HANDBOOK OF

Seafood and Seafood Products Analysis

HANDBOOK OF

Seafood and Seafood Products Analysis
Edited by

LEO M.L. NOLLET FIDEL TOLDRÁ

Boca Raton London New York

CRC Press is an imprint of the Taylor & Francis Group, an informa business

CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2010 by Taylor and Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S. Government works Printed in the United States of America on acid-free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number: 978-1-4200-4633-5 (Hardback) This book contains information obtained from authentic and highly regarded sources. Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume responsibility for the 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

Contents
Preface ..................................................................................................................................ix Editors ..................................................................................................................................xi Contributors ...................................................................................................................... xiii

PART I: CHEMISTRY AND BIOCHEMISTRY 1 Introduction—Importance of Analysis in Seafood and Seafood Products,
Variability and Basic Concepts.....................................................................................3
JÖRG OEHLENSCHLÄGER

2 Peptides and Proteins .................................................................................................11
TURID RUSTAD

3 Proteomics ..................................................................................................................21
HÓLMFRÍÐUR SVEINSDÓTTIR, ÁGÚSTA GUÐMUNDSDÓTTIR, AND ODDUR VILHELMSSON

4 Seafood Genomics ......................................................................................................43
ASTRID BÖHNE, DELPHINE GALIANA-ARNOUX, CHRISTINA SCHULTHEIS, FRÉDÉRIC BRUNET, AND JEAN-NICOLAS VOLFF

5 Nucleotides and Nucleosides ......................................................................................57
M. CONCEPCIÓN ARISTOY, LETICIA MORA, ALEIDA S. HERNÁNDEZ-CÁZARES, AND FIDEL TOLDRÁ

6 Lipid Compounds.......................................................................................................69
SANTIAGO P. AUBOURG

7 Lipid Oxidation ..........................................................................................................87
TURID RUSTAD

8 Volatile Aroma Compounds in Fish ...........................................................................97
GUÐRÚN ÓLAFSDÓTTIR AND RÓSA JÓNSDÓTTIR

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PART II: PROCESSING CONTROL 9 Basic Composition: Rapid Methodologies ...............................................................121
HEIDI NILSEN, KARSTEN HEIA, AND MARGRETHE ESAIASSEN

10 Microstructure .........................................................................................................139
ISABEL HERNANDO, EMPAR LLORCA, ANA PUIG, AND MARÍA-ANGELES LLUCH

11 Chemical Sensors .....................................................................................................153
CORRADO DI NATALE

12 Physical Sensors and Techniques .............................................................................169
RUTH DE LOS REYES CÁNOVAS, PEDRO JOSÉ FITO SUÑER, ANA ANDRÉS GRAU, AND PEDRO FITO-MAUPOEY

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

14 Analytical Methods to Differentiate Farmed from Wild Seafood ............................215
ICIAR MARTÍNEZ, INGER BEATE STANDAL, MARIT AURSAND, YUMIKO YAMASHITA, AND MICHIAKI YAMASHITA

15 Smoke Flavoring Technology in Seafood .................................................................233
VINCENT VARLET, THIERRY SEROT, AND CAROLE PROST

PART III: NUTRITIONAL QUALITY 16 Composition and Calories ........................................................................................257
EVA FALCH, INGRID OVERREIN, CHRISTEL SOLBERG, AND RASA SLIZYTE

17 Essential Amino Acids ..............................................................................................287
M. CONCEPCIÓN ARISTOY AND FIDEL TOLDRÁ

18 Antioxidants .............................................................................................................309
NICK KALOGEROPOULOS AND ANTONIA CHIOU

19 Vitamins ...................................................................................................................327
YOUNG-NAM KIM

20 Minerals and Trace Elements ...................................................................................351
JÖRG OEHLENSCHLÄGER

21 Analysis of n-3 and n-6 Fatty Acids ..........................................................................377
VITTORIO M. MORETTI AND FABIO CAPRINO

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

23 Instrumental Texture ...............................................................................................425
ISABEL SÁNCHEZ-ALONSO, MARTA BARROSO, AND MERCEDES CARECHE

Contents ◾

vii

24 Aroma .......................................................................................................................439
JOHN STEPHEN ELMORE

25 Quality Index Methods ............................................................................................463
GRETHE HYLDIG, EMILÍA MARTINSDÓTTIR, KOLBRÚN SVEINSDÓTTIR, RIAN SCHELVIS, AND ALLAN BREMNER

26 Sensory Descriptors ..................................................................................................481
GRETHE HYLDIG

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

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

29 Detection of Fish Spoilage........................................................................................537
GEORGE-JOHN E. NYCHAS AND E.H. DROSINOS

30 Detection of the Principal Foodborne Pathogens in Seafoods and
Seafood-Related Environments ................................................................................557
DAVID RODRÍGUEZ-LÁZARO AND MARTA HERNANDEZ

31 Parasites....................................................................................................................579
JUAN ANTONIO BALBUENA AND JUAN ANTONIO RAGA

32 Techniques of Diagnosis of Fish and Shellfish Virus and Viral Diseases .................603
CARLOS PEREIRA DOPAZO AND ISABEL BANDÍN

33 Marine Toxins ..........................................................................................................649
CARA EMPEY CAMPORA AND YOSHITSUGI HOKAMA

34 Detection of Adulterations: Addition of Foreign Proteins .......................................675
VÉRONIQUE VERREZ-BAGNIS

35 Detection of Adulterations: Identification of Seafood Species .................................687
ANTONIO PUYET AND JOSÉ M. BAUTISTA

36 Veterinary Drugs ......................................................................................................713
ANTON KAUFMANN

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

38 Spectrochemical Methods for the Determination of Metals in
Seafood .....................................................................................................................751
JOSEPH SNEDDON AND CHAD A. THIBODEAUX

39 Food Irradiation and Its Detection ..........................................................................773
YIU CHUNG WONG, DELLA WAI MEI SIN, AND WAI YIN YAO

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40 Analysis of Dioxins in Seafood and Seafood Products .............................................797
LUISA RAMOS BORDAJANDI, BELÉN GÓMARA, AND MARÍA JOSÉ GONZÁLEZ

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

42 Biogenic Amines in Seafood Products......................................................................833
CLAUDIA RUIZ-CAPILLAS AND FRANCISCO JIMÉNEZ-COLMENERO

43 Residues of Food Contact Materials .........................................................................851
EMMA L. BRADLEY AND LAURENCE CASTLE

44 Detection of GM Ingredients in Fish Feed ...............................................................871
KATHY MESSENS, NICOLAS GRYSON, KRIS AUDENAERT, AND MIA EECKHOUT

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

Preface
There are several seafood and seafood products, which represent some of the most important foods in almost all types of societies, including those in developed and developing countries. The intensive production of fish and shellfish has raised some concerns related to the 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.
ix

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Preface

We would like to thank all the contributors for their excellent work. Their hard work and dedication have resulted in this comprehensive and prized handbook. We wish them all the very best in their academic and/or scientific careers. Leo M.L. Nollet Fidel Toldrá

Editors
Dr. Leo M.L. Nollet is the editor and associate editor of several books. He edited for Marcel Dekker, New York—now CRC Press of Taylor & Francis Group—the first and second editions of Food Analysis by HPLC and the Handbook of Food Analysis. The Handbook of Food Analysis is a three-volume book. He also edited the third edition of the Handbook of Water Analysis, Chromatographic Analysis of the Environment (CRC Press) and the second edition of the Handbook of Water Analysis (CRC Press) in 2007. He coedited two books with F. Toldrá that were published in 2006: Advanced Technologies for Meat Processing (CRC Press) and Advances in Food Diagnostics (Blackwell Publishing). He also coedited Radionuclide Concentrations in Foods and the Environment with M. Pöschl in 2006 (CRC Press). Nollet has coedited several books with Y.H. Hui and other colleagues: the Handbook of Food Product Manufacturing (Wiley, 2007); the Handbook of Food Science, Technology and Engineering (CRC Press, 2005); and Food Biochemistry and Food Processing (Blackwell Publishing, 2005). Finally, he also edited the Handbook of Meat, Poultry and Seafood Quality (Blackwell Publishing, 2007). He has worked on the following five books on analysis methodologies with F. Toldrá for foods of animal origin, all to be published by CRC Press: Handbook of Muscle Foods Analysis Handbook of Processed Meats and Poultry Analysis Handbook of Seafood and Seafood Products Analysis Handbook of Dairy Foods Analysis Handbook of Analysis of Edible Animal By-Products Handbook of Analysis of Active Compounds in Functional Foods He has worked with Professor H. Rathore on the Handbook of Pesticides: Methods of Pesticides Residues Analysis, which was published by CRC Press in 2009. Dr. Fidel Toldrá is a research professor in the Department of Food Science at the Instituto de Agroquímica y Tecnología de Alimentos (CSIC) and serves as the European editor of Trends in Food Science & Technology, the editor-in-chief of Current Nutrition & Food Science, and as a member of the Flavorings and Enzymes Panel at the European Food Safety Authority. In recent years, he has served as an editor or associate editor of several books. He was the editor of Research Advances in the Quality of Meat and Meat Products (Research Signpost, 2002) and the associate editor of the Handbook of Food and Beverage Fermentation Technology and the Handbook of Food Science,
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xii

Editors

Technology and Engineering published in 2004 and 2006, respectively, by CRC Press. He coedited two books with L. Nollet that were published in 2006: Advanced Technologies for Meat Processing (CRC Press) and Advances in Food Diagnostics (Blackwell Publishing). Both he and Nollet are also associate editors of the Handbook of Food Product Manufacturing published by John Wiley & Sons in 2007. Professor Toldrá has edited Safety of Meat and Processed Meat (Springer, 2009) and has also authored Dry-Cured Meat Products (Food & Nutrition Press—now Wiley-Blackwell, 2002). He has worked on the following five books on analysis methodologies with L. Nollet for foods of animal origin, all to be published by CRC Press: Handbook of Muscle Foods Analysis Handbook of Processed Meats and Poultry Analysis Handbook of Seafood and Seafood Products Analysis Handbook of Dairy Foods Analysis Handbook of Analysis of Edible Animal By-Products Handbook of Analysis of Active Compounds in Functional Foods Toldrá was awarded the 2002 International Prize for Meat Science and Technology by the International Meat Secretariat. He was elected as a fellow of the International Academy of Food Science & Technology in 2008 and as a fellow of the Institute of Food Technologists in 2009.

Contributors
M. Concepción Aristoy 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

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Contributors

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

Contributors

xv

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

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Contributors

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

Contributors

xvii

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

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

CHEMISTRY AND BIOCHEMISTRY I .

.

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

and the captured fish. Vietnam (1.6 million tons). Japanese anchovy (1.2 million tons). Russia (3. Indonesia (4.0 million tons). 2000: 40 million tons. including plants) [1]. The major aquaculture (excluding plants) producers (>1 million tons) in 2005 were China (32. Most fish was caught in the Pacific Ocean (Northeast and Southeast) followed by Northeast Atlantic Ocean.4% by quantity. 40% is consumed as wet fish without any further technological processing or preservation.3% and 14.4 ◾ Handbook of Seafood and Seafood Products Analysis number of species and variability.4%.5 million tons).6 million tons). The most important primary product producing countries of marine and inland (freshwater) fisheries in 2005 were China (17. India (3. burden of pollutants. and Norway (2. and sensory properties. Another difference compared with land-living animals is the fact that the quality (size. 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. the United States (4.3 million tons). and Yellowfin tuna (1.4 million tons).1 million tons).000 species. They deteriorate at ambient temperature in a few days. 1970: 4 million tons. state of maturity. Mollusks (bivalves and cephalopods) are the third most important group both by quantity and by value at 22. appearance. Peru (9.1 million tons). Alaska Pollock (2. Chile (4. and Thailand (1. The top 10 species being caught in huge amounts in 2005 were Anchoveta (10. respectively. infestation with parasites.3 million tons). Further. Further. The world’s aquaculture provided 52 million tons (36%).4%. 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).4 million tons). Thailand (2.2% but second by value at 20. 8% is transformed into cured products. fish is the top group in aquaculture at 47. India (2. fish and other seafood are highly perishable products when stored without chilling. Largehead hairtail (1.9 million tons). Aquatic plants that are popular in Southeast Asia are second in quantity at 23.1 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.4 million tons. aquaculture is dramatically growing (1960: 2 million tons. About 75% of the world’s total seafood supply is used for human consumption.) of aquatic animals when captured by fishing techniques is—with few exceptions—completely unknown. 1980: 7 million tons. about 20% is converted into deep frozen products. Indonesia (1. whereas crustaceans are fourth by quantity at 6.3 million tons). However.7 million tons). Atlantic herring (2.000–35. nutritional status. 91 million tons (64%) of the total supply. mostly Pangasius species). . Although land-based animals are today tailor made according to industry’s and consumer’s wishes in weight. whereof the major part are cyprinids like carp). The total world seafood supply for 2007 amounted to 143 million tons.2 million tons).1 million tons). and another 8% into canned products.1 million tons). 25% is converted into fishmeal and other nonfood products. Blue whiting (2.3 million tons). Skipjack tuna (2. Japan (4. 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. etc.8 million tons). Chilean jack mackerel (1. By major groupings. The fish group alone is represented by 25. 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 million tons). 1990: 16 million tons.2%.4 million tons).4 million tons).4 million tons. The stagnation of captured fish is mainly due to fully exploited or partially overfished stocks. body composition. Chub mackerel (2.

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

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

and microbiological methods. . is necessary. workshops. and so forth. it is recommended to store all samples (also solutions) in deep frozen conditions (<−18°C. 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. the whole body may be sampled and analyzed (mussels. and total volatile basic nitrogen (TVB-N). other species or mud. 1. analysis of trimethyl amine.14] that form a very rich source of information about seafood analysis. and only a few samples are taken from big individuals. 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. The first concerted action in this area was “Evaluation of fish freshness” from 1995 to 1997. a careful selection of individuals that have not been mechanically damaged by the catching technique. The objective methods are chemical/biochemical methods. sand. determination of thiobarbituric acid and formaldehyde. and in big fish (tuna. In small specimens that are consumed totally. When sampling is done onboard a vessel. or tail end of fillet). and practical work-ins and allowed on-site measurements. dimethyl amine. always the whole edible part (fillet. These projects brought the scientists together in conferences. After sampling is completed successfully. 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. When sampling for later microbiological analyses. and comparative analyses with different instruments. smaller when medium-sized animals are the target. ammonia.Introduction ◾ 7 The number of individuals should be big when a small specimen has to be analyzed. 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). snails). precaution must be taken not to contaminate the sample by instruments used during manipulation (scissors. physical methods. preferably at −30°C) until analysis. 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. knives) or by protective clothes or gloves. which is based on ATP breakdown products. The analytical methods used for seafood analyses can be divided into objective methods and sensory methods. it has to be made under strict hygienic conditions to avoid any microbial contamination. in medium-sized specimen. tail muscle) must be taken due to intrinsic variations in fillet parts and after homogenization subsamples can be taken. calibrations. trimethyl amine oxide.6]. In addition. and the second concerted action was “Fish quality labeling and monitoring” (FQLM) from 1998 to 2000.6 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. head end. Another method that was developed recently is the two-dimensional gel electrophoresis (2DE). The main results of these projects have been published in books [2–4. and analysis of biogenic amines as histamine or cadaverine. Methods that are still in use are among others k-value. While sampling is done. shark). sprat.

polymerase chain reaction (PCR). are experienced in. reproducible. can be used by untrained personal. The reasons are the relatively complex and difficult handling of the instruments and the need of being applied and maintained by educated personnel. need trained personal. with the same degree and quality of information obtained by sensory assessment. is still missing. Intellectron Fischtester VI. UV and visible light spectroscopy. cheap. low-field (LF) NMR. Frequently used microbiological methods are total viable count (TVC). Warner–Bratzler test. RT Freshness grader. which are noninvasive and nondestructive techniques for the sample. Kramer test. many of them have not graduated from research to seafood industrial application. which is usually not present in seafood industry. Two more systematic methods that involve some analytical methods are the hazard analysis critical control point (HACCP) and traceability. differential scanning calorimetry (DSC). The main problem is that there is no single method existing that can give sufficient information about the quality (freshness) of seafood. Outer inspection is done by the European Union quality-grading scheme and by the quality index method (QIM). and nonpolluting for the environment are. Other methods that are rapid. 1. An instrumental method that is fast. therefore. and time domain spectroscopy (TDR) [7]. Sensory methods. a well-trained sensory panel in which the human senses are used as measuring instruments has been shown to give reliable. 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. expensive. However. determination of specific spoilage organisms (SSO). puncture. and can be applied on many species and also on processed seafood products.8 ◾ Handbook of Seafood and Seafood Products Analysis Physical methods comprise microscopy. are time consuming. analysis of electrical resistance or conductivity by Torrymeter. Further. texture and texture profile analysis (e. Instrumental methods are fast. and oscillatory measurements). and bacterial sensors. tensile. It is. nuclear magnetic resonance (magnetic resonance imaging (MRI). whereas the Torry sensory scheme and the flavor profile analysis are performed on cooked samples. and objective results. comparatively cheap. with the exception of sensory methods. and high-resolution NMR (HR-NMR)). and compression tests. near-infrared spectroscopy (NIR). there are still many problems left in the analysis of seafood and seafood-based products. and are. viscoelastic methods such as stress relaxation. creep. 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. and can be used after a short training period by nonscientific educated personnel. oligonucleotide probes. electronic noses and electronic tongues [14]. however. always a combination of several methods is necessary to give sufficient information equal to sensory assessment [8]. and have used since many years [9]. antibody techniques. image analysis. When using instrumental methods nowadays. we have color measurement. Sensory methods are often considered to be subjective methods. 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. pH measurement. among others. of utmost importance to introduce the newly developed analytical methods into the industry for better product and raw material analysis and quality assurance. therefore.g.7 Analytical Problems Despite the great progress that has been made. not harmful for the operator. ..

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

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

...........13 2..................18 2...............5 Immunoassays ............................6 Electrophoresis-Based Methods .................................................3 Protein Solubility Classes ........................................................................................................... but it is also important to have methods to determine the type and the origin of the proteins present.......................................................................8 Protein Modifications.................................................................................. including the fish industry................................................................................ Both the amino acid composition and the digestibility of fish proteins are excellent....................................2 Total Content of Proteins .......................7 Peptide Characterization .......................... and how these properties are influenced by food additives and other components. 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....... particularly the 11 ........................................... Fish provides about 14% of the world’s need for animal proteins and 4%–5% of the total protein requirement [2]....... Fish are regarded as an excellent source of high-quality protein............................11 2....4 Analysis of Soluble Proteins .... Both the content and the properties of the proteins are important for the value and the quality of the products [1]..................................16 2............1 Introduction Protein analysis is highly important for the food industry............... 12 2.................................17 References .......................1 Introduction ...... 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...........................................Chapter 2 Peptides and Proteins Turid Rustad Contents 2.................14 2......................16 2...................................16 2.....................................................................................

and trapping of ammonia and titration steps. Mariotti and coworkers discuss conversion factors in their critical review and conclude that even if a factor of 6. This method is used as a reference method by many national and international organizations. distillation. nucleotides. 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. which is distilled and trapped in boric acid. . for instance collagen has a nitrogen content of 18%. assuming a nitrogen content of 16% in the proteins. It is important that the methods to analyze food proteins are robust [1]. neutralization.25. The factor can be calculated from the amino acid composition of the proteins.43 to 5.2 Total Content of Proteins The total content of proteins is usually determined by the Kjeldahl or the Dumas method. The advantage of this method is that it gives accurate results for all types of samples. Many proteins have protein contents that deviate from this. The Dumas method is quicker and cheaper and easier to perform and is therefore now considered on equal terms with the Kjeldahl method [1]. it is difficult to start using other and more correct factors [5]. During digestion the nitrogen in the sample is converted to ammonium sulfate. The Kjeldahl method has been automated and several instruments for automated analysis are available. The ammonium sulfate is converted into ammonia. the water-holding capacity and the gelling properties which determine the textural attributes of the products are important quality parameters [3]. both different types of raw materials and processed foods.12 ◾ Handbook of Seafood and Seafood Products Analysis essential amino acids lysine and methionine. For products such as fish mince and surimi. This means that it should be possible to use the method on different types of foods. values from 5. It is also possible to determine the nitrogen content using elemental analysis (C/N analyzers) [4]. and that other components in the food such as lipids and pigments should not interfere with the analysis. The Kjeldahl method was first published in 1883 but has been extensively modified since then. This factor is the amount of nitrogen that contains 1 g of nitrogen. amines. and the amount of protein is calculated by multiplication with a Kjeldahl factor. The disadvantage is that the method requires use of hazardous and toxic chemicals. and textural properties. and urea will also contribute to the calculated protein content. has been shown to be too high for animal proteins. The Kjeldahl method determines the nitrogen content as ammonia. The method includes sample digestion. For fish. but other chemicals such as potassium sulfate and mercury oxide are also used. which has been used for more than 75 years. The method should also require minimal sample pretreatment to decrease analytical error and reduce costs. In addition to the high nutritional value. Originally only sulfuric acid was used for digestion of the samples. emulsification.56 [1]. It is also possible to determine the amount of ammonia by different colorimetric methods [1]. The Technicon AutoAnalyzer uses continuous flow analysis [1]. 2. Tables of conversion factors are given in several papers such as [1.25 is usually used.82 are given.5]. gelling. For animal proteins the value 6. fish proteins also have good functional properties such as water-holding capacity. and the amount of nitrogen is determined by titration [1]. nitrogen from other nitrogen-containing compounds such as free amino acids. When the protein content is calculated based on determination of the nitrogen content. which gives a factor of 5. Retaining the functional properties through preservation and processing operations is therefore of great importance.

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

Since all proteins absorb UV/visible light to varying degrees. The purple complex is relatively stable and has an absorption maximum at 540–560 nm. the method therefore has protein-to-protein variations. This was the acid-soluble collagen. Samples were homogenized in 0. The sensitivity can be increased by measuring absorbance at 310 nm or by increasing the time for the Biuret reaction. Measurement of UV absorption at 280 nm is a simple and popular method to determine protein concentration. However. The Biuret method is based on the formation of complexes between copper salts and peptide bonds under alkaline conditions. the complexes react with the Folin-phenol reagent a mixture of phosphotungstic acid and phosphomolybdic acid in phenol. some of these methods reduce the speed and simplicity of the method [19].5 M acetic acid for 2 days at room temperature and centrifuged as above. The Lowry method [20] is based on a Biuret-type reaction between protein and copper(II) ions under alkaline conditions. The review also discusses many of the modifications that . and a few of them will be treated here. In addition. Peterson have reviewed the Lowry method [21] and listed interfering substances. Methods exist to correct for the influence of light scattering and nucleic acids/ nucleotides [19]. However mg quantities of protein are generally required. giving upper tolerable limits for a long range of these as well as some methods for coping with the effect of these substances. one of the simplest methods is to determine absorbance in the far UV range.1 M NaOH and centrifuged. After extraction. measuring concentrations between 1 and 10 mg/mL. [18]. The precipitate was stirred with 0.4 Analysis of Soluble Proteins There are many indirect colorimetric methods to determine protein content.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]. The extraction in NaOH was repeated five times and the supernatants were pooled for the analysis of alkali-soluble collagen content. The reactions are highly pH dependent. 2. the concentration of the soluble proteins can be analyzed with a wide variety of methods. Absorption at 280 nm is mainly due to tryptophan and tyrosine residues with smaller contributions from phenylalanine and the sulfur-containing amino acids. which is a modification of the method described by Sato et al. Light scattering because of large particles or aggregates can also lead to errors. by absorbance at 205 nm or from knowledge of amino acid composition [19]. The product becomes reduced to molybdenum/tungsten blue and can be measured at 750 nm. Reducing agents and sucrose as well as several common buffers interfere with the Lowry method. but the method is simple and inexpensive. the presence of nonprotein UV-absorbing groups such as nucleic acids and nucleotides which absorb strongly at 260 nm further complicate matters. 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. A standard curve is needed. The method is not very sensitive.

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

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

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

Isaksson. hydrophobicity. Bock. 32: 1–12. molecular flexibility/rigidity in response to external environment (pH. and E. 25: 289–307.E. 2. Hultmann. 3. Time–temperature tolerance and physical-chemical quality tests for frozen Red Hake. in Food Chemistry. Ed. Lanier. 73: R91–R98. net charge. Converting nitrogen into protein—Beyond 6. formation of protein–lipid films. and biological values are sometimes included in the functional properties. 10. 2008. Methods for processing and utilization of low cost fishes: A critical appraisal. moisture and protein in salmon fillets by use of near-infrared diff use spectroscopy. Journal of the Science of Food and Agriculture. Foegeding.. R. p. Nutritional. Mariotti. Automatic methods for the simultaneous determination of carbon. temperature. 96: 491–495. 1995. Marcel Dekker: New York.M. and gelation). thickening. Mirand. and H. cooking. 5: 215–234. Characteristics of edible muscle tissue. 1996.. New York: Marcel Dekker. hydrogen. (2) properties related with the protein structure and rheological characteristics (viscosity. storage. Critical Reviews in Food Science and Nutrition.. T.A. sensory. Non-destructive determination of fat. D. 1968. Functional properties can be divided in several groups. structures (secondary.L. amino acid composition and sequence. 1979. . or interaction with other food constituents. and (3) properties related with the protein surface (emulsifying and foaming activities. 6. 25: 2025–2069. 7. 1992. 1995. 2006. J. 35: 431–435. 9. 879–942. 8. sulphur and sulphur alone in organic and inorganic materials.C. Journal of Food Science & Nutrition. T. F. 2004. tertiary. Ravesi.. Connelly. J. et al. pp.. Journal of Fisheries Research Board Of Canada. 2008. Hultin. and consumption [36]..J. V. Nondestructive determination of water and protein in surimi by near-infrared spectroscopy. 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. 48: 177–184.J. Licciardello. Haard.. N. 87: 31–41. 2002. 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. Analytical chemistry. 69: 95–100. Value added products from underutilised fish species.K. 13. 12. Food Research International. and P. and quaternary). The book edited by Hall [38] gives a good overview of methods to determine protein functionality.R. It is therefore difficult to compare results from different laboratories. and F. 5. elasticity. 51: 1173–1179. Fennema. 4. Food Protein Analysis: Quantitative Eff ects on Processing. Kirsten. Journal of Food Science. V. Venugopal..K.. Venugopal. It is usual to classify them according to mechanism of action into three main groups: (1) properties related with hydration (absorption of water/oil.F. et al. and R. Journal of Food Science & Technology. 1982. Tome. et al.25 and Jones’ factors. 11. Shahidi. O. Anderson. aggregation. Owusu-Apenten. 1995. Food Chemistry. wettability). E. L. solubility. 463. Uddin. Journal of Food Quality. Control of chemical composition and food quality attributes of cultured fish. Iced storage of Atlantic salmon (Salmo salar)—Effects on endogenous enzymes and their impact on muscle proteins and texture.. W. adhesiveness. M. M. whippability). References 1. shape. Relation between protein extractability and free fatty acid production in cod muscle aged in ice.P. hydrophilicity. and T. Food Chemistry. salt concentration). Innovative uses of near-infrared spectroscopy in food processing.18 ◾ Handbook of Seafood and Seafood Products Analysis processing. distribution. Rustad.

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

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

and mass accuracy.4 hold great promise and are deserving of discussion in their own right. the “classic” process of two-dimensional (2D) gel polyacrylamide electrophoresis (2DE) followed by protein identification via peptide mass fingerprinting of trypsin digests (Figure 3.1 Whole Larval Proteomes The production of good quality larvae is still a challenge in marine fish hatcheries. largely because of its high resolution. both on the cellular and tissue-wide levels. during.1 Introduction As with all living matter. .1.2 surface-enhanced laser desorption/ionization3 or protein arrays. that proteome analysis. the cornerstone of most proteomics research.14–18 gill. Selection of tissues for protein extraction is therefore an important issue that needs to be considered before a seafood proteomic study is embarked upon.6–8 liver. While high-throughput. or with the human immune system after consumption.12 kidney.2 Proteome Analysis by 2DE 2DE.2. we present some issues and challenges related to sample matrices of particular interest to the seafood scientist. also known as proteomics. 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. is the simultaneous separation of hundreds. simplicity. fish possess a number of tissues amenable to 2DE-based proteome analysis.22 ◾ Handbook of Seafood and Seafood Products Analysis 3. 3. or even thousands. the construction of the food matrix. the proteome varies from tissue to tissue. 3. This is especially true of fish and meat. for example. growth depression. in the following sections. of proteins on a 2D polyacrylamide slab gel.12. 3. as well as with time and in response to environmental stimuli.9–11 heart. is regulated and brought about by proteins. Like other vertebrates. In the following sections. Proteome analysis allows us to examine the effects of environmental factors on larval global protein expression. gel-free methods. 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. Studies on whole larvae. and after processing or storage.12. Several environmental factors can interfere with the protein expression of larvae leading to poor larval quality like malformations. and low survival rate.1) remains the workhorse of most proteomics work. quality involution within the product before. giving valuable insight into the composition of the raw materials. Proteomics. It stands to reason. This chapter will therefore focus on 2DE. along with some of the main improvements that have developed since.1 Sample Matrix Considerations Unlike the genome.12 and rectal gland12 have been reported. foodstuffs are in large part made up of proteins. Furthermore.13 skeletal muscle. then. where the bulk of the food matrix is constructed from proteins.2. the interactions of proteins with one another or with other food components. based on liquid chromatography tandem mass spectrometry (LC–MS/MS).12 brain.19 intestine. is a tool that can be of great value to the food scientist.

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

3 The Degradome The degradome may be a subproteome of particular interest to the food scientist. Swamping of low-abundance spots by highly abundant ones may not be a problem for applications relating specifically to structural proteins. such as the ubiquitin–proteasome or the lysosome systems.44 It seems likely that the manner. An unfractionated 2DE map of the muscle proteome therefore tends to be dominated by comparatively few high-abundance protein spots.24 ◾ Handbook of Seafood and Seafood Products Analysis proteins were prominent among the identified proteins.2.29. function. is fractionation of the protein sample in order to weed out the high-abundance proteins. as many textural and other quality factors of muscle foods are related to proteolytic activity in the muscle tissue before. Structural proteins. fish skeletal muscle is the main component.42. are of keen interest. then. such as those associated with individual organelles or cell compartments28 or by protein biochemical methods such as affi nity chromatography.5 The remaining option.45. Proteomic analysis on lysosomes has been successfully performed in mammalian (human) systems.46 An exploitable property of proteasome-mediated protein degradation is the phenomenon of polyubiquitination. No amplification method analogous to PCR exists for proteins. rendering analysis of low-abundance proteins difficult or impossible.25. in which protein deposition is regulated. preventing identification of holistic alterations in the analyzed proteomes. However. and biochemistry.2. which include most regulatory proteins and many important metabolic enzymes.30 preparative isoelectrofocusing31 or solubility in the presence of various detergents32 or chaotropes33 have been described.26 3. allowing a larger sample of the remaining proteins to be analyzed. protein turnover is a major regulatory engine of cellular structure. such as actin and tubulin. Various strategies have been presented for the removal of highly abundant proteins24 or enrichment of lowabundant proteins. In addition to having a hand in controlling autolysis determinants. are particularly abundant in the skeletal muscle proteome. The fish muscle proteome is therefore likely to be of comparatively high interest to the seafood scientist. Cellular protein turnover involves at least two major systems: the lysosomal system and the ubiquitin–proteasome system. has profound implications for quality and processability of the fish flesh. as it will give rise to overloading artifacts in the gels. A myriad of methods suitable for subsequent 2DE exist for fractionating the proteome into defined subproteomes. particularly in muscle tissue. during and after processing. Removal of those proteins may increase detection of other proteins present at low concentrations. and simply increasing the amount of sample is usually not an option.34–41 3. but for other applications low-abundance proteins.1. 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. are suitable for rigorous investigation using proteomic methods.43 The 20S proteasome has been found to have a role in regulating the efficiency with which rainbow trout (Oncorhynchus mykiss) deposit protein. it may also result in a loss of other proteins. Protein turnover systems. For example. Fractionation methods for a variety of sample matrices have been reviewed recently. whereby proteins are targeted for destruction by the proteasome by covalent .1.2 Muscle Proteomes In most seafood products.

a general protocol is outlined briefly with some notes of special relevance to the seafood scientist.52–57 3. which proteins are being degraded by the proteasome at a given time or under given conditions.58 Thorough homogenization is essential to ensure complete and reproducible extraction of the proteome. The map can be visualized and individual proteins quantified by radiolabeling or by using any of a host of protein dyes and stains. These strips consist of a dried IPG-containing polyacrylamide gel on a plastic backing. the reader is referred to any of a number of excellent reviews and laboratory manuals. it is possible to observe the ubiquitin–proteasome “degradome. where an electric field is applied to a tube gel on which the protein sample and carrier ampholytes have been deposited.Proteomics ◾ 25 binding to multiple copies of ubiquitin. For more detailed.48–50 Monitoring of the expression levels of these regulatory enzymes.2.1 Sample Extraction and Cleanup For most applications. Activity of matrix metalloproteases is regulated via a complex network of specific proteases.. yielding a two-dimensional map (Figure 3.” i. 3.2) rather than the familiar banding pattern observed in one-dimensional (1D) SDS-PAGE. Ready-made IPG strips are currently available in a variety of linear and .2.51 the procedure remains essentially as outlined earlier. 0.47 By targeting these ubiquitin-labeled proteins. Gygi and coworkers have developed methods to study the ubiquitin–proteasome degradome in the yeast Saccharomyces cerevisiae using multidimensional LC–MS/MS.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 First-Dimension Electrophoresis The extracted proteins are first separated by IEF.22 and Arctic charr (Salvelinus alpinus) liver. 4% (w/v) CHAPS [3-(3-chloramidopropyl)dimethylamino-1-propanesulfonate].2. Although a number of refinements have been made to 2DE since O’Farrell’s paper. up-to-date protocols. 2 M thiourea.2. 0. which is most conveniently performed using commercial dry IPG gel strips. Cleanup of samples using commercial 2D sample cleanup kits may be beneficial for some sample types. such as that of the matrix metalloproteases. silver stains. This separates the proteins according to their molecular charge.3% (w/v) DTT [dithiothreitol]. 3.2 Basic 2DE Methods Overview O’Farrell’s original 2DE method first applies a process called isoelectric focusing (IEF). may be more conveniently carried out using transcriptomic methods. 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). may be less directly amenable to proteomic study.43. In the following sections. sample treatment prior to electrophoresis should be minimal in order to minimize in-sample proteolysis and other sources of experimental artifacts.2. such as Coomassie blue. and how they vary with environmental or dietary variables. most notably the introduction of immobilized pH gradients (IPGs) for IEF. or fluorescent dyes. We have found direct extraction into the gel reswelling buffer (7 M urea.e.2 Some proteolysis systems.

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. Reswelling is normally performed overnight at 4°C. Application of a low voltage current may speed up the reswelling process..000–30. Before electrophoresis. Broad-range linear strips (e. which is then increased stepwise to about 3.2. IEF is normally performed for several hours at high voltage and low current. 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.2. Optimal conditions for reswelling are normally provided by the IPG strip manufacturer.26 ◾ Handbook of Seafood and Seafood Products Analysis MW (kDa) 60 42 30 22 17 4 5 pl 6 7 Figure 3. usually totaling about 10. morhua) larval proteins with pI between 4 and 7 and molecular mass about 10–100 kDa. extraction directly into the reswelling buffer is recommended. Isoelectrofocusing was by pH 4–7 IPG strip and the second dimension was in a 12% polyacrylamide slab gel. sigmoidal pH ranges.1.2 A 2DE protein map of whole Atlantic cod (G.g. 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. The proteins are separated according to their pI in the horizontal dimension and according to their mass in the vertical dimension. pH 3–10) are commonly used for whole-proteome analysis of tissue samples. A recipe for a typical reswelling buffer is presented in Section 3. The appropriate IEF protocol will depend not only on the sample and IPG strip. Typically. but also on .500 V. the dried gel needs to be reswelled to its original volume. the starting voltage is about 150 V. If the protein sample is to be applied during the reswelling process. although this will depend on the IPG gradient and the length of the strip.000 Vh.

0.2. Optimal pore size depends on the size of the target proteins.61 using glycine as the trailing ion and the same buffer (25 mM Tris.2.59 3. This is best performed using a dentist’s tool or other appropriate implement. While some reviewers recommend alternative buffer systems. During the equilibration step.1% SDS) at both electrodes. taking care to put the pressure on the IPG strip’s plastic backing rather than the gel itself. it needs to be equilibrated for 30–45 min in a buffer-containing SDS and a reducing agent such as DTT. 30% glycerol. that the gel side of the IPG strip faces the notched side of the glass plate. trace amount of bromophenol blue.2.8.3 Orientation and placement of an isoelectrofocused IPG strip onto the top of the second-dimension gel.60 the Laemmli method. it is applied to the top edge of an SDS-PAGE slab gel (Figure 3. Figure 3. Ready-made gels suitable for analytical 2DE are available commercially.56 reviewed IEF for 2DE applications. but for most applications gradient gels or gels of about 10% or 12% polyacrylamide are appropriate. and that the strip is pressed gently onto the SDS gel. 1% DTT. thus reducing vertical streaking. This will alkylate thiol groups and prevent their reoxidation during electrophoresis. Care must be taken that the (+) end of the strip is on the same side of all slab gels. .5% iodoacetamide and without DTT (otherwise identical buffer) may be required for some applications. the SDS–polypeptide complex that affords protein-size-based separation will form and the reducing agent will preserve the reduced state of the proteins. A second equilibration step in the presence of 2.2. A typical equilibrationbuffer recipe is as follows: 50 mM Tris–HCl at pH 8. The gel is run at a constant current of 25 mA until the bromophenol blue dye front has reached the bottom of the gel.3) and cemented in place using a molten agarose solution. 3. 192 mM glycine. remains the most popular one. avoiding trapping air bubbles. Görg et al.Proteomics ◾ 27 the equipment used.4 Second-Dimension Electrophoresis Once the gel strip has been equilibrated. 6 M urea. A tracking dye for the second electrophoresis step is also normally added at this point. 2% SDS. The manufacturer’s instructions should be followed.3 Equilibration Before the isoelectrofocused gel strip can be applied to the second-dimension slab gel.

gene expression in several tissues varies considerably among the individuals of the same species.68 where peptides are suspended in a matrix of small. such as ImageMaster (Amersham).62 3. Patton published a detailed review of visualization techniques for proteomics. has improved by leaps and bounds in recent years. the spot of interest is excised from the gel. PDQuest (BioRad). commonly used to assess the significance of observed protein expression differences.2. organic.65–67 3. and followed by destaining for several hours in water.2. Also.2. remains the bottleneck of 2DE-based proteome analysis and still requires a substantial amount of subjective input by the investigator. including protein spot definition. followed by incubation for 1 h in 17% ammonium sulfate/34% methanol/2% ortho-phosphoric acid. or Progenesis (Nonlinear Dynamics). There are. however. spot matching between gels tends to be time-consuming and has proved difficult to automate. 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.6 Analysis Although commercial 2DE image analysis software. Briefly.3 Protein Identification by Peptide Mass Fingerprinting Identification of proteins on 2DE gels is most commonly achieved via mass spectrometry of trypsin digests. such as in difference gel electrophoresis (DIGE).28 ◾ Handbook of Seafood and Seafood Products Analysis 3. and therefore individual variation is a major concern and needs to be accounted for in any statistical treatment of the data.2.64 These difficulties arise from several sources of variation among individual gels. A typical staining procedure includes fi xing the gel for several hours in 50% ethanol/2% ortho-phosphoric acid. UV-absorbing molecules (such as 2. digested with trypsin (or another suitable protease). commercially available colloidal Coomassie staining kits that do not require fi xation or destaining.63 In particular. Pooling samples may also be an option and this depends on the type of experiment. These include radiolabeling.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. The most popular mass spectrometry method is MALDI-TOF mass spectrometry. followed by several 30 min washing steps in water. matching. Multivariate analysis has been successfully used by several investigators in recent years. and the resulting peptide mixture is analyzed by mass spectrometry.1% Coomassie Blue G-250/17% ammonium sulfate/34% methanol/2% ortho-phosphoric acid. such as with [35S] methionine. A great many alternative visualization methods are available. analysis of the 2DE gel image. . such as protein load variability due to varying IPG strip reswelling or protein transfer from strip to slab gel. These multiple sources of variation has led some investigators63–65 to cast doubt on the suitability of univariate tests. followed by staining for several days in 0. 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. and staining with fluorescent dyes.2.5 Staining Visualization of proteins spots is commonly achieved through staining with colloidal Coomassie Blue G-250 due to its low cost and ease of use. such as Student’s t-test. and individual protein quantification. such as the SYPRO or Cy series of dyes.

org/tools) contains links to most of the available software for protein identification and several other tools.50 20 10 0 741. which in many cases is more extensive than the protein sequences available. The solid markers indicate the peaks that were found to correspond to expected b-2 tubulin peptides.60 Peptides identified as those derived from Atlantic cod β-tubulin Trypsin autolysis peaks 1159.4 2212.07 1131.98 1886. it is possible to take advantage of the available nucleotide sequences.96 60 870. this problem is surprisingly acute for species of commercial importance. It is important to realize.90 1822.25 Figure 3. In their work on the rainbow trout liver proteome. to obtain a tentative identity. correlated activity measurements. or transcript abundance).69 1659.54 50 40 1287.06 1575.71 100 90 80  Intensity 1061.801616. The open markers indicate mass peaks corresponding to trypsin self-digestion products and were. however. many with a web-based open-access interface.1.expasy.Proteomics ◾ 29 842.50 30 856.8 1652. As can be seen in Table 3. The resulting spectrum of peptide masses (Figure 3. 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. such as the National Centre for Biotechnology Information (NCBI) nonredundant protein sequences database.43 .61 1272. 100% agreement was observed between the two methods. Martin et al.10 and Vilhelmsson et al. using the appropriate software.36 2564. 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.86 1028. To circumvent this problem. In those cases where both the protein and nucleotide databases yielded results. The ExPASy Tools web site (http://www. 2506.4) is then used for protein identification by searching against expected peptide masses calculated from data in protein sequence databases. excluded from the analysis. How useful this method is will depend on the length and quality of the available nucleotide sequences.00 1196. Several programs are available. identified as b-2 tubulin.54 70 1960.63 1258.56 1974.0 1229.51 1040.6 Mass (m/z) 2108.4 A trypsin digest mass spectrometry fingerprint of an Atlantic cod larval protein spot.2 2798. therefore.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.35 1621.70 1697.

haddock.782. incl. monkfish) Perciformes (perch-likes.489 130.007 911 303 18.344 5.287 8.210 Chondrichthyes (Cartilagenous Fishes) Carcharhiniformes (ground sharks and dogfishes) Lamniformes (mackrel sharks) Rajiformes (skates and rays) 3.008 Mollusca (Mollusks) Bivalvia (mussels.158 20.380 898.086 2. scallops. and wolffish) Pleuronectiformes (flatfishes. turbot.798 81. mackrel.557 .353 287 170.208 2. tuna.284 2.762 726. etc.845 999.656 2. etc.680 1.063 3.933 3. redfish and lumpfishes) 185.896 3. halibut. saithe. whelks and abalone) Cephalopoda (squid and octopi) 32.871 32.442 237 2. incl. sea bass.533 1. sea bream.30 ◾ Handbook of Seafood and Seafood Products Analysis Table 3. sole.626 138 16. and pollock) Lophiiformes (anglerfishes.381 45.237 2.424 2. incl.592 735 179 585 768.751 Crustacea (Crustaceans) Caridea (shrimps.845 10. 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.122 268 26.1 Families of Some Commercially Important Seafood Species and the Availability of Protein and Nucleotide Sequence Data as of March 27. incl.203 467.864 47.) Astacidea (lobsters and crayfishes) Brachyura (short-tailed crabs) 21.585 1. and plaice) Zeiformes (dories) Scorpaeniformes (scorpionfishes.006 121.138 36.) Gastropoda (incl. cod.407 84.245 2.046. incl.

proteomic investigations on fish and seafood products. several short stretches of amino acid sequence will be obtained for each peptide. way of obtaining protein identities is by direct sequence comparison.23. improved reproducibility and resolving power of electrophoretic separation techniques.94 Proteome analysis provides valuable information on the variations that occur within the proteome of organisms.90–92 The morphological and physiological changes that occur during these developmental stages are characterized by differential cellular and organelle functions.76 Nyman. the identified proteins consisted mainly of proteins located in the cytosol. when combined with the peptide and fragment masses obtained. the method of choice is tandem mass spectrometry (MS/MS). physiology. if rather more time-consuming.71.72 Today.88. each peptide mass can potentially represent any of a large number of possible amino acid sequence combinations.Proteomics ◾ 31 A more direct.79 Rappsilber et al.1 Development Fishes go through different developmental stages (embryo. and behavior of the fish.95 resulting in different expression of proteins. Correlating this spectrum with the candidate peptides identified in the first round narrows down the number of candidates. the higher the number of possible combinations. reflect a response to biological perturbations or external stimuli9–11. which. In both these studies. and adult) during their life span that coincide with changes in the morphology. for example.81 Gygi and Aebersold.85 wheat flour baking quality factors. as well as in aquaculture.87 With the lower cost. 3.73–75 Mass spectrometry methods in proteomics have been reviewed.3 Applications of 2DE in Seafood Analysis The two-dimensional electrophoresis has been in use within food science for at least two decades. have gained considerable momentum.83 and Delahunty and Yates..77 Damodaran et al. enhances the specificity of the method even further. Proteome analyses in developing organisms have shown that many . and vastly superior protein spot identification techniques. The larger the mass (and longer the sequence).86 and soybean protein bodies.20 To date few studies on fish development exist in which proteome analysis techniques have been applied. Until recently. this was accomplished by N-terminal or internal (after proteolysis) sequencing by the Edman degradation of eluted or electroblotted protein spots. 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. 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.. for example.3. These variations may. and development.80 Mo and Karger. Furthermore. or redistribution of specific proteins within cells. clearly defined subproteomes and included such applications as the characterization of bovine caseins. posttranslational modifications. and nucleus. fish physiology. cytoskeleton. Early studies focused on relatively small. In the peptide mass fingerprinting discussed earlier.84 3.78 Thiede et al.93 This is reflected in the variations of global protein expression and posttranslational modifications of the proteins that may cause alterations in protein function. yielding a second layer of information.82 Lin et al.. by Yates.89 A brief discussion of a few emerging areas within fish and seafood proteomics is given as follows. larva..

The major obstacle on the use of proteomics in embryonic fish has been the high proportion of yolk proteins. Thus. Furthermore.109. whether they be encoded in structural genes or brought about by posttranslational modification. and pI of the protein present in a tissue.99.3.8. In a recent study on the proteome of embryonic zebrafish. a large number of yolk proteins remained prominently present in the embryonic protein profiles. several commercially important fish muscle processing techniques. usually have different molecular weight or pI and can.21 and dorada (Brycon moorei).21. Link et al. Studies on various proteins have shown that during fish development sequential synthesis of different isoforms appear successively.2 Quality Involution Degradation of proteins during chilled storage. This fact further indicates the importance of the proteome approach to understand cellular mechanisms that underlie fish development.3. such as curing.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.113 . molecular mass. It is also worth noting that protein isoforms other than proteolytic ones. and production of surimi and conserves occur under conditions conducive to endogenous proteolysis. therefore. where differences are expected to occur in the number.99–107 In this context. specific isoforms of myofibrillar proteins. are among persistent quality problems in the seafood industry and have deleterious effects on fish flesh texture. These interfere with any proteomic application that intends to target the cells of the embryo proper. are well suited for investigation using 2DE-based proteomics.2. 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. be distinguished on 2DE gels.88.32 ◾ 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.8.7 Despite this undertaking.21. developmental stage specific muscle protein isoforms have gained a special attention.98 Different isoforms generated by posttranslational modifications are largely overlooked by studies based on RNA expression.108 3.1 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.94. 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.99–107 The developmental changes in the composition of muscle protein isoforms have been tracked by proteome analysis in African catfish (Heterobranchus longifilis). For example.101 These studies demonstrated that the muscle shows the usual sequential synthesis of protein isoforms in the course of development.27 is probably due to dechorionation prior to the deyolking of the embryos. although degradation of myofibrillar proteins by calpains and cathepsins112.110 Problems of this kind. fermentation.111 3. many of which are correlated with specific textural properties in seafood products. the embryos fall out of their chorions facilitating the removal of the yolk. can be observed using 2DE or other proteomic methods. 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.102 common sole (Solea solea). and their oxidation during frozen storage. By dechorionation.

appear to display seasonal variations. etc. 2D-immunoblots and LC–MS/MS to study changes in protein oxidation during frozen storage of rainbow trout. the ubiquitin–proteasome pathway has been shown to be downregulated in response to starvation129 and have a role in regulating protein deposition efficiency. differences that can affect postmortem biochemical processes in the product which.123 found these to comprise several members of the glycolytic and Krebs cycle pathways. We are aware of two recent studies where Atlantic cod muscle proteomes have been compared between farmed and wild fish. is determined by environmental as well as genetic factors. therefore raises the tantalizing prospect of managing quality characteristics of the fish flesh antemortem. and NADPH metabolism.126 in fish fed with the experimental diets.Proteomics ◾ 33 and degradation of the extracellular matrix by the matrix metalloproteases and matrix serine proteases114. where individual physiological characteristics.2. as opposed to wild fish catching. affect the involution of quality characteristics in the fish product. in mammals. For example. In the context of this chapter. The diet was found to have a marked effect on product texture. fatty acid breakdown. With the ever increasing resolving power of molecular techniques.123 Both studies indicated that several proteins are differentially expressed in farmed versus wild cod.115 are thought to be among the main culprits.2 Aquaculture and Antemortem Effects on Quality and Processability It is well known that an organism’s phenotype.112. is thought to be responsible for a large fraction of cellular proteolysis. The practice of rearing fish in aquaculture. in turn. Kjærsgård et al. are optimized.17 used 2DE.128 In rainbow trout. furthermore. Furthermore. They found fish muscle proteins to be differentially carbonylated during frozen storage and were able to identify several carbonylated proteins using LC–MS/MS.111. and the amount and composition of free amino acids in the fish flesh.1 . Indeed. 67.125 and the liver proteome was analyzed9. flesh softening during storage. such as those governing gaping tendency. it is clear.118 Several 2DE studies have been performed on postmortem changes in seafood flesh14–17. The proteasome is a multisubunit enzyme complex that catalyzes proteolysis via the ATP-dependent ubiquitin–proteasome pathway which. Martinez et al.119. In a recent study on the feasibility of substituting fish meal in rainbow trout diets with protein from plant sources. including quality characteristics. such as pathways involved in cellular protein degradation.3. the effects on the proteasome are particularly noteworthy.15. this is fast becoming feasible. the interplay between these physiological parameters and environmental and dietary variables needs to be understood in detail. that these quality changes are species dependent116.117. Whatever may be the mechanism.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. the proteome analysis identified a number of metabolic pathways sensitive to plant protein substitution in rainbow trout feed.117 and.. 3. various quality characteristics of fillet and body were measured124.127. Huss noted in his review122 that product quality differences within the same fish species can depend on feeding and rearing conditions.10. such as proteomics.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. Olsson et al.120 and have demonstrated the importance and complexity of proteolysis and oxidative changes in seafood proteins during storage and processing. To achieve that goal.

14 Unlike the genome. performed a 2DE on crude protein extracts from the tiger prawn. The allergen was identified as a protein with close similarity to arginine kinase. and postmortem treatment. Martinez et al.145 Seafood allergies are caused by an immunoglobulin E-mediated response to particular proteins. found that M.140.3.135–137 which was soon followed by methods to identify species in processed or cooked products. about 0. Mytilus galloprovincialis.142. including structural proteins such as tropomyosin.138. this makes the issue of species authentication an area of increasing economic importance. 3. demonstrating that it had arginine kinase . 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. such as the gadoids or several flat fishes. as different fish species have different market values.5% of young adults are allergic to shrimp. the proteome varies from tissue to tissue and with environmental conditions. 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. trossulus could be distinguished from the other two species on foot extract 2D gels by a difference in a tropomyosin spot. For example. During the 1960s.4 Allergen Identification Allergenic potential is food safety issue of particular concern to the seafood producer.134 recently reviewed proteomic and other methods for species authentication in foodstuffs. The identity was further corroborated by cloning and sequencing the relevant cDNA. These authors.144 Martinez and Jakobsen Friis concluded that the identification of not only the species present.143 Lopez and coworkers. Penaeus monodon. possibly indicating freshness and tissue information in addition to species.34 ◾ Handbook of Seafood and Seafood Products Analysis 3. particularly for addressing questions on the health status of the fish in question. and Mytilus trossulus. Proteome analysis can therefore potentially yield more information than genomic methods. proteomics-based species identification methods are likely to develop rapidly and find commercial uses within this field. 2DE-based methods have been developed to distinguish various closely related species. 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. A final proof was obtained by purifying the protein.3 Species Authentication Processed fish products are increasingly common in the market and.147 at National Taiwan University. From early on. proteomic methods have been recognized as a potential way of fish species identification.3.18. as well as being relevant from a public health standpoint.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. studying three species of European mussels: Mytilus edulis. Allergic reactions to seafood affect a significant part of the population. The allergens were then identified by MALDITOF MS of tryptic digests. 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. They found the difference to be due to a single T to D amino acid substitution.139 These early efforts were reviewed in 1980.141 More recently. 1D electrophoretic techniques were developed to identify the raw flesh of various species. blotted the 2D gel onto a PVDF membrane.143 Indeed. studying the cause of shrimp allergy in humans. the presence of stress-factors or contamination levels at the place of breeding. and probed the membranes with serum from confirmed shrimp allergic patients.

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

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

The overlapping between these clones and their relative arrangement in the genome can be determined through fingerprint analysis (e. useful in the case of regions rich in repetitive sequences posing problems to assembly after whole genome shotgun sequencing.fishbol.Seafood Genomics ◾ 45 A clone-by-clone approach can be used as an alternative to. Large-scale expression studies at the transcriptional level are generally performed using microarrays or other methods of high-throughput expression profiling. A method called “DNA barcoding” should help to identify species and phylogenetic units. for example. This provides a physical map respecting the “real” base pair distance between genes and markers. can be sequenced either to completion or from their ends. The relative position of two contigs can also be estimated cytogenetically using double fluorescent in situ hybridization [8].. Bacterial artificial chromosomes (BACs) accepting inserts from several hundreds of kilobases are frequently used as vectors. Additional approaches are required to study gene expression (transcriptomics. shotgun sequencing. 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. For example. Of particular interest are expressed sequence tags (ESTs). 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]). 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. with novel methods allowing very rapid and much cheaper sequencing of large amounts of DNA [11–13]. Generally. obtained through sequencing of complementary DNA (cDNA) libraries. 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. through the identification of common restriction fragments). hereby contributing to species conservation and management of global fish biodiversity (http://www. zebrafish and medaka are two complementary fish models to study . In addition. 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. cloned in a bacterial vector and constituting a so-called genomic library. see Ref. which can be very useful to precisely determine the relative position of sequence contigs assembled “in silico” from whole genome shotgun sequencing data. 4. for instance. proteomics) and function (functional genomics) as well as interactions with the environment (environmental genomics). Importantly. ESTs can also be used. 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]. or even better in combination with. Such an approach is.org/). 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]). Parts of the genome. Barcoding is based on a sequence of short standard parts of the genome. for SNP detection and phylogenetic reconstructions. [16]).g.3 Genomic Resources and Genome Projects for Aquatic Species Genetic and genomic resources have been generated for many aquatic species of economical interest. a new revolution of large-scale sequencing is ushering in a second era of genomics.

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

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

with possible detection of DNA sequences promoting evolution in their genomes [17]. multiple SNPs have been generated for Atlantic cod. it has been. 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. for which large annual escapees of farmed Atlantic salmon enhance the risk of extinction of wild populations. for example. Genome-wide gene expression profiling can also be used to detect variations in gene expression within and among natural populations [60]. For example. For several species. The effects of stress factors contributing to species collapse and . This approach has already been used to identify adaptive differences between natural populations in several species. conservation efforts could focus on the preservation of genetic diversity allowing biota to adapt to new conditions. Evolutionary genetics and genomics might also help to understand the interplay between fishing and natural selection on population and species targeted by fisheries [65]. resulting in potentially detrimental effects on survival of these populations [67]. Populations of North-East Arctic cod and Norwegian coastal cod have been analyzed. [1]). Beside populations. Genomics and transcriptomics can allow assessing the genetic and functional consequences of interbreeding between farmed and wild fish. Finally. 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]. 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]. 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]. observed that reintroduced steelhead trout presented reduced reproductive capabilities caused by genetic effects of domestication [66]. For example. European eel.62]. brown trout. Genetic monitoring of diversity using polymorphic markers allows monitoring population size and diversity over time. the available population genetic information is insufficient for most other species. and pike. microsatellite data indicated marked genetic changes in declining North Sea cod [57]. SNPs. This type of study has been performed on Atlantic salmon. with poorly represented phylogenetic groups receiving high conservation priorities [52]. 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. thereby identifying loci potentially influenced by natural selection [53]. Population genomics is a form of population genetics extending the analysis of genetic variation in natural populations to the scale of the genome itself. including the European flounder and the brown trout [61. and others (for review. this field will certainly be of major importance in the future of fisheries management and biodiversity conservation. Accordingly. phylogenetics and phylogenomics are of major importance for the recognition of endangered taxa from the systematic point of view. see Ref. sufficient genetic data might be available to provide at least basic information on genetic structure and genetic units for biologically sustainable use [56]. For example.48 ◾ Handbook of Seafood and Seafood Products Analysis RAPD markers.59]. including Atlantic herring. Molecular markers can be used to monitor the efficiency of programs aiming to supplement declining wild populations through individuals reared in captivity. In contrast. heralding a new era in the analysis of adaptive evolution and functional variation [58. Through pedigree reconstruction with microsatellite markers. with the discovery of new groupings and the determination of divergence times and molecular clocks [63]. With the development of much faster and cheaper high-throughput sequencing methods. taxa can also be considered as conservation units. turbot. Atlantic salmon.

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

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

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

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

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

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. although it might be accelerated by the action of different bacteria.1.3 In all cases. IMP is the main nucleotide present in fish species. 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.4 However. and either of the two may be used as freshness indicators. As a result of endogenous enzymes action. which is oxidized to xanthine (Xa) and uric acid in the presence of xanthine oxidase (XO) enzyme. ATP molecule is rapidly degraded to adenosine monophosphate (AMP) and afterward to inosine monophosphate (IMP). This process involves a series of reactions commonly represented according to the sequence shown in Figure 5. and Hx in the flesh of some species of fish during chilled storage. Ino.58 ◾ Handbook of Seafood and Seafood Products Analysis rigor mortis is reached. (2006) published a review of the concentration of IMP.2 Howgate et al. This enzyme is mainly generated in muscle from biochemical processes of microorganisms. the use of a single compound as freshness indicator is not always advisable. Ino and Hx concentrations increased during storage. Inosine is transformed to hypoxanthine (Hx) by the action of the enzyme nucleoside phosphorylase (NP).1 Degradation of ATP in postmortem fish muscle. which is accumulated in postharvest fish. .1 The following IMP dephosphorylation to obtain inosine is mainly autolytic and occurs at a slower rate during the first stage of cold storage. whereas AMP remains major in crustaceans.

6 This value has been used as one of the freshness indexes to evaluate the quality change of postharvest fish. ADP and ATP are derived from the AMP. . 5. the disappearance of the degradation products differs from one species to another3 as mentioned here.15.12 However. and it is important to stop this reaction drastically at the sampling time. Ino.14 Measurement of ATP-related compounds is also useful for the quality control of retorted fishes. and thus. adenosine diphosphate (ADP).16. the knowledge of their molecular structure is important. Some of them are briefly described here.2 Chemical Structure of Main Seafood Nucleosides and Nucleotides To a better understanding of the methods of analysis of these compounds. nucleotides and nucleosides should be extracted and analyzed. The ratio Hx/AMP was considered an adequate alternative to characterize fish freshness due to its constant increment with time.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. a high accumulation of Ino occurs during ATP degradation. it is advisable to collect small tissue samples and immerse them into liquid nitrogen. These cold conditions must be held along the sample preparation. and AMP disappear early postmortem. ATP-chain degradation occurs very fast. This is the main reason for the use of indexes with more than one compound from the ATP-degradation chain. to which one or two additional phosphate groups are attached through pyrophosphate bonds (∼P) (Figure 5. respectively.13. This is achieved by immediately freezing the excised muscle under liquid nitrogen to stop all enzymatic reactions.16 Another suggestion to use nucleotide compounds as a measurement of seafood quality is their relation with sensory attributes. even at refrigeration temperatures. also. for several species. often designed K ′ value or Ki index. a high content of Hx is related with the bitter off taste of spoiled fish. a revised K value.17 5. generally within 1 day of storage in ice after death in all fish species.2. Nucleosides currently analyzed in seafoods are those in which a purine ring.5 and. 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. 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. making K value inadequate as a freshness indicator. consequently.2). In order to achieve this rapid freezing. forming the adenosine or inosine.7–9 Nevertheless. 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. or hypoxanthine is attached to a ribose. whereas IMP evokes a fresh meaty taste sensation. K value is defined as the ratio of Ino and Hx to the sum of ATP and related compounds expressed as a percentage. AMP or adenylic acid is derived from the adenosine in which a phosphate group is attached at the 5-ribose carbon. Nucleosides are glycosylamines that are formed when a nucleobase (purine or pyrimidine base) attaches to a ribose or deoxyribose ring.Nucleotides and Nucleosides ◾ 59 nucleotide degradation such as the type of spoilage bacteria and mechanical handling of fish. adenine. For this reason. Nucleotides are o-phosphoric acid esters of the nucleosides.10. In this way. IMP is derived from the inosine in which a phosphate group is attached to the 5-ribose carbon.18 After this. On the other hand.11 and. ATP. and Hx expressed as percentage.

after centrifugation (27.22 . Once the extract is centrifuged (15. with or without employing an ion-pairing agent. The neutralized extract must be made up to 5 mL with 20 mM phosphate buffer pH 7.5–6.3.2 Structure of adenosine-derived nucleotides.2 μm membrane filter and stored under frozen storage at temperatures below −20°C until analysis. 5.000 g for 15 min).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 5.000 g for 20 min). Ino. is the following: 5 g or less of muscle tissue are excised and quickly frozen with liquid nitrogen.1 Extraction of Nucleotides and Nucleosides A typical extraction procedure for the analysis of fish samples by reversed-phase chromatography. The frozen tissue is minced.8 and then filtered with a 0. cold 0.8 by adding solid potassium carbonate or 1 M potassium hydroxide. they are neutralized with 2 M sodium hydroxide. avoiding any thawing. 3–5 vol.6 M perchloric acid is added. The supernatant is filtered through a 0. and Hx.5 g of fish sample with 10% trichloroacetic acid and.17 Other extraction methods consist in the homogenization of 2. although storage at −18°C has been demonstrated to be enough to preserve fish samples and fish extracts for the analysis of IMP.45 mm membrane.20 and/or spectrophotometers as well as in capillary electrophoresis (CE)21 or ion chromatography (IC). the supernatant is filtered through glass wool and neutralized to pH 6.000 g for 10 min). and the tissue is homogenized with a stomacher-type homogenizer for a few minutes under cold conditions. These fish extracts are used in enzymatic assays with biosensors19. This neutralized extract is kept in an ice bath for 15 min and centrifuged again (15.

radioimmunoassay. AMP). ADP. which may be adsorbed on capillary walls. 5. this technique can present problems in reproducibility.21 Typical conditions to get a good separation of IMP.27 IC. inosine. In this way.3. 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 . Thus. RP-HPLC and ion-paired reverse-phase are the methods of choice for this analysis.24 5.3. 5. both a microwave oven at 500 V for 5 s and heating at 100°C for 60 min have been used. ion-exchange HPLC. Thus.25 thin-layer chromatography (TLC).3. including nuclear magnetic resonance spectroscopy (NMR). some authors have described extraction methods that consisted of heated fish sample. nucleotides will disappear at the rigor mortis state (normally 1 day after catch).Nucleotides and Nucleosides ◾ 61 In the development of biosensor analysis. intact fishes after being submitted to physical and chemical stressors such as hypoxia.2.2. However.2 Nucleotides and Nucleosides Determination Several methods have been used to measure nucleotides and nucleosides. HPLC has been shown to be the most widely used technique to analyze nucleotides and nucleosides. pH 11. In the analysis of complex biological samples.3 Chromatography At present.26 reversed-phase high-performance liquid chromatography (RP-HPLC) with and without ion pair.19 5. high-performance capillary electrophoresis (HPCE). among other chromatographic techniques.3.2. followed by 2 min of the running buffer used. and hypoxanthine would be a potential of 416 V/cm of capillary using 100 mM 3-[cyclohexylamino]-1-propanesulfonic acid (CAPS) buffer.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. Nevertheless. In particular. including fish extract. The mode of separation will depend on the analyte of interest. because these samples usually contain significant amounts of ions. the reconditioning of the capillary surface is ensured by washing 1 min with 1M NaOH.28 Also in vitro 31P-NMR spectroscopy has been applied to both excised tissue and perchloric acid extracts of fish muscle.22 and enzymatic assays. the addition of an ion-pair to the mobile phase greatly improves the separation by increasing the retention time of charged molecules (ATP. Capillary electrophoresis has been used in many nucleotide analysis applications as in the study of nucleotide degradation in fish tissues.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. to analyze nucleotides.23.

.1 Reversed-Phase HPLC The chromatographic analysis should be performed in a liquid chromatograph equipped with an UV detector (254 nm).2. In Figure 5. The column used is an analytical reversed-phase RP-18 column.62 ◾ Handbook of Seafood and Seafood Products Analysis freshness or quality. (5) hypoxanthine. Quantitative analysis can be performed by external or internal standard method.3 RP-HPLC chromatograms of standards (a) and hake (b) ATP-derived compounds. (2) ATP. a simple RP-HPLC with a phosphate buffer as mobile phase will be adequate.17. 5.15 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. There are many approaches to analyze nucleotides and nucleosides by this technique. (4) AMP. (1) IMP.29 With buffer pH 7. (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.3 both chromatograms of standards and hake nucleosides and nucleotides are shown. and (6) inosine.3. 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. The separation was achieved with an RP C-18 column at 35°C and a gradient between phosphate buffer at pH 7 and acetonitrile. which differ mainly in the pH of the mobile phase. Then.3.29 phosphorylated metabolites are also well separated in the chromatogram.

ATP-derived compounds.Nucleotides and Nucleosides ◾ 63 5. The separation is achieved in a reversed-phase column. (4) AMP. due to the ionic nature of the phosphate esters that facilitates strong interactions with the ion-pair reagent at the appropriate pH. and (6) inosine. 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 5.2.3.3. Figure 5.2 Ion-Pair RP-HPLC The most common technique used for the separation of nucleotides is ion-pair RP-HPLC. as well as the resolution. (5) hypoxanthine. Nevertheless. which is especially useful in separating mixtures of charged and uncharged molecules. (3) ADP. (2) ATP. .and tri-nucleotides have to be analyzed.4 shows an ion-paired chromatogram of a 48 h postmortem sardine extract. Thus.30 This ion-paired technique is especially useful when di. (1) IMP. this method is more expensive than the more simple technique previously described. Due to the negative charge of the phosphorylated groups of nucleotides. because the ion pair enhances the retention time and separation. making it less dependant on the type of column. either tetrabutylammonium hydrogen sulfate or phosphate is the ion pair most used.17.4 Ion-paired HPLC chromatograms of salmon (a) and sardine (b). and the key is to add an ion pair (an ion of charge opposite to that of the analyte molecule). the ion pair should be a positive ion with a hydrophobic rest to improve the affinity with the stationary phase.

XO enzyme.64 ◾ Handbook of Seafood and Seafood Products Analysis 5. Ino.3. or sensors have a limited shelf life. all the approaches to date need the sample preparation described earlier.35 The most used is the biosensor based on the measure of hypoxanthine.36 Nevertheless. due to its specificity.4 Enzymatic Analysis The use of enzymatic methods to analyze nucleotides in seafood is widespread due to their high specificity.4.2. although biosensors have shown its utility in some applications such as clinical. IMP. the application in the food industry is still restricted36 mainly due to critical stages such as enzyme immobilization or sample preparation for analysis.39 This procedure was also used to analyze ATP and related compounds in fish sauces with very good results. 5. which will be further quantified by measuring the absorbance at 290 nm and by polarimetry. environmental.2 Enzymatic Methods with Immobilized Enzymes In this case. This sensor has been developed mainly for assessing the freshness of fish meat40.3. the use of commercial kits or disposals presents some problems. oxidizes the hypoxanthine to xanthine and uric acid. agricultural. and IMP may be determined spectrophotometrically by a sequential addition of XO. and 5′-nucleotidase (NT) into a reaction phosphate buffer containing the fish extract sample at pH 7. because the denaturalization of the enzymes with time.31 Another possibility consisted in monitoring the oxygen consumption after these enzymatic reactions with an amperometric-type sensor (oxymeter). and rapid response. and thus test kits. This option offers some advantages in relation to the free enzyme.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. simplicity. inosine. but they remain immobilized in different supports. electrodes. 5. which is immobilized in a membrane fi xed in the sensing area of the electrode.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. although these applications used to be achieved with at least one of these enzymes immobilized as described earlier.3. NP. because no interference of salt in the medium was observed here as was in the case using the HPLC method.8 and 30°C–37°C. in which an enzyme or a group of enzymes are immobilized in a membrane or other supports. Indeed. In addition. These assays may be carried out with the enzymes in solution31. while the depletion of oxygen is measured by a Clark-type elec- .20. which is further coupled to a chemical transducer.2. 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.32 or immobilized.6–7. In these conditions.2.33. and biotechnology. the analysis may be performed with one or more enzymes.41 or for the evaluation of chicken32 and beef meat33 aging. and hypoxanthine will be oxidized to uric acid and H2O2. The depletion of oxygen or the formation of hydrogen peroxide or uric acid may be detected amperometrically. constituting what is known as enzyme sensors o biosensors.1 Enzymatic Methods with the Enzyme in Solution The concentration of Hx. respectively.36–38 The most used biosensors for the nucleotide-related compound analysis are electrochemical sensors. These enzymes act by oxidizing the substrates (analyte) while consuming oxygen or producing hydrogen peroxide or uric acid.4. In this sensor.20. enzyme-coated strips.

uric acid.45 a nafion-coated platinum disc electrode.6 to −0.46 or even in a carbon paste electrode modified with electrodeposited gold nanoparticles.. Mendes. M.47 On the other hand.49 and Ino50 and a multienzymatic sensor to analyze simultaneously AMP. specific biosensors to determine AMP. . P. Food Sci. Gill. Özogul.56 References 1. Kuley. and Nguyen52 and afterward it has been commercialized as a Freshness Meter KV-101 (Oriental Electric Co. Y.33 although this relation should be confirmed in each particular system. Male.L.53. 65: 40–47. Howgate. an Ag/AgCl reference electrode. et al. 3. A review of the kinetics of degradation of inosine monophosphate in some species of fish during chilled storage.. M. Most recent approaches to determine Hx are based on the incorporation of the XO enzyme in a graphite/Teflon matrix. J. Comparable results to that of HPLC were reported. R. IMP. E. thus.. Pacheco-Aguilar. Food Res. 36: 19–22. M. Technol. Soc.53. necessary to obtain K. Sci. Food Biochem.. Ino. K i. which can be present in the sample or formed during the enzymatic reaction. Ino was converted to Hx. Postmortem biochemical and functional characteristic of Monterey sardine muscle stored at 0°C. Postmortem changes in quality indices of ice-stored flounder (Paralichthys patagonicus).E. Surette.51 The use of multienzymatic biosensors to measure fish freshness has been very helpful for the simultaneous determination of AMP. Food Chem. Changes in baseline levels of nucleotides during ice storage of fish and crustaceans from the Portuguese coast. M. In this way.R. R. R.23.. P.E. Ltd. 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. Japan).E... The proposed relation 1 Hx for ½ X for each oxygen molecule formed must be taken into account to quantify the Hx.. 29: 570–590. Fish. Massa. Luong and Male20 used a multienzymatic biosensor system to determine the H value as a fish freshness indicator. A similar application was proposed12. and Hx using a cellulose triacetate membrane have been described. M. cellulose triacetate. Agric. Formed Hx was measured with an amperometric sensor that detected uric acid + hydrogen peroxide in an additive matter. and then after adding a soluble NP. Technol. Quinta. 7. Ino. Food Sci. Eur. Saito.E. Özogul. Lugo-Sánchez. 2005. In the measurement of hypoxanthine.41 preactivated nylon. Paredi. IMP. Nucleotide degradation in sardine (Sardina pilchardus) stored in different storage condition at 4°C.48 IMP. Thus. A new method for estimating the freshness of fish.A.Nucleotides and Nucleosides ◾ 65 trode at a platinum cathode (−0. Biochemical basis of postmortem nucleotide catabolism in cod (Gadus morhua) and its relationship to spoilage. Fish. J. 5. Nunes.18 and a silk fibron membrane in combination with a cellulose acetate membrane42 or a nylon net43 have been used. F. Most of these supports have been developed with the aim of eliminating interferences due to ascorbic acid. 41: 341–353.55. 6.54 In fact. K. 1 mol of Hx would be converted to 1 mol of uric acid and 2 mol of hydrogen peroxide. 2001. J. Int...L. and.. 1959. Matsuyoshi. The consumed oxygen produces a current decrease that can be correlated to the concentration of Hx. An immobilized NT was used for the previous conversion of IMP to Ino. T. 2. LeBlanc. 1: 13–19. Both Hx and X are substrates for the XO action and will be oxidized either simultaneously or sequentially. 2007. J. This method was patented by Luong.. 4.. Arai. T. 24: 749–750. D. and H values. 2000.. 212: 141–146. Bull. and Hx amounts. M. some authors have described this type of biosensor coupled with an oxygen electrode. 2006. Robles-Burgueno. Sci. A.34 to obtain the Ki parameter as a freshness indicator. Jpn. 1988.44 a polyaniline film by electropolimerization. J.J. Palacios. or H2O2.9 V) vs.. different supports have been used for the immobilization of the XO enzyme.

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

...... 77 6.............................3.......................................................................................2 Analysis of Sterols .................................................................. 73 6....1.....4.................2 Stereospecific Analysis of Lipid Classes ..................................3........... Aubourg Contents 6............................3 Column Chromatography................................... 73 6..1 Fatty Acid Methyl Esters Preparation ......................1..............2............1.............................2 GLC Analysis of FAME...............5..........1 Spectrophotometric Assessments of Total Lipid Extract ...................2............1 Acid-Catalyzed Esterification and Transesterification .......................2........................................................................ 72 6............2 Removal of Nonlipid Contaminants .76 6.3 Lipid Manipulation and Storage .....................76 6...............3 Lipid Analysis in Marine Products ..................... 73 6.............. 73 6...2.3...........1 Qualitative Analysis of Fatty Acid Composition .5.......................................................................76 6.1 Isolation of Lipids from Tissues ..............3 Marine Fatty Acid Analysis ............2 First Steps in Marine Lipid Analysis . 70 6....... 79 69 ..................................................................... 72 6......................................2 Quantitative Estimation of Fatty Acid Composition .4 Analysis of Marine Nonsaponifiable Matter ................................................................ 75 6........ 72 6...............................................................................3 Analysis of Ether Lipids .......................................................................................5.........................................................3........1 General Aspects of Lipid Compounds ................................................. 78 6...................1 Introduction .....Chapter 6 Lipid Compounds Santiago P................ 75 6....... 78 6...................76 6............................ 71 6.................2.. 70 6.................................... 70 6................................4 Lipid Quantification ....................................................3.4.....................................2....3..........1 Lipid Saponification.................................... 77 6.................... 73 6..1...............2 Base-Catalyzed Transesterification ...............................................2 Marine Lipid Characteristics ...5 Qualitative and Quantitative Analyses of Marine Lipid Classes .................................................4...................................................... 75 6.................1..............

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

differences according to the type of muscle and its location. In all cases. The approach to the analysis of lipids in a given marine sample will depend on the amount of material in the sample. but mainly the amount of information required.Lipid Compounds ◾ 71 relationship between unsaturated fatty acid content and environmental temperature has been confirmed for many marine fish. With respect to endogenous effects. age. the equipment.1. and instrumentation available. Thus.1 Basic steps to be carried out for the lipid analysis of marine products. sex. 6. .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. 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. lipid matter has been described to exhibit a heterogeneous distribution throughout the body of marine species. content variations have specially been observed in fish locations to be employed as lipid depots. 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. and sexual maturation have been pointed out. A basic protocol procedure is exposed in Figure 6. probably affected by physiological and anatomical factors.1.

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

if not. in general. Their measurement by gas–liquid chromatography (GLC) is the most commonly used method for lipid analysis.16]: acid catalysis and base catalysis. Then. application of nuclear magnetic resonance (NMR). since plasticizers are very easily leached out. Small volumes of solvent can be evaporated by carefully directing a stream of nitrogen onto the surface of the solvent.3 Marine Fatty Acid Analysis 6. the Soxhlet method of extraction has been developed [13].2. 6. leading to selective losses of a proportion of the less polar constituents. a large diethyl ether volume is employed. 6. Conversely. glass is the best choice. provided water absorption onto the dry extract lipid is avoided. Natural tissue antioxidants. afford some protection to lipid extracts. since PUFA will oxidize rapidly in air [2]. and the resulting lipid extract can no more be employed for further analysis. Storage temperature should be −30°C as the highest temperature. This method proved to be accurate in the case of a high lipid content (low complex lipid content). As storage containers. When it is necessary to concentrate lipid extracts. fatty acid methyl esters (FAME) obtained are usually introduced in the GLC system without previous removal of contaminants.3.4 Lipid Quantification For most common purposes. should not exceed about 40°C. In it. a known aliquot of the purified lipid extract is softly heated and the resulting dry lipid matter weighted.2. such as tocopherols.3 Lipid Manipulation and Storage Wherever possible. In addition. Autoxidation of double bonds in marine lipid fatty acids is particularly troublesome.1. Lipid extracts have to be converted into methyl ester derivatives. but it is usually advisable to add further synthetic antioxidants to storage solvents at the level of 50–100 mg/L [2].3.Lipid Compounds ◾ 73 6. relatively important errors are obtained. and Fatmeter measurements is proving to be of increasing interest [14]. fatty acids .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.1 Fatty Acid Methyl Esters Preparation Fatty acids are essential components of lipids. this analysis is more complicated than that with other kinds of living organisms. Two basic strategies can be applied [15. Owing to the wide variety of fatty acid compounds in marine lipids (Table 6. it has been shown that lipids can themselves dissolve in some plastics. and care must be taken at all steps in the analysis of lipids. near-infrared (NIR) spectrometry. According to the special relevance recently acquired by noninvasive technologies. For fast purposes. large volumes of solvents are best removed by means of a rotatory film evaporator at a temperature that.1). 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. Plastic ware of all kinds (other than that made from TeflonTM) can be specially troublesome and is best avoided. 6. lipids should be handled in an atmosphere of nitrogen.

17-Eicosapentaenoic 7.10.14.13.8.” .74 ◾ Handbook of Seafood and Seafood Products Analysis Table 6.16. the double-bond configuration is “cis.12.15-Octadecatrienoic 6.8.12.9.12-Octadecadienoic 9.19-Docosahexaenoic Linoleic Linolenic Stearidonic Araquidonic EPA DPA or clupanodonic DHA or cervonic In all cases.16.7.10.1 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 5.11.14-Eicosatetraenoic 5.11.13.19-Docosapentaenoic 4.

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

such as picolinyl esters.4. On the other hand. nonadecanoic acid is employed and added before the methylation step. In most cases. Problems of measuring this area arise mainly when components are not completely separated. cyclic.1 Lipid Saponification Lipids may be hydrolyzed (saponified) by heating them under reflux with an excess of dilute aqueous ethanolic alkali. the nonsaponifiable layer will contain any long-chain alcohols and sterols originally present in the lipid sample in the esterified form.” 6. [22]. unsaturated. For a complete analysis. 6. the resulting FFA have to be transformed into their corresponding FAME for further analysis by an acid-catalyzed method. within limits. commercially available standard mixtures containing accurately known amounts of methyl esters of saturated. as these are easily prepared and are widely used in chromatographic analysis. Finally. pyridinecontaining derivatives. On the other hand.4. A known quantity of an internal standard should be added to the lipid sample. According to the previous section. branched. and polyenoic fatty acids should be analyzed under the same GLC conditions for checking the quantification results. the fatty acids on one side and diethyl-ether-soluble nonsaponifiable materials on the other side are separately recovered for further analysis [2].2 Analysis of Sterols Sterols are biological compounds.2. or oxygenated chains. as well as the deacylated residues of ether lipid compound. linearly proportional to the amount (by weight) of material eluting from the columns [15. have been shown to be suitable for direct mass spectrometric structural analysis of acids containing straight. However.2 Quantitative Estimation of Fatty Acid Composition With reliable modern gas chromatographs equipped with flame ionization detectors (FID). sterols can be fractionated and analyzed by means of different . Such compounds can be divided into sterols and “ether lipids. 4. the areas under the peaks on the GLC traces are.16].76 ◾ Handbook of Seafood and Seafood Products Analysis In recent years. If necessary. 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]. this is specially relevant for PUFA. calibration factors may have to be calculated for each fatty acid component to correct the areas of the relevant peaks in the mixtures analyzed. A high proportion of the available data has been obtained for the methyl ester derivatives of fatty acids. the basic structure of which includes the cyclopentanophenanthrene ring. Total sterol content can be determined directly and spectrophotometrically from the lipid extract by using the method of Huang et al.4 Analysis of Marine Nonsaponifiable Matter 6.4-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]. based on the Liebermann–Buchardt reaction. monoenoic.3. 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.

Great attention has been accorded to the assessment of cholesterol oxide formation in marine products [29]. such compounds tend to be decomposed during GLC analysis and are best reduced by catalytic hydrogenation to alkylglycerols.4%) in 2M HCl. Although the vinyl ether linkage is unaffected by basic hydrolysis conditions. Adsorption thin-layer chromatography (TLC) on silica gel layers can be used to separate simple alkyl and alkenyl lipids. different analytical . supercritical fluid chromatography (SFC) has been employed for the glycerol ether analysis of liver oils of shark species [34]. such as acetate. Methods for separating and quantifying ether-linked glycerides have been reviewed [31. Although the GLC is normally carried out with cholestane as internal standard. Often. For GLC analysis. and 18:1. 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. information on ether lipid composition in marine PL is less abundant. 6. Concerning alkenylglycerols. but they suffer from the limitation of the lack of a distinctive chromophore in the analyte.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]. batyl. it can be cleaved by acid-catalyzed transmethylation. or they may include alkyl and alkenyl radicals. high-performance liquid chromatography (HPLC) methods can offer a nondestructive alternative. 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]. chimyl. thus. which in turn migrate just in front of TG.32]. no single procedure will achieve the desired analysis. The alkyl groups of 1-alkyl-2. v/v) as a solvent system.” Such compounds are basically found as PL classes (specially in phosphatidylethanolamine). and selachyl alcohols were found to be the most abundant. and its analysis has already been discussed in Section 6.3-diacyl-sn-glycerols are generally saturated or cis-monoenoic even– numbered components (16:0. and combinations of techniques must be used until the required purposes are served. being normally placed as the radical in position 1 and specially abundant in marine invertebrates [5. trifluoroacetate.3 Analysis of Ether Lipids Marine lipids may contain fatty acid residues as the only radicals. or isopropylidene derivatives by GLC. although a great interest has been accorded to their isolation because of their medical and cosmetic applications [30].27]. neutral plasmalogens tend to migrate ahead of alkyldiacylglycerols. The first type is the major one in marine lipids. They can be separated by a double development in a single direction with hexane–diethyl ether (95:5. although invertebrates have shown a significant presence of other sterols [27]. 6.4-dinitrophenyl-hydrazine (0.Lipid Compounds ◾ 77 chromatographic techniques [23. alkenyl compounds have been directly identified and quantified by GLC together with FAME [35]. Accordingly. Neutral plasmalogens may be detected by spraying the TLC plates with 2. The alkyl moieties are usually analyzed in the form of 1-alkylglycerol or as volatile nonpolar derivatives of this compound.3. Chromatographic methods for cholesterol analysis [28] are of relevant importance in foods in relation to human health concerns. Further. In this section.24]. Cholesterol has been shown to be the most abundant sterol in all marine living beings.4. Unlike fatty acids. mostly). 18:0. TMS. whereas no simple spot test is available for the identification of alkyldiacylglycerols. The others are often united into a group called “ether lipids.

The advent of new NMR. although it turned out not to be accurate enough for marine lipids. 6. in it. this including chromatographic separation and further analysis of fatty acids after previous methylation and transmethylation. traditional methodologies are still employed in cases where such advanced technologies are not available and are reviewed in this section.3. An alternative and successful method has been proposed by Raheja et al. . In it. HPLC. such functional groups are made to react with hydroxamic acid and further complexed with Fe (III). preparative TLC on silicic acid impregnated with 5% (w/w) boric acid has been applied to prevent acyl migration during chromatographic separation. where an FFA-cupric acetate-pyridine complex is involved. 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.2 Stereospecific Analysis of Lipid Classes The determination of fatty acid composition at each location in lipid classes has ever since attracted great attention. according to the information provided in following sections. This method can be applied to total lipid extract or to any lipid class after previous isolation [7.5.4. The fatty acid composition of each lipid class can be determined by GLC of the methyl ether derivatives. and GLC technologies combined with MS in the last decades has provided quick and useful procedures for the stereospecific lipid analysis. a method for the quantification of esterified and unesterified total sterols is mentioned in Section 6. More recently. prepared by esterification or transesterification of the purified lipid class. Traditional determination of PL content in lipid extracts has involved the digestion of PL with the release of inorganic phosphate. PL present in the lipid extract are made to react with ammonium molybdate in an organic phase and then measured spectrophotometrically. the Grignard reagent has widely been employed in the case of marine substrates [42]. a rapid NIR spectrometry has been applied for the direct FFA determination in fish oil [37]. In many cases. these enzymes can be isolated and used in simple incubations in vitro as an aid in structural analyses of lipids. giving rise to a tremendous number of species. titrimetric methods were used for many years. A wide use was found for lipase hydrolysis.5. then. Finally.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. although some interference of polar lipids was found. 6. However.41]. according to details explained in Section 6. focused on the qualitative and quantitative analyses of marine lipid classes.78 ◾ Handbook of Seafood and Seafood Products Analysis approaches will be discussed. since the presence of double bonds in the proximity of a carbonyl group of fish PUFA reduces the rate of deacylation of glycerides. This is due to the great complexity of fatty acids present in these oils and fats. Ester linkages can be quantified by the method of Vioque and Holman [40]. the results so far reported for aquatic animals are few. A very popular one is that proposed by Lowry and Tinsley [36]. [39] without previous digestion. this is made to react with ammonium molybdate to form phosphomolybdic acid.2 [22]. Accordingly. For FFA assessment. Additionally. Compared with the data compiled for plant oils and for fats from land animals. Procedures that involve spectrophotometric measurement of highly colored copper complexes are now favored. which is reduced and determined spectrophotometrically [38].

identification.5. Such stereospecific studies have widely been focused onto TG [43.46] classes. The perceived weakness of TLC has been recognized as the quantification aspect. whereas complex lipids are recovered by elution with methanol [41.49]. and quantification [48. and this has led to the evolution of the TLC/FID Iatroscan system. MS. HPLC has undoubtedly been the most widely applied separation technique in the analysis of most simple and complex lipid classes [48. although lengthy conditioning may be necessary before columns can be employed [2. For preparative purposes. The improvement and versatility of TLC enable it to be used for several modern applications.4 Thin-Layer Chromatography Many text books and reviews report TLC application on lipids for routine separations. being simple lipids eluted in a stepwise sequence with hexane containing increasing proportions of diethyl ether. which has been used routinely for lipid analysis in the last decades. diethyl ether.44] and PL [45.5 High-Performance Liquid Chromatography In recent years. HPLC is much more expensive than .47]. 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. Aminopropyl-bonded phase cartridges have been much used for the isolation of simple and complex lipid fractions. In all cases.5 mm thickness [7. coupling of TLC with other techniques such as HPLC.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. Separation can be carried out on silicic acid. 6. although particular care is required to recover the acidic lipids quantitatively. or florisil as adsorbents.41]. The system has been successfully used for marine lipid class analysis [51]. and acetic (or formic) acid in various proportions. 6. 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. Those used most frequently contain hexane. 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. In addition. overpressure TLC (OPTLC). which include highly automated techniques right from sample application and development to detection and quantification. and NMR has increased its analytical power in several applications. 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. infrared (IR) spectrometry. acid-washed florisil. A variety of solvent systems have been used to separate simple lipids on an analytical or semipreparative scale by single or two-dimensional TLC.52].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. 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. However. Such techniques would include high-pressure TLC (HPTLC). 6.5.47]. in spite of the relatively higher costs [50]. precoated plates are much more convenient than laboratory-made plates.

6. On the other hand. 6. high-resolution NMR spectrometry (1H-NMR. quantification and stereospecific analyses have been carried out. . 31P-NMR) has increasingly been applied to the identification of lipid structures to determine patterns of branching.54]. such complexation is favorable for use in chromatography and enables the performance of the various Ag+-chromatographic techniques developed so far. An isocratic and gradient elution procedure with ultraviolet (UV) detection has been employed for marine PL analysis. Thus. HPLC has specially been applied to the most abundant lipid classes. HPLC analysis has been accepted as the most accurate one.7 Nuclear Magnetic Resonance (NMR) Spectrometry In recent years.5. Ag+-TLC is used mostly in the preparative mode.6 Silver Ion Chromatography Silver ions. Thus. 13C-NMR. Both homemade and precoated glass plates are used in Ag+-TLC. Ag+-chromatography has been performed in conjunction with CC. while no oxidation of the unsaturated fatty acid constituents needs to occur during fractionation on an HPLC column. TG separation according to the carbon number or partition number has been achieved [53]. Thus. and HPLC. In the detection. as a complementary separation method to GLC or GLC-MS. However. some of the more impressive separations have made use of FID systems. evaporative light-scattering detection has successfully been applied [16].5. therefore. like the ions of other transition metals. The complexes are usually unstable and exist in equilibrium with the free form of the olefin. The usual supporting materials are silica gel G for FAME and TG and silica gel H for complex lipids. 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].80 ◾ Handbook of Seafood and Seafood Products Analysis TLC in terms of both equipment and running costs. with UV detection at 206 nm both on an analytical and on a preparative scale. Ag+-HPLC and reverse phase (RP)-HPLC applied in complementary ways were effective in the analysis of TG in fish oils [57]. For PL classes. and often the location. interact specifically with the olefinic double bonds of unsaturated compounds to form weak charge transfer complexes. and in particular to the detection. but it can be automated to a considerable degree and gives much cleaner fractions in micropreparative applications. Finally. but others have obtained satisfactory results. The procedure is rapid and nondegradative. In the past 20 years. of the double-bond systems in fatty acid chains. Perona and Ruiz-Gutiérrez [53] were able to resolve a large number of sardine TG molecular species by RP-HPLC. further identification of most peaks was carried out by using preparative Ag+-TLC followed by fatty acid analysis by GLC. or substitution. so it has become an extremely powerful technique for obtaining qualitative and quantitative information of the lipid class profile of a marine tissue extract. by employing both gradients of polar solvents and microparticulate silicic acid [6. It can give better and more consistent separations of minor components. TLC. some important articles and reviews have been published [58]. being successfully applied to all lipid classes in marine species by separating molecules according to unsaturation degree [55].

Over the years. and highly unsaturated fatty acids of lipid extract of Atlantic salmon muscle.and diene-. Among the different food lipids.8 Mass Spectrometry MS has long been used as a powerful tool for the analysis of the molecular weight. A good agreement could be observed between NMR values and those from the GLC analysis. and complete structure of an unknown compound. its intensity should be proportional to the quantity. the high-resolution NMR spectra of four fish oils were recorded. marine lipids have received lesser attention.86 ppm) provided the possibility of proposing this new analytical tool. a rapid and structure-specific method for the determination of ω3-PUFA in fish lipids was presented. This development paralleled the development of atmospheric pressure chemical ionization (APCI). The positional distribution (1. mono.Lipid Compounds ◾ 81 Based on 1H-NMR spectrometry [59]. . Application of 31P-NMR has shown to be far shorter than with 1H and 13C. Thus. EPA. ω6. The different chemical shifts observed for the methyl resonance of ω3-PUFA (δ = 0.65.66]. In a first attempt for 13C-NMR application [60]. Quantitative analysis of fatty acid composition and alpha-beta distribution in TG tuna fish was achieved [62]. Later on [61]. the molecules or their fragments can be separated and identified on the basis of their mass-to-charge ratio (m/z). empirical formula. 2. soft ionization MS techniques such as fast atom bombardment. After different approaches. Following ionization to a negatively or positively charged species (most commonly the later). The first step for any MS method is ionization of the sample molecules in the gas phase. although an increasing importance has been obtained lately for quantitative analysis [20. Some applications concerning the marine lipids’ study will now be mentioned. according to each corresponding resonance. 6. although GLC is conveniently coupled to electron impact ionization (EI) and chemical ionization (CI) sources. 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. Recent developments in MS have been very interesting for complex lipid molecules.5. ω3. The 31P-NMR application has also shown the possibility of analyzing the ether structures within the glycerol backbone of phosphatidylethanolamine and phosphatidylcholine. many of the advances in MS have involved new ionization techniques. Signals in the spectra were assigned. FFA carbonyl resonances were detected at the lower field of the carbonyl region. probably due to their more complicated structure. results obtained using high-resolution 13C-NMR were in good agreement with those obtained by GLC. The information-rich nature of MS makes it the most desirable detector for many explanations. It could be observed that DHA was concentrated in the 2-location of TG in depot fats. and stearidonic acid.95 ppm) with respect to the methyl resonance of all other fatty acids (δ = 0. thus providing a suitable tool for lipolysis analysis. and 3 locations) of ω3 fatty acids in depot fat of several fish species was examined by 13C-NMR [63]. and electrospray have the ability to ionize lipid molecules without causing extensive fragmentation. 13C-NMR was employed for the plasmalogen analysis in fish lipid samples showing a good agreement with the data obtained by GLC [64]. Arpino [67] likened the HPLC-MS union. so little application is specially available for marine lipids [58]. and attention was focused on the identification of specific signals for ω3 fatty acid group and also individually for DHA. 13C-NMR spectrometry was successfully used to determine the proportions of saturated. but. thermospray. the condensed mobile phase used for liquid separations is not readily compatible with high vacuum ionization sources. Finally. This NMR technique can provide a single signal for each PL class. Thus.

eds. the use of SFC can substantially reduce the dependence on organic solvents in solvent extraction or HPLC analysis. 1982. The liver oils of several shark species were analyzed by SFC [34].. p. Nutritional aspects of fish.4. 17. Carbon dioxide is by far the most commonly used SFC mobile phase because of its low critical temperature. The qualitative and quantitative compositions of 1-O-alk-1-enylglycerolipids of albacore tuna (Thunnus alalunga) were studied along the canning process [35]. Purification of PUFA (DHA and EPA) ethyl esters from tuna oil was carried out by SFC [74]. The Physical Chemistry of Lipids.. the method was based on the use of preparative reversed-phase HPLC followed by subsequent identification by APCI HPLC-MS. Plenum Press. D. 2nd edn. J. and Oehlenschläger.. A. whereas its critical pressure and critical density are high enough for good solvation of many potential analytes. The mass spectrometer was operated in the EI mode (70 eV). Christie. U. cholesterol esters. simple classes from marine oils of different species were separated and quantified by capillary SFC [73].. and diacylglycerol ethers required TLC fractionation before SFC analysis. and the four most unsaturated fractions were analyzed by capillary SFC according to their acyl carbon numbers [72].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]. whereas quantification of TG.. analysis was carried out in conjunction with FAME by means of their dimethyl acetal derivatives resulting from the acid transmethylation of lipid extracts. p. New York. W. Integrated Approach to Quality. Simopoulos. Vol. 6. 3. thus. Elsevier Science. the method was capable of direct quantification of squalene and cholesterol. 1997. analytes are eluted from a capillary chromatographic column.. U. in Seafood From Producer to Consumer. carbon dioxide as the mobile phase.K. J. Concerning marine species analysis. Minor fatty acids from mussels (Mytilus galloprovincialis) were enriched by Ag+-TLC and then analyzed by GLC-MS as 2-alkenyl-4. 589. Analytical SFC has been shown to be particularly applicable to the analysis of higher molecular weight lipid moieties. Later on. a nonpolar capillary column. such as mixed glyceride compositions ranging from 200 to 900 in molecular weight. London.9 Supercritical Fluid Chromatography In this advanced technique [10.. A wide range of cholesterol oxides were identified and quantified. . 89. which uses a highly compressed gas above its critical temperature and critical pressure. Small. Luten. and several nonmethylene interrupted fatty acids were singled out.K. 4.71]. Handbook of Lipid Research. References 1. In addition.. T. p.-dimethyloxazoline derivatives [69]. Börrensen. an optimization of process parameters was achieved to obtain a maximal production rate. Lately. 2. An important advantage is that it is compatible with FID. 1986. 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. Oxford.. Pergamon Press.5. and a FID were employed in it.. Lipid Analysis. which has great sensitivity and linearity. in a first attempt Baltic herring flesh TG were separated in eight fractions by Ag+-TLC.

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

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

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

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.... This can lead to 87 ........ 92 7...............4 Instrumental Methods .......................................2......................................... 88 7..2......................... 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].......................................1 Introduction Marine lipids are good and natural sources of polyunsaturated n-3 fatty acids (PUFA) such as docosahexaenoic acid (DHA.....1 Primary Oxidation Products .......3 Stability Methods ........................................................................... due to the high content of long-chain PUFAs...... secondary oxidation products.....2........................................... 89 7...................2]................................... 87 7........................... 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.........2 Analysis of Lipid Oxidation ........2................ 88 7................. However.5 Sensory Analysis of Rancidity ............................................................ 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................ except when microbial processes limit the shelf life...................... 22:6n-3) and eicosapentaenoic acid (EPA......................... 92 7.............................. 20:5n-3)................................ The volatile...... 93 References .....................3 Summary .. 93 7................................................2 Secondary Oxidation Products ......................Chapter 7 Lipid Oxidation Turid Rustad Contents 7..... marine lipids are highly susceptible to oxidation.................................................................................................................2................1 Introduction .............. Reaction products from lipid oxidation have a negative effect on the sensory properties of fish products.............................. 93 7.....

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

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

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

Lipid Oxidation ◾ 91 identified [21]. However. quenching.1 Excitation and Emission Maxima for Chromophores Formed as a Result of Oxidized Lipids. When fluorescence measurements are done on samples in solution. deoxyribonucleic acid (DNA). peptides. and the amount of sample and sampling conditions can be varied according to the needs. or reactions between oxidized lipids and DNA have different excitation and emission maxima as shown in Table 7. Aubourg and Medina [26] extracted fish muscle with a 2/2/1. for assessment of lipid oxidation during fish processing [24–26]. quantification of headspace data. This reaction can lead to formation of brown-colored compounds [22. front-face fluorescence Table 7. Small variations in sampling procedures can give large variations in the data. The fluorescence shift was found to be a more effective index of changes in fish quality than other commonly used methods. The advantages of this method are that it is flexible. nucleic acids. 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. Instead. Analysis of volatiles is discussed by Ólafsdóttir and Jónsdóttir in Chapter 8.23]. the data handling is also difficult. and the results are dependant on the sample material. reactions between oxidized lipids and proteins/ peptides. Fluorescence has traditionally been applied to samples in solution. The different chromophores formed as a result of oxidized lipids. Hydroperoxides (primary lipid oxidation products) and aldehydes (secondary oxidation products) can react with amino groups in proteins. forming Schiff bases. Fluorescence techniques are highly sensitive and 10–100 times more sensitive for detection of MDA than TBARS [3]. 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 . especially from solid matrixes. scatter. Lipid oxidation products can interact with other components in food. and so on. destroy this relationship. and so on. The fluorescence intensities were divided by the fluorescence intensity of quinine sulfate and the fluorescence shift calculated. for example.8 chloroform/methanol/water mixture and measured fluorescence both in the water and in the organic phase.1. the measured intensity follows the Beer–Lambert law. proteins. phospholipids. They measured the fluorescence intensity both at 393/463 nm and 327/415 nm. and the concentration is below a certain level. such as amino acids. and form fluorescent products. is complicated. When the samples are turbid or solid or the concentration is high. as measured by methods such as PV and TBARS. Reactions between lipid oxidation products and other components in seafood or seafood products may lead to underestimation of the degree of lipid oxidation.

4 Instrumental Methods Many instrumental methods have been developed for the determination of oxidation parameters in oils and foods. the Rancimat test [30]. The change in conductivity is measured. and the point where it changes most is called the induction time. Fluorescence spectroscopy has a great potential for on-line or at-line applications. such as different hydroperoxides. and Oxidograph are techniques for measuring the stability of oils toward oxidation. adipose tissue. the oil can be heated to 80°C or more while air is bubbled through it. So far.2.33]. In recent years. and a few minutes of oxidation of docosahexaenoate (DHA) resulted in significant changes in the ESR spectra.92 ◾ Handbook of Seafood and Seafood Products Analysis spectroscopy can be used. new methods have been developed. One challenge is that fluorescence spectra can be very complex and that not only the oxidation products but also connective tissue. active oxygen method (AOM). [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. Using solid-phase fluorescence is a relatively new approach. but it measures the time taken to reach a certain PV. aldehydes. little has been done to study the fluorescence spectra of the different oxidation products that are formed in foods. including near-infrared spectroscopy (NIR). obtaining information . Fluorescence spectroscopy on intact samples has been shown to be a sensitive technique. The level of hydroperoxides in fish oil can be determined using a rapid CL method [14]. and FT-IR spectroscopy methods [16. 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. Lipid oxidation products can produce very weak chemiluminescence (CL). 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]. oil stability index (OSI).32. and additives may contribute to the spectra. It has been shown that sodium hypochlorite-induced decomposition of hydroperoxides gives strong CL [34. Fourier-transform near-infrared (FT-NIR). 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 Oxidograph instrument finds the induction time based on measurement of the decline in pressure caused by the absorption of oxygen in a closed vessel. porphyrins. comparable to sensory analysis and gas chromatography. In Rancimat and OSI instruments.2. In a study of different model systems including fish and meat.3 Stability Methods Several techniques based on accelerated oxidation are used for evaluation of oxidation. The AOM method is performed in a somewhat similar way. Veberg et al. The gas chromatography–mass spectrometry (GC–MS) techniques can be used to determine a wide range of volatile secondary lipid oxidation products [36]. these include the oil stability index method [29]. The liquid chromatography–mass spectrometry (LC–MS) techniques can also determine nonvolatile products—of special interest are the core aldehydes [3. and also cyclic compounds. and oxidative stability measurement by Oxidograph [31] and they are all suitable for analyzing oil systems.37].35]. but studies on the use of this technique in dried fish were published in 1992 [27]. for measuring lipid oxidation [28]. The Rancimat. 7. Free radical assessments by the ESR spin-trapping technique detected the very early stages of lipid oxidation. 7. 1H NMR spectra can be used to study specific lipid oxidation products.

Biol.A. E.. A trained panel can be a very valuable tool for detection of early lipid oxidation of foods containing n-3 fatty acids. Sloan Stanley. and inflammatory disease. C. sensory analysis requires relatively large amounts of samples. 777–795. In addition. their use is limited by the cost of employing a trained panel... Lipid Oxidation.3 Summary Many different methods for the analysis of lipid oxidation exist. 2005.5 Sensory Analysis of Rancidity The ultimate measurement of rancid odor and taste is sensory analysis by a trained panel. Dietary fat. and M. 226: 497–509. Miayshita. Marcel Dekker: New York.01 nM). 2. Folch. 1957.. J. In general. However. Lipids from residual fish raw material. the sensitivity was low (detection levels ∼0.. There is. Int. The sensitivity could be improved by the use of CryoProbe technology. Narayan. 7.Lipid Oxidation ◾ 93 that cannot usually be obtained by single conventional analytical methods [4]. Hosakawa. 2005. even if there are many different methods that are used to determine lipid oxidation. 5. through the nose (nasal) or through the mouth (retronasal).. Chem. Even if sensory methods can give sufficient information. 2nd ed. 3. Chow. Ed. References 1. 4. pp.. intermediary goods. Food Rev. 7. G. Physiological effects of eicosapentanoic acid (EPA) and docosahexanoic acid (DHA)—A review. Lees. Norwegian University of Science and Technology: Trondheim. 2005. in Department of Biotechnology.H. in Fatty Acids in Foods and Their Health Implications. a rapid development in analytical methods to determine lipid oxidation.. The detection of these low levels is not straightforward with classical lipid oxidation measurement methods. B. and G. The Oily Press: Bridgewater. J. 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. the oxidation products from n-3 fatty acids have a lower sensory threshold than those of oxidation products from other fatty acids.N. today it is not possible to use only one method to determine lipid oxidation. M. Boissonneault.K. 206. 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]. 2006.. and the use of chemical and instrumental analyses is recommended to support and complement the sensory analysis [3].K. immunity. However. however. but for many of these methods calibration and verification are needed before they can be used for routine analysis. However. U.. 22: 291–306. A simple method for the isolation and purification of total lipids from animal tissues. It can also be difficult to compare data from different panels using different vocabularies or data from the same panel analyzed at different times. for many of these methods the results obtained vary not only with the method used but also with the analytical procedure that is performed. Frankel. Falch. 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. so care should be taken in standardizing the procedures. . 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. 2000. E. and finished products during seafood processing. K.

9. 46: 3662–3666. D... Ueda. Anal. 79: 1943–1948. Sci.. Microdetermination of thiobarbituric acid values in marine lipids by a direct spectrophotometric method with a monophasic reaction system.. . and M. 160. 17. 106: 279–284. and C..D. 1995. Wold. AOCS. IL. Food Agric. Basics. and W. Food Sci. Ed. Firestone. Bligh.C. Type V collagen in trout (Salmo gairdneri) muscle and its solubility change during chilled storage of muscle. 13. Halliwell. Chem. 32: 497–502. Tironi. Firestone. 16. W. Can.. 78: 441–450. Effect of ascorbic acid in a model food system. Firestone. 19.A. 39: 562–570. 1999.. et al. 1995. 1986... AOCS: Champaign. Woyewoda. 22. Dyer. and K. K.. 37: 911–917. Sci. AOCS: Champaign. J.. Hübschmann. H. M. in Official Methods and Recommended Practices of the American Oil Chemists’ Society. Hayahashi.: New York. J.. Food Agric. in Food Chemistry. in Handbook of GC/MS-Fundamentals and Applications. Oxidative deterioration in dried fi sh model systems assessed by solid sample fluorescence spectrophotometry. Hasegawa. Lipid damage detection during the frozen storage of an underutilized fish species. Rapid assessment of rancidity in complex meat products by front face fluorescence spectroscopy.. Physiol. Lipids. 1996. Technol. Wiley-VCH Verlag GmbH: Weinheim. S. Influence of skinning on lipid oxidation in different horizontal layers of herring (Clupea harengus) during frozen storage. J.W. Biochem. Food Chem. Comparison of wet-chemical methods for determination of lipid hydroperoxides..J. Method Cd 8-53. Veberg. E. J. Food Res. 65: 307–313. M. 21.. AOCS. and M. Y. Gallardo. Ed. of Chemistry. Fourier transform infrared spectra data versus peroxide and anisidine values to determine oxidative stability of edible oils. 1977.M. 1994. Fujimoto. 1999. 39: 1222–1225. Aubourg.. Analysis of early lipid oxidation in foods with n-3 fatty acids. I. Biotechnology and Food Science. E.. 50: 1–7. J. and N. Food Lipids. M. AOCS Official Method Ti 1a-64.94 ◾ Handbook of Seafood and Seafood Products Analysis 6. 10: 35–50... Aubourg. J.P. pp. in Official Methods and Recommended Practices of the American Oil Chemists’ Society. IL. and H. D. 7. J. Food Agric. Olsen. 12. Quality assessment of blue whiting (Micrometistius poutassou) during chilled storage by monitoring lipid damages. 23. 1959. 2001. J. Food Chem. Stading. D. LWT-Food Sci. 2002. Guillen.G. 27.C.. Chemiluminescence of fish oils and its flavour quality. Agric. 26. 28. J. V. Fennema. P. A. Endo...S. J.J... Norway. and J... Acta. M.P. S. 77: 503–510. Cabo. 20. 1998. 24. Medina. 27: 389–393. Ed. Ed.-J. 11. B.. The measurement and mechanism of lipid peroxidation in biological systems. 1990. J. O. M. Pettersen. Agric. Structural and functional changes in myofibrillar proteins of sea salmon (Pseudopercis semifascata) by interaction with malonaldehyde (RI). K. Marcel Dekker Inc. IL. 67: 2397–2404.. 2005. J. AOCS. Food Chem. 1991. 10. 15: 129–135. 1995. 2002. Norwegian University of Life Sciences: Ås. Anon. Aubourg. 1992.P. Agric. in Dept. Interaction of oxidised lipids with protein. Method Cd 18–90. Trends Biochem. and J. Timm-Heinrich. 2003. 25.. Sci. Medina.. 2002. A rapid method of total lipid extraction and purification. Pokorny. et al. AOCS: Champaign. Gutteridge. Namiki.. Ke. Sci. Tomas. N. 1998.. Influence of storage time and temperature on lipid deterioration during cod (Gadus morhua) and haddock (Melanogrammus aeglefinus) frozen storage.. 14. Int. Fluorescence in aldehyde model systems related to lipid oxidation. Jacobsen. pp. Food Sci. Lignert. 15. J. S. 1979. S. and A. Nawar.M. Biol.R. 18.. and I. and J.C. 7–212. Vogt. 2006. 67: 930–935. Sato. Food Sci. 57: 1123–1126. I. La Rivista Italiana Delle Sostanze Grasse. Chim. Undeland. G. Nielsen. 225–319. Wold. 8.D.

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

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

Enhanced oxidation during cooking resulting in off odor development is of concern and an obstacle for application of fish in convenience food. 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. and accumulation of hypoxanthine (Hx). guanidine compounds like creatine.1 Introduction Health and wellness are the main drivers in new product development. extension in shelf life of fresh chilled fish has been achieved. resulting in undesirable texture changes in fish.1.2 Development of Fish Aroma An overview of changes during handling and processing influencing the development of aroma in fish is generalized in Figure 8. The understanding of odor development by chemical. Fish being a valuable source of polyunsaturated fatty acids (PUFA) and other nutrients is a prominent candidate as the healthy choice for consumers. 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. active inosine. biochemical. followed by oxidation processes. Volatile compounds play an important role in the odor quality characteristics and consumer acceptance of fish. valine. including small peptides such as carnosine and anserine. contributing to spoilage changes and thus influencing the freshness and quality of the end product of chilled fish [1–3]. studies on application of natural antioxidants are of prime interest to underpin further utilization of fish in innovative product development as fresh. However. Endogenous enzyme . amino acids. formation of taste. 8. Some of these compounds influence the taste of fish-like peptides (i. As a result. Research over the years has led to improved chilling and packaging technologies aimed at reducing microbial growth.98 ◾ Handbook of Seafood and Seafood Products Analysis 8. Degradation of soluble muscle constituents such as sarcoplasmic proteins and microbial metabolism contributes to changes in the aroma profile of fish during storage. including degradation of nucleotides. 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]. oxidative processes causing odors and texture changes become noticeable during extended storage and limit the shelf life. Proteolysis plays a critical role in postmortem changes. Finally. and nucleotides. TMAO. the changes are dominated by autolytic activity.e.. They are composed of the various nonprotein nitrogenous components (NPN). the proliferation of the specific spoilage organisms (SSO) results in the development of volatile compounds. and glutamic acid are known to contribute to taste together with the degradation components of the nucleotides such as inosine. A prerequisite for increased consumption of fish products is their availability on the market as fresh and high-quality products of delicate flavor. cooked. leading to the formation of secondary oxidation products and off flavors [8]. processed. or hydrolyzed products and as ingredients in functional foods. 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. anserine). lowering of pH and endogenous enzyme activity. Initially. alanine. and the individual amino acids glycine. Other prooxidants like hemeproteins (hemoglobin and myoglobin) are also involved in the initiation of the oxidative processes in fish muscle [7]. The pool of components that are degraded and cause off flavors because of microbial growth are mainly soluble substances in the muscle.

Some components are desirable at low levels. PUFA. putrid. LOX activity on the skin and gills of both freshwater and marine species (rainbow trout. sour. SSO. specific spoilage organisms.. but the mechanism of this activity is not fully elucidated [9]. On the other hand. and 2. malty. mushroom. and cooking Processing smoking. ascorbic acid. 1-octen-3-ol.10–13]. cucumber.15]. 1. stale. hydrolases. polyphenols Lipids phosholipids/PUFA Proteins sarcoplasmic.5-ocatadien-1-ol. lipoxygenase. The compounds that contribute to the characteristic plant-. Soon after harvest. metallic.5-octadien-3-ol. NPN. melon-. 8. [5] summarized the occurrences of volatile compounds in freshwater and saltwater species and concluded that the four common compounds found in saltwater species. amino acids Fresh fish aroma seaweedy. hemoglobin. nonprotein nitrogen-containing compounds. cucumber Figure 8. NPN.14. hexanal. the unsaturated C9 carbonyl compounds such as 2. plant-. Josephson et al. boiled potato. but . The overall perceived odor is dependent on the level of influential compounds and their odor thresholds along with possible synergistic effects. freezing. LOX. and sardines) plays a role in the formation of odorous volatiles.3 Fresh Fish Odors The delicate flavor of fish is mostly contributed by volatile compounds and taste active substances in the aqueous phase. contributing to green. or nine carbon atoms [4. dried fish. phospholipases TMAOase Microbial metabolism Specific spoilage organisms (SSO) Oxidation Prooxidants: metals (Fe. were characteristic for freshwater and euryhaline fish. including calpains (neutral calcium-dependent proteases) and cathepsins (lysosomal proteases). faint odor of saltwater species. spoiled. drying. whereas volatiles generated from fat result in variation in the specific flavor character of different fish species. caramel. Hb. TMAO. malty.5. and mushroom-like odors are unsaturated carbonyl compounds and alcohols with six. neutral Spoilage aroma sweet. Mb. salting. rancid potato. polyunsaturated fatty acids. eight.6-nonadienal. and hydrolysis Endogenous enzymes i. were responsible for the moderate. activity influences the deterioration of fish muscle. Newly caught marine fish contains low levels of volatile compounds and is nearly odorless.Volatile Aroma Compounds in Fish ◾ 99 Handling chilling. myoglobin. LOX proteases. stockfish. pleasant aromas of fish [6. and melon-like odors. popcorn. cucumber-. and processed fish. ammonia-like Oxidized aroma Processed aroma green-like. oxidized.e. nucleotides. cucumber-. river trout. trimethylamineoxide. which have potent green.Cu) Hb.1 Overview of changes in fish influencing the development of characteristic aroma of fresh. Mb Antioxidants: α-tocopherol. peptides Soluble substances.

Purge and trap on Tenax and SPME methods were applied for sampling. 8. where it has been demonstrated by monitoring key volatiles to study changes in different fish products during storage. and sulfur compounds. Environmental conditions and seasonal effects like spawning can influence the odor quality of fish. boiled potato-. The aldehydes contribute most to the spoilage odors because of their low flavor thresholds. cod during storage [21. The volatile pattern changes in mature salmon when migrating from the sea for spawning. An example is the enzymically derived long-chain alcohols and carbonyls that exhibit characteristic fresh. They were suggested as the possible precursors of nine-carbon volatile compounds.22] and in smoked salmon [23]. Table 8. and species of the salmonidae family develop earthy.Z)-2. which has a very characteristic cucumber odor during spawning.21–22. on accumulation of hydroperoxides in fish tissues. Therefore. a saltwater species. amines. they contribute to oxidized and fishy odors in stale fish [16]. Fatty species develop rancid odors and taste. The main classes of compounds detected during storage are alcohols. and sulfur compounds representing the different changes occurring during storage have been suggested by numerous researchers as indicators for freshness and spoilage [22.4 Identification of Quality Indicators Different characteristic odors develop in various fish species during storage. amines. Accumulation of certain hydroperoxide isomers coincided with the period of enhancement of characteristic aroma in sweet smelt. Another example is iodine-like off flavor in prawns associated with bromophenols originating from the feed chain [17]. 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. ketones. but when accumulated in higher levels because of autooxidation.6-nonadienal. Both single compounds such as TMA and ethanol and multicompound indices based on combination of alcohols. GC–MS. C9 LOX-derived compounds have been found in higher levels in spawning euryhaline and freshwater fish [5]. and amine-like odors.30–36]. and GC–O.6-nonadien-1-ol in sweet smelt tissues [20]. iodine-. Some of the influential odor compounds that have very low odor thresholds are often present in low levels. including (E)-2-nonenal. it is useful to monitor the overall pattern of volatile compounds and select indicator compounds. .100 ◾ Handbook of Seafood and Seafood Products Analysis if their concentration increases. indicate their involvement in the development of fresh fish aroma associated with seasonal variation. (E. and sweet odors. Studies performed in Japan. and identification was based on GC–FID. odor.1.6-Nonadienal was identified to be the most characteristic compound for the cucumber-like capelin odor [19]. plant-like notes in fresh fish. which are present in higher levels and can be quantified. they may contribute to off odors. muddy. in nominal levels the bromophenols appear to contribute to natural sea-. lean species typically develop sweet. for example. and marine-like flavors of seafood [18]. 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. 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. However.24–29]. 2. acids. Seasonal effects have also been reported for capelin. and quality changes can be explained in. esters.1 summarizes the occurrence of volatile compounds detected in our studies on cod [22] and haddock fillets [31] and smoked salmon [23]. and 3. This has been the approach in our studies. aldehydes. and these are difficult to detect by analytical techniques. as seen by the detected odors listed in 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. flowery — Ketones 2-Butanone 2. floral Sweet.4-Heptadienal.E)Nonanal Decanal Undecanal × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × Rancid Boiled potato. caramel. earthy Sweet. fish fillet Sweet.3-Butandione × × × × — N/A (continued) . candy × — — Sweet.1 Volatile Compounds Detected in Cod [22]. fatty — Fresh. caramel.Volatile Aroma Compounds in Fish ◾ 101 Table 8. Haddock Fillets [31].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. (E.

ethylester Butanoic acid. caramel — — — Sweet. S-methylester Propanoic acid. ethyl ester Butanoic acid. ethyl ester 2-Butenoic acid. sour Flowery. ethylester Acetic acid. ethylester Hexanoic acid. dried fish Acid Acetic acid × × × — Esters Ethyl acetate Ethanthiocacid. sweet. spicy Amine Trimethylamine × × × TMA-like.1 (continued) Volatile Compounds Detected in Cod [22]. 3-methyl. ethyl ester Propanoicacid-2-methyl. Haddock Fillets [31]. and Smoked Salmon [23] during Chilled Storagea Compound 2-Pentanone 3-Pentanone 2.3-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. 2-methyl. heavy.102 ◾ Handbook of Seafood and Seafood Products Analysis Table 8. ethyl ester × × × × × × × × × × × × × × × — N/A — N/A N/A Sickenly sweet. 2-methylpropyl ester Butanoic acid. vomit N/A N/A N/A N/A Sulfur Compounds Methanethiol Dimethyl sulfide × × × × — — .

and sulfur compounds produced by microbial degradation of fish components. alcohols. N/A. contributing to sweet. and sometimes metallic. The loss of freshness of cod fillets and early spoilage changes were related to the formation of ketones. and malty odors. and finally sour and dirty tablecloth odor. sulfur. not detected by GC–O. and TMA-like smell.5°C) [22].2. and Malty Odors Ketones. The odor descriptors in Table 8.1). Identification of volatile compounds was based on GC–MS analysis (see Table 8. esters. the freshness notes disappear and the odor of the uncooked fish becomes neutral. packaging. alcohols. In general when fish is cooked. and the end of shelf life of cod fillets on day 12 of storage are explained by the presence of TMA. showing results from a storage study of cod fillets packed in styrofoam boxes during chilled storage (0. Haddock Fillets [31].34]. data not available for haddock.4. mainly amino acids. and temperature conditions during storage [33. 8. or geraniumlike odors are characteristic sensory odor descriptors for fresh whole fish. the aroma of the fillet is described as sweet and reminiscent of shellfish. 8.2) were associated with the development of sweet. 3-methyl-1-butanol.3-butandiol were found in the highest levels on day 12 at sensory rejection. development of spoilage odors. cooling. acids. dried fish/stockfish. and 2. which is mostly affected by handling. 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. meat-like. and malty spoilage odors.1 Sweet. and aldehydes.Volatile Aroma Compounds in Fish ◾ 103 Table 8. An example of the spoilage pattern of volatile compounds in chilled fish is illustrated in Figure 8. and quantification of the main classes of compounds was based on the sum of the PAR for respective compounds in each class. Late spoilage changes. the fish is no longer fit for consumption. —. and aldehydes detected on day 4 of storage and their increasing levels on days 7 and 10 (Figure 8. as well as green plant-. and when combined with frozen storage odor. cucumber-. Sweet-milky and vanilla/caramel-like odors are typical in cooked fish. sour.1 (continued) Volatile Compounds Detected in Cod [22].1 Microbial Spoilage Odors The spoilage odors in chilled fish vary depending on the dominant microflora in the products.4. mushroom-.1. The microbially derived alcohols 2-methyl-1-propanol. Seaweedy and marine-like odors.1 based on GC–O analysis of cod and smoked salmon represent most of these overall changes. sour. During prolonged storage boiled potato odor develops. The flavor thresholds . Sour. 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. caramel-like. 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. After several days of storage.

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

The odor of ethyl butanoate.1). ammonia-like odor. dried fish. ammonia-like. At this point there was an increase in the pH value.1. and the incorporation of hydrogen sulfide yields dimethyl trisulfide [38]. Pseudomonas species have also been found responsible for the formation of volatile sulfides. whereas DMA may influence the overall fresh flavor of fish in combination with oxidatively formed aldehydes from long-chain fatty acids in fish. TMA has been noted for intensifying fishiness by a synergistic action with certain volatile unsaturated aldehydes derived from autoxidation of polyunsaturated fatty acids [40]. TMA is a potent odorant with a characteristic fishy. and dimethyl disulfide have been suggested as the main cause of putrid spoilage aromas [41]. Additionally.Volatile Aroma Compounds in Fish ◾ 105 storage. contributed to the sensory rejection of chilled cod fillets on day 12 and suggested the role of Pseudomonas fragi in the development of sweet.2 Dried Fish. volatile sulfur compounds such as hydrogen sulfide. decane. Enzymically produced DMA (dimethylamine). Figure 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. methyl sulfide.4.4 Miscellaneous The concentration of the straight chain alkanes (nonane.4. 8. and ketones (2-butanone). described as sickeningly sweet and nauseous. and undecane) appeared to be similar throughout storage in chilled cod fillets [22].2) indicated that they were not important in the spoilage of chilled cod fillets stored in styrofoam boxes. 8. and Stale Odors The development of dried fish.38. Onion. methyl mercaptan. Dimethyl trisulfide has also been associated with spoilage in fish and associated with the growth of Shewanella putrecfaciens [25. and stale odors by amines during fish spoilage is well known.1. respectively [41]. fruity off odors [37. which contributed to boiled potato-like odors (Table 8. TMA is characteristic for the spoilage odors of fish. Oxidative processes are involved in the formation of dimethyl sulfide from methyl mercaptan and further oxidation of dimethyl disulfide. which forms very early after harvest of fish.2 shows that TMA was detected in high levels on day 12. In whole fish stored in ice. Additionally.38].4. The lipid-derived saturated aldehydes detected on day 12 at sensory rejection also contributed to the overall sweet aroma. 1-penten-3-ol). numerous branched chain . The onset of stale odors can be explained by cis-4-heptenal and heptanal. Ketones can influence the overall odor because of their low odor thresholds. Ammonia-Like. contributing to the stale and putrid off odors in fish because of amino acid and lipid degradation [39]. has been suggested as a freshness indicator along with its precursor TMAO (trimethylamine oxide) [27]. and Cabbage-Like Odors Low levels of sulfur compounds (Figure 8. alcohols (3-methyl-1-butanol. The origin of the sulfur compounds is microbial degradation of cysteine and methionine to form hydrogen sulfide and methyl mercaptan. 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. which may have influenced the overall odor perception leading to the sensory rejection of the fillets.39].3 Putrid.1. but no obvious increase occurred until at the end of shelf-life and during continued storage.

and 6-methyl-5-heptene-2one). Limonene has also been detected in sea bream during storage [29].4. Piperidine levels have been reported to increase in spawning salmon and contribute to off odors [43]. Several odor active terpene derivatives have been identified in fish. ketones. 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. cis-4-heptenal. which are known to be more susceptible to oxidation than triacylglycerols in fat deposits [45]. and 2.4-heptadienal and 2. 8.and potato-like odors contributed by . the feed may have influenced higher levels of aldehydes. Aldehydes generally have low odor thresholds. The influence of other aroma active compounds present in lower levels such as the unsaturated autoxidatively derived aldehydes (2.2.4.7-decadienal. heptanal. their impact was greater than alcohols and ketones. suggesting that it may have an impact on the overall odor of fish fillets [22]. A characteristic earthy odor in many species residing in ponds has been associated with piperidine and its reaction products. Limonene has low odor threshold and a fresh lemon odor was detected by GC–O analysis of cod. and because of their high unsaturation. Oxidative processes occurring during storage of fish result in the accumulation of aldehydes.4. were detected in the fillets throughout the storage time. 2.3 to demonstrate which odors are most dominating in the aroma profile [48].106 ◾ Handbook of Seafood and Seafood Products Analysis alkanes were detected. they are in particular sensitive to oxidation. but the sampling techniques used were not sensitive enough to allow quantification of these compounds. Phospholipids are the main membrane-bound lipids. These oxidation products contributed to the overall characteristic sweet. such as hexanal. although their overall levels were lower. they are not considered of interest as quality indicators. that contribute to the development of rancid cold store flavors [47]. and decanal. in similar or slightly increasing levels. and. Various pro and antioxidants influence the stability of the muscle and have been studied in relation to the oxidative stability of phospholipids [46]. such as hexanal. 3-methyl-butanal. 2-butanone. therefore. These compounds have been associated with rancid and dried fish odors. However. lipid-derived saturated aldehydes.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]. Similarly. 8. but the knowledge of the formation of these compounds is obscure. which is further enhanced by preprocessing and storage of fish. The origin of limonene in fish is most likely related to the diet derived from algae or plant source.4.7-decatrienal) should not be overlooked. fish-like odors of chilled cod fillets in combination with other carbonyls (3-hydroxy-2-butanone. 3-pentanone. and terpenes found in wild sea bream compared with those of its cultured counterpart [29]. aromatics.4-heptadienal.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. Boiled potato. since they are not aroma active. 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]. Piperidine was tentatively identified in chilled cod fillets [22] and has also been suggested as a quality indicator in sea bream [29]. and overall the alkanes showed an increasing trend with storage time.

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

12.5 –0. Interestingly. In boiled trout.4 0. The effect of oxidation induced by cooking and formation of oxidation products such as heptenal and nonanal characterizes the (B-D4) sample.6-nonadienal.4-decadienal from PUFA were determined as character impact odorants of boiled cod [54]. in particular the role of volatile compounds derived from oxidation in heated/boiled samples. The oxidatively formed compounds. in agreement with earlier studies [54].8 R-D17 –0. decanal.4 Principal component analysis of raw and boiled cod. Samples are labeled with R. .1). and 6-methyl-5-hepten-2-one. Sulfur compounds dimethyl sulfide. methional.4). dimethyl disulfide. boiled and storage days. 19% PC1 1. especially in trout [15]. hexanal. methional with a characteristic boiled potato-like odor dominated the odor of the aldehyde fraction of the headspace volatiles. 14. (E. 3-methyl-butanal in combination with acetaldehyde. 10. 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. The PCA demonstrates how volatile compounds can explain the variation in quality of samples according to storage time and handling (raw and boiled).E)-2. 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. D (4.4).0 Figure 8. X-expl: 53%.0 B-D17 1-Penten-3-ol 3-me-butanal Acetic acid 0. and oxidatively derived (Z)-1. 3-methyl-butanal was correlated to the boiled stored cod (B-D17) (Figure 8. However.2 Washed Cod Muscle System Rancid odor development during chilled storage of fish has commonly been associated with fatty species.5-octadien-3-one.4. The malty flavor of 3-methyl butanal was suggested earlier to be mainly responsible for the malty off flavor defect of boiled cod [54].2.6 0. samples after heating (see Table 8. The characteristic pattern or trend in volatiles in raw and boiled fish is clearly different. raw and B. 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).4 –0. Only the spoiled raw samples (R-D14 and R-D17) can be correlated with the freshly boiled (B-D4) sample.108 ◾ Handbook of Seafood and Seafood Products Analysis PC2 Bi-plot 1. and 17 days). Autoxidatively produced unsaturated carbonyl compounds were the most abundant components in boiled and canned fish. On the basis of odor evaluation.2 0. as indicated by the arrows (Figure 8. and dimethyl trisulfide were detected in higher levels in the boiled samples (data not shown).2 Raw and boild c…. oxidation of membrane-bound phospholipids in lean species can cause fishy. 7. that is. increased with time and were pronounced in the spoiled raw samples (R-D14 and R-D17).Z)-2.

g. On the other hand. Sohn et al. These odors were also detected in cod fillets during chilled storage (Table 8. lipid oxidation of muscle phospholipids may be induced by several catalysts. has been studied to understand better the mechanisms of oxidation in the muscle [57.58]. free radical scavenging and chelation) but also on factors such as physical location. The prooxidative effect of hemoglobin was evident by the formation of hexanal in high levels. 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. but the compounds were detected in much lower levels [22]. it is necessary to apply models that take into account the chemical. 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. painty. and color. physical. Preconcentration techniques are necessary for the analysis of unsaturated aldehydes.4heptadienal that contributed to rancid odor caused by oxidation. and the overall odor was an intense dried fish. and environmental conditions (e. 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. . floral. 2. [63]. which is not practical for rapid determination of oxidation. and environmental conditions expected in food products. interaction with other food components. pH) [55.. To accurately evaluate the potential of antioxidants in foods.g. The added hemoglobin was very effective as a prooxidant. Consequently. cucumber-like. and caramel-like odors contributed by 3-methylbutanal. and lemon-like odors were explained by 2.56]. in agreement with TBARS and changes in color [62]. sweet.61].59]. These compounds can be used as indicator compounds for oxidation. soapy. and instrumental color changes. ascorbic acid.3-pentandione.. mushroom odor caused by 1-octen-3-ol. potato-like odor caused by cis-4-heptenal and heptanal. earthy. the concentration and composition of volatile oxidation products analyzed by GC were compared with TBARS measurements. fatty. 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. dried fish-like off odors as discussed before. Furthermore. Washed cod muscle system has been widely used to study oxidation and the influence of prooxidative and antioxidative factors [59. and glutathione peroxidase) and aqueous prooxidants in fish muscle. The most potent odors detected in the model system were malty. grass odor contributed by hexanal. To monitor the development of rancidity. This is because the activity of antioxidants in food systems depends not only on the chemical reactivity of the antioxidant (e. rancid. and spicy and flowery notes exhibited by 6-methyl-5-hepten-2-one. static headspace sampling methods. and a similar trend was observed in the development of cis-4-heptenal (Figure 8. rancid fish oil like. [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. and 1-penten 3-ol. green. it is possible to detect the most volatile oxidation products like propanal and hexanal by rapid.Volatile Aroma Compounds in Fish ◾ 109 rancid.1). including hemoglobin from blood [7. TBARS (thiobarbituric reactive substances). as demonstrated by Boyd et al. Odor development in lean fish studied by hemoglobininduced oxidation in washed cod muscle system showed that sweet. rancid. In lean fish such as cod.5) as well as 2. Similarly. sensory assessments. including blood components like inorganic metals iron (Fe) and copper (Cu). and rancid odors dominated the aroma profile [62]. The role of antioxidants (a-tocopherol.4-heptadienal [62]. we found in our studies on the washed cod muscle system that hexanal could be used as indicator for rancid odor development.

. and in TBARS (Figure 8. citric acid. and Ólafsdóttir. 67.5 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 (-♦-. Some promising results have been reported.. as measured by rapid increase in rancid odor. painty. 2008. Matis Report 08. Aquat. ( Adapted from Jónsdóttir. and EDTA [66]. Hb-Char-II.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. 73. 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. caffeic acid) [65] as well as application of tocopherol.. -▲-. R.e. catechins from tea) and cinnamic acid derivatives (i. Active research is ongoing on the application of various natural antioxidants based on polyphenols like flavonoids (i. Food Prod.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. G. -■-. (From Jónsdóttir.e..6) as well as more rapid loss of red color (not shown) already on the first day of storage. 16. with added hemoglobin (raw and cooked. Blank-II. described as rancid. 2007. 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. HbCod-II). J.) .. R.) Thermal treatment of the cod model system significantly enhanced the oxidation of the model on day 1. et al. where commercially available green tea polyphenols were shown to effectively inhibit the LOX activity of mackerel muscle [67]. With permission. and dried fish odors. respectively) and raw without hemoglobin (blank). Studies on LOX inhibitors are of interest in preventing the initiation of oxidation in fish.

it was verified that selected key volatile compounds performed better as predictors to explain variation in sensory attributes (smoked. [74] analyzed headspace components of cod and swordfish.7 illustrates the main odors that were present in smoked fish samples after 14 days of chilled storage. contributing to mushroom-like odor. 8. whereas carbonyl compounds. 3-methyl-butanal. 2.4.3. sweet/sour rancid. like 3-methyl butanal. ethanol. Other oxidatively derived compounds like 1-penten-3-ol. furan-like compounds have been reported to be responsible for the smoked odor in smoked salmon. associated with spoilage off flavors. Phenolic derivatives like guaiacol (2-methoxyphenol) and syringol (2. hexanal. 2.Volatile Aroma Compounds in Fish ◾ 111 8. thermal degradation. 1-penten-3-ol. and 2. The typical smoked salmon aroma results from a number of chemicals found in the smoke. aldehydes. gave the most intense odors of smoked salmon and contributed to the fish-like earthy odors and fatty and rancid odors (Figure 8. Guillén et al. were characteristic in unsmoked fish. hexanal.4-heptanal. giving rancid. which has a characteristic potato-like odor. but it is mostly attributed to the phenols. and 1-octen-3-ol. plays important roles in the formation of complicated processing flavors.7) (e.72]. and furans have been found in spray-dried shrimp powder and shrimp hydrolysate [69]. 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 8.75]. Lipid-derived aldehydes play an important role in flavor formation and have been reported to contribute to the characteristic fish-like.6-nonadienal. and 2-acetyl-1-pyrroline. Key volatile compounds identified in enzymatically produced seafood flavorants are formed via Maillard reaction and Strecker degradation of amino acids.. Volatile compounds like alkyl-pyrazines and sulfur-containing compounds have been found in cooked crustaceans. 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]. sweet odors of processed seafood like those in smoked salmon [23. and decanal were among key volatiles.72]. Figure 8. and 1-propanol [28.4. 3-hydroxy-2-butanone. Maillard reaction. 3-methyl-1-butanol. and off odor and flavor) than traditional chemical and microbial variables. and alcohols were abundant in the headspace of cold smoked salmon products during storage. potato-like odors. The oxidatively derived compounds cis-4-heptenal and heptanal.Z)-2. which is typical for products on the market [23]. 2-methyl-1-butanol. 2-pentanone. including Strecker degradation. the Strecker aldehyde produced from methionine. Additionally. also contribute to the aroma of seafood flavorants [70].72].7) [23].71. nonanal.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. giving the flesh its typical fishy odor [71. and lipid oxidation. . like cis-4-heptenal.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. In addition to phenolic compounds. 1-octen-3-ol. it is clear that their presence contributes to the characteristic fish odor of smoked salmon products. These are compounds like methional. Microbially produced ketones.6-nonadienal. and 3-methyl-1-butanol) [23]. and although they contributed less to the odors. 2-butanone.4-decadienal. giving a popcorn-like odor that can be thermally generated. where groups of phenol pyrolysis were most noticeable in the smoke flavor volatiles.g. Lipid-derived components. such as heptanal and (E.1 Smoked Fish Odors Degradation compounds from Maillard reactions and lipid oxidation are the main compounds contributing to the aroma of smoked salmon [72].

184. fatty Boiled potato-like Fatty. The rancid. sweet Smoke-like 2.5octadien-3-one were also identified as potent odorants in ripened anchovy [81]. where the ripening of salted cod (Gadus morhua) produced by different salting methods was studied. highly volatile components of ripened anchovy. smoke. Methional and (Z)-1. potato-like odors together with cucumber-like odor [82]. potato-like odor was identified as cis4-heptenal and the boiled potato-like odor. 109. . most of them generated from chemical or enzymatic oxidation of unsaturated fatty acids and further interactions with proteins. and rancid-like odors Burnt. mushroom 2. sweet Flowery. especially those in the Mediterranean. Triqui and Reineccius [80] found that 2. burnt.2 Ripening Odor—Salted and Dried Fish Odor Numerous volatile compounds have been detected in ripened products like dry cured ham. the highest odor scores were given for boiled potato and rancid. earthy. the desired flavor and texture develop as a consequence of protein and fat degradation. (Modified from Jónsdóttir. 4-Heptadienal Sweet. ripened roe products [79] Similarly. and aldehydes such as acetaldehyde. R. 2-methylpropanal and 3-methylbutanal were the key. Similar processes have been reported in ripened seafood products.. [76–78].and 3-Methyl phenol Guaiacol 4-Methyl-guaiacol Sweet and fruity-like odors Wood.7 GC–O evaluation of volatile compounds detected in cold smoked salmon after 14 days of storage at 5°C. In our study. 2008. peptides. as heptanal. sweet Flowery. where methional derived from methionine and 2. and free amino acids. Food Chem.) 8. smoke Mushroom. probably originating from amino acids. smoke 3-Methyl butanal Sweet. they suggested that lipid autoxidation during ripening was primarily responsible for aroma development.112 ◾ Handbook of Seafood and Seafood Products Analysis Smoked salmon odors Characteristic smoke odor Sweet. manufacturers of ripened products have observed that some degree of proteolysis is necessary before flavor can develop. geranium Rancid cis-4-Heptenal Heptanal Earthy-like odors 1-Octen-3-ol Figure 8.3.6-nonadienal from fatty acid oxidation were the main odorants in sugar salted. However. fruity Flowery. caramel Smoke-house. Thus.. Salted cod are traditional products from the North-Atlantic fisheries and are highly regarded as ripened fish products in many countries.4-heptadienal and 3. sweet Wood. During ripening of salted cod. et al. both oxidatively derived compounds.5-octadien-2-one were associated with the development of the typical flavor obtained after anchovy ripening.4.

The cucumber-like odor detected is possibly 2. 1995. Therefore. careful evaluation of the quality of product is needed to ensure acceptable flavor. 1-penten-3-ol. 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. Lipid oxidation during ripening appears to be primarily responsible for desirable aroma development in processed fish.6-nonadienal. 1995. such as ketones. VCH Publishers Inc. Quality and quality changes in fresh fish. No. Volatile compounds as indicators of freshness quality and spoilage can be monitored to determine the quality of fish products. 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. 1–67. were the most intense character impact compounds of salted cod and smoked salmon. 2.5 Conclusions Although aldehydes. derived from methionine and eluting at a similar time as cis-4-heptenal and heptanal. for example. according to retention index (RI) of standard and odor evaluation. and 1-octen-3-ol. Evaluation of Seafood Freshness Quality. hexanal. hemoglobin. cause off odors in fish during storage. FAO.83]. Knowledge of the spoilage pattern of volatile compounds is the basis for the development of rapid techniques like smart sensor technologies. In addition. and myoglobin. 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. and new packaging technologies. FAO Fisheries Technical Paper. proper handling. temperature control.6-nonadienal. esters. and sulfur compounds. Huss.. J. contributing to mushroom-like odor. and in retail for consumers as smart sensors imprinted on packaging. The oxidatively derived compounds cis-4-heptenal and heptanal.Volatile Aroma Compounds in Fish ◾ 113 Methional. Proper handling and application of natural antioxidants to control oxidative processes caused by lipoxygenase. potato-like odors. microbial growth can be limited by effective cooling techniques. References 1. New York. 348. alcohols. Rome.R. Botta. such as heptanal and (E. Other key volatile compounds in salted cod are derived form lipid oxidation. acids. 8. Detection of microbial metabolites originating mainly from soluble aqueous fractions of the muscle can be directly related to the quality of products. aldehydes. However.H. H.Z)-2. exhibiting rancid. and 2-butanone. to increase trust between buyers and sellers in trade. 193 pp. amines. their presence at nominal levels gives the characteristic and desirable fishy odor in fresh and processed fish. although the compound could not be identified by GC–MS. A similar set of sensors with selectivity and sensitivity toward the main quality-indicating classes of compounds. can be used for a variety of fish species that are stored and processed by different techniques. could also be responsible for the boiled potato-like odor. and other prooxidants in combination with mild heating treatment are important factors to maintain the delicate flavor and odor of fish products. . 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.

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

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..........2 Theory..............................1..........................1 Theory....................................................4............ 130 9........................................................ 134 References ........................................... Measurement Principles...........................................................................2................ Karsten Heia.133 Acknowledgment .......................................... and Analysis .. and Data Analysis .....................2 Imaging Spectroscopy ................................... 128 9.........4............1 Determination of Basic Composition................5 Summary ..........................Chapter 9 Basic Composition: Rapid Methodologies Heidi Nilsen.....................2 Analysis of Basic Constituents ..........4 X-Ray Imaging ...............................................132 9................... 130 9.............................................3 Analysis of Basic Constituents ............ 122 9..................................................... 121 ....................1 Near-Infrared Spectroscopy.. 130 9...........132 9........................................2 Theory and Measurement Principles .............................................................................1............................................................ and Margrethe Esaiassen Contents 9......................................131 9............................................................................ 128 9.......................3..........................................2.................... Measurement Principles............132 9.................1......................3 NMR Spectroscopy ............... 128 9..............................................1 Theory and Measurement Principles ...................................................... 124 9..............2 Analysis .................................3.....3 Analysis of Basic Constituents .3.............................................1 Determination of Basic Composition.................... 122 9.................... 122 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............................................................

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

Basic Composition: Rapid Methodologies ◾ 123 and N−H [9] and corresponds mainly to overtones and combinations of fundamental vibrations. placing the light source and the detector at the same side of the sample. . In (a) the transmission setup is shown. If the light source and the detector are placed on the same side of the sample as shown in (b). but focusing the two devices so as to ensure that the light has traversed some region of the sample before detection. 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. enables “transmission” measurements. Different measurement modes for NIR spectroscopy are illustrated in Figure 9. In the context of rapid methodologies. 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. developed toward the facilitation of nondestructive. The setup in (b) displays the reflection setup where light reflected from the sample surface enters the detector.9. nondisruptive. depending on the scattering properties of the medium under investigation.1. where the light passes through the sample from one side to another. For both (a) and (b). traditional chemical determination of the constituents. 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. A setup as shown in (a).1 Different measurement setups for NIR spectroscopy. for example. A common methodology is chemometrics or Detector Shelter Sample Light source (a) (b) (c) Figure 9. NIR spectroscopy is an indirect measurement technique. Finally. In (c) the light source and detector are located to register light that has traversed the sample before detection. the spectral readings must be correlated to a relevant reference method such as. the system is operated in “reflection” mode. we view this in terms of the measurement setup enabled by technology. In order to prevent direct reflection from the surface.10]. (c) illustrates how measurements are performed in “transflection” mode.11]. light from the source penetrates the sample and enters the detector. Over the years there has been a steadily ongoing development of instrumentation for NIR spectroscopy. both with respect to the detectors and the capture of the spectral information [10. and noncontact methods. 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. Hence. a screen is placed between the directly emitted area and the area of inspection. The broad spectral bands may be an indication of the material constituents.

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

and Tuene [24] made use of NIR transmission spectroscopy to assess protein. Torrissen. microwave. (Y–var. [27]. 8) Figure 9. [21. Torry fatmeter.985681 0.600067 0.) Spectral measurements were performed on intact fillets by transflection measurements by use of the fiber optic probe of the instrument NIRS6500 (Perstorp Analytical Inc.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. Isaksson et al. PC): (%fettHS..K. Transmission spectroscopy was also employed for the analysis of fat and dry matter in capelin [25]. (From Nilsen. applying minced samples for the spectral readings. and protein content of European sea bass. Wold et al. In this work they applied a fiber optic measurement setup.22] also conducted studies documenting the efficiency of applying NIR spectroscopy in different measurement modes to assess fat and water content in salmon. The study concluded that NIR is well suited for nondestructive quality evaluation of salmon fillets. in. water. In a recent work by Khodabux et al. fat. Spectroscopic readings obtained on the minced samples correlated better with the reference measurements on fat.603947 0. [26] showed that NIR spectroscopy could be used to estimate lipid.Basic Composition: Rapid Methodologies ◾ 125 salmon. In a research article published in 2004. . whether pre-. Nortvedt. and protein than those made on intact muscles. In both the works of Vogt et al. 1998. Silver Spring).2.. Measured Y Elements: Slope: Offset: Correlation: RMSEP: SEP: 21 Bias: 24 78 0. An example illustrating the use of NIR spectroscopy for assessing fat content in farmed salmon is given in Figure 9. or postspawning. The fat content of herring has also been assessed by the use of NIR spectroscopy. [20] conducted a study in which they compared NIR measurements on intact salmon fillet. Xiccato et al. N. and NIR spectroscopy. [29] one question of interest was the comparison of different methods for measuring fat content. and Sørensen. H. [28] and Nielsen et al. NIR spectroscopy has been used for evaluating the chemical composition of several other fish species as well. This work also emphasized the impact of the conditional state of the fish when making calibration models.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.937575 0. water. NIR spectroscopy was proven to be a useful tool for the evaluation of basic constituents of different types of tuna. and dry matter in halibut fillet. and additionally the spectroscopic measurements could be used for origin identification or authentication of the samples.264047 0. as well as on minced salmon muscle. Unpublished data.

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

. These developments have enabled the use of at-line or online methodology. combining imaging techniques with the spectral information.3 Prototype version of handheld spectroscopic instrument for quality assessment of fish. Another issue is the need for modeling the correlation between the spectroscopic reading and the quality parameter in question. 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. The high price of the instrumentation. The technique has. say. is considered intriguing. fish-quality inspection. may promote the future applicability and usefulness of the information in Figure 9. not yet become an everyday instrumental tool for food-quality control nor.3. As illustrated by the above. 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. The development in recent years in instrumentation. High-cost instrumentation designed for versatile use and flexibility has probably better met the requirements of laboratory use than those of industrial application. however. 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. This instrument was used for the determination of freshness of cod as well as the assessment of frozen storage time of hake. with one reading. on one side.Basic Composition: Rapid Methodologies ◾ 127 the possibility of simultaneously monitoring a number of different issues. There may be several reasons for this.

In addition to what traditional spectroscopy can facilitate. or the result from these techniques can be postprocessed to utilize the spatial information [46]. In order to illustrate the potential parameters to be assessed by imaging spectroscopy. The analytical techniques described in that section are also applied to imaging spectroscopy data. this technique also provides spatial information. 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]. vegetables.2 Imaging Spectroscopy 9. To simplify the concept. Norge). For fruits and vegetables more articles report on determination of chemical constituents such as moisture content. this can be illustrated as simultaneously recording information about shape and color.1 Theory.128 ◾ Handbook of Seafood and Seafood Products Analysis the near-infrared spectra. A novel example of this is the development of the QMonitor (QVision AS. demonstrates the potential of the method in the seafood sector as well. the spectrograph and the object must move relative to each other. It has been shown that NIR hyperspectral imaging techniques are .2 Analysis of Basic Constituents During the last decade several applications within food-quality inspection have been developed based on imaging spectroscopy. and Analysis Imaging spectroscopy. Imaging spectroscopy can be implemented for transmission. an analytical tool for industrial quality control of clipfish and salmon fillets. an imaging spectrograph operates in the following way. Most of them are on foods such as fruits. the feasibility of the method for the analysis of basic composition of foods. is a new technique that has been developed during the last decade [43. As these techniques only use the spectral information. Oslo. In this way an image of the object is built line by line. it uses a two-dimensional sensor. There are still relatively few reports on imaging spectroscopy applied for the analysis of fish and seafood. 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. and each frame captured provides full spectral information for one line across the object to be imaged. the hyperspectral data can be preprocessed based on spatial features before applying analytical spectral techniques. Measurement Principles. as well as transflection measurements. also known as multispectral imaging or hyperspectral imaging. 9. Th is means that for each spatial location it is possible to access the full spectral information. Between each captured frame. and acidity (expressed as pH) [47–49]. in general. Several solutions have also been developed for detection of defects and contaminations on fruits. the spectra may be recorded in the visible and near-infrared region. It has become a widely used technique within fields spanning microscopy to satellite remote sensing. 9. 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. and meat.2. some examples related to the agricultural sector are referred. Typically. Depending on the applied sensor technology. As described in Section 9.1 on NIR spectroscopy. Typically. For instance.44]. However.2. total soluble solids. this method is an indirect measurement technique. improved results can be obtained by combining these techniques with more traditional image processing techniques. reflection.

The parameters included were drip loss. Regarding the determination of basic chemical composition of fish and seafood.4 Fat distribution in salmon fillet measured by the multispectral imaging system QMonitor fabricated by QVision (Oslo. When drying fish.60] where several quality parameters were evaluated by imaging spectroscopy.3%. pH. [61]. 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. QVision (Oslo.55].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. there is one recent publication on assessing water content in salted dried cod by Wold et al.4). [59. The first article addressing analysis of fish or seafood by imaging spectroscopy was published in 2000 by Sigernes et al. The mean fat content for this fillet is 18. Peeling of shrimps and detection of nematodes were mentioned as possible applications for the future. [53]. color. .Basic Composition: Rapid Methodologies ◾ 129 useful for automatic online detection of surface defects and contaminations on apples [50–52]. In this publication the importance of including spatial information is illustrated. and cadaver [54. Fisk: 1 Fettfisk: 18. A thorough review of imaging spectroscopy applications within fruits and vegetables is presented by Nicolai et al. Hence. imaging spectroscopy solutions for detection of contaminants such as fecal and ingesta on poultry carcasses have been studied [56–58]. A recent work on quality assessment of pork has been reported by Qiao et al. septicemic. Norway) has also developed an industrial solution based on multispectral imaging for measuring the fat content in salmon fi llets (see Figure 9. whereas the local fat content varies from approximately 6% up to 43%. the moisture content of the fish varies from the thinner parts to the thicker parts of the fish. The color bar to the right indicates the correspondence between color and fat content in percentage. Norway). [35]. measuring the water content in one spot is not necessarily representative for the whole fish.3055% Share: 21. Since 2000. Inspection systems based on hyperspectral imaging have been tested for poultry carcass inspection focusing on classification of carcasses into normal. and different texture features.

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

[80] used this technique to determine the origin of Atlantic salmon. For example. [74] reported the use of HR-NMR to determine oxidation products in marine lipids. used for seafood authenticity have been provided by Martinez et al. creatine. 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. Rheinstetten. whereas Veliyulin et al. and mobility in herring [67] and oil and water content of salmon and cod [68]. Today. Germany) has been developed to handle such samples. [79] and Masoum et al. 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. anserine. and dimethylamine in extracts . Additionally.and 13C-NMR have been applied to measure the lipid or water content of many different foods including fish. whereas Thomas et al. cod [66]. and there are numerous reports available.3. Aursand et al. Tyl et al.3 Analysis of Basic Constituents For several years 1H. Standal et al. trimethylamine oxide. [77] used NMR to discriminate cod liver oil according to whether the origin was wild/ farmed as well as geographic origin. it was shown that 23Na-NMR has proven useful for quantitative salt determinations in salted cod. 9. fatty acid composition. Low-field (LF) NMR spectroscopy requires little or no sample preparation. [78] demonstrated the use of NMR lipid profiling for classification of gilthead sea bream according to geographic origin. [72] used HR-NMR to measure the content of n-3 polyunsaturated fatty acids in four types of unoxidized fish oils. [75] and Arvanitoyannis et al. Extensive reviews on different techniques. degree of saturated/ unsaturated fatty acids. whereas Siddiqui et al. [70] demonstrated that NMR-MOUSE could also be used for in vivo determination of fat content in Atlantic salmon. As recent examples. Among more recent work. Due to the provision of very detailed information regarding the molecular structure of a food sample. 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. Additionally. [76]. Studies of large objects like whole fish are impossible using most traditional LF-NMR instruments. high-resolution NMR has been applied in many food authenticity studies. water distribution. Martinez et al. and studies of lipid degradation processes in lipid mixtures such as fish oils. [73] used HR-1H. and they may provide different information regarding the food properties. the Bruker Professional MOUSE ® (Bruker Optik GmbH. whereas LF. and some examples of analyses of seafood are given here. Falch et al.Basic Composition: Rapid Methodologies ◾ 131 information regarding the molecular structure of a food sample. Rezzi et al. betaine. Numerous applications of NMR in food analyses have been reported in the literature. different NMR equipments are available. including NMR.and HR-13C-NMR for multicomponent analyses of encapsulated marine oil supplements. [81] showed that it was possible to identify taurine. 1H NMR spectroscopy has been explored to identify the fate of some bioactive compounds during processing of seafood. For analyses of seafood products. [69] demonstrated that this equipment could be applied to determine fat in homogenates from salmon. LF-1H-NMR has been used for studying water distribution in smoked salmon [65]. High-resolution NMR can be used to provide information on lipid classes.1H-NMR seems to correlate to fillet pH and water-holding capacity [71]. in a study focusing on both 23Na-NMR and low-field 1H-NMR spectroscopy. and it has mainly been used for analyses of water in food samples.

and the attenuation will also be influenced by the sample thickness. This is achieved by rotating the x-ray/detector unit around the sample. 9.2 Analysis X-ray imaging provides spatial information in two dimensions (2D) or three dimensions (3D) (CT). smaller. has been that conventional NMR is an expensive technique. but it provides a three-dimensional image of the sample. [82] showed that it was possible to identify single chemical compounds such as hypoxanthine. lactate. more specific information about the sample can be revealed. amino acids. Based on the results obtained the . There are two interactions. The decrease in x-ray intensity inside a sample will be due to absorption by different materials. previously DEXA) and may be implemented using a two-layer detector. a two-dimensional cross section of the sample can be made. the photoelectric effect and the Compton scattering that causes the x-ray attenuation. and lately many low-field. Gribbestad et al. It is not possible to accurately characterize the observed sample by applying only one x-ray energy level. In another study Kolstad et al. [85] tested CT scanning as a tool for estimating the relative size of fat deposits and lean tissue and fat content in Atlantic halibut. Within the field of medicine. Then the third dimension is accomplished by the sample movement. As illustrated here. one layer for each energy level. computed tomographic (CT) scanning is widely used. and some fatty acids in extracts and muscle from salmon using high-resolution 1H NMR spectroscopy.132 ◾ Handbook of Seafood and Seafood Products Analysis from processed cod. 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]. By using two x-ray energy levels. and less sensitive to fluctuations in the environment and thus more applicable in industry as well as in many research fields.4 X-Ray Imaging 9. Such equipment is cheaper. 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. This technique is referred to as dual-energy x-ray absorptiometry (DXA. A more dense material will absorb more x-ray energy. and their relative contributions are energy dependent [83]. the high spectral resolution is not always required.4.4. anserine. however. low-resolution NMR spectrometers have been developed and commercialized. NMR is a versatile tool for the identification and quantification of numerous compounds in fish related to nutritional quality. An objection to the method. This is also an x-ray imaging system. Making profiles from different angles and then combining them by software.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. For online applications this can be implemented as a line-by-line imaging or a frame-by-frame imaging. 9. Typical applications within fish and fish products are related to the detection of bones and bone fragments as well as chemical composition and localization. However. Further on the CT scans gave significant information about dry matter distribution from head to tail of the cod. Th is is a powerful imaging technique that can be used both as a single-energy and a dual-energy module.

9. Iceland).3 mm when operating at industrial speed (see Figure 9. instrumental means capable of objective and rapid determination of basic composition are also available.5 for an example). With respect to bone detection in fish fillets there are commercial solutions available today (Marel Hf.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. 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. [86] showing good results predicting fat content of common carp based on CT scanning. 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. A similar work has been carried out by Hancz et al.Basic Composition: Rapid Methodologies ◾ 133 Figure 9. Iceland). This instrument can detect bones and bone fragments down to a diameter of 0. authors recommended CT scanning as an online technique for carcass evaluation. Throughout development all presented techniques have met the requirements .

. VIS/NIR spectroscopy. Osborne. Acknowledgment The authors would like to thank Dr Jens Petter Wold. using near infrared spectroscopy. the finding of a universal measurement tool to meet with this variety is a challenging task. nearinfrared spectroscopy and low-field H-1 NMR spectroscopy. 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. References 1. T. U. pp.134 ◾ Handbook of Seafood and Seafood Products Analysis of simplicity in sample preparation.. In addition. and x-rays is considerable and. and Williams. Reykjavik. and Fearn. imaging. 2005. also makes it clear that although proven useful and promising in laboratory-scale trials. T... Eds. and x-rays are operated at a speed that makes it possible to perform measurements at or in a processing line. 240–250.. Non-destructive Visible/NIR Spectroscopy for differentiation of fresh and frozenthawed fish. Part of the explanation for this could be the cost level of the equipment in question. J. H. and progress in data processing and analytical tools has facilitated usability and ease of interpretation of measurement results. NMR. 200. Differentiation of frozen and unfrozen beef using near-infrared spectroscopy. et al.. in Near InfraRed Spectroscopy. Nofima Food. Iceland) and QMonitor (QVision AS. U.K. T. C506–C510. 121–128. This chapter.. 6. and Heia. Norway. Hildrum. and Villarroya. M.K.G. Pawlinsky. 103–111. Thyholdt. Ellis Horwood.K. Isaksson. 339–344. T. et al. Thybo. However. Blanco. K. therefore. 73(4). NMR. p. Journal of Near Infrared Spectroscopy. . T. Norway) confirms that these techniques may be applied in commercial and industrial high-speed fish processing applications. NIR spectroscopy: A rapid-response analytical tool. Safety and Authenticity. et al. Longman Scientific & Technical. the technological development exemplified by SensorX (Marel hf. Eds. 33(2). Determination of chemical composition of beef meat by NIRS. 89–104. Wiley-Blackwell Publishing. Uddin. Journal of Food Science. Trends in Analytical Chemistry. M.. Oslo. 1998. and Isakson. A. NIR. Prediction of sensory texture of cooked potatoes using uniaxial compression. Naes. Journal of the Science of Food and Agriculture. 1986. 2009.. B. K.. I. 2002. The price of measurement equipment for NIR. 5. Harlow. Prediction of wheat bread-baking functionality in whole kernels. Nilsen. 7. Rehbein.A. Introducing and applying these methods to industrial applications and enabling production of well-documented. Due to the spread and diversity in fish species and sizes as well as the seasonal difference in bodily composition.J. 2000. P.. and Oehlenschläger.. for providing the example picture used in Figure 9.. K. H. hence allowing for measurements to be performed on large-scale quantities. A. 525–532. De Boever. 21(4). Lebensmittelwissenschaft und Technologie. 70(8). 8. Another issue is the substantial variety and heterogeneity of the material to be analyzed. 3. 2. and Tandberg.I. Near Infrared Spectroscopy in Food Analysis.. in Fishery Products: Quality.. however. 1997. 6.4. not easily applicable for small-scale industries as is often the case in the fish processing industry. 1992. quality seafood products will contribute to retaining the good reputation of fish and seafood in the years to come. 4. J. Oxford. England.

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

Non-destructive texture analysis of farmed Atlantic salmon using visual/ near-infrared reflectance spectroscopy. 68(2). 82.. 2003. 2004.. and Goula. G. 2004.. Wageningen Academic Publishers. et al. Lu. 165–175. Non-destructive measurement of bitter pit in apple fruit using NIR hyperspectral imaging.. 1998. 81(1). et al.. Nielsen. Journal of Food Science.. 52(3). Svensson. Y.M. J. Heia. Eds. 98–107. Lin. the Netherlands. 35. Herrala. E. P. 2005. 2002. Y. 147–157.. 61(1). 38. Nicolai.. Transactions of the ASAE. P. Y. 46(2). Development of hyperspectral imaging technique for the detection of apple surface defects and contaminations. 63. et al.. Oehlenschlager. 10.136 ◾ Handbook of Seafood and Seafood Products Analysis 32. Non-contact transflectance near infrared imaging for representative on-line sampling of dried salted coalfish (bacalao). B.. Hyvarinen. T. Determination of the protein content in brine from salted herring using near-infrared spectroscopy. 34. Nondestructive determination of water and protein in surimi by near-infrared spectroscopy. 2006.S. K. Y. G. Lu. in Digital Solid State Cameras: Designs and Applications. 1821–1826. A. International Journal of Pattern Recognition and Artificial Intelligence. Analytical and Bioanalytical Chemistry. 2001. et al. 43–54. J..B. 49. M. 2003. K. Visible spectroscopy—Evaluation of storage time of ice stored cod and frozen hake. Journal of Food Engineering.. Kohler. A. Proc. in Quality of Fish from Catch to Consumer. Infrared spectroscopic imaging: From planetary to cellular systems. 47. et al. 96.M. Isaksson.P.T. Journal of Food Science.F. 491–495. 45. and Okkonen. 2543–2547. 2001. Adamopoulos.M. 2006. E.. et al. 4161–4167. H. Food Chemistry.. Nilsen. 33. and Olafsdottir. R. Journal of Food Engineering.. 37. Journal of the Science of Food and Agriculture. pp. ElMasry. 48(1)... J. Hyperspectral imaging for nondestructive determination of some quality attributes for strawberry. 199–207. Transactions of the ASAE. 6404–6408. 2003.. R. et al. Wold. Jr. 44. Multivariate image analysis of a set of FTIR microspectroscopy images of aged bovine muscle tissue combining image and design information. 41. R. Application of near-infrared reflectance spectroscopy in the determination of major components in taramosalata. 2004.Technologie.. et al. Imaging spectrograph and camera solutions for industrial applications. et al.. 36. Direct sight imaging spectrograph: A unique add-in component brings spectral imaging to industrial applications. 1996. Herrala.. 803–809. R. and Lu... SPIE 3302. ed.. 389. 52. Postharvest Biology and Technology. 2003. Nondestructive determination of moisture and sodium chloride in cured Atlantic salmon (Salmo salar) (Teijin) Using short-wavelength Near-infrared Spectroscopy (SW-NIR). Nondestructive prediction of moisture and sodium chloride in cold smoked Atlantic salmon (Salmo salar). 1143–1153. G. 46. 42. Journal of Near Infrared Spectroscopy 14(1). Wageningen. Agricultural and Food Chemistry. et al. Huang. Modeling multispectral scattering profiles for prediction of apple fruit firmness. Uddin. Bruise detection in pacific pink Salmon (Oncorhynchus gorbuscha) by visible and shortwavelength Near-Infrared (SW-NIR) Spectroscopy (600–1100 nm). Colarusso. 40. Huang. 2006. A.. 53–60. 49. 39. and Dall’Ava. 67–68. 67(7). 59–66. 48. Peng. M. Detection of bruises on apples using near-infrared hyperspectral imaging. 2007.H. 2004. et al. 50. Luten. et al. 106A–120A. Mehl. 43. . 37. Journal of Food Science. and Bro. Detection of sodium chloride in cured salmon roe by SW-NIR spectroscopy. 523–530. 31(2). Lebensmittel. Williams. 2007. Journal of Food Engineering. 482–486. 67(5).. 235–242. et al. 2002. V. 40(1).G.M. Huang. 1–6. J. 51.Wissenschaft und. Visible/Near-Infrared spectroscopy—A new tool for the evaluation of fish freshness. 51.. H. Postharvest Biology and Technology. Applied Spectroscopy. 1998. T. Multispectral imaging for predicting firmness and soluble solids content of apple fruit. Journal of Agricultural Food Chemistry. 201–209. et al.

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

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

................1 Main Microscopy Techniques for Studying Seafood ....................................................... 149 10..140 10..................................................1 Smoked Salmon..................3 Processed Fish Microstructure ..............................2 Fish Muscle Microstructure......................................................... 148 10..... fats................1 Herring ............................................... (2008) gave an overview of the most important techniques for studying muscle food structure................. Pérez-Munuera et al........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.................. 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........... carbohydrates..................139 10.145 10............................... and María-Angeles Lluch Contents 10........................................................2 Salted Cod....... Ana Puig............................................................................. 151 References ..........2........3..................................3.3....................................................... This chapter 139 .........Chapter 10 Microstructure Isabel Hernando................................................................................ Empar Llorca..148 10.......146 10...2.......................................... 151 10.................... etc.......................) is responsible for their microstructure.............................................. The organization of the chemical components of foods (proteins............................................................150 10...4 Squid Microstructure ................ Several strategies can be used to study food microstructure......................2 Hake ..................................

the sample is frozen in slush nitrogen (Figure 10. In recent years considerable progress has been made in the field of SEM through vitrification techniques. The light microscope (LM) is a very versatile tool that works in different applications such as bright field. The fibers are essentially the same as those of terrestrial animals in terms of the arrangement of the thick and thin filaments.1). They are arranged in concentric circles forming subdivisions of striated muscle (Figure 10. and coating with a conducting metal for SEM imaging or with carbon for x-ray.) before examination in the LM. the samples need to be prepared first. 2008). and observed. When physical fixation is used. light green. backscattered electrons. the sample has to be prepared in semithin sections of about 0. Once the semithin sections are obtained. infiltration and embedding in resin. the sample can be observed with all its constituent water. dehydration in a series of ethanol dilutions of increasing concentration. At each subdivision there are macroscopic collagenous dividing lines (myocommata). in this way. phase contrast or differential interference contrast (Nomarski). etc.4) and quickly transferred under vacuum to a cold stage fit on a microscope where the frozen sample is fractured. Besides the secondary electrons. sudan.0 mm in diameter.. the sample preparation steps are chemical fi xation (with aldehydes and osmium tetroxide. critical point drying.02–1.1–2 mm (Figure 10. The muscle cells are short and 0. They are each surrounded by the sarcolemma membrane and by a thin layer of connective tissue (endomysium). which is contiguous to the myocommata (Ofstad et al. There are two ways of preparing samples for SEM: chemical fi xation and physical fi xation (Figure 10. showing alternate arrangements of .. image analysis relies heavily on computer technology to obtain quantitative results from microscopy observation. other emanations or signals such as x-rays. 2006).3). In this way. For this. may be generated as a result of the electron beam striking the specimen (Pérez-Munuera et al. The most useful application for studying seafood structure is bright field microscopy. the sample is frozen in liquid nitrogen and then freeze-dried before being coated and observed. and so on. In Cryo-SEM. 10. secondary fi xation with osmium tetroxide. Electron microscopy (EM) allows food structures to be studied at higher magnifications than those used in LM. and staining the ultrathin sections with heavy metal solutions such as lead citrate or uranyl acetate. so microanalysis can be carried out by means of x-ray. iodine. The SEM method observes the surface of the sample. 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. polarizing microscopy. ions or molecules can be identified and quantified in situ using specific detectors coupled to the electron microscope. dehydration in a series of ethanol dilutions of increasing concentration. Many of the endomysia are connected to the perymisium. or fluorescence microscopy.140 ◾ Handbook of Seafood and Seafood Products Analysis provides a detailed description of the protocols often followed to obtain information about seafood microstructure.2 Fish Muscle Microstructure Fish muscle consists of myotomes. In both methods. cutting ultrathin sections (5–100 nm) in an ultramicrotome. coated. red oil.2) are primary fi xation with aldehydes such as glutaraldehyde. The steps in preparing samples for TEM observation (Figure 10. these different signals can be captured by the appropriate detector in each case. etched.5). In the former. Two types of microscopes use electron beams as their source of illumination: transmission electron microscopes (TEM) and scanning electron microscopes (SEM). as for TEM). Finally. so there is no need to section it. they are mounted in glass slides and stained with different dyes (toluidine blue.

1% red oil.Microstructure ◾ 141 Food Specimen portions Embedding in paraffin or resin Freezing (CO2. LM observation Figure 10. . ... liquid N2) Preparation for slicing Cold knife CO2 Knife Slice Semithin sections (0.11–2 μm) Cold stub Microtome Cryotome Mounting in glass slides Staining specimen 1% Toluidine blue 1% Lugol.1 Preparation of samples for LM observation.

2 Preparation of samples for TEM observation. 90%. 50%. 70%. 100%) Infiltration and embedding in resine Epoxi resin. Araldite Spurr’s. 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%.142 ◾ Handbook of Seafood and Seafood Products Analysis Food Specimen portions Fixation 2. .

50. . for X-ray) SEM observation Figure 10.3 Preparation of samples for SEM observation. 70. Pd. 90. for SEM imaging) (C. 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.5% Glutaraldehyde 2% Os O4 Ethanol (30.Microstructure ◾ 143 Food Specimen portions Fixation Physical fixation Chemical fixation Quick freezing in liquid N2 2.

.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. vaccuum) Cryo-SEM observation Figure 10.4 Preparation of samples for Cryo-SEM observation. C. . .

The myofibrils are shown in longitudinal section in this sample (Figure 10.6A shows a cross section of herring tissue fi xed with glutaraldehyde and observed by SEM. it is possible to observe ultrastructural details.2. Figure 10.7C shows the inside of a muscle fiber with the myofibrils perfectly bundled.1 Herring Figure 10. When ultrathin sections of herring muscle tissue are studied by TEM. the aggregation of solutes produced during the etching of the sample generates the typical eutectic artifact observed in Figure 10. a: myotome. 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. Figure 10. surrounded by the sarcolemma and by the endomysial connective tissue. fat globules of different sizes are observed occupying the interfibrillar spaces and myofibrils are distinguished inside the cells.6C). 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. 2008). The fiber is composed of myofibrils in which Z disks are distinguished in the areas where the sarcolemma is broken.5 Schematic representation of fish muscle with myotomes. Fixing in osmium tetroxide shows the distribution of fat in the herring tissue. 10. but the total collagen content is lower. with the endomysial connective tissue keeping the muscle fibers firmly attached to one another. Examples of different fresh fish tissues observed by several techniques are described here. which is mainly composed of collagen. When the muscle fibers are observed using the Cryo-SEM technique. The longitudinal section in Figure 10. A and I bands (Pérez-Munuera et al. obtained by the same technique but observed at a higher magnification. At low magnification. since the water in which the fish live lends support for the body (Lampila. In a cross section of the sample fi xed in osmium tetroxide (Figure 10.7A shows a herring sample stained with toluidine blue and observed by LM.6B).. A micrograph cross section of the fibers shows them surrounded by the sarcolemma. The layouts of the Z disks that mark the length of the sarcomere are visible.6E). b: myocommata. The myofibrils are .6D shows the microstructure of herring tissue at a higher magnification. where the Z disks can be distinguished. The typical fish muscle fibers can be seen. 1990). Figure 10. where the fat is viewed as globules on the surface of the fiber. reveals the myofibrils inside each cell.6F. the perymisial connective tissue that surrounds the muscle bundles can be seen. the fat can be observed covering the fibers in a longitudinal section of herring muscle (Figure 10.7B.

7D). for example.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. The main difference is their size: hake fibers are thicker than herring ones. 2007). Z. connected to each other at the Z disk level by the costameres. c. which are the components of the cytoskeletal network that links the myofibrils to one another and to the sarcolemma. can be seen. . (F) Cryo-SEM.. Z disk.8. (2005) after depleting the thick and thin filaments with a potassium iodide treatment. f.9).2. connective tissue.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. a. The cytoskeletal ultrastructure of hake was studied by Pagano et al. pork meat (raw ham) (Larrea et al. the A and I bands. The TEM technique allows images to be obtained at higher magnification and with better resolution than other microscopy techniques (Figure 10. 10. fat globule. and roughly parallel filaments (Figure 10. The structural elements that constitute the sarcomere. along with the M and Z lines.6 Herring tissue. (A–E) SEM. eutectic artifact. The same structure has also been observed in different meat products. Hake fibers surrounded by connective tissue can be observed in Figure 10. TEM and SEM studies demonstrated an extensive network of filaments connecting Z structures that were regularly spaced and connected by sets of longitudinal. continuous.

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

13. the higher the smoking temperature. et al.10B shows a detail of an intercellular space created by the conjunction of three fibers or cells.) 10.10 Smoked salmon. Gudmundsson and (A) 100 μm (B) 10 μm Figure 10. longitudinal filament connecting Z-Z. 2005. The data of the average cross-sectional area of muscle fibers showed that the smoking process produces shrinkage of the fibers. Z-disk. 141. Physiol.. Sigurgisladottir et al. Figure 10. .148 ◾ Handbook of Seafood and Seafood Products Analysis Z LZ IZ DZ 8. the greater the shrinkage.000 X Figure 10. 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. Z. With permission. M. Com.R.9 Cytoskeletal structure of hake observed by TEM.1 Smoked Salmon A cross section of a smoked salmon sample obtained using the Cryo-SEM technique is seen in Figure 10. LZ.3. B.3 Processed Fish Microstructure 10. (A and B) Cryo-SEM. Biochem. (Reprinted from Pagano. (2000) used LM to observe the changes that occurred in the salmon during the smoking process and quantified them by image analysis.10A. The micrograph shows geometrically shaped fibers surrounded by a connective tissue.

and gaps were formed in the tissue structure. (A) SEM.12 Seafood stick (surimi) observed by SEM.11B shows a cross section of salted cod tissue (A) 100 μm (B) 300 μm Figure 10. Figure 10. .Microstructure ◾ 149 Hafsteinsson (2001) studied the effect of pulsed electric fields (PEF) and a combination of PEF and high pressure on smoked salmon microstructure. (B) Cryo-SEM. where two fibers can be observed completely covered by salt deposits. Figure 10. (A) (B) (C) Figure 10. and (C) protein network. A combination of PEF and high pressure had a more detrimental effect on smoked salmon microstructure than PEF treatment alone. (B) cross section. these treatments decreased the cell size compared with fresh salmon.3. (A) longitudinal section.11A shows a longitudinal section of salted cod.11A) in samples that have been obtained using physical fi xing (freeze-drying) instead of chemical fi xing.2 Salted Cod The presence of salt deposits in the cod tissue can be observed by SEM (Figure 10. 10.11 Salted cod.

3 Surimi One of the most common surimi products on the market is artificial crab muscle. Figure 10. 10. after adding different additives. The Chemical and Functional Properties of Food Proteins.3. With permission.150 ◾ Handbook of Seafood and Seafood Products Analysis observed by Cryo-SEM. obtained by SEM.. 2001.A.12A. the paste is shaped and an “artificial fish muscle” is obtained..) . shows a longitudinal section of a crab stick where the “artificial fibers” can be observed. The cross section (Figure 10.” Lean fish meat is minced to a paste. Such a product is often sold as “crab sticks” or “seafood sticks.13 Schematic representation of (A) squid mantle and (B) arrangement of muscle cells. Inc. (From Lluch. Lancaster. Technomic Publishing Co. M.. et al. where the presence of salt makes the etching of the sample for observation difficult and masks the underlying structures. The formation of a new network with the myofibrillar protein (Figure 10. PA.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 10.12B) shows the typical concentric layers of this type of surimi product.

Each fiber is surrounded by a sarcolemma (Llorca et al. 807.14D). . Lampila. Muscle Foods. 1. approximately 3. Circumferential muscle bands (100–200 mm) comprise fibers running about the entire circumference of the mantle cone. 10. respectively.14A and B) (Llorca et al. the intermyofibrillar spaces between these can be observed. 12. This fiber distribution can also be observed by LM in samples stained with toluidine blue (Figure 10. 2001). (C) LM. Eur. Regardless of their orientation. Radial bands (10–15 mm thick) comprise fibers that connect two tunics of connective tissue. 2007). 247–267. J. poultry and fish muscle. m. 2007. Muscle fibers are grouped in bands that are arranged orthogonally. Trends Food Sci.14C). l.13). Comparative microstructure of red meat. The fibers arranged in circumferential and radial bands were observed by SEM in samples fi xed with glutaraldehyde (Figure 10. L... myofibril. With permission. et al. sarcolemma. (A and B) SEM.Microstructure ◾ 151 –200 μm –10 μm (A) 10 μ (B) 3. 2001. 2001).75 μ m s (C) (D) l Figure 10. all the muscle fibers are thin. LM makes it possible to distinguish a peripheral area in blue and a central core in white inside each cell... M. 1990. and Hafsteinsson. Effect of electric field pulses on microstructure of muscle foods and roes. References Gudmundsson.) ties of surimi. E.. The inner and outer tunics are covered by a visceral lining and outer lining.14 Squid.E. H. and (D) TEM. 122–128.. central sarcoplasm. a central sarcoplasm is shown to be surrounded by myofibrils. Food Res. Tech.. s. 225.5 mm in diameter (Lluch et al.4 Squid Microstructure The squid mantle is composed of muscle tissue sandwiched between two tunics of connective tissue (Figure 10.. (From Llorca. When TEM is used to study the ultrastructure of fresh squid (Figure 10. 1990). Technol. Th is gel network structure gives surimi its characteristic elasticity and texture (Sikorski.

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

...157 11.............................................3................................................... The relationship between chemistry and food properties is particularly interesting in the case of fish and seafood in general............165 11.............................3...................... is one of the most used method to assess freshness by both consumers and industries [2].............6 Conclusions ............................................... For these reasons the knowledge of the chemical 153 ................................2 Conducting Polymers and Molecular Aggregates ..... some are of great importance to define overall properties such as freshness or quality [1]...............................................................................1 Introduction Among the thousands of molecules composing food complex mixtures.......................160 11....5 The Application of Electronic Noses for Fish Freshness and Quality Measurement ........3.....1 Metal-Oxide Semiconductors ....................1.157 11...............................160 11.....................1 Introduction ...........................153 11...........2 Sensor Parameters.....157 11......................................... called odor..............1 Sensors Based on Conductance Changes ...159 11...........159 11......................................... for which the human perception of airborne chemicals......4 Field-Effect Transistors ..................................3.............................................................................................................................158 11..................164 References ..................................2 Amperometric Gas Sensors ........Chapter 11 Chemical Sensors Corrado Di Natale Contents 11..................................................5 Color Indicators ...........................3 Mass Transducers .........................................................................154 11..........................................................................................3 Chemical Sensor Technologies .......................158 11..4 Electronic Noses .......................................................................................................................3.....................1...........3...........3..................158 11......

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

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

it is necessary to evaluate the influence of measurement errors.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. the selectivity of a chemical sensor can be obtained only in very limited conditions. and their importance holds for any kind of sensor. the error ΔV is limited by the electronic noise that determines the ultimate uncertainty of any electric signal. For chemical sensors.2b the corresponding sensitivity is shown. The response curve is almost linear at low concentration and tends to saturation corresponding to the complete occupation of available adsorption sites. Lack of selectivity means that the sensor responds with comparable intensity to different species. The previously mentioned quantities are totally general. it is not possible to deduce Saturation Nonlinear region Sensitivity Concentration Signal Linear region Concentration Figure 11.156 ◾ Handbook of Seafood and Seafood Products Analysis Only in case of a linear response function. For chemical sensors. and it tends gradually to zero as the sensor response reaches saturation. and the selectivity can be achieved in many practical applications. In all the other cases. 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. The sensitivity is larger at low concentrations. In the case of physical sensors. Selectivity defines the capability of a sensor to be sensitive only to one quantity rejecting all the others. 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. Simple mathematical considerations lead to the conclusion that given an error ΔVerr affecting the signal V. In order to fully appreciate the importance of sensitivity. With these conditions. the sensitivity is a constant quantity. In Figure 11. Let us consider the generic case of a chemical sensor based on a sensitive material characterized by a limited number of adsorption sites. 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 number of quantities is limited to a dozen.2a. it is a function of the concentration. The amount of adsorbed molecules as a function of the concentration is ruled by the Langmuir isotherm [6]. . 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 worth mentioning that in case of electrical signals. an additional parameter of great importance is selectivity. and with such a sensor.

and a lowering of the potential barrier. which reacts with the bounded oxygen to form carbon dioxide. dissociative adsorption sites of molecular oxygen are active on the oxide surface. These are oxides of transition metals. the number of conductance electrons is limited. The sensitivity can be further modified adding ultrathin amounts of noble catalytic metal atoms on the surface. This is only one of the many interactions taking place on the surface of metal oxides. Sensors are here classified according to the physical intermediate quantity. Metal oxide semiconductor chemoresistors have been used several times in fish freshness applications. The main sensitivity mechanism is related to the role played by oxygen.3. Since the material is a semiconductor.12]. 11.4. 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. in this regard. and as a consequence.1 Sensors Based on Conductance Changes 11. a reduction of the surface conductance band depletion. providing the maximum sensitivity. In practical. 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.1 Metal-Oxide Semiconductors Changes in conductance become appreciable in materials characterized by a limited number of mobile charge carriers. and then the amount of oxygen molecules that can be adsorbed at the surface is also limited. At sufficiently high temperature (above 200°C). semiconductors are subjected to large changes of conductance also in the presence of a modest variation in the number of conductance electrons or holes. Paradigmatic.3 Chemical Sensor Technologies In this section the basic principles of the most popular categories of chemical sensors are illustrated. in any case. the most known and studied of which is SnO2 [7]: a wide band gap n-type semiconductor. although interesting.3. and the sensitivity of these devices is extended to many different kinds of volatile compounds [8]. . an electrically actuated heater is integrated in the device. metal oxide growth in regular shapes such as nanosized belts [9] has shown peculiar properties. It is important to remark that this kind of sensors needs to be operated at high temperature.1. The consequence of the exposure to oxygen is a reduction of the surface conductance. and a surface potential barrier is formed. releasing an electron back to the conductance band. Selectivity will be reconsidered in Section 11. For instance. is the case of carbon monoxide.Chemical Sensors ◾ 157 any reliable information about the chemical composition of the measured sample. The characteristics of these structures. The amount of depletion and the barrier height are proportional to the number of adsorbed molecules. The most popular materials undergoing a conductance change on interaction with gases are metaloxide semiconductors. Metal-oxide semiconductor sensors can be prepared in many different ways. 11. Recently. 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. have not yet resulted in practical improvements of performances. 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.

the mechanical resonance of the crystal is coupled with an electric resonance.3 Mass Transducers The adsorption of molecules into a sorbent layer produces a change of mass.158 ◾ Handbook of Seafood and Seafood Products Analysis 11. QMB coated by sensitive layers was used for many applications. a sensor for ammonia can detect amines. these sensors were properly used to detect fish freshness [16]. sensors designed for CO are found to be sensitive toward alcohols. This property is largely exploited in electronics to build stable oscillators as clock references. the electric resonance is characterized by a very large quality factor (Q).2 Amperometric Gas Sensors Electrolytic cells based on either solid-state or liquid-ionic conductors are used to detect several kinds of gases. with broader scopes related to the possibility of developing a novel sort of electronics based on carbon chemistry [13]. 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. the measurement of these mass shifts can allow the evaluation of the amount of adsorbed molecules. In spite of the claimed properties. aggregates of polypyrrole or polythiophene have a semiconducting character. and a sensor for SO2 can detect volatile sulfides. Since crystal resonance is extremely efficient. and esters. 11. As an example.3. . The measurement of small mass changes is made possible by piezoelectric resonators. With respect to metal oxides these sensors have two important advantages: they are operated at room temperature and.3. most important. and food freshness is among them [15]. the electric frequency decreases linearly with the mass.3. Due these cross-selectivities.1. A typical QMB has a limit of detection around 1 ng. and their conductance can change after exposure to volatile compounds. the possibility of using these sensors to measure fish freshness was demonstrated with metalloporphyrin coating [18].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. The main mechanism is the catalytic reaction occurring on the surface of a noble metal electrode. Chemical sensors based on conducting polymers may be considered as a lateral result of these studies. These are thin slabs of AT cut quartz oscillating at a frequency between 5 and 50 MHz approximately [17]. Due to the piezoelectric effect. Indeed. Thanks to this versatility. If the quartz is connected to an oscillator circuit. Although designed for polluting gases. their chemical sensitivity can be changed at synthesis level modifying the chemical structure of the monomer [14]. aldehydes. The same effect is exploited for chemical sensing adopting particularly shaped crystals such as in quartz microbalances (QMB). A piezoelectric resonator is a piece of piezoelectric crystal properly cut along a well-specified crystalline axis. an amount that is sufficient in many practical applications. More sophisticated mass transducers were proposed by using resonant cantilevers similar to those adopted in atomic force microscopy [19]. these sensors were never demonstrated in practical applications. Piezoelectric effect can also be exploited in other configurations such as those based on surface acoustic waves. conducting polymers sensors can be prepared for different applications. The frequency of the mechanical oscillation decreases almost linearly with the mass gravitating onto the quartz surface. For instance. these sensors demonstrated a good sensitivity for compounds relevant for fish freshness. 11.

PDAs. giving rise to a number of low-cost advanced optical equipments such as digital scanners. organic molecular layers. FET structures were also modified to accommodate. in the last decade we have seen rapid growth in performance in fields such as consumer electronics. Nonetheless. allowing a simultaneous evaluation of absorbance and fluorescence of samples.3. is based on the fact that a computer screen can be easily programmed to display millions of colors. and screens. also appreciable by eye. 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. The feasibility of this approach has been demonstrated using as sensitive layer a film of a sodium salt (bromocresol green) [25]. the colorimetric detection of fish freshness recently received a novel interest. and cellular phones all endowed with color screen. Indeed. known as computer screen photo assisted technique (CSPT). the current flowing in the FET changes revealing the chemical interaction. as a sensing part. In this way sensitivity to ammonia. Furthermore.3.Chemical Sensors ◾ 159 11. standard optical instrumentations are usually expensive. whose sensitivity toward amine was also recently measured [23]. This salt exhibits a rather large change in color. 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]. such as metalloporphyrins [22]. the visual determination limits the performance and may greatly vary between individuals. . This difference can be modulated by a layer of electric dipoles that can reach the metal–oxide interface. 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). On the other hand. 11. and hydrogen atoms can diffuse through the palladium film until they reach the oxide surface. furthermore.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. As a result. an important gas for fish freshness and quality. although under constant bias. was also obtained [21]. cameras. In particular.5 Color Indicators Although known for several years [24]. Due to the large diffusion of portable computers. the chemical practice of this approach is badly balanced by the transducer counterpart. This last technique. and. Compared with the use of digital scanners. H2 molecules dissociate into atomic hydrogen at the palladium surface. The method demonstrated also the possibility to identify a number of different amines [28]. 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. 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]. combining wavelengths in the optical range. This basic structure was successively modified changing the gate metal and thickness to extend the range of measured gases. whose characteristics largely fit the requirements necessary to capture change in optical properties of sensitive layers in many practical applications. where they form an ordered dipoles layer. 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]. The principle was adequately exploited with a palladium gate FET exposed to hydrogen gas [20].

the application of the CSPT concept may be foreseen as greatly extending the analytical capacity worldwide. 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. Receptors were found to be rather unselective. After this conjecture. The most important chemicals involved in the fresh fish odor are long-chain alcohols and carbonyls. and each molecule is sensed by many receptors [33]. and the genes expressed by olfactive receptors are known [31]. CSPT has demonstrated its utility in particular to classify airborne chemicals reading absorbance and fluorescence changes in chemical dyes such as metalloporphyrins [30]. each receptor senses several kinds of molecules. 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]. and acid compounds. The physiology of olfaction has made considerable advances. organic synthetic receptors offer an unlimited number of possibilities to assemble molecules endowed with differentiated sensing features. allowing for electronic nose application oriented optimizations. Investigations about olfaction receptors show that Nature strategies for odor recognition are completely different from those of analytical chemistry. Their concentration and the presence of other compounds are rather typical of each species.” This denomination is currently given to any array of unselective chemical sensor coupled with some multicomponent classifier. 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]. Nonetheless.4 Electronic Noses As discussed above. Among these compounds amines . 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. bromophenols. After this discovery. N-cyclic. it was proposed that arrays of nonselective chemical sensors may show properties similar to those of natural olfaction [34]. 11.5 The Application of Electronic Noses for Fish Freshness and Quality Measurement The composition of fish headspace is a source of information about its freshness. and an even more extended computation capabilities. sulfur compounds. and N-cyclic compounds. amines. To this point of view. 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.160 ◾ Handbook of Seafood and Seafood Products Analysis camera. 11. and aromatic. On the other side. The concentrations of these chemicals are directly correlated to the degree of spoilage. almost all sensor technologies were used to build such systems. 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]. observation of Nature offered a useful suggestion about the use of such devices. Standard optochemical sensors are based either on absorbance or on fluorescence. The features of electronic noses are fundamentally dependent on the sensing properties of the artificial receptors. the possibility of developing artificial olfaction systems became possible. and such systems were soon dubbed as “electronic noses. microbial spoilage produces short-chain alcohols and carbonyls.

the accumulation of some compound. for example. and finally from products of lipid oxidation [37]. An imperfect application of this method was demonstrated with engineered polymer-coated QMB [50]. the interaction between polymers and volatile compounds is often described by the linear sorption energy relationship (LSER) model [51]. from fish processing. quartz microbalance sensors [44. conducting polymer sensors [43]. In order to understand the potential of electronic noses to detect fish freshness. and fresh. but the behavior at the beginning is absolutely nonlinear. five different kinds of interactions are considered: dispersion. amines become instrumentally appreciable only when spoilage processes take place. In addition. Standard analytical methods for volatile amines and also sensors for some specific amines have been used to inspect fish freshness. In gas chromatography. Sensor data can be conveniently represented by a principal component analysis (PCA) scores plot. the progress of spoilage is less linear with respect to Figure 11. with an array of selective sensors it is not possible to distinguish between fresh and flat fishes. nonetheless. This result is rather surprising because fish spoilage is in general expected to be a linear and somewhat straightforward process. As a result. Data are extrapolated from an investigation by Strachan and Nicholson [48]. MOSFET sensors [41]. In LSER. as much as possible. Analyzing the data with PCA the plot of Figure 11. Minor contributions to the fish headspace come from contamination of the environment (e. the data produced by a sensor array can classify samples according to some of their global features. Nevertheless. preserved [49].6 . Instruments based on different sensor technologies have been used. Figure 11. In this regard.5 is obtained. PCA is a data analysis method allowing the representation of a multidimensional dataset in a reduced dimensionality space. hybrid electronic noses were used combining different sensor technologies such as QMB and amperometric sensors [47]. petroleum in sea). The same nonlinearity is observed with electronic noses. In recent years attempts to use electronic nose technology to track the spoilage processes occurring in fishes have been reported in numerous articles.45]. polarity. in case of fishes. When properly analyzed by pattern recognition methods. flat. and acid. let us discuss a simulation of a case study. dipolarity. let us consider an array specific for each LSER interaction and one compound for family. it is more realistic to consider an array of sensors specific for a single interaction mechanism. a plane. and optical indicators [46]. the chemical complexity of the problem. 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. Let us consider the use of an array of sensors absolutely selective for each individual family of compounds mentioned in Figure 11. showing the ability of the electronic nose to track the different spoilage levels occurring at different storage times. The number of compounds whose concentrations are only partially correlated makes this application particularly appealing for sensor arrays of partially selective chemical sensors.4 demonstrate a continuous progress after the 8th day. Since LSER was fruitfully used to model polymer-based chemical sensors [52]. with a super impression of 6th and 1st days.. In Figure 11. and sweet conditions are hardly identified. Each sensor then provides a signal proportional to the concentration of each family. hydrogen bond basic. Results shown in Figure 11.Chemical Sensors ◾ 161 are considered as the typical markers for fish freshness detection. The representation plane is determined as that where the data variance is maximized and then the statistical properties of the dataset are.3.g. such as metal-oxide chemoresistor sensors [38–40]. Most of these are feasibility studies.4. typically according to the freshness or more precisely according to the balance between fresh and spoilage produced compounds. The sensitivity of chemical sensors is not immediately related to the molecular family but rather to the interaction mechanism. and the decrease in others result in a nonlinear problem.3 the time evolution of the major families of volatile compounds found in the headspace of fishes is shown. Apparently. amperometric sensors [42].

The possibility of developing a multisensor device to measure and/or estimate fish freshness with a combination of instrumental techniques (electronic noses. texture meters.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 0.1 0. and olfaction (to smell the gill odor) [54]. 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). The experiment was related to COD fishes. The typical sensorial description is also reported. and original data were previously published [53].5. Nonetheless. humans provide a more reliable identification of fish freshness. . each able to capture different aspects of fishes. This feature that can be interpreted as a failure of the electronic nose is likely due to an intrinsic nonlinearity of the studied problem. and devices measuring electrical properties) has been illustrated in different applications related to cods [56. 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]. As a consequence.57].g. spectroscopic methods. olfaction) provides several errors of evaluation. and groupies [59]. tactile (to test the flesh firmness and elasticity). with a folding back of the spoilage process in the representation plane. in order to measure the quality of fish instrumentally. and the use of only one sense (e. shows the scores plot of a partial least-squares discriminant analysis model related to an array of metalloporphyrin-coated QMBs.01 0 5 10 15 Days 20 25 30 35 Figure 11. color meters. image analyzers. an integration of instruments is necessary..3 Time evolution of the major families of volatile compounds in fish headspace. Results are qualitatively similar to those shown in Figure 11. sardines [58].

39%) Figure 11.76%) 0.5 –2 –2.5 1 PC 2 (15.5 0 –0.5 –6 4 6 –5 –4 –3 –2 PC 1 (80.4 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.Chemical Sensors Scores plot 2 30 1.3 are used. each specific for a single interaction mechanism among those modeled by LSER. Scores plot 1.5 –1 24 10 12 8 28 2 4 6 1 ◾ 163 22 20 18 16 14 –1. Data show the impossibility of distinguishing the spoilage process in the first 6 days and an abrupt change between 6th and 8th days.5 24 PC 2 (19.5 –6 –4 –2 0 2 4 PC 1 (72.93%) 0 –0.5 –1 –1.5 1 0. .5 PCA scores plot of data related to a virtual array of sensors.

one technology may outperform the others. all the actors of the food chain (producers.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 (63.6 PCA score plot of metalloporphyrin-coated QMB. As an example. Each step of the food chain has peculiar needs that a proper chemical sensor approach can in principle contribute to satisfy. 11. These technologies are sometimes equivalent in terms of performances. analyzed.62%) Figure 11. data are related to cod fish fillets. and for some specific applications.164 ◾ Handbook of Seafood and Seafood Products Analysis Scores plot 150 17 17 100 17 17 50 LV 2 (26. sensors are . 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. transmitted and integrated with other data can be performed by several different technologies. Food-related sites are usually highly contaminated from the point of view of odor. and labels indicate the storage days in ice.6 Conclusions The conversion of chemical information into electric signals that can be measured. Chemical sensors are an almost mature technology for many practical applications. In this regard. stored. At the current state of the art. at producer level the increment in quality and yield. All these applications require instruments able to work on-site. and consumers) are potential users of chemical sensor technology. processors. and finally at consumer level. the application of arrays of sensors can greatly improve the performance in terms of prediction of quality and freshness. the control of quality and safety both on the market and at home. at processors level the screening of quality of incoming products to optimize the processing and to sort processed food. In the case of fish and seafood freshness and quality determination.

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

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

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

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.........6 Advantages and Benefits of Microwave Methods ................................................................................................3 New Technologies for Online Control ..172 12...................................3.............................. Pedro José Fito Suñer..2 Visible Spectroscopy ................184 169 ..........5.......................180 12......................................................................5.173 12.................1 Determination of Moisture Content ......................................6 Conclusions ................173 12..................1 Ultrasounds—Acoustic Spectroscopy ........174 12............3.........................................................3....... by Microwaves: Methods and Equipments ..........................4 Overview of Microwave Theory .......5 Microwave Spectroscopy—Dielectric Spectroscopy ....................................................2 Freshness and Salting/Desalting Process Quality Control of Fish and Seafood...................... Ana Andrés Grau...................3............................170 12.................................................................................5 Applications of Microwave Technology in the Assessment or the Control of Processes ..................................................................... 176 12...........................2 The Importance of Quality Control—Advances in the Online Control Techniques ..............182 12................170 12...................................................................................................175 12........179 12.............................................3.............................171 12................Chapter 12 Physical Sensors and Techniques Ruth De los Reyes Cánovas..3......3 IR Spectroscopy .................................................4 RF Spectroscopy—Impedance Spectroscopy ..............184 References ............................................................................................ and Pedro Fito-Maupoey Contents 12............174 12.........................1 Sensors for Quality Assessment ......

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

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. total quality management (TQM). and reduction of production cost and production time (increased throughputs). Thus. which for the consumer include. The great challenge is indeed to focus on the real time and online sensors and data systems surveying processes and products. Consequently. including time/temperature histories that can affect the freshness and quality of the products. This kind of system not only permit an assessment of quality in terms of their properties but also. safety. with the appropriate hardware and software. and product type [3.4]. or chemical properties. and storage techniques. and authentication all require improved control methods. It is necessary to stress that fish quality is a complex concept involving a whole range of factors. in particular through online or at-line quality sensors. 12. In this way many new food safety concepts and key quality parameters have arisen during the last decade: Hazard analysis critical control points (HACCP). convenience and integrity. food inspectors require good manufacturing practices. food producers are increasingly asking for efficient control methods. but online methods are required for industrial quality control. such as water content for drying processes. and compliance with the regulations. they all call for intime and online sensors for control. Further. In addition to the requirements of consumers. consumers. Concretely. warning systems. automation. and low cost in the sensor’s compounds. and much more. new data systems. the safety and quality of fishery products has been of particular concern in recent years. tight feedback loops for automation of the production. an excellence in accuracy. which are commonly structural. 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. nutritional and health information. and regulatory officials have been seeking improved methods for determining freshness and quality [2]. physical. it is able to obtain a final product that will always be within the margins of quality predetermined. is very important for the partners in the chain. therefore it is possible to apply to the product under development the necessary corrective measures while it is still in the manufacturing line. freshness. availability. traceability. With the increasing globalization of fishery product sales. processors. safety. the new sensors’ concept of being easy-to-use. processing. In general.3 New Technologies for Online Control The quality of almost all the industrial processes depends on the modification of a few parameters. 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]. for example. these properties need slow and destructive methods to be controlled. One of the most unique characteristics of fish as food is that it is a highly perishable commodity. and typing the product labels. Information about handling. . These systems will reach three milestones. controlling the automated process and the raw material stream. first to satisfy the consumer and regulatory requirements and second to improve the production feasibility. ISO 9000 Certifications. eating quality. size. labeling. quality sorting. A study performed by Consumers Union found that more than one-quarter of the fish samples tested were on the brink of spoilage [5]. In addition. and so forth. the obvious physical attributes of the species. sensing the final product quality. allow input from the manufacturing line with information obtained from the measurement of quality parameters selected (feedback). nutritional quality.

Ultrasound is a form of energy generated by sound (really pressure) waves of frequencies that are too high to be detected by human ear. when propagated through a biological structure. Depending on the frequency used and the sound wave amplitude applied. and raw meat mixtures can be related to its composition using semiempirical equations [7].19]. NIR measurements are widely used in the food industry to determine the sugar content in fruits [9]. which enable a variety of applications [18. Normally the modification of any quality parameter is macroscopically correlated to the change in any wave parameter that can be controlled. the reason is. it is almost imperative to resort to elastic (sonic) waves such as ultrasounds or to nonionizing electromagnetic radiation. above 16 kHz [17]. structure. below are cited some examples of the use of these new technologies in the quality control of foodstuffs. must act in real time and without producing permanent effects on the food. Thanks to advancing technology. microwave. Highfrequency. Ultrasound attenuation spectroscopy (acoustic spectroscopy) is a method for characterizing properties of fluids and dispersed particles. which. chemical. fulfilling the initial premise. It is also necessary to work at very low power in order to not cause permanent effects such as heating. The salt and water content are related to dielectric properties of cod at microwave frequencies [14–16]. and physical state of foods [20].e.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. a number of physical.13] in foodstuffs. determine the velocity of a moving tissue. Nevertheless. the other techniques that enable online control have been briefly commented on below. and widely used diagnostic tool. 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. The interaction between wave radiation and matter as a function of wavelength or frequency is called spectroscopy. exposing their main disadvantages and highlighting the advances in the field of seafood. we concentrate on electromagnetic methods at microwave frequencies. some of their propagation parameters are modified. Suvanich et al. in the case of Doppler-based modes. impedance measurements (RF) can determine salt and water content in salmon filets [10]. such as radio frequency (RF). Visible (and near UV) transmittance method has been investigated to inspect the internal quality (freshness) of intact chicken egg [8].. and visible. the modification of these parameters can be measured in real time.1 Ultrasounds—Acoustic Spectroscopy Ultrasonic is a rapidly growing field of research. i.3. The spectroscopic techniques use the information found in the spectrum that is emitted for the food to predict certain of its qualities. Ultrasound. 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. well-established. 12. and a high amount of energy can be imparted. in this chapter. When these waves pass through foods (or are refracted by them). It is virtually impossible for . which is finding increasing use in the food industry for the analysis of food products. [21] published a report on how the ultrasonic velocity measurements show potential for analyzing fish composition. The main disadvantage of ultrasound is that the energy propagates poorly through a gaseous medium. moreover. It is impossible to address all these techniques with precision. Ultrasound imaging is a versatile. Ultrasonic velocity in fish tissues. and biochemical effects can be observed. chicken. thermal and near-infrared (NIR). induces compressions and depressions of the medium particles. For fish samples.

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

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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.

12.3.4

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

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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,

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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.

12.4

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.

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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

(12.1)

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.

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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

179

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

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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.

12.5.1

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

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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.)

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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.

12.5.2

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

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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

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].

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12.6

Conclusions

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.

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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).

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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).

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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).

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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
189

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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

13.1

Introduction

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].

13.2

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

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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.

13.2.1

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

respectively) [21]. the QIM is suitable for early stage of storage of fish where other instrumental methods are not accurate [28].dk/QIMRS/qim_0202. Hyldig [29] indicated that the QIM is expected to become the leading reference method for the assessment of fresh fish within the European community. Melanogrammus aelefigus. The average score of 5. Therefore. The most comprehensive scoring scheme to assess fish is the Torry Scheme [36]. This method is considered to be a relatively fast. The scores are given from 10 (very fresh) to 3 (spoiled) (Table 13. . Objective sensory methods are essential for quality control and estimation of shelf life of seafood. farmed Atlantic salmon (Salmo salar) [31].2. panelists evaluate the odor and flavor of cooked fillets.192 ◾ Handbook of Seafood and Seafood Products Analysis quality expressed as a demerit score and storage life on ice.2. nondestructive method based on direct observation of sensory parameters of fish and can also be specific for species. and is nondestructive and can be used as a tool in production planning and quality warranty work [27]. Pandalus borealis. haddock.min. 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). a higher score can be given for a single parameter [27]. redfish shrimp. Rapid PC-based QIM is also available on the Internet at http://www. brill. The advantages of QIM are that it requires short training. Pleuronectes platessa. is rapid and easy to perform. and not always practical for large-scale commercial purposes. herring. and flavor. The Torry Scheme. 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]. Objective terms should be used rather than subjective terms.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. Descriptive words should be carefully selected.htm.2). herring (Clupea harengus) (Table 13.dfu. In QDA. and fat fish species. common octopus (Octopus vulgaris) [33]. pollock. However. the words for describing the odor and flavor of the fish can be categorized into two groups. The method can also be used for texture. and the panelists trained should agree with the terms. During spoilage. and turbot (Scopthalmus rhombus. It has been widely used in its original or modified forms. expensive.5 may be used as the limit for consumption [21]. Solea vulgaris. and redfish by the Danish Institute for Fisheries Research. sensory methods are time consuming. the Torry Scheme was developed at the Torry Research Station for use with expert and trained judges. medium fat. Sebastes mentella marinus. Pollachius virens. positive and negative sensory parameters based on whether fish are fresh fish or fish at the end of the storage period [37].3 The Torry Scheme In contrast to the QIM. instrumental methods are also needed to satisfy the need for quality measurements in fish industry. In addition. odor. 13.1) [34]. fresh cod (Gadus morhua) [32]. often referred to as the Torry scale. is a descriptive 10-point scale and has been developed for lean. QIM Rating system software was developed for cod. trained personnel required. QDA provides a detailed description of all flavor characteristics in a qualitative and quantitative way. plaice. saithe. and Scopthalmus maximus. 13. In this scheme. Recently developed QIM schemes were presented for raw gilthead sea bream (Sparus aurata) [30]. sole. which makes it possible to predict remaining storage life on ice.

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. 2004. S. Nordic Industrial Fund (in Danish). Quality Standards for Fish: Final Report Phase II. With permission. matte.. 975. seaweedy. G. developed by Nielsen.. Int. brown Odor Fresh. Food Res. pp..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. 1992.Methods for Freshness Quality and Deterioration Table 13. 37. and Hyldig. D. 37–59. .

starchy. texture.3 Physical Methods 13.M. wood sap. soapy.1 Texture Analysis Texture analyses for seafood are extremely important in research.. et al. Sci. hake. Among textural attributes.2 The Torrymeter The Torry fish freshness meter “Torrymeter” was developed at Torry Research Station in Aberdeen. blue whiting. natural odor Wood shavings. seaweed.2 Torry Score Sheet for Freshness Evaluation of Cooked Cod Fillets Odor Initially weak odor of sweet. quality control. Baltic herring. These changes occurring at microscopic level are related to alterations in appearance.g. boiled milk. Numerous mechanical methods have been used to measure texture. trace of “off” flavors Slight bitterness. Dielectric properties of fish skin and muscle alter in a systematic way during spoilage as tissue components degrade. Scotland.. 4. Dielectric properties of fish are used for determination of freshness. tallow Flavor Watery. 283. improper handling storage. A linear relationship was found between Torrymeter readings and sensory attributes for cod. which immediately begin to break down the proteins after the harvesting. reminiscent of boiled clothes Lactic acid. rubber. With permission. and product development in the seafood industry [38]. 1953. deciding the commercial value of the meat [41]. slight sulfide Score 10 9 8 7 6 5 4 3 Source: Shewan.40].3. hardness is the most important to the consumer. and chewiness of food. turnip. Fish muscle has higher levels of indigenous proteases. starchy. mackerel. and flavor during spoilage and have been used as quality indicators since the first commercial version of the Torrymeter in 1970 [43]. springiness. 13. sour. metallic. during processing. followed by strengthening of these odors Shellfish. J. 13. vanillin Condensed milk. TMA Strong bitterness. .. Food Agric. Texture includes the most common characteristics such as hardness. J. flounder. however. 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. sour milk.194 ◾ Handbook of Seafood and Seafood Products Analysis Table 13. boiled potato Milk jug odors. Fish muscle may become soft or mushy as a result of autolytic degradation or tough as a result of frozen storage [16]. there is little agreement on which is the best method [42]. “off” flavors.3. acetic or butyric acids) decomposed grass. boiled meat Loss of odor. odor. TMA Lower fatty acids (e. and cooking [39.

[50] found that the Torrymeter readings obtained from six species of different origin were poorly correlated with sensory evaluation.3. [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. The Fischtester readings can be used as an objective criterion for the state of freshness/spoilage together with sensory data across the fish chain. therefore. 13. conductivity. RT-Freshtester. 13. and farmed Senegalese sole [43–49]. Gelman et al. allows automatic grading of 60–70 fish/min. these instruments need calibration depending on sample preparation. works only on whole fish and fillets with skin on. [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. The skin of fish could be affected by osmolarity and contact with electrically charged particles [51]. 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]. They are unsuitable for frozen or thawed fish. partly frozen such as superchilled fish.3 The Intellectron Fischtester VI The basic principles of Torrymeter (the United Kingdom) and the Intellectron Fischtester VI (Germany) are similar. Therefore. This could be explained by seawater containing ions. However. 13. and fish-handling procedures.5 The Cosmos The “Cosmos” instrument developed by Japanese is applied for the evaluation of fish quality by determination of smell intensity. iced gilthead sea bream.3.05) lower in fish washed with seawater than fish washed with tap water or unwashed. and capacitance) of the fish flesh [52].4 The RT-Freshtester Like Torrymeter and the Intellectron Fischtester VI.Methods for Freshness Quality and Deterioration ◾ 195 whole. However. RT-Freshtester reflects dielectrical properties of fish. Gelman et al. and readings from all instruments decrease with storage time. which interfere with the reading of both instruments as they are based on electrical properties of skin. Inácio et al. season. fishing grounds. The method is based on conduction through skin and. fast and nondestructive. and fish chilled in refrigerated seawater [54]. as well as rapid and nondestructive. Mechanical abuse and freezing can affect the readings. . The electric properties of fish can change after death of the fish due to disruption of the cell membranes by autolysis. measuring the electric properties (resistance. portable. Like other instruments. 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.3. it could be used for evaluation of fresh and chilled fish in the seafood industry and on fishing vessels. The Intellectron Fischtester VI gives reliable information about the days in ice and left of iced stored fish. the “Cosmos” instrument is handheld. Fat also has an effect on the dielectric properties of fish and tends to make observed Torrymeter values more variable [47].

NO. These are metal-oxide semiconductor gas sensors. which determines the relation between sensor output patterns and the properties of the sample being analyzed [72]. semiconductor dimethylamine (DMA) gas sensor. chilled modified atmosphere packed (MAP) cod fillets [83]. Since these kinds of analyses are both time consuming and expensive. bromophenols. easy to handle. indicating spoilage of odors in seafood. water-holding capacity of thawed fish muscle [81]. and acid compounds are produced by microbial activity and lipid oxidation during storage of fish [55. The most important chemicals involved in fresh fish odors are long-chain alcohols and carbonyls. an electronic nose called FreshSense was developed and distributed by Element-Bodvaki in Iceland and has been found to be a rapid. and it was found that CO sensor showed the highest response [65]. nondestructive method to measure volatile compounds. 13. H2O.3. nondestructive. On the other hand. The technique is characterized by speed and simplicity. and requires little training of operators [73]. it can be operated on-/at-line. FreshSense is based on a closed. This technique is based on the fact that a computer screen can be easily programmed to show millions of colors. and N-cyclic compounds. The concentrations of these compounds are related to the degree of spoilage.7 Near-Infrared Reflectance Spectroscopy Near-infrared reflectance spectroscopy has been used in various analytical applications. SO2. and NH3). . H2O. thickness shear mode quartz resonators. and partial least-square regression (PLS-R). Data analysis is important in electronic nose measurements. The most frequently used methods are artificial neural networks (ANNs). Fish freshness has also been evaluated by a computer screen photoassisted technique (CSPT)based gas sensor array. Studies on cod fillets and heads also gave similar results. water. that is. [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. and aromatic. which are sensitive to volatile compounds. it requires too much handling of samples.56]. cod caught by long line and gillnet [73]. has been analyzed by sensory panel or gas chromatography (GC). This method has been applied for determination of fat. However. Different electronic noses have been employed for measurement of fish freshness. and protein content in fish [74–78]. chemometric analysis such as principal component analysis (PCA). it has the ability to measure numerous samples within a short time. short-chain alcohols and carbonyls.67–71]. and quality assessment of frozen minced red hake [82]. causing changes in protein and muscle structure.3. sulfur compounds. CSPT evaluates both effects [56.80]. electrochemical sensors (CO. diff use reflectance infrared Fourier transform (DRIFT) spectroscopy has advantages. static sampling system and electrochemical gas sensors. and prototype solid-state–based gas sensor called the FishNose [57–62]. NO. and NH3) results for haddock from different seasons showed a similar trend. the main indicator of fish freshness. 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]. Olafsdottir et al. Previous optics-based electronic noses relied on absorbance and fluorescence. Trggvadottir and Olafsdottir [64] also found that the response of all electronic sensor (CO. amines. However. online industrial production chain. SO2. and it is nondestructive. combining wavelengths in the optical range [56]. it is fast. Fourier transform infrared (FT-IR) spectroscopy is another technology that is a rapid. and thawed. N-cyclic.6 Electronic Nose Odor.196 ◾ Handbook of Seafood and Seafood Products Analysis 13. Compared with FT-IR. free fatty acid (FFA) in fish oils [79.

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

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

124.109. spermidine. putrescine. Cadaverine is derived from lysine.11. spermine.107.4. the presence of decarboxylase-positive microorganisms. Postmortem pH varies from 5. tryptamine from tryptophan. and the concentration of these increases with storage time [91. cadaverine. There are various analytical techniques used to determine the concentration of biogenic amines. Consumption of seafood containing high amounts of these amines can have toxicological effects.0 to 7. 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]. the moment of capture. Food and Drug Administration [117] and the EU [118]. free amino acid content [112]. In addition. and arginine leads to putrescine.108].129]. the enzyme responsible for its detoxification. The most significant biogenic amines produced postmortem in fish and shellfish products are histamine. 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. and stomach contents at death. cadaverine. and tyramine. Among the biogenic amines. their levels are considered as indices of spoilage rather than freshness [112]. The others especially putrescine and cadaverine have been reported to enhance the toxicity of histamine [115].S.2 Biogenic Amines The concentration of biogenic amines has been reported to be a reliable method of measuring the quality of fish. and use of a biosensor [130–132]. reliability.123]. Among these techniques.1 depending on season. By means of decarboxylation reactions.5. and reproducibility. cadaverine.127]. and agmatine. tyramine. including thin-layer chromatography (TLC) [122. histamine is potentially hazardous and the causative agent of histaminic intoxication [114]. and histamine and decreases in spermine and spermidine during storage of fish.125]. Putrescine is also an intermediate of a metabolic pathway that leads to spermidine and spermine [119]. whereas BAI is based on increases in histamine. These problems may be more severe in sensitive consumers who have a reduced mono. HPLC [120. tryptamine. depending on the species being examined [10. [121] for determination of quality of fish. Since the amines are produced by spoilage bacteria toward the end of shelf life of a fish. postmortem temperature. The QI and the biogenic amine index (BAI) were proposed by Mietz and Karmas [120] and Veciana-Nogues et al. since microbial flora vary seasonally [11]. HPLC is mostly performed because of its sensitivity. The formation of biogenic amines results from microbial degradation during the later storage of fish. respectively. 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. tyrosine produces tyramine. and 2-phenylethylamine is derived from phenylalanine. Biogenic amines are generated by microbial decarboxylation of specific free amino acids in fish or shellfish tissue [111]. GC [126.Methods for Freshness Quality and Deterioration ◾ 199 13.4. histidine yields histamine. respectively. 2-phenylethylamine.3 pH The pH is also an important parameter to show depletion in tissue and quality of flesh during storage. 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.110]. putrescine. capillary zone electrophoresis (CZE) [128. The biogenic amine content of fish depends on fish species. species.and diamine oxidase activity [116]. and other factors . and pH [133]. 13. Process technology is influenced by rigor development.

pike perch [146]. bacteria act upon TMAO to produce TMA. Based on the results obtained from the literature. It is well known that determination of TVB-N differs systematically according to the procedures used. such as frozen eel [145].151]. However. The analyses of these indicators are considered unreliable because they reflect later stages of spoilage rather than freshness [140]. produced by spoilage bacteria). affecting light scattering and the appearance of fish.4. Th is is caused by the depletion of energy reserves. and it has been used as an indicator of marine fish spoilage: CH3 CH3 – N=O CH3 TMAO CH3 CH3 – N CH3 TMA .200 ◾ Handbook of Seafood and Seafood Products Analysis [134. Therefore. TVB-N should be used as a chemical check. Atlantic cod [143]. was compared with two routine methods.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. the level of TVB-N was not correlated with the time of storage of some fish species. it could not be regarded as a good indicator of fish freshness and proved to be better as a spoilage index. with the production of lactate. and direct distillation methods have been recommended as a rapid routine method. which is considered to be the main cause of off odors in fish products [58. whereas the second one includes the use of trichloroacetic acid instead of perchloric acid [149]. Low pH also promotes oxidation of myoglobin and lipids [134]. and European eel [13]. It was found that there was a good correlation between three methods. turbot [92]. The EC reference method for TVB-N determination.135]. 13.4 Total Volatile Basic Nitrogen In seafood. TVB-N level correlated with fish quality. The European Commission (Council Regulation No. and hake [148]. ammonia (produced by deamination of amino acids and nucleotide catabolites).59]. 13. The first one includes direct distillation of fish after adding magnesium oxide. 144]. involving preliminary deproteinization with perchloric acid. the levels of 30–35 mg N/100 g muscle are considered the limit of acceptability for icestored cold-water fish [17.136–139]. Therefore. and DMA (produced by autolytic enzymes during frozen storage). Low pH is used as an indicator of stress at the time of slaughtering of many animals. as shown in a variety of fish such as European hake [142]. Since the activity of enzymes depends on pH. 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. However. sardine [12. as shown below: Following death of fish. mainly glycogen. total volatile basic nitrogen (TVB-N) primarily includes trimethylamine (TMA. it affects reactions taking place during storage of fish. farmed gilthead sea bream [147].4. Low initial pH is associated with higher stress before slaughtering [13. A relatively low pH may cause a decrease in water binding to the myofibrils. The level of TVB-N in freshly caught fish is generally between 5 and 20 mg N/100 g muscle.141]. TMA is produced by the decomposition of TMAO due to bacterial spoilage and enzymatic activity [150.

which is converted to TMA by bacteria in iced fish. a capillary electrophoresis method [165]. 13. or TVB-N contents. Fresh fish has a limited shelf life and is prone to deterioration. resulting in rancidity. A close relationship has been found between lipid damage and quality of the final product [173]. fish size. and time. time of year. including steam distillation [158]. especially in gadoid fish. The formation of these products may cause severe quality changes or spoilage during prolonged frozen storage. The TMAO content of seafood varies with species. 164].7 Formaldehyde The formaldehyde content in seafood products is generally considered as nontoxic. GC method [163.156. age. stage of spoilage. causing denaturation and cross-linking of proteins [171]. Fresh fish has a very low amount of TMA with values less than 1. The limiting factor of frozen storage in lean fish species is denaturation of proteins. and environmental factors [152]. its usefulness depends on time of year. DMA can be used as a spoilage index during frozen storage of some species such as frozen hake [170]. Conway microdiff usion and titration [159]. During chilled or frozen storage of fish. flowinjection-gas diff usion method [167]. TMA can be used as a spoilage indicator and not as an index of freshness. HPLC method [162]. The fish is considered stale when the rate of TMA production is higher than 30 mg/100 g cod [155]. fish contain TMAO.5 mg TMA/100 g in fresh cod. DMA.6 Dimethylamine As mentioned earlier. 13. and solid-state sensors based on bromocresol green [169].4. and low-molecular weight compounds. Many analytical methods have been developed for the measurements of TMA. when bacterial growth is inhibited. This reduces the solubility of myofibrillar proteins [172]. terminal amino groups. . 13. lipid oxidation is the limiting factor in fatty fish species. after which the rate of production of TMA parallels the bacterial proliferation pattern [154].150]. TMA is not produced in a significant amount during the early stages of chilled storage of fish.4. whereas freshwater fish generally contain only 5–20 mg% [153].4. type of storage and processing.157]. location of catching. colorimetric method [160]. biosensor using flavin-containing monooxygenase type-3 [168]. and methods employed for analysis. The formaldehyde content of frozen seafood is generally used as a spoilage index. However. However. semiconducting metal–oxide array [166].8 Lipid Oxidation Indicators During processing and storage. The amount of DMA produced depends on species (except gadoid species. but it can react with a number of chemical compounds such as amino acid residues. photometry [161]. Several assays have been described for the determination of TMAOase activity in fish muscle [151. but it appears after 3 or 4 days. other species do not develop adequate amounts of DMA). Seawater fish have 1–100 mg TMAO in every 100 g muscular tissue. enzymatic and nonenzymatic lipid oxidation occurs. this reaction is replaced by a slow conversion by an enzyme to DMA and formaldehyde [16. whereas fish can be stored in a frozen state for several months without severe changes in quality.Methods for Freshness Quality and Deterioration ◾ 201 TMAO appears to be part of the system used for osmoregulation. the storage temperature. which results in a dry and firm texture of the fish muscle [174]. but values increase during spoilage.

There are three steps in autoxidation of unsaturated fatty acids.J.C.175–177]. E. Peroxides are not stable compounds. London. 1994. U. nutritional losses. (From Hamilton. 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 13.180]. produce toxins..K. Initiators (such as light. The major chemical indicators for the determination of the extent of oxidative rancidity . fish and fish oils are highly susceptible to the development of oxidative rancidity. Fish oil contains about 20% of their total fatty acids as long-chain PUFA. resulting in peroxide (ROOH) and new free radical (propagation phase). Chapman & Hall.). thiamine. The hydroperoxide value is generally shortened to peroxide value (PV). propagation. 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. off flavors.. Many factors affect the onset and development of rancidity (oxidative and hydrolytic degradation of lipids). (Eds. and then the quantity of radicals and peroxides decreases. forming stable deterioration products (termination phase) [181. initiation. Under chilled/frozen conditions. B6.202 ◾ Handbook of Seafood and Seafood Products Analysis Initiation: Initiators (heat. light. ketones. R. temperature. pro-oxidants.2). and degree of exposure to light [178–180]. oxygen availability. J. and they break down to aldehydes. unpleasant odors. Allen. the type and concentrations of antioxidants. The peroxide radical can attack another lipid molecule RH. R. and pantothenic acid. 1–22.2 The autoxidation of fatty acids..182]. consequently. moisture content. and alcohols.) Off taste and off odor are usually defined as rancidity. They also destroy pigments. leading to protein denaturation. in Rancidity in Foods. Seafood has highly unsaturated lipid content. The amount of reactive compounds increases gradually. The amount of hydroperoxides can be used as a measure of the extent of oxidation in the early stages. 3rd edn. heat) convert RH to free radicals (initiation phase). Excess free radicals and peroxides in foods cause destruction of essential fatty acids and vitamins A. lipid oxidation compounds interact with proteins. and cause off flavor/odors [183]. and free radicals react with oxygen to produce peroxide radicals (ROO). which are the volatile products causing off flavor in products. and modification of electrophoretic profiles of proteins [172.C. and termination (Figure 13. and taints [179. C. and Hamilton. pp. Peroxides can also react with proteins and result in a decrease in their nutritional value. Free radicals from oxidizing lipids can polymerize with proteins and destroy certain amino acids. including the degree of unsaturation of the oil.

European eel. TOTOX (2VP + AV). or enterococci [195]. It was indicated that the main requirements for shelf life predictions are to collect information about SSO. [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. as oxidation proceeds the PV can start to fall. PV. AV and TBA values measure the secondary products of lipid oxidation. Shewanella putrefaciens . and thiobarbituric acid (TBA). sardine. spoilage domain such as the range of environmental conditions over which a particular SSO is responsible for spoilage and spoilage level [198]. cod [192. 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). since oxidation products are unstable and react with biological amino constituents.188. coliforms. During prolonged storage of seafood. FFAs and their oxidation products would have an effect on muscle texture and functionality. PV measures primary products of lipid oxidation. haddock. Cozzolino et al. Analysis of these interaction products by fluorescence detection as a quality assessment index for frozen-stored sardine was studied by Aubourg et al. Increase in the PV is most useful as an index of the earlier stages of oxidation. It is possible to predict shelf life of seafood based on knowledge of initial numbers and growth of SSO.9 Lipid Hydrolysis Hydrolysis leads to hydrolytic rancidity and involves hydrothermal or enzymic (lipase) hydrolysis to FFA and other products. there are some difficulties with common methods when quality has to be assessed. which break down to secondary products of oxidation or react with proteins. and phospholipids.5 Microbiological Methods Numbers and types of microbes present in foods are important indicators of safety and quality.190. 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.186]. 13. 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 lean fish such as blue whiting. 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. However. horse mackerel [13. Mathematics models have been well established for the growth of spoilage bacteria such as Photobacterium phosphoreum.191]. Many methods have been employed for the measurements of lipid oxidation in foods as a means of determining the degree of damage [20.185]. 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].Methods for Freshness Quality and Deterioration ◾ 203 are anisidine value (AV). and decline [184. reach a peak.193]. 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. causing production of interaction products [187]. since they interact with myofibrillar proteins and promote protein aggregation [189]. 13. peptides. such as proteins. and TBA values may increase. and also freshwater fish [194]. PV. [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.” or numbers of CFU of indicator organisms such as Enterobacteriaceae. AV.

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

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

... In cod............... and caudal peduncles could be used for a total correct classification of wild.... therefore.............................3 it was shown that the morphology of the head.7................................. Standards for organic farming are still under development in many countries.......................... size of the eyes and mouth......................................... analysis of the protein and lipid contents............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. and length of the pectoral fin. fins... salar parr.... 227 References ...2 Morphological Examination There are few publications and no official guidelines for the morphological differentiation of farmed and wild aquatic organisms..... 227 Acknowledgments ............................216 ◾ Handbook of Seafood and Seafood Products Analysis 14..............................1 Sample Preservation .............................. because farmed and wild organisms carry different hazards and are therefore submitted to different regulations and analytical controls........ stable isotope analyses combined with fatty acid (FA) profiles have proven particularly useful when tested. and sea-ranched S.. analytical methods should be made available to confirm it........... 227 14....2 Also in an earlier study............... The later study showed that the environmentally induced phenotypic divergence increased with age and with the numbers of generations under domestication...... genetic analyses.......7.. and these are seldom present in farmed seafood.......... Correct information about the production method of seafood is also important...................7 Trace Element Fingerprint............................ 226 14...... 226 14.......................... shape of the head..... but wild specimens may contain parasites harmful to humans........... 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.................. Although in this work no special mention is made to organic farming.......................8 Other Methods . Several methods have been successfully applied to differentiate farmed from wild seafood........................ and Cosmetic Act and The Fair Packaging and Labeling)......... In particular...... of 1999)............ Drug............... The production method is also part of the information essential to fulfill the traceability of a product and.. and the United States (The Federal Food.....2 ICP-MS .................5 The flesh of farmed cod sometimes presents ......... 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. farmed.............. 100% discrimination between farmed (AquaGen strain) and wild parr was achieved by examining the body form.......... JAS Law............. the most prominent differences are the higher condition factor. as well as examination of the stable isotope and trace element profiles.....1 14...... In small Salmo salar........... 228 14........ larger liver.................................. commercially farmed specimens may contain residues of veterinary drugs whose presence is unlikely in wild seafood.... including morphological examination. 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.... For example..

Analytical Methods to Differentiate Farmed from Wild Seafood ◾ 217 a translucent grayish aspect. Each kit is provided with a detailed description of how to use it. Th is step is not necessary in samples preserved in ethanol. because the enzymes are inactivated by the fi xation. However. and the liver in farmed cod is much bigger than the liver of wild fish. 14. If the sample must be preserved. it would be relatively difficult to find markers for wild and farmed fish. sonication.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.8. The DNA is then separated from the contaminating cellular components by salt precipitation. GeneRelease (Bio Venture Inc. so that all the ethanol is evaporated. The basic steps in all DNA extraction methods include the inactivation of nucleases.11. a normal freezer (−20°C) may also be used.A Stool DNA Isolation Kit (United Bioinformatica Inc. and others. since enzymatic activity also takes place at subzero temperatures. Depending on the type of sample and its use. which is the most common analysis. in most breeding programs the fish are indeed selected based on commercially interesting traits such as growth performance. For very long periods. The DNA .N. Then. in particular if it has a high enzymatic activity (for example if it contains the hepatopancreas in a crustacean).9 Genetic analyses have allowed the differentiation of wild from farmed fish populations in a variety of species.1 Sample Preservation and DNA Extraction Methods The sample should be extracted as soon as possible after sampling. give satisfactory results. Amersham Biosciences (GE Healthcare). Wizard (Promega). E.). classification based on morphological criteria demands the presence of the morphologic diagnostic characters. Dynal (Invitrogen). 14.10 and optimal adaptation to different environments. and then be rehydrated in water or in the extraction buffer.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.). To extract frozen samples we recommend to start the procedure before the sample is completely thawed. thus limiting its application.Z.18 or gel filtration. Many commercial kits. treatment with Chelex. for example by chelating divalent cations using EDTA and EGTA.3. Nucleospin (Clontech). Samples fi xed in ethanol must be allowed to dry completely. which are usually absent in many intermediate products as well as in the ready-to-eat dish. in contrast to the white opaque color of the wild. the best method is to freeze it in liquid nitrogen or in a biofreezer.11–13 and a loss of rare alleles has usually been observed in the farmed populations. chloroform extraction. Delays and the use of preservations methods will diminish the quality and the yield of DNA. If this policy had been followed.3 Genetic Analysis Doyle et al.14–16 Hayes et al. the cells are opened (by heat treatment. and then recovered by ethanol or isopropanol precipitation. we recommend preservation in 96% ethanol. since diversity would be one of the selected traits in the farmed fish.9 resistance to diseases or to stress. One requisite condition for any genetic analysis is the obtention of good quality DNA suitable for PCR amplification. or by the use of Proteinase K) and proteins are removed usually by incubation with Proteinase K. such as Qiagen.6 However. Genomics analyses are dealt with in more detail in Chapter 4 of this handbook.

have started programs to map the whole genome of some species. Three more methods that have reputedly produced good quality DNA suitable for amplification. several countries. protein markers commonly used for genetic analyses have the potential to be used as markers for farmed or wild.17 However. and others.27 tissues between farmed and wild salmon and cod have been reported. trout. such as Norway.21 When using the salt extraction method with heavily degraded samples.). from food matrices include the use of hexadecyl–trimethyl ammonium bromide (CTAB). any marker.4 Analysis of Proteins No clear protein marker has been identified to discriminate farmed from wild seafood. so the next step. and the DNA is reconstituted in double-distilled sterile water or in a slightly alkaline buffer (50 mm Tris–EDTA.18 and the salt extraction method.23 In principle. University of the Basque Country. On other occasions. The outcome of these programs is already producing lists of genetic markers linked to traits of interest. cod. In the future. the alcohol is allowed to evaporate. DNA analyses may be performed using chips that permit the determination in one fast step of many characteristics simultaneously.20. An additional advantage is that it is possible to use robots for many of the steps (DNA preparation. 14. and sea bass. has the potential to be useful to differentiate farmed from wild specimens of a given species. which may be used to identify the strains of the farmed individuals that display an increased frequency of the desired traits. differences in the protein pattern of liver25 and muscle26. including oysters. This method has been successfully used by the authors of this paper (unpublished results) and by Bucklin and Kochert22 with whole individuals of Calanus.15. and also for forensic studies. by using a series of SNP and microsatellite polymorphisms by PCR and by oligonucleotide ligation assay (OLA). In these samples the pellet may be practically invisible.14 In addition. the United States. traits. Arctic charr). cod. Atlantic halibut. etc. pH 8. China.218 ◾ Handbook of Seafood and Seafood Products Analysis pellet is usually washed at least once with 70% ethanol. tilapia. It is worth mentioning. India.24 The method requires that all parent fish of the brood stock are DNA typed as well as all the individuals under examination. sample preparation. catfish. and production method. that the Norwegian company GenoMar has patented a method to trace back farmed individual Atlantic salmon. since some alleles are more frequent in one group than in the other. tilapia.0). must be performed very carefully not to lose the sample. However. Spain. shrimp. which further increases the amount of samples that can be processed. 14. SNPs. however. 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. which is washing the pellet with 50% ethanol.19 the Chelex method. or mitochondrial.3.13. and bass. genomic. salmonids (salmon. Japan. breeding stock. 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. whether microsatellites. personal communication). however. 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.2 DNA Markers In recent years. including the species. .11.

Interestingly. 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.27 also registered the altered expression of five enzymes implicated in the glycolytic pathway and citric acid cycle in farmed cod. which is reflected in the composition of their organs. neutral calcium-activated calpains..4. Some enzymatic systems that may be responsible for the muscle softening are metalloproteases and collagenases. Soft texture. Using proteomic analysis. neither feeds nor breeding conditions may be optimal for farming. Thus. have prompted the development of feed formulations based on vegetable oils and proteins.35. the amino acid profiles of plant proteins do not meet the essential amino acid requirements of fish. indicating increased emphases on catabolism relative to anabolism in the fish fed this diet. and feed diets based on plant protein require supplementation with synthetic amino acids. which were attributed to increased proteolytic activity in the muscle of the farmed compared with the wild cod. several enzymes involved in anabolic metabolism were downregulated in fish fed the diet rich in soybean meal.28–31 The source of protein in teleost fish is very important. and the proteasome. The authors noted a downregulation of some structural proteins in fish-fed soy proteins. Although feeds and breeding conditions need to be developed and optimized for each species. and proteolysis) of the proteins in the sample should be chosen. Johnston et al. and then modify them depending on the results. Thus.37 Martinez et al.26 examined the protein expression in skeletal muscle of farmed and wild cod by high-resolution twodimensional electrophoresis and found differences between the two. several enzymes. and they require high levels of dietary protein (30%–60%). −20°C . Fe. attributed to the fish’s increased requirement for energy metabolism.1 Sample Preservation The optimal case would be when the extraction of proteins can take place on the sample immediately after the experimental treatment. the depletion of the wild stocks of pelagic fish and the high price of feeds based on fish meal and oil. 14. it has been shown that components in fish feeds may contain very high levels of metals (Cu. etc.Analytical Methods to Differentiate Farmed from Wild Seafood ◾ 219 Industrial fish farming is a relatively new activity compared with farming of land animals. lectins. and structural and FA-binding proteins. Optimal methods include fast freezing and frozen storage using temperatures as low as possible.33 Moreover. Texture is an important quality attribute of the fish flesh. and they hypothesized that the greater concentration of insoluble collagen present in wild salmon may contribute to their firmer texture. and Ca)34 and that vegetable meals may contain antinutritional factors (protease inhibitors. loss of functional groups. it is common to apply directly to new species those conditions that have proven successful for other species.33. Proteins and proteomic analyses are dealt with in more detail in a different chapter of this handbook.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. 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.38 found that the reason for the softening in this species did not seem to be the faster growth of the farmed fish.32 Unfortunately. In addition. these authors identified 33 differentially expressed proteins. antigenic proteins. which they use as their preferred energy source. and this may induce stress in the farmed animals. aggregation.)29 that may have adverse effects on fish. Mg. that is. lysosomal cathepsins. For short periods of time. However. including heat shock proteins. which would be a natural diet.25–27 In addition. Olsson et al. is more common in stressed and in farmed than in wild fish. a preservation procedure that minimizes the modifications (denaturation. When this is not possible.36 Martin et al. usually considered negative. with no preservation at all. Zn.

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

42. however. in particular when combined with stable isotope composition (see the following paragraph). and sand eel and C22:6n3 is more abundant (over 10%) in Atlantic and Coho salmon. The FA composition of fish oils is more complex. 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. soybean. herring. The proteins are afterward identified by searching in databases (National Centre for Biotechnology Information. very different staining intensities. has often given correct classification of farmed and wild specimens. C16:0 and C18:1n9 are relatively abundant in all fish oils.). and C18:3n3 were selectively metabolized. lateral flow strip tests permit in situ easy and fast screening of seafood samples. so that the concentrations in flesh were higher than in the diet. However. it has been shown that there was selective deposition and retention of C22:6n3. As in vegetable oils. whereas C18:1n9.43 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. C18:2n6. or menhaden.5 Analysis of the Lipid Content The analysis of the triglyceride (TG) fraction.44 Frequently. In Atlantic salmon for example. the final identification and assignment of the spots in the gels must be performed visually by trained personnel.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. capelin. in control laboratories.55–58 Specific FAs are selectively retained or used. Proteomic analysis is a complicated procedure necessary to identify the biological markers. rapeseed. ProteinLynx Global SERVER). which FA is more abundant is species dependant. 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. and sardines than in capelin. due to frequent errors in the automated spot identification procedure (because of imperfect spot separation and identification caused by overlapping of spots. olive. For example. palm. herring. the FA profile of TGs reflects that of the feed.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. NCBI) using suitable software (MASCOT. and ELISA format will permit the routine analyses of many samples. the total amount of TGs alone may be used as a criterion to differentiate farmed from wild fish.44 and this FA fingerprint has often been successfully used49–52 as a diagnostic to identify the production method. that is. C22:1n11. The whole procedure is described in detail by Martin et al.). and it is seldom that only one of them makes more than 25% of the total. spot cutters. identification of diagnostic spots. there are more FAs present in detectable amounts. sand eel. and often a single FA may account for about 50% of the total FA content in these oils. etc. which uses MS data to identify proteins from primary sequence databases.Analytical Methods to Differentiate Farmed from Wild Seafood ◾ 221 usually performed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS. This is probably the most time consuming step of the whole procedure. 14. because farmed fish usually have a much higher content than wild ones.53.25 and reviewed by Granvogl et al. Development of protein chips will facilitate the simultaneous screening of many targets and samples.45–48 In addition. but C22:1 (several isomers) is relatively more abundant in Coho salmon. cottonseed.60 The FAs C18:1n9 and 18:2n6 may act as markers . for example. Once the diagnostic proteins are identified. linseed. The FA profile of vegetable oils (such as corn. etc.

The most commonly used HR-NMR techniques in wild/farmed classification are 1H NMR and 13C NMR. 10B. 14. including fish and fish products. On the other hand.. 14.52.61 As indicated by Refsgaard et al. The spectrum is often used to obtain information about the number and type of molecules in a mixture.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. 23Na. and an inert atmosphere.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 major advantage of 1H NMR spectroscopy compared with 13C NMR is the higher sensitivity and thereby shorter acquisition times per experiment. it is best to use as low a temperature as possible. 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.5. and even after the levels of C20:5n3 and C22:6n3 were restored to the original high levels. and is the preferred tool in lipid analysis when interpretation of .. the ratio n3/n6 was not fully restored. which. since in a one-dimensional spectrum each peak is produced by those nuclei placed in an identical local chemical environment. For detailed descriptions of the analysis of fish samples. The most commonly measured nucleus is 1H (the most receptive isotope at natural abundance). 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. 14N. 11B.64 may contribute to the difficulty of performing correct classifications as wild/ farmed based only on the FA composition.1 Sample Preservation Due to the high levels of PUFAs. 195Pt). which may be used as a rapid profiling technique. which leads to less overlapping of signals. that is. 13C.52 that give detailed descriptions of the procedure.2 Lipid Extraction and Gas Chromatography Procedures for lipid extraction are described in another book chapter of this series. 17O. but NMR is applicable to any nucleus possessing spin (e.65 NMR gives a fingerprint of the sample analyzed. and in particular.and light-tight containers and stored at low temperatures. 29Si.63. 35Cl. NMR spectroscopy can be used to identify functional groups. 15N. HR-NMR has been particularly valuable in the study of marine lipids. oxidation. Fresh samples should be kept wrapped in air. the reader is directed to several publications46.50.222 ◾ Handbook of Seafood and Seafood Products Analysis for vegetable oils. 13C NMR has a greater range of chemical shifts. the latter seems to be the most persistent after a dietary switch to fish oil diet. 2H. and polymerization. 14. both of which are able to detect a range of metabolites in a nontargeted way.5.g. because it provides multicomponent information and can be applied nondestructively. 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.5. 31P. 19F. −80°C. If freezing is required.

99.76 In some studies. 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. Regions without signals or unwanted signals are removed before multivariate analysis. will increase the sample throughput significantly.77 It is advisable to check that all spectra have acceptable linewidth and lineshape after the NMR analysis. even though the signal intensities within each spectrum are not quantitative. The application of multivariate statistics to NMR spectral data increases the potential of the technique considerably.66 Potential problems about inconsistencies in ppm values between samples in the data analyses should be solved by manual alignment or data pretreatment methods.77 Regarding reproducibility issues. pH.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.75 and it is important that all the samples contain the same volume. Multivariate methods are frequently applied to study differences among NMR spectra.73 which is of value for authentication purposes.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).71 13C NMR gives information about FA composition of fish72 and the positional distribution of PUFAs in triacylglycerols and phospholipids.67 1H NMR has been used to perform quantitative measurements of total n-3 FAs and of the levels of DHA. leaving the sample ready for analysis. although the optimal sample size depends on the instrument. in conjunction with chemometrics.66. Standardized procedures should be followed to ensure repeatability and comparability.78 . such as instabilities in apparatus.0 ppm for 13C NMR.8% CDCl3). which is easily evaporated. they are normally converted to ASCII or JCAMP file formats. due to the fact that quantitative measurements require a considerable longer experimental time. It is expected that in the future the use of flow injection systems. temperature variations.75 Small differences in experimental conditions. The most commonly used solvent in the analysis of neutral lipids is deuterated chloroform (i.28 ppm for 1H NMR and by the triplet of CDCl3 at 77. interactions with metal ions.70. 1H NMR has also been applied to differentiate between wild and farmed salmon and sea bream of different origins.e. 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.68. may lead to erroneous classification. When full spectra are used. a semiquantitative 13C NMR approach has been chosen. Tetramethylsilane (TMS) is usually added as a chemical shift and intensity reference. because it may interfere with the multivariate data analysis. a sample size of 50–100 mg of lipid in 0. However. or differences in relative concentrations of the samples analyzed. the relative intensities for corresponding signals across different spectra are comparable.. but it is not necessary for classification purposes. Both the area/ intensities of peaks and full spectra can be input for multivariate analysis. phasing and baseline correction are applied but no zero fi lling. Normally. although this approach has still not been widely used for authentication purposes.8 mL solvent is used.5–0. inhomogeneities in the applied magnetic field. hydrogen bondings. Factors that affect the exact chemical shift of NMR signals include the type of solvent used.Analytical Methods to Differentiate Farmed from Wild Seafood ◾ 223 spectra is the goal. have allowed the differentiation between wild and farmed salmon74 and cod51 of different origins. Both HR 1H and 13C NMR. the chemical shift scale is referred to the shift of TMS or indirectly to TMS by the peaks from chloroform at 7. ideally for screening many samples with short acquisition time. Typically. and other intermolecular interactions. Typically. The assignment of spectral resonances gives information about the chemical composition of the samples.

such as maize. SD ± 0. resulting in a complete separation of the two groups. nitrogen. d13C of individual FA.52 were able to classify correctly according to the production method. 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. Aursand et al. Since a molecule containing heavier isotopic forms has stronger chemical bonds. exhibit limited variation.89%) and 13C (1. Usually animal products become enriched in the heavier isotope (15N and 13C).46 were equally successful classifying sea bass using the FA profile.037%). For example.6 Stable Isotopes The variation in the abundance of the stable isotopes of carbon.23‰) than the aquaculture specimens. For example. Thus.37%). equilibrium reactions also lead to a fractionation of the isotopic forms. SD ± 0. Differences in the 15N/14N ratio also result essentially from diet.63) and 15N (0. The isotopic abundances in animal tissues and animal food products are the summation of the feeds ingested throughout all their life. N2.204%). because the enzymatic reaction rates on substrates that contain the lighter isotopic forms are faster than in reactions involving the heavier isotopic forms. Moreover. although many broadleaf plants are also C4). Using the d15N of choline and the d18O of total oil. and oxygen as three: 16O (99. In contrast. 171 Atlantic salmon specimens originating from three continents and 15 different geographic regions. C16:1n-9. Samples of muscle tissue of wild salmon were significantly more enriched in nitrogen (d15N: mean = 12. they were also able to correctly classify the fish according to their geographic origin. 17O (0. such as CO2. In addition. 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).75. the higher the proportion of the heavier isotope.79 Carbon exists as two stable isotopes: 12C (abundance 98. and they reflect both significant isotopic fractionation by microbes and the different biological substrates producing these gases. N2O and CH4 exhibit wide isotopic variation.51.759%). Some atmospheric gases. the abundances of the stable isotopes differ between substrate and product. . plus the kinetic fractionations occurring in animal metabolism. C4 plants may have d13C mean values of −12/−14‰. a kinetic fractionation occurs. C18:1n-9. and 18O (0.82 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. and oxygen has been proposed as a method suitable for food authentication.81 This is because the 13C/12C ratio depends almost exclusively on the photosynthetic mechanism used by the plants for CO2 fi xation. the 13C/12C ratio for both milk fat and cheese protein give information on the type of forage fed to the cows. typically tropical grasses.224 ◾ Handbook of Seafood and Seafood Products Analysis 14.11%). depending on their diet and their position in the trophic chain: the higher its position in the trophic chain. since the physical properties of molecules containing heavier isotopic forms are different. and O2. Introducing the percentage of C18:2n6 as a third variable in their model. the abundance of stable isotopes varies among different compounds. Thomas et al.50 were also able to correctly classify Atlantic salmon according to their geographic origin and production method by using four FA compositions (C16:0.80 The natural isotopic abundance largely varies depending on the chemical forms.38‰) but depleted in lipid-corrected carbon (d13C: mean = −20. Bell et al. nitrogen as two: 14N (99. 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. while typical d13C mean values of C3 plants may be −26/−28‰.

respectively (for carbon 13C:12C. Interestingly. organic. nitrogen as N2. where R is the heavy:light isotopic ratio of the sample or standard. and oxygen as CO2. as follows: d = (R sample/R standard − 1) × 1000‰. the ions are finally detected at the detector.84 has helped to understand the nitrogen isotopic variations in fishes.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. it must not be washed in the laboratory after collection (which may alter the O and H profile of the sample).1 were able to differentiate wild. and commercially farmed Atlantic salmon measuring d13C and d15N by IRMS in raw fillets. and the abundance ratios of the heavy and light isotopic species are then calculated. However. ground tissue is used in the simultaneous analysis of stable C and N isotopes. Approximately 1 mg of dried.86 To facilitate comparisons between specimens with differing lipid contents. Usually. since these authors assessed the effects of body size. SNIF–NMR can only be applied to the few isotopomers possessing spin. 14.48 NMR techniques have been described previously.2 SNIF–NMR and IRMS Two methods are used to assess stable isotopes: SNIF–NMR and IRMS. nitrogen. and d15N of the glycerol choline fraction of flesh phospholipids. and oxygen isotopes. 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. The instrument consists of an ionizing source. and so on. whereas IRMS gives only an average value of the isotopic forms in the molecule.6. 14. and stored in glass desiccation vials until analyzed. and environmental conditions on fish tissue.1 Sample Preservation It is very important not to contaminate the sample during handling. and a detector to measure the different isotopic species. the d15N values of heart and liver were also affected by environmental temperature. The C and O isotopic profiles of fish tissues may be altered if CO is used for stunning or killing. whereas IRMS can be applied to all except 12 elements. The ionized gas is then introduced in the flight tube under vacuum or carried by helium. The assumption that fractionation was independent of body mass was upheld for muscle and heart tissue but not for liver. The research of Sweeting et al. are typically determined with a gas isotope rationing mass spectrometer. All international standards are . thus hydrogen is introduced as H2. The light elements. Enriched samples contain relatively more of the heavier isotopes. The element is converted to a gaseous form to be analyzed by the mass spectrometer. d13C values are normalized for lipid content following techniques developed by McConnaughey and McRoy87 and validated by Kline et al.Analytical Methods to Differentiate Farmed from Wild Seafood ◾ 225 d13C and d18O of total muscle oil. for nitrogen 15N:14N). For example. a flight tube with a magnet. 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 collected tissue samples are dried at a constant temperature of approximately 50°C for 48 h. pulverized to a fine powder using a ball mill grinder. such as carbon.6. probably reflecting the metabolic functions of these tissues and their associated turnover rates. Molkeltin et al.85. The gas is introduced in the mass spectrometer and is ionized by removal of an electron in the ion source. experimental duration. carbon as CO.

1 Sample Preservation As for stable isotope analysis. Thirteen elements were shown to be the most diagnostic. cadmium.7 Trace Element Fingerprint Sometimes. lead.226 ◾ Handbook of Seafood and Seafood Products Analysis set at 0‰ by convention. and strontium levels and Factor 3. with the exception that clams from Miyagi had high arsenic content. false labeling problems were encountered in which imported live Japanese eels from Taiwan were illegally sold as being of Japanese origin. in addition to DNA-based species identification techniques. Carbonate rock from the Pee Dee Belemnite formation89 and nitrogen gas in the atmosphere90 are used as the standards for carbon and nitrogen. The first step in the analysis is the digestion of the sample: 0. multiple elemental analysis could also be used in this case to identify imported clams from China and Korea. The same research group (Yamashita et al. quality. and analysis. Therefore. farmed and wild specimens of the same species have different geographic distributions. and it must be accurately weighed with a microbalance. Korea. as well as vitamin K and its metabolites. 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.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.91 In the case of fish. cadmium and arsenic levels in the muscles of clams from China and Korea were higher than those of clams from Japan. Thus. Rare trace elements taken from the environment. All implements and containers should be cleaned with 0. and they found distinct patterns for each of the three origins. and price. Multivariate trace elemental analysis is increasingly used as a technique to differentiate the geographic origins of foodstuff. multivariate trace elemental analysis is expected to be helpful in determining whether the fish was farmed and its geographic distribution. Biochemical analytical techniques using multiple elemental analysis. such as uranium. unpublished data) examined the trace element composition of the muscle and shell of littleneck clams collected in Japan. Multivariate analysis showed that differences in elemental composition in the muscle between Japanese and imported clams were mainly due to two factors: Factor 1. copper. respectively.7. The origins of farmed and wild eel collected from different regions in Japan. and often the geographic origin of both farmed and wild seafood may be of relevance for its safety. handling. attributable to cobalt.92–94 Otolith chemistry is useful for identifying the natal origin and assessing the relative contribution of different nursery areas to mixed adult stocks. Recently. were shown to be of relevance to determine the origin of eels. and China.. in particular since the analysis may detect contaminants at the pM level. and vanadium.95 By using ICP-MS analysis the sensitivity in the determination of rare trace elements can be increased from the nM to pM level. In addition. The sample may be stored in a centrifuge tube at a temperature of −40°C or lower until analyzed. Taiwan. have been used to differentiate the geographical distribution of origin of farmed Japanese eel. Each sample should be separated from the tissues using ceramic knives and scissors and Teflon-coated tweezers to avoid contamination of metals. 14.5 M nitric acid and rinsed with Milli-Q ultrapure deionized water. it is very important to avoid contaminating the sample during sampling. 14. The . otolith chemistry is used as a recorder of time and environmental conditions. attributable to manganese and vanadium levels.

For the determination of mercury. which suffers from severe memory effects. For internal standardization. In the farming of salmon for example. atomization. the Norwegian Research . the use of carotenoids is allowed. Japan).97 using an automatic mercury analyzer (Hiranuma HG-200. To verify that the instrument is properly calibrated on a continuous basis. Canada).2 ICP-MS Multielement determination of trace elements is usually measured by inductively coupled plasma mass spectroscopy (ICP-MS). To initiate the proper operating configuration of the instrument and data system. but although the diet of wild salmon contains astaxanthin. Multiwave 3000 Microwave Oven. and stored at room temperature until use.8 Other Methods Depending on the species. Tb. a calibration blank and calibration standards are used as surrogate test samples after every 10 analyses. five internal standards are used: Sc. and the ion information is processed by a data handling system. If the measured concentration deviates from the true concentration by more than 10%. Afterwards. 14.7. where energy transfer processes cause desolvation. the instrument is recalibrated. Perkin-Elmer). the total mercury concentration is determined by cold vapor atomic absorption spectrometry. 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.Analytical Methods to Differentiate Farmed from Wild Seafood ◾ 227 digestion may be carried out by placing the tubes in a microwave oven (for example. Y.98 making this approach more unreliable than it used to be. In.3′S) that does not occur naturally and can therefore be used as a marker for farmed salmon. and ionization. much of the astaxanthin used in fish feed nowadays is produced from cultured microalgae or from krill. most artificial feeds contain a mixture of canthaxanthin and astaxanthins of different origins (both natural and synthetic). the samples are diluted to a final volume of 50 mL in Digitube (SCP Science. and Bi. Each solution (5 mL) of the microwave samples is applied to the atomic absorption spectrophotometer. and the resulting digest is a clear liquid with a yellow tint.98 However. an internal standard mixture is added. the mass calibration and resolution are checked using diluted metal solutions as standards. 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.42. Ions transmitted through the quadrupole are detected by continuous dynode electron multiplier assembly. 14. and the last 10 samples are analyzed again.96 Samples digested as described above are introduced by pneumatic nebulization into a radio frequency plasma. there might be specific requirements that may be targeted to identify the production method. Analysis by chiral chromatography can be used to identify a chiral form (the meso form 3R.

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249 233 ..2 Smoke Oils ........6..................................1 Role of Volatile Compounds of SF in the Odor .........................................................................2 Extraction and Analysis Methods of PAH in SF and Seafood Treated by SF ..............5 Role of SF Process Parameters in Volatile Compounds Generation .................2..........................................3 Smoke Powders ............2 Role of Volatile Compounds of SF in the Flavor .........................2 Smoke Flavoring Process ... 246 15......9...........................8..9 Legislative Aspects.......................3 Use of Smoke Flavorings ....Chapter 15 Smoke Flavoring Technology in Seafood Vincent Varlet.........1 Introduction ..............1 Properties and Toxicology ......... 248 15...................3 Role of Volatile Compounds of SF in the Texture ..............4 Smoke By-Products ............................................................................................. 240 15.................................................................... 248 15...............................1 Liquid Smokes........ 246 15..................6.......... 234 15.8..........................................6 Organoleptic Roles of Volatile Compounds of SF ...........................................................2......................................................... 238 15.................................................................................. 241 15.............249 15.......241 15........................................................ 245 15...........4 Role of Volatile Compounds of SF in the Aspect and Color ...............2........6..........................7 Role of Volatile Compounds of SF in Preservation .1 European Regulations on PAH Found in SF ......................................................................................4 Chemical Composition of Liquid Smokes ......... 237 15..................................................................................................8 Polycyclic Aromatic Hydrocarbons....................................................................... 236 15........................................... and Carole Prost Contents 15...... 237 15....2................................................... 234 15.....................249 15....................................................247 15........................... 237 15..... 239 15.................................................6..... Thierry Serot..................

.......2 Smoke Flavoring Process The first liquid SF was developed and patented by the Kansas pharmacist Wright in the late of nineteenth century [2]......................10 Conclusion ... After condensation.......... Simultaneously.. between 20% and 30% of European smoked food is treated by liquid smokes........ However............... that is. are recycled and directed also to ......... the crude smoke condensates are separated in three phases: a water insoluble heavy oil by-products phase...... wood smoke imparts desired organoleptic characteristics such as smoky flavor... oils..... a water-soluble phase.........234 ◾ Handbook of Seafood and Seafood Products Analysis 15... allows a better control of PAH in the final product.1 Introduction Smoking is the oldest food preservation technique....... beech.. The smoke is filtered to eliminate particules and condensed........... and their uses in the seafood industry are increasing............... purified.........250 References . and provides a higher diversity of smoked food [1].. hickory....... By comparison with the smoking technology....... and a water insoluble tar phase.. However.... Wood sawdust is pyrolyzed in a furnace with low oxygen content..... It also allows the reduction of the PAH final concentrations. SFs are obtained by the condensation of wood smoke and can be further fractionated.) with a wide range of organoleptic qualities.. wood smoke phenolic components are known to be antioxidants. the chemical composition of soft woods is responsible for the generation of higher quantities of contaminants as PAH.. it allows decreasing microorganism activity....... and reduced risk of accidents due to fire... or concentrated..... 15.. a better preservation of the combustible........ The combustible gases are recycled and directed to the furnace..... and marple.................. liquid smokes are commercialized since the end of the nineteenth century......9...... Moreover.251 15... the use of SF allows an easier storage (SF bottles versus wood logs).... mainly hard woods. This kind of smoke flavoring (SFs) appears as an alternative to the smoking process as it is carried out in Europe...... Indeed.... The gaseous smoke can be cooled down by water or by organic solvents.... which give rise to new perspectives in the food industry [3]. aqueous solutions...1.2 European Regulations on PAH Concentration in Food Treated by SF .... especially thanks to the industrial benefits brought about by their use.. 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.. This industrial process leads to more homogenous products smoked with a repeatable intensity and provides an easier cleaning of the smokehouse.... The fractionation of smoke condensates allows obtaining a high diversity of SFs (powders..... etc...250 15.... Today............... 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..... The main woods used for smoke production are oak............... In United States where 75% of smoked foods are treated by liquid smokes.......................... Coupled to salting and drying steps.. the use of liquid smokes avoids the release of smokes... SFs are widely used in the meat industry...... The heavy oil by-products......... A simplified version of SF processes is presented in Figure 15..... obtained after a settling out time (several days) of the smoke condensates in the settling tank.. Indeed........

◾ 235 .1 Diagram of fabrication of SFs.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.

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

most frequently in 90:10 (v/v) proportions. Consequently. A reaction between the phenolic compounds of SFs and these nitrited salts or powders can lead to a nitrosation and to nitrophenols. Wet salting (or curing if nitrited salts are used) is made with brines spread on food or in which the food is dipped.2. Indeed. These smoke powders can be added to salt used for salting steps or to dehydrated sauces or soups elaborated from seafood products. whereas liquid smokes are used for the characteristic smoky odor and color of smoked products. Today. the organoleptic qualities can vary in a high range changing the food matrix.2. However. Consequently. consequent to the reaction between amino acids and nitrite.4 Smoke By-Products Smoke by-products are constituted by smoke extracts and smoke distillates [6]. These molecules can increase the generation of carcinogenic nitrosamines. 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. Nitrited salts.Smoke Flavoring Technology in Seafood ◾ 237 15. hundreds of smoke by-products are available. 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. are present in the market. 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. smoke powders are mainly used to confer smoky tastes to the final product. As seafood emulsions are not very common. 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 by-products constitute more complex SFs. the liquid smokes and smoke powders can be added to salt or in brine but not smoke oils. Smoke distillates are obtained by the fractionating distillation of PP. We must distinguish dry salting and wet salting. The distillation is commonly performed with steam water at atmospheric pressure. As smoke oils. 15.2 Smoke Oils Smoke oils are made by blending liquid smokes with vegetable oils. 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. and so forth.2. smoke oils can be only used in preparations such as taramas. because smoke oils are especially employed in food preparations such as emulsions. smoke manufacturers can control their products and can create smoke by-products whose uses are recommended for a kind of fish. 15. Dry salting (or dry curing if nitrited salts are used) is made with dry salt deposited directly on food. They are less acidic than aqueous flavors and allow to exhibit more complex smoky tastes. 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. generally forbidden in seafood industry according to the countries.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]. SFs for herring. 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. Therefore. fish sauces. Therefore. or fish oils. salmon. . these SFs are not used much in seafood industry. Smoke powders can also be rehydrated and used in brine as liquid smokes. Therefore. in seafood industry.

mainly liquid smoke concentrates on the products in a smokehouse. Indeed. the composition of liquid smoke mist is not similar to real wood smoke. The mist generated is composed only by small droplets and there is no gaseous phase. etc. Besides. According to the final product. This difference constitutes a critical point in the liquid smoking status. composed entirely of synthetic compounds or partly from a liquid smoke base [7]. Smoke powders are preferred when water use is impossible as in dehydrated mixes. but aqueous liquid smokes are the most used SFs in this technique. SFs can be incorporated directly with the ingredients during the formulation or through injection needles when the shape of the product cannot be modified. taste. Diluted SFs fall by gravity through perforated plates on the hung products. direct addition. Smoke oils are preferred for lipidic emulsions or lipidic sauces. that is. The mist obtained is constituted of small droplets with a similar size as in real wood smoke. from the granulometry point of view. The progress made during the last decades in elucidating the chemical composition of wood smoke gave rise to attempts aiming at producing SF. The final organoleptic qualities (color. 15. and this technique appears as an alternative to the smoking process. Liquid smoke solution is therefore recycled and filtered and the concentration is readjusted. They provide a better water solubility and prevent the heterogeneity of layer formation on the product surface or the product separation during storage. in numerous European countries. which carries a particulate or dispersed phase [8]. and atomization. which can be injected into the product as for the salting step. SF is sprayed with air under pressure through special nozzles and forms a wood smoke mist in the cell of smoking.) 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. vaporized liquid smoke is similar to real wood smoke. Showering is a technique currently used in North America. because to guarantee the homogeneity of the SFs during the treatment and to prevent the settling out of smoke condensates in water. meat treated by this process is considered as smoked but « smoked by liquid smoke » must appear on the package. there are different carriers of SFs. Indeed.238 ◾ Handbook of Seafood and Seafood Products Analysis The development of synthetic SFs must be also noticed. an emulsifier must be added in the SFs. However. between 15 and 20 mm. Finally.3 Use of Smoke Flavorings There are four techniques to incorporate or deposit SFs in or on seafood products: showering. In France. Drenching/soaking is the opposite of showering technique. wood smoke is composed by a gaseous phase formed by the most volatile compounds. especially used for meat products. From a physical point of view. especially in the labeling of the smoked products. Soluble aqueous flavors or buffered aqueous flavors are mainly used. because the products are immersed in the SFs solution instead of pouring the SFs solution on the products. but it is also employed in the seafood industry. liquid smokes are also employed in the curing brine. Other devices have been optimized in order to generate a similar physical composition of . products treated by liquid smoke atomization are considered as flavored and not smoked. Water-based composed SFs such as soluble aqueous flavors or buffered aqueous flavors are commonly used in this technique. 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. Products are dipped in SFs solution for short periods (from 5 to 60 s). the synthetic SFs created are not sufficiently similar to real wood smoke or to SFs. However. Direct addition consists in the incorporation of SFs during the fabrication of the food products. Therefore. atomization of SFs consists in the vaporization of liquid smokes. drenching/soaking.

liquid smoke atomization is the most used technique of SF. and sometimes small quantities of furans and phenolic compounds. The pyrolysis of lignin can also lead to alkyls aryls . hence the high acidity of liquid smokes. and various anhydroglucopyranoses (mostly levoglucosans) [11]. The moisture control is essential. furfural and homologues. but the optimization of the parameters to have a similar particulate or dispersed phase is not easy. the methods of production and the possibilities of applications of SF are very wide. A high knowledge of the biochemical composition of the wood used and the parameters of the combustion are essential to generate SF. water. predominant in softwoods) and in para position (syringol derivatives predominant in hardwoods). The main characteristics that permit the differentiation of hardwoods and softwoods are the guaiacol:syringol (G/S) and guaiacol:phenol (G/P) ratios. In seafood industry. hydroxyacetaldehyde. 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. Indeed. The wood polysaccharides lead to methanol. and air moisture are also essential in the SF final composition. ventilation must be planned in order to reduce the moisture. 15. The surface must present a beginning of protein coagulation.5 and 2.6-anhydroglucose (betaglucosan) and finally to acetic acid and its homologues. Hemicellulose pyrolysis leads to furan and its derivatives and aliphatic carboxylic acids. of 1. wood moisture. The smokehouse must be hermetically closed during atomization. Finally. acetaldehyde. which favors the vaporization of SF [9]. lignin thermal decomposition provides compounds considered as most important for the smoke flavor. to favor the deposition of smoke components. The SF composition can be complexified by the addition of spices and aromatic herbs [10]. In fish smoking. furanones.Smoke Flavoring Technology in Seafood ◾ 239 wood smoke with liquid smoke atomization. Similarly. The compounds generated from hemicellulose pyrolysis depend on the nature of the wood. respectively. The pyrolysis of cellulose initiates the hydrolysis of glucose followed by dehydration to 1. The role of pyrolysis parameters as pyrolysis temperature. the drying step is necessary to prepare the surface of the fillets. The volume of liquid smoke mist is controlled by the number of nozzles and the smokehouse size. The SF is sprayed on a surface at high temperature. formic acid. whereas weak moisture gives to the product a good color but a weaker smoky taste. Important moisture favors the smoke penetration and strong smoky organoleptic characteristics. hemicellulose in hardwood (nonconiferous woods) is mainly constituted by pentosans whereas hemicellulose in softwood (coniferous woods) is mainly composed by hexosans. 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. airflow. which confers a subtle glossy and sticky aspect. Therefore. methanal. According to the liquid smoke used. Glucuronic acids decompose to carboxylic acids. The thermal decomposition of pentosans provides a higher amount of furans than hexosans. which decompose to form alpha cellulose and provide a higher amount of PAH. it creates a gaseous phase. Hardwoods lead to G/S and G/P ratios. This step must take into account the initial water rate of the raw material and the composition of the final product. acetic acid. Therefore. such as alkyl phenolic compounds and derivatives like phenolic ethers with methoxy groups in ortho position (guaiacol derivatives.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. hence the limitation of the use of softwoods for smoking. first to control the drying of the product and second.

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

These observations have been recently corrected [14. A much more complex mixture of compounds is responsible for the characteristic aroma of smoked fishes [35]. which gather furannic and enolone derivatives. the diversity of SF causes diverse consequences on the texture. Phenolic compounds of low-boiling fraction (60°C–90°C) composed mainly of phenol. 15.33. Phenolic compounds of medium volatility have been considered as the most important odorant molecules. odor.Smoke Flavoring Technology in Seafood ◾ 241 chemical composition.34] (Table 15. They are the major compounds in SF with a wide range of odorant thresholds (Table 15.34].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] . phenolic compounds are not sufficient to explain the SF role in smoked fish odors.29]. Phenolic compounds are known to constitute the odorant “smoky” skeleton of wood smoke and smoked fish. syringol. Some of them have very low odorant thresholds. The medium-boiling fraction (91°C–132°C) composed of isoeugenols. color. Many studies have indicated that phenolic compounds present in the vapor phase of smoke may be odor-active compounds [30–32]. and preservation of the product. and alkylguaiacol may also contribute to imparting a smoky flavor to smoked fishes [13. Table 15. guaiacol. cresols. and methylsyringol has a pure and characteristic smoky flavor [10]. making them odorant at low concentrations.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. but it may not be the main contributor to wood smoke flavor.6.28. two main classes of odor-impact molecules can be defined: phenolic compounds and carbonyl compounds. taste.6 Organoleptic Roles of Volatile Compounds of SF 15.2).1) [14. Similarly.

55 ± 39. LRI. milk Smoke. chemical Green. potato Cooked potato. STD MS. STD MS.62 74.44 17. burnt.12 42. LRI.37 ± 3. LRI.94 49. green.24 ± 63. vanilla. LRI MS. LRI. LRI. earthy. STD MS.53) 8. milk Cooked/soup. 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. STD MS.90 (1. spicy Spicy. spicy/ woody (3) (2) 5 6 (2) 4 4 3 5 6 7 (2) 4 4. Animal.55 ± 9. fatty Roasty. roasty Chemical. LRI.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.17 ± 26. STD MS.18 ± 37.3-Dimethyl-2cyclopentenone 1052 o-Cresol 1068 p-Cresol 1093 Guaiacol 1110 2. burnt.74 ± 27.2 Odorant Characteristics and Concentrations of the Most Potent Odorant Volatile Compounds in Salmon Fillets Treated by Liquid Smoke Means of Identificationa Mean ± SDe 124.25 6 (5) 6 7 (4) 7 7 6 8 8 8 Chemical. LRI. LRI.98 20.6-Dimethylphenol 1130 . LRI MS. ink Marine. LRI MS. STD MS.07 (1.04 7 4 16.53 360.6-Dimethylpyridine 890 2.27 ± 0. STD MS. LRI MS.63 ± 13.34 (24. spicy.22 ± 9.75) MS. spicy.27 ± 2.64 ± 18. chemical.97 23.50) 65.Table 15. wood fire. mushroom Cooked. STD MS. burnt Smoked. STD MS. LRI. green Cooked vegetable. metallic. STD MS.45 ± 172. green Cooked vegetable. LRI.48 ± 8. STD Cooked.

spicy Oily.26 ± 1.86 (2. LRI. STD MS. spicy Spicy.2-Dimethoxybenzene 1147 2.25 ± 4.36 1132 MS Cooked.13 6. LRI.87 ± 1. STD MS. violet. smoked Candy. green. fatty. LRI. vanilla.15) 86. green.97 2. STD MS. spicy.2. rotten. spicy 6 5 17.21 ± 7. LRI MS.72 44. LRI.82 ± 6.85 ± 40.46 Solvent.2.61 ± 22.95) 8.50 18.79 (6. green.3. spicy. LRI. STD MS.24 ± 1.3-Trimethoxy-5methylbenzene 243 .15 ± 1.25 ± 10. green Spicy. LRI MS. LRI.51 ± 18. LRI. clove Sawdust. LRI 1160–1180 4-Methylguaiacol 1192 482.15) (continued) 2. fatty Burnt rubber.35 3-Ethyl-2-hydroxy-2cyclopentenone 1140 1. green 6 3 11. smoked Cooked vegetable. spicy. LRI MS.and 2.91 36. chemical Green. STD MS.15 ± 243. milk Burnt. vanilla Cooked.17 15.5Dimethylphenol/ (E)-2-nonenal MS.3-Dimethoxytoluene 1247 (E)-2-Decenal 1266 3. burnt Smoke. green.12 4. LRI MS. STD MS. STD Cucumber. smoke.E)-2. STD MS.96 ± 10.4-Decadienal 1330 Syringol 1365 Eugenol 1370 Smoke Flavoring Technology in Seafood 4-Propylguaiacol 1382 1400 ◾ 1.5-Dimethoxytoluene 1282 4-Ethylguaiacol 1287 Indanone 1307 4-Vinylguaiacol 1330 (E.16 ± 5. smoke.4Trimethylcyclopenten-1one MS MS. LRI.49 ± 6. clove Green.4.62 ± 4. medicinal 7 4 10. LRI MS. green.71 (3. 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.

linear retention index (when the LRI of the identified compound corresponds to the LRI in the literature). Number of judges (out of eight) who have detected an odor. STD. 4518. STD MS.68 MS. roasty. LRI. In micrograms equivalents of dodecane per 100 g of smoked salmon. a b c Handbook of Seafood and Seafood Products Analysis d e Means of identification: MS.77 24. green. . and odor description of the injected standard of this compound).40 ± 3. V. roasty 7 4 Burnt rubber. spectrum. 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. Agric. standard (when the retention time.. 2007.5 and 6 is rounded to 6 (1 = very weak odor.39 6. STD MS. An intensity between 5 and 5. J. it must be considered as an attempt of identification. LRI Animal.48) 1.. spectrum.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 7.55 ± 8. Average intensity of the eight judges is rounded to the nearest whole number.3.35 (20. rotten 7 4 Spicy. When only MS is available for identification. and odor description of an identified compound correspond to the retention time. Means of three fillets. mass spectrum (identified using the mass spectra of the compounds). and quantities of odor-active compounds detected by fewer than six judges are indicated in parenthesis. LRI MS. et al.Table 15. odor intensity. Food Chem. chemical 6 4 Smoke.5-Trimethoxytoluene 1527 4-Allylsyringol 1615 8-Heptadecene 1680 Source: Varlet. LRI MS.81 ± 11.23 ± 0. woody (4) (2) Clove. LRI. 55. Note: Frequency of detection.5 is rounded to 5 and an intensity between 5.87 ± 2. LRI. 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. 9 = very strong odor intensity).

Synergic or masking effects are possible and make the final odor complex. early works performed on individual phenolic compounds have identified the impact of guaiacol on the smoky flavor. However. As for the assessment of the odor.3. Then. The taste thresholds of some phenolic compounds were determined [40] and showed a high diversity between the molecules. the physical state of SF can also influence the aroma. it was commonly admitted that syringol derivatives impart a smoky odor and guaiacol derivatives contribute to a smoky flavor. 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 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]. as the other minor odor-active compounds. whereas syringol and 4-methylguaiacol showed the same but lower effect than guaiacol [40]. . if they do not have a strong individual influence.Smoke Flavoring Technology in Seafood ◾ 245 Carbonyl compounds have also been reported as contributors to the smoky aroma of wood smoke. A polyfunctional carbonyl subfraction was isolated from wood smoke and possessed a caramellic/burnt sugar aromatic note [36]. they may contribute in mixture to the overall odor.6.39]. 4-methylguaiacol perception was superior to that of syringol and guaiacol. Moreover. 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. Recently. Furthermore. and seem to contribute little to overall aroma. and isoeugenol in spicy/sweet flavor [41]. because they are not the compounds mainly detected in SF and seafood treated by SF odors by sensory analysis [37]. Concerning the bitter taste. 4-methylguaiacol. the same compounds responsible for the odor should be involved in the flavor that SF confers to seafood. 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. reactions between liquid smoke compounds and the components of the matrix can occur through Maillard and Strecker reactions. For several decades. liquids. Two categories of carbonyl compounds can be differentiated: furannic compounds and enolone derivatives. and very little information is available. phenolic compounds are not the only flavor-active compounds. or oils. Enolone derivatives are compounds derived from cyclopentenone. sometimes cooked. More recently. but taste was not investigated as much as odor.2 Role of Volatile Compounds of SF in the Flavor Phenolic compounds were shown as the major contributors of the smoky flavor [35]. However. furannic compounds were found to play a role in cold smoke odors of liquid smoke or fishes treated by liquid smoke [14. and the odorant contribution of odor-active compounds cannot be the same if the SF is in the form of powders. Furfural and homologues exhibit cooked/roasty aromatic notes.38].34]. the determination of the effects of compounds of SF on the flavor is complex. Furannic compounds were thought to contribute to soften the heavy smoky aromas associated with phenolic compounds [37. The results of this fractionation are given in Table 15. The oils used in smoke oils can soften the smoke aroma. They were isolated early from wood smoke and described as grassy. 15. 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. Sensory analysis performed on standards confirmed the importance of guaiacol and o-cresol in the smoky flavor and dimethylphenol.

246 ◾ Handbook of Seafood and Seafood Products Analysis Table 15. These . Their physical deposition of SF on seafood can confer its color to the product. distilled at 67°C–90°C.3 Role of Volatile Compounds of SF in the Texture The texture of smoked products is due to coagulation of proteins. Formaldehyde. because they are added in the product during its fabrication. which can vary from golden yellow to dark brown according to the nature of the wood. 3. furannic compounds could also have an effect on the flavor [43]. Thus. Other compounds such as enolone derivatives could also play a role in the SF flavor. 5. SFs under powder or oil forms do not act on the surface texture.6. melanoidines could be created by polymerization through aldolic condensations. A brief drying after smoke absorption can cause a higher level of dehydration and lead to higher amounts of Maillard products. However. which has been reported in wood smoke and smoked meat [41] but not in smoked fishes until now. which have brown/yellow characteristic color. However. Formaldehyde was shown to react with the amino group of the N-terminal amino acid residue and the side chains of arginine. can react with proteins. In the liquid smoking process. Studies on standards have shown that cyclotene was a flavor-active compound [41]. 15. distilled at 133°C– 200°C. 4. 11 = highest value. After scission and dehydration. Maillard and Strecker compounds can also be responsible of the color of the smoked product [45]. According to their concentrations found in the different SF. the eventual dilutions of SF. 2. and lysine residues [44]. 1. smoke condensates are colored mainly due to phenolic compounds. cysteine. 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]. 6.3 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. the product must also be placed in a dry and hot ambient atmosphere for short periods in order to favor color formation. histidine. terpene subfraction. whole liquid smoke.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. they could play a role in the inner texture of the product. and the intensity of the process. Indeed.6. 15. distilled at 91°C–132°C. the deposition of Maillard compounds leads to a darker color of fish flesh [46]. The acidic aqueous SF can also increase the coagulation of proteins and act on the texture. phenolic subfraction.

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

8 15.8. because their catabolism leads to poly-hydroxy-epoxy-PAH suitable for binding to DNA adducts. Therefore.8-dihydrodiol OH B[a]P glutathion conjugate OH OH B[a]P 7. 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. PAHs comprise fused aromatic rings made up of carbon and hydrogen atoms: up to four fused benzene rings.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. PAHs are formed by the incomplete burning of carbon-containing material. they are considered as heavy PAHs.10-epoxyde Glutathion S OH OH OH O B[a]P 7. PAHs are considered as carcinogenic contaminants of processed food [56]. hence their toxicity (Figure 15. more toxic than light PAHs.8-dihydrodiol-9. PAHs can cross the biological membranes and accumulate in tissues.3 Benzo[a]pyrene metabolization. However.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].8 epoxyde OH B[a]P 7. They are considered as carcinogenic contaminants. As they can be absorbed by animals. which are constituted by less than four benzene rings. PAHs are generated during smoke production by wood pyrolysis.3). Therefore. 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 7.248 ◾ Handbook of Seafood and Seafood Products Analysis 15. it is used as the leading substance to illustrate PAH contamination.54]. Hundreds of individual PAHs may be formed and released during the process of incomplete burning of the wood. In SF.58]. . home cooking and industrial food processes represent the major source of human contamination [55]. These compounds have been studied for several years. Owing to their lipophilic properties (log Kow between 4 and 7). they are considered as environmental pollutants and can contaminate the human feed raw material [53. particularly smoked food [57.

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

The content of PAH in SFs and in the final product can be better controlled than during traditional smoking.78].63] which justifies controls and regulations. that is. Moreover. very small amounts. Finally. whereas food is treated by SF and not directly by PP. that is. SFs are used in higher quantities than those employed in flavoring processes.69.2 European Regulations on PAH Concentration in Food Treated by SF In 1988. All these benefits could help to reconsider the status of atomization of liquid smoke and the maximum PAH contents related. The main criticism that can be formulated against SF is the lack of control of the final organoleptic qualities of such processed food. Moreover. the liquid smoking process decreases the emissions of PAH compounds to the environment.03 mg/kg. Nevertheless. 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]. excluding bivalve molluscs. the PAH contamination was only set for B[a]P [77]. Therefore. Therefore. As for SF. the vaporization of SF in a smokehouse causes a loophole in the legislation. a European regulation set the maximum content of B[a]P in foodstuffs treated by SF at 0. Thus. However. the PAH contamination reached in SF is largely below the values authorized in PP [60. However. This value is the result of the necessary harmonization between the national laws of European countries [79]. 15. Indeed. it is paradoxical to apply flavoring regulations to the smoking process. Therefore. Indeed. but it could also initiate an international consideration of labeling of smoked and flavored food. for meat industry. Moreover. 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. if it is considered as smoking technique. this process can also be considered as a flavoring of the surface of the product.250 ◾ Handbook of Seafood and Seafood Products Analysis However. 0. brown meat of crab. leading to less PAH contaminated food by comparison with the traditional smoking techniques [80]. atomization of liquid smoke must be lower than 0. as drenching or showering. it can lead to problems of labeling. Indeed. 15. 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. atomization of liquid smoke would constitute the smoking technique. and head and thorax meat of lobster and similar large crustaceans [76. the smoking regulations set a maximum B[a]P value of 5 mg/kg of smoked fishery products and smoked crustaceans. This value is very low compared to those authorized in PP. important differences in PAH concentrations are noticeable [57. However. . because these values were set for PP and not SF. the respective legal B[a]P amount is 5 mg/kg. Th is fact can be understood by the use of smoke condensates in flavoring quantities.10 Conclusion A wide range of SFs and uses of SFs are now available to flavor seafood products. according to the origin of SF and industrial manufacturers. In this case. The high maximum values authorized in PP do not seem well adjusted with the weak final PAH contamination of SF. it leads to lower PAH contents.03 mg/kg. it is necessary to better control the composition of SFs and to improve knowledge about the influence of the pyrolysis parameters (wood nature. In certain countries such as France. SFs appear as a safe alternative to smoking techniques.9. the 2003 maximum values must be reviewed again.03 mg/kg and leads often to noncompliant smoked products. Indeed.75].

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

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

B. 218. Suñen. D. 27.. Volatile aldehydes in smoked fishes: Analysis methods. J. Guillén. Food Chem.D. Tilgner. Single-laboratory validation of a gas chromatography–mass spectrometry method for quantitation of 15 European priority polycyclic aromatic hydrocarbons in spiked smoke flavourings. fumage. Hooven. M. Food Microbiol. Technol. 1103. 18. Yersinia enterocolitica. Sainclivier. Jira. Changes of benzo(a)pyrene contents in smoked fish during storage. 2000. 1993.. Food Res. 57. 770. Scientific Committee on Food (SCF).. Antibacterial activity of smoke wood condensates against Aeromonas hydrophila. Müller. 1160. Food Addit. P. P. 111.Smoke Flavoring Technology in Seafood ◾ 253 45. 2000. C. Carcinogenic polycyclic aromatic hydrocarbonDNA adducts and mechanism of action. 58. M. and Partearroyo. C. 45.. 47.. 49. and Turesky. V. and Mahadevan. J. Baird.. 219. 36. cold-smoked rainbow trout stored at 4°C.. Simon. et al. 2004. 2006..J.J. B. 48. 71(1). Agric. B. 2007. 2003..P. 876.A. 2005. 64.. E. 106. 54. and Listeria monocytogenes at low temperature. 49. E. marinage. Food Addit. P. 1536.. 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. 307. Food Chem. 52.. Paris. 61. 59. M. C.. Mutagen. Simon. 1977. Pure Appl.. 50. Food Chem. Girard. R. Curing and smoking. B.. Determination of polycyclic aromatic hydrocarbons in commercial liquid smoke flavorings of different compositions by gas chromatography–mass spectrometry. P. 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. occurrence and mechanisms of formation. Evaluation of analysis of polycyclic aromatic hydrocarbons in meat products by liquid chromatography. 60.A. 51. 208. Palme.. and Anklam. hydrolysats. 171.D. 2007.. L. 56. R. Determination of polycyclic aromatic hydrocarbons in smoked meat products and smoke flavourings additives... 2005. 126. R. 46. Guillén. W. Opinion of the Scientific Committee on Food on the risks to human health of polycyclic aromatic hydrocarbons in food (expressed on 4 December 2002). Mol. in Technologie de la viande et des produits carnés. 44. A GC/MS method for the determination of carcinogenic polycyclic aromatic hydrocarbons (PAH) in smoked meat products and liquid smokes... 2244. séchage. Bulletin scientifique et technique de l’Ecole Nationale Supérieure Agronomique Centre de Recherches de Rennes. . P... Environ...M. 2000. Lavoisier... Polycyclic aromatic hydrocarbons in smoked fish—A critical review. 55. 10(5). Int. P. J.. 3. Eur. V.. 1988. T.. Parisod..D. E. L’industrie alimentaire halieutique. 53. Sopelana. 2002.. 91(2). Suñen. B. Nyman. Food Chem.Y..P. Z. C.. The phenomena of quality in the smoke curing process.. 104(2). Sopelana.. Chromatogr. 105. Fernandez-Galian. and Anklam. Šimko. S.H. Food Chem. and Chiu. and Aristimuño. Wang. Contam. 48. 40. Chem... and Sérot. W. Contam. Quantitative determination of polycyclic aromatic hydrocarbons in barbecued meat sausages by gas chromatography coupled to mass spectrometry.. 1629. Aristimuño. 17(1).. J. Stołyhwo. Agric. 303. Des techniques ancestrales à leurs réalisations contemporaines: salage. and Sikorski. Prost. Palme. J. 1991. Varlet. Fleischwirtsch. 293.387. Agric. C.A.. La fumaison.. S.. Study of several aspects of a general method for the determination of polycyclic aromatic hydrocarbons in liquid smoke flavourings by gas chromatography-mass spectrometry.. 1996.. SCF/ CS/CNTM/PAH/29 final. and Partearroyo. Šimko... A.. Food Chem. 1991. 2001. 2002. W.J.. E. 62. M.E. Mottier. Food Chem. J. Chromatogr. M.. A. Food Res. Activity of smoke wood condensates against Aeromonas hydrophila and Listeria monocytogenes in vacuum-packaged.. 63.. 489. 61. 1985. 48.. and Fernandez-Galian. Chen.

et al. J.-Technol. et al. J. Analytical methods for polycyclic aromatic hydrocarbons (PAHs) in food and the environment needed for new food legislation in the European Union. 1161. A. 1. 66. . A. 19(9). 2006. EC 1881/2006. Off.. J. 716. L. T. Determination of polycyclic aromatic hydrocarbons in water by solid-phase microextraction–gas chromatography–mass spectrometry. 169.. U. 1999. 69. Contam. 304. 75. 1988.. W. 2001.254 ◾ Handbook of Seafood and Seafood Products Analysis 65. Popp. Eur. Accelerated solvent extraction and gas chromatography/mass spectrometry for determination of polycyclic aromatic hydrocarbons in smoked food samples. Moret. 259.. Arch. et al. Eur. Environ. L.. A.. Off. 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. Liq. Italian Law Decree. Agric..S.-Wiss. Wenzl. Union. Dos Santos Barbosa. Chem. 68. 71. 78. and Santiago-Silva. Use of supercritical fluid extraction-high performance chromatography in the determination of polynuclear aromatic hydrocarbons from smoked and broiled fish. Commission Regulation of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs. Union. Huopalahti. E. 67. 2007. L. 38. Chromatogr. and Dean. Evaluation of acute toxicity and genotoxicity of liquid products from pyrolysis of Eucalyptus grandis wood. Contam.M. J.L. EC 88/388. 1996. Food Chem.. Determination of PAH profiles by GC-MS/MS in salmon muscle processed according to four different smoking techniques. Acta.. Eur. Chromatogr. 1. 2006. Technol. M. 2003. T. 25(7). Lebensm.. Polycyclic aromatic hydrocarbons from wood pyrolysis in charcoal production furnaces. Relat. et al. Off. 153. Decreto Legislativo N°107 del 25/01/1992.. P. J. Res. 2004. 1999. Trends Anal. 2003. 47. Union.J. A.. Anal. D.. 79... Ré-Poppi. 1367. 521. Eur. L. Food Addit. and Tapanainen. 47.. Union. Readman. G. EC 2005/108. J. 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.. 77. Wang. Järvenpää. Determination of polycyclic aromatic hydrocarbons in wastewater by off-line coupling of solid-phase microextraction with column liquid chromatography.. Environ. 34.. 80. 523(2). Hattula. 309. R. L 34: 43. 2000. Allegato III. 1062. 2007. J. Conte. 364: 5.. 744.. S. 284.. 70.. Toxicol. Food Chem. 73. J. Determination of polycyclic aromatic hydrocarbons in vegetable oils using solidphase microextraction—Comprehensive two-dimensional gas chromatography coupled with time-offlight mass spectrometry. Chim. Chromatogr. Use of liquid smoke flavouring as an alternative to traditional flue gas smoking of rainbow trout fillets (Oncorhyncus mykiss). 24(7). Assessment of polycyclic aromatic hydrocarbon content of smoked fish by means of a fast HPLC/HPLC method.. 74. 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. 2006. 76. 897... 72.. et al. Commission Recommendation of 4 February 2005 on the further investigation into the levels of polycyclic aromatic hydrocarbons in certain foods. 184. N. EC 2065/2003. et al. King. 101. Purcaro. 1473. P. G. J. Varlet et al. J. Agric. J. and Zhou. 1–2. Pimenta. 2005. Off.

NUTRITIONAL QUALITY III .

.

............7 Calories ........ 269 16..............4 Comparison of Methods ..................................................... 269 16.................................................................270 16....................................7.......................... and Rasa Slizyte Contents 16........................................267 16......4.3...4............................................................................................................. 273 16.............1 Nutritional Aspects .......................................2 Methods for Determination of Total Lipids ............ 258 16.....................................................................4..........5 Nondestructive Analysis of Proteins .........................3 Nondestructive Methods ......3 Food Composition Tables and Databases ..................................... Ingrid Overrein................................................267 16........................3.............................. Christel Solberg................1 Nutritional Aspects .....................4 Direct Methods for Soluble Protein Determination ...........................................2 Nondestructive Analysis of Total Proximate Composition..............................................................274 16..........................................................7..............Chapter 16 Composition and Calories Eva Falch................................................................2 Indirect Measurements of Energy................275 16................................270 16..........................276 References .2 Methods for Protein Determination ...........276 257 ........274 16.. 273 16.......................................... 268 16....................1 Direct Measurement of Energy ........................................................ 269 16.....................270 16......................3......................................4 Proteins .............................6 Determination of Water Content ...274 16...........................267 16..............7.................1 Introduction .....5 Determination of Carbohydrate Content ..........................................................................4.............................................................................. 258 16.................3....................................................................................................................4.............3 Determination of Total Nitrogen ......3 Lipids ..........

258 ◾ Handbook of Seafood and Seafood Products Analysis 16. the range 1100–2500 nm. The NIR spectrum is defined between the wavelength 800 and 2500 nm. making it difficult to use this spectral range before the development of multivariate calibration technology. Therefore. 600. and then one can perform a linear regression on the principal components. When Williams was running the program for the Canadian Grain Commission. or reflected depending on the interaction with NIR wavelength and physical status of the sample as transparent or nontransparent. The development of NIR in food analysis started with the development of analysis of cereal grains and oilseeds in Canada [2]. The first instruments on the market were filter instruments measuring in reflectance mode. the chemical composition of fish generally varies due to seasons.1 and further discussed in the text below. and the other minor constituents include carbohydrates. stages of maturity. . rapid.7 deals with the different methods to determine and calculate calories in fish and shellfish. which involves concentrated sulfuric acid and heavy metal catalysts. analysis should be performed on the specific samples. The spectral data will be reduced by principal component analysis. and so on. to ensure obtaining data on the exact proximate composition. and a lead sulfide. The end result is a calibration equation from which the constituent of interest is calculated from a linear combination of spectral data. Section 16. 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. the adoption of NIR testing resulted in a total cost saving of CAN$ 2. NIR has been found to be a reliable. and sizes. Nearinfrared spectroscopy (NIR) is the most common method for such analysis and is therefore comprehensively presented in this chapter. such as the introduction of partial-least squares (PLS) by Martens in 1982 [3]. Proximate data on different fish species are collected in databases such as the FishBase (www.org). However. the silicon detector covers the range 400–1100 nm. proteins. geographical locations.1 Introduction The proximate composition in most fish and shellfish is primarily water. in this spectral region the spectrum of the transmitted light is very compact and no single peaks are visible. vitamins.000 Kjeldahl analyses were conducted per year and incidentally producing 47 ton of caustic waste in the process. and lipids. an indium gallium arsenic covers the range 800–1700 nm.2 Nondestructive Analysis of Total Proximate Composition Analysis of each nutrient separately is time-consuming and requires a diverse set of equipments. and easy to perform nondestructive analysis for simultaneous determination of the major components in fish. where the weak absorptions enable useful data to be obtained using sample thickness of 1–2 cm of samples such as meat. cheese. however. and minerals [1]. by the chemical-free NIR method. 16. Methods for simultaneous determination of the major components are therefore valuable.fishbase. In fish meat these constituents make up about 98% of the total mass. transmitted. As well as increased efficiency of the Canadian wheat segregation program. or whole grain.5 m per year and a saving for the environment by replacing the Kjeldahl system. making it possible to measure over the whole NIR spectrum and not only on a small number of selected filters. During the 1980s monochromator instruments were developed. Diff use transmittance measurements are usually carried out in the 800–1100 nm region of the spectrum. but the available detectors cover a smaller range. The NIR radiation interacting with a sample may be absorbed.

or transmission of nearinfrared light (850–1700 nm) Ultrasound Measurement of ultrasonic velocity Rapid. can be used on live fish Samples are placed in an electromagnetic field and electric conductivity is measured Specific for different species.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. physiological and physical states can affect values of conductivity. fully automated. nondestructive. Calibrations need to be made against reference methods. 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. expensive Few articles on fish composition Need more research Composition and Calories [21. and can be performed online [17–20. and protein. Nondestructive and can be used on live fish.133] NIR/NIT Reflection. water. precise. Calibrations require skilled personnel. [4–6.173] Total body electrical conductivity (TOBEC) May obtain data on water. and protein content noninvasively.Table 16. transflection. can be nonsensitive. displacement of reflectance spectrum by moisture content. 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.

location of lipids. nondestructive (See under NMR below) [13–15. (SoxTech. Fosslet.) Microwave drying The sample is dried and from the water content found.22.55.57] Excellent for determination of fat and water content or even distinguish lipid classes and water properties.Table 16. fexICA). 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. processing) [46] Automatic. lipid content. the fat content can be calculated theoretically by the formula Fat% = 80 − water % . 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. etc.1 (continued) Advantages Rapid.

and portable Allows in vivo measurements Nondestructive and rapid Broad range of applications. location of lipids. rapid. easy. nondestructive. and nondestructive and allows in vivo measurements (continued) 261 .Nondestructive Methods: No laboratory facilities are required Precision level may be dependent on sample (maturity stages of the fish. portable (small size). Low-field NMR See NMR above Composition and Calories NMR mouse ◾ Nondestructive and rapid.22.50. NIR/NIT Transmitted or transflected Near Infrared light (800–1700 nm). processing) Needs to be calibrated for the individual species Most suitable for neutral lipid determination See NIR/NIT above [4–6.8] [46.55] Fat meters Determination of water by analyzing the dielectric properties using a microwave strip (calculation of lipids as for the drying method). lipid content. 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.51–52] Relatively inexpensive. requires specific calibrations Traditional low-field instruments require withdrawal of homogeneous samples for analysis (invasive) [14–16.

high precision and good reproducibility. 74. standard method for comparison.133] Direct Protein Determination on Soluble Proteins Rapid. 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 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. independent of physical state of sample Does not give a measure of the true protein. Absorbance depends on the type of protein analyzed . safe (no chemical exposure). interference by nonprotein nitrogen compounds. easy to perform. and environmentally friendly High initial costs.67. low sensitivity.120. inexpensive to use. time-consuming. distillation.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. Difficult to obtain the accurate protein concentration [53.Table 16. trapping of ammonia. hazardous. since all nitrogen in foods is not in the form of protein.133] Kjeldahl Sample digestion followed by neutralization. potentially toxic chemicals are used Rapid.

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

low interference from nucleic acids and nucleotides Interference by oxygen and UV-absorbing compounds (buffer.97. might interfere [90–92. option to quantify free amino acids. quantifies amino acids. high sensitivity.133] Faster than ion exchange chromatography. chromatographic separation.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. FMOC: less soluble. no addition of reagents required. and detection of amino acids with UV absorbance. low dependency of signal response on amino acid composition.Table 16. value for net protein. Derivatization agents: OPA: no derivatization with secondary amino acids. nondestructive.1 (continued) Advantages Rapid. multicomponent analysis See NIR/NIT above Strong interference by water.96. salts) [132. fluorescence Handbook of Seafood and Seafood Products Analysis Nondestructive Determination Rapid. influence by lipids and sample particle size. hydrolysis destroys some of the amino acids. 108. nondestructive. 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) . sensitive Most methods do not include all amino acids. derivatization.

178] [151] Long analysis time. 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.Water Determination Simple to use and inexpensive equipments required [151] Shorter analysis time compared with air and vacuum drying. Air drying (101°C) may lead to thermal damage. 12 or 24 h) and water evaporated is determined. 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 . 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.g..

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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.

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16.3

Lipids

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].

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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

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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

Proteins

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].

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16.4.2

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.

16.4.3

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].

16.4.4

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

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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].

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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].

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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].

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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].

16.7

Calories

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].

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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

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[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.

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........... especially in all the essential amino acids necessary for physical and mental well-being..........................................2 Reversed-Phase High-Performance Liquid Chromatography .......3 Capillary Zone Electrophoretic Methods ...3............2..........1 Sample Preparation for Free Essential Amino Acid Analysis .... 298 17......... not all proteins have the same nutritional value..................... 300 17......................................................................... because protein quality strongly depends on its amino acid composition and digestibility.........3 Seafood Essential Amino Acid Analysis..... 287 17........3....................................1..... seafood proteins are considered as highquality proteins because of their balanced content in amino acids...3 by generation of volatile 287 ..3....1 Introduction ...................Chapter 17 Essential Amino Acids M....................... 289 17......2 Sample Preparation for Total or Hydrolyzed Essential Amino Acid Analysis.........................................................................1 Cation Exchange Chromatography . 300 References ............1 High-Performance Liquid Chromatographic Methods.........1 Fish and....291 17...............................................3.................2 Gas Liquid Chromatographic Methods ......... 288 17............................... 292 17.....291 17........................1...4 Mass Spectrometry ............. Amino acids may also be found in free form.........................................4 Conclusions ................2................... However..........2 Sample Preparation for the Analysis of Seafood Essential Amino Acids ...................3.3........ 299 17...... 290 17...............................................................................1 Introduction Amino acids are the basic components of the muscle protein structure of seafood.... in general.......................... 298 17........................................ 288 17. which contribute to fish taste and indirectly to aroma 2..................................................... Concepción Aristoy and Fidel Toldrá Contents 17.............................

the elderly.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. or diluted phosphate buffers. Although classified as nonessential.14 6% of perchloric acid. or (3) its Maillard reaction with sugars yielding characteristic flavors.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. concentrated strong acid solutions such as 4% of 5-sulfosalicylic acid. then. and leucine). and aromatic amino acids (phenylalanine and tyrosine) are the most important from this point of view. Polytron.12. Sample cleanup is necessary to eliminate proteins and polypeptides by means of the deproteinization process.13 5% of trichloroacetic acid. in rare cases. It is usually achieved by homogenization of the ground sample in an appropriate solvent by using a Stomacher. which can be achieved through different chemical or physical procedures.288 ◾ Handbook of Seafood and Seafood Products Analysis compounds through Maillard reactions and Strecker degradations. or by means of a simple stirring in warm solvent.20 have been successfully used as extraction solvents. are described. Free amino acids initiate important changes at early postmortem and during storage and can be very useful as quality indices of processing and storage. the analysis of essential amino acids in seafood is important for the evaluation of both the nutritive value and the sensory quality of seafood.11 17. with the additional advantage that proteins are not extracted and. 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. especially of those considered essentials. In this chapter. methods for the analysis of amino acids in seafood.23–25 and picric .4 Branched-chain essential amino acids (valine. Sample preparation will depend on whether free or total essential amino acids have to be analyzed. and individuals with certain metabolic disease or who suffer from malabsorption syndromes.16. and so forth. there is no need for further cleaning up of the sample.2. A more detailed description of amino acid methods of analysis may be found in the work of Aristoy and Toldrá.21 perchloric (PCA).22 trichloroacetic (TCA). In some cases.18.1 N hydrochloric acid solution. 17.000 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. Once homogenized. 0. sulfosalicylic (SSA).15 or a rich alcohol-containing solution (>75%) such as ethanol16–18 or methanol19. The extraction solvent can be hot water.5–10 Thus. sulfur-containing amino acids (methionine and cystine/cysteine).13. cysteine may be essential for infants.01–0. the sample is centrifuged at more than 10. isoleucine. which confers numerous biological functions to this amino acid (precursor to the antioxidant glutathione). which increases the protein stability in the harsh extracellular environment by conferring proteolytic resistance. The extraction consists in the separation of the free amino acid fraction from the insoluble portion of the matrix (fish muscle). Several chemical methods include the use of concentrated strong acids such as phosphotungstic (PTA). (2) its ability to cross-link proteins.

Therefore. also disposes of an oven to accomplish the hydrolysis.).22 Under these conditions. recovery of amino acids.22.41 The use of microwave technology for the hydrolysis has been assayed by some authors. the vapor-phase hydrolysis method is preferred to minimize contaminants coming from aqueous 6 N hydrochloric acid. Typically.34 17. Proteins must be hydrolyzed into their constituent amino acids before the analysis. whereas free amino acids remain in solution. addition of constant boiling hydrochloric acid and additives. and performance under vacuum) is similar to that of a conventional oven. 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.000. A good choice may be the use of 0.30 All these methods give a sample solution rich in free amino acids but free of proteins. or separation method (interferences in the chromatogram. only the acid vapor comes into contact with the sample. is well suited to hydrolyze large amounts or complex samples.35–38 These temperatures in such acidic and oxidative medium may degrade some amino acids. compatibility with derivatization (pH. 30.40. Differences among all these chemical and physical methods are caused by several aspects such as differences in the cutoff molecular weight. which permits the alternative air evacuating/inert gas purging. proteins precipitate by denaturation. In the vapor-phase hydrolysis method. liquid phase or vapor phase.2. Hydrolysis may be improved by optimizing the temperature and time of incubation41 or with the addition of amino acid oxidation protective compounds. all of them . by mixing two or three volumes of organic solvent with one volume of extract. or acetonitrile.39 Some commercial systems are available.2 Sample Preparation for Total or Hydrolyzed Essential Amino Acid Analysis The total essential amino acid profile is usually requested. which is easily separated by centrifugation. The most common method used for complete hydrolysis of proteins is acid digestion. oxygen is removed and substituted by nitrogen or other inert gas. samples are treated with constant boiling 6 N hydrochloric acid in an oven at around 110°C for 20–96 h. The hydrolysis may be accomplished using either liquid-phase or vapor-phase methods. resulting in a very simple deproteinization procedure with no interferences. Liquid-phase. thus excluding nonvolatile contaminants. the tubes containing the samples are located inside large vessels containing the acid. When limited amounts of sample are available. In both cases.31–33 with amino acid recoveries around 100% for all them. etc. but the duration of the treatment is shorter (less than 20 min). creating an appropriate atmosphere inside the vessels to ensure low amino acid degradation. and so forth. An additional advantage is the easy evaporation to concentrate the sample.29.39. The presence of appropriate antioxidants/scavengers during hydrolysis can prevent losses of the most labile amino acids. ethanol. a system capable of alternative air evacuating/inert gas purging to get a correct deaeration inside is valuable. which is easily neutralized by the addition of KOH or potassium bicarbonate. has also given very good results.000. 10.Essential Amino Acids ◾ 289 (PA)26–28 acids or organic solvents such as methanol. 5. where the hydrochloric acid contacts the sample directly. Some comparative studies have been published on these deproteinization techniques. Upon heating. to rend insoluble potassium perchlorate. etc.000. Some physical methods consist in centrifugation through cutoff membrane filters (1.6 N PCA. The use of organic solvents.18.42 Sample manipulation (sample evaporation to dryness. Nitrogen atmosphere and sealed vials are required during the hydrolysis to minimize the degradation.). because it gives information on the nutritional value of fish meat.000 Da) that allow free amino acids through while retaining large compounds. Digestion at 145°C for 4 h has also been proposed. presence of salts.

38 Alkaline hydrolysis instead of acid hydrolysis is also proposed (see below). or BaOH. The use of alkylating agents to stabilize the previous hydrolysis of cysteine constitutes a valid alternative. This option is chosen to analyze specific amino acid sequences or single amino acids because of their specific and well-defined activity. carboxypeptidase. The choice mainly depends on the equipment available or personal preferences. or pronase. chymotrypsin.62 An alternative to acid hydrolysis is the alkaline hydrolysis with 4. Derivatization is a usual practice in amino acid analysis.1% sodium sulfite.3 Seafood Essential Amino Acid Analysis The analysis of individual amino acids needs a previous separation of all others. cysteine sulfinic acid. which is recommended by many authors47.43–50 improves cysteine (and methionine) recoveries.54 or 3. threonine. a compromise of conditions offers the best overall estimation for the largest number of amino acids. cysteine.2 M of either NaOH. The previous performic acid oxidation of cysteine to cysteic acid. serine.30.61.67–69 17. 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. In fact. Before or after this separation.63–66 for a better tryptophan determination. being enough for the requirements of any food industry. has been extensively reported in papers35. and so on. (2) give a quantitative and reproducible reaction. Tryptophan is often completely destroyed by hydrochloric acid hydrolysis.58.59. thermolysin. adequate hydrolysis procedure as the performic acid oxidation before the hydrolysis is a good alternative. and tryptophan. methionine. In general. with or without the addition of 1% (w/v) thiodiglycol for 18 h at 110°C. such as tyrosine.60 and books. such as chromatographic (liquid or gas chromatography (GC)) or capillary electrophoresis (CE) techniques. When high sensitivity is required. Additionally. although considerable recoveries have been found if no oxygen is present.52 4-vinyl pyridine53. The optimization of conditions for hydrolysis based on the study of hydrolysis time and temperature.55. and cysteic acid making its analysis rather difficult. KOH. Good recoveries have been achieved by using 3-bromopropionic acid. up to 1% phenol or 0. 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. unless a very selective way of detection is used.36. 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. because each possible methodology has advantages and drawbacks. amino acids used to be derivatized to allow their separation or to enhance their detection. . presence and concentration of oxidation protective agents.290 ◾ Handbook of Seafood and Seafood Products Analysis essential amino acids. making the posterior analysis easier. papain. no single set of conditions will yield the accurate determination of all essential amino acids.36. A third way to hydrolyze proteins is enzymatic hydrolysis by proteolytic enzymes such as trypsin. improve the recovery of nearly all of these amino acids except tryptophan and cysteine. acid-to-protein ratio. yields acceptable results for the majority of amino acids.51 3-bromopropylamine. 41. protective agents currently used. importance of a correct deaeration. Cyst(e)ine is partially oxidized during acid hydrolysis yielding several adducts: cystine. LiOH. Thus. in which methionine is also oxidized to methionine sulfone. when the analysis of cyst(e)ine would be necessary. Some additives have been proposed to protect tryptophan against oxidation as is the case of thioglycolic acid.3¢-dithiodipropionic acid.56 As can be observed in this section. The separation of the individual amino acids in a mixture requires very efficient separation.

17. because reagent-consuming amines. the spectroscopic detection of amino acids requires their previous derivatization to obtain an UV absorbing or fluorescent molecule.1 and 17. tyrosine. Amino acids.3. or 4-fluoro-7-nitrobenzo-2. recent improvements of the ninhydrin derivatization method71–73 .1 High-Performance Liquid Chromatographic Methods HPLC is the preferred technique to analyze amino acids. and those with more than one primary amino group or possessing a guanidyl residue elute at the end of the chromatogram.1 Cation Exchange Chromatography This methodology is based on the amino acid charge. are always present. and they include derivatives for spectroscopic or for electrochemical detection. (4) have mild and simple reaction conditions. Tryptophan also possesses native fluorescence (l ex = 295 nm. The classical procedure has been improved with a new polystyrene matrix that offers better resolution power due to smaller particle size. although unidentified.3.12 Two types of derivatives are obtained depending on the chosen separation and/or detection technique.3-oxadiazole postcolumn derivatization to obtain highly fluorescent derivatives with enhanced sensitivity.1.70 fluorescamine. Although postcolumn techniques should be run online for maximum accuracy. l em = 345 nm). absorb at 210 nm and thus cannot be used for spectroscopic detection.3. in their native form. amino acids were converted into colored ninhydrin derivatives for spectrophotometric (colorimetric) detection. because their spectral (high-ultraviolet (UV) absorbing or fluorescence properties) or electrochemical characteristics will affect the sensitivity and selectivity of detection. The first type are derivatives that enhance amino acid detection in liquid media. 17. which facilitates a more selective detection. The derivatization reaction can be performed after separation of the amino acids (postcolumn derivatization) or before separating them (precolumn derivatization). It must be remarked that the use of sufficient amount of reagent is of special importance when dealing with biological samples. pellicular packaging. Under these conditions. After separation. speed.1. and better detection systems. 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. (5) have the possibility of automation.15.3. Only three amino acids (phenylalanine. 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. permitting 5–10 pmol sensitivity as standard. as it is a very unspecific detection wavelength. precolumn techniques can be run either offline or online. The elution involves a stepwise increase in both pH and sodium or lithium ion concentration. The HPLC techniques to analyze amino acids are cation exchange and reversed-phase (RP) chromatography and are described in Sections 17.1.38. The original method required two separate columns and needed about 4 h to achieve a complete analysis. Nevertheless. the more acidic amino acids elute first.2.68 Thus. 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. (6) have good stability of the derivatization products. The latest generation of Moore and Stein amino acid analyzers also use o-phthaldialdehyde (OPA).Essential Amino Acids ◾ 291 (3) yield a single derivative of each amino acid. 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).1.

74 Nowadays. Precolumn amino acid derivatization may be necessary to confer hydrophobicity to the amino acid molecule. some difficulties to analyze some essential or sulfur-containing amino acid derivatives).) who offer integrated commercial systems including the column. each new methodology must contrast its results with those obtained by cation exchange chromatography (CEC). or the stability of formed derivatives. Although this broadening may not affect when using standard-bore columns with flow rates above 1 mL/min. Another disadvantage is the peak broadening produced by the dead volume introduced behind the column. There are many manufacturers (Beckman. biological fluids. All PTC-amino acids have similar response factors.77 After separation.70. followed by a reaction coil. 17. 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. and tissues). but. the derivatizing reagent is pumped into the effluent from the column system. 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. feed. Dionex. 66. which makes it a reference method for amino acid analysis. and the long time of analysis.1. plants. To choose the most appropriate method some aspects must be taken into account such as the following: the disposable detector (fluorescence or UV).75.. also. especially in a dry condition. Biotronik. which are detectable at UV (254 nm) with detection limits around 5–50 pmol. In this way. The advantage of this method is the accurate results for all known sample types (food. which constitutes an advantage. The PTC-amino acids are moderately stable at room temperature for 1 day and much longer when kept under frozen storage.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. the formed molecule improves sensitivity and selectivity at the detection by allowing the spectroscopic (UV or fluorescent) detection of amino acids. tissues. plants). . The most usual derivatizing agents for tissue amino acids are described below. Pickering. and an optimized methodology with the advantage of ease of use and reliability. etc. 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.e. through a mixing manifold. buffer system. The main drawbacks of this methodology are the high cost of the ion exchange amino acid analyzer and its maintenance.3. with which many of them have been marketed. 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. Amersham Biosciences.42. possibility of automation of the derivatization reaction (in the autosampler). because it requires only a standard equipment that can be shared by different types of analysis. postcolumn derivatization is not suitable for narrow-bore HPLC. LKB. time for sample preparation and amino acids separation. This method has been employed in the classical Moore and Steintype commercial amino acid analyzers. Kontron. Phenylisothiocyanate (PITC): This methodology involves the conversion of primary and secondary amino acids to their phenylthiocarbamyl (PTC) derivatives. the analysis requirements for free or hydrolyzed amino acids or required sensibility. Hitachi. the highly complex mobile phase composition. making it adequate for partition based on chromatography.76 There are other reports of applying this technique to the amino acid analysis in food and tissues. (i. and finally the derivatized amino acids reach an online detector system. Obviously.2 Reversed-Phase High-Performance Liquid Chromatography RP-HPLC has been widely used.

Milford. The only limitation is the determination of PTC cystine that gives a poor linearity. It is important to ensure a basic pH to get adequate derivatization recoveries.82 The selection of the column is critical to get a good resolved separation especially when the analysis of physiological amino acids is involved. Moreover.78–80 Sample preparation is quite tedious: it requires a basic medium (pH = 10. IS.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).5).29.78–80 The chromatographic separation takes around 20 min for hydrolyzed amino acids and 60 min for physiological. Both examples applied to the analysis of total amino acids from hake and free amino acids from salmon are shown in Figures 17. standards. Sarwar et al. respectively.1 Reversed-phase HPLC chromatogram of PTC amino acids from hydrolyzed hake muscle. This method is available as a commercially prepackaged system named Pico-Tag (Waters Associates.2. which is more critical when amino acids from acid hydrolysis are analyzed. because no buffer is used during the reaction. Massachusetts). the last one being the elimination of the excess of reagent that may cause some damage to the chromatographic column.Essential Amino Acids ◾ 293 The methodology is well described in the literature. The reaction time is less than 10 min even though 20 min are recommended for a complete reaction. 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 17.1 and 17. which includes the analytical column.29. the residual PITC reagent left after evaporation will cause damage to the column package. which makes the quantitation of free cystine nonfeasible with this method. and solvents. as some columns are more resistant than others. which is achieved by the addition of triethylamine and includes several drying steps. internal standard nor-Leucine. .

Reaction efficiency is highly matrix dependent and variable for different amino acids.58. and only needs a basic pH.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. taurine. internal standard nor-Leucine. IS. the reaction conditions .5. 4-Dimethyl-aminoazobenzene-4′-sulfonyl chloride (Dabsyl-Cl): This reagent was first described in 1975 for use in amino acid analysis. Detection limits are in the low picomole range.83. because it is especially affected by the presence of high levels of some chloride salts. Tau.2 Reversed-phase HPLC chromatogram of PITC-free amino acids from salmon muscle extract. Stocchi et al. standard amino acid solution should be derivatized under similar conditions. Palo Alto California.84 Derivatives are very stable (weeks) and can be formed from both primary and secondary amino acids. around 9. Nevertheless. anserine.33 To overcome this problem and obtain an accurate calibration. By-products originating from an excess of reagent absorb at the same wavelength and thus they appear in the chromatogram.87 or even 2 min at 100°C. Ans.84 Detection is by absorption in the visible range. The dansylated amino acids are stable for 1 day85 or until 7 days when kept at −4°C86 and protected from light. l em = 510 nm) although UV (l = 250 nm) detection may also be used. 15 min at 60°C. United States). However. presenting a maximum from 448 to 468 nm. The sample derivatization is rather simple. 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. The reaction time is around 15 min at 70°C and takes place in a basic medium with an excess of reagent.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. The high wavelength of absorption makes the baseline chromatogram very stable with a large variety of solvents and gradient systems. and a reaction time of 1 h at room temperature (in the dark). Commercial System Gold/Dabsylation Kit™ uses this technique (Beckman Instruments.

In order to obtain reliable and precise results. as well as reaction time. because it guarantees the repeatability of parameters. reaction conditions. l em = 315 nm) and is detected at the femtomole range. and 3-mercaptopropionic acid are the most frequently used. OPA derivatives can be detected by UV absorption (338 nm) as well. On the contrary. chromatographic selectivity.86.94–96 2-mercaptoethanol. several methods have been proposed before derivatization. this will commonly form multiple derivatives with histidine. Nowadays. itself or hydrolyzed. Tryptophan adducts do not fluoresce and histidine and cyst(e)ine adducts fluoresce weakly. One of the main disadvantages of this procedure is the inability of OPA to react with secondary amines.Essential Amino Acids ◾ 295 (pH. 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. An automated precolumn derivatization routine.97.82. 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. this methodology reveals excellent linearity for cystine and also cystine-containing short-chain peptides.94 into the automatic sample preparation protocol described by Schuster. using 3. The addition of detergents like Brij 35 to the derivatization reagent seems to increase the fluorescence response of lysine. such as FMOC/amino acid ratio.32 In these methods. This is relatively easy because the reaction is fast and no heating is necessary. many automatic injectors are programmable and able to achieve automatic derivatizations. The yield with lysine and cysteine is low and variable. and tyrosine. Some reports have been published proposing several ways of automation. The derivatization is fast (1–3 min) and is performed at room temperature in alkaline (pH 9.101 or the formation of the mixed disulfide S-2-carboxyethylthiocysteine (Cys-MPA) from cysteine and cystine. temperature. This excess is hydrolyzed to dansyl sulfonic acid.3′-dithiodipropionic acid55 and incorporated by Godel et al. OPA amino acids are not stable. which includes the addition of ADAM.92. The derivative is fluorescent (l ex = 265 nm. ethanethiol. 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. is of great advantage.32 .82 Another problem is the large excess of reagent needed to assure a quantitative reaction. this problem is overcome by standardizing the time between sample derivatization and column injection by automation.5) medium. and the reagent itself is not fluorescent. lysine. Histidine gives a very poor fluorescence response (10% of the other amino acids). The first option was included in the automated AminoTag method90 developed by Varian (Varian Associates Limited).99 In the case of cysteine. 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. which is not the case with any essential amino acid.88 Even so.91 This method is preferred because the addition of ADAM is more easily automatized. and fluorescent intensity. as it is highly fluorescent and then. The choice of the mercaptan can affect derivative stability. The reaction time is fast (45–90 s) and does not require any heating. The major disadvantage is due to the reagent. cysteine and cystine are quantified together. reinforcing the poor reproducibility of its results. have to be optimized very carefully. the reaction of the excess of reagent with a very hydrophobic amine as 1-adamantylamine (ADAM) gives a late-eluting noninterfering peak. which is present in excess as it is highly fluorescent and probably interferes into the chromatogram as a huge peak.89 9-Fluorenylmethyl chloroformate (FMOC): This reagent yields stable derivatives (days) with primary and secondary amines.98 and some of them have been patented and commercially marketed (AutoTag OPA from Waters Associates).93 The fluorescence is recorded at 455 or 470 nm after excitation at 230 or 330 nm. and excess of reagent) must be carefully fi xed to optimize the product yield and to minimize secondary reactions. In the second option.

The most used column packaging consists of alkyl-bonded silica particles. and the separation of physiological amino acids is improved. . Massachusetts. It means that when transferring a published method to a particular set of samples. The chromatographic separation of these derivatives has been optimized for the amino acids from hydrolyzed proteins. 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.296 ◾ Handbook of Seafood and Seafood Products Analysis 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC): It reacts with primary and secondary amines from amino acids. the optimum pH for the reaction is in a broad range. can cause unwanted tailing of peaks (especially for the basic amino acids).2 to 10. because the resulting CisH is well separated inside the chromatogram. UV detection (254 nm) may also be used.3A shows the separation of hydrolyzed amino acids from salmon. or charge. Furthermore. Reaction time is short. even those made by the same manufacturer. lipids. and other compounds naturally occurring in biological samples and foods. Sensitivity is in the femtomole range.and di-derivatives are the initial adducts from tyrosine. Milford. Cystine and cysteine may be analyzed after their conversion to cysteic acid (CisH) by performic acid oxidation. and proteins. Both facts facilitate sample preparation. such as current. In these cases. but 10 min at 55°C would be necessary if a tyrosine monoderivative is required. detergents. columns are more carefully manufactured with these silanol groups blocked or inaccessible by steric impediment avoiding the tailing. Only amino acids with aromatic rings or sulfur-containing side chains are sufficiently electrochemically active to be detected by this method.. In this case. United States).50). is related to the analyte concentration. it will be necessary to readjust the chromatographic conditions to get a good separation of all amino acids. peptides. Figure 17.3B shows the same sample but submitted to a performic acid oxidation before the hydrolysis in which the CisH peak appears by 7. different selectivity. from 8. Any electrical measure. different selectivity may be found among same columns. However. which are separated by RP-HPLC. OPA/mercaptoethanol or OPA/sulfite104. Indeed. The presence of residual uncapped silanol groups on the silica surface. whereas Figure 17.105 in addition to fluorescent properties possesses electroactivity (750 mV) and PITC106 has again the advantage of reacting with secondary amines. accessible to sample molecules.e. 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 addition of a strong cation (i. and UV detection at 254 nm may be used for its analysis. mainly octadecylsilane. because both mono. The methodology has been marketed as a prepackaged AccQ Tag kit (Waters Corporation. l em = 395 nm).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. 1 min.107. conductance. as a consequence. Nowadays. the choice of the RP column is not an easy subject because of the great variability of commercially available RP columns. triethylamine) to the mobile phase can overcome the problem. which is only weakly fluorescent at the amino acid derivatives detection conditions and does not interfere in the chromatogram. because they are molecules with electroactive functional groups. making them very adequate for biochemical research. Due to these variables.108 If the choice of the derivative reaction is a challenge. Electrochemical detection consists in one electrode or an array of electrodes mounted in a cell with an applied potential difference. yielding very stable derivatives (1 week at room temperature) with fluorescent properties (l ex = 250 nm. The fluorescence of tryptophan derivative is very poor. The excess of reagent is consumed during the reaction to form aminoquinoline (AMQ). potential. Only columns manufactured in the same batch are guaranteed to give the same selectivity if the rest of parameters are fi xed.5 min. Some of these derivatives are also susceptible to electrochemical detection.

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. aAba. Mobile-phase requirements consist in the ability to dissolve the sample while keeping it transparent to the detection system. methiomine sulfone. Typical analytical column dimensions are 15 cm (for hydrolyzed amino acids) or 25–30 cm (for physiological amino acids). MeS. cysteic acid. CysH. 1-methylhistidine. a aminobutyric acid used as internal standard. packed with 5 mm particle size or shorter columns (10 or 15 cm length) when packed with less than 3 mm particle size. 1MeHis.3 Reversed-phase HPLC chromatogram of AQC amino acids from hydrolyzed salmon muscle (A) without and (B) after performic acid oxidation. Mobile-phase composition combines an aqueous buffered phase .

298 ◾ Handbook of Seafood and Seafood Products Analysis with an organic phase constituted by acetonitrile and/or methanol and/or tetrahydrofuran. United States). although applications on meat samples are scarcely described. which is universal and the most widely used. the amino acids must be converted to volatile and thermostable molecules.2 Gas Liquid Chromatographic Methods The extremely high-resolution capacity is the main advantage of GC. 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.121 The high efficiency. 17. in comparison with liquid chromatographic techniques. and urine matrices but not in tissues in which the presence of natural dipeptides.116 to analyze phosphoserine. especially. Reactions consist of two stages: an esterification with an acidified alcohol followed by N-acylation with an acid anhydride in an anhydrous medium. 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. nuts.113 and cheese. This methodology has been patented as EZ:faast and commercialized by Phenomenex (Torrance. The NPD was used by Buser and Erbersdobler115 and FPD by Kataoka et al. The difficulty of separating amino acids by this technique relies on their structure.114 In their analysis by GLC. a very highly efficient technique adequate for the amino acid analysis. speed. has been developed. A finely adjusted binary (most used) or ternary gradient elution is often necessary when the overall amino acid profile from hydrolyzed and. especially in the analysis of D isomers. GLC has been combined with mass spectrometry (MS) for detection and identification. In many cases. and the equipment is very versatile and usually available in any analytical laboratory.3. Gas liquid chromatography (GLC) is not often used for the determination of amino acids from tissues or foods.109. a very fast GC analysis of physiological amino acids. Recently. neutral. plasma. and even though a particular pH can significantly . carnosine. 17. California. or GC and LC with MS detection.112 milk. phosphothreonine. Described applications are available for the analysis of physiological amino acids in blood. which are much more sensitive than FID for such compounds. The main advantages of these detectors are their high sensitivity and wide linear range. GLC is. especially since the capillary columns appeared. and acidic constituents. including amino acids.3. GC/NPD. The buffer may be constituted by less than 100 mM concentration of acetate or phosphate. and balenine may complicate the amino acid analysis. GLC is not very expensive because no solvent is used. physiological amino acids has to be analyzed. The detector used is the flame ionization detector (FID). anserine. and beans111 or other results obtained in honey.110 comparing GLC with cation exchange chromatography reported different conclusions when analyzing some hydrolyzed food samples. and amines. capable of separating 50 compounds. Protein removal is not required. whereas thermionic-N-P (NPD) or flame photometric detector (FPD) are selective toward organic compounds containing phosphorous and nitrogen. in summary.120. Nevertheless. the technique is very efficient and it is worth mentioning the separation of 32 nonprotein amino acids from edible seeds.117–119 where the separation was achieved by using chiral-GC stationary phases. Some applications67.3 Capillary Zone Electrophoretic Methods Capillary zone electrophoretic technique is extremely efficient for the separation of charged solutes.113. Amino acids constitute a mix of basic. and phosphotyrosine. dipeptides.

showing that higher efficiency is obtained by the MECC methods with sodium dodecyl sulfate (SDS) as micelle-forming substance. Some reviews covering high-sensitivity detection following CE have been published. which can be resolved on the basis on their mass-to-charge ratios that are characteristic of each ion and allow its identification. the high cost of purchase and maintenance of mass spectrometers has inhibited their more widespread use in the food industry and/or food control. A good compatibility between both techniques.146. biomedical or pharmaceutical research. in particular when capillary columns were available.124 Basic theoretical considerations on this technique125 and its food applications126 are described elsewhere. Unfortunately. methanol. reports in the literature of its applications are increasing rapidly.118. compatible with MS. which constitutes an important limitation of this technique.138 and OPA139 compared with the separation of OPA-amino acid derivatives by CZE with normal and micellar solutions.Essential Amino Acids ◾ 299 improve the resolution of one kind. it is relatively easy to analyze low picomol levels of OPA derivatives in micellar solutions by using a conventional fluorometric detector. although other additives such as dodecyltrimethylammonium bromide. etc. Application in foods such as in the identification of nonprotein amino acids.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.127–131 derivatization is used to improve separation. 19 amino acids were analyzed by CE-ESI-MS in only 17 min with a minimal sample preparation and no matrix interference.).137. and so forth. etc.) and instrumentation (CE. nonprotein amino acids. Under the conditions of electro-osmotic flow in CE. Good separations have been reported for precapillary derivatized amino acids with dansyl-Cl.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. the species with different charge can be simultaneously analyzed but with serious doubts in their adequate resolution. The effect of these additives on the electro-osmotic mobility and electrophoretic mobility of the micelle has been studied. With few exceptions.136.and l-isomer mixtures.125.147 17.131 and thus. Terabe et al.138 Tween 20. although this detector may be used for more complex identifications as in d.133–135 PITC. allowed a rapid development and the onset of these complementary techniques. or to allow fluorescence or electrochemical132 detection of amino acids.113 o-tyrosine in chicken148 or pork149 tissues.21 chiral amino acids.136 phenylthiohydantoin. o-tyrosine analysis.139 which is usually enough for food analysis or an LIF (laser-induced fluorescence) detector.3. . and the composition and flow rate of the sheath liquid to obtain the best sensitivity. tetrahydrofurane. isobutanol. The identification of the 22 protein amino acids may not be a problem. Nevertheless.144.119 have been reported.4 Mass Spectrometry MS is based on the conversion of components of a sample into rapidly moving gaseous ions. and others. etc. This report includes the optimization of important parameters like the choice of a volatile electrolyte (1 M formic acid) for the electrophoresis. SDS is indeed the most used additive to form micelles in this kind of analysis.122. Other additives commonly used in this analysis are organic modifiers (acetonitrile. to enhance UV detection. microcolumn liquid chromatography.). This technique has also been termed micellar electrokinetic capillary chromatography (MECC or MEKC). When sensitivity is the target. CZE shows poor ability for the separation of neutral compounds. Mass spectrometer detectors were first connected to GC equipments.141–143 The CE coupled to electrospray ionization (ESI) MS (CE-ESI-MS) allows direct amino acid analysis without derivatization.140 or even urea141 have been assayed. it is likely to cause overlap with the others.

the first decision is the choice of the hydrolysis method. once again. Shimada. The best results were obtained by using AP-MIPI in conjunction with a dual oscillating capillary nebulizer.2. but tedious and time-consuming sample derivatization is required. Fisheries Sci. the convenience of purchasing commercially available prepackaged kits should be considered. atmospheric pressure chemical ionization (APCI). were compared by Kwon and Moini150 in relation to sensitivity. and the technique is widespread although it is still expensive.. 1996. Res. Yamanaka. T. The requirements in resolution are not so exigent as those for physiological amino acids. Particular hydrolysis problems related with certain amino acids are described in Section 17. and many applications can be found in other matrices like cheese or meat. References 1. 17. 62.152 A very careful control of the derivatization reactions and chromatographic conditions are necessary for a consistent and reproducible analysis. 479–483. S. Protein digestibility: In vitro methods of assessment. such as phenol. due to its high specificity. Food Nutr. Catignani. J. 185–236. may appear in the chromatogram. G.300 ◾ Handbook of Seafood and Seafood Products Analysis MS has also been used as a spectroscopic detector after HPLC or CZE. The highest resolution is obtained by GC with the capillary column technique. vacuum). Konosu. One of the main requirements for samples to be analyzed by MS is that analytes. Therefore..L. cation exchange and postcolumn derivatization or RP-HPLC precolumn derivatization techniques are the preferred methods. must be ionized. The connection of HPLC and MS detector is much more problematic than with GLC because of the incompatibility between both techniques (solvents from chromatography. In general. 3. the analytical technique for a determined sample must be carefully chosen based on the literature. and. K. The convenience of purchasing commercially available kits must be evaluated. Sensory analysis of taste-active components in the extract of boiled snow crab meat. 1991. acid hydrolysis with HCl 6 N (110°C for 22 h or 145°C for 4 h) with an oxidation protective agent. However. nucleosides. Three types of ionization modes. Hayashi. and ESI.. .. is enough for the majority of purposes. Adv.2. RP-HPLC methods with precolumn OPA or PITC derivatization are very convenient methods to use. which means a minor sample manipulation. Yamaguchi. a complete resolution of the whole peaks is really difficult. the most important factors to take into account are the resolution power and selectivity. 35. atmospheric pressure microwave-induced plasma ionization (AP-MIPI). 821–824. 1981. When amino acids from seafood proteins have to be analyzed. Any separation strategy may give good results. Post-mortem biochemical changes in the muscle of Japanese spiny lobster during storage. and. high mobile-phase flow rate vs. H. Since many peaks corresponding to protein and nonprotein amino acids. The majority of published reports in which seafood amino acids are analyzed have used the cation exchange method. and so on. In general.E. Swaisgood. and taking care of avoiding the presence of oxygen with vacuum and nitrogen purging. 46. nowadays these difficulties have been overcome with the development of new interfaces. Nevertheless. which may be consulted.151. H.4 Conclusions To obtain the total essential amino acids profile of a given seafood. offering the additional advantage of analyzing the amino acids without derivatization. because fewer peaks appear in the chromatogram. reduced problems related with matrix interferences or poor resolution between peaks. R. Food Sci. amino acids in this case. 2. small peptides.

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..1 Antioxidant and Other Functions of Carotenoids ..............................1........1..................2 Carotenoid Determination ................................................................................2 Determination of Antioxidants and Antioxidant Capacity in Biological and Food Systems .................................310 18.....................2........1 Introduction ...................................................317 18............... 315 18....................Chapter 18 Antioxidants Nick Kalogeropoulos and Antonia Chiou Contents 18.3 Marine Lipid Oxidation ..........313 18.........317 18.....................317 18.......................................................................3.311 18...........................3..3 Occurrence of Carotenoids ...1 Vitamin E as an Antioxidant.......................1.............2........1 Oxidation and Its Implications.................3...............................1........................................310 18..........316 18...............3..310 18......314 18...................3 Occurrence of Ascorbic Acid in Marine Organisms ..311 18............. 315 18....313 18......................................316 18.....3.....................2.........2 Ascorbic Acid .......3...............3.......................................................3....3 Vitamin E ........3...............1....................4....................1 Antioxidant Enzymes ..................3..3 Antioxidants in Seafood and Seafood Products .....................313 18...........3................................................2 Ascorbic Acid Analysis ................................................1..3.............................................................................................................................................314 18.......................3.....3...................................318 18....2 Lipid Peroxidation.....................................2 Antioxidants........................4........................................3 Occurrence of Vitamin E .......4......................1 Ascorbic Acid Functions .................311 18....................................................................1.........................................1...........1 Oxidative Stress and Its Implications ......3...........................................................318 309 .........2 Vitamin E Determination .............314 18................4 Carotenoids ........3....

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

Autoxidation occurs through a three-phase process.2 Lipid Peroxidation A very damaging effect of oxidant reactive intermediates is lipid peroxidation. antioxidants often act via more than one mechanism that combines different types of antioxidant activity. Lipid oxidation of omega-3 polyunsaturated fatty acids (PUFA)-rich food products results in the development of particularly unpleasant off flavors. that is. and termination.1. antioxidants are .11 and unhealthy compounds that reduce their shelf life and nutritive value. and they stall the propagation phase. chain-breaking antioxidants. protect the cellular components from oxidative damage. free radicals. in which polyunsaturated fatty acids on lipid molecules are attacked and oxidized.).1. marine lipids are relatively more susceptible to oxidation. and (3) enzymatic oxidation. Moreover. (2) nonenzymatic and nonradical photooxidation. This can be especially damaging to lipid-rich cell membranes. Nonradical photooxidation seems to be a minor reaction compared with the 3O2-induced radical chain autoxidation. 18. propagation. heat.12 In biological systems various biochemical defense mechanisms. carcinogenic. mutagenic. antioxidants are molecules that protect macromolecules from being oxidized. for which the human sensory apparatus has a low threshold. For food systems. and storage. are essential for counteracting oxidative stress.13 Antioxidants counteract oxidation in two different ways: They protect lipids from oxidation initiators. Three reaction pathways have been proposed: (1) nonenzymatic chain autoxidation. In the first two cases a combination of reactions involving 3O2 and 1O2 occurs. significantly delays or prevents oxidation of that substrate. The major components of the antioxidant defense system together with their proposed mechanisms of action are presented in Table 18. Chain-breaking antioxidants intercept •) or participate in halting radical chain propagation. antioxidants supplied by foods.Antioxidants ◾ 311 loss or oxidation product development. There is increasing evidence that oxidative stress is implicated in the pathogenesis of many inflammatory and degenerative diseases and conditions.8 Studies on the pathological significance of dietary lipid oxidation products have indicated that some lipid oxidation products have cytotoxic. In general.1.2 Antioxidants Antioxidants are defined as any substance that when present at low concentrations compared with those of an oxidizable substrate.3 Marine Lipid Oxidation Compared with other food lipids. 1O2.10 Lipids deteriorate in seafood products during processing. Nevradical oxidation propagators (LOO ertheless.9 Preventive antioxidants hinder ROS formation or scavenge spe• cies responsible for oxidation initiation (O2−. and metal ions.7 18.1. mainly of plant origin.9 18. initiation. because of their high degree of unsaturation.3. Lipid oxidation is a complex procedure induced by oxygen in the presence of initiators such as light. including enzymatic systems and nonenzymatic antioxidants. atherogenic. The health implications of tissue lipid oxidation are numerous and well documented.1. and angiotoxic effects.1. handling. 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. preventive antioxidants.

chain-breaking antioxidants Synergistic to vitamin E Scavenges NO2 Transient metal chelators. Rev. J. synergistic to vitamin E Transient metal chelators Transient metal chelators (the ones with o-diphenolic structure). EDTA. 2004. chain-breaking antioxidants. scavenges peroxyradicals. Crit. lipoic acid. ROS detoxification (hydroperoxides). 44. 275. Cu) Transient metal chelators (chelates Cu) Transient metal chelators (chelates Fe) Transient metal chelators (chelates Fe) Transient metal chelators (chelates Cd. Zn. et al. citric acid Polyphenols (exogenous) Chain-breaking antioxidant. Food Sci. regenerates oxidized vitamin E 1 O2 quenchers.. melanins (endogenous) Source: Adapted from Willcox.K. anserine. but uncertain in vivo Vitamin E (exogenous) Bilirubin. carnosine.312 ◾ Handbook of Seafood and Seafood Products Analysis Table 18.1 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. . regenerate oxidized vitamin E Chain-breaking antioxidant. Cu) Low Molecular Mass Ascorbic acid (exogenous) Carotenoids (exogenous) Coenzyme Q (endogenous) Urate (endogenous) Phospholipids (endogenous) Polyphosphates. 1O2 quencher Compounds with proven antioxidant activity in vitro.. sex hormones melatonin. 2-oxo acids.

9 and 9. the latter including paper. coenzyme Q. which act as primary preventive inhibitors and catalyze the dismutation of superoxide anion • • (O2−) by reducing one O2− to H2O2 and oxidizing another one to O2. tocopherols.18 In reviewing methods and tests for the assessment of lipid oxidation. oxidative damage to macromolecules is controlled by two types of antioxidant systems.19 concluded that. kidneys. whereas Fe SOD were purified from red algae. These antioxidants act in a concerted way to protect sensitive molecules such as the unsaturated fatty acids from oxidation. bilirubin. Kolanowski et al. Catalase activities ranged between 386 and 1523 mmol/ min/g tissue in several Atlantic fish and was higher in liver. carotenoids. and heart. and high-performance liquid chromatography (HPLC) alone or combined with mass spectroscopy. 18.. 18. and uric acid.7 U/mg of protein. Cu. together with traces of phenolic compounds.20 Superoxide dismutases (SOD): SODs are metalloproteins. and algae.16 and Laguerre et al. thiols.3.3. or Fe in their active site. one represented by enzymes and the second represented by low molecular mass compounds.Antioxidants ◾ 313 effective at very low concentration levels.20 . polarographic. and in red muscle compared with that in white.1 Cu/Zn SOD were purified from marine fish tissues.2 and 18.1 Antioxidant Enzymes Catalases are metal-containing enzymes. cephalopods. and crustaceans from the Mediterranean sea. such as ascorbic acid. blue-green algae. with Mn. for quality control of fish oil and fish oil-containing foods.17 and Wood et al.2 Determination of Antioxidants and Antioxidant Capacity in Biological and Food Systems Analytical techniques developed for some of the common endogenous or exogenous antioxidants range from qualitative detection by color-developing reactions to semiquantitative and quantitative determinations by means of spectroscopic.15 Griffiths et al. and chromatographic methods. widely distributed in aerobic cells that help in preventing the accumulation of H2O2 within cells. spleen. Zn.3. At higher levels most of them behave as prooxidants possibly due to their involvement in the initiation reactions. glutathione (GSH). Cu/Zn-SOD activities ranged between 1.9 The determination of specific waterand fat-soluble antioxidants is discussed in Sections 18.3 Antioxidants in Seafood and Seafood Products In living organisms. voltammetric. 18. The available methods have been reviewed by Rajalakshmi and Narasimhan. thin-lay