast Atlantic Ocean. The major aquaculture (excluding plants) producers (>1 million tons) in 2005 were China (32.4 million tons, whereof the major part are cyprinids like carp), India (2.8 million tons), Viet- nam (1.4 million tons, mostly Pangasius species), Indonesia (1.1 million tons), and Thailand (1.1 million tons). By major groupings, fish is the top group in aquaculture at 47.4% by quantity. Aquatic plants that are popular in Southeast Asia are second in quantity at 23.4%, whereas crustaceans are fourth by quantity at 6.2% but second by value at 20.4%. Mollusks (bivalves and cephalo- pods) are the third most important group both by quantity and by value at 22.3% and 14.2%, respectively. About 75% of the world’s total seafood supply is used for human consumption, 25% is con- verted into fishmeal and other nonfood products, 40% is consumed as wet fish without any fur- ther technological processing or preservation, about 20% is converted into deep frozen products, 8% is transformed into cured products, and another 8% into canned products. Introduction ◾ 5 1.2 Variability of Aquatic Animals The variability of aquatic animals can be described and explained in many different ways. Based on taxonomic criteria, we have different groups such as bony and cartilaginous fishes, crustaceans, and mollusks, which are very different from each other in appearance, composition, and nutritive properties. When concentrating on fish as the major group contributing to the world’s fish supply, we arrange them in order according to their shape into round fish, flat fishes, eellike fishes, and so forth, or according to their occurrence in the ocean’s water column into pelagic fish, bottom fish, demersal fish, and ground fish. We can also group them according to their fat content into three groups: lean fish species (1% to 10% fat). However, these are all very rough classifications. In addition, the main difficulty in the analysis of fish and other seafood is that there is not only a big variation between groups of species and species but also within a given species. Not only weight and length are varying with age but also other factors such as proximate composition, mineral, and trace element content, which are subject to variations based on state of maturity, fishing area, season, pollution of water, and so on. This means that each fish can be different and unique, and before analyzing fish, a careful consider- ation has to be made if the variation is important and if it is worth or essential knowing (leading to analysis of individuals) or if a more general impression about the target component is sufficient (pooled samples). A drastic example illustrating the variability in fish is the Atlantic mackerel. The prespawning fish can have a fat content in fillet up to 35%, and the spawned fish can exhibit fillet fat contents of down to 5%. Mackerel is a typical pelagic swarm fish occurring in big schools. When captured during the spawning season, in one haul specimen of 5% fat and 35% fat are present. This can lead to extreme problems not only in processing but also in analysis, since parallel with fat content, other parameters such as organic pollutant concentrations vary. Also within the fish body, a certain degree of variability is found. Components like water, fat, and protein are not even distributed in the edible part and also trace element concentrations vary from head to tail or back to belly. With all these variations in the raw seafood material before the analysis of any components, decisions must be made where the results should be used and how detailed an analysis must be. 1.3 Special Problems with Aquatic Animals The main problem with aquatic animals is the fact that from the moment that they are caught or harvested, a change in properties starts, which continues until a state of spoilage is reached. After catch and harvest, not only spoilage and freshness parameters are changing due to metabolic (autolytic) and microbiological processes but also the microbial flora is changing. Besides this more general aspect, some groups offer special problems to which a lot of atten- tion has to be given: aquatic animals may contain parasites (e.g., nematodes, cestodes) that can be harmful to humans when they enter live and intact into the human body. Predatory fish species such as sharks, which are at the end of the marine food web, can accumu- late mercury during their long life span to quantities that exceed legal limits. Toxins from dinofla- gellates can accumulate in bivalve mollusks, leading to several diseases such as diarrhetic shellfi sh poisoning (DSP), paralytic shellfish poisoning (PSP), neurotoxic shellfish poisoning (NSP), and 6 ◾ Handbook of Seafood and Seafood Products Analysis amnesic shellfish poisoning (ASP), and in fish, leading to ciguatera or maitotoxin poisoning. In the digestive glands of mollusks (hepatopancreas) such as cephalopods and mussels, cadmium is accumulated to amounts that exceed any legal limits by far. When not eviscerated immediately after catch, cadmium from hepatopancreas penetrates into the edible part (mantle) during storage, leading to elevated cadmium concentrations also in this body compartment. In products that have not undergone thermal treatment and that are offered to the consumer as ready to eat (e.g., cold smoked products, gravad products, sushi, and sashimi), there is an inherent microbial risk. Fish and other aquatic animals from areas that are polluted (rivers, inshore waters, estuaries, seas with no or limited water exchange with world oceans such as Baltic Sea, Mediterranean Sea, Caspian Sea, or Black Sea) can carry a high burden of environmental pollutants, especially in their organs responsible for detoxification such as liver and kidney. Aquatic animals from some areas of the world can carry viruses and microorganisms (e.g., Vibrio sp.) that are harmful to human health and must be destroyed or removed before marketing of the products. 1.4 Benefits and Risks Seafood is a rich source for a great number of nutritive and important components. The high amount of long-chain polyunsaturated fatty acids of the n-3 series such as eicosapentanoic acid (20:5) and docosahexanoic acid (22:6); the vitamins A, D, E, and B12; the well-balanced content of essential amino acids; the high amount of taurine; the presence of antioxidants such as tocoph- erols; the exceptional concentrations of essential elements such as selenium and iodine; and the good digestibility of fish protein due to low amounts of connective tissue are some examples of the many benefits seafood offers when consumed. On the other hand, we have the risk of viruses and microorganisms; we are confronted with toxins in mussels and fish; we have sometimes a parasitical problem; we may find high amounts of inorganic toxic elements and organic pollutants (POP, persistent organic pollutants), and residues of pharmaceuticals and hormones used in aquaculture can be detected and more. All of these parameters and substances have to be carefully analyzed and quantified to allow a risk benefit analysis, which can give reliable advice and guidance for wise and responsible seafood consump- tion. Unfortunately, only few quantitative analytical data have entered these assessments, with the consequence that recommendations are mostly restricted to a few factors being appropriately analyzed but not based on all factors. Considering the great variability of seafood described here, a tremendous amount of analytic work in seafood has to be done. 1.5 Sampling Sampling, which means here the selection of an appropriate number and part of aquatic animals under well-defined conditions, is very often underestimated. Most errors and most erroneous results arising from analytical methods are based on poor or even wrong sampling plans and prac- tices. Before starting the sampling procedure, a sampling plan has to be developed describing the numbers of samples to be taken, the body compartments to be dissected, and the measures to be taken to avoid any contamination as well as the storage and transport conditions of the samples after sample preparation. Introduction ◾ 7 The number of individuals should be big when a small specimen has to be analyzed, smaller when medium-sized animals are the target, and only a few samples are taken from big individu- als. In small specimens that are consumed totally, the whole body may be sampled and analyzed (mussels, sprat, snails); in medium-sized specimen, always the whole edible part (fillet, tail muscle) must be taken due to intrinsic variations in fillet parts and after homogenization subsamples can be taken; and in big fish (tuna, shark), it is advisable to concentrate on a muscle part that is simple to identify and can be dissected without destroying the fish completely (examples are muscle below gill cover, head end, or tail end of fillet). While sampling is done, precaution must be taken not to contaminate the sample by instru- ments used during manipulation (scissors, knives) or by protective clothes or gloves. When sam- pling is done onboard a vessel, a careful selection of individuals that have not been mechanically damaged by the catching technique, other species or mud, sand, and so forth, is necessary. When sampling for later microbiological analyses, it has to be made under strict hygienic con- ditions to avoid any microbial contamination. After sampling is completed successfully, it is recommended to store all samples (also solutions) in deep frozen conditions (0.3. The connective tissue proteins are often called the insoluble proteins and can be extracted using alkali or acid. The methods for extraction are not standardized so the amount of proteins extracted will vary with the method used. However, changes in solubility can be used to measure changes in protein structure caused by denaturation during storage and processing. Fish muscle proteins are more sensitive and less stable than proteins from mammals. A few examples of methods to extract proteins from fish muscle are given here. Hultmann and Rustad used a modification of the method by Anderson and Ravesi and Licciardello and coworkers. Four grams of muscle was homogenized for 20 s in 80 mL 50 mM phosphate buffer, pH 7. After centrifugation, the supernatant was decanted and the volume made up to 100 mL—this was the water-soluble fraction. The precipitate was homogenized in 80 mL phos- phate buffer with 0.5 M KCl and centrifuged as above. The volume of the supernatant was made up to 100 mL. This was the salt-soluble fraction. Martinez-Alvarez and Gomez-Guillen used a modification on the method of Stefansson and Hultin. The soluble protein was extracted in distilled water (low ionic strength), and in 0.86 M NaCl solution (high ionic strength). Two grams of minced muscle was homogenized at low temperature for 1 min in 50 mL of distilled water. The homogenates of these solutions were stirred constantly for 30 min at 2°C, then centri- fuged (6000 g) for 30 min at 3°C. Kelleher and Hultin compared the use of NaCl, KCl, and LiCl for extraction of protein from fish muscle and concluded that LiCl was a better extractant of fish muscle proteins over a wider range of conditions than NaCl or KCl. 14 ◾ Handbook of Seafood and Seafood Products Analysis Solubility of collagen can be determined by extraction in alkali or acid as described by Eckhoff and coworkers , which is a modification of the method described by Sato et al.. Samples were homogenized in 0.1 M NaOH and centrifuged. The extraction in NaOH was repeated five times and the supernatants were pooled for the analysis of alkali-soluble collagen content. The precipitate was stirred with 0.5 M acetic acid for 2 days at room temperature and centrifuged as above. This was the acid-soluble collagen. After extraction, the concentration of the soluble proteins can be analyzed with a wide vari- ety of methods. Since all proteins absorb UV/visible light to varying degrees, one of the simplest methods is to determine absorbance in the far UV range. The protein concentration can then be calculated from the Lambert–Beer law: A = ε cl where A is the absorption at a given wavelength c is the molar protein concentration l is the path length for the light (cm) e is the molar absorption or extinction coefficient (M−1 cm−1) The molar absorptivity can be determined by dry weight estimation of a purified protein, by absorbance at 205 nm or from knowledge of amino acid composition. Measurement of UV absorption at 280 nm is a simple and popular method to determine protein concentration. However mg quantities of protein are generally required. Absorption at 280 nm is mainly due to tryptophan and tyrosine residues with smaller contributions from phenylalanine and the sulfur-containing amino acids, the method therefore has protein-to-protein variations. In addition, the presence of nonprotein UV-absorbing groups such as nucleic acids and nucleotides which absorb strongly at 260 nm further complicate matters. Light scattering because of large particles or aggregates can also lead to errors. Methods exist to correct for the influence of light scattering and nucleic acids/ nucleotides. 2.4 Analysis of Soluble Proteins There are many indirect colorimetric methods to determine protein content, and a few of them will be treated here. The Biuret method is based on the formation of complexes between copper salts and peptide bonds under alkaline conditions. The purple complex is relatively stable and has an absorption maximum at 540–560 nm. A standard curve is needed, but the method is simple and inexpensive. The method is not very sensitive, measuring concentrations between 1 and 10 mg/mL. The sensitivity can be increased by measuring absorbance at 310 nm or by increas- ing the time for the Biuret reaction. However, some of these methods reduce the speed and sim- plicity of the method. The Lowry method is based on a Biuret-type reaction between protein and copper(II) ions under alkaline conditions, the complexes react with the Folin-phenol reagent a mixture of phosphotungstic acid and phosphomolybdic acid in phenol. The product becomes reduced to molybdenum/tungsten blue and can be measured at 750 nm. The reactions are highly pH dependent. Peterson have reviewed the Lowry method and listed interfering substances, giving upper tolerable limits for a long range of these as well as some methods for cop- ing with the effect of these substances. Reducing agents and sucrose as well as several common buffers interfere with the Lowry method. The review also discusses many of the modifications that Peptides and Proteins ◾ 15 have been suggested for the Lowry method. Finally he compares the Lowry method with other methods to determine protein concentration and concludes that the advantages of the Lowry method are simplicity, sensitivity, and precision, the disadvantages are interfering substances and time—compared to some of the dye-binding methods such as the Coomassie Blue methods. Use of bicinchonic acid (BCA) was introduced as an easier way to determine protein; it uses only one reagent instead of two as in the Lowry procedure. Sensitivity is similar to the Lowry procedure, but detergents, buffer salts, and denaturing agents such as urea and guanidine hydro- chloride cause less interference. However, for lipids, reducing agents, chelators such as EDTA, and acids and alkali cause interference. There are different dye-binding methods, and one of the most widely used is the Biorad method based on binding of Coomassie Brilliant Blue G (CBBG). This method is based on the color change taking place when CBBG binds to proteins under acidic conditions. Th is method is faster to perform than the Lowry procedure (5 min development compared to 30–45 min), and stable reagents and kits are available. The method is compatible with a wide range of buffers/substances. The Coomassie Brilliant Blue method is also used for visualizing proteins in electrophoretic gels. The ability of proteins to bind silver has also been used as a very sensitive method to visual- ize proteins in gel electrophoresis. The silver staining methods are 100 times more sensitive than the CBBG staining. Silver binding is also being used as a method to analyze concentration of soluble proteins. All the methods discussed above are highly protein dependent and this should be kept in mind when applying these methods for analysis of the protein content. It would be best if the protein being analyzed could be used as the standard protein; however, this is often not possible or practical. The Lowry method determines both proteins, small peptides and free amino acids, while methods such as Biuret and Biorad only determine peptide chains above a certain length. However, as different amino acids and peptides give different colors in the Lowry method, the method is highly protein dependent (Table 2.1). The amount of collagen can be determined by analysis of the hydroxylysine content by the Neuman and Logan method as modified by Leach. Hydroxylysine is an amino acid that is almost exclusively found in collagen. However, an accurate determination requires that the amount of hydroxylysine residues per 100 residues in the collagen is known. Th is figure varies for different collagen types such as collagen from fish skins from different fish species. Table 2.1 Comparison of Useful Range for Methods to Determine Protein Concentration Method Range (μg) Kjeldahl 500–30,000 Biuret 1,000–10,000 Lowry 10–300 Biorad (Coomassie Brilliant Blue) 20–140 Biorad (Coomassie Brilliant Blue)—micro 1–20 Bicinchonic acid 1–50 Absorption at 280 nm 100–300 16 ◾ Handbook of Seafood and Seafood Products Analysis 2.5 Immunoassays The amount of a specific protein in a mixture can be determined by enzyme-linked immunosorbent assays (ELISA). It is then necessary to have the antibody of the protein that one seeks to quantify. A polyclonal or monoclonal antibody against the protein of interest is then bound to a film through the Fc region of the antibody. Bovine serum albumin (BSA) is then added to block nonspecific bind- ing sites. After washing, a second antibody is bound to the protein bound to the primary antibody. The amount of secondary antibody bound is proportional to the amount of the specific protein in the sample. The secondary antibody is usually linked to peroxidase or alkaline phosphatase. These enzymes can convert a colorless substrate to a colored product which can then be detected. The method is very sensitive but requires available antibodies. 2.6 Electrophoresis-Based Methods The molecular weight of proteins and peptides is often of interest and this can be determined by several different methods. In native gel filtration chromatography, the proteins are separated based on their size and shape (Stokes’ radii). For low- and medium-pressure chromatography, beads are made of open, cross-linked three-dimensional polymer networks such as agarose, dextrans, cellulose, polyacrylamide, and combinations of these. For high-pressure systems, macroporous silica, porous glass, or inorganic–organic composites are used as support media. Small proteins can enter all the pores in the beads, while larger proteins can only enter the larg- est pores. As the protein solution moves down the column, smaller proteins will, on the average, spend more time inside the beads and the larger proteins will emerge from the column first. How a certain protein behaves in a gel filtration column can be described by the coefficient Kav which defines the proportion of pores that are accessible to that molecule. Kav = (Ve − V0)/(Vt − V0), where Ve is the elution volume of the molecule, V0 is the void volume of the column, and Vt is the total volume of the column. By using standard proteins of known molecular weight, a standard curve can be made allowing determination of the molecular weight distribution in a protein mixture. Molecular weight can also be determined by electrophoresis. One of the most commonly used methods is SDS-PAGE, using gels of polyacrylamide and denaturing the samples by boiling in a solution of sodium dodecyl sulfate (SDS). SDS binds to proteins in a weight ratio of 1:1.4, which gives one SDS molecule for every two amino acids. Since SDS is charged, this results in a charged complex where the charge is proportional to the molecular weight of the protein. Dithiothreitol (DTT) or mercaptoethanol is often added to reduce disulfide bonds. The most commonly used system is that of Laemmli. The denatured proteins are applied to the gel and an electric current is applied, causing the negatively charged proteins to migrate across the gel toward the anode. The proteins will migrate based on their size; smaller proteins will travel farther down the gel, while larger ones travel a shorter distance. By using markers of known molecular weight, a standard curve can be made in the same way as for gel chromatography and the weight of the unknown proteins determined. 2.7 Peptide Characterization Studying the composition and properties of peptides in seafood is often of interest, for instance after enzymatic hydrolysis of proteins or during processing and storage of seafood. Many peptides are bioactive and have physiological properties, such as immunostimulating or antihypertensive Peptides and Proteins ◾ 17 properties. For characterization of mixtures of peptides, especially after enzymatic degradation/ hydrolysis, the term degree of hydrolysis describes the extent to which peptide bonds are broken by the enzymatic hydrolysis reaction. The measurement shows the number of specific peptide bonds broken in hydrolysis as a percent of the total number of peptide bonds present in the intact protein. Several methods to determine this value exist. One of these is the determination of free amino groups after reaction with trinitrobenzene-sulfonic acid (TNBS) ; this is spectrophotometric method determining the amount of the chromophore formed when TNBS reacts with primary amines. The reaction takes place under slightly alkaline conditions and is stopped by lowering the pH in the solution. Another widely used method is the determination of free amino groups after titration with formaldehyde. Formaldehyde reacts with unprotonated primary amine groups resulting in loss of protons. The amount of liberated protons can be determined by titration. Studying the peptide fraction can give a lot of useful information as peptides may have several functions in the food. The peptides may also give valuable information about the quality of the food, such as provide information about the enzymes that are active during storage. For deter- mination of the amount of peptides below a certain chain length, selective precipitation using ethanol, methanol, or trichloroacetic acid can be used. The amount of peptides soluble in different concentrations of ethanol was found to be dependent on the chain length as well as on the hydrophobicity of the peptides. Precipitation of the proteins makes it possible to study pep- tides which are found in lower concentrations using different chromatographic methods such as LC–MS or electrophoretic methods. Mass spectroscopy can be used to determine the molecular mass of the peptides, and by using tandem mass spectroscopy detailed information of the struc- ture of the peptides can be found. Bauchart and coworkers studied the peptides in rainbow trout using precipitation with perchloric acid followed by electrophoresis and MS-analysis in order to study proteolytic degradation. 2.8 Protein Modifications During storage and processing of marine raw materials, changes take place in the proteins and it is often of interest to quantify these changes. In addition to lipids and pigments, muscle proteins are also vulnerable to oxidative attack during processing and storage of muscle foods. Oxidation can occur at both the protein backbone and on the amino acid side chains, and can result in major physical changes in protein structure ranging from fragmentation of the backbone to oxidation of the side chains. Oxidation of protein side chains can give rise to unfolding and conformational changes in protein and also to dimerization or aggregation. Oxidative modification often leads to alterations in the functional, nutritional, and sensory properties of the muscle proteins, including gelation, emulsification, viscosity, solubility, and water-holding capacity. Several meth- ods are used to determine protein oxidation, the most used are determination of formation of carbonyl groups [32,33] and reduction in SH-groups. The content of sulfhydryl groups can be determined using DTNB by the method of with the modification of. Formation of dityrosine is also used to determine the degree of protein oxidation. In addition oxidation can be measured as loss of functional properties such as loss of solubility, loss of water-holding capacity, gelling and emulsification properties, and formation of aggregates. However, these properties are not only dependent on the oxidation state of the proteins, and changes in these properties may be due to other factors. Changes in proteins during storage and processing will often result in changes in the func- tional properties of the proteins. One much used definition of functional properties is this: Those physical and chemical properties that influence the behavior of proteins in food systems during 18 ◾ Handbook of Seafood and Seafood Products Analysis processing, storage, cooking, and consumption. A description of the properties of the proteins important for functional properties was given by Damodaran : The physicochemical properties that influence functional behavior of proteins in food include their size, shape, amino acid composition and sequence, net charge, distribution, hydrophobicity, hydrophilicity, struc- tures (secondary, tertiary, and quaternary), molecular flexibility/rigidity in response to external environment (pH, temperature, salt concentration), or interaction with other food constituents. Nutritional, sensory, and biological values are sometimes included in the functional properties. Functional properties can be divided in several groups. It is usual to classify them according to mechanism of action into three main groups: (1) properties related with hydration (absorption of water/oil, solubility, thickening, wettability), (2) properties related with the protein structure and rheological characteristics (viscosity, elasticity, adhesiveness, aggregation, and gelation), and (3) properties related with the protein surface (emulsifying and foaming activities, formation of protein–lipid films, whippability). Methods to determine functional properties are often developed for a particular use in a specific food system resulting in a vast number of different methods. It is therefore difficult to compare results from different laboratories. The book edited by Hall gives a good overview of methods to determine protein functionality. References 1. Owusu-Apenten, R.K., Food Protein Analysis: Quantitative Eff ects on Processing. New York: Marcel Dekker. 2002, p. 463. 2. Venugopal, V., Methods for processing and utilization of low cost fishes: A critical appraisal. Journal of Food Science & Technology, 1995. 32: 1–12. 3. Venugopal, V. and F. Shahidi, Value added products from underutilised fish species. Journal of Food Science & Nutrition, 1995. 35: 431–435. 4. Kirsten, W.J., Automatic methods for the simultaneous determination of carbon, hydrogen, sulphur and sulphur alone in organic and inorganic materials. Analytical chemistry, 1979. 51: 1173–1179. 5. Mariotti, F., D. Tome, and P.P. Mirand, Converting nitrogen into protein—Beyond 6.25 and Jones’ factors. Critical Reviews in Food Science and Nutrition, 2008. 48: 177–184. 6. Isaksson, T. et al., Non-destructive determination of fat, moisture and protein in salmon fillets by use of near-infrared diff use spectroscopy. Journal of the Science of Food and Agriculture, 1995. 69: 95–100. 7. Uddin, M. et al., Nondestructive determination of water and protein in surimi by near-infrared spectroscopy. Food Chemistry, 2006. 96: 491–495. 8. Bock, J.E. and R.K. Connelly, Innovative uses of near-infrared spectroscopy in food processing. Journal of Food Science, 2008. 73: R91–R98. 9. Foegeding, E.A., T.C. Lanier, and H. Hultin, Characteristics of edible muscle tissue, in Food Chemistry, O.R. Fennema, Ed., Marcel Dekker: New York. 1996, pp. 879–942. 10. Haard, N.F., Control of chemical composition and food quality attributes of cultured fish. Food Research International, 1992. 25: 289–307. 11. Hultmann, L. and T. Rustad, Iced storage of Atlantic salmon (Salmo salar)—Effects on endogenous enzymes and their impact on muscle proteins and texture. Food Chemistry, 2004. 87: 31–41. 12. Anderson, M.L. and E.M. Ravesi, Relation between protein extractability and free fatty acid produc- tion in cod muscle aged in ice. Journal of Fisheries Research Board Of Canada, 1968. 25: 2025–2069. 13. Licciardello, J.J. et al., Time–temperature tolerance and physical-chemical quality tests for frozen Red Hake. Journal of Food Quality, 1982. 5: 215–234. Peptides and Proteins ◾ 19 14. Martinez-Alvarez, O. and M.C. Gomez-Guillen, Effect of brine salting at different pHs on the functional properties of cod muscle proteins after subsequent dry salting. Food Chemistry, 2006. 94: 123–129. 15. Stefansson, G. and H.O. Hultin, On the solubility of cod muscle proteins in water. Journal of Agricultural and Food Chemistry, 1994. 42: 2656–2664. 16. Kelleher, S.D. and H.O. Hultin, Lithium chloride as a preferred extractant of fish muscle proteins. Journal of Food Science, 1991. 56: 315–317. 17. Eckhoff, K.M. et al., Collagen content in farmed Atlantic salmon (Salmo salar L.) and subsequent changes in solubility during storage on ice. Food Chemistry, 1998. 62: 197–200. 18. Sato, K. et al., Isolation of types I and V collagen from carp muscle. Comparative Biochemistry & Physiology, 1988. 90B: 155–158. 19. Yada, R.Y. et al., Analysis: Quantitation and physical characterization, in Food Proteins: Properties and Characterization, S. Nakai and H.W. Modler, Eds., VCH: New York, 1996. 20. Lowry, O.H. et al., Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry, 1951. 193: 265–275. 21. Peterson, G.L., Review of the Folin Phenol protein quantitation method of Lowry, Rosebrough, Farr and Randall. Analytical Biochemitry, 1979. 100: 201–220. 22. Bradford, M.M., A rapid and sensitive method for the determination of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 1976. 72: 248–254. 23. Leach, A.A., Notes on a modification of the Neuman & Logan method for the determination of the hydroxyproline. Biochemistry Journal, 1960. 74: 70–71. 24. Almås, K.A., Muskelcellehylsteret hos torsk: Ultrastruktur og biokjemi, in Dep. Technicla Biochemistry. Norges Tekniske høgskole: Trondheim. 1981, p. 175. 25. Laemmli, U.K., Cleavage and structural proteins during assembly of the head of bacteriophage T4. Nature, 1970. 227: 680–685. 26. Adler-Nissen, J., Determination of the degree of hydrolysis of food protein hydrolysates by trinitrobenzenesulfonic acid. Journal of Agricultural and Food Chemistry, 1979. 27(6): 1256–1262. 27. Taylor, W.H., Formol titration: An evaluation of its various modifications. Analyst, 1957. 82: 488–498. 28. Rohm, H. et al., Comparison of ethanol and trichloracetic acid fractionation for measurement of proteolysis in Emmental cheese. International Dairy Journal, 1996. 6: 1069–1077. 29. Bauchart, C. et al., Peptides in rainbow trout (Oncorhynchus mykiss) muscle subjected to ice storage and cooking. Food Chemistry, 2007. 100: 1566–1572. 30. Choe, E. and D.B. Min, Mechanisms and factors for edible oil oxidation. Comprehensive Reviews in Food Science and Food Safety, 2006. 5: 169–186. 31. Davies, M.J., The oxidative environment and protein damage. Biochimica Biophysica Acta, 2005. 1703: 93–109. 32. Baron, C.P. and H.J. Andersen, Myoglobin-induced lipid oxidation. A review. Journal of Agricultural and Food Chemistry, 2002. 50: 3887–3897. 33. Baron, C.P. et al., Protein and lipid oxidation during frozen storage of rainbow trout (Oncorhynchus mykiss). Journal of Agricultural and Food Chemistry, 2007. 55: 8118–8125. 34. Ellman, G.L., Tissue sulfhydryl groups. Archives in Biochemistry & Biophysics, 1959. 82: 70–78. 35. Sompongse, W., Y. Itoh, and A. Obtake, Effect of Cryoprotectants and a reducing reagent on the stability of actomyosin during ice storage. Fisheries Science, 1996. 62: 73–79. 36. Kinsella, J.E., Functional properties of food proteins: A review. CRC Critical Reviews Food Science & Nutrition, 1976, 7: 219–280. 37. Damodaran, S., Food Proteins: An Overview, in Food Proteins and their applications, S. Damodaran and A. Paraf, Eds., Marcel Dekker, Inc.: New York. 1997, pp. 1–24. 38. Hall, G.M., Ed., Methods for Testing Protein Functionality. Blackie Academic and Professional: London, U.K., 1996, p. 265. Chapter 3 Proteomics Hólmfríður Sveinsdóttir, Ágústa Guðmundsdóttir, and Oddur Vilhelmsson Contents 3.1 Introduction..................................................................................................................... 22 3.2 Proteome Analysis by 2DE............................................................................................... 22 3.2.1 Sample Matrix Considerations.............................................................................. 22 3.2.1.1 Whole Larval Proteomes......................................................................... 22 3.2.1.2 Muscle Proteomes................................................................................... 24 3.2.1.3 The Degradome...................................................................................... 24 3.2.2 Basic 2DE Methods Overview...............................................................................25 3.2.2.1 Sample Extraction and Cleanup..............................................................25 3.2.2.2 First-Dimension Electrophoresis..............................................................25 3.2.2.3 Equilibration.......................................................................................... 27 3.2.2.4 Second-Dimension Electrophoresis......................................................... 27 3.2.2.5 Staining.................................................................................................. 28 3.2.2.6 Analysis.................................................................................................. 28 3.2.3 Protein Identification by Peptide Mass Fingerprinting.......................................... 28 3.3 Applications of 2DE in Seafood Analysis..........................................................................31 3.3.1 Development.........................................................................................................31 3.3.2 Quality Involution................................................................................................ 32 3.3.2.1 Protein Autolysis and Oxidation during Storage and Processing............. 32 3.3.2.2 Aquaculture and Antemortem Effects on Quality and Processability........33 3.3.3 Species Authentication.......................................................................................... 34 3.3.4 Allergen Identification.......................................................................................... 34 Acknowledgments......................................................................................................................35 References..................................................................................................................................35 21 22 ◾ Handbook of Seafood and Seafood Products Analysis 3.1 Introduction As with all living matter, foodstuffs are in large part made up of proteins. This is especially true of fish and meat, where the bulk of the food matrix is constructed from proteins. Furthermore, the construction of the food matrix, both on the cellular and tissue-wide levels, is regulated and brought about by proteins. It stands to reason, then, that proteome analysis, also known as proteomics, is a tool that can be of great value to the food scientist, giving valuable insight into the composition of the raw materials, quality involution within the product before, during, and after processing or storage, the interactions of proteins with one another or with other food components, or with the human immune system after consumption. Proteomics, succinctly defined as “the study of the entire proteome or a subset thereof”1 is currently a highly active field possessing a wide spectrum of analytical methods that continue to be developed at a brisk pace. While high-throughput, gel-free methods, for example, based on liquid chromatography tandem mass spectrometry (LC–MS/MS),2 surface-enhanced laser desorption/ionization3 or protein arrays,4 hold great promise and are deserving of discussion in their own right, the “classic” process of two-dimensional (2D) gel polyacrylamide electrophoresis (2DE) followed by protein identification via peptide mass fingerprinting of trypsin digests (Figure 3.1) remains the workhorse of most proteomics work, largely because of its high resolution, sim- plicity, and mass accuracy. This chapter will therefore focus on 2DE. 3.2 Proteome Analysis by 2DE 2DE, the cornerstone of most proteomics research, is the simultaneous separation of hundreds, or even thousands, of proteins on a 2D polyacrylamide slab gel. The method most commonly used was originally developed by Patrick O’Farrell and is described in his seminal and thorough 1975 paper5 and briefly outlined, along with some of the main improvements that have developed since, in the following sections. 3.2.1 Sample Matrix Considerations Unlike the genome, the proteome varies from tissue to tissue, as well as with time and in response to environmental stimuli. Selection of tissues for protein extraction is therefore an important issue that needs to be considered before a seafood proteomic study is embarked upon. Like other vertebrates, fish possess a number of tissues amenable to 2DE-based proteome analysis. Studies on whole larvae,6–8 liver,9–11 heart,12 kidney,12,13 skeletal muscle,14–18 gill,12 brain,12,19 intestine,12 and rectal gland12 have been reported. In the following sections, we pres- ent some issues and challenges related to sample matrices of particular interest to the seafood scientist. 3.2.1.1 Whole Larval Proteomes The production of good quality larvae is still a challenge in marine fish hatcheries. Several environmental factors can interfere with the protein expression of larvae leading to poor lar- val quality like malformations, growth depression, and low survival rate. Proteome analysis allows us to examine the effects of environmental factors on larval global protein expression, Proteomics ◾ 23 2D PAGE Trypsin digestion MS fingerprinting MS/MS sequencing Figure 3.1 An overview over the “classic approach” in proteomics. First, a protein extract (crude or fractionated) from the tissue of choice is subjected to 2D PAGE. Once a protein of interest has been identified, it is excised from the gel, subjected to degradation by trypsin (or other suitable protease) and the resulting peptides analyzed by mass spectrometry, yielding a peptide mass fingerprint. In many cases this is sufficient for identification purposes, but if needed, peptides can be dissociated into smaller fragment and small partial sequences obtained by MS/MS. See text for further details. posttranslational modifications and redistribution of specific proteins within cells,20 all impor- tant information for controlling factors influencing the aptitude to continue a normal develop- ment until adult stages. Only a few proteome analysis studies on fish larvae have been published.6,7,21,22 Three of these publications have focused on the whole larval proteomes in Atlantic cod (Gadus morhua)6,22 and zebrafish (Danio rerio).7 These studies provided protocols for the production of high-resolution 2D gels. Peptide mass mapping using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry was performed only on the cod larval proteins, allowing iden- tification of ca. 85% of the of the selected protein spots.6,22 The advantage of working with whole larvae versus distinct tissues is the ease of keeping the sample handling to a minimum in order to avoid loss or modification of the proteins. Nevertheless, there are several drawbacks when working with the whole larval proteome, like the overwhelm- ing presence of muscle and skin proteins. These proteins may mask subtle changes in proteins expressed in other tissues or systems, such as the gastrointestinal tract or the central nervous system. The axial musculature is the largest tissue in larval fishes as it constitutes approximately 40% of their body mass.23 This is reflected in our studies on whole cod larval proteome, where the majority of the highly abundant proteins were identified as muscle proteins.6,22 Also, cytoskeletal 24 ◾ Handbook of Seafood and Seafood Products Analysis proteins were prominent among the identified proteins. Removal of those proteins may increase detection of other proteins present at low concentrations. However, it may also result in a loss of other proteins, preventing identification of holistic alterations in the analyzed proteomes. Various strategies have been presented for the removal of highly abundant proteins24 or enrichment of low- abundant proteins.25,26 3.2.1.2 Muscle Proteomes In most seafood products, fish skeletal muscle is the main component. The fish muscle pro- teome is therefore likely to be of comparatively high interest to the seafood scientist. Structural proteins, such as actin and tubulin, are particularly abundant in the skeletal muscle proteome. An unfractionated 2DE map of the muscle proteome therefore tends to be dominated by com- paratively few high-abundance protein spots, rendering analysis of low-abundance proteins dif- ficult or impossible. Swamping of low-abundance spots by highly abundant ones may not be a problem for applications relating specifically to structural proteins, but for other applications low-abundance proteins, which include most regulatory proteins and many important metabolic enzymes, are of keen interest. No amplification method analogous to PCR exists for proteins, and simply increasing the amount of sample is usually not an option, as it will give rise to over- loading artifacts in the gels.5 The remaining option, then, is fractionation of the protein sample in order to weed out the high-abundance proteins, allowing a larger sample of the remaining proteins to be analyzed. A myriad of methods suitable for subsequent 2DE exist for fractionat- ing the proteome into defined subproteomes, such as those associated with individual organelles or cell compartments28 or by protein biochemical methods such as affi nity chromatography,29,30 preparative isoelectrofocusing31 or solubility in the presence of various detergents32 or chao- tropes33 have been described. Fractionation methods for a variety of sample matrices have been reviewed recently.34–41 3.2.1.3 The Degradome The degradome may be a subproteome of particular interest to the food scientist, as many tex- tural and other quality factors of muscle foods are related to proteolytic activity in the muscle tissue before, during and after processing. In addition to having a hand in controlling autolysis determinants, protein turnover is a major regulatory engine of cellular structure, function, and biochemistry. Cellular protein turnover involves at least two major systems: the lysosomal system and the ubiquitin–proteasome system.42,43 The 20S proteasome has been found to have a role in regulating the efficiency with which rainbow trout (Oncorhynchus mykiss) deposit protein.44 It seems likely that the manner, in which protein deposition is regulated, particularly in muscle tissue, has profound implications for quality and processability of the fish flesh. Protein turnover systems, such as the ubiquitin–proteasome or the lysosome systems, are suitable for rigorous investigation using proteomic methods. For example, lysosomes can be iso- lated and the lysosome subproteome queried to answer the question whether and to what extent lysosome composition varies among fish expected to yield flesh of different quality characteris- tics. Proteomic analysis on lysosomes has been successfully performed in mammalian (human) systems.45,46 An exploitable property of proteasome-mediated protein degradation is the phenomenon of polyubiquitination, whereby proteins are targeted for destruction by the proteasome by covalent Proteomics ◾ 25 binding to multiple copies of ubiquitin.43,47 By targeting these ubiquitin-labeled proteins, it is possible to observe the ubiquitin–proteasome “degradome,” i.e., which proteins are being degraded by the proteasome at a given time or under given conditions. Gygi and coworkers have developed methods to study the ubiquitin–proteasome degradome in the yeast Saccharomyces cerevisiae using multidimensional LC–MS/MS.2 Some proteolysis systems, such as that of the matrix metalloproteases, may be less directly amenable to proteomic study. Activity of matrix metalloproteases is regulated via a complex net- work of specific proteases.48–50 Monitoring of the expression levels of these regulatory enzymes, and how they vary with environmental or dietary variables, may be more conveniently carried out using transcriptomic methods. 3.2.2 Basic 2DE Methods Overview O’Farrell’s original 2DE method first applies a process called isoelectric focusing (IEF), where an electric field is applied to a tube gel on which the protein sample and carrier ampholytes have been deposited. This separates the proteins according to their molecular charge. The tube gel is then transferred onto a polyacrylamide slab gel and the isoelectrically focused proteins are further sepa- rated according to their molecular mass by conventional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), yielding a two-dimensional map (Figure 3.2) rather than the familiar banding pattern observed in one-dimensional (1D) SDS-PAGE. The map can be visual- ized and individual proteins quantified by radiolabeling or by using any of a host of protein dyes and stains, such as Coomassie blue, silver stains, or fluorescent dyes. Although a number of refine- ments have been made to 2DE since O’Farrell’s paper, most notably the introduction of immobi- lized pH gradients (IPGs) for IEF,51 the procedure remains essentially as outlined earlier. In the following sections, a general protocol is outlined briefly with some notes of special relevance to the seafood scientist. For more detailed, up-to-date protocols, the reader is referred to any of a number of excellent reviews and laboratory manuals.52–57 3.2.2.1 Sample Extraction and Cleanup For most applications, sample treatment prior to electrophoresis should be minimal in order to minimize in-sample proteolysis and other sources of experimental artifacts. We have found direct extraction into the gel reswelling buffer (7 M urea, 2 M thiourea, 4% (w/v) CHAPS [3-(3-chloramidopropyl)dimethylamino-1-propanesulfonate], 0.3% (w/v) DTT [dithiothreitol], 0.5% Pharmalyte ampholytes for the appropriate pH range) supplemented with a protease inhibi- tor cocktail to give good results for proteome extraction from whole Atlantic cod larvae6,22 and Arctic charr (Salvelinus alpinus) liver.58 Thorough homogenization is essential to ensure complete and reproducible extraction of the proteome. Cleanup of samples using commercial 2D sample cleanup kits may be beneficial for some sample types. 3.2.2.2 First-Dimension Electrophoresis The extracted proteins are first separated by IEF, which is most conveniently performed using commercial dry IPG gel strips. These strips consist of a dried IPG-containing polyacrylamide gel on a plastic backing. Ready-made IPG strips are currently available in a variety of linear and 26 ◾ Handbook of Seafood and Seafood Products Analysis MW (kDa) 60 42 30 22 17 4 5 6 7 pl Figure 3.2 A 2DE protein map of whole Atlantic cod (G. morhua) larval proteins with pI between 4 and 7 and molecular mass about 10–100 kDa. The proteins are separated according to their pI in the horizontal dimension and according to their mass in the vertical dimension. Isoelectrofocusing was by pH 4–7 IPG strip and the second dimension was in a 12% polyacryl- amide slab gel. sigmoidal pH ranges. This method is thus suitable for most 2DE applications and has all but completely replaced the older and less reproducible method of IEF by carrier ampholytes in tube gels. Broad-range linear strips (e.g., pH 3–10) are commonly used for whole-proteome analysis of tissue samples, but for many applications narrow-range and/or sigmoidal IPG strips may be more appropriate as these will give a better resolution of proteins in the fairly crowded pI 4–7 range. Narrow-range strips also allow for higher sample loads (since part of the sample will run off the gel) and thus may yield improved detection of low-abundance proteins. Before electrophoresis, the dried gel needs to be reswelled to its original volume. A recipe for a typical reswelling buffer is presented in Section 3.2.2.1. Reswelling is normally performed over- night at 4°C. Application of a low voltage current may speed up the reswelling process. Optimal conditions for reswelling are normally provided by the IPG strip manufacturer. If the protein sample is to be applied during the reswelling process, extraction directly into the reswelling buffer is recommended. IEF is normally performed for several hours at high voltage and low current. Typically, the starting voltage is about 150 V, which is then increased stepwise to about 3,500 V, usually totaling about 10,000–30,000 Vh, although this will depend on the IPG gradient and the length of the strip. The appropriate IEF protocol will depend not only on the sample and IPG strip, but also on Proteomics ◾ 27 the equipment used. The manufacturer’s instructions should be followed. Görg et al.56 reviewed IEF for 2DE applications. 3.2.2.3 Equilibration Before the isoelectrofocused gel strip can be applied to the second-dimension slab gel, it needs to be equilibrated for 30–45 min in a buffer-containing SDS and a reducing agent such as DTT. During the equilibration step, the SDS–polypeptide complex that affords protein-size-based sepa- ration will form and the reducing agent will preserve the reduced state of the proteins. A tracking dye for the second electrophoresis step is also normally added at this point. A typical equilibration- buffer recipe is as follows: 50 mM Tris–HCl at pH 8.8, 6 M urea, 30% glycerol, 2% SDS, 1% DTT, trace amount of bromophenol blue. A second equilibration step in the presence of 2.5% iodoacetamide and without DTT (otherwise identical buffer) may be required for some applica- tions. This will alkylate thiol groups and prevent their reoxidation during electrophoresis, thus reducing vertical streaking.59 3.2.2.4 Second-Dimension Electrophoresis Once the gel strip has been equilibrated, it is applied to the top edge of an SDS-PAGE slab gel (Figure 3.3) and cemented in place using a molten agarose solution. Optimal pore size depends on the size of the target proteins, but for most applications gradient gels or gels of about 10% or 12% polyacrylamide are appropriate. Ready-made gels suitable for analytical 2DE are available com- mercially. While some reviewers recommend alternative buffer systems,60 the Laemmli method,61 using glycine as the trailing ion and the same buffer (25 mM Tris, 192 mM glycine, 0.1% SDS) at both electrodes, remains the most popular one. The gel is run at a constant current of 25 mA until the bromophenol blue dye front has reached the bottom of the gel. Figure 3.3 Orientation and placement of an isoelectrofocused IPG strip onto the top of the second-dimension gel. Care must be taken that the (+) end of the strip is on the same side of all slab gels, that the gel side of the IPG strip faces the notched side of the glass plate, and that the strip is pressed gently onto the SDS gel, avoiding trapping air bubbles. This is best performed using a dentist’s tool or other appropriate implement, taking care to put the pressure on the IPG strip’s plastic backing rather than the gel itself. 28 ◾ Handbook of Seafood and Seafood Products Analysis 3.2.2.5 Staining Visualization of proteins spots is commonly achieved through staining with colloidal Coomassie Blue G-250 due to its low cost and ease of use. A typical staining procedure includes fi xing the gel for several hours in 50% ethanol/2% ortho-phosphoric acid, followed by several 30 min wash- ing steps in water, followed by incubation for 1 h in 17% ammonium sulfate/34% methanol/2% ortho-phosphoric acid, followed by staining for several days in 0.1% Coomassie Blue G-250/17% ammonium sulfate/34% methanol/2% ortho-phosphoric acid, and followed by destaining for sev- eral hours in water. There are, however, commercially available colloidal Coomassie staining kits that do not require fi xation or destaining. A great many alternative visualization methods are available, many of which are more sensitive than colloidal Coomassie and thus may be more suitable for applications where the visualization of low-abundance proteins is important. These include radiolabeling, such as with [35S] methion- ine, and staining with fluorescent dyes, such as the SYPRO or Cy series of dyes. Multiple staining with dyes fluorescing at different wavelengths offers the possibility of differential display allowing more than one proteome to be compared on the same gel, such as in difference gel electrophoresis (DIGE). Patton published a detailed review of visualization techniques for proteomics.62 3.2.2.6 Analysis Although commercial 2DE image analysis software, such as ImageMaster (Amersham), PDQuest (BioRad), or Progenesis (Nonlinear Dynamics), has improved by leaps and bounds in recent years, analysis of the 2DE gel image, including protein spot definition, matching, and individual protein quantification, remains the bottleneck of 2DE-based proteome analysis and still requires a sub- stantial amount of subjective input by the investigator.63 In particular, spot matching between gels tends to be time-consuming and has proved difficult to automate.64 These difficulties arise from several sources of variation among individual gels, such as protein load variability due to varying IPG strip reswelling or protein transfer from strip to slab gel. Also, gene expression in several tissues varies considerably among the individuals of the same species, and therefore individual variation is a major concern and needs to be accounted for in any statistical treatment of the data. Pooling samples may also be an option and this depends on the type of experiment. These multiple sources of variation has led some investigators63–65 to cast doubt on the suitability of univariate tests, such as Student’s t-test, commonly used to assess the significance of observed protein expres- sion differences. Multivariate analysis has been successfully used by several investigators in recent years.65–67 3.2.3 Protein Identification by Peptide Mass Fingerprinting Identification of proteins on 2DE gels is most commonly achieved via mass spectrometry of trypsin digests. Briefly, the spot of interest is excised from the gel, digested with trypsin (or another suitable protease), and the resulting peptide mixture is analyzed by mass spectrometry. The most popular mass spectrometry method is MALDI-TOF mass spectrometry,68 where peptides are suspended in a matrix of small, organic, UV-absorbing molecules (such as 2,5-dihydroxybenzoic acid) followed by ionization by a laser at the excitation wavelength of the matrix molecules and acceleration of the ionized peptides in an electrostatic field into a flight tube where the time of flight of each peptide is measured and this gives its expected mass.

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