Seafood and Seafood Products Analysis


Seafood and Seafood Products Analysis
Edited by


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 validity of all materials or the consequences of their use. The authors and publishers have attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged please write and let us know so we may rectify in any future reprint. Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright.com (http:// www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Library of Congress Cataloging-in-Publication Data Handbook of seafood and seafood products analysis / editors, Leo M.L. Nollet, Fidel Toldrá. p. cm. Includes bibliographical references and index. ISBN 978-1-4200-4633-5 (hardcover : alk. paper) 1. Seafood--Analysis--Handbooks, manuals, etc. I. Nollet, Leo M. L., 1948- II. Toldrá, Fidel. III. Title. TX385.H36 2010 641.3’92--dc22 Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com 2009034833

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

2 Peptides and Proteins .................................................................................................11

3 Proteomics ..................................................................................................................21

4 Seafood Genomics ......................................................................................................43

5 Nucleotides and Nucleosides ......................................................................................57

6 Lipid Compounds.......................................................................................................69

7 Lipid Oxidation ..........................................................................................................87

8 Volatile Aroma Compounds in Fish ...........................................................................97


vi ◾


PART II: PROCESSING CONTROL 9 Basic Composition: Rapid Methodologies ...............................................................121

10 Microstructure .........................................................................................................139

11 Chemical Sensors .....................................................................................................153

12 Physical Sensors and Techniques .............................................................................169

13 Methods for Freshness Quality and Deterioration...................................................189

14 Analytical Methods to Differentiate Farmed from Wild Seafood ............................215

15 Smoke Flavoring Technology in Seafood .................................................................233

PART III: NUTRITIONAL QUALITY 16 Composition and Calories ........................................................................................257

17 Essential Amino Acids ..............................................................................................287

18 Antioxidants .............................................................................................................309

19 Vitamins ...................................................................................................................327

20 Minerals and Trace Elements ...................................................................................351

21 Analysis of n-3 and n-6 Fatty Acids ..........................................................................377

PART IV: SENSORY QUALITY 22 Quality Assessment of Fish and Fishery Products by Color Measurement ..............395

23 Instrumental Texture ...............................................................................................425

Contents ◾


24 Aroma .......................................................................................................................439

25 Quality Index Methods ............................................................................................463

26 Sensory Descriptors ..................................................................................................481

27 Sensory Aspects of Heat-Treated Seafood.................................................................499

PART V: SAFETY 28 Assessment of Seafood Spoilage and the Microorganisms Involved.........................515

29 Detection of Fish Spoilage........................................................................................537

30 Detection of the Principal Foodborne Pathogens in Seafoods and
Seafood-Related Environments ................................................................................557

31 Parasites....................................................................................................................579

32 Techniques of Diagnosis of Fish and Shellfish Virus and Viral Diseases .................603

33 Marine Toxins ..........................................................................................................649

34 Detection of Adulterations: Addition of Foreign Proteins .......................................675

35 Detection of Adulterations: Identification of Seafood Species .................................687

36 Veterinary Drugs ......................................................................................................713

37 Differentiation of Fresh and Frozen–Thawed Fish ...................................................735

38 Spectrochemical Methods for the Determination of Metals in
Seafood .....................................................................................................................751

39 Food Irradiation and Its Detection ..........................................................................773

viii ◾


40 Analysis of Dioxins in Seafood and Seafood Products .............................................797

41 Environmental Contaminants: Persistent Organic Pollutants .................................817

42 Biogenic Amines in Seafood Products......................................................................833

43 Residues of Food Contact Materials .........................................................................851

44 Detection of GM Ingredients in Fish Feed ...............................................................871

Index .................................................................................................................................889

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 nutritional 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 methodologies 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 descriptors 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 analytical 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.



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

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,



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.

M. Concepción Aristoy Instituto de Agroquímica y Tecnología de Alimentos Consejo Superior de Investigaciones Científicas Burjassot, Valencia, Spain Santiago P. Aubourg Instituto de Investigaciones Marinas Consejo Superior de Investigaciones Científicas Vigo, Spain Kris Audenaert Department of Plant Production Faculty of Biosciences and Landscape Architecture University College Ghent Ghent, Belgium Marit Aursand SINTEF Fisheries and Aquaculture Trondheim, Norway Juan Antonio Balbuena Cavanilles Institute of Biodiversity and Evolutionary Biology University of Valencia Valencia, Spain Isabel Bandín Departamento de Microbiología y Parasitología Instituto de Acuicultura Universidad de Santiago de Compostela Santiago de Compostela, Spain Marta Barroso Instituto del Frío Consejo Superior de Investigaciones Científicas Madrid, Spain José M. Bautista Faculty of Veterinary Sciences Department of Biochemistry and Molecular Biology IV Universidad Complutense de Madrid Ciudad Universitaria Madrid, Spain Astrid Böhne Institut de Génomique Fonctionnelle de Lyon Ecole Normale Supérieure de Lyon University of Lyon Lyon, France Luisa Ramos Bordajandi Instrumental Analysis and Environmental Chemistry Department General Organic Chemistry Institute Consejo Superior de Investigaciones Científicas Madrid, Spain




Emma L. Bradley Food and Environment Research Agency York, United Kingdom Allan Bremner Allan Bremner and Associates Mount Coolum, Queensland, Australia Frédéric Brunet Institut de Génomique Fonctionnelle de Lyon Ecole Normale Supérieure de Lyon University of Lyon Lyon, France Cara Empey Campora Department of Pathology John A. Burns School of Medicine University of Hawaii Honolulu, Hawaii Fabio Caprino Dipartimento de Scienze e Technologie Veterinari per la Sicurezza Alimentare Università degli Studi di Milano Milan, Italy Mercedes Careche Instituto del Frío Consejo Superior de Investigaciones Científicas Madrid, Spain Laurence Castle Food and Environment Research Agency York, United Kingdom Antonia Chiou Department of Science of Dietetics-Nutrition Harokopio University Athens, Greece Ruth De los Reyes Cánovas Institute of Food Engineering for Development Polytechnic University of Valencia Valencia, Spain

Corrado Di Natale Department of Electronic Engineering University of Rome Tor Vergata Rome, Italy Carlos Pereira Dopazo Departamento de Microbiología y Parasitología Instituto de Acuicultura Universidad de Santiago de Compostela Santiago de Compostela, Spain E.H. Drosinos Laboratory of Food Quality Control and Hygiene Department of Food Science & Technology Agricultural University of Athens Athens, Greece Mia Eeckhout Department of Food Science and Technology Faculty of Biosciences and Landscape Architecture University College Ghent Ghent University Association Ghent, Belgium John Stephen Elmore Department of Food Biosciences University of Reading Reading, United Kingdom Margrethe Esaiassen Nofima Marked Tromsø, Norway Eva Falch Mills DA Trondheim, Norway Pedro Fito-Maupoey Institute of Food Engineering for Development Polytechnic University of Valencia Valencia, Spain



Delphine Galiana-Arnoux Institut de Génomique Fonctionnelle de Lyon Ecole Normale Supérieure de Lyon University of Lyon Lyon, France Belén Gómara Instrumental Analysis and Environmental Chemistry Department General Organic Chemistry Institute Consejo Superior de Investigaciones Científicas Madrid, Spain María José González Instrumental Analysis and Environmental Chemistry Department General Organic Chemistry Institute Consejo Superior de Investigaciones Científicas Madrid, Spain Ana Andrés Grau Institute of Food Engineering for Development Polytechnic University of Valencia Valencia, Spain Nicolas Gryson Department of Food Science and Technology Faculty of Biosciences and Landscape Architecture University College Ghent Ghent University Association Ghent, Belgium Ágústa Guðmundsdóttir Department of Food Science and Nutrition School of Health Sciences Science Institute University of Iceland Reykjavik, Iceland Karsten Heia Nofima Marine Tromsø, Norway

Marta Hernandez Molecular Biology and Microbiology Laboratory Instituto Tecnologico Agrario de Castilla y León Valladolid, Spain Aleida S. Hernández-Cázares Instituto de Agroquímica y Tecnología de Alimentos Consejo Superior de Investigaciones Científicas Burjassot, Valencia, Spain Isabel Hernando Department of Food Technology Universidad Polite ′cnica de Valencia Valencia, Spain Yoshitsugi Hokama Department of Pathology John A. Burns School of Medicine University of Hawaii Honolulu, Hawaii Grethe Hyldig Aquatic Process and Product Technology National Institute of Aquatic Resources (DTU Aqua) Technical University of Denmark Kongens Lyngby, Denmark Francisco Jiménez-Colmenero Department of Meat and Fish Science and Technology Instituto del Frío Consejo Superior de Investigaciones Científicas Ciudad Universitaria Madrid, Spain Rósa Jónsdóttir Matís Icelandic Food Research Reykjavik, Iceland Nick Kalogeropoulos Department of Science of Dietetics-Nutrition Harokopio University Athens, Greece



Anton Kaufmann Kantonales Labor Zurich Zurich, Switzerland Young-Nam Kim Department of Nutrition and Health Sciences Duksung Women’s University Seoul, South Korea Robert E. Levin Department of Food Science University of Massachusetts Amherst, Massachusetts Empar Llorca Departamento de Tecnología de Alimentos Universidad Politécnica de Valencia Valencia, Spain María-Angeles Lluch Department of Food Technology Universidad Politécnica de Valencia Valencia, Spain Iciar Martínez Instituto de Investigaciones Marinas (CSIC) Consejo Superior de Investigaciones Científicas Vigo, Spain Emilía Martinsdóttir Matís Iceland Food Research Reykjavík, Iceland Kathy Messens Department of Food Science and Technology Faculty of Biosciences and Landscape Architecture University College Ghent Ghent University Association Ghent, Belgium Leticia Mora Instituto de Agroquímica y Tecnología de Alimentos Consejo Superior de Investigaciones Científicas Burjassot, Valencia, Spain

Vittorio M. Moretti Dipartimento de Scienze e Technologie Veterinari per la Sicurezza Alimentare Università degli Studi di Milano Milan, Italy Heidi Nilsen Nofima Marine Tromsø, Norway George-John E. Nychas Laboratory of Microbiology and Biotechnology of Foods Department of Food Science and Technology Agricultural University of Athens Athens, Greece Jörg Oehlenschläger Max Rubner-Institute Federal Research Centre for Nutrition and Food Hamburg, Germany Guðrún Ólafsdóttir Syni Laboratory Services and University of Iceland Reykjavik, Iceland Ingrid Overrein SINTEF Fisheries and Aquaculture and Department of Biotechnology Norwegian University of Science and Technology Trondheim, Norway Yesim Ozogul Department of Seafood Processing Technology Faculty of Fisheries Cukurova University Adana, Turkey Monia Perugini Department of Food Science University of Teramo Teramo, Italy



Carole Prost Food Aroma Quality Group LBAI—ENITIAA Rue de la Géraudière Nantes, France Ana Puig Department of Food Technology Universidad Politécnica de Valencia Valencia, Spain Antonio Puyet Faculty of Veterinary Sciences Department of Biochemistry and Molecular Biology IV Universidad Complutense de Madrid Ciudad Universitaria Madrid, Spain Juan Antonio Raga Cavanilles Institute of Biodiversity and Evolutionary Biology University of Valencia Valencia, Spain David Rodríguez-Lázaro Food Safety and Technology Research Group Instituto Tecnologico Agrario de Castilla y León Valladolid, Spain Claudia Ruiz-Capillas Department of Meat and Fish Science and Technology Instituto del Frío Consejo Superior de Investigaciones Científicas Ciudad Universitaria Madrid, Spain Turid Rustad Department of Biotechnology Norwegian University of Science and Technology Trondheim, Norway

Isabel Sánchez-Alonso Instituto del Frío Consejo Superior de Investigaciones Científicas Madrid, Spain Rian Schelvis Wageningen IMARES Institute for Marine Resources & Ecosytem Studies IJmuiden, the Netherlands Reinhard Schubring Department of Safety and Quality of Milk and Fish Products Federal Research Institute for Nutrition and Food Max Rubner-Institut Hamburg, Germany Christina Schultheis Institut de Génomique Fonctionnelle de Lyon Ecole Normale Supérieure de Lyon University of Lyon Lyon, France Thierry Serot Food Aroma Quality Group LBAI—ENITIAA Rue de la Géraudière Nantes, France Della Wai Mei Sin Analytical and Advisory Services Division Government Laboratory Hong Kong, People’s Republic of China Rasa Slizyte SINTEF Fisheries and Aquaculture Trondheim, Norway Joseph Sneddon Department of Chemistry McNeese State University Lake Charles, Louisiana

Japan Wai Yin Yao Analytical and Advisory Services Division Government Laboratory Hong Kong. Louisiana Fidel Toldrá Instituto de Agroquímica y Tecnología de Alimentos Consejo Superior de Investigaciones Científicas Burjassot.xviii ◾ Contributors Christel Solberg Faculty of Biosciences and Aquaculture Bodø University College Bodø. People’s Republic of China . France Oddur Vilhelmsson Department of Science University of Akureyri Akureyri. Spain Musleh Uddin Corporate Quality Assurance Albion Fisheries Ltd. France Véronique Verrez-Bagnis Ifremer Nantes. Norway Inger Beate Standal SINTEF Fisheries and Aquaculture Trondheim. Iceland Kolbrún Sveinsdóttir Matís Iceland Food Research Reykjavik. Iceland Chad A. People’s Republic of China Michiaki Yamashita Food Biotechnology Section National Research Institute of Fisheries Science Yokohama. France Yiu Chung Wong Analytical and Advisory Services Division Government Laboratory Hong Kong. Vancouver. Valencia. Norway Pedro José Fito Suñer Institute of Food Engineering for Development Polytechnic University of Valencia Valencia. Spain Hólmfríður Sveinsdóttir Division of Biotechnology and Biomolecules Matís Iceland Food Research SauđárkrÓkur. British Columbia. Japan Yumiko Yamashita Food Biotechnology Section National Research Institute of Fisheries Science Yokohama. Canada Vincent Varlet Food Aroma Quality Group LBAI—ENITIAA Rue de la Géraudière Nantes. Thibodeaux Department of Chemistry McNeese State University Lake Charles. Iceland Jean-Nicolas Volff Institut de Génomique Fonctionnelle de Lyon Ecole Normale Supérieure de Lyon University of Lyon Lyon.



..................................................... 7 1...................4 Benefits and Risks .............................................. goat....... 9 References .......... 8 1...........................................8 Trends and Outlook ....... 6 1......... Variability and Basic Concepts Jörg Oehlenschläger Contents 1.............................Chapter 1 Introduction—Importance of Analysis in Seafood and Seafood Products.........1 World Catch and Harvest Seafood has by far the greatest variety of all animal-based foods........... 6 1...........................5 Sampling ...................... fishes and other aquatic animals show an abundant 3 ............................................................ turkey............................................................................................................................................ 5 1............ Whereas the species consumed as warm-blooded mammals (beef............................................. 3 1........................................................................................................... and donkey) or poultry (hen. pork................................................................................................................................1 World Catch and Harvest ............................................7 Analytical Problems ................. 5 1.................................... lamb..... geese..............2 Variability of Aquatic Animals ....................3 Special Problems with Aquatic Animals .6 Analytical Methodologies...................... and duck) are represented by very few species............10 1.......

Most fish was caught in the Pacific Ocean (Northeast and Southeast) followed by Northeast Atlantic Ocean.8 million tons). 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. Blue whiting (2. and sensory properties. 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.2 million tons).) of aquatic animals when captured by fishing techniques is—with few exceptions—completely unknown.1 million tons). The fish group alone is represented by 25.4 million tons.8 million tons).4 million tons. Mollusks (bivalves and cephalopods) are the third most important group both by quantity and by value at 22. Largehead hairtail (1.2 million tons). The total world seafood supply for 2007 amounted to 143 million tons.2% but second by value at 20. about 20% is converted into deep frozen products. 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. body composition. Atlantic herring (2. Peru (9. whereof the major part are cyprinids like carp). Further. state of maturity.1 million tons).0 million tons).000–35.4 ◾ Handbook of Seafood and Seafood Products Analysis number of species and variability. burden of pollutants. 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. 8% is transformed into cured products. They deteriorate at ambient temperature in a few days. The stagnation of captured fish is mainly due to fully exploited or partially overfished stocks.4 million tons).6 million tons). Vietnam (1.4%.4 million tons).3 million tons). 40% is consumed as wet fish without any further technological processing or preservation.3 million tons).3 million tons).1 million tons). Another difference compared with land-living animals is the fact that the quality (size. Skipjack tuna (2. Russia (3. whereas crustaceans are fourth by quantity at 6. and Thailand (1. and Norway (2. aquaculture is dramatically growing (1960: 2 million tons. infestation with parasites.6 million tons).1 million tons).7 million tons). 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). Alaska Pollock (2. However. Chile (4. India (2.000 species. The top 10 species being caught in huge amounts in 2005 were Anchoveta (10.3% and 14. The major aquaculture (excluding plants) producers (>1 million tons) in 2005 were China (32.4%. Further. Indonesia (4. and the captured fish.4 million tons). nutritional status. . Although land-based animals are today tailor made according to industry’s and consumer’s wishes in weight. 1970: 4 million tons.3 million tons). About 75% of the world’s total seafood supply is used for human consumption. 25% is converted into fishmeal and other nonfood products. 2000: 40 million tons. By major groupings.1 million tons).4 million tons). India (3. 91 million tons (64%) of the total supply. Japan (4. fish is the top group in aquaculture at 47. Thailand (2. 1980: 7 million tons.9 million tons). and Yellowfin tuna (1.4% by quantity. The most important primary product producing countries of marine and inland (freshwater) fisheries in 2005 were China (17. the United States (4. The world’s aquaculture provided 52 million tons (36%). Japanese anchovy (1.5 million tons). and another 8% into canned products. appearance. etc. Indonesia (1. 1990: 16 million tons. Chub mackerel (2. Chilean jack mackerel (1. respectively.2%. mostly Pangasius species). fish and other seafood are highly perishable products when stored without chilling. Aquatic plants that are popular in Southeast Asia are second in quantity at 23. including plants) [1].

and protein are not even distributed in the edible part and also trace element concentrations vary from head to tail or back to belly. demersal fish. When captured during the spawning season. paralytic shellfish poisoning (PSP). Components like water. some groups offer special problems to which a lot of attention has to be given: aquatic animals may contain parasites (e. and nutritive properties. flat fishes. which are very different from each other in appearance. fat. and before analyzing fish. Not only weight and length are varying with age but also other factors such as proximate composition. and so on.g. other parameters such as organic pollutant concentrations vary. 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. a careful consideration 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). and the spawned fish can exhibit fillet fat contents of down to 5%. Also within the fish body. or according to their occurrence in the ocean’s water column into pelagic fish. mineral. medium fatty fish species (>1% to <10% fat). fishing area. Based on taxonomic criteria. which continues until a state of spoilage is reached. since parallel with fat content.Introduction ◾ 5 1. cestodes) that can be harmful to humans when they enter live and intact into the human body. a change in properties starts. pollution of water. we arrange them in order according to their shape into round fish. season. With all these variations in the raw seafood material before the analysis of any components. and trace element content. not only spoilage and freshness parameters are changing due to metabolic (autolytic) and microbiological processes but also the microbial flora is changing. and mollusks. and ground fish. Besides this more general aspect. eellike fishes. 1. After catch and harvest. bottom fish.2 Variability of Aquatic Animals The variability of aquatic animals can be described and explained in many different ways.. and so forth. we have different groups such as bony and cartilaginous fishes. neurotoxic shellfish poisoning (NSP). which are at the end of the marine food web. The prespawning fish can have a fat content in fillet up to 35%. This means that each fish can be different and unique. 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. We can also group them according to their fat content into three groups: lean fish species (<1% fat). Predatory fish species such as sharks. However. When concentrating on fish as the major group contributing to the world’s fish supply.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. nematodes. which are subject to variations based on state of maturity. Mackerel is a typical pelagic swarm fish occurring in big schools. decisions must be made where the results should be used and how detailed an analysis must be. and fatty fish species (>10% fat). crustaceans. leading to several diseases such as diarrhetic shellfi sh poisoning (DSP). A drastic example illustrating the variability in fish is the Atlantic mackerel. composition. Toxins from dinoflagellates can accumulate in bivalve mollusks. can accumulate mercury during their long life span to quantities that exceed legal limits. a certain degree of variability is found. and . In addition. these are all very rough classifications.

) that are harmful to human health and must be destroyed or removed before marketing of the products. is very often underestimated. 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. Considering the great variability of seafood described here. especially in their organs responsible for detoxification such as liver and kidney. leading to elevated cadmium concentrations also in this body compartment. and sashimi). we have the risk of viruses and microorganisms. with the consequence that recommendations are mostly restricted to a few factors being appropriately analyzed but not based on all factors. Before starting the sampling procedure. the vitamins A. Mediterranean Sea. Aquatic animals from some areas of the world can carry viruses and microorganisms (e. a tremendous amount of analytic work in seafood has to be done. inshore waters. sushi. 1. the exceptional concentrations of essential elements such as selenium and iodine. and the measures to be taken to avoid any contamination as well as the storage and transport conditions of the samples after sample preparation. . cold smoked products. only few quantitative analytical data have entered these assessments.5 Sampling Sampling. which can give reliable advice and guidance for wise and responsible seafood consumption. and in fish. On the other hand. and B12. 1. 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). estuaries. cadmium is accumulated to amounts that exceed any legal limits by far. which means here the selection of an appropriate number and part of aquatic animals under well-defined conditions. In the digestive glands of mollusks (hepatopancreas) such as cephalopods and mussels. seas with no or limited water exchange with world oceans such as Baltic Sea.g.. we are confronted with toxins in mussels and fish..g. Fish and other aquatic animals from areas that are polluted (rivers. leading to ciguatera or maitotoxin poisoning. the well-balanced content of essential amino acids. Vibrio sp. the high amount of taurine. we may find high amounts of inorganic toxic elements and organic pollutants (POP. cadmium from hepatopancreas penetrates into the edible part (mantle) during storage.6 ◾ Handbook of Seafood and Seafood Products Analysis amnesic shellfish poisoning (ASP). the body compartments to be dissected. or Black Sea) can carry a high burden of environmental pollutants. Caspian Sea. we have sometimes a parasitical problem. All of these parameters and substances have to be carefully analyzed and quantified to allow a risk benefit analysis. persistent organic pollutants). there is an inherent microbial risk. D. In products that have not undergone thermal treatment and that are offered to the consumer as ready to eat (e. the presence of antioxidants such as tocopherols. gravad products. When not eviscerated immediately after catch. Most errors and most erroneous results arising from analytical methods are based on poor or even wrong sampling plans and practices. a sampling plan has to be developed describing the numbers of samples to be taken. E. and residues of pharmaceuticals and hormones used in aquaculture can be detected and more.4 Benefits and Risks Seafood is a rich source for a great number of nutritive and important components. Unfortunately.

and so forth. calibrations. knives) or by protective clothes or gloves. determination of thiobarbituric acid and formaldehyde. and total volatile basic nitrogen (TVB-N). snails). it has to be made under strict hygienic conditions to avoid any microbial contamination.6]. These projects brought the scientists together in conferences. always the whole edible part (fillet. trimethyl amine oxide. and the second concerted action was “Fish quality labeling and monitoring” (FQLM) from 1998 to 2000. in medium-sized specimen. When sampling for later microbiological analyses. While sampling is done.Introduction ◾ 7 The number of individuals should be big when a small specimen has to be analyzed. In addition. preferably at −30°C) until analysis.6 Analytical Methodologies The improvement and further development of analytical methods in the field of seafood research in Europe were initiated and brought forward by a number of research projects and concerted actions (CA) financed by the European Union within the research and technological development (RTD) framework programs 3 to 6. analysis of trimethyl amine. When sampling is done onboard a vessel. and in big fish (tuna. or tail end of fillet). The analytical methods used for seafood analyses can be divided into objective methods and sensory methods. 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. is necessary. and comparative analyses with different instruments. precaution must be taken not to contaminate the sample by instruments used during manipulation (scissors. dimethyl amine. two books shall be mentioned that have been published earlier but still contain a significant amount of basic knowledge about analytical methods for seafood quality determination [5. The main results of these projects have been published in books [2–4. workshops. smaller when medium-sized animals are the target. physical methods. The chemical/biochemical methods are mostly traditional methods that were developed earlier than the physical (instrumental) methods and have been mostly applied as methods for freshness/spoilage determinations. and analysis of biogenic amines as histamine or cadaverine. 1. sand. it is recommended to store all samples (also solutions) in deep frozen conditions (<−18°C. The first concerted action in this area was “Evaluation of fish freshness” from 1995 to 1997. The objective methods are chemical/biochemical methods. In small specimens that are consumed totally. head end. Methods that are still in use are among others k-value. More chemical methods have been developed for differentiation between fresh and frozen/thawed products (see Chapter 48) and for species identification and authenticity (see Chapters 37 and 38). the whole body may be sampled and analyzed (mussels. shark). and microbiological methods. other species or mud. sprat. tail muscle) must be taken due to intrinsic variations in fillet parts and after homogenization subsamples can be taken. and practical work-ins and allowed on-site measurements. which is based on ATP breakdown products.14] that form a very rich source of information about seafood analysis. a careful selection of individuals that have not been mechanically damaged by the catching technique. To be also mentioned are the research project “Multisensor techniques for monitoring the quality of fish” (MUSTEC) from 1999 to 2002 and the research project SEQUID “A new method for measurement of the quality of seafood” from 2001 to 2003. . After sampling is completed successfully. ammonia. Another method that was developed recently is the two-dimensional gel electrophoresis (2DE). and only a few samples are taken from big individuals.

therefore. and nonpolluting for the environment are. . Two more systematic methods that involve some analytical methods are the hazard analysis critical control point (HACCP) and traceability. and time domain spectroscopy (TDR) [7]. image analysis. a well-trained sensory panel in which the human senses are used as measuring instruments has been shown to give reliable. are time consuming. texture and texture profile analysis (e.7 Analytical Problems Despite the great progress that has been made. analysis of electrical resistance or conductivity by Torrymeter. whereas the Torry sensory scheme and the flavor profile analysis are performed on cooked samples. tensile. and can be used after a short training period by nonscientific educated personnel. electronic noses and electronic tongues [14]. and bacterial sensors. creep. and can be applied on many species and also on processed seafood products. which is usually not present in seafood industry. The main problem is that there is no single method existing that can give sufficient information about the quality (freshness) of seafood. The reasons are the relatively complex and difficult handling of the instruments and the need of being applied and maintained by educated personnel. Sensory methods. pH measurement. can be used by untrained personal. The sensory methods can also be divided into two principal methodologies: methods based on outer inspection of the sample (without cooking) and methods based on assessing the cooked sample. Frequently used microbiological methods are total viable count (TVC). of utmost importance to introduce the newly developed analytical methods into the industry for better product and raw material analysis and quality assurance. When using instrumental methods nowadays. 1. among others. with the exception of sensory methods. and are. many of them have not graduated from research to seafood industrial application. Sensory methods are often considered to be subjective methods. however. Warner–Bratzler test. It is. antibody techniques. Instrumental methods are fast. cheap. However. Kramer test. there are still many problems left in the analysis of seafood and seafood-based products. viscoelastic methods such as stress relaxation. RT Freshness grader. low-field (LF) NMR. determination of specific spoilage organisms (SSO). need trained personal.8 ◾ Handbook of Seafood and Seafood Products Analysis Physical methods comprise microscopy. Although many instrumental analytical methods have been developed and have been intensively tested and proven in research to be working sufficiently and reliably on seafood and seafood products. puncture. Further. near-infrared spectroscopy (NIR).. and objective results. with the same degree and quality of information obtained by sensory assessment. and oscillatory measurements). expensive. Other methods that are rapid. Outer inspection is done by the European Union quality-grading scheme and by the quality index method (QIM). not harmful for the operator. The European seafood sector where the majority of enterprises are small and medium sized (SMEs) hesitate to apply new instrumentation and prefer to rely on the methods they know. we have color measurement. is still missing. which are noninvasive and nondestructive techniques for the sample. nuclear magnetic resonance (magnetic resonance imaging (MRI). and compression tests. oligonucleotide probes. An instrumental method that is fast. reproducible. differential scanning calorimetry (DSC). polymerase chain reaction (PCR). and high-resolution NMR (HR-NMR)). Intellectron Fischtester VI. therefore. and have used since many years [9]. always a combination of several methods is necessary to give sufficient information equal to sensory assessment [8]. are experienced in. comparatively cheap. UV and visible light spectroscopy.g.

more research is needed to make them simpler to apply and to increase the speed of analysis. own standards. There are many seafood products on our markets that have not been characterized by analytical methods at all. Analytical instruments that are simple to use. pharmaceuticals. the protein and peptides are analyzed to a much lesser extent. however. all progress in analytical methods and instrumentation needs an analyst who is responsible and follows the guidelines and advice for analytical quality assurance. it is necessary that the schemes for the QIM as the quality method of the future are extended to all species on the market (about 100). and virus contamination in seafood. journals will in the near future no longer accept manuscripts in this field. remarkably developments have occurred very recently [10–12]. This will shorten delays in seafood trade. and have a wide range of applicability will be built. Recent findings show that seafood contains important functional proteins and peptides [13]. Without a well-documented and traceable analytical quality assurance (reference materials. food additives. This holds for all methods for species differentiation. most chemical and biochemical analytical methods that use a huge amount of chemicals and manpower will be substituted by instrumental methods that are more reliable. More research and development of analytical methodology will be initiated by these new findings. inorganic and organic residues. In the area of sensory methods.Introduction ◾ 9 For some analytical methods. Some methods that are well known such as k-value or TVB-N will disappear. bacterial pathogens. 1. their spoilage characteristics and shelf life. Th is is a large area where a significant amount of analytical input is needed. toxins such as ciguatera. sensory characteristics.8 Trends and Outlook In the future. for almost all microbiological methods. and for many methods of trace element and residue analysis. QIM will be digitalized and will work in combination with image analysis and electronic nose without sensory experts involved. and QIM schemes will also be developed for exotic species on our markets and for processed products. Many exotic fish. . justification of methods used. PCR-based methods will soon take the place of the traditional microbiological methods and will enable the checking of microbiologic status of samples in minutes or hours. and more environmental friendly. crustacean. the presence of parasites. and contents of all the beneficial components. New methods are also urgently needed for the reduction of microbial risk. proficiency tests. Although the lipids in seafood are analyzed very intensively. The method of the future will analyze a well-homogenized sample without any other sample preparatory steps except homogenizing. The QIM will be further developed. and mollusk species from tropical and subtropical countries enter our markets in large quantities or as single fish specimen and are not thoroughly investigated for their microbiological status including viruses. However. In this field. robust. more cost efficient. sampling strategy) showing that the results obtained are accurate and correct. Almost all analytical methods for seafood analysis will be developed further to avoid time and chemicals and to minimize sample preparation and digestion steps. allergens. This next generation of instruments will then also find its way into the fish industry and fish inspection.

9. Børresen. Bosch. 3. Cambridge. Detecting virus contamination in seafood... 2006. in Improving Seafood Products for the Consumer.. Thorkelsson.). Woodhead Publishing Limited.. 227p. J. in Improving Seafood Products for the Consumer. J. Pommepuy. Kent. V. 212p.).).). Rome. 8. 1995. Martinsdottir. Woodhead Publishing Limited.. (Eds. U.B. T. and Olafsdottir. Rehbein.K. 2008.). Oehlenschläger. Methods to Determine the Freshness of Fish in Research and Industry. G. 2004.. (Ed. R. Surrey. Børresen. (Eds. Lee. and Oehlenschläger. Multisensor for fish quality determination. SEQUID: A New Method for Measurement of the Quality of Seafood..K. Trends Food Sci. Verrez-Bagnis. Shaker Verlag GmbH. and Heia. 2008.B. Control of Fish Quality (3rd edn..). Wageningen... (Ed. (Ed. 134p.. 216p. 2003.. Wageningen. (Eds. Barr. Jørgensen. Aachen.. FAO. Oehlenschläger. Anon. Connell. Bacterial pathogens in seafood. Sæbø. B. Bekaert. in Improving Seafood Products for the Consumer. P. M. A study of the attitudes of the European fish sector towards quality monitoring and labeling. E. A. Tejada. 363p. Wageningen Academic Publishers. J.).. Børresen.). 13. T. 180p. G. Cambridge.. Fishery Products—Quality. .. Knöchel..J. Cambridge.. (Eds. 477p. No.. J.-K.). Cambridge. Nunes. 57p. et al. M. 12. VCH. FAO Fisheries Department.. WileyBlackwell. R.. H. Quality of Fish from Catch to Consumer—Labelling. 2003. 14. K. Reducing microbial risk associated with shellfish in European countries. in Improving Seafood Products for the Consumer. Seafood Research from Fish to Dish. State of world aquaculture 2006. 7.K..10 ◾ Handbook of Seafood and Seafood Products Analysis References 1. J. Paris. J. (Eds. in Quality of Fish from Catch to Consumer—Labelling. Monitoring and Traceability. G. 2009. J. 2008. Børresen. J. 1998. 6.K.). Monitoring and Traceability.R. J... U. New York. T. 2... (Ed. 2005. FAO Fisheries Technical Paper.. L. 567p.).M. Safety and Authenticity. 10. J. 396p. Luten. 5.. G. U. et al. Evaluation of Seafood Freshness Quality. A. U. 500. Dalgaard. Luten. 86. J. International Institute of Refrigeration. Woodhead Publishing Limited. Farnham. Olafsdottir. Woodhead Publishing Limited. 15. 2008. 1990. Olafsdottir. and Oehlenschläger. Botta. Careche. Wageningen Academic Publishers. T. 2006. (Eds.. 11. M. G.. U. Fishing News Books. Jacobsen C. Mild processing techniques and development of functional marine protein and peptide ingredients. Oehlenschläger. et al.. K. et al. 4. Wageningen. and Olafsdottir... 247p. et al. M.B.J. Wageningen Academic Publishers. 456p.. et al. Luten. Technol. Luten. 194p.

...... Both for quality control and food labeling it is therefore important to have methods to determine not only the total content of proteins in a raw material or a product................................16 2..................13 2..................................................................16 2........................................................................................................................... Fish provides about 14% of the world’s need for animal proteins and 4%–5% of the total protein requirement [2]........... 12 2..........3 Protein Solubility Classes ......................................................................... and how these properties are influenced by food additives and other components........................................14 2................................ particularly the 11 ...............................7 Peptide Characterization .................. Fish are regarded as an excellent source of high-quality protein........................................................................17 References ........................ Both the amino acid composition and the digestibility of fish proteins are excellent.............. Both the content and the properties of the proteins are important for the value and the quality of the products [1]......1 Introduction Protein analysis is highly important for the food industry...............4 Analysis of Soluble Proteins .......................................1 Introduction ..... but it is also important to have methods to determine the type and the origin of the proteins present..............................5 Immunoassays ..18 2.................. For product and process development it is important to have methods to determine the properties of the proteins and how these change during processing and storage................................ including the fish industry................16 2..................................................................................Chapter 2 Peptides and Proteins Turid Rustad Contents 2.........................2 Total Content of Proteins .......................................6 Electrophoresis-Based Methods ...............................11 2...................8 Protein Modifications....................................

The advantage of this method is that it gives accurate results for all types of samples. it is difficult to start using other and more correct factors [5]. It is important that the methods to analyze food proteins are robust [1]. The method includes sample digestion. The method should also require minimal sample pretreatment to decrease analytical error and reduce costs.5]. Originally only sulfuric acid was used for digestion of the samples. When the protein content is calculated based on determination of the nitrogen content. but other chemicals such as potassium sulfate and mercury oxide are also used. The Kjeldahl method was first published in 1883 but has been extensively modified since then. Tables of conversion factors are given in several papers such as [1. For products such as fish mince and surimi. gelling. The factor can be calculated from the amino acid composition of the proteins. and urea will also contribute to the calculated protein content. This means that it should be possible to use the method on different types of foods. both different types of raw materials and processed foods. Mariotti and coworkers discuss conversion factors in their critical review and conclude that even if a factor of 6. and the amount of protein is calculated by multiplication with a Kjeldahl factor. which has been used for more than 75 years. nucleotides. has been shown to be too high for animal proteins. the water-holding capacity and the gelling properties which determine the textural attributes of the products are important quality parameters [3]. values from 5. The Technicon AutoAnalyzer uses continuous flow analysis [1]. and trapping of ammonia and titration steps. The Kjeldahl method determines the nitrogen content as ammonia. and that other components in the food such as lipids and pigments should not interfere with the analysis. 2. For animal proteins the value 6. The Dumas method is quicker and cheaper and easier to perform and is therefore now considered on equal terms with the Kjeldahl method [1]. For fish. for instance collagen has a nitrogen content of 18%.82 are given. and the amount of nitrogen is determined by titration [1]. emulsification. assuming a nitrogen content of 16% in the proteins. nitrogen from other nitrogen-containing compounds such as free amino acids. This factor is the amount of nitrogen that contains 1 g of nitrogen. Many proteins have protein contents that deviate from this. neutralization. The Kjeldahl method has been automated and several instruments for automated analysis are available. In addition to the high nutritional value. fish proteins also have good functional properties such as water-holding capacity. . It is also possible to determine the amount of ammonia by different colorimetric methods [1]. which gives a factor of 5.25 is usually used. The disadvantage is that the method requires use of hazardous and toxic chemicals. which is distilled and trapped in boric acid. Retaining the functional properties through preservation and processing operations is therefore of great importance. amines. The Kjel-Foss® instrument mechanizes the entire micro-Kjeldahl procedure while the Kjel-Tec® instrument uses a digestion block together with an apparatus for automated distillation and titration. The ammonium sulfate is converted into ammonia. This method is used as a reference method by many national and international organizations. During digestion the nitrogen in the sample is converted to ammonium sulfate. and textural properties.25.43 to 5. It is also possible to determine the nitrogen content using elemental analysis (C/N analyzers) [4].56 [1].12 ◾ Handbook of Seafood and Seafood Products Analysis essential amino acids lysine and methionine.2 Total Content of Proteins The total content of proteins is usually determined by the Kjeldahl or the Dumas method. distillation.

and calculation of the amount of different amino acids. The advantages of this method are that it is rapid. However. determination of the amino acid profile. Today accurate combustion nitrogen analyzers are used. It is easy to perform. and in 0. 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 [16]. SO2. The volume of the supernatant was made up to 100 mL. KCl. Martinez-Alvarez and Gomez-Guillen [14] used a modification on the method of Stefansson and Hultin [15]. The connective tissue proteins are often called the insoluble proteins and can be extracted using alkali or acid. also called the salt-soluble proteins can be extracted in buffers with an ionic strength of >0. The homogenates of these solutions were stirred constantly for 30 min at 2°C. The combustion method has been calibrated with the Kjeldahl method and this has led to approval of the method by Association of Official Analytical Chemists (AOAC). After centrifugation. and several other organizations [1]. The sample is put in a furnace (950°C–1050°C). nondestructive. pH 7. Hultmann and Rustad [11] used a modification of the method by Anderson and Ravesi [12] and Licciardello and coworkers [13]. and water are removed. based on differences in solubility [9. The principle of quantitative amino acids is described in Owusu-Apenten [1]. Four grams of muscle was homogenized for 20 s in 80 mL 50 mM phosphate buffer. 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. The methods for extraction are not standardized so the amount of proteins extracted will vary with the method used. the NO2 is reduced to N2 and measured with a thermal conductivity meter [1]. Two grams of minced muscle was homogenized at low temperature for 1 min in 50 mL of distilled water. Quantitative amino acid analysis is one of the most reliable methods for quantification of food proteins. Near infrared spectroscopy can also be used to determine protein content. American Oil Chemists’ Society (AOCS). and connective proteins. The myofibrillar proteins. changes in solubility can be used to measure changes in protein structure caused by denaturation during storage and processing.5 M KCl and centrifuged as above. . The precipitate was homogenized in 80 mL phosphate buffer with 0. myofibrillar. It has been successfully used to determine protein and water content of salmon fillets [6] as well as of surimi [7]. Kelleher and Hultin compared the use of NaCl.10].86 M NaCl solution (high ionic strength). It can also be used to determine the properties of food proteins [8] and has been used to detect adulteration of beef with animal and plant proteins as well as classify tenderness of beef in two categories. 2. but the instrumentation is expensive and the method requires calibration. After cooling of the gas mixture. O2. This procedure involves hydrolyzation of the food sample using concentrated hydrochloric acid. This was the salt-soluble fraction. and can be used online.Peptides and Proteins ◾ 13 The Dumas method was first published in 1831 and the first instruments used were not user friendly.3 Protein Solubility Classes Fish muscle proteins can be divided into three groups. The sarcoplasmic proteins consist mainly of enzymes and can be extracted using water or buffers with low ionic strength such as for instance 50 mM phosphate buffer. It is also described in Chapter 16. the CO2. and filled with pure oxygen. The soluble protein was extracted in distilled water (low ionic strength). then centrifuged (6000 g) for 30 min at 3°C.3. the supernatant was decanted and the volume made up to 100 mL—this was the water-soluble fraction. sarcoplasmic. purged free of atmospheric gas.

5 M acetic acid for 2 days at room temperature and centrifuged as above. measuring concentrations between 1 and 10 mg/mL. The Lowry method [20] is based on a Biuret-type reaction between protein and copper(II) ions under alkaline conditions. and a few of them will be treated here. one of the simplest methods is to determine absorbance in the far UV range. Light scattering because of large particles or aggregates can also lead to errors. 2. This was the acid-soluble collagen. Absorption at 280 nm is mainly due to tryptophan and tyrosine residues with smaller contributions from phenylalanine and the sulfur-containing amino acids.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 [17]. Reducing agents and sucrose as well as several common buffers interfere with the Lowry method. which is a modification of the method described by Sato et al.4 Analysis of Soluble Proteins There are many indirect colorimetric methods to determine protein content. The sensitivity can be increased by measuring absorbance at 310 nm or by increasing the time for the Biuret reaction. However mg quantities of protein are generally required.1 M NaOH and centrifuged. The method is not very sensitive. 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. some of these methods reduce the speed and simplicity of the method [19]. The review also discusses many of the modifications that . the complexes react with the Folin-phenol reagent a mixture of phosphotungstic acid and phosphomolybdic acid in phenol. Since all proteins absorb UV/visible light to varying degrees. The precipitate was stirred with 0. After extraction. the concentration of the soluble proteins can be analyzed with a wide variety of methods. Peterson have reviewed the Lowry method [21] and listed interfering substances. [18]. The extraction in NaOH was repeated five times and the supernatants were pooled for the analysis of alkali-soluble collagen content. The Biuret method is based on the formation of complexes between copper salts and peptide bonds under alkaline conditions. Samples were homogenized in 0. A standard curve is needed. the presence of nonprotein UV-absorbing groups such as nucleic acids and nucleotides which absorb strongly at 260 nm further complicate matters. the method therefore has protein-to-protein variations. The product becomes reduced to molybdenum/tungsten blue and can be measured at 750 nm. Methods exist to correct for the influence of light scattering and nucleic acids/ nucleotides [19]. The purple complex is relatively stable and has an absorption maximum at 540–560 nm. Measurement of UV absorption at 280 nm is a simple and popular method to determine protein concentration. by absorbance at 205 nm or from knowledge of amino acid composition [19]. The reactions are highly pH dependent. In addition. giving upper tolerable limits for a long range of these as well as some methods for coping with the effect of these substances. but the method is simple and inexpensive. However.

and acids and alkali cause interference.1 Comparison of Useful Range for Methods to Determine Protein Concentration Method Kjeldahl Biuret Lowry Biorad (Coomassie Brilliant Blue) Biorad (Coomassie Brilliant Blue)—micro Bicinchonic acid Absorption at 280 nm Range (μg) 500–30. This method is based on the color change taking place when CBBG binds to proteins under acidic conditions. The amount of collagen can be determined by analysis of the hydroxylysine content by the Neuman and Logan method as modified by Leach [23]. The silver staining methods are 100 times more sensitive than the CBBG staining. The Lowry method determines both proteins. However. Hydroxylysine is an amino acid that is almost exclusively found in collagen. and precision. Th is figure varies for different collagen types such as collagen from fish skins from different fish species [24]. The method is compatible with a wide range of buffers/substances. It would be best if the protein being analyzed could be used as the standard protein. However. this is often not possible or practical. buffer salts.1). reducing agents. and denaturing agents such as urea and guanidine hydrochloride cause less interference. however.Peptides and Proteins ◾ 15 have been suggested for the Lowry method. it uses only one reagent instead of two as in the Lowry procedure [1].000 10–300 20–140 1–20 1–50 100–300 . Sensitivity is similar to the Lowry procedure. sensitivity. and stable reagents and kits are available. Table 2. Th is method is faster to perform than the Lowry procedure (5 min development compared to 30–45 min). There are different dye-binding methods. as different amino acids and peptides give different colors in the Lowry method. The ability of proteins to bind silver has also been used as a very sensitive method to visualize proteins in gel electrophoresis. Use of bicinchonic acid (BCA) was introduced as an easier way to determine protein. However. Finally he compares the Lowry method with other methods to determine protein concentration and concludes that the advantages of the Lowry method are simplicity. small peptides and free amino acids. while methods such as Biuret and Biorad only determine peptide chains above a certain length. chelators such as EDTA. for lipids. The Coomassie Brilliant Blue method is also used for visualizing proteins in electrophoretic gels. the method is highly protein dependent (Table 2. 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. and one of the most widely used is the Biorad method based on binding of Coomassie Brilliant Blue G (CBBG) [22]. the disadvantages are interfering substances and time—compared to some of the dye-binding methods such as the Coomassie Blue methods.000–10.000 1. but detergents. Silver binding is also being used as a method to analyze concentration of soluble proteins [19]. an accurate determination requires that the amount of hydroxylysine residues per 100 residues in the collagen is known.

Molecular weight can also be determined by electrophoresis. and Vt is the total volume of the column. or inorganic–organic composites are used as support media [1]. beads are made of open.and medium-pressure chromatography. while larger proteins can only enter the largest pores. a standard curve can be made in the same way as for gel chromatography and the weight of the unknown proteins determined. The proteins will migrate based on their size. the proteins are separated based on their size and shape (Stokes’ radii). The secondary antibody is usually linked to peroxidase or alkaline phosphatase. For high-pressure systems. Dithiothreitol (DTT) or mercaptoethanol is often added to reduce disulfide bonds. The denatured proteins are applied to the gel and an electric current is applied. Kav = (Ve − V0)/(Vt − V0). polyacrylamide. 2. macroporous silica. As the protein solution moves down the column. on the average. One of the most commonly used methods is SDS-PAGE. cross-linked three-dimensional polymer networks such as agarose. spend more time inside the beads and the larger proteins will emerge from the column first. dextrans.16 ◾ Handbook of Seafood and Seafood Products Analysis 2. Bovine serum albumin (BSA) is then added to block nonspecific binding sites. causing the negatively charged proteins to migrate across the gel toward the anode. Many peptides are bioactive and have physiological properties. Since SDS is charged. and combinations of these. 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. V0 is the void volume of the column. a second antibody is bound to the protein bound to the primary antibody. 2. In native gel filtration chromatography. a standard curve can be made allowing determination of the molecular weight distribution in a protein mixture. smaller proteins will travel farther down the gel.7 Peptide Characterization Studying the composition and properties of peptides in seafood is often of interest.4. The most commonly used system is that of Laemmli [25]. where Ve is the elution volume of the molecule. By using standard proteins of known molecular weight. smaller proteins will.5 Immunoassays The amount of a specific protein in a mixture can be determined by enzyme-linked immunosorbent assays (ELISA). for instance after enzymatic hydrolysis of proteins or during processing and storage of seafood. Small proteins can enter all the pores in the beads. while larger ones travel a shorter distance. It is then necessary to have the antibody of the protein that one seeks to quantify. By using markers of known molecular weight. which gives one SDS molecule for every two amino acids. such as immunostimulating or antihypertensive . this results in a charged complex where the charge is proportional to the molecular weight of the protein. The amount of secondary antibody bound is proportional to the amount of the specific protein in the sample. The method is very sensitive but requires available antibodies. For low. SDS binds to proteins in a weight ratio of 1:1. After washing. porous glass. cellulose.6 Electrophoresis-Based Methods The molecular weight of proteins and peptides is often of interest and this can be determined by several different methods. A polyclonal or monoclonal antibody against the protein of interest is then bound to a film through the Fc region of the antibody. These enzymes can convert a colorless substrate to a colored product which can then be detected. using gels of polyacrylamide and denaturing the samples by boiling in a solution of sodium dodecyl sulfate (SDS).

Several methods to determine this value exist. One much used definition of functional properties is this: Those physical and chemical properties that influence the behavior of proteins in food systems during . or trichloroacetic acid can be used [28]. The peptides may also give valuable information about the quality of the food. The content of sulfhydryl groups can be determined using DTNB by the method of [34] with the modification of [35]. In addition oxidation can be measured as loss of functional properties such as loss of solubility. 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. Oxidation of protein side chains can give rise to unfolding and conformational changes in protein and also to dimerization or aggregation [31]. solubility. selective precipitation using ethanol.8 Protein Modifications During storage and processing of marine raw materials. emulsification. For determination of the amount of peptides below a certain chain length. this is spectrophotometric method determining the amount of the chromophore formed when TNBS reacts with primary amines. methanol. and water-holding capacity.Peptides and Proteins ◾ 17 properties. loss of water-holding capacity. Formaldehyde reacts with unprotonated primary amine groups resulting in loss of protons. Changes in proteins during storage and processing will often result in changes in the functional properties of the proteins. Oxidative modification often leads to alterations in the functional. Formation of dityrosine is also used to determine the degree of protein oxidation. the term degree of hydrolysis describes the extent to which peptide bonds are broken by the enzymatic hydrolysis reaction. Precipitation of the proteins makes it possible to study peptides which are found in lower concentrations using different chromatographic methods such as LC–MS or electrophoretic methods. Another widely used method is the determination of free amino groups after titration with formaldehyde [27]. muscle proteins are also vulnerable to oxidative attack during processing and storage of muscle foods [30]. The amount of liberated protons can be determined by titration. The reaction takes place under slightly alkaline conditions and is stopped by lowering the pH in the solution. Mass spectroscopy can be used to determine the molecular mass of the peptides. 2. and by using tandem mass spectroscopy detailed information of the structure of the peptides can be found. these properties are not only dependent on the oxidation state of the proteins. gelling and emulsification properties. In addition to lipids and pigments. For characterization of mixtures of peptides. Studying the peptide fraction can give a lot of useful information as peptides may have several functions in the food. nutritional. One of these is the determination of free amino groups after reaction with trinitrobenzene-sulfonic acid (TNBS) [26]. changes take place in the proteins and it is often of interest to quantify these changes. the most used are determination of formation of carbonyl groups [32. including gelation. Oxidation can occur at both the protein backbone and on the amino acid side chains. viscosity. Several methods are used to determine protein oxidation. such as provide information about the enzymes that are active during storage. 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. and changes in these properties may be due to other factors. However.33] and reduction in SH-groups. and can result in major physical changes in protein structure ranging from fragmentation of the backbone to oxidation of the side chains. and formation of aggregates. and sensory properties of the muscle proteins. especially after enzymatic degradation/ hydrolysis. Bauchart and coworkers [29] studied the peptides in rainbow trout using precipitation with perchloric acid followed by electrophoresis and MS-analysis in order to study proteolytic degradation.

pp. 2008. 1992. A description of the properties of the proteins important for functional properties was given by Damodaran [37]: The physicochemical properties that influence functional behavior of proteins in food include their size. 1996. Value added products from underutilised fish species. Venugopal. 87: 31–41. 463. T. Journal of Food Science & Technology. Converting nitrogen into protein—Beyond 6. Nutritional. Owusu-Apenten. 48: 177–184. J. W. 2. 2004.C. Food Chemistry.. wettability). Iced storage of Atlantic salmon (Salmo salar)—Effects on endogenous enzymes and their impact on muscle proteins and texture. Uddin. Control of chemical composition and food quality attributes of cultured fish. Haard. Food Protein Analysis: Quantitative Eff ects on Processing. cooking. 25: 289–307. adhesiveness. thickening. 7.18 ◾ Handbook of Seafood and Seafood Products Analysis processing.25 and Jones’ factors. Journal of Food Science. sensory. et al. J. V. M. 1982. The book edited by Hall [38] gives a good overview of methods to determine protein functionality.F. E. 879–942. and quaternary). It is therefore difficult to compare results from different laboratories. 2006... Hultin.. solubility. distribution. 25: 2025–2069. moisture and protein in salmon fillets by use of near-infrared diff use spectroscopy. et al. and gelation). or interaction with other food constituents.R. 2002. Ravesi. 9. Bock. F. Journal of Food Quality. Methods for processing and utilization of low cost fishes: A critical appraisal. whippability). 35: 431–435. N. Anderson. Isaksson. 13. Journal of the Science of Food and Agriculture. and R. 1968. Food Research International. R. sulphur and sulphur alone in organic and inorganic materials. Ed. Lanier.. Characteristics of edible muscle tissue. New York: Marcel Dekker.M. Innovative uses of near-infrared spectroscopy in food processing. 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. 69: 95–100. and E.. 32: 1–12. aggregation. 8. Journal of Fisheries Research Board Of Canada. D. Automatic methods for the simultaneous determination of carbon. Mirand. 5. T. 73: R91–R98. amino acid composition and sequence. and T. molecular flexibility/rigidity in response to external environment (pH. Journal of Food Science & Nutrition. Relation between protein extractability and free fatty acid production in cod muscle aged in ice.. and biological values are sometimes included in the functional properties. hydrophobicity. (2) properties related with the protein structure and rheological characteristics (viscosity. V. et al. O. storage. salt concentration). Food Chemistry. structures (secondary. 96: 491–495.E. Rustad. L. and consumption [36].. 51: 1173–1179. p.A.. Foegeding. 1995. Non-destructive determination of fat. Shahidi. Critical Reviews in Food Science and Nutrition. in Food Chemistry. 1995.K. hydrogen.J. hydrophilicity. formation of protein–lipid films.L. 5: 215–234. Connelly. Kirsten. 2008. Analytical chemistry.J. 6.. Functional properties can be divided in several groups. Marcel Dekker: New York. 4. net charge. 1979. Mariotti.P.K. 10. 1995. 12. 11. elasticity. temperature. Licciardello. M. Fennema. Venugopal. References 1. It is usual to classify them according to mechanism of action into three main groups: (1) properties related with hydration (absorption of water/oil. Nondestructive determination of water and protein in surimi by near-infrared spectroscopy. and (3) properties related with the protein surface (emulsifying and foaming activities. tertiary. Hultmann. and F. 3. Tome. . shape. and P. Time–temperature tolerance and physical-chemical quality tests for frozen Red Hake. and H.

. Food Chemistry. 82: 70–78.H. M. Kelleher. and D. Food Proteins: An Overview. K. 38. Hultin. Analyst. International Dairy Journal. 32. 21. 56: 315–317. 1996. Ed.L.M. 34. Biochimica Biophysica Acta. Stefansson. Review of the Folin Phenol protein quantitation method of Lowry. On the solubility of cod muscle proteins in water. G. Norges Tekniske høgskole: Trondheim. 26. Rohm. S. O. Y. pp. Blackie Academic and Professional: London. Paraf. Protein and lipid oxidation during frozen storage of rainbow trout (Oncorhynchus mykiss).. 1976. 23. Ellman. 55: 8118–8125. R. 18. 1996. and M. 27(6): 1256–1262. G. 1957. Bradford. Taylor. in Food Proteins: Properties and Characterization.. 29. A. S. S.. Hultin. U. 37. C. Journal of Biological Chemistry. Journal of Food Science. K. G. A rapid and sensitive method for the determination of microgram quantities of protein utilizing the principle of protein-dye binding.. 1994.. Eckhoff. Formol titration: An evaluation of its various modifications. 17. Food Chemistry. Itoh.. Adler-Nissen.. Isolation of types I and V collagen from carp muscle. The oxidative environment and protein damage. H.Peptides and Proteins ◾ 19 14.L.. Myoglobin-induced lipid oxidation.A.M. 19. Comparative Biochemistry & Physiology.. 2005. 30.K. 33.B. Kinsella. 5: 169–186. 1951. Baron. 2007. Peptides in rainbow trout (Oncorhynchus mykiss) muscle subjected to ice storage and cooking. Collagen content in farmed Atlantic salmon (Salmo salar L. 74: 70–71.. Journal of Agricultural and Food Chemistry. in Food Proteins and their applications.. CRC Critical Reviews Food Science & Nutrition. 2007. Fisheries Science. p. 1960. 20. K. 100: 201–220. Mechanisms and factors for edible oil oxidation. 15.J. Protein measurement with the Folin phenol reagent. Bauchart. 31. Sompongse.Y. 62: 197–200. Analytical Biochemistry. Martinez-Alvarez. 7: 219–280. et al. J. 94: 123–129. et al. Analysis: Quantitation and physical characterization. Lowry. et al. Functional properties of food proteins: A review. Sato. Technicla Biochemistry.. Analytical Biochemitry. 25. G. 1997. 1996. et al.W.. . 2002.M. E. Comprehensive Reviews in Food Science and Food Safety. Baron. 6: 1069–1077. 1979. 72: 248–254..D.: New York..E. Davies. 36.. VCH: New York. 28. C. Obtake. Nature.P. J.. Marcel Dekker. 265. 100: 1566–1572. O. Eds. Inc. 35. Cleavage and structural proteins during assembly of the head of bacteriophage T4. U. W.) and subsequent changes in solubility during storage on ice. 90B: 155–158. Min. Eds.. Andersen.. Lithium chloride as a preferred extractant of fish muscle proteins. Biochemistry Journal.H. 16. p. Laemmli. 1976. Tissue sulfhydryl groups. Journal of Agricultural and Food Chemistry. C. 1979. and H. 27. and H. S. et al. 1981. 62: 73–79. Methods for Testing Protein Functionality. 227: 680–685. Almås.A. Comparison of ethanol and trichloracetic acid fractionation for measurement of proteolysis in Emmental cheese. Yada. Farr and Randall. Choe. Journal of Agricultural and Food Chemistry. Notes on a modification of the Neuman & Logan method for the determination of the hydroxyproline. 24. Food Chemistry. Rosebrough. Gomez-Guillen..O. Effect of brine salting at different pHs on the functional properties of cod muscle proteins after subsequent dry salting. 1703: 93–109.. 1–24.P. 2006.J. et al.C. 22. W. Damodaran and A. Effect of Cryoprotectants and a reducing reagent on the stability of actomyosin during ice storage. and H. Peterson. Muskelcellehylsteret hos torsk: Ultrastruktur og biokjemi. Modler. Journal of Agricultural and Food Chemistry. et al. 1998. 50: 3887–3897. M. 2006. Archives in Biochemistry & Biophysics. and A. 193: 265–275. 1959.. Hall. 82: 488–498. 1991. 1988.K. 42: 2656–2664. 1970. in Dep.O. Leach.. Damodaran. 175. 1996. A review. Determination of the degree of hydrolysis of food protein hydrolysates by trinitrobenzenesulfonic acid. Nakai and H.


.....3.25 3............3......................31 3..........................................................5 Staining ......2 Proteome Analysis by 2DE ..1 Sample Matrix Considerations ...............................................................................................................................33 3..................................................................Chapter 3 Proteomics Hólmfríður Sveinsdóttir............4 Second-Dimension Electrophoresis ...................3 Equilibration ......2............6 Analysis .............2................................35 References ...................... 32 3.1 Introduction .............................. 34 3.................2.3..............2........................................................................ 27 3. 28 3......2.......... 22 3.. 22 3....................1.....................2...................................... 28 3...........2........................................................2 First-Dimension Electrophoresis ...2 Aquaculture and Antemortem Effects on Quality and Processability ....... 28 3...2..... 24 3...............................................1 Whole Larval Proteomes......1 Development .................................................31 3..........3 Applications of 2DE in Seafood Analysis .................. 22 3...........................................2......25 3..................3 Species Authentication ..........................1 Protein Autolysis and Oxidation during Storage and Processing .....2.......................35 21 ...............................................................................................................2 Muscle Proteomes .................................................. 34 Acknowledgments ............. and Oddur Vilhelmsson Contents 3......................................2................3 Protein Identification by Peptide Mass Fingerprinting ....................... Ágústa Guðmundsdóttir..................................................2......................1 Sample Extraction and Cleanup ..................... 24 3....2.2................................................1.......................................................1.........2.........2......2 Basic 2DE Methods Overview ...................3...... 27 3...2.................................................................4 Allergen Identification ........................................................................................25 3............................ 32 3......................................................................3 The Degradome ............................3.......................................2.............2 Quality Involution ......2..............................................2.....3......... 22 3............

we present some issues and challenges related to sample matrices of particular interest to the seafood scientist. and after processing or storage. and low survival rate. This is especially true of fish and meat. based on liquid chromatography tandem mass spectrometry (LC–MS/MS).12.2 Proteome Analysis by 2DE 2DE.4 hold great promise and are deserving of discussion in their own right. the interactions of proteins with one another or with other food components.1) remains the workhorse of most proteomics work. This chapter will therefore focus on 2DE. for example. the cornerstone of most proteomics research.13 skeletal muscle. the proteome varies from tissue to tissue. gel-free methods. growth depression. in the following sections.2.14–18 gill. where the bulk of the food matrix is constructed from proteins. as well as with time and in response to environmental stimuli.6–8 liver. then.22 ◾ Handbook of Seafood and Seafood Products Analysis 3. simplicity.1 Sample Matrix Considerations Unlike the genome. 3. 3. the “classic” process of two-dimensional (2D) gel polyacrylamide electrophoresis (2DE) followed by protein identification via peptide mass fingerprinting of trypsin digests (Figure 3. 3. or even thousands. also known as proteomics.1 Introduction As with all living matter. fish possess a number of tissues amenable to 2DE-based proteome analysis. largely because of its high resolution.12. 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. .2. Proteome analysis allows us to examine the effects of environmental factors on larval global protein expression. is a tool that can be of great value to the food scientist. While high-throughput. of proteins on a 2D polyacrylamide slab gel. Selection of tissues for protein extraction is therefore an important issue that needs to be considered before a seafood proteomic study is embarked upon. both on the cellular and tissue-wide levels. Proteomics.9–11 heart. foodstuffs are in large part made up of proteins.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 larval quality like malformations. is the simultaneous separation of hundreds. In the following sections.12 and rectal gland12 have been reported. is regulated and brought about by proteins.12 brain. Studies on whole larvae. Like other vertebrates.12 kidney. during.1.2 surface-enhanced laser desorption/ionization3 or protein arrays. giving valuable insight into the composition of the raw materials. and mass accuracy. It stands to reason. or with the human immune system after consumption. the construction of the food matrix. that proteome analysis. along with some of the main improvements that have developed since.19 intestine. quality involution within the product before. Furthermore. 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.

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.22 Three of these publications have focused on the whole larval proteomes in Atlantic cod (Gadus morhua)6. where the majority of the highly abundant proteins were identified as muscle proteins. subjected to degradation by trypsin (or other suitable protease) and the resulting peptides analyzed by mass spectrometry.22 Also. allowing identification of ca. See text for further details. 85% of the of the selected protein spots.Proteomics ◾ 23 2D PAGE Trypsin digestion MS fingerprinting MS/MS sequencing Figure 3. peptides can be dissociated into smaller fragment and small partial sequences obtained by MS/MS. a protein extract (crude or fractionated) from the tissue of choice is subjected to 2D PAGE. yielding a peptide mass fingerprint. First.7. These proteins may mask subtle changes in proteins expressed in other tissues or systems. posttranslational modifications and redistribution of specific proteins within cells.6. it is excised from the gel. In many cases this is sufficient for identification purposes.22 and zebrafish (Danio rerio). The axial musculature is the largest tissue in larval fishes as it constitutes approximately 40% of their body mass. cytoskeletal . Only a few proteome analysis studies on fish larvae have been published. like the overwhelming presence of muscle and skin proteins. but if needed.21. Peptide mass mapping using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry was performed only on the cod larval proteins. there are several drawbacks when working with the whole larval proteome. Once a protein of interest has been identified.6.6. such as the gastrointestinal tract or the central nervous system. Nevertheless.23 This is reflected in our studies on whole cod larval proteome.7 These studies provided protocols for the production of high-resolution 2D gels.1 An overview over the “classic approach” in proteomics.20 all important information for controlling factors influencing the aptitude to continue a normal development until adult stages.

30 preparative isoelectrofocusing31 or solubility in the presence of various detergents32 or chaotropes33 have been described.44 It seems likely that the manner. No amplification method analogous to PCR exists for proteins. Removal of those proteins may increase detection of other proteins present at low concentrations. which include most regulatory proteins and many important metabolic enzymes. preventing identification of holistic alterations in the analyzed proteomes. Swamping of low-abundance spots by highly abundant ones may not be a problem for applications relating specifically to structural proteins.42.1. such as the ubiquitin–proteasome or the lysosome systems.2. A myriad of methods suitable for subsequent 2DE exist for fractionating the proteome into defined subproteomes.2. are of keen interest. whereby proteins are targeted for destruction by the proteasome by covalent . Protein turnover systems. Proteomic analysis on lysosomes has been successfully performed in mammalian (human) systems. are suitable for rigorous investigation using proteomic methods. is fractionation of the protein sample in order to weed out the high-abundance proteins.5 The remaining option.25. Structural proteins. are particularly abundant in the skeletal muscle proteome. as it will give rise to overloading artifacts in the gels. in which protein deposition is regulated. such as those associated with individual organelles or cell compartments28 or by protein biochemical methods such as affi nity chromatography. The fish muscle proteome is therefore likely to be of comparatively high interest to the seafood scientist. An unfractionated 2DE map of the muscle proteome therefore tends to be dominated by comparatively few high-abundance protein spots. it may also result in a loss of other proteins. but for other applications low-abundance proteins. and biochemistry. rendering analysis of low-abundance proteins difficult or impossible.24 ◾ Handbook of Seafood and Seafood Products Analysis proteins were prominent among the identified proteins.2 Muscle Proteomes In most seafood products. In addition to having a hand in controlling autolysis determinants. allowing a larger sample of the remaining proteins to be analyzed. protein turnover is a major regulatory engine of cellular structure. and simply increasing the amount of sample is usually not an option. Fractionation methods for a variety of sample matrices have been reviewed recently. Cellular protein turnover involves at least two major systems: the lysosomal system and the ubiquitin–proteasome system. lysosomes can be isolated 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 characteristics.1. fish skeletal muscle is the main component. Various strategies have been presented for the removal of highly abundant proteins24 or enrichment of lowabundant proteins. However. during and after processing.26 3. then. For example.34–41 3.46 An exploitable property of proteasome-mediated protein degradation is the phenomenon of polyubiquitination. particularly in muscle tissue.45. such as actin and tubulin.3 The Degradome The degradome may be a subproteome of particular interest to the food scientist. has profound implications for quality and processability of the fish flesh.29. function.43 The 20S proteasome has been found to have a role in regulating the efficiency with which rainbow trout (Oncorhynchus mykiss) deposit protein. as many textural and other quality factors of muscle foods are related to proteolytic activity in the muscle tissue before.

may be more conveniently carried out using transcriptomic methods.1 Sample Extraction and Cleanup For most applications. it is possible to observe the ubiquitin–proteasome “degradome.2 Basic 2DE Methods Overview O’Farrell’s original 2DE method first applies a process called isoelectric focusing (IEF). The map can be visualized and individual proteins quantified by radiolabeling or by using any of a host of protein dyes and stains.” i. 3. such as Coomassie blue. which proteins are being degraded by the proteasome at a given time or under given conditions.2.22 and Arctic charr (Salvelinus alpinus) liver. Gygi and coworkers have developed methods to study the ubiquitin–proteasome degradome in the yeast Saccharomyces cerevisiae using multidimensional LC–MS/MS. These strips consist of a dried IPG-containing polyacrylamide gel on a plastic backing. such as that of the matrix metalloproteases. 4% (w/v) CHAPS [3-(3-chloramidopropyl)dimethylamino-1-propanesulfonate]. most notably the introduction of immobilized pH gradients (IPGs) for IEF.52–57 3. For more detailed. where an electric field is applied to a tube gel on which the protein sample and carrier ampholytes have been deposited. Ready-made IPG strips are currently available in a variety of linear and .51 the procedure remains essentially as outlined earlier.Proteomics ◾ 25 binding to multiple copies of ubiquitin. 0. a general protocol is outlined briefly with some notes of special relevance to the seafood scientist.48–50 Monitoring of the expression levels of these regulatory enzymes. or fluorescent dyes. 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. yielding a two-dimensional map (Figure 3. may be less directly amenable to proteomic study.2. The tube gel is then transferred onto a polyacrylamide slab gel and the isoelectrically focused proteins are further separated according to their molecular mass by conventional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). 0. and how they vary with environmental or dietary variables. Although a number of refinements have been made to 2DE since O’Farrell’s paper.43. 2 M thiourea.3% (w/v) DTT [dithiothreitol]..2 Some proteolysis systems. which is most conveniently performed using commercial dry IPG gel strips. up-to-date protocols.2. the reader is referred to any of a number of excellent reviews and laboratory manuals. This separates the proteins according to their molecular charge. In the following sections.2.2 First-Dimension Electrophoresis The extracted proteins are first separated by IEF. Activity of matrix metalloproteases is regulated via a complex network of specific proteases.47 By targeting these ubiquitin-labeled proteins.e. 3.5% Pharmalyte ampholytes for the appropriate pH range) supplemented with a protease inhibitor cocktail to give good results for proteome extraction from whole Atlantic cod larvae6.2. silver stains. Cleanup of samples using commercial 2D sample cleanup kits may be beneficial for some sample types.58 Thorough homogenization is essential to ensure complete and reproducible extraction of the proteome.2) rather than the familiar banding pattern observed in one-dimensional (1D) SDS-PAGE.

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. usually totaling about 10. 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. Broad-range linear strips (e. Application of a low voltage current may speed up the reswelling process. Optimal conditions for reswelling are normally provided by the IPG strip manufacturer. Reswelling is normally performed overnight at 4°C. 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.500 V.2.2 A 2DE protein map of whole Atlantic cod (G.2.26 ◾ Handbook of Seafood and Seafood Products Analysis MW (kDa) 60 42 30 22 17 4 5 pl 6 7 Figure 3.g.000 Vh. The proteins are separated according to their pI in the horizontal dimension and according to their mass in the vertical dimension. which is then increased stepwise to about 3. pH 3–10) are commonly used for whole-proteome analysis of tissue samples. extraction directly into the reswelling buffer is recommended. A recipe for a typical reswelling buffer is presented in Section 3. Before electrophoresis. the dried gel needs to be reswelled to its original volume. Typically. morhua) larval proteins with pI between 4 and 7 and molecular mass about 10–100 kDa.1. IEF is normally performed for several hours at high voltage and low current. although this will depend on the IPG gradient and the length of the strip. Isoelectrofocusing was by pH 4–7 IPG strip and the second dimension was in a 12% polyacrylamide slab gel. sigmoidal pH ranges. If the protein sample is to be applied during the reswelling process. The appropriate IEF protocol will depend not only on the sample and IPG strip.. but also on .000–30. the starting voltage is about 150 V.

This is best performed using a dentist’s tool or other appropriate implement.4 Second-Dimension Electrophoresis Once the gel strip has been equilibrated. the SDS–polypeptide complex that affords protein-size-based separation will form and the reducing agent will preserve the reduced state of the proteins.56 reviewed IEF for 2DE applications. avoiding trapping air bubbles. 0. . Figure 3. During the equilibration step. and that the strip is pressed gently onto the SDS gel. A tracking dye for the second electrophoresis step is also normally added at this point. but for most applications gradient gels or gels of about 10% or 12% polyacrylamide are appropriate.3 Equilibration Before the isoelectrofocused gel strip can be applied to the second-dimension slab gel. A second equilibration step in the presence of 2.Proteomics ◾ 27 the equipment used. 2% SDS. 30% glycerol. 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. taking care to put the pressure on the IPG strip’s plastic backing rather than the gel itself.8. A typical equilibrationbuffer recipe is as follows: 50 mM Tris–HCl at pH 8.2.59 3.5% iodoacetamide and without DTT (otherwise identical buffer) may be required for some applications. 192 mM glycine.2.1% SDS) at both electrodes.3 Orientation and placement of an isoelectrofocused IPG strip onto the top of the second-dimension gel. This will alkylate thiol groups and prevent their reoxidation during electrophoresis. The gel is run at a constant current of 25 mA until the bromophenol blue dye front has reached the bottom of the gel. thus reducing vertical streaking. 3. The manufacturer’s instructions should be followed. Care must be taken that the (+) end of the strip is on the same side of all slab gels.60 the Laemmli method. that the gel side of the IPG strip faces the notched side of the glass plate. remains the most popular one. trace amount of bromophenol blue. Görg et al. While some reviewers recommend alternative buffer systems.2.61 using glycine as the trailing ion and the same buffer (25 mM Tris. 1% DTT.2. 6 M urea. it needs to be equilibrated for 30–45 min in a buffer-containing SDS and a reducing agent such as DTT. Ready-made gels suitable for analytical 2DE are available commercially.

There are.68 where peptides are suspended in a matrix of small.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. followed by several 30 min washing steps in water. A typical staining procedure includes fi xing the gel for several hours in 50% ethanol/2% ortho-phosphoric acid. and individual protein quantification. and therefore individual variation is a major concern and needs to be accounted for in any statistical treatment of the data. UV-absorbing molecules (such as 2.6 Analysis Although commercial 2DE image analysis software.65–67 3. Pooling samples may also be an option and this depends on the type of experiment. A great many alternative visualization methods are available. The most popular mass spectrometry method is MALDI-TOF mass spectrometry. These multiple sources of variation has led some investigators63–65 to cast doubt on the suitability of univariate tests. PDQuest (BioRad). such as in difference gel electrophoresis (DIGE). such as with [35S] methionine.64 These difficulties arise from several sources of variation among individual gels. digested with trypsin (or another suitable protease). or Progenesis (Nonlinear Dynamics).28 ◾ Handbook of Seafood and Seafood Products Analysis 3. and staining with fluorescent dyes. matching. such as the SYPRO or Cy series of dyes. and followed by destaining for several hours in water. organic. including protein spot definition. however. analysis of the 2DE gel image. followed by incubation for 1 h in 17% ammonium sulfate/34% methanol/2% ortho-phosphoric acid. the spot of interest is excised from the gel. 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.3 Protein Identification by Peptide Mass Fingerprinting Identification of proteins on 2DE gels is most commonly achieved via mass spectrometry of trypsin digests. These include radiolabeling.62 3. 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. spot matching between gels tends to be time-consuming and has proved difficult to automate. Patton published a detailed review of visualization techniques for proteomics.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. commonly used to assess the significance of observed protein expression differences. such as ImageMaster (Amersham).2.2. Multivariate analysis has been successfully used by several investigators in recent years. gene expression in several tissues varies considerably among the individuals of the same species. such as protein load variability due to varying IPG strip reswelling or protein transfer from strip to slab gel.2.1% Coomassie Blue G-250/17% ammonium sulfate/34% methanol/2% ortho-phosphoric acid. has improved by leaps and bounds in recent years. such as Student’s t-test. Also.2. commercially available colloidal Coomassie staining kits that do not require fi xation or destaining. . Briefly. remains the bottleneck of 2DE-based proteome analysis and still requires a substantial amount of subjective input by the investigator.63 In particular. and the resulting peptide mixture is analyzed by mass spectrometry. followed by staining for several days in 0.2.

2506.Proteomics ◾ 29 842. however. To circumvent this problem.25 Figure 3.43 .4 2212.06 1575.51 1040.4 A trypsin digest mass spectrometry fingerprint of an Atlantic cod larval protein spot.9 were able to attain an identification rate of about 80% using a combination of search algorithms that included the open-access Mascot program69 and a licensed version of Protein Prospector MS-Fit70 by searching against both protein databases and a database containing all salmonid nucleotide sequences.69 1659.801616. that an identity obtained in this manner is less reliable than that obtained through protein sequences and should be regarded only as tentative in the absence of corroborating evidence (such as 2D immunoblots.36 2564. The solid markers indicate the peaks that were found to correspond to expected b-2 tubulin peptides.00 1196.org/tools) contains links to most of the available software for protein identification and several other tools. such as the National Centre for Biotechnology Information (NCBI) nonredundant protein sequences database.54 50 40 1287.54 70 1960. which in many cases is more extensive than the protein sequences available. As can be seen in Table 3.71 100 90 80  Intensity 1061.07 1131. The resulting spectrum of peptide masses (Figure 3.56 1974.expasy. The open markers indicate mass peaks corresponding to trypsin self-digestion products and were.4) is then used for protein identification by searching against expected peptide masses calculated from data in protein sequence databases.2 2798.98 1886. identified as b-2 tubulin. using the appropriate software. In those cases where both the protein and nucleotide databases yielded results.6 Mass (m/z) 2108.35 1621. many with a web-based open-access interface. therefore.61 1272. excluded from the analysis.10 and Vilhelmsson et al.90 1822.1.0 1229. In their work on the rainbow trout liver proteome. correlated activity measurements. it is possible to take advantage of the available nucleotide sequences. It is important to realize.50 20 10 0 741.50 30 856. or transcript abundance). to obtain a tentative identity.86 1028. The ExPASy Tools web site (http://www.63 1258.96 60 870. How useful this method is will depend on the length and quality of the available nucleotide sequences. this problem is surprisingly acute for species of commercial importance. Several programs are available. Martin et al. 100% agreement was observed between the two methods.8 1652.60 Peptides identified as those derived from Atlantic cod β-tubulin Trypsin autolysis peaks 1159.70 1697. Attaining a high identification rate is problematic in fish and seafood proteomics due to the relative paucity of available protein sequence data for these animals.

138 36. incl.864 47.533 1. and plaice) Zeiformes (dories) Scorpaeniformes (scorpionfishes.344 5.) Gastropoda (incl.284 2.30 ◾ Handbook of Seafood and Seafood Products Analysis Table 3.680 1. incl. cod.353 287 170. halibut.896 3.845 10.122 268 26.798 81.006 121. turbot.086 2.585 1.782.063 3. sole. mackrel. incl. whelks and abalone) Cephalopoda (squid and octopi) 32.656 2.557 . redfish and lumpfishes) 185. etc.424 2.381 45.845 999. saithe.751 Crustacea (Crustaceans) Caridea (shrimps.158 20.008 Mollusca (Mollusks) Bivalvia (mussels.287 8. tuna.007 911 303 18. haddock.871 32. 2008 Protein Sequences Nucleotide Sequences Actinopterygii (Ray-Finned Fishes) Anguilliformes (eels and morays) Clupeiformes (herrings) Cypriniformes (carps) Siluriformes (catfishes) Salmoniformes (salmons and trout) Gadiformes (cod-likes. sea bass. incl.626 138 16.380 898. sea bream.592 735 179 585 768. and pollock) Lophiiformes (anglerfishes.762 726.489 130. etc. incl.) Astacidea (lobsters and crayfishes) Brachyura (short-tailed crabs) 21. monkfish) Perciformes (perch-likes.208 2.933 3.407 84.237 2.1 Families of Some Commercially Important Seafood Species and the Availability of Protein and Nucleotide Sequence Data as of March 27.210 Chondrichthyes (Cartilagenous Fishes) Carcharhiniformes (ground sharks and dogfishes) Lamniformes (mackrel sharks) Rajiformes (skates and rays) 3. scallops.203 467.442 237 2.046. and wolffish) Pleuronectiformes (flatfishes.245 2.

85 wheat flour baking quality factors.20 To date few studies on fish development exist in which proteome analysis techniques have been applied.. Until recently.73–75 Mass spectrometry methods in proteomics have been reviewed. and vastly superior protein spot identification techniques.95 resulting in different expression of proteins. physiology. Correlating this spectrum with the candidate peptides identified in the first round narrows down the number of candidates.76 Nyman.80 Mo and Karger.94 Proteome analysis provides valuable information on the variations that occur within the proteome of organisms. this was accomplished by N-terminal or internal (after proteolysis) sequencing by the Edman degradation of eluted or electroblotted protein spots. larva. if rather more time-consuming. and behavior of the fish.87 With the lower cost. Recent studies on global protein expression during early developmental stages of zebrafish7 and Atlantic cod6 revealed that distinctive protein profiles characterize the developmental stages of these fishes even though abundant proteins are largely conserved during the experimental period.77 Damodaran et al. Proteome analyses in developing organisms have shown that many . for example.71. reflect a response to biological perturbations or external stimuli9–11.93 This is reflected in the variations of global protein expression and posttranslational modifications of the proteins that may cause alterations in protein function.84 3. and adult) during their life span that coincide with changes in the morphology.82 Lin et al.79 Rappsilber et al.89 A brief discussion of a few emerging areas within fish and seafood proteomics is given as follows. improved reproducibility and resolving power of electrophoretic separation techniques.3.1 Development Fishes go through different developmental stages (embryo. which. In the peptide mass fingerprinting discussed earlier. for example.86 and soybean protein bodies. and development.3 Applications of 2DE in Seafood Analysis The two-dimensional electrophoresis has been in use within food science for at least two decades.23.. by Yates. cytoskeleton. yielding a second layer of information. In MS/MS one or several peptides are separated from the mixture and dissociated into fragments that are then subjected to a second round of mass spectrometry. fish physiology. proteomic investigations on fish and seafood products.88.. have gained considerable momentum. These variations may. way of obtaining protein identities is by direct sequence comparison. posttranslational modifications. In both these studies.90–92 The morphological and physiological changes that occur during these developmental stages are characterized by differential cellular and organelle functions. the method of choice is tandem mass spectrometry (MS/MS). the identified proteins consisted mainly of proteins located in the cytosol.72 Today.81 Gygi and Aebersold. Furthermore. or redistribution of specific proteins within cells. clearly defined subproteomes and included such applications as the characterization of bovine caseins. and nucleus.78 Thiede et al. Early studies focused on relatively small. 3. as well as in aquaculture. the higher the number of possible combinations.Proteomics ◾ 31 A more direct. several short stretches of amino acid sequence will be obtained for each peptide. each peptide mass can potentially represent any of a large number of possible amino acid sequence combinations..83 and Delahunty and Yates. when combined with the peptide and fragment masses obtained. The larger the mass (and longer the sequence). enhances the specificity of the method even further.

101 These studies demonstrated that the muscle shows the usual sequential synthesis of protein isoforms in the course of development.7 Despite this undertaking.111 3. specific isoforms of myofibrillar proteins.99.2 Quality Involution Degradation of proteins during chilled storage.2. and their oxidation during frozen storage. The major obstacle on the use of proteomics in embryonic fish has been the high proportion of yolk proteins.108 3.27 published a method to efficiently remove the yolk from large batches of embryos without losing cellular proteins. The success in the removal of yolk proteins by Link et al. can be observed using 2DE or other proteomic methods. many of which are correlated with specific textural properties in seafood products. the embryos were deyolked to enrich the pool of embryonic proteins and to minimize ions and lipids found in the yolk prior to 2D gel analysis. be distinguished on 2DE gels.27 is probably due to dechorionation prior to the deyolking of the embryos. and production of surimi and conserves occur under conditions conducive to endogenous proteolysis.109. In a recent study on the proteome of embryonic zebrafish. molecular mass. such as curing. fermentation. Thus. several commercially important fish muscle processing techniques.1 Protein Autolysis and Oxidation during Storage and Processing The specifics of fish muscle protein autolysis during storage and processing still remain in large part to be elucidated.110 Problems of this kind.8. Proteomic techniques have thus been shown to be applicable for investigating cellular and molecular mechanisms involved in the morphological and physiological changes that occur during fish development.99–107 In this context. are among persistent quality problems in the seafood industry and have deleterious effects on fish flesh texture. whether they be encoded in structural genes or brought about by posttranslational modification.21 and dorada (Brycon moorei). usually have different molecular weight or pI and can. By dechorionation.94. although degradation of myofibrillar proteins by calpains and cathepsins112. a large number of yolk proteins remained prominently present in the embryonic protein profiles.98 Different isoforms generated by posttranslational modifications are largely overlooked by studies based on RNA expression. It is also worth noting that protein isoforms other than proteolytic ones. therefore.88. the embryos fall out of their chorions facilitating the removal of the yolk. and pI of the protein present in a tissue. These interfere with any proteomic application that intends to target the cells of the embryo proper. Furthermore.21. Link et al.8.3.32 ◾ Handbook of Seafood and Seafood Products Analysis of the identified proteins have multiple isoforms96 that reflect either different gene products97 or posttranslationally modified forms of these proteins. in the common sole 2DE revealed two isoforms (larval and adult) of myosin light chain 2 and likewise in dorada larval and adult isoforms of troponin I were sequentially expressed during development.113 . This fact further indicates the importance of the proteome approach to understand cellular mechanisms that underlie fish development.21.3. where differences are expected to occur in the number. are well suited for investigation using 2DE-based proteomics. Studies on various proteins have shown that during fish development sequential synthesis of different isoforms appear successively. developmental stage specific muscle protein isoforms have gained a special attention.99–107 The developmental changes in the composition of muscle protein isoforms have been tracked by proteome analysis in African catfish (Heterobranchus longifilis).102 common sole (Solea solea). For example.

121 used a 2DE approach to demonstrate different protein composition of surimi made from prerigor versus postrigor cod and found that 2DE could distinguish between the two. 2D-immunoblots and LC–MS/MS to study changes in protein oxidation during frozen storage of rainbow trout. differences that can affect postmortem biochemical processes in the product which.2 Aquaculture and Antemortem Effects on Quality and Processability It is well known that an organism’s phenotype. such as those governing gaping tendency.117.. Whatever may be the mechanism.127.117 and. the interplay between these physiological parameters and environmental and dietary variables needs to be understood in detail.1 .115 are thought to be among the main culprits.120 and have demonstrated the importance and complexity of proteolysis and oxidative changes in seafood proteins during storage and processing.3. and the amount and composition of free amino acids in the fish flesh. Kjærsgård et al.17 used 2DE. furthermore. etc.123 found these to comprise several members of the glycolytic and Krebs cycle pathways.10. is determined by environmental as well as genetic factors.118 Several 2DE studies have been performed on postmortem changes in seafood flesh14–17. various quality characteristics of fillet and body were measured124.2. is thought to be responsible for a large fraction of cellular proteolysis. To achieve that goal. are optimized. in turn. the proteome analysis identified a number of metabolic pathways sensitive to plant protein substitution in rainbow trout feed.119. 67.112. With the ever increasing resolving power of molecular techniques. fatty acid breakdown. in mammals. Furthermore.15. it is clear.125 and the liver proteome was analyzed9. Olsson et al. Indeed. They found fish muscle proteins to be differentially carbonylated during frozen storage and were able to identify several carbonylated proteins using LC–MS/MS.126 in fish fed with the experimental diets.123 Both studies indicated that several proteins are differentially expressed in farmed versus wild cod. flesh softening during storage. as opposed to wild fish catching. Martinez et al.111. therefore raises the tantalizing prospect of managing quality characteristics of the fish flesh antemortem. this is fast becoming feasible. 3.44 The results led the authors to speculate that the difference in texture and postmortem amino acid-free pool development are affected by antemortem proteasome activity. where individual physiological characteristics. appear to display seasonal variations. In a recent study on the feasibility of substituting fish meal in rainbow trout diets with protein from plant sources. including quality characteristics. affect the involution of quality characteristics in the fish product. In the context of this chapter. The practice of rearing fish in aquaculture. The proteasome is a multisubunit enzyme complex that catalyzes proteolysis via the ATP-dependent ubiquitin–proteasome pathway which. and NADPH metabolism. the effects on the proteasome are particularly noteworthy. such as pathways involved in cellular protein degradation. We are aware of two recent studies where Atlantic cod muscle proteomes have been compared between farmed and wild fish. For example. the ubiquitin–proteasome pathway has been shown to be downregulated in response to starvation129 and have a role in regulating protein deposition efficiency. The diet was found to have a marked effect on product texture. that these quality changes are species dependent116.Proteomics ◾ 33 and degradation of the extracellular matrix by the matrix metalloproteases and matrix serine proteases114.128 In rainbow trout. Huss noted in his review122 that product quality differences within the same fish species can depend on feeding and rearing conditions. such as proteomics.

Proteome analysis can therefore potentially yield more information than genomic methods. demonstrating that it had arginine kinase . about 0. as different fish species have different market values. Mytilus galloprovincialis.5% of young adults are allergic to shrimp. These authors.18. The allergen was identified as a protein with close similarity to arginine kinase. possibly indicating freshness and tissue information in addition to species. Allergic reactions to seafood affect a significant part of the population. as well as being relevant from a public health standpoint. A final proof was obtained by purifying the protein. the proteome varies from tissue to tissue and with environmental conditions. 2DE-based methods have been developed to distinguish various closely related species.146 Proteome analysis can be a valuable tool for the identification and the characterization of allergens as exemplified by the study of Yu et al.143 Indeed.138. For example. this makes the issue of species authentication an area of increasing economic importance. 1D electrophoretic techniques were developed to identify the raw flesh of various species. During the 1960s. While DNA-based species identification130–132 and isotope distribution techniques for determining geographical origin133 are powerful tools in this area and likely to remain the methods of choice in the near term. Piñeiro and coworkers have found that Cape hake (Merluccius capensis) and European hake (Merluccius merluccius) can be distinguished on 2D gels from other closely related species by the presence of a particular protein spot identified as corresponding to nucleoside diphosphate kinase.3 Species Authentication Processed fish products are increasingly common in the market and. Martinez et al. The allergens were then identified by MALDITOF MS of tryptic digests. proteomic methods have been recognized as a potential way of fish species identification.141 More recently. blotted the 2D gel onto a PVDF membrane. They found the difference to be due to a single T to D amino acid substitution.147 at National Taiwan University.144 Martinez and Jakobsen Friis concluded that the identification of not only the species present. 3.34 ◾ Handbook of Seafood and Seafood Products Analysis 3.134 recently reviewed proteomic and other methods for species authentication in foodstuffs.140. such as the gadoids or several flat fishes.142. proteomics-based species identification methods are likely to develop rapidly and find commercial uses within this field. the proteomes of even closely related fish species are be easily distinguishable by eye from one another on 2D gels1 indicating that diagnostic protein spots may be used to distinguish closely related species. trossulus could be distinguished from the other two species on foot extract 2D gels by a difference in a tropomyosin spot.3. studying the cause of shrimp allergy in humans. but also their relative ratios in mixtures of several fish species and muscle types14 would become viable once a suitable number of markers have been identified. Penaeus monodon. studying three species of European mussels: Mytilus edulis. From early on. including structural proteins such as tropomyosin.143 Lopez and coworkers. found that M. and Mytilus trossulus.145 Seafood allergies are caused by an immunoglobulin E-mediated response to particular proteins.14 Unlike the genome. The identity was further corroborated by cloning and sequencing the relevant cDNA. performed a 2DE on crude protein extracts from the tiger prawn. the presence of stress-factors or contamination levels at the place of breeding.139 These early efforts were reviewed in 1980. particularly for addressing questions on the health status of the fish in question.135–137 which was soon followed by methods to identify species in processed or cooked products. and postmortem treatment.3.4 Allergen Identification Allergenic potential is food safety issue of particular concern to the seafood producer. and probed the membranes with serum from confirmed shrimp allergic patients.

L. Eds. 17–29. S. Kaushik. 921–926. Cash. and Kültz.. Proteomics 7(13). P. 2. 10.. Blackwell Publishing.. Proteomic identification of processes and pathways characteristic of osmoregulatory tissues in spiny dogfish shark (Squalus acanthias).. and Glover. 401–421. Vandewalle. Tay.. Focant. J. Mohanty. H.. F.. D. Paliyath. Comparative Biochemistry and Physiology B 133. Tan. 2004. and Houlihan. 2275–2286. T.. and Simpson. B.. the rainbow trout.. 6. M. Vilhelmsson. 69–75... 8. P. T. S. Kaushik. and Gudmundsdóttir. 2008. and Gong.. 4. 2003. Martin. C.. .. Hui.. Lin.. B. J. C.. J. S.. and Houlihan. 13. T. Comparative Biochemistry and Physiology D 1. induced skin reactions in sensitized shrimp allergic patients. C. S. 9. H. N. L. Protein microarrays and their applications. Peng... K. K. P. Acknowledgments This work was supported by grants from the Icelandic Graduate Research Fund. A. Seow. furthermore. and Nagamune.. Lee. Nollet. C. S. Blaney. 2007. Proteomic sensitivity to dietary manipulations in rainbow trout.. O. 269–278. P. N. 7. Cheng. S. Proteomics: Methodology and application in fish processing.. Médale. S. and Gygi. and the AVS Research Fund. 2003.-K. High resolution two-dimensional electrophoresis of proteins. 5. F. Houlihan. Médale. J.. D. F. Proteome analysis of abundant proteins in two age groups of early Atlantic cod (Gadus morhua) larvae. C. Martin. E... B. A. and Houlihan. K.. 328–343.. 71–80. S... Roelofs.. Danio rerio. Poli. 259–270. Nip. Vilhelmsson. 2006. and Secombes. M. G. British Journal of Nutrition 92. H.. P. 12. A. Balesaria. F. 2000. Dietary plant protein substitution affects hepatic metabolism in rainbow trout (Oncorhynchus mykiss). Martin. Cash. O’Farrell.. Proteomic analysis of protein profiles during early development of the zebrafish.. Finley.. D. Valkova.... Biotechnology and Bioprocess Engineering 9. 11. Application of genomics and proteomics for study of the integrated response to zinc exposure in a non-model fish species..Proteomics ◾ 35 activity and reacted to serum IgE from shrimp allergic patients and. 2001. W. 523–535. D.. Sveinsdóttir.. and Houlihan. in Food Biochemistry and Food Processing. A. M. L. Journal of Biological Chemistry 250. 2006. T. Martin. D. Vilhelmsson.. M. H.. Amsterdam. O. Á. 2006. M. Q. B. Proteomics 6. S. Basic and Applied Myology 10.. S.. Watt. References 1.. D. 243–250.. L... F. Schwartz.. D. F. Proteome analysis of the Atlantic salmon (Salmo salar) cell line SHK-1 following recombinant IFN-gamma stimulation. Marsischky. Y.. Expression of myofibrillar proteins and parvalbumin isoforms in white muscle of the developing turbot Scophthalmus maximus (Pisces. P. M.. 3176–3188.. 2002. Hogstrand. M. Thoreen. 2004. Hew. 1975. C. O. A proteomics approach to understanding protein ubiquitination.. and Huriaux. H. A. the University of Iceland Research Fund. P. Lee. D.. P. Martin. White. F. S. Nature Biotechnology 21. Collin. F. J.. the University Research Fund of Eimskipafélag Íslands. Biochimica et Biophysica Acta 1651. pp. Vilhelmsson. G. O. M. Pleuronectiformes). Proteome analysis of rainbow trout (Oncorhynchus mykiss) liver proteins during short term starvation. J.. 4007–4021. 3. Elias. T.. D. Comparative Biochemistry and Physiology D 3. B. Fish Physiology and Biochemistry 24.. Z.

2003. 2003. development. Garfin. K. 67–85. Comparative Biochemistry and Physiology B 135. Gibson. Kjærsgård. 2003.. Journal of Agricultural and Food Chemistry 54(25). G. G. 81–90. J. F. H. Háskólaútgáfan.. and Huriaux. J. N. R. A. . Oda. Link.. Kjærsgård. NJ. Link.. and Roberts. 2007. 18. 24. Journal of Agricultural and Food Chemistry 51. T. Enrichment analysis of phosphorylated proteins as a tool for probing the phosphoproteome.. 28. 9437–9446. and Mann. Proteomics 2. From genomics to proteomics. G. R. Barros-Velázquez.... C. Šližyté. and Jessen. T. Ahmed. Proteomics 3.. H. Martinez. Neville. 1995. Totowa.. J. Sotelo. 425–440.. 677–696... pp. Q. Ed. Corthals. Nørrelykke. 23. Yates. C. W. Nature 422.. Proteomics 6.. Y. Baron. C.. Rýnt í próteinmengi lirfa Atlantshafsþorsks (Gadus morhua). I.. 422–432.. Tyers. L..... Klose.. 26... Quinn. D. and Makowski. J. A. Identification of carbonylated protein in frozen rainbow trout (Oncrhynchus mykiss) fillets and development of protein oxidation during frozen storage. 2000. and Chait. Wu.. C.. R. 504–510. Haraldsson. Proteome analysis elucidating post-mortem changes in cod (Gadus morhua) muscle proteins. R. M. 2963–2975. G. and Steiner. H.. Subcellular shotgun proteomics in plants: Looking beyond the usual suspects. 1998. T. M. K. Traffic 1. V. and Jessen. Talmadge. Journal of Agricultural and Food Chemistry 46.. Pérez-Martín. 2005. Proteomics 4. M. Link. 2004. A. 2006. Journal of Chromatography B: Analytical Tools for Proteomics 849(1–2). M.. 2006. 17. T. and Vandenbussche. B. G. 3985–3991. G. L. G. 20. and van den Boogaart.. A. NJ. L. Ramsby. C. 39–50. and Heisenberg. 379–382. N. E. in Vísindin heilla: afmælisrit til heiðurs Sigmundi Guðbjarnasyni 75 ára. Twodimensional electrophoretic study of the water-soluble protein fraction in white muscle of gadoid fish species. 2001.. F.. Huet. 1999. I. allometric growth. pp. 53–66. C... Oliva. N.. Piñeiro.. and Rice. J. C... Baeyens-Volant. Journal of Chromatography B 815. J. Looze. T. and Sveinsdóttir. Nature Biotechnology 19. Proteomics 3. Gatlin. H. C.. Haynes. S. H. Y. ICES Marine Science Symposia 201. 1–9. H. J. Strategies for revealing lower abundance proteins in two-dimensional protein maps. 30. Fractionated extraction of total tissue proteins from mouse and human for 2-D electrophoresis. D. 2006. M.. 27. in 2-D Proteome Analysis Protocols.. U. Locke. and Daukšas. Vandewalle. Wellbrock. Humana Press. 19. I. M. J. 21... K. L. 1254–1260. M. P.. 493–502. Guðmundsdóttir. M. G. 2003. I. T. Huang. G. Schevchenko.. Differential detergent fractionation of eukaryotic cells. Reykjavík.. Ahmed. B. M. BMC Developmental Biology 6. the common sole Solea solea: Comparison with the turbot Scophthalmus maximus.. and Rylatt. 21–34. V. M. 2002. Su. 2003. Georgiou. 32. An approach to remove albumin for the proteomic analysis of low abundance biomarkers in human serum. Proteomics of early zebrafish embryos.. Gradiflow as a prefractionation tool for two-dimensional electrophoresis.. Proteomic analysis of two functional states of the Golgi complex in mammary epithelial cells. Expression of myofibrillar proteins and parvalbumin isoforms during the development of a flatfish... G. V. 33. M. 34. 347–354. V. Humana Press. 2007. M. 1980–1987. Martinez. Food Chemistry 102.. Affinity chromatography: A useful tool in proteomics studies.. and Jakobsen Friis. 25.. pp. S.. Nagasu.. 769–782... Á. Azarkan. and the aquatic environment... Multi-component immunoaffinity subtraction chromatography: An innovative step towards a comprehensive survey of the human plasma proteome. 3991–3997. M. 193–197. K. Pieper. Proteome analysis identifies novel protein candidates involved in regeneration of the cerebellum of teleost fish. P. J. Barker. E. T. B. 1999.. 16. 22. and Howell.. Ed. 31. and Zupanc. 2007. S. Anderson. Fish larvae. E. P.. Osse.. L. High resolution two-dimensional electrophoresis as a tool to differentiate wild from farmed cod (Gadus morhua) and to assess the protein composition of klipfish. 2006. and Rice. M. Zupanc. Ed. and Gallardo. S.-P. Focant.. F. D.. E. 15. in 2-D Proteome Analysis Protocols. Application of proteome analysis to seafood authentication. R. 29. Thomas. Proteomics 7.. Totowa.36 ◾ Handbook of Seafood and Seafood Products Analysis 14. G.

. 13–21. 49–70.. 57.. 50. Günther. and Lardeaux. A. Progress in Nucleic Acid Research and Molecular Biology 33. Proteomics 19. p. Jarzebinska. K. Application of proteomics to understand the molecular mechanisms behind meat quality. R. W. Görg. F. Electrophoresis 9. and Garin. Dreger. The Protein Protocols Handbook. and Krull.. Strahler. 2000. Electrophoresis 8. Sweden. 1994. Dobly. P. and Stetler-Stevenson. Y.. Current two-dimensional electrophoresis technology for proteomics. The current state of two-dimensional elelctrophoresis with immobilized pH gradients. Diabetes Metabolism Review 5. Weinheim. López-Ferrer. W. Walker. Weiss. and Dunn.. . G. Subcellular proteomics.. and Lakatta... J. J. X. Towards a human repertoire of monocytic lysosomal proteins.. 531–546. and Boschetti. A. and Veenstra. T. W. 49. 40. 865–873.. Efficiency of conversion of ingested proteins into growth. 3411–3419. Martin. and Gallardo. A. Chapel.. 1–31. A. E. Noga. and Kido. Totowa. Uppsala.. 2007. 56. M.. Righetti. 46. p. Mass Spectrometry Reviews 22. I. A. NJ.. A.. 47. Harder. 53. B. 1997. R. 51. 2007. Altered regulation of matrix metalloproteinase-2 in aortic remodeling during aging. Hanash. and Weiss. 43. Brown. Comparative Biochemistry and Physiology A 137. 2195–2224. 44. Prefractionation techniques in proteome analysis: The mining tools of the third millennium. Hershko. 2007. A. B. A. Journal of Chromatography B 849. Wang. Kieffer... 2-D proteome Analysis Protocols.. G. E.. S. 1993.. Electrophoresis 26... M.. 1998. Link.. and Hildrum.. Seiki.. Wildgruber. and Houlihan. O.. Cañas. 1026–1040. Journet. M. Kieffer. D. T. Unsworth. Mechanism and regulation of protein degradation in liver. Conrads. B. M. T. 54... 1999. Hollung. and Stenstedt. F. E. A. 19–56. M. J. G. Postel. Methods for fractionation. 2002. 2000. Pösö. Coe. D. K. Görg. Scheibe. Castagna. Roux. Subproteomics in analytical chemistry: Chromatographic fractionation techniques in the characterization of proteins and peptides. A.. T. Kleiner. 1988. Dylag. Electrophoresis 23.. Meat Science 77. 48.. P.. Electrophoresis 21. J. J. p. 2005. 2002.. S. T.. Electrophoresis 21. 181–184. W. 2002. J. H. E.. Obermaier. D. Trends in sample preparation for classical and second generation proteomics.. The ubiquitin-proteasome proteolytic pathway. G. 3048–3061. Færgestad. Cellular activation of the 72 kDa type IV procollagenase/TIMP-2 complex. FEBS Letters 402. H. 2002. S. 1989.. 45. C. Ciechanover. Proteomics in Practice. and Vilhelmsson. E.. 27–56. A. G. 37. and Silberring. E.. separation and profiling of proteins and peptides. 59. 2003. S. J.. Boguth. Methods for samples preparation in proteomic research.. 55. E. T. The current state of two-dimensional electrophoresis with immobilized pH gradients. D. P. A.. 41. J. Proteolytic activation of the precursor of membrane type 1 matrix metalloproteinase by human plasmin. Antonioli. M. 297–319. Kidney International 43. J. Berkelman.. 1986. L. and Somerlot. A. M. S. 58.. 601.. Chapel. Proteomic analysis of human lysosomes: application to monocytic and breast cancer cells. C. Oncorhynchus mykiss. Mortimore... G.. R. 75–85. 36.. Drabik. 97–104.. Wiley-VCH. S. 2002.. Amersham Biosciences.. Journal of Chromatography A 1153. The ubiquitin pathway for the degradation of intracellular proteins. 122–124. Westermeier.. A. 2004. Okumura. E. Janini. Issaq. Journal of Liquid Chromatography and Related Technologies 26. Hypertension 39.. M. 1987.. Suder. unpublished results. 3665–3685... Blaney.. 39. Journet. A. Louwagie. 1037–1053.. and Naven. 163–170. Luche.. Calvo... D. Cell 79. S. 2nd edn... Proteomics 2.. Bodzon-Kulakowska. Millea... P. R. I. M. and Gunther. and Garin. 235–258. M. M. J. Veiseth. 52. 1176. 38. Weser.. 42. Jia... protein degradation assessed by 20S proteasome activity in rainbow trout. and Ciechanover. A possible cell surface activator. P. A. S. 2003. M. Humana Press.. 2-D Electrophoresis Using Immobilized pH Gradients. H.. Görg. Humana Press. E. R. A. Görg. W. J.. Piñeiro. J. J. S... Principles and Methods.Proteomics ◾ 37 35. Totowa. Elimination of point streaking on silver stained two-dimensional gels by addition of iodoacetamide to the equilibration buffer.. K. M. A. NJ. 318. Bierczynska-Krysik. Postel. A. 2004. Sato..... A..

. Pappin. Link. 1999. J. 2006.. Ed.. J. 680–685... 70. Nature 227... M. Humana Press. 64. Characterizing proteins from 2-DE gels by internal sequence analysis of peptide fragments. pp. Courchesne. S. Global protein identification and quantification technology using two-dimensional liquid chromatography nanospray mass spectrometry. J. L. R. D. Y. M.. 2003... Perkins. Nature 379(6564). 221–227. P.. Link. J. L. Chelius. B. K.... 152–157. in 2-D Proteome Analysis Protocols. 245–253. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Y.. J. T. A. Karp..... NJ. J. 75... Breit. 3–31. M.. Link. R. H. Identification of proteins by matrix-assisted laser desorption/ ionization mass spectrometry using peptide and fragment ion masses. L. D. and Tsugita. 1999. Fotsis. L. D. Schweigerer.. P. H.. J. Z. J. V. R. M. A. and Rudemo. Wilm. D. Proteomics and Bioinformatics 5. NJ. 2005. R. W. Brophy. G. 2007. Gustafson.. Detection technologies in proteome analysis. Ceasar. Totowa.. Humana Press. A. H... Nagarajan. Mattow. Electrophoresis 20. S. M. Role of accurate measurement (+/− 10 ppm) in protein identification strategies employing MS or MS/MS and database searching. Biomolecular Engineering 18. T. Houthaeve. 237–247. Analysing proteomic data. W. M.. 487–511.. Nørrelykke. Expert Review of Proteomics 3(1). Wang.. and Herbert..... and Yang. International Journal of Mass Spectrometry 226.. Proteomic studies of macrophagederived foam cell from human U937 cell line using two-dimensional gel electrophoresis and tandem mass spectrometry. M. N. J. pp. Clauser. 63. T. 1606–1618. . 61. Blomberg. A. 1999. 1999. Ed. C. in 2-D Proteome Analysis Protocols. and Lilley. Ed. Wood. Wheelock. Changes in cod muscle proteins during frozen storage revealed by proteome analysis and multivariate data analysis. J. Schmid. pp. M. Nyman. Analytical Chemistry 71. 67... 461–466. 2005. Proteomics 5.. pp. 129–142. and Mann. and Jungblut. 65. P. A. International Journal for Parasitology 35. Experiences and perspectives of MALDI MS and MS/MS in proteomic research.. P... 66. 2001. M. L... Humana Press. Walsh. Proteomics 4.. B. R. Effects of post-electrophoretic analysis on variance in gel-based proteomics. and Tempst. Moniatte. in 2-D Proteome Analysis Protocols. Journal of Cardiovascular Pharmacology 42. Journal of Mass Spectrometry 33. Thiede. N-terminal amino acid sequencing of 2-DE spots. S. Totowa. 466–469. T. Nielsen. 62.. Barrett. V. Shevchenko. K. Totowa. U. Journal of Chromatography B 771. F.. R. T. I. Y. Totowa. Baker. 1999. 2871–2882. R. Probability-based protein identification by searching sequence databases using mass spectrometry data.. 78.. R... B. Patton. Analytical Chemistry 75. Yang... M... 467–472. J. A. P. and Jessen. 71. S. Casting and running vertical slab-gel electrophoresis for 2D-PAGE. Yu.. C. 69. and Shen.. A. P. Lacomis. Application of partial least squared discriminant analysis to two-dimensional difference gel studies in expression proteomics. A. A.. 80.. 223–237. S. A. 77.. and Burlingame. and Patterson. 81–90. 1999. Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry. E. 3791–3799. Ed. 1996. M. Proteomics 6(5). Y. F. Yates. 76. NJ. and Cottrell. L. Evaluating peptide mass fingerprinting-based protein identification.. 1970. Peptide mass fingerprinting. K. and Mann. 2006. Statistical exploration of variation in quantitative two-dimensional gel electrophoresis data. Huang.. 74. 3551–3567. 782–789. Mass spectrometry and the age of the proteome.. 68. Zhang.. L.. Podtelejnikov. Rui. in 2-D Proteome Analysis Protocols. S. Hohenwarter. V. 2002. S. M. Schmidt. M. and Hamilton. C. Griffin. F. Krah.. 2003. P. NJ. 73.. 6658–6665. 2005. 2003. Creasy. 2004.. Lui. Link. Kjærsgård. Genomics. A. A. R. Glasbey. D. D. Y. 1–19. A. Methods 35. Damodaran. Rappsilber. P. J. J. J.. and Goto. A. A. Kamo... 543–553.. The role of mass spectrometry in proteome studies. N.. Erdjument-Bromage. Humana Press.38 ◾ Handbook of Seafood and Seafood Products Analysis 60. and Rabin. 79. 1998. 72. Laemmli.

1987. Silva. M.. A. 90. 82. 404–415.. Figueras. Skiftesvik. Baras. Huriaux.. F. High-resolution two-dimensional electrophoresis of bovine caseins. 1091–1097. L... B. Morin. J. Zeece. A. A. Baras. Legendre. D. 2000.. and Focant. Fish Physiology and Biochemistry 21. L. 2005. G. D. N. C. Parrington. M.. S. Huriaux. Piñeiro. and Gallardo... A. O. and Focant. 1996. R. Vandewalle. E. 2003. 1998. 2006. 666–675. Anderson. Wehling.. Journal of Fish Biology 62.. Óskarsson.Proteomics ◾ 39 81. and Power. L. and Bush. and Craik... 1999. E. Jensen.. Govoni. 86.. P. and Focant. G. 378–383.. Gygi. S. 774–792. 1–10. new concepts. L.... The physiology of digestion in fish larvae. Vandewalle. Bjarnason. N. and del Maso. B.. M. M. J. 89.. K. 296–300. M. Changes in behaviour at onset of exogenous feeding in marine fish larvae. Proteome and proteomics: New technologies. W. F. E. Parvalbumin isotypes in white muscle from three teleost fish: Characterization and their expression during development. J. W. Isolation and characterization of cDNAs from Atlantic cod encoding two different forms of trypsinogen. Wehling.. Myofibrillar proteins in white muscle of the developing catfish Heterobranchus longifilis (Siluriforms.. C. Methods 35. Clariidae). Lin.. 4751–4767. N. Anatomy and Embryology 211.. M. Development of organ systems in the northern anchovy Engraulis mordax and other teleosts. L. 193–196. Holt. M. Guðmundsdóttir.. Eakin. 1990. Delahunty. B. P. Aquatic Living Resources 15. 92. P. R. 475–484.. G.. Journal of Agricultural and Food Chemistry 35. L. T. 2002. 2006.. I. and Coward. 1992. B. Zeece. 429–446. Collin. M. Journal of Proteome Research 2. Canadian Journal of Fisheries and Aquatic Sciences 49. Sweeney. 2003. G. Melot. Analytical aspects of mass spectrometry and proteomics. Large-scale protein identification using mass spectrometry... 83. R. J. Smáradóttir. 94. C.. M. 1981. Journal of Agricultural and Food Chemistry 37. 2002.. American Zoologist 21. D. R. P. K. . B.. Boehlert. B. 96. Thyroid and pituitary gland development from hatching through metamorphosis of a teleost flatfish.. Dougherty.... Cereal Chemistry 67. Campinho. 2006. C. and Aebersold. E. 248–255. 88. D. Vandewalle. Biochimica et Biophysica Acta 1646. J. 95... J. 2004. Comparative Biochemistry and Physiology D 1. 47–60. 93.. and Yates.. and Partridge. Mo.. 2003.. Comparative Biochemistry and Physiology B 113. 84. 100. and Anderson.. and Björnsson.. and Yates. 1986. P. Evaluation of selected baking quality factors of hard red winter wheat flours by two-dimensional electrophoresis. 97. Environmental Biology of Fishes 16. J... Lei. Current Opinion in Chemical Biology 6. Y.. Vázquez. Liewen. J.. Journal of Experimental Biology 209.. L. 85. 1570–1572... R. Current Opinion in Chemical Biology 4. R. Paz. 1993. G. Barros-Velázquez. A. B. F... 1989. 564–569. 91. European Journal of Biochemistry 217. Huriaux. 1853–1861. S... Modification-specific proteomics: Characterization of post-translational modifications by mass spectrometry. Expression of myofibrillar proteins and parvalbumin isoforms in white muscle of dorada during development. 489–494. G. Current Opinion in Chemical Biology 8. Mass spectrometry and proteomics. 98. Use of emerging genomic and proteomic technologies in fish physiology. E. G. H. S. Á. Two dimensional electrophoretic analysis of isolated soybean protein bodies and of the glycosylation of soybean proteins. 59–77. J. and Karger. M. O’Connell. 99. Proteomics as a tool for the investigation of seafood and other marine products. and new words. D. and Reeck. E. Power. Einarsdóttir. the Atlantic halibut. Electrophoresis 19. Guðmundsdóttir. 101. 287–301.. 87. Regulation of troponin T expression during muscle development in sea bream Sparus auratus Linnaeus: The potential role of thyroid hormones. 33–41. J. Tabb. and Watanabe. R. J.. F. B. D. Proteome profile changes during mouse testis development. 127–135. M. Protein identification using 2D-LC-MS/MS..

. Bogason. H. G. T. K. 109. N. Comparative Biochemistry and Physiology B 125... Roldán. H. Collin. 499–504. Melot..) muscle: Polymorphism and biochemical studies. 1031–1036.. and Johnston. 1998. 832–851. Papa. Kjørsvik.) embryos incubated at different temperatures: Transient asymmetric expression of MyoD. Food Chemistry 77. 1999... 117. I. M. Thorarinsdottir.. Verrez-Bagnis.. and Ishihara. J. S. Alvarez.. Post mortem release of fish white muscle a-actinin as a marker of disorganisation. Y. 110. J. 2000. Bardal. Chaplet.. E. Cell and Tissue Research 327. myogenin and myosin in Atlantic halibut (Hippoglossus hippoglossus L.. A. V. 111.. and Fleurence. F. 108. K. 267–284.. Journal of Experimental Biology 206. 240–242. 119. 113. Journal of the Science of Food and Agriculture 72. 106... and Huriaux. 104. Noël.. A Guide for the Laboratory Use of Zebrafish (Danio rerio).. Use of two-dimensional electrophoresis to evaluate proteolysis in salmon (Salmo salar) muscle as affected by a lactic fermentation. 3187–3200.. Noël.. Kvam. 2006. and Kryvi... 112... Westerfield. Roura. 2432–2441. Proteolytic degradation of myofibrillar components by carp cathepsin L.and post-spawning hake (Merluccius hubbsi Marini) after frozen storage.... 63–70. Sautereau. 2003. W. Journal of the Science of Food and Agriculture 83. Molecular. F. FASEB Journal 5.. Journal of Fish Biology 55. M... Woessner.. E.. . S. I.. V. F. 115. G. Somite formation and expression of MyoD. Morzel. T. and Anderson. J... Dahle. 114. C. Kjørsvik. Matrix metalloproteases and their inhibitors in connective-tissue remodelling. Gelatinolytic activities in muscle of Atlantic cod (Gadus morhua). Martinez. 103. Journal of Food Science 64. Journal of Agricultural and Food Chemistry 48. S. Journal of Experimental Biology 209. 2002. K. Electrophoretic study of myosin isoforms in white muscles of some teleost fishes. Chikou. Verrez-Bagnis.. Journal of Aquatic Food Product Technology 7. 325–330.. I. 107. Muscle growth in yolk sac larvae of the Atlantic halibut as influenced by temperature in the egg and yolk sac stage. Montecchia. 377–385. 1998.. 2007. 118. H. R. and Fleurence. T. A.... S.. M. Comparative Biochemistry and Physiology B 96. Galloway. Arason. E. 1999. C.. M. E. I. Ingólfsdóttir. and Olsen. 2002.. Effect of temperature on viability and axial muscle development in embryos and yolk sac larvae of the Northeast Atlantic cod (Gadus morhua). spotted wolffish (Anarhichas minor) and Atlantic salmon (Salmo salar). Sweeney. B.. 105. Eugene. and Crupkin. N. T. 1990. and Kristbergsson.40 ◾ Handbook of Seafood and Seafood Products Analysis 102. 1991. Vandewalle. J. and Kristbergsson.... B. L. J. Verrez-Bagnis. E. 1999. A. S. Lødemel. M. and Kryvi. Neutral calcium-activated proteases from European sea bass (Dicentrachus labrax L. K. Verrez-Bagnis. Proteolytic activity of muscle in pre.. G. Aranishi. 2145–2154.. Campinho.. Galloway.. O. K. 239–244. cellular and histological changes in skin from a larval to an adult phenotype during bony fish metamorphosis. I. and Power. 26–43. and expression during development. H. Journal of Fish Biology 54. and Olsen. 2000... D. F. Heterobranchus longifilis and Chrysichthys auratus: Isolation. Hall.. Pérez-Borla. P. J. M. Nesse. J. Changes in myofibrillar proteins during processing of salted cod (Gadus morhua) as determined by electrophoresis and differential scanning calorimetry. K. Stefánsson. G. Ofstad. H. and Benyamin.. J. V. Muscle parvalbumin isoforms of Clarias gariepinus. 4th edn.. Desmin degradation in postmortem fi sh muscle. M. Kjørsvik. Arendt. Fleurence. M.. A. Hara.. Temperature and the expression of seven muscle-specific protein genes during embryogenesis in the Atlantic cod Gadus morhua L.. C.. R. T. R. 1998. Galloway. Osatomi.. 39–61.. Ogata. Focant.. 2003. Seasonal variations in physicochemical and textural properties of North Atlantic cod (Gadus morhua) mince. characterization.. L. Geirsdottir. S. T.. 116. 83–95. F. Cole. 221–227.. Randøl. C. Ladrat. N. V. Silva. 547–557. F. Marine Biology 132. University of Ohio Press. Lebensmittelwissenschaft und -Technologie 35. 2000. 1996. The Zebrafish Book. and Fleurence.. Ø. F.. Journal of the Science of Food and Agriculture 76. J. S.

Aquaculture 236. 1995. M.. Rey-Mendez.. and Cooper.. F. I. 1968. P. A. 10850–10855.. R. D. Fish species identification in seafood products.. 2001. Eds. Cell 78. S.. M. Journal of the Fisheries Research Board of Canada 23. Electrophoresis 22. Stein. I. and Poli. H.. M.. Parisi. L. M. Martinez. and Poli. Salvelinus namaycush. W.. 132.. M. Souffrant.. R. H... Hwang. .. Two distinct proteolytic processes in the generation of a major histocompatibility complex class I-presented peptide. J.. and Seppola. Identification of fish species by a modified polyacrylamide disc electrophoresis technique. 257–66. and Pérez-Martín. S. K.. Blaney.. Effect of long-term feeding with a plant protein mixture based diet on growth and body/fillet quality traits of large rainbow trout (Oncorhynchus mykiss). pp.. F. Mackie. S. R. and Houlihan. C. 125. C. Veliyulin.. Dick... Identification of fish species by thin slab polyacrylamide gel electrophoresis. E. I.. Quinteiro. S. C. Clark. Metabolic disorders in muscle of farmed Atlantic cod (Gadus morhua). Bowman. and Rock. S. Aursand. T. Martinez. C.. W.. Gonzales-Sotelo. P.. D. 126. S. S. 1199–1208. malma and S. G. and van den Zwaag. L... 201–206. S.. W.. 413–429. C. B. S. FAO. Destructive and non-destructive analytical techniques for authentication and composition analyses of foodstuffs. I. 2004. Pryde. Ubiquitin-proteasome-dependent proteolysis in rainbow trout (Oncorhynchus mykiss): Effect of food deprivation. Two-dimensional electrophoretic analyses of cod (Gadus morhua L. Journal of the Science of Food and Agriculture 19. The Netherlands. Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class I molecules. 30–38. Vilhelmsson. Veterinary Research Communications 28.. Trends in Food Science and Technology 4. Journal of the Association of Public Analysts 5. and Thorrold. Mackie. Jakobsen Friis. and Rehbein.. Huss.. Comparative electropherograms of Coregonis clupeoformis. 127. 2002. Sotelo. T.. 136. Kaushik.. L. 2001.. 489–498. C. De Francesco. 129. 1992. 2003.. fontinalis from the family Salmonidae. De Francesco. E.. Pérez-Martín..) whole muscle proteins. Campana. Trends in Food Science and Technology 14. J. 137. 2004. A. 135. in Progress in Research on Energy and Protein Metabolism. 130. M. Martin.. 226–229.. 1994. E. 133. Jensen.. and Goldberg.. alpinus. Rock. Applied and Theoretical Electrophoresis 2.. Trends in Food Science and Technology 10.. M. C. A. Solberg. K. 2007. 1969.. 122. S. M.. Scappini. 2001. F. Martin. Rome. M. Médale. M. A. F. I. Wageningen.. T. Wageningen Academic Publishers. H. I. Quality and Quality Changes in Fresh Fish. Parisi.. A. L. Effect of total replacement of dietary fish meal by plant protein sources on early post mortem changes in the biochemical and physical parameters of rainbow trout. M. J. 121.. C. J.. R... Jakobsen Friis. Gramm. M. Mecatti. T. D. Rainbow trout liver proteome—Dietary manipulation and protein metabolism. G. and Careche. 1599–1606. Erikson. Singstad. and Houlihan. Challenges in the identification of species of canned fish. L. F. E..Proteomics ◾ 41 120. B. Lupi. 395–401. 2003.. and Ofstad. M.. 123.. increments.. G. 134.. M. Otoliths. Piñeiro. K. an elements: Keys to a comprehensive understanding of fish populations? Canadian Journal of Fisheries and Aquatic Sciences 58. Craiu. Gallardo. 1966. H. and Metges. Kaushik. T. Uthe. Roberts. Martinez. G. Lauritzen.. Martinez. 237–240. 9–14.. S. Effect of the addition of CaCl 2 and MgCl2 during the washing procedure. O. Post mortem muscle protein degradation during ice-storage of Arctic (Pandalus borealis) and tropical (Penaeus japonicus and Penaeus monodon) shrimps: A comparative electrophoretic and immunological study. 1999. 1997. Pflügers Archives European Journal of Physiology 445 (2). Rothstein. R. I.. 131. Olsson. A. B. Proceedings of the National Academy of Science USA 94. E.. 57–60. 1223–1227.. Friis. water soluble fraction and surimi. 83–87. Médale... B. Requirements for the application of protein sodium dedecyl sulfate-polyacrylamide gel electrophoresis and randomly amplified polymorphic DNA analyses to product speciation.. C.. 1993.. 124. Aquaculture Research 38. 1526–1533. F. I. Tsuyuki. 128. 761–771. Akopian.. Cowie.. J. U. Goldberg. Medina... and Clarke. E. Journal of the Science of Food and Agriculture 81... L. J.

1972. C.-F. C. 139. L. S.. and Vazquez.. Lehrer. 609–611. Raven.K. Seafood allergy and allergens: A review. J.. and eczema. H. 2002. 141. Alvarez. Proteomics and immunological analysis of a novel shrimp allergen. J. 2002. Fishing News Books Ltd. Marina. Yu. K. Thien. Application of proteomics for fast identification of species-specific peptides from marine species... J.. 1980. G. Identification of species of heat-sterilized canned fish by polyacrylamide disc electrophoresis. Piñeiro. Electrophoresis 22. Fishing News Books Ltd.. Y. Chiang...... Some improvements in the polyacrylamide disc electrophoretic method of identifying species of cooked fish. Ed. 143.. 145.. M.. J... Piñeiro. 142.. 1545–1552. The use of electrophoresis of the water-soluble muscle proteins in the quantitative analysis of the species components of a fish mince mixture. 2003. . U. Sotelo.. Hume.-J. Connell... J. Woods. A. R. I.. I.. M. and Gallardo. M... U. 2003. B. Ayuso. A. Marine Biotechnology 5.. J.42 ◾ Handbook of Seafood and Seafood Products Analysis 138. Lin. 140.. J. and Mackie. L. and Taylor. Walters.. 1972.. Aberdeen. Vázquez. 18–20. and Abramson. Annals of Allergy Asthma and Immunology 88. Mackie. 2001. The use of two-dimensional electrophoresis for the identification of commercial flat fish species. Analyst 97. I.. I.. and Chow.. pen m 2. Ed... 183–189. in Advances in Fish Science and Technology.K. Barros-Velázquez. 146..-L. 1658–1665. A. R. Journal of the Association of Public Analysts 8. 445–453. J. Proteomics 2. and Gallardo. Prevalence of food allergies in young adults and their relationship to asthma. J. C.. Mackie. Aberdeen. 147. T. Zeitschrift für Lebensmitteluntersuchung und -forschung 208. E.-P. Marina. 339–348. F. in Advances in Fish Science and Technology. Mackie. B. J. Connell. nasal allergies.. 144. 342–348. Lopez. G. The Journal of Immunology 170. A review of some recent applications of electrophoresis and isoelectric focusing in the identification of species of fish in fish and fish products. M. 1999. J. I. C. Characterization and partial sequencing of species-specific sarcoplasmic polypeptides from commercial hake species by mass spectrometry following two-dimensional electrophoresis. and Reese. M. G. Barros-Velázquez. 1980. J.

..................................... 43 ...........................................* Delphine Galiana-Arnoux.................52 There the nets brought up beautiful specimens of fish: Some with azure fins and tails like gold.....1 Introduction ...............4 Genomics.............................. others....................................................... Jules Verne...... 49 4................... all fish that would be of use to us......................................2 Genetics and Genomics................... but of exquisite flavour....... which has revolutionized biology............... the flesh of which is unrivalled...6 Concluding Remarks ................................. and Jean-Nicolas Volff Contents 4....................................47 4.................................................................................5 Genomics and Aquaculture . and biotechnology over the last decade....... as good as bonitos..................................................45 4....* Christina Schultheis.............................1 Introduction The development of high-throughput DNA sequencing methods has opened the era of genomics........................ Fisheries........... some nearly destitute of scales...51 Acknowledgments ................. 43 4. medicine..........................52 References .................... 44 4........Chapter 4 Seafood Genomics Astrid Böhne.... with bony jaws..... The rise of * Equal contributors.................................. and yellow-tinged gills.......................* Frédéric Brunet...........3 Genomic Resources and Genome Projects for Aquatic Species ............................. and the Management of Biodiversity .. Twenty Thousand Leagues under the Sea 4..............

called genetic mapping. comparative mapping provides important information on the structure and evolution of genomes in different species. which are reviewed in this chapter. which are carried by chromosomes. such as restriction fragment length polymorphisms (RFLPs. in population genetics. providing an estimation of their localization in the genome. Molecular markers are not only useful for genome mapping but also represent important tools in other domains. generally orthologous sequences [3]. that is. increasingly used for phylogenetic reconstructions [4]. In the field of biotechnology. genomics is principally used to identify molecular markers. sequenced. DNA fragments are amplified enzymatically using primers matching both adaptor and restriction site. and therefore of their linkage. 4. Gene regulatory and coding sequences are then predicted through bioinformatic analysis involving sequence prediction and database comparisons. polymorphic insertions of retrotransposable elements. Genetic markers must be polymorphic to allow the analysis of their segregation. and subsequently assembled in “contigs” in silico. arrangement. The development of efficient methods in bioinformatics is a condition sine qua non for progresses in the field of genomics. Finally. Most genes are located in the nucleus. This generates a genetic linkage map. There are different but complementary ways to analyze genomes. The science dealing with the analysis of genomes as a whole is called genomics. In order to investigate gene content. Genomics has important applications for fisheries and aquaculture [1]. Random amplified polymorphic DNA (RAPD) markers are amplified enzymatically by polymerase chain reaction (PCR) using short arbitrary oligonucleotide primers. Since SNPs can occur not only in noncoding but also in coding sequences. Different types of DNA markers are used for mapping. genes and alleles of zootechnical interest for the genetic improvement of economically important species. which themselves constitute the genome. they are likely to be less neutral than other markers from the functional point of view.2 Genetics and Genomics Genetics can be defined as the science of heredity and variation in organisms. Heredity is based on genes. caused by sequence polymorphisms at restriction sites) [2]. Other important markers are single nucleotide polymorphisms (SNPs). the latter being of wide use in genotyping and mapping experiments. . and structure. as done for the human genome [6. which have genomes with very diverse transposable elements [5]. with randomly sheared pieces of DNA massively cloned. Traditionally. Amplified fragment length polymorphism (AFLP) markers combine the principle of RFLP with PCR: fragments cut with restriction enzymes are ligated with adaptors. DNA markers with a polymorphic number of tandem repeats are called minisatellites (repeat units up to 25 bp in length) and microsatellites (shorter repeat units. for example. In addition. SNP analysis can therefore uncover genes and residues that are targeted by evolution and lead to the identification of disease-associated genes. one nucleotide differences within otherwise identical. consists in delineating intervals on the genome with genetic markers. Genetic loci and genes of interest can then be mapped relative to these markers.7]. usually dinucleotides or tetranucleotides). and evolution. Massive analysis of functional gene variability in many organisms has allowed to better understand the molecular basis of biodiversity and disease. and to contribute to the management of biodiversity. Such markers might be further developed in fish. genomes are sequenced using the “shotgun” strategy. function. but organelles (mitochondria and chloroplasts) have their own genome too. can also be used for mapping purposes. One of them.44 ◾ Handbook of Seafood and Seafood Products Analysis genomics has generated an impressive wave of novel information concerning genome structure. with the distance between markers being directly proportional to the frequency of recombination between them. nuclear and organelle genomes can be sequenced to (almost) completion.

This provides a physical map respecting the “real” base pair distance between genes and markers. hereby contributing to species conservation and management of global fish biodiversity (http://www. 4. For example. The relative position of two contigs can also be estimated cytogenetically using double fluorescent in situ hybridization [8]. zebrafish and medaka are two complementary fish models to study . Parts of the genome. Such an approach is. for SNP detection and phylogenetic reconstructions. which can be very useful to precisely determine the relative position of sequence contigs assembled “in silico” from whole genome shotgun sequencing data. A method called “DNA barcoding” should help to identify species and phylogenetic units. These fragments are either integrated in the genome of a host cell line from a different organism in radiation hybrid (RH) mapping [9] or diluted to give aliquots containing approximately one haploid genome equivalent (HAPPY mapping [10]). useful in the case of regions rich in repetitive sequences posing problems to assembly after whole genome shotgun sequencing. a new revolution of large-scale sequencing is ushering in a second era of genomics. with novel methods allowing very rapid and much cheaper sequencing of large amounts of DNA [11–13]. Additional approaches are required to study gene expression (transcriptomics. see Ref. for instance. Of particular interest are expressed sequence tags (ESTs).. shotgun sequencing.Seafood Genomics ◾ 45 A clone-by-clone approach can be used as an alternative to. EST analysis not only provides important data on genes expressed in particular tissues/ organs or at specific stages of development but also allows the characterization of gene structure through comparison with genomic sequences. aquatic model organisms of insignificant importance such as seafood have been developed for other scientific purposes and have been targeted for whole genome-sequencing projects [17]. The overlapping between these clones and their relative arrangement in the genome can be determined through fingerprint analysis (e. Bacterial artificial chromosomes (BACs) accepting inserts from several hundreds of kilobases are frequently used as vectors. Physical maps can also be constructed by analyzing the segregation of genomics markers (also called STSs for sequence-tagged sites) in randomly fragmented parts of the genome. Importantly. proteomics) and function (functional genomics) as well as interactions with the environment (environmental genomics). for example. or even better in combination with. Sequence data can be used among others to identify similarities and differences between species and study genome evolution (comparative genomics [14]) or to infer reliable phylogenetic relationships between organisms (molecular phylogenetics and phylogenomics [15]).3 Genomic Resources and Genome Projects for Aquatic Species Genetic and genomic resources have been generated for many aquatic species of economical interest. Barcoding is based on a sequence of short standard parts of the genome.fishbol. obtained through sequencing of complementary DNA (cDNA) libraries. [16]). Generally. In addition. through the identification of common restriction fragments). cloned in a bacterial vector and constituting a so-called genomic library. can be sequenced either to completion or from their ends.g.org/). Large-scale expression studies at the transcriptional level are generally performed using microarrays or other methods of high-throughput expression profiling. a 650 bp fragment of the 5′ end of the mitochondrial gene cytochrome c oxidase I is used as a global standard in fish and other animals (for review. Probes specific to each contig marked with different fluorochromes are cohybridized on chromosome preparations to test if they are located on the same or on different chromosomes. ESTs can also be used.

Particularly.ensembl. and hagfish. physical maps are available for species such as Nile tilapia.org/Danio_rerio/).ncbi. assignment of linkage groups to specific chromosomes has been performed through fluorescent in situ hybridization [29]. and others) (for review. Atlantic salmon genome should be sequenced soon.46 ◾ Handbook of Seafood and Seafood Products Analysis vertebrate development [18]. possibly followed by the genome of the rainbow trout. has been sequenced [41]. and gene content of fish genomes. see Refs. evolution. including the amphipod . shrimp. and other salmonids.org/research/skategenome. [1.org/Gasterosteus_ aculeatus/). and channel catfish [22–28]. the three-spined stickleback Gasterosteus aculeatus (http://www. these sequencing projects have provided valuable general information on the structure. which occupy strategic taxonomic positions within and relative to vertebrates (http://www. the purple sea urchin Strongylocentrotus purpuratus. (http://www. providing useful information on gene sequence and expression in different tissues and organs or at different stages of development (http://www. Both species have an extremely compact genome with low repeat content and short intronic and intergenic sequences and have been useful to identify conserved genes and noncoding sequences in the human genome [36]. genomic studies on aquatic species are relatively recent. For some species like the rainbow trout. for the Atlantic cod (Cod Genomics and Broodstock Development Project. For cartilaginous fish. the sequencing of the genome of other crustaceans is planned. shrimp.gov/10002154). and others) and invertebrates (oyster. http:// www. for example. Aquatic invertebrate species with well-developed EST resources include scallop and oyster (mollusks) as well as blue/green crabs. Beside the genome of the zooplankton Daphnia pulex (water flea.fugu-sg. rainbow trout. A genome project is in the pipeline for another cartilaginous fish. gar. Further projects aim to sequence the genome of coelacanth. http://wfleabase. Most genome drafts available so far are for aquatic model species without any real economic importance (for review. sea bass. A variety of genomic libraries. carp. see Ref.20]. Atlantic salmon.ca/grasp/). For Atlantic salmon and other salmonids.imcb. which is relatively compact. [17]).nlm. The genome of an echinoderm. Expressed sequence tags are also available for many fish species. Other species with advanced or completed genome projects include the medaka Oryzias latipes [37. abalone.a-star. an aquaculture species of high economical value. org/).ca/index. and lobster (crustaceans). mussel. skate. Compared with agricultural plants and terrestrial livestock. http:// codgene. Other projects aim to enhance genomic resources for economically important species. including fish (sea bream.mdibl. Atlantic salmon. such as the high diversity of transposable elements and presence of numerous duplicated genes that are remnants of an ancestral whole genome duplication [30–33].gov/10002154).40]. but many other genomic resources have been developed.gov/dbEST/). they have revealed some evolutionary peculiarities possibly linked to biodiversity.genome. SNPs and other polymorphic markers as well as linkage maps have now been generated for many aquaculture species.21]). particularly by the Genomics Research on All Salmon Project consortium (cGRASP) (http://web.uvic. Fishes with sequenced genomes include the pufferfish species Takifugu rubripes ([34].38]. However. particularly BAC libraries.org/) and Tetraodon nigroviridis [35]. the little skate Leucoraja erinacea.edu. the genome of the elephant shark Callorhinchus milii. and the zebrafish Danio rerio (http://www. scallop. in association with low-coverage sequencing projects for three additional cichlids (http://www.nih. has been sequenced at low coverage [39. lamprey. no draft genome is available now. A genomesequencing project is underway for the tilapia Oreochromis niloticus. tilapia.php). as well as RH panels and cDNA microarrays have been constructed for aquatic organisms.shtml).sg/). Japanese flounder. sea urchin.ensembl. catfish.genome. http://esharkgenome. These models are nevertheless useful to decipher gene content in species targeted by fisheries and aquaculture through comparative genomics [19.

50]. the loss of marine biodiversity impairs the ability of ocean to provide food. and brown algae). with a major role for genomics. reproductive structure and behavior.home. Genetic monitoring. Nuclear and mitochondrial molecular markers can be used to identify units of management for fisheries and priorities for the conservation of biodiversity.gov/sequencing). leading to a reduction of fisheries’ yield [49. the diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum.jgi. Important demographic and evolutionary parameters to be considered include organism abundance and vital rates.metazome. Fisheries. fisheries targeting large individuals will select for early maturation at smaller sizes. and the Management of Biodiversity Many aquatic populations have been overexploited through overfishing or collapsed and even become extinct through other factors such as pollution. population structure and interactions. site occupancy. the marine picoeukaryote Ostreococcus tauri. the green algae or chlorophytes Chlamydomonas reinhardtii and Volvox carteri. Consequently.html).gov/10002154) as well as the genome of the Atlantic horseshoe crab (chelicerate) (http://www.4 Genomics.kazusa. and the definition of conservation units and priorities for sustainable fishery management. the quantification of temporal changes in populations using molecular markers. micro/minisatellites. and perturbations of ocean biogeochemistry [44–47]. as well as with a decrease in water quality. that is. Population genetics is determined using various polymorphic genetic markers. and conservation of biodiversity of aquatic organisms are now high priorities. with their particular adaptations and contributions to biodiversity. including mitochondrial DNA polymorphisms.jp/en/plant/porphyra/EST/). Organelle genome sequences and EST resources are available for many algal species. for example. Genome drafts have been generated for the red alga Cyanidioschyzon merolae. Characterization of minimum viable population size is required to assess if they are facing a risk of extinction [45]. and the haptophyte Emiliania huxleyi (for review. AFLP and . http://genome. [43]).or. see Ref. and hybridization. can be considered as conservation units [52]. exploitation can act as a selective pressure and induce phenotypical shifts as evolutionary responses. the Pacific oyster [42]. For example. In addition.net/. monitoring. Genome sequencing should follow for many other aquatic animal species of economical interest. description. is used as food by coastal populations. climate change. Harvesting and other forms of stress can cause strong alterations in population structure as well as a reduction in biodiversity. for the red alga Porphyra yezoensis (http://est. for example.org/Nemve1/Nemve1. green algae. pedigrees and social structure. and to recover from perturbations [48]. and invasion of disease and invasive species [51.doe. 4. Seaweed.jgi-psf. habitat degradation and loss. introduction of exogenous species. Biodiversity decline is associated with a collapse of seafood resource and a reduction in species stability and recovery potential.Seafood Genomics ◾ 47 crustacean Jassa slatteryi (http://www. it has been predicted that all commercial fish and seafood species will have done so by 2048 [48]. provides information relevant to both the ecological and evolutionary time frame [51]. About 30% of seafood stocks available in 1950 have already collapsed. particularly in East Asia. Restoration of biodiversity increases fisheries productivity.genome. to maintain water quality. particularly in the assessment and follow-up of biodiversity in wild stocks. Hence.52]. gene flow. the estimation of fisheries-induced evolution. constituted by several groups of multicellular algae (red algae. Populations and ecosystems. Genome projects are performed for the cnidarian species Hydra magnipapillata (green hydra) and Nematostella vectensis (sea anemone) (http://hydrazome.

[1]).48 ◾ Handbook of Seafood and Seafood Products Analysis RAPD markers. Population genomics is a form of population genetics extending the analysis of genetic variation in natural populations to the scale of the genome itself. species-rich groups such as the East African cichlids [64] might be preserved with priority since their evolution potential might predispose them to serve as progenitors of future biodiversity [52]. and pike. This approach has already been used to identify adaptive differences between natural populations in several species. Beside populations. observed that reintroduced steelhead trout presented reduced reproductive capabilities caused by genetic effects of domestication [66].62]. for example. For example. Genome-wide gene expression profiling can also be used to detect variations in gene expression within and among natural populations [60]. it has been. with poorly represented phylogenetic groups receiving high conservation priorities [52]. SNPs. For example. Genetic monitoring of diversity using polymorphic markers allows monitoring population size and diversity over time. thereby identifying loci potentially influenced by natural selection [53]. resulting in potentially detrimental effects on survival of these populations [67]. The effects of stress factors contributing to species collapse and . With the development of much faster and cheaper high-throughput sequencing methods. Accordingly.59]. Different types of markers have been used for the estimation of natural population and the determination of conservation genetic parameters in salmonids [54] and to estimate quantitative genetic parameters under wild conditions [55]. This type of study has been performed on Atlantic salmon. including the European flounder and the brown trout [61. European eel. see Ref. Evolutionary genetics and genomics might also help to understand the interplay between fishing and natural selection on population and species targeted by fisheries [65]. multiple SNPs have been generated for Atlantic cod. conservation efforts could focus on the preservation of genetic diversity allowing biota to adapt to new conditions. with the discovery of new groupings and the determination of divergence times and molecular clocks [63]. the available population genetic information is insufficient for most other species. including Atlantic herring. Through pedigree reconstruction with microsatellite markers. for which large annual escapees of farmed Atlantic salmon enhance the risk of extinction of wild populations. with possible detection of DNA sequences promoting evolution in their genomes [17]. Genomics and transcriptomics can allow assessing the genetic and functional consequences of interbreeding between farmed and wild fish. In contrast. DNA barcoding and other methods have applications not only for species identification and molecular phylogenies but also in the field of population genetics to describe genetic diversity within species [16]. sufficient genetic data might be available to provide at least basic information on genetic structure and genetic units for biologically sustainable use [56]. and others (for review. heralding a new era in the analysis of adaptive evolution and functional variation [58. Quantitative genetics as well as evolutionary genetics and genomics can help to identify such groups of high evolvability and to study the mechanisms driving their adaptability and speciation. Molecular markers can be used to monitor the efficiency of programs aiming to supplement declining wild populations through individuals reared in captivity. turbot. For several species. Gene transcription profiling suggested that interbreeding of fugitive farmed salmon and wild individuals can substantially modify gene transcription in natural populations exposed to high migration from fish farms. this field will certainly be of major importance in the future of fisheries management and biodiversity conservation. Finally. Populations of North-East Arctic cod and Norwegian coastal cod have been analyzed. brown trout. phylogenetics and phylogenomics are of major importance for the recognition of endangered taxa from the systematic point of view. Atlantic salmon. For example. microsatellite data indicated marked genetic changes in declining North Sea cod [57]. taxa can also be considered as conservation units.

a gene conferring disease resistance into a strain selected for production. sexual development. These methods are particularly useful when classical individual tagging is difficult or when individual tanks are not available to separate families. Linkage analysis allows determining the segregation of a trait of interest relative to polymorphic molecular markers. and/or are expressed late in development. as well as in growth-related traits in the Pacific abalone [83]. The efficiency of the method depends on the predictability provided by the marker. and others must be analyzed to allow efficient breeding and management programs. as well as the development of resistance mechanisms by the targeted species can be studied using transcriptomics [25. [2]). pollution (ecotoxicogenomics). is also feasible with this method (for review. such as resistance to viral and bacterial diseases. Genomic sequences. for example. individuals backcrossed with the “production” parent will be selected for the presence of a molecular marker linked to the resistance locus. exhibit low heritability.5 Genomics and Aquaculture Fish consumption has doubled over the past 50 years and would need to double again over the next 25 years ([69] and references therein). especially in developing countries. Molecular methods have contributed to the significant increase in aquaculture production worldwide. can be used for parental assignment and construction of DNA pedigrees to analyze the heritability of zootechnical parameters and reproductive success or to avoid inbreeding and estimate genetic diversity [71]. but genetics and genomics remain poorly developed for aquaculture species compared with crops and livestocks [70]. The genetic basis of important zootechnical traits. This method also allows monitoring the transfer of genes that control desired phenotypes between breeds. texture. disease resistance and thermal tolerance in salmonids [72–78]. innate immunity. Selection against an allele. response to stress. diversification and genetic improvement of cultivated species should lead to both a reduction in production costs and an increase in fish production. Linkage maps are used to map onto genomes genetic loci such as quantitative trait loci (QTLs) influencing traits of economical interest in aquaculture fish species. Aquaculture needs to be further developed in the future. body weight and length in the Kuruma prawn [85]. for example. aquaculture including marine aquaculture (mariculture) has increased its production by a 20-fold factor over the last 30 years. MAS can be performed at early stages of development and is particularly appropriate for traits that are difficult to measure. Examples include the mapping of QTLs involved in development rate. Significant improvements have been obtained through efficient breeding programs for several species such as farmed salmon and trout. In this case. Accordingly. and virus resistance in shrimp [86]. A variation of MAS using markers covering the whole genome to assess the status of multiple QTLs is called genomic selection . on its linkage with the locus of interest. see Ref.Seafood Genomics ◾ 49 extinction. that is. 4. DNA markers linked to a locus of zootechnical interest can subsequently be used to perform marker-assisted selection (MAS). the most effective markers to perform this method of selection are the functional mutations within the trait genes (“direct” markers). biochemical parameters of blood and fish size in tilapia [79–81] and growth-related traits in sea bass [82]. cold tolerance. fillet quality (color. body weight and size. conferring for example a disease.68]. and fat deposition). particularly polymorphic DNA markers such as microsatellites. disease resistance in oyster [84]. In order to reduce the ecological disaster of overfishing and contribute to solve the problem of global feeding. growth and feed efficiency. Marker-assisted selection is an indirect process based on the selection of a DNA marker linked to a trait of interest to choose animals for selective breeding programs instead of selecting on the trait itself.

and African catfish [90–97]. sex determination is hypervariable in fish [88]. flesh quality. behavior. A better knowledge of sex determination is also required for environment-friendly manipulation of phenotypic sex.99]. sequencing can be performed on the tilling path. Sequencing and sequence comparison of the different versions of the gene in individuals polymorphic for the phenotypes studied can allow the identification of the sequence variation at the origin of phenotype differences. dmrt1bY from the medaka fish Oryzias latipes [98. Such monosex populations can be obtained with parents sex-reversed through hormone treatment or produced by androgenesis or gynogenesis. with the hope of revealing a colocalization with the locus itself. thus reflecting a frequent switching between sex determination systems during evolution. Molecular sexing of individuals at early stages of their development using sex-specific markers would allow the early selection of breeders of a chosen genotype for the production of monosex populations and the rapid analysis of breeding. Genes identified through sequencing can be chosen for further analysis according to their described function or their pattern of expression. particularly due to the lack of high-resolution genetic maps [1]. When a physical map is available. When a genomic library is available. is not present in any fish species of economical interest. Gene candidates with potentially interesting functions can be also directly sequenced in different families without . the gene itself and the sequence polymorphism involved in phenotypic variation can be identified through positional cloning. For the great majority of aquaculture species. Sex-specific molecular markers linked to the master sex-determining gene on the sex chromosomes have been identified in many aquaculture fish species. Synchronous hermaphrodites also exist in fish.). Once DNA markers linked to a locus controlling a trait of economical interest have been identified. in contrast to the situation observed for example in birds and mammals.50 ◾ Handbook of Seafood and Seafood Products Analysis [87]. all possible forms of genetic sex determination have been observed. In numerous species. Interestingly. Interestingly. depending on the species) are frequently used in fish farming. from male and female heterogamety with or without influence of autosomal loci to more complicated systems involving several loci but without sex chromosomes (polyfactorial sex determination) or more than two sex chromosomes and even several pairs of sex chromosomes. the minimal set of overlapping clones covering the region of interest. etc. a method largely used in aquaculture to control fish reproduction. androgenesis. Due to this variability. gene candidates with described functions related to the trait of interest can be directly mapped on the linkage map. tilapia. Several hundreds of fish species are sequential hermaphrodites and develop either first as a male and subsequently as a female (protandrous) or vice versa (protogynous). sex determination can be influenced by temperature and other environmental factors such as the pH of water and even social parameters [89]. Sequencing of genomic clones covering a region of interest can also provide new DNA markers that can be used to refine the mapping of the locus. MAS has not been used so far. A trait of particular interest for aquaculture is sex determination. including salmonids. as an alternative to exogenous hormone treatment. thereby reducing the number of genes to be tested. Further characterization can be performed at the functional level in vitro or in vivo. sex-linked markers for molecular sexing at early stages of development are generally restricted to a single species or are even population-specific within a same species. Phenotypic sex can frequently be fully reversed by hormone treatment. Alternatively. for example. even closely related fish species can have very different mechanisms of sex determination. In gonochoristic (with distinct sexes) species. monosex cultures (either all-male or all-female populations. for example through temperature. and gynogenesis products. a BAC library. genomic clones containing markers linked to the locus can be isolated from the library and sequenced to determine their gene content. In order to avoid overcrowding and stress induced by sexual maturation and exploit advantageous sex-linked traits (growth rate. The only master sex-determining gene identified so far in fish.

and evolutionary perspectives. Finally. see Wenne et al. Importantly. Genes expressed in response to infection with white spot syndrome virus have been identified in shrimp [111]. genomics will boost the discovery of new bioactive molecules in aquatic organisms [113. For example. In this domain. The effects of hormone treatments can be also monitored using microarrays [105–107]. In aquaculture. genomics has important applications in biodiversity analysis. wolf fish. ecological. 4.Seafood Genomics ◾ 51 mapping in order to test for associations between sequence and phenotype variation. The detection of genes of zootechnical interest can also be performed through large-scale transcriptional analysis (transcriptomics). but see Ref. with the potential of increasing growth. and conservation. since information on resource status and extinction risk is available for only a minority of marine fish species [45]. seafood genetics and genomics might revolutionize fisheries management and aquaculture development. much work is still to be done. and grouper for marine species. dolphin fish.114] and will be further developed for the identification/authentication of the composition of sea food products put on the market [115]. with strong consequences on fisheries productivity. selection methods based on molecular makers remain extremely underdeveloped for aquatic species and will require further exploration based on denser genetic maps. Immune response genes downregulated in the gills of amoebic gill disease-affected Atlantic salmons have been found through transcriptome analysis [108]. exploitation.6 Concluding Remarks In the future. Genomics will also help to improve and control transgenesis and other methods of modification of gene expression. One example is the identification of associations between SNPs in candidate genes and the growth rate in Arctic charr [100]. Microarray analysis of gene expression changes in catfish liver after infection with the gram-negative bacterium Edwardsiella ictaluri indicated a strong upregulation of several pathways involved in the inflammatory immune response and potentially in innate disease resistance [110]. Comparative genomics will need to be further developed to increase the transfer of knowledge from models to aquaculture. bream. and Australian Murray cod for fresh water species [69]. Transcriptomics is frequently used to analyze disease and other stress response gene expression and identify resistance gene candidates.and microarray-based transcription profiling for specific tissues. organs and stages of development has been performed in a variety of aquaculture species (for review. EST. The effect of dietary fish oil and fishmeal replacement by vegetable oils and plant proteins on farmed fish metabolism has been investigated in juvenile rainbow trout through hepatic gene expression profiling (nutrigenomics [103]). jack. Transcriptomics is useful to detect genes differentially expressed in different genetic backgrounds or conditions. Such new species might include halibut. environmental tolerance. and disease resistance ([69]. [1]). genes differentially expressed in progenies exhibiting opposed susceptibility to summer mortality have been identified by suppression subtractive hybridization in oyster [101]. flounder. cobia. . hybrid striped bass. a better knowledge of genes involved in the control of economically important traits will contribute to improve the production and reduce the costs for current aquaculture species and to identify and develop new potential target species for aquaculture. Phosphorus-responsive genes have been identified through transcriptomics in rainbow trout [104]. [112]). From systematic. cod. and stress response genes have been investigated in the gilthead sea bream [109]. and Arctic char. The effect of artificial selection on gene expression has been monitored through transcriptome analysis in Atlantic salmon [102].

Shedlock. 24. and spreading of high-throughput approaches for the investigation of the biology of marine organisms (http:// www.vitamib. Acknowledgments Our work is supported by grants from the Association pour la Recherche contre le Cancer (ARC). Diversity of retrotransposable elements in compact pufferfish genomes. SINEs of speciation: Tracking lineages with retroposons. 19. Living Resour.. SNPs in disease gene mapping. 860. et al. 3. The first full human genome to be sequenced using next generation rapid-sequencing technology has been already published [116]. 6. Genomics is a fast evolving discipline. 2001. 2004. Tech. 379. the Fondation de la Recherche Médicale (FRM). K. 3. 19. and the Institut National de la Recherche Agronomique (INRA). Science. Volff. Trends Ecol. J. 241. 2007. 52. Importantly.org/index.R. Cox. 5. Sci. “AquaGenome” aims to coordinate the ongoing and future national and international research projects in the field of genomics in fish and shellfish European aquaculture and support diff usion of genomic approaches within research laboratories. 2005. with major applications in genome sequencing.. 1990. J.. 2001. and most other aspects of genomics. New sequencing platforms allow rapid and much cheaper sequencing of large amounts of DNA. et al. J. Application of fluorescence in situ hybridization (FISH) to fish genetics and genome mapping. et al.com/). “Bridgemap” (http://www. 2007. and Okada. Evol. recent impressive progresses in large-scale DNA sequencing technology are currently re-revolutionizing the field of genomics (next generation rapid sequencing technology. SNP analysis. 2. Phillips. The use of marker-assisted selection in animal breeding and biotechnology. 871. Rev.M. 545. 7. 8.-N.C. et al. Radiation hybrid mapping–a somatic-cell genetic method for constructing high-resolution maps of mammalian chromosomes.52 ◾ Handbook of Seafood and Seafood Products Analysis Accordingly. [11–13]). medicinal drug development and evolution. R. References 1. 250.. Takahashi. the European Union supports different projects. Science. R. Mar. Wenne. 409. Hum. “Marine Genomics” is a network of excellence devoted to the development. 674.L.. D. N. Venter.bridgemap..gr/) develops an integrated genomic approach toward the improvement of aquacultured fish species. for review.B. J. . with a strong potential impact of such new technologies on seafood production for the future..marine-genomics-europe. Trends Genet. 2001. Williams. 2003. 1304. What role for genomics in fisheries management and aquaculture? Aquat. Shastry.. The sequence of the human genome. see Refs.. Nature.aquaculture-europe.S. “AquaFunc” wants to generate an integrated knowledge on functional genomics in sustainable aquaculture (http://genomics. Finally. Genet. A. International Human Genome Sequencing Consortium.. 245. “Aquafirst” aims to combine genetic and functional genomic approaches for stress and disease resistance MAS in fish and shellfish (http://aquafirst. the Centre National de la Recherche Scientifique (CNRS). 9... 291.tuc.. S145... Biotechnol. B.. utilization.php?id = 3).org/). many collaborative projects dealing with marine and aquaculture genomics have been or are currently funded by various agencies. For example. 4. 20. Initial sequencing and analysis of the human genome.

2003. Trends Genet. Gene loss and evolutionary rates following whole-genome duplication in teleost fishes.. 2005.. 32. 588. von Bubnoff. G. Mech. 2007. 280. P. M. 30. et al. R. 89. The first radiation hybrid map of a perch-like fish: The gilthead seabream (Sparus aurata L). 946. Telford. H. Curr.. 1301. 21. F. 89. 2007.. molecular phylogenetics and population genetics.. Genome evolution and biodiversity in teleost fish. Phylogenomics. 28. 22.. 21. 233. Transposable elements as drivers of genomic and biological diversity in vertebrates. S. an evolutionary twin study. Genome Res. S. 6. Mar. 138. 40.. Dev. 16. BMC Genomics. Curr. and Chu. Trends Genet. Curr. E. 124.R. 44. 2005. 721. Genet. Sasaki. The DNA sequence of medaka chromosome LG22. Next-generation sequencing: The race is on. 1453. 5. Evolution and diversity of fish genomes. 380.H. 15. Lond. 18. et al. O. Genome mapping in aquatic animals: Progress and future perspectives. 13. 2005. BMC Genomics. et al.H. Genet. 37. Biotechnol. 2008. J. Happy mapping: Linkage mapping using a physical analogue of meiosis. 2008..T. 11. Brunet. Quiniou. Venkatesh. 8. Genomics. 174. 13. 1675. Genome duplication in the teleost fish Tetraodon nigroviridis reveals the early vertebrate proto-karyotype. Roest Crollius. 35. Whole-genome shotgun assembly and analysis of the genome of Fugu rubripes. 40. 121. 23.. et al. 25. Fish genomics and biology. 2006. 2007. 19. Mol. 23. et al. 39. C Toxicol.C. A. Linking the genomes of nonmodel teleosts through comparative genomics. 2008. 1661. 10.. 2007. Survey sequencing and comparative analysis of the elephant shark (Callorhinchus milii) genome. Copley. A.M.. 2005. A compact cartilaginous fish model genome. 1808. 2004. Trends Genet. Genomics. Ng. Evol. Dev. et al. 227. Senger. B. The impact of next-generation sequencing technology on genetics. 86.. 94..B. R82. B Biol. 363. J. Opin. Salmo salar. 31. T... Hajibabaei. E. Miyake. Biochem. et al.. 545. A physical map of the genome of Atlantic salmon.. M.. 34. and Amemiya. Heredity. 2004. Venkatesh. 87. 16. 2007. Microarray studies of gene expression in fish. 13. M. 447. 15. K. Curr. S. 2006. Assignment of rainbow trout linkage groups to specific chromosomes..-N. Bentley. J. 629.. and Duke. 481. B... P. 2007. 20. 297. Whole-genome re-sequencing. 15. Medaka and zebrafish. 24. P. Dear... Nature. and Wittbrodt. DNA barcoding: How it complements taxonomy. 90.. Physiol. Biol. 17.. F.. T. et al. Opin. 36. 2007. Kasahara.E. D... et al. Waldbieser. 38. 27. 12. 23. Furutani-Seiki.. 133. Science. Philos.. R Soc. Xu.. 2002. et al. 10. Phillips.H. J. PLoS Biol.. BAC libraries and comparative genomics of aquatic chordate species. The animal in the genome: Comparative genomics and evolution. 20. Douglas.. 2006. Genetics. 33. 474. 8. B. Volff. 2008. Nucleic Acids Res.. 16. Biol. 2004. M. 167. Subfunction partitioning. 714. Sarropoulou. R. The medaka draft genome and insights into vertebrate genome evolution.. Postlethwait. 26. 203. Jaillon. Genet. Cell. 1993. Aparicio. et al.R... 29.. 2006. Trans. Katagiri. R945.. 431.R.. Genomics.J. M. 24.. and Cook. OMICS.Seafood Genomics ◾ 53 10. et al.. A BAC-based physical map of the channel catfish genome.. Pharmacol. T. 17. 612. C. . Böhne. A gene-based radiation hybrid map of the gilthead sea bream Sparus aurata refines and exploits conserved synteny with Tetraodon nigroviridis.. 2006. 38. et al. Biol. et al. Tong.. 396. Mardis. and Weissenbach. et al... e101. 14. 2005. 2008. et al. Sci. 793. A BAC-based physical map of the Nile tilapia genome. 2002. E. J. Genomics. Comp. Dev.. Sarropoulou. the teleost radiation and the annotation of the human genome. 2007.R.V. Nature. A first generation BAC-based physical map of the channel catfish genome. 2004. Russ. J.. 132. BMC Genomics. Venkatesh. S. et al. Chromosome Res.

. Evol. Mol.. In the grip of algal genomics. et al. K. Acad...F.. 9. 43. R.F. D. N... Evol. 98. 4.. Science. 111. T. 49. Population genomics: Whole-genome analysis of polymorphism and divergence in Drosophila simulans. 435. W. et al. Evol. Churchill. R. Trends Ecol.. 2006.. A. 981. 23. Global patterns of predator diversity in the open oceans. 63. Transcriptomic analysis of growth heterosis in larval Pacific oysters (Crassostrea gigas). e310. Luikart. 53. PLoS Biol. 62. 56. 18. 22. Natl. 58. Genetic effects of captive breeding cause a rapid. 2007. U. 65. Ryynänen.. The genome of the sea urchin. 279. Nat. and Crawford. Biology of extinction risk in marine fishes. 2008. E. 61. 2007.. et al. 64.. Trait changes in a harvested population are driven by a dynamic tug-of-war between natural and harvest selection. et al. M. 2008. Salzburger.. Genetic consequences of interbreeding between farmed and wild Atlantic salmon: Insights from the transcriptome. Ambio.E. 2006. et al. Acad.54 ◾ Handbook of Seafood and Seafood Products Analysis 41. D. G. 1046. 25. Proc.J. 2003. and Merilä. G... 2007. 2007. 15799. 16.L. 270.J. 314.. Nat. 327. 2313. Science. and Ryman. 2002. J.. Adv. Natl. 48. M. Gozlan.. Allendorf. 67. Acad.W. Hedgecock.. Science. et al. Sci. 47. Reynolds. 2005. Mol. 42. Evol. G. Proc. Strongylocentrotus purpuratus. 941. Brander. B.. Worm. F.M. 2007.... 60. J. Ecol. 32. 2004. 104.. Palm. et al. 2007.S. Begun. cumulative fitness decline in the wild. Ecol. 277.S.). 1989... Trends Ecol. J... H.. 2008. Evol. 51. Naturwissenschaften. 2007.F. species or ecosystems? Healing the fractured foundations of conservation policy. B. 1999.A. 104. Biol.. Oleksiak. 692. Proc. Science. et al. Temporal analysis of archived samples indicates marked genetic changes in declining North Sea cod (Gadus morhua). 91.. Biol. Thériault. 50.. Genetic monitoring as a promising tool for conservation and management. 2007. Mol.R. 616. 2005.. 22.. 2003. H. 2005.C. et al. 2007. 4674. et al. S. J. A comparison of biallelic markers and microsatellites for the estimation of population and conservation genetic parameters in Atlantic salmon (Salmo salar).. Genetic population structure of fishes: Implications for coastal zone management. Sci. Biodiversity: Disease threat to European fish. BMC Genet. 59. 57.. and Blouin. 9. A. B. Nature. M. Worm. Detecting and managing fisheries-induced evolution. 17. Moen. 45. Impacts of biodiversity loss on ocean ecosystem services. BMC Genet. 318. 54. 104.. 66. 52. Larsen. L. 20.. Biol. Ecol. P. J. 2337. Grossman. 34. 46... 4. 2005. Variation in gene expression within and among natural populations.. Preserving genes. Laikre... 787. Larsen.... et al. The species flocks of East African cichlid fishes: Recent advances in molecular phylogenetics and population genetics. et al. 272. 314. 5. 44.F. Edeline. 314. 54. et al. The power and promise of population genomics: From genotyping to genome typing. Adaptive differences in gene expression in European flounder (Platichthys flesus). Luikart. Interpopulation differences in expression of candidate genes for salinity tolerance in winter migrating anadromous brown trout (Salmo trutta L.W. Genet.S. Sci. B. 2266. Proc.A. and Waples. Biol. Sci.D. Med.A.K. U. V. 2007. et al. Hutchinson. Natl.A. 2007. .. 100. Sci. S5. Proc. Sea Urchin Genome Sequencing Consortium. Heritability of life-history tactics and genetic correlation with body size in a natural population of brook charr (Salvelinus fontinalis). and Meyer. 261. et al. Kuparinen. P.. 55. 8.. Rev. Bowen. Genet. Identification and characterization of novel SNP markers in Atlantic cod: Evidence for directional selection. 2125.. Schwartz. Exp. W. U. D. Araki. Cooper. 2008. Global fish production and climate change. A.. 19709. et al. 12. Genetic effects of harvest on wild animal populations. Hered.S.S. 652. 1365. Trends Ecol. A role for molecular genetics in the recognition and conservation of endangered species. C. Trends Ecol. Avise. 309. Roberge.

Trans. 86. 2008. Composite interval mapping reveals a major locus influencing embryonic development rate in rainbow trout (Oncorhynchus mykiss). et al. 2005.D. 53. A genome scan for quantitative trait loci affecting growth-related traits in an F1 family of Asian seabass (Lates calcarifer).J. et al. GH-psi. X. C. 191. Genetica.. et al. QTL for body weight and condition factor in Atlantic salmon (Salmo salar): Comparative analysis with rainbow trout (Oncorhynchus mykiss) and Arctic charr (Salvelinus alpinus). 84. 2005. 2005. 2007.. R. 96.A.J. Aquaculture. BMC Genomics. R. Breeding new strains of tilapia: Development of an artificial center of origin and linkage map based on AFLP and microsatellite loci.. and Hew.G. 198. Jones. 1109. et al.. Z. Heredity. et al. Quantitative trait loci x maternal cytoplasmic environment interaction for development rate in Oncorhynchus mykiss. D. 115. Genome-scan analysis for quantitative trait loci in an F2 tilapia hybrid. Biochem. 82. 77.. Genome. RAPD markers as predictors of infectious hypodermal and hematopoietic necrosis virus (IHHNV) resistance in shrimp (Litopenaeus stylirostris). M. Dev. 7. Bull. 16. 8. 204. 2007. Aquaculture. C. C.H. 83. Genetics. 327. 78. W. Identification of quantitative trait loci for growth-related traits in the Pacific abalone Haliotis discus hannai Ino.F. 1993. Perry. 255. 86. 81. and Ardren. Devlin.. K. 88. Genet. Major quantitative trait loci affect resistance to infectious pancreatic necrosis in Atlantic salmon (Salmo salar). 2002. and a malespecific pseudogene. 89. H.M. 2001. Fisher M. J. 130. 45.. B. J. 12. and Smailus.E. 2004.. 91. Lond. Aquac. J. Genetics. 80. X. Mapping of a quantitative trait locus for resistance against infectious salmon anemia in Atlantic salmon (Salmo salar): Comparing survival analysis with analysis on affected/resistant data. BMC Genet. et al. Soc. Anim. Hered. Du. X. 399. S.J. Hizer. 2007. 124. 8. Li..D. 1. 272. 175. Charo-Karisaa.A. Aquaculture. 1. B Biol. 90. The potential impact of modern biotechnology on fish aquaculture... 76. A. 85. 162.. 360. 38. Sex. Liu..L. and D’Cotta. 2511. P. 2006. Agresti. Comp. 2006. and Hayes. 97. Perry. GH-I and GH-II. Mol. J.. Breed. QTL detection of production traits for the Kuruma prawn Penaeus japonicus (Bate) using AFLP makers.. B. 2007. Sci... 2003. 108. 2006. Environment and sex determination in farmed fish. 249. C Toxicol. Hered.. et al. Genomic selection. 2001. 178. BMC Genomics. 92. 204. Wang. et al. J. Ecol.. 2001. 2002. 185. 2001.. Reid. Volff. Res. Moen.. 71. Quantitative trait loci for upper thermal tolerance in outbred strains of rainbow trout (Oncorhynchus mykiss). 74.. Devlin. Genomic structure of growth hormone genes in chinook salmon (Oncorhynchus tshawytscha): Presence of two functional genes.. Melamed. and Oleksiak.. Heritability of cold tolerance in Nile tilapia. juveniles.. 2007. 2005. Houston. Genetic mapping of Y-chromosomal DNA markers in Pacific salmon.M. Liu.M.. A. DNA Cell Biol. Methods of parentage analysis in natural populations.. Physiol. 12.Seafood Genomics ◾ 55 68. Cnaani. 323. Genetic linkage map of the Eastern oyster Crassostrea virginica Gmelin. Genet. Governing sex determination in fish: Regulatory putsches and ephemeral dictators.. Baroiller. 333. 43. Managing to harvest? Perspectives on the potential of aquaculture.F. et al. Aquaculture.. et al. 79. 94. 75. Heredity. Robison. Biagi. Philos. 789. 111.. S..E. 43. 87. 69. G. 739. and Guo. Biol. 73. G. and Zhang.. Muir.-N. R. Mol.. H. 274.. 166. 72. Nichols. R. 85. 258. J. Goddard. .M. T. D. M. et al. Sex-linked quantitative trait loci for thermotolerance and length in the rainbow trout. et al. Pharmacol.E. G...R.H.. J. Oreochromis niloticus. 70. 2007.. et al. 335. et al. Genomics. Convergence and divergence in gene expression among natural populations exposed to pollution. Y. Yu.P... 2000.

S. 543.. 2008. Heredity. C. Physiol. 2008. The complete genome of an individual by massively parallel DNA sequencing. S. 108.. D. et al. 94. 2. Panserat. Effects of short-term growth hormone treatment on liver and muscle transcriptomes in rainbow trout (Oncorhynchus mykiss). 781. Growth of domesticated transgenic fish. Aquaculture. D.. 110.G. et al. 211. et al.. 102. 109. E. Trends Biotechnol.. 93... Genet. 182. Gene expression profi ling of gilthead sea bream during early development and detection of stress-related genes by the application of cDNA microarray technology. Endocrinol.. 343. M. and environmental influences.T.. 23. Res. Kovács.S. Associations between single nucleotide polymorphisms in candidate genes and growth rate in Arctic charr (Salvelinus alpinus L.Y. et al.. 156. 43. D. Mol. Mol. G.H. Baron. 45. 97. Microarray analysis of gene expression in the blue catfish liver reveals early activation of the MHC class I pathway after infection with Edwardsiella ictaluri.D. 380. 417. et al. 52. Physiol. The identification of genes from the oyster Crassostrea gigas that are differentially expressed in progeny exhibiting opposed susceptibility to summer mortality. 2003. E. Sci. Oryzias latipes.). C.. 11778.] 104. 60. 92. 2007. Nagahama. 8. 95. et al. 2004. 35. Food and forensic molecular identification: Update and challenges. 2002. Natl.D. 2008. 13.. Maudet. 267.A. Rasmussen. Nutr.. U. Roberge. Teletchea. 319. 96. et al. and Kocher. Dev. 111.. Nature. Wheeler. 208. Physiol. 32. 559. Gene.. Proc. Two unlinked loci controlling the sex of blue tilapia (Oreochromis aureus). et al. Peatman.. 2004. J.. 2008. Robalino. 101. 2003. Sex. M. Biotechnol.J. et al. Anim. and Boulding. 409. and Morrissey. T.. Salmonid microarrays identify intestinal genes that reliably monitor P deficiency in rainbow trout aquaculture. Immunol. Expression profiling of candidate genes during ovary-to-testis trans-differentiation in rainbow trout masculinized by androgens. et al. 98. Hulata. et al. Heredity. et al. Lee.. 2002. Baron. Coordinated down-regulation of the antigen processing machinery in the gills of amoebic gill disease-affected Atlantic salmon (Salmo salar L. 2001. Mar.. 435. Nat. Sarropoulou. BMC Genomics. Isolation and physical mapping of sex-linked AFLP markers in nile tilapia (Oreochromis niloticus L. A. Adv.A. 23. 9. Marine natural products. and Hänni. B. 100. A duplicated copy of DMRT1 in the sex-determining region of the Y chromosome of the medaka. Young. Rapid parallel evolutionary changes of gene transcription profiles in farmed Atlantic salmon. . 99. R. Immunol. 2008. 106. S.... Comp.). 2000. M. 2005. 2004. E. A.. 99. 116. W. Genomics. 6. 15. 2005. 2008. Food Nutr. Gen. Tao. I. et al. Matsuda. 44. et al. 2007.S... 369. Ezaz. Genomics. Ecol.. 2008. et al. Rep. J. 115. 107. C.. N.A. 2002. 25. 112. Acad. Huvet. et al.T. Nature. 237. Androgen-induced masculinization in rainbow trout results in a marked dysregulation of early gonadal gene expression profiles. 114. Kirchner. et al.. Genomics. et al. 359. Genome Res. 872.. 29. R. Nanda. 91. 2007. et al.W. Y. F. [Epub ahead of print. Cnaani.. Blunt.).. Nature. Br. 191. 105. Devlin. 553. Male-specific DNA markers from African catfish (Clarias gariepinus). Gahr. Sex determination and sex differentiation in fish: An overview of genetic. Woram. 357.. Marine biotechnology for production of food ingredients.. 452. R.. 272. 110.. 45.H. Prod. 103. 2008.. 2007. 113.56 ◾ Handbook of Seafood and Seafood Products Analysis 92.. Genetica. 2581. et al. Mol. physiological. Devlin. J. B.A.. Genetics of sex determination in tilapiine species. DMY is a Y-specific DM-domain gene required for male development in the medaka fish. R. Hepatic gene expression profiles in juvenile rainbow trout (Oncorhynchus mykiss) fed fishmeal or fish oil-free diets.. Insights into the immune transcriptome of the shrimp Litopenaeus vannamei: Tissue-specific expression profiles and transcriptomic responses to immune challenge... 2006. Comparative genome analysis of the primary sex-determining locus in salmonid fishes.

............2...............................2 Capillary Electrophoresis .... Nevertheless............. beheaded...65 5... the autolytic process derived from tissue enzymatic activity and lipid oxidations also contributes to fish maturation and subsequent spoilage..4 Enzymatic Analysis................................ 64 References .............61 5...1 Introduction ............... Thus...................................... and Fidel Toldrá Contents 5..... the adenosine triphosphate (ATP) regeneration that occurs in vivo stops and ATP is degraded until 57 .................................................2 Chemical Structure of Main Seafood Nucleosides and Nucleotides .......Chapter 5 Nucleotides and Nucleosides M...........................2...........................................3..................57 5........... Sensory methods to evaluate fish quality are subjective and difficult to use in the evaluation of processed (fillets.........3 Chromatography................2.....3............ Concepción Aristoy...................3 Analysis of ATP-Related Compounds ......3..................................................1 Extraction of Nucleotides and Nucleosides ..................2.......................1 31Phosphorous-Nuclear Magnetic Resonance Spectroscopy ......3................ Hernández-Cázares......3..................... The first autolytic process taking place in fish affects carbohydrates and nucleotides................................. 60 5............ Aleida S...........2 Nucleotides and Nucleosides Determination ................61 5......... objective methods for freshness determination are required and the determination of the biochemical changes occurring in early postmortem in fish constitute a helpful tool.....61 5......61 5.59 5........................................ After death............. or eviscerated fish) or canned fish.........59 5..3................. Leticia Mora...........................1 Introduction Bacterial growth is the main factor limiting fish commercial life by producing its alteration and unpleasant flavor.....................................

although it might be accelerated by the action of different bacteria. IMP is the main nucleotide present in fish species. and either of the two may be used as freshness indicators. which is oxidized to xanthine (Xa) and uric acid in the presence of xanthine oxidase (XO) enzyme. whereas AMP remains major in crustaceans. This enzyme is mainly generated in muscle from biochemical processes of microorganisms.1 The following IMP dephosphorylation to obtain inosine is mainly autolytic and occurs at a slower rate during the first stage of cold storage.58 ◾ Handbook of Seafood and Seafood Products Analysis rigor mortis is reached.1 Degradation of ATP in postmortem fish muscle. the use of a single compound as freshness indicator is not always advisable.3 In all cases. which is accumulated in postharvest fish. (2006) published a review of the concentration of IMP. because many factors can affect O N H 2N N N N O O HO ATP OH HO P O P O P OH O OH O ATP ase N Pi HO ADP Pi Myokinase OH N N H2N N O O HO P O P OH O O OH OH N HO N N N O OH HO Ino Pi Nucleosidase phosphorilase Ribose 1-phosphate O HN N Hx N N H O2 Xanthine oxidase OH Nucleotidase Pi HO N N N N HO IMP O O OH O P OH AMP deaminase OH NH3 H2N N N N N HO AMP O O OH O P OH OH O HN O H2O2 N H Xa N N H O2 Xanthine oxidase O H2O2 HN O H N O N H UA N H Figure 5. IMP degradation to inosine (Ino) and its disappearance have been correlated with lack of freshness in some fish species. Inosine is transformed to hypoxanthine (Hx) by the action of the enzyme nucleoside phosphorylase (NP).4 However. ATP molecule is rapidly degraded to adenosine monophosphate (AMP) and afterward to inosine monophosphate (IMP). and Hx in the flesh of some species of fish during chilled storage. Ino. .2 Howgate et al. As a result of endogenous enzymes action. Ino and Hx concentrations increased during storage.1. This process involves a series of reactions commonly represented according to the sequence shown in Figure 5. The speed of each step in this reaction chain and especially in the Ino to Hx and Hx to Xa conversion depends on the fish species.

ATP. even at refrigeration temperatures. In this way. making K value inadequate as a freshness indicator. K value is defined as the ratio of Ino and Hx to the sum of ATP and related compounds expressed as a percentage. 5. consequently.11 and. Some of them are briefly described here. to which one or two additional phosphate groups are attached through pyrophosphate bonds (∼P) (Figure 5. the disappearance of the degradation products differs from one species to another3 as mentioned here. for several species. For this reason. Nucleosides currently analyzed in seafoods are those in which a purine ring. forming the adenosine or inosine. as shown when comparing high-temperature short-time process at 125°C for 9 min with a common retort process at 115°C for 90 min. or hypoxanthine is attached to a ribose.3 Analysis of ATP-Related Compounds The correct analysis of ATP-related compounds must take into account that early postmortem fish muscle is very sensitive to temperature.5 and. ADP and ATP are derived from the AMP. These cold conditions must be held along the sample preparation. nucleotides and nucleosides should be extracted and analyzed. a high content of Hx is related with the bitter off taste of spoiled fish. and it is important to stop this reaction drastically at the sampling time.Nucleotides and Nucleosides ◾ 59 nucleotide degradation such as the type of spoilage bacteria and mechanical handling of fish. also. The ratio Hx/AMP was considered an adequate alternative to characterize fish freshness due to its constant increment with time. the knowledge of their molecular structure is important. respectively. This is the main reason for the use of indexes with more than one compound from the ATP-degradation chain. and thus. adenine.2).15.7–9 Nevertheless. a revised K value. On the other hand.16. whereas IMP evokes a fresh meaty taste sensation.6 This value has been used as one of the freshness indexes to evaluate the quality change of postharvest fish.2. This is achieved by immediately freezing the excised muscle under liquid nitrogen to stop all enzymatic reactions. and AMP disappear early postmortem. In order to achieve this rapid freezing.2 Chemical Structure of Main Seafood Nucleosides and Nucleotides To a better understanding of the methods of analysis of these compounds. Nucleosides are glycosylamines that are formed when a nucleobase (purine or pyrimidine base) attaches to a ribose or deoxyribose ring. it is advisable to collect small tissue samples and immerse them into liquid nitrogen. adenosine diphosphate (ADP).16 Another suggestion to use nucleotide compounds as a measurement of seafood quality is their relation with sensory attributes. a hypoxanthine ratio or H value (Hx/(IMP + Ino + Hx) × 100) was considered as a better indicator of fish freshness in this type of species.18 After this. generally within 1 day of storage in ice after death in all fish species.12 However.14 Measurement of ATP-related compounds is also useful for the quality control of retorted fishes. AMP or adenylic acid is derived from the adenosine in which a phosphate group is attached at the 5-ribose carbon. is more often considered as monitoring the loss of IMP and is defined as the ratio of Ino and Hx to the sum of IMP. ATP-chain degradation occurs very fast. a high accumulation of Ino occurs during ATP degradation.10.13.17 5. Nucleotides are o-phosphoric acid esters of the nucleosides. IMP is derived from the inosine in which a phosphate group is attached to the 5-ribose carbon. Ino. often designed K ′ value or Ki index. and Hx expressed as percentage. .

000 g for 15 min). after centrifugation (27.3. and Hx. they are neutralized with 2 M sodium hydroxide. although storage at −18°C has been demonstrated to be enough to preserve fish samples and fish extracts for the analysis of IMP.5–6. avoiding any thawing. The neutralized extract must be made up to 5 mL with 20 mM phosphate buffer pH 7. is the following: 5 g or less of muscle tissue are excised and quickly frozen with liquid nitrogen. with or without employing an ion-pairing agent.2 Structure of adenosine-derived nucleotides. These fish extracts are used in enzymatic assays with biosensors19. Ino. and the tissue is homogenized with a stomacher-type homogenizer for a few minutes under cold conditions. 5.8 and then filtered with a 0.1 Extraction of Nucleotides and Nucleosides A typical extraction procedure for the analysis of fish samples by reversed-phase chromatography.000 g for 10 min).17 Other extraction methods consist in the homogenization of 2. The frozen tissue is minced.2 μm membrane filter and stored under frozen storage at temperatures below −20°C until analysis.60 ◾ Handbook of Seafood and Seafood Products Analysis Adenosine nucleoside N N NH2 OH HO P O O OH P O O O P O OH HO O N N OH Ribose Adenine purine base AMP ADP ATP Adenosine nucleotide Figure 5.8 by adding solid potassium carbonate or 1 M potassium hydroxide.45 mm membrane. This neutralized extract is kept in an ice bath for 15 min and centrifuged again (15.6 M perchloric acid is added. 3–5 vol.000 g for 20 min).20 and/or spectrophotometers as well as in capillary electrophoresis (CE)21 or ion chromatography (IC). cold 0.5 g of fish sample with 10% trichloroacetic acid and. the supernatant is filtered through glass wool and neutralized to pH 6. Once the extract is centrifuged (15. The supernatant is filtered through a 0.22 .

this technique can present problems in reproducibility. However.2. The mode of separation will depend on the analyte of interest.3. the addition of an ion-pair to the mobile phase greatly improves the separation by increasing the retention time of charged molecules (ATP.22 and enzymatic assays. both a microwave oven at 500 V for 5 s and heating at 100°C for 60 min have been used. HPLC has been shown to be the most widely used technique to analyze nucleotides and nucleosides. In particular. 5. radioimmunoassay. and hypoxanthine would be a potential of 416 V/cm of capillary using 100 mM 3-[cyclohexylamino]-1-propanesulfonic acid (CAPS) buffer. inosine. AMP). because these samples usually contain significant amounts of ions. Capillary electrophoresis has been used in many nucleotide analysis applications as in the study of nucleotide degradation in fish tissues. RP-HPLC and ion-paired reverse-phase are the methods of choice for this analysis.21 Typical conditions to get a good separation of IMP. Thus.3 Chromatography At present. to analyze nucleotides. among other chromatographic techniques.3. including nuclear magnetic resonance spectroscopy (NMR).2. 5. the reconditioning of the capillary surface is ensured by washing 1 min with 1M NaOH. followed by 2 min of the running buffer used. In the analysis of complex biological samples.26 reversed-phase high-performance liquid chromatography (RP-HPLC) with and without ion pair.28 Also in vitro 31P-NMR spectroscopy has been applied to both excised tissue and perchloric acid extracts of fish muscle. intact fishes after being submitted to physical and chemical stressors such as hypoxia.23. including fish extract.27 IC. ADP.3. which may be adsorbed on capillary walls. ion-exchange HPLC. some authors have described extraction methods that consisted of heated fish sample. in vivo 31P-NMR spectroscopy has been used as a powerful technique to characterize the biochemical changes that occur in live. and the K′ or K i index will be usually enough to characterize fish .1 31 31 Phosphorous-Nuclear Magnetic Resonance Spectroscopy The phosphorous nuclear magnetic resonance spectroscopy (31P-NMR) technique makes it possible to perform multiple determinations of high-energy phosphates in vivo in the same muscle sample.2.2 Nucleotides and Nucleosides Determination Several methods have been used to measure nucleotides and nucleosides. pH 11.19 5.24 5. Nevertheless.3.2 Capillary Electrophoresis CE is a powerful separation technique that can provide high separation efficiency and high sample throughput with minimal sample volume and buffer consumption.Nucleotides and Nucleosides ◾ 61 In the development of biosensor analysis.25 thin-layer chromatography (TLC). high-performance capillary electrophoresis (HPCE). In this way. nucleotides will disappear at the rigor mortis state (normally 1 day after catch). Thus.

(4) AMP. The separation was achieved with an RP C-18 column at 35°C and a gradient between phosphate buffer at pH 7 and acetonitrile. (2) ATP. 1000 (a) 6 800 600 400 Absorbance at 254 nm (mAU) 1 2 3 4 5 200 0 1200 1000 800 600 400 200 0 0 2 4 6 8 10 2 3 6 (b) 1 5 4 Retention time (min) Figure 5.3. There are many approaches to analyze nucleotides and nucleosides by this technique.15 The identification of the chromatographic peaks can be performed by comparing the peak retention times and spectral characteristics (if a diode array detector is available) with those of standards. a simple RP-HPLC with a phosphate buffer as mobile phase will be adequate. which differ mainly in the pH of the mobile phase. Quantitative analysis can be performed by external or internal standard method. and (6) inosine. (3) ADP. All of them use a phosphate buffer as the mobile phase and a gradient with methanol or acetonitrile should be accomplished to improve the Ino resolution and reduce the chromatogram time.17.3 both chromatograms of standards and hake nucleosides and nucleotides are shown. (5) hypoxanthine. Then.2. The column used is an analytical reversed-phase RP-18 column.29 With buffer pH 7.3 RP-HPLC chromatograms of standards (a) and hake (b) ATP-derived compounds.1 Reversed-Phase HPLC The chromatographic analysis should be performed in a liquid chromatograph equipped with an UV detector (254 nm). In Figure 5.3. (1) IMP.62 ◾ Handbook of Seafood and Seafood Products Analysis freshness or quality. 5. .29 phosphorylated metabolites are also well separated in the chromatogram.

3. (1) IMP. (4) AMP.and tri-nucleotides have to be analyzed. as well as the resolution.3. because the ion pair enhances the retention time and separation. The separation is achieved in a reversed-phase column. this method is more expensive than the more simple technique previously described.4 shows an ion-paired chromatogram of a 48 h postmortem sardine extract. ATP-derived compounds.2 Ion-Pair RP-HPLC The most common technique used for the separation of nucleotides is ion-pair RP-HPLC.30 This ion-paired technique is especially useful when di. Due to the negative charge of the phosphorylated groups of nucleotides.17. (5) hypoxanthine. and (6) inosine. making it less dependant on the type of column.2. either tetrabutylammonium hydrogen sulfate or phosphate is the ion pair most used. and the key is to add an ion pair (an ion of charge opposite to that of the analyte molecule). Thus.Nucleotides and Nucleosides ◾ 63 5. (3) ADP. Nevertheless. Figure 5. which is especially useful in separating mixtures of charged and uncharged molecules. the ion pair should be a positive ion with a hydrophobic rest to improve the affinity with the stationary phase. 1400 1200 1000 800 600 Absorbance at 254 nm (mAU) 400 200 0 1200 5 1 (a) 6 2 3 4 6 1000 800 600 400 5 200 2 0 0 5 10 Retention time (min) 15 3 1 (b) 4 20 Figure 5. .4 Ion-paired HPLC chromatograms of salmon (a) and sardine (b). (2) ATP. due to the ionic nature of the phosphate esters that facilitates strong interactions with the ion-pair reagent at the appropriate pH.

in which an enzyme or a group of enzymes are immobilized in a membrane or other supports. 5. The depletion of oxygen or the formation of hydrogen peroxide or uric acid may be detected amperometrically.1 Enzymatic Methods with the Enzyme in Solution The concentration of Hx. XO enzyme. environmental.64 ◾ Handbook of Seafood and Seafood Products Analysis 5.36–38 The most used biosensors for the nucleotide-related compound analysis are electrochemical sensors. because no interference of salt in the medium was observed here as was in the case using the HPLC method. although biosensors have shown its utility in some applications such as clinical. Ino. because the denaturalization of the enzymes with time. the application in the food industry is still restricted36 mainly due to critical stages such as enzyme immobilization or sample preparation for analysis.34 A biosensor is a system composed of a biological recognition element and a biochemical or physical transducer in intimate contact or in close proximity with each other in order to relate the concentration of an analyte to a measurable signal.3. This option offers some advantages in relation to the free enzyme. Prodomidis and Karayannis85 reported a review on enzyme-based amperometric sensors applied to food analysis in which the principles and materials commonly used for the construction of the electrodes are described. although these applications used to be achieved with at least one of these enzymes immobilized as described earlier.4.20. These enzymes act by oxidizing the substrates (analyte) while consuming oxygen or producing hydrogen peroxide or uric acid. These assays may be carried out with the enzymes in solution31. which will be further quantified by measuring the absorbance at 290 nm and by polarimetry.2 Enzymatic Methods with Immobilized Enzymes In this case. This sensor has been developed mainly for assessing the freshness of fish meat40.35 The most used is the biosensor based on the measure of hypoxanthine. inosine. and hypoxanthine will be oxidized to uric acid and H2O2. which is further coupled to a chemical transducer.8 and 30°C–37°C. or sensors have a limited shelf life. enzyme-coated strips.31 Another possibility consisted in monitoring the oxygen consumption after these enzymatic reactions with an amperometric-type sensor (oxymeter).20. and 5′-nucleotidase (NT) into a reaction phosphate buffer containing the fish extract sample at pH 7.2.36 Nevertheless. and IMP may be determined spectrophotometrically by a sequential addition of XO. the analysis may be performed with one or more enzymes. agricultural. oxidizes the hypoxanthine to xanthine and uric acid. IMP. while the depletion of oxygen is measured by a Clark-type elec- . 5. the use of commercial kits or disposals presents some problems. which is immobilized in a membrane fi xed in the sensing area of the electrode.41 or for the evaluation of chicken32 and beef meat33 aging.39 This procedure was also used to analyze ATP and related compounds in fish sauces with very good results. Indeed. and rapid response. due to its specificity.33. NP.6–7.4. respectively.35 Some details about the use of different biomaterials in order to select the best recognition elements and the most adequate methods for the enzyme immobilization have been described.3.3. In these conditions. and thus test kits.32 or immobilized. In addition. constituting what is known as enzyme sensors o biosensors.4 Enzymatic Analysis The use of enzymatic methods to analyze nucleotides in seafood is widespread due to their high specificity.2. and biotechnology. simplicity. all the approaches to date need the sample preparation described earlier. electrodes.2. but they remain immobilized in different supports. In this sensor.

The proposed relation 1 Hx for ½ X for each oxygen molecule formed must be taken into account to quantify the Hx. 65: 40–47.. or H2O2.44 a polyaniline film by electropolimerization.53. The consumed oxygen produces a current decrease that can be correlated to the concentration of Hx. In the measurement of hypoxanthine. 1988. Technol. thus. An immobilized NT was used for the previous conversion of IMP to Ino. In this way.55. D.. and Hx using a cellulose triacetate membrane have been described. an Ag/AgCl reference electrode.56 References 1. E. specific biosensors to determine AMP. Fish.6 to −0. R. Paredi. 36: 19–22. Sci. 2000. 1: 13–19..49 and Ino50 and a multienzymatic sensor to analyze simultaneously AMP. J. A similar application was proposed12. Saito. M. different supports have been used for the immobilization of the XO enzyme. J. K i.E.E. M.J. some authors have described this type of biosensor coupled with an oxygen electrode. Ino was converted to Hx.L. 2001. Comparable results to that of HPLC were reported. cellulose triacetate. 2.46 or even in a carbon paste electrode modified with electrodeposited gold nanoparticles. . 2006. and Hx amounts.Nucleotides and Nucleosides ◾ 65 trode at a platinum cathode (−0. LeBlanc. Ltd. 24: 749–750. 3. Jpn. Food Biochem. Fish. Male. Howgate. A review of the kinetics of degradation of inosine monophosphate in some species of fish during chilled storage. Formed Hx was measured with an amperometric sensor that detected uric acid + hydrogen peroxide in an additive matter. K. 212: 141–146.L. Int. Özogul.. which can be present in the sample or formed during the enzymatic reaction.41 preactivated nylon. necessary to obtain K. and. Y. Changes in baseline levels of nucleotides during ice storage of fish and crustaceans from the Portuguese coast. Food Chem. Thus. IMP. A new method for estimating the freshness of fish.E..9 V) vs. Postmortem changes in quality indices of ice-stored flounder (Paralichthys patagonicus). Postmortem biochemical and functional characteristic of Monterey sardine muscle stored at 0°C. Ino. J. 1 mol of Hx would be converted to 1 mol of uric acid and 2 mol of hydrogen peroxide. 41: 341–353.33 although this relation should be confirmed in each particular system. Gill. Özogul. Bull... Most recent approaches to determine Hx are based on the incorporation of the XO enzyme in a graphite/Teflon matrix. P.34 to obtain the Ki parameter as a freshness indicator. 2007. Lugo-Sánchez. uric acid. and then after adding a soluble NP. 7.. Matsuyoshi. M. 1959. 6. T. Most of these supports have been developed with the aim of eliminating interferences due to ascorbic acid.48 IMP. M. Robles-Burgueno. J. Arai. Sci.A.. R.23. Quinta.18 and a silk fibron membrane in combination with a cellulose acetate membrane42 or a nylon net43 have been used.. Food Sci. Agric. Ino.. M. 5.51 The use of multienzymatic biosensors to measure fish freshness has been very helpful for the simultaneous determination of AMP. and H values. T. et al. IMP.45 a nafion-coated platinum disc electrode.R. Mendes. Both Hx and X are substrates for the XO action and will be oxidized either simultaneously or sequentially. Japan). Biochemical basis of postmortem nucleotide catabolism in cod (Gadus morhua) and its relationship to spoilage. A. 4. Palacios. Surette.. P.. Nucleotide degradation in sardine (Sardina pilchardus) stored in different storage condition at 4°C. Technol.54 In fact. Flow injection analysis (FIA) has been widely used in the development of these multienzymatic biosensors constituting different types of reactors in which different enzyme combinations can be immobilized as well as introduced as soluble enzyme. Luong and Male20 used a multienzymatic biosensor system to determine the H value as a fish freshness indicator. Food Sci. 2005. Soc. F. Pacheco-Aguilar.E. Food Res.47 On the other hand. Massa. J. This method was patented by Luong. Eur. 29: 570–590. M.. Kuley. R..53. and Nguyen52 and afterward it has been commercialized as a Freshness Meter KV-101 (Oriental Electric Co. Nunes.

Food Sci. B. LWT-Food Sci. Paredi. E.. Biochem. D.B. J. S. 95(4): 789–795. J. Food Sci. H.H. Marquez-Rios. 22. K. Nguyen. 2007.. 57–1: 77–81.). Bioelectron. trawled cod (Gadus callarius). J. P. 32: 314–319.A.. M. Goto. Hypoxanthine ratio determination in fish extract using capillary electrophoresis and immobilized enzymes. 1990a.. Technol. 2007. Homarus americanus. Biochem. F. LWT-Food Sci.. Lugo-Sanchez. Dingle. Physiol. Nucleotides and its derived compounds.. 41: 469–473. Borgese. Aristoy.. Food Chem. Effects of retort conditions on ATP-related compounds in pouched fish muscle. Technol. Food Chem. Agric. J. Fujita. 13. Quantick.. M. Watanabe. 1969. 14: 125–130. Male.T. M. Technol. Taylor. H. Male. Roberts. Fraser. 2002. J..C.F. Pacheco-Aguilar. J.T. 279–288.. Van der Thillart. Food Agric.. A. J.R. E. 10. Hines. J.. 2006. Boca Raton. Muller. M.L.R...N. Toldra. 11. Comp. 17. M. Roth. 1990. et al. Handbook of Muscle Foods Analysis. Development of a new biosensor system for the determination of the hypoxanthine ratio. 1962. 19. an indicator of fish freshness. Tamada. 18. Gao.66 ◾ Handbook of Seafood and Seafood Products Analysis 8. Mol. Luong. 12. J. A 1118: 278–280.E. et al. Enzyme Microb. L. 2008. Larrain. 29... 1997. Technol. Nucleotide catabolism in cold stored adductor muscle of scallop (Zygochlamys patagonica). H. 1977. Izquierdo-Pulido. Kuda. 72: C356–C362. Yuan. CRC Press.. 1992a. Fujita. C. Özogul.C. (Eds. 21: 534–538. Freshness loss in sierra fish (Scomberomorus sierra) muscle stored in ice as affected by postcapture handling practices. Food Chem. J. Postmortem biochemical behavior of giant squid (Dosidicus gigas) mantle muscle stored in ice and its relation with quality parameters. Determination of ATP related compounds in fresh and canned tuna fish by HPLC.. J. pp. Chem. A.J. E. et al.A. B.. Comp. Luong.H. Clifford. M.D.T.. 16. et al. Biol. M.C. Int..R. et al.L. 25.I. R. 2000. Xue. 21. Suzuki. 1984.. Post-mortem degradation of adenine nucleotides in muscle of the lobster. E. C. 2005. K.. Guanosine triphosphate (GTP): The major organic phosphate in the erythrocytes of the elasmobranch Mustelus canis (smooth dogfish). Bioelectron.. Sci. R.H. Luong.. Comparison of radioimmunoassay and spectrophotometric analysis for the quantitation of hypoxanthine in fish muscle.P. 13: 475–480. N. Morris. Castillo-Yanez.. Kuda. M.L. . 31: 56–67. J. J. Part B: Biochem. 42: 1–17. 33: 100–103. et al. 14. 104: 369–375.M.J. J. Food Biochem. M. S. 1968. Masson. 24. Vidal-Carou. K. Agric. Van Waarde. J. Okuma. Masson. In Nollet L.A.. J. Effects of freshness on ATP-related compounds in retorted chub mackerel Scomber japonicus. 2009. Development of quality evaluation sensor for fish freshness control based on K1 value.. 1992b.. 9. T. Degradation of adenine.E. et al. Food Chem. et al. V. 35: 549–554.B. Determination of high-energy phosphate compounds in fish muscle: 31P-NMR spectroscopy and enzymatic methods. ´ ´. C. Food Sci. 60: 317–321. Aubourg. Meat and fish flavors—Significance of ribomononucleotides and their metabolites. et al. Márquez-Rios. Goto. A rapid HPLC-determination of ATP-related compounds and its application to herring stored under modified atmosphere. Biosens. 23. Y. Nagel. G. Toldra F. 27. Karube. Anal. 1991.. 59: 467–472. 17: 367–372. Jones. E.. H. N.. Determination of fish freshness with an enzyme sensor system. 15. Quitral. A. Matsuoka.and hypoxanthine nucleotide in the muscle of chillstored. 26: 295–305. et al. Chromatogr. FL. Food Chem.T. E. Flow system for freshness determination based on double multi-enzyme reactor electrodes. N. Moran-Palacio. Jones. 26. Veciana-Nogués.. Food Biochem. T.E. F...A. 2007. Autolytic degradation and microbiological activity in farmed Coho salmon (Oncorhynchus kisutch) during chilled storage. F. Food Sci.. Crupkin. et al.. T. I. Hamada-Sato. 2007.A. H.. 17: 712. Biosens. M. Massa. Murray. 20. 28. 62: 2490–2493. Determination of the big head carp myofibrillar (Aristichthys nobilis) adenosine triphosphatase activity by ion chromatography. R. 40: 1186–1190.. Determination of nucleosides in fish tissues using capillary electrophoresis. Physiol. 2002. M. Watanabe.

Xu. Food Sci. J. Otani.. Technol. Agüí.Nucleotides and Nucleosides ◾ 67 30. I. I. Anal. 33. N. 35. Biotechnol. Analyst 123: 371–377. 1996. S. 37. A. 36. 34. 1983. Appl. M. 41. S. H. Kubota. Enzymatic method for measuring ATP related compounds in fish sauces. Toyama. Y. 48: 114–116. Chim. J. N. Talanta 43: 283–289. Biosensor for detection of hypoxanthine based on xanthine oxidase immobilized on chemically modified carbon paste electrode. 48: 496–500. 46. Karube. 47. Enzyme sensors for determination of fish freshness. Watanabe. P.. 6: 207–211. 40. 38..com/5288613.B. E. 17: 147–157. K. G. A.. E.. 32: 385–390. Karube.. Viera dos Santos. M. 1984b.. 1984c. inosine-5′ phosphate and adenosine 5′-phosphate with a multielectrode enzyme sensor.T.L. Chem.T. Reviejo. 52: 107–112. 49.. Silk fibroin/cellulose acetate membrane electrodes incorporating xanthine oxidase for the determination of fish freshness. Ogura. Actuators B 113: 272–280. A. J. Biosensor based on xanthine oxidase for monitoring hypoxanthine in fish meat. Kataho. Application of polarography for monitoring the fish post-mortem metabolite transformation. 11: 277–282. Yano. Korean Fish. Enzyme Microb. 39. T. 1989. Review of the use of biosensors as analytical tools in the food and drink industries. Acta 412: 55–61. Amperometric determination of fish freshness by hypoxanthine biosensor. Luong. Enzyme based amperometric biosensors for food analysis. 1990b. Peña. Im. E. 43.. Watanabe. A. M. 2005. E. Prodomidis. 53.. L. Biosens. Development of a bioenzymatic graphite-Teflon composite electrode for the determination of hypoxanthine in fish. 1984a. S.. 37: 729–735. Tokimatsu. K. Biochem. Determination of a system with double enzyme reactors for the determination of fish freshness. 1996. T. S. 50. et al..I. Agric. Hu.. 1999.. Anal.. 19(1): 18. Electroanalysis 14: 214–261.. 42. Nguyen. J. 1992.H.. I. 3: 307–316. I.. Anal... Anal. Cho. 2006.. 1(2): 85–892. Pelegrine. Applicability of the K0 value as an index of freshness for porcine and chicken muscles. Male.. Curr. 1995. Chim. 52. Karube. S. V.. Tuzhi.html. H. . Simultaneous separation of nucleotides and nucleotide sugars using an ion-pair reversed-phase HPLC: Application for assaying glycosyltransferase activity. 44. Nakatani. Ando. K. Manso. Y. http://www. Shen.. et al. et al. Toyama.. D. M. Food Sci. J. Hori. T.S. 51. L. Kumar.M. Watanabe. 2003. H.. Luo. Acta 260: 93–98. C. Biotechnol. Nguyen. Paquet.. N. 52: 439–445. Venugopal.S. H. Sharma. Bioelectron. Cayuela.. Soc. P. Chim... Watanabe. Toyama. Watanabe. Q. T. Simultaneous determination of hypoxanthine. Fujita.. Kim. Microbiol.D. 1995. J.B.. K. Biomolecules for development of biosensors and their applications. 2002.K. Acta 164: 139–146. Yazawa. Male. T. et al. 1994.M. Determination of inosine-5-monophosphate in fish tissue with an enzyme sensor. T. 67: 1627–1631. Biosensor in fish production and quality control. E. 45. Yacynych. Chim. Evaluation of beef ageing by determination of hypoxanthine contents: Application of a xanthine sensor. Combes.I. L. et al.L. Technol. Determination of adenosine 5′-monophosphate in fish and shellfish using an enzyme sensor. Biotech. et al. Determination of hypoxanthine in fish meat with an enzyme sensor. 1988. V. 31. Sens.. L.. H.. Biol. et al. Retention of enzyme by electropolymerized film: A new approach in developing a hypoxanthine biosensors. Watanabe.freepatentsonline. et al. Takahashi. Karayannis. A. J. Y. Enzyme Microb. Nguyen.. Anal..J. Am. Volpe.M. 2000.. Sci Food Agric. Okuma. Pingaron. et al. L. K. Acta 369: 245–251. et al... Sehgal. Meynial. Toyama. Luong. Y. Luong. Bioeng. Mello.J. 48.. 2002.P. G. Appl.T.L. Food Chem. Chem.. 1998. 77: 237–256. A. C. Y. 1984d. Mascini. 1998. 2002.. J. J.T. 70: 298–302. 32. Yang. L. Peng. Yañez-Sedeño. Phys. Qiong. Liju. Enzyme sensor for hypoxanthine and inosine determination in edible fish. Food Chem. Amperometric biosensor for hypoxanthine based on immobilized xanthine oxidase on nanocrystal gold-carbon paste electrodes..

Kim.. I. 1997. Acta 394: 201–221. 56. Chim. Simultaneous determination of hypoxanthine.-J. Y. I. G.. 55.. . Volpe. Mascini.-S. Characterization of meat freshness application of a serial three-enzyme reactor system measuring ATP-degradative compounds. Park. M. Acta 404: 75–81. inosine and inosine 5′-monophosphate with serially connected three enzyme reactors. Kim. 1999. Talanta 44: 2151–2159.. Park. N. N. Cho. Anal. Amperometric detection of uric acid and hypoxanthine with xanthine oxidase immobilized and carbon based screen-printed electrode. Application for fish freshness determination. 2000. M.-A. Carsol.-S. Anal.68 ◾ Handbook of Seafood and Seafood Products Analysis 54. Chim..

.......1 Fatty Acid Methyl Esters Preparation .....................3.................................................................5 Qualitative and Quantitative Analyses of Marine Lipid Classes .....3.........................3.................................3...4.......1................................................................................................................................3 Column Chromatography...2.......................1 Isolation of Lipids from Tissues ....................................................................... 72 6.....................................................................2...........................4....3..........................................................3 Lipid Analysis in Marine Products ....................................... 73 6.................1................................................ 73 6..3 Lipid Manipulation and Storage ..........1................................................. 73 6.....................2..............1.............3.5...1 Acid-Catalyzed Esterification and Transesterification ... 72 6..............................................2 First Steps in Marine Lipid Analysis ........... 79 69 ........................... 75 6.....................................3 Marine Fatty Acid Analysis ................... 70 6.... 71 6..................................1..............Chapter 6 Lipid Compounds Santiago P.... 75 6.............................................................................................................4 Analysis of Marine Nonsaponifiable Matter ...................76 6....... 77 6.......... 77 6.........................................................................................................4 Lipid Quantification ......................2........................................2..............................1 Introduction .................2....76 6.....2 Analysis of Sterols ..................76 6......................2 Base-Catalyzed Transesterification ......1 Spectrophotometric Assessments of Total Lipid Extract .. 75 6..........................................1 Qualitative Analysis of Fatty Acid Composition ....... 70 6..........2 Stereospecific Analysis of Lipid Classes ............................................... Aubourg Contents 6.................5....76 6................... 73 6... 78 6....2 Quantitative Estimation of Fatty Acid Composition ...1 General Aspects of Lipid Compounds ............2 Marine Lipid Characteristics ...................5.... 73 6........................................................4..............2 Removal of Nonlipid Contaminants .......1 Lipid Saponification................................ 78 6......... 70 6...........2 GLC Analysis of FAME....3 Analysis of Ether Lipids ........................... 72 6.............................

......6 Silver Ion Chromatography .... and a larger proportion of highly unsaturated fatty acids.......5 High-Performance Liquid Chromatography ... Such diverse compounds as hydrocarbons.... Most animal and plant lipids from terrestrial and marine sources are similar in that they contain mainly even-numbered saturated and unsaturated fatty acids combined with glycerol (glycerides and glyceryl ethers). steroids.............5......7 Nuclear Magnetic Resonance (NMR) Spectrometry ....1................... phospholipids (PL)..... however..............1... 6....... glycoglycerolipids... soaps.... in it............................................. Marine species have shown large variations in lipid content and composition as a result of endogenous and exogenous effects [5–7]...................... Because of their structural and functional variety... the catching season has been shown to play a key role regarding temperature and feeding availability......70 ◾ Handbook of Seafood and Seafood Products Analysis 6.. particularly C20:5ω3 (eicosapentaenoic acid.. gangliosides..81 6............. such as nutritional lipid-soluble vitamins (namely A and D) and essential and ω3 polyunsaturated fatty acids (PUFA) that have shown a positive role in preventing certain human diseases..... sterols (sterol esters)..... chloroform...................................1 Introduction 6....................... although no satisfactory or widely accepted definition exists... 80 6...................................... longer-chain fatty acids... Related to exogenous effects...... Seafood lipids are known to provide high contents of important components for the human diet.... 79 6.....5.5................. and lipopolysaccharides would be included.......4 Thin-Layer Chromatography . and amines (PL).................................. An alternative division into two broad classes has been shown to be convenient for lipid analysts [2]...... indeed.... such as hexane.................8 Mass Spectrometry .....1 General Aspects of Lipid Compounds Lipids are found in all living organisms and have been shown to play two critical roles: (1) maintaining the integrity of plants and animals as structural compounds by forming a barrier separating the living cell from the outside world and (2) being a major source of cellular energy and function in living organisms where they can be stored.. phosphoric acid..9 Supercritical Fluid Chromatography ..................... toluene............... EPA) and C22:6ω3 (docosahexaenoic acid.. triacylglycerols (TG)........ a widely accepted division has been difficult.......... Different attempts have been carried out to define what is meant by the term lipid....... differ from the other sources in that they contain a wider range of fatty acids............ lipid is usually the second largest biochemical constituent after protein......... ethers.. Most textbooks describe lipids as a group of naturally occurring compounds............. an inverse ....5.............. including cardiovascular ones [3]. carotenoids............ 80 6....... Marine lipids.. DHA) [4].2 Marine Lipid Characteristics In many marine organisms.... “simple lipids” (fatty acid and alcohol components) would be those that yield on hydrolysis at most two types of different products per mol...... which have in common a ready solubility in organic solvents.. whereas “complex lipids” (glycerophospholipids... 79 6............... 82 6.5... 82 References . and alcohols..... A simple physicochemical classification that empirically groups lipid molecules according to the hydrophilic–lipophilic balance has been proposed [1].... and sphingolipids) would yield three or more types of products per mol... fatty alcohols (wax esters).............5....

6.1. sex. age. With respect to endogenous effects. .3 Lipid Analysis in Marine Products Researchers are required to analyze the lipid composition and its changes that arose during processing of food material from marine sources. the equipment. differences according to the type of muscle and its location. A basic protocol procedure is exposed in Figure 6. and sexual maturation have been pointed out. probably affected by physiological and anatomical factors. lipid matter has been described to exhibit a heterogeneous distribution throughout the body of marine species. In all cases. Marine products Lipid isolation from tissues Removal of nonlipid contaminants Frozen storage Fatty acid analysis Lipid classes analysis Traditional and advanced analytical methodology Figure 6. Thus.Lipid Compounds ◾ 71 relationship between unsaturated fatty acid content and environmental temperature has been confirmed for many marine fish. content variations have specially been observed in fish locations to be employed as lipid depots. but mainly the amount of information required. The approach to the analysis of lipids in a given marine sample will depend on the amount of material in the sample.1. The present chapter is focused on describing the available traditional and advanced analytical methodology to assess the lipid composition of marine species on the basis of a food technologist and nutritionist requirements.1 Basic steps to be carried out for the lipid analysis of marine products. and instrumentation available.

or lyso-phosphatidylglycerols in lipid extracts. Where large amounts of tissue have to be extracted. the tissue should be kept frozen (about −60°C or less) as rapidly as possible. peptides. but many of these are not suitable for extracting lipids from tissues as they are not sufficiently polar to overcome the strong forces of association between tissue lipids and the other cellular constituents. The second problem is endogenous lipolytic enzymes that can lead to large amounts of unesterified fatty acids. Its employment has recently been reviewed [10]. this method applies a single-phase solubilization of the lipids using chloroform–methanol (1:1) in a solvent–tissue ratio of 4:1. In addition. First. supercritical fluid extraction shows an increasing demand.2 Removal of Nonlipid Contaminants Most polar organic solvents used to extract lipids from tissues also extract significant amounts of nonlipid contaminants such as sugars. all solvents can contain contaminants.. most workers in the field appear to accept two basic routines currently in general use. At the same time. a major driving force being the environmental concern regarding the use of organic solvents. may on occasion be extracted by these when they are in the presence of large amounts of simple lipids such as TG. The ideal solvent or solvent mixture for extracting lipids from tissues should be sufficiently polar to remove all lipids from their association with cell membranes or with lipoproteins but should not react chemically with those lipids. it is advisable to include an additional antioxidant at a level of 50–100 mg/L to the solvents.1 Isolation of Lipids from Tissues Ideally. and salts. diacylglycerides. which yield essentially quantitative extractions of the major lipid classes when applied to homogenates of whole marine tissue extractions. When this is not feasible. However. Pure single lipid classes are soluble in a wide variety of organic solvents.72 ◾ Handbook of Seafood and Seafood Products Analysis 6. which do not normally dissolve readily in nonpolar solvents. phosphatidic acid. A more elegant and complete.88% KCl) [8]. although endogenous tissue antioxidants can provide some protection. amino acids. . Although there are limitations to its use and alternatives are frequently suggested. 0. As an advanced alternative. polar complex lipids. urea. marine tissues should be extracted from the living organism as soon as possible after catching or slaughtering [2]. Two main problems can arise with lipid fraction when employing inconvenient storage conditions.2. PUFA can autoxidize as a result of endogenous oxidant enzymes. any such impurities can be troublesome.g. it should not be so polar that TG and other nonpolar simple lipids do not dissolve and are left adhered to the tissues. 6. the procedure of Bligh and Dyer [9] offers some advantages as it does not use large volumes of solvent. method of removing nonlipid contaminants is to carry out the washing procedure by liquid–liquid partition chromatography on a column of a dextran gel such as Sephadex G-25. The most popular is the method of Folch et al. though more time-consuming. This type of washing procedure was first developed by Wells and Dittmer [11] and simplified later by Wuthier [12] for large numbers of samples.2. For all extraction methods. which employs chloroform–methanol (2:1) in a solvent–tissue ratio of 20:1. such as proteins. Most of the contaminating compounds can be removed from the lipid extract mixtures simply by shaking the combined solvents with one-quarter their total volume of a dilute salt solution (e. [8]. and as large volumes of solvents may be used to obtain small amounts of lipids.2 First Steps in Marine Lipid Analysis 6.

Conversely. but it is usually advisable to add further synthetic antioxidants to storage solvents at the level of 50–100 mg/L [2]. large volumes of solvents are best removed by means of a rotatory film evaporator at a temperature that. Natural tissue antioxidants. Then. 6. relatively important errors are obtained. 6. Storage temperature should be −30°C as the highest temperature. Owing to the wide variety of fatty acid compounds in marine lipids (Table 6.1.2. the Soxhlet method of extraction has been developed [13]. and Fatmeter measurements is proving to be of increasing interest [14]. a known aliquot of the purified lipid extract is softly heated and the resulting dry lipid matter weighted.2. it has been shown that lipids can themselves dissolve in some plastics. According to the special relevance recently acquired by noninvasive technologies. Two basic strategies can be applied [15. such as tocopherols. Their measurement by gas–liquid chromatography (GLC) is the most commonly used method for lipid analysis. should not exceed about 40°C. Small volumes of solvent can be evaporated by carefully directing a stream of nitrogen onto the surface of the solvent.1 Acid-Catalyzed Esterification and Transesterification Free fatty acids (FFA) are methylated and O-acyl lipids transmethylated by heating them with a large excess of anhydrous methanol in the presence of an acidic catalyst.Lipid Compounds ◾ 73 6. near-infrared (NIR) spectrometry. lipids should be handled in an atmosphere of nitrogen. application of nuclear magnetic resonance (NMR). fatty acids . since PUFA will oxidize rapidly in air [2]. 6.1 Fatty Acid Methyl Esters Preparation Fatty acids are essential components of lipids. leading to selective losses of a proportion of the less polar constituents. In it.4 Lipid Quantification For most common purposes. Lipid extracts have to be converted into methyl ester derivatives. When it is necessary to concentrate lipid extracts. and care must be taken at all steps in the analysis of lipids. fatty acid methyl esters (FAME) obtained are usually introduced in the GLC system without previous removal of contaminants. For fast purposes.3 Lipid Manipulation and Storage Wherever possible. this analysis is more complicated than that with other kinds of living organisms. In addition.16]: acid catalysis and base catalysis. if not. Lipids should not be left for any time in the dry state and should be stored in an inert nonalcoholic solvent such as chloroform from which air is excluded by flushing with a stream of nitrogen. a large diethyl ether volume is employed. afford some protection to lipid extracts.3. Plastic ware of all kinds (other than that made from TeflonTM) can be specially troublesome and is best avoided.3. As storage containers.3 Marine Fatty Acid Analysis 6. provided water absorption onto the dry extract lipid is avoided. glass is the best choice. and the resulting lipid extract can no more be employed for further analysis. This method proved to be accurate in the case of a high lipid content (low complex lipid content).1). since plasticizers are very easily leached out. Autoxidation of double bonds in marine lipid fatty acids is particularly troublesome. in general.

19-Docosahexaenoic Linoleic Linolenic Stearidonic Araquidonic EPA DPA or clupanodonic DHA or cervonic In all cases. Fatty Acids Commonly Present in Marine Speciesa Systematic Name Trivial Name Abbreviated Name Saturated Fatty Acids 14:0 15:0 16:0 17:0 18:0 20:0 22:0 24:0 Tetradecanoic Pentadecanoic Hexadecanoic Heptadecanoic Octadecanoic Eicosanoic Docosanoic Tetracosanoic Myristic — Palmitic Margaric Stearic Arachidic Behenic Lignoceric Monounsaturated Fatty Acids 16:1 ω7 18:1 ω9 18:1 ω7 20:1 ω11 20:1 ω9 22:1 ω11 22:1 ω9 24:1 ω9 9-Hexadecenoic 9-Octadecenoic 11-Octadecenoic 9-Eicosenoic 11-Eicosenoic 11-Docosenoic 13-Docosenoic 15-Tetracosenoic Palmitoleic Oleic Vaccenic Gadoleic Gondoic Cetoleic Erucic Nervonic Polyunsaturated Fatty Acids 18:2 ω6 18:3 ω3 18:4 ω3 20:4 ω6 20:5 ω3 22:5 ω3 22:6 ω3 a 9.15-Octadecatetraenoic” .17-Eicosapentaenoic 7.14-Eicosatetraenoic 5.12-Octadecadienoic 6.7.16. the double-bond configuration is “cis.74 ◾ Handbook of Seafood and Seafood Products Analysis Table 6.10.

In order to guarantee complete solution of nonpolar lipid classes. an additional solvent is necessary to solubilize nonpolar lipids such as cholesterol esters or TG. The reagent has a limited shelf life unless refrigerated.1 Qualitative Analysis of Fatty Acid Composition During the previous decades. This reagent has been applied directly to fish muscle to obtain FAME without previous lipid extraction [17]. 6. under base catalysis. a further solvent such as toluene should be employed. This is simply prepared by adding acetyl chloride slowly to cooled dry methanol. . known commercial FAME have been employed for the provisional identification of fatty acids by direct comparison of their retention times and those of the unknown esters on the same columns under identical conditions. a PUFA loss.3. A different possibility consists of employing a solution of 1%–2% (v/v) concentrated sulfuric acid in methanol. FFA are not esterified. aldehydes are not liberated from plasmalogens and amide-bound fatty acids are not affected. The retention times of the unknown acids should be measured under identical operating conditions. 6. Boron trifluoride in methanol is also used as a transmethylation catalyst and in particular as a rapid esterifying reagent for FFA.3. parameters known as equivalent chain lengths (ECLs) or carbon numbers have considerably been employed. so most performances have been carried out for qualitative and quantitative analysis [16]. although potassium methoxide or hydroxide have also been used as catalysts. accordingly. and the use of old or too concentrated solutions may result in the production of methoxy-substituted acids from unsaturated fatty acids and. ECL values can be calculated from an equation similar to that for Kovats’ indices or found by reference to the straight line obtained by plotting the logarithms of the retention times of a homologous series of straight-chain saturated FAME against the number of carbon atoms in the aliphatic chain of each acid. The commonest and mildest reagent used for the purpose is anhydrous hydrogen chloride in methanol. Initially. the application of wall-coated open tubular (WCOT) columns to the analysis of fatty acids has provided a better knowledge of the complexity of marine fatty acids [19]. Transesterification is carried out in the same manner and at much the same rate as with methanolic hydrogen chloride. glasspacked columns were widely employed [18]. As with acid-catalyzed procedures.1. The commonest reagent used for this purpose has been sodium methoxide in anhydrous methanol. FAME are obtained by heating the reaction mixture in a stoppered tube at 50°C overnight. prepared simply by dissolving fresh clean sodium in dry methanol. and the ECL values are read directly from the graph. 6. Parallel to ECL value employment. whereas aldehydes are liberated from plasmalogens under acidic conditions.Lipid Compounds ◾ 75 from amide-bound lipids (sphingolipids) are also transesterified.3.2. However. However. Later on.2 Base-Catalyzed Transesterification O-acyl lipids are transesterified very rapidly in anhydrous methanol in the presence of a basic catalyst.2 GLC Analysis of FAME The advent of GLC revolutionized the analysis of the fatty acid components of lipids.

within limits. as well as the deacylated residues of ether lipid compound. In most cases. and there is no way of overcoming this difficulty entirely. Results can be expressed as weight percentages of the fatty acids present or as molar amounts of each fatty acid. quantitative results would first be calculated on its basis.4.” 6.76 ◾ Handbook of Seafood and Seafood Products Analysis In recent years. Total sterol content can be determined directly and spectrophotometrically from the lipid extract by using the method of Huang et al.4-dimethyloxazoline derivatives of fatty acids have been found to show several advantages and have been applied successfully to the structural determination of PUFA and cyclopropenoid fatty acids [21].2. the fatty acids on one side and diethyl-ether-soluble nonsaponifiable materials on the other side are separately recovered for further analysis [2]. pyridinecontaining derivatives. the nonsaponifiable layer will contain any long-chain alcohols and sterols originally present in the lipid sample in the esterified form. According to the previous section. If necessary. On the other hand.1 Lipid Saponification Lipids may be hydrolyzed (saponified) by heating them under reflux with an excess of dilute aqueous ethanolic alkali. On the other hand. However. branched. and polyenoic fatty acids should be analyzed under the same GLC conditions for checking the quantification results.16].4 Analysis of Marine Nonsaponifiable Matter 6. commercially available standard mixtures containing accurately known amounts of methyl esters of saturated.4. the areas under the peaks on the GLC traces are. based on the Liebermann–Buchardt reaction.2 Quantitative Estimation of Fatty Acid Composition With reliable modern gas chromatographs equipped with flame ionization detectors (FID). this is specially relevant for PUFA. Finally. cyclic. Problems of measuring this area arise mainly when components are not completely separated. sterols can be fractionated and analyzed by means of different . A high proportion of the available data has been obtained for the methyl ester derivatives of fatty acids. such as picolinyl esters. the basic structure of which includes the cyclopentanophenanthrene ring.3. Such compounds can be divided into sterols and “ether lipids. 4. 6. GLC/mass spectrometry (MS) has been widely accepted as one of the most valuable techniques for the identification of fatty acids and their derivatives [20]. For a complete analysis. calibration factors may have to be calculated for each fatty acid component to correct the areas of the relevant peaks in the mixtures analyzed. have been shown to be suitable for direct mass spectrometric structural analysis of acids containing straight. [22]. 6. linearly proportional to the amount (by weight) of material eluting from the columns [15. A known quantity of an internal standard should be added to the lipid sample. nonadecanoic acid is employed and added before the methylation step.2 Analysis of Sterols Sterols are biological compounds. or oxygenated chains. the resulting FFA have to be transformed into their corresponding FAME for further analysis by an acid-catalyzed method. unsaturated. as these are easily prepared and are widely used in chromatographic analysis. monoenoic.

Accordingly. and 18:1. batyl.4-dinitrophenyl-hydrazine (0. Great attention has been accorded to the assessment of cholesterol oxide formation in marine products [29]. high-performance liquid chromatography (HPLC) methods can offer a nondestructive alternative.3 Analysis of Ether Lipids Marine lipids may contain fatty acid residues as the only radicals.3-diacyl-sn-glycerols are generally saturated or cis-monoenoic even– numbered components (16:0. marine sterols have to be converted into more volatile compounds such as acetate [25] and trimethylsilyl (TMS) [26] derivatives. which generates dimethyl acetals from the liberated fatty aldehydes. Often. v/v) as a solvent system. and combinations of techniques must be used until the required purposes are served.Lipid Compounds ◾ 77 chromatographic techniques [23. 6. Adsorption thin-layer chromatography (TLC) on silica gel layers can be used to separate simple alkyl and alkenyl lipids. trifluoroacetate. Although the vinyl ether linkage is unaffected by basic hydrolysis conditions. alkenyl compounds have been directly identified and quantified by GLC together with FAME [35]. Further. The alkyl moieties are usually analyzed in the form of 1-alkylglycerol or as volatile nonpolar derivatives of this compound. which in turn migrate just in front of TG. Unlike fatty acids.3.24]. They can be separated by a double development in a single direction with hexane–diethyl ether (95:5.32]. or they may include alkyl and alkenyl radicals. or isopropylidene derivatives by GLC. Concerning alkenylglycerols. such as acetate. thus. such compounds tend to be decomposed during GLC analysis and are best reduced by catalytic hydrogenation to alkylglycerols. supercritical fluid chromatography (SFC) has been employed for the glycerol ether analysis of liver oils of shark species [34]. it can be cleaved by acid-catalyzed transmethylation. In this section. no single procedure will achieve the desired analysis. different analytical . TMS. although invertebrates have shown a significant presence of other sterols [27]. The alkyl groups of 1-alkyl-2. The first type is the major one in marine lipids.4. Cholesterol has been shown to be the most abundant sterol in all marine living beings. although a great interest has been accorded to their isolation because of their medical and cosmetic applications [30]. Methods for separating and quantifying ether-linked glycerides have been reviewed [31.5 Qualitative and Quantitative Analyses of Marine Lipid Classes Lipid samples obtained from extraction of biological material are complex mixtures of individual lipid classes [16]. 6. information on ether lipid composition in marine PL is less abundant. Chromatographic methods for cholesterol analysis [28] are of relevant importance in foods in relation to human health concerns. 18:0. The others are often united into a group called “ether lipids. The determination of double-bond positions in long alkyl chains has been carried out by means of picolinyl and nicotinylidene derivatives by GLC-MS [33]. and selachyl alcohols were found to be the most abundant. For GLC analysis. neutral plasmalogens tend to migrate ahead of alkyldiacylglycerols.4%) in 2M HCl. but they suffer from the limitation of the lack of a distinctive chromophore in the analyte.27]. mostly). Neutral plasmalogens may be detected by spraying the TLC plates with 2. Although the GLC is normally carried out with cholestane as internal standard. chimyl. being normally placed as the radical in position 1 and specially abundant in marine invertebrates [5. and its analysis has already been discussed in Section 6. whereas no simple spot test is available for the identification of alkyldiacylglycerols.” Such compounds are basically found as PL classes (specially in phosphatidylethanolamine).

A very popular one is that proposed by Lowry and Tinsley [36]. this including chromatographic separation and further analysis of fatty acids after previous methylation and transmethylation. Most living organisms have developed lipolytic enzyme systems that are able to distinguish between bonds to the various positions of glycerol or between certain types of bonds in specific lipids. then. Compared with the data compiled for plant oils and for fats from land animals. prepared by esterification or transesterification of the purified lipid class. In it. Accordingly. In many cases. Procedures that involve spectrophotometric measurement of highly colored copper complexes are now favored. since the presence of double bonds in the proximity of a carbonyl group of fish PUFA reduces the rate of deacylation of glycerides. such functional groups are made to react with hydroxamic acid and further complexed with Fe (III). and GLC technologies combined with MS in the last decades has provided quick and useful procedures for the stereospecific lipid analysis. Traditional determination of PL content in lipid extracts has involved the digestion of PL with the release of inorganic phosphate. However. this is made to react with ammonium molybdate to form phosphomolybdic acid. 6.1 Spectrophotometric Assessments of Total Lipid Extract Some classical methods are available for the analysis of lipid classes or lipid class groups when applied directly onto the lipid extract without prior separation. a rapid NIR spectrometry has been applied for the direct FFA determination in fish oil [37].2 Stereospecific Analysis of Lipid Classes The determination of fatty acid composition at each location in lipid classes has ever since attracted great attention. A wide use was found for lipase hydrolysis. Additionally. a method for the quantification of esterified and unesterified total sterols is mentioned in Section 6. An alternative and successful method has been proposed by Raheja et al. Finally.2 [22]. focused on the qualitative and quantitative analyses of marine lipid classes. although some interference of polar lipids was found. Ester linkages can be quantified by the method of Vioque and Holman [40]. HPLC. preparative TLC on silicic acid impregnated with 5% (w/w) boric acid has been applied to prevent acyl migration during chromatographic separation. according to the information provided in following sections. . the results so far reported for aquatic animals are few.78 ◾ Handbook of Seafood and Seafood Products Analysis approaches will be discussed. More recently.4. titrimetric methods were used for many years.3. This method can be applied to total lipid extract or to any lipid class after previous isolation [7. where an FFA-cupric acetate-pyridine complex is involved.5. according to details explained in Section 6. The advent of new NMR.41]. PL present in the lipid extract are made to react with ammonium molybdate in an organic phase and then measured spectrophotometrically. although it turned out not to be accurate enough for marine lipids. these enzymes can be isolated and used in simple incubations in vitro as an aid in structural analyses of lipids. [39] without previous digestion. The fatty acid composition of each lipid class can be determined by GLC of the methyl ether derivatives. 6. traditional methodologies are still employed in cases where such advanced technologies are not available and are reviewed in this section. giving rise to a tremendous number of species. which is reduced and determined spectrophotometrically [38].5. the Grignard reagent has widely been employed in the case of marine substrates [42]. For FFA assessment. in it. This is due to the great complexity of fatty acids present in these oils and fats.

infrared (IR) spectrometry. 6. and tubular TLC (TTLC).Lipid Compounds ◾ 79 Traditional research accounts for consecutive series of methods combining chemical reactions and enzymatic releases of fatty acids in different positions for resolution of the molecular species. Those used most frequently contain hexane. HPLC has undoubtedly been the most widely applied separation technique in the analysis of most simple and complex lipid classes [48.5 High-Performance Liquid Chromatography In recent years.5. The system has been successfully used for marine lipid class analysis [51]. and quantification [48. For preparative purposes. 6. In addition. Such stereospecific studies have widely been focused onto TG [43.5. lipid classes can be detected by any of the nonspecific available reagents and identified by their migration characteristics relative to authentic standards chromatographed simultaneously alongside the samples under investigation.49].41]. 20–50 mg of marine lipid may be applied with ease as a band on a 20×20 cm plate coated with a layer of silica gel of 0. coupling of TLC with other techniques such as HPLC.5 mm thickness [7. A variety of solvent systems have been used to separate simple lipids on an analytical or semipreparative scale by single or two-dimensional TLC.4 Thin-Layer Chromatography Many text books and reviews report TLC application on lipids for routine separations. or florisil as adsorbents. whereas complex lipids are recovered by elution with methanol [41. and this has led to the evolution of the TLC/FID Iatroscan system.46] classes. in spite of the relatively higher costs [50]. and NMR has increased its analytical power in several applications. MS. Separation can be carried out on silicic acid. It combines the separation capabilities of conventional TLC with the quantification power of the FID and has application in the quantitative analysis of all substances separable by conventional TLC.5. In all cases. precoated plates are much more convenient than laboratory-made plates. Aminopropyl-bonded phase cartridges have been much used for the isolation of simple and complex lipid fractions. although particular care is required to recover the acidic lipids quantitatively. identification. diethyl ether. and acetic (or formic) acid in various proportions. The perceived weakness of TLC has been recognized as the quantification aspect.3 Column Chromatography Normal-pressure or low-pressure column chromatography (CC) was widely employed in the past and is now mostly used as a way of preliminary fractionation of lipid classes. being simple lipids eluted in a stepwise sequence with hexane containing increasing proportions of diethyl ether.47]. Such techniques would include high-pressure TLC (HPTLC). acid-washed florisil. overpressure TLC (OPTLC).44] and PL [45. although lengthy conditioning may be necessary before columns can be employed [2. 6.52]. However. Column chromatography on diethylaminoethyl (DEAE)-cellulose has shown to be a valuable method for the isolation of particular groups of complex lipids in comparatively large amounts. The principal advantages of the method are the ease of preparation of a column and the comparatively large amount of lipid that can be separated. which include highly automated techniques right from sample application and development to detection and quantification. which has been used routinely for lipid analysis in the last decades.47]. The improvement and versatility of TLC enable it to be used for several modern applications. HPLC is much more expensive than .

However. but others have obtained satisfactory results. In the past 20 years. The complexes are usually unstable and exist in equilibrium with the free form of the olefin. high-resolution NMR spectrometry (1H-NMR. Ag+-TLC is used mostly in the preparative mode.54]. Thus. The usual supporting materials are silica gel G for FAME and TG and silica gel H for complex lipids. 6. An isocratic and gradient elution procedure with ultraviolet (UV) detection has been employed for marine PL analysis. quantification and stereospecific analyses have been carried out. and in particular to the detection. Thus.80 ◾ Handbook of Seafood and Seafood Products Analysis TLC in terms of both equipment and running costs. such complexation is favorable for use in chromatography and enables the performance of the various Ag+-chromatographic techniques developed so far. Both homemade and precoated glass plates are used in Ag+-TLC. and often the location. but it can be automated to a considerable degree and gives much cleaner fractions in micropreparative applications. Perona and Ruiz-Gutiérrez [53] were able to resolve a large number of sardine TG molecular species by RP-HPLC. or substitution. being successfully applied to all lipid classes in marine species by separating molecules according to unsaturation degree [55]. It can give better and more consistent separations of minor components. further identification of most peaks was carried out by using preparative Ag+-TLC followed by fatty acid analysis by GLC. while no oxidation of the unsaturated fatty acid constituents needs to occur during fractionation on an HPLC column. of the double-bond systems in fatty acid chains.6 Silver Ion Chromatography Silver ions. the complementary employment of GLC or GLC-MS together with Ag+-TLC is considered one of the most powerful tools for elucidation of fatty acid composition in complex lipid samples [56]. HPLC analysis has been accepted as the most accurate one. TG separation according to the carbon number or partition number has been achieved [53]. Ag+-HPLC and reverse phase (RP)-HPLC applied in complementary ways were effective in the analysis of TG in fish oils [57]. In the detection. some of the more impressive separations have made use of FID systems. TLC.5.5. so it has become an extremely powerful technique for obtaining qualitative and quantitative information of the lipid class profile of a marine tissue extract. as a complementary separation method to GLC or GLC-MS. some important articles and reviews have been published [58]. 6. with UV detection at 206 nm both on an analytical and on a preparative scale. For PL classes. therefore. Finally. . 31P-NMR) has increasingly been applied to the identification of lipid structures to determine patterns of branching. Thus. and HPLC. HPLC has specially been applied to the most abundant lipid classes. The procedure is rapid and nondegradative. interact specifically with the olefinic double bonds of unsaturated compounds to form weak charge transfer complexes. evaporative light-scattering detection has successfully been applied [16].7 Nuclear Magnetic Resonance (NMR) Spectrometry In recent years. by employing both gradients of polar solvents and microparticulate silicic acid [6. On the other hand. Ag+-chromatography has been performed in conjunction with CC. like the ions of other transition metals. 13C-NMR.

many of the advances in MS have involved new ionization techniques. Arpino [67] likened the HPLC-MS union. FFA carbonyl resonances were detected at the lower field of the carbonyl region. so little application is specially available for marine lipids [58].5. but. The first step for any MS method is ionization of the sample molecules in the gas phase. the molecules or their fragments can be separated and identified on the basis of their mass-to-charge ratio (m/z). Finally. the condensed mobile phase used for liquid separations is not readily compatible with high vacuum ionization sources. ω3. although GLC is conveniently coupled to electron impact ionization (EI) and chemical ionization (CI) sources. This development paralleled the development of atmospheric pressure chemical ionization (APCI).66].95 ppm) with respect to the methyl resonance of all other fatty acids (δ = 0. Following ionization to a negatively or positively charged species (most commonly the later). results obtained using high-resolution 13C-NMR were in good agreement with those obtained by GLC. 6. soft ionization MS techniques such as fast atom bombardment. and complete structure of an unknown compound. The 31P-NMR application has also shown the possibility of analyzing the ether structures within the glycerol backbone of phosphatidylethanolamine and phosphatidylcholine. fragmentation of the molecular ion species produced by soft ionization processes can further be achieved in a second mass spectrometer (MS/MS) by collision-induced dissociation. although an increasing importance has been obtained lately for quantitative analysis [20.8 Mass Spectrometry MS has long been used as a powerful tool for the analysis of the molecular weight. 2. It could be observed that DHA was concentrated in the 2-location of TG in depot fats. and electrospray have the ability to ionize lipid molecules without causing extensive fragmentation.86 ppm) provided the possibility of proposing this new analytical tool. marine lipids have received lesser attention. Later on [61]. 13C-NMR spectrometry was successfully used to determine the proportions of saturated.Lipid Compounds ◾ 81 Based on 1H-NMR spectrometry [59]. EPA. Recent developments in MS have been very interesting for complex lipid molecules. The information-rich nature of MS makes it the most desirable detector for many explanations. Quantitative analysis of fatty acid composition and alpha-beta distribution in TG tuna fish was achieved [62]. Thus. This NMR technique can provide a single signal for each PL class. In a first attempt for 13C-NMR application [60]. the high-resolution NMR spectra of four fish oils were recorded. and 3 locations) of ω3 fatty acids in depot fat of several fish species was examined by 13C-NMR [63]. and attention was focused on the identification of specific signals for ω3 fatty acid group and also individually for DHA. The positional distribution (1. thermospray. ω6. A good agreement could be observed between NMR values and those from the GLC analysis. empirical formula. a rapid and structure-specific method for the determination of ω3-PUFA in fish lipids was presented.65. and stearidonic acid. The different chemical shifts observed for the methyl resonance of ω3-PUFA (δ = 0. Signals in the spectra were assigned. 13C-NMR was employed for the plasmalogen analysis in fish lipid samples showing a good agreement with the data obtained by GLC [64]. Among the different food lipids. Some applications concerning the marine lipids’ study will now be mentioned. Over the years. Thus. and highly unsaturated fatty acids of lipid extract of Atlantic salmon muscle. its intensity should be proportional to the quantity. according to each corresponding resonance.and diene-. probably due to their more complicated structure. Application of 31P-NMR has shown to be far shorter than with 1H and 13C. After different approaches. thus providing a suitable tool for lipolysis analysis. . mono.

J. References 1. analysis was carried out in conjunction with FAME by means of their dimethyl acetal derivatives resulting from the acid transmethylation of lipid extracts. 1986. cholesterol esters. a nonpolar capillary column. U. and Oehlenschläger.. 4. London. Vol.. 2. Carbon dioxide is by far the most commonly used SFC mobile phase because of its low critical temperature. A.K. Small.. Plenum Press. T.. 3. Concerning marine species analysis.-dimethyloxazoline derivatives [69]. which has great sensitivity and linearity.82 ◾ Handbook of Seafood and Seafood Products Analysis The oxidative decomposition of cholesterol in different fish products was investigated by means of MS analysis of cholesterol oxide TMS derivatives with a quadrupole mass spectrometer fitted with an EI source [68]. The mass spectrometer was operated in the EI mode (70 eV). New York. simple classes from marine oils of different species were separated and quantified by capillary SFC [73]. The liver oils of several shark species were analyzed by SFC [34]. analytes are eluted from a capillary chromatographic column. such as mixed glyceride compositions ranging from 200 to 900 in molecular weight. p. 89. 1997. J. The Physical Chemistry of Lipids.. an optimization of process parameters was achieved to obtain a maximal production rate. An important advantage is that it is compatible with FID.71].K. 2nd edn.. . p. p. thus. carbon dioxide as the mobile phase. the method was based on the use of preparative reversed-phase HPLC followed by subsequent identification by APCI HPLC-MS. the method was capable of direct quantification of squalene and cholesterol. in Seafood From Producer to Consumer.. 17. Börrensen. The qualitative and quantitative compositions of 1-O-alk-1-enylglycerolipids of albacore tuna (Thunnus alalunga) were studied along the canning process [35]. whereas quantification of TG. W. A wide range of cholesterol oxides were identified and quantified. Nutritional aspects of fish. Lately... and several nonmethylene interrupted fatty acids were singled out. Simopoulos. and the four most unsaturated fractions were analyzed by capillary SFC according to their acyl carbon numbers [72]. which uses a highly compressed gas above its critical temperature and critical pressure. Handbook of Lipid Research. D. Analytical SFC has been shown to be particularly applicable to the analysis of higher molecular weight lipid moieties. Later on. 6. and diacylglycerol ethers required TLC fractionation before SFC analysis. U. Luten. Minor fatty acids from mussels (Mytilus galloprovincialis) were enriched by Ag+-TLC and then analyzed by GLC-MS as 2-alkenyl-4. the use of SFC can substantially reduce the dependence on organic solvents in solvent extraction or HPLC analysis.. Christie. Oxford. in a first attempt Baltic herring flesh TG were separated in eight fractions by Ag+-TLC. Lipid Analysis. whereas its critical pressure and critical density are high enough for good solvation of many potential analytes. Integrated Approach to Quality. 589.4. 1982. Rezanka [70] described a method for the enrichment of long-chain fatty acids from fatty acids of a green freshwater alga and their identification as picolinyl esters by means of HPLCMS with APCI. eds. Purification of PUFA (DHA and EPA) ethyl esters from tuna oil was carried out by SFC [74].5. and a FID were employed in it. In addition. Pergamon Press.9 Supercritical Fluid Chromatography In this advanced technique [10. Elsevier Science.

. Vol. New York. Biol. Elsevier Applied Science. 2003. Wuthier. eds. IL. p. Food Res. Supercritical fluid chromatography (SFC)-Global perspective and applications in lipid technology. H. 1989. Food Sci. in New Trends in Lipid and Lipoprotein Analyses. Biochem. R. The use of sephadex for the removal of nonlipid contaminants from lipid extracts. 49. Wells. Technol. Fats. 6. and Panunzio. E. J. Patterson. 7. G..Lipid Compounds ◾ 83 4. Chromatographic separation of cholesterol in foods. Vaskowski. 21..... 113. 911. Cholesterol and fatty acids in several species of shrimp. 497.. Boca Raton. 108. 17.. R.. Fatty Acids and Glycerides. 27. Kuksis. R. 11. and Dyer. Champaign. 1994. Elsevier Applied Science. London. CRC Press.. 23. . p. J. Pérez-Martín.. poultry and fish.. Medina.. 1961. U. Ackman. E. and Garrido.. Chrom. 1. 54. American Oil Chemists’ Society Press. ed. Fatty Acids.K. ed. 624.K. Adv. Distribution and composition of lipids in marine invertebrates. M. G. S.. 1978. One-step conversion of fatty acids into their 2-alkenyl-4. Plenum Press. in Analysis of Oils and Fats. J. WCOT (capillary) Gas–liquid chromatography.. 37. 1995. and Nielsen H. and Raftery. C. Eur. p. Separation and determination of structure of fatty acids. 137. D. Evaluation of soxhlet’s and Bligh and Dyer’s methods in the determination of fat in meat. 2003. Lees. 1989. Packed-column gas chromatography. 1989. R.. 341. Lipid Analysis. Ackman. 8.... and Wrebiakowski. and Oils. King.. 193. and Perkins. Fatmeter.. FL. J. Fats. 1. 20.. Boca Raton. Chromatographic methods in the analysis of cholesterol and related lipids. and Oils. 22. Bligh. 7. 1989. J.. FL. 2. Hamilton. “Warmed-over” flavor in meat. M.. and Rocha. and New York. 16.). 25.. U. S. 1986. p. Ackman. Ackman. Lipid Res. 24.4. Aubourg. 12.. J. J. Fats and Oils... 103.. ed. Bridgwater. J. and New York. Z. 191. E. G. 26. 101. Joseph. Teshima. 2005. J.... 1990. 9. M. 229. Influence of biological factors and comparison of different methods of analyses: Solvent extraction. 199. U. in Marine Biogenic Lipids.. J. CRC Press Inc. L. J. 1959. Huang. V. 28. Technol. eds.. J. 1986. Le Quéré... Ackman. 15.. 673. Christie. Nielsen. Can. Food Sci. Nielsen. R. A. p. 479. Purification of lipids from nonlipid contaminants on sephadex bead columns. 24. Lipid Res. 3rd edn. FL.K.. 237. W. Stability of lipids of frozen albacore (Thunnus alalunga) during steam cooking. J. eds. 1986. in Marine Biogenic Lipids. Direct transesterification of all classes of lipids in a one step reaction. 114.. E. in Analysis of Oils and Fats. Hammond. 1957. J. 1963. A simple method for the isolation and purification of total lipids from animal tissue. A stable reagent for the Liebermann-Buchardt reaction. Adlof.. Forsch. p. Chem. in Physiology and Biochemistry of Sterols. 1966.. in Marine Biogenic Lipids. and Nes.. B. 341. 2. 38. Int. V. 5. G. Phospholipids. J. and Dittmer. 1995.K. 23. Biochemistry. F. Hoving. Vol. 226. J. 18. Gas chromatography-mass spectrometry and tandem mass spectrometry in the analysis of fatty acids. NIR and NMR. Chen... 1972.-dimethyloxazoline derivatives directly from total lipids. A.. 671. 1405. A. R. Anal. E. Chem. The Oily Press. J. AOCS Press. Physiol. 14. Sterols and crustaceans. and Shorland. 1259. 558. 1977.. Seasonal study of the lipid composition in different tissues of the common octopus (Octopus vulgaris). C. 149. R.. Kuksis.. and Gallardo. Aubourg. Folch. T.. 537. CRC Press.. 37. J. Love. Lepage. A. Boca Raton. Sieiro. Chrom.. 2.. Pearson.. W. Unters. 301. Hyldig. J. Food Sci. England. S.. R. Lebensm. Krzynowek. p. Technol. 27. in Advances in Lipid Methodology—Five. and Rossell.. 1. U. P. 33. mollusks and fish. ed. Prost. and Stanley. Hamilton. 1992. R. London.. Fenton. p. p. I. Sebedio... Lipid content in herring (Clupea harengus L.. A. Champaign. and Rossell.. J... Bridgwater.. Vol. R.. IL. J. 13. The Oily Press. in Handbook of Lipid Research. 2006. 1989. J.. ed.. 369. eds.. and Roy.. Chrom.. Lipid Sci. A rapid method of total extraction and purification. W. 19. J. p. 10.. F. Wefler..

33.. 175... American Oil Chemists’ Society Press.... G.. A. Lipids. London. p. New colorimetric method for the quantitative determination of phospholipids without digestion.. 1973. T.. 1383. Nicotinylidene derivatives for the structural elucidation of glycerol mono-ethers and mono-esters by gas chromatography/mass spectrometry. J. Food Chem. 35. W.. 43. 38. Hamilton... Hemming. Christie. Capillary supercritical fluid chromatographic analysis of shark liver oils. Singh. eds.. Magnussen. R. Estimation of polyunsaturated fatty acid content in lipids of aquatic organisms using thin-layer chromatography on a plain silica gel plate. IL.. 255. Chem. AOCS Press. Urata.. 44. 2001. 1986. 5. Lowry. 39. Thin layer chromatography and high-performance liquid chromatography.. Stereospecific analysis of triacylglycerols rich in long-chain polyunsaturated fatty acids. A. 37. 44. Agric. future potential. J. Quantitative estimation of esters by thin-layer chromatography. and Tinsley. 695. Stereospecific analysis of triacyl-sn-glycerols.. ed.. Lipid Res. 20. J. D. S.. p. 36. 1996. 31... J. Am. J.. Ohshima.. 186. G. J.. Lipid Analysis. 1989. IL. Aubourg.. Sargent. 1.K. Polyunsaturated fatty acids in tuna phospholipids: Distribution in the sn-2 location and changes during cooking. eds.. Agric. Sebedio... Kuksis. Food Chem. and Takaishi. R. Methods Enzymol. C. Zhang. and Perkins. Codony. 31.. 315. J. 53. I. T. R. Park. S. 1995. Agric.84 ◾ Handbook of Seafood and Seafood Products Analysis 29. 585. 1997. J.. Hamilton. 93. Sotelo. 1959.. Borch-Jensen. CRC Press Inc. K. 1996. 1995. Elsevier Applied Science. 73. 45.. p. .. Hamilton. and Rossell. R. and Tanaka. p. J. Raheja... Am.. G. Shantha. A.. 47.. 1991.. K.. in New Trends in Lipid and Lipoprotein Analyses. Occurrence of diacyl glycerol ethers in liver lipids of gonatid squid Gonatopsis borealis. Agric. 45. 35. Zonal distribution of fatty acids in albacore (Thunnus alalunga) triglycerides and their changes during cooking. in Marine Biogenic Lipids. 1996. Aubourg. 63. and Diersen-Schade. P. 46. Analysis of 1-O-alk-1-enyl glycerophospholipids of albacore tuna (Thunnus alalunga) and their alterations during thermal processing. and Mollerup. p. 497. 3515. R. London. 1. U. 40. Biol. 819. Nakamura. 41. 1989. Ether lipids based on the glyceryl ether skeleton: Present state. Determination of the positional distribution of fatty acids in glycerolipids. and Gallardo. 87. Food Chem... in Cholesterol and Phytosterol Oxidation Products. in Lipid Analysis in Oils and Fats.. R. R. V. Am. J. E. 1989. Guardiola. and Lee. Fats and Oils... Soc.. Champaign. Oil Chem. 207. Formation and content of cholesterol oxidation products in seafood and seafood products. ed. Vioque... R. and Bhatia.. 42. Gallardo. T. 37. K. and Holman. J. Rapid colorimetric determination of free fatty acids. and Pérez-Martín.. Thin-layer chromatography of lipids. J. 55. D. 1997. Oil Chem. eds.. Phosphorus assay in column chromatography.K. Myher. I. 39. Champaign. Hayashi.. 38. 48. Aubourg.. H. Nippon Suisan Gakkaishi. Fukuda.. 2395. Soc. and Pérez-Martín. Oil Chem.. 2002. M.. and Pérez-Martín. J. Soc. 1962. 1996. Medina. N. Harvey. Geher. 427. Editorial Acribia. H. IL. Zaragoza (Spain).. Ether-linked glycerides in marine animals. 234. 466. R. Champaign.. Brockerhoff. 17. 74. Kaur.. R. P. I. S.. Vol. Ackman. 1976. I. Blackie Academic and Professional.. J.. 51. 470. M. S. and Hawthorne. 1993. Sebedio. 1060. 32. 34. R. 41. Am.. Shukla. 49. FL. 30. 31. and Napolitano. S. Soc.. E.. Agric. J. F. Barlett. U. Oil Chem. C. Aubourg.. J. 1998. 1975. Boca Raton.. p. Biol.. Mass Spectrom. and Ackman. C.. F. J. Food Chem. J.. and Savage. in New Trends in Lipid and Lipoprotein Analyses. N. 50. J. Lipids. in Analysis of Oils and Fats. Dutta. 1990. Food Chem. 14.. Lipid analysis using thin-layer chromatography and the Iatroscan. eds. Rapid near-infrared spectroscopic method for the determination of free fatty acid in fish and its application in fish quality assessment.. p. 243. Lipid classes and their fatty acids at different loci of albacore (Thunnus alalunga): Effects of the pre-cooking. J. Medina. AOAC Press.. R. R.

K. Popov. Phys.. Diehl. 25. 409. Addeo. Joh. W.. Demirbuker. and Ruiz-Gutiérrez. 70. 1995. R. Mass spectrometry of complex lipids. Positional distribution of ω3 fatty acids in marine lipid triacylglycerols by high-resolution 13C nuclear magnetic resonance spectroscopy. Sacchi.K. Lipid analysis by silver ion chromatography. M. Soc. S. Bridgwater. Aursand. Food Chem. 1332. T. p. Composition of phospholipids of white muscle of six tuna species.. Amsterdam (Holland). in Advances in Lipid Methodology—Five. 1998... 1699. R. Am. H. 1998. Chrom. Arpino. in Advances in Lipid methodology—Five. High resolution NMR studies of fish oils. Oil Chem. N. 59. Adlof. Aursand. 64.. . Champaign... Am. and Christie. U. Blackie Academic and Professional.. 54. Huss.. Am.. 1998. 2002. APCI-MS in lipid analysis.. V..K. Lipids. R. 1247. Combination of silver ion and reversed-phase high-performance liquid chromatography in the fractionation of herring oil triacylglycerols.. 1995... R. p. W.. ed. 213. Medina. Soc. Sacchi. 68. I. Chem. J. in Lipid Analysis in Oils and Fats... England. Oil Chem. Hamilton. R. Li. Hamilton. 407.. England. P.. 41. London.... 56. AOCS Press. 1995. T. I. Chem. 57. Lipids. 43. Lipids. M.. 67. Aubourg. J. p. 1995.. ed. W. Chem. Adlof. 1997. R. I. and Grasdalen. Oxidative decomposition of cholesterol in fish products. Chim. 53. and Christie. Technol. The Oily Press.. 595.Lipid Compounds ◾ 85 52.. 65. Laakso. P.. J. and Paolillo. 69. Perona.. J.. Elsevier Science Publishers B. ed. 32. L.. Garrido. Acta. J. L. 30. U. G. in New Trends in Lipid and Lipoprotein Analyses. and Grasdalen.. F. in Lipid Analysis in Oils and Fats. R. S. A. B. London. Characterization of lipids by supercritical fluid chromatography and supercritical fluid extraction. p. One and two-dimensional NMR study of plasmalogens (alk-1-enyl-phosphatidylethanolamine).. 201. 1127. 70.. Kuksis. Multinuclear high-resolution nuclear magnetic resonance. reverse-phase detection methodology. 62. 63. L. C. London. 465. Medina... tri. Rainuzzo. Identification of minor fatty acids in mussels (Mytillus galloprovincialis) by GC-MS of their 2-alkenyl-4.K. 34.. 66. 22. Rezanka.. Hamilton. Blackie Academic and Professional.. I.. Oil Chem.. Addeo. Gunstone. and Koizumi. Sebedio. Medina. Sacchi. J. M.. 1982. 76. ed.. S.. High-performance liquid chromatography: Normal-phase. and Paolillo. Rel. and Andersson... Anal. Nikolova-Damyanova. Jørgensen. R. L.. U. 1991.4-dimethyloxazoline derivatives.. Aubourg. Sep. U. L. 2003. 1991. 68. V.. H. 70. 38.. R. Aubourg. Blomberg. p. and Medina.. On-line liquid chromatography/mass spectrometry? An odd couple! Trends Anal.. M.. IL. J.. V. Y. R. 55. 171. p.. Stefanov. 58.and tetraenoic fatty acids with bis-methylene-interrupted double-bond systems from the sponge Haliclona cinerea. ed.. 1993.. Quantitative high resolution 13C-NMR analysis of lipids extracted from the white muscle of Atlantic tuna.. Identification of very long chain fatty acids by atmospheric pressure chemical ionization liquid chromatography-mass spectrometry from green alga Chlorella kesslerri.. F. 1992.. Novel di-. 1999. Shukla. Elenkov. 1. 71.. Bridgwater. J. Proton nuclear magnetic resonance rapid and structure-specific determination of ω-3 polyunsaturated fatty acids in fish lipids. 61.. and Paolillo. Oshima. H. Soc. 72. Agric. 293. 1993.K... Byrdwell.. I. and Pérez-Martín. Giudicianni. Blackie Academic and Professional. S. Studies of fatty acids in Atlantic salmon (Salmo salar) by 13C and 1H nuclear magnetic resonance (NMR) spectroscopy. 2002. Sci. eds. Liq. England. 59. p. Soc. B. R. Characterization of the triacylglycerol molecular species of fish oil by reversed-phase high performance liquid chromatography. I.... 60. 87. and Ackman. J. in Quality Assurance in the Fish Industry. U.. in Lipid Analysis in Oils and Fats. Oil Chem. Phys. 154. J. 83. ed. Dobson. 1993..K. 13. The Oily Press. J. Medina. Am.. 2003. 225. Lipids.. I.. 181.

and Mollerup... Kallio. and Aaltonen. 39. K.. Purification of polyunsaturated fatty acid esters from tuna oil with supercritical fluid chromatography.. S. Food Chem. M. C. 39. H. Alkio. T. 315.. J.. 1991. 77. R. Jäntti.... 74. Soc. 697. J. Staby. 1994. Chromatographia. and Linko.. 73.86 ◾ Handbook of Seafood and Seafood Products Analysis 72. Gonzales. J. Agric. O.. Thin-layer silver ion chromatography and supercritical fluid chromatography of Baltic herring (Clupea harengus membras) triacylglycerols. 2000. Balchen. . Oil Chem. Quantitative analysis of marine oils by capillary supercritical fluid chromatography. Borch-Jensen... A. Vauhkonen. C. Am. 1573.

............1 Primary Oxidation Products ................................................... This can lead to 87 ......... except when microbial processes limit the shelf life....................................................................... The volatile............................ secondary oxidation products................................ 20:5n-3)...................... due to the high content of long-chain PUFAs.......... 92 7........................................ 88 7.....................Chapter 7 Lipid Oxidation Turid Rustad Contents 7................2............................................. Reaction products from lipid oxidation have a negative effect on the sensory properties of fish products.... These fatty acids have beneficial health effects and are reported to prevent coronary heart diseases and have a positive effect on the brain and nervous system as well as stimulating the immune system [1................................... marine lipids are highly susceptible to oxidation..................3 Summary ..................2 Analysis of Lipid Oxidation ........1 Introduction Marine lipids are good and natural sources of polyunsaturated n-3 fatty acids (PUFA) such as docosahexaenoic acid (DHA................................2......... 92 7................. However. especially those that originate from n-3 PUFAs are components that have a low threshold and therefore have a negative impact on the sensory quality of the food even in low concentrations [3]...............2 Secondary Oxidation Products ........ 93 7......... Lipid oxidation is the most important factor limiting the shelf life of marine oils and is also an important factor determining the shelf life of seafood products............ 87 7................... 93 References ...2]...................2............................. 22:6n-3) and eicosapentaenoic acid (EPA.............5 Sensory Analysis of Rancidity .....................................3 Stability Methods ................................................2......................................... 88 7.................... 93 7.........................1 Introduction .......2... 89 7.................................................................................4 Instrumental Methods .........................

and also more complex reaction products such as epoxy and polymeric compounds are formed during the propagation and termination steps [4]. acids. The peroxides are easily broken down to alkoxy radicals. The sensitivity is about 0. but it is important to keep in mind that the results for PV measurements will vary both according to the method used and how the procedure is performed [3]. volatile compounds and nonvolatile components with a relatively high molecular weight. If this contains a terminal carbonyl group. This also makes the determination of the degree of oxidation a challenging task. and the liberated iodine is titrated with sodium thiosulfate with starch as an indicator. Free radicals are formed when hydrogen ions are extracted from the fatty acids. These include nonradical species such as aldehydes. resulting in a wide variety of degradation products. methods that determine the primary oxidation products and methods that measure the secondary oxidation products. and enzymatic oxidation.88 ◾ Handbook of Seafood and Seafood Products Analysis loss of products. and reduced sales.2. 7. ketones. autoxidation. the molecule is called a core aldehyde. making it even more difficult to determine the degree of rancidity. The fatty acids and the lipid oxidation products in foods can also react with other components in the food such as proteins. The radicals react with oxygen forming peroxy radicals and hydroperoxides. leading to a wide variety of reaction products. the parent triglyceride is left with a shorter fatty acid.5 meq/kg. the lipid can be extracted using the methods of Ref. When the decomposition of a hydroperoxide has resulted in the formation of a low-molecular weight volatile compound. Autooxidation of lipids takes place when the unsaturated fatty acids are exposed to oxygen and proceeds through an autocatalytic chain reaction [3]. 7. Some of the reaction products from lipid oxidation may also have negative health effects. The secondary oxidation products can also react further. Methods to determine the degree of lipid oxidation can be divided into two main groups. Lipid oxidation can be divided into three types. it is both one of the oldest and one of the most used methods. the influence of these compounds has been little studied [3]. and alcohols. This method requires a sample of 5 g if the PV is below 10 and about 1 g if the PV is higher [3]. complaints from the consumers. this is oxidized by the hydroperoxides or other components present in the sample. A simple titration method where the sample is dissolved in chloroform–acetic acid (or isooctane–acetic acid) is often used for fats and oils. Several analytical procedures are available. PV is one of the classical methods for determination of oxidative status. Potassium iodide is added. However.2 Analysis of Lipid Oxidation Many different methods have been implemented both by the industry and in research to determine the degree of lipid oxidation both in marine oils and in seafood. which makes it difficult to find where the components originated. The secondary oxidation products include both low molecular weight. [5] or [6] before analysis. The PV is expressed in milliequivalent of iodine per kilogram of lipid or as millimolar of peroxide per kilogram of lipid [7].1 Primary Oxidation Products The most common methods to determine primary oxidation products are peroxide value (PV) and conjugated dienes. For determination of PVs in foods. and water. but this can be improved by determining the endpoint colorimetrically or by . photooxidation. carbohydrates.

Oxygen in the air. Small changes in quality of ethanol can give widely different standard curves and thereby influence the results. Conjugated dienes have a strong absorption maximum at 230–235 nm [12]. and the modified IDF method. These methods are therefore most useful as a measure of lipid oxidation for lipids with a low level of oxidation. high-performance liquid chromatography (HPLC) methods can be used [3]. and use a low amount of solvent. and determinations of PV have to be combined with the determination of secondary products such as thiobarbituric acid-reactive substances (TBARS) and . One of these is the colorimetric ferric thiocyanate method. The sensitivity and specificity can be increased by using second derivative spectra [12]. which react with ammonium thiocyanate forming ferric thiocyanate. The different methods gave different PVs for the same sample. light. the IDF method was chosen as the best of these methods. However. Based on the fact that the methods chosen should have a large linear range. For use on tissue extracts. the micromethod determining oxidation of iodide to free iodine. A known amount of sample is diluted in methanol (esters). and absorption of iodine by the unsaturated fatty acids in the oil may interfere and cause variations in the results. a high reproducibility. which is a red complex with an absorption maximum of 500 nm [3]. This method is more sensitive and requires smaller samples. In this procedure ferrous ions are oxidized to ferric ions. [10]—requires a low amount of sample (less than 10 mg). extraction and separation techniques are necessary. or hexane [13]. In order to determine individual peroxides. PV is reported to be an unreliable indicator of lipid peroxidation in fish [4]. the FOX2 method determining oxidation of ferrous salts to ferric ions and reaction with xylenol orange. and there was no consistency in the levels of PV determined by the different methods. how these are stored. the colorimetric ferro method. Peroxides are unstable and are rapidly transformed into secondary [14] oxidation products. the level of primary oxidation products increases and passes through a maximum.2. isooctane. [9] and Undeland et al. Using PV as a sole determination of oxidation level can therefore be misleading. The method of The International Dairy Federation—often called the IDF method [8] as modified by Ueda et al.Lipid Oxidation ◾ 89 determining the liberated iodine electrometrically using a platinum electrode.2 Secondary Oxidation Products Development of peroxides and conjugated dienes follows the same process and can be reduced after a certain oxidation level. Even if new instrumental methods now have been developed for determination of PVs. with regard to chemicals used. and how the procedure is performed. also for this method care should be taken in standardizing the procedure. After the initiation phase. Frankel [3] suggests measuring the absorbance of conjugated dienes at 243 nm. and it is important to know the history of the oil or the seafood to interpret the measurement of PV. it is often desirable to use a method that either does not require instruments or requires only a spectrophotometer. Nielsen et al. Conjugated dienes are useful for bulk lipids. The fatty acid chain then contains a structure with alternating simple and double bonds. and the AOCS method requires a sample size of around 10 mg. Care should therefore be taken in standardizing how the procedure is performed. Due to rapid polymerization of EPA and DHA compared with the formation of stable peroxides of these fatty acids. compared five different methods for determination of PVs [11]—the titration method. Conjugated diene hydroperoxides are formed when polyunsaturated fatty acids oxidize. 7. Several colorimetric methods for determination of PV values are used.

time of heating. Purge and trap techniques.3. nitrite. and the absorbance at 350 nm is determined after 10 min [15]. Of these methods. reaction products from browning reactions. In another method. This determines the amount of aldehydes (mainly 2-alkenals and 2. p-anisidine dissolved in acetic acid is added. These methods determine the presence of aldehydes. which is formed as a decomposition product from lipid hydroperoxides under the acidic test conditions [3]. sulfite. Many factors influence the color in the TBA test—temperature. and the optical density of the water phase is determined at 538 nm. In addition.13]. and identified using different gas sensors. Some of the MDA detected in this test is formed during the peroxidation of the lipids. the colored complex was ascribed to the condensation of two moles of TBA and one mole of malonaldehyde (MDA). in addition H2O2. alkenals. but most of it is formed during the decomposition of the lipid peroxides during the acid heating stage.4-dienals). AnV can also be determined using Fourier transform infrared (FT-IR) [16]. Protein. antioxidants.1. amino acids. Different types of headspace analyses can be used. TBARS values have been found to correlate with sensory scores within the same materials [19]. Many variations of this test are being used. The Totox value is still one of the most commonly used oxidation parameters used in commercial laboratories and laboratories in the edible oil industry. However.90 ◾ Handbook of Seafood and Seafood Products Analysis anisidine value (AnV). There are many published methods to determine TBARS. and trace metals can influence the result [3. This value is a combination of the PV and the AV. antioxidants. Dienals also give a red pigment absorbing at 530 nm. the reaction is not specific. This process is accelerated by metal ions [12]. All the methods are based on the pink color absorbance formed by reaction between TBA and oxidation products of polyunsaturated lipids. many other components in foods can react with TBA or interfere with the measurements. but as for the determination of PV.18]. static headspace and solid-phase microextraction (SPME) are the least sensitive. are highly sensitive. metal ions. and the absorbance of the solution is read at 530 nm. different methods give different results. which is generated by acid hydrolysis of 1. For determination of secondary oxidation products. the lipids are dissolved in a solution of thiobarbituric acid in butanol.4-dienals also react with TBA. After sampling. the volatiles can be thermally desorbed into a gas chromatograph for separation.3-tetraethoxypropane [3]. Originally. In other variations. However. The AnV of freshly deodorized oils is caused by core aldehydes. the sample is incubated at 95°C for 2 h. and antioxidants. the TBARS are separated by steam distillation or HPLC to increase selectivity. The sample is dissolved in isooctane. forming a yellow pigment absorbing light at 450 nm. The volatile compounds formed as a result of lipid oxidation can be analyzed using electronic noses/gas-sensor array systems [20]. hence the name TBARS. and the color is formed by many different secondary oxidation products. However. the AnV is a common method. alkanals. sucrose and other sugars.13. In the micromethod of Ke and Woyewoda [17]. where the samples are flushed or purged with nitrogen and the volatiles in the gas flow are trapped on a solid absorber. The TBA test can be standardized using MDA. which are secondary oxidation products. where the headspace volatiles over the samples are sampled. separated. the lipids are boiled for 45 min with a mixture of TBA. The determination of TBARS (or TBA) is a common method to determine secondary oxidation products. and 2. nucleic acids. the oxidation products are extracted in trichloroacetic acid (TCA) before the reaction with TBA. pH. and chloroform before adding TCA. The Totox value is given as 2*PV + AnV. and chelating agents may also influence the peroxide decomposition during the assay. The TBARS values for different foods with the same level of oxidation (based on flavor scores) can vary significantly [3. In the AOCS method [13]. The mass spectra of the compounds can also be compared with spectra of pure standard compounds and .

proteins. Hydroperoxides (primary lipid oxidation products) and aldehydes (secondary oxidation products) can react with amino groups in proteins.Lipid Oxidation ◾ 91 identified [21]. Fluorescence has traditionally been applied to samples in solution. When fluorescence measurements are done on samples in solution. and the results are dependant on the sample material. Small variations in sampling procedures can give large variations in the data. The different chromophores formed as a result of oxidized lipids. and so on. peptides. and the concentration is below a certain level. Aubourg and Medina [26] extracted fish muscle with a 2/2/1. and so on. The fluorescence intensities were divided by the fluorescence intensity of quinine sulfate and the fluorescence shift calculated. destroy this relationship. The advantages of this method are that it is flexible. front-face fluorescence Table 7. Reactions between Oxidized Lipids and Proteins/Peptides or Reactions between Oxidized Lipids and DNA Chromophore Oxidized phospholipids/oxidized fatty acids + phospholipids MDA + phospholipids Oxidized arachidonic acid + dipalmityl phosphatidylethanolamine Oxidized arachidonic acid + DNA Peroxides/secondary oxidation products + DNA in the presence of metal ions or reducing agents Excitation Maxima (nm) 365 400 360–390 315 320 Emission Maxima (nm) 435–440 475 430–460 325 420 . Fluorescence techniques are highly sensitive and 10–100 times more sensitive for detection of MDA than TBARS [3]. and form fluorescent products. nucleic acids.23]. Analysis of volatiles is discussed by Ólafsdóttir and Jónsdóttir in Chapter 8. scatter. as measured by methods such as PV and TBARS. the measured intensity follows the Beer–Lambert law.1. quenching.1 Excitation and Emission Maxima for Chromophores Formed as a Result of Oxidized Lipids. This reaction can lead to formation of brown-colored compounds [22. especially from solid matrixes. quantification of headspace data. They measured the fluorescence intensity both at 393/463 nm and 327/415 nm. the data handling is also difficult. phospholipids. or reactions between oxidized lipids and DNA have different excitation and emission maxima as shown in Table 7.8 chloroform/methanol/water mixture and measured fluorescence both in the water and in the organic phase. deoxyribonucleic acid (DNA). The fluorescence shift was found to be a more effective index of changes in fish quality than other commonly used methods. However. Instead. Reactions between lipid oxidation products and other components in seafood or seafood products may lead to underestimation of the degree of lipid oxidation. is complicated. for example. Lipid oxidation products can interact with other components in food. When the samples are turbid or solid or the concentration is high. reactions between oxidized lipids and proteins/ peptides. The fluorescent compounds formed from lipids are the result of oxidation of phospholipids or are formed from oxidized fatty acids in the presence of phospholipids. such as amino acids. for assessment of lipid oxidation during fish processing [24–26]. and the amount of sample and sampling conditions can be varied according to the needs. forming Schiff bases.

The AOM method is performed in a somewhat similar way. So far. comparable to sensory analysis and gas chromatography. the oil can be heated to 80°C or more while air is bubbled through it. for measuring lipid oxidation [28]. little has been done to study the fluorescence spectra of the different oxidation products that are formed in foods. In recent years. 7. but it measures the time taken to reach a certain PV. obtaining information . The levels of free radicals trapped in cod liver oil and salmon oil during the first hours of oxidation were in accordance with the oxidative stability measured by conventional methods [4]. Veberg et al. active oxygen method (AOM).37].33]. Fourier-transform near-infrared (FT-NIR). including near-infrared spectroscopy (NIR). and a few minutes of oxidation of docosahexaenoate (DHA) resulted in significant changes in the ESR spectra. and Oxidograph are techniques for measuring the stability of oils toward oxidation. adipose tissue. The level of hydroperoxides in fish oil can be determined using a rapid CL method [14]. and FT-IR spectroscopy methods [16. This results in the formation of low molecular weight acids that are flushed out with the air and collected in vessels containing distilled water. The change in conductivity is measured. The gas chromatography–mass spectrometry (GC–MS) techniques can be used to determine a wide range of volatile secondary lipid oxidation products [36]. oil stability index (OSI). and the point where it changes most is called the induction time. aldehydes. The Rancimat. One challenge is that fluorescence spectra can be very complex and that not only the oxidation products but also connective tissue. Fluorescence spectroscopy on intact samples has been shown to be a sensitive technique. and oxidative stability measurement by Oxidograph [31] and they are all suitable for analyzing oil systems. 7.4 Instrumental Methods Many instrumental methods have been developed for the determination of oxidation parameters in oils and foods.92 ◾ Handbook of Seafood and Seafood Products Analysis spectroscopy can be used. 1H NMR spectra can be used to study specific lipid oxidation products. Free radical assessments by the ESR spin-trapping technique detected the very early stages of lipid oxidation. the Rancimat test [30]. and additives may contribute to the spectra. Fluorescence spectroscopy has a great potential for on-line or at-line applications. Using solid-phase fluorescence is a relatively new approach. new methods have been developed. these include the oil stability index method [29]. The Oxidograph instrument finds the induction time based on measurement of the decline in pressure caused by the absorption of oxygen in a closed vessel. In a study of different model systems including fish and meat. It has been shown that sodium hypochlorite-induced decomposition of hydroperoxides gives strong CL [34.3 Stability Methods Several techniques based on accelerated oxidation are used for evaluation of oxidation. but studies on the use of this technique in dried fish were published in 1992 [27]. and these include assessment of free radicals using electron spin resonance (ESR) spectroscopy and use of different chromatographic methods to determine both primary and secondary oxidation products. The liquid chromatography–mass spectrometry (LC–MS) techniques can also determine nonvolatile products—of special interest are the core aldehydes [3.32. In Rancimat and OSI instruments.2.35].2. such as different hydroperoxides. porphyrins. Lipid oxidation products can produce very weak chemiluminescence (CL). and also cyclic compounds. [28] concluded that fluorescence spectroscopy may be able distinguish between different oxidation products formed but that this would require using the whole spectrum and not only the intensity at the maximum wavelength.

intermediary goods. Falch.. however. A trained panel can be a very valuable tool for detection of early lipid oxidation of foods containing n-3 fatty acids. 22: 291–306. today it is not possible to use only one method to determine lipid oxidation. Chow. 1957. in Fatty Acids in Foods and Their Health Implications. 206. Marcel Dekker: New York.K. E. Odor threshold values vary both with the chemical structure of the carbonyl compounds and with the food matrix and based on how the sensory detection is performed. Dietary fat. References 1. pp. 2005. Norwegian University of Science and Technology: Trondheim.. Boissonneault. 7. 5. J. However. However.5 Sensory Analysis of Rancidity The ultimate measurement of rancid odor and taste is sensory analysis by a trained panel. Multivariate data analysis is a valuable tool in elucidating changes in spectra during storage and showed the resonances that came from n-3 fatty acids during oxidation. Hosakawa. The detection of these low levels is not straightforward with classical lipid oxidation measurement methods. 3. G.Lipid Oxidation ◾ 93 that cannot usually be obtained by single conventional analytical methods [4].3 Summary Many different methods for the analysis of lipid oxidation exist. 777–795. The ultimate wish from the food industry would be a rapid nondestructive method that can be applied on-line to analyze the oxidative or sensory quality in raw materials. 2. and the use of chemical and instrumental analyses is recommended to support and complement the sensory analysis [3].. However. . Chem. but for many of these methods calibration and verification are needed before they can be used for routine analysis. Lipid Oxidation. E. It can also be difficult to compare data from different panels using different vocabularies or data from the same panel analyzed at different times. Sloan Stanley. the oxidation products from n-3 fatty acids have a lower sensory threshold than those of oxidation products from other fatty acids. through the nose (nasal) or through the mouth (retronasal). Frankel. 2005. for many of these methods the results obtained vary not only with the method used but also with the analytical procedure that is performed. sensory analysis requires relatively large amounts of samples. Lipids from residual fish raw material.. Int. the sensitivity was low (detection levels ∼0. C. and finished products during seafood processing. and M.. and G. immunity. J. In addition. There is. and inflammatory disease. K. 2005. 2nd ed.K. Folch.. Some of the degradation products from long-chain n-3 PUFAS have a profound effect on odor and flavor in concentrations as low as in the parts per billion range [3]. Miayshita. their use is limited by the cost of employing a trained panel. 4. a rapid development in analytical methods to determine lipid oxidation.A. Narayan. M. in Department of Biotechnology. Ed. U. Food Rev.H.. 2000.. B. Lees. Even if sensory methods can give sufficient information. Biol. 7. 2006. so care should be taken in standardizing the procedures.N. The Oily Press: Bridgewater.. The sensitivity could be improved by the use of CryoProbe technology. 226: 497–509.2.01 nM). In general. A simple method for the isolation and purification of total lipids from animal tissues. Physiological effects of eicosapentanoic acid (EPA) and docosahexanoic acid (DHA)—A review.. even if there are many different methods that are used to determine lipid oxidation.

J. B. 10: 35–50. Undeland. Food Chem. Ed. IL. Nielsen. 1977.C. AOCS: Champaign.M. Type V collagen in trout (Salmo gairdneri) muscle and its solubility change during chilled storage of muscle. Wiley-VCH Verlag GmbH: Weinheim. and M. IL.. G. AOCS Official Method Ti 1a-64. 67: 2397–2404. 8. Tironi. 2002. 1995. Food Agric. 1998.. V. 28. and J. Nawar. Ed. Food Sci..94 ◾ Handbook of Seafood and Seafood Products Analysis 6. J. Food Sci. Jacobsen. D. 32: 497–502. E. Bligh.. M. 46: 3662–3666. 2006. Ke.. Quality assessment of blue whiting (Micrometistius poutassou) during chilled storage by monitoring lipid damages. Firestone. Medina.P. K. 106: 279–284. Oxidative deterioration in dried fi sh model systems assessed by solid sample fluorescence spectrophotometry. Agric. Chem.. and J. Cabo. Influence of storage time and temperature on lipid deterioration during cod (Gadus morhua) and haddock (Melanogrammus aeglefinus) frozen storage. J. Food Lipids. Anal... Sci. 22. J. Wold. Food Sci. Ed. of Chemistry. Wold. 1994. 160. A. The measurement and mechanism of lipid peroxidation in biological systems. 2001.P. Effect of ascorbic acid in a model food system. et al. Y. 1992. AOCS. Timm-Heinrich. in Food Chemistry. Food Res. and C.R. Vogt. Endo. pp. N.. Food Agric. S. Biol..S. 50: 1–7. in Official Methods and Recommended Practices of the American Oil Chemists’ Society. 1995. Hasegawa. Gallardo. Norwegian University of Life Sciences: Ås. Guillen. 12.. 26.-J. 11. 7–212. Food Chem. J. in Official Methods and Recommended Practices of the American Oil Chemists’ Society. H. in Dept. M.. 1991. J. Structural and functional changes in myofibrillar proteins of sea salmon (Pseudopercis semifascata) by interaction with malonaldehyde (RI). Method Cd 18–90. Physiol. I. pp. Chemiluminescence of fish oils and its flavour quality. Sci. 1998. AOCS: Champaign.. 39: 1222–1225. J. 7. AOCS: Champaign. Aubourg. .. 23. Food Chem. 2005. 24. Method Cd 8-53... 77: 503–510. P. Influence of skinning on lipid oxidation in different horizontal layers of herring (Clupea harengus) during frozen storage.A. and J. Fennema.. Lipid damage detection during the frozen storage of an underutilized fish species. Veberg. Hayahashi. and W. O.W. and K. 17. 1996. 1999. Can. 13.. S. W. Basics.J. Lignert. J. 37: 911–917.P. Marcel Dekker Inc. Firestone. 15: 129–135. 20. Halliwell. 65: 307–313. 16. 1959. Norway. I. 57: 1123–1126.. Rapid assessment of rancidity in complex meat products by front face fluorescence spectroscopy. J. Ueda. Tomas.. Acta..D.C. Aubourg.J. D. 15.: New York. 2003. 27: 389–393. S. 39: 562–570. and H. Food Agric. 79: 1943–1948. IL. Aubourg. Agric. Biochem. J. 25. 14. E. and A. Trends Biochem. K. 10. Anon. Fourier transform infrared spectra data versus peroxide and anisidine values to determine oxidative stability of edible oils. Sci. J. M. 1979.. 1995... 67: 930–935.... Hübschmann.C. 21. Dyer.. Microdetermination of thiobarbituric acid values in marine lipids by a direct spectrophotometric method with a monophasic reaction system. Namiki. Technol. Fujimoto. Woyewoda. M. S. AOCS. 1986. A rapid method of total lipid extraction and purification.. 2002. 9. Ed. Fluorescence in aldehyde model systems related to lipid oxidation. D. 18. 225–319. Firestone. Chim. and M.D. La Rivista Italiana Delle Sostanze Grasse. 1999. Pettersen. M.M. Int. 78: 441–450.. AOCS. J.. Olsen. Biotechnology and Food Science. Lipids. Stading.G. Sci. Comparison of wet-chemical methods for determination of lipid hydroperoxides. J. LWT-Food Sci. 27. Analysis of early lipid oxidation in foods with n-3 fatty acids. Pokorny. and I. 2002.. Interaction of oxidised lipids with protein. 19. in Handbook of GC/MS-Fundamentals and Applications.. Medina. Gutteridge.. et al. and N. 1990. Sato. Agric.

and biological significance.. Matthäus. J.-G. in Scandinavian Symposium of Lipids (Lipidforum) 16th. H. Fat Sci. natural occurrence. 96: 95–99. 32. AOCS Press: Champaign. Oil Chem. A. 160–162. Matlock..H. Olafsdottir. Glycerophospholipid core aldehydes: Mechanism of formation.Lipid Oxidation ◾ 95 29. T. J. M. Oil Chem. A. Moh. C. Aquat. Jonsdottir. Kuksis. Kamido. J. Kamal-Eldin. Y. Technol. H. Study of oxidation by chemiluminescence. B... 16: 67–86. Soc. Fast chemiluminescence method for detection of oxidized lipids. Sleeter... The Oxidograph. R. Comparison of Rancimat evaluation modes to assess oxidative stability in fi sh oils. Ravandi. 30.A. Determination of peroxide value in thermally oxidized crude palm oil by near infrared spectroscopy. Determination of peroxide value by Fourier transform near-infrared spectroscopy.. methods of detection.. M. Soc. Am. H. Technol. and A. Bragadottir. et al. Li. J. IV. 36. Detection of low levels of lipid hydroperoxides by chemiluminescence. Eichner. Am. Soc.. Collaborative study of the oil stability index analysis. Am. in Lipid Oxidation Pathways. M. 1991. Soc. J. IL. Yamamoto. 31.. Mendez. 70: 1055–1061. 76: 19–23. and G.. Oil Chem. Am. 2000. Oil Chem. Ed. Oil Chem.. 34. and R. 1985. 77: 137–142. Wiezorek. 1994. 1999. 2007. J.. et al. 33. E. Am.. Jebe.T.. 37. 62: 1248–1250. . 2003. 138–189. pp. 1997. pp. 1993. and K. 74: 331–332. 35. Vinter. Soc. The role of volatile compounds in odor development during hemoglobin-mediated oxidation of cod muscle membrane lipids. et al. A development within accelerated measurement of stability. Food Prod. et al..


......2 Washed Cod Muscle System ...........................................................4...............................................................................3.......................................................Chapter 8 Volatile Aroma Compounds in Fish Guðrún Ólafsdóttir and Rósa Jónsdóttir Contents 8.............................................................. Onion........108 8..................................................................... 111 8..............2 8. 100 8..........................................................4......................................................................113 97 ......................1..1 Sweet..... Ammonia-Like........4.......................................112 8.............................................2 Oxidatively Derived Odors .... 98 Development of Fish Aroma..............................4 Miscellaneous ...........103 8...............4...... and Stale Odors ..........105 8........ 98 Fresh Fish Odors .105 8..............4..................2...............1...103 8......4............................................................................3 Processing Odors ...............4...3 8............ Sour.......... 111 8............................1 Cooked Odor—Boiled Potato and Rancid Odors ................2 Dried Fish..........106 8............113 References ..1 Smoked Fish Odors ..............1 8.....................................................................105 8............4...........................4...5 Conclusions ...........4............................2 Ripening Odor—Salted and Dried Fish Odor............4......................3 Putrid..........106 8.............................................................. 99 Identification of Quality Indicators ............3.......1..........1.............................4 Introduction .. and Malty Odors ..........................1 Microbial Spoilage Odors ................................................................. and Cabbage-Like Odors ....2......................................................

Proteolysis plays a critical role in postmortem changes. cooked. processed. Initially. valine.98 ◾ Handbook of Seafood and Seafood Products Analysis 8.e. anserine). including degradation of nucleotides. followed by oxidation processes. Finally. resulting in undesirable texture changes in fish. Some of these compounds influence the taste of fish-like peptides (i. studies on application of natural antioxidants are of prime interest to underpin further utilization of fish in innovative product development as fresh. As a result.1. However. leading to the formation of secondary oxidation products and off flavors [8]. amino acids. and the individual amino acids glycine. or hydrolyzed products and as ingredients in functional foods. Fish being a valuable source of polyunsaturated fatty acids (PUFA) and other nutrients is a prominent candidate as the healthy choice for consumers. and accumulation of hypoxanthine (Hx).. biochemical. contributing to spoilage changes and thus influencing the freshness and quality of the end product of chilled fish [1–3]. They are composed of the various nonprotein nitrogenous components (NPN). alanine. Endogenous enzyme . TMAO. Research over the years has led to improved chilling and packaging technologies aimed at reducing microbial growth. and glutamic acid are known to contribute to taste together with the degradation components of the nucleotides such as inosine. It is well established that enzyme lipoxygenase (LOX)-mediated conversions of polyunsaturated fatty acids (PUFA) to volatile aroma compounds initiates the development of plant-like aroma of fresh fish [4–6]. and microbiological processes in fish postharvest is of importance to be able to control the various extrinsic factors that influence the formation of volatile degradation products and consequently the quality of fish products. Consequently. 8. Improved understanding of the role of oxidation of polyunsaturated fatty acids in the development of off odors in fish products has directed research efforts to search for effective means to control oxidative processes. lowering of pH and endogenous enzyme activity. Volatile compounds play an important role in the odor quality characteristics and consumer acceptance of fish. formation of taste. including small peptides such as carnosine and anserine. Degradation of soluble muscle constituents such as sarcoplasmic proteins and microbial metabolism contributes to changes in the aroma profile of fish during storage. extension in shelf life of fresh chilled fish has been achieved. oxidative processes causing odors and texture changes become noticeable during extended storage and limit the shelf life. and nucleotides.1 Introduction Health and wellness are the main drivers in new product development. the changes are dominated by autolytic activity. Research has aimed at strengthening the marine-based food industry in the development of fish products of acceptable quality to meet new trends in lifestyles.2 Development of Fish Aroma An overview of changes during handling and processing influencing the development of aroma in fish is generalized in Figure 8. guanidine compounds like creatine. Other prooxidants like hemeproteins (hemoglobin and myoglobin) are also involved in the initiation of the oxidative processes in fish muscle [7]. Enhanced oxidation during cooking resulting in off odor development is of concern and an obstacle for application of fish in convenience food. the proliferation of the specific spoilage organisms (SSO) results in the development of volatile compounds. The pool of components that are degraded and cause off flavors because of microbial growth are mainly soluble substances in the muscle. The understanding of odor development by chemical. active inosine. A prerequisite for increased consumption of fish products is their availability on the market as fresh and high-quality products of delicate flavor.

Newly caught marine fish contains low levels of volatile compounds and is nearly odorless. dried fish. cucumber Figure 8.5. including calpains (neutral calcium-dependent proteases) and cathepsins (lysosomal proteases).Volatile Aroma Compounds in Fish ◾ 99 Handling chilling. or nine carbon atoms [4. popcorn. stockfish. nonprotein nitrogen-containing compounds. LOX. but the mechanism of this activity is not fully elucidated [9]. Soon after harvest. PUFA. and 2. drying. cucumber-. NPN. 1-octen-3-ol. and cooking Processing smoking. [5] summarized the occurrences of volatile compounds in freshwater and saltwater species and concluded that the four common compounds found in saltwater species. polyphenols Lipids phosholipids/PUFA Proteins sarcoplasmic. salting.. putrid. TMAO. LOX activity on the skin and gills of both freshwater and marine species (rainbow trout. phospholipases TMAOase Microbial metabolism Specific spoilage organisms (SSO) Oxidation Prooxidants: metals (Fe.6-nonadienal. plant-. Some components are desirable at low levels. specific spoilage organisms. malty. pleasant aromas of fish [6. oxidized. hemoglobin. cucumber-. whereas volatiles generated from fat result in variation in the specific flavor character of different fish species. stale. and hydrolysis Endogenous enzymes i. peptides Soluble substances. Mb Antioxidants: α-tocopherol. LOX proteases. 8. sour. cucumber. eight.3 Fresh Fish Odors The delicate flavor of fish is mostly contributed by volatile compounds and taste active substances in the aqueous phase.10–13]. ammonia-like Oxidized aroma Processed aroma green-like. amino acids Fresh fish aroma seaweedy. On the other hand. and sardines) plays a role in the formation of odorous volatiles. The compounds that contribute to the characteristic plant-. were responsible for the moderate. lipoxygenase. metallic. SSO.5-octadien-3-ol. Josephson et al. caramel. 1. malty. and mushroom-like odors are unsaturated carbonyl compounds and alcohols with six. spoiled. contributing to green.e. Hb.1 Overview of changes in fish influencing the development of characteristic aroma of fresh. river trout. were characteristic for freshwater and euryhaline fish. but . melon-.5-ocatadien-1-ol. which have potent green. faint odor of saltwater species. nucleotides. polyunsaturated fatty acids. ascorbic acid. the unsaturated C9 carbonyl compounds such as 2. hexanal. and melon-like odors. myoglobin. NPN. trimethylamineoxide. mushroom. hydrolases. freezing. The overall perceived odor is dependent on the level of influential compounds and their odor thresholds along with possible synergistic effects. neutral Spoilage aroma sweet. rancid potato. boiled potato. Mb. and processed fish. activity influences the deterioration of fish muscle.14.15].Cu) Hb.

for example. However. Studies performed in Japan. Some of the influential odor compounds that have very low odor thresholds are often present in low levels. and marine-like flavors of seafood [18]. and species of the salmonidae family develop earthy. and 3. C9 LOX-derived compounds have been found in higher levels in spawning euryhaline and freshwater fish [5].1. 2. acids. Fatty species develop rancid odors and taste.24–29]. boiled potato-. as seen by the detected odors listed in Table 8. where it has been demonstrated by monitoring key volatiles to study changes in different fish products during storage. which are present in higher levels and can be quantified. aldehydes. they may contribute to off odors.22] and in smoked salmon [23].21–22. Rapid methods can then be applied to detect indicators or alternatively classes of compounds if the pattern of the volatile compounds is known and a connection has been verified between the indicator compounds and the compounds that are responsible for the odors and quality changes. which has a very characteristic cucumber odor during spawning. GC–MS. . They were suggested as the possible precursors of nine-carbon volatile compounds. Accumulation of certain hydroperoxide isomers coincided with the period of enhancement of characteristic aroma in sweet smelt. Table 8.6-nonadien-1-ol in sweet smelt tissues [20]. (E. and these are difficult to detect by analytical techniques. esters.6-nonadienal.1 summarizes the occurrence of volatile compounds detected in our studies on cod [22] and haddock fillets [31] and smoked salmon [23]. amines. in nominal levels the bromophenols appear to contribute to natural sea-. Another example is iodine-like off flavor in prawns associated with bromophenols originating from the feed chain [17]. indicate their involvement in the development of fresh fish aroma associated with seasonal variation. Seasonal effects have also been reported for capelin.Z)-2. Purge and trap on Tenax and SPME methods were applied for sampling. cod during storage [21. but when accumulated in higher levels because of autooxidation. Therefore.100 ◾ Handbook of Seafood and Seafood Products Analysis if their concentration increases. it is useful to monitor the overall pattern of volatile compounds and select indicator compounds. lean species typically develop sweet. they contribute to oxidized and fishy odors in stale fish [16]. including (E)-2-nonenal. and amine-like odors. Environmental conditions and seasonal effects like spawning can influence the odor quality of fish. and GC–O. The main classes of compounds detected during storage are alcohols. The aldehydes contribute most to the spoilage odors because of their low flavor thresholds. iodine-. and identification was based on GC–FID.30–36]. ketones. odor. and sweet odors.4 Identification of Quality Indicators Different characteristic odors develop in various fish species during storage. Both single compounds such as TMA and ethanol and multicompound indices based on combination of alcohols. This has been the approach in our studies. and sulfur compounds. An example is the enzymically derived long-chain alcohols and carbonyls that exhibit characteristic fresh. amines. and sulfur compounds representing the different changes occurring during storage have been suggested by numerous researchers as indicators for freshness and spoilage [22. The volatile pattern changes in mature salmon when migrating from the sea for spawning. Volatile compounds formed by microbial metabolism and oxidation contributing to these odors have been identified by gas chromatography methods and suggested as indicators of quality. 8. and quality changes can be explained in. on accumulation of hydroperoxides in fish tissues. Volatile degradation compounds as quality indicators can be detected by rapid techniques such as electronic nose to monitor and predict quality changes in various fish species and in smoked salmon [19. plant-like notes in fresh fish. a saltwater species.6-Nonadienal was identified to be the most characteristic compound for the cucumber-like capelin odor [19]. muddy.

earthy Sweet.E)Nonanal Decanal Undecanal × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × Rancid Boiled potato. caramel.4-Heptadienal.1 Volatile Compounds Detected in Cod [22]. flowery — Ketones 2-Butanone 2.Volatile Aroma Compounds in Fish ◾ 101 Table 8. and Smoked Salmon [23] during Chilled Storagea Compound Raw Cod Boiled Cod Raw Haddock Smoked Salmon Odor Description (GC–O) Alcohols Ethanol 2-Methyl-1-propanol/pentane 1-Penten-3-ol 3-Methyl-1-butanol 2-Methyl-1-butanol 2. Haddock Fillets [31]. (E.3-Butandiol 1-Octen-3-ol 2-Ethyl-1-hexanol 1-Octanol × × × × × × × × × × × × × × × × × × × × × × × × — — — — — — Mushroom — — Aldehydes Acetaldehyde 2-Methyl-propanal 2-Methyl-butanal 3-Methyl-butanal Hexanal cis-4-Heptenal Heptanal 2. fatty — Fresh. fish fillet Sweet. candy × — — Sweet. caramel. floral Sweet.3-Butandione × × × × — N/A (continued) .

sweet. ethylester Hexanoic acid. ethyl ester × × × × × × × × × × × × × × × — N/A — N/A N/A Sickenly sweet.1 (continued) Volatile Compounds Detected in Cod [22]. ethyl ester Butanoic acid. 2-methyl. sour Flowery. heavy.3-Pentanedione 3-Hexanone 3-Methyl-2-butanone 3-Hydroxy-2-butanone 6-Methyl-5-hepten-2-one × × × × × × × × × × × Raw Cod Boiled Cod Raw Haddock Smoked Salmon × Odor Description (GC–O) — Sweet. ethyl ester 2-Butenoic acid. caramel — — — Sweet. Haddock Fillets [31]. ethylester Butanoic acid. dried fish Acid Acetic acid × × × — Esters Ethyl acetate Ethanthiocacid. and Smoked Salmon [23] during Chilled Storagea Compound 2-Pentanone 3-Pentanone 2. ethyl ester Propanoicacid-2-methyl. 3-methyl. ethylester Acetic acid. 2-methylpropyl ester Butanoic acid. vomit N/A N/A N/A N/A Sulfur Compounds Methanethiol Dimethyl sulfide × × × × — — .102 ◾ Handbook of Seafood and Seafood Products Analysis Table 8. spicy Amine Trimethylamine × × × TMA-like. S-methylester Propanoic acid.

34]. The aim was to screen for potential quality indicators and determine which compounds and classes of compounds were most abundant in the headspace and also to identify the most influential spoilage odors contributing to sensory rejection.4. dried fish/stockfish. and the end of shelf life of cod fillets on day 12 of storage are explained by the presence of TMA.1 based on GC–O analysis of cod and smoked salmon represent most of these overall changes.2. sour.2) were associated with the development of sweet. and Malty Odors Ketones. and temperature conditions during storage [33. During prolonged storage boiled potato odor develops. An example of the spoilage pattern of volatile compounds in chilled fish is illustrated in Figure 8. the aroma of the fillet is described as sweet and reminiscent of shellfish. Haddock Fillets [31]. packaging. Sweet-milky and vanilla/caramel-like odors are typical in cooked fish. not detected by GC–O. cabbage Volatiles in boiled cod were analyzed in samples of raw chilled cod fillets [22] by heating corresponding samples at 80°C for 60 min. After several days of storage. and quantification of the main classes of compounds was based on the sum of the PAR for respective compounds in each class. Identification of volatile compounds was based on GC–MS analysis (see Table 8. 8. the freshness notes disappear and the odor of the uncooked fish becomes neutral.1 Microbial Spoilage Odors The spoilage odors in chilled fish vary depending on the dominant microflora in the products.5°C) [22]. which is mostly affected by handling. sulfur.1 Sweet. as well as green plant-. In general when fish is cooked. and aldehydes. and malty spoilage odors. The microbially derived alcohols 2-methyl-1-propanol. cooling.4. and malty odors. showing results from a storage study of cod fillets packed in styrofoam boxes during chilled storage (0. Late spoilage changes. —. meat-like. Seaweedy and marine-like odors. and aldehydes detected on day 4 of storage and their increasing levels on days 7 and 10 (Figure 8. or geraniumlike odors are characteristic sensory odor descriptors for fresh whole fish. alcohols. esters. the fish is no longer fit for consumption.1 (continued) Volatile Compounds Detected in Cod [22]. 8. Sour. caramel-like. The loss of freshness of cod fillets and early spoilage changes were related to the formation of ketones. The flavor thresholds . and finally sour and dirty tablecloth odor.1. and 2. N/A. 3-methyl-1-butanol. sour. mushroom-. and TMA-like smell. mainly amino acids. development of spoilage odors. cucumber-. and sulfur compounds produced by microbial degradation of fish components. and Smoked Salmon [23] during Chilled Storagea Compound Dimethyl disulfide Dimethyl trisulfide a Raw Cod × × Boiled Cod × × Raw Haddock × × Smoked Salmon Odor Description (GC–O) Onion like Rotten.Volatile Aroma Compounds in Fish ◾ 103 Table 8.3-butandiol were found in the highest levels on day 12 at sensory rejection. and sometimes metallic. alcohols. data not available for haddock. and when combined with frozen storage odor.1). acids. contributing to sweet. The odor descriptors in Table 8.

caramel. and 3-methyl-1-butanol as potential indices of refrigerated fish spoilage based on studies of freshwater whitefish. and acetic acid were identified as spoilage indicators [29]. PhD thesis. The initial high levels of ethanol in spoilage of fish has been related to the utilization of carbohydrate sources. Ethanol was detected in high levels initially (on days 4 and 7) and then declined. 3-methyl-1-butanol. and they did not contribute to the odor of the fillets as evaluated by GC–O (Table 8. 3-methyl-1-butanol. The concentration of acetoin was much higher than the lipid derived ketones detected. therefore. 3-hydroxy-butanone. University of Iceland.104 ◾ Handbook of Seafood and Seafood Products Analysis 120 100 Peak area ratio (PAR) 80 60 40 20 0 0 2 4 6 8 10 Days of storage 12 14 Alcohols Aldehydes Ketones TMA Aceticacid Esters Figure 8. Reykjavík. TMA. Volatile compounds as quality indicators in fish during chilled storage: Evaluation of microbial metabolites by an electronic nose.5°C until sensory rejection on day 12. methanethiol. Levels of acetoin increased earlier than those of TMA. piperidine. In chilled haddock fillets stored in styrofoam boxes. (Modified from Ólafsdóttir. that were present in cod fillets throughout . 1-penten-3-ol. respectively. dimethyl trisulfide. and butanoic acid ethyl ester were found in the highest amounts and increased with storage.1) [22]. TMA. butanol. it is more useful to monitor the loss of freshness as an early indicator of spoilage. The branched chain aldehyde. whereas the formation of branched-chain alcohols and aldehydes such as 2-methyl-1-propanol. such as 2-butanone. 3-pentanone. The formation of acetoin (3-hydroxy-2-butanone) was characteristic for the spoilage of chilled cod fillets packed in styrofoam boxes and was attributed to the growth of Photobacterium phosphoreum [22]. and fish-fillet-like odors by GC–O in our study. and the carotenoid-derived 6-methyl-5-heptene-2-one. 2-methyl-1-propanol. whereas dimethyl sulfide was detected initially and throughout storage [31]. In cultured and wild sea bream stored in ice for 23 days. G.. ethyl acetate. Dimethyl disulfide and dimethyl trisulfide were detected at the end of storage time when samples were spoiled. 3-methyl-butanal.) of alcohols are higher than those of carbonyls. and 3-methyl-butanal probably originate from degradation of valine and leucine.2 GC–MS analysis of volatile compounds showing changes in the levels (PAR. Propanol was suggested as a potential indicator when using modified atmosphere packaging techniques. dimethyl disulfide. Lindsay [8] suggested using short-chain alcohols such as ethanol. peak area ratio) of the main classes of compounds contributing to spoilage in cod fillets packed in styrofoam boxes during storage at 0. was characterized by sweet. 2005. and. 3-methyl-1-butanol.

TMA is a potent odorant with a characteristic fishy. and ketones (2-butanone). Dimethyl trisulfide has also been associated with spoilage in fish and associated with the growth of Shewanella putrecfaciens [25. ammonia-like odor. methyl mercaptan. and Cabbage-Like Odors Low levels of sulfur compounds (Figure 8.39]. which forms very early after harvest of fish. The onset of stale odors can be explained by cis-4-heptenal and heptanal.1). Additionally. 1-penten-3-ol). numerous branched chain .1. contributed to the sensory rejection of chilled cod fillets on day 12 and suggested the role of Pseudomonas fragi in the development of sweet. The odor of ethyl butanoate.2) indicated that they were not important in the spoilage of chilled cod fillets stored in styrofoam boxes. Pseudomonas species have also been found responsible for the formation of volatile sulfides.1. Additionally. In whole fish stored in ice. but no obvious increase occurred until at the end of shelf-life and during continued storage. At this point there was an increase in the pH value. contributing to the stale and putrid off odors in fish because of amino acid and lipid degradation [39]. fruity off odors [37. respectively [41].2 Dried Fish.4.38].1. and undecane) appeared to be similar throughout storage in chilled cod fillets [22]. has been suggested as a freshness indicator along with its precursor TMAO (trimethylamine oxide) [27]. described as sickeningly sweet and nauseous. and dimethyl disulfide have been suggested as the main cause of putrid spoilage aromas [41]. 8. ammonia-like. alcohols (3-methyl-1-butanol. which contributed to boiled potato-like odors (Table 8. Milo and Grosch [42] evaluated the headspace of boiled cod by gas chromatography olfactometry (GC–O) and found that dimethyl trisulfide was the most potent odorant contributing to off odors in cod formed when the raw material was inappropriately stored. Oxidative processes are involved in the formation of dimethyl sulfide from methyl mercaptan and further oxidation of dimethyl disulfide. TMA is characteristic for the spoilage odors of fish. The lipid-derived saturated aldehydes detected on day 12 at sensory rejection also contributed to the overall sweet aroma. 8. and stale odors by amines during fish spoilage is well known.Volatile Aroma Compounds in Fish ◾ 105 storage. Ammonia-Like.4 Miscellaneous The concentration of the straight chain alkanes (nonane. volatile sulfur compounds such as hydrogen sulfide.3 Putrid. TMA has been noted for intensifying fishiness by a synergistic action with certain volatile unsaturated aldehydes derived from autoxidation of polyunsaturated fatty acids [40]. decane. whereas DMA may influence the overall fresh flavor of fish in combination with oxidatively formed aldehydes from long-chain fatty acids in fish. Enzymically produced DMA (dimethylamine). which may have influenced the overall odor perception leading to the sensory rejection of the fillets. and the incorporation of hydrogen sulfide yields dimethyl trisulfide [38]. Ketones can influence the overall odor because of their low odor thresholds. dried fish. methyl sulfide. Figure 8. and measurements of volatile amines such as TMA or total volatile bases (TVB-N) have been used in the fish industry as indicators of quality for fish and fish products.2 shows that TMA was detected in high levels on day 12.4. 8. and Stale Odors The development of dried fish.4. The origin of the sulfur compounds is microbial degradation of cysteine and methionine to form hydrogen sulfide and methyl mercaptan.38. Onion.

Several odor active terpene derivatives have been identified in fish. they are in particular sensitive to oxidation. Oxidative processes occurring during storage of fish result in the accumulation of aldehydes. and overall the alkanes showed an increasing trend with storage time. such as hexanal. suggesting that it may have an impact on the overall odor of fish fillets [22]. 3-methyl-butanal. These oxidation products contributed to the overall characteristic sweet. and 6-methyl-5-heptene-2one). Phospholipids are the main membrane-bound lipids. Our studies on the development of volatile compounds in chilled cod fillets packed in styrofoam boxes during storage at 0°C showed that oxidatively formed. that contribute to the development of rancid cold store flavors [47]. since they are not aroma active. although their overall levels were lower.4. However. therefore.7-decadienal. heptanal. Piperidine levels have been reported to increase in spawning salmon and contribute to off odors [43]. their impact was greater than alcohols and ketones. but the sampling techniques used were not sensitive enough to allow quantification of these compounds. The influence of other aroma active compounds present in lower levels such as the unsaturated autoxidatively derived aldehydes (2. lipid-derived saturated aldehydes. 2.106 ◾ Handbook of Seafood and Seafood Products Analysis alkanes were detected. Piperidine was tentatively identified in chilled cod fillets [22] and has also been suggested as a quality indicator in sea bream [29]. 6-Methyl-5-heptene-2-one derived from carotenoids was described as spicy and flowery by GC–O and suggested to contribute along with other ketones and aldehydes to the characteristic sweet odor of cod fillets [22].2 Oxidatively Derived Odors Initiation of lipid oxidation in fish is generally associated with the polyunsaturated fatty acids in phospholipids of muscle cell membranes [44]. fish-like odors of chilled cod fillets in combination with other carbonyls (3-hydroxy-2-butanone. These compounds have been associated with rancid and dried fish odors. Limonene has also been detected in sea bream during storage [29]. Limonene has low odor threshold and a fresh lemon odor was detected by GC–O analysis of cod. ketones.1 Cooked Odor—Boiled Potato and Rancid Odors Characteristic odors and key volatile compounds in boiled cod stored in closed plastic bags for 22 days compared with fresh boiled cod are shown in Figure 8. A characteristic earthy odor in many species residing in ponds has been associated with piperidine and its reaction products. they are not considered of interest as quality indicators.2. and 2. and terpenes found in wild sea bream compared with those of its cultured counterpart [29]. Various pro and antioxidants influence the stability of the muscle and have been studied in relation to the oxidative stability of phospholipids [46].3 to demonstrate which odors are most dominating in the aroma profile [48]. the feed may have influenced higher levels of aldehydes.and potato-like odors contributed by . aromatics. but the knowledge of the formation of these compounds is obscure. in similar or slightly increasing levels. and decanal. and. such as hexanal. 2-butanone. Similarly. 8.4.4-heptadienal and 2. which is further enhanced by preprocessing and storage of fish.7-decatrienal) should not be overlooked.4-heptadienal. which are known to be more susceptible to oxidation than triacylglycerols in fat deposits [45]. 3-pentanone.4. 8. Aldehydes generally have low odor thresholds. and because of their high unsaturation. cis-4-heptenal. were detected in the fillets throughout the storage time. Boiled potato. The origin of limonene in fish is most likely related to the diet derived from algae or plant source.4.

2-methylbutanal. this aldehyde does not exhibit a fishy-type aroma by itself.. however. In fact. In fresh baked herring (200°C. Fatty. sweet. (From Jónsdóttir. as well as boiled potato-like [51. Ideally. pop-like Earthy-like odors Figure 8.3 Odor profile (GC–O analysis) of boiled cod stored in plastic bags (-♦-) after 22 days of refrigerated storage (3°C) compared with freshly boiled cod (---▲---). and octadienes also increased many-fold during further storage. 1-penten-3-ol. microbial metabolites such as 3-methyl1-butanol and cresol were identified [53].Volatile Aroma Compounds in Fish DMS Sulfur 5 4 3 2 1 Fishy odors Fishy 3-Pentanone 1-Penten-3-ol Flowery 2-Penten-1-ol Flowery Fatty. and rancid odors contributed by 2-nonenal and 2. green-like. sweet. and Ólafsdóttir. and after 8 days of storage at 6°C. Principal component analysis (PCA) was performed (Figure 8. paint-like [50]. quality indicators should demonstrate clear increasing or decreasing levels with storage time. and fish oil notes in the same study. R. The occurrence of cis-4-heptenal has been associated with the “cold storage flavor” of cod [47]. Hexanal. rancid odors Flowery 2. but it rather participates in the expression of the overall fishy odor. green-like. 20 min) 3-methylbutanal. although the level of the compounds may vary and explain the differences in the characteristic odor of these species. Taking into account the complexity of the spoilage processes. The fresh raw salmon odor was characterized as cucumber-like with weak sweet. and green-like odors were associated with oxidatively derived 3-pentanone. earthy Potato-like Boiled potato cis-4-Heptenal Heptanal ◾ 107 Cucumber. sourish. Overall earthy. multivariate data analysis is useful to explore the overall trend of the main quality indicators.4) on data from our studies on volatiles in cod [22] during prolonged storage for 17 days and compared with corresponding .4-Heptadienal Rancid Geranium-like 1-Octen-3-ol Mushroom Earthy. this is not always the trend for dynamic microbial and oxidative changes and the formation of volatiles in fish during storage [22]. 2004. and hexanal were abundant in headspace. melon 2-Nonenal Cucumber Fatty Fatty. Unpublished data. and fish oil notes were characteristic for fresh cooked salmon. and octatriene increased significantly.4-heptadienal. sweet. Baltic herring has been reported to have a similar development of volatiles. green-like odors Grass Hexanal Heavy Mushroom. sour. flowery.) heptanal and cis-4-heptenal were the most potent odors. 2-heptanone. However. and the most pronounced attribute was a boiled potato odor [49]. Its odor has been described both as cardboardy.3) were fatty. some confusion exists about the role of cis-4heptenal as the “cold-storage compound” [8]. heptanal. Other pronounced odors detected in boiled cod (Figure 8. 1-penten-3-ol and hexanal. and after storage for 3 days the proportions of 4-heptenal. G.52].

3-methyl-butanal in combination with acetaldehyde. especially in trout [15]. Other oxidatively formed compounds like 2-butanone and aldehydes were in higher levels in the B-D4 sample compared with the corresponding raw sample (R-D4). The malty flavor of 3-methyl butanal was suggested earlier to be mainly responsible for the malty off flavor defect of boiled cod [54]. In boiled trout. and 17 days).4). 12. samples after heating (see Table 8. (E.Z)-2.5 –0. boiled and storage days. dimethyl disulfide. On the basis of odor evaluation. that is. in agreement with earlier studies [54].4).2 Washed Cod Muscle System Rancid odor development during chilled storage of fish has commonly been associated with fatty species. . 19% PC1 1. D (4.8 R-D17 –0. 14.E)-2.4 0.108 ◾ Handbook of Seafood and Seafood Products Analysis PC2 Bi-plot 1. The characteristic pattern or trend in volatiles in raw and boiled fish is clearly different.2 0. methional with a characteristic boiled potato-like odor dominated the odor of the aldehyde fraction of the headspace volatiles. hexanal. Interestingly. 3-methyl-butanal was correlated to the boiled stored cod (B-D17) (Figure 8. Autoxidatively produced unsaturated carbonyl compounds were the most abundant components in boiled and canned fish.1). and (E.5 Undecanal Ethanol 3-me-1-butanol B-D10 0 R-D4 R-D12 R-D7 R-D10 Ethylbutanoate B-D4 Heptanal Nonanal Acetaldehyde Ethylacetate 2-Butanone 3-HO-2-Butanone 2-me-1-propanol TMA Decanal R-D14 6-me-5-h-2-one Hexanal 0 0. 8.6-nonadienal. in particular the role of volatile compounds derived from oxidation in heated/boiled samples.4 Principal component analysis of raw and boiled cod. However. raw and B. It is in particular interesting to demonstrate that the influence of heating gives a very different volatile profile compared with that of the raw samples that are all clustered on the left of the PCA plot. The oxidatively formed compounds.0 Figure 8. X-expl: 53%. The effect of oxidation induced by cooking and formation of oxidation products such as heptenal and nonanal characterizes the (B-D4) sample. and dimethyl trisulfide were detected in higher levels in the boiled samples (data not shown). as indicated by the arrows (Figure 8.0 B-D17 1-Penten-3-ol 3-me-butanal Acetic acid 0. Only the spoiled raw samples (R-D14 and R-D17) can be correlated with the freshly boiled (B-D4) sample. 10.4 –0.5-octadien-3-one. oxidation of membrane-bound phospholipids in lean species can cause fishy.4.2 Raw and boild c…. 7. The PCA demonstrates how volatile compounds can explain the variation in quality of samples according to storage time and handling (raw and boiled). Samples are labeled with R. Sulfur compounds dimethyl sulfide. increased with time and were pronounced in the spoiled raw samples (R-D14 and R-D17). and 6-methyl-5-hepten-2-one.4-decadienal from PUFA were determined as character impact odorants of boiled cod [54]. methional. and oxidatively derived (Z)-1.2. decanal.6 0.

free radical scavenging and chelation) but also on factors such as physical location. we found in our studies on the washed cod muscle system that hexanal could be used as indicator for rancid odor development. 2. Odor development in lean fish studied by hemoglobininduced oxidation in washed cod muscle system showed that sweet. pH) [55. earthy.1). Consequently. has been studied to understand better the mechanisms of oxidation in the muscle [57. soapy. green. [63]. and the overall odor was an intense dried fish. physical. To monitor the development of rancidity. as demonstrated by Boyd et al. dried fish-like off odors as discussed before. Preconcentration techniques are necessary for the analysis of unsaturated aldehydes. lipid oxidation of muscle phospholipids may be induced by several catalysts. In lean fish such as cod. grass odor contributed by hexanal. and caramel-like odors contributed by 3-methylbutanal. mushroom odor caused by 1-octen-3-ol.56]. sensory assessments. fatty. the concentration and composition of volatile oxidation products analyzed by GC were compared with TBARS measurements. potato-like odor caused by cis-4-heptenal and heptanal. Studies on the development of the odorous degradation compounds of phospholipid oxidation can lead to a better understanding of the kinetics and reaction pathways of oxidation in lean fish.. cucumber-like. Washed cod muscle system has been widely used to study oxidation and the influence of prooxidative and antioxidative factors [59. and 1-penten 3-ol. it is necessary to apply models that take into account the chemical. Similarly. rancid.59]. They showed that direct analysis of propanal can provide a quick and economical method for the determination of oxidation of n-3 fatty acids and pentane and hexanal analysis can give an indication of the oxidation of linoleic acid. including blood components like inorganic metals iron (Fe) and copper (Cu). The role of antioxidants (a-tocopherol. Furthermore. it is possible to detect the most volatile oxidation products like propanal and hexanal by rapid.. and rancid odors dominated the aroma profile [62]. but the compounds were detected in much lower levels [22]. in agreement with TBARS and changes in color [62].g.5) as well as 2.g. The effect of thermal treatment on hemoglobin-mediated oxidation in the phospholipid model system from cod muscle was studied by monitoring oxidative changes during chilled storage on ice by sensory analysis. On the other hand. The added hemoglobin was very effective as a prooxidant. This is because the activity of antioxidants in food systems depends not only on the chemical reactivity of the antioxidant (e. TBARS (thiobarbituric reactive substances). static headspace sampling methods. sweet.3-pentandione. this may facilitate the selection of preventive measures to limit oxidation and guide new technological developments with the aim to ensure the delicate taste and nutritional value of lean fish products. and instrumental color changes. These compounds can be used as indicator compounds for oxidation. ascorbic acid. rancid fish oil like. To accurately evaluate the potential of antioxidants in foods. which is not practical for rapid determination of oxidation. These odors were also detected in cod fillets during chilled storage (Table 8. Sohn et al. rancid. and a similar trend was observed in the development of cis-4-heptenal (Figure 8. and lemon-like odors were explained by 2. interaction with other food components. The most potent odors detected in the model system were malty. . and environmental conditions (e. and spicy and flowery notes exhibited by 6-methyl-5-hepten-2-one. The prooxidative effect of hemoglobin was evident by the formation of hexanal in high levels.58]. and glutathione peroxidase) and aqueous prooxidants in fish muscle. painty. and environmental conditions expected in food products. [60] studied lipid oxidation and rancid odor during the early stage of ice storage of ordinary and dark muscle of yellowtail and concluded that myoglobin was the main cause in the development of the unpleasant color and undesirable odor during ice storage of fish muscle. floral.4heptadienal that contributed to rancid odor caused by oxidation.61]. including hemoglobin from blood [7. and color.4-heptadienal [62].Volatile Aroma Compounds in Fish ◾ 109 rancid.

Matis Report 08.e. R. and Ólafsdóttir. (From Jónsdóttir. with added hemoglobin (raw and cooked. caffeic acid) [65] as well as application of tocopherol. Food Prod. Aquat. painty.) Thermal treatment of the cod model system significantly enhanced the oxidation of the model on day 1. Blank-II. With permission. 2007.. described as rancid.) . -■-. J.. ( Adapted from Jónsdóttir. -▲-. Some promising results have been reported. 100 90 80 70 60 50 40 30 20 10 0 40 35 Odor score (rancidity) TBARS (μmol/kg) 0 1 Blank 2 Raw 3 Cooked 4 30 25 20 15 10 5 0 0 1 Blank 2 Raw 3 Cooked Figure 8. and EDTA [66].110 ◾ 1000 800 ng/g Handbook of Seafood and Seafood Products Analysis Hexanal 30 25 20 ng/g 15 10 5 0 0 1 Blank-II 2 Hb-Char-II 3 Hb-Cod-II 4 0 1 Blank-II 2 Hb-Char-II 3 4 cis-4-Heptenal 600 400 200 0 Hb-Cod-II Figure 8. The studies on the washed cod muscle system verify the importance of oxidation in off odor development in fish muscle and consequently the benefit of being able to control oxidation to prevent the formation of the aldehydes. G. 73.e. HbCod-II). 2008.. respectively) and raw without hemoglobin (blank). and in TBARS (Figure 8. R... citric acid. Active research is ongoing on the application of various natural antioxidants based on polyphenols like flavonoids (i. 67. et al. catechins from tea) and cinnamic acid derivatives (i. and dried fish odors.5 Gas chromatography analysis (FID) of characteristic volatile compounds contributing to rancid odor (hexanal and cis-4-heptenal) in hemoglobin (from Arctic char and cod) mediated oxidation in washed cod model stored at 0°C for 4 days (-♦-. Studies on LOX inhibitors are of interest in preventing the initiation of oxidation in fish.6 Sensory analysis of rancid odor (odor score) and TBARS measurements in raw and cooked washed cod model stored at 0°C for 4 days. Hb-Char-II.6) as well as more rapid loss of red color (not shown) already on the first day of storage. as measured by rapid increase in rancid odor. where commercially available green tea polyphenols were shown to effectively inhibit the LOX activity of mackerel muscle [67]. 16.

6-nonadienal. Other oxidatively derived compounds like 1-penten-3-ol. Figure 8. Thermally generated aroma-active compounds via the Maillard reaction such as pyrazines are characteristic for enzymatically hydrolyzed seafood products like crayfish processing by-products [68]. Guillén et al. 2-butanone. like cis-4-heptenal. Phenolic derivatives like guaiacol (2-methoxyphenol) and syringol (2. giving a popcorn-like odor that can be thermally generated. which is typical for products on the market [23].6-dimethoxyphenol) have been identified as the most characteristic smoke-related compounds in smoked fish-like herring (Clupea harengus) [73] and in smoked salmon (Salmo salar) [23. 3-methyl-butanal. giving the flesh its typical fishy odor [71. nonanal. These are compounds like methional. and 2-acetyl-1-pyrroline. hexanal. sweet odors of processed seafood like those in smoked salmon [23. The typical smoked salmon aroma results from a number of chemicals found in the smoke.71. 3-methyl-1-butanol. 3-hydroxy-2-butanone.6-nonadienal. [74] analyzed headspace components of cod and swordfish. associated with spoilage off flavors. 1-octen-3-ol. and although they contributed less to the odors.4. Microbially produced ketones.72]. where groups of phenol pyrolysis were most noticeable in the smoke flavor volatiles. sweet/sour rancid.72]. contributing to mushroom-like odor. Maillard reaction.Volatile Aroma Compounds in Fish ◾ 111 8. were characteristic in unsmoked fish.4-heptanal. and lipid oxidation. Some of these compounds were selected as key spoilage indicators for smoked salmon based on their high levels and contribution to sweet and fruity spoilage off odors in our study on smoked salmon (Figure 8. 8. hexanal. such as heptanal and (E. it was verified that selected key volatile compounds performed better as predictors to explain variation in sensory attributes (smoked. furan-like compounds have been reported to be responsible for the smoked odor in smoked salmon.4. 2. and 1-octen-3-ol.7) [23].3.4-decadienal. gave the most intense odors of smoked salmon and contributed to the fish-like earthy odors and fatty and rancid odors (Figure 8. Volatile compounds like alkyl-pyrazines and sulfur-containing compounds have been found in cooked crustaceans. and off odor and flavor) than traditional chemical and microbial variables. and 2.72]. and furans have been found in spray-dried shrimp powder and shrimp hydrolysate [69]. and decanal were among key volatiles. plays important roles in the formation of complicated processing flavors. . 1-penten-3-ol. potato-like odors. Lipid-derived components. 2-methyl-1-butanol.7) (e. and 3-methyl-1-butanol) [23]. whereas carbonyl compounds. including Strecker degradation. In addition to phenolic compounds. The oxidatively derived compounds cis-4-heptenal and heptanal. thermal degradation. 2-pentanone. 2. ethanol. Lipid-derived aldehydes play an important role in flavor formation and have been reported to contribute to the characteristic fish-like. it is clear that their presence contributes to the characteristic fish odor of smoked salmon products.g. Key volatile compounds identified in enzymatically produced seafood flavorants are formed via Maillard reaction and Strecker degradation of amino acids.3 Processing Odors Flavor development in processed seafood is a result of complex proteolytic and lipolytic reactions induced by different processing parameters like enzymes and temperature. the Strecker aldehyde produced from methionine. which has a characteristic potato-like odor.. like 3-methyl butanal. Additionally.75]. also contribute to the aroma of seafood flavorants [70].7 illustrates the main odors that were present in smoked fish samples after 14 days of chilled storage. aldehydes. giving rancid.1 Smoked Fish Odors Degradation compounds from Maillard reactions and lipid oxidation are the main compounds contributing to the aroma of smoked salmon [72].Z)-2. but it is mostly attributed to the phenols. and 1-propanol [28. and alcohols were abundant in the headspace of cold smoked salmon products during storage.

potato-like odors together with cucumber-like odor [82]. especially those in the Mediterranean.6-nonadienal from fatty acid oxidation were the main odorants in sugar salted. 4-Heptadienal Sweet. and rancid-like odors Burnt. 2008.) 8. both oxidatively derived compounds. most of them generated from chemical or enzymatic oxidation of unsaturated fatty acids and further interactions with proteins. During ripening of salted cod. fruity Flowery. as heptanal.3. the desired flavor and texture develop as a consequence of protein and fat degradation.4-heptadienal and 3. manufacturers of ripened products have observed that some degree of proteolysis is necessary before flavor can develop.7 GC–O evaluation of volatile compounds detected in cold smoked salmon after 14 days of storage at 5°C. highly volatile components of ripened anchovy. 184.5octadien-3-one were also identified as potent odorants in ripened anchovy [81]. where methional derived from methionine and 2. Methional and (Z)-1. ripened roe products [79] Similarly. sweet Smoke-like 2. where the ripening of salted cod (Gadus morhua) produced by different salting methods was studied. . probably originating from amino acids. geranium Rancid cis-4-Heptenal Heptanal Earthy-like odors 1-Octen-3-ol Figure 8. sweet Wood. sweet Flowery. Triqui and Reineccius [80] found that 2.. caramel Smoke-house. 2-methylpropanal and 3-methylbutanal were the key.2 Ripening Odor—Salted and Dried Fish Odor Numerous volatile compounds have been detected in ripened products like dry cured ham. (Modified from Jónsdóttir. et al. smoke. peptides. and aldehydes such as acetaldehyde. the highest odor scores were given for boiled potato and rancid.4. [76–78].and 3-Methyl phenol Guaiacol 4-Methyl-guaiacol Sweet and fruity-like odors Wood. burnt. fatty Boiled potato-like Fatty. earthy. smoke Mushroom. and free amino acids. potato-like odor was identified as cis4-heptenal and the boiled potato-like odor. smoke 3-Methyl butanal Sweet. they suggested that lipid autoxidation during ripening was primarily responsible for aroma development. Food Chem. R.112 ◾ Handbook of Seafood and Seafood Products Analysis Smoked salmon odors Characteristic smoke odor Sweet. sweet Flowery. In our study. Thus.. The rancid. Similar processes have been reported in ripened seafood products. Salted cod are traditional products from the North-Atlantic fisheries and are highly regarded as ripened fish products in many countries. mushroom 2. However. 109.5-octadien-2-one were associated with the development of the typical flavor obtained after anchovy ripening.

FAO. and in retail for consumers as smart sensors imprinted on packaging. their presence at nominal levels gives the characteristic and desirable fishy odor in fresh and processed fish. and sulfur compounds. proper handling. Detection of microbial metabolites originating mainly from soluble aqueous fractions of the muscle can be directly related to the quality of products. 1995. No. J. were the most intense character impact compounds of salted cod and smoked salmon. Volatile compounds as indicators of freshness quality and spoilage can be monitored to determine the quality of fish products. such as ketones. and other prooxidants in combination with mild heating treatment are important factors to maintain the delicate flavor and odor of fish products. and 1-octen-3-ol. Rome. A similar set of sensors with selectivity and sensitivity toward the main quality-indicating classes of compounds. Other key volatile compounds in salted cod are derived form lipid oxidation. FAO Fisheries Technical Paper. Lipid oxidation during ripening appears to be primarily responsible for desirable aroma development in processed fish. Studies on hemoglobin-induced oxidation in the washed cod model system and enhanced oxidation after heating verified the role of the oxidatively derived compounds contributing to off odors in chilled stored and boiled cod. hexanal. 193 pp. according to retention index (RI) of standard and odor evaluation. and new packaging technologies. can be used for a variety of fish species that are stored and processed by different techniques. temperature control. for example. contributing to mushroom-like odor. Knowledge of the spoilage pattern of volatile compounds is the basis for the development of rapid techniques like smart sensor technologies. cause off odors in fish during storage. The cucumber-like odor detected is possibly 2. In addition. Proper handling and application of natural antioxidants to control oxidative processes caused by lipoxygenase. 8. 348. . derived from methionine and eluting at a similar time as cis-4-heptenal and heptanal. Therefore. Development of smart sensor technologies like the electronic nose to detect microbial metabolites and oxidation products is of interest to verify the quality of products to facilitate process management. and myoglobin. aldehydes. exhibiting rancid.83]..Volatile Aroma Compounds in Fish ◾ 113 Methional. although the compound could not be identified by GC–MS. 1995. careful evaluation of the quality of product is needed to ensure acceptable flavor. 2. microbial growth can be limited by effective cooling techniques. References 1. esters.R.Z)-2. Evaluation of Seafood Freshness Quality. Quality and quality changes in fresh fish.6-nonadienal. to increase trust between buyers and sellers in trade. New York. amines. alcohols.H. careful control of handling and processing conditions should open up possibilities for fish to become a favored choice in new product development of convenience food and in functional food because of its health beneficial properties. H. A certain degree of lipid oxidation is both necessary and desirable for sufficient ripening of the products but the process should be controlled to obtain a desirable degree of ripening based on consumer preferences [82. 1-penten-3-ol. 1–67. Botta. potato-like odors.5 Conclusions Although aldehydes.6-nonadienal. Huss. acids. The oxidatively derived compounds cis-4-heptenal and heptanal. However. such as heptanal and (E. hemoglobin. and 2-butanone. could also be responsible for the boiled potato-like odor. VCH Publishers Inc.

Josephson. Seasonal variation of phosphatidylcholine hydroperoxides in blood of sweet smelt Plecoglossus altivelis. Josephson. German. J. Acta. Jónsdóttir. Zhang. Enzymatically generated specific volatile compounds in ayu tissues. and Kinsella. Ushio. Comparative Biochemistry and Physiology Part B 130. and Shirai.6-dibromophenol: The cause of an iodoform-like off-flavour in some Australian crustacea.P. C..L. German. P. K.. Agric. and Ólafsdóttir. G. R. 10140. Identification of compounds characterizing the aroma of fresh Whitefish (Coregonus clupeaformis).. Morishita. J. Variation in the occurence of volatile compounds in cold smoked salmon (Salmo salar) during storage. Reykjavík. 2004.B. 1344.B. 32.H. 29. Nippon Suisan Gakkaishi. Agric.C. 58. 31. and Shirai. Lipoxygenase in fish tissue: Some properties of the 12-lipoxygenase from trout gill..O. Technol. E. in Proceedings of the International Symposium on Seafood Quality Determinations. 184. J. Lipoxygenase in trout gill tissue acting on arachidonic.. Occurrence and properties of flavour-related bromophenols found in the marine environment: A review. Ólafsdóttir. 50. 2005. D. PhD Thesis. T. T. J. J. 1988. 507. G. 279.. 33.. 11. Measurement of volatile aroma constituents as a means for following sensory deterioration of fresh fish and fishery products.. J. 1983. Suzuki. J.. 13. J. and Kinsella..R. 4. Int. R. D.E. 8. Food Chem. Chanie. 2.. 1985. and Ohshima... T.. H.L.B. 15. Josephson. 27. H. T. T. Agric. 875. 1993. 179. R. . Kramer. Biophys.. 1992.. J.J. 559. J. and Dalgaard. 24. and Stuiber. Luten. G. Whitfield. T. 58. Hsieh. Food Chem.. Shaw. and Grosch. Biotechnol. Sci. J. 18. L. F. Agric. 23. German.. 2002. 1986. 10. Gas sensor and GC measurements of volatile compounds in capelin (Mallotus villosus). J. eds. Luten. M. Jónsdóttir. 326. 33. Boyle. Enzymatic initiation via lipoxygenase. and Oehlenschläger. Fish flavours. 680. Food Chem. and Jónsson. C. 1997.B. Marcel Dekker.B..B. K. eds.. G. 36. J. Lindsay.. Relative contribution of calpain and cathepsins to protein degradation in muscle of sea bass (Dicentrarchus labrax). p. T. Josephson. Elsevier. Agric. the Netherlands.. R. 88. Food Agric. Food Chem. J. Food Rev. R. 46. and Hultin. Faculty of Science. Maarse.C. C. 75. 9.. J. 1993.B.. J. University of Iceland.E. and Stuiber. T. J.A. Food Chem. Amsterdam. New York. 262. Ed. 2005. Zhang.C. Agric. R. and Kinsella.. J. Food Chem. R.. D. Lipid oxidation in fish tissue.C.C. Ólafsdóttir. Biochim. and Kinsella..E. 2076. Richards. 13.. 53. 2002.. Curr.J.. 12. 1991.. in Seafood from Producer to Consumer.... 437. Noël. Hirano.E. Agric. H. Changes in the odourants of boiled trout (Salmo fario) as affected by the storage of the raw material. 41.. Hirano. Delbarre-Ladrat.. Verrez-Bagnis. 1986. 109. Lauzon. and Tindale...C. 2008.and freshwater fish. G. V. Lipoxygenase generation of specific volatile flavour carbonyl compounds in fish tissue. Fish spoilage bacteria–problems and solutions. Aquat. 1989. D. Buckner. 14.H. Food Prod. 2001.. J. 20.H. Food Chem.. Identification of volatile compounds in ayu fish and its feeds.. and Stuiber. Lindsay. 12.E. 7. eicosapentaenoic and docosahexaenoic acids. 37. Martinsdóttir E.. D. A.B.C.. W. 19. Food Chem. 555.B. 6. Nippon Suisan Gakkaishi. 22. Volatile compounds as quality indicators in fish during chilled storage: Evaluation of microbial metabolites by an electronic nose.. H. Food Chem.. J. Elsevier Science Publishers BV. Ólafsdóttir. J. and Kristbergsson. 389. 5. and Liston. Ólafsdóttir. 6. 21.. Iceland. Hsieh. 1990. Contributions of blood and blood components to lipid oxidation in fish muscle. Milo. E.B. Amsterdam. Variations in the occurrences of enzymically derived volatile aroma compounds in salt. R. D. 1992. Börresen.. Integrated Approach to Quality. 680. C. G.. R. 1984. the Netherlands. in Volatile Compounds in Foods and Beverages. Agric. Suzuki..J. Seafood. and Fleurence. Lindsay. Lindsay. Kaewsrithong.. J. J. J. 1988.114 ◾ Handbook of Seafood and Seafood Products Analysis 3... Lindsay. 2. J. Gram.. Food Chem.. D. D.... 17. Characterization of volatile compounds in chilled cod (Gadus morhua) fillets by gas chromatography and detection of quality indicators by an electronic nose. and Haugen.E. Opin. 547. C. J.. 16.

J. p. Nesvadba. R. J. Oehlenschläger.. Dalgaard. 1973. J... J. Food Chem. Appl. A. J. 26.. in Rapid and Online Instrumentaiton for Food Quality Assurance.. Westad. Evaluation of some well established and some underrated indices for the determination of freshness and/or spoilage of ice stored wet fish.. G. and Heia. Henehan. Int.. 2006. 1957. 35. 32.H. Fish. E. 953. Jónsdóttir. Tryggvadóttir.. R. G. Labreche.. S.. and Dalgaard. 563. 1989. H. Technol. Comparison of volatiles of cultured and wild sea bream (Sparus aurata) during storage in ice by dynamic headspace analysis gas chromatography mass spectrometry. Trends Food Sci. Actuators B. Ólafsdóttir. R. Chanie. G. and Kristbergsson.S.. and Liston.H. P. Woodhead Publishing Ltd. 1997. E. et al. G. 29. International Institute of Refrigeration. Castell. K.. Methods to evaluate fish freshness in research and industry. 53. Martinsdóttir. Bazzo. Huss. Lee. Lauzon. J. L. S. Soc. 86. G. Undeland. J.. in Methods to Determine the Freshness of Fish in Research and Industry. Food Sci. G. Agric. P. 339. Verrez-Bagnis. H. J. 26.. eds. 112. K.. Oehlenschläger. 18. Haugen. Microbiol. Food Sci. E. I. Evaluation of compounds contributing fi shy flavors in fish oils.. and Nilsen.. 2006.H. M. C. 70.. Huss.. and Ólafsdóttir... Lindsay.. P. 55.. R. 25. the Netherlands. 66. Careche.. Volatile compounds produced by Pseudomonas putrefaciens.A. Prediction of microbial and sensory quality of cold smoked Atlantic salmon (Salmo salar) by electronic nose. F. Tothill. and Kristbergsson.A. Thalman. Oehlenschläger. Significance of volatile compounds produced by spoilage bacteria in vacuum-packed cold-smoked salmon (Salmo salar) analyzed by GC-MS and multivariate regression.. Ólafsdóttir.E... C. J. 49. Jónsdóttir.Volatile Aroma Compounds in Fish ◾ 115 25. Martinsdóttir. Schubring. Alasalvar. Chanie.. Careche. 8. F. J. J. E. Trends Food Sci. D.. 111. The action of Pseudomonas on fish muscle: 1. 37. 36.F. H.M. Lundby.. G.. 2001. Oehlenschläger. 2003..C.. Oil Chem.. 2006... 1998. Elsevier Science Publishers. Appl. 2616. 38. Am.K. Lee. B. U.V. and Haugen. Josephson. P.A. J. Agric. L.. in Proceedings of the International Symposium on Seafood Quality Determinations. Amsterdam... 39. P. J.. 2005. Jørgensen. I.. 2004. 1973.. Board Can. G.. K. Sens..B. 2005. J. 31.. 12. 40. and Ólafsdóttir. Di Natale. 97. H.. Rapid control of smoked Atlantic salmon quality by electronic nose: Correlation with classical evaluation methods. Chemical and biochemical indices for assessing the quality of fish packaged in controlled atmospheres. R. D. J. Ólafsdóttir. Organisms responsible for odour produced during incipient spoilage of chilled fish muscle. and Greenough.. C. Ed. J. Mackie.. 30. and Liston.S.D. eds.. Identification of the volatile compounds produced in sterile fish muscle (Sebastes melanops) by Pseudomonas fragi.. Pseudomonas fluorescens and an Achromobacter species.. and Lindsay. 2376. M.. Paris.. F. H. the Netherlands.M. E.. S.. Ólafsdóttir. Labreche. J.. 221. 339.. in Quality Assurance in the Fish Industry. J. Scanlan. Nielsen. 72. M. Jakobsen.. Dalgaard. 952. Miller III.. Amsterdam. Karahadian. Evaluation of shelf-life of superchilled cod (Gadus morhua) fillets and influence of temperature fluctuations on microbial and chemical quality indicators. Food Microbiol. M. Ólafsdóttir.. Ibtisam E. C. A. 27. and Fleurence. 1986. P... Elsevier Science Publisher. Evaluation of fish freshness using volatile compounds—classification of volatile compounds in fish.C.. Multisensor for fish quality determination. Res. S. Marcq. Taylor. Miller III... 71.258.M.. Influence of storage temperature on microbial spoilage characteristics of haddock fillets (Melanogrammus aeglefinus) evaluated by multivariate quality prediction... G. Microbiol. K. Lundby. Cambridge... 34. Marcq.V. Food Chem. Martinsdóttir..E. S.. and Shahidi. J. R.. C. G. 617. and Libbey. Bazzo. 15. F. eds. and Libbey. J. 33. 116... Lauzon. Proceedings of the final meeting of the concerted action “Evaluation of Fish Freshness” AIR3 CT94 2283. Luten.. 28. E. Ólafsdóttir. G. L... F.. Ólafsdóttir. Martinsdóttir. Developing rapid olfaction arrays for determining fish quality. Technol. V. Westad. R.. Jensen. . Ólafsdóttir. Kramer. Scanlan. 1992.

. A. Hardy.. M.. Bragadóttir. M. 524. Rancidity development in a fish model system as affected by phospholipids.. C. 25. and Jensen. 76. Jónsdóttir. 43. Decker. T. F. R.F.. L. 52. Koizumi. Agric. 53.. Taki. Kohata. 1989. 2007. Food Res. Unpublished data. M. 52.. I. Burt. in Odour of Marine Products. W.O. M. Lipid oxidations in ordinary and dark muscles of fish: Influences on rancid off-odor development and colour darkening of yellowtail flesh during ice storage. 2003. and Botta. T.S. and Hultin. Sci.. Detection of defects in boiled cod and trout by gas chromatographyolfactometry of headspace samples. 47. J.. 1186.C... 67. Food Chem.F. 19. 1998. Glasgow. Agric. Technol.. Herbert.. E.B. 1998. Antioxidant strategies for preventing oxidative flavour deterioration of foods enriched with n-3 polyunsaturated lipids: A comparative evaluation. Agric.D.. 303. 55. Lipid and autoxidative changes in cold stored cod (Gadus morhua). G. Food Chem. J.. K. 2004. eds. 3473. R. 44. 46. H. M. Koseisha-Koseikaku. and Ólafsdóttir. 459.S. Food Chem. 490. 70. and Ohshima. H. 1987. Oxidation of lipids in seafoods. Tahvonen. 45. Refsgaard. L. 53. F.W. Lipid Oxidation. 1975. A.K. 42. Trends Food Sci. 4444. Dundee. 325.. Hall. Shahidi... 2001. Brockhoff. Sci.O.. J. 57. C. W. 1996. J. J.J. Ed.. Food Sci. and Gunstone. Technol. 61. 328. J.. C. J. Richards.. Oil Chem. Y. Kristinsson. R. N.. and Lindsay. 1998.. Aro. 483.. and Grosch. and Gunstone.. Japan.. H... A. T. J. 2005. Am. Eur. Tokyo.. Trends Food Sci. D. Agric. H. J. Frankel. J. E. 1987..B. 1992.. Food Chem.H.H. Measuring antioxidant effectiveness in food. Hardy. B. J. 1979. Richards.. in Seafoods: Chemistry. Scotland. 46. c4-Heptenal: An influential volatile compound in boiled potato flavour.M.C. T.F. New York. D. Retro-aldol degradations of unsaturated aldehydes: Role in the formation of c4-heptenal from t2. Marcel Dekker Inc.c6-nonadienal in fish. R. 2000.. p. 49. Soc. Ed. Processing Technology and Quality.P. P. Agric. Lipid oxidation in fillets of herring (Clupea harengus) during ice storage. A rapid method for determining the oxidation of n-3 fatty acids. I. Undeland. 2005. R. F.. U. 8..D. H. 49. 50.. King. The Oily Press.B. 9. J. . Hemoglobin-mediated oxidation of washed minced cod muscle phospholipids: Effect of pH and hemoglobin source. Food Agric. Technol. and Sheldon. and Meyer. Food Agric. 52. 28.C. 1974. and Shahidi. 64. McGill.O. 48.B. R. Sci.. 1195. H. Agric. M. 1999. 53. B. 1994. Josephson. Minimizing rancidity in muscle foods. Z. Food Chem. Food Sci. 2004.. J...A.. Hultin. Changes in the odourants of boiled salmon and cod as affected by the storage of the raw material. C. Yamanaka. I. 63. 44. T. and Grosch. J. Jónsdóttir. 241. Jacobsen. and Shewan. J. 216. J.. Koskinen. H. Milo. and Ólafsdóttir... Let. J. Park. McGill. Food Lipids..116 ◾ Handbook of Seafood and Seafood Products Analysis 41. Isolation and identification of the volatile sulphides produced during chill-storage of North sea cod (Gadus morhua).. 7. Sohn. and Kelleher. G. Milo. Ellis. Shioya.. Decker.S. and Xu. 26.A. Offensive odour of fish and shellfish. and Lingnert. in Surimi and Surimi Seafood. Hept-cis-4-enal and its contribution to the off-flavour of cold-stored cod. Volatile compounds of Baltic herring analysed by dynamic headspace sampling-gas chromatography-mass spectrometry. Hultin.S.A. Blackie Academic and Professional. 56. Boyd. Nielsen. 14. H... and Kallio H. 60. G.. Ushio. 43. 62. 47.. Sensory and chemical changes in farmed Atlantic salmon (Salmo salar) during frozen storage... Undeland.P. J. 4303. J.D. Surimi processing from dark muscle fish. and Lindsay. 2008. 69.G. Josephson. 59. 51.. J. 215. and other flavours. 59. Warner. and Hultin. Food Prod. E. oyster. 58. Food Chem. 999. 54. Food Agric. R.. Aquat.R. 1995. p. 2366. R. S.O. The role of volatile compounds in odor development during hemoglobin-mediated oxidation of cod muscle membrane lipids. Food Sci. 1477. F.

1997. Martinsdóttir. Inhibition of mackerel (Scomber scombrus) muscle lipoxygenase by green tea polyphenols. ACS Symposium Series 674 American Chemical Society. Food Res. 94. G. K. Characterisation of volatile compounds produced by bacteria isolated from the spoilage flora of cold-smoked salmon. 81. 2007.. Food Research International.. 67.L.. Blackie Academic and Professional. Headspace volatile components of smoked swordfish (Xiphias gladius) and cod (Gadus morhua) detected by means of solid phase microextraction and gas chromatography–mass spectrometry.. Gallardo. R. and Serot. C. 33. J.. M. Food Chem. in Seafoods: Chemistry. J. Joffraud. Food Chem. Agric. F. Baron. pro-oxidants.. Proteolysis and lipolysis in flavour development of dry-cured meat products. Lois. Flavorants from seafood byproducts. NJ. Int. 85. Milk. Volatile compounds in flavour concentrates produced from crayfish-processing byproducts with and without protease treatment. Comparison of odour-active volatile compounds of fresh and smoked salmon. Crit. 1996. and Serot. Processing.L. J.. and Stefánsson. B. 76. and Flores.J. 65.. F. R. Agric. Y. R. R. The role of muscle proteases and lipases in flavour development during the processing of dry-cured ham. Varlet. Food Chem.H. J. Iceland. 931. Food Agr.S. R. occurrence and mechanisms of formation.. Reykjavík. Jónsdóttir. Sci. 2006. M. Meat. Toldrá.) by aroma extract dilution analysis and by gas chromatography-olfactometry of headspace samples.. 2006. Ed. 3262. Int. 73.. Glasgow. 82. Flavour of shellfish and kamaboko flavourants. 70. 2004.. and Ólafsdóttir.Volatile Aroma Compounds in Fish ◾ 117 64. C. N.. Salmerón. G. Guillén. 1995. R. 68..C. Effect of smoke processes on the content of 10 major phenolic compounds in smoked fillets of herring (Cuplea harengus)..J.. K.. Shahidi. antioxidants and the effect of heating. F. and Berdagué. Changes in flavour profiles with ripening of anchovy (Engraulis encrasicholus)... Food Lipids. in Flavour and Lipid Chemistry of Seafoods. Gonzalez. Flavour characterization of ripened cod roe by gas chromatography. K. Toldrá. and Kuo..L. G. and Hedges. 2004. Bragadóttir. 79. Oxidation in fish muscle: The role of phosholipids. Food Chem. U. Food Microbiol.C.. Vol 2. Shahidi. J. in Handbook of Food Products Manufacturing: Health.. Roy. Knockaert. 66. Prost. Food Chem. I. 101. Effects of antioxidants on copper induced lipid oxidation during salting of cod (Gadus morhua).. M. 52.R. 1. 1883. 2007. Lauritzesen. Washington. 78. 49. 181. N. 151. 43. 70. 99. V.. J. Ólafsdóttir...E. 105. Food Chem.H. Toldrá. John Wiley & Sons. Poultry. and Cadwallader. 1999. S. M. C... Determination of potent odourants in ripened anchovy (Engraulis encrasicholus L. and Flores. 69. Jónsdóttir... Lauritzesen. Ólafsdóttir. Medina. J. R.. 80. M. 72. 77. H. and Thórarinsdóttir.. 1994. J. V. Matis report 08. Ushio.. Rev. Jónsdóttir. Aristoy. Contribution of muscle aminopeptidases to flavour development in dry-cured ham. 1998. and Botta. 39. and Einarsson. and Vegetables.. sensory analysis and electronic nose. M. 486. 31. Food Sci.. 2004. 105. Jónsdóttir. R. 2000. 2001. 175.. 2007. 83. 2006.R.. 44. proteins. Knockaert. Effects of EDTA and a combined use of nitrite and ascorbate on lipid oxidation in cooked Japanese sardine (Sardinops melanostictus) during refrigerated storage. M. Agric. and Casas.D. Volatile aldehydes in smoked fish: Analysis methods. Jittrepotch. Varlet. J. H... Food Chem. Seafood. 71. J. T. Hui. G. 2008. Prost. 73. Sérot. 85. C. Agric. G. Banerjee. Effect of molecular structure of phenolic families as hydroxycinnamic acids and catechins on their antioxidant effectiveness in minced fish muscle. 1536. 2007. and Vallet.K. Leroi. Baek. F. F. J. Eds. 55. Hauksson. J... 111.A. Food Chem. 75. Hoboken. T.. 3889. Triqui. S. T.R.. S. and Olsen. and Ohshima. 2006. Triqui. E. 74. . and Reineccius.M. 331.. Unpublished data. C. Pan. K... 3391. 66. 54. J. 38. C.. 6250.. Errecalde. T. Meat Science. Technology and Quality.. K. F. DC. Eds. H. and Guth.M. J. and Cadwallader.. J....




.......................................................3..........................3.........................................................132 9.......... 121 ......5 Summary ...1 Determination of Basic Composition.......2 Theory....................................................1..........................................................4 X-Ray Imaging ...................................................132 9................................................................................ 134 References ............................................... 128 9...3 Analysis of Basic Constituents ........................................................................3 Analysis of Basic Constituents .....................................4............................1 Determination of Basic Composition...... and Analysis .3.................................132 9..2 Analysis of Basic Constituents .............................. 122 9....... 122 9.....................2 Imaging Spectroscopy .................................................1....................2..............................................................................2.........................133 Acknowledgment ...... 130 9.................... 128 9.................. Measurement Principles...........................................1 Near-Infrared Spectroscopy....................131 9................................................ and Data Analysis ........ 124 9.....1......1 Theory and Measurement Principles .... and Margrethe Esaiassen Contents 9...........................................4................................................................................................................3 NMR Spectroscopy . 130 9.................. 128 9.......................... 122 9................2 Analysis ...2 Theory and Measurement Principles ..................................Chapter 9 Basic Composition: Rapid Methodologies Heidi Nilsen.................. 130 9...... 134 Fish and seafood consumption has gained increased attention during the last years as a consequence of increased focus on nutritional quality as well as aspects related to healthy living........ Karsten Heia........1 Theory..................................................... Measurement Principles.....................................................................................................................

The basic principles of the techniques are described as well as a presentation of the use and applicability of quality measures of fish. C−H. In this chapter. to analysis related to the environment and the petrochemical sector [1]. There are several reasons why NIR as a food analytical tool has caught attention and approval during the last decennia.2 Theory. pharmaceutical applications. throughout the years the method has also proven useful for the analysis of seafood and seafood products [8]. which is a prerequisite for a methodology to be applied along a production line. Another aspect to be considered is the increased consumer awareness regarding the quality of their food. frequently consumers want readily accessible information about nutritional parameters and food quality. The work in food analysis tends to have a focus within the agricultural sector [1]. 9. and so the need for measurement and documentation of such parameters is both a consumer requirement and also issued by law. . we review some of the most relevant methods for assessing the basic composition of fish and seafood as presented in scientific literature. Measurement Principles. The measurements are based on light interaction with material. requirements for such a method would preferably be that it is rapid and nondestructive. followed by a presentation of the usage of NIR measurements for the rapid determination of basic constituents in fish and seafood products. imaging techniques. and additionally the method may be applied with little or no obtrusion to the material sample. these techniques may be applied in or at a production line. The documentation of basic nutritional composition of foods is a legal requirement in many countries. During the last 30 years the use of NIR spectroscopy has gained increased importance in the evaluation of a number of different food quality parameters [2–7]. However. In this perspective there is an obvious need for objective methods for evaluating and documenting the basic composition of fish and seafood. The absorption of light is due to the response of the molecular bonds O−H. These methods are near-infrared (NIR) spectroscopy.1 Near-Infrared Spectroscopy Determination of Basic Composition The development and usage of near-infrared spectroscopy (NIR) as an analytical tool has proven useful in areas varying from food quality. we give a short introduction to the principles of NIR spectroscopy. facilitating a rapid response.122 ◾ Handbook of Seafood and Seafood Products Analysis Compared with the production and distribution of meat from the agricultural sector. seafood is considered highly fragile and perishable with a short shelf life and delicate texture. Another benefit is the potential of simultaneous measurements of more parameters.1. as well as x-rays. In this context seafood is particularly challenging as it comprises a vast number of different species with their own characteristics and qualities.1. The four methods presented fit with the requirements of speed and nonobtrusiveness. C−O. 9. hence. A food sample exposed to emission in this wavelength range will absorb certain parts of the energy depending on the chemical composition of the sample. and Data Analysis The electromagnetic range applied in NIR spectroscopy spans from 700 to 2500 nm. magnetic resonance. Regarding industrialized food production. and hence these issues must be considered during the processing and characterization of the material.1 9. In the following section. comprising the frequencies just below those of visible light.

the spectral readings must be correlated to a relevant reference method such as. If the light source and the detector are placed on the same side of the sample as shown in (b). but an immediate look at an NIR spectrum is not sufficient to quantify the different substances. the transmission and reflection may be either direct or diff use. Different measurement modes for NIR spectroscopy are illustrated in Figure 9. nondisruptive. light from the source penetrates the sample and enters the detector.11]. A setup as shown in (a). The amount of light entering the detector unit depends on the scattering and absorption features of the sample as well as the sample thickness and lamp characteristics. traditional chemical determination of the constituents. Over the years there has been a steadily ongoing development of instrumentation for NIR spectroscopy. A thorough theoretical description of the NIR theory as well as the designation of numerous bands of absorptions may be found in Osborne and Fearn [2] and reviews on the subject [1. depending on the scattering properties of the medium under investigation. the system is operated in “reflection” mode. NIR spectroscopy is an indirect measurement technique. In (c) the light source and detector are located to register light that has traversed the sample before detection. (c) illustrates how measurements are performed in “transflection” mode. . Hence.9. for example. enables “transmission” measurements. placing the light source and the detector at the same side of the sample. In the context of rapid methodologies. and noncontact methods.10]. In (a) the transmission setup is shown. developed toward the facilitation of nondestructive.Basic Composition: Rapid Methodologies ◾ 123 and N−H [9] and corresponds mainly to overtones and combinations of fundamental vibrations. The setup in (b) displays the reflection setup where light reflected from the sample surface enters the detector. both with respect to the detectors and the capture of the spectral information [10. a screen is placed between the directly emitted area and the area of inspection. we view this in terms of the measurement setup enabled by technology.1 Different measurement setups for NIR spectroscopy. Finally. NIR spectroscopy would not have had such an impact as an analytical tool had it not been for the development of mathematical tools for spectral analysis. A common methodology is chemometrics or Detector Shelter Sample Light source (a) (b) (c) Figure 9. The broad spectral bands may be an indication of the material constituents.1. In order to prevent direct reflection from the surface. where the light passes through the sample from one side to another. For both (a) and (b). but focusing the two devices so as to ensure that the light has traversed some region of the sample before detection.

the measurement locations for obtaining the best calibration results were also addressed. partial least square (PLS) regression.124 ◾ Handbook of Seafood and Seafood Products Analysis multivariate data analysis. namely. fat. demonstrating the possibility to determine fat content in live fish. fat. water. mackerel. a model based on several wavelengths is required to extract useful information from the spectroscopic data.3 Analysis of Basic Constituents As found in NIR analysis of foods in general. Darwish and others [15] used the technique in 1989 to measure fat. The prospect of measuring the chemical composition of intact fish could facilitate the use of the method in connection with selection in breeding programs [17] as well as for quality grading in terms of nutritional quality [19]. and water. As early as in 1992 Lee and others [17] showed how NIR spectroscopy could be used noninvasively to estimate the lipid content of small-sized.1. protein. we give several examples of the use of NIR spectroscopy for the determination of basic food constituents in fish and seafood and how the method has been applied and developed over the last 20 years. and protein in cod. Among the most used multivariate techniques are principal component analysis (PCA). This measurement setup clearly displayed how NIR spectroscopy could be used in a nondestructive way. and. and protein content in rainbow trout. the reference method may be replaced by the spectral reading and the analytical model. In both studies reflectance measurements were performed. For the measurement of fat and protein. [14] reported the use of NIR spectroscopy to determine lipid and protein content in freshwater fish. an easy. The measurements were performed by use of fiber optic bundles conveying the light to and from the sample site. This could account for the many studies relating to the rapid analysis of the basic chemical composition of . The earliest reports of NIR spectroscopy to measure chemical components in fish appeared more than two decades ago. and tuna. Downey [18] applied a similar spectroscopic setup to measure fat and water content of intact farmed salmon. In the following paragraphs. and rapid method for the assessment and quantification of these constituents is considered a valuable tool in the quality evaluation of any foodstuff. the study concluded that the method could be a useful tool for rapid quality control. Sollid and Solberg [16] measured the fat content in salmon by transmission spectroscopy on raw minced muscle. Farmed salmon is of high commercial value and a worldwide favorable product. 9. Typically. whereas water determination was made on the water extracted from the fish mince. Consecutive research articles proved the feasibility of the tool in developing the method to apply with simpler procedures of sample preparation. by use of NIR in connection with fiber optics Solberg et al. reliable. [19] performed a study on live anesthetized farmed salmon. In addition. In 1987 Gjerde and Martens [13] demonstrated the applicability of NIR to predict water. Both of these early reports concluded that the method was promising in terms of speed and efficiency when measuring a large number of samples. fingerling Arctic charr and rainbow trout. Based on measurements through scales and skin. The same year Mathias et al. a substantial part of the work related to NIR analysis of fish and food from fish concerns the quantification of the chemical constituents. and soft independent modeling of class analogies (SIMCA) [12]. If there is good correlation between the spectral measurements and the method of reference. In spite of the rather cumbersome sampling procedure. it was possible to estimate the lipid content of the intact muscle. [17]. intact rainbow trout. Being the basic nutritional components of any food. and the sample preparation included mincing and freeze drying of the material to be evaluated. as in the work of Lee et al. the samples were minced and dissolved in a milk-like emulsion.

as well as on minced salmon muscle.2. Xiccato et al. and Tuene [24] made use of NIR transmission spectroscopy to assess protein. Silver Spring). Spectroscopic readings obtained on the minced samples correlated better with the reference measurements on fat. PC): (%fettHS. Nortvedt. [29] one question of interest was the comparison of different methods for measuring fat content.K. and protein than those made on intact muscles. Torry fatmeter.2 The plot shows the predicted versus measured fat content in farmed salmon based on multivariate analysis of 78 spectra from salmon fillets and the respective chemical analyses of the fillets.264047 0.600067 0. and protein content of European sea bass. NIR spectroscopy was proven to be a useful tool for the evaluation of basic constituents of different types of tuna. 1998.603947 0. . Unpublished data. water. An example illustrating the use of NIR spectroscopy for assessing fat content in farmed salmon is given in Figure 9. Wold et al.937575 0.Basic Composition: Rapid Methodologies ◾ 125 salmon. fat. Measured Y Elements: Slope: Offset: Correlation: RMSEP: SEP: 21 Bias: 24 78 0.985681 0. In this work they applied a fiber optic measurement setup. or postspawning. [20] conducted a study in which they compared NIR measurements on intact salmon fillet. 8) Figure 9. in. microwave. The fat content of herring has also been assessed by the use of NIR spectroscopy.. [28] and Nielsen et al. In both the works of Vogt et al. This work also emphasized the impact of the conditional state of the fish when making calibration models. In a research article published in 2004. [21. and NIR spectroscopy.22] also conducted studies documenting the efficiency of applying NIR spectroscopy in different measurement modes to assess fat and water content in salmon. (Y–var. and Sørensen. and additionally the spectroscopic measurements could be used for origin identification or authentication of the samples.. applying minced samples for the spectral readings. H. and dry matter in halibut fillet. Transmission spectroscopy was also employed for the analysis of fat and dry matter in capelin [25]. (From Nilsen. [27]. water. Isaksson et al.002328 61 66 54 55 168 17 16 62 72 18 2 4 77 23 6 53 6373 25343 31 13 74 80 22 15 78 41 27 44 18 36 38 46 70 9 65 20 12 67 19 49 60 52 26 83 48 51 100 48 69 676 32 35 20 24 37 56 40 50 28 30 39 5 15 Predicted Y 18 20 22 24 14 16 Hsfett1. Torrissen. N. NIR spectroscopy has been used for evaluating the chemical composition of several other fish species as well. [26] showed that NIR spectroscopy could be used to estimate lipid. In a recent work by Khodabux et al. whether pre-. The study concluded that NIR is well suited for nondestructive quality evaluation of salmon fillets.) Spectral measurements were performed on intact fillets by transflection measurements by use of the fiber optic probe of the instrument NIRS6500 (Perstorp Analytical Inc.

Moisture and sodium chloride in cured Atlantic salmon were measured nondestructively by NIR diff use reflectance spectroscopy [34]. A work by Adamopoulos and Goula [37] showed that the chemical composition could be assessed with a high degree of accuracy in addition to the obvious benefit of the ease and simplicity of the measurement method. A few years later the same group used NIR to assess the fat content in frozen skipjack [31]. alter the physical and chemical properties as well as the textural properties of the fish muscle. respectively. [39]. the nonintrusive method would still be an interesting alternative for rapid testing of high-value food products.42]. lipids. either intact fish/muscle or minced muscle. NIR spectroscopy has also been applied for the analysis of basic chemical constituents in other types of fish products. [30] used NIR spectroscopy in connection with an interactance probe as a means of determining the fat content in frozen horse mackerel nonintrusively. and exposure to elevated temperatures. also commented on the cost aspect of the different methods as part of the feasibility of the methods. Similar findings were made on hot smoked portions of salmon fillets by Lin et al. [32] performed a study to show that moisture and salt content in cold smoked salmon could be evaluated using NIR measurements. although the assessment of salt did not prove as effective as that of water content. [36] presented a study where NIR spectroscopy was used for the investigation of salt content in cured salmon roe. enzymes. however. and NMR. however. combine the NIR technique with imaging—further described later in this chapter—which facilitates a novel way of measuring and analyzing fish quality. NIR spectroscopy was applied to determine water and protein content [38]. about 63°C for the hot smoking process. Smoked and cured fish have also been subject to investigation by the use of NIR spectroscopy. Shimamoto et al. refined fish-based products made by washing mechanically deboned fish to remove constituents such as blood. and the detection of bruises in the fish muscle [33]. It was argued that the sensitivity of the method could have been better. They did. The broadbanded spectra contain information about several parameters. They addressed the sampling/measurement location and the method of performing measurements in a representative way. The salting. The use and results described above were all on raw fish samples. In this work it was demonstrated how NIR spectroscopy could be used to assess the protein content in brine from salted herring and thus indirectly be a measure of the maturity and ripening of the salted herring. For surimi products. and protein content in another roe-based product. [33]. In addition to the many studies assessing the basic chemical constituents in fish and seafood. fat. however. namely. Examples of these are nondestructive texture analysis of farmed salmon [40]. [35] applying the NIR technique to determine water content in salted dried cod—clipfish. differentiation between fresh and frozen-thawed fish [7]. Of the most recent studies in the field is work by Wold et al. NIR spectroscopy has proven applicable also for the analysis of frozen products as well as processed and refined products. In addition to the analysis on raw fish and processed fish material. Vogt et al. and certain proteins. the Greek dish taramosalata. Huang et al. In both studies NIR spectroscopy resulted in favorable outcomes with respect to speed and accuracy. In 2001 Huang et al. still proved viable for assessing the chemical constituents of the samples. storage time of frozen fish [41]. The versatility of the method is one reason for its relevance and growing popularity during the recent years. the spectroscopic method has confirmed its applicability for the evaluation of several other quality issues in fish.126 ◾ Handbook of Seafood and Seafood Products Analysis and Distell fatmeter. [28] however. evaluation of freshness or storage time of fresh fish [41. and . The spectroscopic method has been used to assess moisture. NIR. NIR spectroscopy. A further use of NIR measurements for the evaluation of basic food constituents was suggested by Svensson et al. smoking.

As illustrated by the above. . The development in recent years in instrumentation. the ease of use of the methodology has increased through instruments facilitating little or no sample preparation as well as measurement setups for rapid and nonintrusive registration. with one reading. has been a reason for the method not gaining a broader range of applicability. Instrument development has come from the grand-size laboratory desktop versions to portable or handheld instruments as illustrated in Figure 9.3.Basic Composition: Rapid Methodologies ◾ 127 the possibility of simultaneously monitoring a number of different issues. There may be several reasons for this. The high price of the instrumentation. These developments have enabled the use of at-line or online methodology.3 Prototype version of handheld spectroscopic instrument for quality assessment of fish. on one side. fish-quality inspection. is considered intriguing. however. not yet become an everyday instrumental tool for food-quality control nor. The technique has. High-cost instrumentation designed for versatile use and flexibility has probably better met the requirements of laboratory use than those of industrial application. Another issue is the need for modeling the correlation between the spectroscopic reading and the quality parameter in question. This instrument was used for the determination of freshness of cod as well as the assessment of frozen storage time of hake. combining imaging techniques with the spectral information. Th is is a challenging task in view of the variety and the heterogeneity of the material and so may have contributed to the reluctance in investing in and developing this technology to a commercial tool for assessment of fish quality. may promote the future applicability and usefulness of the information in Figure 9. say.

This implies that this technique is a powerful tool for segmentation and classification and that it may also map the chemical composition into the spatial domain [45]. Measurement Principles. the spectrograph and the object must move relative to each other. Depending on the applied sensor technology.1 Theory. In addition to what traditional spectroscopy can facilitate. and Analysis Imaging spectroscopy. However.2 Analysis of Basic Constituents During the last decade several applications within food-quality inspection have been developed based on imaging spectroscopy. Between each captured frame. Norge). Imaging spectroscopy can be implemented for transmission.1 on NIR spectroscopy. The analytical techniques described in that section are also applied to imaging spectroscopy data. 9.44]. the feasibility of the method for the analysis of basic composition of foods. Most of them are on foods such as fruits. or the result from these techniques can be postprocessed to utilize the spatial information [46]. As these techniques only use the spectral information. an analytical tool for industrial quality control of clipfish and salmon fillets. total soluble solids.2 Imaging Spectroscopy 9. As described in Section 9. reflection. as well as transflection measurements. demonstrates the potential of the method in the seafood sector as well. and acidity (expressed as pH) [47–49]. There are still relatively few reports on imaging spectroscopy applied for the analysis of fish and seafood. also known as multispectral imaging or hyperspectral imaging. Typically. A novel example of this is the development of the QMonitor (QVision AS. It has been shown that NIR hyperspectral imaging techniques are . It has become a widely used technique within fields spanning microscopy to satellite remote sensing. it uses a two-dimensional sensor. To simplify the concept. Typically. an imaging spectrograph operates in the following way. some examples related to the agricultural sector are referred. For instance. Th is means that for each spatial location it is possible to access the full spectral information. In this way an image of the object is built line by line. this method is an indirect measurement technique. the hyperspectral data can be preprocessed based on spatial features before applying analytical spectral techniques. and meat. improved results can be obtained by combining these techniques with more traditional image processing techniques.2. and each frame captured provides full spectral information for one line across the object to be imaged. is a new technique that has been developed during the last decade [43. The realization of a commercial processing analytical tool for the simultaneous analysis of several parameters makes the technology interesting for a broad range of fish and seafood processing industries. 9. the spectra may be recorded in the visible and near-infrared region. the relative motion is accomplished by mounting the imaging spectrograph above a conveyer belt where each captured frame images a line perpendicular to the direction of motion.128 ◾ Handbook of Seafood and Seafood Products Analysis the near-infrared spectra. this can be illustrated as simultaneously recording information about shape and color. in general. In order to illustrate the potential parameters to be assessed by imaging spectroscopy.2. this technique also provides spatial information. Oslo. vegetables. Several solutions have also been developed for detection of defects and contaminations on fruits. For fruits and vegetables more articles report on determination of chemical constituents such as moisture content.

color.4 Fat distribution in salmon fillet measured by the multispectral imaging system QMonitor fabricated by QVision (Oslo. . the moisture content of the fish varies from the thinner parts to the thicker parts of the fish.4). imaging spectroscopy solutions for detection of contaminants such as fecal and ingesta on poultry carcasses have been studied [56–58]. Fisk: 1 Fettfisk: 18. Inspection systems based on hyperspectral imaging have been tested for poultry carcass inspection focusing on classification of carcasses into normal. Since 2000. The color bar to the right indicates the correspondence between color and fat content in percentage.3055% Share: 21.55]. there is one recent publication on assessing water content in salted dried cod by Wold et al. Norway) has also developed an industrial solution based on multispectral imaging for measuring the fat content in salmon fi llets (see Figure 9.Basic Composition: Rapid Methodologies ◾ 129 useful for automatic online detection of surface defects and contaminations on apples [50–52]. Regarding the determination of basic chemical composition of fish and seafood. septicemic.3348 50 45 50 40 100 35 30 150 25 200 20 15 250 10 300 5 10 20 30 40 50 60 0 Figure 9. pH. The first article addressing analysis of fish or seafood by imaging spectroscopy was published in 2000 by Sigernes et al.60] where several quality parameters were evaluated by imaging spectroscopy. The parameters included were drip loss. [53]. [61]. Peeling of shrimps and detection of nematodes were mentioned as possible applications for the future. A recent work on quality assessment of pork has been reported by Qiao et al. whereas the local fat content varies from approximately 6% up to 43%. When drying fish. A thorough review of imaging spectroscopy applications within fruits and vegetables is presented by Nicolai et al. In this publication the importance of including spatial information is illustrated. QVision (Oslo. Further on. the main activities within imaging spectroscopy and fish analysis have been focused on online solutions for assessing chemical composition and detection of quality defects in fish products. The mean fat content for this fillet is 18. [35]. and different texture features. measuring the water content in one spot is not necessarily representative for the whole fish. Norway). and cadaver [54.3%. [59. Hence.

The most commonly measured nuclei are 1H and 13C. Hence. NMR active nuclei absorb at a frequency characteristic of the isotope. this is a relatively new field. 9. Still the number of imaging spectroscopy applications with fish and seafood is low. and the methods that are feasible by spot measurements may also be implemented using imaging spectroscopy. and skin remnants in whitefish fillets [62–64].3 NMR Spectroscopy 9. The energy absorptions of the atomic nuclei are also affected by the nuclei of neighboring atoms within the same molecule as well as nuclei in surrounding molecules. imaging spectroscopy of fish has been applied to address other quality issues. Norway) in fish.3. With respect to commercial implementation of imaging spectroscopy. if blood oxidation should be quantified spectra from blood-infested area of a fillet can easily be extracted for analysis based on imaging spectroscopy data. NMR techniques use electromagnetic radiation and magnetic fields to obtain chemical information. Additionally. NMR spectroscopy may provide detailed . black lining. but during the last few years magnetic resonance imaging (MRI) has also been explored for its usefulness in food analyses. and they are based on the magnetic properties of atomic nuclei. since it is possible to use spectra from dedicated relevant areas on the sample. With imaging spectroscopy this is not a problem since spectra are available for all spatial locations. blood spots.2 Theory and Measurement Principles NMR provides a large amount of information regarding composition and structure of components in food.1 Determination of Basic Composition Nuclear magnetic resonance (NMR) has evolved from being an expensive and academic analytical technique into being a technique applicable for the food industry in both size and price of the equipment as well as speed of analyses. but looking at reported applications within other areas the potential for new applications is high. experience with NIR spectroscopy shows that more than one attribute can be estimated based on one recording. In addition to this a high-resolution prototype imaging spectrograph has been developed for detection of defects as well as determination of chemical constituents in fish fillets as reported by Heia et al. For detection of flaws or defects in fish. For instance. When an external magnetic field is applied. Even more important is that for some applications imaging spectroscopy can provide better results. For NIR spectroscopy several applications within fish and seafood are reported. measurements may be performed at high speed as well as in noncontact mode. 31P-NMR and 23Na-NMR have also been used for food analyses. [63]. Using the interaction between light and the sample object. Oslo.130 ◾ Handbook of Seafood and Seafood Products Analysis In addition to the measurement and documentation of basic composition. a lot of effort has been invested in the detection of nematodes. A low-resolution (spectral and spatial) instrument is available for industrial assessment of chemical composition such as fat and water content (QMonitor.3. All nuclei that contain odd numbers of protons or neutrons have an intrinsic magnetic moment and angular momentum. QVision. Imaging spectroscopy is well suited for application in the fish processing industry as an online technique. 9. The main technique used is NMR spectroscopy. Furthermore. and currently there are a limited number of equipment suppliers. but this requires that the same spot be used.

different NMR equipments are available. As recent examples. whereas Veliyulin et al. whereas Siddiqui et al. creatine. [73] used HR-1H.and HR-13C-NMR for multicomponent analyses of encapsulated marine oil supplements. [74] reported the use of HR-NMR to determine oxidation products in marine lipids. whereas LF. cod [66]. [70] demonstrated that NMR-MOUSE could also be used for in vivo determination of fat content in Atlantic salmon. Studies of large objects like whole fish are impossible using most traditional LF-NMR instruments. low-resolution NMR (LR-NMR) and high-resolution NMR (HR-NMR) spectroscopy as well as MRI and NMR-mobile universal surface explores (NMR-MOUSE) have been used. and mobility in herring [67] and oil and water content of salmon and cod [68].1H-NMR seems to correlate to fillet pH and water-holding capacity [71]. [75] and Arvanitoyannis et al. Low-field (LF) NMR spectroscopy requires little or no sample preparation. [72] used HR-NMR to measure the content of n-3 polyunsaturated fatty acids in four types of unoxidized fish oils. and dimethylamine in extracts . Germany) has been developed to handle such samples. Among more recent work. 1H NMR spectroscopy has been explored to identify the fate of some bioactive compounds during processing of seafood. [78] demonstrated the use of NMR lipid profiling for classification of gilthead sea bream according to geographic origin. Additionally. Aursand et al. used for seafood authenticity have been provided by Martinez et al. Due to the provision of very detailed information regarding the molecular structure of a food sample. it was shown that 23Na-NMR has proven useful for quantitative salt determinations in salted cod. For analyses of seafood products. fatty acid composition. Tyl et al. whereas Thomas et al. and it has mainly been used for analyses of water in food samples. Falch et al. Standal et al. degree of saturated/ unsaturated fatty acids. 9.3. in a study focusing on both 23Na-NMR and low-field 1H-NMR spectroscopy. [79] and Masoum et al. Extensive reviews on different techniques. Today. [81] showed that it was possible to identify taurine. [69] demonstrated that this equipment could be applied to determine fat in homogenates from salmon. Numerous applications of NMR in food analyses have been reported in the literature. and there are numerous reports available. Martinez et al. For example. but the technique has been applied in the recent years for determination of both fat and water content in different food products and also seafood. and some examples of analyses of seafood are given here. High-resolution NMR can be used to provide information on lipid classes. Rheinstetten. high-resolution NMR has been applied in many food authenticity studies. Rezzi et al. Additionally. [80] used this technique to determine the origin of Atlantic salmon. the Bruker Professional MOUSE ® (Bruker Optik GmbH. [76]. A new type of LF-NMR instrument. HR-NMR has been used in many studies and has the advantage over LR-NMR that it is possible to obtain detailed information regarding the molecular structure.and 13C-NMR have been applied to measure the lipid or water content of many different foods including fish. and studies of lipid degradation processes in lipid mixtures such as fish oils. LF-1H-NMR has been used for studying water distribution in smoked salmon [65]. and they may provide different information regarding the food properties. trimethylamine oxide. [77] used NMR to discriminate cod liver oil according to whether the origin was wild/ farmed as well as geographic origin. betaine. anserine. water distribution. including NMR.3 Analysis of Basic Constituents For several years 1H.Basic Composition: Rapid Methodologies ◾ 131 information regarding the molecular structure of a food sample.

A study has been conducted on the applicability of CT scanning as a nondestructive and rapid way of measuring muscle dry matter content and liquid leakage in cod fillets [84].4. amino acids. The decrease in x-ray intensity inside a sample will be due to absorption by different materials. the high spectral resolution is not always required. Based on the results obtained the . NMR is a versatile tool for the identification and quantification of numerous compounds in fish related to nutritional quality. more specific information about the sample can be revealed. [85] tested CT scanning as a tool for estimating the relative size of fat deposits and lean tissue and fat content in Atlantic halibut.1 Theory and Measurement Principles X-ray imaging is a technique based on the emission of x-rays through a sample and recording the amount of attenuation. one layer for each energy level. This is also an x-ray imaging system.4. However. has been that conventional NMR is an expensive technique. [82] showed that it was possible to identify single chemical compounds such as hypoxanthine.2 Analysis X-ray imaging provides spatial information in two dimensions (2D) or three dimensions (3D) (CT). Gribbestad et al. Such equipment is cheaper. computed tomographic (CT) scanning is widely used. There are two interactions. but it provides a three-dimensional image of the sample. and their relative contributions are energy dependent [83]. Within the field of medicine. the photoelectric effect and the Compton scattering that causes the x-ray attenuation. a two-dimensional cross section of the sample can be made. and some fatty acids in extracts and muscle from salmon using high-resolution 1H NMR spectroscopy. In another study Kolstad et al. Typical applications within fish and fish products are related to the detection of bones and bone fragments as well as chemical composition and localization. As illustrated here. lactate. This technique is referred to as dual-energy x-ray absorptiometry (DXA. Th is is a powerful imaging technique that can be used both as a single-energy and a dual-energy module. An objection to the method. 9. For online applications this can be implemented as a line-by-line imaging or a frame-by-frame imaging. Making profiles from different angles and then combining them by software. A more dense material will absorb more x-ray energy. It is not possible to accurately characterize the observed sample by applying only one x-ray energy level. This is achieved by rotating the x-ray/detector unit around the sample.4 X-Ray Imaging 9. smaller. previously DEXA) and may be implemented using a two-layer detector. and lately many low-field. 9. and less sensitive to fluctuations in the environment and thus more applicable in industry as well as in many research fields. however. By using two x-ray energy levels. low-resolution NMR spectrometers have been developed and commercialized. anserine. The results obtained showed that CT scanning could be used as a rapid method for the assessment of these attributes and would add valuable information to be used in genetic studies and breeding programs. Then the third dimension is accomplished by the sample movement. Further on the CT scans gave significant information about dry matter distribution from head to tail of the cod.132 ◾ Handbook of Seafood and Seafood Products Analysis from processed cod. and the attenuation will also be influenced by the sample thickness.

9.5 for an example). Iceland). A similar work has been carried out by Hancz et al. Iceland). instrumental means capable of objective and rapid determination of basic composition are also available. authors recommended CT scanning as an online technique for carcass evaluation. This instrument can detect bones and bone fragments down to a diameter of 0. Throughout development all presented techniques have met the requirements . With respect to bone detection in fish fillets there are commercial solutions available today (Marel Hf.Basic Composition: Rapid Methodologies ◾ 133 Figure 9. [86] showing good results predicting fat content of common carp based on CT scanning. Marel developed an X-ray-based bone detection unit (SensorX) that was commercially available on the market in 2003 [87].5 Detection of pin bones in fish fillets by x-ray imaging using the SensorX instrumentation (Marel.3 mm when operating at industrial speed (see Figure 9.5 Summary The methods and applications presented in the above clearly illustrate that there are more tools and techniques that could serve as an easy and useful way of rapid quality determination of fish and seafood. To the left is the original x-ray image of one cod fillet and to the right is the processed image where only the bones identified in the fillet are shown.

Thyholdt. T.. Near Infrared Spectroscopy in Food Analysis. for providing the example picture used in Figure 9.. imaging. 6. Journal of the Science of Food and Agriculture. 70(8). 2000. K. also makes it clear that although proven useful and promising in laboratory-scale trials. 525–532. 1986. not easily applicable for small-scale industries as is often the case in the fish processing industry. 89–104. This chapter. in Fishery Products: Quality. NIR spectroscopy: A rapid-response analytical tool. and progress in data processing and analytical tools has facilitated usability and ease of interpretation of measurement results. therefore. References 1. 1997. U..I.G. Eds. quality seafood products will contribute to retaining the good reputation of fish and seafood in the years to come. the finding of a universal measurement tool to meet with this variety is a challenging task.134 ◾ Handbook of Seafood and Seafood Products Analysis of simplicity in sample preparation. 2002..K. using near infrared spectroscopy.. however. and Villarroya.K. Another issue is the substantial variety and heterogeneity of the material to be analyzed. and Heia..4. 1998. NMR. 103–111. U. 5. Blanco. 121–128. these techniques have—with a few commercial exceptions—still not been shown to be commercially valid for quality determination in the fish and seafood processing industry.. Nofima Food. Nilsen. However. Reykjavik. Hildrum. pp. Longman Scientific & Technical. Differentiation of frozen and unfrozen beef using near-infrared spectroscopy. Due to the spread and diversity in fish species and sizes as well as the seasonal difference in bodily composition. and x-rays is considerable and. M. I. J. Uddin. Harlow. Journal of Near Infrared Spectroscopy. VIS/NIR spectroscopy.K. Non-destructive Visible/NIR Spectroscopy for differentiation of fresh and frozenthawed fish. T. T. K. B. T. T.. A. Isaksson. A. 6. K.. Acknowledgment The authors would like to thank Dr Jens Petter Wold. 240–250... Oxford. H. Ellis Horwood. Osborne. H. 33(2). and Fearn. 21(4). Thybo. Safety and Authenticity. .A. 200. 73(4). England. Oslo. Trends in Analytical Chemistry. Determination of chemical composition of beef meat by NIRS. et al. The price of measurement equipment for NIR. 2005. Prediction of sensory texture of cooked potatoes using uniaxial compression. 1992. 2009. and Tandberg. p. and x-rays are operated at a speed that makes it possible to perform measurements at or in a processing line. et al. Iceland) and QMonitor (QVision AS. 7. 339–344. and Oehlenschläger.. J. De Boever. NIR. Introducing and applying these methods to industrial applications and enabling production of well-documented. the technological development exemplified by SensorX (Marel hf. Journal of Food Science. 3. Naes. nearinfrared spectroscopy and low-field H-1 NMR spectroscopy. Norway) confirms that these techniques may be applied in commercial and industrial high-speed fish processing applications. In addition. Lebensmittelwissenschaft und Technologie. and Isakson. Pawlinsky. 8. et al. Norway. Eds. 2. in Near InfraRed Spectroscopy. Prediction of wheat bread-baking functionality in whole kernels. Rehbein. M. NMR. Part of the explanation for this could be the cost level of the equipment in question.J. P. 4.. C506–C510.. and Williams. hence allowing for measurements to be performed on large-scale quantities.. Wiley-Blackwell Publishing.

. M. J. 19. Journal of Animal Breeding and Genetics–Zeitschrift für Tierzuchtung und Zuchtungsbiologie. 1987. Non-destructive determination of fat and moisture in whole atlantic salmon by near-infrared diff use spectroscopy. 26. Non-destructive determination of fat. 1989. Gjerde.P. Food Chemistry. Proximate analysis of fish tissue by mid-infrared transmission spectroscopy. D. 30. D. Darwish. H.K. Mathias. 205–215. Sollid. 734–736. Vogt. Journal of Food Science. 102.. 717–722. Rapid non-destructive determination of fat content in frozen skipjack using a portable near infrared spectrophotometer. Food Chemistry. H.. 23. 11. 38.. 2003. 1987. 2006. 20. 29. J. T.. 69. Xiccato.R. 305–311. Pasquini. U. 24. 199–207. 644–649. 2001.. Torrisen. J. 2003. 17. Lee. C. Chichester. 104(1–2). Solberg. 2001. 198–219. Solberg. and Isaksson. Fatmeter. Salmon fat content estimation by near infrared transmission spectroscopy. 13. 221–228... 18.. Journal of Food Science.. Chemometrics and Intelligent Laboratory Systems. A comparison of selected rapid methods for fat measurement in fresh herring (Clupea harengus).C.. Journal of the Science of Food and Agriculture. Fisheries Science. Noninvasive short-wavelength near-infrared spectroscopic method to estimate the crude lipid content in the muscle of intact rainbow trout.)–influence of biological factors and comparison of different methods of analysis: Solvent extraction. 86.. 669–675. 83. NIR and NMR. 1992. Williams. 62(4). 1992. and Sobering. D. 275–281. W. et al.. Martens. Y. et al. Trends in Food Science and Technology. G. 1996. 18. Lipid content in herring (Clupea harengus L. 55(3). H. and He. 2006. and Smith. 1998. and Solberg. Unpublished data. Shimamoto.. protein and dry matter in Atlantic halibut fillet. C. R. Non-invasive and non-destructive percutaneous analysis of farmed salmon flesh by near infra-red spectroscopy. 27. 61(1).. N. fat and protein in tuna fishes.J. C. T. 2002. Canadian Journal of Fisheries and Aquatic Sciences.H. 692–696. McClure.. 15. 419. Near infrared spectroscopy: Fundamentals. 1998. et al. G.. 12.S.A. 57(3). et al. H. and Fredriksen. moisture and protein in salmon fillets by use of near-infrared diff use spectroscopy. Khodabux.. P. Food Chemistry. Journal of Food Science. Isaksson. Nippon Suisan Gakkaishi 67(4). et al. and Krane.F. Shimamoto. John Wiley & Sons Ltd. 1996. Determination of fat in live farmed Atlantic salmon using non-invasive NIR techniques. Journal of Near Infrared Spectroscopy. 31.. Journal of Agricultural and Food Chemistry. G. 2003.P. G. Nortvedt.P. Chemical and near-infrared determination of moisture. 2004. . F. 22. 95–100.. 9. Van de Voort. 21. 487–518. Analysis of fat and dry matter in capelin by near infrared transmission spectroscopy. Wold. J. 137–148. and Tuene.A. Nilsen. J. Atlantic salmon average fat content estimated by near-infrared transmittance spectroscopy. 46. and Rasco. 40. S. p.Basic Composition: Rapid Methodologies ◾ 135 9.. 204 years of near infrared technology: 1800–2003. Application of near-infrared transmittance spectroscopy in the determination of fat. 537–548. 25. 856–860. T. 1997. 14. J. O. Aquaculture. 792–793.. Wold. The determination of lipid and protein in fresh-water fish using near-infrared reflectance spectroscopy. Predicting carcass composition of rainbow trout by near-infrared reflectance spectroscopy. Cen.. Journal of the Science of Food and Agriculture. Journal of Near Infrared Spectroscopy. practical aspects and analytical applications. and Næs. 16. Mulitvariate Calibration.K. 28... Downey. Nondestructive determination of the fat content in raw and frozen horse mackerel by Near Infrared Spectroscopy. 1989. 42. K. Jakobsen. 72–83. T. Theory and application of near infrared reflectance spectroscopy in determination of food quality. L. and Sørensen. 15.. Journal of Food Composition and Analysis. 303–311.) by near infrared reflectance spectroscopy (NIRS).C. C. 14(2). 74–77. 1995. and Martens H. Journal of the Brazilian Chemical Society. B. Prediction of chemical composition and origin identification of European sea bass (Dicentrarchus labrax L.. 2005. 2176–2181. 69. et al.. Nielsen. Lebensmittel-Wissenschaft und-Technologie.. 61(3–4). et al. A. et al. B. 10. Cavinato. 11.. 2003.

Lu. et al. G. 41.. et al.M. 61(1). 49. Development of hyperspectral imaging technique for the detection of apple surface defects and contaminations..B... et al.S. ed. Isaksson. and Goula. T. Food Chemistry.M. 2002. Non-contact transflectance near infrared imaging for representative on-line sampling of dried salted coalfish (bacalao). 2001. Multivariate image analysis of a set of FTIR microspectroscopy images of aged bovine muscle tissue combining image and design information. 40(1). P. 482–486. Modeling multispectral scattering profiles for prediction of apple fruit firmness. International Journal of Pattern Recognition and Artificial Intelligence. Journal of Food Engineering. and Dall’Ava. Postharvest Biology and Technology. 59–66... 4161–4167. 42.. 35. Peng. 34.G. E. 2006. 67(5). J. Y. 39. R. Lin. M. 199–207. J. Journal of Food Science. G. Heia. 37. Nondestructive determination of moisture and sodium chloride in cured Atlantic salmon (Salmo salar) (Teijin) Using short-wavelength Near-infrared Spectroscopy (SW-NIR)..F. 43. in Digital Solid State Cameras: Designs and Applications. J. 49. Williams. . Detection of sodium chloride in cured salmon roe by SW-NIR spectroscopy. 98–107.. Mehl.. Wageningen. 33. Detection of bruises on apples using near-infrared hyperspectral imaging. 2006. Svensson. 147–157. Non-destructive texture analysis of farmed Atlantic salmon using visual/ near-infrared reflectance spectroscopy. 52(3). pp. B.Technologie. ElMasry.. 50. Application of near-infrared reflectance spectroscopy in the determination of major components in taramosalata. 48. Direct sight imaging spectrograph: A unique add-in component brings spectral imaging to industrial applications. Nielsen. et al. Nondestructive prediction of moisture and sodium chloride in cold smoked Atlantic salmon (Salmo salar). H. 235–242. Huang. Visible spectroscopy—Evaluation of storage time of ice stored cod and frozen hake. T. R. K... 37. Determination of the protein content in brine from salted herring using near-infrared spectroscopy. 10. 1143–1153. Transactions of the ASAE. Nondestructive determination of water and protein in surimi by near-infrared spectroscopy.. 1998. 1821–1826. 2003. 81(1). Non-destructive measurement of bitter pit in apple fruit using NIR hyperspectral imaging. H. A. M. 2004. R. Bruise detection in pacific pink Salmon (Oncorhynchus gorbuscha) by visible and shortwavelength Near-Infrared (SW-NIR) Spectroscopy (600–1100 nm). Hyperspectral imaging for nondestructive determination of some quality attributes for strawberry. et al. Y. 2543–2547. Infrared spectroscopic imaging: From planetary to cellular systems. 491–495...P.H. 40. 67(7). 45. A. 48(1). Y. et al. et al. Uddin. et al. Herrala. Analytical and Bioanalytical Chemistry. J. 2004. and Bro. the Netherlands.. Eds. Nicolai. 2002. 31(2).. Colarusso. Proc.. and Okkonen. Herrala. et al. et al. 82. 52. Adamopoulos. R.. Oehlenschlager. Journal of Near Infrared Spectroscopy 14(1). 2004.. Journal of Food Engineering. Wold. V. 2004. 67–68. 43–54. 38. 51. 2007. 2005. Lebensmittel. 36. et al. 2006.. 47. Postharvest Biology and Technology. Lu. K.Wissenschaft und. Applied Spectroscopy. Journal of the Science of Food and Agriculture. Hyvarinen. et al. 46(2). 68(2). Journal of Food Engineering.. and Lu. E.. Wageningen Academic Publishers. A.136 ◾ Handbook of Seafood and Seafood Products Analysis 32. Visible/Near-Infrared spectroscopy—A new tool for the evaluation of fish freshness. Y. 44. 53–60. Journal of Food Science. 2001. 201–209. 63. Kohler.M. Multispectral imaging for predicting firmness and soluble solids content of apple fruit. Agricultural and Food Chemistry. 1998. 389. and Olafsdottir. 106A–120A.M.T. et al. Journal of Agricultural Food Chemistry.. Transactions of the ASAE. Jr. Huang. 803–809.. Imaging spectrograph and camera solutions for industrial applications. 6404–6408. 1996. Luten. in Quality of Fish from Catch to Consumer. 165–175. 2003. 96. 2003. 2003. SPIE 3302. 523–530. Nilsen.. Huang. 51. 2007. 46. 1–6. Journal of Food Science. G. et al. P.

T. Lawrence. Park. 83(1). 1259–1267. Correlation between H-1 NMR and traditional methods for determining lipid oxidation of ethyl docosahexaenoate. 1793–1802. Journal of Food Engineering. J. Water distribution and mobility in herring muscle in relation to lipid content. Journal of the Science of Food and Agriculture. 107–114. 2005. et al. Hyperspectral imaging for detecting fecal and ingesta contaminants on poultry carcasses. U. et al. Park.K. 67. and color for pork using a hyperspectral imaging technique. et al. et al.. low-field H-1 NMR. Destructive and non-destructive analytical techniques for authentication and composition analyses of foodstuffs. 36(8). 59.. 1999.. 185–192. Veliyulin. 2006. et al. Chao. Chen. Dordrecht. Salting and desalting of fresh and frozen-thawed cod (Gadus morhua) fillets: A comparative study using Na-23 NMR. Jensen. 2003.. E11–E15. et al. 55. Brecker. season..R. and Huffman. S. Journal of Near Infrared Spectroscopy. Qiao. Ed.B. 56. H. 76(1). 39(18). 60. K.. and Engelsen.. Transactions of the ASAE. Journal of Food Science. et al. Jepsen. 807–812. Sigernes. E. Journal of the Science of Food and Agriculture. 54. 72.H. 57. 64.. K. Detection of nematodes in cod (Gadus morhua) fillets by imaging spectroscopy. Webb GA. W.. et al. Falch. and Erikson.. 2007. 2406–2427. Prediction of drip-loss. Pedersen. and Wagner. Effects of single wavelength selection for anisakid roundworm larvae detection through multispectral imaging. Windham. Integration of visible/NIR spectroscopy and multispectral imaging for poultry carcass inspection. 13. et al.G. In vivo determination of fat content in Atlantic salmon (Salmo salar) with a mobile NMR spectrometer. Postharvest Biology and Technology 46. 110(2). 10–16. Multipurpose spectral imager. J. On-line inspection of poultry carcasses by a dual-camera system. C. 65. 55(8). 51(3).. Journal of Food Engineering. 2002. Journal of the Science of Food and Agriculture. 11(4). et al. 58. 62. 212–217.. 1996. 85(8). Veliyulin. 69(3). Tyl. . Na-23 MRI. Qiao. Transactions of the ASAE. 70(8). B. Trends in Food Science and Technology. et al.. 2007. 3143–3153. A hyperspectral imaging system for identification of faecal and ingesta contamination on poultry carcasses... 61. et al...M. Meat Science. 30(1–2). Aursand. 2002. K. Wold. 81(12)... 2007. Water distribution in smoked salmon. 73. et al. 74... 63. 1025–1034.. Siddiqui.M. Applied Spectroscopy. 2001.. 85(5). Loje. H. Journal of Food Engineering. fishing ground and biological parameters. C. 68. Detection of parasites in cod fillets by using SIMCA classification in multispectral images in the visible and NIR region. Journal of Food Science 72. 2017–2026. 2004. I. Y.. 46(6). et al. 66. Westad. R. E. Journal of the Science of Food and Agriculture. LWT-Food Science and technology. 79(13).E.M. K. Stormo. 1890–1895.Basic Composition: Rapid Methodologies ◾ 137 53. H-1 NMR spectroscopy as tool to follow changes in the fatty acids of fish oils. Algorithm development with visible/near-infrared spectra for detection of poultry feces and ingesta.N. N. and Heia. 69. Journal of Food Protection. 87(2). 2007. 45(6).. J. 75. 269–281.. 141–148. B. A. Journal of the American Oil Chemists Society.C. Andersen. 1105–1110. 44(12). 2004. L. Application of chemometrics to low-field H-1 NMR relaxation data of intact fish flesh..W. Applied Optics.. F. E. U. 2003. in Modern Magnetic Resonance. Multicomponent analysis of encapsulated marine oil supplements using highresolution H-1 and C-13 NMR techniques. 197–207. Springer. 2007. F. B.. 489–498. 1–8. European Journal of Lipid Science and Technology. 2003.. K. Low Field NMR Studies of Atlantic Salmon (Salmo salar). Heia. et al. 2002. the Netherlands.. 2007. 70. 99–118. K. 71. 2003. et al.R. I. S.. S. 1299–1304.P. 1733–1738. Nicolai. 2005. Pork quality and marbling level assessment using a hyperspectral imaging system... Distribution of water in fresh cod. Eriksson. et al. pH. Martinez. and Rinnan. Nondestructive measurement of fruit and vegetable quality by means of NIR spectroscopy: A review. 2008. 2000. and physicochemical analytical methods. Journal of Lipid Research.

B. 2008. 209–216. Journal of Agricultural and Food Chemistry. 34(12). and Martinez.. Implementation of quality control methods (physicochemical. Tsitsika. Luten. Oehlenschlager.. E. Morkore. Hancz. 87. 255–264. et al. 2004.. and Panagiotaki. I. et al. 6889–6895. Aquaculture. V. Bioactive compounds in cod (Gadus morhua) products and suitability of 1H NMR metabolite profiling for classification of the products using multivariate data analyses. et al.. 589–594.S. 2005. Masoum. 84.. 78. in Quality of Fish from Catch to Consumer. K. et al. 77. 9963–9968. J. 83.. Journal of the American Oil Chemists Society. and Dinten. I..M. 82. 79. Standal. S. High resolution 1H magnetic spectroscopy of whole fish. Aquaculture Research. J. M. 81. Rezzi. Wageningen Academic Publishers.138 ◾ Handbook of Seafood and Seafood Products Analysis 76. Andersen. Kolstad.S. Aquaculture. Discrimination of cod liver oil according to Wild/Farmed and geographical origins by GC and C-13 NMR. F. and Olafsdottir. 105–112. 53(17). K. Insight. and Thomassen. 2007.. 86. 85. M. 445–447. P. 2007. et al. 250. International Journal of Food Science and Technology. I. Thomas. 2008.V.B. C. Rebuffel. G... Journal of Agricultural and Food Chemistry. 989–997. Classification of gilthead sea bream (Sparus aurata) from H-1 NMR lipid profiling combined with principal component and linear discriminant analysis. Analytical and Bioanalytical Chemistry. 40. I. Arvanitoyannis. X-ray techniques for quality assessment. 229(1–4). 85(2). 387(4). Aursand. Determination of origin of Atlantic salmon (Salmo salar): The use of multiprobe and multielement isotopic analyses in combination with fatty acid composition to assess wild or farmed origin. K. Kolstad. microbiological and sensory) in conjunction with multivariate analyses towards fish authenticity. 55(24). J.. Wageningen. 49(10). I.) using computerised X-ray tomography (CT).. 1499–1510. 56.S. 275(1–4). Gribbestad. 237–263. Quantification of fat deposits and fat distribution in Atlantic halibut (Hippoglossus hippoglossus L. Eds. 2005.. 283–286.. S. Quantification of dry matter % and liquid leakage in Atlantic cod (Gadus morhua) using computerised X-ray tomography (CT).. 2003... 2008. Aquaculture. Measurement of total body composition changes of common carp by computer tomography. et al.. . 2005... et al.. fillets and extracts from farmed Atlantic salmon (Salmo salar) for quality assessment and compositional analyses. Martinez. 2007. Journal of Agricultural and Food Chemistry. the Netherlands. T. Application of support vector machines to H-1 NMR data of fish oils: Methodology for the confirmation of wild and farmed salmon and their origins. 991–997. 2003. 80. Dual-energy X-ray imaging: Benefits and limits.

149 10................................. carbohydrates.....3 Processed Fish Microstructure .............................. (2008) gave an overview of the most important techniques for studying muscle food structure...............146 10...Chapter 10 Microstructure Isabel Hernando...................................1 Herring ...........................................................................................................140 10...........................2 Fish Muscle Microstructure........148 10.....................2 Salted Cod..............3........................ Ana Puig..................2.......................................150 10..1 Smoked Salmon............ and María-Angeles Lluch Contents 10........ This chapter 139 ..............) is responsible for their microstructure..........................................3 Surimi .......................1 Main Microscopy Techniques for Studying Seafood The microstructure of foods forms a link between the molecular and macroscopic levels and constitutes a key factor for studying the properties of foods and for improving and optimizing food processes.......................4 Squid Microstructure ............................................. so any chemical or enzymatic change that takes place in the chemical components has an effect on the microstructural organization of the food matrices and their functionality.................139 10....................................... fats......................................................1 Main Microscopy Techniques for Studying Seafood ............... Several strategies can be used to study food microstructure................... 151 10. 151 References ...145 10......2.... The organization of the chemical components of foods (proteins................... Pérez-Munuera et al..3........................... etc.......................................................................3............................. Empar Llorca........................................................................................................ 148 10........2 Hake ..................................................

red oil. sudan. the samples need to be prepared first. The sections are obtained using a microtome after embedding the food in paraffin or resin or using a cryotome after freezing the sample with CO2 or liquid N2. Besides the secondary electrons. 2006). The light microscope (LM) is a very versatile tool that works in different applications such as bright field. phase contrast or differential interference contrast (Nomarski). The muscle cells are short and 0. the sample is frozen in liquid nitrogen and then freeze-dried before being coated and observed. the sample has to be prepared in semithin sections of about 0. There are two ways of preparing samples for SEM: chemical fi xation and physical fi xation (Figure 10.140 ◾ Handbook of Seafood and Seafood Products Analysis provides a detailed description of the protocols often followed to obtain information about seafood microstructure. critical point drying. Electron microscopy (EM) allows food structures to be studied at higher magnifications than those used in LM. showing alternate arrangements of . The steps in preparing samples for TEM observation (Figure 10. Once the semithin sections are obtained. other emanations or signals such as x-rays.1–2 mm (Figure 10. the sample preparation steps are chemical fi xation (with aldehydes and osmium tetroxide. 10. so there is no need to section it. dehydration in a series of ethanol dilutions of increasing concentration. In the former.0 mm in diameter. backscattered electrons.3). In this way.1). and so on. iodine.2 Fish Muscle Microstructure Fish muscle consists of myotomes. image analysis relies heavily on computer technology to obtain quantitative results from microscopy observation. so microanalysis can be carried out by means of x-ray. 2008). they are mounted in glass slides and stained with different dyes (toluidine blue.02–1.5). In both methods. these different signals can be captured by the appropriate detector in each case. dehydration in a series of ethanol dilutions of increasing concentration.4) and quickly transferred under vacuum to a cold stage fit on a microscope where the frozen sample is fractured. They are arranged in concentric circles forming subdivisions of striated muscle (Figure 10. Many of the endomysia are connected to the perymisium. The fibers are essentially the same as those of terrestrial animals in terms of the arrangement of the thick and thin filaments. The SEM method observes the surface of the sample. etched. cutting ultrathin sections (5–100 nm) in an ultramicrotome. and staining the ultrathin sections with heavy metal solutions such as lead citrate or uranyl acetate. In recent years considerable progress has been made in the field of SEM through vitrification techniques. polarizing microscopy.2) are primary fi xation with aldehydes such as glutaraldehyde. The most useful application for studying seafood structure is bright field microscopy. may be generated as a result of the electron beam striking the specimen (Pérez-Munuera et al. Two types of microscopes use electron beams as their source of illumination: transmission electron microscopes (TEM) and scanning electron microscopes (SEM). At each subdivision there are macroscopic collagenous dividing lines (myocommata). In Cryo-SEM. ions or molecules can be identified and quantified in situ using specific detectors coupled to the electron microscope.. Finally. infiltration and embedding in resin. the sample can be observed with all its constituent water. in this way. secondary fi xation with osmium tetroxide. and observed. the sample is frozen in slush nitrogen (Figure 10. coated. as for TEM). which is contiguous to the myocommata (Ofstad et al. They are each surrounded by the sarcolemma membrane and by a thin layer of connective tissue (endomysium). etc. light green. When physical fixation is used.. For this. or fluorescence microscopy. and coating with a conducting metal for SEM imaging or with carbon for x-ray.) before examination in the LM.

...Microstructure ◾ 141 Food Specimen portions Embedding in paraffin or resin Freezing (CO2. 1% red oil.1 Preparation of samples for LM observation. LM observation Figure 10. .11–2 μm) Cold stub Microtome Cryotome Mounting in glass slides Staining specimen 1% Toluidine blue 1% Lugol. liquid N2) Preparation for slicing Cold knife CO2 Knife Slice Semithin sections (0.

LR white Cured resin Glass or diamond knife Ultrathin sectioning (5–100 nm) Ultramicrotome Specimen block Trimmed block Tweezers Specimen block face Grid Knife 4% Lead citrate 2% Uranyl acetate Section collection Ultrathin section staining TEM observation Figure 10. .5% Glutaraldehyde 2% Os O4 Dehydration Ethanol (30%. 70%. Araldite Spurr’s. 100%) Infiltration and embedding in resine Epoxi resin.142 ◾ Handbook of Seafood and Seafood Products Analysis Food Specimen portions Fixation 2.2 Preparation of samples for TEM observation. 90%. 50%.

100%) Sublimated H2O P T Dehydration (To vaccum) Freeze dryer CO2 Critical point dryer (To transformer) (To pumps) Sputter metal coater (or evaporation coater) Coating (Au. for X-ray) SEM observation Figure 10.3 Preparation of samples for SEM observation. 50.Microstructure ◾ 143 Food Specimen portions Fixation Physical fixation Chemical fixation Quick freezing in liquid N2 2. 70. for SEM imaging) (C. .5% Glutaraldehyde 2% Os O4 Ethanol (30. 90. Pd.

.144 ◾ Handbook of Seafood and Seafood Products Analysis Food Specimen portions Quick freezing in slush N2 (T < –210°C) Freezing Specimen transfer Transfer to Cryo-SEM Knife Specimen fracturing (into Cryo-SEM) Specimen fracturing (–180°C... . vaccuum) Etched H2O Etching (into Cryo-SEM) Specimen fracturing (–90°C. vaccuum) Au deposition Coating (Au.) (into Cryo-SEM) (–130°C.4 Preparation of samples for Cryo-SEM observation. C. vaccuum) Cryo-SEM observation Figure 10.

Figure 10. where the Z disks can be distinguished. A and I bands (Pérez-Munuera et al. 2008). fat globules of different sizes are observed occupying the interfibrillar spaces and myofibrils are distinguished inside the cells. b: myocommata. The fiber is composed of myofibrils in which Z disks are distinguished in the areas where the sarcolemma is broken. In a cross section of the sample fi xed in osmium tetroxide (Figure 10. the perymisial connective tissue that surrounds the muscle bundles can be seen. The longitudinal section in Figure 10. Examples of different fresh fish tissues observed by several techniques are described here.6E).1 Herring Figure 10. Figure 10. with the endomysial connective tissue keeping the muscle fibers firmly attached to one another.6C). The typical fish muscle fibers can be seen. When ultrathin sections of herring muscle tissue are studied by TEM.7A shows a herring sample stained with toluidine blue and observed by LM. but the total collagen content is lower.7C shows the inside of a muscle fiber with the myofibrils perfectly bundled. the aggregation of solutes produced during the etching of the sample generates the typical eutectic artifact observed in Figure 10. which is mainly composed of collagen.6B). reveals the myofibrils inside each cell. The layouts of the Z disks that mark the length of the sarcomere are visible. surrounded by the sarcolemma and by the endomysial connective tissue. where the fat is viewed as globules on the surface of the fiber. a: myotome. 1990). obtained by the same technique but observed at a higher magnification. A micrograph cross section of the fibers shows them surrounded by the sarcolemma. 10.2. When the muscle fibers are observed using the Cryo-SEM technique. the myofibrils at the cell edges have a less rounded section than the central myofibrils and are arranged like a palisade.Microstructure ◾ 145 a b Figure 10.6A shows a cross section of herring tissue fi xed with glutaraldehyde and observed by SEM..6F. Fixing in osmium tetroxide shows the distribution of fat in the herring tissue. Figure 10. The separation that can be observed between the muscle cells is usually attributed to the effect produced by chemical fixation and dehydration during preparation for SEM.6D shows the microstructure of herring tissue at a higher magnification.5 Schematic representation of fish muscle with myotomes. The myofibrils are shown in longitudinal section in this sample (Figure 10. it is possible to observe ultrastructural details.7B. The myofibrils are . since the water in which the fish live lends support for the body (Lampila. At low magnification. the fat can be observed covering the fibers in a longitudinal section of herring muscle (Figure 10.

eutectic artifact. The cytoskeletal ultrastructure of hake was studied by Pagano et al. a. Hake fibers surrounded by connective tissue can be observed in Figure 10. The structural elements that constitute the sarcomere. pork meat (raw ham) (Larrea et al. 10. and roughly parallel filaments (Figure 10. along with the M and Z lines. The same structure has also been observed in different meat products. connected to each other at the Z disk level by the costameres. The main difference is their size: hake fibers are thicker than herring ones. the A and I bands. (F) Cryo-SEM.2.9). Z disk. can be seen. connective tissue. (2005) after depleting the thick and thin filaments with a potassium iodide treatment. for example. (A–E) SEM. . continuous. which are the components of the cytoskeletal network that links the myofibrils to one another and to the sarcolemma.6 Herring tissue. TEM and SEM studies demonstrated an extensive network of filaments connecting Z structures that were regularly spaced and connected by sets of longitudinal.. Z.8.2 Hake The observation of hake muscle by SEM after fi xing with glutaraldehyde allows distinguishing that the fibers of hake muscle tissue are very similar to those of herrings.7D). c. f. The TEM technique allows images to be obtained at higher magnification and with better resolution than other microscopy techniques (Figure 10. fat globule. 2007).146 ◾ Handbook of Seafood and Seafood Products Analysis Z (A) 300 μm (B) 6 μm I (C) f 40 μm f (D) a 10 μm f (E) 60 μm (F) c 30 μm Figure 10.

I band. . perymisial connective tissue. I. Z disks. costameres. Z.7 Herring tissue. 100 μm Figure 10. M line. (C and D) TEM. P. A. (A and B) LM. A band. myofibrils in a “palisade” ringing the edge.8 Hake tissue observed by SEM.Microstructure ◾ 147 p m m 30 μm (A) c M 10 μm (B) m m A Z (C) (D) Figure 10. m. M. c.

148 ◾ Handbook of Seafood and Seafood Products Analysis Z LZ IZ DZ 8.10 Smoked salmon. (A and B) Cryo-SEM. Z-disk. The micrograph shows geometrically shaped fibers surrounded by a connective tissue. (2000) used LM to observe the changes that occurred in the salmon during the smoking process and quantified them by image analysis. The data of the average cross-sectional area of muscle fibers showed that the smoking process produces shrinkage of the fibers. longitudinal filament connecting Z-Z.1 Smoked Salmon A cross section of a smoked salmon sample obtained using the Cryo-SEM technique is seen in Figure 10. et al. Sigurgisladottir et al. Com. 141. Z. Figure 10. Physiol.000 X Figure 10. Biochem.10A. B. M. the higher the smoking temperature. 13. Gudmundsson and (A) 100 μm (B) 10 μm Figure 10.R. LZ.3 Processed Fish Microstructure 10.10B shows a detail of an intercellular space created by the conjunction of three fibers or cells.9 Cytoskeletal structure of hake observed by TEM. . The fiber shrinkage and the space between the fibers both increased to a greater extent in the muscle from the salmon that were frozen before smoking than in muscle smoked from fresh salmon. the greater the shrinkage.. With permission.3. 2005.) 10. (Reprinted from Pagano.

and gaps were formed in the tissue structure.11A shows a longitudinal section of salted cod. (A) SEM.11A) in samples that have been obtained using physical fi xing (freeze-drying) instead of chemical fi xing. and (C) protein network. (B) cross section. these treatments decreased the cell size compared with fresh salmon. (A) longitudinal section. where two fibers can be observed completely covered by salt deposits. 10. Figure 10. (A) (B) (C) Figure 10. (B) Cryo-SEM.11B shows a cross section of salted cod tissue (A) 100 μm (B) 300 μm Figure 10.3. Figure 10.11 Salted cod.2 Salted Cod The presence of salt deposits in the cod tissue can be observed by SEM (Figure 10.12 Seafood stick (surimi) observed by SEM. A combination of PEF and high pressure had a more detrimental effect on smoked salmon microstructure than PEF treatment alone. .Microstructure ◾ 149 Hafsteinsson (2001) studied the effect of pulsed electric fields (PEF) and a combination of PEF and high pressure on smoked salmon microstructure.

10. where the presence of salt makes the etching of the sample for observation difficult and masks the underlying structures. obtained by SEM. the paste is shaped and an “artificial fish muscle” is obtained. (From Lluch.. With permission. The formation of a new network with the myofibrillar protein (Figure 10. Such a product is often sold as “crab sticks” or “seafood sticks. Figure 10.3 Surimi One of the most common surimi products on the market is artificial crab muscle. The cross section (Figure 10. Lancaster.12C) is responsible for the water-holding capacity and functional properOuter lining Outer tunic Muscle tunic Inner tunic Visceral lining Radial fibers Circumferential fibers (A) Radial fibers Circumferential fibers (B) Figure 10. The Chemical and Functional Properties of Food Proteins..” Lean fish meat is minced to a paste.. M. after adding different additives.12B) shows the typical concentric layers of this type of surimi product.3.13 Schematic representation of (A) squid mantle and (B) arrangement of muscle cells. PA.150 ◾ Handbook of Seafood and Seafood Products Analysis observed by Cryo-SEM. 2001. et al.A. Inc.) . shows a longitudinal section of a crab stick where the “artificial fibers” can be observed. Technomic Publishing Co.12A.

2007.. . the intermyofibrillar spaces between these can be observed. 1990. Tech. H. References Gudmundsson. E.E..14C)..75 μ m s (C) (D) l Figure 10. Effect of electric field pulses on microstructure of muscle foods and roes..5 mm in diameter (Lluch et al. 2001. 12. The fibers arranged in circumferential and radial bands were observed by SEM in samples fi xed with glutaraldehyde (Figure 10. Muscle Foods. 10. 122–128.14D).4 Squid Microstructure The squid mantle is composed of muscle tissue sandwiched between two tunics of connective tissue (Figure 10. 225. J. 2007). sarcolemma. 1.13). m. With permission. Lampila. l.. s. When TEM is used to study the ultrastructure of fresh squid (Figure 10.. The inner and outer tunics are covered by a visceral lining and outer lining. (From Llorca. a central sarcoplasm is shown to be surrounded by myofibrils. This fiber distribution can also be observed by LM in samples stained with toluidine blue (Figure 10. Food Res. Comparative microstructure of red meat. and (D) TEM. 2001). Technol. LM makes it possible to distinguish a peripheral area in blue and a central core in white inside each cell. L. et al. central sarcoplasm. myofibril. Regardless of their orientation. Circumferential muscle bands (100–200 mm) comprise fibers running about the entire circumference of the mantle cone. respectively. all the muscle fibers are thin.Microstructure ◾ 151 –200 μm –10 μm (A) 10 μ (B) 3. (C) LM. approximately 3.. (A and B) SEM.14A and B) (Llorca et al. 247–267. Th is gel network structure gives surimi its characteristic elasticity and texture (Sikorski.) ties of surimi. Muscle fibers are grouped in bands that are arranged orthogonally. Radial bands (10–15 mm thick) comprise fibers that connect two tunics of connective tissue. 807. poultry and fish muscle..14 Squid. 1990). Trends Food Sci. M. 2001). and Hafsteinsson. Each fiber is surrounded by a sarcolemma (Llorca et al. Eur.

and Crupkin.. 2001. Quiles. Ofstad. Z. E. Food Res. M. PA. Effect of frying on the microstructure of frozen battered squid rings. and Lluch. 857–865. H. I..A. 2008. Llorca. R. S. I.. Paredi. Effects of freezing/ thawing on the microstructure and the texture of smoked Atlantic salmon (Salmo salar). Meat Sci. 2. and Hernando.. FL. Pérez-Munuera. Sigurgisladottir.. Quiles. Fiszman. F. V. 2007. in The Chemical and Functional Properties of Food Proteins. M. chap.. Physiol. R.. Food Res. A.and post spawned female hake (Merluccius hubbsi). Quiles. in Handbook of Muscle Foods Analysis. Nutritional Composition and Preservation.. 807–813. K. I.. Biochem. M. Seafood: Resources. CRC Press. Pérez-Munuera. Protein breakdown during the preparation of frozen batter-coated squid rings. Tech. Lluch. 39. Technol. Olsen.A. Taylor.. 1. M. Pérez-Munuera. Hernando. and Hafsteinsson. 2001. I.. M. and Hanneson.. 448–455.). 33.E. Technomic Publishing Co...... Part B 141.. A. I.. S. Eur. . Lancaster.. O. Technol... V. 225(5–6). E. 213(6).. 2000.A. Food Res.....) related to gaping. R. Pérez-Munuera. Z.J. and Lluch. and Lluch... I. I. 1143–1154. Lebens. Larrea. Cardinal. M. M.A. 2007. I. Boca Raton. 574–582.E. Sikorsi. Larrea.L. Nollet. (Eds. V.... 13–21. Eur... Boca Raton. M. 2006. M. Llorca. and Lluch. Breakdown of intramuscular connective tissue in cod (Gadus morhua L. and Toldrá...) and spotted wolfish (Anarhichas minor O. Torrisen. chap. Hernando.R. Inc.. E. Wiss. (Ed. CRC Press.. Proteins in food structures. Microstructure.E.. Pérez-Munuera.. H. Int.)..... Ingvarsdottir. L. Microstructural changes in Teruel dry-cured ham during processing.A.M. 1990. Pagano. 76. Cytoskeletal ultrastructure and lipid composition of I-Z-I fraction in muscle from pre. I. Com. A. Llorca. 2005. Hernando.152 ◾ Handbook of Seafood and Seafood Products Analysis Larrea. FL. Sikorski.O.

......................159 11............................ For these reasons the knowledge of the chemical 153 ............................................................................................................4 Electronic Noses ..........................................4 Field-Effect Transistors ..................................................................157 11.......................2 Amperometric Gas Sensors ............159 11.1 Metal-Oxide Semiconductors ...............................................................................158 11......157 11.....3.........2 Conducting Polymers and Molecular Aggregates .........3..........................................................................158 11.........................................5 Color Indicators .............3 Chemical Sensor Technologies ...........157 11....................................165 11.. called odor........153 11..........................................................................................................................3..158 11...........2 Sensor Parameters............................................160 11............................................3...........................................1 Introduction ................................1 Sensors Based on Conductance Changes ......................1..........3 Mass Transducers ..................................................3................ The relationship between chemistry and food properties is particularly interesting in the case of fish and seafood in general................................................................160 11................................................................. is one of the most used method to assess freshness by both consumers and industries [2].............5 The Application of Electronic Noses for Fish Freshness and Quality Measurement .........164 References .Chapter 11 Chemical Sensors Corrado Di Natale Contents 11..........3........1 Introduction Among the thousands of molecules composing food complex mixtures.....1...........................................................6 Conclusions ....................................154 11..... for which the human perception of airborne chemicals........3.... some are of great importance to define overall properties such as freshness or quality [1]..

Global perceptions may be enough in many cases to detect freshness or edibility. capacitors. in order to achieve chemical sensors. according to this definition there are resistors whose resistance is a function of external temperature (thermistors) or diodes whose current–voltage relationship is strongly altered once they are illuminated by light (photodiodes). and a number of methods and protocols for different food are available. These methods require in some cases complex sample treatments and instrumentation such as gas chromatography or spectrophotometers. Figure 11. natural senses are not analytical. As an example. a complex structure is necessary. and they are sometimes called “basic devices. The device is composed of two parts. mass. whose electrical parameters may depend on the chemical composition of the environment in which they are in contact. and it resulted in a class of chemical analyzers that have the advantage of interacting directly with samples and of providing signals bearing the notion of the chemical composition of a sample being a liquid or gas. do not require any sample treatment. The interaction with a target molecule (hereafter called analyte) and a solid-state layer is a chemical event that. in the sense that the interaction of human senses with complex mixtures provides a global perception rather than a list of compounds. Differently from analytical instrumentations. Analytical chemistry is naturally based on “separation” approaches: namely. it is known that Nature provides living beings chemical senses that. Chemical analysis of foodstuff is a large part of the analytical chemistry discipline.” The matching between sensitive material and transducer is not univocal: a single sensitive material can be coupled with many different transducers and vice versa. In practice. and the more utilized are adsorption and reaction phenomena. Electronic properties of materials may hardly be directly influenced by the ambiental chemistry. work function. In the rest of this chapter an overview of the technologies related to these devices is provided together with examples of their use for fish freshness and quality determination. it develops methods to decompose complex mixtures (foods contains thousands of different molecules) in order to target either a single molecular species or a molecular family. The first is a chemically interactive material.1 shows what can be considered as the general structure of a chemical sensor. and ultimately they are of paramount importance to determine the acceptance of foodstuff [3]. These analyzers are chemical sensors. . as a consequence.2 Sensor Parameters A sensor is an electronic device whose parameters depend on some external quantity of whatever nature [4]. 11. can modify the physical properties of the sensing layer.154 ◾ Handbook of Seafood and Seafood Products Analysis profile of food is considered of great value. there are many possibilities of assembling a chemical sensor. The optimal matching between a sensitive layer and transducer is fundamental to achieving a well-performing sensor. Properties such as conductivity. These interactions can be of different nature. These transducers are the second component of a chemical sensor. or even field effect transistors. A sort of combination of natural and analytical approaches has been pursued since decades. namely a solid-state layer of molecules that can interact with the molecules in the environment. and the development of rapid and reliable chemical analyzers has been pursued since decades. In the same way there are devices that from the electronic point of view are resistors. or optical absorbance are among those that can be transduced into an electric signal by suitable transducers. in order to be reliable. On the other hand.

Chemical Sensors ◾ 155 Environment Quantity to be measured (concentration) Chemically interactive material ΔT Δm Δσ Δn ΔΦ Intermediate quantity Basic device Δi Δv Δf ΔΦ Electrical or optical signal Figure 11. These quantities can be the temperature (DT). As a consequence of the interaction. and many others.1 Schematic representation of a generic chemical sensor. The sensitivity expresses the capability of a sensor to modify its signal as a consequence of a change in concentration. of these quantities. electric conductance (Ds). Before illustrating the technological basis of chemical sensors. resolution. V = f (C ) where V is a generic signal C is the analyte concentration The knowledge of the response function is necessary to estimate from the sensor signal the amount of concentration. Besides response function. The fundamental action of a chemical sensor is the conversion of the information about the concentration of a chemical species into an electric signal. once properly connected in an electric circuit. For each. One of these quantities is sensitivity. Targeted molecules interact with a chemically interactive material. provide an electrical signal that is a function of the quantity of interactions occurring at the interface between the sensor and the environment. there are a number of devices that. mass (Dm). it is important to introduce the fundamental parameters that allow a correct interpretation of the performance of any sensor. other important quantities are necessary to be known to appreciate sensor performances [5]. it is the first derivative of the response function S= dV df (C ) = dC dC . Analytically. one or more physical properties of the interactive material change. and in more general cases. and selectivity. The relationship between the signal and the chemical concentration can be represented by an analytical function that embodies the sensor operation. the estimation may require the solution of a nonlinear equation. refraction index (Dn). This estimation is straightforward if the response function is linear. or work function (DF). These parameters are sensitivity.

it is not possible to deduce Saturation Nonlinear region Sensitivity Concentration Signal Linear region Concentration Figure 11. The knowledge of the signal V is affected by an error and this error is propagated in an error on the estimation of the concentration. the selectivity of a chemical sensor can be obtained only in very limited conditions. the sensitivity is a constant quantity. In order to fully appreciate the importance of sensitivity. and their importance holds for any kind of sensor. it is a function of the concentration.2b the corresponding sensitivity is shown. Selectivity defines the capability of a sensor to be sensitive only to one quantity rejecting all the others. With these conditions. . Lack of selectivity means that the sensor responds with comparable intensity to different species. It is worth mentioning that in case of electrical signals. For chemical sensors. the error ΔV is limited by the electronic noise that determines the ultimate uncertainty of any electric signal. In Figure 11. and it tends gradually to zero as the sensor response reaches saturation. and the selectivity can be achieved in many practical applications. In the case of physical sensors. Simple mathematical considerations lead to the conclusion that given an error ΔVerr affecting the signal V.156 ◾ Handbook of Seafood and Seafood Products Analysis Only in case of a linear response function. The response curve is almost linear at low concentration and tends to saturation corresponding to the complete occupation of available adsorption sites. the number of quantities is limited to a dozen. it is important to consider that the number of chemical compounds is in millions and that the structural differences among them may be extremely subtle. and with such a sensor.2a. the error ΔC on the estimated concentration is given by the following relationship: ΔC = ΔVerr S The error in concentration is then inversely proportional to the sensitivity. it is necessary to evaluate the influence of measurement errors. The sensitivity is larger at low concentrations. The previously mentioned quantities are totally general. A sensor containing such a sensing material and a basic transducer simply providing a signal proportional to the number of adsorbed molecules is represented by the response curve shown in Figure 11. For chemical sensors. In all the other cases. an additional parameter of great importance is selectivity.2 Typical response curve (left) and sensitivity (right) of a generic chemical sensor based on adsorption of target molecules in a sensing layer characterized by a limited amount of adsorption sites. Let us consider the generic case of a chemical sensor based on a sensitive material characterized by a limited number of adsorption sites. The amount of adsorbed molecules as a function of the concentration is ruled by the Langmuir isotherm [6].

In practical. Sensors are here classified according to the physical intermediate quantity. Metal oxide semiconductor chemoresistors have been used several times in fish freshness applications. . and a lowering of the potential barrier.3 Chemical Sensor Technologies In this section the basic principles of the most popular categories of chemical sensors are illustrated. For instance. releasing an electron back to the conductance band. Recently. Since the material is a semiconductor. The characteristics of these structures. which reacts with the bounded oxygen to form carbon dioxide. and then the amount of oxygen molecules that can be adsorbed at the surface is also limited. although interesting.1 Metal-Oxide Semiconductors Changes in conductance become appreciable in materials characterized by a limited number of mobile charge carriers. the most known and studied of which is SnO2 [7]: a wide band gap n-type semiconductor. The main sensitivity mechanism is related to the role played by oxygen. metal oxide growth in regular shapes such as nanosized belts [9] has shown peculiar properties. the sensitivity to trimethylamine and dimethylamine of aluminum-doped ZnO films was demonstrated [10] as well as the sensitivity to trimethylamine of SnO2 and CuO [11. These are oxides of transition metals. Selectivity will be reconsidered in Section 11. in this regard. The sensitivity can be further modified adding ultrathin amounts of noble catalytic metal atoms on the surface. dissociative adsorption sites of molecular oxygen are active on the oxide surface.1. Paradigmatic. and as a consequence. The most popular materials undergoing a conductance change on interaction with gases are metaloxide semiconductors. and a surface potential barrier is formed. It is important to remark that this kind of sensors needs to be operated at high temperature. The amount of depletion and the barrier height are proportional to the number of adsorbed molecules. a reduction of the surface conductance band depletion. 11. At sufficiently high temperature (above 200°C).3.1 Sensors Based on Conductance Changes 11. in any case. the number of conductance electrons is limited. and the sensitivity of these devices is extended to many different kinds of volatile compounds [8]. is the case of carbon monoxide. Metal-oxide semiconductor sensors can be prepared in many different ways.Chemical Sensors ◾ 157 any reliable information about the chemical composition of the measured sample. 11. an electrically actuated heater is integrated in the device. have not yet resulted in practical improvements of performances. the general advice is to produce a nanocrystalline material in such a way that the modulation of the surface conductance band population becomes dominant in the whole sensor. semiconductors are subjected to large changes of conductance also in the presence of a modest variation in the number of conductance electrons or holes. providing the maximum sensitivity.12]. The exposure to any molecule interacting on the sensor surface with adsorbed oxygen atoms may result in a release of electrons back to the conductance band. This is only one of the many interactions taking place on the surface of metal oxides. The consequence of the exposure to oxygen is a reduction of the surface conductance.3. A charge transfer occurs between the material and the adsorbed oxygen atom with the consequence that the conductance band in proximity of the surface becomes depleted.4.

QMB coated by sensitive layers was used for many applications. a sensor for ammonia can detect amines. the measurement of these mass shifts can allow the evaluation of the amount of adsorbed molecules. This property is largely exploited in electronics to build stable oscillators as clock references. and esters. these sensors were properly used to detect fish freshness [16]. and their conductance can change after exposure to volatile compounds. Thanks to this versatility. . Chemical sensors based on conducting polymers may be considered as a lateral result of these studies. and a sensor for SO2 can detect volatile sulfides. with broader scopes related to the possibility of developing a novel sort of electronics based on carbon chemistry [13]. these sensors were never demonstrated in practical applications.1. One of the drawbacks of these sensors is the instability mainly due to the degradation of doping radicals that are added to increase the conductance. A piezoelectric resonator is a piece of piezoelectric crystal properly cut along a well-specified crystalline axis. For instance. The measurement of small mass changes is made possible by piezoelectric resonators. Indeed.3. These are thin slabs of AT cut quartz oscillating at a frequency between 5 and 50 MHz approximately [17]. The main mechanism is the catalytic reaction occurring on the surface of a noble metal electrode. conducting polymers sensors can be prepared for different applications. sensors designed for CO are found to be sensitive toward alcohols.3 Mass Transducers The adsorption of molecules into a sorbent layer produces a change of mass. and food freshness is among them [15]. Piezoelectric effect can also be exploited in other configurations such as those based on surface acoustic waves. their chemical sensitivity can be changed at synthesis level modifying the chemical structure of the monomer [14]. the electric resonance is characterized by a very large quality factor (Q). If the quartz is connected to an oscillator circuit. the mechanical resonance of the crystal is coupled with an electric resonance. A typical QMB has a limit of detection around 1 ng. The frequency of the mechanical oscillation decreases almost linearly with the mass gravitating onto the quartz surface. In spite of the claimed properties.2 Amperometric Gas Sensors Electrolytic cells based on either solid-state or liquid-ionic conductors are used to detect several kinds of gases. As an example. Although designed for polluting gases. Due these cross-selectivities.2 Conducting Polymers and Molecular Aggregates The conductance properties of organic materials based either on polymers or on molecular aggregates have been studied since several years. More sophisticated mass transducers were proposed by using resonant cantilevers similar to those adopted in atomic force microscopy [19]. these sensors demonstrated a good sensitivity for compounds relevant for fish freshness. most important. an amount that is sufficient in many practical applications. The same effect is exploited for chemical sensing adopting particularly shaped crystals such as in quartz microbalances (QMB). 11.158 ◾ Handbook of Seafood and Seafood Products Analysis 11. Since crystal resonance is extremely efficient. aldehydes. Due to the piezoelectric effect. the electric frequency decreases linearly with the mass.3.3. With respect to metal oxides these sensors have two important advantages: they are operated at room temperature and. the possibility of using these sensors to measure fish freshness was demonstrated with metalloporphyrin coating [18]. aggregates of polypyrrole or polythiophene have a semiconducting character. 11.

is based on the fact that a computer screen can be easily programmed to display millions of colors. was also obtained [21].3.3. allowing a simultaneous evaluation of absorbance and fluorescence of samples. as a sensing part. giving rise to a number of low-cost advanced optical equipments such as digital scanners. Indeed. The principle was adequately exploited with a palladium gate FET exposed to hydrogen gas [20].5 Color Indicators Although known for several years [24]. known as computer screen photo assisted technique (CSPT). the importance of amines as spoilage markers leads to consider their reducing role and then the possibility to detect them with functional layers sensitive to pH changes. also appreciable by eye. 11. the visual determination limits the performance and may greatly vary between individuals. and cellular phones all endowed with color screen. combining wavelengths in the optical range. Chemical sensing based on optical sensitive layers is a captivating strategy due to the strong influence of target chemicals on the absorption and fluorescence spectra of chosen indicators [26]. Nonetheless. PDAs. the colorimetric detection of fish freshness recently received a novel interest. As a result. FET structures were also modified to accommodate. the chemical practice of this approach is badly balanced by the transducer counterpart. whose sensitivity toward amine was also recently measured [23]. whose characteristics largely fit the requirements necessary to capture change in optical properties of sensitive layers in many practical applications. the use of pH indicators is limited by the fact that mainly amines are considered (limiting the detection not to freshness but rather to spoilage).4 Field-Effect Transistors Most of the properties of field-effect transistors (FET) depend on the difference between the work function of electrons in the metal gate and in the semiconductor. to probe the sample with a variable combination of wavelengths instead of using the white light of scanners gives the possibility of performing an optical fingerprint measurement. Nonetheless. and. standard optical instrumentations are usually expensive. In particular. This basic structure was successively modified changing the gate metal and thickness to extend the range of measured gases. organic molecular layers. where they form an ordered dipoles layer. although under constant bias.Chemical Sensors ◾ 159 11. and hydrogen atoms can diffuse through the palladium film until they reach the oxide surface. Due to the large diffusion of portable computers. In this way sensitivity to ammonia. This difference can be modulated by a layer of electric dipoles that can reach the metal–oxide interface. The method demonstrated also the possibility to identify a number of different amines [28]. such as metalloporphyrins [22]. The feasibility of this approach has been demonstrated using as sensitive layer a film of a sodium salt (bromocresol green) [25]. Compared with the use of digital scanners. Lundström and Filippini proved that it is possible to assemble a sort of spectrophotometer using the computer screen monitor as a programmable source and a web camera as detector [29]. furthermore. cameras. an important gas for fish freshness and quality. . and screens. in the last decade we have seen rapid growth in performance in fields such as consumer electronics. The first demonstration in this direction was given by Suslick and colleagues when they showed that a digital scanner has enough sensitivity to detect the color changes in chemical dyes due to the adsorption of volatile compounds [27]. H2 molecules dissociate into atomic hydrogen at the palladium surface. Furthermore. the current flowing in the FET changes revealing the chemical interaction. This last technique. On the other hand. This salt exhibits a rather large change in color.

each receptor senses several kinds of molecules. and aromatic. 11. After this discovery. The concentrations of these chemicals are directly correlated to the degree of spoilage. To this point of view. Their concentration and the presence of other compounds are rather typical of each species. and such systems were soon dubbed as “electronic noses. organic synthetic receptors offer an unlimited number of possibilities to assemble molecules endowed with differentiated sensing features. amines.5 The Application of Electronic Noses for Fish Freshness and Quality Measurement The composition of fish headspace is a source of information about its freshness. Standard optochemical sensors are based either on absorbance or on fluorescence. Odor classification properties of artificial systems were tested on several different fields proving that electronic noses could be in principle used to replace human olfaction in practical applications such as food quality and medical diagnosis [35]. Among these compounds amines . and the genes expressed by olfactive receptors are known [31]. The physiology of olfaction has made considerable advances. After this conjecture. Investigations about olfaction receptors show that Nature strategies for odor recognition are completely different from those of analytical chemistry. and acid compounds. Since the 1980s. the lack of selectivity of many chemical sensors was considered as one of the main problems limiting their diff usion for practical applications. and an even more extended computation capabilities. whereas CSPT arrangement gives the possibility of evaluating at the same time both the effects. models of receptor mechanisms explaining the sensitivity to volatile compounds are now available. allowing for electronic nose application oriented optimizations. Nonetheless. The most important chemicals involved in the fresh fish odor are long-chain alcohols and carbonyls. CSPT has demonstrated its utility in particular to classify airborne chemicals reading absorbance and fluorescence changes in chemical dyes such as metalloporphyrins [30]. microbial spoilage produces short-chain alcohols and carbonyls. 11.4 Electronic Noses As discussed above.” This denomination is currently given to any array of unselective chemical sensor coupled with some multicomponent classifier. the application of the CSPT concept may be foreseen as greatly extending the analytical capacity worldwide. The features of electronic noses are fundamentally dependent on the sensing properties of the artificial receptors. and each molecule is sensed by many receptors [33]. Recent studies are also beginning to unveil the signal pathways leading from the generation of olfactory neuron signal to the conscious identification of odors [32]. the possibility of developing artificial olfaction systems became possible. observation of Nature offered a useful suggestion about the use of such devices. it was proposed that arrays of nonselective chemical sensors may show properties similar to those of natural olfaction [34]. On the other side. almost all sensor technologies were used to build such systems. Receptors were found to be rather unselective. Previous investigations evidenced that the headspace composition is a result of the balance between the “fresh fish” odor and the microbial spoilage produced compounds [36].160 ◾ Handbook of Seafood and Seafood Products Analysis camera. sulfur compounds. bromophenols. and N-cyclic compounds. The possibility having some versatile tool to tailor the sensitivity and selectivity of sensors is of primary importance to make arrays capable of capturing either large or narrow ranges of chemicals. N-cyclic.

the interaction between polymers and volatile compounds is often described by the linear sorption energy relationship (LSER) model [51]. Standard analytical methods for volatile amines and also sensors for some specific amines have been used to inspect fish freshness. from fish processing. Minor contributions to the fish headspace come from contamination of the environment (e. petroleum in sea). The same nonlinearity is observed with electronic noses. Nevertheless. the progress of spoilage is less linear with respect to Figure 11. An imperfect application of this method was demonstrated with engineered polymer-coated QMB [50]. the chemical complexity of the problem. Let us consider the use of an array of sensors absolutely selective for each individual family of compounds mentioned in Figure 11. for example. Data are extrapolated from an investigation by Strachan and Nicholson [48]. it is more realistic to consider an array of sensors specific for a single interaction mechanism. Figure 11. quartz microbalance sensors [44. hybrid electronic noses were used combining different sensor technologies such as QMB and amperometric sensors [47]. showing the ability of the electronic nose to track the different spoilage levels occurring at different storage times. Apparently. dipolarity.4 demonstrate a continuous progress after the 8th day. as much as possible. When properly analyzed by pattern recognition methods.g. but the behavior at the beginning is absolutely nonlinear. flat.45]. with a super impression of 6th and 1st days. PCA is a data analysis method allowing the representation of a multidimensional dataset in a reduced dimensionality space. Sensor data can be conveniently represented by a principal component analysis (PCA) scores plot. and finally from products of lipid oxidation [37]. In this regard. This result is rather surprising because fish spoilage is in general expected to be a linear and somewhat straightforward process. a plane. conducting polymer sensors [43]. five different kinds of interactions are considered: dispersion. Most of these are feasibility studies.4.Chemical Sensors ◾ 161 are considered as the typical markers for fish freshness detection. typically according to the freshness or more precisely according to the balance between fresh and spoilage produced compounds. Results shown in Figure 11. Analyzing the data with PCA the plot of Figure 11. Each sensor then provides a signal proportional to the concentration of each family. polarity. Such sensor arrangement consists in the application of a number of sensors characterized by a broad sensitivity toward species that are relevant for a certain application. In LSER. In Figure 11.5 is obtained. and acid.. The number of compounds whose concentrations are only partially correlated makes this application particularly appealing for sensor arrays of partially selective chemical sensors.3 the time evolution of the major families of volatile compounds found in the headspace of fishes is shown.3. The sensitivity of chemical sensors is not immediately related to the molecular family but rather to the interaction mechanism. nonetheless. MOSFET sensors [41]. such as metal-oxide chemoresistor sensors [38–40]. The representation plane is determined as that where the data variance is maximized and then the statistical properties of the dataset are. In gas chromatography.6 . Instruments based on different sensor technologies have been used. hydrogen bond basic. the accumulation of some compound. preserved [49]. in case of fishes. amines become instrumentally appreciable only when spoilage processes take place. let us discuss a simulation of a case study. In recent years attempts to use electronic nose technology to track the spoilage processes occurring in fishes have been reported in numerous articles. and the decrease in others result in a nonlinear problem. Since LSER was fruitfully used to model polymer-based chemical sensors [52]. and optical indicators [46]. In addition. the data produced by a sensor array can classify samples according to some of their global features. let us consider an array specific for each LSER interaction and one compound for family. amperometric sensors [42]. with an array of selective sensors it is not possible to distinguish between fresh and flat fishes. As a result. In order to understand the potential of electronic noses to detect fish freshness. and sweet conditions are hardly identified. and fresh.

The possibility of developing a multisensor device to measure and/or estimate fish freshness with a combination of instrumental techniques (electronic noses. The typical sensorial description is also reported. and the use of only one sense (e. Results are qualitatively similar to those shown in Figure 11. humans provide a more reliable identification of fish freshness. each able to capture different aspects of fishes.3 Time evolution of the major families of volatile compounds in fish headspace. Nonetheless. spectroscopic methods. sardines [58].. As a consequence.162 ◾ Handbook of Seafood and Seafood Products Analysis Amines Aromatics Fresh fish alcohols Fresh fish carbonyls Short-chain alcohols Sulfides 100 Fresh Sweet Flat Stale Putrid Concentration (ppm) 10 1 0. tactile (to test the flesh firmness and elasticity). It is important to consider that sensorial methods of freshness appraisal involve the use of sight (to evaluate the skin appearance and the color and the global aspect of eyes). image analyzers.5.01 0 5 10 15 Days 20 25 30 35 Figure 11. olfaction) provides several errors of evaluation. shows the scores plot of a partial least-squares discriminant analysis model related to an array of metalloporphyrin-coated QMBs. The fusion of multi-instrumental information can then be treated as the descriptors provided by a trained panel providing a sort of artificial quality index [55]. and devices measuring electrical properties) has been illustrated in different applications related to cods [56.57]. texture meters. This feature that can be interpreted as a failure of the electronic nose is likely due to an intrinsic nonlinearity of the studied problem. color meters. and groupies [59]. and original data were previously published [53]. with a folding back of the spoilage process in the representation plane. The experiment was related to COD fishes. and olfaction (to smell the gill odor) [54]. .g.1 0. in order to measure the quality of fish instrumentally. an integration of instruments is necessary.

76%) 0.5 0 –0.Chemical Sensors Scores plot 2 30 1.5 –1 –1.5 –6 4 6 –5 –4 –3 –2 PC 1 (80.5 24 PC 2 (19.5 –2 –2. each specific for a single interaction mechanism among those modeled by LSER.5 1 PC 2 (15.3 are used.4 PCA scores plot of a simulated experiment where sensors selective for the compound family in Figure 11.03%) –1 0 1 2 30 2 28 22 20 10 10 12 14 16 8 1 Figure 11.5 1 0. Scores plot 1.5 –1 24 10 12 8 28 2 4 6 1 ◾ 163 22 20 18 16 14 –1.5 –6 –4 –2 0 2 4 PC 1 (72.5 PCA scores plot of data related to a virtual array of sensors. .93%) 0 –0.39%) Figure 11. Data show the impossibility of distinguishing the spoilage process in the first 6 days and an abrupt change between 6th and 8th days.

stored. These technologies are sometimes equivalent in terms of performances. and for some specific applications. one technology may outperform the others. Food-related sites are usually highly contaminated from the point of view of odor. analyzed.31%) 17 17 3 15 3 44 3 4 4 3 11 11 3 11 11 2 11 3 11 2 2 2 2 9 7 9 1 1 91 11 1 9 7797 9 7 7 7 17 11 11 11 99 15 15 15 15 15 15 0 4 3 2 2 4 2 4 15 2 –50 –100 –150 –100 –50 0 1 50 9 100 150 200 LV 1 (63. Each step of the food chain has peculiar needs that a proper chemical sensor approach can in principle contribute to satisfy. All these applications require instruments able to work on-site. and finally at consumer level.6 Conclusions The conversion of chemical information into electric signals that can be measured. 11. sensors are . In the case of fish and seafood freshness and quality determination. the application of arrays of sensors can greatly improve the performance in terms of prediction of quality and freshness. transmitted and integrated with other data can be performed by several different technologies. Chemical sensors are an almost mature technology for many practical applications.6 PCA score plot of metalloporphyrin-coated QMB. all the actors of the food chain (producers. As an example. At the current state of the art. data are related to cod fish fillets. at processors level the screening of quality of incoming products to optimize the processing and to sort processed food.164 ◾ Handbook of Seafood and Seafood Products Analysis Scores plot 150 17 17 100 17 17 50 LV 2 (26. at producer level the increment in quality and yield. processors. and labels indicate the storage days in ice. In this regard.62%) Figure 11. It is important in any application to design the optimal sensor array to determine quality and quantity of the relevant chemical species and to select sensors optimizing sensitivity and resolution. the control of quality and safety both on the market and at home. and consumers) are potential users of chemical sensor technology.

Methods to evaluate fish freshness in research and industry. Bremner. 2006.Chemical Sensors ◾ 165 not able to distinguish between background and relevant odor. 40. T. RSC Press. CA. From this perspective. U. Chemical Sensing with Solid State Devices. Shimizu. measuring the odor of a fish in a typical storage room among dozens of stacks of fish crates.. 11. For this a strong cooperation between sensor developers and end users is necessary in order to optimize practical solutions. M. Let us imagine.P. Electron. 84. Fraden. Cambridge.. synesthetic action among the senses is required to form a full judgment over a certain food sample. Handbook of Modern Sensors. Actually. and firmness. References 1. 1989. 8. tactile.. S. there are applications. M. for instance. Alberty. 40. Ed. K. 183.. and Takao. 2001. it is important to consider that sensory analysis is almost never confined to only olfactory perception. J. 4. 2. Actuators B. Food: The Chemistry of its Components. This opens a further novel investigation direction involving again researchers from different areas. IEEE Sens. 87. Y. H. Metal oxide nano crystals for gas sensing. Sens. Food Sci. in order to fulfill user requirement. et al. At this level a correct and careful analysis of user needs and expectations and an education effort toward the users are important to disseminate the intrinsic novelty carried by sensor systems such as those widely belonging to the class of artificial olfaction. Trends Food Sci. ZnO thin film sensors for detecting dimethyl.. It is also important that developers and users are aware of the intrinsic limit of information that is carried by the volatile part of a food. 6. 1997. Physical Chemistry. Mater. 321. This suggests that to fully reproduce the perceptions of humans with artificial sensors. Semiconducting and metallic polymers (Nobel lecture). 14. and olfactory perceptions. Koziej. 38. John Wiley & Sons. D’Amico. Roy. the electronic nose has to be compared and integrated with instruments providing information about visual aspects. in fish analysis. Acta. confirming that interdisciplinarity is the most strong added value for food analysis.. 18. 2002. Olafsdottir. Toward practical definitions of quality for food science. Actuators B. For instance. 2004. Sens. Angew.and trimethyl-amine vapors. Madou.K. Persaud. 2000. is calculated considering at the same time visual. . A contribution on some basic definitions of sensors properties. J. 12. 83. Polymers for chemical sensing. Today. A. 108. U. AIP Press. Mater. San Diego. 10. where existing chemical sensors can be specialized. Trimethylamine sensor based on semiconductive metaloxides for detection of fish freshness. Sci. Heeger. quality index. Metal oxide based gas sensor research: How to? Sens. A. and Basu. 3. Crit. 7. New York.A. Int. in terms of sampling and data presentations. Coultate. G.. D. Comini. As an example. S. J. S. Academic Press. 8. Anal. 568.. and Di Natale. 28. E. so that the performance of the sampling of an application is difficult. 2004. A semiconducting metal-oxide array for monitoring fish freshness. 113. 2591. Y. 13. 121. 2001. and Weimar. New York. R. texture. 1982. Chim. interesting at industrial level.. 1990. and Morrison. Tech. 1. 258. Chem.J. 2004. Rev. portable systems without any conditioning of sample are of limited use for fish inspection. 15. linearly correlated with the days in ice. et al. Barsan.. 1. Hammond. 5. On the other hand. N. Actuators B. 9. 2007.. Egashira. C. Mater. 2005. J.

Ólafsdóttir. R. Talanta. 283. 839. J. Martinsdóttir. 102.. Röck.. 2006.. 45. Trends Anal. 299. 77. San Diego. 44. E. 1969. Monitoring of fish freshness using tin oxide sensors.. Anal. 36. CA.. Neurobiol. Cell. 34. A hydrogen sensitive MOS field effect transistor. Brain Res. Phys.. November 1986. Chem. Evaluation of fish freshness using volatile compounds: Classification of volatile compounds in fish. Ballantine. in Sensors and Sensory Systems for an Electronic Nose. Rapid gas sensor measurements to predict the freshness of capelin (Mallotus villosus). Nature. G. Recent dynamics in olfactory population coding. 21. 1996. 23.S. 31. Chem. 352. Paquit. K. 1992. 28. A colorimetric sensor array for odour visualization.. Lett. Takulapalli. M. 1997. the Netherlands. and electronic nose evaluations of yellowfin tuna under various storage conditions. et al.. 43. Ólafsson. Winquist. 567. 175. Halifax (Canada). Food. 1982. Opin. 25. 406. Modified palladium metal-oxide semiconductor structure with increased ammonia gas sensitivity.. 1975. Agric. 292. 130. 30. and Lundström. N. Food Chem. 710. 29. Du. 2226. 1997. 16. 33. and Fleurence.. 25.. Lett. Chem. Ed. et al. 22.. et al. Angew. et al. 1986. L. 108. . Sens. 1991. A chemical sensor based on a microfabricated cantilever array with simultaneous resonance-frequency and bending readout. Electrical detection of amine ligation to a metalloporphyrin via a hybrid SOIMOSFET. Chem. and Holley. 55. Acta. Persaud. Kluwer. 257. et al. 2000. Kramer. in Seafood Quality Determination Symposium. Proceedings of the Final meeting of the Concerted Action “Evaluation of Fish Freshness” AIR3 CT94 2283. 24. Computer screen as a programmable light source for visible absorption characterization of (bio)chemical assays. Gardner. G. Josephson. 2002. Development of a smart packaging for the monitoring of fish spoilage. (eds. Elsevier. et al.). 15–25 July. and Amano. G. and Suslick. et al. D. Battiston. et al. et al. 45. 1997. Filippini. Curr. K. F.. W. Proposed modification of dyer’s method for trimethylamine determination in cod fish. 4458. sensory..166 ◾ Handbook of Seafood and Seafood Products Analysis 15. D. Chim. 325.M. et al.. 32. 122. P. 2008.X. 2001. Actuators B. Gauglitz. Nature. Phys. Prot. R. Analysis of discrimination mechanisms in the mammalian olfactory system using a model nose. Tozawa. Measurement of volatile aroma constituents as a means for following sensory deterioration of fresh fish and fishery products. I. Angew. and Jónsson. Dordrecht. 1984. Rev. 38. Development of a ChemFET sensor with molecular films of porphyrins as sensitive layers. A. the Netherlands. Soc. 20. Acoustic Wave Sensors. 2001.. Filippini. Int. Buck. R. 748. 19. Lindsay. J. Lundstrom. November 12–14. Nantes. in Methods to Determine the Freshness of Fish in Research and Industry. Chem. 2005. Sicard. and Dodds. and Weimar. 2007. 77. 37. J. R. 2001. Molecular recognition and discrimination of amines with a colorimetric array. 240. 2006. Chemical sensing with familiar devices. Ed. 705. and Axel. L. G. et al.. B. J. Brunink. Microbiological. 55–69.. Technical Conference on Fish Inspection and Quality Control. D. Academic Press. S.H. 2654.N. 53. Rakow. and Olafsdottir.. 3800. 26.. 705. Commun. D. International Institute of Refrigeration. 1983. H. Int. J. Svensson. D. and Stopfer. K. K. Liston (eds.. 65. F. 468. F. A novel multigene family may encode odorant receptors: A molecular basis for odor recognition. Friedrich.. G. 10–14. 11. 18. Amsterdam. Andersson. 2008. 26. Actuators B. p. 17. 2027. Appl. 35. U. 466. Receptor cell responses to odorants: Similiarities and differences among odorants. Electronic nose: Current status and future trends. G. Barsan. 64. The application of metalloporphyrins as coating material for quartz microbalance based chemical sensors.W. Olafsdottir. I. J. M.. 27.W. Bartlett. A. Chem. Optical sensing looks to new field. Am. Sens. 2003. Rakow et al.. N.). 108. Enokihara. Appl. E.

April 10–14. Vaihinger. 282.B. 582. Agric. C. 273. et al. J. N. E. A. et al. 85. 1997. 293. Potential application of the electronic nose for quality assessment of salmon fillets under various storage conditions. and Martinsdottir. 2007. 87. 1995.. Solubility interactions and the design of chemically selective sorbent coatings for chemical sensors and arrays. 27. Houser. W. and Abrahams. and Undeland. 2004. J. C. Haugen.. 752. G. 320.Chemical Sensors ◾ 167 39. R. Polymer based sensor array and mulicomponent analysis for the detection of hazardous organic vapours in the environment. 2005. Actuators B. and Nicholson. Prentice Hall Inc. A model to predict fish quality from instrumental features. 49. 2002. Ólafsdóttir. Johnson. Rational materials design of sorben coatings for explosives: Applications with chemical sensors. Wageningen Academic Publishers. Sens. Olafsdottir. C. 53. Food Chem. Sens. 55. J. and Wichern. 48. Proceedings of the Final Meeting of the Concerted Action “Evaluation of Fish Freshness” AIR3 CT94 2283. Monitoring and Traceability. Olafsdottir. Hierlemann. QIM an European tool for fish freshness evaluation in the fishery chain. 67. J. 469. Measurements of quality of fish by electronic noses. E.. Tech. 261. C. Luten. W. 45. et al. 3.. Di Natale. Rome. Di Natale. 86. 50. Lipid oxidation in herring fillets (Clupea harengus) during ice storage measured by a commercial hybrid gas-sensor array system. 1996. Acta. Gill air analysis as an indicator of cod freshness and spoilage.B. 46. Technol.. Recognition of fish storage time by a metalloporphyrins-coated QMB sensor array.. Meas. Actuators B.. 51. J.. Anal. Agric. G.X. 70. D. et al. et al. Nantes. Sens. Oehlenschlager. J. Di Natale. Englewood Cliffs. et al. November 12–14. 111. 57. G. 225. Food Sci. et al. Food Chem... 58.. J.. Ubiquitous chemical sensing and optical imaging for ubiquitous environments. and Göpel. 531. in Methods to Determine the Freshness of Fish in Research and Industry. Strachan. 40. 2001. 59. (eds. 2003. J. 2001.E. 287. G. J. C. Wageningen Academic Publishers. 26. 15. 42. 1994. Du. 572. 1991.. and Olafsdottir. Food Chem. A. Luten. Characterisation of food freshness with sensor arrays. 41. G. Chim. F. Wageningen.. Comparison and integration of different electronic noses for freshness evaluation of cod-fish fillets. Sens. NJ. (eds. Di Natale. J. Actuators. 54. 77. A. 47. 1992.B. 2654. et al. H.Z. and Macagnano. C. Assay of fish freshness using trimethylamine vapor probe based on a sensitive membrane on piezoelectric quartz crystal. 45. Martinsdóttir. 563.). M. and Jónsson. Actuators B. 18. 307. Macagnano.. E. the Netherlands. Sci. Food Sci. Applied Multivariate Statistical Analysis. Rapid gas sensor measurements to predict the freshness of capelin (Mallotus villosus). et al.. . IEEE International Conference on Robotics and Automation. Luten. Technol. Fish freshness detection by a computer screen photoassisted based gas sensor array. 52. Ólafsdóttir. in Quality of Fish from Catch to Consumer: Labeling. Talanta. 1103. Kent. 81.. J. Data fusion in Mustec: Towards the definition of an artificial quality index. Trends Food Sci. 43. Actuators B. Sens.). Zhao. Sens.. 2002. Actuators B. Alimelli. 2005. 56. 126. G. Di Natale. and Olafsdottir. J. P. 51. Monitoring and Traceability. I. 54. A new multivariate approach to the problem of fish quality estimation. in Quality of Fish from Catch to Consumer: Labeling. et al. 2004. Wageningen. 2003. et al. 218. A. Int. 1982. Food Sci. Schweizer-Berberich. 2003. 44. Multisensor for fish quality determination. et al. the Netherlands.. 7.. 2006. Grate. Prediction of microbial and sensory quality of cold smoked Atlantic salmon (Salmo salar) by electronic nose.M. E. S. International Institute of Refrigeration. Oehlenschlager..H. 1997.


................................................... Pedro José Fito Suñer........................4 RF Spectroscopy—Impedance Spectroscopy ............................................2 The Importance of Quality Control—Advances in the Online Control Techniques ..........1 Determination of Moisture Content .................. by Microwaves: Methods and Equipments ............170 12...........4 Overview of Microwave Theory .................................................5 Applications of Microwave Technology in the Assessment or the Control of Processes .................175 12........184 References ...................5 Microwave Spectroscopy—Dielectric Spectroscopy ..1 Ultrasounds—Acoustic Spectroscopy .....................................................180 12... 176 12................6 Advantages and Benefits of Microwave Methods ...................................................3.....174 12...................................3.................... and Pedro Fito-Maupoey Contents 12.....................................................................172 12............174 12..................3........................3.......2 Visible Spectroscopy .......6 Conclusions ...................173 12....3..179 12..........................................................5...............................3..182 12.........................................173 12............................3 New Technologies for Online Control ................................................................5..........................................................................1 Sensors for Quality Assessment ......2 Freshness and Salting/Desalting Process Quality Control of Fish and Seafood................................................Chapter 12 Physical Sensors and Techniques Ruth De los Reyes Cánovas.........................................170 12........................................................184 169 ........... Ana Andrés Grau.........................................................................................3 IR Spectroscopy ...................................................................171 12.....

as a result of not being able to perform online nondestructive measures that would correct the manufacturing process in real time. However. In this way.e.2 The Importance of Quality Control—Advances in the Online Control Techniques Quality control is essential in the food industry. Existing techniques in food quality assessment. often an electric signal. This was due to the absence of nondestructive technologies that would allow the product classification by its properties (internal properties). different physical and chemical parameters related to the quality of foodstuffs have been selected [1]. traditionally. This signal provides direct information about the quality factor(s) to be measured or may have a known relation to the quality factors. sensors are classified according to their mode of use: online. Since consumers expect good shelf life and high-safety products with an adequate ratio of quality–price. can provide reliable information about food quality. these techniques are destructive. at-line. etc. quality control in manufacturing lines was limited to destructive off-line analyses that determine the acceptance or disposal of much of the production of the day. size. which relates to the quality factors. . such as color.). focusing on the seafood sector advances. timeconsuming. color. Therefore. and unsuitable for online application. and the internal properties were determined off-line by destructive and time-consuming technologies. size. and they give a real time signal. The acquisition of these parameters that characterize the abstract concept of “quality perceived by the consumer” leads to the development of the necessary technology for application in the classification of products.) that can be measured by a simple balance or by a sophisticated video camera. taste. or flavor. Traditionally the on/at-line quality control was restricted to external properties (weight. ease of consumption. the calibration lines for fruit processing). 12. At-line sensors are devices to be used for instance in split-flow measurements. and efficient quality assurance is becoming increasingly important. responding within hours or days.170 ◾ Handbook of Seafood and Seafood Products Analysis 12. Consumers perceive the quality of a product on the basis of a feeling of satisfaction that some sensory properties produce in them. an online sensor has the advantage of giving an immediate quality measurement and provides possibilities for regulating the process by adjustments.1 Sensors for Quality Assessment A food quality sensor is a device that can respond to some physical or chemical property or properties of food and transform the response(s) into a signal. research. however. Usually. Online sensors operate directly in the process. quality in food products is very difficult to define. Thus.. This chapter tries to show the increasing growth of new and efficient online and at-line control methods that can provide important information about the internal quality of foods. Th is perception is used to choose the product one wishes to buy. the development of in-line calibrators was restricted to external properties (weight. and development. the food industry is progressively investing more and more capital in quality control. etc. Because of that. either instrumental or sensory evaluation. or off-line. They often have short-response times (minutes or seconds) and also allow process corrections. Off-line sensors are laboratory devices. as well as in machinery for the separation of products by their varying degrees of quality (i. requiring reagent additions or equilibrations/reaction times.

and regulatory officials have been seeking improved methods for determining freshness and quality [2]. nutritional and health information. food inspectors require good manufacturing practices. first to satisfy the consumer and regulatory requirements and second to improve the production feasibility. the new sensors’ concept of being easy-to-use. controlling the automated process and the raw material stream. Thus.4]. which are commonly structural. they all call for intime and online sensors for control. . 12. With the increasing globalization of fishery product sales. in particular through online or at-line quality sensors. warning systems. automation. Further. and much more. these techniques can provide new quality control systems of the internal (and external) properties of foods that act in real time and in a nondestructive way. these properties need slow and destructive methods to be controlled. processing. with the appropriate hardware and software.3 New Technologies for Online Control The quality of almost all the industrial processes depends on the modification of a few parameters. eating quality. It is necessary to stress that fish quality is a complex concept involving a whole range of factors. and compliance with the regulations. and authentication all require improved control methods. and so forth. food producers are increasingly asking for efficient control methods. new data systems. sensing the final product quality. allow input from the manufacturing line with information obtained from the measurement of quality parameters selected (feedback). consumers. the safety and quality of fishery products has been of particular concern in recent years. quality sorting. it is able to obtain a final product that will always be within the margins of quality predetermined. for example. but online methods are required for industrial quality control. therefore it is possible to apply to the product under development the necessary corrective measures while it is still in the manufacturing line. In this way many new food safety concepts and key quality parameters have arisen during the last decade: Hazard analysis critical control points (HACCP). and product type [3. nutritional quality. This kind of system not only permit an assessment of quality in terms of their properties but also. safety. physical. traceability. ISO 9000 Certifications. In addition. and storage techniques. such as water content for drying processes. These systems will reach three milestones. In general. A study performed by Consumers Union found that more than one-quarter of the fish samples tested were on the brink of spoilage [5]. or chemical properties. which for the consumer include. and low cost in the sensor’s compounds. freshness. availability. Consequently.Physical Sensors and Techniques ◾ 171 New analytical techniques have been (and they are still being) developed to study the quality of complex food materials and to monitor the properties of foods during processing. convenience and integrity. the obvious physical attributes of the species. is very important for the partners in the chain. and typing the product labels. The great challenge is indeed to focus on the real time and online sensors and data systems surveying processes and products. One of the most unique characteristics of fish as food is that it is a highly perishable commodity. time passed after catch and the temperature “history” of fish are very often the key factor determining the final quality characteristics of a fish product [6]. an excellence in accuracy. Concretely. Information about handling. safety. total quality management (TQM). including time/temperature histories that can affect the freshness and quality of the products. processors. labeling. tight feedback loops for automation of the production. In addition to the requirements of consumers. size. and reduction of production cost and production time (increased throughputs).

The main disadvantage of ultrasound is that the energy propagates poorly through a gaseous medium.. some of their propagation parameters are modified. above 16 kHz [17]. determine the velocity of a moving tissue. and raw meat mixtures can be related to its composition using semiempirical equations [7]. when propagated through a biological structure. must act in real time and without producing permanent effects on the food. Suvanich et al. The interaction between wave radiation and matter as a function of wavelength or frequency is called spectroscopy. [21] published a report on how the ultrasonic velocity measurements show potential for analyzing fish composition. It is also necessary to work at very low power in order to not cause permanent effects such as heating. Ultrasound imaging is a versatile. Normally the modification of any quality parameter is macroscopically correlated to the change in any wave parameter that can be controlled. fulfilling the initial premise. moreover. Highfrequency. low-energy diagnostic ultrasounds are used as a nondestructive analytical technique for quality assurance and process control with particular reference to physicochemical properties such as composition. structure. we concentrate on electromagnetic methods at microwave frequencies. This technique encompasses a wide range of imaging modes and techniques that use the interaction of sound waves with living tissues to produce an image of the tissues or. it is almost imperative to resort to elastic (sonic) waves such as ultrasounds or to nonionizing electromagnetic radiation. exposing their main disadvantages and highlighting the advances in the field of seafood. Depending on the frequency used and the sound wave amplitude applied. and widely used diagnostic tool. such as radio frequency (RF). which is finding increasing use in the food industry for the analysis of food products. and physical state of foods [20]. induces compressions and depressions of the medium particles. Ultrasonic velocity in fish tissues. chicken. Thanks to advancing technology. and a high amount of energy can be imparted. It is virtually impossible for . the other techniques that enable online control have been briefly commented on below.13] in foodstuffs. the reason is. NIR measurements are widely used in the food industry to determine the sugar content in fruits [9]. microwave. Ultrasound attenuation spectroscopy (acoustic spectroscopy) is a method for characterizing properties of fluids and dispersed particles. impedance measurements (RF) can determine salt and water content in salmon filets [10].172 ◾ Handbook of Seafood and Seafood Products Analysis Given the premise that online control requires a nondestructive method. and dielectric measurements at microwave frequencies can be used to analyze water activity [11] and water content [12. Ultrasound is a form of energy generated by sound (really pressure) waves of frequencies that are too high to be detected by human ear. in this chapter. and visible. in the case of Doppler-based modes. For fish samples. It is impossible to address all these techniques with precision. which enable a variety of applications [18. The spectroscopic techniques use the information found in the spectrum that is emitted for the food to predict certain of its qualities. The salt and water content are related to dielectric properties of cod at microwave frequencies [14–16]. which. a number of physical.e. thermal and near-infrared (NIR). Nevertheless. and biochemical effects can be observed. Ultrasound.19]. When these waves pass through foods (or are refracted by them). 12.3.1 Ultrasounds—Acoustic Spectroscopy Ultrasonic is a rapidly growing field of research. i. chemical. well-established. the modification of these parameters can be measured in real time. Visible (and near UV) transmittance method has been investigated to inspect the internal quality (freshness) of intact chicken egg [8]. below are cited some examples of the use of these new technologies in the quality control of foodstuffs.

000 nm). Thermal infrared imagers translate the energy transmitted in the infrared wavelength into data that can be processed . the main disadvantage of this method is that only the surface of the sample is examined.3 IR Spectroscopy In the recent years. The far IR. and it is able to provide thermal information. A rapid. Other information should be used in conjunction with visible spectra in determining the specific properties of interest. [26] using the visible wavelengths only. 12.3. Karoui et al.3. a multispectral imaging NIR transflectance system was developed for online determination of moisture content in dried salted codfish [29].2 Visible Spectroscopy In recent years. but their use is limited by their low penetration in the product (it depends on the wave length. The region of the electromagnetic spectrum under consideration in Raman spectroscopy is similar to that in MIR. the usefulness of visible spectroscopy/near infrared spectroscopy (VS/NIRS) has been researched for many quality aspects [23–25]. The most popular IR spectroscopy is the NIR one. but it involves a scattering process. [33] applied MIR spectroscopy combined with chemometric tools to determine whether fish has been frozen–thawed. thus. NIR spectroscopy is based on the absorption of electromagnetic radiation at wavelengths in the range 780–2500 nm. All these techniques have been gradually implemented as monitoring systems in food processing [31]. This technique measures the reflectance of light from the product in the visible and NIR wavelength range.000 and 400 cm−1 (2. the energy at defined frequencies can be partially absorbed. Marquardt and Wold [34] concluded that Raman spectroscopy might be a useful tool for rapid and nondestructive analysis of fish quality. This complicates the noncontact measurements. 12. O–H. Raman spectroscopy is based on the shift of an excited incident beam of radiation that results from inelastic interactions between the photons and the sample molecules. NIR spectra of foods comprise broad bands arising from overlapping absorptions corresponding mainly to overtones and combinations of vibration modes involving C–H. For example. In the fish sector. This makes it very feasible for measurements to be made in organic and biological systems. but it is not the only one. the visible spectrum is a function of the entire structure of the compound rather than specific bonds.5 and 20 micrometers. [28] applied NIR spectroscopy to assess the end point temperature (EPT) of heated fish and shellfish meats. When radiation with energy corresponding to the MIR range interacts with a molecule. NIR spectroscopic method has been developed by Zhang and Lee [30] to directly determine free fatty acids (FFA) in fish oil and for the assessment of mackerel quality. the freshness of cod was estimated by Heia et al.500–25. and N–H chemical bonds [27]. ultrasound transducers must have airless contact with the sample during examinations [22]. Mid-infrared (MIR) and Raman spectroscopy have high structural selectivity and contain more of the type of information needed in structural elucidation studies.Physical Sensors and Techniques ◾ 173 ultrasound to pass through air. which is also called thermal infrared (TIR) refers to electromagnetic waves with a wavelength of between 3. Focusing on fish products. NIR technology has been widely developed as an analytical tool. MIR spectroscopy concerns the region of the spectrum lying between 4. Uddin et al. but it is measured in terms of tenths of a millimeter [32] and is dependent on less-precise reference methods [27]. Most industrial processes require the measurement of temperature.


Handbook of Seafood and Seafood Products Analysis

into a visible light spectrum video display. Thermography (infrared; thermal scans) uses specially designed infrared video or still cameras to make images (called thermograms) that show surface heat variations. This technology has a number of applications, for example, recent studies conducted by Fito et al. [35] lay the groundwork for the use of TIR image for the control of the optimum drying time in a citrus line. Focusing on fish industry, Jacobsen and Pedersen [36] developed a method based on infrared measurement of temperature changes in cold-water prawns during the glazing process studied in a small-scale controlled experiment. The method is thus remote and physically based on the heat transfer between prawns and glazing water.


RF Spectroscopy—Impedance Spectroscopy

Radio frequency is an electromagnetic radiation within the range of 3 Hz to 300 GHz. This range corresponds to the frequency of alternating current electrical signals used to produce and detect radio waves. Different techniques have been developed for quality control based on the response of foods to waves in the RF region. The technique called “bioelectrical impedance analysis” (BIA) is highly effective for measuring human body composition such as fat content, lean muscle, or total water [37] and nutritional status [37,38] and there is abundant supporting literature from medical studies demonstrating the effectiveness of the approach. This technique works at 50 kHz and is also an accurate predictor of the composition of fish [39,40] as the amount of water or proportion of fat tissue to lean tissue is correlated to BIA measurements through regression equations built on multiple measurements of control groups [41]. Impedance spectroscopy measures the dielectric properties (see Section 12.4) of a “food material” as a function of frequency; this term usually applies to the range of RF frequencies, sometimes extended to low microwaves. Impedance spectroscopy has been widely used to estimate the physiological state of various biological tissues [42,43]. In studies of a biological tissue, it is of great importance to establish an appropriate equivalent circuit model to relate the measured data to the physical and physiological properties. A number of spectroscopic methods in RF have been used quite recently to measure the quality-determining properties of frozen fish [44,45]. Haddock muscle showed significant changes in its dielectric properties during rigor mortis at frequencies between 1 Hz and 100 kHz [46]. In quality control of fish, the principal method of data analysis of impedance results has been to calculate indices with the measurements conducted at one or two frequencies [44,47]. With living tissues and in the postmortem period, impedance data have been analyzed by regression at each measured frequency and at several selected frequencies, by Cole-Cole analysis, and so on [48], but multivariate techniques of data analysis are still not widely used. The main disadvantages of RF for online monitoring are related to the physical size of its hardware, which is very voluminous and difficult to manage; moreover, interactions with metals and other materials can be problematic, and ionic conduction effects (i.e., due to dissolved salts) are highly significant (masking other effects).

12.3.5 Microwave Spectroscopy—Dielectric Spectroscopy
The actual state of art of microwave technology permits measuring in real time and in a nondestructive way most of the parameters that are related to quality control. For instance, in the late

Physical Sensors and Techniques


sixties, microwave sensors emerged as a plausible solution for real-time, nondestructive sensing of moisture content in a variety of materials [49–51]. Moreover, in recent years, the price of microwave components has dropped drastically because of a surge in demand from the wireless telecommunications sector. This, with new developments in solid-state and planar circuit technologies, provides an opportunity to develop reasonably priced microwave/RF sensors. Therefore, the application of microwave technologies to food quality control is a growing interest for the industry. Until recently, the interest of the food industry in microwave applications had been fi xed mainly in dielectric heating. These applications appeared in the years following the end of the Second World War, but the development of microwaves stopped due to technological reasons and the high cost of investment. At the beginning of the 1980s, the possibilities of microwave applications and their considerable advantages were recognized, and microwave ovens become more popular. This increase in the use of domestic microwave ovens gave rise to a reduction in the cost of the relatively high-power magnetron. However, the cost of these elements increases exponentially when the power is on an industrial scale [35]. Presently, domestic microwave ovens are universally accepted by consumers, and other microwave heating applications are widely used in industry; baking, drying, blanching, thawing, tempering, and packaging are the most important. Therefore, considerable experience has now been accumulated in this field and can be used in the design of sensor systems based on microwaves. These sensors are viable and affordable for online control in food industrial processes. Dielectric spectroscopy measures the dielectric properties (see Section 12.4) of a “material” as a function of frequency; this term usually applies to the range of microwave frequencies, sometimes extended to high RF. Dielectric spectroscopy is considered to be a very useful tool in food quality determinations, because, as will be explained in Sections 12.4 and 12.5, dielectric properties of biological tissues are closely correlated with water content and the aggregation state of it. Furthermore, the dielectric properties depend not only on water binding in foods but also on its composition. The interplay between molecular composition, presence of ions, electrical charges on proteins, and pH variations leads to a complex dielectric spectrum regulated by several phenomena. Dielectric properties are also related to structure, and the structural organization and composition of a muscle makes it a highly anisotropic dielectric material. This dielectric anisotropy was modeled by Felbacq et al. [52] to provide insight into microwave–muscle interactions. It tends to decrease during ageing or process-related cellular degradation. The main theoretical aspects of microwaves are treated in Section 12.4. In Section 12.5 some interesting applications of microwave technology in quality control are cited.

12.3.6 Advantages and Benefits of Microwave Methods
A very important benefit of microwave sensing is that the bulk property (i.e., moisture or density) is determined, in contrast to surface determination provided, for example, with infrared (IR) or NIR techniques. This is particularly important in monitoring operations, for example, drying, where moisture gradients exist in the material; variations in moisture can exist within a few microns of the surface, but their effects are substantially reduced or insignificant at microwave frequencies. Another decided advantage is logistical flexibility in installation. With a wide variety of sensors from which to choose, placement can be on conveyors or in hoppers, shakers, pipes,


Handbook of Seafood and Seafood Products Analysis

chutes, and so on. Installation is generally minimally intrusive. Moreover, results can be obtained almost in real time, because the measurement time ranges from a few milliseconds to one second. A further advantage is that microwave radiation is noncontaminating and environmentally safe at power levels typically used for online sensing. Human exposure is usually less than that from common consumer electronic devices such as cordless and cellular telephones. Finally, microwave sensors are insensitive to environmental conditions such as dust, color, or ambient light, vapors, and machine vibrations, in contrast to IR and NIR techniques.


Overview of Microwave Theory

Microwaves are a common designation for electromagnetic waves at frequencies between 300 MHz and 300 GHz. These waves travel through the free space with a given energy (E) and propagation parameters, which are mainly magnitude (A) and phase (q). When they find a different “dielectric material” (in this case, food), one part of the radiation is refracted and another one passes through it (see Figure 12.1). The amount of radiation refracted or transmitted by food as well as its new propagation parameters are governed by the dielectric properties of the material. Therefore, the measurement of these properties allows both the characterization of food and the control of the process (see Figure 12.1). In the communications argot, “materials” are usually divided into the categories of conductors, insulators, and dielectrics. “Dielectric materials” cover the whole spectrum of anything between conductors and insulators. Therefore, dielectrics can consist of polar molecules or nonpolar molecules, or very often both. According to this classification, foods are “dielectric materials” (or really an addition of dielectric materials) susceptible to be defined by their dielectric properties. Complex permittivity (e r) (Equation 12.1) is the dielectric property that describes food behavior under an electromagnetic field [53].

E1, A1, θ1

Material permittivity εr1 = ε΄ –j.ε˝ r1 r1 Natural or industrial process

E2, A2, θ2

, θ3 E 3, A 3

Product characterization

E1, A1, θ1

, θ5 E 5, A 5

Modified material permittivity εr2 = ε΄ –j.ε˝ r2 r2

E4, A4, θ4 Processes control (or monitoring)

Figure 12.1 Scheme of the possibilities of the measurement of dielectric properties in quality control applications.

Physical Sensors and Techniques


The real part of complex permittivity is called the dielectric constant (e′), and the imaginary ′′ part is called the effective loss factor ( ε eff ). The subscript r indicates that values are related to vacuum, and the variable is therefore dimensionless:
′′ εr = ε ′ − j ε eff


Under a microwave field, the charges of certain food components (water, salts, etc.) try to displace from their equilibrium positions to orientate themselves following the field, storing microwave energy that is released when the applied field stops. This behavior is called polarization; e′ denotes the material’s ability to store this electromagnetic energy (or the ability to be polarized). Only a ′′ perfect dielectric can store and release wave energy without absorbing it. The parameter ε eff is related to absorption and dissipation of the electric energy from the field. Such energy absorptions are caused by different factors that depend on structure, composition, and measurement ′′ frequency, thus ε eff can be expressed by Equation 12.2 [53]: ε ′eff = ε ′′ + ε ′′ + ε ′′ + ε ′′ + σ/ε o ω e a MW d (12.2)

In this equation the last term is called ionic losses. The symbols s, e o, and w refer to material conductivity, vacuum permittivity, and angular frequency, respectively. Subscripts d, MW, e, and a indicate dipolar, Maxwell–Wagner, and electronic and atomic losses, respectively. The different contributing mechanisms to the loss factor of a moist material are schematically represented in Figure 12.2.

ε˝ i + – + + – MW + – + – dw

+ + – – + –

a da e log f (Hz) 3E14 V nm UV

1.8E10 3E8 3E11 Radio frequency Microwaves IR AC L–M–K VHF dm wave cm mm μm

Figure 12.2 Schematic representation of the different effects that contribute to effective loss factor (e″ff ) along the electromagnetic spectrum (logarithmic scale). i, ionic losses; MW, e Maxwell–Wagner effect; dw, dipolar losses of water; da, dipolar losses of isopropyl alcohol; a, atomic losses; e, electronic losses.


Handbook of Seafood and Seafood Products Analysis

Under a microwave field, molecules with an asymmetric charge distribution (permanent dipoles such as water) rotate trying to align themselves with the electric field, storing part of the wave energy [54]. The dipolar contribution to total losses is one of the most important at microwave frequencies due to the fact that water is an abundant and common component in foods. Otherwise, as frequency is increased (the highest microwave frequencies and above them), the electromagnetic field can affect smaller particles, inducing dipoles even in neutral molecules (atomic polarization) and neutral atoms (electronic polarization). Atomic and electronic losses have behavior similar to that of permanent dipolar losses. At RF and the lowest microwave frequencies, charged atoms and molecules (ions) are affected by the field. Such ions move trying to follow the changes in the electric field. In case ions do not find any impediment (aqueous solutions, conducting materials), ionic conductivity gives rise to an increment in effective losses. At these frequencies, the ionic losses are the main contributors to the loss factor (supposing ions to be present in the material). Foods are complex systems and usually present conducting regions surrounded by nonconducting regions, for example, foods with a cellular structure have cytoplasm (conducting region) surrounded by the membrane (nonconducting region). In these cases, ions are trapped by the interfaces (nonconducting regions) and, as the ion movement is limited, the charges are accumulated, increasing the overall capacitance of the food [55] and the dielectric constant (Maxwell– Wagner Polarization). This phenomenon is produced at low frequencies at which the charges have enough time to accumulate at the borders of the conducting regions. The Maxwell–Wagner losses curve vs. frequency has the same shape as the dipolar losses curve (see Figure 12.2). At higher frequencies, the charges do not have enough time to accumulate and the polarization of the conducting region does not occur. At frequencies above the Maxwell– Wagner relaxation frequency, both ionic losses and the Maxwell–Wagner effect are difficult to distinguish due to the fact that both effects exhibit the same slope (1/f ). Foods are multicomponent and multiphase systems; therefore, more than one mechanism contributes to the combined effects. Figure 12.3 shows different shape variations in effective loss factor curves vs. frequency for the case of combined dipolar and ionic losses. Type_0 represents a typical pure dipolar loss factor curve (without ionic contribution), s increases between type_0 and type_4 curves (the corresponding ionic contribution is marked in discontinuous trace), ″ ε d max is the highest value of dipolar losses, and relaxation frequency is the inverse of relaxation time [53,16]. In general, foods are dielectric materials with high losses and, under a microwave field, they can absorb part of the wave energy. The power that can be dissipated in a given material volume ′′ (Pv) is related to ε eff by Equation 12.3, in which E is the electric field strength [53]: Pv = 2π f ε0 ε eff ·E 2 (W/cm3 ) (12.3)

The high-power dissipation in foods has given rise to numerous high-power heating applications that have been developed since the fi fties. The interest in improving heating applications has provided a great deal of knowledge on dielectric properties and wave parameter measurements. Th is detailed knowledge has been very useful in further research into new lowpower online sensors, which relate these properties or parameters to process variables of food industry.

Physical Sensors and Techniques
ε˝ 4


3 σ/ωε0 + – + + – 1 εd ˝ 0 log ( f ) 2 + – + –

+ –

Figure 12.3 Influence of salt content in systems with different proportions of dipoles (water) and ions (salts) in the shape of effective loss factor curve. Salt content increases in curves from 0 (water) and 4 (saturation). (Adapted from De los Reyes, R. et al., Medida de propiedades dieléctricas en alimentos y su aplicación en el control de calidad de productos y procesos, ProQuest (Ed.), 2007.)

12.5 Applications of Microwave Technology in the Assessment or the Control of Processes
The applications of electromagnetic radiation in the microwave band are varied and cover broad fields, from the radar [56] and radiometry [57], to medical applications, such as the diagnosis of breast cancer [58] and other image applications. In addition, industrial applications have been developed, such as rubber vulcanization [59], soils, wood, and animal products disinfection [60–62], or food processing [63,64]. They are so many that some frequency bands have been reserved especially for industrial, scientific, and medical applications (ISM). These frequencies are detailed in Table 12.1. Microwave applications that are better known within the food industry are related to energy absorption and, therefore, are made at high power and usually at 2.45 GHz, which is the frequency often reserved in Europe for industrial applications. These applications are mainly used for heating, pasteurization, sterilization, dehydration, thawing, and scalding [65–67]. Recently, the application of microwaves in combination with warm air in drying of foods has been also studied, either during the whole drying process or in part of it [68,69]. Within this field, applications to the drying of fruits and vegetables are notable for their interest to the food industry [70,71]. However, as noted above, the development of the technology that brings this large number of applications has allowed the onslaught of new applications such as the assessment or the control of


Handbook of Seafood and Seafood Products Analysis Table 12.1 Frequency Bands Reserved for Industrial, Scientific, and Medical Applications (ISM)
Frequency (MHz) 433.92 ± 8 915 ± 13 2,450 ± 50 5,800 ± 75 24,125 ± 125 Wave Longitude (cm) 69.14 32.75 12.24 5.17 1.36

processes by microwaves in a nondestructive way (MNDT or MNDE) which is receiving a growing interest in the food industry. In these applications, very low power is used to avoid permanent effects in foods. As a result of that, the methods for determining dielectric properties have experienced a spectacular expansion within the field of the analysis of materials by microwaves, which until relatively recently, was exclusively associated with the design of electronic equipment. As has been explained before, the measurement of the dielectric properties can provide important information during industrial processes due to the relationships between food properties and electromagnetic parameters. This is because low-power microwaves change their parameters (amplitude, phase) according to the food properties, and this change can be measured in real time. This is the basic principle on which food-quality microwave sensors are based. Complex permittivity can be correlated with structural, physical, and chemical properties such as humidity, soluble solids content, porosity, characteristics of solid matrix, and density [16]. The changes in these properties are usually related with the treatments applied to foods throughout the industrial process; for instance, water losses in drying processes [72] or salt losses in desalting processes [14,15]. In addition, the structural changes produced in macromolecules, such as protein denaturalization, can occur during processing, leading to a modification of the dielectric properties [73]. For all these reasons, the measurement of dielectric properties can be used as a tool for online food process control. This section provides an overview of the most important microwave applications as techniques in food control.


Determination of Moisture Content

Water represents the main component of foods influenced by microwave energy and, therefore, nowadays most methods of determining moisture content are based on electrical properties. The determination of moisture based on electromagnetic parameters has been used in agriculture for at least 90 years and has been in common use for 50 years [12,74,75]. Diverse studies have been carried out relating the dielectric constant and loss factor with moisture in foods [76,74]. Further researches in this field have occurred during recent years. Trabelsi and Nelson [77] studied a method of moisture sensing in grains and seeds by measuring their dielectric properties. The reliability of the method was tested for soybean, corn, wheat, sorghum, and barley. The frequency used was 7 GHz with the free space technique. In the same year, the authors used the same technique at 2–18 GHz to determine the dielectric properties of cereal grains and oilseeds in order to predict the moisture content by microwave measurements [78]. This article presents a unified

Physical Sensors and Techniques


grain moisture algorithm, based on measurements of the real part of the complex permittivity of grain at 149 MHz using the transmission line method. Trabelsi and Nelson [79] reported the moisture in unshelled and shelled peanuts using the free space method at a frequency of 8 GHz. In 2005, Joshi [80] reported a technique for online, time domain, nondestructive microwave aquametry (US Patent numbers 6,204,670 and 6,407,555); this technique was used for determining moisture levels in substances such as seeds, soil, tissue paper, and milk powder. Plaza-González et al. [81] have published a report about a microwave sensor intended for online measurements of paper moisture. Since most efforts have been directed to the moisture determination of different materials, commercial meters for online moisture measurements have already been developed. These moisture meters are based on automatic online calculations of the reflected wave and dielectric permittivity, yielding physicochemical properties, such as moisture, chemical composition, and density, without affecting the product. For instance, Keam Holdem® Industry (Auckland, New Zealand) provides online moisture testing and analyzing systems. This manufacturer provides devices for measuring moisture in processed cheese, moisture and salt in butter, moisture and density in dried lumber and whole kernel grain, and fat-to-lean ratio in pork middles. A microwave moisture meter has also been developed for continuous control of moisture in grains, sugar, and dry milk in technological processes [82]. A consortium of companies from different countries, Microradar®, produces a commercial microwave moisture meter for measuring moisture in fluids, solids, and bulk materials based on this method. The enterprise KDC Technology Corporation (www.kdctech.com) provides microwave sensors for monitoring industrial processes and quality control. KDC sensors work in a wide range of applications such as monitoring moisture and density of manufactured wood and wood-based products, construction, and agricultural and processed food products. Patented contact (MDA1000) and noncontact (MMA-2000) sensors are used for online, continuous process monitoring of solids, particulates, and liquids or for in situ nondestructive testing/inspection. Another interesting application for online moisture measurement is a sensor for green tea developed by Okamura and Tsukamoto [72], which can measure moisture as high as 160%–300% on dry basis by use of microwaves at 3 GHz with a microstripline (Figure 12.4). A Guided Microwave Spectrometer (Thermo Electron Corporation, Waltham, MA) has been developed for online measurements of multiphase products. This guide is used to measure
Microwave source Receiver Microstripline Electric field

Tea leaves

Figure 12.4 Schema of a microstripline used for tea leaves moisture measurement. (Adapted from Okamura, S. and Tsukamoto, S., New sensor for high moist leaves in green tea production, in Proceedings of ISEMA 2005, Kupfer, K. (Ed.), MFPA an der Bauhaus-Universität Weimar, Weimar, Germany, 2005, 340–346.)


Handbook of Seafood and Seafood Products Analysis

moisture in raw materials such as corn, rice, soybeans, and in processed materials such as tomato paste and ground meat. It can also measure content of soluble solids, pH, viscosity, and acidity in orange juice, soft drinks, mayonnaise, and tomato products; fat in ground meats, peanut butter, and milk and other dairy products; salt in mashed potatoes and most vegetable products and, lastly, alcohol in beverages.


Freshness and Salting/Desalting Process Quality Control of Fish and Seafood, by Microwaves: Methods and Equipments

The dielectric properties of fish products have been measured by different authors [83–86]; nevertheless, the electromagnetic determination of quality parameters in muscle tissues is still a complex challenge due to its complex matrix, heterogeneous composition, and anisotropic disposition. It is important to point out that the limitation of most dielectric probes is the volume of the sample that interacts with the field. The volume has to be representative of the whole piece of fish, due to the fact that the electromagnetic parameters in this kind of tissue vary in a heterogeneous way. It has been reported that it is possible to predict the fat composition in fish using electromagnetic measurements [87]; this is because it is clearly related to the water content of the product, so that if one is known the other can be determined; this is the knowledge base of the “Torrymeter” mentioned later. Moreover, this author [88,89] has studied the determination of added water in fish using microwave dielectric spectra measurements. Measurements of dielectric properties have been tested and used during almost 40 years for quality grading and remaining shelf life determination of various fish. These investigations have been mainly focused on freshness and self-life evaluation and detecting fishes previously thawed. However, a number of research studies have been carried out to control or monitor the processing of fish products. In this field, De los Reyes et al. [14,15] verified the viability of an online measurement system using low-power microwaves to determine the desalting point of salted cod. Dielectric spectroscopy was performed on cod samples at different desalting stages and on its desalting solutions in order to find the appropriate measurement frequency. Figure 12.5 shows the dielectric spectra (e′ and e″) from cod loin samples (2 cm/side parallelepipeds) at desalting times (t) yielding from 15 min to 48 h. Optimum frequencies were selected from the spectrum, and dielectric properties data were related to other physicochemical properties of cod samples measured at the same desalting stages, such as moisture and salt content. Good correlations were found between salt content in cod samples and their loss factor values at 200 and 300 MHz. These results indicated the viability of developing an online control system for a cod desalting process. Polarimetric measurements, that is, with a linearly polarized electric field, make it possible to evaluate anisotropy. This method has been applied to assess fish freshness [90]. This is because, after death, muscle is not able to use energy by the respiratory system. Postmortem changes lead to a temporary rigidity of muscles, decreasing the water-holding capacity [91]. The level of glycogen stored in the animal at the time of slaughter affects the texture of the future marketed meat. For all these reasons, during rigor mortis the dielectric properties are expected to change. The “Intellectron Fishtester” [92], the “Torrymeter” (Distell.com), and the “RT-Freshtester” (RT rafagnatækni), represent instruments with increasing degrees of sophistication invented for fish-quality evaluation. Readings from all these instruments are based in the reflected dielectric properties of fish, because they decrease with storage time, almost following a straight line. Based on these rapid and nondestructive measurements, the “RT-Freshtester” allows automatic grading of 60–70 fish per min. Nevertheless, electrical properties of fish are not directly responsible for

Physical Sensors and Techniques


ε΄, ε˝ 800 700 600 500 400 300 200 100 0

0.2 GHz 0.3 GHz

0.9 GHz 1.8 GHz 2.45 GHz

10 GHz

ε˝ t



1E + 08

1E + 09 Frequency

1E + 10

Figure 12.5 Dielectric spectra from cod samples at desalting times (t) yielding from 15 min to 48 h. The arrows beside t indicate the growth of the desalting time. Frequency axis is in the logarithmic scale, and broken lines mark the selected frequencies (0.2, 0.3, 0.9, 1.8, 2.45, and 10 GHz). (Adapted from De los Reyes, R. et al., Dielectric spectroscopy studies of “salted cod-water” systems during the desalting process, in Proceedings of the IMPI’s 40th Annual Symposium, 2006.)

sensory spoilage and it is, therefore, to be expected that numerous factors influence the relationship between such measurements and seafood spoilage. In fact, these instruments need calibration depending on the season and fish handling procedures, and they are unsuitable for grading frozen–thawed fish, partially frozen, that is, superchilled fish, fish chilled in refrigerated seawater, or for fish fillets. This and the high cost of the instruments limit their practical use in the seafood sector for freshness evaluation. However, electrical measurements can also be used to test if fish was previously frozen [2]. Kent et al. [93] studied the effect of storage time and temperature on the dielectric properties of thawed–frozen cod (Gadus morhua) in order to estimate the quality of this product. The same year, Kent et al. [94] developed a combination of dielectric spectroscopy and multivariate analysis to determine the quality of chilled Baltic cod (Gadus morhua). These researches yielded a prototype developed by SEQUID [95,96] for measuring and analyzing the quality of different seafood. The SEQUID project concentrated on the measurement of the dielectric properties of fish tissue as a function of time both in frozen and chilled storage. This project has shown that it is possible, using a combination of time domain reflectometry and multivariate analysis, to predict certain quality-related variables, both sensory and biochemical, with an accuracy comparable to existing methods. Kent et al. [97] have also reported a way to determine the quality of frozen hake (Merluccius capensis) by analyzing its changes in microwave dielectric properties. The above mentioned “Torrymeter” has been successfully improved as a sensor for measuring fish freshness as a result of these investigations. In further investigations, the SEQUID project has shown that it is possible to predict certain quality-related variables (with comparable accuracy to existing methods) using a combination of time-domain reflectometry at microwave and RF frequencies and multivariate analysis [98].


Handbook of Seafood and Seafood Products Analysis



It is possible to implant reliable online sensors in fish industry both for determining the freshness as well as for monitoring processes (salting/desalting, thawing, etc.). The future of control in fish processing is the analysis of the physical and chemical properties using the dielectric signal at different frequencies, using multisensors. Multivariable knowledge of the process yields a modeling of the product.

1. Moltó, E. Investigación sobre sensores electrónicos para la medida objetiva de la calidad postcosecha. Agrícola Vergel, 219, 193–198 (2000). 2. Dalgaard, P. Freshness, quality, and safety in seafoods. Flair-Flow Europe Technical Manual F-FE 380A/00. The National Food Centre, Dublin, Ireland (2000). 3. Bisogni, C.A., Ryan, G.J., and Regenstein, J.M. What is fish quality? Can we incorporate consumer perceptions? In: Seafood Quality Determination, Elsevier Science Publishers, BV, Amsterdam, the Netherlands, pp. 547–563 (1987). 4. Botta, J.R. Evaluation of Seafood Freshness Quality, VCH Publishers Inc, New York, (1995). 5. Consumer Reports. America’s fish: Fair or foul? Consumer Reports. Fe. 25–31 (2001). 6. Olafsdottir, G., Nesvadbab, P., Di Natalec, C., Careched, M., Oehlenschlägere, J., Tryggvadóttira, S.V., Schubringe, R. et al. Multisensor for fish quality determination. Trends Food Sci. Technol., 15(2), 86–93 (2004). 7. Simal, S., Benedito, J., Clemente, G., Femenia, A., Rosselló, C. Ultrasonic determination of the composition of a meat-based product. J. Food Eng., 58, 253–257 (2003). 8. Liu, Y., Ying, Y., Ouyang, A., and Li, Y. Measurement of internal quality in chicken eggs using visible transmittance spectroscopy technology. Food Control., 18, 18–22 (2007). 9. Bittner, D.R. and Norris, K.H. Optical properties of selected fruits vs maturity. Trans. ASAE., 11(4), 534–536 (1968). 10. Chevalier, D., Ossart, F., and Ghommidh, C. Development of a non-destructive salt moisture measurement method in salmon (Salmo salar) fillets using impedance technology. Food control, 17, 342– 347 (2006). 11. Clerjon, S., Daudin, J.D., and Damez, J.L. Water activity and dielectric properties of gels in the frequency range 200 MHz–6 GHz. Food Chemistry, 82, 87–97 (2003). 12. Nelson, S.O. Use of electrical properties for grain-moisture measurement. J. Microwave Power, 12(1), 67–72 (1977). 13. Nelson, S.O. Dielectric properties measurement techniques and applications. ASAE Annual Int. Meeting, Orlando, paper 98–3067 (1998). 14. De los Reyes, R., Haas, C., Andrés, A. Changes in the dielectric properties of “salted cod–water” system during the desalting process and their relation with other physical properties. In Proceedings of EFFOST, Valencia, Spain (2005). 15. De los Reyes, R., Haas, C., Andrés, A., Fito, P., and De los Reyes, E. Dielectric spectroscopy studies of “Salted Cod–Water” systems during the desalting process. In Proceedings of the IMPI’s 40th Annual Symposium (2006). 16. De los Reyes, R., Fito, P., and De los Reyes E. Medida de propiedades dieléctricas en alimentos y su aplicación en el control de calidad de productos y procesos. ed., ProQuest (2007). 17. Jayasooriya, S.D., Bhandari, B.R., Torley, P., and D’Arcy, B.R. Effect of high power ultrasound waves on properties of meat: a review. Int. J. Food Prop. 7, 2, 301–319 (2004). 18. Got, F., Culioli, J., Berge, P., Vignon, X., Astruc, T., Quideau, J.M., and Lethiecq, M. Effects of high-intensity high frequency ultrasound on ageing rate, ultrastructure and some physicochemical properties of beef. Meat Sci. 51, 35–42 (1999).

Physical Sensors and Techniques


19. Knorr, D., Zenker, M., Heinz, V., and Lee, D.-U. Applications and potential of ultrasonics in food processing. Trends Food Sci. Technol., 15, 261–266 (2004). 20. Dolatowski, Z.J., Stadnik, J., and Stasiak, D. Applications of ultrasound in food technology. Acta Sci. Pol., Technol. Aliment., 6(3), 89–99 (2007). 21. Suvanich, V., Ghaedian, R., Chanamai, R., Decker, E.A., and Mcclements, D.J. Prediction of proximate fish composition from ultrasonic properties: Catfish, cod, flounder, mackerel and salmon. J. Food Sci., 63(6), 966–968 (1998). 22. Dove, E.L. Notes on Ultrasound—Echocardiography. 51:060 Fundamentals of Bioimaging (2003). 23. Chen, H. and Marks, B.P. Evaluation previous thermal treatment of chicken patties by visible/nearinfrared spectroscopy. J. Food Sci., 62, 753–756, 780 (1997). 24. Chen, H. and Marks, B.P. Visible/near-infrared spectroscopy for physical characteristics of cooked chicken patties. J. Food Sci., 63, 279–282 (1998). 25. McElhinney, J., Downey, G., and Fearn, T. Chemometric processing of visible and near infrared reflectance spectra for species identification in selected raw homogenized meats. J. Near Infrared Spec., 7, 145–154 (1999). 26. Heia, K., Sigernes, F., Nilsen, H., Oehlenschläger, J., Schubring, R., Borderias, J., Nilsson, K., Jørgensen, B.M., and Nesvadba, P. Evaluation of fish freshness by physical measurement techniques. In: Methods to determine the freshness of fish in research and industry. Proceedings of the final meeting of the concerted action “evaluation of fish freshness” AIR3CT94 2283, Institut International du Froid, Paris, France, pp. 347–354 (1998). 27. Osborne, B.G. Near-infrared spectroscopy in food analysis. In: Encyclopedia of Analytical Chemistry. ed., Robert A. Meyers. John Wiley & Sons Ltd, Chichester, U.K. (2000). 28. Uddin, M., Ishizaki, S., Okazaki, E., and Tanaka, M. Near-infrared reflectance spectroscopy for determining end-point temperature of heated fish and shellfish meats. J. Sci. Food Agri., 82(3), 286– 292 (2002). 29. Wold, J.P., Johansen, I.R., Haugholt, K.H., Tschudi, J., Thielemann, J., Segtnan, V.H., Narum, B., and Wold, E. Non-contact transflectance near infrared imaging for representative on-line sampling of dried salted coalfish (bacalao). J. Near Infrared Spec., 14, 59–66 (2006). 30. Zhang, H. and Lee, T. Rapid near-infrared spectroscopic method for the determination of free fatty acid in fish and its application in fish quality assessment. J. Agr. Food Chem., 45, 3515–3521 (1997). 31. Huang, H., Yu, H., Xu, H., and Ying, Y. Near infrared spectroscopy for on/in-line monitoring of quality in foods and beverages: A review. J. Food Eng., 87, 303–313 (2008). 32. Benson, I. B. Near infrared absorption technology for analysing food. In: Food Authenticity and Traceability. ed., Lees, M. Woodhead Publishing, Cambridge, U.K. (2003). 33. Karoui, R., Lefur, B., Grondin, C., Thomas, E., Demeulemester, C., De Baerdemaeker, J., and Guillard, A. Mid-infrared spectroscopy as a new tool for the evaluation of fish freshness. Int. J. Food Sci. Technol., 42(1), 57–64 (2007). 34. Marquardt, B. Wold, J.P. Raman analysis of fish: A potential method for rapid quality screening. Lebensmittel-Wissenschaft + Technologie, 37, 1–8 (2004). 35. Fito, P.J., Ortolá, M.D., De los Reyes, R., Fito, P., and De los Reyes, E. Control of citus surface drying by image analysis of infrared thermography. J. Food Eng., 61, 287–290 (2004). 36. Jacobsen, S. and Pedersen, W. Noncontact determination of cold-water prawn ice-glaze content using radiometry. Lebensmittel - Wissenschaft + Technologie, 30(6), 578–584 (1997). 37. Dittmar, M. Reliability and variability of bio-impedance measures in normal adults: Effects of age, gender, and body mass. Am. J. Phys. Anthropol., 122, 361–370 (2003). 38. Barbosa-Silva, M., Barros, A., Post, C., Waitzberg, D., and Heymsfield, S. Can bioelectrical impedance analysis identify malnutrition in preoperative nutrition assessment? Nutrition, 19, 422–426 (2003); Wirth, R. and Miklis, P. Bioelectric impedance analysis in the diagnosis of malnutrition. Z. Gerontol. Geriatr. 38, 315–321 (2005). 39. Bosworth, B.G. and Wolters, W.R. Evaluation of bioelectric impedance to predict carcass yield, carcass composition, and fi llet composition in farm-raised catfish. J. World Aquacult. Soc., 32, 72–78 (2001).


Handbook of Seafood and Seafood Products Analysis

40. Duncan, M., Craig, S.R., Lunger, A.N., Kuhn, D.D., Salze, G., and McLean, E. Bio-impedance assessment of body composition in cobia Rachycentron canadum (L. 1766). Aquaculture, 271, 432– 438 (2007). 41. Barbosa-Silva, M. and Barros, A. Bioelectric impedance and individual characteristics as prognostic factors for post-operative complications. Clin. Nutr., 24, 830–838 (2005). 42. Cole, K.S. Electric phase angle of cell membranes. J. Gen. Physiol., 15, 641–649 (1932). (Full Text via CrossRef.) 43. Damez, J.-L., Clerjon, S., Abouelkaram, S., and Lepetit, J. Dielectric behavior of beef meat in the 1 kHz to 1500 kHz range. Simulation with the Fricke/Cole–Cole Model. Meat Sci., doi: 10.1016/j. meatsci.2007.04.028 (2007). 44. Yu, T.H., Liu, J., and Zhou, Y.X. Using electrical impedance detection to evaluate the viability of biomaterials subject to freezing or thermal injury. Anal. Bioanal. Chem., 378, 1793–1800 (2004). 45. Vidačeka, S., Medića, H., Botka-Petrakb, K., Nežakc, J., and Petraka, T. Bioelectrical impedance analysis of frozen sea bass (Dicentrarchus labrax). J. Food Eng., 88, 263–271 (2008). 46. Martisen, O.G., Grimnes, S., and Mirtaheri, P. Noninvasive measurements of post-mortem changes in dielectric properties of haddock muscle–A pilot study. J. Food Eng., 43, 189–192 (2000). 47. Hennings, C. The “Interelectron Fish Tester V”–A new electronic method and device for the rapid measurement of the degree of freshness of “wet” fish. In: The Technology of Fish Utilization, R. Kreutzer, ed., Fishing News Ltd., London, U.K., pp. 154–157 (1964). 48. Thomas, B.J. Ward, L.C., and Cornish, B.H. Bioimpedance spectrometry in the determination of body water compartments: Accuracy and clinical significance. Appl. Radiat. Isotopes, 49, 447–455 (1998). 49. Taylor, H.B. Microwave moisture measurements. Ind. Electron., 3, 66–70 (1965). 50. Kraszewski, A. Microwave Aquametry, IEEE Press, Piscataway, NJ (1996). 51. Busker, L.H. Microwave moisture measurement, I & CS, 41, 89–92 (1968). 52. Felbacq, D., Clerjon, S., Damez, J.L., and Zolla, F. Modeling microwave electromagnetic field absorption in muscle tissues. Eur. Phys. J.–Appl. Phys., 19(1), 25–27 (2002). 53. Metaxas, A.C. and Meredith, R.J. Industrial Microwave Heating, IEE Power Engineering series 4, Peter Peregrinus Ltd., London, U.K. (1993). 54. Datta, A.K. and Anantheswaran, R.C. Handbook of Microwave Technology for Food Applications, eds., Datta, A.K. and Anantheswaran, R.C., Series of Food Science and Technology, Marcel Dekker, New York (2001). 55. Hewlett-Packard. Basic of measuring the dielectric properties of materials. Application note 1217–1. Hewlett-Packard Company, Palo Alto, CA (1992). 56. De los Reyes, E., Imágenes radar para el estudio de superficies agrícolas, 113, Dcbre. 1981, pp. 111–116 (1981). 57. Sempere, L. Radiometría interferométrica de microondas para la monitorización del contenido en humedad del suelo. Tesis doctoral de la Universidad Politécnica de Valencia. Director Elías De los Reyes (1999). 58. Fear, E.C., Hagness, S.C., Meaney, P.M., Okoniewski, M., and Stuchly, M.A. Enhancing Breast tumor detection with Near-Field Imaging. IEEE Microwave Magazine, 3(1), 48–56 (2002). 59. Catalá-Civera, J.M., Sánchez-Hernández, D., and y de los Reyes, E. Rubber vulcanisation for the footwear industry using microwave energy in a pressure-aided cavity. International Conference on Microwave Chemistry, Prague, Czech Republic (1998). 60. Plaza, P.J., Zona, A.T., Sanchís, R., Balbastre, J.V., Martínez, A., Muñoz, E.M., Gordillo, J., and de los Reyes, E. Microwave disinfestation of bulk timber. J. Microwave Power E.E., 41(3), 21–36 (2007). 61. Zona, A.T., Balbastre, J.V., Nuno, L., de los Reyes, E., Calderon, O., Perez, E., and Vivancos, M.V. Procedure to exterminate woodworm in wood timbers by microwave-power application. In Proceedings of Global Congress on Microwave Energy Applications GCMEA 2008 MAJIC 1st (2008). 62. WO/2005/009122. Microwave method of controlling mites In A Food Product Of Animal Origin (2005).

Physical Sensors and Techniques


63. Catalá-Civera, J.M. and de los Reyes, E. Enzyme inactivation analysis for industrial blanching applications: Comparison of microwave, conventional and combination heat treatments on mushroom polyphenoloxidase activity. ed., Acs., J. Agric. Food Chem., 47, 4506–4511 (1999) (ISSN 0021-8561). 64. Andrés, A., Bilbao, C., and Fito, P. Drying kinetics of apple cylinders under combined hot air-microwave dehydration. J. Food Eng., 63, 71–78 (2004). 65. Schiffmann, R.F. Microwave processes for the food industry. In: Handbook of Microwave Technology for Food Applications, Datta, A.K., and Anantheswaran, R.C., Cap. 9, 299–337. Marcel Dekker, Inc., New York (2001). 66. Anon, G. Tempers frozen fish blocks inside a cold storage warehouse, Quick frozen foods, 43(11), 64 (1981). 67. Ohlsson, T. Industrial uses of dielectric properties of foods. In: Physical Properties of Foods. 2. COST 90bis final seminar proceedings. eds., Jowitt, R., Escher, F., Kent, M., McKenna, B., and Roques, M., Elsevier Applied Science. London, U.K., pp. 199–211 (1987). 68. Catalá-Civera, J.M. Combined Microwave and air drying of apple (var. Granny Smith). In Proceedings of European Research towards Safer and Better Food, 74, 383–387 (1998). 69. Martín, M.E., Fito, P., Martínez-Navarrete, N., and Chiralt, A. Combined air-microwave drying of fruit as affected by vacuum impregnation treatments. In Proceedings of the 6th Conference of Food Engineering (CoFE’99), 465–470 (1999). 70. Bilbao, C, Albors, A, Gras, M.L., Andrés, A., and Fito, P. Shrinkage during apple tissue air-drying: macro and microstructural changes. Proceedings of the 12th International Drying Symposium IDS2000, Paper No. 330 (2000). 71. Sharma, G.P. and Prasad, S. Drying of garlic (Allium sativum) cloves by microwave-hot air combination. J. Food Eng., 50(2), 99–105 (2001). 72. Okamura, S., Tsukamoto, S. New sensor for high moist leaves in green tea production. In Proceedings of ISEMA 2005, ed., Kupfer, K., pp. 340–346. MFPA an der Bauhaus-Universität Weimar, Weimar, Germany (2005). 73. Bircan, C. and Barringer, S.A. Determination of protein denaturation of muscle foods using dielectric properties, J. Food Sci., 67(1), 202–205 (2002). 74. Nelson, S.O. Dielectric properties of agricultural products–Measurements and applications. Digest of Literature on Dielectrics, ed. A. de Reggie. IEEE Trans. Electr. Insul., 26(5), 845–869 (1991). 75. Nelson, S.O. Dielectric properties measurement techniques and applications. Trans. ASAE, 42(2), 523–529 (1999). 76. Nelson, S.O. Radio frequency and microwave dielectric properties of shelled corn. J. Microwave Power, 13, 213–218 (1978). 77. Trabelsi, S. and Nelson, S.O. Universal Microwave Moisture Sensor. In Proceedings of ISEMA 2005, ed., Kupfer, K., pp. 232–235. MFPA an der Bauhaus-Universität Weimar. May 29–June 1, Weimar, Germany (2005). 78. Trabelsi, S. and Nelson, S.O. Microwave dielectric properties of cereal grain and oilseed. In Proceedings of the American Society of Agricultural Engineers, St. Joseph, MI, Paper No. 056165 (2005). 79. Trabelsi, S. and Nelson, S.O. Microwave dielectric methods for rapid, nondestructive moisture sensing in unshelled and shelled peanuts. In Proceedings of the American Society of Agricultural Engineers, St. Joseph, MI, Paper No. 056162 (2005). 80. Joshi, K. High resolution, non-destructive and in-process time domain aquametry for FMCG and other products using microstrip sensors. In Proceedings of ISEMA 2005, ed. Kupfer, K., pp. 384–390. MFPA an der Bauhaus-Universität Weimar, Weimar, Germany (2005). 81. Plaza-González, P.J., Canós, A.J., Catalá-Civera, J.M., and Peñaranda-Foix, F. Microwave non-contact sensor for on-line moisture measurement of laminate paper. International Conference on Sensor Technologies and Applications, pp. 52–55 (2007). 82. Lisovsky, V.V. Automatic Control of Moisture in Agricultural Products by Methods of Microwave Aquametry. In Proceedings of ISEMA 2005, ed. Kupfer, K., pp. 375–383. MFPA an der BauhausUniversität Weimar. May 29–June 1, Weimar, Germany (2005).


Handbook of Seafood and Seafood Products Analysis

83. Kent, M. Microwave dielectric properties of fish meal. J. Microwave Power, 7, 109–116 (1972). 84. Kent, M. Complex permittivity of fish meal: A general discussion of temperature, density, and moisture dependence. J. Microwave Power, 12, 341–345 (1977). 85. Wu, H., Kolbe, E., Flugstad, B., Park, J.W., and Yongsawatdigul, J. Electrical properties of fish mince during multifrequency ohmic heating. J. Food Sci., 63, 1028–1032 (1988). 86. Zheng, M., Huang, Y.W., Nelson, S.O., Bartley, P., and Gates, K.W. Dielectric properties and thermal conductivity of marinated shrimp and channel catfish, J. Food Sci., 63, 668–672 (1998). 87. Kent, M. Hand-held instrument for fat/water determination in whole fish, Food Control, 1, 47–53 (1990). 88. Kent, M., MacKenzie, K., Berger, Knöchel, R., and Daschner, F. Determination of prior treatment of fish and fish products using microwave dielectric spectra. Eur. Food Res. Technol., 210, 427–433 (2000). 89. Kent, M., Knöchel, R., Daschner, F., and Berger, U. Composition of foods including added water using microwave dielectric spectra, Food Control, 12, 467–482 (2001). 90. Clerjon, S., and Damez, J.L. Microwave sensing for food structure evaluation. In Proceedings of ISEMA 2005, ed. Kupfer, K., pp. 357–364. MFPA an der Bauhaus-Universität Weimar. May 29–June 1, Weimar, Germany (2005). 91. Hullberg, A. Quality of Processed Pork. Influence of RN genotype and processing conditions, P.H.G, Swedish University of Agricultural Sciences, Uppsala, Sweden (2004). 92. Oehlenschläger, J. The intellectron fishtester VI an almostforgotten powerful tool for freshness/spoilage determination of fish on inspection level. 5th World Fish Inspection & Quality Control Congress, The Hague, the Netherlands, 20.10.–22.10 (2003) 93. Kent, M., Oehlenschlager, J., Mierke-Klemeyer, S., Knöchel, R., Daschner, F., and Schimmer, O. Estimation of the quality of frozen cod using a new instrumental method. Eur. Food Res. Technol., 219, 540–544 (2004). 94. Kent, M., Oehlenschlager, J., Mierke-Klemeyer, S., Manthey-Karl, M., Knöchel, R., Daschner, F., and Schimmer, O. A new multivariate approach to the problem of fish quality estimation. Food Chemistry, 87, 531–535 (2004). 95. Knöchel, R., Barr, U.K., Tejada, M., Nunes, M.L., Oehlenschläger, J., and Bennink, D. Newsletter of the SEQUID (Seafood Quality Identification) project. European Commission Framework Programme V Quality of Life and Management of Living Resources RTD Project QLK 1-200101643 (2004). 96. Kent, M., Knöchel, R., Daschner, F., Schimmer, O., Albrechts, C., Oehlenschläger, J., Mierke-Klemeyer, S. et al. Intangible but not Intractable: The prediction of food ‘quality’ variables using dielectric spectroscopy. In Proceedings of ISEMA 2005, ed. Kupfer, K., pp. 347–356. MFPA an der Bauhaus-Universität Weimar, Weimar, Germany (2005). 97. Kent, M., Knöchel, R., Daschner, F., Schimmer, O., Tejada, M., Huidobro, A., Nunes, L., Batista, I., Martins, A. Determination of the quality of frozen hake using its microwave dielectric properties. Int. J. Food Sci. Technol., 40, 55–65 (2005). 98. Kent, M., Knöchel, R., Daschner, F., Schimmer, O., Oehlenschläger, J., Mierke-Klemeyer, S., Kroeger, M. et al. Intangible but not intractable: The prediction of fish ‘quality’ variables using dielectric spectroscopy. IOP Publ. Meas. Sci. Technol., 18, 1029–1037 (2007).

Chapter 13

Methods for Freshness Quality and Deterioration
Yesim Ozogul Contents
13.1 Introduction ..................................................................................................................190 13.2 Sensory Methods ...........................................................................................................190 13.2.1 The European Union Freshness Grading (EU or EC Scheme) ..........................191 13.2.2 The Quality Index Method ..............................................................................191 13.2.3 The Torry Scheme ............................................................................................192 13.2.4 The Quantitative Descriptive Analysis .............................................................192 13.3 Physical Methods ..........................................................................................................194 13.3.1 Texture Analysis ...............................................................................................194 13.3.2 The Torrymeter ................................................................................................194 13.3.3 The Intellectron Fischtester VI .........................................................................195 13.3.4 The RT-Freshtester ...........................................................................................195 13.3.5 The Cosmos .....................................................................................................195 13.3.6 Electronic Nose ................................................................................................196 13.3.7 Near-Infrared Reflectance Spectroscopy...........................................................196 13.4 Chemical and Biochemical Methods .............................................................................197 13.4.1 ATP and Its Breakdown Products ....................................................................197 13.4.2 Biogenic Amines ..............................................................................................199 13.4.3 pH....................................................................................................................199 13.4.4 Total Volatile Basic Nitrogen........................................................................... 200 13.4.5 Trimethylamine .............................................................................................. 200 13.4.6 Dimethylamine ................................................................................................201


Handbook of Seafood and Seafood Products Analysis

13.4.7 Formaldehyde ..................................................................................................201 13.4.8 Lipid Oxidation Indicators ...............................................................................201 13.4.9 Lipid Hydrolysis .............................................................................................. 203 13.5 Microbiological Methods ............................................................................................. 203 References ............................................................................................................................... 204



Seafood is generally considered to be a high-protein food, low in fat and saturated fat when compared with other protein-rich animal foods. It is well known that fish oil is the major and the best source of polyunsaturated fatty acids (PUFA), called omega-3 fatty acids, especially eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). Scientific evidence suggests that omega-3 fatty acids are essential for normal growth and development throughout the life cycle and inhibit the formation of atherosclerotic plaques, prevent arrhythmias, and contribute to the prevention or amelioration of autoimmune disorders, Crohn’s disease, breast, colon and prostate cancers, rheumatoid arthritis, and particularly cardiovascular diseases [1–6]. The Nutrition Committee of the American Heart Association recommends consumption of any type of fish two or three times a week. Therefore, it is important to prevent their loss due to oxidation. Freshness is the most important attribute when assessing the quality of seafood and is of great concern in the seafood sector [7]. The quality of seafood degrades after death due to the chemical reactions [changes in protein and lipid fractions, the formation of biogenic amines and hypoxanthine (Hx)] and microbiological spoilage. As a result of these events, sensory quality of seafood deteriorates [8–13]. Seafoods are rich in PUFAs, which are susceptible to lipid oxidation. It leads to the development of off flavor and off odors in edible oils and fat-containing foods called oxidative rancidity [14,15]. Because of their high degree of unsaturation, they are less resistant to oxidation than other animal or vegetable oils [14]. This chapter summarizes methods used for evaluation of freshness and spoilage of seafood. As it is well known, no single instrumental method is reliable for assessment of the freshness and spoilage of seafood. However, chemical, microbiological methods along with sensory methods have been applied by commercial seafood companies and many researchers to ensure that the seafood products meet expectations of consumers. The current regulation of the European Community (1996) establishes principles based on sensory, chemical, and microbiological analysis to control and certify the quality warranty in the seafood field (Council Regulation No.: 2406/96). The shelf life of fish is affected by many factors such as handling, storage condition from catch to the consumers, the kind of fishing gear, bleeding, gutting methods, season, catching ground, age, and life cycle of fish affecting the nutritional quality, freshness, and safety of seafood. Therefore, estimation of remaining shelf life of fish should be made with caution [7].


Sensory Methods

Sensory evaluation is the most important method in freshness assessments. Sensory evaluation is defined as the scientific discipline used to evoke, measure, analyze, and interpret reactions to characteristics of food as perceived through the senses of sight, smell, taste, touch, and hearing [16]. Sensory evaluation provides rapid measurements of freshness of seafood. There has been a trend to standardize sensory evaluation as an objective assessment of freshness. Sensory characteristics of

Methods for Freshness Quality and Deterioration


whole fish are clearly visible to consumers, and sensory methods are still the most satisfactory way of assessing the freshness quality since they give the best idea of consumer acceptance [17]. Freshness declines as storage life progresses until the product is no longer acceptable to consumers. The most appropriate method to assess freshness is a sensory panel. There are many factors affecting the measurement of sensory quality, including the sample under investigation, the assessment method, and the judges [18]. There are two types of sensory methods, subjective and objective. Subjective assessments of fish have been used for acceptability. They are often estimated generally using adjectives such as like/dislike or good/bad, which require subjective decisions. Fish freshness is most commonly determined by objective scoring based on organoleptic changes that occur as fish storage time is extended [19]. Objective scoring schemes require trained, expert judges, but the advantage is that panels can be small. These assessors individually use their appropriate senses (sight, smell, taste, and touch) to determine the level of each sensory characteristic in the defined grade standard appropriate for the seafood examined [20]. Subjective assessment, where the response is based on the assessor’s preference for a product, can be applied in the fields like market research and product development where the reaction of consumers is needed. Assessment in quality control must be objective [16]. Assessors must be trained and have clear and descriptive guidelines and standards to get reliable results for sensory analyses [21]. Sensory methods are also fast and nondestructive unless fish is cooked.


The European Union Freshness Grading (EU or EC Scheme)

The EU Freshness Grading was introduced for the first time in the Council Regulation No. 103/76 (for fish) and 104/76 (for crustaceans) and updated by decision No. 2406/96 (for some fish, some crustaceans, and only one cephalopod, the cuttlefish). The EU scheme is commonly accepted in the EU countries for freshness grading to market fish within the Union and generally carried out by trained personnel in auctions. Whole and gutted fish are assessed in terms of appearance of skin, eyes, gills, surface slime, belly cavity, odor, and texture of fish. There are four quality levels in the EC scheme, E (extra), A (good quality), B (satisfactory quality), where E is the highest quality and below level B (called Unfit or C) is the level where fish is discarded or rejected for human consumption. However, there are still some disadvantages; trained and experienced persons are required, since the scheme uses only general parameters for iced fish [16,22,23]. It does not take differences between species into account. In addition, it does not give information on the remaining shelf life of fish. A suggestion for renewal of the EU scheme can be seen in the Multilingual Guide to EU Freshness Grades for Fishery Products [24], in which special schemes for some fish species (whitefish, dogfish, herring, and mackerel) were developed.

13.2.2 The Quality Index Method
The quality index method (QIM) has been suggested as an alternative to the EU scheme. The QIM, originally developed by the Tasmanian Food Research Unit in Australia [25] and improved further, is considered to be rapid and reliable to measure the freshness of whole fish stored in ice [21,22]. This method is based on significant sensory parameters (skin, slime, eyes, belly, odor, gills, etc.) for raw fish [25,26], and the characteristics listed on the sheet are assessed and appropriate demerit point score is recorded (from 0 to 3). The scores for all characteristics are summed to give the overall sensory score. Quality index (QI) is close to 0 for very fresh fish, whereas higher scores are obtained as the fish deteriorates [16,26]. There is a linear correlation between the sensory

and redfish by the Danish Institute for Fisheries Research. the words for describing the odor and flavor of the fish can be categorized into two groups.2). often referred to as the Torry scale.2. and the panelists trained should agree with the terms. Pandalus borealis. herring (Clupea harengus) (Table 13. redfish shrimp.3 The Torry Scheme In contrast to the QIM.4 The Quantitative Descriptive Analysis Quantitative descriptive analysis (QDA) is used by a trained sensory panel to analyze the sensory attributes of products such as texture. and is nondestructive and can be used as a tool in production planning and quality warranty work [27]. which makes it possible to predict remaining storage life on ice.192 ◾ Handbook of Seafood and Seafood Products Analysis quality expressed as a demerit score and storage life on ice. respectively) [21]. and fat fish species.dfu. In QDA. medium fat. plaice. odor. common octopus (Octopus vulgaris) [33]. Objective sensory methods are essential for quality control and estimation of shelf life of seafood. Pollachius virens. the Torry Scheme was developed at the Torry Research Station for use with expert and trained judges. Objective terms should be used rather than subjective terms. During spoilage. Recently developed QIM schemes were presented for raw gilthead sea bream (Sparus aurata) [30]. It has been widely used in its original or modified forms. In addition. sole.htm. herring. and Scopthalmus maximus. brill. and flavor. positive and negative sensory parameters based on whether fish are fresh fish or fish at the end of the storage period [37]. instrumental methods are also needed to satisfy the need for quality measurements in fish industry. and turbot (Scopthalmus rhombus. Hyldig [29] indicated that the QIM is expected to become the leading reference method for the assessment of fresh fish within the European community. Rapid PC-based QIM is also available on the Internet at http://www. However. The most comprehensive scoring scheme to assess fish is the Torry Scheme [36]. pollock. panelists evaluate the odor and flavor of cooked fillets. QDA provides a detailed description of all flavor characteristics in a qualitative and quantitative way. The Torry Scheme. Sebastes mentella marinus. The average score of 5.5 may be used as the limit for consumption [21]. In this scheme. QIM Rating system software was developed for cod. haddock. is a descriptive 10-point scale and has been developed for lean. The scores are given from 10 (very fresh) to 3 (spoiled) (Table 13. This method is considered to be a relatively fast. The trained panel is handed a broad selection of reference samples and use the samples for creating terminology that describes all aspects of the product [16]. trained personnel required. fresh cod (Gadus morhua) [32]. is rapid and easy to perform. the QIM is suitable for early stage of storage of fish where other instrumental methods are not accurate [28]. expensive. Melanogrammus aelefigus. and not always practical for large-scale commercial purposes.1) [34].dk/QIMRS/qim_0202. QIM Eurofish published a manual [21] containing QIM schemes for 12 fish species and information about how to use the QIM schemes (QIM-Eurofish 2004). farmed Atlantic salmon (Salmo salar) [31].min. Solea vulgaris. The advantages of QIM are that it requires short training. 13. a higher score can be given for a single parameter [27]. sensory methods are time consuming. . 13.2. The method can also be used for texture. Pleuronectes platessa. saithe. nondestructive method based on direct observation of sensory parameters of fish and can also be specific for species. Therefore. Descriptive words should be carefully selected.

With permission.Methods for Freshness Quality and Deterioration Table 13. and Hyldig... pp. 975. metallic Neutral Some off odor Strong off odor Score 0 1 2 0 1 2 3 0 1 2 3 0 1 2 0 1 2 3 0 1 0 1 2 0 1 0 1 2 3 ◾ 193 Sources: Modified by Jónsdóttir. Quality Standards for Fish: Final Report Phase II.1 QIM Scheme for Sensory Evaluation of Herring Quality Parameter Whole fish Appearance of skin Description Very shiny Shiny Matte Blood on gill cover None Very little (10%–30%) Some (30%–50%) Much (50%–100%) Texture on loin Hard Firm Yielding Soft Texture of belly Firm Soft Burst Odor Fresh sea odor Neutral Slight off odor Strong off odor Eyes Appearance Bright Somewhat lusterless Shape Convex Flat Sunken Gills Color Characteristic red Somewhat pale. 37. Int. seaweedy. 2004.. . 37–59. brown Odor Fresh. Nordic Industrial Fund (in Danish). 1992. developed by Nielsen. Food Res. D. G. S. matte.

hake. blue whiting. Dielectric properties of fish skin and muscle alter in a systematic way during spoilage as tissue components degrade. sour milk. odor. 283. deciding the commercial value of the meat [41]. and product development in the seafood industry [38]. Sci. slight sulfide Score 10 9 8 7 6 5 4 3 Source: Shewan. metallic. Fish muscle has higher levels of indigenous proteases. Among textural attributes. texture. Dielectric properties of fish are used for determination of freshness.2 Torry Score Sheet for Freshness Evaluation of Cooked Cod Fillets Odor Initially weak odor of sweet.2 The Torrymeter The Torry fish freshness meter “Torrymeter” was developed at Torry Research Station in Aberdeen. hardness is the most important to the consumer. .3 Physical Methods 13. Numerous mechanical methods have been used to measure texture. 13. Scotland. wood sap. springiness. seaweed.. starchy.1 Texture Analysis Texture analyses for seafood are extremely important in research. and cooking [39. quality control. sour.3. followed by strengthening of these odors Shellfish. trace of “off” flavors Slight bitterness. Initially no sweetness but meaty flavors with slight sweetness may develop Sweet and meaty characteristic Sweet and characteristic flavors but reduced in intensity Neutral Insipid Slight sourness. starchy.40]. and flavor during spoilage and have been used as quality indicators since the first commercial version of the Torrymeter in 1970 [43]. TMA Strong bitterness. reminiscent of boiled clothes Lactic acid. 1953.g. soapy. J. during processing.3. boiled meat Loss of odor. boiled milk. boiled potato Milk jug odors. “off” flavors. Fish muscle may become soft or mushy as a result of autolytic degradation or tough as a result of frozen storage [16]. mackerel.. These changes occurring at microscopic level are related to alterations in appearance. which immediately begin to break down the proteins after the harvesting. Baltic herring. however. turnip. vanillin Condensed milk. tallow Flavor Watery. natural odor Wood shavings. flounder.. 13.M. With permission. acetic or butyric acids) decomposed grass. improper handling storage. Food Agric. J. A linear relationship was found between Torrymeter readings and sensory attributes for cod. and chewiness of food.194 ◾ Handbook of Seafood and Seafood Products Analysis Table 13. Texture includes the most common characteristics such as hardness. TMA Lower fatty acids (e. rubber. et al. 4. there is little agreement on which is the best method [42].

Gelman et al. 13. 13.3. The skin of fish could be affected by osmolarity and contact with electrically charged particles [51]. The Fischtester readings can be used as an objective criterion for the state of freshness/spoilage together with sensory data across the fish chain. It has also reported that there is a linear correlation between the instrument readings obtained on the day of harvest/catch and the date of spoilage [53]. and fish-handling procedures. the “Cosmos” instrument is handheld. partly frozen such as superchilled fish. The method is based on conduction through skin and. iced gilthead sea bream. measuring the electric properties (resistance. RT-Freshtester reflects dielectrical properties of fish. However.Methods for Freshness Quality and Deterioration ◾ 195 whole. 13. and capacitance) of the fish flesh [52]. . Gelman et al. However. Inácio et al. RT-Freshtester. [51] also studied the effect of washing with tap and treated seawater on the quality of whole scad (Trachurus trachurus) and found that Torrymeter and RT-Freshmeter readings were significantly (P < 0. and fish chilled in refrigerated seawater [54]. as well as rapid and nondestructive. This could be explained by seawater containing ions. and readings from all instruments decrease with storage time. allows automatic grading of 60–70 fish/min. and farmed Senegalese sole [43–49].3. Like other instruments. The electric properties of fish can change after death of the fish due to disruption of the cell membranes by autolysis. fast and nondestructive. Therefore. The Intellectron Fischtester VI gives reliable information about the days in ice and left of iced stored fish. portable. works only on whole fish and fillets with skin on.5 The Cosmos The “Cosmos” instrument developed by Japanese is applied for the evaluation of fish quality by determination of smell intensity. season. [50] found strong correlation between the organoleptic and Cosmos results for six species of fish and concluded that application of the “Cosmos” instrument for objective quantitative evaluation of fresh and chilled fish quality by determination of smell intensity appears to be practicable.05) lower in fish washed with seawater than fish washed with tap water or unwashed.3 The Intellectron Fischtester VI The basic principles of Torrymeter (the United Kingdom) and the Intellectron Fischtester VI (Germany) are similar.3. Fat also has an effect on the dielectric properties of fish and tends to make observed Torrymeter values more variable [47]. Mechanical abuse and freezing can affect the readings. therefore. [50] found that the Torrymeter readings obtained from six species of different origin were poorly correlated with sensory evaluation.4 The RT-Freshtester Like Torrymeter and the Intellectron Fischtester VI. conductivity. which interfere with the reading of both instruments as they are based on electrical properties of skin. it could be used for evaluation of fresh and chilled fish in the seafood industry and on fishing vessels. these instruments need calibration depending on sample preparation. They are unsuitable for frozen or thawed fish. The loss of skin and muscle integrity and deterioration of the skin caused by bruising during harvesting and packing operations would result in more variable Torrymeter values. fishing grounds.

SO2. chemometric analysis such as principal component analysis (PCA). and thawed.56]. cod caught by long line and gillnet [73]. chilled modified atmosphere packed (MAP) cod fillets [83]. and protein content in fish [74–78]. NO. SO2. online industrial production chain. Different electronic noses have been employed for measurement of fish freshness. which are sensitive to volatile compounds. . It has been indicated that a combination of electronic nose systems based on different sensor technologies improved the performances compared with the single technology for the codfish fillets [66]. and acid compounds are produced by microbial activity and lipid oxidation during storage of fish [55. Trggvadottir and Olafsdottir [64] also found that the response of all electronic sensor (CO. and partial least-square regression (PLS-R). static sampling system and electrochemical gas sensors. the main indicator of fish freshness.80].67–71]. Fourier transform infrared (FT-IR) spectroscopy is another technology that is a rapid.3. FreshSense is based on a closed. which determines the relation between sensor output patterns and the properties of the sample being analyzed [72]. The most important chemicals involved in fresh fish odors are long-chain alcohols and carbonyls. and quality assessment of frozen minced red hake [82]. NO. The concentrations of these compounds are related to the degree of spoilage. and N-cyclic compounds. sulfur compounds. The most frequently used methods are artificial neural networks (ANNs). water. This method has been applied for determination of fat.7 Near-Infrared Reflectance Spectroscopy Near-infrared reflectance spectroscopy has been used in various analytical applications. it is fast. Data analysis is important in electronic nose measurements.196 ◾ Handbook of Seafood and Seafood Products Analysis 13. Since these kinds of analyses are both time consuming and expensive. it has the ability to measure numerous samples within a short time. combining wavelengths in the optical range [56]. diff use reflectance infrared Fourier transform (DRIFT) spectroscopy has advantages. causing changes in protein and muscle structure. These are metal-oxide semiconductor gas sensors. and requires little training of operators [73]. and NH3). amines. easy to handle. The technique is characterized by speed and simplicity. and NH3) results for haddock from different seasons showed a similar trend. short-chain alcohols and carbonyls. This technique is based on the fact that a computer screen can be easily programmed to show millions of colors. semiconductor dimethylamine (DMA) gas sensor. Fish freshness has also been evaluated by a computer screen photoassisted technique (CSPT)based gas sensor array. Compared with FT-IR. H2O. 13. that is. On the other hand. However. and it is nondestructive. However. Previous optics-based electronic noses relied on absorbance and fluorescence.3. thickness shear mode quartz resonators. it requires too much handling of samples. N-cyclic. bromophenols. indicating spoilage of odors in seafood. water-holding capacity of thawed fish muscle [81]. an electronic nose called FreshSense was developed and distributed by Element-Bodvaki in Iceland and has been found to be a rapid. free fatty acid (FFA) in fish oils [79. Studies on cod fillets and heads also gave similar results. nondestructive. electrochemical sensors (CO. and it was found that CO sensor showed the highest response [65]. and aromatic. Olafsdottir et al.6 Electronic Nose Odor. has been analyzed by sensory panel or gas chromatography (GC). it can be operated on-/at-line. CSPT evaluates both effects [56. H2O. and prototype solid-state–based gas sensor called the FishNose [57–62]. nondestructive method to measure volatile compounds. [63] studied the freshness of iced redfish and found that there was a good correlation between the response of CO sensor and QIM method for both air and modified atmosphere storage of redfish.

sensitive. The sequences of nucleotide catabolism proceed as shown in Figure 13. and it has been indicated that this spectroscopic technique is useful in assessing the freshness and quality of sardine during iced storage [84].1. cheap. since they eliminate personal opinions on the product quality. which is the main energy source for metabolic activity. 13. The initial stage of the reaction catalyzed by endogenous enzymes takes place quickly. 13. and the chemical compound that is determined should increase or decrease as microbial spoilage or autolysis progresses [16]. The traceability system can also be used for the determination of fish freshness. In postmortem fish muscle.4. For the first time. This alternative method could be cost effective and definitely more reliable.Methods for Freshness Quality and Deterioration ◾ 197 its use is simple. The oxidation Adenosine triphosphate (ATP) Adenosine diphosphate (ADP) Adenosine monophosphate (AMP) Inosine monophosphate (IMP) Inosine (Ino) Hypoxanthine (Hx) Xanthine (Xa) Uric acid (Uric) Figure 13. this technique has been applied to sardine muscle during iced storage. It has been indicated that there is a correlation between nucleotide catabolism and loss of freshness. This process results from breakdown of adenosine triphosphate (ATP).4 Chemical and Biochemical Methods Chemical and biochemical methods for the evaluation of seafood quality are more reliable and accurate. Traceability is becoming a method of providing safer food supplies and of connecting producers and consumers.1 shown. leading to accumulation of adenosine diphosphate (ADP) and inosine monophosphate (IMP). degradation of ATP proceeds according to the sequence . the most used method to evaluate fish freshness is to combine several measurements obtained from different methods and correlate the findings with sensory analysis [59]. The most used procedures for the objective measurements of seafood quality are given in the next sections. and requires a small amount of sample.1 ATP and Its Breakdown Products Rigor mortis occurs in postmortem muscle tissue and is associated with stiffness of muscle or flesh. Currently. These objective methods should correlate with sensory quality. recording the product temperature from the moment of catch. Nucleotide breakdown reflects both action of autolytic enzymes and bacterial action [16]. Traceability can be defined as the history of a product in terms of the direct properties of that product and/or properties that are associated with that product once these products have been subject to particular value-adding processes [85].

However. Burns et al.198 ◾ Handbook of Seafood and Seafood Products Analysis of Hx to xanthine and uric acid is slower and is the result of endogenous enzyme activity or microbial activity [86].9. [102] also proposed Fr value for yellow fin tuna. The K value includes intermediate breakdown products. H. Shahidi et al. [103]. G. Since adenosine nucleotides are almost converted to IMP within 24 h postmortem [96]. and it varies within species of fish [94. Determination of G and P values are useful with lean fish. The rate of nucleotide degradation varies with species. The formulas are as follows: lno + Hx ⎡ ⎤ K (%) = ⎢ × 100 ATP + ADP + AMP + IMP + lno + Hx ⎥ ⎣ ⎦ lno + Hx ⎡ ⎤ K i (%) = ⎢ × 100 IMP + lno + Hx ⎥ ⎣ ⎦ lno + Hx ⎡ ⎤ G (%) = ⎢ × 100 ⎣ AMP + IMP + lno ⎥ ⎦ lno + Hx ⎡ ⎤ P (%) = ⎢ × 100 AMP + IMP + lno + Hx ⎥ ⎣ ⎦ Hx ⎡ ⎤ H (%) = ⎢ × 100 IMP + lno + Hx ⎥ ⎣ ⎦ IMP ⎡ ⎤ Fr (%) = ⎢ × 100 IMP + lno + Hx ⎥ ⎣ ⎦ . body location (dark or white muscle). Karube et al. [101] as an index of freshness quality. However. ADP. These results showed that measuring the concentration of single nucleotide degradation product to determine freshness quality of seafood is not appropriate. Karube et al. [103]. [100]. and sea bream [104]. [97] proposed the Ki value.13. whereas inosine and Hx reflect poor quality [87]. in some species ATP. which excludes ATP. The concentrations of ATP and its breakdown products have been used as indicators of freshness in many fish species [8. although it was observed to decrease during the first 2 or 3 days of iced storage. The K. Ki. [100] was found to be superior to Ki value for iced Atlantic cod. Therefore. the Ki value has been shown to increase very rapidly and then remain constant even though freshness quality continues to decrease greatly [98. European eel [13]. but measuring the concentration of ATP and its degradation products can be useful in determining freshness quality [20]. In addition. ADP. and Gill et al. respectively. season. but the high-performance liquid chromatography (HPLC) method is the most reliable among them. and Fr values are calculated by the procedures described by Saito et al. Luong et al. before its subsequent increase.99]. and storage conditions [105. handling. [93]. The G value proposed by Burns et al. The IMP is associated with fresh fish flavor. [93] is a biochemical index for fish quality assessment based on nucleotide degradation. [97]. P. The H value of iced Pacific cod was observed to increase steadily. stress during capture.95]. Several methods have been proposed for the analysis of single or a combination of nucleotide catabolites. it is difficult to obtain meaningful G and P values since fatty fish deteriorate due to rancidity [103]. indicating its superiority to Ki value [101]. It was reported that K and related values increased linearly (except Fr value) with storage time in turbot [91]. With some species. and AMP remain even after 2 weeks [97]. [102]. The K value proposed by Saito et al. Gill et al.88–92]. and adenosine monophosphate (AMP).106]. P value has been described by Shahidi et al. [101]. the K value can be superior to the other values. H values have been described by Luong et al.

and stomach contents at death.4. The most significant biogenic amines produced postmortem in fish and shellfish products are histamine. HPLC is mostly performed because of its sensitivity. The formation of biogenic amines results from microbial degradation during the later storage of fish.127]. The biogenic amine content of fish depends on fish species. HPLC [120.5. and pH [133]. The QI and the biogenic amine index (BAI) were proposed by Mietz and Karmas [120] and Veciana-Nogues et al. GC [126. the disadvantages of using biogenic amines as an index of freshness quality are that their absence does not necessarily indicate a high-quality product [113]. 2-phenylethylamine. capillary zone electrophoresis (CZE) [128. and other factors . tryptamine.129]. Since the amines are produced by spoilage bacteria toward the end of shelf life of a fish. tyramine. cadaverine. These problems may be more severe in sensitive consumers who have a reduced mono. depending on the species being examined [10.124. putrescine. Consumption of seafood containing high amounts of these amines can have toxicological effects.0 to 7. spermidine. There are various analytical techniques used to determine the concentration of biogenic amines. and agmatine. free amino acid content [112]. [121] for determination of quality of fish.4. respectively. Putrescine is also an intermediate of a metabolic pathway that leads to spermidine and spermine [119].S. The formulas used were as follows: QI = (histamine + putrescine + cadaverine)/1 + (spermidine + spermine) BAI = (histamine + putrescine + cadaverine + tyramine) QI is based on the increases in putrescine. spermine. putrescine.11.110]. tyrosine produces tyramine. since microbial flora vary seasonally [11].109. the moment of capture. the enzyme responsible for its detoxification. species. Food and Drug Administration [117] and the EU [118]. 13. respectively. In addition. tryptamine from tryptophan. and the concentration of these increases with storage time [91. Among these techniques. and histamine and decreases in spermine and spermidine during storage of fish. cadaverine. The importance of estimating the concentration of biogenic amines in fish and fish products is related to their impact on human health and food quality. whereas BAI is based on increases in histamine. Among the biogenic amines. cadaverine. including thin-layer chromatography (TLC) [122. and use of a biosensor [130–132]. their levels are considered as indices of spoilage rather than freshness [112]. the presence of decarboxylase-positive microorganisms. Biogenic amines are generated by microbial decarboxylation of specific free amino acids in fish or shellfish tissue [111].108].2 Biogenic Amines The concentration of biogenic amines has been reported to be a reliable method of measuring the quality of fish. Process technology is influenced by rigor development. and arginine leads to putrescine.Methods for Freshness Quality and Deterioration ◾ 199 13. histamine is potentially hazardous and the causative agent of histaminic intoxication [114].3 pH The pH is also an important parameter to show depletion in tissue and quality of flesh during storage. The others especially putrescine and cadaverine have been reported to enhance the toxicity of histamine [115]. postmortem temperature.and diamine oxidase activity [116]. and 2-phenylethylamine is derived from phenylalanine.1 depending on season. histidine yields histamine.123]. Postmortem pH varies from 5. and tyramine.107. and reproducibility. Cadaverine is derived from lysine. By means of decarboxylation reactions. reliability.125]. The hazardous concentrations of histamine are 5 mg/100 g and 20 mg/100 g fish—the legal limit for histamine set by the U.

5 Trimethylamine The one type of spoilage caused by microorganisms often detected as a fishy odor is due to the decomposition of trimethylamine oxide (TMAO) via the enzyme TMAOase demethylase. such as frozen eel [145]. and it has been used as an indicator of marine fish spoilage: CH3 CH3 – N=O CH3 TMAO CH3 CH3 – N CH3 TMA . Low initial pH is associated with higher stress before slaughtering [13. The level of TVB-N in freshly caught fish is generally between 5 and 20 mg N/100 g muscle. the level of TVB-N was not correlated with the time of storage of some fish species. total volatile basic nitrogen (TVB-N) primarily includes trimethylamine (TMA. The analyses of these indicators are considered unreliable because they reflect later stages of spoilage rather than freshness [140]. TVB-N level correlated with fish quality.151]. bacteria act upon TMAO to produce TMA. Low pH also promotes oxidation of myoglobin and lipids [134]. 13. The EC reference method for TVB-N determination. However. with the production of lactate. Atlantic cod [143]. it affects reactions taking place during storage of fish.135]. The first one includes direct distillation of fish after adding magnesium oxide. and hake [148].200 ◾ Handbook of Seafood and Seafood Products Analysis [134. However. it could not be regarded as a good indicator of fish freshness and proved to be better as a spoilage index. the levels of 30–35 mg N/100 g muscle are considered the limit of acceptability for icestored cold-water fish [17. mainly glycogen. involving preliminary deproteinization with perchloric acid. ammonia (produced by deamination of amino acids and nucleotide catabolites). pike perch [146]. Based on the results obtained from the literature. Therefore. produced by spoilage bacteria). It was found that there was a good correlation between three methods.4. turbot [92].4 Total Volatile Basic Nitrogen In seafood.141]. farmed gilthead sea bream [147]. and European eel [13]. Since the activity of enzymes depends on pH. A relatively low pH may cause a decrease in water binding to the myofibrils. which is considered to be the main cause of off odors in fish products [58. and DMA (produced by autolytic enzymes during frozen storage). Th is is caused by the depletion of energy reserves. TVB-N should be used as a chemical check. whereas the second one includes the use of trichloroacetic acid instead of perchloric acid [149]. It is well known that determination of TVB-N differs systematically according to the procedures used. 144].59]. 95/149/EEC of March 1995) on fish hygiene specifies that if the organoleptic examination indicates any doubt as to the freshness of the fish. affecting light scattering and the appearance of fish. Therefore. The European Commission (Council Regulation No. TMA is produced by the decomposition of TMAO due to bacterial spoilage and enzymatic activity [150. and direct distillation methods have been recommended as a rapid routine method. was compared with two routine methods. as shown below: Following death of fish. sardine [12. as shown in a variety of fish such as European hake [142].4. 13. Low pH is used as an indicator of stress at the time of slaughtering of many animals.136–139].

causing denaturation and cross-linking of proteins [171]. and time. Seawater fish have 1–100 mg TMAO in every 100 g muscular tissue. 13. The formaldehyde content of frozen seafood is generally used as a spoilage index. stage of spoilage. TMA can be used as a spoilage indicator and not as an index of freshness. which is converted to TMA by bacteria in iced fish. whereas freshwater fish generally contain only 5–20 mg% [153]. During chilled or frozen storage of fish. semiconducting metal–oxide array [166]. enzymatic and nonenzymatic lipid oxidation occurs. colorimetric method [160]. 164]. fish contain TMAO. Conway microdiff usion and titration [159]. its usefulness depends on time of year. this reaction is replaced by a slow conversion by an enzyme to DMA and formaldehyde [16. the storage temperature. or TVB-N contents.6 Dimethylamine As mentioned earlier. DMA. . including steam distillation [158]. TMA is not produced in a significant amount during the early stages of chilled storage of fish. The limiting factor of frozen storage in lean fish species is denaturation of proteins.4. age. and environmental factors [152]. type of storage and processing. Many analytical methods have been developed for the measurements of TMA. Several assays have been described for the determination of TMAOase activity in fish muscle [151. and solid-state sensors based on bromocresol green [169].Methods for Freshness Quality and Deterioration ◾ 201 TMAO appears to be part of the system used for osmoregulation. photometry [161].5 mg TMA/100 g in fresh cod.8 Lipid Oxidation Indicators During processing and storage. fish size. biosensor using flavin-containing monooxygenase type-3 [168].156. The fish is considered stale when the rate of TMA production is higher than 30 mg/100 g cod [155]. DMA can be used as a spoilage index during frozen storage of some species such as frozen hake [170]. The TMAO content of seafood varies with species. lipid oxidation is the limiting factor in fatty fish species.4. However. other species do not develop adequate amounts of DMA). 13. resulting in rancidity.150]. especially in gadoid fish. GC method [163. but it can react with a number of chemical compounds such as amino acid residues. which results in a dry and firm texture of the fish muscle [174]. a capillary electrophoresis method [165]. HPLC method [162]. However. time of year. but it appears after 3 or 4 days. flowinjection-gas diff usion method [167]. whereas fish can be stored in a frozen state for several months without severe changes in quality. when bacterial growth is inhibited. A close relationship has been found between lipid damage and quality of the final product [173]. Fresh fish has a limited shelf life and is prone to deterioration. location of catching.157]. and methods employed for analysis. The amount of DMA produced depends on species (except gadoid species. Fresh fish has a very low amount of TMA with values less than 1. and low-molecular weight compounds. 13.7 Formaldehyde The formaldehyde content in seafood products is generally considered as nontoxic. after which the rate of production of TMA parallels the bacterial proliferation pattern [154]. This reduces the solubility of myofibrillar proteins [172]. terminal amino groups. The formation of these products may cause severe quality changes or spoilage during prolonged frozen storage. but values increase during spoilage.4.

light.. off flavors.) Off taste and off odor are usually defined as rancidity. The major chemical indicators for the determination of the extent of oxidative rancidity . Many factors affect the onset and development of rancidity (oxidative and hydrolytic degradation of lipids). Free radicals from oxidizing lipids can polymerize with proteins and destroy certain amino acids.2 The autoxidation of fatty acids. trace of heavy metals) RH Propagation: O2 R RO2 + RH ROOH 2ROOH Termination: R+R R + ROO ROO + ROO RR ROOH ROOH + O2 RO2 ROOH + R RO + OH ROO + ROO + H2O R+H Figure 13.C. nutritional losses. produce toxins. E.182]. R. and they break down to aldehydes. The amount of reactive compounds increases gradually. consequently. Chapman & Hall.2). and termination (Figure 13. and free radicals react with oxygen to produce peroxide radicals (ROO). The amount of hydroperoxides can be used as a measure of the extent of oxidation in the early stages. C. They also destroy pigments. and cause off flavor/odors [183]. and taints [179. including the degree of unsaturation of the oil. There are three steps in autoxidation of unsaturated fatty acids.J. and then the quantity of radicals and peroxides decreases. the type and concentrations of antioxidants.. in Rancidity in Foods.. 3rd edn. Peroxides are not stable compounds. J. and modification of electrophoretic profiles of proteins [172. moisture content. lipid oxidation compounds interact with proteins. fish and fish oils are highly susceptible to the development of oxidative rancidity. which are the volatile products causing off flavor in products. 1994. heat) convert RH to free radicals (initiation phase). leading to protein denaturation. and Hamilton. (Eds. and alcohols. B6. The peroxide radical can attack another lipid molecule RH. Initiators (such as light. temperature. U. The hydroperoxide value is generally shortened to peroxide value (PV). London. Under chilled/frozen conditions. Fish oil contains about 20% of their total fatty acids as long-chain PUFA.). and pantothenic acid. Several chemical and physical techniques applied alone or together have been used to determine the degree of oxidation and hydrolytic degradation of lipids in edible oils. (From Hamilton.K.180]. unpleasant odors. Excess free radicals and peroxides in foods cause destruction of essential fatty acids and vitamins A. ketones. pro-oxidants. pp. R. thiamine.202 ◾ Handbook of Seafood and Seafood Products Analysis Initiation: Initiators (heat. 1–22. forming stable deterioration products (termination phase) [181. propagation. and degree of exposure to light [178–180]. resulting in peroxide (ROOH) and new free radical (propagation phase). Seafood has highly unsaturated lipid content.175–177]. Allen.C. Peroxides can also react with proteins and result in a decrease in their nutritional value. initiation. oxygen availability.

since they interact with myofibrillar proteins and promote protein aggregation [189]. cod [192. and lean fish such as blue whiting. such as proteins. which break down to secondary products of oxidation or react with proteins. coliforms. Microbial assessments have been carried out to monitor the numbers of various groups of microorganisms during the production process as part of food safety objectives and also hazard analysis critical control point (HACCP) systems [196]. 13. spoilage domain such as the range of environmental conditions over which a particular SSO is responsible for spoilage and spoilage level [198]. PV.185]. It was indicated that the main requirements for shelf life predictions are to collect information about SSO. AV. Spoilage of fish and fish products is a result of the production of off odors and flavors mainly caused by bacterial metabolites [197]. free amino acids. and thiobarbituric acid (TBA). there are some difficulties with common methods when quality has to be assessed. Microbiological analyses of seafood involve testing for presence or absence of pathogens such as salmonellas and determination of numbers of colony-forming units (CFU) named “total viable counts (TVC)” or “aerobic plate count (APC).190. However.9 Lipid Hydrolysis Hydrolysis leads to hydrolytic rancidity and involves hydrothermal or enzymic (lipase) hydrolysis to FFA and other products. sardine. Shewanella putrefaciens . A gradual increase in FFA formation was obtained for all kinds of samples as a result of the frozen storage time for fatty fish such as tuna.4.193]. reach a peak. and TBA values may increase.188. Analysis of these interaction products by fluorescence detection as a quality assessment index for frozen-stored sardine was studied by Aubourg et al. [188] and it was found that fluorescence detection of interaction compounds can provide an accurate method to assess quality differences during frozen storage of sardine. Increase in the PV is most useful as an index of the earlier stages of oxidation. or enterococci [195]. AV and TBA values measure the secondary products of lipid oxidation. 13. It is possible to predict shelf life of seafood based on knowledge of initial numbers and growth of SSO. since oxidation products are unstable and react with biological amino constituents. Microbial growth models can be used to determine the effect of various time/temperature combinations on shelf life of fish in production and distribution chain. European eel. The numbers of specific spoilage organisms (SSOs) and the concentration of their metabolites can be used as objective quality indicators for determination of shelf life of seafood. and also freshwater fish [194]. PV. as oxidation proceeds the PV can start to fall.” or numbers of CFU of indicator organisms such as Enterobacteriaceae. causing production of interaction products [187]. PV measures primary products of lipid oxidation.186]. and phospholipids. horse mackerel [13. and decline [184. Cozzolino et al. TOTOX (2VP + AV). haddock.Methods for Freshness Quality and Deterioration ◾ 203 are anisidine value (AV). peptides. Many methods have been employed for the measurements of lipid oxidation in foods as a means of determining the degree of damage [20.191]. Mathematics models have been well established for the growth of spoilage bacteria such as Photobacterium phosphoreum. FFAs and their oxidation products would have an effect on muscle texture and functionality.5 Microbiological Methods Numbers and types of microbes present in foods are important indicators of safety and quality. During prolonged storage of seafood. [80] also reported that PLS-R and near-infrared (NIR) spectroscopy to monitor both oxidation and hydrolytic degradation of lipids in fish oil can be successfully employed.

J. Woodhead Publishing Limited. 1986. also lack in sensitivity. Listeria monocytogenes [200].K. Mathematical models along with impedance technique may provide reliable information on shelf life of seafood within 24 h. which have more charges than the substrate itself [207]. and are costly. Background. U. 171S–175S. These methods are laborious and time consuming. In: Safety and Quality Issues in Fish Processing. Branden.. pp. 107(21). 5. Arnesen. Am. The decrease in impedance (or increase in conductance) is due to the breakdown of the substrate molecules in the media to smaller molecules (e. because it varies from batch to batch due to season. Roggero. an accelerating change in impedance (or conductance) will occur in the growth media. These methods are also not appropriate for online processing of seafood. J. Clin. However. Quality management of stored Wsh. 2005.. P. Rasmussen. A82. epidemiology.E. Food Technol. modern microbiological techniques [such as polymerase chain reaction (PCR). Marine n-3 polyunsaturated fatty acids and coronary heart disease: Part I. 34(1). A.e.. E. reverse transcriptase PCR (RT-PCR)].. Food components with potential therapeutic benefits: The n-3 polyunsaturated fatty acids in fish oils..g.M. 285–288. and storage after catch [202]. Bremner (Ed. 3. 115(3). handling. and biochemical and serological identification. 146. Circulation. 360–378. The principle of the impedance measurement is based on the phenomenon that at a time point (i. C.. C. effects on risk factors and safety. W. Conner. 7.H. Romano. catching method.). Sferlazzas....B. A. L. Schmidt.. S. Martinsdóttir.A. References 1. coliforms [205]... Among the microbiological methods for determination of bacterial counts in a short time. Cucchiara. Nutr. and Carroll.D. Cambridge. 21. H. V. enzyme-linked immunomagnetic chemiluminescence (ELIMCL)]. 17(1). G. de Caterina. On the other hand. 4.. feeding. impedance is the most promising [203]. and oligonucleotide probes give results in 1 day or even less [209–213]. Kinsella. Lipids. 6. J. animal data... requiring a minimum of 1 or 5 days to recognize. . R. 2003. Clinical prevention of sudden cardiac death by n-3 polyunsaturated fatty acids and mechanism of prevention of arrththmias by n-3 fish oils.. Liver Dis. K. antibody techniques [such as enzyme-linked immunosorbent assay (ELISA).E. detection time—DT) at which bacteria have grown to a population of approximately 107 CFU/mL or higher. acids). 40. Brochothrix thermosphacta [199].. followed by isolation. E. 22 PS omega-3 fatty acids supplementation in pediatric Crohn’s disease Italian multicentric study.. 2002..X.204 ◾ Handbook of Seafood and Seafood Products Analysis [198].E. 2. H. and Clostridium perfringens [201].K.. Barabino. L. Prediction of the remaining shelf life of seafood requires reliable estimates of the initial population of SSO. which were shown to correlate with remaining shelf life of product and also correlated better than classical TVC measurements.. these methods have limitations in performing quantitative analyses. Current microbiological culture methods rely on growth in culture media. The change in electrical properties (impedance. Leaf.F. and Kristensen.. 2002. [206]. Importance of n-3 fatty acids in health and disease. Dietary polyunsaturated fats in relation to mammary carcinogenesis in rats. 89–97.. Kang.. 2632–2634. and Annese.. Thromb. 163–170. and capacitance) due to the growth of microorganisms in the culture media has been used for the rapid estimation of total bacterial counts [204]. S. 2000. Xiao. conductance. and Billman. Dig.. 1986. and Salmonella spp. Y. Res.

L. 17. Bull. K..Methods for Freshness Quality and Deterioration ◾ 205 8. pp. Freshness assessment of European eel (Anguilla anguilla) by sensory. pp.. Technol. Food Technol.K. F. G. Food Sci. 19.). M. 2001. Cambridge.H. 9–23. and Pascual. Fish Process.. J. pp. O. Hanna.... Allen and R. G. A..A. In: Methods to determine the freshness of fish in research and industry. H.W. Rapid microbial methods and fresh fish quality assessment. Surrey. Verrez-Bagnis. Polat. 26.. In: Fish Processing Technology.R. K. Food Agric..” AIR3CT942283 (FAIR Programme of the EU) Nantes Conference. Fishing News Books Ltd. Ijmuiden. 682–686. Heia (Eds. 37(8). 1997. 279–286. Paris. 85. 352–355. Kyrana.. 319–328. November 12–14. J. France. and Polat. Ozyurt. T. Yamanaka. France.Y. Heia (Eds.. Nielsen.J. Proceedings of the Final Meeting of the Concerted Action “Evaluation of Fish Freshness. 287–296. Shiomi.. J... Connel. and Whittle. . 30. Martinsdóttir. 13. A. and K. FAO. London. Verrez-Bagnis. Scotland. Chang. J.). Y.J. R... I. 1996. 18. Fishing News Books Ltd. 225–320. Food Chem. G. In: Methods to determine the freshness of fish in research and industry.. A. and Kikuchi. K.C. J.” AIR3CT942283 (FAIR Programme of the EU) Nantes Conference. 79.. 1473–1480. J. Int. the Netherlands. 10. E. QIM-Eurofish.. Ozogul. J. 49–57. Bringing fish inspection into the computer age. 14. 1994. 15.P. Dalgaard. H. U. Lipids. V... Howgate. 1995. U. Food Chem.).B. 195 p. B. Olafsdóttir. chemical and microbiological assessment of farm-raised European sea bass (Dicentrarchus labrax) stored in melting ice.. A. Cadaverine as a potential index for decomposition of salmonid fishes. Sensory evaluation of fish freshness. Shiau.. 1985.C. Luten. H. E.. C. 245 p.. V. V. Agric. J. Chapman & Hall. Quality and Quality Changes in Fresh Fish. In: Reference Manual for the Fish Sector. 745–751.. Bremner.C. sensory and microbiological changes of sardines (Sardina pilchardus).M. R. Ozogul. International Institute of Refrigeration.. Rome. 25. Luten.K. Rodríguez. 24.J. Food Hyg. 56–65. E.K. C. Branch. chemical and microbiological methods.D. Chang. pp. In: Rancidity in Foods. 1989.J.A. pp. U. 20.. Nawar. E. G. 275–300. Food Chem. Botta. 1992. New York. 1985. Farnham. In: Advances in Fish Science and Technology. P. Johnston. G. Connell (Ed. 3rd Edn. Blackie Academic and Professional.R. Sensory... pp. C.. microbiological and sensory changes of sea bass (Lateolabrax japonicus) under partial freezing and refrigerated storage. and Lougovois. 1980.). Multilingual Guide to EC Freshness Grades for Fishery Products. A convenient.. Connel.. P. Schelvis-Smit.. Biochemical. Careche. Hamilton (Eds. Huss. 7. 37. E. International Institute of Refrigeration.R. 22.. Surrey.S.). J. 46. 16. 2004. Ozogul. 1999. pp.. Marcel Dekker. 59–70.J.J.B.. Paris. 1995. Hall (Ed. Kuley. 23.... Sci. 9. Farnham. Sensory analysis in fish. Olafsdóttir.). P. Fennema (Ed. Careche.M. Dalgaard. Control of Fish Quality. Glasgow. In: Food Chemistry. Martinsdóttir. 1995. A. and Shewan. J. 1997. 170–174. and K. F. J. Soc. 2002. J. 11.. Luten.. Y.. J. Biochemical changes in freshwater rainbow trout (Oncorhynchus mykiss) during chilled storage. and Vail. J. easy to use system for estimating the quality of chilled seafood. Evaluation of Seafood Freshness Quality. VCH Publishers Ltd. and Hyldig. 1998. November 12–14.B. Tommy Research Station. Martinsdóttir. M.. Sveinsdóttir. The effects of modified atmosphere packaging and vacuum packaging on chemical. 92. and Martinsdóttir. V. 1–22. Aberdeen. and Ozogul.. Sensory and nonsensory assessment of fish. 21. W... J.. U...K.. A. J. Australia.. 1992. 12. 2005. and Pan. Hamilton. Besteiro. Luten... J. QIM: A European tool for fish freshness evaluation in the fishery chain. Proceedings of the Final Meeting of the Concerted Action “Evaluation of Fish Freshness.

A.L... J.. Hyldig.. J. D. A. The development of a numerical scoring system for the sensory assessment of the spoilage of wet white fish stored in ice.. R. 42. 89–105. Development of quality index method (QIM) scheme for fresh cod (Gadus morhua) fillets and application in shelf life study.. International Institute of Refrigeration. 1990. J. L. 1993. 41... 161–168. Karakoltsidis.. Olafsdóttir. 49. C. and Hyldig. Trucco. and Manikantan. 2007. and Tejada. R. Food Sci. 1990. J. 209–221. Aksnes. 1997. 117–122. Barassi. J Sci.. Heia (Eds. Martinsdottir. B. 11.. Food Sci... R. Lupin. G. H. A. Quality index method (QIM): Development of a sensorial scheme for common octopus (Octopus vulgaris). Res.206 ◾ Handbook of Seafood and Seafood Products Analysis 27. Food Control.. A rapid method for quality management. Food Eng. A. H. Food Sci. Evaluation of texture parameters of Rohu fish (Labeo rohita) during iced storage.H. P.... M. 1992. Verrez-Bagnis. 37. 2003. Comparison of methods of freshness assessment of wet fish. and Ehrenberg.M.. Pastor. 65(7). Jason. Toyohara.. and Lupín.). Davidovich. Bonilla. Nielsen. B. 32. Giannini. E. Food Technol.. 15(3).A. G. 283–298. Sakata..R. J. Influence of handling procedures and biological factors on the QIM evaluation of whole herring (Clupea harengus L. 35. Nielsen. 18. 1202–1205. Tucker. and Kristbergsson. Soule. J. D. 81. M. 31. In: Methods to determine the freshness of fish in research and industry. Z. M. 23(3–4). M. E... 1976. T. Quality index method (QIM) scheme developed for farmed Atlantic salmon (Salmo salar).. and Boeri. Hyldig. Sci.M. 28.M. Huidobro. J..A.). Food Technol.. Food Agric. J. Laboratory assessments of commercial fish.. Food Qual. 44.. E. pp.. D. Food Control.. Hyldig. 55(1).E. 1570–1579.L. 13(4). Kazantzis.. G. S. Y. Jorgensen. Pathare. the Netherlands. Effects of proteinase inhibitors from potato on the quality of stored herring.. Hattula. Martinsdóttir. K.. Barbosa.. AIR3CT942283 (FAIR Programme of the EU) Nantes Conference. Quality index method developed for raw gilthead seabream (Sparus aurata). and Rand. Kostiainen. 2004.. November 12–14. The storage life of iced southern blue whiting (Micromesistius australis).N.G. D. Proceedings of the Final Meeting of the Concerted Action “Evaluation of Fish Freshness”. In: FAR Meeting. G.A. Jónsdóttir.C. 46.. T. and Suuronen P... 55. Mackintosh. K... Jørgensen. E. H. S. K... Food Res.. Chambers.R.. A. Pavlisko. 1989. Jain. Hansen. 37(10). Development of methods for quality index of fresh fish. K.. 975–983.. Fish. Davidovich. Giannini.. 1980. A. In: Quality Standards for Fish: Final Report Phase II. J. 116–120.S.. L.T... J. 237–245. G. Kimoshita.P. M..C. A. 15(3). K. Sveinsdottir.. J... 11(1). 1995.. 34. 37–59. Noordwijkerhout.. Kallio. P. 1953. Quality standards for herring. J...C. J. An objective tool for determination of sensory quality. Jhaveri. S.K.G. France. 67(4).. C. V.. . R. 39. Pivarnik.L. J. 297–305.. E. 16(2).. Luoma. Quality Index Method. and Larsen.G... Part III. 40. M. J.N. 352–358. Jouko Poutanen. 71–80.. Sveinsdóttir. 225–234. 79–82. Martinsdóttir. Luten. J.. 14. 364–368. G. Effects of catching method on different quality parameters of Baltic herring (Clupea harengus L. C. C. Food Agric. Aquatic Food Product Technol. Food Technol.. 1981. and Nielsen. Burt. 2002.L. 285–300. Texture measurements in fish and fish products. Int..). Coppes. P. 336–340. S. Sveinsdottir. 36.. Consumer perception of sensory quality in muscle foods. 2007. Soule4... Constantinides.F. 47(11).. Freshness assessment of six New England fish species using the Torrymeter. 29. Jonsdóttir. Application of quality index method (QIM) scheme in shelf life study of farmed Atlantic salmon (Salmo salar). Hydilg. H.R. Storage life of chilled Patagonian hake (Merluccius hubbsi). 2004. Food Sci.A. 33. J. D. 47. Preference. 2000. A. Boeri. E. 2002. 185–197.. Shewan. K. and Martinsdottir. A. 30. P. Dalgaard.. L. 38.. 45. Crupkin. R.. and K. Careche. and De Vecchi. 2004. 4.. R. Food Technol. and Kristbergsson. Gibson. 43. Degradation of oval-filefi sh meat gel caused by myofibrillar proteinases..B..H. Paris.. T.M. Yamashita. 1992.. and Shimizu. S.. Aquatic Food Product Technol.. and Sanders. Nordic Industrial Fund (in Danish). D. and Bowers. and Vaz-Pires. pp. J.

Int. I. Salimbeni. Martinsdóttir. J. 63.. 320–328. E. and Kent. and D’Amico. C. G. Acta. G. T. M. Mackie. R. C. C. S. D. 7. June 10–14. 54. E: Sci.. 65. A. M. Meas.. Technol. G.. Reykjavik. Eur. B. 72–77. Di Natale. Bungaro.. RT-freshmeter and sensory analysis. P... E... 45. 225–232.. 229–249. P. Martinelli. 67. D’Amico. E. Porphyrins-based opto-electronic nose for the volatile compounds detection.. Food Res. S. P..A. 2654–2659.. Dublin. Paolesse. C. Quality and Safety in Seafoods F-Fe 380a/00 (May 2000). The development of an electronic fish freshness meter. R. 2001. Food Agric.. D. Precision and application of electronic nose for freshness monitoring of whole redfish (Sebastes marinus) stored in ice and modified atmosphere bulk storage.. 2353–2359. J.R. R.... Alimelli. M. X. Characterisation of food freshness with sensor array. G. F. Bazzo. and Jónsson. Project Report 02-01 FAIR CT-98-4076. 1995.. Olafsdottir.. J. G. 62.. B.P. Trends Food Sci. Comparision and integration of different electronic noses for freshness evaluation of cod-fillets. Multisensor for fish: Questionnaire on quality attributes and control methods. texture and electronic nose to evaluate fish freshness. and Di Natale... F.. and Ferri. Anal. 77. S. I. D’Amico. Fish freshness detection by a computer screen photoassisted based gas sensor array. and Richards. 1103–1114. Di Natale... Boschi. 2000. Inácio... 61.. Gelman. Hognadottir. Jason.. Shimizu. Rapid gas sensor measurements to predict the freshness of capelin (Mallotus villosus)... E. In: TAFT Trans Atlantic Fisheries Technologies Conference. Olafsdottir. 2003. 49. G.... A... L. Olafsdottir. 11(3/4). The Intellectron Fischtester VI—A neglected and underestimated powerful instrument for fish freshness/spoilage determination. Kyranas. Santonico. Methods to evaluate fish freshness in research and industry. Actuat. Bernando. W.M.. 2007. Teagasc. 220–226. Sens. Henehan. B. Li. and Vaz-Pires. Sci. Dalgaard. Effect of washing with tap and treated seawater on the quality of whole scad (Trachurus trachurus). Ólafsdóttir. A. 65. B: Chem. 2007.. electronic nose. Actuat.. Sci. Semiconductor dimethylamine gas sensors with high sensitivity and selectivity. 59. J Phys.. C. and Jonsdottir.. Iceland. 8.Methods for Freshness Quality and Deterioration ◾ 207 48. R. Olafsdottir. Martindottir. Sens. Multisensor for fish: Storage studies of cod in Reykjavik and Tromso. Food Chem. A.. Trggvadottir.. J. Filippini. Actuat. 225.. 64.. Eur. S. 58.. A. F. Sens. 2003.. texture... 580–585. 2002. 258–266(9).. Y. Lundby... Sens. F. 582(2). Haugen.V.. Food Res. 217.. Flair-Flow EUROPE Technical Manual.. Changes in the quality indices during ice storage of farmed Senegalese sole (Solea senegalensis).. 48(6). Actuat.. and Jonsdottir. Ólafsdóttir.C... A. The National Food Centre. Dalgaard. Oehlenschläger. H. Brunink. F. 406–411. Aquatic Food Product Technol.H.. Technol. Food Res. V.J. Tajeda. H. and Göpel. Labreche. 282–290. S. M. FIGD analysis. Martinsdottir. Marcq. Chanie. G. Agric. J.. G. A. Instrum. and Olafsdottir. Takao. 1994. V... Drabkin. J. Lauzon. 1996.. Nielsen. Kawaguchi.. 1975. and Glatman. A. Y. Paolesse. J. 36. Oehlenschlager. 2001. V... 55. Di Natale. Food Chem. Westad... 18(19). G. T. Recognition of fish storage time by a metallophorphyrins coated QMB sensor array. J... B. 1997.. E. I. Ireland. A. 53. M. 83(6). Paolesse.. M.. 1997. Faccio. 52. Chim. 2003. A rapid non-destructive method for fish quality control by determination of smell intensity... P.. 25. 51. 2002. Nakanishi. 60. 572–578. M. G. For fish meal production. 551–560. S.V. and Kyrana. E. 56.E.. Davide.. Pennazza.R. and Olafsdottir... Lundström.S. Agric. and Einarsson. G. Paolesse.. Einarsson. S. Application of an electronic nose to predict total volatile bases in capeline (Mallotus villosus). Actuat. Rapid control of smoked Atlantic salmon (Salmo salar) quality by electronic nose: Correlation with classical evaluation methods. P.. 2006. J. 826–830. Comparison of selected methods of assessing freshness quality and remaining storage life of iced gilthead sea bream (Sparus aurata). Undeland. and Egashira.. Macagnano. B. De las Heras... Schweizer-Berberich. 66. 8(8). 116(1–2). J.. 57.. Freshness. Technol. 2000. R. J. Jonsdottir. and D’Amico. E. 50. Project Report 04-00 FAIR CT-98-4076. Trggvadottir. . 380–382.. R. Technol. 2000. R. Sens..C.. Jensen.. Vahinger. and Nilsen. Lougovoisa. 2000..

R. and Tuene. 3515–3521. 209 p. Food Sci.. P.K. Jensen. Actuat. 1987. J. Jhaumeer-Laulloo. K.. and Careche. 1996. Evaluation of the quality of frozen minced red hake: Use of Fourier transform infrared spectroscopy. and Reond... Lab.M. and Manzini R. 70.. Rakow.S.. and Isaksson. J. 80.E. Nortvedt.J. and Lee.M. O. Vogt.. Dickinson.R. 75. 2005. 82.. LWT-Food Sci. and Jørgensen. Carmona. 382. A.. Technol...H. fat and protein in tuna fishes. R. D. 3667–3672. 71. J... Food Chem. Gamberi.. J. J. and Rondeau. 1024–1030. J. Sinesio..I.Z. Anal. L’Omelette. 69. D. and Mann... protein and dry matter in Atlantic halibut filet. Drew. 1997. S.. Nature. Ramasami. Moreno. 1997. P. Babbitt.. Int. 697–700. Electronic nose and sensorial analysis: Comparison of performances in selected cases. D.. C. 1998. Pink. Rapid near-infrared spectroscopic method for the determination of free fatty acid in fish and its application in fish quality assessment..208 ◾ Handbook of Seafood and Seafood Products Analysis 68. M. Suslick. A. Bucarelli. J. 73. Dublin. Howgate. 579–588. B: Chem. 42. 84. Bøknæs. Naczk. 246–252. and Esaiassen. 1996. Paolesse. 1998.. 347–356. 55(3). Technol.. 35. K. 8414–8415. 76.. Application of near-infrared transmittance spectroscopy in the determination of fat. 87. 103(3)..M.. J. LWT-Food Sci. 85. Janzen. Food Chem. D.. IEEE Sens. A.. 79. and Walt. A. A. 2005.. 2007. Non destructive determination of fat and moisture in whole Atlantic salmon by near-infrared diff use spectroscopy..R. C. Di Natale. G.. C. 734–736. Macagnano. Freshness assessment of thawed and chilled cod fillets packed in modified atmosphere using near-infrared spectroscopy. Macagnano..F Kramer and J..M.. 15. 2004. Chemometr.. G. 648–652. N. Syst.. Moneta.. D.A. M. 86. Greene.. 45(9). Food Sci. Rodriguez-Casado. K. and Somersw. Food Eng. Technol.. 628–634.. D’Amico. Torrissen. P.. D.. 50(3). K. 40.S. P. 2007... E.. Sánchez-González. 199–207. J. A. E. C. Sens. 821–828. Food Compos. J. 83. 11133–11138. LWT-Food Sci. 2002. and D’Amico. S.. 2001. 5. Murraya. D. Agric. 81. Chem. Wold. H. 62. Mantini.... J. Chreeb.. Bechmann. Scaife. A. 1165–1174. J. 2002. Colorimetric sensors array for molecular recognition. Food Chem. A. J. 1236–1238. Mencaglia.. T... Paolesse. 78.N. 305–311. F. P.M. Predicting sensory score of cod (Gadus morhua) from visible spectroscopy. 205–215... 2005. C. In: Seafood Quality Determination. New York.. Macagnano. Kinetics of degradation of adenosine triphosphate in chill-stored rainbow trout (Oncorhynchus mykiss).B. E... 669–675. 123. R. A. M. N. Liston (Eds. 1998. Chemical and near-infrared determination of moisture. 60. A.D. MacEwan.. Di Natale. and Sen. T. Technol. Downey. 38(1). R. Tarizzo.. Food Sci. Nilsen. H. 102. Gerard Downey. Am... Array of opto-chemical sensors based on fiber optic spectroscopy. A. 74.C... and Pink. 1998. Technol. Multivariate determination of free fatty acids and moisture in fish oils by partial least-squares regression and near-infrared spectroscopy.E. Food Chem. Anelectronic nose transducer array of vapoluminescent platinum(II) double salt. and Martens.. Regattieri. Food Chem..... 1990.. B. G. Buss. I. A comparison of selected rapid methods for fat measurement in fresh herring (Clupea harengus). Elsevier Science Publishers. K. 31(7–8). 2005. 95–99. Khodabux.. T. Zhang. P. A chemical detecting system based on a cross-reactive optical sensor array. A.. E.. Rapid assessment of quality parameters for frozen cod using near infrared spectroscopy. Traceability of food products: General framework and experimental evidence. 38. and Kramer.. 77. LWT-Food Sci.S. H. 72. S.. Kennish.. 46. Tetrahedron. A.G. I. 55(5). F.. M. T.. Agric.. J.. Andersen..). J. M.M... 88. Cozzolino.. Patterns of nucleotide catabolism as freshness indicators in flatfish from the Gulf of Alaska. Structural changes in sardine (Sardina pilchardus) muscle during iced storage: Investigation by DRIFT spectroscopy. 2007. Soc. Review of high-pressure lipid chromatographic methods for measuring nucleotide degradation in fish muscle... Gormley. . K. Di Natale. Non-invasive and non-destructive percutaneous analysis of farmed salmon flesh by near infra-red spectroscopy. J. Kauer. and Quaglia.. 81(2). Mignani..R. Intell.. J.. White.

E. and Male. 52.D. Gill. M.D.. London... 14. Taylor. Int. da Silva. P. Technol.Methods for Freshness Quality and Deterioration ◾ 209 89.. 36. Biogenic amines formation in Atlantic herring (Clupea harengus) stored under modified atmosphere packaging using a rapid HPLC method. 1978. Suzuki. Saito. Agric..K. 752–758. 2006. 106. P. 99.. Gill. 314–319... 139–145. S. Determination of fish freshness with an enzyme sensor system. D. S. Jpn. Karube.. Male.. Thompson. and Sigholt. 37. 1994. 107.T... Enzyme Microb. F. U. F. B.. Development of a new biosensor system for the determination of the hypoxanthine ratio. 93. T. Effects of freshness on ATP-related compounds in retorted chub mackerel. 55.. J.C. Food Sci.. H. Luong. Food Chem. J. J. Adenosine triphosphate catabolites as flavor compounds and freshness indicators in Pacific cod (Gadus macrocephalus) and Pollock (Theragra chalcogamma). and Karmas.. D. 515–522.. E.. 1986... D. E.. Chong. Surette. and Dunajski.. and Yano...N. and Kose. 574–578. 40. Sci. J. Characterization of quality deterioration of yellow fin tuna. 96. 1984. 92. T. Özogul. Suzuki.. Jones. 56. O. G. 100.A.A. Food Sci. Can.. T. J. Masson. Y. Report Fish. J. Fish.D. Fujita. Biochemical basis of postmortem nucleotide catabolism in cod (Gadus morhua) and its relationship to spoilage.. Scott.. Huynh..B. Burns. 1186–1190. 457–461. 50. 868–872..H.J. 2002. J. during chilled storage. Kee. 57.. S.). pp. and Sherwood. J.. 2007. Muscle high-energy phosphate and stress affect K-values during ice storage of Atlantic salmon (Salmo salar). Objective procedure for fish freshness evaluation based on nucleotide changes using a HPLC system. 580–583. Anal. and Gibbs. L.. I. J. Agric. A. T. H. 335–340. A new method for estimating the freshness of fish. 2002.H... Food Sci. 105. 51.. Bull. Soc. Biogenic amine content and biogenic amine quality indices of sardines (Sardina pilchardus) stored in modified atmosphere packaging and vacuum packaging..A. N. and Sakaguchi M. Fishing News Books Ltd. salmon. E.B.. 101.. 749–750.. 2006. 62. 13.A. 1965. K...I. Food Chem.. Agustini. Luong. and Özogul. Agric. X. 257–258. Applications of polarography for assessment of fish freshness. Y.. J. 306–313. Assoc.. Gould. A.. and Toyama. 125–130. 2039–2045. P.. Buisson. P. R.. T. 67. Hypoxanthine ratio determination in fish extract using capillary electrophoresis and immobilized enzymes... K. Aquat. J. 42. and Matsuyoshi. Özogul. M. 1997. S..T. Gokbulut. Y. U. 77–81.W. K. Food Chem.B. Chem. 1477. 98. Goto....R. C. Hagiwara. Biochemical. J. Hypoxanthine and other purine-containing fractions in fish muscle as indices of freshness. Arai. and Fletcher. J. Food Sci. 43–47. Technol.. J. Mietz. I. 102. Polyamine and histamine content of rockfish... Ishizaki. Technol. 2001. F. T. 1987. Pinho. E.H. J. 1373. Ryder. 35. Food Sci.. Change of K value and water state of yellowfin tuna Thunnus albacares meat stored in a wide temperature range (20 to 84°C). Kreuzer (Ed. 110. 49. Food Control. J.. T. and Bernatt-Byrne. Kuda. 321–326. Luong. Sci. Scomber japonicus.. and Takai. Watanabe. K..J. T. an indicator of fish freshness.H. 1990.V. In: The Technology of Fish Utilisation.. Food Chem. M. K. 32. A. Ozogul. Greene..L. and Nguyen. 2002. from the Black Sea. Taylor. Shahidi. 19–22.S. 109. 1453–1456... Sci.. Beyer. Ozkutuk. 1988. Food Chem.B. M. 99. Kuley. Quantick. 179–183...M. 1992. 91. lobster and shrimp as an indicator of decomposition. Storage of New Zealand Jack mackerel (Trachurus novaezelandiae) in ice: Chemical.. T. Male. C. F.. Tech. 90... 61(1). 24. sensory and microbiological attributes of wild turbot (Scophthalmus maximus). LeBlanc. J. 94... Plestilová. 1984.. Freshness quality of harp seal (Phoca groenlandica) meat. M. 103. Erikson.L.. J. LWT-Food Sci. Fish. 1985. Comparative quality assessment of cultured and wild sea bream (Sparus aurata) stored in ice. F.. J. Murata. Off. 99(3). Tanaka. 1959.. Food Chem.R.. . K. Ferreira.. and Irvine. Storage of yellowtail (Seriola quinqueradiata) white and dark muscle in ice: Changes in content of adenine nucleotides and related compounds. M. and Shahidi.A. 1991... Food Sci.W..T. and Özogul. Food Sci. Production of histamine and tyramine by bacteria isolated from Portuguese vacuum-packed cold-smoked fish. Ozogul. Alasalvar. 1992. 95. 104.. D.. Agric. microbiological and sensory assessment.. M.. 108. R. T.. Food Sci.H.B. Matsuoka. 97. C. G..

A. and Yamanaka. Food Chem. A.. L. pp. Determination of biogenic amines in fish and fish products—HPLC method. FDA. J. 716–720. Agric. July.) Elsevier Science Publisher. H.. Life Chem. Agric. A. M... 120.. 1993.. 44(6). Virginia Tech. 22. Liston (Eds. G. 1996. Evidence that histamine the causative toxin of scombroid fish poisoning.. Objective analysis of seafood quality. 296–300. Determination of histamine. Food Chem. 11. 112. Chang. H. semiquantitative method for determining biogenic amines in foods.... 1996. M. In: Seafood Quality Determination. 2000. K. Mackie. Decomposition and histamine in raw... Inter.. Multidetection. and Arakawa. Med. 335–348. S. Chromatogr... 45. pp.M. Rios. Chromatogr.. 1994. 1991. J. M. 129. Effects of processing on the amine content of pork bellies. Aspen Publication. T. sec. C. 236–246. 693. Pirie. and Flick. Shalaby. Otsuka. H. Effects of canning on biogenic amine contents.. 127. Taylor. L. Compliance Policy Guides 7108.. Chemical quality index of canned tuna as determined by high-pressure liquid chromatography. Enriquez-Ibarra.. 59..T. G. 113....A. Adachi.. L. 1999.M. Shiomi. 118. 1–47.. Rowland. Res. M. . Food Chem. Marine-Font... 1997.. 367–372 1995..L. A. Chromatographia. Amsterdam. and Goetsch. A. Food Sci. and Macsini. and Wasserman. 125. Histamine Poisoning Associated with Fish. Rep..R. Morrow. 155–158. Shimakura. and Flick. S..-H. 116. 1026–1029. Rawles. Gill. Feier. and Chai. Biosensor for food freshness.G....210 ◾ Handbook of Seafood and Seafood Products Analysis 111. 1994.. and Karmas. 2041–2044.. 46(3/4). 235–245. Biosensors applied to biochemical fish quality indicators in refrigerated and frozen sea bass reared in aerated or hyperoxic conditions.S. Taylor. Veciana-Nogues.... 4324–4328. S.M. 121. Food Sci. Food Rev. 1997. 1996. D. Kikuchi. McNair... 123. Analysis of Biogenic Amines in Finfish.. A. Food Nutr. Selective and rapid determination of biogenic amines by capillary zone electrophoresis. and Conte. and Valcarcel.. T.. U. Hwang. An analysis of different methods of sample preparation in relation to food characteristics. 540. Greaser. 130. J. Spinelli.1. G. J. 6(4). 52. Blacksburg. 1990.S. 729. 23–30. B. VPH/FOS/85. 1999. and Sumner. B.. M. N. 114. and Vidal-Carou. 48. Aquacult. and Kneubühl. K. 53.. 1997.D. Wang. World Health Organization.. 1985. 134–135.R.. 291–295. M. Iida. Res. 170–176. 131. Lakritz. Archiv für Lebensmittelhygiene. Ohashi. frozen tuna and mahi-mahi. Imark. pp. A. L. N Eng.. Mascini.. Ritchie. Flesh foods and their analogues..J. O. A.M. Biogenic amines in fish and shellfish. Kramer and J. 133. Y. Biogenic amines in food. Histamine and food processing. I. F. M. Inter-laboratory studies on precision characteristics of analytical methods. 681. A... High-performance liquid chromatographic evaluation of biogenic amines in foods.240. Concentrations of polyamines in fresh water fishes. Adv.-F. H. B. Bodmer. E. Moret. Council Directive (EEC) 91/493/EEC. In: Food Texture Measurement and Perception.V. 363–369. J. M. canned tuna and related species. 119. M.L. Nihon Suisan Gakkaishi. L.. 1977.525. 126. T.H. 39. Proceeding of an International Symposium.. Cheese and Other Foods. Oxygensensorbased simple assay of histamine in fish using purified amine oxidase. G..). Shiua.C. EEC.J. The formation of non-volatile amines in relation to concentrations of free basic amino acids during postmortem storage of the muscle of scallop (Pecten maximus). 117. 115. J. 124. G. A. herring (Clupea harengus) and mackerel (Scomber scombrus). Margolis.L.-Y. and Pearson. K. D. S. 1994. Mietz. 8.J. M.J.. 128. Suzuki. L.. J. Poli. 519–522.. Rosenthal (Ed.E. 289–301. 1997.. 132. P. High-pressure liquid chromatographic determination of biogenic amines in fish implicated in food poisoning.. 60(3). VA. the Netherlands. Int. 42. S. 1987... S. 1974. C. 1986.L. and Roberts. Inflamm.H. J.-J. Poli.. 324.. putrescine and cadaverine. 1991. D. Nomura. and Nakamara. 122.H. Zampacavallo. 329–365. Laying down the health conditions for the production and the placing on the market of fishery products.. A Progress Report.. Arce. J. Parisi. Yamanaka.E... Bonilla. Gaithersburg. Biogenic amines in fresh and canned tuna. MD.. J. Food Chem. M.D.

S. 73. R. Özoğul.R. C. Woodhead Publishing Limited. 147. 2002. Perez-Villarreal. Westport. 143. Int. Rhaliby.. Weinzierl.. 347–356. K. Food Microbiol. Quality of farmed gilthead sea bream (Sparus aurata) during ice storage related to the slaughter method and gutting. Hebard. Stansby. Rehbein. M. H. Hebard.D. Özyurt. Determination of the quality parameters of pike perch Sander lucioperca caught by gillnet.A. Havemeister. C. and Schelvis-Smit. 139. 1989..V.. Int. Merluccius.J.. Ouadhi.. J. 1984.. W. Warriss (Eds. . 1998. 2007. H..F. Lauder. Effect of methodology on total volatile basic nitrogen (TVB-N) determination as an index of quality of fresh Atlantic cod (Gadus morhua). Y.. Cambridge. 1963.. 151. J. 750.K. Tejada. Archiv für Lebensmittelhygiene. Mackie. Haard. 80. Özogul.M. Govaris. 138. K. Food Sci. 34. 1–7.A. 2003. and Vareltzis. and Jewer.. A. J.. B. Eur. 1996.E.. J. Özogul. Simeonidou. Oehlenschläger.H..E.R. Morzel M. 33–37. Chemical and sensory changes in Mediterranean hake (Merluccius merluccius) under refrigeration (6°C–8°C) and stored in ice. L. A. 224.. Ward (Eds. M. 412–420.. and Oehlenschlager. J.K. K. S. 2002. 50. pp.E. longline and harpoon in Turkey. and Jørgensen. 221–254.. Food Chem.. Comparison of the official EC method for the determination of total volatile bases in fish with routine methods. J. 49. Z Lebensm Unters Forsch A. 215. 479–484. 123–132. F. pp. 30(7). 1997. Fish. S. 69–79. W. 148. E. B. 1996.. A. Sci. H... 204.. Marx H. Eur. Technol. J. Sci. 197. 2001. and Vidal-Carou. 6504–6510...H. 136. CO. Food Res. Occurrence and significance of trimethylamine oxide and its derivatives in fish and shellfish. J. Azam. Simeonidou. R. 33. 140. 2006. Chapter 26. M. Fishing News Book. A. Sotelo.S..T. D. 110–112. Food Res. W. K.. Robb.. Food Agric. 1987. Zeitschrift fur Lebensmittel-Untersuchung Und-Forschung. A. and Stolle. Botta. Flick. Bremner (Ed.. F. M.H. 234–248. Food Sci. 145.. 153. Gökbulut... New York. Effect of commercial and experimental slaughter of eels (Anguilla anguilla L. A. 1982. In: Industrial Fishery Technology. J.J. C. G.A.). Fresh Fish: Quality and Quality Changes.).. M.. p. In: Chemistry and Biochemistry of Marine Food Products. G. N. G.. pp. M.Methods for Freshness Quality and Deterioration ◾ 211 134. Effect of the slaughter method on the quality of raw and smoked eels (Anguilla anguilla L. 132.. G. and Smith.S. A sensitive trimethylamine-N-oxide aldolase assay in two steps without deproteinisation. 1997. Özogul.... Food Agric. In: Safety and Quality Issues in Fish Processing.. 734–736. 135.. Quality changes in sardines (Sardina pilchardus) stored in ice and at ambient temperatures.K. Nielsen. H. Methods of stunning freshwater fish: Impact on meat quality and aspects of animal welfare. H.. Aquacult. 282–286.. Food and Agriculture Organization (FAO) of the United Nations.. The effect of slaughter method on the quality of rainbow trout (Salmo gairdneri) during storage on ice. and D.. 152. Zur zusammensetzung der TVB-N fraktion in sauren extrakten und alkalischen destillaten von seefishfillet. 47. 141.F.. E. van de Vis.. 40...E. Res. 150... Kuhlmann.. Reinhold Pub. Ababouch. R. 137.. and Küley. 405–410. and Huidobro. Sci. Y... and Vareltzis. flavor and texture. 1–11.. Bover-Cid. H. Agric.M. Composition of fish.E. Technol. A. Archiv für Lebensmittelhygiene.). H. Brunner B. I. 144. Baixas-Nogueras. U. Composition of European hake. Özkütük. and Olcott. 2000... and Howgate.. 1988. The role of enzymes in determining seafood color.. Effect of frozen storage on the quality of whole fish and fillets of horse mackerel (Trachurus trachurus) and Mediterranean hake (Merluccius mediterraneus). 1982. Quality assessment of seven Mediterranean fish species during storage on ice. B. Özyurt... 44–48. Technol. Italy. Ersoy.. Souibri. 13. Food Res.. Govaris. Vyncke.. and Küley. P. Polat. F.. Hydrolysis and oxidation of European eel oil during frozen storage for 48 weeks. Kestin and P. J. Avi. 149–304.G.. Veciana-Nogués. J.. Oxford. 2002..M. C.C. In: Farmed Fish Quality. Rome. L. Martin.). and Martin. 204A.. Flick.) on quality and welfare.. M.. and van de Vis. O.E. Battal.. Hofmann. W. 24. 146. Huss... and Busta. 149.. Münkner. U. Rehbein. 142. S. S.

A.. Talanta. 172.A. Sikorski. pp. K. and Nielsen.K. 57.. P..K. Frisby. Shahidi (Eds. 159. Influence of frozen storage and defrosting on the chemical and nutritional quality of sardine (Clupea pilchardus). 1994. 164.. 569–573. VA. P.. 1996. 103. Adv.. and inhibition with emphasis on fish. 3814–3822. Food Protect.. Jørgensen. 7. Technol. M. New York. 1982. 103(3).. J. 1994.C. J. Actuat. 75–82. In: Seafoods Chemistry. Albala-Hurtado. Food Agric. Flavour of fish. A semiconducting metal-oxide array for monitoring fish freshness. 1994. 2002. and Vida-Carou. T. Biochem. A. B. Hanching. AOAC Official Method 971. 2001. Hashimoto. J...T.. 74–75. Quality Assurance in Seafood Processing: A Practical Guide. R. Quilty. C.). 49–56. Anal.. 871–877. B. Direct determination of trimethylamine in fi sh in the flow-reversal injection mode using a gas extraction sampling device. P. 161.... Comp. Sikorski. Validation of a flow-injection-gas diff usion method for total volatile basic nitrogen determination in seafood products. 29–34.C. T.. S. Int. J. Malle. B. Food Chem. Lindsay. Crosby Lockwood and Sons. Vrbanac. M.. J.. Veciana-Nogues.). and Kinsella. 575–610. Sadok.. U. and Navarro. B. 1987.. Great Britain. 43–49. K. Anal. and Zeisel. Oickle. J. Hammond.. Dhaouadi. M. 173. Sens. The effects of freezing on flesh proteins. Mitsubayashi. Rehbein. A. Development of a volatile amine sensor for the monitoring of fish spoilage. A. Validation of a gas-chromatographic method for volatile amine determination in fish samples. 509–518.. N. characterization and partial purification of TMAO-ase. Food Agric. 1992. M.. Bo M.K. Joly. 1989.. and Nakakura. Food. and Paulson. 70.. Segee. L. J.14. . 157. 9. 2004. trimethylamine and trimethylamine-imageoxide in fish extracts by capillary electrophoresis with indirect UV detection. Biochem. S. 160.. The measurement of dimethylamine. 1996.. and trimethylamine N-oxide using capillary gas chromatography-mass spectrometry.E. 170.. U. 1947. Marquis. 155. Bonnell. Conway. 162. 171... AOAC. Off. Trimethlyamine N-oxide demethylase (TMAO-ase) of saithe (Pollachius-virens) kidney-a study of some physicochemical and enzymatic properties. R.M. Monser.. Anal. J.. 99–112. A. and Jørgensen... products. Assoc. and Vetelino. Del M. J.. and Ducastaing. 158. London. Botta (Eds. Sens. Quantitative relationship between trimethylamine oxide aldolase activity and formaldehyde accumulation in white muscle gadiform fish during frozen storage..J. Micheals. 187(2).. Chapman and Hall.T. M.. Shahidi and J. Pacquit. Localization.. Processing Technology and Quality. Food Rev... 67. Microdiffusion Analysis and Volumetric Error. 43–47. S. Localization of formaldehyde production during frozen storage of European hake (Merluccius merluccius).. 33.... 2004. 233–341. 1995.E.. pp. 50. Z. B. Assay of biogenic amines involved in fish decomposition. Kubotera. Sci. Cottin.. Havemeister. Sci. Simultaneous determination of ammonia. Nielsen. B. 69. Aubourg. 463–467. 1993. Z.. Yano. B. Rapid quantitative determination of trimethylamine using steam distillation. S.K. trimethylamine. B..G.. E. 52. P. M. Rey-Mansilla. Chem.L. Agric. Pan.. In: AOAC Official Methods of Analysis.. Bo M... pp. A. Chapman and Hall. Mackie. 163. Diamonda.. 234–239. A. 1995. 1996. Kon. M. Oxidation of polyunsaturated fatty acids: Mechanisms.. 515–520..D...H... S. 169. Lau. 168. and F. 1990. p. 71B. Food Chem. Ann daCosta. 2007.T. Nutr. Changes in protein in frozen stored fish. 84. Chem. and Valcàrcel. 261–267.. Arlington. and Tao. W.S. L.R. and Adhoum. 113–122. 1049–1053. Food Res. Physiol. A. 165. and Valle.. Gill. Izquierdo-Pulido. Hsieh. Actuat. D. H..T. 79. 166. Res. Trimethylamine nitrogen in seafood..S. dimethylamine. 76. A. and Jørgensen.. 756–760. 2002. 213.. Y. M.. R. I.. Trimethylamine biosensor with flavin-containing monooxygenase type 3 (FMO3) for fish-freshness analysis. M. K. and Kolakowska.. Zhi. 167. McLaughlin. Z. 175. J. García.P. 156.. Chapman & Hall. K. Food Chem. Y. Castrillón. Rios.. Malle. F. 59. Sotelo. Álvarez-Pontes. 2006. Chem. M. M. H.. Food Res. Timm. 174.212 ◾ Handbook of Seafood and Seafood Products Analysis 154. In: Seafood Proteins. London. Eur.

179... 189. and D.. Quality loss related to rancidity development during frozen storage of horse mackerel (Trachurus trachurus). and Sotelo.. Modelling of microbial activity and prediction of shelf life of packed fresh fish. R. 198. Lingnert. and Ugliano. H. J. 2075–2081. C. R.. C. and Spie.. 2nd Edn. H. 2004. J. Sotelo. Benjakul. B. M. Jones. United States.P. 30. Series of Monographs. Food Microbiol. S. 2007. 193–199.. F...K. Kim. M. C. Int.E. E. 1999.. 671–678. 2000. In: Autoxidation of Unsaturated Lipids. Kitts (Eds. Tucker. Lugo-Sánchez. 189–193. 192. Effect of lipid oxidation and frozen storage on muscle proteins of atlantic mackerel (Scomber scombrus). 195.. JAOCS. J. T. 1997. Food Microbiol. 75(5). 1995. Prisk. and Robles-Burgueňo. I. Microbiological Testing in Food Safety Management.. Measurement uncertainty of the EU methods for microbiological examination of red meat.W.. 652–657. In: Seafood Safety.D. 196.. 208.W. 5–10.. S.. 289–294. 1995. and Medina. Assessment of quality hanges in frozen sardine (Sardina pilchardus) by fluorescence detection. 1110–1117. Wolf. L. Int.. 1983. Chan. Kolle. Lipid components and enzymatic hydrolysis of lipids in muscle of Chinese freshwater fish. J. . Miao. R. The mechanism of autoxidation. 184. Yuan.. Food Agric. Food Sci. Aubourg.. Hamilton. 305–318. Oil Chem. 187. Sci. Influence of prefreezing storage. E. Lida. Chantachum. 1985. 117–136. C. P. T. Methodology for following lipid oxidation in muscle foods.. 19. J.. R. 199. 111–116. Dalgaard.B. 197.. Nature of fluorescent compounds generated by exposure of protein to oxidizing lipids.. 2002.Methods for Freshness Quality and Deterioration ◾ 213 176. Chemistry of free radicals in lipids. 1998.. Kaneniwa. 502–507. Effect of brine pre-treatment on lipid stability of frozen horse mackerel (Trachurus trachurus). Undeland. S. A predictive model for the combined effect of pH. Saeed.. 1–16. 82. H. Y.. 825–831. Food Agric. 2000.. M. 40–47. Int. 121–137. Kluwer. Soc. 33. J. and Karel.. Aubourg. 38–80. 575–580. Gram. 1943–1948. Deman. H. 1987. McClure.J.. Corry.. Separation and quality of fish oil from pre-cooked and non pre-cooked tuna heads.. H. N. Borquez. 1987.J.. Food Agric. B. M. Principles of Food Chemistry. 161–178.. Food Science and Technology. J. Aubourg. and H. Aubourg... and Sriwirat. Agric. L. 180. Piñeiro.. An AVI Book. Food Technol. A rapid method to determine the oxidation kinetics of n−3 fatty acids in fish oil. Padley.. S. 1997. Technomic Publishing Company. Food Sci. and Pérez-Martín. PA. J. 69. Micro-organisms in Foods 7.S.J. Sacchi.. Food Res. 47. 188. 28. 2002. and Jarvisc.. Overview of current seafood nutritional issues: Formation of potentially toxic products. 2007. 1999. P.. W.G. Eur.. Kalu. 194.. and Pierce.). W. F.P. 1990.. Food Res. Food Biochem. Chan (Ed. M..W. 65(1). Leake. 19. pp. and González. Processing and Biotechnology.). M. ICMSF. 579–586. Medina I.. Kelly. Technol. Lipid oxidation in fillets of herring (Clupea harengus) during frozen storage. Shahidi.. C.... Am.M. Differential lipid damage in various muscle zones of frozen hake. Melton. 60(2). F. A. 2002. 190. and Aubourg. Technol.. R. 1999. 37(7).K... Eur. 182. J.. Food Microbiol. 1993. 81(7). 191. and Labella. 185. S. W..A. and Fukuda. I.... 77(8). Microbiological spoilage of fish and fish products.. J. 79. Influence of storage time and temperature on lipid deterioration during cod (Gadus morhua) and haddock (Melanogrammus aeglefinus) frozen storage. N. Postmortem biochemical and functional characteristic of monterey sardine muscle stored at 0°C. 215. 177. 193. R.S. Lipid Res. 24. S. Hedgesb. Food Microbiol. S. pp..P. A 13C-NMR study of lipid alterations during fish canning: Effect of filling medium. Food Chem.L. 69. 9.E. 1996. J. and Roberts. the Netherlands. Sci.. Rey-Mansilla. Food Chem. Baranyi. Lancaster.. Technol. pp... J. Sci. S. and Huss.D.. Food Chem.L.. 183. Y... Amsterdam. 445–450. sodium chloride and storage temperature on the growth of Brochothrix thermosphacta. 91–95. Academic Press.J.. Boogard.H. Pacheco-Aguilar. and Howell. J. 1997. M. Aubourg. Comparison of analytical methods for monitoring autoxidation profiles of authentic lipids.. JAOCS. S. U. J. 181. S.M. London.E. S. 178. 186..

66. J.. G... I.214 ◾ Handbook of Seafood and Seafood Products Analysis 200. J.C. 1986.. Environ. Rapid detection of oxacillin-resistant staphylococcus aureus in blood cultures by an impedance method. 2007. Applicability of an Arrhenius model for the combined effect of temperature and CO2 packaging on the spoilage microflora of fish. Microbiol.K. Rivas. 110. Lida. S. FEMS Microbiol. Nowak. 209. M. H. J. 202. T.S.. G. 263–268. Food Microbiol. S.. Landete. 1137–1142. A. 211.. V. S.. and Klein. Lee. Abe. 53. H. 1983.. P. and Muñoz. Müffling. Danbara. J. Ogden. Lambropoulou. 85–92. 117.. 114(2). de las. S.. Taoukis. and Magnani.. Irwin. June 1997. L. Juneja. 2007.J.. P... 2006.. C. Int. K. 66. J. Food Microbiol. Int.. 2007. 2004. J.. Lett. K.. 48. Huang. C..P. 201. 2000. Clin. . Carrasco. Brandi. J.E. 61. R. and Handley... R. Marcobal. Omiccioli. Food Microbiol..-J. K. A... E. 63–74.. H.. A. Predictive model for growth of Clostridium perfringens in cooked cured pork. Int. G. A. Amagliani.. Methods. Drosinos. 1989. Bacteriol.. 167–172... and Thippareddi. and Nychas. K. G.M. 204. B. J. Food Microbiol. Management of microbiological safety of ready-to-eat meat products by mathematical modelling: Listeria monocytogenes as an example.. J. 49(1–2). Microbiol. Chaunchom... Firstenberg-Eden...D.L. Appl. H.H. R. Koutsoumanis. Matsui. Int.G. 213. 1999. Andreotti.S.-J.E. Immunol. Evaluation of a rapid impedimetric procedure for the quantitative estimation of coliforms.H. J.. Wakayama. García-Gimeno. 114. E. Use of conductance methods to predict bacterial counts in fish...H. B.. 1460–1464. E. Appl. 259–267. 206. 221–226. 1307–1311. Bullock.K. T. Reed..M....Y. K. and Nychas. Appl. R.. Panicker. and Bej..H. J..E. 199–209. Bacteriol. Giammarini. 210.D. Valero. 205... J. 3528–3534. 1993. R. A. Food Control. and Zurera. Food Microbiol. J. Rapid impedance detection of salmonella in confectionery using modified LICNR broth. Int. Molecular methods for the detection of biogenic amine-producing bacteria on foods.. and Kawahara. 115. Microbiol. G. 212. P. Dai. and Frodsham.. 2007..A.. 97–106. 18.. G. C. Methods. Enzyme-linked immunomagnetic chemiluminescent detection of Escherichia coli O157:H7. Salmonella contamination in pigs at slaughter and on the farm: A field study using an antibody ELISA test and a PCR technique. Tu. Food Sci. and Chang.V. 258–269. Gehring. Pérez-Rodríguez. F. Rapid and sensitive method for detection of Salmonella strains using a combination of polymerase chain reaction and reverse dot-blot hybridization. and Hartung.. 2003. Akhavan-Tafti. 208.. D. Koutsoumanis. 203. A. A predictive model for the non-thermal inactivation of Salmonella enteritidis in a food model system supplemented with a natural antimicrobial.. 207. Wu.. Huang.. 293. 385–391. Detection of pathogenic bacteria in shellfish using multiplex PCR followed by CovaLinkk NH microwell plate sandwich hybridization. J. J.. Detection of Listeria monocytogenes using a commercial PCR kit and different DNA extraction methods.

................................................................................2 Morphological Examination.. 222 14......... 224 14................................... Marit Aursand.............................2 Protein Extraction ......................... Yumiko Yamashita.......................................................................... 225 215 .....................6 Stable Isotopes ................ Inger Beate Standal.....221 14......................... 225 14..............3.......1 Sample Preservation and DNA Extraction Methods ...................................... 220 14...................................4..........4 Analysis of Proteins .......1 Sample Preservation...................................216 14. 219 14.................. 220 14....................3.....................................................2 SNIF–NMR and IRMS ............217 14....................................................................................217 14...........218 14.....................................3 Genetic Analysis ............... 222 14.......................Chapter 14 Analytical Methods to Differentiate Farmed from Wild Seafood Iciar Martínez..................................1 Sample Preservation .. 222 14.................................................1 Introduction ...................3 Analysis of Proteins ............................................................218 14.........................................216 14.......................................5..4.............................................................................................2 Lipid Extraction and Gas Chromatography ..........................2 DNA Markers ............................. and Michiaki Yamashita Contents 14...........5 Analysis of the Lipid Content ..........................4.........................6..................................6................3 1H NMR and 13C NMR Analyses ......1 Sample Preservation .......................................................................................5..............................................................................5..........

. but wild specimens may contain parasites harmful to humans.................... and Cosmetic Act and The Fair Packaging and Labeling).. 227 Acknowledgments ........ and these are seldom present in farmed seafood................................ Although in this work no special mention is made to organic farming. 228 14. because farmed and wild organisms carry different hazards and are therefore submitted to different regulations and analytical controls....................... JAS Law................... farmed.... analysis of the protein and lipid contents...............7........................2 Also in an earlier study....................1 14... genetic analyses............. The later study showed that the environmentally induced phenotypic divergence increased with age and with the numbers of generations under domestication...2 Morphological Examination There are few publications and no official guidelines for the morphological differentiation of farmed and wild aquatic organisms............ and the United States (The Federal Food.......................5 The flesh of farmed cod sometimes presents .......... and length of the pectoral fin.. Standards for organic farming are still under development in many countries............. In particular.......................... salar parr.7.......7 Trace Element Fingerprint....1 Sample Preservation ... 100% discrimination between farmed (AquaGen strain) and wild parr was achieved by examining the body form................ including morphological examination. Farmed cod often present unattractive black lines consisting of layers of melanin-filled cells associated with blood vessels due to overabundance of copper in commercial feeds........216 ◾ Handbook of Seafood and Seafood Products Analysis 14... Correct information about the production method of seafood is also important................. 227 14........ the most prominent differences are the higher condition factor........................... Several methods have been successfully applied to differentiate farmed from wild seafood......... stable isotope analyses combined with fatty acid (FA) profiles have proven particularly useful when tested....... analytical methods should be made available to confirm it.............2 ICP-MS .................... 2001 laying down detailed rules for the application of CR EC No 104/2000 regarding informing consumers about fishery and aquaculture products) and similar laws apply in Japan (Law on Standardization and Proper Labeling of Agricultural and Forestry Products..................3 it was shown that the morphology of the head....................... In cod.... and caudal peduncles could be used for a total correct classification of wild.................1 Introduction The implementation of analytical methods to differentiate farmed from wild-produced seafood is important to ensure correct consumer information and avoid fraud: Information about the production method of seafood is obligatory in the EU (CR EC No 2065/2001 of October 22........ therefore...........8 Other Methods ..... The production method is also part of the information essential to fulfill the traceability of a product and................. Drug............. For example........ size of the eyes and mouth....... shape of the head........... of 1999)...... 227 References . 226 14.. commercially farmed specimens may contain residues of veterinary drugs whose presence is unlikely in wild seafood............... In small Salmo salar...... and sea-ranched S............................. as well as examination of the stable isotope and trace element profiles....... larger liver...................... and smaller head4 as well as backbone malformations in farmed specimens...... the set of technologies to apply are basically the same as those described here.... fins........................ 226 14........

which is the most common analysis. If this policy had been followed.).8.18 or gel filtration. Depending on the type of sample and its use.1 Sample Preservation and DNA Extraction Methods The sample should be extracted as soon as possible after sampling. in particular if it has a high enzymatic activity (for example if it contains the hepatopancreas in a crustacean). so that all the ethanol is evaporated. Samples fi xed in ethanol must be allowed to dry completely. For very long periods.3. give satisfactory results. E. Dynal (Invitrogen). chloroform extraction. in most breeding programs the fish are indeed selected based on commercially interesting traits such as growth performance.17 suggested that it was possible to assign accurately a fish sampled from the market place to either the farmed population or the wild using either microsatellite or single nucleotide polymorphism (SNP) markers.11–13 and a loss of rare alleles has usually been observed in the farmed populations. and then recovered by ethanol or isopropanol precipitation.7 proposed that the genetic diversity of aquacultured stocks of fish should be maintained and their genetic impact on wild stocks minimized by using breeding programs designed to generate genetic diversity. a normal freezer (−20°C) may also be used. thus limiting its application. To extract frozen samples we recommend to start the procedure before the sample is completely thawed. If the sample must be preserved.9 Genetic analyses have allowed the differentiation of wild from farmed fish populations in a variety of species. 14. treatment with Chelex. classification based on morphological criteria demands the presence of the morphologic diagnostic characters. it would be relatively difficult to find markers for wild and farmed fish.14–16 Hayes et al. Amersham Biosciences (GE Healthcare). Delays and the use of preservations methods will diminish the quality and the yield of DNA. which are usually absent in many intermediate products as well as in the ready-to-eat dish.9 resistance to diseases or to stress. 14.6 However. and then be rehydrated in water or in the extraction buffer. One requisite condition for any genetic analysis is the obtention of good quality DNA suitable for PCR amplification.A Stool DNA Isolation Kit (United Bioinformatica Inc.N. such as Qiagen. The basic steps in all DNA extraction methods include the inactivation of nucleases. Genomics analyses are dealt with in more detail in Chapter 4 of this handbook.).Analytical Methods to Differentiate Farmed from Wild Seafood ◾ 217 a translucent grayish aspect. the cells are opened (by heat treatment. and others. Th is step is not necessary in samples preserved in ethanol. GeneRelease (Bio Venture Inc. because the enzymes are inactivated by the fi xation. Nucleospin (Clontech). The DNA is then separated from the contaminating cellular components by salt precipitation. since enzymatic activity also takes place at subzero temperatures.11. or by the use of Proteinase K) and proteins are removed usually by incubation with Proteinase K. Each kit is provided with a detailed description of how to use it. sonication. Then. The DNA . for example by chelating divalent cations using EDTA and EGTA. Wizard (Promega). in contrast to the white opaque color of the wild.10 and optimal adaptation to different environments.Z. and the liver in farmed cod is much bigger than the liver of wild fish.3 Genetic Analysis Doyle et al. since diversity would be one of the selected traits in the farmed fish. we recommend preservation in 96% ethanol. Many commercial kits. However. the best method is to freeze it in liquid nitrogen or in a biofreezer.

On other occasions. tilapia. tilapia. has the potential to be useful to differentiate farmed from wild specimens of a given species. the United States.3.13. from food matrices include the use of hexadecyl–trimethyl ammonium bromide (CTAB). genomic.11. Japan. that the Norwegian company GenoMar has patented a method to trace back farmed individual Atlantic salmon. Three more methods that have reputedly produced good quality DNA suitable for amplification. we have found it helpful to leave the tubes after the first precipitation of DNA with isopropanol in a freezer at −20°C or at −80°C for a few hours before centrifugation (Marian Martinez de Pancorbo. however. This method has been successfully used by the authors of this paper (unpublished results) and by Bucklin and Kochert22 with whole individuals of Calanus. China. 14. by using a series of SNP and microsatellite polymorphisms by PCR and by oligonucleotide ligation assay (OLA).). DNA analyses may be performed using chips that permit the determination in one fast step of many characteristics simultaneously. which markers and how many of them are necessary to differentiate a wild from a farmed specimen are completely dependant on the species and the breeding stock and need to be examined on an individual basis. the alcohol is allowed to evaporate. cod. SNPs. pH 8. any marker. traits.24 The method requires that all parent fish of the brood stock are DNA typed as well as all the individuals under examination. and bass. and sea bass. cod. including the species. breeding stock. India. . several countries. 14.0).15.14 In addition. In the future.17 However. which is washing the pellet with 50% ethanol.2 DNA Markers In recent years.23 In principle.21 When using the salt extraction method with heavily degraded samples. have started programs to map the whole genome of some species. protein markers commonly used for genetic analyses have the potential to be used as markers for farmed or wild. which may be used to identify the strains of the farmed individuals that display an increased frequency of the desired traits. including oysters. such as Norway. it is possible to amplify the DNA of a sample by simply dehydrating it and placing a small amount directly into the PCR amplification mixture. Atlantic halibut. shrimp. salmonids (salmon.27 tissues between farmed and wild salmon and cod have been reported. and production method. personal communication). which further increases the amount of samples that can be processed.20. so the next step.218 ◾ Handbook of Seafood and Seafood Products Analysis pellet is usually washed at least once with 70% ethanol. and also for forensic studies. An additional advantage is that it is possible to use robots for many of the steps (DNA preparation. However. sample preparation. and others.4 Analysis of Proteins No clear protein marker has been identified to discriminate farmed from wild seafood. Arctic charr). It is worth mentioning. catfish.18 and the salt extraction method. must be performed very carefully not to lose the sample. differences in the protein pattern of liver25 and muscle26.19 the Chelex method. however. Spain. whether microsatellites. University of the Basque Country. since some alleles are more frequent in one group than in the other. or mitochondrial. trout. and the DNA is reconstituted in double-distilled sterile water or in a slightly alkaline buffer (50 mm Tris–EDTA. etc. The outcome of these programs is already producing lists of genetic markers linked to traits of interest. In these samples the pellet may be practically invisible.

optimal freezing would be achieved immediately after excision by submersion in liquid nitrogen and storage at −80°C or by freezing and storing directly at −80°C. which they use as their preferred energy source.32 Unfortunately. aggregation. Thus. Some enzymatic systems that may be responsible for the muscle softening are metalloproteases and collagenases. Optimal methods include fast freezing and frozen storage using temperatures as low as possible. and then modify them depending on the results. and they require high levels of dietary protein (30%–60%).35. etc. The authors noted a downregulation of some structural proteins in fish-fed soy proteins. and structural and FA-binding proteins. When this is not possible. Interestingly. Zn.. is more common in stressed and in farmed than in wild fish. several enzymes. have prompted the development of feed formulations based on vegetable oils and proteins. lectins. these authors identified 33 differentially expressed proteins. Olsson et al. Johnston et al. −20°C . Proteins and proteomic analyses are dealt with in more detail in a different chapter of this handbook. it has been shown that components in fish feeds may contain very high levels of metals (Cu. which would be a natural diet. and feed diets based on plant protein require supplementation with synthetic amino acids.28–31 The source of protein in teleost fish is very important. which is reflected in the composition of their organs.33. with no preservation at all. neither feeds nor breeding conditions may be optimal for farming. and they hypothesized that the greater concentration of insoluble collagen present in wild salmon may contribute to their firmer texture. lysosomal cathepsins.25 attributed the alteration in the protein expression in the liver of rainbow trout to the presence of antinutritional factors in feeds containing soy protein. attributed to the fish’s increased requirement for energy metabolism.Analytical Methods to Differentiate Farmed from Wild Seafood ◾ 219 Industrial fish farming is a relatively new activity compared with farming of land animals. For short periods of time. Soft texture. that is. Mg. a preservation procedure that minimizes the modifications (denaturation.4. several enzymes involved in anabolic metabolism were downregulated in fish fed the diet rich in soybean meal. and proteolysis) of the proteins in the sample should be chosen.37 Martinez et al. the depletion of the wild stocks of pelagic fish and the high price of feeds based on fish meal and oil. and Ca)34 and that vegetable meals may contain antinutritional factors (protease inhibitors.36 Martin et al.38 found that the reason for the softening in this species did not seem to be the faster growth of the farmed fish.27 also registered the altered expression of five enzymes implicated in the glycolytic pathway and citric acid cycle in farmed cod. Thus. which were attributed to increased proteolytic activity in the muscle of the farmed compared with the wild cod. 14.1 Sample Preservation The optimal case would be when the extraction of proteins can take place on the sample immediately after the experimental treatment. indicating increased emphases on catabolism relative to anabolism in the fish fed this diet. However. loss of functional groups. including heat shock proteins. neutral calcium-activated calpains. Using proteomic analysis. usually considered negative. In addition. antigenic proteins. Although feeds and breeding conditions need to be developed and optimized for each species.33 Moreover. and the proteasome. Fe. and this may induce stress in the farmed animals.)29 that may have adverse effects on fish. Texture is an important quality attribute of the fish flesh. no study has identified yet the main system/s responsible for the soft texture in farmed fish or the spots that may be used as markers to discriminate farmed from wild fish. the amino acid profiles of plant proteins do not meet the essential amino acid requirements of fish. it is common to apply directly to new species those conditions that have proven successful for other species.26 examined the protein expression in skeletal muscle of farmed and wild cod by high-resolution twodimensional electrophoresis and found differences between the two.25–27 In addition.

The choice of method depends on the protein and the property one wishes to examine. tributylphosphine) to solubilize the widest possible spectrum of proteins. cooking. as well as the different degrees of processing to which the sample may have been submitted (freezing.2 Protein Extraction There are many methods for extraction of proteins. However. and. thiourea). the strip containing the proteins separated by their pI is loaded on top of the second-dimension SDS-PAGE gel. It is common to use several buffers with increasing concentration of chaotropic agents (urea. Proteomic techniques have a clear advantage in this field. and reducing agents (b-mercaptoethanol. usually with trypsin. and peptide mass fingerprinting of the digests is then . detergents (CHAPS. We therefore recommend not to use such enzymes. silver (high sensitivity. It should be noted that any preservation procedure will alter the protein profile in the sample. the optimal extraction procedure for any given sample must be determined empirically.).3 Analysis of Proteins As already mentioned. The tryptic fragments are cleaned from contaminants. 14.220 ◾ Handbook of Seafood and Seafood Products Analysis may be acceptable. The use of protease inhibitors should always be considered: use of some inhibitors and cocktails may help to preserve the sample during the extraction procedure. therefore. one should be very careful when comparing samples preserved and stored under different conditions. The first step in proteomic analyses is to extract as many proteins as possible from the sample.4. the gels can be stained by Coomassie Blue (low sensitivity but compatible with mass spectrometry (MS) analysis necessary for subsequent peptide fingerprinting and sequencing). dithiothreitol (DTT). depending on the proteins one wishes to examine. Since current studies are still trying to identify markers. and fluorescent labeling or staining (of intermediate sensibility and also compatible with MS). The optimal pH range to choose depends on the sample. therefore. its application is widespread in many fields. In our experience.25–27 BioRad39 and GE Healthcare Amersham have some excellent manuals about protein extraction and analysis. and. Their use should be evaluated for each particular study. Afterward. for a wide screening. Triton X-100. and digested. 14. alkylated. After separation. The pictures of the gels containing similar samples of wild and farmed specimens obtained after scanning are compared using adequate software (such as Bionumerics or PDQuest) to identify differentially expressed spots that are then excised from the gels. but 3–10 are commonly used for wide screenings. destained. Both first and second-dimension gels can be purchased as precast. ready to use gels from several companies. we focus on the use of techniques with the potential to identify such markers. due to the great diversity and properties of the proteins contained in the edible tissues of seafood. but not all protocols are compatible with MS). Some authors have claimed that the use of DNase I and RNase in the extraction buffer increases the number of spots in the gels. there are many methods suitable for protein analyses. but they will hamper the study of protease activities that may be relevant in some other works. etc.4. The proteins are separated first according to their pI in 3% polyacrylamide gels in which a pH gradient is created using a mixture of ampholytes. and there are special protocols for each application. Proteomics permits the separation of many proteins (often thousands) from a complex protein mixture in one step. this may be because some commercial preparations of these enzymes are contaminated with proteases. SDS). reduced. usually 8%–20% or 12% PAGE. In addition to published works.

C16:0 and C18:1n9 are relatively abundant in all fish oils. Once the diagnostic proteins are identified. however. very different staining intensities. sand eel.44 Frequently. or menhaden. herring.45–48 In addition. For example.44 and this FA fingerprint has often been successfully used49–52 as a diagnostic to identify the production method. for example. so that the concentrations in flesh were higher than in the diet. and sunflower) used as partial substitutes for marine oils in fish feeds53–57 have in common a very low or undetectable amount of the long-chain omega-3 polyunsaturated fatty acids (PUFAs) C20:5n3 (EPA) and C22:6n3 (DHA) characteristic from fish oils.). capelin.42. has often given correct classification of farmed and wild specimens. C22:1n11. olive. cottonseed. and ELISA format will permit the routine analyses of many samples.43 The changes in the FA composition of the TG fraction following changes in the composition of the diet have been explained using a dilution model. which FA is more abundant is species dependant. herring. and C18:3n3 were selectively metabolized. the whole process can be greatly simplified by targeting only the biomarkers: raising or synthesizing antibodies targeting those proteins in order to use them in several formats. etc. the final identification and assignment of the spots in the gels must be performed visually by trained personnel.53. but C22:1 (several isomers) is relatively more abundant in Coho salmon. The proteins are afterward identified by searching in databases (National Centre for Biotechnology Information. and often a single FA may account for about 50% of the total FA content in these oils. and it is seldom that only one of them makes more than 25% of the total. In Atlantic salmon for example. linseed. soybean. Proteomic analysis is a complicated procedure necessary to identify the biological markers. The FA composition of fish oils is more complex.).55–58 Specific FAs are selectively retained or used. palm. and sand eel and C22:6n3 is more abundant (over 10%) in Atlantic and Coho salmon. spot cutters. the total amount of TGs alone may be used as a criterion to differentiate farmed from wild fish.Analytical Methods to Differentiate Farmed from Wild Seafood ◾ 221 usually performed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS.5 Analysis of the Lipid Content The analysis of the triglyceride (TG) fraction. due to frequent errors in the automated spot identification procedure (because of imperfect spot separation and identification caused by overlapping of spots.25 and reviewed by Granvogl et al. 14. rapeseed. the FA profile of TGs reflects that of the feed. there are more FAs present in detectable amounts. in control laboratories. in particular when combined with stable isotope composition (see the following paragraph). etc. The whole procedure is described in detail by Martin et al. and sardines than in capelin. The FA profile of vegetable oils (such as corn. However.60 The FAs C18:1n9 and 18:2n6 may act as markers . NCBI) using suitable software (MASCOT. whereas C18:1n9.41 The workload can be reduced by using precast gels and automated procedures with suitable software and robotic stations (sample and gel handling and staining. lateral flow strip tests permit in situ easy and fast screening of seafood samples. that is. identification of diagnostic spots. C18:2n6. which uses MS data to identify proteins from primary sequence databases. ProteinLynx Global SERVER).59 Other studies in the same species showed that the flesh had higher levels of C18:1n9 and C22:6n3 and less C20:5n3 than the feeds. Development of protein chips will facilitate the simultaneous screening of many targets and samples. This is probably the most time consuming step of the whole procedure. As in vegetable oils. it has been shown that there was selective deposition and retention of C22:6n3. because farmed fish usually have a much higher content than wild ones.

Fresh samples should be kept wrapped in air. which leads to less overlapping of signals. 29Si. it is best to use as low a temperature as possible..52 that give detailed descriptions of the procedure. 31P. oxidation. it is particularly important to exercise care when working with marine lipids: it is recommended to use low temperature (work in ice or in a cold room) and avoid or minimize exposition to air and light in order to prevent lipid hydrolysis. 14. The major advantage of 1H NMR spectroscopy compared with 13C NMR is the higher sensitivity and thereby shorter acquisition times per experiment. the latter seems to be the most persistent after a dietary switch to fish oil diet. and even after the levels of C20:5n3 and C22:6n3 were restored to the original high levels. since in a one-dimensional spectrum each peak is produced by those nuclei placed in an identical local chemical environment.64 may contribute to the difficulty of performing correct classifications as wild/ farmed based only on the FA composition. which. 14.5.62 one must always take into account the very wide variation in the concentrations of lipid components that can be found in apparently homogeneous populations of farmed salmon. the ratio n3/n6 was not fully restored. The spectrum is often used to obtain information about the number and type of molecules in a mixture.52. 2H. and polymerization. 14. 35Cl.1 Sample Preservation Due to the high levels of PUFAs. 17O.g. 23Na. which may be used as a rapid profiling technique. including fish and fish products. together with the special feed formulations used for organic farming and the fact that escaped farmed fish and wild fish eating around farms may display intermediate lipid profiles. 11B. −80°C.61 As indicated by Refsgaard et al. NMR spectroscopy exploits the magnetic properties of certain nuclei: nuclei that contain odd numbers of protons or neutrons have an intrinsic magnetic moment and angular momentum. and is the preferred tool in lipid analysis when interpretation of .65 NMR gives a fingerprint of the sample analyzed. For detailed descriptions of the analysis of fish samples. HR-NMR has been particularly valuable in the study of marine lipids.222 ◾ Handbook of Seafood and Seafood Products Analysis for vegetable oils. If freezing is required.63. 195Pt)..3 1 H NMR and 13C NMR Analyses High-resolution nuclear magnetic resonance spectroscopy (HR-NMR) has emerged as a popular technique in the analysis of foodstuff. NMR spectroscopy can be used to identify functional groups.5. 10B. both of which are able to detect a range of metabolites in a nontargeted way. and an inert atmosphere.50. 19F. and in particular.and light-tight containers and stored at low temperatures. that is. but NMR is applicable to any nucleus possessing spin (e.5. On the other hand. the reader is directed to several publications46. because it provides multicomponent information and can be applied nondestructively. The most commonly measured nucleus is 1H (the most receptive isotope at natural abundance). The most commonly used HR-NMR techniques in wild/farmed classification are 1H NMR and 13C NMR.2 Lipid Extraction and Gas Chromatography Procedures for lipid extraction are described in another book chapter of this series. 13C NMR has a greater range of chemical shifts. 13C. 15N. 14N.

76 In some studies. 1H NMR has also been applied to differentiate between wild and farmed salmon and sea bream of different origins.71 13C NMR gives information about FA composition of fish72 and the positional distribution of PUFAs in triacylglycerols and phospholipids.Analytical Methods to Differentiate Farmed from Wild Seafood ◾ 223 spectra is the goal. in conjunction with chemometrics. even though the signal intensities within each spectrum are not quantitative. 13C NMR and 1H NMR spectra are fi rst obtained by Fourier transformation of the resulting free-induction decay (FID) function after applying a prospective line-broadening function. the whole procedure from sample preparation to analysis by a data exploration technique can be affected by factors unrelated to the sample characteristic of interest. the chemical shift scale is referred to the shift of TMS or indirectly to TMS by the peaks from chloroform at 7. Factors that affect the exact chemical shift of NMR signals include the type of solvent used. they are normally converted to ASCII or JCAMP file formats.75 and it is important that all the samples contain the same volume. a sample size of 50–100 mg of lipid in 0. such as instabilities in apparatus.73 which is of value for authentication purposes. a semiquantitative 13C NMR approach has been chosen. Multivariate methods are frequently applied to study differences among NMR spectra. because it may interfere with the multivariate data analysis. although this approach has still not been widely used for authentication purposes. 99.8% CDCl3). pH. may lead to erroneous classification. ideally for screening many samples with short acquisition time.69 This analysis can be carried out with a high degree of automation and gives a rapid fingerprint (2–5 min) of the lipid profile.71 Another technique that in the future may be used more often is the analysis of intact tissue by high-resolution magic angle spinning (HR-MAS). interactions with metal ions. The application of multivariate statistics to NMR spectral data increases the potential of the technique considerably. It is expected that in the future the use of flow injection systems.75 Small differences in experimental conditions. Regions without signals or unwanted signals are removed before multivariate analysis. or differences in relative concentrations of the samples analyzed. which is easily evaporated. inhomogeneities in the applied magnetic field.77 It is advisable to check that all spectra have acceptable linewidth and lineshape after the NMR analysis. have allowed the differentiation between wild and farmed salmon74 and cod51 of different origins.67 1H NMR has been used to perform quantitative measurements of total n-3 FAs and of the levels of DHA.68.28 ppm for 1H NMR and by the triplet of CDCl3 at 77.5–0. hydrogen bondings. Tetramethylsilane (TMS) is usually added as a chemical shift and intensity reference. However. the relative intensities for corresponding signals across different spectra are comparable. Both HR 1H and 13C NMR. will increase the sample throughput significantly. The most commonly used solvent in the analysis of neutral lipids is deuterated chloroform (i. and other intermolecular interactions.0 ppm for 13C NMR. Both the area/ intensities of peaks and full spectra can be input for multivariate analysis. Normally. When full spectra are used. Typically.66.78 . leaving the sample ready for analysis. The assignment of spectral resonances gives information about the chemical composition of the samples.77 Regarding reproducibility issues. although the optimal sample size depends on the instrument. but it is not necessary for classification purposes. Standardized procedures should be followed to ensure repeatability and comparability.8 mL solvent is used. due to the fact that quantitative measurements require a considerable longer experimental time.e. temperature variations..66 Potential problems about inconsistencies in ppm values between samples in the data analyses should be solved by manual alignment or data pretreatment methods.70. Typically. phasing and baseline correction are applied but no zero fi lling.

037%). For example. Since a molecule containing heavier isotopic forms has stronger chemical bonds.80 The natural isotopic abundance largely varies depending on the chemical forms. SD ± 0. equilibrium reactions also lead to a fractionation of the isotopic forms. although many broadleaf plants are also C4).204%).23‰) than the aquaculture specimens. Using the d15N of choline and the d18O of total oil. Usually animal products become enriched in the heavier isotope (15N and 13C).82 Dempson and Power83 examined the potential of using stable isotopes of carbon and nitrogen 13C and d15N) by isotope ratio mass spectrometry (IRMS) to identify escaped farmed Atlantic (d salmon. the higher the proportion of the heavier isotope.6 Stable Isotopes The variation in the abundance of the stable isotopes of carbon. For example. resulting in a complete separation of the two groups. Bell et al.38‰) but depleted in lipid-corrected carbon (d13C: mean = −20. mostly broadleaf plants and plants in the temperate zones) shows a higher degree of 13C depletion than the C4 plants (where the CO2 is converted first into a four-carbon organic acid: these plants are mostly found in warm sunny regions. and 18O (0.37%). while typical d13C mean values of C3 plants may be −26/−28‰.50 were also able to correctly classify Atlantic salmon according to their geographic origin and production method by using four FA compositions (C16:0. Introducing the percentage of C18:2n6 as a third variable in their model. 17O (0.759%). the 13C/12C ratio for both milk fat and cheese protein give information on the type of forage fed to the cows.52 were able to classify correctly according to the production method. nitrogen as two: 14N (99.224 ◾ Handbook of Seafood and Seafood Products Analysis 14. N2. Samples of muscle tissue of wild salmon were significantly more enriched in nitrogen (d15N: mean = 12. and they reflect both significant isotopic fractionation by microbes and the different biological substrates producing these gases. Thomas et al. Thus. C4 plants may have d13C mean values of −12/−14‰. depending on their diet and their position in the trophic chain: the higher its position in the trophic chain. A significant kinetic fractionation is already found in the initial fi xation of carbon dioxide in photosynthesis: the isotopic signature of C3 plants (plants that form a three-carbon compound as the first stable intermediate in the incorporation of CO2. In addition. 171 Atlantic salmon specimens originating from three continents and 15 different geographic regions. Some atmospheric gases.75. Moreover. N2O and CH4 exhibit wide isotopic variation. and oxygen has been proposed as a method suitable for food authentication. and O2. and C22:1n-9) together with the overall isotope ratio 2H/1H of the fish oils and three deuterium molar fractions obtained by site-specific natural isotope fraction studied by NMR (SNIF–NMR). SD ± 0. nitrogen.46 were equally successful classifying sea bass using the FA profile.79 Carbon exists as two stable isotopes: 12C (abundance 98. Differences in the 15N/14N ratio also result essentially from diet. Aursand et al.63) and 15N (0. d13C of individual FA. the abundances of the stable isotopes differ between substrate and product. C16:1n-9. they were also able to correctly classify the fish according to their geographic origin.11%). and oxygen as three: 16O (99. since the physical properties of molecules containing heavier isotopic forms are different.51. such as maize.81 This is because the 13C/12C ratio depends almost exclusively on the photosynthetic mechanism used by the plants for CO2 fi xation. The isotopic abundances in animal tissues and animal food products are the summation of the feeds ingested throughout all their life. exhibit limited variation. because the enzymatic reaction rates on substrates that contain the lighter isotopic forms are faster than in reactions involving the heavier isotopic forms. In contrast. a kinetic fractionation occurs. C18:1n-9. such as CO2.89%) and 13C (1. typically tropical grasses. the abundance of stable isotopes varies among different compounds. plus the kinetic fractionations occurring in animal metabolism. .

and stored in glass desiccation vials until analyzed. The light elements. it must not be washed in the laboratory after collection (which may alter the O and H profile of the sample).Analytical Methods to Differentiate Farmed from Wild Seafood ◾ 225 d13C and d18O of total muscle oil. are typically determined with a gas isotope rationing mass spectrometer. thus hydrogen is introduced as H2. 14. and a detector to measure the different isotopic species. such as carbon. The instrument consists of an ionizing source. d13C values are normalized for lipid content following techniques developed by McConnaughey and McRoy87 and validated by Kline et al. The gas is introduced in the mass spectrometer and is ionized by removal of an electron in the ion source. pulverized to a fine powder using a ball mill grinder. for nitrogen 15N:14N). and environmental conditions on fish tissue.48 NMR techniques have been described previously. probably reflecting the metabolic functions of these tissues and their associated turnover rates. The assumption that fractionation was independent of body mass was upheld for muscle and heart tissue but not for liver. The ionized gas is then introduced in the flight tube under vacuum or carried by helium. All international standards are . experimental duration. the collected tissue samples are dried at a constant temperature of approximately 50°C for 48 h.1 were able to differentiate wild. However. Molkeltin et al. nitrogen as N2.1 Sample Preservation It is very important not to contaminate the sample during handling. the d15N values of heart and liver were also affected by environmental temperature. Enriched samples contain relatively more of the heavier isotopes. Approximately 1 mg of dried. and oxygen isotopes.2 SNIF–NMR and IRMS Two methods are used to assess stable isotopes: SNIF–NMR and IRMS. ground tissue is used in the simultaneous analysis of stable C and N isotopes.85. For example. since these authors assessed the effects of body size. respectively (for carbon 13C:12C.88 Stable isotope ratios are expressed in delta (d) notation with measurements consisting of parts per thousand difference (‰) between the isotopic ratio of a sample relative to an international standard. the ions are finally detected at the detector. a flight tube with a magnet. and so on. The C and O isotopic profiles of fish tissues may be altered if CO is used for stunning or killing. Interestingly. An advantage of SNIF–NMR over IRMS is that it produces a distinct isotopic fingerprint giving information on the frequency of each isotope in a given molecule and the position of the isotope in the molecule. as follows: d = (R sample/R standard − 1) × 1000‰. SNIF–NMR can only be applied to the few isotopomers possessing spin. The element is converted to a gaseous form to be analyzed by the mass spectrometer.86 To facilitate comparisons between specimens with differing lipid contents.6. the paths of isotopic species are deflected by the magnet by an angle that is a direct function of their mass over charge ratio. The research of Sweeting et al.84 has helped to understand the nitrogen isotopic variations in fishes. organic. where R is the heavy:light isotopic ratio of the sample or standard. and oxygen as CO2. nitrogen. whereas IRMS can be applied to all except 12 elements. whereas IRMS gives only an average value of the isotopic forms in the molecule.6. 14. Usually. and d15N of the glycerol choline fraction of flesh phospholipids. and commercially farmed Atlantic salmon measuring d13C and d15N by IRMS in raw fillets. carbon as CO. and the abundance ratios of the heavy and light isotopic species are then calculated.

attributable to cobalt. and vanadium. All implements and containers should be cleaned with 0. as well as vitamin K and its metabolites. In addition. The same research group (Yamashita et al. farmed and wild specimens of the same species have different geographic distributions. Recently. unpublished data) examined the trace element composition of the muscle and shell of littleneck clams collected in Japan. and they found distinct patterns for each of the three origins. Therefore. and analysis. 14. with the exception that clams from Miyagi had high arsenic content.91 In the case of fish. 14. Rare trace elements taken from the environment. The first step in the analysis is the digestion of the sample: 0.226 ◾ Handbook of Seafood and Seafood Products Analysis set at 0‰ by convention. quality. and often the geographic origin of both farmed and wild seafood may be of relevance for its safety.95 By using ICP-MS analysis the sensitivity in the determination of rare trace elements can be increased from the nM to pM level. multivariate trace elemental analysis is expected to be helpful in determining whether the fish was farmed and its geographic distribution. and strontium levels and Factor 3. multiple elemental analysis could also be used in this case to identify imported clams from China and Korea. Thirteen elements were shown to be the most diagnostic. Taiwan.. copper. Korea. cadmium and arsenic levels in the muscles of clams from China and Korea were higher than those of clams from Japan. were shown to be of relevance to determine the origin of eels. false labeling problems were encountered in which imported live Japanese eels from Taiwan were illegally sold as being of Japanese origin.7 Trace Element Fingerprint Sometimes. Multivariate analysis showed that differences in elemental composition in the muscle between Japanese and imported clams were mainly due to two factors: Factor 1. Thus. Each sample should be separated from the tissues using ceramic knives and scissors and Teflon-coated tweezers to avoid contamination of metals. Multivariate trace elemental analysis is increasingly used as a technique to differentiate the geographic origins of foodstuff. The sample may be stored in a centrifuge tube at a temperature of −40°C or lower until analyzed. and it must be accurately weighed with a microbalance. Carbonate rock from the Pee Dee Belemnite formation89 and nitrogen gas in the atmosphere90 are used as the standards for carbon and nitrogen. otolith chemistry is used as a recorder of time and environmental conditions. cadmium. have been used to differentiate the geographical distribution of origin of farmed Japanese eel. in addition to DNA-based species identification techniques. Biochemical analytical techniques using multiple elemental analysis. and price. handling. such as uranium.1 Sample Preservation As for stable isotope analysis. lead.92–94 Otolith chemistry is useful for identifying the natal origin and assessing the relative contribution of different nursery areas to mixed adult stocks. it is very important to avoid contaminating the sample during sampling. and China were compared by analyzing the trace and heavy metal contents in the muscles to determine the differences among the fish farms for cultured eels and also to identify the river where wild eels had been caught.7.1–1 g of tissue samples are placed into 50 mL Teflon tubes and 8–16 sample volumes of a mixture of concentrated trace-metal-grade nitric acid/hydroperoxide mixture (5:3) is added. in particular since the analysis may detect contaminants at the pM level. The . respectively.5 M nitric acid and rinsed with Milli-Q ultrapure deionized water. The origins of farmed and wild eel collected from different regions in Japan. and China. attributable to manganese and vanadium levels.

and Bi. Multiwave 3000 Microwave Oven.97 using an automatic mercury analyzer (Hiranuma HG-200. For the determination of mercury.98 However. In the farming of salmon for example. Y. Acknowledgments This work was carried out with the financial support of the EU-STREP Project Sigma Chain: “Developing a Stakeholders’ Guide on the Vulnerability of Food and Feed Chains to Dangerous Agents and Substances” Contract No FOOD-CT-2004-506359. Tb. there might be specific requirements that may be targeted to identify the production method. Ions transmitted through the quadrupole are detected by continuous dynode electron multiplier assembly. Each solution (5 mL) of the microwave samples is applied to the atomic absorption spectrophotometer. 14. the mass calibration and resolution are checked using diluted metal solutions as standards. most artificial feeds contain a mixture of canthaxanthin and astaxanthins of different origins (both natural and synthetic). the instrument is recalibrated. The ions are extracted from the plasma through a differentially pumped vacuum interface and are separated on the basis of mass-to-charge ratio by a quadrupole mass spectrometer that has a minimum resolution capability of 1 atomic mass unit (amu) peak width at 5% peak height. and the ion information is processed by a data handling system. and the last 10 samples are analyzed again.Analytical Methods to Differentiate Farmed from Wild Seafood ◾ 227 digestion may be carried out by placing the tubes in a microwave oven (for example. much of the astaxanthin used in fish feed nowadays is produced from cultured microalgae or from krill.42. In. 14. If the measured concentration deviates from the true concentration by more than 10%. an internal standard mixture is added. Japan). the Norwegian Research . a calibration blank and calibration standards are used as surrogate test samples after every 10 analyses. and ionization. Perkin-Elmer). To verify that the instrument is properly calibrated on a continuous basis. the total mercury concentration is determined by cold vapor atomic absorption spectrometry. Canada). Analysis by chiral chromatography can be used to identify a chiral form (the meso form 3R.2 ICP-MS Multielement determination of trace elements is usually measured by inductively coupled plasma mass spectroscopy (ICP-MS).98 making this approach more unreliable than it used to be.3′S) that does not occur naturally and can therefore be used as a marker for farmed salmon. To initiate the proper operating configuration of the instrument and data system. the samples are diluted to a final volume of 50 mL in Digitube (SCP Science. and the resulting digest is a clear liquid with a yellow tint. the use of carotenoids is allowed.96 Samples digested as described above are introduced by pneumatic nebulization into a radio frequency plasma. For internal standardization.7. five internal standards are used: Sc. Afterwards. where energy transfer processes cause desolvation. but although the diet of wild salmon contains astaxanthin. and stored at room temperature until use.8 Other Methods Depending on the species. atomization. which suffers from severe memory effects.

224. 148.E. and Nakamura. Selective diversification of aquaculture stocks: A proposal for economically sustainable genetic stock conservation. Genetic improvement of fanned tilapias: Composition and genetic parameters of a synthetic base population of Oreochromis niloticus for selective breeding. Jobling. Genetic differences between five major domesticated strains of Atlantic salmon and wild salmon. Gildberg. 7. A. 18.J. Mar. M. 10. Aquat. Aquaculture. Berg. 238.. 67. Aquaculture. Aquacult. 213. Aquaculture. 2006. Kang. 52. and Gunnes. C.. Microsatellite DNA variation in wild populations and farmed strains of turbot from Ireland and Norway: A preliminary study. 250. et al. J. 131. Food Res. 69. 261. Evaluation of three methods for effective extraction of DNA from human hair. A. Genetic comparison of experimental farmed strains and wild Icelandic populations of Atlantic cod (Gadus morhua L. I. Inter.P. D.. 16.. G. 48. Ø. and Martinez. farmed and wild salmon. Roed..J.M.. Fleming. R.. Aljanabi. 3. 67.. NAR. J. Can. 2005.. Hayes. Sci. 63. Microsatellite analysis in domesticated and wild Atlantic salmon (Salmo salar L. Gadus morhua L. Applications of selection for multiple traits in cagereared Atlantic salmon (Salmo salar). Atlantic cod (Gadus morhua). and by grants from the Fisheries Research Agency and the Ministry of Agriculture. B. Fish Biol. B. 43. 2005.. et al. 273. A simple salting put procedure fore extracting DNA from human nucleated cells.): Allelic diversity and identification of individuals.H.and intra-population morphological differences between wild and farmed Atlantic salmon juveniles. and Gjerde. Skaala. and Gross.. Coughlan. K. 1466.. 20. 16. Forestry and Fisheries of Japan.K.M.... Aquacult. Doyle. 6. Food Control. 240. 2005. 2007.. Paralichthys olivaceus (Temminck and Schlegel) using microsatellite markers. et al. H. 17. 15. 1991. Fevolden. 916. Genetic characteristics of broodstock collected from four Norwegian coastal cod (Gadus morhua) populations. 11. Dahle.. 1988. 2006.B. Fish Biol. Miller.. Solem. O. 535. K. 4. Aquaculture. A. References 1. Ø. . M.K.. B. Res. 70. Ø. Chapela. J. Eknath. Chromatogr. K. I.. Can. 2006. 2004.. 2006.A. Eur. J. 18. et al...F. 2808. NAR. and Fjalestad. J.. Fish. Digestive enzyme activities in starved pre-slaughter farmed and wild-captured. Pampoulie. 9. 137. J... with particular reference to growth under farmed condition. 2003. A review of the physiological and nutritional energetics of cod. 820. 209.. Dykes. 137. Sonesson. J. Identification of organically farmed Atlantic salmon by analysis of stable isotopes and fatty acids. et al. M. Bailey. 15. Skaala. 1. 14. S...Ø.R. M. J.. Aquaculture. et al. Phenotypic divergence of sea-ranched. G. 1. et al. and Polesky. J. F. et al. 1995. Technol.W..D.. 2007. Universal and rapid salt-extraction of high quality genomic DNA for PCR-based techniques.. 8. H. ICES J. 51. E. 2007. Aquaculture. 1211. and O’Flynn. Comparison of DNA extraction methods from muscle of canned tuna for species identification. Sci. and Midling K. Fish. 1215. 4692.H.W. 70.). Taggart. 556. 13. 2..A. 5. S.228 ◾ Handbook of Seafood and Seafood Products Analysis Council. S. 1994. Jonsson. J. 12. Aquaculture. 19. B. 25. Genetic relationship between broodstocks of olive flounder. 216. Aquaculture. Friars. Suenaga. Fish Biol..K. Blood vessel melanosis: A physiological detoxification mechanism in Atlantic cod (Gadus morhua). 1997. A. et al. 343. J. Molkentin. Evaluation of three strategies using DNA markers for traceability in aquaculture species.E. SINTEF Fisheries and Aquaculture. Int. 2006. 1998. and Kjønes.. 1988. A combined salt and confinement stress enhances mortality in rainbow trout (Oncorhynchus mykiss) selected for high stress responsiveness. 37. 701. Cooper. 118. 21. Sci. Aquat. 2004.

40. J. B. E.. G. Appl.. Bio-Rad. 102. Principles and Methods. D.I.. Deep Sea Res. Partial or total replacement of fish meal by soybean protein on growth. Anal. C. et al. 31. 38. 1995. http://www. J. H. et al. Biochim. 2001. Eds. Verification of food origin based on nucleic acid pattern recognition. and Westgaard. 34. 25. Carter.html. Roth. 38. Makkar. Salmo salar L. 37. 32. Quality. EP1472366. 11. Aquacult.pdf. the Netherlands. 299. 107A. 39.. 36. 2-D Electrophoresis. Slizyte. and Arildsen. M. 5. E.. 2-D Electrophoresis for Proteomics: A Methods and Product Manual. I. Delghandi. Simultaneous analysis of six microsatellite markers in Atlantic cod (Gadus morhua): A novel multiplex assay system for use in selective breeding studies.. Influence of dietary soy and phytase levels on performance and body composition of large rainbow trout (Oncorhynchus mykiss) and algal availability of phosphorus load.. and Kochert. Technol. and Hauler.B. Fast growth was not associated with an increased incidence of soft flesh and gaping in two strains of Atlantic salmon (Salmo salar) grown under different environmental conditions. J.. K... Aquaculture. 2001. Garfin. cholesterolemia and flesh quality in rainbow trout.. Protein and amino acid requirements: A critique of methods.M... 257. L. Handbook 80-6429-60AC. 199. 31. L. 2004. Physiol.pdf. 30. and Heerdet. 2007.Analytical Methods to Differentiate Farmed from Wild Seafood ◾ 229 22. 1996. Water Sci. 41. Mar. and Eichacker.. Aquaculture. et al. 205.freepatentsonline. 215. Aquaculture. protein utilization. 35. Antinutritional factors present in plant-derived alternate fish feed ingredients and their effects in fish.. Aquaculture. gelifesciences. 504. Olsson. A. Burel. et al. Chem.. S. 161.B. C.. Biophys... Kaushik. Gelatinolytic activity in muscle of farmed and wild Atlantic cod (Gadus morhua) related to muscle softening..com/aptrix/upp00919. Gomes.D. The effect on rigor mortis and the physical properties of flesh.141. Eds. Replacement of fish meal by plant-proteins in diets for rainbow trout (Oncorhynchus mykiss)-effect of the quality of the fish-meal based control diets on digestibility and nutrient balance.J.. 265..C. 2003. Cowey. Johnston. Francis.. R.. E. Acta.B. Metabolic disorders in muscle of farmed Atlantic cod (Gadus morhua). 1665. Martin.. 2006. I. and Olli. 133. J.biorad.. 363. 24. Safety and Processing of Wild and Farmed Fish. Genomar. http://www. Mortensen. Asa (NO). 1995. Vielma. Proteomic sensitivity to dietary manipulations in rainbow trout. O.. 389. Granvogl. 197. 651. Res. T. 188.. Krogdahl. 2004.. Aquaculture.G. Food Chem. 257. Soybean proteinase inhibitors affect intestinal trypsin activities and amino acid digestibilities in rainbow trout (Oncorhynchus mykiss). Bucklin. 23... 26. 349. (http://www1.. Luten. 183.A. 185.S. Ploscher. Source regions for recruitment of Calanus finmarchicus to Georges Bank: Evidence from molecular population genetic analysis of mtDNA. 199. S.. .F.. and Becker.. 2000. R. II. 29. 28. et al. A. B.nsf/Content/2A3643B6787 885E0C 12570BE000DC671/$file/ 80642960. et al. 2007. 991. potential estrogenic or antigenic effects. High resolution two-dimensional electrophoresis as a tool to differentiate wild from farmed cod (Gadus morhua) and to assess the protein composition of klipfish. J. 27. Oncorhynchus mykiss. 504. G. J.A. M..com/EP1472366A2.P. Wageningen Academic Publishers. et al. Lie. A. in Seafood from Fish to Dish. et al.. et al.. et al.A. Slinde. 1994. 2007. C. Bioanal. nutrient utilisation and thyroid status. Pre or post mortem muscle activity in Atlantic salmon (Salmo salar). 2003. and Dauksas. 43. Sample preparation by in-gel digestion for mass spectrometry-based proteomics. Olsson. Comp. T. Biotechnol. Aquaculture.. Ichthyol. Martinez. 2000. Lea. Aquaculture. Biochem.L. 148. 2007. G. 2000.. 2006. GE Healthcare. Fish meal replacement by plant meals in extruded feeds for Atlantic salmon. Potential o f plant-protein sources as fish meal substitutes in diets for turbot (Psetta maxima): Growth.com/LifeScience/pdf/Bulletin_2651. 17.B. 33. 1995. 1223..B.

311. Biochem. Eds. 1995. 2008. Discrimination of wild and cultured European sea bass (Dicentrarchus labrax) using chemical and isotopic analyses. 52 p. Food Sci. Food Chem.. Olsson.K.. 232. Aquacult. U. D. in Fishery Products: Quality. J. Aquaculture.) pineal organ: Modification by diet and effect on prostaglandin production.D. 2007.. N. and Lie. Henderson.. Aquaculture.. 105.G. 1535. 58. Safety and Authenticity. Aursand. Dosanjh. Safety and Processing of Wild and Farmed Fish.. 217. F. 55. Australia. Froyland. et al. Agric.G. Evaluation of the profile of lipids as a tool to discriminate wild from farmed salmon. 2000.. .G. 60. Characterization of farmed and wild salmon (Salmo salar)... 46.. 55.. F. 77... Agric. F. J. Harwood. Torstensen..T. B.D. Acta. 59. JAOCS. High-resolution C-13 nuclear magnetic resonance spectroscopy pattern recognition of fish oil capsules. Nichols. Gunstone.D. F. p. Martinez. Hobart. and Martin.G. 2002. I. Influence of dietary blends of menhaden oil and canola oil on growth. 1996. M. et al. 85...230 ◾ Handbook of Seafood and Seafood Products Analysis 42. Food Chem. Compositional and organoleptic quality of farmed and wild gilthead sea bream (Sparus aurata) and sea bass (Dicentrarchus labrax) and factors affecting it: A review. Quality. 57.. 60.. Grigorakis..G.. 218.3. Agric. Aquaculture. 61.. Chen.B... and Park. 10. Bell. R. 50. et al... J.. Tasmania. Are modifications in tissue fatty acid profiles following a change in diet the results of dilution? Test of a simple dilution model. Bell. 2007. Gunstone. Nutr. Replacing dietary fish oil with increasing levels of rapeseed oil and olive oil—Effects on Atlantic salmon (Salmo salar L. Polyunsaturated fatty acid composition of the salmon (Salmo salar L.) tissue and lipoprotein lipid composition and lipogenic enzyme activities. F. Bell. et al. et al. 55. I. 551. 659. B. B. 2007.. Revision of analytical methodologies to verify the production method of fish. 989. M. 2004. SEAFOODplus report 6.L. Fish Physiol.. 2002. Bell. J. et al. Standal.. 175. 123. J.. I. Jobling. Ø. 388.. 5934. 51. and thyroidal status of Atlantic salmon (Salmo salar) in sea water. G. London. J. 1994. 56. 631.E. I.. Martinez. Wageningen Academic Publishers. ISBN 1-876-996-07-2. J. 48.. 38. JAOCS. Influence of high contents of dietary soybean oil on growth. stable isotopes and trace elements. and Padley. 131.. J. 55.. 2001. 191. Aursand. and Elliot. 19. 2nd ed. Martinez.. H. J. K.D. Blackwell Publishing. 515. heart histology and standard oxygen consumption of Atlantic salmon (Salmo salar) raised at two temperatures. 2004. 1299. tissue fatty acid composition. Determination of origin of Atlantic salmon (Salmo salar): The use of multiprobe and multielement isotopic analyses in combination with fatty acid composition to assess wild or farmed origin.B.. 56. Authenticity assessment based on other principles: Analysis of lipids. 52...B. Biophys. the Netherlands. 207. 289. Eds.B. 1998. p. J. 198pp. Thomas.. Standal. P.. Chapman & Hall. Eds.. Nutr. Rapeseed oil as an alternative to marine fish oil in diets of post-smolt Atlantic salmon (Salmo salar): Changes in flesh fatty acid composition and effectiveness of subsequent fish oil “wash out. 272. Mabon. 2008. and Rehbein. B.” Aquaculture. Food Chem.J. 541. et al. Oehlenschläger. 49. 43. in Seafood from Fish to Dish.. Seasonal variations in chemical and sensory characteristics of farmed and wild Atlantic halibut (Hippoglossus hippoglossus). Biochim.. Replacement of fish oil with rapeseed oil in diets of Atlantic salmon (Salmo salar) affects tissue lipid compositions and hepatocyte fatty acid metabolism. 53.. Aquaculture.B. I. 2003. et al.S. 44. 47. and Axelson. 2003. M. Nutritional Value of Australian Seafood II. J. muscle lipid composition. 2006.. The Lipid Handbook. feed utilization.. Luten. et al. Mooney. J.J. 45.. I. Discrimination of cod liver oil according to wild/farmed and geographical origins by GC and 13C NMR. 54. ISBN 978-87-7075-001-1. Grisdale-Helland. CSIRO Marine Research and FRDC. Differentiation of cultured and wild sturgeon based on fatty acid composition.8. G. et al. 2009. L.E. M. 2006..

H. Chem. Jørgensen. 1. 63.. 73. fillets and extracts of farmed Atlantic salmon (Salmo salar) for quality assessment and compositional analyses. Aursand. D. 34. RSC Books. M. M. M. Steindachner. Classification of gilthead sea bream (Sparus aurata) from 1H NMR lipid profiling combined with principal component and linear discriminant analysis. 39. Biological and Marine Sciences. Aquaculture Res. 65.. Chemometric analysis of NMR spectroscopy data: A review. and Alam. J. Soc.. Fernandez-Jover. Sacchi. Biological variation of lipid constituents and distribution of tocopherols and astaxanthin in farmed Atlantic salmon (Salmo salar).. K. Aursand.. Am. Am. J.Analytical Methods to Differentiate Farmed from Wild Seafood ◾ 231 62. Fan. M.... 2004. Metabolite profiling by one-and two-dimensional NMR analysis of complex mixtures. 1996... Agric. 999. in Handbook of Modern Magnetic Resonance Modern Magnetic Resonance. Skog. et al. Prog. 293. Application of multielement stable isotope ratio analysis to the characterization of French. Aursand. Brockhoff. Springer.. 2005. De Niro. et al... 69. Eds. Soc. J. Pattern recognition methods and applications in biomedical magnetic resonance. the Netherlands. 225. P. Bioanal..A. 2003. D. Application of support vector machines to 1H NMR data of fish oils: Methodology for the confirmation of wild and farmed salmon and their origins. I. and Colquhoun I. Gribbestad..... S. Defernez. and Grasdalen. Camin. Magn. S... Holmes. Aursand..F. Reson.E.. M. M. 55. and Epstein.. Omega-3 fatty acid content of intact muscle of farmed Atlantic salmon (Salmo salar) examined by 1H MAS NMR spectroscopy. et al. 2003. Res. Alam. Refsgaard. 2001. 75. et al. Lindon.P. J. High resolution 1H magnetic resonance spectroscopy of whole fish. I.E. Science... H. J. Aursand. Phys. U. Positional distribution of n-3 fatty acids in marine lipid triacylglycerols by high-resolution 13C nuclear magnetic resonance spectroscopy. et al. T. 2007.. 63. 70. Interpretation of the 13C NMR spectra of omega-3 fatty acids and lipid extracted from the white muscle of Atlantic salmon (Salmo salar). 123. Webb. Anal. K.M. Peak alignment of NMR signals by means of a genetic algorithm. 1993.. M... 161. Aursand. 41.. and Nicholson. 1868) associated to sea cage fish farms. 1. NMR Spectrosc. and Spanish cheese. Rainuzzo. Chim. 189. 1009. Origin recognition of wild and farmed salmon (Norway and Scotland) using 13C NMR spectroscopy in combination with pattern recognition techniques. 71. Food Chem. Aquaculture.B. et al.. 2003. M. and Martinez. et al. T. M. G. C. 70.H. 70. Forshed... R.. Part 1: Applications in Chemistry. 906. Masoum.S. G. J. Soc. 67. Annu Rep. Recent developments in food authentication. 62. Lipids. London.S. Analyst. H. Anal. and Martinez. Food Chem.. Prog. and Grasdalen. 66. Schuppe-Koistinen.W. 2005.. Carbon isotopic evidence for different feeding patterns in two hyrax species occupying the same habitat. J. I.A. Ed. M. 9963..J.. Magn. Acta 487. 6592. Agric. 971. and Jensen. 931. I. Mar. 151R. 239..J. L. 64. Factors affecting the robustness of metabolite fingerprinting using 1H NMR spectra.. Oil Chem. 78. J. 227. J. and Grasdalen. and Jacobsson. Phytochemistry. I. 54. Food Chem. M.. T.. 1978. 1993. Nucl. Oil Chem. 1992. 2007.. in Magnetic Resonance in Food Science: A View to the Future. 1998. and Axelson. 1995. Proton nuclear magnetic resonance rapid and structure-specific determination of w-3 polyunsaturated fatty acids in fish lipids. 808.. 74. F. Webb. Gribbestad. Rezzi. Quantitative high-resolution 13C and 1H nuclear magnetic resonance of fatty acids from white muscle of Atlantic salmon (Salmo salar). 52.. 1499. 1998. 250. H. 68. 80. p. Salmon farming affects the fatty acid composition and taste of wild saithe Pollachius virens L. J. S. Italian. Amsterdam. 2001. Reson. Chem. Oil Chem.. Dennis.. Changes in body condition and fatty acid composition of wild Mediterranean horse mackerel (Trachurus mediterraneus. 77. 79.. Spectrosc. E. B. 445. 72. Environ. 201. 387.. 81. 46. Agric. 72. S. Nucl. Am. 2006. 2007.K.. . 62. 76. Spectrosc. 28.

Biol. Fish. Ecol. Sci. 98. Omura. Fish Biol.1998. 87. 1993. First action. 55. Isotopic standards for carbon and oxygen and correction factors for mass spectrometric analysis of carbon dioxide.J. Distinct regional profiles of trace element content in muscle of Japanese eel Anguilla japonica from Japan. 1983. Fac. Salmo salar. M. 93. Trace elements in waters and wastewaters. 84.. Effects of body size and environment on diet-tissue d15N fractionation in fishes.. Oceanogr.. Y. Ann. 2003. 12.. Kline. et al. et al. 622. Ser..J. J. J.. et al. Ihnat.. 685. 89. T. 72. 92. 2007. et al. Committee on residues and related topics—Metals and other elements.. J.. 188. Rapid liquid chromatographic method to distinguish wild salmon from aquacultured salmon fed synthetic astaxanthin. Mar. et al. Limnol. mechanisms and applications..C. Food Res. Mariotti. J. Y.. Identification of northern bluefin tuna stocks from putative nurseries in the Mediterranean Sea and western Atlantic Ocean using otolith chemistry. W. 96. Di Parma. Dempson.M. 80. 263. Can.14. and Okazaki. Sweeting. Inductively coupled plasma-mass spectrometric method. S. 97. 193. Acta. 2006. Atmospheric nitrogen is a reliable standard for natural 15N abundance measurements.J. Taiwan. K. 2005. M. Exp. Sci.. Stable isotopes determination in food authentication: A review. A. Franke.E. 2002. et al. 1957. Alaska.A. AOAC Official Method 993. Oceanogr. 257.. Guiguer. Doucett.R.. 1998.. 1979. 83. Fish. Using stable isotopes to confirm the trophic ecology of Arctic charr morphotypes from Lake Hazen. 94.. J. H.. 2006. Fish. 43. 133. Vet. 53. Eur. Chemistry and composition of fish otoliths: Pathways.elements of an analytical approach to its authentication. XXVI. Geographic origin of meat . 221. 84. E. AOAC Int. McConnaughey. 88.. 176. 1494. Prog. Use of stable isotopes to distinguish farmed from wild Atlantic salmon. et al. Canada.. Cosmochim. 1997. Technol... 2004. Trace element signatures in otoliths record natal river of juvenile American shad (Alosa sapidissima).. J. Nunavut. Albert. Aquat. and Goering. 493. Nature 303. 1. Rooker. 85. R.. R. J. 89. 90. Natural isotope indicators of fish migration at Prudhoe Bay.. 1999.. Wilson.. . Mar.P. Geochim. Evidence for anadromy in a southern relict population of Arctic charr from North America. Ghidini.B.. and Power.R. Fish Biol. Food-web structure and the fractionation of carbon isotopes in the Bering Sea. C. 290.R. 91. 55. 348. and McRoy. C.. 86. Ecol. Medic. B. T.R.R. 1109.232 ◾ Handbook of Seafood and Seafood Products Analysis 82. 12. et al. 75. 340. Campana. 1999... 95.. 60. Craig. J. Biol. AOAC Int. S. Mar. Ecol. 2006. Yamashita. Freshwater Fish. and China. S. J. 13. Thorrold.. 1826.

................................... 239 15..........2 Smoke Flavoring Process ...3 Role of Volatile Compounds of SF in the Texture ...8......... Thierry Serot.......2 Role of Volatile Compounds of SF in the Flavor ....2....................249 15...................................Chapter 15 Smoke Flavoring Technology in Seafood Vincent Varlet......... 241 15.................249 15...................................... 246 15............................................................................................................................................................. 245 15....................6.......... and Carole Prost Contents 15.......................2.................................................................................... 246 15...................................................................................... 237 15.... 240 15.......1 Liquid Smokes..1 Introduction .............................................................7 Role of Volatile Compounds of SF in Preservation ..4 Smoke By-Products .......2 Smoke Oils ...........................1 Role of Volatile Compounds of SF in the Odor ............................ 237 15......................3 Use of Smoke Flavorings ............................9 Legislative Aspects.............................. 248 15.................. 236 15... 234 15..................................... 234 15...................2...............................5 Role of SF Process Parameters in Volatile Compounds Generation ....................................3 Smoke Powders .....................................................................249 233 ...................241 15..............4 Role of Volatile Compounds of SF in the Aspect and Color ............6........ 237 15........ 238 15.....8 Polycyclic Aromatic Hydrocarbons........6...1 European Regulations on PAH Found in SF .........................................................................................................6.....................................247 15.....................................................1 Properties and Toxicology ..4 Chemical Composition of Liquid Smokes ......................................2..........................................................................9........ 248 15......................................6 Organoleptic Roles of Volatile Compounds of SF .................2 Extraction and Analysis Methods of PAH in SF and Seafood Treated by SF .........................8.................................

. After condensation... Indeed...10 Conclusion ...........) with a wide range of organoleptic qualities......... the crude smoke condensates are separated in three phases: a water insoluble heavy oil by-products phase......... obtained after a settling out time (several days) of the smoke condensates in the settling tank... allows a better control of PAH in the final product.. and provides a higher diversity of smoked food [1].. or concentrated. aqueous solutions....... wood smoke imparts desired organoleptic characteristics such as smoky flavor. the legislation and the organoleptic quality of SF and products treated by SF constitute critical points that show the necessity of better improvement and harmonization of this technology.. which give rise to new perspectives in the food industry [3]...250 15........... A simplified version of SF processes is presented in Figure 15.... 15..... and reduced risk of accidents due to fire..... etc........250 References ........ This kind of smoke flavoring (SFs) appears as an alternative to the smoking process as it is carried out in Europe........... In United States where 75% of smoked foods are treated by liquid smokes.. Coupled to salting and drying steps. The fractionation of smoke condensates allows obtaining a high diversity of SFs (powders. a water-soluble phase... Wood sawdust is pyrolyzed in a furnace with low oxygen content...9....... However........ the use of SF allows an easier storage (SF bottles versus wood logs)......... It also allows the reduction of the PAH final concentrations...... This industrial process leads to more homogenous products smoked with a repeatable intensity and provides an easier cleaning of the smokehouse..1 Introduction Smoking is the oldest food preservation technique............... By comparison with the smoking technology........... SFs are widely used in the meat industry. Today............. SFs are obtained by the condensation of wood smoke and can be further fractionated. the use of liquid smokes avoids the release of smokes......... purified. are recycled and directed also to . The smoke is filtered to eliminate particules and condensed.. and their uses in the seafood industry are increasing..234 ◾ Handbook of Seafood and Seafood Products Analysis 15.... The heavy oil by-products... The main woods used for smoke production are oak. and marple................ The gaseous smoke can be cooled down by water or by organic solvents...... liquid smokes are commercialized since the end of the nineteenth century.. that is. the smoking process has two main inconveniences: the production of carcinogenic contaminants—polycyclic aromatic hydrocarbons (PAHs)—during the incomplete pyrolysis of wood used to produce smoke and the release of smokes in the atmosphere................. hickory.. mainly hard woods....... the chemical composition of soft woods is responsible for the generation of higher quantities of contaminants as PAH. between 20% and 30% of European smoked food is treated by liquid smokes.. Indeed................... Moreover.. Simultaneously..... it allows decreasing microorganism activity. a better preservation of the combustible........................... The combustible gases are recycled and directed to the furnace... oils.... beech............... However........ wood smoke phenolic components are known to be antioxidants....2 Smoke Flavoring Process The first liquid SF was developed and patented by the Kansas pharmacist Wright in the late of nineteenth century [2]........1.... especially thanks to the industrial benefits brought about by their use.......251 15.. and a water insoluble tar phase.......2 European Regulations on PAH Concentration in Food Treated by SF ......

1 Diagram of fabrication of SFs. ◾ 235 .Condensing tower Filter stage two Settling tank Oil exchange system Filter stage one Dryer/blender Further processing Aqueous smokes Recycled combustible gases Smoke oils Patented furnace Recycled heavy oil by-products Smoke powders Wood dust by-products Smoke Flavoring Technology in Seafood Figure 15.

They are employed to confer smoky organoleptic qualities (tastes. or drying/blending of these PP. These products have a pH greater than 4 and can also be added to the brine. Different SFs (liquid smokes. due to their low pH. This form of SF is especially used for the smoky taste that it confers to the food. separation. They can be employed in sauces or marinades of seafood products. Concentrates of liquid smokes consist of concentrated versions of aqueous flavors and require lower usage quantities. smoke oils. smoke condensates obtained from PTF and PSC are named primary smoke products (PP). can be used directly or diluted for applications requiring lower concentrations [4].1 Liquid Smokes Different kinds of liquid smokes are available: aqueous flavors. smoke powders.236 ◾ Handbook of Seafood and Seafood Products Analysis Smoke extracts Smoke distillates Primary products Liquid smokes Smoke oils Aqueous flavours Soluble aqueous flavours Concentrates of liquid smoke Buffered aqueous flavours Smoke powders Figure 15. They are used when intermediate product dispersion is required as in brine. Finally. and buffered aqueous flavors. soluble aqueous flavors. Soluble aqueous flavors are aqueous flavors that contain an emulsifier such as polysorbates. Therefore. allowing better water solubility. a purified extract of the high-density water insoluble tar phase can be used for the production of SFs and is called primary tar fraction (PTF).2. buffered aqueous flavors are partially neutralized or buffered aqueous flavors. 15. concentrates of liquid smokes. However.2 SFs from primary products. Aqueous flavors. or smoke by-products) can be obtained after different steps of filtration. They are presented in Figure 15.2. They are especially used when the final water rate in the treated food must be low. . flavors) and also the characteristic aspect and color of smoked food. The water-soluble phase leads to primary smoke condensates (PSC). the furnace because they cannot be used for human consumption.

Wet salting (or curing if nitrited salts are used) is made with brines spread on food or in which the food is dipped. the organoleptic qualities can vary in a high range changing the food matrix. in seafood industry. The distillation is commonly performed with steam water at atmospheric pressure. . most frequently in 90:10 (v/v) proportions. can be added to the smoke powders used in the meat industry to improve simultaneously the storage of food and to confer smoky characteristics to the final product. 15. Indeed. Nitrited salts. Dry salting (or dry curing if nitrited salts are used) is made with dry salt deposited directly on food. Smoke by-products constitute more complex SFs. Smoke powders can also be rehydrated and used in brine as liquid smokes. These smoke powders can be added to salt used for salting steps or to dehydrated sauces or soups elaborated from seafood products. Consequently. Today. SFs for herring. We must distinguish dry salting and wet salting. However.4 Smoke By-Products Smoke by-products are constituted by smoke extracts and smoke distillates [6]. Therefore. smoke manufacturers can control their products and can create smoke by-products whose uses are recommended for a kind of fish. Smoke distillates are obtained by the fractionating distillation of PP. They are less acidic than aqueous flavors and allow to exhibit more complex smoky tastes. are present in the market. whereas liquid smokes are used for the characteristic smoky odor and color of smoked products.2 Smoke Oils Smoke oils are made by blending liquid smokes with vegetable oils. and so forth. As seafood emulsions are not very common.Smoke Flavoring Technology in Seafood ◾ 237 15. fish sauces. but their uses are specific to a food: smoke aromatic preparations can be produced to treat certain kinds of meat and cannot be used for fishes for example.2. because smoke oils are especially employed in food preparations such as emulsions. Smoke extracts are produced by way of more or less selective extraction of smoke constituents directly from the smoke aerosol (by countercurrent circulation of water or organic solvents) or from the PP. or fish oils. These molecules can increase the generation of carcinogenic nitrosamines. generally forbidden in seafood industry according to the countries. Therefore. salmon. the liquid smokes and smoke powders can be added to salt or in brine but not smoke oils.2. Therefore.3 Smoke Powders Smoke powders are obtained by blending liquid smokes and dry powder carriers such as maltodextrines or barley and corn flours and drying them [5]. As smoke oils. consequent to the reaction between amino acids and nitrite. it is very important to consider the salting step made with common salt mainly authorized for seafood and the curing technology made with a salt treated by nitrite and nitrate authorized for meat.2. their uses are really characteristic of a product and cannot be employed for a wide variety of food due to their typical organoleptic qualities. smoke powders are mainly used to confer smoky tastes to the final product. A reaction between the phenolic compounds of SFs and these nitrited salts or powders can lead to a nitrosation and to nitrophenols. The final composition of smoke powders must be known in order to avoid the presence of allergens or other nonrequired additives such as nitrited salts. these SFs are not used much in seafood industry. smoke oils can be only used in preparations such as taramas. hundreds of smoke by-products are available. smoke powders used in the meat industry should be different from those used in seafood industry in order to avoid nitrited salts in seafood treated with smoke powders. Consequently. 15.

Diluted SFs fall by gravity through perforated plates on the hung products. In France. They provide a better water solubility and prevent the heterogeneity of layer formation on the product surface or the product separation during storage. because the products are immersed in the SFs solution instead of pouring the SFs solution on the products. vaporized liquid smoke is similar to real wood smoke. Showering is a technique currently used in North America. SFs are so easy to produce that it would not be profitable to create synthetic SFs when natural ones are available at a cheap price. in numerous European countries. direct addition. Indeed.3 Use of Smoke Flavorings There are four techniques to incorporate or deposit SFs in or on seafood products: showering. Water-based composed SFs such as soluble aqueous flavors or buffered aqueous flavors are commonly used in this technique. 15.238 ◾ Handbook of Seafood and Seafood Products Analysis The development of synthetic SFs must be also noticed. between 15 and 20 mm. Liquid smoke solution is therefore recycled and filtered and the concentration is readjusted.) are dependant of the dilution of SFs in water according to proportions varying between 20% and 25% for SFs and 75% and 80% for water. Smoke powders are preferred when water use is impossible as in dehydrated mixes. the composition of liquid smoke mist is not similar to real wood smoke. SF is sprayed with air under pressure through special nozzles and forms a wood smoke mist in the cell of smoking. Drenching/soaking is the opposite of showering technique. Finally. there are different carriers of SFs. taste. composed entirely of synthetic compounds or partly from a liquid smoke base [7]. and atomization. From a physical point of view. However. because to guarantee the homogeneity of the SFs during the treatment and to prevent the settling out of smoke condensates in water. but aqueous liquid smokes are the most used SFs in this technique. liquid smokes are also employed in the curing brine. especially used for meat products. wood smoke is composed by a gaseous phase formed by the most volatile compounds. SFs can be incorporated directly with the ingredients during the formulation or through injection needles when the shape of the product cannot be modified. which carries a particulate or dispersed phase [8]. Indeed. Besides. that is. products treated by liquid smoke atomization are considered as flavored and not smoked. etc. Other devices have been optimized in order to generate a similar physical composition of . Therefore. Soluble aqueous flavors or buffered aqueous flavors are mainly used. This difference constitutes a critical point in the liquid smoking status. The final organoleptic qualities (color. The mist generated is composed only by small droplets and there is no gaseous phase. atomization of SFs consists in the vaporization of liquid smokes. The mist obtained is constituted of small droplets with a similar size as in real wood smoke. The progress made during the last decades in elucidating the chemical composition of wood smoke gave rise to attempts aiming at producing SF. Direct addition consists in the incorporation of SFs during the fabrication of the food products. but it is also employed in the seafood industry. from the granulometry point of view. mainly liquid smoke concentrates on the products in a smokehouse. Smoke oils are preferred for lipidic emulsions or lipidic sauces. and this technique appears as an alternative to the smoking process. meat treated by this process is considered as smoked but « smoked by liquid smoke » must appear on the package. Products are dipped in SFs solution for short periods (from 5 to 60 s). which can be injected into the product as for the salting step. especially in the labeling of the smoked products. However. According to the final product. drenching/soaking. an emulsifier must be added in the SFs. the synthetic SFs created are not sufficiently similar to real wood smoke or to SFs.

it creates a gaseous phase. which decompose to form alpha cellulose and provide a higher amount of PAH. predominant in softwoods) and in para position (syringol derivatives predominant in hardwoods). hemicellulose in hardwood (nonconiferous woods) is mainly constituted by pentosans whereas hemicellulose in softwood (coniferous woods) is mainly composed by hexosans. Glucuronic acids decompose to carboxylic acids.4 Chemical Composition of Liquid Smokes The chemical composition of SF depends on the composition of the wood raw material used and especially the relative amounts and structure of its main components: two polysaccharides namely cellulose and hemicellulose and lignin.Smoke Flavoring Technology in Seafood ◾ 239 wood smoke with liquid smoke atomization. Hardwoods lead to G/S and G/P ratios. respectively. hence the high acidity of liquid smokes.6-anhydroglucose (betaglucosan) and finally to acetic acid and its homologues. The pyrolysis of cellulose initiates the hydrolysis of glucose followed by dehydration to 1. The volume of liquid smoke mist is controlled by the number of nozzles and the smokehouse size. hydroxyacetaldehyde. The smokehouse must be hermetically closed during atomization. The surface must present a beginning of protein coagulation. which confers a subtle glossy and sticky aspect. and various anhydroglucopyranoses (mostly levoglucosans) [11]. The SF composition can be complexified by the addition of spices and aromatic herbs [10]. In seafood industry. hence the limitation of the use of softwoods for smoking. to favor the deposition of smoke components. the drying step is necessary to prepare the surface of the fillets. whereas weak moisture gives to the product a good color but a weaker smoky taste. first to control the drying of the product and second. furanones. The pyrolysis of lignin can also lead to alkyls aryls . Finally. but the optimization of the parameters to have a similar particulate or dispersed phase is not easy. which favors the vaporization of SF [9]. liquid smoke atomization is the most used technique of SF. In fish smoking. The thermal decomposition of pentosans provides a higher amount of furans than hexosans. acetic acid. Indeed. a good knowledge of the food matrix to be treated is required to apply SF in the best conditions and to reach the expected organoleptic qualities controlled by SF chemical composition. The SF is sprayed on a surface at high temperature. The wood polysaccharides lead to methanol. The moisture control is essential. methanal. ventilation must be planned in order to reduce the moisture. The role of pyrolysis parameters as pyrolysis temperature. This step must take into account the initial water rate of the raw material and the composition of the final product. lignin thermal decomposition provides compounds considered as most important for the smoke flavor. acetaldehyde. Therefore. and sometimes small quantities of furans and phenolic compounds. The main characteristics that permit the differentiation of hardwoods and softwoods are the guaiacol:syringol (G/S) and guaiacol:phenol (G/P) ratios. and air moisture are also essential in the SF final composition. Similarly. Therefore. Hemicellulose pyrolysis leads to furan and its derivatives and aliphatic carboxylic acids. Important moisture favors the smoke penetration and strong smoky organoleptic characteristics. water. 15. of 1. The compounds generated from hemicellulose pyrolysis depend on the nature of the wood.5 and 2. According to the liquid smoke used. formic acid. The adjustments are carried out on the flow of liquid smoke from the tank owing to a temporization on the liquid admission and on the flow of air under pressure. wood moisture. A high knowledge of the biochemical composition of the wood used and the parameters of the combustion are essential to generate SF. furfural and homologues. the methods of production and the possibilities of applications of SF are very wide. such as alkyl phenolic compounds and derivatives like phenolic ethers with methoxy groups in ortho position (guaiacol derivatives. airflow.

the pyrolysis temperature. From 200°C to 600°C. whereas syringol quantity is tripled. The air velocity indirectly influences the SF composition by the modification of pyrolysis temperature or smoke temperature [21. the quantity of phenolic compounds increases with a maximum close to 500°C and decreases after 500°C. A step of filtration is almost obligatory to avoid these contaminants. Therefore. between 280°C and 320°C for cellulose. Air moisture is also very important and must be set in adequation with air velocity to keep the water rate constant in the air during the combustion. and humidity of air constitute key parameters of SF composition. Then. phenol amount is multiplied by two between 450°C and 650°C. and phenolic compounds [10. because their contents in smoke or in food increase from 400°C to 1000°C. The wood moisture appears as the second important parameter [20]. known as the smoky skeleton of SF. As the best pyrolysis temperature to obtain the required volatile compounds are between 380°C and 500°C. Lower concentrations of oxygenated compounds have been found to be caused by an oxygen depletion during combustion [20]. 15. The generation of volatile compounds is dependent on the wood pyrolysis temperature [16]. lignin dimers. and 400°C for lignin [17]. Therefore. exothermic reactions of pyrolysis of wood components occur between 200°C and 250°C for hemicellulose.19].22]. it seems difficult to generate the desired organoleptic volatile compounds without PAH contaminants. because it plays a role in the pyrolysis temperature. Wood granulometry can also influence SF composition. the diversity of settings of pyrolysis parameters can explain the diversity of organoleptic volatile compounds and the diversity of qualities of SF. The use of hardwood. is recommended because it burns slower.24]. The manufacturer can choose SF according to the required result on the organoleptic characteristics of the final product. For example.18. and the enolones derivatives [14]. The rate of acids is higher for temperature lower than 300°C and decreases after 300°C with the increase in temperature. the velocity. PAH must also be surveyed. The combustion is faster when the granulometry of the wood raw material is important [23. whereas a rate between 20% and 30% has been reported as optimal to reduce the emission of particules [11]. and trimers [12]. the main organoleptically active volatile compounds generated during the pyrolysis process can be sorted in three groups of molecules: the phenolic compounds. A slow combustion is reached with weak air velocity. but they have a weak impact on the smoky flavor of SF and food processed with SF [13]. However.5 Role of SF Process Parameters in Volatile Compounds Generation Except the wood type that influences the smoke quality strongly [15]. with a lower water rate than that in softwood. A temperature of 450°C–500°C was reported to lead to the best composition for the creation of carbonyls. differences can be observed depending on the molecules. After the water release (close to 120°C–150°C). The rate of carbonyl compounds increases gradually with the temperature from 200°C to 600°C. An optimal moisture is planned in the industry between 17% and 20%. Due to their . steps of SF purification through filters or apolar solvent washes are often required to decrease the PAH levels. The high moisture allows to reduce the wood combustion efficiency. Indeed. the wood granulometry and moisture. According to the pyrolysis process. furannic compounds. the furannic derivatives. a lower temperature is reached and allows increasing the generation of smoke volatile compounds and minimizing PAH formation.240 ◾ Handbook of Seafood and Seafood Products Analysis ethers from lignans. different groups of compounds are formed.

guaiacol. and alkylguaiacol may also contribute to imparting a smoky flavor to smoked fishes [13. phenolic compounds are not sufficient to explain the SF role in smoked fish odors. cresols.1 Odorant Thresholds of Various Phenolic Compounds Phenolic Compounds Phenol o-Cresol m-Cresol p-Cresol Guaiacol 4-Methylguaiacol 4-Ethylguaiacol 4-Vinylguaiacol Vanilline Syringol Eugenol Ethylvanilline Odorant Thresholds in Water (μg/L) 5900 650 680 55 3–21 90 50 3 20–200 1850 6–30 100 References [25] [25] [25] [26] [25] [25] [27] [26] [25] [25] [26] [25] . Many studies have indicated that phenolic compounds present in the vapor phase of smoke may be odor-active compounds [30–32].Smoke Flavoring Technology in Seafood ◾ 241 chemical composition.33. Phenolic compounds of medium volatility have been considered as the most important odorant molecules. Table 15. They are the major compounds in SF with a wide range of odorant thresholds (Table 15. taste. Phenolic compounds are known to constitute the odorant “smoky” skeleton of wood smoke and smoked fish.29]. odor.2). but it may not be the main contributor to wood smoke flavor.1) [14. two main classes of odor-impact molecules can be defined: phenolic compounds and carbonyl compounds. making them odorant at low concentrations. A much more complex mixture of compounds is responsible for the characteristic aroma of smoked fishes [35]. and methylsyringol has a pure and characteristic smoky flavor [10].6. 15. syringol.34]. The medium-boiling fraction (91°C–132°C) composed of isoeugenols. which gather furannic and enolone derivatives.6 Organoleptic Roles of Volatile Compounds of SF 15. color. Phenolic compounds of low-boiling fraction (60°C–90°C) composed mainly of phenol. Similarly.1 Role of Volatile Compounds of SF in the Odor Even if the concentrations of odorant volatile compounds in SF can be various. The role of syringol is important.28. and preservation of the product. Some of them have very low odorant thresholds. These observations have been recently corrected [14.34] (Table 15. the diversity of SF causes diverse consequences on the texture.

LRI. LRI. STD Cooked.94 49.27 ± 2. STD MS. STD MS.90 (1.Table 15. STD MS. mushroom Cooked.98 20. wood fire.25 6 (5) 6 7 (4) 7 7 6 8 8 8 Chemical.2 Odorant Characteristics and Concentrations of the Most Potent Odorant Volatile Compounds in Salmon Fillets Treated by Liquid Smoke Means of Identificationa Mean ± SDe 124.53 360. LRI.53) 8.24 ± 63. STD MS. green. LRI.48 ± 8. potato Cooked potato. green Cooked vegetable.07 (1. STD MS. LRI MS.37 ± 3. milk Cooked/soup. ink Marine. metallic. spicy. LRI.22 ± 9. STD MS. LRI. STD MS.04 7 4 16.50) 65.3-Dimethyl-2cyclopentenone 1052 o-Cresol 1068 p-Cresol 1093 Guaiacol 1110 2. LRI MS.12 42. fatty Roasty.45 ± 172. burnt. STD MS. LRI.97 23. LRI MS.62 74.75) MS. roasty Chemical. spicy/ woody (3) (2) 5 6 (2) 4 4 3 5 6 7 (2) 4 4. green Cooked vegetable. spicy.18 ± 37. LRI.34 (24. vanilla.44 17. LRI. Animal. green Odorant Attributes Given by the Judgesb Number of Judgesc Average Intensity d 242 ◾ Compounds LRI (DB5) Furfural 859 4-Methylpyridine 865 Furfuryl alcohol 875 2.33 ± 1. chemical.55 ± 39.17 ± 26.63 ± 13. STD MS. spicy Spicy.6-Dimethylpyridine 890 2. LRI. earthy.4-Hexadienal 904 2-Methyl-2-cyclopenten1-one 920 2-Acetylfuran 925 5-Methylfurfural 970 Phenol 992 Handbook of Seafood and Seafood Products Analysis 2-Hydroxy-3-methyl-2cyclopenten-1-one 1036 2. LRI. milk Smoke. LRI MS.64 ± 18. burnt.74 ± 27. chemical Green.6-Dimethylphenol 1130 . burnt Smoked. STD MS.27 ± 0.55 ± 9.

61 ± 22. LRI. STD MS.96 ± 10. fatty.36 1132 MS Cooked.5-Dimethoxytoluene 1282 4-Ethylguaiacol 1287 Indanone 1307 4-Vinylguaiacol 1330 (E.46 Solvent. fatty Burnt rubber. LRI. milk Burnt.17 15. spicy 6 5 17. spicy. LRI MS.4-Decadienal 1330 Syringol 1365 Eugenol 1370 Smoke Flavoring Technology in Seafood 4-Propylguaiacol 1382 1400 ◾ 1. green.85 ± 40.4Trimethylcyclopenten-1one MS MS.16 ± 5. green. burnt Smoke.3.50 18.25 ± 4.35 3-Ethyl-2-hydroxy-2cyclopentenone 1140 1.15) 86.71 (3.2.24 ± 1. medicinal 7 4 10. STD MS. LRI MS.2. LRI MS. clove Green. LRI 1160–1180 4-Methylguaiacol 1192 482. spicy Spicy. LRI MS.86 (2. LRI. LRI.21 ± 7.15) (continued) 2. LRI. STD MS.82 ± 6. LRI MS.4.26 ± 1.3-Dimethoxytoluene 1247 (E)-2-Decenal 1266 3. green Spicy.5Dimethylphenol/ (E)-2-nonenal MS. spicy Oily. green.87 ± 1. rotten. chemical Green. spicy.51 ± 18. STD MS. green 6 3 11.25 ± 10.3-Trimethoxy-5methylbenzene 243 .15 ± 243. STD MS. spicy. vanilla. smoke. smoke. green.12 4.72 44. earthy 6 (5) 8 7 (3) 7 8 8 8 (5) 7 7 5 4 3 (3) 6 4 (2) 5 5 5 5 (2) 8 6 Ashes.62 ± 4.79 (6.15 ± 1. green. LRI. vanilla Cooked.49 ± 6. violet.13 6.95) 8. smoked Cooked vegetable. LRI.97 2.E)-2. STD MS.91 36. LRI.and 2. STD MS. smoked Candy. STD Cucumber. clove Sawdust.2-Dimethoxybenzene 1147 2.

.68 MS.35 (20.40 ± 3. LRI MS. spicy 6 3 Odorant Attributes Given by the Judgesb Number of Judgesc Average Intensityd 244 ◾ Compounds LRI (DB5) (Z)-Isoeugenol 1423 (E)-Isoeugenol 1473 2. Food Chem.39 6.87 ± 2.55 ± 8. LRI. rotten 7 4 Spicy. 9 = very strong odor intensity). spectrum. In micrograms equivalents of dodecane per 100 g of smoked salmon. V. green.5-Trimethoxytoluene 1527 4-Allylsyringol 1615 8-Heptadecene 1680 Source: Varlet. 55. it must be considered as an attempt of identification. Means of three fillets. woody (4) (2) Clove.2 (continued) Odorant Characteristics and Concentrations of the Most Potent Odorant Volatile Compounds in Salmon Fillets Treated by Liquid Smoke Means of Identificationa Mean ± SDe 7. When only MS is available for identification. J. STD. spectrum.23 ± 0. standard (when the retention time.Table 15.. LRI.81 ± 11. LRI MS.5 is rounded to 5 and an intensity between 5. Average intensity of the eight judges is rounded to the nearest whole number. . An intensity between 5 and 5. Number of judges (out of eight) who have detected an odor. Agric. odor intensity. and quantities of odor-active compounds detected by fewer than six judges are indicated in parenthesis. STD MS. LRI. and odor description of an identified compound correspond to the retention time. 2007. linear retention index (when the LRI of the identified compound corresponds to the LRI in the literature). a b c Handbook of Seafood and Seafood Products Analysis d e Means of identification: MS. roasty. mass spectrum (identified using the mass spectra of the compounds). et al. The odor given corresponds to the odor detected by the judges during olfactometric analysis for its retention time but not surely due to the compound that we try to identify. STD MS. and odor description of the injected standard of this compound). Note: Frequency of detection.48) 1. chemical 6 4 Smoke.3.77 24. roasty 7 4 Burnt rubber. LRI Animal. 4518.5 and 6 is rounded to 6 (1 = very weak odor.

The fractionation of a commercial liquid smoke preparation evaluated by a sensory panel concluded that the phenol fraction was essential but not complete from a sensory standpoint [42]. The high-boiling fraction of phenolic compounds (133°C–200°C) was described with an acid and chemical property that was judged of poor quality. and the odorant contribution of odor-active compounds cannot be the same if the SF is in the form of powders. 15. they may contribute in mixture to the overall odor. the same compounds responsible for the odor should be involved in the flavor that SF confers to seafood. early works performed on individual phenolic compounds have identified the impact of guaiacol on the smoky flavor. sometimes cooked. Recently. and seem to contribute little to overall aroma.6.39]. reactions between liquid smoke compounds and the components of the matrix can occur through Maillard and Strecker reactions. The oils used in smoke oils can soften the smoke aroma. . liquids. guaiacol derivatives and more generally the phenolic compounds of low-boiling fraction molecules (60°C–90°C) have been shown to cause the odor. However. They were isolated early from wood smoke and described as grassy. Enolone derivatives are compounds derived from cyclopentenone.3. but taste was not investigated as much as odor. However. However. Sensory analysis performed on standards confirmed the importance of guaiacol and o-cresol in the smoky flavor and dimethylphenol.Smoke Flavoring Technology in Seafood ◾ 245 Carbonyl compounds have also been reported as contributors to the smoky aroma of wood smoke. Furannic compounds were thought to contribute to soften the heavy smoky aromas associated with phenolic compounds [37. The amino acids from the seafood matrix and the carbonyl compounds from the SF can generate furannic compounds and nitrogen-containing compounds with roasty/smoky aromatic notes [19.38]. The taste thresholds of some phenolic compounds were determined [40] and showed a high diversity between the molecules. Concerning the bitter taste. As for the assessment of the odor. Furthermore. 4-methylguaiacol. the determination of the effects of compounds of SF on the flavor is complex. and very little information is available. Furfural and homologues exhibit cooked/roasty aromatic notes. whereas syringol and 4-methylguaiacol showed the same but lower effect than guaiacol [40]. A polyfunctional carbonyl subfraction was isolated from wood smoke and possessed a caramellic/burnt sugar aromatic note [36]. phenolic compounds are not the only flavor-active compounds. if they do not have a strong individual influence. For several decades. 4-methylguaiacol perception was superior to that of syringol and guaiacol. and isoeugenol in spicy/sweet flavor [41]. as the other minor odor-active compounds.34]. The determination of the role of SF components in the final product odor is complex due to the odorant interactions that can occur between the odor-active compounds. The results of this fractionation are given in Table 15. Then. the physical state of SF can also influence the aroma. or oils. Two categories of carbonyl compounds can be differentiated: furannic compounds and enolone derivatives. Synergic or masking effects are possible and make the final odor complex. because they are not the compounds mainly detected in SF and seafood treated by SF odors by sensory analysis [37]. furannic compounds were found to play a role in cold smoke odors of liquid smoke or fishes treated by liquid smoke [14. Moreover. More recently. it was commonly admitted that syringol derivatives impart a smoky odor and guaiacol derivatives contribute to a smoky flavor.2 Role of Volatile Compounds of SF in the Flavor Phenolic compounds were shown as the major contributors of the smoky flavor [35].

histidine.246 ◾ Handbook of Seafood and Seafood Products Analysis Table 15.6. 2. and lysine residues [44]. A brief drying after smoke absorption can cause a higher level of dehydration and lead to higher amounts of Maillard products. 5. SFs under powder or oil forms do not act on the surface texture. According to their concentrations found in the different SF. because they are added in the product during its fabrication. furannic compounds could also have an effect on the flavor [43]. Formaldehyde seems to be involved in the texture of smoked fishes and to be responsible for the layer at the dried surface of fishes [45].3 Sensory Taste Intensities of Liquid Smoke Fractions Fractionb Taste Property a 1 6 3 1 1 2 7 1 1 2 3 3 2 3 3 4 11 0 0 0 5 4 6 1 0 6 10 1 0 0 Smoke taste intensity Tarry taste intensity Chemical taste intensity Acidulous taste intensity a b Intensity scale: 0 = below threshold. smoke condensates are colored mainly due to phenolic compounds. the product must also be placed in a dry and hot ambient atmosphere for short periods in order to favor color formation. 15. whole liquid smoke. and the intensity of the process.3 Role of Volatile Compounds of SF in the Texture The texture of smoked products is due to coagulation of proteins. which have brown/yellow characteristic color. However. they could play a role in the inner texture of the product. 1.6. which has been reported in wood smoke and smoked meat [41] but not in smoked fishes until now. distilled at 67°C–90°C. which can vary from golden yellow to dark brown according to the nature of the wood. cysteine. can react with proteins. Thus. the eventual dilutions of SF. After scission and dehydration. Other compounds such as enolone derivatives could also play a role in the SF flavor. melanoidines could be created by polymerization through aldolic condensations.4 Role of Volatile Compounds of SF in the Aspect and Color The color of seafood treated by SF can derive from physical and chemical reactions. 3. Indeed. The acidic aqueous SF can also increase the coagulation of proteins and act on the texture. Maillard and Strecker compounds can also be responsible of the color of the smoked product [45]. 6. distilled at 133°C– 200°C. 15. terpene subfraction. 11 = highest value. However. the deposition of Maillard compounds leads to a darker color of fish flesh [46]. In the liquid smoking process. Formaldehyde was shown to react with the amino group of the N-terminal amino acid residue and the side chains of arginine. These . 4. distilled at 91°C–132°C. Their physical deposition of SF on seafood can confer its color to the product. Studies on standards have shown that cyclotene was a flavor-active compound [41]. phenolic subfraction. Formaldehyde.

the most prevalent essential amino acid in fish. Protein-bound lysine. Carbonyl-amino reactions as Maillard reaction could play a main role in smoked food. and hydrocarbons are not influential. A synergic effect has been shown between high-boiling point phenolic compounds and oxidized phenolic compounds and it prolongs the antioxidant action [44]. and 2-oxopropanal are considered to be important color precursors [6. the activity of compounds must take into account the synergic or antagonist effects in mixture. and the degrees of reticulation of the molecule vary as a function of time [49]. or 4-propenylsyringol. phenolic compounds can easily support the lack of electrons. is considered as a major source of the amino components in such reactions. which could contribute to product safety by controlling the growth of foodborne pathogens. Glycolic aldehyde. Carbonyl compounds and esters are nearly not implied. that is why some researchers have concluded the absence of relation between the inhibitory effect of essential oils and their phenolic content. there is a critical concentration that must not be overcome to avoid an inversion of antioxidant effect that can become prooxidant. could be responsible for most of the antimicrobial properties. but a loss in arginine and histidine is also observed. 15. Concerning the antimicrobial effect of wood smoke condensates. Studies on the antimicrobial activity of some smoke condensates have revealed very variable effects on the growth of microorganisms [50]. Among monohydroxyphenolic compounds.7 Role of Volatile Compounds of SF in Preservation Smoking process is the oldest preservation technique because of the antimicrobial and antioxidants properties of wood smoke. The polymerization is favored by the heat. Finally. the antioxidant properties depend on the radical located in the para position from the hydroxy group as in 4-methylguaiacol. The phenolic compounds can give an electron to stabilize the oxidant molecule and with their ringlike structure and mesomeric forms. phenolic compounds and carboxylic acids. because of its terminal amino group. Coniferaldehyde and syringaldehyde are considered to be irreversibly bound to proteins and to contribute orange tints to the products [24]. it seems that phenolic compounds and carboxylic acids play an inhibitory role. Food industries are working to develop new applications of smoke condensates.24]. An oxidant molecule acts by electronic capture and can trouble the preservation of the product by the initiation of lipid oxidation. The antioxidant compounds of wood smoke condensates are those with an active phenolic function. 4-methylsyringol. the glossy aspect noticeable on certain smoked products is the result of reactions between phenolic compounds and aldehydes [48]. A part of the fi nal color could derive from phenolic compounds with aldehyde function. methylglyoxal. syringol. The antioxidant activity of guaiacol. alone or in synergy. especially against bacteria [51]. As in odor and flavor. Therefore. . and 4-vinylsyringol is lower.Smoke Flavoring Technology in Seafood ◾ 247 compounds give to the final product a brown color. They lead to resinous substances (phenoplasts). The most active compounds are polyhydroxyphenolic compounds such as pyrogallol and resorcinol. However. 4-vinylguaiacol. but no information is available concerning this pathway [47]. The antioxidant behavior is increasing with the temperature of the boiling point of the phenolic compounds [44].

8-catechol O OH S O COOH O O OH OH OH OH OH O B[a]P sulfo-conjugate B[a]P glucuronide Detoxification products Figure 15.54]. hence their toxicity (Figure 15.8 15.8. they are considered as heavy PAHs.8-dihydrodiol OH B[a]P glutathion conjugate OH OH B[a]P 7. They are considered as carcinogenic contaminants. PAHs are considered as carcinogenic contaminants of processed food [56].3 Benzo[a]pyrene metabolization. because their catabolism leads to poly-hydroxy-epoxy-PAH suitable for binding to DNA adducts. However. PAHs are formed by the incomplete burning of carbon-containing material. Therefore. Therefore. PAHs are generated during smoke production by wood pyrolysis. the uses of SF in food industrial processes must be ruled out in order to guarantee food O DNA adducts OH Benzo[a]pyrene:B[a]P OH B[a]P 7. Hundreds of individual PAHs may be formed and released during the process of incomplete burning of the wood.8-dihydrodiol-9. they are considered as environmental pollutants and can contaminate the human feed raw material [53. In SF. PAHs comprise fused aromatic rings made up of carbon and hydrogen atoms: up to four fused benzene rings.58].3).8 epoxyde OH B[a]P 7. which are constituted by less than four benzene rings. Owing to their lipophilic properties (log Kow between 4 and 7). These compounds have been studied for several years.248 ◾ Handbook of Seafood and Seafood Products Analysis 15. it is used as the leading substance to illustrate PAH contamination. PAHs can cross the biological membranes and accumulate in tissues. particularly smoked food [57. home cooking and industrial food processes represent the major source of human contamination [55]. B[a]P is the first PAH whose toxicity and carcinogenicity was assessed from the observations of Sir Percival Plott in 1775 at St Bartholomew hospital of London about cancer of the scrotum of the chimney sweepers.1 Polycyclic Aromatic Hydrocarbons Properties and Toxicology PAHs are well known as being food contaminants and carcinogens [52]. . especially benzo[a]pyrene (B[a]P) [59]. As they can be absorbed by animals. more toxic than light PAHs.10-epoxyde Glutathion S OH OH OH O B[a]P 7.

supercritical fluid extraction (SFE) [66] or solid-phase microextraction (SPME) [67]. gas chromatography and liquid chromatography are the most used techniques [55]. but. cause chromatographic coelutions. which combines a separation step and a detection step. For the separation of the PAHs extracted from SF or seafood treated by SF. and stir bar sorptive extraction (SBSE) [68] but not on liquid smokes or seafood treated by SF.1 European Regulations on PAH Found in SF In 2003. a European regulation set the maximum contents of PAH in the primary products (PP) of smoke condensates used for the production of SF. that is. in Italy. Gas chromatography is coupled to mass spectrometry [58. The eight heavy PAHs left were shown as being carcinogenic or mutagenic contaminants and gave rise to serious health concern.9 Legislative Aspects 15. PSC and PTF. the analysis step is the most critical point. Although all steps are important. purification and/or delipidation steps such as saponification are often applied to reduce the fat matter rate of samples [64]. In both condensates. to our knowledge. respectively [74]. Purification was especially performed on an alumina or silica column. eight light PAHs were considered as environmental contaminants. Environmental Protection Agency (US-EPA) identified a list of 16 PAHs as the most frequently found [60]. such as bidimensional chromatography at the gaseous phase (GC/GC) [71] or liquid phase (LC/LC) [72]. This harmonization was necessary to homogenize the legislation about SFs. 15. Moreover.9. or tandem mass spectrometry [70].Smoke Flavoring Technology in Seafood ◾ 249 safety avoiding PAH contamination. Apolar solvents or mixes of apolar and semipolar solvents are used to extract the maximum of PAHs. Therefore. they have not been applied to SF or seafood treated by SF. Parameters of the chromatographic separation and detection must be adjusted to avoid coelutions with interferences from lipids. PAHs are often coextracted with fat matter. the maximum levels of B[a]A and B[a]P were set at 20 and 10 mg/kg of liquid smoke. these PAHs were considered as toxic at low levels. which can disturb the extraction.8. For example. the concentrations of benzo[a]anthracene (B[a]A) and benzo[a]pyrene (B[a]P) must not exceed 20 and 10 mg/kg of liquid smoke. The extraction step must integrate the composition of the matrix. . and liquid chromatography is coupled to ultraviolet or fluorimetric detector [59–63]. respectively [73]. flame ionization detector (FID) [60]. but solid-phase extraction (SPE) cartridges are now more frequently employed. solid–liquid extraction can be carried out. even if they were found in weak quantities.2 Extraction and Analysis Methods of PAH in SF and Seafood Treated by SF The quantification of PAHs in SF and seafood treated by SFs is performed in two steps: an extraction step and the analysis step. the chromatography must be sufficiently efficient to separate isomers of PAH. In the case of solid seafood treated by liquid smoke. the U. Among them. Other extraction devices have been developed to investigate PAH in smoked food such as accelerated solvent extraction (ASE) [65]. it is essential to quantify only the toxictargeted compounds. Indeed. with a weak toxicity but high concentrations in the samples analyzed. Several devices are therefore developed to optimize the analysis. In the case of liquid matrices as liquid smoke. and lead to mistakes in the identification. because they do not have the same toxicity. a liquid–liquid solvent extraction is often used [61–63]. In the 1980s.S. 15. However.69]. Thus. The nature of the SPE cartridge phase is linked to the extraction method and the biochemical composition of the initial matrix.

the smoking regulations set a maximum B[a]P value of 5 mg/kg of smoked fishery products and smoked crustaceans. the respective legal B[a]P amount is 5 mg/kg. Therefore. important differences in PAH concentrations are noticeable [57. 15. Indeed. the vaporization of SF in a smokehouse causes a loophole in the legislation. However. Thus. it is necessary to better control the composition of SFs and to improve knowledge about the influence of the pyrolysis parameters (wood nature. The high maximum values authorized in PP do not seem well adjusted with the weak final PAH contamination of SF. 15.78]. atomization of liquid smoke would constitute the smoking technique. Indeed. The main criticism that can be formulated against SF is the lack of control of the final organoleptic qualities of such processed food. and head and thorax meat of lobster and similar large crustaceans [76. that is. Moreover. leading to less PAH contaminated food by comparison with the traditional smoking techniques [80].63] which justifies controls and regulations. Therefore. Moreover. according to the origin of SF and industrial manufacturers. this process can also be considered as a flavoring of the surface of the product. SFs are used in higher quantities than those employed in flavoring processes. it leads to lower PAH contents. As for SF. Finally. the liquid smoking process decreases the emissions of PAH compounds to the environment.10 Conclusion A wide range of SFs and uses of SFs are now available to flavor seafood products. This value is very low compared to those authorized in PP. brown meat of crab. All these benefits could help to reconsider the status of atomization of liquid smoke and the maximum PAH contents related.03 mg/kg.75].2 European Regulations on PAH Concentration in Food Treated by SF In 1988. as drenching or showering. Moreover. the PAH contamination reached in SF is largely below the values authorized in PP [60. it can lead to problems of labeling. Indeed.250 ◾ Handbook of Seafood and Seafood Products Analysis However. a European regulation set the maximum content of B[a]P in foodstuffs treated by SF at 0. atomization of liquid smoke must be lower than 0.9. SFs appear as a safe alternative to smoking techniques. it is paradoxical to apply flavoring regulations to the smoking process. However. atomization of liquid smoke in a smokehouse is considered as a smoking process but the maximum level of PAH must not overcome that of flavoring legislation. Indeed. whereas food is treated by SF and not directly by PP. This value is the result of the necessary harmonization between the national laws of European countries [79]. the 2003 maximum values must be reviewed again. for meat industry. In certain countries such as France. very small amounts. In this case. because these values were set for PP and not SF.69. However. if it is considered as smoking technique. that is. the toxicity of other heavy PAHs was recently demonstrated and the monitoring of these PAHs was recommended by a European regulation published in 2005 [76]. Nevertheless. Therefore. Th is fact can be understood by the use of smoke condensates in flavoring quantities.03 mg/kg and leads often to noncompliant smoked products. excluding bivalve molluscs. The content of PAH in SFs and in the final product can be better controlled than during traditional smoking.03 mg/kg. the PAH contamination was only set for B[a]P [77]. . 0. it is legitimate to wonder if the exclusive monitoring of the B[a]A and B[a]P in PP is adequate to illustrate the PAH contamination of SF. but it could also initiate an international consideration of labeling of smoked and flavored food.

I. Guillén..M.. and Manzanos. Agric. Guillén. The problem can come from the emulsifiers that are sometimes added in SFs. 1302. P.. et al.. Varlet. L.. and Preservation. 11. Boca Raton. the traceability of SF must be improved. 1995. Agric. 21. Principles of Smokeless Smoke Curing. Appl.. Anal..J. R. SFs are forbidden for the smoking of organic products from aquaculture. 637. M. Finally. Hollenbeck. Pyrolysis-gas chromatography/mass spectrometry of Quercus sp.. 19. Appl. Food Chem. 18. Factors affecting elimination of polycyclic aromatic hydrocarbons from smoked meat foods and liquid smoke flavourings.. Guillén. Simon. Food Agric. J.. Study of the volatile composition of an aqueous oak smoke preparation. Food Agric.E.Smoke Flavoring Technology in Seafood ◾ 251 wood size. Indeed. 14. 3(1–2). References 1. 79... D.. Manzanos. Study of a commercial liquid smoke flavoring by means of gas chromatography/mass spectrometry and Fourier transform infrared spectroscopy. Studies of the smoking process for foods.... and Zabala. 1996. 4.. 2005. Food Res..L. J. 2. 8. 4. J. 1996. Relationships between the maximum temperature reached in the smoke generation processes from Vitis vinifera L... in France. T. 503.J. shoot sawdust and composition of the aqueous smoke flavoring preparations obtained. M. Z... E. 6. 9. Šimko.. 283. Pyrol. Volatiles composition and flavour profile identity of smoke flavourings. Sep.M.. and Sikorski. Pure Appl. Besides. and Campbell..). FL. Alén. 1995. Tour highlights production and uses of smoke-based flavors. and Manzanos. 1267. 4518. 49.J. 2005. and Ibargoitia. according to allergic people and religious groups. CRC Press. V. Food Addit. Sci.B. Smoke and liquid smoke. 1987. Guillén.D. Polycyclic aromatic hydrocarbons in smoked food products and commercial liquid smoke flavourings. et al. 49.. 1990. 2005. J. Study of an aqueous smoke flavouring from the aromatic plant Thymus vulgaris L.. M... 17. Contam. N. 55. W. whereas SFs are produced from natural wood. wood.. 871. .. 1961. Guillén. 139.A.. Jira. et al. 55. Nonier. and Oesch. which could contribute to give to the SF a less processed characteristic. E. Sci. 10. Z. M. January. Pszczola. Food Rev.. 9. 16. 251. the processed food cannot be consumed. 1999. 463. Smoking. P. 163. and Manzanos. Simpson. 2005.. 635. W. J. M. Foster. 17(1–2). 1977. 44. Food Chem. Kurko. Food Technol. 15. Chem.. moisture.W. Pref. 28. Composition and analysis of liquid smoke flavouring primary products. Agric. the optimization of SFs effects on food products must be done avoiding PAH generation. Miler.E. 13. 1993. E. in Seafood: Resources. M. Olfactometric determination of the most potent odor-active compounds in salmon muscle (Salmo salar) smoked by using four smoke generation techniques. Food Chem. J. Kuoppala. M. Chemical reactions of smoking. Mol. 75(2). Ed. Legkaja i Pishchevaja Promyshlennost.D. Int. 5. 70. and today no information is available.. D. Fleischwirtschaft Int. Sci. Food Qual. Gomaa. Kostyra. M. 85. 7.E...D. 2007. The flavor chemistry of wood smoke. M. 3. Moscow. Study of the components of a solid smoke flavouring preparation.. Anal. M. 137. 1996. Food Chem. Food Chem. J. M. However. Sikorski. etc. 10(5).J. Application to structural elucidation of macromolecules and aromatic profiles of different species.. Novel concepts in technology and design of machinery for production and application of smoke in the food industry.D.. C. Nutr. 43... V. 181. 79. K. temperature. J..A.H. 36. and Baryłko-Pikielna.D. R. 2002. 1687. Maga.F. 12. 2006. 1984. J. Pyrol. The role of smoke particles.. et al. p. Formation of the main degradation compound groups from wood and its components during pyrolysis. Nutritional Composition.

. Baryłko-Pikielna. Kurata. Pol. Food Chem. 40. Agric. Sérot. 433. M. Pure Appl. 85. Radecki. Effect of smoking processes on the contents of 10 major phenolic compounds in smoked fillets of herring (Cuplea harengus). and Ling.252 ◾ Handbook of Seafood and Seafood Products Analysis 20. Food Sci. J.. 1201. 4126. 1972. 37. 137. Bratzler... F...D. Food Chem. 1977. Eur. 1988. 25. Res.G.. 240.. 2395. PA. R. 1966. Contribution of smoke compounds to sensory. 36(5) 1006. J. 27. Fish. L.L... F 7:1.. M. Rusz. Thesis. 41.. and Eyo. Food Chem.. Flavor effects of different woods on whitefish smoked in a kiln with controlled temperature. Meet.. 22. Food Sci. 1977. L. Tang. Fazzalari. 1980.. M. Chemical aspects of the smoking of meat and meat products. 2002. Chem. W. Lantz.. Rev. 22. Chem..T. Food Chem.G. 102. and air velocity. CRC Press LLC. V. 1969. A. 1639. and Ibargoitia. Agric.A. 1978. Agric.. M. Contribution of volatiles to rice aroma. et al. . Agric.M. M. 49. and Toledo. Burdock. 27(7).. 42. Soc.M. 43. 23. Wasserman. 1977. wood. Meat Res. L. J. Turnbaugh. 6235. 1984. J. Acta Aliment. ASTM Data Series DS 48A.. 8. 2004. 3. 1005. et al. 49.C. Ojeda. U. J. A. 2006..Z. June/July. Agric. Carbohydrate and nitrogenated compounds in liquid smoke flavorings. Chem. 87. Wasserman.. Sensory properties of phenolic compounds isolated from curing smoke as influenced by its generation parameters.. 28. and Potthast. T. Buttery. et al. Boca Raton. 1655. 44. Biol. 38. 18(5). Chem. bacteriostatic and antioxidative effects in smoked foods. 29.C.. and Burtles... Pure Appl. 1975. 34. Physical and chemical processes involved in the production and application of smoke... K. Biol.E. Hamm. 36. 1999. Analysis of smoke and smoke products. et al. Influence of the moisture content on the composition of the liquid moke produced in the pyrolysis process of Fagus sylvatica L. Chemical references in sensory analysis of smoke flavourings. Fiddler.. Daun..S. W.. B. Food Chem.. 26. Organoleptic evaluation of three phenols present in wood smoke. 35. Toth. J. M. The development of flavour in potable spirits.. Lebensm. 96.W. humidity and air flow on smoke penetration into fish muscle. Food Sci. 111. Manzanos. Adv. 47. Smoke flavor as related to phenol.L. Caractérisation des composes volatils responsables des qualities odorants du saumon fume (Salmo salar) et evaluation des contaminants du fumage (Hydrocarbures Aromatiques Polycycliques). carbonyl and acid content of bologna. Kim. Identification of flavour constituents in carbonyl. T. 2007. 279. H. and Miler. Philadelphia. Effects of the smoking process on odour characteristics of smoked herring (Cuplea harengus) and relationships with phenolic compound content. S. 24..N. G... 49. et al...-Wiss. R. J. 32. A. humidity. 30.J. J. Board Can. J. Effect of smokehouse temperature. and Doerr. Workers. 1978. 2002. M. Chem. 1970. 1977. 39. 2001..A. 49. and Vaisey.. Proc.. American Society for Testing and Materials. 1974.. 203. N. Identification of formaldehyde-induced modifications in proteins: reactions with model peptides. R. Isolation and identification of some components of the lower-boiling fraction of commercial smoke flavourings. M. 31. A. Chan. B. J. Cardinal. A “smoke” flavor fraction of a liquid smoke solution. Process Biochem. and Ibargoitia... 34. Compilation of odor and taste threshold values data... 146. 40. and Fujimaki. 78(4)... Smoking of foods.. 934. Chem. 38. FL.L. Swan. 7(2). 33.. J. 5(3).. 1970.. A. Food Chem. S. Varlet. Food Chem. Pure Appl.E. Clifford. M. et al. 201. Chemical composition and application of smoke flavor. K.. Guillén. 1667. K. 53.S. 31. Feranoli’s Handbook of flavor Ingredients.. Olsen. 21.-Technol. University of Sciences of Nantes. Metz. C. J. noncarbonyl neutral and basic fractions of aqueous smoke condensates. 29.D. 1976. M..B.J. Guillén. 2004. Food Res..A.

2007.. P.H. 104(2). and Partearroyo. and Anklam. 1977. 126. Determination of polycyclic aromatic hydrocarbons in smoked meat products and smoke flavourings additives. 2002. W.. 106. Sopelana. and Sérot.. P. 47.. P. 50. marinage. Chem. 46. Food Res. E. Single-laboratory validation of a gas chromatography–mass spectrometry method for quantitation of 15 European priority polycyclic aromatic hydrocarbons in spiked smoke flavourings. Girard. Contam. Eur. 770. P. J. 1985. 111. 303.. 2006.. J. E.J. Mottier.387. 1536. Müller. Sainclivier.. Z. Šimko.Smoke Flavoring Technology in Seafood ◾ 253 45. E.. Agric. S. 48. Changes of benzo(a)pyrene contents in smoked fish during storage. 2005. M. S. P. cold-smoked rainbow trout stored at 4°C. Comparison of two clean-up methodologies for the gas chromatographic/ma ss spectrometric determination of low nanogram/gram levels of polynuclear aromatic hydrocarbons in seafood. M.J.. R. 2003. L’industrie alimentaire halieutique. Validation (in-house and collaborative) of a method based on liquid chromatography for the quantitation of 15 European-priority polycyclic aromatic hydrocarbons in smoke flavourings: HPLC-method validation for 15 EU priority PAH in smoke condensates. M.. 171. Contam. Guillén. 2000. 10(5). 48.. Opinion of the Scientific Committee on Food on the risks to human health of polycyclic aromatic hydrocarbons in food (expressed on 4 December 2002). 52. 57. 56.. 17(1). 59. . 61. Int. C..... 219. Des techniques ancestrales à leurs réalisations contemporaines: salage.. Determination of polycyclic aromatic hydrocarbons in commercial liquid smoke flavorings of different compositions by gas chromatography–mass spectrometry. Hooven. J.. 1993. Bulletin scientifique et technique de l’Ecole Nationale Supérieure Agronomique Centre de Recherches de Rennes. 307. 2005. and Anklam. 49. Parisod. and Turesky.. Food Res. 1160.D. Varlet. A.. M. B. 1629. 63. W. 45. Technol. C. P. 62. Paris.A. 2000. R.. Carcinogenic polycyclic aromatic hydrocarbonDNA adducts and mechanism of action. and Chiu. Food Chem. 49.. 1991.... 1991.. E. 54. 51. Aristimuño.P. Jira. in Technologie de la viande et des produits carnés.. 18. and Partearroyo.E.D. 36.. and Sikorski.. Food Chem. Fleischwirtsch. 1996. Chromatogr. Environ. C. 876. and Fernandez-Galian. B. Food Addit. C. Agric. Evaluation of analysis of polycyclic aromatic hydrocarbons in meat products by liquid chromatography. Chromatogr. Palme. V.. 2004. 61. 64. 2000.. V. 71(1).M. Volatile aldehydes in smoked fishes: Analysis methods. 293. 60. 105. 58. SCF/ CS/CNTM/PAH/29 final. L. J. 55.A. 218. R. Fernandez-Galian. Suñen. Mutagen. Chen.. Agric. A GC/MS method for the determination of carcinogenic polycyclic aromatic hydrocarbons (PAH) in smoked meat products and liquid smokes. W. B. Pure Appl. 208. Food Chem.. 489. Study of several aspects of a general method for the determination of polycyclic aromatic hydrocarbons in liquid smoke flavourings by gas chromatography-mass spectrometry.. Food Microbiol.... Quantitative determination of polycyclic aromatic hydrocarbons in barbecued meat sausages by gas chromatography coupled to mass spectrometry. La fumaison. 2001.. Simon. fumage. and Mahadevan.. and Listeria monocytogenes at low temperature..Y. Baird. 2002.. J... and Aristimuño.. séchage. C. Food Chem. Polycyclic aromatic hydrocarbons in smoked fish—A critical review. Stołyhwo. B. Nyman.A.. et al. Antibacterial activity of smoke wood condensates against Aeromonas hydrophila.. Food Chem. Yersinia enterocolitica.. 1103. 2007. T. M. Activity of smoke wood condensates against Aeromonas hydrophila and Listeria monocytogenes in vacuum-packaged. Tilgner. occurrence and mechanisms of formation.D. 40. Suñen. Guillén. Food Chem.. A. 1988. Sopelana. Wang. Food Addit.. 2244. Šimko. Prost. 3. hydrolysats.. Scientific Committee on Food (SCF). B. 27. 91(2). The phenomena of quality in the smoke curing process. Curing and smoking.J. J. 48.P. Mol. D. 53.. Simon. Lavoisier.. Palme. Food Chem. 44..

. Contam. 2003. et al. Readman. 24(7).. 38.. 79. Agric. 47. Chem. Moret. S. Contam. 2003. L. 259. 66.-Technol. Res. Ré-Poppi.M. 716. Pimenta. Commission Recommendation of 4 February 2005 on the further investigation into the levels of polycyclic aromatic hydrocarbons in certain foods. Analytical methods for polycyclic aromatic hydrocarbons (PAHs) in food and the environment needed for new food legislation in the European Union.254 ◾ Handbook of Seafood and Seafood Products Analysis 65.. 73. 47. Evaluation of acute toxicity and genotoxicity of liquid products from pyrolysis of Eucalyptus grandis wood. T. Chromatogr. Use of supercritical fluid extraction-high performance chromatography in the determination of polynuclear aromatic hydrocarbons from smoked and broiled fish. R. 2007. Eur. Toxicol. et al. EC 2065/2003. 1988. A. Off. N. 69. Conte. Popp.. J. Wenzl. 309. 153. Eur. 521. 1–2. 2007. EC 1881/2006.. 364: 5. and Tapanainen. Environ. 284.. M. J. Off. 34. Food Chem. Anal. Council Directive of 22 June 1988 on the approximation of the laws of the Member States relating to flavourings for use in foodstuffs and to source materials for their production. Regulation (EC) No 2065/2003 of the European Parliament and of the Council of 10 November 2003 on smoke flavourings used or intended for use in or on foods.. U.. A. and Santiago-Silva. 76. et al. 1473. Varlet et al. 1999. 70. J. Relat. Eur. 25(7). Agric.. Environ. 897. . Union.. Determination of PAH profiles by GC-MS/MS in salmon muscle processed according to four different smoking techniques.. Determination of polycyclic aromatic hydrocarbons in vegetable oils using solidphase microextraction—Comprehensive two-dimensional gas chromatography coupled with time-offlight mass spectrometry. 67. EC 88/388. Lebensm. Chromatogr. 75.. Trends Anal. et al. G.. 2000. L. 1..L. Chromatogr. Polycyclic aromatic hydrocarbons from wood pyrolysis in charcoal production furnaces. et al. Wang. 77. 2006. attuazione delle direttive 88/388/CEE e 91/71/CEE relative agli aromi destinati ad essere impiegati nei prodotti alimentari ed ai materiali di base pere la loro preparazione. 523(2). Allegato III. 1062. Food Addit. 744. Järvenpää. Off. 78. 1367. D. Purcaro.. J. J. 19(9). Italian Law Decree. 1161. Union. L.S. Union. Off. Determination of polycyclic aromatic hydrocarbons in water by solid-phase microextraction–gas chromatography–mass spectrometry. Liq. Technol.. EC 2005/108. 2006. J. G. 2006. E.. et al. and Dean. 71. and Zhou. 74. P. 101.-Wiss. 80. Use of liquid smoke flavouring as an alternative to traditional flue gas smoking of rainbow trout fillets (Oncorhyncus mykiss).. 169. Acta. 2004.J. Union. J. W. Dos Santos Barbosa. L 34: 43. J. Decreto Legislativo N°107 del 25/01/1992. 1999. Assessment of polycyclic aromatic hydrocarbon content of smoked fish by means of a fast HPLC/HPLC method..... 1. L. T. A. Commission Regulation of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs. 184. Eur. J. A. Food Chem. Hattula. J... 304. J. Huopalahti. 68. 2005. King. 72. Determination of polycyclic aromatic hydrocarbons in wastewater by off-line coupling of solid-phase microextraction with column liquid chromatography. Chim.. Arch. P. 2001. Accelerated solvent extraction and gas chromatography/mass spectrometry for determination of polycyclic aromatic hydrocarbons in smoked food samples. 1996..



.....................4 Comparison of Methods ........................................................................2 Nondestructive Analysis of Total Proximate Composition...........................................7......................270 16.......................5 Nondestructive Analysis of Proteins .270 16....................................................... Christel Solberg..................4................................3 Lipids ...............................................3 Nondestructive Methods ........................................................4........................276 257 .......................................274 16.........6 Determination of Water Content ................................................... 269 16.1 Nutritional Aspects ................ and Rasa Slizyte Contents 16.................2 Methods for Protein Determination .................................... 258 16......2 Indirect Measurements of Energy.....................................275 16....................................3.........1 Nutritional Aspects .....4 Direct Methods for Soluble Protein Determination ..........1 Introduction .3 Determination of Total Nitrogen ..................................................................................................................267 16.............. 269 16.......... 269 16......267 16......................................... Ingrid Overrein...................................................................... 273 16....................................................................................7.....................................4 Proteins ......4...............................Chapter 16 Composition and Calories Eva Falch.................4........276 References .................................5 Determination of Carbohydrate Content .......................1 Direct Measurement of Energy ...............................3........4..3.......................... 268 16................274 16................................ 258 16.............................................2 Methods for Determination of Total Lipids .................7.....................................................................267 16..............................................................................................3 Food Composition Tables and Databases .......274 16........................................270 16.................7 Calories ... 273 16.........3.......................................................................

the range 1100–2500 nm.5 m per year and a saving for the environment by replacing the Kjeldahl system. however. or whole grain.1 Introduction The proximate composition in most fish and shellfish is primarily water. NIR has been found to be a reliable. In fish meat these constituents make up about 98% of the total mass. Nearinfrared spectroscopy (NIR) is the most common method for such analysis and is therefore comprehensively presented in this chapter. to ensure obtaining data on the exact proximate composition. The NIR spectrum is defined between the wavelength 800 and 2500 nm.1 and further discussed in the text below. Section 16. During the 1980s monochromator instruments were developed. making it possible to measure over the whole NIR spectrum and not only on a small number of selected filters. There are several methods available to analyze the major components in seafood and the main methods along with their advantages and limitations are presented in Table 16. analysis should be performed on the specific samples. cheese. the adoption of NIR testing resulted in a total cost saving of CAN$ 2. 600.000 Kjeldahl analyses were conducted per year and incidentally producing 47 ton of caustic waste in the process. transmitted. rapid. by the chemical-free NIR method. and a lead sulfide. The first instruments on the market were filter instruments measuring in reflectance mode. such as the introduction of partial-least squares (PLS) by Martens in 1982 [3].7 deals with the different methods to determine and calculate calories in fish and shellfish. When Williams was running the program for the Canadian Grain Commission. in this spectral region the spectrum of the transmitted light is very compact and no single peaks are visible. The end result is a calibration equation from which the constituent of interest is calculated from a linear combination of spectral data. . The NIR radiation interacting with a sample may be absorbed. an indium gallium arsenic covers the range 800–1700 nm.258 ◾ Handbook of Seafood and Seafood Products Analysis 16. but the available detectors cover a smaller range. Proximate data on different fish species are collected in databases such as the FishBase (www. Diff use transmittance measurements are usually carried out in the 800–1100 nm region of the spectrum. However. 16. or reflected depending on the interaction with NIR wavelength and physical status of the sample as transparent or nontransparent. and sizes. Therefore. and then one can perform a linear regression on the principal components.fishbase.2 Nondestructive Analysis of Total Proximate Composition Analysis of each nutrient separately is time-consuming and requires a diverse set of equipments. and minerals [1]. making it difficult to use this spectral range before the development of multivariate calibration technology. As well as increased efficiency of the Canadian wheat segregation program. The spectral data will be reduced by principal component analysis. and so on. and the other minor constituents include carbohydrates. stages of maturity.org). the silicon detector covers the range 400–1100 nm. vitamins. where the weak absorptions enable useful data to be obtained using sample thickness of 1–2 cm of samples such as meat. and lipids. which involves concentrated sulfuric acid and heavy metal catalysts. and easy to perform nondestructive analysis for simultaneous determination of the major components in fish. Methods for simultaneous determination of the major components are therefore valuable. proteins. geographical locations. The development of NIR in food analysis started with the development of analysis of cereal grains and oilseeds in Canada [2]. the chemical composition of fish generally varies due to seasons.

precise. or transmission of nearinfrared light (850–1700 nm) Ultrasound Measurement of ultrasonic velocity Rapid. [4–6. and disturbance by particle size in samples Ultrasonic properties of tissue depend on composition and temperature [11. Different calibrations for different species and organs. lipids. water. and protein. nondestructive.172–174] (continued) ◾ 259 .8] For reflectance instruments (surface analysis) some drawbacks such as interference by starch and lipids. The equipments are relatively expensive.1 Overview of the Most Common Methods for Analysis of Proximate Composition in Fish and Fish Products Advantages Drawbacks Selected References Methods Principle Total Proximate Composition Rapid method simultaneously analyzing fat. and can be performed online [17–20. fully automated. and protein content noninvasively.173] Total body electrical conductivity (TOBEC) May obtain data on water. displacement of reflectance spectrum by moisture content. can be used on live fish Samples are placed in an electromagnetic field and electric conductivity is measured Specific for different species.133] NIR/NIT Reflection.Table 16. can be nonsensitive. expensive Few articles on fish composition Need more research Composition and Calories [21. nondestructive. Calibrations need to be made against reference methods. Nondestructive and can be used on live fish. transflection. Calibrations require skilled personnel. physiological and physical states can affect values of conductivity.

Fosslet. (SoxTech. lipid content.Table 16. less exposure to chemicals (compared with manual solvent extraction) No laboratory facilities are required No use of chemicals A simple and inexpensive method [43] Possibilities to further characterize the lipids extracted Requires laboratory facilities [26–31] Handbook of Seafood and Seafood Products Analysis Manual solvent extraction Extraction of minced samples generally using chloroform and methanol as solvent Gravimetric determination Automatic solvent extraction Extraction of minced samples by solvents in automatic systems (Soxhlet. nondestructive (See under NMR below) [13–15. etc.1 (continued) Advantages Rapid. the fat content can be calculated theoretically by the formula Fat% = 80 − water % .22.) Microwave drying The sample is dried and from the water content found. Need of sample specific calibrations Weaknesses due to quantification of proteins without combining with destructive methods Drawbacks Selected References Overview of the Most Common Methods for Analysis of Proximate Composition in Fish and Fish Products 260 ◾ Methods Principle Nuclear magnetic resonance (NMR) Nuclei of atoms in a sample provide spectra when the sample is exposed to a magnetic field Total Lipid Determination Chemical Extractions: Provides high total lipid yield Time-consuming Use of health hazard chemicals Destructive technique Requires well-trained laboratory personnel May discriminate structured fat (such as phospholipids) Requires laboratory facilities Physical and chemical changes might occur during examination Precision level may be dependent on sample (maturity stages of the fish.55. processing) [46] Automatic. location of lipids. fexICA).57] Excellent for determination of fat and water content or even distinguish lipid classes and water properties.

Low-field NMR See NMR above Composition and Calories NMR mouse ◾ Nondestructive and rapid. location of lipids.55] Fat meters Determination of water by analyzing the dielectric properties using a microwave strip (calculation of lipids as for the drying method). processing) Needs to be calibrated for the individual species Most suitable for neutral lipid determination See NIR/NIT above [4–6. rapid. and portable Allows in vivo measurements Nondestructive and rapid Broad range of applications.8] [46. portable (small size).51–52] Relatively inexpensive. requires specific calibrations Traditional low-field instruments require withdrawal of homogeneous samples for analysis (invasive) [14–16.Nondestructive Methods: No laboratory facilities are required Precision level may be dependent on sample (maturity stages of the fish. The NMR mouse is rapid. may also provide other nutrient data in the same analysis Some portable instruments are available Allows in vivo measurements Expensive.50. easy. lipid content. nondestructive.22. NIR/NIT Transmitted or transflected Near Infrared light (800–1700 nm). and nondestructive and allows in vivo measurements (continued) 261 .

time-consuming. trapping of ammonia.133] Direct Protein Determination on Soluble Proteins Rapid. potentially toxic chemicals are used Rapid.1 (continued) Advantages Drawbacks Selected References Overview of the Most Common Methods for Analysis of Proximate Composition in Fish and Fish Products 262 ◾ Methods Principle Protein Determination Proteins Total Nitrogen Determination Widely used internationally. interference by nonprotein nitrogen compounds. hazardous. 74. since all nitrogen in foods is not in the form of protein. independent of physical state of sample Does not give a measure of the true protein. inexpensive to use and sensitive to low concentrations of proteins Limited to soluble proteins Most samples must undergo steps of sample preparation before they can be analyzed. and environmentally friendly High initial costs. high precision and good reproducibility. easy to perform.Table 16. distillation. low sensitivity. Difficult to obtain the accurate protein concentration [53. Absorbance depends on the type of protein analyzed . standard method for comparison. safe (no chemical exposure).67.120. inexpensive to use.133] Kjeldahl Sample digestion followed by neutralization. and titration with acid Handbook of Seafood and Seafood Products Analysis Dumas combustion method High temperature combustion and detection of N by thermal conductivity detector [53.

175] High sensitivity and easy to perform Standard curve is nonlinear. Unstable reagents are used. easy to perform. High amounts of endogenous proteases may cause errors. variation of binding capacity for different batches of commercial grade dyes [68. interference from ammonia. nondestructive. buffers salts. and reaction products are detected between 500 and 750 nm Rapid. and internationally accepted Dye-binding (Bradford) method The protein and dye complex causes a shift in the absorption maximum of the dye from 465 to 595 nm. interference by UV-absorbing compounds (nucleic acids and nucleotides). Absorbance at 540 nm Lowry protein assay Copper(II) ions in alkaline solution react with protein to form complexes. which react with the Folinphenol reagent. high sensitivity. no addition of reagents required Low sensitivity. technique is less sensitive to protein type: it utilizes absorption involving peptide bonds that are common to all proteins.177] Composition and Calories Near-UV absorption Measurement of UV absorption (280 nm) [133] ◾ Rapid.133. detergents [133.Biuret method (Alkaline copper reagent test) Negligible interference from materials that absorb at lower wavelengths. depends on amino acid composition 263 (continued) . interference from common laboratory chemicals.133] A violet-purplish color is produced when copper(II) ions interact with peptide bonds under alkaline conditions.176. Other compounds can interfere. easy to perform Relatively low sensitivity compared with other UV-visible methods.131. color development depends on amino acid composition [67. protein-dye complex adsorbs on glass surface. The amount of absorption is proportional to the protein present Color formation and binding depend on proteins present.

salts) [132. complex calibration See NIR/NIT above [133] Infrared absorption Absorption at 780–2500 nm NIR/NIT Transmitted or transflected Near-infrared light (800–1700 nm) .178] Drawbacks Selected References Overview of the Most Common Methods for Analysis of Proximate Composition in Fish and Fish Products 264 ◾ Methods Principle Far-UV absorption Measurement of UV absorption Highperformance chromatography (HPLC) Hydrolysis. nondestructive.Table 16. derivatization. no addition of reagents required. low interference from nucleic acids and nucleotides Interference by oxygen and UV-absorbing compounds (buffer. nondestructive. chromatographic separation. value for net protein. 108.96. option to quantify free amino acids. quantifies amino acids. Derivatization agents: OPA: no derivatization with secondary amino acids. and detection of amino acids with UV absorbance. might interfere [90–92. multicomponent analysis See NIR/NIT above Strong interference by water.97.133] Faster than ion exchange chromatography. FMOC: less soluble.1 (continued) Advantages Rapid. influence by lipids and sample particle size. hydrolysis destroys some of the amino acids. high sensitivity. fluorescence Handbook of Seafood and Seafood Products Analysis Nondestructive Determination Rapid. sensitive Most methods do not include all amino acids. low dependency of signal response on amino acid composition.

Air drying (101°C) may lead to thermal damage. Vacuum drying: may be difficult to keep uniform temperature distribution in the oven Air or vacuum drying The sample is dried until constant weight (e. Infrared drying Microwave drying Drying by irradiation Dean and Stark method Volumetric analysis of water after boiling in toluene See NIR/NIT above Possible to distinguish between free and bounded water NIR/NIT See NIR/NIT above NMR See NMR above Composition and Calories ◾ 265 . 12 or 24 h) and water evaporated is determined.g. For the microwave method it is possible to analyze many samples simultaneously Risk of overheating [40] Faster than oven drying methods Requires laboratory facilities Uses health hazard chemicals (toluene) See NIR/NIT above Calibrations are needed and knowledge on chemometry is an advantage [152..178] [151] Long analysis time.Water Determination Simple to use and inexpensive equipments required [151] Shorter analysis time compared with air and vacuum drying.


Handbook of Seafood and Seafood Products Analysis

Near-infrared transmittance (NIT) instruments are particularly suitable to the analysis of fish. Generally, the sample has to be minced, and it is usually possible to run several subsamples. The results are averaged to obtain more representative spectral data from the sample. The spectral data are then used to perform multivariate calibrations against the chemical or physical data. The same spectral data will be used against the different selected variables, so one can simultaneously predict, for example, water, fat, and protein content from the same spectral data as accurately as the traditional “wet” chemical methods [4]. To analyze directly on a fillet one needs an interactance probe; this involves illumination and detection at laterally separated points on the sample’s surface. It is normally accomplished using a fiber-optic probe in which one set of fiber-optic bundles carries the incident radiation and another carries the reflected radiation. Due to the striped structure of fish muscle, it is necessary to have a large interactance probe, usually two times 2 cm. With this type of probe it is possible to make analysis directly on the fillet, without previous mincing, but with a slightly lower accuracy [4–6]. Portable instruments are now available [7], and successful results are also obtained for whole fish [5] and for live fish [8]. Instead of a conventional monochromator, instruments are now also made with diode arrays, making it possible to measure the whole spectrum at the same time and in that way reducing the time for measurement, making online analysis possible [8]. NIR absorption will change with temperature and calibration, and NIR measurements must therefore be made on samples with approximately the same temperature [9]. Moreover, the measurements are affected by texture and whether the sample has been frozen and thawed [10,11]. Due to the requirement of extensive sample specific calibrations, the analysis should be performed by skilled personnel [12]; however, once calibrated the analysis is easy to perform. Nuclear magnetic resonance (NMR) is another nondestructive technique that enables determination of fat and water, and recent studies have shown that it might be possible to also gain data on protein levels in dried samples [13]. The low-field NMR instruments commonly in use require withdrawal of cylindrical samples of 10–40 mm diameter for analysis [14,15]. The method is fast, accurate, and easy to use when the calibrations are performed. A new handheld portable NMR instrument (NMR mouse) has recently been developed [16,14], and it enables an analysis time of less than 20 s and can even be used in vivo on living fish [14]. Less common methods for nondestructive analysis of proximate composition in fi sh are ultrasound techniques [17–20], the total body electrical conductivity (TOBEC) technique [21], and magnetic resonance imaging (MRI) [22]. The ultrasound method is rapid, automated, and can be used online, and empirical equations have been developed to relate the ultrasonic velocity to composition [17]. A weakness in this method is the variations in ultrasonic properties of fi sh tissue due to temperature [17]. For nonfatty fi sh, the solid nonfat content can be determined from a single measurement; however at least two temperatures are suggested during analysis of fat and solid nonfat in fatty tissue [17]. In the TOBEC method the live fish is placed in a low-frequency electromagnetic field, and the distinct electrical characteristics of body fat and fat free tissue provide the proximate data [21]. MRI can provide valuable information on proximate composition and distribution of chemical constituents in fish samples [14]; however, these imaging instruments are expensive and are used primarily in certain research laboratories. Calculation of fat content by measuring the water content is possible with cheap, robust instruments (see below), but they can be used only when the protein content is stable.

Composition and Calories




16.3.1 Nutritional Aspects
Marine lipids contain the omega-3 fatty acids such as C20:5n-3 (EPA) and C22:6n-3 (DHA) with well-documented beneficial health effects [23–25]. These fatty acids are found in all parts of the fish and are constituents of different lipid classes such as phospholipids, triacylglycerols, lysophospholipids, partial glycerides, esters, and free fatty acids. Marine lipids are the only source of EPA and DHA, and extraction and utilization of these fatty acids is a major industry. The market shares for higher value applications such as food ingredients, health care products, and medicine are increasing owing to the supply to aquaculture business.

16.3.2 Methods for Determination of Total Lipids
The lipid content in fish can be determined by several different methods varying in efficiency, total lipid yield, accuracy, skill requirement, and cost. The main methods are shown in Table 16.1 ranging from organic solvent extraction, microwave drying, to nondestructive techniques. Fish lipids are generally composed of polar and neutral lipid compounds. Although the triacylglycerols dominate in the lipid classes of fatty fish such as the pelagic species, the phospholipids are the main lipid class in lean white fish species. In addition, other derivatives of fatty acids (partial glycerides, free fatty acids, esters etc.), sterols, fat-soluble vitamins, and carotenoids are found in fish and comprise the large group called total lipids. Chemical methods: Traditional methods for determination of total lipids are generally based on solvent extraction followed by gravimetric determination. The lipid yield obtained is highly dependent on the solvent system, and using a combination of polar and nonpolar solvents it is possible to extract the total lipids and not only the free lipids such as triacylglycerols. Differences in lipid yield among the methods are claimed to correlate with the extraction efficiency of the more tightly bounded polar lipids such as phospholipids [26]. A combination of chloroform, methanol, and water is most often used for manual extraction of total lipids in fish [27,28]. The methanol penetrates the tissue while the chloroform dissolves the fat. The samples are first homogenized and after several extraction steps, followed by evaporation of solvents, the total lipids are gravimetrically determined. The Bligh & Dyer method (B&D) was originally used on fish muscle and less solvent volumes were used compared with the Folch method. A comparison between the Folch and B&D method has previously shown that the B&D method underestimates the lipid yield when the lipid content in fish muscle is above 2%, whereas no significant differences are found at lower levels [29]. Modifications of the B&D method are widely reported in the literature [30,31], although these specific modifications are rarely described in detail [29]. One recent study demonstrated that a modified B&D method using NaCl and electrolyzed cathode water gave higher lipid yield compared with the conventional method [32]. Generally, the crude lipids extracted by B&D compose a broad range of lipid classes, and the method demonstrates a high efficiency in extracting both polar and neutral lipids. However, parameters such as solvent ratio, order of solvent addition, and number of extraction steps are important parameters that affect the lipid yield and might be individually suited for specific sample material differing in lipid class composition. An example is the increased lipid yield obtained when using higher amounts of methanol, which was explained by a better extraction of phospholipids in a study by Smedes and Askland [31].


Handbook of Seafood and Seafood Products Analysis

Due to the high lipid yield generally obtained by the B&D method, it has been widely used as a reference to test the efficiency of other methods, and it is particularly used in research laboratories. Additionally, this extraction allows the successive characterization of lipids such as lipid classes (tri-, di-, and monoacylglycerols, free fatty acids, phospholipids etc.), lipid oxidation products, and fatty acid composition. Hence, manual extraction is relatively time-consuming, requires laboratory facilities, and the solvents used are toxic to humans and environment. Less toxic solvents are used in some studies [31,33–37] without achieving the same lipid yield as that obtained by using the traditional solvents. Solvent extraction of animal tissues in general and procedures for preparation of samples are comprehensively discussed by Christie [38] and by the same author in the Lipid Library Website (http://www.lipidlibrary.co.uk/topics/extract2/index.htm). Another commonly used method for solvent extraction of fatty fish species is the ethylacetate method [39] without the use of expensive equipment. The method even specifies what part of the fish should be included in the analysis. Ethylacetate has replaced the health-harmful benzene that was used in the early extractions. Among the automatic solvent extraction techniques, the Soxhlet method [40] and modifications of this method have been most widely used for determination of total lipids in fish. The sample is lyophilized before solvent extractions, removal of solvents, and gravimetric determination [41]. Petroleum ether and diethyl ether are the most common solvent used but the use of hexane and acetone are also reported in some studies [41,26]. The original Soxhlet method was developed by Soxhlet in 1879. This was originally a time-consuming method (16 h); however, today, there are more rapid methods available based on the same principle with commercial instrumentation such as the SoxTec equipment. New developments in this field are continuously reducing the analysis time, and a new microwave-integrated Soxhlet may run samples in less than an hour [42]. Lipid content can also be determined without the use of chemicals such as in the microwave drying method. This is a simple and inexpensive method that indirectly calculates the lipid content from the water content analyzed [43]. The principle behind this method is a reported reverse intercorrelation between water and lipid content in clupeid fish [43–45] calculated from the following formula: Fat content% = 80% − water content % [43]. Limitations in this method lie particularly in the lack of fitness of the intercorrelation between water and lipids during different maturity stages for the fish [46] and also variations between different locations in the fish [46–50]. Furthermore, this intercorrelation is affected by processing, particularly heat treatment, that might reduce the water content.

16.3.3 Nondestructive Methods
The intercorrelation between water and lipids in fish is also applied as the principle for the nondestructive portable Fat Meters developed by Kent [44,51–52]. The sample is irradiated by microwaves with a microwave strip, the water is measured by the dielectric properties, and the lipid content is then calculated. These instruments (Fish Fat Meters and Torry Fat Meters) are calibrated for a range of fish species [45], and they are simple to use. However, these methods share some of the same limitations as those in the microwave drying method such as the lack of fitness during spawning, and additionally, the accuracy of the Fat Meters has also been reported to be dependent on the lipid content in the fish [46]. Although the Fat Meter is limited to determining fat and water content, methods such as NIR spectroscopy may simultaneously determine the content of lipids, proteins, and water from the surface of the sample in a few seconds [4,53]. The NMR technique has particularly been applied

Composition and Calories


in quantification of lipids in fish [15,46,54–56], and the low-field NMR can distinguish between different lipid classes [57]. When increasing the field strength to high-resolution NMR, a range of different lipid constituents can be detected [58,59]. The ultrasound velocity technique has provided data that enable classification of salmon muscle into low, medium, and high fat [20]. See earlier section in this chapter for further information on these methods.

16.3.4 Comparison of Methods
Nondestructive and rapid techniques are of particular importance for fatty fish such as herring, mackerel, and some farmed fish species. The lipid content in these species usually shows large variation, and analysis results are valuable on board the fishing vessel or processing plant for sorting into groups based on their lipid content. Vogt et al. [43] who compared the lipid yield obtained by Torry Fat Meter, NIR, the microwave method and a modified Soxhlet, found that the NIR- and microwave methods were closest to the reference solvent extraction (R 2 = 0.90). A high correlation (R 2 = 0.96) has been found between ethyl acetate extraction and NIT analysis of whole minced capelin [60], and another study [46] demonstrated a good correlation between NIR and solvent extraction in specific locations of the fish (middle part of fish and fi llet skin side) (R 2 = 0.80–0.93). NMR measurements, in the same study, showed a good correlation with the solvent extraction when the analysis was performed on minced samples. Generally, the solvent extraction techniques obtain the higher yield, which might be explained by the contribution of other lipid classes than triacylglycerols, such as polar lipids and sterols that are not always included in the rapid analyses. However, readings from the Fat Meter have been reported to show higher yield than reference values in samples of herring [61], which might be explained by the variation in the intercorrelation between water and lipids. Th is same study demonstrated a bigger difference between the methods at higher lipid content in the samples. Higher variation between methods are reported when analyzing lean fish compared with fatty fish high in unpolar lipids [26]. The statement of what is the most suitable method for lipid determination is highly dependent on the applicability and what criteria are the most important for the analysis such as accuracy, robustness, time of analysis, use of solvents, and portability, and so on.



16.4.1 Nutritional Aspects
Due to its favorable content and balance of essential and nonessential amino acids, fish protein is regarded to be of high nutritive value. Seafood proteins are also highly digestible, which adds to the understanding that digestibility of raw fish meat is in the range 90%–98% and that of shellfish about 85% [62]. Protein and amino acid requirements vary through life and are generally higher among young growing children compared with adults [63,64]. These nutritional aspects are more comprehensively described in other chapters in this book. Fish and marine invertebrate tissue contains from about 11%–24% (ww) crude protein depending on species, nutritional conditions, and the type of muscle. Although amino acid composition might vary among different types of tissue, there is a high similarity in the same tissue among species as pointed out by Mambrini and Kaushik [65]. The total body composition of amino acids shows high similarity among various cultured fish species [66].

270 ◾

Handbook of Seafood and Seafood Products Analysis


Methods for Protein Determination

Several of the most important methods for protein determination in food date from the late 1800s (Dumas, Nessler’s reagent, Biuret, Kjeldahl, Folin-Ciocalteau, and Dye binding) [67]. Quantification of total protein in fish and fish products can be determined by total organic nitrogen followed by conversion into crude protein or by a set of direct methods.


Determination of Total Nitrogen

Determination of proteins by analysis of total nitrogen (N) multiplied by a specific factor is a common procedure in fish analysis [68]. The N content of food is commonly determined using the Kjeldahl [69] or the Dumas [70] methods. Kjeldahl includes digestion of material and quantifies only N that is transformable to NH4+ using titration, colorimetry, or an ion-specific electrode [71]. In the Dumas method, all N is converted to N2 through combustion using a nitrogen element analyzer. Generally, the Dumas method gives higher N values than the Kjeldahl method [72–74], and a Kjeldahl-N to Dumas-N ratio of 0.80 for fish has been calculated [71]. The conversion factor for N was originally 6.25, based on average nitrogen content in different proteins of 16%, which might not be suitable for all protein sources, as they vary in amino acid composition. Generally, studies on fish have shown lower values with a more specific conversion factor of 5.8 presented for fish filet [75,76], and a factor of 4.94 (nitrogen to net protein) for protein estimates for fish and fish products are suggested by Salo-Väänänen and Koivistoinen [77]. More specific conversion factors based on the N content in isolated proteins are frequently applied for different categories of food [78]. Salo-Väänänen and Koivistoinen [77] showed that the true conversion factor was 5%–20% lower than the general 6.25 in a line of food products. Moreover, up to 40% variations were found in a comparison study of the 6.25 factor against foodspecific factors or sum of amino acids [79]. These differences indicate a significant contribution of nitrogen from other than amino acids or protein structures. Large amounts of those compounds are found in fish and fish products, probably due to both natural composition and degradation products [77]. These other N contributions might originate from nucleic acids, nucleotides, trimethylamine n-oxide (TMAO), free amino acids, or others. Contributions of N from products such as urea might appear in sharks, skates, and rays. There are, however, options to separate protein N from nonprotein N by precipitation and filtration after solvent extraction if required [80]. The nitrogenous compounds that do not originate from proteins can also be separated using methods such as ion-exchange chromatography (IEC), gas chromatography (GC), thin-layer chromatography (TLC), and high-performance liquid chromatography (HPLC) [81,82].


Direct Methods for Soluble Protein Determination

Protein is amino acids linked together via peptide bonds, and quantification of these amino acids might give more accurate values for protein estimates [68,77,83]. The term “net protein” is often used for those values that are corrected for added water during analysis. There are options to exclude or include the free amino acids during sample preparations, or they have also been analyzed separately using HPLC methods [84–86]. A more extensive description of various methods and techniques used in protein analyses are covered by Owusu-Apenten [67]. Acid hydrolysis followed by amino acid quantification such as by HPLC [87–90] or the more traditional IEC [89,91–93] are direct and specific methods for protein determination. During IEC, the derivatization of amino acids takes place postcolumn in most methods using, for

Composition and Calories


example, ninhydrin [94,89] or O-phthalaldehyde (OPA) [95]. Common derivatization reagents for quantification of amino acids in HPLC methods are OPA [90,96] and 9-fluorenylmethyl chloroformate (FMOC) [96], which are often used in combination with 2-mercaptoethanol, ethanethiol [90], or 3-mercaptopropionic acid [90,96]. An additional derivatization agent 2-(9-anthryl)ethyl chloroformate showed good correlation with the use of FMOC and lower detection limits for amino acids when analyzed in UV absorbance due to better spectral properties of the produced chromophore [97]. Other derivatization reagents are discussed in Sarwar and Botting [91] and in Fekkes [92]. In HPLC methods both pre- and postcolumn derivatizations are used with variable mobile phases based on methanol and acetonitrile. The reaction time, choice of solvents, and the concentration of 2-mercaptoethanol determine the efficiency of the reaction between OPA and amino acids with influence on quantification of the amino acids [90] (generally, 2-mercaptoethanol should be kept in the lower concentration range for optimization of the method [90]). OPA does not react with secondary amino acids, and FMOC is, among others, less soluble and might create interference reactions, but by combining those both, the primary and secondary amino acids can be detected [98]. Further optimization of this approach and adding an online dialysis step have improved the method with separation of 25 amino acids, and quantification of most of them [96]. Hyp (hydroxyproline), which is primarily found in connective collagenous tissue [99], might otherwise be quantified through derivatization with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole [100,101] or N2-(5-fluoro-2,4-dinitrophenyl)-l-valine amide [102]. Alternative methods are the spectrophotometric determination of Hyp as a measure of collagen [103] or collagen/gelatin in fish skin [104], the latter using a modified spectrophotometric method for Hyp determination by Bergman and Loxley [105]. The destruction of Trp (tryptophan) during hydrolysis in hydrochloric acid can be omitted by replacing with a line of others, including methane sulfonic acid containing 3-(2-aminoethyl) indole [106,107]. Enhanced signal of tyrosine, phenylalanine, and Trp has also been obtained using online photolysis with chemoluminescence methods in the HPLC system [108]. A more comprehensive overview of alternative methods for quantification of Trp is otherwise reviewed by Molnar-Pearl [109] and includes both alkali hydrolyses along with more complex derivatization and detection methods. During amino acid determination with the HPLC methods, detection of Cys (cysteine/ cysteine) might require special procedures during extract preparations such as iodoacetic acid [110] or 3,3′-dithiodipropionic acid as used in Glencross et al. [111]. Some nitrogenous compounds such as nucleic acids and amines, the latter originating mainly from microbial decarboxylation of amino acids in food such as putrescine, cadaverine, spermidine, spermine, tyramine, and histamine [112], can also be separated using methods such as HPLC [113,114] and reverse-phase HPLC [115,116]. Amino acid determination is often used in nutritional studies on fish, and requirements are frequently determined after analysis using IEC or HPLC methods [111,117–119], or alternatively 13C-NMR after extraction has been applied in such studies [120]. Quantification of the individual amino acids in HPLC methods is based on standards (amino acids) and use of an internal analytical standard such as a-butyric acid (ABA), responses to those, and molecular weight make the basis for calculating the amino acids. The protein values are calculated as the sum of all amino acids corrected for water added during hydrolyses, and the free amino acids might be removed through the extraction procedure or analyzed separately. Proteins can also be determined by a number of spectrophotometric methods. Some of these analyses are based on the ability of proteins to absorb (or scatter) light, whereas in other analyses, proteins are chemically or physically modified to absorb (or scatter) light. Due to variation of amino acid composition in proteins, most of these methods give results that can be different from absolute protein concentrations [83].

272 ◾

Handbook of Seafood and Seafood Products Analysis

Methods where proteins are chemically or physically modified for determination (colorimetric assays) can also be divided in to two groups: dye-binding reaction and redox reaction with proteins [121]. In the redox spectrophotometric methods, analyses are based on reaction with Folin reagent, and the following methods could be mentioned: Biuret reaction [122], Lowry protein method [123], and bicinchoninic acid (BCA) assay [124]. In the Biuret reaction Cu(II) with proteins in alkaline medium is reduced to Cu(I), which binds to protein forming a Cu(I)–peptide complex with purplish-violet color [121]. The same principle is used in BCA assay, where Cu(I) is detected by reaction with BCA, which gives an intense purple color [125]. One of the most popular methods in this group is the Lowry protein method [123], which is initially based on the Biuret reaction, where peptide bonds react with Cu(II) in alkaline medium to produce Cu(I). Later Cu(I) reacts with the Folin reagent. The reaction gives a strong blue color [83]. The intensity of color partly depends on the amount of Tyr and Trp in samples but can also be influenced by other components such as N-containing buffer or carbohydrates [121]. The amounts of proteins in sardine determined by the Lowry method were comparable to those determined by Kjeldahl method [121]. The Lowry method is suitable for protein extracts such as actomyosin, which is an important component in surimi-based products [126]. However, the BCA assay is shorter compared with the Lowry method (where two steps are needed), more flexible and stable in alkaline conditions, and has a broad linear range. The BSA assay can also be interpreted by the usual chemical components such as EDTA, thiols, reducing sugars, hydrogen peroxide, or phospholipids [121,125]. The dye-binding spectrophotometric assay is based on the reaction between acid dye and positively charged amino acid residues in proteins [121]. In acidic conditions, the created insoluble complexes are removed and the unbound dye is determined by measuring its absorbance. The amount of protein is proportional to the amount of bound dye. Coomassie dye in acidic conditions binds to proteins and creates complexes that influence a color shift from a maximum from 465 nm to 595 nm, using the Bradford method [127]. Absorbance of Coomassie dye-protein complex is measured at 595 (575–615) nm, because the difference between the two forms of the dye is greatest in this area. Within the linear range of the assay (∼5–25 mg/mL), the protein amount is proportional to bounded Coomassie [127]. This method is suitable for determination of extractability of proteins [128] or protein content in extracts [129–131]. Th is technique is simple, sensitive, and uses shorter analysis time compared with the Lowry method. Moreover, the dye-binding assay is less affected by reagents and nonprotein components from biological samples [132]. Proteins in solution can be quantified in a simple spectrophotometric analysis by near- or farUV absorbance [133,134]. Absorption in the near UV by proteins depends mostly on the content of Tyr and Trp and less on the amount of phenylalanine (Phe) and disulfide bonds. This absorbance measurement is simple, sensitive, needs no reagents, and the sample is recoverable [133,134] Crude protein extracts or individual fractions of proteins [135] can be measured at 280 nm. Disadvantages of the method include interference with other components such as nucleic acid, which absorbs in the same wavelength region [133]. Far-UV absorption can also be used for determination of protein content: peptide bonds absorb in the area with the maximum at about 190 nm. Different proteins give a small variation in absorbance, and the method can be considered as accurate for protein determination. However, oxygen also absorbs at these wavelengths, and to avoid interference, measurements at 205 nm is used. It should also be mentioned that components such as carbohydrates, salts, lipids, amides, phosphates, and detergents interfere [133,134].

Composition and Calories


16.4.5 Nondestructive Analysis of Proteins
Recently, other advanced and nondestructive methods have become more common for determining protein. NIR is one of these [4,53], and it was originally developed for protein analysis and has since that time been developed and calibrated for a range of fish species. Low-field NMR is generally not suitable for protein determination in a nondestructive manner. See earlier text for more information on the nondestructive techniques.

16.5 Determination of Carbohydrate Content
Carbohydrates are often classified into three broad groups: sugars (mono- and disaccharides), oligosaccharides (three to nine monosaccharides) and, polysaccharides (more than nine). The content of carbohydrates in fish muscle is low [136,137] and is further influenced by conditions experienced before and during capture, which may lead to depletion of glycogen stores and thereby a decrease in the carbohydrate level. Under anoxic conditions postmortem, glycogen will continue to be metabolized, resulting in increased lactic acid along with reduced pH and eventually a gradual loss of the sweet, meaty character of fresh fish. Some marine invertebrates on the other hand are characterized by a high content of carbohydrates; up to 10.2% and 12.5% total sugars can be found in subcuticular tissue of spiny lobster and blue crab, respectively, with the highest amounts of glucose followed by galactose and mannose [138]. Glycogen stores of scallops are highly dependent on season (temperature, food availability, and lifecycle), and highest levels are usually reached after the summer period [139], showing levels up to 23%–25% glycogen of dry weight of adductor muscle [139,140]. Seasonal variations of glycogen content in mussels (Mytilus edulis) are also high, showing values in the range 4%–37% of tissue dry weight [141,142]. Among the line of methods suitable for seafood, the amount of total carbohydrates in shellfish can be determined by using the phenol-sulfuric acid procedures described by Dubois et al. [143] as used for scallop (Pecten maximus) in Maguire et al. [144] and silver carp in Gnaiger and Bitterlich [144]. This method is based on hydrolysis of polysaccharides and does not measure all sugar molecules in the materials equally accurately, because the carbohydrates are absorbed at different maximum wavelengths and in addition differ in the ability to form the chromogenes formed in the method. If measurements are performed at 488 nm and a standard curve is prepared using glucose, this will lead to a possible underestimation in the case of chemical characteristics of monosaccharides deviant from glucose. This relatively simple method is often used, because it gives a good estimate of total carbohydrates in tissue that contain 10% or more of hexose polymers [145]. Glycogen from seafood can also be determined after preparation of solution of glucose units using a range of assay kits for glucose followed by colorimetric determination (Boehringer Mannheim, Cayman chemicals, Biovision or others), as described for Abalone tissue using a combination lipid and glucose extraction method in studies of Allen et al. [146]. Glycogen levels in small amounts of tissue can additionally be analyzed using the anthrone methods with spectrophotometric determinations [147–149], which have been demonstrated as useful for scallop [150]. Carbohydrates are frequently calculated and expressed as total carbohydrates by difference, which is the remainder after subtraction of moisture, crude protein, total fat, and ash and includes fibers if present in the analyzed material. An excellent overview of definitions and internationally used carbohydrate tag names along with applicable analytical procedures for food in general is given by Munro and Burlingame [151].

274 ◾

Handbook of Seafood and Seafood Products Analysis

16.6 Determination of Water Content
Water content in fish can be determined by simple drying methods. Using conventional air ovens, a common practice has been to dry the sample at 105°C for 12 h, which by experience has shown satisfactory drying of fish and fish products. To ensure complete drying, the sample can be dried to constant weight. Other methods [40] refer to 101°C for 24 h by conventional ovens and 70°C for 24 h using vacuum ovens. The sample is weighed in a container, and after heating the sample is cooled and weighed again. The water content is determined by the following formula: Water content (%) = (Weight of wet material − weight of dried material) × 100 Weight of wet material

Infrared and some microwave ovens may allow an analysis time of 1–2 h [152]. Further, the new nondestructive methods such as NIR/NIT, NMR, or Fatmeter, which are described previously in this chapter, may be used for fast determination of water, and the low-field NMR technique can even distinguish between free and bounded water [15,153]. In a volumetric method (Dean & Stark), the samples are boiled in toluene before measuring the volume of water. This method is relatively fast but uses toluene, which is hazardous to health [152].



The energy content of food is generally given in kilocalories (kcal) and kilojoules (kJ), which have a conversion factor of 1 kcal = 4.184 kJ. Seafood show variable composition of proteins and fat, and energy content is dependent on this distribution, which often might also be highly influenced by seasonal variations. In a seasonal study of 35 fish and shellfish species, Soriguer et al. [154] found a substantial variation in biochemical composition, where even mackerel known as fatty type of fish, in parts of the year could be classified within the lean fish category. The lipid level in particular has high significance for the calorie content of fish, with implications for calculations in dietary studies and databases; this is important to bear in mind when these are used.

16.7.1 Direct Measurement of Energy
The gross energy content of food (measured as heat of combustion, kcal/g) may be determined directly by using a bomb calorimeter (micro- or macromethods), which includes burning food with oxygen in an insulated container of constant volume [155,156]. The heat is adsorbed in water, and the energy is determined from the mass of water, its temperature rise, and its specific heat. Dichromate wet oxidation with potassium dichromate is also sometimes used as a direct method, giving rise to slightly lower energy values in fish samples than when measured by bomb calorimetric methods [157,158]. Food composition databases are not based on direct measurements of gross energy, because those are not equal to energy requirements [159]. Instead the metabolizable food energy is used, which accounts for the energy in food remaining after losses through the feces, gas, urea, and the body surface [160].

Composition and Calories


16.7.2 Indirect Measurements of Energy
The energy released by oxidation of protein, fat, and carbohydrate is the basis for sets of conversion factors. The Atwater general factor system is the foundation for the most frequently used systems for energy conversion [161], which originates from combustion with adjustments for losses in digestion, absorption, and excretion of urea. The Atwater general energy conversion values are 4.0 kcal/g for proteins, 9.0 kcal/g for lipids, and 4.0 kcal/g for carbohydrates (calculated by difference, i.e., subtracting water, ash, proteins, and lipids). Originally no differences were determined between the fiber and available digestive carbohydrates, but exploring more specific heat of combustion led to factors of 3.75 kcal/g when used for monosaccharides and 4.2 kcal/g for polysaccharides, with application in the Atwater system [162]. However, the specific conversion factor used for carbohydrates in shellfish is 4.11 kcal/g [163]. For other food material, energy factors for dietary fiber have been developed, taking into account availability, provided also by the microorganisms in the colon giving values recommended by FAO [164] of 8.0 kJ/g (2.0 kcal/g). A more specific set of factors for energy conversion were developed due to different combustion rates and digestibility of various sources of proteins and fats and additional impact caused by processing. The specific set of factors presented in Merrill and Watt [163,165] arrived at 4.27 kcal/g for protein and 9.02 kcal/g for fat in meat and fish. It is, however, important to consider the choice of analytical methods regarding conversion of proteins to calories. Both the variable nonprotein N and the variations in amino acid composition in different protein sources might have implications on the calculated energy levels if based on N analysis (see above). When energy contributions from proteins are set, the most accurate method will be as the sum of amino acids (free and protein bound). Alternatively, Kjeldahl or Dumas techniques are used with more source-specific conversion factors such as those used by Jones [166] or others, when these are known. In terms of conversion to energy, the more specific conversion factor of 5.65 kcal/g for protein was suggested [167] and tested in combination with direct energy measurements for use with fish tissue, resulting in slightly higher values compared with bomb calorimetric methods [157]. Calculation of energy contribution from fat might include analysis of fatty acids with total fat calculated as triacylglycerol equivalents [160]. For fatty fish muscle the factor 0.90 is used in conversion of total fat to total fatty acids, whereas 0.70 is used for white fish muscle [169]. Gravimetric methods are also used for energy calculations, which (depending on methods used; see above) would include weight of the additional lipid components that are not transformed to energy, per se. The calorie content of extracted lipids (methanol/chloroform extraction) from fish tissue as found by microcalorimetric methods suggests the use of a lower energy conversion factor such as 8.49 kcal/g [157]. Gross energy levels obtained from bomb calorimetry might deviate from energy when based on analysis and conversion factors due to the lipid calculations. A high level of lipids in tissue is usually accompanied with high energetic content by both methods. However, with high levels of sterols, the gross energy by bomb calorimeter can be higher than the metabolic energy level calculated from the analysis by use of conversion factors. Th is method deviation was pointed out for low-lipid squid samples by Krishnamoorthy et al. [169]. In the study of feed, fish, and feces by Henken et al. [158] three different methods for calculating energy content were compared (I, dichromate wet oxidation; II, bomb calorimeter; or III, chemical analyses followed by conversion factors 5.65, 9.45, 4.2 kcal/g [proteins:fat:carbohydrates]). Proteins were calculated with N*6.25, fat analyzed by Soxhlet with hexane extraction, and carbohydrates calculated by difference. Agreements were obtained in methods II and III and lower energy values were obtained with method I. Inadequate protein oxidation by dichromate method

276 ◾

Handbook of Seafood and Seafood Products Analysis

[170] was solved by correction factors but still resulted in lower values in fish, feed, and feces compared with bomb calorimetry or direct analyses followed by conversion factors. In recent years the field of nutrition has become highly complex due to developments in both analytical and physiological methods. A variety of different analytical methods are in use along with various sets of conversion factors, which again are based on their own specific analytical methods. In scientific work it is particularly important to specify methods and calculations made in the presented results. Standardization of analytical methods and energy conversion factors might improve the use of nutrient databases for energy calculation.

16.7.3 Food Composition Tables and Databases
Food composition databases are practical tools providing a line of useful information on foodrelated subjects. For the users it is convenient to find further links, reports, published works, nutrient composition tables, and so forth, through a database. Researchers are requested to make relevant publications available through these pages, adding to the up-front knowledge in the area. When food databases contain original analytical results, the values can be trusted to represent more accurate levels and are more useful for governmental and research purposes. There are several general databases available to the public both on international, regional, and national levels such as those of The International Network of Food Data Systems (FAO/INFOODS), United States Department of Agriculture (USDA), Pacific Island Food Composition Tables (PIFCT), and German Nutrient Database (BSL). The user groups for food databases are among others found within the groups of food researchers and industry, dieticians, epidemiological and health researchers, and national and governmental authorities. National and regional food composition tables are important, because they may reveal specific dietary traits of subpopulations important for health and epidemiological research. Differing nutritional definitions are also common as with different sets of energy conversion factors, which is important to be aware of when food tables are used. Databases as such FishBase provide specific tables for seafood such as proximate data and energy levels of different organs and ecological data of harvested species in specific regions. However, the databases might have a potential for improvement with regard to expected variability in the composition of food items, which might be due to seasonal variations, variations experienced during the growth, production phase, or as influenced by storage or processing conditions. Additionally, processed food with many ingredients is complex, some nutrients are labile, and constituents such as fat and moisture might be added and/or removed during food preparations. As it might be practically impossible to obtain the full detailed composition, there is selection of constituents in food tables. Most databases contain 10–25 food groups [160], but some also contain more than 100 nutrients and food components such as the Nutrition Data System for Research (NDS-R) in the United States [171]. Skills and knowledge in the analytical methods on which the values are based on, advantages, and drawbacks in the table values are required.

1. Sikorski, Z.E., Kolakowska, A., and Sun Pan, B., The nutritive composition of the major groups of marine food organisms. In: Seafood: Resources, Nutritional Composition and Preservation, Ed., Sikorski, Z.E., CRC Press, 29–54. Boca Raton, FL, 1990. 2. Williams, P.C., Application of near infrared reflectance spectroscopy to analysis of cereal grains and oilseeds, Cereal Chem., 52, 561–576, 1975.

Composition and Calories


3. Martens, H. and Russwurm, H., Food Research and Data Analysis, Applied Science Publisher, London, U.K., 1982. 4. Solberg, C., NIR–A rapid method for quality control. In: Seafood from Producer to Consumer, Integrated Approach to Quality, Eds., Luten, J.B., Børresen, T., and Oehlenschläger, J., Elsevier Science, Amsterdam, the Netherlands, pp. 529–534, 1997. 5. Wold, J.P. and Isaksson, T., Non-destructive determination of fat and moisture in whole Atlantic Salmon by near infrared diff use reflectance spectroscopy, J. Food Sci., 62, 734–736, 1997. 6. Downey, G., Non-invasive and non-destructive percutaneous analysis of farmed salmon flesh by near infrared spectroscopy, Food Chem., 55, 305–311, 1999. 7. Shimamoto, J., Hiratsuka, S., Hasegawa, K., Sato, M., and Kawano, S., Rapid non-destructive determination of fat content in frozen skipjack using a portable near infrared spectrophotometer, Fish. Sci., 69(4), 856–860, 2003. 8. Solberg, C., Saugen, E., Swenson, L.-P., Bruun, L., and Isaksson, T., Determination of fat in alive farmed Atlantic salmon using non-invasive NIR techniques, J. Sci. Food Agr., 83, 692–696, 2003. 9. Solberg, C., Rapid on line non-destructive measurement of fat in live and filleted salmon. In Near Infrared Spectroscopy: Proceedings of the 11th International Conference, Ed., Davies, A.M.C. and GarridoVaro, A., NIR Publications, Sussex, U.K., pp. 471–474, 2004. 10. Uddin, M., Okazaki, E., Turza, S., Yumiko, Y., Tanaka, M., and Fukuda, Y., Non-destructive visible/NIR spectroscopy for differentiation of fresh and frozen thawed fish, Food Chem. Toxicol., 70(8), 506–510, 2005. 11. Uddin, M., Okazaki, E., Ahmad, M.U., Fukuda, Y., and Tanaka, M., NIR spectroscopy: A nondestructive fast technique to verify heat treatment of fish-meat gel, Food Control, 17, 660–664, 2006. 12. Osborn, B.G., Fearn, T., and Hindle, P.H., Practical NIR Spectroscopy with Application in Food and Beverage Analysis. Longman Scientific and Technical, Essex, U.K., 90–144, 1993. 13. Lundby, F., Sørland, G.H., and Eilertsen, S., Determination of fat, moisture and protein in fish powder within 30 min, by combining low resolution NMR techniques and microwave technology, Abstract from the 8th International Conference on the Applications of Magnetic Resonance in Food Science, 2006, Nottingham, U.K. 14. Veliuylin, E., Van der Zwaag, C., Burk, W., and Erikson, U., In vivo determination of fat content in Atlantic salmon (Salmo salar) with a mobile NMR spectrometer, J. Sci. Food Agr., 85, 1299–1304, 2005. 15. Aursand, I., Veliuylin, E., and Erikson, U., Low-field NMR studies of Atlantic salmon (Salmo salar), In: Modern Magnetic Resonance, Modern Magnetic Resonance. Part 1: Applications in Chemistry, Biological and Marine Sciences, Ed., Webb, G.A., 895–903, 2006, Springer, Dordrecht, the Netherlands. 16. Blumich, B., Blumer, P., Eidman, G., Guthausen, A., Haken, R., Schmitz, U., Saito, K., and Zimmer, G., The NMR MOUSE: Construction, excitation and applications, Magn. Reson. Imaging, 16, 479–484, 1998. 17. Ghaedian, R., Coupland, J.N., Decker, E.A., and McClements, D.J., Ultrasonic determination of fish composition, J. Food Eng., 35(3), 323–337, 1998. 18. Suvanich, V., Ghaedian, R., Chanami, R., Decker, E.A., and McClements, D.J., Prediction of proximate fish composition from ultrasonic properties: Catfish, Cod, Flounder, Mackerel, and Salmon, J. Food Sci., 63(6), 966–968, 1998. 19. Sigfusson, H., Decker, E.A., and McClements, D.J., Ultrasonic characterisation of Atlantic mackerel (Scomber scombrus), Food Res. Int., 43, 15–23, 2001. 20. Shannon, R.A., Probert-Smith, P.J., Lines, J., and Mayia, F., Ultrasound velocity measurement to determine lipid content in salmon muscle; the effect of myosepta, Food Res. Int., 37, 611–620, 2004. 21. Jaramillo, F., Bai, S.C., Murphy, B.R., and Gatlin, D.M., Application of electrical-conductivity for nondestructive measurement of channel Catfish, Ictalurus punctatus, body-composition, Aquat. Living Resour., 7(2), 87–91, 1994.


Handbook of Seafood and Seafood Products Analysis

22. Veliuylin, E., Borge, A., Singstad, T., Gribbestad, I., and Erikson, U., Post-mortem studies of fish using magnetic resonance imaging. In: Modern Magnetic Resonance, Modern Magnetic Resonance. Part 1: Applications in Chemistry, Biological and Marine Sciences, Ed., Webb, G.A., 949–956, Springer, Dordrecht, the Netherlands, 2006. 23. Dyerberg, J., Bang, H.O., Stofferson, E., Monkada, S., and Vane, J.R., Eicosapentaenoic acid and prevention of thrombosis and atherosclerosis? Lancet, 117, 1978. 24. Uauy, R. and Valenzuela, A., Marine oils: The health benefits of n-3 fatty acids, Nutrition, 16(7–8), 680–684, 2000. 25. Vanshoonbecek, K., de Maat, M.P., and Heemssterk, J.W., Fish oil consumption and reduction of arterial disease, J. Nutr., 133, 657–660, 2003. 26. Ewald, G., Bremle, G., and Karlsson, A., Difference between Bligh and Dyer and Soxhlet extractions of PCBs and lipids from fat and lean fish muscle: Implications for data evaluations, Mar. Pollut. Bull., 36(3), 222–230, 1998. 27. Folch, J., Lees, M., and Stanley, G.H.S. Preparation of lipid extracts from brain tissue, J. Biol. Chem., 226, 497–509, 1957. 28. Bligh, E.G. and Dyer, W.J., A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol., 37, 911–917, 1959. 29. Iverson, S., Lang, S.L.C., and Cooper, M.H., Comparison of the Bligh and Dyer and Folch method for total lipid determination of a broad range of marine tissue, Lipids, 36(11), 1283–1287, 2001. 30. Lee, M.C., Trevino, B., and Chaiyawat, M., A simple and rapid solvent extraction method for determination of total lipids in fish, J. AOAC Int., 79(2), 487–492, 1996. 31. Smedes, F. and Askland, T.K., Revisiting the Bligh and Dyer total lipid determination method, Mar. Pollut. Bull., 38(3), 193–201, 1999. 32. Toge, Y. and Miyashita, K., Lipid extraction with electrolyzed cathode water from marine products, J. Oleo Sci., 52(2), 1–6, 2003. 33. Hara, A. and Radin, N.S., Lipid extraction of tissue with a low toxicity solvent, Anal. Biochem., 90, 420–426, 1978. 34. Burton, G.W., Webb, A., and Ingold, K.U., A mild, rapid and efficient method of lipid extraction for use in determining vitamin E/lipid ratios, Lipids, 20(1), 29–39, 1985. 35. Undeland, I., Harrod, M., and Lignert, H., Comparison between methods using low-toxicity solvents for the extraction of lipids from herring (Clupea harengus), Food Chem., 61(3), 355–365, 1998. 36. Woitke, P., Haarich, M., and Harms, U., Co-factors in biota: Results of a German interlaboratory exercise on the determination of total lipids in fi sh tissue, Accredit. Qual. Assur., 5, 499–503, 2000. 37. Jensen, S., Heggberg, L., Jorundsdottir, H., and Odham, G., A quantitative lipid extraction method for residue analysis of fish involving nonhalogenated solvents, J. Agr. Food Chem., 51(19), 5607–5611, 2003. 38. Christie, W.W., Ed., in Advances in Lipid Methodology–Two, Oily Press, Dundee, U.K., pp. 195–213, 1993. 39. Norwegian Standard (NS 9402), Atlantic Salmon: Measurement of Colour and Fat, 1st edn., Oslo, Norway, 1994. 40. AOAC Official methods, 960.39. In: Official Methods of Analysis, 15th edn., Association of Official Analytical Chemists, Arlington, VA, 1990. 41. Bremle, G., Okla, L., and Larsson, P., Uptake of PCBs in a contaminated river system: Bio concentration factors measured in the field, Environ. Sci. Technol., 29(8), 2010–2015, 1995. 42. Virot, M., Tomao, V., Colnagui, G., Visinoni, F., and Chemat, F., New microwave-integrated Soxhlet extraction. An advantageous tool for the extraction of lipids from food products, J. Chrom., 1174, 138–144, 2007. 43. Vogt, A., Gormley, T.R., Downey, G., and Somers, J., A comparison of selected rapid methods for fat measurement in fresh herring (Clupea harengus), J. Food Compos. Anal., 15, 205–215, 2002.

Composition and Calories


44. Kent, M., Measurement of dielectric properties of herring flesh using transmission time domain spectroscopy, Int. J. Food Sci. Technol., 25, 26–38, 1990a. 45. Clerjon, S. and Damez, J.L., Microwave sensing for food structure evaluation, Meas. Sci. Technol., 18, 1038–1045, 2007. 46. Nielsen, D., Hylding, G., Nielsen, J., and Hauch Nielsen, H., Lipid content in herring (Clupea harengus L.)–influence of biological factors and comparison of different method of analyses: Solvent extraction, Fatmeter, NIR, and NMR, Lebensm Wiss. U. Technol.-Food. Sci. Technol., 38, 537–548, 2005. 47. Aursand, M., Bleivik, B., and Rainuzzo, J., Lipid distribution and composition in commercially farmed Atlantic salmon (Salmo salar), J. Sci Food Agr., 64, 239–248, 1994. 48. Undeland, I., Hall, G., and Lignert, H., Lipid oxidation in fillets of herring (Clupea harengus) during ice storage, J. Agr. Food Chem., 47(2), 524–532, 1999. 49. Katikou, P., Hughes, S I., and Robb, H.D.F., Lipid distribution within Atlantic salmon (Salmo salar) fillets, Aquaculture, 202(1–2), 89–99, 2001. 50. Toussaint, C., Fauconneau, B., Medale, F., Collewet, G., Akoka, S., Haff ray, P., and Davenel, A., Description of the heterogeneity of lipid distribution in the flesh of brown trout (Salmo trutta) by MR imaging, Aquaculture, 243, 255–267, 2005. 51. Kent, M., Hand held instrument for fat/water determination in whole fish, Food Control, 1, 47–53, 1990b. 52. Kent, M., Seasonal variation in the calibration of microwave fat: Water content meter for fish flesh, Int. J. Food Sci. Technol., 27, 137–143, 1992. 53. Khodabux, K., L’Omelette, M.S.S., Jhaumeer-Laulloo, S., Ramasami, P., and Rondeau, R., Chemical and near-infrared determination of moisture, fat and protein in tuna fishes, Food Chem., 102(3), 669–675, 2007. 54. Jebsen, S.M., Pedersen, H.T., and Engelsen, S. B., Application of chemometrics to low-field 1H NMR relaxation data of fish flesh, J. Sci. Food Agric., 79, 1793–1802, 1999. 55. Toussaint, C.A., Medale, F., Davenel, A., Fauconneau, B., Haff ray, P., and Akoka, S., Determination of the lipid content in fish muscle by a self-calibrated NMR relaxometry method: Comparison with classical chemical extraction methods, J. Sci. Food Agric., 82, 173–178, 2001. 56. Shimamoto, J., Hasagawa, K., Sato, M., and Kawano, S., Non-destructive determination of fat content in frozen and thawed mackerel by near infrared spectroscopy, Fish. Sci., 70, 345–347, 2004. 57. Sørland, G.H., Larsen, P.M., Lundby, F., Anthonsen, H.W., and Foss, B.J., On the use of low-field NMR methods for the determination of total lipid content in marine products. In: Magnetic Resonance in Food Science, Royal Society of Chemistry, Cambridge, U.K., 299, pp. 20–27, 2005. 58. Aursand, M., Rainuzzo, J., and Grasdalen, H., Quantitative high resolution 13C and1H nuclear magnetic resonance of fatty acids from white muscle of atlantic salmon (Salmo salar), J. Am. Oil. Chem. Soc., 70(10), 971–981, 1993. 59. Falch, E., Størseth, T.R., and Aursand, M., Multi-component analysis of marine lipids in fish gonads with emphasis on phospholipids using high resolution NMR spectroscopy, Chem. Phys. Lipids, 144(1), 2006. 60. Solberg, C. and Fredriksen G., Analysis of fat and dry matter in capelin by near infrared transmission spectroscopy, J. Near Infrared Spec., 9, 221–228, 2001. 61. McAdams, D., Measuring fat by meter, Seafood Int., 11(9), 35, 1996. 62. Acton, J.C. and Rudd, C.L., Protein quality methods for seafoods. In: Seafood Quality determination, Eds., Kramer, D.E. and Liston, J., Elsevier, Amsterdam, the Netherlands, 1983. 63. Pellett, P.L. and Young, V.R., Nutritional Evaluation of Protein Foods. Publication no. WHTR-3/ UNUP-129, United Nations University, Tokyo, Japan, 1980. 64. FAO/WHO/UNU Expert Consultation, energy and protein requirements, World Health Organization Technical Report Series 724, 1985. 65. Mambrini, M. and Kaushik, S.J., Indispensable amino acid requirements of fish: Correspondence between quantitative data and amino acid profiles of tissue proteins, J. Appl. Ichthyol.-Z. Angew. Ichtyol., 11, 240–247, 1995.


Handbook of Seafood and Seafood Products Analysis

66. Wilson, R.P., Amino acids and proteins. In: Fish Nutrition, 3rd edn., Eds., Halver, J.E. and Hardy, R.W. Academic press, San Diego, California, 144–179, 2002. 67. Owusu-Apeten, R.K., Food Protein Analysis. Marcel Dekker, Inc., New York, 2002. 68. Greenfield, H. and Southgate, D.A.T., Food composition data. production, management and use, Food and Agriculture Organization of the United Nations, Rome, 2003. 69. Kjeldahl, J., A new method for the determination of nitrogen in organic matter, Z. Anal. Chem., 22, 366–382, 1883. 70. Dumas, J.B.A., Procedes de l’analyse organique. Ann. Chim. Phys., 247, 198–213, 1831. 71. Simonne, A.H., Simonne, E.H., Eitenmiller, R.R., Mills, H.A., and Cresman, III C.P., Could the Dumas method replace the Kjeldahl digestion for nitrogen and crude protein determinations in foods? J. Sci. Food Agr., 73(1), 39–45, 1997. 72. Thompson, M., Owen, L., Wilkinson, K., Wood, R., and Damant, A., A comparison of the Kjeldahl and Dumas methods for the determination of protein in foods, using data from a proficiency testing scheme, Analyst, 127(12), 1666–1668, 2002. 73. Jung, S., Rickert, D.A., Deak, N.A., Aldin, E.D., Recknor, J., Johnson, L.A., and Murphy, P.A., Comparison of Kjeldahl and Dumas methods for determining protein contents of soybean products, J. Am. Oil Chem. Soc., 80(12), 1169–1173, 2003. 74. Miller, E.L., Bimbo, A.P., Barlow, S.M., and Sheridan, B., Repeatability and reproducibility of determination of the nitrogen content of fishmeal by the combustion (Dumas) method and comparison with the Kjeldahl method: Interlaboratory study, J. AOAC Int., 90(1), 6–20, 2007 75. Sosulski, F.W. and Imafidon, G.I., Amino acid composition and nitrogen to protein conversion factors for animal and plant foods, J. Agric. Food. Chem., 38, 1351–1356, 1990. 76. Gnaiger, E. and Bitterlich, G., Proximate biochemical composition and caloric content calculated from elemental CHN analysis: A stochiometric concept, Oecologia, 62, 289–298, 1984. 77. Salo-Väänänen, P.P. and Koivistoinen, P.E., Determination of protein in foods: Comparison of net protein and crude protein (N × 6.25) values, Food Chem., 57(1), 27–31, 1996. 78. Jones, D.B., Munsey, V.E., and Walker, L.E., Report of committee on protein factors, J. Assoc. Off. Agr. Chem., 25, 118–120, 1942. 79. Heidelbaugh, N.D., Huber, S.C., Bednavzk, J.F., Smith, M.C., Rambaut, P.C., and Wheeler, H.O., Comparison of three methods of calculating protein content of foods, J. Agr. Food Chem., 23, 611–613, 1975. 80. Orban, E., Nevigato, T., Lena, G.D., Masci, M., Casini, I., Gambelli, L., and Caproni, R., New trends in the seafood market. Sutchi catfish (Pangasius hypophthalmus) fillets from Vietnam: Nutritional quality and safety aspects, Food Chem., 110, 383–389, 2008. 81. Cinquina, A.L., Cali, A., Longo, F., De Santis, L., Severoni, A., and Abballe, F., Determination of biogenic amines in fish tissues by ion-exchange chromatography with conductivity detection, J. Chromatogr. A, 1032(1–2), 73–77, 2004. 82. Karovicova, J. and Kohajdova, Z., Biogenic amines in food, Chem. Pap., 59(1), 70–79, 2005. 83. Waterborg, J.H., The Lowry method for protein quantitation. In: The Protein Protocols Handbook, 2nd edn, Ed., Walker, J.M., Humana Press Inc., Totowa, NJ, 2002. 84. Flynn, K.J., Some measurements of dissolved free amino acids in natural waters and within microalgae by the use of HPLC, Chem. Ecol., 3, 269–293, 1988. 85. Quereshi, G.A. and Qureshi, A.R. Determination of free amino acids in biological samples: Problems of quantification, J. Chromatogr., 491, 281–289, 1989. 86. Duun, A. and Rustad, T. Quality changes during superchilled storage of cod (Gadus morhua) fillets, Food Chem., 105, 1067–1075, 2007. 87. Ishida, Y., Fujita, T., and Asai, K., New detection and separation method for amino acids by highperformance liquid chromatography, J. Chromatogr., 204, 204, 143–148, 1981. 88. Blundell, G. and Brydon, W.G., High performance liquid chromatography of plasma aminoacids using orthophthalaldehyde derivatisation, Clin. Chim. Acta, 170, 79–84, 1987.

Composition and Calories


89. Bütikofer, U., Fuchs, D., Bosset, J.O., and Gmur, W., Automated HPLC-amino acid determination of protein hydrolysates by precolumn derivatisation with OPA and FMOC and comparison with classical ion exchange chromatography, Chromatographia, 31, 9/10, 441–447, 1991. 90. Dorresteijn, R.C., Berwald, L.G., Zomer, G., Goojijer, C.D.de., Wieten, G., and Beuvery, E.C., Determination of amino acids using o-phthalaldehyde-2-mercaptoethanol derivatisation, effect of reaction conditions, J. Chromatogr. Part A, 724, 159–167, 1996. 91. Sarwar, G. and Botting, H.G., Evaluation of liquid chromatographic analysis of nutritionally important amino acids in the food and physiological samples, J. Chromatogr., 615, 1–22, 1993. 92. Fekkes, D., State-of-the-art of high-performance liquid chromatographic analysis of amino acids in physiological samples, J. Chromatogr. Part B, 682, 3–22, 1996. 93. Wu, T. and Mao, L., Influences of hot air drying and microwave drying on nutritional and odourous properties of grass carp (Ctenopharyngodon idellus) filets, Food Chem., 110, 647–653, 2008. 94. Hamilton, P.B. and Anderson, R.A, Ion-exchange chromatography of amino acids-semi-automatic method of operation with cationic-exchange resin columns, Anal. Chem., 31, 1504–1512, 1959. 95. Benson, J.R. and Hare, P.E., O-Phthalaldehyde: Fluorogenic detection of primary amines in the picomole range. Comparison with fluorescamine and ninhydrin, Proc. Nat. Acad. Sci. USA., 72, 1, 619–622, 1975. 96. Heems, D., Luck, G., Fraudeau, C., and Verette, E., Fully automated precolomn derivatisation, on line dialysis and high performance liquid chromatographic analysis of amino acids in food, beverages and feedstuff, J. Chrom. Part A, 798, 9–17, 1998. 97. Björklund, J., Einarsson, S., Engström, A., Grzegorczyk, A., Becker, H-D., and Josefsson, B., Automated amino acid determination by high-performance liquid chromatography with 2-(9-antryl)ethyl chloroformate as precolumn reagent, J. Chromatogr. Part A, 798, 1–8, 1998. 98. Schuster, R., Determination of amino acids in biological, pharmaceutical, plant and food samples by automated precolumn derivatization and high-performance liquid chromatography, J. Chromatogr., 431, 271–284, 1988. 99. Adams, E. and Frank, L., Metabolism of proline and the hydroxyprolines, Ann. Rev. Biochem., 49, 1005–1061, 1980. 100. Welch, R.W., Acworth, I., and Levine, M., Coulometric electrochemical detection of hydroxyproline using 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, Anal. Biochem., 210, 1, 199–205, 1993. 101. Dugan, M.E.R., Thacker, R.D., Aalhus, J.N., Jeremiah, L.E., and Lien, K.A., Analysis of 4-hydroxyproline using 4-chloro-7-nitrobenzo-2-oxa-1,3-diazol derivatization and micellar electrokinetic chromatography combined with laser-induced fluorescence detection, J. Chromatogr. Part B, Biomed. Sci. Appl., 744, 1, 195–199, 2000. 102. Langrock, T., Garcia-Villar, N., and Hoff mann, R., Analysis of hydroxyproline isomers and hydroxylysine by reverse-phase HPLC and mass spectrometry, J. Chromatogr. Part B, 847, 282–288, 2007. 103. Wold, J.P., Lundby, F., and Egelandsdal, B., Quantification of connective tissue (hydroxyproline) in ground beef by autofluorescense spectroscopy, J. Food Sci., 64, 3, 377–383, 1999. 104. Intarasirisawat, R., Benjakul, S., Visessanguan, W., Prodpran, T., Tanaka, M., and Howell, N.K., Autolysis study of big eye snapper (Priacanthus macracanthus) skin and its effects on gelatine. Food Hydrocolloids, 21, 537–544, 2007. 105. Bergman, I. and Loxley, R., Two improved and simplified methods for the spectrophotometric determination of hydroxyproline, Anal. Chem., 35, 1961–1965, 1963. 106. Simpson, R.J., Neuberger, M.R., and Liu, T-Y., Complete amino acid analysis of proteins from a single hydrolysate, J. Biol. Chem., 251, 7, 1936–1940, 1976. 107. Chiou, S.H. and Wang, K.T., Simplified protein hydrolysis with methanesulphonic acid at elevated temperature for the complete amino acid analysis of protein, J. Chromatogr. Part A, 448, 404–410, 1988. 108. Bolden, M.E. and Danielson, N.D., Liquid chromatography of aromatic amines with photochemical derivatization and tris(bipyridine)ruthenium(III) chemiluminescense detection, J. Chromatogr. A, 828, 421–430, 1998.


Handbook of Seafood and Seafood Products Analysis

109. Molnar-Perl, I., Tryptophan analysis in peptides and proteins, mainly by liquid chromatography, Review. J. Chromatogr, A, 763, 1–10. 1997. 110. Campanella, L., Crescentini, G., and Avino, P., Simultaneous determination of cysteine, cystine and 18 other amino acids in various metrices by high performance liquid chromatography, J. Chromatogr. A, 833, 137–145, 1999. 111. Glencross, B., Hawkins, W., Evans, D., Rutherford, N., McCafferty, P., Dods, K. and Sipsas, S., Assessing the implications of variability in the digestible protein and energy value of lupin kernel meals when fed to rainbow trout, Oncorhynchus mykiss, Aquaculture, 277, 251–262, 2008. 112. Vacha, F., Krizek, M., and Pavlieck, T., Content of biogenic amines in common carp (Cyprinus carpio). In: Towards a Predictable Quality. Aquaculture Europe ‘99. Trondheim, Norway, August 7–10, 249–250, 1999. 113. Ozogul, F., Taylor, K.D.A., Quantick, P., and Ozogul, Y., Biogenic amine formation in Atlantic herring (Clupea herengus) stored under modified atmosphere packing using a rapid HPLC method, Int. J. Food. Sci. Tech., 37, 515–522, 2002. 114. Korös, A, Hanczkó, R., Jámbor, A., Qian, Y., Perl, A., and Molnár-Perl, I., Analysis of amino acids and biogenic amines in biological tissues as their o-phthalaldehyde/ethanethiol/fluorenylmethyl chloroformate derivatives by high-performance liquid chromatography A deproteinization study, J. Chromatogr. A, 1149, 46–55, 2007. 115. Krause, I., Bockhardt, A. Neckermann, H., Henle, T., and Klostermeyer, H., Simultaneous determination of amino acids and biogenic amines by reversed-phase high-performance liquid chromatography of the dabsyl derivatives, J. Chromatogr. A, 715, 67–79, 1995. 116. Pereira, V., Pontes, M., Camara, J.S., and Marques, J.C., Simultaneous analysis of free amino acids and biogenic amines in honey and wine samples using in loop orthophthalaldehyde derivatization procedure, J. Chromatogr. A, 1189, 435–443, 2008. 117. Berge, G.E., Sveier, H., and Lied, E., Nutrition on Atlantic salmon (Salmon salar); the requirements and metabolic effect of lysine, Comp. Biochem. Physiol. Part A, 120, 477–485, 1998. 118. Refstie, S., Bakke-McKellop, A.M., Penn, M.H., Sundby, A., Shearer, K.D., and Krogdahl, Å., Capacity for digestive hydrolysis and amino acid absorption in Atlantic salmon (Salmo salar) fed diets with soybean meal or inulin with or without addition of antibiotics, Aquaculture, 261, 392–406, 2006. 119. Espe, M., Lemme, A., Petri, A., and El-Mowafi, A., Assessment of lysine requirement for maximal protein accretion in Atlantic salmon usin plant protein diets, Aquaculture, 263, 168–178, 2007. 120. Conceicao, L.E.C., Grasdalen, H., and Dinis, M.T., A new method to estimate the relative bioavailability of individual amino acids in fish larvae using C-NMR spectroscopy, Comp. Biochem. Physiol. Part B, 103–109, 2003. 121. Sozgen, K., Cekic, S.D., Tutem, E., and Apak, R., Spectrophotometric total protein assay with copper(II)-neocuproine reagent in alkaline medium, Talanta, 68(5), 1601–1609, 2006. 122. Noll, J.S., Simmonds, D.H., and Bushuk, W.C., A modified biuret reagent for the determination of protein, Cereal Chem., 52, 610–616, 1974. 123. Lowry, G.H., Rosenbraugh, R.J., Farr, A.L., and Randall, R.J., Protein measurements with the Folin phenol reagent, J. Biol. Chem., 193, 263–275, 1951. 124. Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J., and Klenk, D.C., Measurement of protein using bicinchoninic acid, Anal. Biochem., 150, 76–85, 1985. 125. Walker, J.M., The bicinchoninic acid (BCA) assay for protein quantitation, In: The Protein Protocols Handbook, 2nd edn., Ed., Walker, J.M., Humana Press Inc., Totowa, NJ, 2002. 126. Liu, R., Zhao, S.-M., Xiong, S.-B., Qui, C.-G., and Xie, B.-J., Rheological properties of fish actomyosin and pork actomyosin solutions, J. Food Eng., 85, 173–179, 2008. 127. Bradford, M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 72, 248–254, 1976.

Composition and Calories


128. Sarkardei, S. and Howell, N.K., The effects of freeze-drying and storage on the FT-Raman spectra of Atlantic mackerel (Scomber scombrus) and horse mackerel (Trachurus trachurus), Food Chem., 103, 62–70, 2007. 129. Benjakul, S. and Bauer, F., Physicochemical and enzymatic changes of cod muscle proteins subjected to different freeze-thaw cycles, J. Sci. Food Agr., 80, 1143–1150, 2000. 130. Hultmann, L. and Rustad, T., Iced storage of Atlantic salmon (Salmo salar)–effects on endogenous enzymes and their impact on muscle proteins and texture, Food Chem., 87, 31–41, 2004. 131. Sovik, S. L. and Rustad, T., Effect of season and fishing ground on the activity of lipases in byproducts from cod (Gadus morhua). LWT-Food Sci. Technol., 38, 867–876, 2005. 132. Kruger, N.J., The Bradford method for protein quantitation. In: The Protein Protocols Handbook, 2nd edn, Ed., Walker, J.M., Humana Press Inc., Totowa, NJ, 2002. 133. Aitken, A. and Learmonth, M.P., Protein determination by UV absorption. In: The Protein Protocols Handbook, 2nd edn., Ed., Walker, J.M., Humana Press Inc., Totowa, NJ, 2002. 134. Van Camp, J. and Dierckx, S., Proteins. In: Handbook of Food Analysis, Physical Characterisation and Nutrient Analysis, Volume I, 2nd edn., Revised and expanded, Ed., Leo, M.L., Nollet, F. Marcel Dekker, Inc., New York, 2004. 135. Watts, M., Munday, B.L., and Burke, C.M., Isolation and partial characterisation of immunoglobulin from southern bluefin tuna Thunnus maccoyii Castelnau, Fish Shellfish Immun., 11, 491–503, 2001. 136. Mendel, B., Kemp, A., and Myers, O.K., A colorimetric micro-method for the determination of glucose, Biochem. J., 56, 639–646, 1954. 137. Schultz, M., Liese, A.D., Mayer-Davis, E.J., D’Agostino, R.B., Fang, F., Sparks, K.C., and Wolever, T.M., Nutritional correlates of dietary glycaemic index: New aspects from a population perspective, Br. J. Nutr., 94, 397–406, 2005. 138. Kimura, S., Studies on marine invertebrate collagens. V. The neutral sugar composition and glucosylated hydroxylysine contents of several collagens, Bull. Jpn. Soc. Sci. Fish., 38, 1153, 1972. 139. Ansell, A.D., Storage and utilization of reserves in Pectinid bivalves with particular reference to the adductor muscle. Proceedings of the Scallop Workshop, Brest, France 8–13 May, 17pp, 1978. 140. Epp, J., Bricelj, V.M., and Malouf. R.E., Seasonal partitioning and utilization of energy reserves in two age classes of the bay scallop Argopecten irradians (Lamark), J. Exp. Mar. Biol. Ecol., 121, 113–136, 1988. 141. Zwann, A. de. and Zandee, D.I., Body distribution and seasonal changes in the glycogen content of the common sea mussel Mytilus edulis, Comp. Biochem. Physiol. A: Physiology, 43(1), 53–58, 1972. 142. Dare, P.J. and Edwards, D.B., Seasonal changes in flesh weight and biochemical composition of mussels (Mytilus edulis L.) in the Conwy estuary, North Wales, J. Exp. Mar. Biol. Ecol., 18(2), 89–97, 1975. 143. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., and Smith, F., Colorimetric method for determination of sugars and related substances, Anal. Chem., 18, 350–356, 1956. 144. Maguire, J.A., Fleury, P.G., and Burnell, G.M., Some methods for quantifying quality in the scallop Pecten maximus (L.), J. Shellfish Res., 18(1), 59–66, 1999. 145. Gerhardt, P., Murray, R.G.E., Wood, W.A., and Krieg, N.R., Chemical analysis. In: Methods for General and Molecular Bacteriology, 2nd edn., Am. Soc. Microbiol., pp. 518–520, 1994. 146. Allen, V.J., Marsden, I.D., Ragg, N.L.C., and Gieseg, S., The effects of tactile stimulants on feeding, growth, behaviour, and meat quality of cultures Blackfoot abalone, Haliotis iris, Aquaculture, 257, 294–308, 2006. 147. Carroll, N.V., Longley, R.W., and Roe, J.H., The determination of glycogen in liver and muscle by use of anthrone reagent, Department of Biochemistry, School of Medicine, George Washington University, Washington, D.C., J. Biol. Chem., 583–593, 1955. 148. Templeton, M.C., Microdetermination of glycogen with anthrone reagent, Northwestern University, Dept. Medical Anatomy, Publn no. 658, 670–672, 1961. 149. Van Handel, E., Estimation of glycogen in small amounts of tissue, Anal. Biochem., 11, 256–265, 1965.

152. 56. FAO. Newell. Merrill. 158. Seasonal variations in chemical. Erikson. U. 1993. Agriculture Handbook No. Pareja. and uses.. An evaluation of the wet oxidation technique for use in determining the energy content of seston samples.. Lucas.. Tijssen. shellfish. Miller. 17. F. An experimental reassessment of the factors used in the calculation of the energy value of human diets. 501–508.org/es/ESN/nutrition/requirements_pub_en. 1973.T. and Nolasco-Soria. Agric. Martin-Reyes. 1896. C.74. Aquaculture. Food Sci. 160. 2003.A. 29–33. 1996. 1985. FAO Food and Nutrition Paper 66. J. 163. J. Carbohydrates in human nutrition. L. Atwater...E. A ballistic bomb calorimeter.. A. 162. Burlingame. FAO/WHO. Tinahones. 9.K. 168. 517–535. Maeda-Martinez. G.J. Munro. Beltran-Lugo. and Lees. 1970.N. D. 170. New York. J. Eur. Food and nutritional paper 77. R. A comparison between methods used to determine the energy content of feed. S... J. 1955. 2004. Int. 1997. Nutr.R.. United States Department of Agriculture.V.stm.. J. Anal. A. Merrill. Energy value of foods. P. Oil Chem. protein.K. Food. 13. B. DC.. T.O.. 157.. J. Esteva. P. 8... Lipids and fatty acids of important finfish. S.. R. 58.. 166. A. J. 1998.. and molluscs commonly eaten in the south of Spain. Reinhold Publishing Corporation. 164.. Lipid. J. Soriguer. Kenley. Food energy-methods of analysis and conversion factors. Department of Agriculture..I. 117–127. and Watt. D. The chemical composition of American food materials. United States Department of Agriculture Office of Experiment Stations. B. basis and derivation.. Calorie conversion factors. Washington DC. Nutr. A.L. B. Kent.G. Serna... Food Comp.C.M.M. Krishnamoorthy. Food.L. Report of a joint FAO/WHO expert consultation. Br.A. D. 1986. Gallart-Jornet. Factors for converting percentages of nitrogen in foods and feeds into percentages of proteins. 27(5). and Durnin. Craig. Marroni. Anal.R. Impact of different macronutrient definitions and energy conversion factors on energy supply estimations.. Brown J. 13..A. J. 1125–1127. Bulletin 28. 1979. W.. Food Technology International (Europe). B. meanings. G. updated in 1941.. 2006. Carbohydrates and related food components: INFOODS tagnames. Bioenergetics and Growth.284 ◾ Handbook of Seafood and Seafood Products Analysis 150. A. and Machiels. Freshwater Biol. textural.S. J. Washington. J. M.. 100–118.. fish and faeces samples. Venkataramiah.V. 154–159. S. 451–463. Hernando.. 167. Chevassus-Agnes. F. 1975. Lakshmi. and Watt. 155.Fish.F..A. .. M. R. Axelson. D. Developing an international food energy system. http://www. 1383–1388... Pacheco-Aguilar R. Jones. J. R. 619–632. DC. Microwave and infra-red drying versus conventional oven drying methods for moisture determination in fish flesh. 156. 1997. J. B.. Can. Southgate.K.. 154. Water distribution in brine salted cod (Gadus morhua) and salmon (Salmo salar): A low field NMR study. Valverde. and microstructural properties of adductor muscles of Pacific lions-paw scallop (Nodipecten subnodosus). E.. 24.. M.B.. Caloric densities of shellfish meat and fats. Br. Rome... Broody.. Exler. Christie. DC. physical. M. 151.. A. 39. New data for nutrient tables. H. Soc. J. 195–201.J.G. Washington. Comparative estimations of the energy content of fish tissue from bomb calorimetry. Rome. Aquat. 183. 153. A. and Biesiot. wet oxidation and proximate analysis.. United States Department of Agriculture Circular No. 165.. S. 161.. and Rustad. 339–360. 20(2).I. Agric... Soriguer.. J.T. J.. 2008.E. 585–590. and Talling.S.. A. I. Energy value of foods. 6252–6260. Henken. and Watt.. 1978. Government Printing Office.. Chem. Agriculture Handbook. Comp.. U.. 1945. 159.. Washington. and Livesey.. 1931. and calorie content of different Atlantic and Mediterranean fish... Aquaculture. Kinsella. No. 74. P. Technol. and Payne.. 52. U. Epidemiology. Food Chem.F. 1959.D. 169.. H. Charrondiere. Report of a technical workshop. Am. I. Sci.fao. J. Faulks. A. Aursand. Rome. 258. 1982. and Burlingame.M. and Woods. basis and derivation.G.

5(2). C. Food Comp.M. Arch. J.. J. Freese. Southgate..F. 176. 913. and Jensen. M. Lantry. Turgut. 4. 315–322. Ed.. A. Hancz. B. advantages and disadvantages. Part 1: Applications in Chemistry. Shono. 2003..I.. Bull. Food Chem. Failure of total body electrical conductivity to predict lipid content of brook trout.) by electrical conductivity.L. S. 179.J.. 14. Anal. 19(4).. In: Modern Magnetic Resonance. Jørgensen.. Biological and Marine Sciences. Publisher IEEE.T.. and Baskaeva. 1983. Schakel.. Fish. Lab Delo. Maintaining a nutrient database in a changing marketplace: Keeping pace with changing food products–A research perspective. B. and Mills. the Netherlands. D. 161–165. 175. Lipid content determination in whole fish using ultrasonic pulse backscatter. G. 178. C. 1974.. Tierzucht–Archives of Animal Breeding 46. Ed. H.A. 1979. Bradford’s method of determining protein: Application. 397–402. G. Springer. Derivatisation and chromatographic behaviour of the o-phthalaldehyde amino acid derivatives obtained with various SH-group containing additives.. D. Webb.. 173. M... 1999. 1999. 1989. 2001. 283–302. P. and Martinez Del Rio.. Molnar-Perl. 180. Stewart. and Horn. 71–79. D.. C.K. 30–39. Novinger. North Am. E.. 4–7. 4.A. Rand. 97..Composition and Calories ◾ 285 171.N. De Klerk. Food and Nutr. Manag. I.F. 905–908. 69–76... N. K. 177. Drawbacks in the use of the Biuret method for determination of the same protein in differently treated fish samples. 2006. P.A. Water distribution and mobility in fish products in relation to quality. Availability of and needs for reliable analytical methods for the assay of foods. 3. 174.S.. Chromatogr. . In: Ultrasonic Symposium Conference. and Hamid. Fish.. Dordercht. In vivo measurement of total body lipid content of common carp (Cyprinus carpio L. J. Milisits. Bull. E. 2001. Evaluation of total body electrical conductivity to estimate whole body water content of yellow perch (Perca flavescens) and alewife (Alosa pseudoharengu). J.. 172.M.


.................Chapter 17 Essential Amino Acids M...................... 298 17........................ 288 17....................................4 Mass Spectrometry .........3.......................... which contribute to fish taste and indirectly to aroma 2........................ 300 17.2 Gas Liquid Chromatographic Methods .........291 17...............1 Introduction Amino acids are the basic components of the muscle protein structure of seafood.2..................1......2 Sample Preparation for Total or Hydrolyzed Essential Amino Acid Analysis...2....................3 by generation of volatile 287 ...........................................3................................................................................1 Sample Preparation for Free Essential Amino Acid Analysis ............. 299 17..... Concepción Aristoy and Fidel Toldrá Contents 17......... 298 17...................... 290 17................................ However...1.... 288 17.................................................................... 292 17.............3....................2 Sample Preparation for the Analysis of Seafood Essential Amino Acids ..... 287 17...... not all proteins have the same nutritional value...............................................1 Cation Exchange Chromatography ..2 Reversed-Phase High-Performance Liquid Chromatography .......1 High-Performance Liquid Chromatographic Methods.4 Conclusions .........3........3.....................3 Seafood Essential Amino Acid Analysis............................ in general....................... 289 17.... seafood proteins are considered as highquality proteins because of their balanced content in amino acids........................... Amino acids may also be found in free form...291 17.1 Introduction ........ 300 References ............... because protein quality strongly depends on its amino acid composition and digestibility........3....... especially in all the essential amino acids necessary for physical and mental well-being................................3 Capillary Zone Electrophoretic Methods ..................................................................1 Fish and.........................................

21 perchloric (PCA). and so forth.18. Polytron. The extraction consists in the separation of the free amino acid fraction from the insoluble portion of the matrix (fish muscle).4 Branched-chain essential amino acids (valine.2. in rare cases.20 have been successfully used as extraction solvents.16. Once homogenized. (2) its ability to cross-link proteins. with the additional advantage that proteins are not extracted and. or diluted phosphate buffers. isoleucine. sulfosalicylic (SSA). methods for the analysis of amino acids in seafood. which confers numerous biological functions to this amino acid (precursor to the antioxidant glutathione). and leucine). or (3) its Maillard reaction with sugars yielding characteristic flavors. which can be achieved through different chemical or physical procedures. 17.13 5% of trichloroacetic acid. the elderly. the sample is centrifuged at more than 10. or by means of a simple stirring in warm solvent. Although classified as nonessential.14 6% of perchloric acid.12. which increases the protein stability in the harsh extracellular environment by conferring proteolytic resistance. the analysis of essential amino acids in seafood is important for the evaluation of both the nutritive value and the sensory quality of seafood.5–10 Thus.2 Sample Preparation for the Analysis of Seafood Essential Amino Acids Free or total essential amino acids are analyzed from the whole amino acid profile. there is no need for further cleaning up of the sample.11 17.13. The extraction solvent can be hot water. are described.000 g under refrigeration (4°C) to separate the supernatant from the nonextracted materials (pellet) and filtered through glass wool to retain any fat material remaining on the surface of the supernatant. Sample preparation will depend on whether free or total essential amino acids have to be analyzed. then. sulfur-containing amino acids (methionine and cystine/cysteine). concentrated strong acid solutions such as 4% of 5-sulfosalicylic acid.01–0. Sample cleanup is necessary to eliminate proteins and polypeptides by means of the deproteinization process. It is usually achieved by homogenization of the ground sample in an appropriate solvent by using a Stomacher.22 trichloroacetic (TCA). and aromatic amino acids (phenylalanine and tyrosine) are the most important from this point of view. In some cases. 0.288 ◾ Handbook of Seafood and Seafood Products Analysis compounds through Maillard reactions and Strecker degradations. Several chemical methods include the use of concentrated strong acids such as phosphotungstic (PTA). especially of those considered essentials. and individuals with certain metabolic disease or who suffer from malabsorption syndromes.23–25 and picric . A more detailed description of amino acid methods of analysis may be found in the work of Aristoy and Toldrá.1 Sample Preparation for Free Essential Amino Acid Analysis Sample preparation for free essential amino acids includes their extraction and the cleanup or deproteinization of the extract. Special attention is also devoted to the analysis of the sulfur amino acid cysteine for several reasons: (1) the high reactivity of its thiol group.1 N hydrochloric acid solution. cysteine may be essential for infants. In this chapter.15 or a rich alcohol-containing solution (>75%) such as ethanol16–18 or methanol19. Free amino acids initiate important changes at early postmortem and during storage and can be very useful as quality indices of processing and storage.

all of them . 10. The presence of appropriate antioxidants/scavengers during hydrolysis can prevent losses of the most labile amino acids. thus excluding nonvolatile contaminants.30 All these methods give a sample solution rich in free amino acids but free of proteins. A good choice may be the use of 0.2 Sample Preparation for Total or Hydrolyzed Essential Amino Acid Analysis The total essential amino acid profile is usually requested. proteins precipitate by denaturation. When limited amounts of sample are available. Upon heating. creating an appropriate atmosphere inside the vessels to ensure low amino acid degradation. and performance under vacuum) is similar to that of a conventional oven.000 Da) that allow free amino acids through while retaining large compounds. In the vapor-phase hydrolysis method. has also given very good results. 30. recovery of amino acids. addition of constant boiling hydrochloric acid and additives.6 N PCA. which is easily neutralized by the addition of KOH or potassium bicarbonate. by mixing two or three volumes of organic solvent with one volume of extract. is well suited to hydrolyze large amounts or complex samples.34 17.000. the vapor-phase hydrolysis method is preferred to minimize contaminants coming from aqueous 6 N hydrochloric acid. because it gives information on the nutritional value of fish meat. or acetonitrile. a system capable of alternative air evacuating/inert gas purging to get a correct deaeration inside is valuable. etc. to rend insoluble potassium perchlorate.2.Essential Amino Acids ◾ 289 (PA)26–28 acids or organic solvents such as methanol. Some comparative studies have been published on these deproteinization techniques.22 Under these conditions. Differences among all these chemical and physical methods are caused by several aspects such as differences in the cutoff molecular weight. the tubes containing the samples are located inside large vessels containing the acid. presence of salts.29. One of them is the Pico-Tag Workstation that includes special vessels (flat-bottom glass tubes) fitted with a heat-resistant plastic screw cap equipped with a Teflon valve. Therefore. compatibility with derivatization (pH. Hydrolysis may be improved by optimizing the temperature and time of incubation41 or with the addition of amino acid oxidation protective compounds. The most common method used for complete hydrolysis of proteins is acid digestion. liquid phase or vapor phase. where the hydrochloric acid contacts the sample directly. samples are treated with constant boiling 6 N hydrochloric acid in an oven at around 110°C for 20–96 h.000. ethanol.39 Some commercial systems are available. etc. Liquid-phase.). but the duration of the treatment is shorter (less than 20 min). or separation method (interferences in the chromatogram. which is easily separated by centrifugation. Some physical methods consist in centrifugation through cutoff membrane filters (1. Proteins must be hydrolyzed into their constituent amino acids before the analysis. The hydrolysis may be accomplished using either liquid-phase or vapor-phase methods.42 Sample manipulation (sample evaporation to dryness. which permits the alternative air evacuating/inert gas purging. and so forth.31–33 with amino acid recoveries around 100% for all them.).39. oxygen is removed and substituted by nitrogen or other inert gas.35–38 These temperatures in such acidic and oxidative medium may degrade some amino acids. only the acid vapor comes into contact with the sample. Digestion at 145°C for 4 h has also been proposed.000. also disposes of an oven to accomplish the hydrolysis.41 The use of microwave technology for the hydrolysis has been assayed by some authors. whereas free amino acids remain in solution. Nitrogen atmosphere and sealed vials are required during the hydrolysis to minimize the degradation. In both cases. 5.22. resulting in a very simple deproteinization procedure with no interferences. The use of organic solvents. Typically.40. An additional advantage is the easy evaporation to concentrate the sample.18.

67–69 17. LiOH.55. protective agents currently used. cysteine sulfinic acid. being enough for the requirements of any food industry.30. no single set of conditions will yield the accurate determination of all essential amino acids. Thus.56 As can be observed in this section. This option is chosen to analyze specific amino acid sequences or single amino acids because of their specific and well-defined activity.63–66 for a better tryptophan determination. such as chromatographic (liquid or gas chromatography (GC)) or capillary electrophoresis (CE) techniques. chymotrypsin. when the analysis of cyst(e)ine would be necessary. importance of a correct deaeration. The use of alkylating agents to stabilize the previous hydrolysis of cysteine constitutes a valid alternative. thermolysin. methionine.60 and books. When high sensitivity is required. or pronase. in which methionine is also oxidized to methionine sulfone.38 Alkaline hydrolysis instead of acid hydrolysis is also proposed (see below).3¢-dithiodipropionic acid.59. or BaOH.58. serine. carboxypeptidase. and tryptophan.36. Tryptophan is often completely destroyed by hydrochloric acid hydrolysis. Before or after this separation. .51 3-bromopropylamine. threonine. The separation of the individual amino acids in a mixture requires very efficient separation.36. and so on. a compromise of conditions offers the best overall estimation for the largest number of amino acids. Cyst(e)ine is partially oxidized during acid hydrolysis yielding several adducts: cystine.2 M of either NaOH. In general. and cysteic acid making its analysis rather difficult. unless a very selective way of detection is used. the 22–24 h acid hydrolysis at 110°C (vapor-phase or liquid-phase hydrolysis) with the addition of a protective agent like 1% phenol.61. which is recommended by many authors47. papain. presence and concentration of oxidation protective agents. The optimization of conditions for hydrolysis based on the study of hydrolysis time and temperature. because each possible methodology has advantages and drawbacks.54 or 3. A third way to hydrolyze proteins is enzymatic hydrolysis by proteolytic enzymes such as trypsin. up to 1% phenol or 0.52 4-vinyl pyridine53. cysteine. making the posterior analysis easier.3 Seafood Essential Amino Acid Analysis The analysis of individual amino acids needs a previous separation of all others. although considerable recoveries have been found if no oxygen is present. the pyrolysis from 500°C for 3 h57 to 600°C overnight58 of all glass material in contact with the sample is advisable as well as the analysis of some blank samples to control the level of background present. KOH.290 ◾ Handbook of Seafood and Seafood Products Analysis essential amino acids. In fact. Some additives have been proposed to protect tryptophan against oxidation as is the case of thioglycolic acid. has been extensively reported in papers35. improve the recovery of nearly all of these amino acids except tryptophan and cysteine. The choice mainly depends on the equipment available or personal preferences. (2) give a quantitative and reproducible reaction.1% sodium sulfite. The effect of a derivatizing agent is evaluated based on the following aspects: (1) It must be able to react with both primary and secondary amino acids.43–50 improves cysteine (and methionine) recoveries. adequate hydrolysis procedure as the performic acid oxidation before the hydrolysis is a good alternative. Derivatization is a usual practice in amino acid analysis. yields acceptable results for the majority of amino acids. 41. Additionally. acid-to-protein ratio. The previous performic acid oxidation of cysteine to cysteic acid. amino acids used to be derivatized to allow their separation or to enhance their detection. Good recoveries have been achieved by using 3-bromopropionic acid.62 An alternative to acid hydrolysis is the alkaline hydrolysis with 4. with or without the addition of 1% (w/v) thiodiglycol for 18 h at 110°C. such as tyrosine.

68 Thus.1. are always present. The first type are derivatives that enhance amino acid detection in liquid media. 17. and better detection systems. Tryptophan also possesses native fluorescence (l ex = 295 nm. absorb at 210 nm and thus cannot be used for spectroscopic detection.2. (6) have good stability of the derivatization products.1 and 17. which facilitates a more selective detection.15. The original method required two separate columns and needed about 4 h to achieve a complete analysis. and thus the underivatized amino acids are separated using sulfonated polystyrene beads as the stationary phase and aqueous sodium citrate buffers as the mobile phase. although unidentified.1. because their spectral (high-ultraviolet (UV) absorbing or fluorescence properties) or electrochemical characteristics will affect the sensitivity and selectivity of detection.3-oxadiazole postcolumn derivatization to obtain highly fluorescent derivatives with enhanced sensitivity. (4) have mild and simple reaction conditions. in their native form. speed. recent improvements of the ninhydrin derivatization method71–73 .3. amino acids were converted into colored ninhydrin derivatives for spectrophotometric (colorimetric) detection. the spectroscopic detection of amino acids requires their previous derivatization to obtain an UV absorbing or fluorescent molecule. permitting 5–10 pmol sensitivity as standard. tyrosine.1. and they include derivatives for spectroscopic or for electrochemical detection. The derivatization reaction can be performed after separation of the amino acids (postcolumn derivatization) or before separating them (precolumn derivatization).1.1 Cation Exchange Chromatography This methodology is based on the amino acid charge.70 fluorescamine.3. as it is a very unspecific detection wavelength. Although postcolumn techniques should be run online for maximum accuracy.3. The latest generation of Moore and Stein amino acid analyzers also use o-phthaldialdehyde (OPA).12 Two types of derivatives are obtained depending on the chosen separation and/or detection technique. because reagent-consuming amines. Under these conditions. Only three amino acids (phenylalanine. or 4-fluoro-7-nitrobenzo-2. It must be remarked that the use of sufficient amount of reagent is of special importance when dealing with biological samples. 17. After separation. precolumn techniques can be run either offline or online. The HPLC techniques to analyze amino acids are cation exchange and reversed-phase (RP) chromatography and are described in Sections 17.38. the more acidic amino acids elute first. Nevertheless. The elution involves a stepwise increase in both pH and sodium or lithium ion concentration. The second type are derivatives that allow gas chromatographic amino acid separation by increasing their volatility and temperature stability. and (7) have no interferences due to by-products or excess of reagent.3. Amino acids. pellicular packaging. and tryptophan) have a chromophore moiety that confers a suitable maximum absorbance for more specific UV detection (280 nm for tyrosine and tryptophan and 254 nm for phenylalanine). The classical procedure has been improved with a new polystyrene matrix that offers better resolution power due to smaller particle size.1 High-Performance Liquid Chromatographic Methods HPLC is the preferred technique to analyze amino acids. l em = 345 nm). and those with more than one primary amino group or possessing a guanidyl residue elute at the end of the chromatogram.Essential Amino Acids ◾ 291 (3) yield a single derivative of each amino acid. The formed derivatives will be separated by high-performance liquid chromatography (HPLC) or capillary zone electrophoresis (CZE) as it is important to choose the most adequate derivative. (5) have the possibility of automation.

time for sample preparation and amino acids separation. The resulting system is simpler and cheaper compared with the combination of cation-exchange plus postcolumn derivatization and permits choosing among a great number of possible methodologies. through a mixing manifold. biological fluids. Although this broadening may not affect when using standard-bore columns with flow rates above 1 mL/min. In this way. which are detectable at UV (254 nm) with detection limits around 5–50 pmol. The advantage of this method is the accurate results for all known sample types (food. Hitachi. plants. the formed molecule improves sensitivity and selectivity at the detection by allowing the spectroscopic (UV or fluorescent) detection of amino acids.42. and an optimized methodology with the advantage of ease of use and reliability. and finally the derivatized amino acids reach an online detector system. tissues. also. making it adequate for partition based on chromatography. buffer system.292 ◾ Handbook of Seafood and Seafood Products Analysis together with the low sensitivity requirements of fish amino acid analysis still make this method the most used. All PTC-amino acids have similar response factors. the analysis requirements for free or hydrolyzed amino acids or required sensibility. especially in a dry condition.1. There are many manufacturers (Beckman. Obviously. To choose the most appropriate method some aspects must be taken into account such as the following: the disposable detector (fluorescence or UV). Pickering. Phenylisothiocyanate (PITC): This methodology involves the conversion of primary and secondary amino acids to their phenylthiocarbamyl (PTC) derivatives. . some difficulties to analyze some essential or sulfur-containing amino acid derivatives).e. This fact and the proliferation of precolumn derivatizing agents have stimulated the development of RP-HPLC methods to analyze amino acids in all kind of matrices (food. which makes it a reference method for amino acid analysis. or the stability of formed derivatives. The most usual derivatizing agents for tissue amino acids are described below. This method has been employed in the classical Moore and Steintype commercial amino acid analyzers. the main drawback of this type of derivatization method is the required additional equipment: another pump to introduce the reagent as well as mixing and sometimes heating devices. Dionex. followed by a reaction coil. The PTC-amino acids are moderately stable at room temperature for 1 day and much longer when kept under frozen storage. feed. Amersham Biosciences. (i. LKB.2 Reversed-Phase High-Performance Liquid Chromatography RP-HPLC has been widely used. 66. and the long time of analysis.) who offer integrated commercial systems including the column. but. postcolumn derivatization is not suitable for narrow-bore HPLC. and tissues). each new methodology must contrast its results with those obtained by cation exchange chromatography (CEC). with which many of them have been marketed.. the highly complex mobile phase composition.76 There are other reports of applying this technique to the amino acid analysis in food and tissues. The main drawbacks of this methodology are the high cost of the ion exchange amino acid analyzer and its maintenance.77 After separation. Another disadvantage is the peak broadening produced by the dead volume introduced behind the column. Kontron. possibility of automation of the derivatization reaction (in the autosampler). Biotronik. the separation times for the 20 amino acids naturally occurring in fish proteins take around 1 h and somewhat longer (2 h) for physiological amino acids. biological fluids. because it requires only a standard equipment that can be shared by different types of analysis.3.75. 17.70. etc. which constitutes an advantage. the derivatizing reagent is pumped into the effluent from the column system. plants). Precolumn amino acid derivatization may be necessary to confer hydrophobicity to the amino acid molecule.74 Nowadays.

This method is available as a commercially prepackaged system named Pico-Tag (Waters Associates. which is achieved by the addition of triethylamine and includes several drying steps.81 reported a modification of the method in which the analysis of 27 physiological amino acids could be performed in 22 min (30 min including equilibration). 700 Ala 600 Gly 500 Absorbance at 254 nm (mAU) Glu 400 lS Lys Asp 200 Ser 100 OHpro 300 Arg Thr Leu Pro Tyr Val Met lle Phe His 0 0 2 4 6 8 10 12 14 16 18 Retention time (min) Figure 17. internal standard nor-Leucine. and solvents. which includes the analytical column. because no buffer is used during the reaction.29. Massachusetts).2. which makes the quantitation of free cystine nonfeasible with this method. Both examples applied to the analysis of total amino acids from hake and free amino acids from salmon are shown in Figures 17.82 The selection of the column is critical to get a good resolved separation especially when the analysis of physiological amino acids is involved. The reaction time is less than 10 min even though 20 min are recommended for a complete reaction. Sarwar et al. IS.1 Reversed-phase HPLC chromatogram of PTC amino acids from hydrolyzed hake muscle. the residual PITC reagent left after evaporation will cause damage to the column package.1 and 17.78–80 Sample preparation is quite tedious: it requires a basic medium (pH = 10. The only limitation is the determination of PTC cystine that gives a poor linearity.Essential Amino Acids ◾ 293 The methodology is well described in the literature. standards. Moreover. the last one being the elimination of the excess of reagent that may cause some damage to the chromatographic column. Milford.78–80 The chromatographic separation takes around 20 min for hydrolyzed amino acids and 60 min for physiological.5).29. It is important to ensure a basic pH to get adequate derivatization recoveries. . as some columns are more resistant than others. which is more critical when amino acids from acid hydrolysis are analyzed. respectively.

1-Dimethylamino-naphthalene-5-sulfonyl chloride (Dansyl-Cl): Dansyl-Cl reacts with both primary and secondary amines to give a highly fluorescent derivative (l ex = 350.2 Reversed-phase HPLC chromatogram of PITC-free amino acids from salmon muscle extract. the reaction conditions .58 obtained a good separation of 35 dabsyl-amino acids and by-products in a 15 cm C18 column packed with 3 mm particle size. standard amino acid solution should be derivatized under similar conditions. and only needs a basic pH. around 9. anserine. Ans. because it is especially affected by the presence of high levels of some chloride salts.58. United States). The reaction time is around 15 min at 70°C and takes place in a basic medium with an excess of reagent. The dansylated amino acids are stable for 1 day85 or until 7 days when kept at −4°C86 and protected from light. IS. The high wavelength of absorption makes the baseline chromatogram very stable with a large variety of solvents and gradient systems. Stocchi et al. internal standard nor-Leucine. By-products originating from an excess of reagent absorb at the same wavelength and thus they appear in the chromatogram. Detection limits are in the low picomole range.294 ◾ Handbook of Seafood and Seafood Products Analysis 1400 1200 Glu Ans Absorbance at 254 nm (mAU) 1000 800 Gly 600 Tau βAla His Ala 400 Asp 200 OHpro Ser Lys Pro Thr Arg Tyr Val Met lle lS Leu Trp Phe Orn 0 0 Asn Gln 10 20 30 Retention time (min) 40 50 Figure 17. and a reaction time of 1 h at room temperature (in the dark).83.33 To overcome this problem and obtain an accurate calibration. 15 min at 60°C. presenting a maximum from 448 to 468 nm. The sample derivatization is rather simple. However.84 Detection is by absorption in the visible range. taurine. 4-Dimethyl-aminoazobenzene-4′-sulfonyl chloride (Dabsyl-Cl): This reagent was first described in 1975 for use in amino acid analysis. Reaction efficiency is highly matrix dependent and variable for different amino acids. Commercial System Gold/Dabsylation Kit™ uses this technique (Beckman Instruments.5. l em = 510 nm) although UV (l = 250 nm) detection may also be used.87 or even 2 min at 100°C. Nevertheless.84 Derivatives are very stable (weeks) and can be formed from both primary and secondary amino acids. Tau. Palo Alto California.

Another proposal102 consists of a slight modification in the OPA derivatization method by using 2-aminoethanol as a nucleophilic agent and altering the order of the addition of reagents in the automated derivatization procedure. using 3. The choice of the mercaptan can affect derivative stability. the reaction of the excess of reagent with a very hydrophobic amine as 1-adamantylamine (ADAM) gives a late-eluting noninterfering peak. reinforcing the poor reproducibility of its results.82 Another problem is the large excess of reagent needed to assure a quantitative reaction. An automated precolumn derivatization routine. and excess of reagent) must be carefully fi xed to optimize the product yield and to minimize secondary reactions. which includes the addition of ADAM. The reaction time is fast (45–90 s) and does not require any heating. which is not the case with any essential amino acid. The first option was included in the automated AminoTag method90 developed by Varian (Varian Associates Limited).89 9-Fluorenylmethyl chloroformate (FMOC): This reagent yields stable derivatives (days) with primary and secondary amines. which is present in excess as it is highly fluorescent and probably interferes into the chromatogram as a huge peak. The derivative is fluorescent (l ex = 265 nm.92. lysine. and the reagent itself is not fluorescent.86. This is relatively easy because the reaction is fast and no heating is necessary.93 The fluorescence is recorded at 455 or 470 nm after excitation at 230 or 330 nm. In order to obtain reliable and precise results. Tryptophan adducts do not fluoresce and histidine and cyst(e)ine adducts fluoresce weakly.101 or the formation of the mixed disulfide S-2-carboxyethylthiocysteine (Cys-MPA) from cysteine and cystine. l em = 315 nm) and is detected at the femtomole range. One of the main disadvantages of this procedure is the inability of OPA to react with secondary amines. On the contrary. Some reports have been published proposing several ways of automation. OPA derivatives can be detected by UV absorption (338 nm) as well.32 .97. is of great advantage. In the second option. itself or hydrolyzed.82. chromatographic selectivity. several methods have been proposed before derivatization. ethanethiol. This excess is hydrolyzed to dansyl sulfonic acid. this problem is overcome by standardizing the time between sample derivatization and column injection by automation.98 and some of them have been patented and commercially marketed (AutoTag OPA from Waters Associates). and tyrosine. because it guarantees the repeatability of parameters. Histidine gives a very poor fluorescence response (10% of the other amino acids). have to be optimized very carefully.3′-dithiodipropionic acid55 and incorporated by Godel et al. The major disadvantage is due to the reagent. reaction conditions.94 into the automatic sample preparation protocol described by Schuster. The yield with lysine and cysteine is low and variable. such as FMOC/amino acid ratio. OPA amino acids are not stable. cysteine and cystine are quantified together. and fluorescent intensity. as it is highly fluorescent and then.5) medium.32 In these methods. this will commonly form multiple derivatives with histidine. The addition of detergents like Brij 35 to the derivatization reagent seems to increase the fluorescence response of lysine. Nowadays. and 3-mercaptopropionic acid are the most frequently used. o-Phthaldialdehyde (OPA): This reagent reacts with primary amino acids in the presence of a mercaptan cofactor to give highly fluorescent 1-alkylthio-2-alkyl-substituted isoindols.Essential Amino Acids ◾ 295 (pH.91 This method is preferred because the addition of ADAM is more easily automatized. These methods include the conversion of cysteine and cystine to cysteic acid by oxidation with performic acid or carboxymethylation of the sulfhydryl residues with iodoacetic100. this methodology reveals excellent linearity for cystine and also cystine-containing short-chain peptides. temperature.99 In the case of cysteine. as well as reaction time.94–96 2-mercaptoethanol.88 Even so. many automatic injectors are programmable and able to achieve automatic derivatizations. the excess may interfere in the chromatogram and for this reason it must be extracted (with pentane or diethyl ether) or converted into noninterfering adduct before injection. The derivatization is fast (1–3 min) and is performed at room temperature in alkaline (pH 9.

OPA/mercaptoethanol or OPA/sulfite104.296 ◾ Handbook of Seafood and Seafood Products Analysis 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC): It reacts with primary and secondary amines from amino acids. making them very adequate for biochemical research. In these cases. even those made by the same manufacturer. Nowadays. but 10 min at 55°C would be necessary if a tyrosine monoderivative is required. UV detection (254 nm) may also be used. potential. It means that when transferring a published method to a particular set of samples. whereas Figure 17.3A shows the separation of hydrolyzed amino acids from salmon.2 to 10. yielding very stable derivatives (1 week at room temperature) with fluorescent properties (l ex = 250 nm. or charge.50).. different selectivity. Indeed. The fluorescence of tryptophan derivative is very poor. Only columns manufactured in the same batch are guaranteed to give the same selectivity if the rest of parameters are fi xed. Cystine and cysteine may be analyzed after their conversion to cysteic acid (CisH) by performic acid oxidation. detergents. the AMQ peak is very large at the beginning of the chromatogram and may interfere with the first eluting peaks (see Bosch et al. the selectivity obtained with each trademark column is different due to the particular chemistry employed in their manufacture rendering different density of bonded-phase coverage on the silica particle and hydrophobic behavior and. Sensitivity is in the femtomole range.and di-derivatives are the initial adducts from tyrosine. Any electrical measure. because they are molecules with electroactive functional groups. United States). and other compounds naturally occurring in biological samples and foods. columns are more carefully manufactured with these silanol groups blocked or inaccessible by steric impediment avoiding the tailing.3B shows the same sample but submitted to a performic acid oxidation before the hydrolysis in which the CisH peak appears by 7. mainly octadecylsilane.107. is related to the analyte concentration. Electrochemical detection consists in one electrode or an array of electrodes mounted in a cell with an applied potential difference. Both facts facilitate sample preparation. can cause unwanted tailing of peaks (especially for the basic amino acids). and UV detection at 254 nm may be used for its analysis.5 min. the addition of a strong cation (i. from 8. conductance. and proteins. Milford. Due to these variables. Furthermore. such as current. The chromatographic separation of these derivatives has been optimized for the amino acids from hydrolyzed proteins. and the separation of physiological amino acids is improved. which are separated by RP-HPLC. Figure 17.108 If the choice of the derivative reaction is a challenge. The methodology has been marketed as a prepackaged AccQ Tag kit (Waters Corporation. accessible to sample molecules. different selectivity may be found among same columns. l em = 395 nm).105 in addition to fluorescent properties possesses electroactivity (750 mV) and PITC106 has again the advantage of reacting with secondary amines. Some of these derivatives are also susceptible to electrochemical detection. In this case. as a consequence. it will be necessary to readjust the chromatographic conditions to get a good separation of all amino acids. which is only weakly fluorescent at the amino acid derivatives detection conditions and does not interfere in the chromatogram. because the resulting CisH is well separated inside the chromatogram. .e. the optimum pH for the reaction is in a broad range. peptides. The most used column packaging consists of alkyl-bonded silica particles. because both mono. However.103 The main advantage of this reagent is that the yield and reproducibility of the derivatization reaction are scarcely interrupted by the presence of salts. Massachusetts. Reaction time is short. The presence of residual uncapped silanol groups on the silica surface. lipids. triethylamine) to the mobile phase can overcome the problem. the choice of the RP column is not an easy subject because of the great variability of commercially available RP columns. 1 min. The excess of reagent is consumed during the reaction to form aminoquinoline (AMQ). Only amino acids with aromatic rings or sulfur-containing side chains are sufficiently electrochemically active to be detected by this method.

methiomine sulfone.Essential Amino Acids 200 (A) ◾ 297 Leu 150 Val lle Phe 100 Ala NH3 Arg Asp Thr Gly Ser His Glu Met Tyr Pro αAba Lys 50 Fluorescence (% FS) 0 1MeHis 200 (B) βAla 150 100 MeS 50 CysH 0 0 5 10 15 20 25 30 35 Retention time (min) Figure 17. CysH.3 Reversed-phase HPLC chromatogram of AQC amino acids from hydrolyzed salmon muscle (A) without and (B) after performic acid oxidation. MeS. aAba. a aminobutyric acid used as internal standard. Mobile-phase composition combines an aqueous buffered phase . packed with 5 mm particle size or shorter columns (10 or 15 cm length) when packed with less than 3 mm particle size. Mobile-phase requirements consist in the ability to dissolve the sample while keeping it transparent to the detection system. 1MeHis. 1-methylhistidine. cysteic acid. Typical analytical column dimensions are 15 cm (for hydrolyzed amino acids) or 25–30 cm (for physiological amino acids).

phosphothreonine. A finely adjusted binary (most used) or ternary gradient elution is often necessary when the overall amino acid profile from hydrolyzed and. Amino acids constitute a mix of basic. The difficulty of separating amino acids by this technique relies on their structure.120. The method yields a full amino acid profile (33 amino acids) in 15 min including a 7 min extraction-derivatization step plus 8 min for the gas chromatographic separation. carnosine. United States). and balenine may complicate the amino acid analysis. and the equipment is very versatile and usually available in any analytical laboratory. speed. nuts. anserine. whereas thermionic-N-P (NPD) or flame photometric detector (FPD) are selective toward organic compounds containing phosphorous and nitrogen. and beans111 or other results obtained in honey. GLC is.298 ◾ Handbook of Seafood and Seafood Products Analysis with an organic phase constituted by acetonitrile and/or methanol and/or tetrahydrofuran. The buffer may be constituted by less than 100 mM concentration of acetate or phosphate. 17. which are much more sensitive than FID for such compounds. especially since the capillary columns appeared. or GC and LC with MS detection.109. Protein removal is not required. This methodology has been patented as EZ:faast and commercialized by Phenomenex (Torrance. has been developed.121 The high efficiency. Reactions consist of two stages: an esterification with an acidified alcohol followed by N-acylation with an acid anhydride in an anhydrous medium.114 In their analysis by GLC. a very highly efficient technique adequate for the amino acid analysis. The main advantages of these detectors are their high sensitivity and wide linear range. capable of separating 50 compounds. California. and phosphotyrosine. in summary. Nevertheless. Some applications67. GLC is not very expensive because no solvent is used.117–119 where the separation was achieved by using chiral-GC stationary phases. GC/NPD.112 milk. Recently. physiological amino acids has to be analyzed. the amino acids must be converted to volatile and thermostable molecules. GLC has been combined with mass spectrometry (MS) for detection and identification. in comparison with liquid chromatographic techniques. 17.113 and cheese.110 comparing GLC with cation exchange chromatography reported different conclusions when analyzing some hydrolyzed food samples. Described applications are available for the analysis of physiological amino acids in blood. and acidic constituents. dipeptides. In many cases. especially in the analysis of D isomers. The detector used is the flame ionization detector (FID). and even though a particular pH can significantly .3. Gas liquid chromatography (GLC) is not often used for the determination of amino acids from tissues or foods.113.2 Gas Liquid Chromatographic Methods The extremely high-resolution capacity is the main advantage of GC. The NPD was used by Buser and Erbersdobler115 and FPD by Kataoka et al. and urine matrices but not in tissues in which the presence of natural dipeptides. including amino acids. which is universal and the most widely used. plasma.3. neutral.116 to analyze phosphoserine. and low amount of sample make this technique very interesting when compared with classical electrophoresis and chromatographic techniques. and the derivatives are stable and ready for GC/FID. and amines. a very fast GC analysis of physiological amino acids. the technique is very efficient and it is worth mentioning the separation of 32 nonprotein amino acids from edible seeds.3 Capillary Zone Electrophoretic Methods Capillary zone electrophoretic technique is extremely efficient for the separation of charged solutes. although applications on meat samples are scarcely described. especially.

Unfortunately.138 Tween 20. Some reviews covering high-sensitivity detection following CE have been published. showing that higher efficiency is obtained by the MECC methods with sodium dodecyl sulfate (SDS) as micelle-forming substance.21 chiral amino acids. Other additives commonly used in this analysis are organic modifiers (acetonitrile.140 or even urea141 have been assayed. and the composition and flow rate of the sheath liquid to obtain the best sensitivity. in particular when capillary columns were available. although other additives such as dodecyltrimethylammonium bromide. Good separations have been reported for precapillary derivatized amino acids with dansyl-Cl. to enhance UV detection.118. although this detector may be used for more complex identifications as in d. Application in foods such as in the identification of nonprotein amino acids.). When sensitivity is the target.). The effect of these additives on the electro-osmotic mobility and electrophoretic mobility of the micelle has been studied. With few exceptions. SDS is indeed the most used additive to form micelles in this kind of analysis.144. isobutanol.147 17. the species with different charge can be simultaneously analyzed but with serious doubts in their adequate resolution. The identification of the 22 protein amino acids may not be a problem. etc.122. This report includes the optimization of important parameters like the choice of a volatile electrolyte (1 M formic acid) for the electrophoresis.127–131 derivatization is used to improve separation.Essential Amino Acids ◾ 299 improve the resolution of one kind.) and instrumentation (CE.125.145 when looking for more selective and sensitive detectors with a wide linear dynamic range (3 orders of magnitude) to cover new high-sensitivity applications (chiral analysis. This technique has also been termed micellar electrokinetic capillary chromatography (MECC or MEKC). and so forth. biomedical or pharmaceutical research. etc.146. and others. it is relatively easy to analyze low picomol levels of OPA derivatives in micellar solutions by using a conventional fluorometric detector. Mass spectrometer detectors were first connected to GC equipments.136. CZE shows poor ability for the separation of neutral compounds.123 introduced a modified version of CZE in which surfactant-formed micelles were included in the running buffer to provide a two-phase chromatographic system for separating neutral compounds together with charged ones in a CE system. or to allow fluorescence or electrochemical132 detection of amino acids. which can be resolved on the basis on their mass-to-charge ratios that are characteristic of each ion and allow its identification. Terabe et al. nonprotein amino acids.3. compatible with MS. microcolumn liquid chromatography. Nevertheless.131 and thus.137. reports in the literature of its applications are increasing rapidly. Under the conditions of electro-osmotic flow in CE. which constitutes an important limitation of this technique. the high cost of purchase and maintenance of mass spectrometers has inhibited their more widespread use in the food industry and/or food control. tetrahydrofurane.and l-isomer mixtures. 19 amino acids were analyzed by CE-ESI-MS in only 17 min with a minimal sample preparation and no matrix interference. o-tyrosine analysis.133–135 PITC. methanol.138 and OPA139 compared with the separation of OPA-amino acid derivatives by CZE with normal and micellar solutions.4 Mass Spectrometry MS is based on the conversion of components of a sample into rapidly moving gaseous ions.124 Basic theoretical considerations on this technique125 and its food applications126 are described elsewhere.113 o-tyrosine in chicken148 or pork149 tissues.139 which is usually enough for food analysis or an LIF (laser-induced fluorescence) detector. allowed a rapid development and the onset of these complementary techniques.141–143 The CE coupled to electrospray ionization (ESI) MS (CE-ESI-MS) allows direct amino acid analysis without derivatization. . it is likely to cause overlap with the others. etc. A good compatibility between both techniques.136 phenylthiohydantoin.119 have been reported.

4 Conclusions To obtain the total essential amino acids profile of a given seafood. 1991. the first decision is the choice of the hydrolysis method. 46. However. The convenience of purchasing commercially available kits must be evaluated. 1981. 1996. a complete resolution of the whole peaks is really difficult. small peptides. but tedious and time-consuming sample derivatization is required.. The requirements in resolution are not so exigent as those for physiological amino acids. amino acids in this case.. high mobile-phase flow rate vs. reduced problems related with matrix interferences or poor resolution between peaks. such as phenol. K.. Shimada. 479–483. and taking care of avoiding the presence of oxygen with vacuum and nitrogen purging. 35. References 1.E. Nevertheless..151. Hayashi. is enough for the majority of purposes. the convenience of purchasing commercially available prepackaged kits should be considered. S. Res. Particular hydrolysis problems related with certain amino acids are described in Section 17.L. . 185–236. Fisheries Sci. Sensory analysis of taste-active components in the extract of boiled snow crab meat. Yamaguchi. Food Sci. 2. Since many peaks corresponding to protein and nonprotein amino acids. and the technique is widespread although it is still expensive. atmospheric pressure chemical ionization (APCI). and so on. 3. The majority of published reports in which seafood amino acids are analyzed have used the cation exchange method. J. Swaisgood. In general. 821–824. RP-HPLC methods with precolumn OPA or PITC derivatization are very convenient methods to use. atmospheric pressure microwave-induced plasma ionization (AP-MIPI). vacuum). 17. nowadays these difficulties have been overcome with the development of new interfaces. The connection of HPLC and MS detector is much more problematic than with GLC because of the incompatibility between both techniques (solvents from chromatography. the analytical technique for a determined sample must be carefully chosen based on the literature. R.2. G. which means a minor sample manipulation. Post-mortem biochemical changes in the muscle of Japanese spiny lobster during storage. Yamanaka. the most important factors to take into account are the resolution power and selectivity. due to its high specificity. Three types of ionization modes. may appear in the chromatogram. H. Therefore. and ESI. must be ionized. Konosu. which may be consulted. One of the main requirements for samples to be analyzed by MS is that analytes. T. nucleosides. Protein digestibility: In vitro methods of assessment. offering the additional advantage of analyzing the amino acids without derivatization. because fewer peaks appear in the chromatogram. Adv.2. The best results were obtained by using AP-MIPI in conjunction with a dual oscillating capillary nebulizer. and many applications can be found in other matrices like cheese or meat. H. Catignani. In general.152 A very careful control of the derivatization reactions and chromatographic conditions are necessary for a consistent and reproducible analysis. 62. were compared by Kwon and Moini150 in relation to sensitivity. When amino acids from seafood proteins have to be analyzed. cation exchange and postcolumn derivatization or RP-HPLC precolumn derivatization techniques are the preferred methods. and. acid hydrolysis with HCl 6 N (110°C for 22 h or 145°C for 4 h) with an oxidation protective agent. once again. The highest resolution is obtained by GC with the capillary column technique. Food Nutr. and. Any separation strategy may give good results.300 ◾ Handbook of Seafood and Seafood Products Analysis MS has also been used as a spectroscopic detector after HPLC or CZE.

Ordóñez. 575–582. 17. 5–83 to 5–123. 2002. Culbertson.. J.M.. Int.C.H.. 81. 295–306. Food Chem. 72–77. 14. Toldrá.I. et al. P. Goncalves. Food Sci. urine and foodstuffs with electrochemical and fluorometric detections.. 18. et al. J. I. 1.M. Biotech. C. 1662–1666. Vol..Y. Vol. M. A. Bergström.. Food Sci. 15. Food Chem. Xiong.. 297. Ludiwg. 11.. 1998. 1279–1286. Moral. 6. 24. 10. Toldra. Nollet.C.C. Mendes. Chromatogr. tissues. T.R. et al. C. I.... Toldrá. 1993. 14. Grings. Measurement of free amino acids in human biological fluids by high-performance liquid chromatography. R.R. S. Eur. 22–25. Inc. Torre.. Ohshima. J. A 212. Food Agric. Food Res. 1984. Y. F. Otsuka Y. J.. A. Bull.. Y.. Nguyen. Sakaguchi. M. Biochem. 1. (Ed. 16. et al. Agric. Boca Raton.C. 7–11. Changes in free amino acids and creatine contents in yellowtail (Seriola quinqueradiata) muscle during ice storage. Sci.H. Amino acids.. Quantitative determination of free amino acids in cheese. Nishigaki K. Sanz.A. J.G. Food Chem. Y. Meat: Chemistry and biochemistry. 21. G. 8.I..H. 1475–1481. 47. Büetikofer. 2001.. 1992. 12. Chang. C.D.. J. J. Technol. Dairy Sci. 49–61. S. Fohlin. C. 55.L. Li-Chan. Ruiz-Capillas. 24–32. Application of high-performance liquid chromatography to the determination of free amino acids in physiological fluids. Tanaka S. 23. 1999. A. Ruiz-Capillas. 13. Effect of controlled and modified atmospheres on the production of biogenic amines and free amino acids during storage of hake. Antoine. Changes in free amino acids and biogenic amines during ripening of fresh and frozen sardine. 1994.R. Determination of amino acids after precolumn derivatisation using 9-fluorenylmethylchloroformate. E. M.. In: Handbook of Food Science.S.. Arnold. Food Microbiol. et al. Agric.I. Food Chem.. 337. Littell. Food Control 11. 1984. P. Forsch. Y. C. Analysis of free amino acids in green coffee beans.. F. C. Changes in composition of lipids. 9.M. A. 1989.. Biosci. Deproteinization techniques for HPLC amino acid analysis in fresh pork muscle and dry-cured ham. F.L.. A. Comparison of the amino acid composition and connective tissue protein contents of selected bovine skeletal muscles. M. Free amino acids in muscle of Norway lobster (Nephrops novergicus L. Food Biochem. K. U... 1792–1795. Part 2. C. Barcina. 302–307. Change in the contents of arginine. L. 1997.. Changes in free amino acids during chilled storage of hake (Merluccius merluccius L.C. Foldi. Z. Hagen. M. 1999. et al. 25. J. 476–481. Food Res. R. Unters. 1992. Aihara. Murata. . F. 521–528. T.. Augustin. Toldrá. Nollet. 2004. Aristoy. 863–866. 59. 56. 1991. L. Duncan. C. Shibata... 2006. and urea in the muscle of marine invertebrates stored in ice. H.. 28–1 to 28–18. CRC Press. Hui.. Precolumn phenylisothiocyanate derivatization and liquid chromatography of free amino acids in biological samples. L. R. Marcel Dekker.C. 2089–2095. (Eds. Godel. Lebensm. J.). Dairy Fed. 5100–5107. C. A 214. HPLC method for analysis of free amino acids in fish using o-phthaldialdehyde precolumn derivatization. Aristoy. J. 39. Ma.. 1982. Agric.... Y.. Kawai.and white-muscle fish as determined by o-phthaldialdehyde precolumn derivatization. free amino acids and organic acids in rice bran fermented sardine (Etrumeus teres) during processing and subsequent storage. 20. 199. Littell. C. Ibañez. Ruiz-Capillas. R. Koizumi.. J Chromatogr.Essential Amino Acids ◾ 301 4. T.. F. Agric. Q. Flores. Wei.. 66. Nunes. Toldrá.. Characterization of the casein hydrolysis of Idiazabal cheese manufactured from ovine milk. 85–91... Technology and Engineering. Technol.. 1999. et al. Zarkadas. F. 16. E. Manley. Effect of pre-ripening on microbial and chemical changes in dry fermented sausages. S. Biol. 91–100. 319–323. Kuhn. M.K. 2004. U.L. Onodera.. 2000. Eur. F. Rahman. Ali Qureschi. Graser. McMeekin. Moral. A. Izco. 7. 37. Moral. M. P. 22. Food Chem. Torre.Y. Nip. Chem. 47. Antoine.) in controlled atmospheres and their use as a quality control index. High-performance liquid chromatographic measurement of tryptophan in blood. In: Handbook of Food Analysis. 19. J.. et al. 23. J. Guerrero-Legarreta.) in controlled and modified atmospheres during chilled storage. 5.. J. 86. Ripening of Ossau-Iratzy cheese: Determination of free amino acids by RP-HPLC and of total free amino acids by the TNBS method. ornithine.M. 1991.). Ardö. New York. 2001. Wei. W.L. 297. Free amino acids in dark. E. FL. F.

Assoc. 36. Chem. Rosa. A 708. Chromatographia 36. origin and nutritional aspects. R. Itoh. Gilman. Evaluation of the relative efficacy of various techniques for deproteinizing plasma samples prior to high-performance liquid chromatographic analysis. 235–237. J.R. J.. E. 84. Biomed.H. T. 29. J.. Kim. Quantitative analysis of cystine. 811–821. d-amino acids in dairy products: Detection. 61. 40. R. Simultaneous determination of amino acids and biogenic amines by reversed-phase high-performance liquid chromatography of the dabsyl derivatives. Barbera. Leffler. H. M.. 1987. Chem.. C. 131–141. et al. Anal. On the determination of cystine and cysteic acid. Hydrolysate preparation for analysis of amino acids in sorghum grains: Effect of oxidative pre-treatment. Parapenaeus longirostris (Lucas). K... 41. Chem. Khalifa. 243. Meredith. plant and food samples by automated precolumn derivatization and high-performance liquid chromatography. Gehrke.H. Milchwissenschaft. I. 42. 357–360. Vandenberg. fresh cheese and acid curd cheese... 47. pharmaceutical. 2004. Anal. Wall. Zootech. Sample preparation for chromatography of amino acids: Acid hydrolysis of proteins. 43. 27. 1963. and Norway lobster. L. Griffith. 123–133. C. M. MacDonald. Cohen. 1172–1175. foods. J. Off. Hausch. A 715. Strydon. 70. S. et al. 1985. Kim. and heart) by liquid chromatography of precolumn phenylisothiocyanate. Khalifa. 349–356. Hirs. Appl. H. 11. Nutritional quality of red shrimp.L. C. Chromatogr. 1988. Off. J.. J.H. Anal. C. R. 48.E. 1984. Tryptophan analysis simultaneously with other amino acids in gas phase hydrochloric acid hydrolyzates using the Pico-Tag™ Work Station. 1995. Sugawara. et al. L. 70.. nuts and beans for non-protein and protein amino acids. lysine. Assoc. 45. J. 1132–1135. Milk. Farre. 1993. 826–829. J.J. brain. M. 1982. Performic acid oxidation. I. et al.. Sarwar.L. R.. 32. 35. Biochem. methionine. Amino acid determination in pure proteins. Oxidation and hydrolysis determination of sulfur amino acids in food and feed ingredients: Collaborative study. Lucas. 45. F.. Chem. 1985.. 37. Anal. Assignment of an unknown peak on the high performance liquid chromatogram of free amino acids isolated from fresh chicken egg albumen using picric acid as the deproteinizing agent. Methods Enzymol. 1990. Jansen.W.M.. 892–893. R. 46. . R. 1981.B. 1996. A.H. J. 43–46. H. 248–252.. 68. HPLC method for cyst(e)ine and methionine in infant formulas. Biochem. I. H.G.I. J. Amino acid concentrations and comparison of different hydrolysis procedures for American and foreign chestnuts. J. 226. 44. Absheer. and feeds using two different acid hydrolysis methods. Bockhardt. Keller. Food Agric. 36.. fermented milk. 1170. Assoc. 7. Rapid analysis of nutritionally important free amino acids in serum and organs (liver.W. Chem.. Neckermann. Ion-exchange separation of amino acids with postcolumn orthophthalaldehyde detection. 28.. J. Elkin. 271–284. Biol. 31.A. 36.. Chromatogr. Determination of amino acids in biological. Ashworth.H. Jap. S. Anal. Botting. 1988.W. 67–79. 1–16. 33. Agric.. 89–94. Chem. Bothmiedema. Gehrke. R. J. 1991.G. 1985. 123. Anal.M.. 171–174.W. 39. 1994. Sept. Rapid gas chromatographic screening of edible seeds. J. Off. LC-GC Int. McCarthy. Sci. Assoc. J. 470–475. 1117–1121. Schisla. 1995. 1988. Amino acid analysis utilizing phenylisothiocyanate derivatives. 1967. Int Lab. Off. S. J. Chromatogr. Food Chem. G. 1987a. and nine other amino acids by a single oxidation-4 h hydrolysis method. Molnár-Perl. 1990. J. M.. Oh. Krueger. Schuster.G. Anal.. J. 38. Off. C. Chromatogr. Food Sci. A. 73. Sci. M.. 1990. Aristeus antennatus (Risso). 174. Sotelo. P.A.. Moore. Engelhart W. J. 431. Anal. et al. A. Advantages and limitations of precolumn derivatization of amino acids with dabsyl chloride. Chem. 34. Molnár-Perl.B. R. 55.. J. R. pink shrimp. Chromatogr. M. J.. 553. et al. Analysis of foodstuff amino acids using vapour-phase hydrolysis. Assoc. Nephrops norvegicus (Linnaeus). Blanchard. I. Nunes. Assoc. J. R. 30.S. Woodward. B. Krause.H. 455–460. 68.302 ◾ Handbook of Seafood and Seafood Products Analysis 26. 40–45. 395–398. Brüeckner. Rexroad.R. 197–199. An evaluation of microwave heating for the vapor phase hydrolysis of proteins. J.L. Off. Alegría. Adachi.

3535–3540. 47. Jandasek. R. Biochem. Yu. J. 1981. Anal.G. C. Kaiser. Magnani. Farré. J.. A. Milerski. J. 1972. 2828–2834. García. J. Anal. C. 63. 48.. Acid hydrolysis of proteins for chromatographic analysis of amino acids.E. Determination of the tryptophan content of proteins by ion-exchange chromatography of alkaline hydrolysates. 1973. Morel.and dimethylaminoazobenzene thiohydantoinamino acid derivatives for amino acid analysis and microsequencing studies at the picomole level. Proc.E. Anal. Rattenbury.S. Gabert. Alegria. 2003. 65. (Ed. 43. 1112–1119. Wubben. J.M. J. Moore. Anal. 1989.M. 1987. Food Chem.P.. 301–306.. Int. Anal. 131. Hale.and sorghum-based diet and digesta samples.. Amino acid analysis: Determination of cysteine plus half-cystine in proteins after hydrochloric acid hydrolysis with a disulfide compound as additive. Zarkadas. 1999. Chichester. et al. Biochem. 493–496. Use of 7-chloro-4-nitrobenzo-2-oxa-1.B. pp. Determination of the number of cysteine residues in high molecular weight subunits of wheat glutenin. Zumwalt.E. Nutritional composition of soluble and insoluble fractions obtained by enzymatic hydrolysis of fish-raw materials. R.F. D. 367–372. Anal. 178. Beidler. R.W. S. 1997. Chang. Agric.. Standards and accuracy in amino acid analysis. Optimized determination of cystine/cysteine and acid-stable amino acids from a single hydrolysate of casein. Comparison of the contents of intramuscular amino acids in different lamb hybrids. Piccoli. 70. 1996. Bosch.C. Hugli. Assoc. pp.. 2375–2379. 2006. Bradbury. Chem.E. Assoc.. The use of 3-bromopropionic acid for the determination of protein thiol groups. Williams. Liquid chromatographic determination of amino acids after gas-phase hydrolysis and derivatization with (dimethylamino)azobenzenesulfonyl chloride. 51. Determination of amino acids and peptides. B. Cz. Off. Assoc. L. In: Amino Acid Analysis. Absheer.. Jue. J. V. 64. 2008. Biochem. 637–642.K. Ellis Horwood. 176–183. V. Biol.M.. 2000. Sci. 55. 77–83. Anal. et al. 1995.. 36.H. Animal Sci. Academic Press. J. A. Biochem.. 52. Chromatogr. U. 53. Chem. Albin. Quantitative gas-liquid chromatography of amino acids in enzymic hydrolysates of food proteins. 56. M. Agric. J.. A.G.H. 66.Essential Amino Acids ◾ 303 49. Agric. Electrophoresis 17.. Smith. 1986. M. J. V. A. 285–311. 177. J. Zarkadas. 1989. Stocchi. 60. Ambler.. R.D.. Assessment of the protein quality of beefstock bone isolates for use as an ingredient in meat and poultry products. 45. J. Liaset. M. 1985. J.. Alegría. 119–137. O. 1992.E. A..I.. Ihekoronye A. Int. Chem. (Ed. 70. Chem. McCrae. 67. Identification of cysteine-containing peptides in protein digests by high-performance liquid chromatography. J. 1987b. Food Agric. Determination of tryptophan content in infant formulas and medical nutritionals. Amino acid profile of milk-based infant formulas.. 59. J. R. Effect of hydrolysis time on the determination of amino acids in samples of soybean products with ion-exchange chromatography or precolumn derivatization with phenyl isothiocyanate. 42. S. Kracmar. . R. 58.. Fullmer. 57. Biochem... D. et al.Y. Reversed-phase high-performance liquid chromatography separation of dymethylaminoazobenzne sulfonyl. 58. Okogun. Nutr. Viadel. New York. 62. 51.L. Application of the 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) reagent to the RP-HPLC determination of amino acids in infant formula. 54. Jensen. 1004–1012. Espe.. 61. Tuan. D. Anal. 147–151. 247.P. F. 80–85. et al. Off. Faboya. et al.P. A. Chem. 70. M.I. Food Chem. 107–117.S. Z. Quantitation of cysteine residues alkylated with 3-bromopropylamine by amino acid analysis. B.H. Y. 75.E. In HPLC in Food Analysis. J. G.. Phillips.F. J. T.). Ashworth. 318–322. 1984..3-diazole for determining cysteine and cystine in cereal and legume seeds. S.. 216. R. Amino acids analysis for meat protein evaluation. 2000. 1684–1691. R. J. 50. Food Chem. 142. J. Barkholt. Food Sci.. Knecht. J. 1994. Bonicel. 336–339. 68. Food Chem. J. Biochem. 69. 2303–2307. Off.). Agric.A.R. 48. 43. Akinyele. G. Farre. 1982. Baxter. 61–66. J. B 831.

47. 41–48.. 85.. Bëtner. Ardo. et al. Anal. Physiol. formation of 1-alkyl(and aryl)thio-2-alkylisoindoles.M. 1988. Liq. 82. et al. 336. Sci.F. Lab. Nahrung 43. C. 1470–1474. R. Chromatogr. Heinrikson. J. Polo.A. An evaluation of the usefulness of pre-column Dns derivatization.S. Amino acid analysis: Reduction of ninhydrin by sodium borohydride. B.. et al. T. L. S. G. 84.. Pseudosciaena crocea (large yellow croaker). Chromatogr.. Anal. 12.. J. J. E. J. Assoc. 76. Jing. P. 1982. 1978. 83. 19. 53–59. L. J. Reversed-phase high-performance liquid chromatographic determination of biochemical changes in free amino acids during wheat flour mixing and bread baking. Swiader. J. 79. Y.L. Hughes. J.A.. Bidlingmeyer. D. Buetikofer. J. J. 72. Burton. 1988. Quantitative determination of free amino acids in cheese. Chromatogr. Off... Rosa. Braun. & Mol. 2886–2891.A. 1995.A. Amino acids composition difference and nutritive evaluation of the muscle of five species of marine fish. Standara. 1172–1175. wines and wine vinegar. Andrade. 1983. C. Lin. Chromatogr. 345–350. Meredith. 1999. Reaction of o-phtalaldehyde and thiols with primary amines. Ardo. Knecht. 65–74. Grunau. Chromophore labelling of amino acids with 4-dimethyl-aminoazobenzene-4′sulfonyl chloride. Chem. P.C..Y. D. Calado. 337. R. C. Tapuhi. Chang. 140. Simons. 1984. Chem. Int. 90. Biochem. Johnson..Y. Chem. J. Nutr. 1988. 70.. ..38. J. Nutr. et al. 1989. 241. S. Rapid analysis of amino acids using pre-column derivatization.. Chromatography of 99 amino acids and other ninhydrin-reactive compounds in the Pickering lithium gradient system. 43. 89. Dairy Res.A... 2733–2760. M. 7. M. C. Meth.J. 2006.K. 136. A. 6. S. Cabezudo. U. 73. J.A. Anal. Anal. Cohen S. Martín. Bull. Dejong. R... 88. 12. 28. 1634–1638. Homarus gammarus (Crustacea: Decapoda). J. 241–247.D.. Seriola dumerili (Dumeril’s amberjack) and Hapalogenys nitens (black grunt) from Xiamen Bay of China. W.. J. R. Off.. J. Vesela. Liq. Pollack. 77.. Amino acid analysis by reverse-phase high-performance liquid chromatography. S. Albers. 1992. 92. Y.36. Y. 115. Vanwieringen. Int.L. et al. 62. Collar. 594. Dairy Fed..C. 539–558.A. Comp.L.. J. 87. 1984. Dansylation of amino acids for high-performance liquid chromatography analysis. 81. 24–32.C. Lateolabrax japonicus (common sea perch). Anal. B-Biochem.. Amino acid analysis in the picomole range by precolumn derivatization and high-performance liquid chromatography. Y. Efficiency improvements on ninhydrin method for amino acid quantification.. Feng.. H. Comparative study of peptidolysis in some semi-hard round-eyed cheese varieties with different fat contents. Foldi. The FMOC-ADAM approach to amino acid analysis.. Chemical methods for evaluating proteolysis in cheese maturation (part 2). P. Chem. J. J. X. Peace.. Graser. precolumn derivatization with phenylisothiocyanate. 832–840. 71. Dansyl amino-acids behavior on a Radial Pak C-18 column. 86. Enzymol. Anal. Y. Derivatization of grape wine musts. et al. Botting. 410–413. I. Complete amino acid analysis in hydrolysates of foods and feces by liquid chromatography of precolumn phenylisothiocyanate derivatives.W. 1981. Limin. 241–249.E. Biochem. 74. Benedito de Barber. 572–577... 1975. Org.. 75. Sarwar. Cohen. Fully automated amino acid analysis using precolumn derivatization. Pollack. Fürst. Bidlingmeyer..304 ◾ Handbook of Seafood and Seafood Products Analysis 71. Lindner..M. 1987.. Assoc. S. Chromatogr. 2005. C. B. J. Gripon. 93–104. S. LC-GC Int. L. Amino acid analysis by high-performance liquid chromatography. D. Schmidt. Sun. Changes in amino acids and lipids during embryogenesis of European lobster. 2006. Liu. Chrom. 1984. M. 91.W.. HPLC analysis of free amino acids in biological material—An appraisal of four pre-column derivatization methods.M.G. 91. et al. 80. Biochem. Weng. Stehle. Food Comp. 1990. New rapid high sensitivity analysis of amino acids in food type samples. 165–171. P. 112–117. 78. Aq. R. H. G. 1999. 543–547. Tarvin T. 1986. 123–129.. Drdak.G. et al. 32–34. 118. Pagrosomus major (red seabream). Tarvin. In vivo utilization of cystine-containing synthetic short chain peptides after intravenous bolus injection in the rat. T. Prieto. 30. Chang.

A. K.. L. and proteins using high-performance liquid chromatography with photolytic electrochemical detection..C. I. Enzymol. first applications. M. Cereal Chem. G. A.A. Sherwood. van. 347–354. J.M. et al. et al.. Fremaut. Eur. M.. Gas chromatographic detection of d-amino acids in natural and thermally treated bee honeys and studies on the mechanism of their formation as result of the Maillard reaction. 223. 16.. B.. 68. 104. Jarret. 94.V. J.. J. Chromatogr A 828. C.C. H. Camanas R. Dou. 398–405. H. 378–382. 10. B. R. Measurement of plasma and urine amino acids by high-performance liquid chromatography with electrochemical detection using phenylisothiocyanate derivatization.S. Seitz. Jones.R. J.L. Chem. Gurd.. J. 2001. F. 424–438. Automated pre-column derivatization of amino acids with o-phthalaldehyde by a reagent sandwich technique. .. Wang. Chem. J. 96. Meth. 25.M. Titheradge.M. 1991. 97.R. J. Chromatogr.Essential Amino Acids ◾ 305 93. 44–49.. Tcherkas.J. 270. 5774–5778. 2001. D. 153.L. High-performance liquid chromatography analysis of amino acids at the picomole level. D..A. B. 109. 97–102. 1990. Simultaneous gas chromatographic analysis of non-protein and protein amino acids as N(O. Cerevisia Biotechnol.G.J. Godel. Paetzold. Formation and instability of ortho-phtalaldehyde derivatives of amino acids.D. Automated amino acid analysis using combined OPA and FMOC-Cl precolumn derivatization. Glutathione and cyst(e)ine profiles of vegetables using high performance liquid chromatography with dual electrochemical detection. Ogunsua. 100. 303–308. R. A. Amino acid determination in conophor nut by gas-liquid chromatography. 95.. Chatrathi. 106. C. 125–137. 1997. 1989..A.V. Food Agric. Anal. 1983. Schrijver. Pripis-Nicolau. Alvarez-Coque. Sanuda-Pena. 102. Kim.C. et al. Harvey-White. 103.. H... J. J. Richards. The separation of ortho-phthalaldehyde derivatives of amino acids by reversed-phase chromatography on octylsilica columns..B. Lookhart. Oh. 1994. Liu. 1988. 99. 189–198.K. 1972.H. 528.. M. 98. de Revel. 1998.. Chromatogr. Carboxymethylation. 383–395..C. 2000.. Tian. H. 475–480. 28. Anal. 101. Anal. LC-GC Int. Automated HPLC method for the measurement of free amino acids including cysteine in musts and wines. 1990. S. 1986. Hernandez. 5. Automated HPLC analysis of glutathion and thiol-containing compounds in grape juice and wine using pre-column derivatization with fluorescence detection. 731–738. Eenaeme. M. 293–303. Food Chem. Effect of differing thiols on the reversed-phase high-performance liquid chromatographic behaviour of o-phthaldialdehyde-thiol-amino acids. 62.. 62. 217–225. peptides.C. Winspear. G. Euerby. M. Mills. Verhoef. Ellis. Biochem. Chromatogr.. Kartsova. 81. M. Analysis of amino acids in human serum by isocratic reversed-phase high-performance liquid chromatography with electrochemical detection.. P. 26–37. 110. J. 178. Food Comp. 287–298. A 669. 112. Anal. D. 108. et al. Liu. Marchand. M. Chromatogr.D.S)-isobutyloxycarbonyl tert-butyldimethylsilyl derivatives. et al. M. Hydrolysate preparation and comparative amino acid determination by cation-exchange and gas-liquid chromatography in diet ingredients. et al.H. Biochem. de. I. Kim. Boulton. Anal.N.. Krull. Park. S.W.T. Determination of aromatic and sulfur-containing amino acids..P. 2599–2606. 1992. 1987. 2000. 1988. 105. A. 107. J. C. R. B. 1–7.R.. Noble. Cooksy K. J. Oaks. 454. L. Willis. Micromachined separation chips with a precolumn reactor and end-column electrochemical detector. Automated pre-column amino acid analyses by reversed-phase highperformance liquid chromatography. A 913. Brüeckner. 90–101. Stinson.. Sci. 408. 1985. et al. R. Determination of submicromolar concentrations of neurotransmitter amino acids by fluorescence detection using a modification of the 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate method for amino acid analysis.J. 72. Food Chem. 111. Y. Chromatogr. L. Food Res. Krasnova. Chromatogr.E.O. 2006. Technol.

347–355. Corradini. Determination of free intracellular amino acids in single mouse peritoneal macrophages after naphthalene-2. Clin. Kataoka. Free-zone electrophoresis in glass capillaries.. Jorgenson.. Int. 1985. 130. Brüeckner. Terabe. S. Chem. 2216–2218. sugar alcohols. . 266–272. Kitamori. G. K. Determination of free D-amino acids in Mammalia by gas chromatography-mass spectrometry. Simultaneous determination of sugars.. Determination of amino acid ratios in natural products by micellar ˆ electrokinetic chromatography. J. 134. Science 222(4621). Anal. 117. Sepaniak. 119.. et al. Analysis of B6 vitamers by micellar electrokinetic capillary chromatography with laser-excited fluorescence detection. Chromatogr. 131. C. D. Starke. Chim. J. Sass. 207. Chiral amino acid analysis of vinegars using gas chromatography-selected ion monitoring mass spectrometry.J. 123. 509–513. 1995. T. Anal. J. 129. Kamm.. Application of capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) in food analysis.W. 128. Burton. Determination of amino acids by gas-liquid chromatography and nitrogen selective detection.. 118. Lukacs. Bertacco. and quantification of amino acids in L-lysine fermentation potato juices by gas chromatography-mass spectrometry. E. Separation.W. H.. 1992. Buser. 1551–1553. Turner. 186. Sci. Chromatographia 39. 57. Sandra. 91–102.F. 1991. P. 7–10. 311–315. Matsubara.. Lercker. Anal. M. J. 1981. Molnar-Perl. 1986.. 1998. K. Unters. J. Chem. 56. 125. Cavazza. S. Baldwin. S. 126. A 847. Chem.3-dicarboxaldehyde derivatization by capillary zone electrophoresis with electrochemical detection. Odake.. Chromatogr. Electrophoresis 22.P.. 1998. Fresen. M.. Electrokinetic chromatography with micellar solution and open-tubular capillary. A 680. F. et al.W. Separation of 23 dansylated amino acids by micellar electrokiˆ netic chromatography at low temperature. Klampfl. M. K. For. 3324–3329. 1983. M. Heiger.. J. Vindevogel. 2000... B.. Sakiyama. 10. Amino acid analysis by capillary electrophoresis electrospray ionisation mass spectrometry. 24.. Anal. identification. 1988. Chromatogr. 380–384. J. I. Lebensm. 2000. Lauria. Otsuka. P. Brüeckner. Terabe. W. J.. 1999.. Analysis of organic acids and inorganic anions in different types of beer using capillary electrophoresis. A. et al. A. Ultrasensitive detection for capillary zone electrophoresis using laser-induced capillary vibration. acids and amino acids in apricots by gas chromatography-mass spectrometry. Lebensm.. 577–580.. G. 1991. Wu. Schieber. A 804. Erbersdobler. Unters. 2001... Lukacs. 2001. Capillary zone electrophoresis. Ichikawa. 1984.W. 1998. Agric. Skoc ir. 1994. 400–409. A. Milchwissenschaft. 66. H. 109. 47. Z. Cavazza. H. Zs. T. Jorgenson. 299–316. D. et al. 133. 122. Otsuka. Chem. J.. Food Chem. 121. O. Q. Skoc ir. Ye. 1236–1241. K. 2000. Food Sci.. K.. 347–351.306 ◾ Handbook of Seafood and Seafood Products Analysis 113. 48. 638–644. M. 348–350. Buchberger. W. Biochem. 72. 1999.. 135..N. 111–113. 834–841. J. Boschelle. Food Chem. Anal. 1994. 23. Agric. T. H.E. J. 63. 371.. Chem. Chem. Soga. 2669–2674. I. Distribution and contents of free o-phosphoamino acids in animal-tissues. T. 115. Makita. C. 116. J. 127. Determination of amino acids and peptides by capillary electrophoresis and electrochemical detection at a copper electrode. Anal. Erbe. J Chromatogr. Gas chromatographic determination of free amino acids in cheese. Klampfl. 114. Jin.. R. C. E. Terabe. N. 987–990.D. HRC. Determination of underivatized amino acids in beverage samples by capillary electrophoresis. Maskarinec. Katona.. 576–582. 27. 124. 2797–2803. A. W. Prosek. Rapid analysis of essential and branched-chain amino acids in nutraceutical products by micellar electrokinetic capillary chromatography.D. 1994. N. For. Separation of 24 dansylamino acids by capillary electrophoresis with a non-ionic surfactant. J.P. Ando. 120. Z. Chromatographia 41. Kleinpeter. E. T.. Electrokinetic separations with micellar solutions and open-tubular capillaries. Corradini.. 47. C. Weng. 132.

In: Handbook of Dairy Foods Analysis. peptides and amino acids. K.. Anal. 467–480. (Eds. T. S. Boca Raton. Ando. Electrophoresis 16. T. et al. L. 1995. T. low molecular peptide and amino acid impurities of biotechnologically produced amino acids by means of CE. Am.3benzoxadiazoles. 149. et al. J..C. Meth Enzymol. Aristoy. 1990. 213–218. D.L. Chem. Terabe. Ulrike. Otsuka. Novotny. Essential amino acids.. 1996.. Kwon. 9–32. M. Toldra. 11581–11585.. 138. 146.Y. Chen. et al. New LASER fluorometric detection for ortho-tyrosine in gamma-irradiated phenylalanine solution and pork. Chromatogr. F. Terabe. P. Nollet. H.Essential Amino Acids ◾ 307 136. 124– 132.. J. Terabe. Separation and detection of amino acids and their enantiomers by capillary electrophoresis: A review. Moini. Food Irrad. Sci. Analysis of underivatized amino acid mixtures using high performance liquid chromatography/dual oscillating nebulizer atmospheric pressure microwave induced plasma ionisation-mass spectrometry. N. Part A. L..V.. Karam. ´ . FL. (Eds. J. Chromatogr. Soc. J. N.. Spectrom.. Terabe. Simeon.G. 1997. 468. N.). 144. Electrokinetic chromatography with micellar solutions separation of phenylthiohydantoin-amino acids. Aristoy. et al. Cassiano. Matsubara. 265..A. J. 1457–1462.. 270. 385–397. 475–486. L. Simic. A quantitative study on the effect of organic modifiers in micellar electrokinetic chromatography. M. Liu.. K. FL.. 1990.. Toldra. In: Handbook of Muscle Foods Analysis. 1993. M. 142. 2100–2103. Rossetti. 141. Novotny. ´ 152..1. Nagasawa.R. K. R(−)and S(+)-a-(3-isothiocyanatopyrrolidin-1-yl)-7-(N. 148. S. 1995. 139. K. 137. Issaq..V. ´ Toldra. 2001. Chan. L. Cobb. 3–8. 1995. S. Vigne Vin. M. Separation of pre-column ortho-phthalaldehyde-derivatized amino acids by capillary zone electrophoresis with normal and micellar solutions in the presence of organic modifiers.N-dimethylaminosulfonyl)-2. Int. 145. 117–122.L. 12. Miyahara. Oe. 143. 147. Effect of organic modifier concentrations in micellar electrokinetic chromatography. Castagnola. Izumi.. Determination of d-amino acids labelled with fluorescent chiral reagents. Optimization of phenylthiohydantoinamino acid separation by micellar electrokinetic capillary chromatography. Biomed.A..C. CRC Press. Nakagawa. Biol.R. H. T. Cobb. 1999. Pharm. 28. Otsuka. Miyahara. S. K. M.. D. Electrophoresis 11. Novatchev. J.. amino acids and biogenic amines. N. 2009. Quantitation of organic and inorganic acids.M. Electrophoresis 16. 735–749. Chromatogr. 150. J. 151.V. Arellano. F. 319–341. Jin. Boca Raton. J. in biological and food samples by liquid chromatography. S. Several applications of capillary electrophoresis for wines analysis. 269. J. Nollet. K. 1989. F. Puig. M. D. F. 1999. M.. N. ´ Toldra. 55–65. Biochem. 31. Electrophoresis 16.M. inorganic cations. 332. Anal. M. et al.J. Liu. 34. Recent advances in capillary electrophoresis of proteins. Electrophoresis 11. 982–984. Essential amino acids. T.. CRC Press.C. 327–333. Effect of methanol and urea on optical resolution of phenylthiohidantoinDL-amino acids by micellar electrokinetic chromatography with sodium N-dodecanoyl-l-valinate. Chen. Evaluation of amino sugar. Jap.. 1990. J. 2002. Terabe.. Formation of o-tyrosine by radiation and organic solvents in chicken tissue. 140. Micellar electrokinetic chromatography. 638. 1985.). Mass... 219–226. 2009.


.1..........................................1...............................................3...................................................... 315 18................1.....310 18.......................................................................3....3.................................316 18...................3..............................310 18..........2 Antioxidants...........................313 18....3......318 18...2.........317 18...................311 18..............................1.........................2 Determination of Antioxidants and Antioxidant Capacity in Biological and Food Systems ........................................2...............1 Oxidative Stress and Its Implications ...........................1.............................................2.............................3 Occurrence of Vitamin E ................313 18..................1.........3...............................3..316 18.....................3........4..1...3..............4...............3.............................1 Antioxidant and Other Functions of Carotenoids ...........2 Ascorbic Acid Analysis ..............314 18..3....................310 18...........3 Vitamin E .................................1 Vitamin E as an Antioxidant.......................................1 Introduction ..........................................................1 Oxidation and Its Implications................................3......3..............................................................................................................................................317 18..............Chapter 18 Antioxidants Nick Kalogeropoulos and Antonia Chiou Contents 18..........2 Lipid Peroxidation...................................... 315 18................................................4...........317 18...2 Vitamin E Determination ...........................3....................................3 Antioxidants in Seafood and Seafood Products ..1 Ascorbic Acid Functions ....318 309 ............311 18.....................313 18.....................314 18............................3 Marine Lipid Oxidation ......................1 Antioxidant Enzymes .................................3.3..........3 Occurrence of Carotenoids ...................4 Carotenoids ..1............314 18..2 Carotenoid Determination .........3 Occurrence of Ascorbic Acid in Marine Organisms ...........311 18..................2 Ascorbic Acid ..............................

............ 18...............6 have been reported to cause oxidative stress to fish or bivalves.........2 Determination of Ubiquinone ...3 pollution.310 ◾ Handbook of Seafood and Seafood Products Analysis 18..... generation of free radicals occurs.......4........................321 References ..3 Occurrence of Ubiquinone...... 320 18....................... thousands of free radical reactions may occur within a few seconds.............. nitric oxide (NO (OH•) radicals.................. This is also followed in the marine environment............. they may oxidize several cell components..1...... polyphenols................4.. and hydroxyl hydrogen peroxide (H2O2). that is.......321 18.............................5 Ubiquinone ..319 18... more than 2 billion years ago.4 Added Antioxidants ............319 18.. peroxyl (ROO •).......... Aging...1 Synthetic Antioxidants ......................319 18.. lipids.....1.............................................. Humans and most animals cannot synthesize the majority of these antioxidants and depend on the dietary intake from plant consumption.............6 Other Endogenous Antioxidants ..................... When the electron flow becomes uncoupled (transfer of unpaired single electrons)...... and proteins may be damaged by reactive oxidants.......1 Oxidation and Its Implications Oxidation is the transfer of electrons from one atom to another and represents an essential part of aerobic life...... another free radical is generated in the process........3... Although the initial attack causes the neutralization of the free radical..... Under conditions of oxidative stress... •). since oxygen is the final electron acceptor in the electron flow system that produces energy................. 320 18......... ROS production in organisms is related to both the basal metabolism and the influence of environmental factors2.... resulting in a chain reaction.... and the so-called reactive nitrogen species (RNS)....5.......3....................1 Function of Ubiquinone ......... and carotenoids.5......... alkoxyl (RO •)................... if ROS are not immediately intercepted by antioxidants........ are rich in antioxidants such as vitamins C and E............2 Natural Antioxidants .... Common free radicals in biological systems are the so• called reactive oxygen species (ROS)................ 320 18...........................................3..... Until subsequent free radicals are deactivated.................3................... such as peroxynitrite (ONOO−)...319 18........................... electrically charged compounds that seek out and capture electrons from other compounds in order to neutralize themselves......... resulting in an increased antioxidant activity and antioxidants ..... which include among others superoxide anion radical (O2−)............................ nucleic acids.1 Indeed cyanobacteria and the laterevolved green plants....4 and environmental stress5..................... 18......1.1 Oxidative Stress and Its Implications Oxidative stress occurs when the prooxidant–antioxidant balance becomes too favorable to the prooxidants..... as a defense against oxygen toxicity.......................5...........................................3.....1 Introduction Antioxidants evolved together with the emergence of photosynthesis by cyanobacteria.......... where antioxidants are mainly produced by photosynthetic organisms and are consequently transported through the trophic web..... being exposed to the oxygen they produce......

and angiotoxic effects.Antioxidants ◾ 311 loss or oxidation product development.9 18. and they stall the propagation phase. significantly delays or prevents oxidation of that substrate. in which polyunsaturated fatty acids on lipid molecules are attacked and oxidized. antioxidants often act via more than one mechanism that combines different types of antioxidant activity. mainly of plant origin. 1O2. heat.1. carcinogenic. including enzymatic systems and nonenzymatic antioxidants. for which the human sensory apparatus has a low threshold. protect the cellular components from oxidative damage.). and termination. and (3) enzymatic oxidation. and metal ions.1.8 Studies on the pathological significance of dietary lipid oxidation products have indicated that some lipid oxidation products have cytotoxic. Lipid oxidation is a complex procedure induced by oxygen in the presence of initiators such as light.7 18. There is increasing evidence that oxidative stress is implicated in the pathogenesis of many inflammatory and degenerative diseases and conditions. antioxidants are . antioxidants are molecules that protect macromolecules from being oxidized.12 In biological systems various biochemical defense mechanisms. Chain-breaking antioxidants intercept •) or participate in halting radical chain propagation. marine lipids are relatively more susceptible to oxidation. antioxidants supplied by foods. In the first two cases a combination of reactions involving 3O2 and 1O2 occurs. that is.11 and unhealthy compounds that reduce their shelf life and nutritive value. In general.13 Antioxidants counteract oxidation in two different ways: They protect lipids from oxidation initiators. The major components of the antioxidant defense system together with their proposed mechanisms of action are presented in Table 18. atherogenic. preventive antioxidants. handling.1.2 Antioxidants Antioxidants are defined as any substance that when present at low concentrations compared with those of an oxidizable substrate. Lipid oxidation of omega-3 polyunsaturated fatty acids (PUFA)-rich food products results in the development of particularly unpleasant off flavors. propagation. This can be especially damaging to lipid-rich cell membranes. chain-breaking antioxidants.1. Three reaction pathways have been proposed: (1) nonenzymatic chain autoxidation. initiation. Autoxidation occurs through a three-phase process. mutagenic. Nonradical photooxidation seems to be a minor reaction compared with the 3O2-induced radical chain autoxidation.9 Preventive antioxidants hinder ROS formation or scavenge spe• cies responsible for oxidation initiation (O2−. Nevradical oxidation propagators (LOO ertheless.3. Moreover. free radicals.2 Lipid Peroxidation A very damaging effect of oxidant reactive intermediates is lipid peroxidation. are essential for counteracting oxidative stress. and storage. being the major cause of the development of off-flavor compounds and rancidity as well as a number of other reactions that reduce the shelf life and nutritive value of food products. etc. (2) nonenzymatic and nonradical photooxidation. 18. because of their high degree of unsaturation.3 Marine Lipid Oxidation Compared with other food lipids. The health implications of tissue lipid oxidation are numerous and well documented. For food systems.1.10 Lipids deteriorate in seafood products during processing.1.

lipoic acid. Rev. 275. citric acid Polyphenols (exogenous) Chain-breaking antioxidant.1 Major Components of Antioxidant Defense System and Proposed Mechanism of Action Antioxidant Species Mechanism of Action Enzymes Catalase Glutathione peroxidase Superoxide dismutase (SOD) Thioredoxin ROS detoxification (reduction of H2O2 to water) ROS detoxification (reduction of H2O2 to water) ROS detoxification (removal of superoxide radical) ROS detoxification (reduction of peroxides) Metal Ion Sequestration Transferrin Albumin Ceruloplasmin Ferritin Lactalbumin Phytochelatins Transient metal chelators (chelates Fe) Transient metal chelators (chelates Fe. 1O2 quencher Compounds with proven antioxidant activity in vitro. ROS detoxification (hydroperoxides). anserine. carnosine.312 ◾ Handbook of Seafood and Seafood Products Analysis Table 18. sex hormones melatonin. Cu) Low Molecular Mass Ascorbic acid (exogenous) Carotenoids (exogenous) Coenzyme Q (endogenous) Urate (endogenous) Phospholipids (endogenous) Polyphosphates. J. Cu) Transient metal chelators (chelates Cu) Transient metal chelators (chelates Fe) Transient metal chelators (chelates Fe) Transient metal chelators (chelates Cd. Food Sci. 2004. regenerate oxidized vitamin E Chain-breaking antioxidant.. 44. et al.. Crit. synergistic to vitamin E Transient metal chelators Transient metal chelators (the ones with o-diphenolic structure). scavenges peroxyradicals. EDTA. chain-breaking antioxidants Synergistic to vitamin E Scavenges NO2 Transient metal chelators. . but uncertain in vivo Vitamin E (exogenous) Bilirubin. regenerates oxidized vitamin E 1 O2 quenchers.K. Zn. 2-oxo acids. chain-breaking antioxidants. melanins (endogenous) Source: Adapted from Willcox.

spleen.1 Cu/Zn SOD were purified from marine fish tissues. and heart.21 In several species of teleosts.9 and 9. for quality control of fish oil and fish oil-containing foods.1 Antioxidant Enzymes Catalases are metal-containing enzymes. whereas Fe SOD were purified from red algae..17 and Wood et al. widely distributed in aerobic cells that help in preventing the accumulation of H2O2 within cells.3. coenzyme Q. Zn.3. such as ascorbic acid. one represented by enzymes and the second represented by low molecular mass compounds. bilirubin. the correlation of instrumental and sensory methods with multivariate data analysis should be followed.9 The determination of specific waterand fat-soluble antioxidants is discussed in Sections 18. and algae. with Mn.22 whereas in nine Atlantic fish species total SOD values ranged between 157 and 796 U/g fish and Mn-SOD ranged between 45 and 751 U/g. cephalopods.7 U/mg of protein.3 Antioxidants in Seafood and Seafood Products In living organisms. Cu. thin-layer. glutathione (GSH).3. kidneys. and uric acid. and high-performance liquid chromatography (HPLC) alone or combined with mass spectroscopy. Cu/Zn-SOD activities ranged between 1. carotenoids.19 concluded that. These antioxidants act in a concerted way to protect sensitive molecules such as the unsaturated fatty acids from oxidation. the latter including paper. polarographic. thiols. 18. 18.Antioxidants ◾ 313 effective at very low concentration levels. or Fe in their active site.16 and Laguerre et al.3. The available methods for monitoring the antioxidant capacity in biological and food systems in vitro or in vivo were recently reviewed by MacDonald-Wicks et al. Catalase activities ranged between 386 and 1523 mmol/ min/g tissue in several Atlantic fish and was higher in liver. and in red muscle compared with that in white. voltammetric. and crustaceans from the Mediterranean sea.15 Griffiths et al. oxidative damage to macromolecules is controlled by two types of antioxidant systems.18 In reviewing methods and tests for the assessment of lipid oxidation. and chromatographic methods. Kolanowski et al. The available methods have been reviewed by Rajalakshmi and Narasimhan.20 Superoxide dismutases (SOD): SODs are metalloproteins. At higher levels most of them behave as prooxidants possibly due to their involvement in the initiation reactions. tocopherols.2 Determination of Antioxidants and