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

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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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Contents

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

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

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

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

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

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

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

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

CHEMISTRY AND BIOCHEMISTRY I .

.

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

About 75% of the world’s total seafood supply is used for human consumption.7 million tons). 1990: 16 million tons. and sensory properties. Although land-based animals are today tailor made according to industry’s and consumer’s wishes in weight.4 million tons). including plants) [1]. 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). and another 8% into canned products. However. the United States (4. Chile (4.3% and 14. burden of pollutants.) of aquatic animals when captured by fishing techniques is—with few exceptions—completely unknown. Further.1 million tons). body composition. 40% is consumed as wet fish without any further technological processing or preservation.4%. aquaculture is dramatically growing (1960: 2 million tons. Mollusks (bivalves and cephalopods) are the third most important group both by quantity and by value at 22. respectively. Largehead hairtail (1. . Vietnam (1.6 million tons). and Norway (2. mostly Pangasius species).1 million tons). 1980: 7 million tons.2 million tons). They deteriorate at ambient temperature in a few days. The major aquaculture (excluding plants) producers (>1 million tons) in 2005 were China (32. etc. and Yellowfin tuna (1. Most fish was caught in the Pacific Ocean (Northeast and Southeast) followed by Northeast Atlantic Ocean. whereof the major part are cyprinids like carp). Alaska Pollock (2. 2000: 40 million tons. Peru (9. and the captured fish. 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). The most important primary product producing countries of marine and inland (freshwater) fisheries in 2005 were China (17. whereas crustaceans are fourth by quantity at 6. 91 million tons (64%) of the total supply.4 million tons).4 million tons). and Thailand (1. 8% is transformed into cured products. Another difference compared with land-living animals is the fact that the quality (size. Chilean jack mackerel (1. fish and other seafood are highly perishable products when stored without chilling.2 million tons). state of maturity.2% but second by value at 20. nutritional status. India (2. Russia (3. The world’s aquaculture provided 52 million tons (36%). about 20% is converted into deep frozen products. The total world seafood supply for 2007 amounted to 143 million tons.6 million tons). fish is the top group in aquaculture at 47. By major groupings.000–35. Thailand (2.3 million tons).4%. 1970: 4 million tons. 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. Indonesia (4. Aquatic plants that are popular in Southeast Asia are second in quantity at 23.1 million tons). Chub mackerel (2. Japan (4. India (3.1 million tons). infestation with parasites.5 million tons). Japanese anchovy (1.4 million tons). 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.8 million tons).3 million tons).2%. The top 10 species being caught in huge amounts in 2005 were Anchoveta (10. Further.4% by quantity.000 species.4 million tons.1 million tons).4 million tons. Skipjack tuna (2. 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.9 million tons). The fish group alone is represented by 25. The stagnation of captured fish is mainly due to fully exploited or partially overfished stocks. 25% is converted into fishmeal and other nonfood products. Blue whiting (2. Atlantic herring (2.3 million tons).4 ◾ Handbook of Seafood and Seafood Products Analysis number of species and variability. appearance.3 million tons). Indonesia (1.0 million tons).

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

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

These projects brought the scientists together in conferences. and in big fish (tuna. and practical work-ins and allowed on-site measurements. which is based on ATP breakdown products. 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. smaller when medium-sized animals are the target. After sampling is completed successfully. and total volatile basic nitrogen (TVB-N). In small specimens that are consumed totally. 1. The analytical methods used for seafood analyses can be divided into objective methods and sensory methods. sand. In addition. snails). 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. or tail end of fillet). shark). sprat. and comparative analyses with different instruments. Methods that are still in use are among others k-value. head end. other species or mud. trimethyl amine oxide.14] that form a very rich source of information about seafood analysis. preferably at −30°C) until analysis.6]. ammonia. and microbiological methods. tail muscle) must be taken due to intrinsic variations in fillet parts and after homogenization subsamples can be taken. the whole body may be sampled and analyzed (mussels. in medium-sized specimen. 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. When sampling is done onboard a vessel. While sampling is done. Another method that was developed recently is the two-dimensional gel electrophoresis (2DE). calibrations. it has to be made under strict hygienic conditions to avoid any microbial contamination. The main results of these projects have been published in books [2–4. and so forth. and analysis of biogenic amines as histamine or cadaverine. The objective methods are chemical/biochemical methods. is necessary. always the whole edible part (fillet. 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. a careful selection of individuals that have not been mechanically damaged by the catching technique.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. knives) or by protective clothes or gloves.Introduction ◾ 7 The number of individuals should be big when a small specimen has to be analyzed. precaution must be taken not to contaminate the sample by instruments used during manipulation (scissors. The first concerted action in this area was “Evaluation of fish freshness” from 1995 to 1997. it is recommended to store all samples (also solutions) in deep frozen conditions (<−18°C. and only a few samples are taken from big individuals. and the second concerted action was “Fish quality labeling and monitoring” (FQLM) from 1998 to 2000. physical methods. . determination of thiobarbituric acid and formaldehyde. When sampling for later microbiological analyses. workshops. analysis of trimethyl amine. dimethyl amine. 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).

nuclear magnetic resonance (magnetic resonance imaging (MRI). Sensory methods.. and objective results. cheap. low-field (LF) NMR. expensive. and oscillatory measurements). and can be used after a short training period by nonscientific educated personnel. tensile. and high-resolution NMR (HR-NMR)). and bacterial sensors. Further. a well-trained sensory panel in which the human senses are used as measuring instruments has been shown to give reliable. image analysis. 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. need trained personal. An instrumental method that is fast. can be used by untrained personal. Other methods that are rapid. Instrumental methods are fast. many of them have not graduated from research to seafood industrial application. are experienced in. comparatively cheap. and nonpolluting for the environment are. polymerase chain reaction (PCR). 1. analysis of electrical resistance or conductivity by Torrymeter. Two more systematic methods that involve some analytical methods are the hazard analysis critical control point (HACCP) and traceability. 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. therefore. 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. among others. there are still many problems left in the analysis of seafood and seafood-based products. determination of specific spoilage organisms (SSO). are time consuming. The reasons are the relatively complex and difficult handling of the instruments and the need of being applied and maintained by educated personnel. not harmful for the operator. puncture. reproducible. and are. and compression tests. 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. pH measurement. oligonucleotide probes. which are noninvasive and nondestructive techniques for the sample.8 ◾ Handbook of Seafood and Seafood Products Analysis Physical methods comprise microscopy. When using instrumental methods nowadays. Kramer test. and have used since many years [9]. therefore. and can be applied on many species and also on processed seafood products. antibody techniques. near-infrared spectroscopy (NIR). viscoelastic methods such as stress relaxation. with the same degree and quality of information obtained by sensory assessment. texture and texture profile analysis (e. and time domain spectroscopy (TDR) [7]. of utmost importance to introduce the newly developed analytical methods into the industry for better product and raw material analysis and quality assurance. always a combination of several methods is necessary to give sufficient information equal to sensory assessment [8]. creep. is still missing. RT Freshness grader. . Intellectron Fischtester VI. Outer inspection is done by the European Union quality-grading scheme and by the quality index method (QIM). however. UV and visible light spectroscopy.7 Analytical Problems Despite the great progress that has been made. Frequently used microbiological methods are total viable count (TVC).g. with the exception of sensory methods. Warner–Bratzler test. we have color measurement. whereas the Torry sensory scheme and the flavor profile analysis are performed on cooked samples. Sensory methods are often considered to be subjective methods. However. It is. electronic noses and electronic tongues [14]. which is usually not present in seafood industry.

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

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

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

43 to 5. and textural properties. for instance collagen has a nitrogen content of 18%. The ammonium sulfate is converted into ammonia. neutralization. nitrogen from other nitrogen-containing compounds such as free amino acids. distillation. It is also possible to determine the nitrogen content using elemental analysis (C/N analyzers) [4]. The Kjeldahl method determines the nitrogen content as ammonia. It is also possible to determine the amount of ammonia by different colorimetric methods [1]. The Kjeldahl method was first published in 1883 but has been extensively modified since then. Mariotti and coworkers discuss conversion factors in their critical review and conclude that even if a factor of 6. The method should also require minimal sample pretreatment to decrease analytical error and reduce costs. When the protein content is calculated based on determination of the nitrogen content. nucleotides. The Kjeldahl method has been automated and several instruments for automated analysis are available. Originally only sulfuric acid was used for digestion of the samples. This means that it should be possible to use the method on different types of foods. For fish. it is difficult to start using other and more correct factors [5]. Tables of conversion factors are given in several papers such as [1. and trapping of ammonia and titration steps. and the amount of protein is calculated by multiplication with a Kjeldahl factor. the water-holding capacity and the gelling properties which determine the textural attributes of the products are important quality parameters [3]. For products such as fish mince and surimi. . The disadvantage is that the method requires use of hazardous and toxic chemicals. During digestion the nitrogen in the sample is converted to ammonium sulfate. which is distilled and trapped in boric acid. For animal proteins the value 6. The method includes sample digestion. The factor can be calculated from the amino acid composition of the proteins. and urea will also contribute to the calculated protein content. which gives a factor of 5. fish proteins also have good functional properties such as water-holding capacity. and the amount of nitrogen is determined by titration [1].5]. This method is used as a reference method by many national and international organizations. 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 has been used for more than 75 years. emulsification. gelling.82 are given. The advantage of this method is that it gives accurate results for all types of samples.56 [1]. has been shown to be too high for animal proteins. both different types of raw materials and processed foods. Many proteins have protein contents that deviate from this.12 ◾ Handbook of Seafood and Seafood Products Analysis essential amino acids lysine and methionine. The Technicon AutoAnalyzer uses continuous flow analysis [1]. Retaining the functional properties through preservation and processing operations is therefore of great importance. assuming a nitrogen content of 16% in the proteins. It is important that the methods to analyze food proteins are robust [1]. but other chemicals such as potassium sulfate and mercury oxide are also used. 2. and that other components in the food such as lipids and pigments should not interfere with the analysis. values from 5. In addition to the high nutritional value. The Dumas method is quicker and cheaper and easier to perform and is therefore now considered on equal terms with the Kjeldahl method [1].25 is usually used.25.2 Total Content of Proteins The total content of proteins is usually determined by the Kjeldahl or the Dumas method. This factor is the amount of nitrogen that contains 1 g of nitrogen. amines.

but the instrumentation is expensive and the method requires calibration. This procedure involves hydrolyzation of the food sample using concentrated hydrochloric acid. O2. The myofibrillar proteins. Hultmann and Rustad [11] used a modification of the method by Anderson and Ravesi [12] and Licciardello and coworkers [13]. Quantitative amino acid analysis is one of the most reliable methods for quantification of food proteins. and connective proteins. It is easy to perform. and several other organizations [1]. and can be used online. . It has been successfully used to determine protein and water content of salmon fillets [6] as well as of surimi [7].5 M KCl and centrifuged as above. Today accurate combustion nitrogen analyzers are used. Four grams of muscle was homogenized for 20 s in 80 mL 50 mM phosphate buffer. The sample is put in a furnace (950°C–1050°C). myofibrillar. 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. 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). the NO2 is reduced to N2 and measured with a thermal conductivity meter [1]. 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]. and water are removed. The soluble protein was extracted in distilled water (low ionic strength). pH 7. and filled with pure oxygen. changes in solubility can be used to measure changes in protein structure caused by denaturation during storage and processing. American Oil Chemists’ Society (AOCS). After centrifugation. also called the salt-soluble proteins can be extracted in buffers with an ionic strength of >0. The advantages of this method are that it is rapid.Peptides and Proteins ◾ 13 The Dumas method was first published in 1831 and the first instruments used were not user friendly. nondestructive. SO2. The homogenates of these solutions were stirred constantly for 30 min at 2°C. Martinez-Alvarez and Gomez-Guillen [14] used a modification on the method of Stefansson and Hultin [15]. the supernatant was decanted and the volume made up to 100 mL—this was the water-soluble fraction. This was the salt-soluble fraction. A few examples of methods to extract proteins from fish muscle are given here. determination of the amino acid profile. After cooling of the gas mixture. The connective tissue proteins are often called the insoluble proteins and can be extracted using alkali or acid. 2. Near infrared spectroscopy can also be used to determine protein content. purged free of atmospheric gas. The principle of quantitative amino acids is described in Owusu-Apenten [1].3. based on differences in solubility [9. 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. The volume of the supernatant was made up to 100 mL. Kelleher and Hultin compared the use of NaCl.10]. sarcoplasmic.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. It is also described in Chapter 16. KCl. The precipitate was homogenized in 80 mL phosphate buffer with 0.3 Protein Solubility Classes Fish muscle proteins can be divided into three groups. Two grams of minced muscle was homogenized at low temperature for 1 min in 50 mL of distilled water. Fish muscle proteins are more sensitive and less stable than proteins from mammals. and calculation of the amount of different amino acids. the CO2. then centrifuged (6000 g) for 30 min at 3°C. and in 0. However.

the complexes react with the Folin-phenol reagent a mixture of phosphotungstic acid and phosphomolybdic acid in phenol. This was the acid-soluble collagen. Since all proteins absorb UV/visible light to varying degrees.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]. Measurement of UV absorption at 280 nm is a simple and popular method to determine protein concentration. Methods exist to correct for the influence of light scattering and nucleic acids/ nucleotides [19]. 2. The reactions are highly pH dependent. The extraction in NaOH was repeated five times and the supernatants were pooled for the analysis of alkali-soluble collagen content. one of the simplest methods is to determine absorbance in the far UV range. The method is not very sensitive. [18]. the concentration of the soluble proteins can be analyzed with a wide variety of methods. 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. Light scattering because of large particles or aggregates can also lead to errors. The product becomes reduced to molybdenum/tungsten blue and can be measured at 750 nm.4 Analysis of Soluble Proteins There are many indirect colorimetric methods to determine protein content. In addition. some of these methods reduce the speed and simplicity of the method [19].1 M NaOH and centrifuged. However mg quantities of protein are generally required. The purple complex is relatively stable and has an absorption maximum at 540–560 nm. which is a modification of the method described by Sato et al. Peterson have reviewed the Lowry method [21] and listed interfering substances. However. The precipitate was stirred with 0.5 M acetic acid for 2 days at room temperature and centrifuged as above. 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. Reducing agents and sucrose as well as several common buffers interfere with the Lowry method. and a few of them will be treated here. by absorbance at 205 nm or from knowledge of amino acid composition [19]. measuring concentrations between 1 and 10 mg/mL. Absorption at 280 nm is mainly due to tryptophan and tyrosine residues with smaller contributions from phenylalanine and the sulfur-containing amino acids. The review also discusses many of the modifications that . A standard curve is needed. Samples were homogenized in 0. giving upper tolerable limits for a long range of these as well as some methods for coping with the effect of these substances. The Biuret method is based on the formation of complexes between copper salts and peptide bonds under alkaline conditions. the method therefore has protein-to-protein variations. After extraction. The Lowry method [20] is based on a Biuret-type reaction between protein and copper(II) ions under alkaline conditions. The sensitivity can be increased by measuring absorbance at 310 nm or by increasing the time for the Biuret reaction.

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

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

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

and consumption [36]. W. Functional properties can be divided in several groups. 463.. hydrophobicity.. net charge. 25: 289–307. Ed. 1992. and E. and H. salt concentration). Journal of Food Science & Nutrition. and quaternary).. 1982. 2002. Food Research International. hydrophilicity. M. 11. Uddin. Journal of the Science of Food and Agriculture. formation of protein–lipid films. adhesiveness. Automatic methods for the simultaneous determination of carbon.P. Haard. whippability). Venugopal. 2008. Non-destructive determination of fat. E. amino acid composition and sequence. 1979. 10. Venugopal. T. 879–942. T. and biological values are sometimes included in the functional properties. cooking. L. 1968. It is usual to classify them according to mechanism of action into three main groups: (1) properties related with hydration (absorption of water/oil. 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.E. 7. and F. 3. 2006.K. Innovative uses of near-infrared spectroscopy in food processing. shape. Bock.A. 87: 31–41. moisture and protein in salmon fillets by use of near-infrared diff use spectroscopy.. 35: 431–435. References 1. 4. and P. It is therefore difficult to compare results from different laboratories. 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. D. Tome.J. R. . Food Protein Analysis: Quantitative Eff ects on Processing. N. 5. tertiary.F.. 1996. Owusu-Apenten. Hultmann. and gelation). 13. elasticity. Food Chemistry. Shahidi. Journal of Food Quality. in Food Chemistry. V. et al. New York: Marcel Dekker. Hultin. Analytical chemistry. thickening. 5: 215–234. and R. 2008. 2004. 51: 1173–1179. or interaction with other food constituents. Marcel Dekker: New York. Methods for processing and utilization of low cost fishes: A critical appraisal.25 and Jones’ factors. Relation between protein extractability and free fatty acid production in cod muscle aged in ice. distribution. F. Control of chemical composition and food quality attributes of cultured fish. hydrogen. (2) properties related with the protein structure and rheological characteristics (viscosity. temperature.M. Mirand. M. O. Foegeding.R. 2. molecular flexibility/rigidity in response to external environment (pH. aggregation. 48: 177–184. J. sulphur and sulphur alone in organic and inorganic materials.. Rustad. 6. and T. Licciardello. 73: R91–R98. Connelly. Iced storage of Atlantic salmon (Salmo salar)—Effects on endogenous enzymes and their impact on muscle proteins and texture. structures (secondary.. et al. J. storage. 1995.18 ◾ Handbook of Seafood and Seafood Products Analysis processing. 8. Converting nitrogen into protein—Beyond 6. Value added products from underutilised fish species. Isaksson. Journal of Fisheries Research Board Of Canada. p. Journal of Food Science. Anderson. V. 1995.. Journal of Food Science & Technology. 9. et al. solubility.L. 12. 1995. pp. sensory. Nondestructive determination of water and protein in surimi by near-infrared spectroscopy. Time–temperature tolerance and physical-chemical quality tests for frozen Red Hake. The book edited by Hall [38] gives a good overview of methods to determine protein functionality. Lanier.J. 32: 1–12.. Critical Reviews in Food Science and Nutrition. wettability).. Kirsten. 69: 95–100. Nutritional. Mariotti. Characteristics of edible muscle tissue. Fennema. 96: 491–495.C. 25: 2025–2069. Food Chemistry.K. and (3) properties related with the protein surface (emulsifying and foaming activities. Ravesi.

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

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

as well as with time and in response to environmental stimuli. It stands to reason. is a tool that can be of great value to the food scientist. based on liquid chromatography tandem mass spectrometry (LC–MS/MS). the proteome varies from tissue to tissue.14–18 gill. in the following sections. This chapter will therefore focus on 2DE.13 skeletal muscle. we present some issues and challenges related to sample matrices of particular interest to the seafood scientist. during. the interactions of proteins with one another or with other food components. or even thousands.12. foodstuffs are in large part made up of proteins. 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. and after processing or storage. quality involution within the product before.2. the construction of the food matrix. Proteome analysis allows us to examine the effects of environmental factors on larval global protein expression. 3.19 intestine. growth depression. Furthermore. and low survival rate. of proteins on a 2D polyacrylamide slab gel.1 Whole Larval Proteomes The production of good quality larvae is still a challenge in marine fish hatcheries. where the bulk of the food matrix is constructed from proteins.12 brain.2 Proteome Analysis by 2DE 2DE. Several environmental factors can interfere with the protein expression of larvae leading to poor larval quality like malformations.12 and rectal gland12 have been reported.1. Like other vertebrates. .1 Sample Matrix Considerations Unlike the genome.1 Introduction As with all living matter.9–11 heart.1) remains the workhorse of most proteomics work. This is especially true of fish and meat. giving valuable insight into the composition of the raw materials. is the simultaneous separation of hundreds.2 surface-enhanced laser desorption/ionization3 or protein arrays. the cornerstone of most proteomics research. that proteome analysis. also known as proteomics.4 hold great promise and are deserving of discussion in their own right. The method most commonly used was originally developed by Patrick O’Farrell and is described in his seminal and thorough 1975 paper5 and briefly outlined.2. fish possess a number of tissues amenable to 2DE-based proteome analysis. or with the human immune system after consumption.12 kidney. is regulated and brought about by proteins. along with some of the main improvements that have developed since. Selection of tissues for protein extraction is therefore an important issue that needs to be considered before a seafood proteomic study is embarked upon. the “classic” process of two-dimensional (2D) gel polyacrylamide electrophoresis (2DE) followed by protein identification via peptide mass fingerprinting of trypsin digests (Figure 3. 3. for example. simplicity.6–8 liver.22 ◾ Handbook of Seafood and Seafood Products Analysis 3. Proteomics. Studies on whole larvae. While high-throughput. In the following sections.12. largely because of its high resolution. then. 3. both on the cellular and tissue-wide levels. and mass accuracy. gel-free methods.

like the overwhelming presence of muscle and skin proteins. posttranslational modifications and redistribution of specific proteins within cells. 85% of the of the selected protein spots. These proteins may mask subtle changes in proteins expressed in other tissues or systems. there are several drawbacks when working with the whole larval proteome. yielding a peptide mass fingerprint. Only a few proteome analysis studies on fish larvae have been published. See text for further details.7 These studies provided protocols for the production of high-resolution 2D gels. such as the gastrointestinal tract or the central nervous system.6. where the majority of the highly abundant proteins were identified as muscle proteins.23 This is reflected in our studies on whole cod larval proteome.22 and zebrafish (Danio rerio).22 Three of these publications have focused on the whole larval proteomes in Atlantic cod (Gadus morhua)6. Peptide mass mapping using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry was performed only on the cod larval proteins. peptides can be dissociated into smaller fragment and small partial sequences obtained by MS/MS. The axial musculature is the largest tissue in larval fishes as it constitutes approximately 40% of their body mass.Proteomics ◾ 23 2D PAGE Trypsin digestion MS fingerprinting MS/MS sequencing Figure 3. allowing identification of ca.1 An overview over the “classic approach” in proteomics. cytoskeletal .22 Also.6. a protein extract (crude or fractionated) from the tissue of choice is subjected to 2D PAGE.21.6. Once a protein of interest has been identified. In many cases this is sufficient for identification purposes. but if needed. subjected to degradation by trypsin (or other suitable protease) and the resulting peptides analyzed by mass spectrometry.20 all important information for controlling factors influencing the aptitude to continue a normal development until adult stages. it is excised from the gel.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.7. Nevertheless. First.

it may also result in a loss of other proteins.2. Removal of those proteins may increase detection of other proteins present at low concentrations. An unfractionated 2DE map of the muscle proteome therefore tends to be dominated by comparatively few high-abundance protein spots. as many textural and other quality factors of muscle foods are related to proteolytic activity in the muscle tissue before. Various strategies have been presented for the removal of highly abundant proteins24 or enrichment of lowabundant proteins. such as the ubiquitin–proteasome or the lysosome systems. such as actin and tubulin. fish skeletal muscle is the main component. are of keen interest. No amplification method analogous to PCR exists for proteins. However. preventing identification of holistic alterations in the analyzed proteomes. are particularly abundant in the skeletal muscle proteome. and simply increasing the amount of sample is usually not an option.45. such as those associated with individual organelles or cell compartments28 or by protein biochemical methods such as affi nity chromatography. has profound implications for quality and processability of the fish flesh.42.43 The 20S proteasome has been found to have a role in regulating the efficiency with which rainbow trout (Oncorhynchus mykiss) deposit protein. A myriad of methods suitable for subsequent 2DE exist for fractionating the proteome into defined subproteomes.30 preparative isoelectrofocusing31 or solubility in the presence of various detergents32 or chaotropes33 have been described. are suitable for rigorous investigation using proteomic methods. whereby proteins are targeted for destruction by the proteasome by covalent . function. in which protein deposition is regulated. protein turnover is a major regulatory engine of cellular structure. allowing a larger sample of the remaining proteins to be analyzed. is fractionation of the protein sample in order to weed out the high-abundance proteins. Cellular protein turnover involves at least two major systems: the lysosomal system and the ubiquitin–proteasome system.2 Muscle Proteomes In most seafood products.3 The Degradome The degradome may be a subproteome of particular interest to the food scientist. In addition to having a hand in controlling autolysis determinants. 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. then. as it will give rise to overloading artifacts in the gels.26 3. during and after processing.2.46 An exploitable property of proteasome-mediated protein degradation is the phenomenon of polyubiquitination.24 ◾ Handbook of Seafood and Seafood Products Analysis proteins were prominent among the identified proteins.1.34–41 3.5 The remaining option.25. Fractionation methods for a variety of sample matrices have been reviewed recently.1. particularly in muscle tissue. Structural proteins. Swamping of low-abundance spots by highly abundant ones may not be a problem for applications relating specifically to structural proteins. The fish muscle proteome is therefore likely to be of comparatively high interest to the seafood scientist. and biochemistry. Proteomic analysis on lysosomes has been successfully performed in mammalian (human) systems.44 It seems likely that the manner. rendering analysis of low-abundance proteins difficult or impossible. but for other applications low-abundance proteins. Protein turnover systems. For example.29. which include most regulatory proteins and many important metabolic enzymes.

silver stains. such as Coomassie blue. 4% (w/v) CHAPS [3-(3-chloramidopropyl)dimethylamino-1-propanesulfonate].47 By targeting these ubiquitin-labeled proteins.51 the procedure remains essentially as outlined earlier. We have found direct extraction into the gel reswelling buffer (7 M urea. Activity of matrix metalloproteases is regulated via a complex network of specific proteases.e. These strips consist of a dried IPG-containing polyacrylamide gel on a plastic backing. up-to-date protocols.3% (w/v) DTT [dithiothreitol]. which proteins are being degraded by the proteasome at a given time or under given conditions. 3. In the following sections. Cleanup of samples using commercial 2D sample cleanup kits may be beneficial for some sample types. most notably the introduction of immobilized pH gradients (IPGs) for IEF.43. Although a number of refinements have been made to 2DE since O’Farrell’s paper. may be less directly amenable to proteomic study.2. 0. yielding a two-dimensional map (Figure 3. or fluorescent dyes. such as that of the matrix metalloproteases. may be more conveniently carried out using transcriptomic methods.22 and Arctic charr (Salvelinus alpinus) liver.. 3. Ready-made IPG strips are currently available in a variety of linear and . it is possible to observe the ubiquitin–proteasome “degradome.” i.Proteomics ◾ 25 binding to multiple copies of ubiquitin.2. This separates the proteins according to their molecular charge.2 Some proteolysis systems.2.2.5% 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. where an electric field is applied to a tube gel on which the protein sample and carrier ampholytes have been deposited.1 Sample Extraction and Cleanup For most applications.2. sample treatment prior to electrophoresis should be minimal in order to minimize in-sample proteolysis and other sources of experimental artifacts.52–57 3. 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). which is most conveniently performed using commercial dry IPG gel strips. 2 M thiourea.2) rather than the familiar banding pattern observed in one-dimensional (1D) SDS-PAGE. and how they vary with environmental or dietary variables. 0. Gygi and coworkers have developed methods to study the ubiquitin–proteasome degradome in the yeast Saccharomyces cerevisiae using multidimensional LC–MS/MS. The map can be visualized and individual proteins quantified by radiolabeling or by using any of a host of protein dyes and stains.48–50 Monitoring of the expression levels of these regulatory enzymes. a general protocol is outlined briefly with some notes of special relevance to the seafood scientist.58 Thorough homogenization is essential to ensure complete and reproducible extraction of the proteome. For more detailed. the reader is referred to any of a number of excellent reviews and laboratory manuals.2 Basic 2DE Methods Overview O’Farrell’s original 2DE method first applies a process called isoelectric focusing (IEF).

2.26 ◾ Handbook of Seafood and Seafood Products Analysis MW (kDa) 60 42 30 22 17 4 5 pl 6 7 Figure 3. Reswelling is normally performed overnight at 4°C. but also on . the starting voltage is about 150 V. usually totaling about 10. morhua) larval proteins with pI between 4 and 7 and molecular mass about 10–100 kDa. 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. 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.. although this will depend on the IPG gradient and the length of the strip. sigmoidal pH ranges. A recipe for a typical reswelling buffer is presented in Section 3.000–30. 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. pH 3–10) are commonly used for whole-proteome analysis of tissue samples. extraction directly into the reswelling buffer is recommended. Application of a low voltage current may speed up the reswelling process.000 Vh. The appropriate IEF protocol will depend not only on the sample and IPG strip.2 A 2DE protein map of whole Atlantic cod (G. Broad-range linear strips (e.500 V.2. which is then increased stepwise to about 3.g. Typically.1. The proteins are separated according to their pI in the horizontal dimension and according to their mass in the vertical dimension. If the protein sample is to be applied during the reswelling process. the dried gel needs to be reswelled to its original volume. Before electrophoresis. IEF is normally performed for several hours at high voltage and low current. Isoelectrofocusing was by pH 4–7 IPG strip and the second dimension was in a 12% polyacrylamide slab gel. Optimal conditions for reswelling are normally provided by the IPG strip manufacturer.

56 reviewed IEF for 2DE applications.3 Orientation and placement of an isoelectrofocused IPG strip onto the top of the second-dimension gel. 3.60 the Laemmli method. it is applied to the top edge of an SDS-PAGE slab gel (Figure 3.2. and that the strip is pressed gently onto the SDS gel. but for most applications gradient gels or gels of about 10% or 12% polyacrylamide are appropriate. Care must be taken that the (+) end of the strip is on the same side of all slab gels. While some reviewers recommend alternative buffer systems.2. 6 M urea.3) and cemented in place using a molten agarose solution. remains the most popular one.4 Second-Dimension Electrophoresis Once the gel strip has been equilibrated.8. Görg et al. A second equilibration step in the presence of 2. 30% glycerol. 0. thus reducing vertical streaking. The manufacturer’s instructions should be followed. Optimal pore size depends on the size of the target proteins. Figure 3.61 using glycine as the trailing ion and the same buffer (25 mM Tris. Ready-made gels suitable for analytical 2DE are available commercially. it needs to be equilibrated for 30–45 min in a buffer-containing SDS and a reducing agent such as DTT. During the equilibration step. avoiding trapping air bubbles.1% SDS) at both electrodes. A typical equilibrationbuffer recipe is as follows: 50 mM Tris–HCl at pH 8. taking care to put the pressure on the IPG strip’s plastic backing rather than the gel itself. This is best performed using a dentist’s tool or other appropriate implement. 2% SDS. trace amount of bromophenol blue.2. 1% DTT. 192 mM glycine.3 Equilibration Before the isoelectrofocused gel strip can be applied to the second-dimension slab gel. This will alkylate thiol groups and prevent their reoxidation during electrophoresis. A tracking dye for the second electrophoresis step is also normally added at this point.5% iodoacetamide and without DTT (otherwise identical buffer) may be required for some applications. The gel is run at a constant current of 25 mA until the bromophenol blue dye front has reached the bottom of the gel.Proteomics ◾ 27 the equipment used. the SDS–polypeptide complex that affords protein-size-based separation will form and the reducing agent will preserve the reduced state of the proteins. .59 3.2. that the gel side of the IPG strip faces the notched side of the glass plate.

followed by incubation for 1 h in 17% ammonium sulfate/34% methanol/2% ortho-phosphoric acid.6 Analysis Although commercial 2DE image analysis software. such as Student’s t-test.65–67 3. or Progenesis (Nonlinear Dynamics). such as the SYPRO or Cy series of dyes. Multiple staining with dyes fluorescing at different wavelengths offers the possibility of differential display allowing more than one proteome to be compared on the same gel. These include radiolabeling. A typical staining procedure includes fi xing the gel for several hours in 50% ethanol/2% ortho-phosphoric acid. A great many alternative visualization methods are available. Also.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. . spot matching between gels tends to be time-consuming and has proved difficult to automate. remains the bottleneck of 2DE-based proteome analysis and still requires a substantial amount of subjective input by the investigator.28 ◾ Handbook of Seafood and Seafood Products Analysis 3.68 where peptides are suspended in a matrix of small. and staining with fluorescent dyes. PDQuest (BioRad). 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. the spot of interest is excised from the gel.3 Protein Identification by Peptide Mass Fingerprinting Identification of proteins on 2DE gels is most commonly achieved via mass spectrometry of trypsin digests. These multiple sources of variation has led some investigators63–65 to cast doubt on the suitability of univariate tests. There are.1% Coomassie Blue G-250/17% ammonium sulfate/34% methanol/2% ortho-phosphoric acid. followed by staining for several days in 0.2. Patton published a detailed review of visualization techniques for proteomics. and the resulting peptide mixture is analyzed by mass spectrometry. and followed by destaining for several hours in water. however.2. and individual protein quantification.63 In particular. such as in difference gel electrophoresis (DIGE).2. and therefore individual variation is a major concern and needs to be accounted for in any statistical treatment of the data. Pooling samples may also be an option and this depends on the type of experiment. including protein spot definition. such as with [35S] methionine. UV-absorbing molecules (such as 2.2.2. such as protein load variability due to varying IPG strip reswelling or protein transfer from strip to slab gel. matching. has improved by leaps and bounds in recent years.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. such as ImageMaster (Amersham). commonly used to assess the significance of observed protein expression differences. commercially available colloidal Coomassie staining kits that do not require fi xation or destaining. organic. Briefly. gene expression in several tissues varies considerably among the individuals of the same species.64 These difficulties arise from several sources of variation among individual gels. The most popular mass spectrometry method is MALDI-TOF mass spectrometry. digested with trypsin (or another suitable protease).62 3. Multivariate analysis has been successfully used by several investigators in recent years. followed by several 30 min washing steps in water. analysis of the 2DE gel image.

71 100 90 80  Intensity 1061.69 1659.4 A trypsin digest mass spectrometry fingerprint of an Atlantic cod larval protein spot.61 1272.54 50 40 1287. To circumvent this problem. The resulting spectrum of peptide masses (Figure 3.10 and Vilhelmsson et al. to obtain a tentative identity.90 1822. many with a web-based open-access interface. excluded from the analysis. In those cases where both the protein and nucleotide databases yielded results.org/tools) contains links to most of the available software for protein identification and several other tools.98 1886. however.96 60 870.2 2798.Proteomics ◾ 29 842.8 1652. The ExPASy Tools web site (http://www.25 Figure 3.43 .60 Peptides identified as those derived from Atlantic cod β-tubulin Trypsin autolysis peaks 1159.36 2564.6 Mass (m/z) 2108. As can be seen in Table 3.07 1131.54 70 1960.4 2212. 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. correlated activity measurements.4) is then used for protein identification by searching against expected peptide masses calculated from data in protein sequence databases. Martin et al. The solid markers indicate the peaks that were found to correspond to expected b-2 tubulin peptides.0 1229. such as the National Centre for Biotechnology Information (NCBI) nonredundant protein sequences database.1.86 1028.56 1974.00 1196. this problem is surprisingly acute for species of commercial importance. it is possible to take advantage of the available nucleotide sequences. using the appropriate software. which in many cases is more extensive than the protein sequences available. or transcript abundance).70 1697.801616.35 1621. The open markers indicate mass peaks corresponding to trypsin self-digestion products and were. 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. 2506. In their work on the rainbow trout liver proteome. 100% agreement was observed between the two methods. It is important to realize. identified as b-2 tubulin.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.expasy. therefore. Several programs are available.50 20 10 0 741. How useful this method is will depend on the length and quality of the available nucleotide sequences.63 1258.51 1040.50 30 856.06 1575.

533 1.287 8. and wolffish) Pleuronectiformes (flatfishes. incl.585 1.122 268 26. sea bream.063 3.933 3.680 1.489 130. tuna. scallops.208 2.407 84.008 Mollusca (Mollusks) Bivalvia (mussels. incl. incl. sea bass.284 2. saithe.782.592 735 179 585 768.845 999.) Gastropoda (incl.30 ◾ Handbook of Seafood and Seafood Products Analysis Table 3.203 467.344 5.381 45.656 2.442 237 2. 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.210 Chondrichthyes (Cartilagenous Fishes) Carcharhiniformes (ground sharks and dogfishes) Lamniformes (mackrel sharks) Rajiformes (skates and rays) 3. whelks and abalone) Cephalopoda (squid and octopi) 32.845 10.046.380 898.762 726.751 Crustacea (Crustaceans) Caridea (shrimps.798 81.158 20.) Astacidea (lobsters and crayfishes) Brachyura (short-tailed crabs) 21. haddock. monkfish) Perciformes (perch-likes. etc. halibut.626 138 16.245 2.864 47. and plaice) Zeiformes (dories) Scorpaeniformes (scorpionfishes. cod. incl.138 36.871 32. incl. mackrel.557 .1 Families of Some Commercially Important Seafood Species and the Availability of Protein and Nucleotide Sequence Data as of March 27. sole.353 287 170. etc. redfish and lumpfishes) 185.006 121. and pollock) Lophiiformes (anglerfishes.896 3.424 2.237 2. turbot.086 2.007 911 303 18.

each peptide mass can potentially represent any of a large number of possible amino acid sequence combinations. Furthermore.71. Correlating this spectrum with the candidate peptides identified in the first round narrows down the number of candidates. for example. cytoskeleton.72 Today. Until recently. which.89 A brief discussion of a few emerging areas within fish and seafood proteomics is given as follows.88.3. enhances the specificity of the method even further. yielding a second layer of information.. if rather more time-consuming. larva. the identified proteins consisted mainly of proteins located in the cytosol. or redistribution of specific proteins within cells.76 Nyman. the method of choice is tandem mass spectrometry (MS/MS)..82 Lin et al. improved reproducibility and resolving power of electrophoretic separation techniques.86 and soybean protein bodies. In both these studies. have gained considerable momentum.77 Damodaran et al. proteomic investigations on fish and seafood products.79 Rappsilber et al.94 Proteome analysis provides valuable information on the variations that occur within the proteome of organisms. and behavior of the fish.3 Applications of 2DE in Seafood Analysis The two-dimensional electrophoresis has been in use within food science for at least two decades. Proteome analyses in developing organisms have shown that many . by Yates. and adult) during their life span that coincide with changes in the morphology. and nucleus.90–92 The morphological and physiological changes that occur during these developmental stages are characterized by differential cellular and organelle functions.23. and vastly superior protein spot identification techniques. several short stretches of amino acid sequence will be obtained for each peptide. reflect a response to biological perturbations or external stimuli9–11.20 To date few studies on fish development exist in which proteome analysis techniques have been applied.87 With the lower cost. These variations may. The larger the mass (and longer the sequence).93 This is reflected in the variations of global protein expression and posttranslational modifications of the proteins that may cause alterations in protein function.83 and Delahunty and Yates.95 resulting in different expression of proteins.73–75 Mass spectrometry methods in proteomics have been reviewed. as well as in aquaculture.80 Mo and Karger. 3. posttranslational modifications. the higher the number of possible combinations.84 3.85 wheat flour baking quality factors. physiology. Early studies focused on relatively small.Proteomics ◾ 31 A more direct. this was accomplished by N-terminal or internal (after proteolysis) sequencing by the Edman degradation of eluted or electroblotted protein spots. when combined with the peptide and fragment masses obtained. clearly defined subproteomes and included such applications as the characterization of bovine caseins. 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.. In the peptide mass fingerprinting discussed earlier. fish physiology. 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..81 Gygi and Aebersold.1 Development Fishes go through different developmental stages (embryo. way of obtaining protein identities is by direct sequence comparison. for example. and development.78 Thiede et al.

many of which are correlated with specific textural properties in seafood products. several commercially important fish muscle processing techniques. a large number of yolk proteins remained prominently present in the embryonic protein profiles. It is also worth noting that protein isoforms other than proteolytic ones. where differences are expected to occur in the number.2 Quality Involution Degradation of proteins during chilled storage.27 published a method to efficiently remove the yolk from large batches of embryos without losing cellular proteins.99.102 common sole (Solea solea). Link et al. be distinguished on 2DE gels.99–107 In this context.2. Furthermore. molecular mass. such as curing. fermentation.94. developmental stage specific muscle protein isoforms have gained a special attention. the embryos fall out of their chorions facilitating the removal of the yolk. although degradation of myofibrillar proteins by calpains and cathepsins112.109. This fact further indicates the importance of the proteome approach to understand cellular mechanisms that underlie fish development. 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. 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. These interfere with any proteomic application that intends to target the cells of the embryo proper. are among persistent quality problems in the seafood industry and have deleterious effects on fish flesh texture. The major obstacle on the use of proteomics in embryonic fish has been the high proportion of yolk proteins.99–107 The developmental changes in the composition of muscle protein isoforms have been tracked by proteome analysis in African catfish (Heterobranchus longifilis). 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. Thus.21 and dorada (Brycon moorei).21. and their oxidation during frozen storage.21.113 .8.1 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. The success in the removal of yolk proteins by Link et al. whether they be encoded in structural genes or brought about by posttranslational modification.110 Problems of this kind.32 ◾ Handbook of Seafood and Seafood Products Analysis of the identified proteins have multiple isoforms96 that reflect either different gene products97 or posttranslationally modified forms of these proteins. In a recent study on the proteome of embryonic zebrafish. specific isoforms of myofibrillar proteins.98 Different isoforms generated by posttranslational modifications are largely overlooked by studies based on RNA expression.3. By dechorionation.108 3. Studies on various proteins have shown that during fish development sequential synthesis of different isoforms appear successively. can be observed using 2DE or other proteomic methods.88. therefore.3.8. For example.7 Despite this undertaking.111 3.101 These studies demonstrated that the muscle shows the usual sequential synthesis of protein isoforms in the course of development. and pI of the protein present in a tissue.27 is probably due to dechorionation prior to the deyolking of the embryos. usually have different molecular weight or pI and can. and production of surimi and conserves occur under conditions conducive to endogenous proteolysis. are well suited for investigation using 2DE-based proteomics.

2 Aquaculture and Antemortem Effects on Quality and Processability It is well known that an organism’s phenotype.118 Several 2DE studies have been performed on postmortem changes in seafood flesh14–17.44 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. is determined by environmental as well as genetic factors. In the context of this chapter. affect the involution of quality characteristics in the fish product. various quality characteristics of fillet and body were measured124. fatty acid breakdown. where individual physiological characteristics. that these quality changes are species dependent116. With the ever increasing resolving power of molecular techniques. the ubiquitin–proteasome pathway has been shown to be downregulated in response to starvation129 and have a role in regulating protein deposition efficiency.128 In rainbow trout. In a recent study on the feasibility of substituting fish meal in rainbow trout diets with protein from plant sources.120 and have demonstrated the importance and complexity of proteolysis and oxidative changes in seafood proteins during storage and processing. appear to display seasonal variations.126 in fish fed with the experimental diets. Whatever may be the mechanism. is thought to be responsible for a large fraction of cellular proteolysis. it is clear. Olsson et al. the interplay between these physiological parameters and environmental and dietary variables needs to be understood in detail.117. 67.115 are thought to be among the main culprits.125 and the liver proteome was analyzed9.119.121 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. such as pathways involved in cellular protein degradation. the effects on the proteasome are particularly noteworthy. The proteasome is a multisubunit enzyme complex that catalyzes proteolysis via the ATP-dependent ubiquitin–proteasome pathway which. 2D-immunoblots and LC–MS/MS to study changes in protein oxidation during frozen storage of rainbow trout. Indeed. such as proteomics. Martinez et al. We are aware of two recent studies where Atlantic cod muscle proteomes have been compared between farmed and wild fish.Proteomics ◾ 33 and degradation of the extracellular matrix by the matrix metalloproteases and matrix serine proteases114. and the amount and composition of free amino acids in the fish flesh.1 ..17 used 2DE. Furthermore.117 and. They found fish muscle proteins to be differentially carbonylated during frozen storage and were able to identify several carbonylated proteins using LC–MS/MS. such as those governing gaping tendency.10.123 found these to comprise several members of the glycolytic and Krebs cycle pathways. are optimized. Huss noted in his review122 that product quality differences within the same fish species can depend on feeding and rearing conditions. and NADPH metabolism. as opposed to wild fish catching. Kjærsgård et al. flesh softening during storage.3. differences that can affect postmortem biochemical processes in the product which. The diet was found to have a marked effect on product texture. this is fast becoming feasible.111. furthermore. in mammals.15. therefore raises the tantalizing prospect of managing quality characteristics of the fish flesh antemortem. 3. the proteome analysis identified a number of metabolic pathways sensitive to plant protein substitution in rainbow trout feed. To achieve that goal. For example.123 Both studies indicated that several proteins are differentially expressed in farmed versus wild cod. in turn. including quality characteristics.112. The practice of rearing fish in aquaculture.2. etc.127.

34 ◾ Handbook of Seafood and Seafood Products Analysis 3. and postmortem treatment. Penaeus monodon. blotted the 2D gel onto a PVDF membrane.18. studying the cause of shrimp allergy in humans.5% of young adults are allergic to shrimp. the presence of stress-factors or contamination levels at the place of breeding.14 Unlike the genome. Martinez et al. 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.140. The allergen was identified as a protein with close similarity to arginine kinase. For example. demonstrating that it had arginine kinase . Proteome analysis can therefore potentially yield more information than genomic methods.144 Martinez and Jakobsen Friis concluded that the identification of not only the species present. performed a 2DE on crude protein extracts from the tiger prawn. 1D electrophoretic techniques were developed to identify the raw flesh of various species. During the 1960s. Mytilus galloprovincialis. about 0.3.147 at National Taiwan University.3. From early on. the proteome varies from tissue to tissue and with environmental conditions. trossulus could be distinguished from the other two species on foot extract 2D gels by a difference in a tropomyosin spot.134 recently reviewed proteomic and other methods for species authentication in foodstuffs. proteomic methods have been recognized as a potential way of fish species identification. These authors. including structural proteins such as tropomyosin. and Mytilus trossulus. and probed the membranes with serum from confirmed shrimp allergic patients. possibly indicating freshness and tissue information in addition to species. studying three species of European mussels: Mytilus edulis. 3. as different fish species have different market values.143 Lopez and coworkers. as well as being relevant from a public health standpoint. 2DE-based methods have been developed to distinguish various closely related species. this makes the issue of species authentication an area of increasing economic importance.145 Seafood allergies are caused by an immunoglobulin E-mediated response to particular proteins. They found the difference to be due to a single T to D amino acid substitution.138. Allergic reactions to seafood affect a significant part of the population. A final proof was obtained by purifying the protein. such as the gadoids or several flat fishes.139 These early efforts were reviewed in 1980. particularly for addressing questions on the health status of the fish in question.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.4 Allergen Identification Allergenic potential is food safety issue of particular concern to the seafood producer.141 More recently. proteomics-based species identification methods are likely to develop rapidly and find commercial uses within this field. found that M. The allergens were then identified by MALDITOF MS of tryptic digests. 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.135–137 which was soon followed by methods to identify species in processed or cooked products.3 Species Authentication Processed fish products are increasingly common in the market and.143 Indeed.142. 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. The identity was further corroborated by cloning and sequencing the relevant cDNA. 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.

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............................ The rise of * Equal contributors..........................................................* Delphine Galiana-Arnoux.............52 References ..... 43 ........................ Jules Verne..1 Introduction The development of high-throughput DNA sequencing methods has opened the era of genomics...............................6 Concluding Remarks .............................. some nearly destitute of scales.... the flesh of which is unrivalled...............4 Genomics......... all fish that would be of use to us.......................................................................1 Introduction . with bony jaws....................... but of exquisite flavour............................................................................51 Acknowledgments .. 43 4..................... which has revolutionized biology.......................... and Jean-Nicolas Volff Contents 4. as good as bonitos......... and the Management of Biodiversity .......47 4.........Chapter 4 Seafood Genomics Astrid Böhne.................................45 4.. others........ 49 4........................5 Genomics and Aquaculture ......... and biotechnology over the last decade..............2 Genetics and Genomics................................................................................................ and yellow-tinged gills.................52 There the nets brought up beautiful specimens of fish: Some with azure fins and tails like gold..............................* Frédéric Brunet............... Fisheries......... Twenty Thousand Leagues under the Sea 4........* Christina Schultheis.. 44 4.3 Genomic Resources and Genome Projects for Aquatic Species .... medicine.............................................

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

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

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

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

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

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

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

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

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

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Wheeler.. The complete genome of an individual by massively parallel DNA sequencing. Growth of domesticated transgenic fish. 116. 781. Mol. Nature. W. Y..56 ◾ Handbook of Seafood and Seafood Products Analysis 92. Microarray analysis of gene expression in the blue catfish liver reveals early activation of the MHC class I pathway after infection with Edwardsiella ictaluri. Kovács.). E. J. M. Lee. 32.. et al. 319. 559.. Two unlinked loci controlling the sex of blue tilapia (Oreochromis aureus)..A. et al.. and Hänni.. Br. 107. Endocrinol. 343.).. Insights into the immune transcriptome of the shrimp Litopenaeus vannamei: Tissue-specific expression profiles and transcriptomic responses to immune challenge. R. Ezaz. 359. Comp.. 156. G. 9. Robalino. D. Marine natural products. A. 211.D.. Hulata. and Kocher. 2008. Androgen-induced masculinization in rainbow trout results in a marked dysregulation of early gonadal gene expression profiles. 435. Blunt. 52.. 2008. Food Nutr. E. 95. 2002. Marine biotechnology for production of food ingredients. 110. Oryzias latipes. 109. 267. Sex determination and sex differentiation in fish: An overview of genetic. Nanda. 191. Acad. 2007. et al.S. Res.H. 2007. Physiol. 15. 11778.. 543. Devlin.. Maudet. M.. 94. 2004. 2004. and environmental influences. 97. et al. Sex. Proc. 25. et al. 98. Genet. et al. Nature. 2000. et al. Physiol. E.. R. 182. Roberge. U. et al. Genomics.. 237. 2004. Biotechnol. et al.. et al. 2005. F. Rasmussen. Sci. J. 409.A. A duplicated copy of DMRT1 in the sex-determining region of the Y chromosome of the medaka. 2. N. Expression profiling of candidate genes during ovary-to-testis trans-differentiation in rainbow trout masculinized by androgens. 108. Kirchner. 6. Heredity. Tao. Young. 100. 369. 112. 2007. Genomics. et al. 99..D. Prod. Sarropoulou. Food and forensic molecular identification: Update and challenges. 115. R. M. 2581. Devlin. 96.S. 2001. S. 2003... physiological. 2006. 102. S. 553. Dev. Nutr.. 2005. 110. Effects of short-term growth hormone treatment on liver and muscle transcriptomes in rainbow trout (Oncorhynchus mykiss). et al. 2008. Isolation and physical mapping of sex-linked AFLP markers in nile tilapia (Oreochromis niloticus L. B. Nat. Gene expression profi ling of gilthead sea bream during early development and detection of stress-related genes by the application of cDNA microarray technology. Anim. Immunol. 99. et al. Physiol. 113. DMY is a Y-specific DM-domain gene required for male development in the medaka fish. Nagahama.] 104. Baron.. Associations between single nucleotide polymorphisms in candidate genes and growth rate in Arctic charr (Salvelinus alpinus L.T. Trends Biotechnol. B.. 43..... . Nature. C. 103. Peatman. 2008. 452. The identification of genes from the oyster Crassostrea gigas that are differentially expressed in progeny exhibiting opposed susceptibility to summer mortality. 23. C. 106. 2002.. D. S. 44. 2003.. Panserat. 872. Coordinated down-regulation of the antigen processing machinery in the gills of amoebic gill disease-affected Atlantic salmon (Salmo salar L. Salmonid microarrays identify intestinal genes that reliably monitor P deficiency in rainbow trout aquaculture. Male-specific DNA markers from African catfish (Clarias gariepinus). Matsuda. BMC Genomics.J. Mol. 417. 208...G. Comparative genome analysis of the primary sex-determining locus in salmonid fishes. 2002. 93.. 45. Genomics. Woram. et al. Rapid parallel evolutionary changes of gene transcription profiles in farmed Atlantic salmon. et al. 60. 2008.. Genome Res. and Morrissey. D. 29. I.. 91. Gahr. et al.. 13. Ecol. Gene. 8. Mol. [Epub ahead of print. Baron. Genetica. 272.Y. 35. Aquaculture. Immunol. Genetics of sex determination in tilapiine species. 380.. 101.).H. A. et al.W. Adv. 357. C. et al. Huvet. 114. Rep. Gen.T. Teletchea. 105.. Hepatic gene expression profiles in juvenile rainbow trout (Oncorhynchus mykiss) fed fishmeal or fish oil-free diets. 2008..A. R. Mar.. and Boulding. T. 2008. et al. Natl. 111. 23. et al... 92. 2008. 45.A. Heredity.. J. Cnaani. 2007.

........................................................... or eviscerated fish) or canned fish..................................... beheaded.1 Introduction Bacterial growth is the main factor limiting fish commercial life by producing its alteration and unpleasant flavor................... the adenosine triphosphate (ATP) regeneration that occurs in vivo stops and ATP is degraded until 57 ...................1 Introduction .................................57 5.......................1 31Phosphorous-Nuclear Magnetic Resonance Spectroscopy ...........59 5...... Aleida S............... Nevertheless........................................................................2. The first autolytic process taking place in fish affects carbohydrates and nucleotides.... 60 5.....................................3 Analysis of ATP-Related Compounds ..3................ and Fidel Toldrá Contents 5..............................2....... Thus.......3........... Leticia Mora.......................... objective methods for freshness determination are required and the determination of the biochemical changes occurring in early postmortem in fish constitute a helpful tool.............4 Enzymatic Analysis............ After death..............................3...3 Chromatography..............1 Extraction of Nucleotides and Nucleosides .....3....... the autolytic process derived from tissue enzymatic activity and lipid oxidations also contributes to fish maturation and subsequent spoilage.................... Concepción Aristoy.............59 5.....61 5.....3......2 Chemical Structure of Main Seafood Nucleosides and Nucleotides ....................2 Capillary Electrophoresis ...........................3..............................2.............................................2 Nucleotides and Nucleosides Determination ...........Chapter 5 Nucleotides and Nucleosides M...........61 5............ Sensory methods to evaluate fish quality are subjective and difficult to use in the evaluation of processed (fillets...................61 5.... Hernández-Cázares.......61 5.65 5.... 64 References .....2.........

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. and either of the two may be used as freshness indicators. which is oxidized to xanthine (Xa) and uric acid in the presence of xanthine oxidase (XO) enzyme. and Hx in the flesh of some species of fish during chilled storage. .1 Degradation of ATP in postmortem fish muscle. Inosine is transformed to hypoxanthine (Hx) by the action of the enzyme nucleoside phosphorylase (NP).1 The following IMP dephosphorylation to obtain inosine is mainly autolytic and occurs at a slower rate during the first stage of cold storage. the use of a single compound as freshness indicator is not always advisable.4 However. As a result of endogenous enzymes action. (2006) published a review of the concentration of IMP.3 In all cases. ATP molecule is rapidly degraded to adenosine monophosphate (AMP) and afterward to inosine monophosphate (IMP). Ino and Hx concentrations increased during storage. IMP degradation to inosine (Ino) and its disappearance have been correlated with lack of freshness in some fish species.2 Howgate et al.1. Ino. whereas AMP remains major in crustaceans. This enzyme is mainly generated in muscle from biochemical processes of microorganisms. This process involves a series of reactions commonly represented according to the sequence shown in Figure 5. although it might be accelerated by the action of different bacteria. IMP is the main nucleotide present in fish species. which is accumulated in postharvest fish. 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 5.58 ◾ Handbook of Seafood and Seafood Products Analysis rigor mortis is reached.

This is the main reason for the use of indexes with more than one compound from the ATP-degradation chain.13. ATP-chain degradation occurs very fast. and Hx expressed as percentage.10. Ino. In this way. also. Nucleotides are o-phosphoric acid esters of the nucleosides. a revised K value.14 Measurement of ATP-related compounds is also useful for the quality control of retorted fishes. AMP or adenylic acid is derived from the adenosine in which a phosphate group is attached at the 5-ribose carbon. making K value inadequate as a freshness indicator. 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.Nucleotides and Nucleosides ◾ 59 nucleotide degradation such as the type of spoilage bacteria and mechanical handling of fish. generally within 1 day of storage in ice after death in all fish species. respectively. often designed K ′ value or Ki index.5 and. for several species.11 and. This is achieved by immediately freezing the excised muscle under liquid nitrogen to stop all enzymatic reactions.17 5. a high accumulation of Ino occurs during ATP degradation. and thus.18 After this. Nucleosides are glycosylamines that are formed when a nucleobase (purine or pyrimidine base) attaches to a ribose or deoxyribose ring. In order to achieve this rapid freezing.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.6 This value has been used as one of the freshness indexes to evaluate the quality change of postharvest fish. 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. For this reason. and it is important to stop this reaction drastically at the sampling time. K value is defined as the ratio of Ino and Hx to the sum of ATP and related compounds expressed as a percentage. The ratio Hx/AMP was considered an adequate alternative to characterize fish freshness due to its constant increment with time. a high content of Hx is related with the bitter off taste of spoiled fish. whereas IMP evokes a fresh meaty taste sensation. adenine. nucleotides and nucleosides should be extracted and analyzed. the knowledge of their molecular structure is important.2. IMP is derived from the inosine in which a phosphate group is attached to the 5-ribose carbon.2). the disappearance of the degradation products differs from one species to another3 as mentioned here. These cold conditions must be held along the sample preparation. . ATP. and AMP disappear early postmortem. Some of them are briefly described here.7–9 Nevertheless. 5. or hypoxanthine is attached to a ribose. it is advisable to collect small tissue samples and immerse them into liquid nitrogen. Nucleosides currently analyzed in seafoods are those in which a purine ring. forming the adenosine or inosine.2 Chemical Structure of Main Seafood Nucleosides and Nucleotides To a better understanding of the methods of analysis of these compounds. consequently.16.12 However. ADP and ATP are derived from the AMP.16 Another suggestion to use nucleotide compounds as a measurement of seafood quality is their relation with sensory attributes.15. even at refrigeration temperatures. 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. On the other hand. to which one or two additional phosphate groups are attached through pyrophosphate bonds (∼P) (Figure 5. adenosine diphosphate (ADP).

6 M perchloric acid is added.1 Extraction of Nucleotides and Nucleosides A typical extraction procedure for the analysis of fish samples by reversed-phase chromatography.8 and then filtered with a 0.45 mm membrane. the supernatant is filtered through glass wool and neutralized to pH 6. and Hx. 5.2 μm membrane filter and stored under frozen storage at temperatures below −20°C until analysis.3.000 g for 20 min). The frozen tissue is minced. is the following: 5 g or less of muscle tissue are excised and quickly frozen with liquid nitrogen.2 Structure of adenosine-derived nucleotides. Ino.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. and the tissue is homogenized with a stomacher-type homogenizer for a few minutes under cold conditions.20 and/or spectrophotometers as well as in capillary electrophoresis (CE)21 or ion chromatography (IC).5 g of fish sample with 10% trichloroacetic acid and. The supernatant is filtered through a 0. This neutralized extract is kept in an ice bath for 15 min and centrifuged again (15. Once the extract is centrifuged (15. they are neutralized with 2 M sodium hydroxide.000 g for 15 min).22 . cold 0. with or without employing an ion-pairing agent. 3–5 vol.000 g for 10 min). avoiding any thawing.17 Other extraction methods consist in the homogenization of 2.8 by adding solid potassium carbonate or 1 M potassium hydroxide.5–6. These fish extracts are used in enzymatic assays with biosensors19. The neutralized extract must be made up to 5 mL with 20 mM phosphate buffer pH 7. after centrifugation (27. although storage at −18°C has been demonstrated to be enough to preserve fish samples and fish extracts for the analysis of IMP.

inosine.3. this technique can present problems in reproducibility.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. AMP).2 Nucleotides and Nucleosides Determination Several methods have been used to measure nucleotides and nucleosides. some authors have described extraction methods that consisted of heated fish sample. The mode of separation will depend on the analyte of interest. the reconditioning of the capillary surface is ensured by washing 1 min with 1M NaOH. nucleotides will disappear at the rigor mortis state (normally 1 day after catch). including fish extract.3. However. because these samples usually contain significant amounts of ions. to analyze nucleotides. RP-HPLC and ion-paired reverse-phase are the methods of choice for this analysis. and the K′ or K i index will be usually enough to characterize fish . both a microwave oven at 500 V for 5 s and heating at 100°C for 60 min have been used.24 5.3.25 thin-layer chromatography (TLC). In this way.26 reversed-phase high-performance liquid chromatography (RP-HPLC) with and without ion pair. intact fishes after being submitted to physical and chemical stressors such as hypoxia.23. which may be adsorbed on capillary walls. 5. In the analysis of complex biological samples. 5.19 5. Capillary electrophoresis has been used in many nucleotide analysis applications as in the study of nucleotide degradation in fish tissues.2. and hypoxanthine would be a potential of 416 V/cm of capillary using 100 mM 3-[cyclohexylamino]-1-propanesulfonic acid (CAPS) buffer. Thus. Nevertheless. radioimmunoassay.27 IC.3 Chromatography At present. HPLC has been shown to be the most widely used technique to analyze nucleotides and nucleosides.3.2 Capillary Electrophoresis CE is a powerful separation technique that can provide high separation efficiency and high sample throughput with minimal sample volume and buffer consumption. high-performance capillary electrophoresis (HPCE).22 and enzymatic assays.28 Also in vitro 31P-NMR spectroscopy has been applied to both excised tissue and perchloric acid extracts of fish muscle. ADP. in vivo 31P-NMR spectroscopy has been used as a powerful technique to characterize the biochemical changes that occur in live.2. the addition of an ion-pair to the mobile phase greatly improves the separation by increasing the retention time of charged molecules (ATP.Nucleotides and Nucleosides ◾ 61 In the development of biosensor analysis.21 Typical conditions to get a good separation of IMP. pH 11. In particular. ion-exchange HPLC. Thus.2. among other chromatographic techniques. followed by 2 min of the running buffer used. including nuclear magnetic resonance spectroscopy (NMR).

The column used is an analytical reversed-phase RP-18 column. (5) hypoxanthine. which differ mainly in the pH of the mobile phase. 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. . 5. There are many approaches to analyze nucleotides and nucleosides by this technique. (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. The separation was achieved with an RP C-18 column at 35°C and a gradient between phosphate buffer at pH 7 and acetonitrile.3. (4) AMP.1 Reversed-Phase HPLC The chromatographic analysis should be performed in a liquid chromatograph equipped with an UV detector (254 nm).2.29 phosphorylated metabolites are also well separated in the chromatogram. and (6) inosine.62 ◾ Handbook of Seafood and Seafood Products Analysis freshness or quality. Then. (1) IMP. a simple RP-HPLC with a phosphate buffer as mobile phase will be adequate.17. (2) ATP. In Figure 5.29 With buffer pH 7.3.15 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. Quantitative analysis can be performed by external or internal standard method.3 RP-HPLC chromatograms of standards (a) and hake (b) ATP-derived compounds.

which is especially useful in separating mixtures of charged and uncharged molecules. (4) AMP.4 Ion-paired HPLC chromatograms of salmon (a) and sardine (b). (2) ATP.3. ATP-derived compounds. 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. The separation is achieved in a reversed-phase column. because the ion pair enhances the retention time and separation. .17.Nucleotides and Nucleosides ◾ 63 5.2.2 Ion-Pair RP-HPLC The most common technique used for the separation of nucleotides is ion-pair RP-HPLC. either tetrabutylammonium hydrogen sulfate or phosphate is the ion pair most used. Nevertheless. Thus. 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. and (6) inosine.4 shows an ion-paired chromatogram of a 48 h postmortem sardine extract. (1) IMP.and tri-nucleotides have to be analyzed. Due to the negative charge of the phosphorylated groups of nucleotides. this method is more expensive than the more simple technique previously described. due to the ionic nature of the phosphate esters that facilitates strong interactions with the ion-pair reagent at the appropriate pH. Figure 5. (3) ADP. making it less dependant on the type of column. as well as the resolution.30 This ion-paired technique is especially useful when di.3. (5) hypoxanthine.

although these applications used to be achieved with at least one of these enzymes immobilized as described earlier. because no interference of salt in the medium was observed here as was in the case using the HPLC method. and thus test kits. but they remain immobilized in different supports.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. while the depletion of oxygen is measured by a Clark-type elec- . Ino. inosine.20.36 Nevertheless.3. although biosensors have shown its utility in some applications such as clinical. all the approaches to date need the sample preparation described earlier.6–7. the application in the food industry is still restricted36 mainly due to critical stages such as enzyme immobilization or sample preparation for analysis. the analysis may be performed with one or more enzymes. Indeed. oxidizes the hypoxanthine to xanthine and uric acid.2. In addition. electrodes.2 Enzymatic Methods with Immobilized Enzymes In this case. simplicity. NP.3. which is immobilized in a membrane fi xed in the sensing area of the electrode. which is further coupled to a chemical transducer. constituting what is known as enzyme sensors o biosensors.4 Enzymatic Analysis The use of enzymatic methods to analyze nucleotides in seafood is widespread due to their high specificity. due to its specificity. XO enzyme. These assays may be carried out with the enzymes in solution31. because the denaturalization of the enzymes with time. In these conditions. agricultural.41 or for the evaluation of chicken32 and beef meat33 aging. or sensors have a limited shelf life.2. enzyme-coated strips.64 ◾ Handbook of Seafood and Seafood Products Analysis 5.39 This procedure was also used to analyze ATP and related compounds in fish sauces with very good results. which will be further quantified by measuring the absorbance at 290 nm and by polarimetry. In this sensor. 5. This sensor has been developed mainly for assessing the freshness of fish meat40. 5. The depletion of oxygen or the formation of hydrogen peroxide or uric acid may be detected amperometrically. in which an enzyme or a group of enzymes are immobilized in a membrane or other supports.32 or immobilized.4. and rapid response.36–38 The most used biosensors for the nucleotide-related compound analysis are electrochemical sensors. the use of commercial kits or disposals presents some problems.1 Enzymatic Methods with the Enzyme in Solution The concentration of Hx.20. and biotechnology. This option offers some advantages in relation to the free enzyme.31 Another possibility consisted in monitoring the oxygen consumption after these enzymatic reactions with an amperometric-type sensor (oxymeter). respectively. and IMP may be determined spectrophotometrically by a sequential addition of XO. IMP.3. These enzymes act by oxidizing the substrates (analyte) while consuming oxygen or producing hydrogen peroxide or uric acid. environmental.4.35 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.2. and 5′-nucleotidase (NT) into a reaction phosphate buffer containing the fish extract sample at pH 7.8 and 30°C–37°C. and hypoxanthine will be oxidized to uric acid and H2O2. 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.33.35 The most used is the biosensor based on the measure of hypoxanthine.

D. 36: 19–22. Changes in baseline levels of nucleotides during ice storage of fish and crustaceans from the Portuguese coast. Fish. 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. T. 4. and H values. J.. Nunes. Robles-Burgueno. Lugo-Sánchez. Agric. cellulose triacetate..6 to −0. and Nguyen52 and afterward it has been commercialized as a Freshness Meter KV-101 (Oriental Electric Co. Thus..55.9 V) vs. Gill. Bull. and Hx amounts. different supports have been used for the immobilization of the XO enzyme. Surette. Fish. Ino was converted to Hx. Formed Hx was measured with an amperometric sensor that detected uric acid + hydrogen peroxide in an additive matter. Sci. 2006.L.. Ino. Comparable results to that of HPLC were reported. J. T. The consumed oxygen produces a current decrease that can be correlated to the concentration of Hx.L. 1 mol of Hx would be converted to 1 mol of uric acid and 2 mol of hydrogen peroxide. In this way. 1959. Eur. Ino. This method was patented by Luong. 1: 13–19. Japan). IMP.E. M. Both Hx and X are substrates for the XO action and will be oxidized either simultaneously or sequentially.23. Jpn. an Ag/AgCl reference electrode. or H2O2.54 In fact.41 preactivated nylon. Food Chem.33 although this relation should be confirmed in each particular system. P. 29: 570–590. Paredi. . uric acid. 1988. 3. Özogul.. Palacios. Male. which can be present in the sample or formed during the enzymatic reaction.Nucleotides and Nucleosides ◾ 65 trode at a platinum cathode (−0. Most of these supports have been developed with the aim of eliminating interferences due to ascorbic acid. A similar application was proposed12. R...51 The use of multienzymatic biosensors to measure fish freshness has been very helpful for the simultaneous determination of AMP. 41: 341–353. In the measurement of hypoxanthine. 6. Kuley. M.47 On the other hand. Food Sci. J. and. Luong and Male20 used a multienzymatic biosensor system to determine the H value as a fish freshness indicator. 2001...44 a polyaniline film by electropolimerization. R. P.46 or even in a carbon paste electrode modified with electrodeposited gold nanoparticles. K. Technol. 212: 141–146. 2007. some authors have described this type of biosensor coupled with an oxygen electrode.. thus. Postmortem biochemical and functional characteristic of Monterey sardine muscle stored at 0°C. Food Sci. Özogul. Mendes.J. necessary to obtain K. 5. Y.A.E. Pacheco-Aguilar.53. A new method for estimating the freshness of fish. Postmortem changes in quality indices of ice-stored flounder (Paralichthys patagonicus).48 IMP. E. Howgate.53. Technol.56 References 1. M. M. 7.. 2.34 to obtain the Ki parameter as a freshness indicator. A review of the kinetics of degradation of inosine monophosphate in some species of fish during chilled storage. specific biosensors to determine AMP..45 a nafion-coated platinum disc electrode. IMP. M.. 2000. An immobilized NT was used for the previous conversion of IMP to Ino. 2005. Massa. Int. Ltd. Most recent approaches to determine Hx are based on the incorporation of the XO enzyme in a graphite/Teflon matrix. Food Res.E. 24: 749–750. et al. M. Quinta. LeBlanc. Soc. Arai. Nucleotide degradation in sardine (Sardina pilchardus) stored in different storage condition at 4°C. R.E. and Hx using a cellulose triacetate membrane have been described. K i. A.49 and Ino50 and a multienzymatic sensor to analyze simultaneously AMP. Matsuyoshi. The proposed relation 1 Hx for ½ X for each oxygen molecule formed must be taken into account to quantify the Hx. Food Biochem.R. J. Sci.. J. and then after adding a soluble NP. Saito. Biochemical basis of postmortem nucleotide catabolism in cod (Gadus morhua) and its relationship to spoilage. F.18 and a silk fibron membrane in combination with a cellulose acetate membrane42 or a nylon net43 have been used.. 65: 40–47.

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

..............................................................1 Acid-Catalyzed Esterification and Transesterification .............2 GLC Analysis of FAME...............1.....3 Lipid Analysis in Marine Products ........ 79 69 .. 73 6.................1 General Aspects of Lipid Compounds ...........................................2 Stereospecific Analysis of Lipid Classes ............. 75 6.......Chapter 6 Lipid Compounds Santiago P...............5 Qualitative and Quantitative Analyses of Marine Lipid Classes .............................................3 Column Chromatography....... 70 6.......3 Lipid Manipulation and Storage ................................1 Qualitative Analysis of Fatty Acid Composition .............................................................................................2......................................1..1 Introduction ............................. 73 6........................................ 70 6..............................................1 Isolation of Lipids from Tissues .............3......................................................................... 72 6.....................1 Spectrophotometric Assessments of Total Lipid Extract .......................................................................... 78 6.................76 6.....................................................1.5.....................................................................3... 73 6........1..2........................4....3 Marine Fatty Acid Analysis .....................2....................................................................1 Fatty Acid Methyl Esters Preparation ..........2 Marine Lipid Characteristics ....2 Base-Catalyzed Transesterification ..............1......2..................3......................3....................2 Quantitative Estimation of Fatty Acid Composition ..76 6........................2.2......................... 77 6..2 Analysis of Sterols ... Aubourg Contents 6........... 73 6......................3.............................3 Analysis of Ether Lipids .5....................................................... 73 6.....4..............3........................ 70 6.......................2 First Steps in Marine Lipid Analysis ....................................................76 6.......................... 75 6. 78 6........................ 72 6...........76 6........4 Lipid Quantification .......4 Analysis of Marine Nonsaponifiable Matter ................. 75 6.....................1 Lipid Saponification........................................... 71 6............ 72 6.....................................5................................... 77 6...........2 Removal of Nonlipid Contaminants .......................................................................................................................4............

. ethers...... 79 6.......... particularly C20:5ω3 (eicosapentaenoic acid..... chloroform............. in it. longer-chain fatty acids..........5. glycoglycerolipids...... and amines (PL).............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..........70 ◾ Handbook of Seafood and Seafood Products Analysis 6... although no satisfactory or widely accepted definition exists.....5........8 Mass Spectrometry ... Related to exogenous effects.......1........ 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 sphingolipids) would yield three or more types of products per mol.. Marine lipids....... fatty alcohols (wax esters).................... Most textbooks describe lipids as a group of naturally occurring compounds.... Seafood lipids are known to provide high contents of important components for the human diet.......... toluene... indeed........9 Supercritical Fluid Chromatography ........... gangliosides.............. and alcohols.....1 Introduction 6........ the catching season has been shown to play a key role regarding temperature and feeding availability. 6....... which have in common a ready solubility in organic solvents.... triacylglycerols (TG).........81 6...... differ from the other sources in that they contain a wider range of fatty acids............. Marine species have shown large variations in lipid content and composition as a result of endogenous and exogenous effects [5–7]..................... an inverse ........ and lipopolysaccharides would be included..... sterols (sterol esters)......... such as hexane... EPA) and C22:6ω3 (docosahexaenoic acid.... “simple lipids” (fatty acid and alcohol components) would be those that yield on hydrolysis at most two types of different products per mol. 82 6.... carotenoids...... steroids......5 High-Performance Liquid Chromatography .... phosphoric acid...4 Thin-Layer Chromatography .. 79 6.6 Silver Ion Chromatography .........................5................... whereas “complex lipids” (glycerophospholipids............. a widely accepted division has been difficult...... 82 References . 80 6.......... 80 6...............7 Nuclear Magnetic Resonance (NMR) Spectrometry ...5.....5................................................. An alternative division into two broad classes has been shown to be convenient for lipid analysts [2]....... 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............ Such diverse compounds as hydrocarbons.......... Different attempts have been carried out to define what is meant by the term lipid.......... including cardiovascular ones [3]...................... and a larger proportion of highly unsaturated fatty acids... A simple physicochemical classification that empirically groups lipid molecules according to the hydrophilic–lipophilic balance has been proposed [1].. DHA) [4]............ however...........................2 Marine Lipid Characteristics In many marine organisms.....5. phospholipids (PL)................ soaps..1................... lipid is usually the second largest biochemical constituent after protein....... Because of their structural and functional variety....

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

The second problem is endogenous lipolytic enzymes that can lead to large amounts of unesterified fatty acids. Although there are limitations to its use and alternatives are frequently suggested. the procedure of Bligh and Dyer [9] offers some advantages as it does not use large volumes of solvent. Its employment has recently been reviewed [10]. 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. it is advisable to include an additional antioxidant at a level of 50–100 mg/L to the solvents. 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. and salts.88% KCl) [8]. 6. most workers in the field appear to accept two basic routines currently in general use. Two main problems can arise with lipid fraction when employing inconvenient storage conditions. In addition. phosphatidic acid. may on occasion be extracted by these when they are in the presence of large amounts of simple lipids such as TG. First.72 ◾ Handbook of Seafood and Seafood Products Analysis 6. or lyso-phosphatidylglycerols in lipid extracts. which employs chloroform–methanol (2:1) in a solvent–tissue ratio of 20:1. The most popular is the method of Folch et al. Pure single lipid classes are soluble in a wide variety of organic solvents. At the same time. although endogenous tissue antioxidants can provide some protection. though more time-consuming. and as large volumes of solvents may be used to obtain small amounts of lipids.1 Isolation of Lipids from Tissues Ideally.2 First Steps in Marine Lipid Analysis 6. amino acids. a major driving force being the environmental concern regarding the use of organic solvents. marine tissues should be extracted from the living organism as soon as possible after catching or slaughtering [2].2. For all extraction methods. As an advanced alternative. which yield essentially quantitative extractions of the major lipid classes when applied to homogenates of whole marine tissue extractions. the tissue should be kept frozen (about −60°C or less) as rapidly as possible.g.. 0. which do not normally dissolve readily in nonpolar solvents. such as proteins. any such impurities can be troublesome. supercritical fluid extraction shows an increasing demand. peptides.2.2 Removal of Nonlipid Contaminants Most polar organic solvents used to extract lipids from tissues also extract significant amounts of nonlipid contaminants such as sugars. 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. urea. A more elegant and complete. This type of washing procedure was first developed by Wells and Dittmer [11] and simplified later by Wuthier [12] for large numbers of samples. 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. When this is not feasible. polar complex lipids. . it should not be so polar that TG and other nonpolar simple lipids do not dissolve and are left adhered to the tissues. PUFA can autoxidize as a result of endogenous oxidant enzymes. However. all solvents can contain contaminants. this method applies a single-phase solubilization of the lipids using chloroform–methanol (1:1) in a solvent–tissue ratio of 4:1. diacylglycerides. [8]. Where large amounts of tissue have to be extracted.

fatty acid methyl esters (FAME) obtained are usually introduced in the GLC system without previous removal of contaminants. application of nuclear magnetic resonance (NMR). Lipid extracts have to be converted into methyl ester derivatives. provided water absorption onto the dry extract lipid is avoided. but it is usually advisable to add further synthetic antioxidants to storage solvents at the level of 50–100 mg/L [2]. Then. Storage temperature should be −30°C as the highest temperature. a large diethyl ether volume is employed. relatively important errors are obtained. As storage containers. Plastic ware of all kinds (other than that made from TeflonTM) can be specially troublesome and is best avoided.3 Lipid Manipulation and Storage Wherever possible. a known aliquot of the purified lipid extract is softly heated and the resulting dry lipid matter weighted.1. glass is the best choice. In it.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. and care must be taken at all steps in the analysis of lipids. When it is necessary to concentrate lipid extracts.3.4 Lipid Quantification For most common purposes. large volumes of solvents are best removed by means of a rotatory film evaporator at a temperature that. Two basic strategies can be applied [15.16]: acid catalysis and base catalysis. Owing to the wide variety of fatty acid compounds in marine lipids (Table 6. this analysis is more complicated than that with other kinds of living organisms. lipids should be handled in an atmosphere of nitrogen. 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. since plasticizers are very easily leached out.2. if not. For fast purposes. 6. Natural tissue antioxidants. fatty acids .1). Conversely. leading to selective losses of a proportion of the less polar constituents.3. According to the special relevance recently acquired by noninvasive technologies. 6. and Fatmeter measurements is proving to be of increasing interest [14].3 Marine Fatty Acid Analysis 6. 6. it has been shown that lipids can themselves dissolve in some plastics. This method proved to be accurate in the case of a high lipid content (low complex lipid content).1 Fatty Acid Methyl Esters Preparation Fatty acids are essential components of lipids. and the resulting lipid extract can no more be employed for further analysis. such as tocopherols.2. in general. the Soxhlet method of extraction has been developed [13]. Small volumes of solvent can be evaporated by carefully directing a stream of nitrogen onto the surface of the solvent. Their measurement by gas–liquid chromatography (GLC) is the most commonly used method for lipid analysis. In addition. should not exceed about 40°C. Autoxidation of double bonds in marine lipid fatty acids is particularly troublesome. since PUFA will oxidize rapidly in air [2]. afford some protection to lipid extracts.Lipid Compounds ◾ 73 6. near-infrared (NIR) spectrometry.

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-Octadecatrienoic 6.12-Octadecadienoic 9.10.8.74 ◾ Handbook of Seafood and Seafood Products Analysis Table 6.12.16.13.12.8.17-Eicosapentaenoic 7.19-Docosahexaenoic Linoleic Linolenic Stearidonic Araquidonic EPA DPA or clupanodonic DHA or cervonic In all cases.” .10.19-Docosapentaenoic 4. the double-bond configuration is “cis.7.9.11.15-Octadecatetraenoic 5.14.14-Eicosatetraenoic 5.11.16.13.

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

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].16]. branched.4 Analysis of Marine Nonsaponifiable Matter 6. commercially available standard mixtures containing accurately known amounts of methyl esters of saturated. However. as these are easily prepared and are widely used in chromatographic analysis.” 6.1 Lipid Saponification Lipids may be hydrolyzed (saponified) by heating them under reflux with an excess of dilute aqueous ethanolic alkali.4.2 Quantitative Estimation of Fatty Acid Composition With reliable modern gas chromatographs equipped with flame ionization detectors (FID). or oxygenated chains. the nonsaponifiable layer will contain any long-chain alcohols and sterols originally present in the lipid sample in the esterified form. 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]. If necessary. On the other hand. Finally. based on the Liebermann–Buchardt reaction. linearly proportional to the amount (by weight) of material eluting from the columns [15.2 Analysis of Sterols Sterols are biological compounds. within limits. Such compounds can be divided into sterols and “ether lipids. the basic structure of which includes the cyclopentanophenanthrene ring. A high proportion of the available data has been obtained for the methyl ester derivatives of fatty acids. monoenoic. and there is no way of overcoming this difficulty entirely. In most cases. cyclic. Problems of measuring this area arise mainly when components are not completely separated. as well as the deacylated residues of ether lipid compound. 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. According to the previous section. pyridinecontaining derivatives. the fatty acids on one side and diethyl-ether-soluble nonsaponifiable materials on the other side are separately recovered for further analysis [2]. Results can be expressed as weight percentages of the fatty acids present or as molar amounts of each fatty acid. unsaturated. On the other hand. and polyenoic fatty acids should be analyzed under the same GLC conditions for checking the quantification results. 4. Total sterol content can be determined directly and spectrophotometrically from the lipid extract by using the method of Huang et al. the resulting FFA have to be transformed into their corresponding FAME for further analysis by an acid-catalyzed method. 6. 6.2.4. the areas under the peaks on the GLC traces are. have been shown to be suitable for direct mass spectrometric structural analysis of acids containing straight. such as picolinyl esters. A known quantity of an internal standard should be added to the lipid sample. sterols can be fractionated and analyzed by means of different .3. [22]. nonadecanoic acid is employed and added before the methylation step. For a complete analysis.76 ◾ Handbook of Seafood and Seafood Products Analysis In recent years. quantitative results would first be calculated on its basis.

Adsorption thin-layer chromatography (TLC) on silica gel layers can be used to separate simple alkyl and alkenyl lipids. neutral plasmalogens tend to migrate ahead of alkyldiacylglycerols. TMS.4%) in 2M HCl.3-diacyl-sn-glycerols are generally saturated or cis-monoenoic even– numbered components (16:0. which in turn migrate just in front of TG. information on ether lipid composition in marine PL is less abundant. which generates dimethyl acetals from the liberated fatty aldehydes. it can be cleaved by acid-catalyzed transmethylation. Neutral plasmalogens may be detected by spraying the TLC plates with 2. such as acetate. being normally placed as the radical in position 1 and specially abundant in marine invertebrates [5. but they suffer from the limitation of the lack of a distinctive chromophore in the analyte. high-performance liquid chromatography (HPLC) methods can offer a nondestructive alternative. Unlike fatty acids. such compounds tend to be decomposed during GLC analysis and are best reduced by catalytic hydrogenation to alkylglycerols. Although the GLC is normally carried out with cholestane as internal standard. no single procedure will achieve the desired analysis. batyl. Accordingly. or isopropylidene derivatives by GLC.27]. 18:0. The first type is the major one in marine lipids. Often. v/v) as a solvent system. alkenyl compounds have been directly identified and quantified by GLC together with FAME [35]. and its analysis has already been discussed in Section 6. and selachyl alcohols were found to be the most abundant. Cholesterol has been shown to be the most abundant sterol in all marine living beings. Methods for separating and quantifying ether-linked glycerides have been reviewed [31.5 Qualitative and Quantitative Analyses of Marine Lipid Classes Lipid samples obtained from extraction of biological material are complex mixtures of individual lipid classes [16]. The alkyl groups of 1-alkyl-2. and 18:1. different analytical . supercritical fluid chromatography (SFC) has been employed for the glycerol ether analysis of liver oils of shark species [34]. Further.Lipid Compounds ◾ 77 chromatographic techniques [23.4. whereas no simple spot test is available for the identification of alkyldiacylglycerols. although invertebrates have shown a significant presence of other sterols [27]. The others are often united into a group called “ether lipids. 6. Great attention has been accorded to the assessment of cholesterol oxide formation in marine products [29]. and combinations of techniques must be used until the required purposes are served. The alkyl moieties are usually analyzed in the form of 1-alkylglycerol or as volatile nonpolar derivatives of this compound. Although the vinyl ether linkage is unaffected by basic hydrolysis conditions. In this section. They can be separated by a double development in a single direction with hexane–diethyl ether (95:5.3 Analysis of Ether Lipids Marine lipids may contain fatty acid residues as the only radicals. Chromatographic methods for cholesterol analysis [28] are of relevant importance in foods in relation to human health concerns. thus. trifluoroacetate.4-dinitrophenyl-hydrazine (0. chimyl.3. or they may include alkyl and alkenyl radicals.24]. For GLC analysis.” Such compounds are basically found as PL classes (specially in phosphatidylethanolamine). 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]. marine sterols have to be converted into more volatile compounds such as acetate [25] and trimethylsilyl (TMS) [26] derivatives. although a great interest has been accorded to their isolation because of their medical and cosmetic applications [30]. mostly). 6. Concerning alkenylglycerols.32].

This is due to the great complexity of fatty acids present in these oils and fats. 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.5. traditional methodologies are still employed in cases where such advanced technologies are not available and are reviewed in this section. [39] without previous digestion.41]. In many cases. where an FFA-cupric acetate-pyridine complex is involved. Compared with the data compiled for plant oils and for fats from land animals. this is made to react with ammonium molybdate to form phosphomolybdic acid. PL present in the lipid extract are made to react with ammonium molybdate in an organic phase and then measured spectrophotometrically. For FFA assessment. which is reduced and determined spectrophotometrically [38]. then. Finally. However.78 ◾ Handbook of Seafood and Seafood Products Analysis approaches will be discussed. This method can be applied to total lipid extract or to any lipid class after previous isolation [7. these enzymes can be isolated and used in simple incubations in vitro as an aid in structural analyses of lipids. 6. a rapid NIR spectrometry has been applied for the direct FFA determination in fish oil [37]. the Grignard reagent has widely been employed in the case of marine substrates [42]. according to details explained in Section 6. and GLC technologies combined with MS in the last decades has provided quick and useful procedures for the stereospecific lipid analysis. Procedures that involve spectrophotometric measurement of highly colored copper complexes are now favored. 6. An alternative and successful method has been proposed by Raheja et al. In it.5. 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. Additionally. The fatty acid composition of each lipid class can be determined by GLC of the methyl ether derivatives. according to the information provided in following sections.3.2 [22]. The advent of new NMR.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. Ester linkages can be quantified by the method of Vioque and Holman [40]. HPLC. giving rise to a tremendous number of species. although some interference of polar lipids was found.2 Stereospecific Analysis of Lipid Classes The determination of fatty acid composition at each location in lipid classes has ever since attracted great attention.4. the results so far reported for aquatic animals are few. Traditional determination of PL content in lipid extracts has involved the digestion of PL with the release of inorganic phosphate. a method for the quantification of esterified and unesterified total sterols is mentioned in Section 6. . although it turned out not to be accurate enough for marine lipids. focused on the qualitative and quantitative analyses of marine lipid classes. titrimetric methods were used for many years. A wide use was found for lipase hydrolysis. this including chromatographic separation and further analysis of fatty acids after previous methylation and transmethylation. Accordingly. in it. since the presence of double bonds in the proximity of a carbonyl group of fish PUFA reduces the rate of deacylation of glycerides. More recently. A very popular one is that proposed by Lowry and Tinsley [36]. prepared by esterification or transesterification of the purified lipid class.

whereas complex lipids are recovered by elution with methanol [41. Such techniques would include high-pressure TLC (HPTLC).44] and PL [45. Such stereospecific studies have widely been focused onto TG [43.5 High-Performance Liquid Chromatography In recent years. overpressure TLC (OPTLC). diethyl ether.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. A variety of solvent systems have been used to separate simple lipids on an analytical or semipreparative scale by single or two-dimensional TLC. or florisil as adsorbents. 6. 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.47]. The perceived weakness of TLC has been recognized as the quantification aspect. precoated plates are much more convenient than laboratory-made plates. and tubular TLC (TTLC). acid-washed florisil.5. coupling of TLC with other techniques such as HPLC. In all cases.5 mm thickness [7. identification. and NMR has increased its analytical power in several applications. The improvement and versatility of TLC enable it to be used for several modern applications. 6.3 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. and this has led to the evolution of the TLC/FID Iatroscan system. The system has been successfully used for marine lipid class analysis [51]. Separation can be carried out on silicic acid. 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.52]. which include highly automated techniques right from sample application and development to detection and quantification. although particular care is required to recover the acidic lipids quantitatively. 6.4 Thin-Layer Chromatography Many text books and reviews report TLC application on lipids for routine separations. which has been used routinely for lipid analysis in the last decades. and acetic (or formic) acid in various proportions. 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.46] classes. For preparative purposes. However. MS.41]. In addition.5. infrared (IR) spectrometry. 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.5. Aminopropyl-bonded phase cartridges have been much used for the isolation of simple and complex lipid fractions.49]. and quantification [48. 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. in spite of the relatively higher costs [50]. HPLC has undoubtedly been the most widely applied separation technique in the analysis of most simple and complex lipid classes [48. HPLC is much more expensive than . Those used most frequently contain hexane.47]. being simple lipids eluted in a stepwise sequence with hexane containing increasing proportions of diethyl ether. although lengthy conditioning may be necessary before columns can be employed [2.

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

and complete structure of an unknown compound. soft ionization MS techniques such as fast atom bombardment. Application of 31P-NMR has shown to be far shorter than with 1H and 13C. Later on [61]. EPA.86 ppm) provided the possibility of proposing this new analytical tool.and diene-. and 3 locations) of ω3 fatty acids in depot fat of several fish species was examined by 13C-NMR [63]. Quantitative analysis of fatty acid composition and alpha-beta distribution in TG tuna fish was achieved [62].Lipid Compounds ◾ 81 Based on 1H-NMR spectrometry [59]. 2. It could be observed that DHA was concentrated in the 2-location of TG in depot fats. and electrospray have the ability to ionize lipid molecules without causing extensive fragmentation. mono. Finally. and highly unsaturated fatty acids of lipid extract of Atlantic salmon muscle. but. the condensed mobile phase used for liquid separations is not readily compatible with high vacuum ionization sources. a rapid and structure-specific method for the determination of ω3-PUFA in fish lipids was presented. Some applications concerning the marine lipids’ study will now be mentioned. Thus. This NMR technique can provide a single signal for each PL class. ω6. This development paralleled the development of atmospheric pressure chemical ionization (APCI). 6. The different chemical shifts observed for the methyl resonance of ω3-PUFA (δ = 0. thermospray. A good agreement could be observed between NMR values and those from the GLC analysis.66]. and attention was focused on the identification of specific signals for ω3 fatty acid group and also individually for DHA. the high-resolution NMR spectra of four fish oils were recorded. The positional distribution (1. 13C-NMR was employed for the plasmalogen analysis in fish lipid samples showing a good agreement with the data obtained by GLC [64]. The 31P-NMR application has also shown the possibility of analyzing the ether structures within the glycerol backbone of phosphatidylethanolamine and phosphatidylcholine. marine lipids have received lesser attention. In a first attempt for 13C-NMR application [60]. Among the different food lipids. empirical formula. although an increasing importance has been obtained lately for quantitative analysis [20.95 ppm) with respect to the methyl resonance of all other fatty acids (δ = 0. 13C-NMR spectrometry was successfully used to determine the proportions of saturated. Signals in the spectra were assigned. results obtained using high-resolution 13C-NMR were in good agreement with those obtained by GLC. many of the advances in MS have involved new ionization techniques. The information-rich nature of MS makes it the most desirable detector for many explanations. 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. FFA carbonyl resonances were detected at the lower field of the carbonyl region. probably due to their more complicated structure. ω3. Over the years. The first step for any MS method is ionization of the sample molecules in the gas phase. thus providing a suitable tool for lipolysis analysis. . Arpino [67] likened the HPLC-MS union. although GLC is conveniently coupled to electron impact ionization (EI) and chemical ionization (CI) sources. according to each corresponding resonance. Recent developments in MS have been very interesting for complex lipid molecules. Following ionization to a negatively or positively charged species (most commonly the later).8 Mass Spectrometry MS has long been used as a powerful tool for the analysis of the molecular weight. and stearidonic acid. its intensity should be proportional to the quantity. After different approaches. the molecules or their fragments can be separated and identified on the basis of their mass-to-charge ratio (m/z). Thus.65.5. so little application is specially available for marine lipids [58].

Luten. 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. 1997.-dimethyloxazoline derivatives [69]. Nutritional aspects of fish. Minor fatty acids from mussels (Mytilus galloprovincialis) were enriched by Ag+-TLC and then analyzed by GLC-MS as 2-alkenyl-4. U.. p. carbon dioxide as the mobile phase. cholesterol esters. a nonpolar capillary column. whereas its critical pressure and critical density are high enough for good solvation of many potential analytes. which uses a highly compressed gas above its critical temperature and critical pressure. London. Pergamon Press. such as mixed glyceride compositions ranging from 200 to 900 in molecular weight. analysis was carried out in conjunction with FAME by means of their dimethyl acetal derivatives resulting from the acid transmethylation of lipid extracts. References 1.. p. and a FID were employed in it. Concerning marine species analysis. and several nonmethylene interrupted fatty acids were singled out. 6. Lately. Integrated Approach to Quality. J. J. whereas quantification of TG. The liver oils of several shark species were analyzed by SFC [34]. simple classes from marine oils of different species were separated and quantified by capillary SFC [73]. T.. 589.. In addition. The Physical Chemistry of Lipids.. A wide range of cholesterol oxides were identified and quantified. Vol. Börrensen. Purification of PUFA (DHA and EPA) ethyl esters from tuna oil was carried out by SFC [74]. thus. 3. 17. 2. analytes are eluted from a capillary chromatographic column. and Oehlenschläger. . Handbook of Lipid Research. and the four most unsaturated fractions were analyzed by capillary SFC according to their acyl carbon numbers [72]. eds.9 Supercritical Fluid Chromatography In this advanced technique [10. 4.. Plenum Press. the method was capable of direct quantification of squalene and cholesterol. the use of SFC can substantially reduce the dependence on organic solvents in solvent extraction or HPLC analysis. An important advantage is that it is compatible with FID.. in Seafood From Producer to Consumer. Small. The qualitative and quantitative compositions of 1-O-alk-1-enylglycerolipids of albacore tuna (Thunnus alalunga) were studied along the canning process [35]. New York. 89. 1986.K.. Later on. 2nd edn. Elsevier Science. and diacylglycerol ethers required TLC fractionation before SFC analysis. the method was based on the use of preparative reversed-phase HPLC followed by subsequent identification by APCI HPLC-MS. Simopoulos.. Lipid Analysis.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].K. D. p. The mass spectrometer was operated in the EI mode (70 eV). which has great sensitivity and linearity. W.4..5. an optimization of process parameters was achieved to obtain a maximal production rate. Oxford.71]. Carbon dioxide is by far the most commonly used SFC mobile phase because of its low critical temperature. in a first attempt Baltic herring flesh TG were separated in eight fractions by Ag+-TLC. Christie. A. 1982. U. Analytical SFC has been shown to be particularly applicable to the analysis of higher molecular weight lipid moieties.

K. 1989. 1. Aubourg.. 113. Food Sci. Krzynowek. England. 54. V. D. J. G. Wefler. Sebedio. 24.. AOCS Press. 369. Love. “Warmed-over” flavor in meat.). R.K.. Chromatographic methods in the analysis of cholesterol and related lipids. 1990. J. J. 226.. NIR and NMR. J. and Garrido. 12. R. J. 17.. 18. B. 27... 2003. p. 108. ed... Biol. W. F. S. and Wrebiakowski. J. Anal.. E. The use of sephadex for the removal of nonlipid contaminants from lipid extracts. Folch. 1978. and Nielsen H. Vaskowski. and Gallardo. Ackman. p. ed. Christie. 237.. 149.K. eds. S.. and Nes.. 1.. J. 1959. 3rd edn... J. Fenton... 1957. 27. FL. Biochemistry.. R. p. 49. 21. Chrom. Fats. Champaign. 1989. Vol. U. 624... p. J.. U.... R. Purification of lipids from nonlipid contaminants on sephadex bead columns. 11. 479. Nielsen. Hammond.. in New Trends in Lipid and Lipoprotein Analyses.. M. Influence of biological factors and comparison of different methods of analyses: Solvent extraction. J. 911. and Oils. 1989. p. I. CRC Press. 22. Separation and determination of structure of fatty acids. R. FL. 1259. R. 1986. 19. 2003.. . Lebensm. poultry and fish. in Marine Biogenic Lipids. Chrom.. Chen. J. G. in Handbook of Lipid Research. 671.. Fats and Oils. U. Forsch. American Oil Chemists’ Society Press.. 7.. Ackman. 37. 1. and Dittmer. 1989. Plenum Press. 2. Bridgwater.. Stability of lipids of frozen albacore (Thunnus alalunga) during steam cooking.. 193. J. Bridgwater. R. A... 2. Adv. 199. 1961. FL.. Z... A. Lipid Res. ed. and Raftery. Evaluation of soxhlet’s and Bligh and Dyer’s methods in the determination of fat in meat. P. 1405. Wuthier. One-step conversion of fatty acids into their 2-alkenyl-4. Ackman. mollusks and fish. Fats. C. 497. Kuksis. WCOT (capillary) Gas–liquid chromatography. in Marine Biogenic Lipids. p... Chem. Huang.. Elsevier Applied Science. Eur. Vol. 1972. and Shorland. 341. 23. 9. 25. M. H. Chromatographic separation of cholesterol in foods. and Rossell. 23.. Phospholipids. Physiol. 2006. 2. Hyldig. Distribution and composition of lipids in marine invertebrates. G. J. Kuksis... Technol. and New York. and Rossell. L. New York. J. IL. J. Fatty Acids.. V.K. 33. 6.. J. p. Lipid Sci. 301. The Oily Press. Prost... R. 191.. 20. E. Lipid Res.. 1992.. A.. Boca Raton. eds. Sterols and crustaceans.. in Advances in Lipid Methodology—Five. 673.. 1995. and New York. in Physiology and Biochemistry of Sterols. F. 101. Supercritical fluid chromatography (SFC)-Global perspective and applications in lipid technology.-dimethyloxazoline derivatives directly from total lipids. Fatmeter. Vol. Chem. Lepage. 558. Pérez-Martín.. Hamilton. 38. J. p. and Rocha. Pearson. Champaign. J. J. R. W.. E. ed. and Panunzio. p.. The Oily Press. J. CRC Press Inc. Boca Raton. 8. 137. J. in Analysis of Oils and Fats. A. Ackman. A rapid method of total extraction and purification. Food Res.. Hoving. 37. Lipid Analysis. eds. 103. Lipid content in herring (Clupea harengus L. 341. Lees. p. Sieiro.. 10. R. 1963. Fatty Acids and Glycerides. 15. and Stanley. T.. 1994.. Technol. A simple method for the isolation and purification of total lipids from animal tissue. 16... Boca Raton.Lipid Compounds ◾ 83 4. Direct transesterification of all classes of lipids in a one step reaction. J. 1995. London. Nielsen. and Roy. 7.4. 114. Technol. 229. and Perkins. Food Sci. Can. Aubourg. Joseph. Hamilton. 5. Ackman. U. ed. W. Biochem. Unters. King. 2005. E. G. Packed-column gas chromatography. S. in Marine Biogenic Lipids. Teshima... Cholesterol and fatty acids in several species of shrimp. Food Sci. Int. Medina. CRC Press. 1966. in Analysis of Oils and Fats. and Dyer. Le Quéré. London. 537. Chrom. 14. 1986. Wells. Adlof. and Oils. eds. M. 1989. 1986. 13. Seasonal study of the lipid composition in different tissues of the common octopus (Octopus vulgaris). 26. J.. 24.. E. 1977.. A. A stable reagent for the Liebermann-Buchardt reaction.. 28.. Elsevier Applied Science. IL. C. Bligh.. Gas chromatography-mass spectrometry and tandem mass spectrometry in the analysis of fatty acids. Patterson.

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

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

5 meq/kg. and also more complex reaction products such as epoxy and polymeric compounds are formed during the propagation and termination steps [4]. and water. [5] or [6] before analysis. Several analytical procedures are available. Autooxidation of lipids takes place when the unsaturated fatty acids are exposed to oxygen and proceeds through an autocatalytic chain reaction [3]. leading to a wide variety of reaction products. Potassium iodide is added. If this contains a terminal carbonyl group. These include nonradical species such as aldehydes. the influence of these compounds has been little studied [3]. However. and enzymatic oxidation. acids. carbohydrates. Methods to determine the degree of lipid oxidation can be divided into two main groups. and alcohols. The peroxides are easily broken down to alkoxy radicals. ketones. Lipid oxidation can be divided into three types. the lipid can be extracted using the methods of Ref. A simple titration method where the sample is dissolved in chloroform–acetic acid (or isooctane–acetic acid) is often used for fats and oils. Free radicals are formed when hydrogen ions are extracted from the fatty acids. photooxidation. resulting in a wide variety of degradation products. but this can be improved by determining the endpoint colorimetrically or by . For determination of PVs in foods.88 ◾ Handbook of Seafood and Seafood Products Analysis loss of products. This method requires a sample of 5 g if the PV is below 10 and about 1 g if the PV is higher [3]. PV is one of the classical methods for determination of oxidative status. and reduced sales. 7. volatile compounds and nonvolatile components with a relatively high molecular weight. complaints from the consumers. The radicals react with oxygen forming peroxy radicals and hydroperoxides.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. 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]. autoxidation. The fatty acids and the lipid oxidation products in foods can also react with other components in the food such as proteins.1 Primary Oxidation Products The most common methods to determine primary oxidation products are peroxide value (PV) and conjugated dienes. Some of the reaction products from lipid oxidation may also have negative health effects. which makes it difficult to find where the components originated. methods that determine the primary oxidation products and methods that measure the secondary oxidation products. making it even more difficult to determine the degree of rancidity. The secondary oxidation products can also react further. The secondary oxidation products include both low molecular weight. and the liberated iodine is titrated with sodium thiosulfate with starch as an indicator. 7.2. When the decomposition of a hydroperoxide has resulted in the formation of a low-molecular weight volatile compound. The PV is expressed in milliequivalent of iodine per kilogram of lipid or as millimolar of peroxide per kilogram of lipid [7]. the molecule is called a core aldehyde. The sensitivity is about 0. it is both one of the oldest and one of the most used methods. This also makes the determination of the degree of oxidation a challenging task. the parent triglyceride is left with a shorter fatty acid. this is oxidized by the hydroperoxides or other components present in the sample.

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

and 2. The Totox value is still one of the most commonly used oxidation parameters used in commercial laboratories and laboratories in the edible oil industry. In other variations. which is formed as a decomposition product from lipid hydroperoxides under the acidic test conditions [3]. p-anisidine dissolved in acetic acid is added. Purge and trap techniques. However. and identified using different gas sensors. Protein. time of heating. the sample is incubated at 95°C for 2 h. pH.3. The volatile compounds formed as a result of lipid oxidation can be analyzed using electronic noses/gas-sensor array systems [20]. forming a yellow pigment absorbing light at 450 nm. This determines the amount of aldehydes (mainly 2-alkenals and 2. and antioxidants. Of these methods. and chloroform before adding TCA. Originally. but most of it is formed during the decomposition of the lipid peroxides during the acid heating stage. Some of the MDA detected in this test is formed during the peroxidation of the lipids. in addition H2O2. and the color is formed by many different secondary oxidation products. which are secondary oxidation products. This value is a combination of the PV and the AV. AnV can also be determined using Fourier transform infrared (FT-IR) [16]. The determination of TBARS (or TBA) is a common method to determine secondary oxidation products. nucleic acids. different methods give different results. TBARS values have been found to correlate with sensory scores within the same materials [19]. alkanals. After sampling. sucrose and other sugars. and chelating agents may also influence the peroxide decomposition during the assay. are highly sensitive. Many variations of this test are being used. metal ions. alkenals.13. reaction products from browning reactions. All the methods are based on the pink color absorbance formed by reaction between TBA and oxidation products of polyunsaturated lipids. and the absorbance of the solution is read at 530 nm. separated.4-dienals). However. The TBARS values for different foods with the same level of oxidation (based on flavor scores) can vary significantly [3. static headspace and solid-phase microextraction (SPME) are the least sensitive. hence the name TBARS. The Totox value is given as 2*PV + AnV. the lipids are dissolved in a solution of thiobarbituric acid in butanol. antioxidants. antioxidants. Different types of headspace analyses can be used. the TBARS are separated by steam distillation or HPLC to increase selectivity. These methods determine the presence of aldehydes.4-dienals also react with TBA. In the AOCS method [13]. where the samples are flushed or purged with nitrogen and the volatiles in the gas flow are trapped on a solid absorber. but as for the determination of PV. and the optical density of the water phase is determined at 538 nm. Dienals also give a red pigment absorbing at 530 nm. which is generated by acid hydrolysis of 1. sulfite. However. In addition. the lipids are boiled for 45 min with a mixture of TBA. the volatiles can be thermally desorbed into a gas chromatograph for separation.1. The sample is dissolved in isooctane.3-tetraethoxypropane [3]. the colored complex was ascribed to the condensation of two moles of TBA and one mole of malonaldehyde (MDA). The TBA test can be standardized using MDA. There are many published methods to determine TBARS. In the micromethod of Ke and Woyewoda [17]. The mass spectra of the compounds can also be compared with spectra of pure standard compounds and . nitrite. and the absorbance at 350 nm is determined after 10 min [15]. This process is accelerated by metal ions [12]. where the headspace volatiles over the samples are sampled.18]. the reaction is not specific. The AnV of freshly deodorized oils is caused by core aldehydes.90 ◾ Handbook of Seafood and Seafood Products Analysis anisidine value (AnV). many other components in foods can react with TBA or interfere with the measurements. Many factors influence the color in the TBA test—temperature. In another method. the AnV is a common method. For determination of secondary oxidation products. amino acids. and trace metals can influence the result [3.13]. the oxidation products are extracted in trichloroacetic acid (TCA) before the reaction with TBA.

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

The Rancimat. It has been shown that sodium hypochlorite-induced decomposition of hydroperoxides gives strong CL [34. The change in conductivity is measured. 7. but it measures the time taken to reach a certain PV. for measuring lipid oxidation [28]. So far. The liquid chromatography–mass spectrometry (LC–MS) techniques can also determine nonvolatile products—of special interest are the core aldehydes [3. Veberg et al. Fluorescence spectroscopy on intact samples has been shown to be a sensitive technique.4 Instrumental Methods Many instrumental methods have been developed for the determination of oxidation parameters in oils and foods. 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.33]. oil stability index (OSI). but studies on the use of this technique in dried fish were published in 1992 [27]. In Rancimat and OSI instruments. the Rancimat test [30]. these include the oil stability index method [29].32. Using solid-phase fluorescence is a relatively new approach. and also cyclic compounds. new methods have been developed. This results in the formation of low molecular weight acids that are flushed out with the air and collected in vessels containing distilled water. including near-infrared spectroscopy (NIR). and Oxidograph are techniques for measuring the stability of oils toward oxidation. The gas chromatography–mass spectrometry (GC–MS) techniques can be used to determine a wide range of volatile secondary lipid oxidation products [36]. The level of hydroperoxides in fish oil can be determined using a rapid CL method [14]. obtaining information .3 Stability Methods Several techniques based on accelerated oxidation are used for evaluation of oxidation. In recent years. 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. [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. 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]. and oxidative stability measurement by Oxidograph [31] and they are all suitable for analyzing oil systems.2. and a few minutes of oxidation of docosahexaenoate (DHA) resulted in significant changes in the ESR spectra. In a study of different model systems including fish and meat. Lipid oxidation products can produce very weak chemiluminescence (CL).35]. 7. Fourier-transform near-infrared (FT-NIR). One challenge is that fluorescence spectra can be very complex and that not only the oxidation products but also connective tissue.92 ◾ Handbook of Seafood and Seafood Products Analysis spectroscopy can be used.2. and additives may contribute to the spectra. porphyrins. aldehydes. and the point where it changes most is called the induction time. little has been done to study the fluorescence spectra of the different oxidation products that are formed in foods. Fluorescence spectroscopy has a great potential for on-line or at-line applications. comparable to sensory analysis and gas chromatography. adipose tissue. active oxygen method (AOM). Free radical assessments by the ESR spin-trapping technique detected the very early stages of lipid oxidation.37]. such as different hydroperoxides. the oil can be heated to 80°C or more while air is bubbled through it. The AOM method is performed in a somewhat similar way. and FT-IR spectroscopy methods [16. 1H NMR spectra can be used to study specific lipid oxidation products.

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

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

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

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........3 Processing Odors ..........................................................................................108 8.4.................................4.............. 98 Fresh Fish Odors ....1.........................................................................................................105 8.106 8........1....................................................................................112 8.....................................................1................1 Sweet....... 111 8..........................4........................... Onion............4...............................................3..................2 8....................1 Microbial Spoilage Odors .............................4 Introduction ....................................4...... and Stale Odors .5 Conclusions ... 100 8...............................4....... 98 Development of Fish Aroma...........2 Dried Fish....4............... Sour..1 Cooked Odor—Boiled Potato and Rancid Odors ..............2............. and Cabbage-Like Odors ............ 99 Identification of Quality Indicators ..............................................4...3 8............................................................................3 Putrid.................103 8................................2............113 97 ..................105 8....................................4......2 Washed Cod Muscle System ...................................................105 8...........................4.... and Malty Odors .......1...........................113 References ..... 111 8.....................................................1 Smoked Fish Odors ....................Chapter 8 Volatile Aroma Compounds in Fish Guðrún Ólafsdóttir and Rósa Jónsdóttir Contents 8...........2 Oxidatively Derived Odors .......................4...3......2 Ripening Odor—Salted and Dried Fish Odor...................................................4 Miscellaneous .........103 8................................ Ammonia-Like............................................1 8.........106 8....................

TMAO. and nucleotides. resulting in undesirable texture changes in fish. The understanding of odor development by chemical. alanine. valine. Other prooxidants like hemeproteins (hemoglobin and myoglobin) are also involved in the initiation of the oxidative processes in fish muscle [7]. 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]. Volatile compounds play an important role in the odor quality characteristics and consumer acceptance of fish. including degradation of nucleotides. anserine). 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. The pool of components that are degraded and cause off flavors because of microbial growth are mainly soluble substances in the muscle. lowering of pH and endogenous enzyme activity. However.2 Development of Fish Aroma An overview of changes during handling and processing influencing the development of aroma in fish is generalized in Figure 8.1 Introduction Health and wellness are the main drivers in new product development. leading to the formation of secondary oxidation products and off flavors [8]. As a result.1. extension in shelf life of fresh chilled fish has been achieved. Finally. Some of these compounds influence the taste of fish-like peptides (i. Initially. cooked. processed. Enhanced oxidation during cooking resulting in off odor development is of concern and an obstacle for application of fish in convenience food. guanidine compounds like creatine. A prerequisite for increased consumption of fish products is their availability on the market as fresh and high-quality products of delicate flavor. Proteolysis plays a critical role in postmortem changes. They are composed of the various nonprotein nitrogenous components (NPN). the changes are dominated by autolytic activity. Fish being a valuable source of polyunsaturated fatty acids (PUFA) and other nutrients is a prominent candidate as the healthy choice for consumers. or hydrolyzed products and as ingredients in functional foods. Research over the years has led to improved chilling and packaging technologies aimed at reducing microbial growth. 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. 8. and glutamic acid are known to contribute to taste together with the degradation components of the nucleotides such as inosine. contributing to spoilage changes and thus influencing the freshness and quality of the end product of chilled fish [1–3]. biochemical. and accumulation of hypoxanthine (Hx).. oxidative processes causing odors and texture changes become noticeable during extended storage and limit the shelf life. followed by oxidation processes. active inosine.98 ◾ Handbook of Seafood and Seafood Products Analysis 8. Endogenous enzyme . studies on application of natural antioxidants are of prime interest to underpin further utilization of fish in innovative product development as fresh. including small peptides such as carnosine and anserine. Degradation of soluble muscle constituents such as sarcoplasmic proteins and microbial metabolism contributes to changes in the aroma profile of fish during storage. and the individual amino acids glycine. 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.e. formation of taste. the proliferation of the specific spoilage organisms (SSO) results in the development of volatile compounds. Consequently. amino acids.

stale. malty. Josephson et al.e. and melon-like odors. NPN. LOX activity on the skin and gills of both freshwater and marine species (rainbow trout. neutral Spoilage aroma sweet. which have potent green. spoiled. specific spoilage organisms. pleasant aromas of fish [6. ammonia-like Oxidized aroma Processed aroma green-like. cucumber-.3 Fresh Fish Odors The delicate flavor of fish is mostly contributed by volatile compounds and taste active substances in the aqueous phase. were responsible for the moderate. Mb.5. SSO. metallic. the unsaturated C9 carbonyl compounds such as 2. drying. eight. mushroom. whereas volatiles generated from fat result in variation in the specific flavor character of different fish species. were characteristic for freshwater and euryhaline fish. trimethylamineoxide. freezing.10–13]. cucumber-. NPN. melon-. 1. but the mechanism of this activity is not fully elucidated [9]. but . myoglobin. and hydrolysis Endogenous enzymes i. salting. cucumber. Soon after harvest. phospholipases TMAOase Microbial metabolism Specific spoilage organisms (SSO) Oxidation Prooxidants: metals (Fe. peptides Soluble substances. nonprotein nitrogen-containing compounds. The overall perceived odor is dependent on the level of influential compounds and their odor thresholds along with possible synergistic effects.5-octadien-3-ol. dried fish. nucleotides. rancid potato. cucumber Figure 8. boiled potato. Hb. and sardines) plays a role in the formation of odorous volatiles. TMAO. hydrolases. 1-octen-3-ol. hemoglobin. The compounds that contribute to the characteristic plant-.1 Overview of changes in fish influencing the development of characteristic aroma of fresh. Newly caught marine fish contains low levels of volatile compounds and is nearly odorless. oxidized. and 2. [5] summarized the occurrences of volatile compounds in freshwater and saltwater species and concluded that the four common compounds found in saltwater species. contributing to green. LOX proteases..15]. polyunsaturated fatty acids. putrid. popcorn.Volatile Aroma Compounds in Fish ◾ 99 Handling chilling. LOX. On the other hand. or nine carbon atoms [4. Mb Antioxidants: α-tocopherol. lipoxygenase. ascorbic acid. hexanal. caramel. Some components are desirable at low levels. including calpains (neutral calcium-dependent proteases) and cathepsins (lysosomal proteases). sour. faint odor of saltwater species. malty.14. and cooking Processing smoking. PUFA. activity influences the deterioration of fish muscle. amino acids Fresh fish aroma seaweedy. river trout. and mushroom-like odors are unsaturated carbonyl compounds and alcohols with six.5-ocatadien-1-ol. and processed fish. 8.6-nonadienal. plant-. stockfish. polyphenols Lipids phosholipids/PUFA Proteins sarcoplasmic.Cu) Hb.

including (E)-2-nonenal. a saltwater species. plant-like notes in fresh fish.24–29].1 summarizes the occurrence of volatile compounds detected in our studies on cod [22] and haddock fillets [31] and smoked salmon [23]. indicate their involvement in the development of fresh fish aroma associated with seasonal variation. Studies performed in Japan. (E.22] and in smoked salmon [23]. on accumulation of hydroperoxides in fish tissues. The volatile pattern changes in mature salmon when migrating from the sea for spawning. The main classes of compounds detected during storage are alcohols.4 Identification of Quality Indicators Different characteristic odors develop in various fish species during storage. which has a very characteristic cucumber odor during spawning. Fatty species develop rancid odors and taste. as seen by the detected odors listed in Table 8.Z)-2. and sulfur compounds.6-Nonadienal was identified to be the most characteristic compound for the cucumber-like capelin odor [19]. they contribute to oxidized and fishy odors in stale fish [16]. 8. lean species typically develop sweet.6-nonadien-1-ol in sweet smelt tissues [20]. However. odor. Another example is iodine-like off flavor in prawns associated with bromophenols originating from the feed chain [17]. The aldehydes contribute most to the spoilage odors because of their low flavor thresholds. boiled potato-. iodine-. . aldehydes. Therefore. and GC–O. They were suggested as the possible precursors of nine-carbon volatile compounds. but when accumulated in higher levels because of autooxidation. 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. and 3.30–36]. and marine-like flavors of seafood [18]. amines. and quality changes can be explained in. for example. and species of the salmonidae family develop earthy. they may contribute to off odors. Table 8. 2. ketones. Both single compounds such as TMA and ethanol and multicompound indices based on combination of alcohols. muddy. and sulfur compounds representing the different changes occurring during storage have been suggested by numerous researchers as indicators for freshness and spoilage [22. and sweet odors.1. which are present in higher levels and can be quantified. 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. acids. GC–MS. Seasonal effects have also been reported for capelin.21–22. An example is the enzymically derived long-chain alcohols and carbonyls that exhibit characteristic fresh. in nominal levels the bromophenols appear to contribute to natural sea-. Accumulation of certain hydroperoxide isomers coincided with the period of enhancement of characteristic aroma in sweet smelt.6-nonadienal. amines. esters. where it has been demonstrated by monitoring key volatiles to study changes in different fish products during storage. C9 LOX-derived compounds have been found in higher levels in spawning euryhaline and freshwater fish [5]. Environmental conditions and seasonal effects like spawning can influence the odor quality of fish.100 ◾ Handbook of Seafood and Seafood Products Analysis if their concentration increases. Purge and trap on Tenax and SPME methods were applied for sampling. cod during storage [21. Some of the influential odor compounds that have very low odor thresholds are often present in low levels. 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. and amine-like odors. it is useful to monitor the overall pattern of volatile compounds and select indicator compounds. and identification was based on GC–FID. and these are difficult to detect by analytical techniques. This has been the approach in our studies.

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. caramel. 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. earthy Sweet. fatty — Fresh. flowery — Ketones 2-Butanone 2.4-Heptadienal. floral Sweet. candy × — — Sweet.1 Volatile Compounds Detected in Cod [22]. caramel.3-Butandione × × × × — N/A (continued) .Volatile Aroma Compounds in Fish ◾ 101 Table 8. fish fillet Sweet. (E.E)Nonanal Decanal Undecanal × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × Rancid Boiled potato.

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

and TMA-like smell.Volatile Aroma Compounds in Fish ◾ 103 Table 8.1). cucumber-. and when combined with frozen storage odor.3-butandiol were found in the highest levels on day 12 at sensory rejection. and Malty Odors Ketones. dried fish/stockfish. 8. and quantification of the main classes of compounds was based on the sum of the PAR for respective compounds in each class. 3-methyl-1-butanol. which is mostly affected by handling. 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. The flavor thresholds . mainly amino acids. The microbially derived alcohols 2-methyl-1-propanol. Identification of volatile compounds was based on GC–MS analysis (see Table 8. Sweet-milky and vanilla/caramel-like odors are typical in cooked fish. the aroma of the fillet is described as sweet and reminiscent of shellfish. sour. The loss of freshness of cod fillets and early spoilage changes were related to the formation of ketones. acids. Seaweedy and marine-like odors. or geraniumlike odors are characteristic sensory odor descriptors for fresh whole fish. Late spoilage changes. and sulfur compounds produced by microbial degradation of fish components. —.1. esters. sulfur. alcohols.4. development of spoilage odors. During prolonged storage boiled potato odor develops. Haddock Fillets [31].4.1 (continued) Volatile Compounds Detected in Cod [22]. data not available for haddock. 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. 8. and sometimes metallic. and finally sour and dirty tablecloth odor.1 based on GC–O analysis of cod and smoked salmon represent most of these overall changes. contributing to sweet.2) were associated with the development of sweet. and temperature conditions during storage [33. N/A. the fish is no longer fit for consumption. Sour.34]. packaging. as well as green plant-.5°C) [22]. In general when fish is cooked.1 Microbial Spoilage Odors The spoilage odors in chilled fish vary depending on the dominant microflora in the products. showing results from a storage study of cod fillets packed in styrofoam boxes during chilled storage (0. and malty spoilage odors. not detected by GC–O. mushroom-. meat-like. and malty odors. The odor descriptors in Table 8. and aldehydes. and the end of shelf life of cod fillets on day 12 of storage are explained by the presence of TMA. cooling. and aldehydes detected on day 4 of storage and their increasing levels on days 7 and 10 (Figure 8.2. An example of the spoilage pattern of volatile compounds in chilled fish is illustrated in Figure 8. and 2. alcohols.1 Sweet. 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. sour. the freshness notes disappear and the odor of the uncooked fish becomes neutral. After several days of storage.

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

3 Putrid. and the incorporation of hydrogen sulfide yields dimethyl trisulfide [38]. The lipid-derived saturated aldehydes detected on day 12 at sensory rejection also contributed to the overall sweet aroma. ammonia-like. which may have influenced the overall odor perception leading to the sensory rejection of the fillets. Oxidative processes are involved in the formation of dimethyl sulfide from methyl mercaptan and further oxidation of dimethyl disulfide. TMA is characteristic for the spoilage odors of fish. At this point there was an increase in the pH value.4 Miscellaneous The concentration of the straight chain alkanes (nonane. which contributed to boiled potato-like odors (Table 8. and stale odors by amines during fish spoilage is well known.Volatile Aroma Compounds in Fish ◾ 105 storage. 8.4. and Stale Odors The development of dried fish.39]. fruity off odors [37. Additionally. TMA is a potent odorant with a characteristic fishy. In whole fish stored in ice.38. 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. Dimethyl trisulfide has also been associated with spoilage in fish and associated with the growth of Shewanella putrecfaciens [25. and dimethyl disulfide have been suggested as the main cause of putrid spoilage aromas [41]. Ketones can influence the overall odor because of their low odor thresholds. decane. 1-penten-3-ol).1. ammonia-like odor. but no obvious increase occurred until at the end of shelf-life and during continued storage. 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.1. alcohols (3-methyl-1-butanol. whereas DMA may influence the overall fresh flavor of fish in combination with oxidatively formed aldehydes from long-chain fatty acids in fish. Figure 8. dried fish.2 shows that TMA was detected in high levels on day 12. described as sickeningly sweet and nauseous.2 Dried Fish. volatile sulfur compounds such as hydrogen sulfide. methyl mercaptan. has been suggested as a freshness indicator along with its precursor TMAO (trimethylamine oxide) [27]. The onset of stale odors can be explained by cis-4-heptenal and heptanal.1. respectively [41]. and Cabbage-Like Odors Low levels of sulfur compounds (Figure 8. The odor of ethyl butanoate.4. 8.1). Enzymically produced DMA (dimethylamine). contributing to the stale and putrid off odors in fish because of amino acid and lipid degradation [39]. methyl sulfide. Additionally. TMA has been noted for intensifying fishiness by a synergistic action with certain volatile unsaturated aldehydes derived from autoxidation of polyunsaturated fatty acids [40]. which forms very early after harvest of fish. and undecane) appeared to be similar throughout storage in chilled cod fillets [22]. Pseudomonas species have also been found responsible for the formation of volatile sulfides.2) indicated that they were not important in the spoilage of chilled cod fillets stored in styrofoam boxes. Onion.38]. numerous branched chain . The origin of the sulfur compounds is microbial degradation of cysteine and methionine to form hydrogen sulfide and methyl mercaptan.4. contributed to the sensory rejection of chilled cod fillets on day 12 and suggested the role of Pseudomonas fragi in the development of sweet. Ammonia-Like. and ketones (2-butanone).

and decanal. Piperidine levels have been reported to increase in spawning salmon and contribute to off odors [43]. and terpenes found in wild sea bream compared with those of its cultured counterpart [29]. These compounds have been associated with rancid and dried fish odors. which is further enhanced by preprocessing and storage of fish. cis-4-heptenal.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. although their overall levels were lower. 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. Limonene has low odor threshold and a fresh lemon odor was detected by GC–O analysis of cod. they are in particular sensitive to oxidation.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]. However. Limonene has also been detected in sea bream during storage [29].106 ◾ Handbook of Seafood and Seafood Products Analysis alkanes were detected. 2.4. suggesting that it may have an impact on the overall odor of fish fillets [22]. and 2. but the knowledge of the formation of these compounds is obscure.4-heptadienal and 2. Aldehydes generally have low odor thresholds.4. and because of their high unsaturation. lipid-derived saturated aldehydes. 3-pentanone. which are known to be more susceptible to oxidation than triacylglycerols in fat deposits [45]. since they are not aroma active. fish-like odors of chilled cod fillets in combination with other carbonyls (3-hydroxy-2-butanone.7-decatrienal) should not be overlooked. and overall the alkanes showed an increasing trend with storage time. 8. and 6-methyl-5-heptene-2one). heptanal. ketones. The origin of limonene in fish is most likely related to the diet derived from algae or plant source. that contribute to the development of rancid cold store flavors [47]. 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]. were detected in the fillets throughout the storage time.7-decadienal. 8. Oxidative processes occurring during storage of fish result in the accumulation of aldehydes.4. Boiled potato.2. but the sampling techniques used were not sensitive enough to allow quantification of these compounds. The influence of other aroma active compounds present in lower levels such as the unsaturated autoxidatively derived aldehydes (2. Phospholipids are the main membrane-bound lipids. Several odor active terpene derivatives have been identified in fish. their impact was greater than alcohols and ketones. such as hexanal. 2-butanone.4-heptadienal. Similarly. Piperidine was tentatively identified in chilled cod fillets [22] and has also been suggested as a quality indicator in sea bream [29]. the feed may have influenced higher levels of aldehydes. aromatics. These oxidation products contributed to the overall characteristic sweet. A characteristic earthy odor in many species residing in ponds has been associated with piperidine and its reaction products.3 to demonstrate which odors are most dominating in the aroma profile [48].4. Various pro and antioxidants influence the stability of the muscle and have been studied in relation to the oxidative stability of phospholipids [46].and potato-like odors contributed by . they are not considered of interest as quality indicators. therefore. and. in similar or slightly increasing levels. such as hexanal. 3-methyl-butanal.

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

Autoxidatively produced unsaturated carbonyl compounds were the most abundant components in boiled and canned fish. in particular the role of volatile compounds derived from oxidation in heated/boiled samples. However. 14. dimethyl disulfide. The characteristic pattern or trend in volatiles in raw and boiled fish is clearly different.6 0. 12. The effect of oxidation induced by cooking and formation of oxidation products such as heptenal and nonanal characterizes the (B-D4) sample. 19% PC1 1. The oxidatively formed compounds.5 –0. and oxidatively derived (Z)-1.6-nonadienal. increased with time and were pronounced in the spoiled raw samples (R-D14 and R-D17). that is. 7.4-decadienal from PUFA were determined as character impact odorants of boiled cod [54]. 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). Sulfur compounds dimethyl sulfide.0 B-D17 1-Penten-3-ol 3-me-butanal Acetic acid 0.5 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. 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. 3-methyl-butanal in combination with acetaldehyde. On the basis of odor evaluation. 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.Z)-2.2 Raw and boild c….2.108 ◾ Handbook of Seafood and Seafood Products Analysis PC2 Bi-plot 1. and dimethyl trisulfide were detected in higher levels in the boiled samples (data not shown).2 0. Interestingly. especially in trout [15]. Samples are labeled with R. and (E.8 R-D17 –0. (E. samples after heating (see Table 8. X-expl: 53%.4 0.0 Figure 8.4 Principal component analysis of raw and boiled cod.5-octadien-3-one. methional with a characteristic boiled potato-like odor dominated the odor of the aldehyde fraction of the headspace volatiles.4 –0.4. decanal. Only the spoiled raw samples (R-D14 and R-D17) can be correlated with the freshly boiled (B-D4) sample. as indicated by the arrows (Figure 8.1). 8. in agreement with earlier studies [54]. methional. and 17 days). . 10. D (4.4). hexanal.4).2 Washed Cod Muscle System Rancid odor development during chilled storage of fish has commonly been associated with fatty species. 3-methyl-butanal was correlated to the boiled stored cod (B-D17) (Figure 8. The malty flavor of 3-methyl butanal was suggested earlier to be mainly responsible for the malty off flavor defect of boiled cod [54]. boiled and storage days. oxidation of membrane-bound phospholipids in lean species can cause fishy. raw and B. and 6-methyl-5-hepten-2-one. In boiled trout.

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

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

and furans have been found in spray-dried shrimp powder and shrimp hydrolysate [69]. 3-methyl-butanal. potato-like odors. 1-octen-3-ol. Phenolic derivatives like guaiacol (2-methoxyphenol) and syringol (2. Microbially produced ketones. plays important roles in the formation of complicated processing flavors. which is typical for products on the market [23]. gave the most intense odors of smoked salmon and contributed to the fish-like earthy odors and fatty and rancid odors (Figure 8.6-nonadienal. Key volatile compounds identified in enzymatically produced seafood flavorants are formed via Maillard reaction and Strecker degradation of amino acids. like cis-4-heptenal.4-heptanal. Figure 8. 2-methyl-1-butanol. giving a popcorn-like odor that can be thermally generated. such as heptanal and (E.72]. [74] analyzed headspace components of cod and swordfish. Other oxidatively derived compounds like 1-penten-3-ol.7 illustrates the main odors that were present in smoked fish samples after 14 days of chilled storage. . In addition to phenolic compounds. 2-pentanone. it is clear that their presence contributes to the characteristic fish odor of smoked salmon products.4.6-nonadienal. and 1-octen-3-ol. hexanal. 2-butanone. 1-penten-3-ol. sweet odors of processed seafood like those in smoked salmon [23. giving rancid. where groups of phenol pyrolysis were most noticeable in the smoke flavor volatiles. the Strecker aldehyde produced from methionine. furan-like compounds have been reported to be responsible for the smoked odor in smoked salmon. sweet/sour rancid. Additionally. 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]. 3-methyl-1-butanol. it was verified that selected key volatile compounds performed better as predictors to explain variation in sensory attributes (smoked. associated with spoilage off flavors. also contribute to the aroma of seafood flavorants [70].g.4-decadienal. and 3-methyl-1-butanol) [23].1 Smoked Fish Odors Degradation compounds from Maillard reactions and lipid oxidation are the main compounds contributing to the aroma of smoked salmon [72]. 3-hydroxy-2-butanone. hexanal. nonanal. thermal degradation. and lipid oxidation. aldehydes. These are compounds like methional.72]. and 2. 2. which has a characteristic potato-like odor. Guillén et al. 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. The typical smoked salmon aroma results from a number of chemicals found in the smoke. and 2-acetyl-1-pyrroline. Lipid-derived components. including Strecker degradation.Z)-2. but it is mostly attributed to the phenols. ethanol. 8. and alcohols were abundant in the headspace of cold smoked salmon products during storage. The oxidatively derived compounds cis-4-heptenal and heptanal..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.72]. Maillard reaction.71.75]. giving the flesh its typical fishy odor [71. like 3-methyl butanal. and decanal were among key volatiles. were characteristic in unsmoked fish.7) [23].6-dimethoxyphenol) have been identified as the most characteristic smoke-related compounds in smoked fish-like herring (Clupea harengus) [73] and in smoked salmon (Salmo salar) [23. and 1-propanol [28.4. 2.Volatile Aroma Compounds in Fish ◾ 111 8. contributing to mushroom-like odor.7) (e. whereas carbonyl compounds. Lipid-derived aldehydes play an important role in flavor formation and have been reported to contribute to the characteristic fish-like. and off odor and flavor) than traditional chemical and microbial variables.3. Volatile compounds like alkyl-pyrazines and sulfur-containing compounds have been found in cooked crustaceans. and although they contributed less to the odors.

as heptanal. fruity Flowery. Similar processes have been reported in ripened seafood products. During ripening of salted cod. earthy. most of them generated from chemical or enzymatic oxidation of unsaturated fatty acids and further interactions with proteins. et al. Thus.4-heptadienal and 3. 184. ripened roe products [79] Similarly. where methional derived from methionine and 2. 4-Heptadienal Sweet.) 8. potato-like odors together with cucumber-like odor [82]. fatty Boiled potato-like Fatty. mushroom 2. the highest odor scores were given for boiled potato and rancid. especially those in the Mediterranean.5octadien-3-one were also identified as potent odorants in ripened anchovy [81]. 109. potato-like odor was identified as cis4-heptenal and the boiled potato-like odor. and rancid-like odors Burnt. In our study.. geranium Rancid cis-4-Heptenal Heptanal Earthy-like odors 1-Octen-3-ol Figure 8. smoke 3-Methyl butanal Sweet. Salted cod are traditional products from the North-Atlantic fisheries and are highly regarded as ripened fish products in many countries. Triqui and Reineccius [80] found that 2. sweet Smoke-like 2.and 3-Methyl phenol Guaiacol 4-Methyl-guaiacol Sweet and fruity-like odors Wood.4.7 GC–O evaluation of volatile compounds detected in cold smoked salmon after 14 days of storage at 5°C.. sweet Flowery. sweet Wood. and free amino acids. Methional and (Z)-1. However.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. 2-methylpropanal and 3-methylbutanal were the key. probably originating from amino acids. R. 2008. they suggested that lipid autoxidation during ripening was primarily responsible for aroma development.6-nonadienal from fatty acid oxidation were the main odorants in sugar salted. [76–78]. smoke Mushroom. and aldehydes such as acetaldehyde. . burnt. caramel Smoke-house.3. highly volatile components of ripened anchovy. where the ripening of salted cod (Gadus morhua) produced by different salting methods was studied. (Modified from Jónsdóttir. sweet Flowery. peptides. Food Chem. The rancid. smoke. the desired flavor and texture develop as a consequence of protein and fat degradation. both oxidatively derived compounds.2 Ripening Odor—Salted and Dried Fish Odor Numerous volatile compounds have been detected in ripened products like dry cured ham.5-octadien-2-one were associated with the development of the typical flavor obtained after anchovy ripening.

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

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

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

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PROCESSING CONTROL II .

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..1 Near-Infrared Spectroscopy......................... and Analysis .1 Determination of Basic Composition......................... and Margrethe Esaiassen Contents 9....132 9....................3 NMR Spectroscopy ............ 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..5 Summary .. Measurement Principles....................................... and Data Analysis . 134 References ............................................................ 122 9.................................4.................................. 128 9.......................................... 122 9............................................ 128 9. 122 9.........................132 9..............................4...................................................................... 124 9.............................2 Imaging Spectroscopy ............3......................2 Theory..............133 Acknowledgment ........2........................ Measurement Principles.................................2.132 9...........................................................2 Theory and Measurement Principles ................................................................................................................2 Analysis .................................................................Chapter 9 Basic Composition: Rapid Methodologies Heidi Nilsen............................................ Karsten Heia.........................1...........................3............2 Analysis of Basic Constituents ...............1 Determination of Basic Composition.........................4 X-Ray Imaging ............1 Theory and Measurement Principles ............................................. 121 ..3........... 130 9.....................................................................................1................................................................1 Theory..........................................................3 Analysis of Basic Constituents ..................................................... 128 9. 130 9.......................................131 9...........................3 Analysis of Basic Constituents ......................................1..... 130 9..................................................................

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

For both (a) and (b).10]. The setup in (b) displays the reflection setup where light reflected from the sample surface enters the detector. 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.1 Different measurement setups for NIR spectroscopy. Different measurement modes for NIR spectroscopy are illustrated in Figure 9.11]. both with respect to the detectors and the capture of the spectral information [10. A setup as shown in (a). traditional chemical determination of the constituents. If the light source and the detector are placed on the same side of the sample as shown in (b).1. (c) illustrates how measurements are performed in “transflection” mode. we view this in terms of the measurement setup enabled by technology. The broad spectral bands may be an indication of the material constituents. the spectral readings must be correlated to a relevant reference method such as. where the light passes through the sample from one side to another. Over the years there has been a steadily ongoing development of instrumentation for NIR spectroscopy. In (a) the transmission setup is shown.Basic Composition: Rapid Methodologies ◾ 123 and N−H [9] and corresponds mainly to overtones and combinations of fundamental vibrations. In the context of rapid methodologies. enables “transmission” measurements. A common methodology is chemometrics or Detector Shelter Sample Light source (a) (b) (c) Figure 9. . but focusing the two devices so as to ensure that the light has traversed some region of the sample before detection. the system is operated in “reflection” mode. In (c) the light source and detector are located to register light that has traversed the sample before detection. the transmission and reflection may be either direct or diff use. and noncontact methods. depending on the scattering properties of the medium under investigation. for example. a screen is placed between the directly emitted area and the area of inspection. developed toward the facilitation of nondestructive. Finally. 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. but an immediate look at an NIR spectrum is not sufficient to quantify the different substances. placing the light source and the detector at the same side of the sample. Hence. 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.9. nondisruptive. NIR spectroscopy is an indirect measurement technique. light from the source penetrates the sample and enters the detector. In order to prevent direct reflection from the surface.

124 ◾ Handbook of Seafood and Seafood Products Analysis multivariate data analysis. Being the basic nutritional components of any food. Among the most used multivariate techniques are principal component analysis (PCA). [19] performed a study on live anesthetized farmed salmon. For the measurement of fat and protein. and the sample preparation included mincing and freeze drying of the material to be evaluated. The earliest reports of NIR spectroscopy to measure chemical components in fish appeared more than two decades ago. The measurements were performed by use of fiber optic bundles conveying the light to and from the sample site. In 1987 Gjerde and Martens [13] demonstrated the applicability of NIR to predict water. a model based on several wavelengths is required to extract useful information from the spectroscopic data. 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]. a substantial part of the work related to NIR analysis of fish and food from fish concerns the quantification of the chemical constituents. the reference method may be replaced by the spectral reading and the analytical model. Darwish and others [15] used the technique in 1989 to measure fat. fat. and protein in cod. 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 water.1. as in the work of Lee et al. If there is good correlation between the spectral measurements and the method of reference. The same year Mathias et al. and rapid method for the assessment and quantification of these constituents is considered a valuable tool in the quality evaluation of any foodstuff. intact rainbow trout. the study concluded that the method could be a useful tool for rapid quality control. it was possible to estimate the lipid content of the intact muscle. and soft independent modeling of class analogies (SIMCA) [12]. the measurement locations for obtaining the best calibration results were also addressed. protein. namely. whereas water determination was made on the water extracted from the fish mince. In spite of the rather cumbersome sampling procedure. In both studies reflectance measurements were performed. Farmed salmon is of high commercial value and a worldwide favorable product. Downey [18] applied a similar spectroscopic setup to measure fat and water content of intact farmed salmon. This could account for the many studies relating to the rapid analysis of the basic chemical composition of . Sollid and Solberg [16] measured the fat content in salmon by transmission spectroscopy on raw minced muscle. the samples were minced and dissolved in a milk-like emulsion. 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. water. Both of these early reports concluded that the method was promising in terms of speed and efficiency when measuring a large number of samples. Consecutive research articles proved the feasibility of the tool in developing the method to apply with simpler procedures of sample preparation. [17]. an easy. In the following paragraphs. partial least square (PLS) regression. 9.3 Analysis of Basic Constituents As found in NIR analysis of foods in general. mackerel. by use of NIR in connection with fiber optics Solberg et al. fingerling Arctic charr and rainbow trout. In addition. This measurement setup clearly displayed how NIR spectroscopy could be used in a nondestructive way. reliable. [14] reported the use of NIR spectroscopy to determine lipid and protein content in freshwater fish. and. demonstrating the possibility to determine fat content in live fish. Typically. Based on measurements through scales and skin. fat. and tuna. and protein content in rainbow trout.

water. Torrissen. and protein than those made on intact muscles.. Nortvedt. and additionally the spectroscopic measurements could be used for origin identification or authentication of the samples. microwave. as well as on minced salmon muscle.) Spectral measurements were performed on intact fillets by transflection measurements by use of the fiber optic probe of the instrument NIRS6500 (Perstorp Analytical Inc. and protein content of European sea bass. and NIR spectroscopy. Silver Spring).600067 0. NIR spectroscopy has been used for evaluating the chemical composition of several other fish species as well. and Tuene [24] made use of NIR transmission spectroscopy to assess protein. Torry fatmeter. In this work they applied a fiber optic measurement setup. water. (From Nilsen.22] also conducted studies documenting the efficiency of applying NIR spectroscopy in different measurement modes to assess fat and water content in salmon. applying minced samples for the spectral readings. [26] showed that NIR spectroscopy could be used to estimate lipid.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. and dry matter in halibut fillet. Unpublished data. Xiccato et al.Basic Composition: Rapid Methodologies ◾ 125 salmon. Measured Y Elements: Slope: Offset: Correlation: RMSEP: SEP: 21 Bias: 24 78 0.002328 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.. whether pre-.K. Transmission spectroscopy was also employed for the analysis of fat and dry matter in capelin [25]. In a research article published in 2004. In a recent work by Khodabux et al. N.264047 0. (Y–var. Isaksson et al. or postspawning. Spectroscopic readings obtained on the minced samples correlated better with the reference measurements on fat. In both the works of Vogt et al. The study concluded that NIR is well suited for nondestructive quality evaluation of salmon fillets. and Sørensen. 8) Figure 9. . The fat content of herring has also been assessed by the use of NIR spectroscopy. [29] one question of interest was the comparison of different methods for measuring fat content. fat.603947 0.937575 0. An example illustrating the use of NIR spectroscopy for assessing fat content in farmed salmon is given in Figure 9.2. [21. in. [27]. [20] conducted a study in which they compared NIR measurements on intact salmon fillet. NIR spectroscopy was proven to be a useful tool for the evaluation of basic constituents of different types of tuna. [28] and Nielsen et al. H. Wold et al. This work also emphasized the impact of the conditional state of the fish when making calibration models. 1998. PC): (%fettHS.985681 0.

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

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

In addition to what traditional spectroscopy can facilitate. or the result from these techniques can be postprocessed to utilize the spatial information [46]. demonstrates the potential of the method in the seafood sector as well. and Analysis Imaging spectroscopy. an analytical tool for industrial quality control of clipfish and salmon fillets. For instance. the spectra may be recorded in the visible and near-infrared region. As these techniques only use the spectral information. Depending on the applied sensor technology. To simplify the concept. in general. Measurement Principles. also known as multispectral imaging or hyperspectral imaging. Oslo.1 on NIR spectroscopy. 9. improved results can be obtained by combining these techniques with more traditional image processing techniques. this can be illustrated as simultaneously recording information about shape and color. Most of them are on foods such as fruits. As described in Section 9. Several solutions have also been developed for detection of defects and contaminations on fruits. Th is means that for each spatial location it is possible to access the full spectral information. total soluble solids.44]. and acidity (expressed as pH) [47–49]. is a new technique that has been developed during the last decade [43. some examples related to the agricultural sector are referred. the hyperspectral data can be preprocessed based on spatial features before applying analytical spectral techniques. 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. 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]. an imaging spectrograph operates in the following way. this technique also provides spatial information. It has been shown that NIR hyperspectral imaging techniques are . In this way an image of the object is built line by line. and each frame captured provides full spectral information for one line across the object to be imaged. this method is an indirect measurement technique. Norge).2. Between each captured frame. In order to illustrate the potential parameters to be assessed by imaging spectroscopy. reflection. Typically. and meat. it uses a two-dimensional sensor.2. A novel example of this is the development of the QMonitor (QVision AS. The analytical techniques described in that section are also applied to imaging spectroscopy data. vegetables.1 Theory. It has become a widely used technique within fields spanning microscopy to satellite remote sensing. Typically. 9. 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. Imaging spectroscopy can be implemented for transmission. the spectrograph and the object must move relative to each other. For fruits and vegetables more articles report on determination of chemical constituents such as moisture content. the feasibility of the method for the analysis of basic composition of foods.128 ◾ Handbook of Seafood and Seafood Products Analysis the near-infrared spectra.2 Imaging Spectroscopy 9.2 Analysis of Basic Constituents During the last decade several applications within food-quality inspection have been developed based on imaging spectroscopy. There are still relatively few reports on imaging spectroscopy applied for the analysis of fish and seafood. as well as transflection measurements. However.

color. QVision (Oslo. The mean fat content for this fillet is 18. A thorough review of imaging spectroscopy applications within fruits and vegetables is presented by Nicolai et al. pH. A recent work on quality assessment of pork has been reported by Qiao et al. Peeling of shrimps and detection of nematodes were mentioned as possible applications for the future.60] where several quality parameters were evaluated by imaging spectroscopy.Basic Composition: Rapid Methodologies ◾ 129 useful for automatic online detection of surface defects and contaminations on apples [50–52]. whereas the local fat content varies from approximately 6% up to 43%. imaging spectroscopy solutions for detection of contaminants such as fecal and ingesta on poultry carcasses have been studied [56–58]. [35]. [53]. Since 2000. measuring the water content in one spot is not necessarily representative for the whole fish.3%. Norway).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. [61]. and cadaver [54. the moisture content of the fish varies from the thinner parts to the thicker parts of the fish.55]. and different texture features. The first article addressing analysis of fish or seafood by imaging spectroscopy was published in 2000 by Sigernes et al. The parameters included were drip loss. Inspection systems based on hyperspectral imaging have been tested for poultry carcass inspection focusing on classification of carcasses into normal. When drying fish. . Further on.3055% Share: 21. 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. Fisk: 1 Fettfisk: 18. there is one recent publication on assessing water content in salted dried cod by Wold et al.4). septicemic. [59.4 Fat distribution in salmon fillet measured by the multispectral imaging system QMonitor fabricated by QVision (Oslo. The color bar to the right indicates the correspondence between color and fat content in percentage. Hence. Norway) has also developed an industrial solution based on multispectral imaging for measuring the fat content in salmon fi llets (see Figure 9. In this publication the importance of including spatial information is illustrated. Regarding the determination of basic chemical composition of fish and seafood.

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. QVision. With imaging spectroscopy this is not a problem since spectra are available for all spatial locations.3 NMR Spectroscopy 9.3. Furthermore. The most commonly measured nuclei are 1H and 13C. NMR active nuclei absorb at a frequency characteristic of the isotope. Additionally. and they are based on the magnetic properties of atomic nuclei. and the methods that are feasible by spot measurements may also be implemented using imaging spectroscopy. For detection of flaws or defects in fish. For NIR spectroscopy several applications within fish and seafood are reported. 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. 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. but during the last few years magnetic resonance imaging (MRI) has also been explored for its usefulness in food analyses. 9. NMR spectroscopy may provide detailed . but this requires that the same spot be used. All nuclei that contain odd numbers of protons or neutrons have an intrinsic magnetic moment and angular momentum. this is a relatively new field. since it is possible to use spectra from dedicated relevant areas on the sample. Still the number of imaging spectroscopy applications with fish and seafood is low. and skin remnants in whitefish fillets [62–64]. measurements may be performed at high speed as well as in noncontact mode. The main technique used is NMR spectroscopy. Oslo. NMR techniques use electromagnetic radiation and magnetic fields to obtain chemical information. and currently there are a limited number of equipment suppliers. [63].3. Using the interaction between light and the sample object. With respect to commercial implementation of imaging spectroscopy. 9. black lining. imaging spectroscopy of fish has been applied to address other quality issues. blood spots.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. Even more important is that for some applications imaging spectroscopy can provide better results. When an external magnetic field is applied. For instance.2 Theory and Measurement Principles NMR provides a large amount of information regarding composition and structure of components in food. a lot of effort has been invested in the detection of nematodes. experience with NIR spectroscopy shows that more than one attribute can be estimated based on one recording.130 ◾ Handbook of Seafood and Seafood Products Analysis In addition to the measurement and documentation of basic composition. Hence. Imaging spectroscopy is well suited for application in the fish processing industry as an online technique. Norway) in fish. but looking at reported applications within other areas the potential for new applications is high. A low-resolution (spectral and spatial) instrument is available for industrial assessment of chemical composition such as fat and water content (QMonitor. 31P-NMR and 23Na-NMR have also been used for food analyses.

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

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

instrumental means capable of objective and rapid determination of basic composition are also available. Iceland).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. Iceland).5 for an example). 9. 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.Basic Composition: Rapid Methodologies ◾ 133 Figure 9. Marel developed an X-ray-based bone detection unit (SensorX) that was commercially available on the market in 2003 [87]. This instrument can detect bones and bone fragments down to a diameter of 0.5 Detection of pin bones in fish fillets by x-ray imaging using the SensorX instrumentation (Marel. A similar work has been carried out by Hancz et al. [86] showing good results predicting fat content of common carp based on CT scanning. Throughout development all presented techniques have met the requirements . authors recommended CT scanning as an online technique for carcass evaluation. With respect to bone detection in fish fillets there are commercial solutions available today (Marel Hf.3 mm when operating at industrial speed (see Figure 9.

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

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

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...........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.............. 151 10...........................................1 Smoked Salmon.......2 Salted Cod............ etc...................140 10......148 10...................... 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.............................................................................3 Processed Fish Microstructure ..............2...............................145 10..................................................... 148 10........................150 10............................................................................ fats....... and María-Angeles Lluch Contents 10......146 10................................................... Empar Llorca............. This chapter 139 ..................3 Surimi ........) is responsible for their microstructure..................................139 10......... The organization of the chemical components of foods (proteins...........................................................2 Hake ..............................2 Fish Muscle Microstructure................... 149 10..............................1 Herring .............1 Main Microscopy Techniques for Studying Seafood .......................... Several strategies can be used to study food microstructure.......3........................... 151 References .3... (2008) gave an overview of the most important techniques for studying muscle food structure....................4 Squid Microstructure .........................................................................Chapter 10 Microstructure Isabel Hernando............. Ana Puig.............3...... Pérez-Munuera et al.......................................................................... carbohydrates....................................2...........

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

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. . LM observation Figure 10..1 Preparation of samples for LM observation.Microstructure ◾ 141 Food Specimen portions Embedding in paraffin or resin Freezing (CO2.. 1% red oil. .

Araldite Spurr’s. 70%. 100%) Infiltration and embedding in resine Epoxi resin.5% Glutaraldehyde 2% Os O4 Dehydration Ethanol (30%. 50%.2 Preparation of samples for TEM observation. 90%.142 ◾ Handbook of Seafood and Seafood Products Analysis Food Specimen portions Fixation 2. 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. .

50. 90. . for X-ray) SEM observation Figure 10. Pd. 70. for SEM imaging) (C.3 Preparation of samples for SEM observation. 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..4 Preparation of samples for Cryo-SEM observation.. vaccuum) Cryo-SEM observation Figure 10. C.) (into Cryo-SEM) (–130°C. vaccuum) Au deposition Coating (Au. vaccuum) Etched H2O Etching (into Cryo-SEM) Specimen fracturing (–90°C.

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. the myofibrils at the cell edges have a less rounded section than the central myofibrils and are arranged like a palisade. it is possible to observe ultrastructural details. since the water in which the fish live lends support for the body (Lampila.Microstructure ◾ 145 a b Figure 10. The layouts of the Z disks that mark the length of the sarcomere are visible.1 Herring Figure 10. When ultrathin sections of herring muscle tissue are studied by TEM. At low magnification. When the muscle fibers are observed using the Cryo-SEM technique. fat globules of different sizes are observed occupying the interfibrillar spaces and myofibrils are distinguished inside the cells.6E). the perymisial connective tissue that surrounds the muscle bundles can be seen.6B).7C shows the inside of a muscle fiber with the myofibrils perfectly bundled. In a cross section of the sample fi xed in osmium tetroxide (Figure 10. which is mainly composed of collagen.5 Schematic representation of fish muscle with myotomes. A and I bands (Pérez-Munuera et al. a: myotome. Figure 10.7A shows a herring sample stained with toluidine blue and observed by LM. 2008). reveals the myofibrils inside each cell.2. 10. The longitudinal section in Figure 10. The fiber is composed of myofibrils in which Z disks are distinguished in the areas where the sarcolemma is broken.7B.6D shows the microstructure of herring tissue at a higher magnification. where the Z disks can be distinguished.6F.6C). The typical fish muscle fibers can be seen. A micrograph cross section of the fibers shows them surrounded by the sarcolemma. Figure 10. Fixing in osmium tetroxide shows the distribution of fat in the herring tissue. b: myocommata.6A shows a cross section of herring tissue fi xed with glutaraldehyde and observed by SEM. where the fat is viewed as globules on the surface of the fiber. but the total collagen content is lower.. surrounded by the sarcolemma and by the endomysial connective tissue. obtained by the same technique but observed at a higher magnification. the aggregation of solutes produced during the etching of the sample generates the typical eutectic artifact observed in Figure 10. Figure 10. Examples of different fresh fish tissues observed by several techniques are described here. with the endomysial connective tissue keeping the muscle fibers firmly attached to one another. The myofibrils are . The myofibrils are shown in longitudinal section in this sample (Figure 10. the fat can be observed covering the fibers in a longitudinal section of herring muscle (Figure 10. 1990).

(F) Cryo-SEM. (A–E) SEM. Hake fibers surrounded by connective tissue can be observed in Figure 10. continuous. can be seen. f. connective tissue. and roughly parallel filaments (Figure 10. for example. along with the M and Z lines.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. which are the components of the cytoskeletal network that links the myofibrils to one another and to the sarcolemma. the A and I bands.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 10.7D)..2. 10. TEM and SEM studies demonstrated an extensive network of filaments connecting Z structures that were regularly spaced and connected by sets of longitudinal. c. (2005) after depleting the thick and thin filaments with a potassium iodide treatment. The main difference is their size: hake fibers are thicker than herring ones.8. . connected to each other at the Z disk level by the costameres.9).6 Herring tissue. eutectic artifact. Z. Z disk. The cytoskeletal ultrastructure of hake was studied by Pagano et al. The structural elements that constitute the sarcomere. pork meat (raw ham) (Larrea et al. fat globule. a. The TEM technique allows images to be obtained at higher magnification and with better resolution than other microscopy techniques (Figure 10. 2007). The same structure has also been observed in different meat products.

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

148 ◾ Handbook of Seafood and Seafood Products Analysis Z LZ IZ DZ 8.R.1 Smoked Salmon A cross section of a smoked salmon sample obtained using the Cryo-SEM technique is seen in Figure 10. the greater the shrinkage.3 Processed Fish Microstructure 10. (A and B) Cryo-SEM. Gudmundsson and (A) 100 μm (B) 10 μm Figure 10. With permission.10A. B. 141. 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. Sigurgisladottir et al..) 10. 13. 2005. Z. . Figure 10. Z-disk. longitudinal filament connecting Z-Z. (2000) used LM to observe the changes that occurred in the salmon during the smoking process and quantified them by image analysis. M. (Reprinted from Pagano.10B shows a detail of an intercellular space created by the conjunction of three fibers or cells. The data of the average cross-sectional area of muscle fibers showed that the smoking process produces shrinkage of the fibers. Com.000 X Figure 10. the higher the smoking temperature. LZ. The micrograph shows geometrically shaped fibers surrounded by a connective tissue. Biochem.3.9 Cytoskeletal structure of hake observed by TEM. Physiol. et al.10 Smoked salmon.

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

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 10.3.12B) shows the typical concentric layers of this type of surimi product. Lancaster. The Chemical and Functional Properties of Food Proteins.150 ◾ Handbook of Seafood and Seafood Products Analysis observed by Cryo-SEM.13 Schematic representation of (A) squid mantle and (B) arrangement of muscle cells. shows a longitudinal section of a crab stick where the “artificial fibers” can be observed. where the presence of salt makes the etching of the sample for observation difficult and masks the underlying structures.” Lean fish meat is minced to a paste.12A. Inc.3 Surimi One of the most common surimi products on the market is artificial crab muscle. after adding different additives.. M.. The cross section (Figure 10. 10.A.) . The formation of a new network with the myofibrillar protein (Figure 10. Figure 10.. 2001. Technomic Publishing Co. Such a product is often sold as “crab sticks” or “seafood sticks. With permission. PA. (From Lluch. et al. the paste is shaped and an “artificial fish muscle” is obtained. obtained by SEM.

central sarcoplasm. References Gudmundsson.75 μ m s (C) (D) l Figure 10. a central sarcoplasm is shown to be surrounded by myofibrils. m. 247–267.) ties of surimi.14D).. Food Res. Comparative microstructure of red meat. all the muscle fibers are thin. H.. and (D) TEM. L. and Hafsteinsson. With permission. . The inner and outer tunics are covered by a visceral lining and outer lining. approximately 3. Effect of electric field pulses on microstructure of muscle foods and roes. 2001. J.. E. (A and B) SEM.. 10. 2007.14 Squid.Microstructure ◾ 151 –200 μm –10 μm (A) 10 μ (B) 3.. 225. 12. Trends Food Sci.. Muscle Foods. (From Llorca. Circumferential muscle bands (100–200 mm) comprise fibers running about the entire circumference of the mantle cone. LM makes it possible to distinguish a peripheral area in blue and a central core in white inside each cell.5 mm in diameter (Lluch et al. respectively. M. 2007). Muscle fibers are grouped in bands that are arranged orthogonally. 2001). Lampila. Regardless of their orientation. Eur.14C). myofibril. (C) LM. l..13). Radial bands (10–15 mm thick) comprise fibers that connect two tunics of connective tissue. Th is gel network structure gives surimi its characteristic elasticity and texture (Sikorski.. the intermyofibrillar spaces between these can be observed. s. 1990.4 Squid Microstructure The squid mantle is composed of muscle tissue sandwiched between two tunics of connective tissue (Figure 10. 2001).E. poultry and fish muscle. 122–128. Tech. Each fiber is surrounded by a sarcolemma (Llorca et al. Technol. The fibers arranged in circumferential and radial bands were observed by SEM in samples fi xed with glutaraldehyde (Figure 10. 807. 1990). et al. sarcolemma. 1.14A and B) (Llorca et al. When TEM is used to study the ultrastructure of fresh squid (Figure 10. This fiber distribution can also be observed by LM in samples stained with toluidine blue (Figure 10.

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

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

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

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

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

For instance. Selectivity will be reconsidered in Section 11. and then the amount of oxygen molecules that can be adsorbed at the surface is also limited.3. in any case. the number of conductance electrons is limited. At sufficiently high temperature (above 200°C). 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. In practical. The most popular materials undergoing a conductance change on interaction with gases are metaloxide semiconductors.4.3 Chemical Sensor Technologies In this section the basic principles of the most popular categories of chemical sensors are illustrated. which reacts with the bounded oxygen to form carbon dioxide. although interesting. The characteristics of these structures. These are oxides of transition metals. and a surface potential barrier is formed. and as a consequence. Since the material is a semiconductor. The consequence of the exposure to oxygen is a reduction of the surface conductance.12].3. a reduction of the surface conductance band depletion. Sensors are here classified according to the physical intermediate quantity. semiconductors are subjected to large changes of conductance also in the presence of a modest variation in the number of conductance electrons or holes.Chemical Sensors ◾ 157 any reliable information about the chemical composition of the measured sample. Metal-oxide semiconductor sensors can be prepared in many different ways. 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. metal oxide growth in regular shapes such as nanosized belts [9] has shown peculiar properties. 11. The amount of depletion and the barrier height are proportional to the number of adsorbed molecules. Recently. The main sensitivity mechanism is related to the role played by oxygen. . and a lowering of the potential barrier. have not yet resulted in practical improvements of performances. 11. This is only one of the many interactions taking place on the surface of metal oxides. Paradigmatic. in this regard.1 Metal-Oxide Semiconductors Changes in conductance become appreciable in materials characterized by a limited number of mobile charge carriers. releasing an electron back to the conductance band.1. an electrically actuated heater is integrated in the device. the most known and studied of which is SnO2 [7]: a wide band gap n-type semiconductor. 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. 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. providing the maximum sensitivity.1 Sensors Based on Conductance Changes 11. dissociative adsorption sites of molecular oxygen are active on the oxide surface. and the sensitivity of these devices is extended to many different kinds of volatile compounds [8]. It is important to remark that this kind of sensors needs to be operated at high temperature. is the case of carbon monoxide. The sensitivity can be further modified adding ultrathin amounts of noble catalytic metal atoms on the surface. Metal oxide semiconductor chemoresistors have been used several times in fish freshness applications.

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

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

and aromatic. Their concentration and the presence of other compounds are rather typical of each species. To this point of view. N-cyclic. the lack of selectivity of many chemical sensors was considered as one of the main problems limiting their diff usion for practical applications. 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]. 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]. bromophenols. 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. observation of Nature offered a useful suggestion about the use of such devices. allowing for electronic nose application oriented optimizations. The most important chemicals involved in the fresh fish odor are long-chain alcohols and carbonyls. Among these compounds amines . each receptor senses several kinds of molecules. Receptors were found to be rather unselective. and each molecule is sensed by many receptors [33]. and the genes expressed by olfactive receptors are known [31].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. On the other side. organic synthetic receptors offer an unlimited number of possibilities to assemble molecules endowed with differentiated sensing features. After this conjecture. and such systems were soon dubbed as “electronic noses. CSPT has demonstrated its utility in particular to classify airborne chemicals reading absorbance and fluorescence changes in chemical dyes such as metalloporphyrins [30]. After this discovery. The features of electronic noses are fundamentally dependent on the sensing properties of the artificial receptors. the application of the CSPT concept may be foreseen as greatly extending the analytical capacity worldwide. and acid compounds. 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]. models of receptor mechanisms explaining the sensitivity to volatile compounds are now available.” This denomination is currently given to any array of unselective chemical sensor coupled with some multicomponent classifier. the possibility of developing artificial olfaction systems became possible. whereas CSPT arrangement gives the possibility of evaluating at the same time both the effects. microbial spoilage produces short-chain alcohols and carbonyls. and N-cyclic compounds. amines. The physiology of olfaction has made considerable advances. almost all sensor technologies were used to build such systems. it was proposed that arrays of nonselective chemical sensors may show properties similar to those of natural olfaction [34]. Nonetheless. Standard optochemical sensors are based either on absorbance or on fluorescence. 11. Investigations about olfaction receptors show that Nature strategies for odor recognition are completely different from those of analytical chemistry. sulfur compounds.4 Electronic Noses As discussed above. Since the 1980s. The concentrations of these chemicals are directly correlated to the degree of spoilage.160 ◾ Handbook of Seafood and Seafood Products Analysis camera. 11.

Analyzing the data with PCA the plot of Figure 11. amperometric sensors [42]. MOSFET sensors [41]. The representation plane is determined as that where the data variance is maximized and then the statistical properties of the dataset are. In gas chromatography. petroleum in sea). In LSER. Data are extrapolated from an investigation by Strachan and Nicholson [48]. showing the ability of the electronic nose to track the different spoilage levels occurring at different storage times. Instruments based on different sensor technologies have been used. Minor contributions to the fish headspace come from contamination of the environment (e.. Results shown in Figure 11.4 demonstrate a continuous progress after the 8th day.4. amines become instrumentally appreciable only when spoilage processes take place. the data produced by a sensor array can classify samples according to some of their global features. PCA is a data analysis method allowing the representation of a multidimensional dataset in a reduced dimensionality space. and sweet conditions are hardly identified. a plane. The sensitivity of chemical sensors is not immediately related to the molecular family but rather to the interaction mechanism. hybrid electronic noses were used combining different sensor technologies such as QMB and amperometric sensors [47]. the accumulation of some compound.Chemical Sensors ◾ 161 are considered as the typical markers for fish freshness detection. Since LSER was fruitfully used to model polymer-based chemical sensors [52]. such as metal-oxide chemoresistor sensors [38–40]. from fish processing. When properly analyzed by pattern recognition methods. the chemical complexity of the problem. Nevertheless. Sensor data can be conveniently represented by a principal component analysis (PCA) scores plot. quartz microbalance sensors [44. nonetheless. This result is rather surprising because fish spoilage is in general expected to be a linear and somewhat straightforward process. polarity.5 is obtained. but the behavior at the beginning is absolutely nonlinear. it is more realistic to consider an array of sensors specific for a single interaction mechanism. dipolarity. in case of fishes. with an array of selective sensors it is not possible to distinguish between fresh and flat fishes. with a super impression of 6th and 1st days. As a result. and acid. Standard analytical methods for volatile amines and also sensors for some specific amines have been used to inspect fish freshness. 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. flat. typically according to the freshness or more precisely according to the balance between fresh and spoilage produced compounds.g. five different kinds of interactions are considered: dispersion. The number of compounds whose concentrations are only partially correlated makes this application particularly appealing for sensor arrays of partially selective chemical sensors. In Figure 11. let us discuss a simulation of a case study.45]. In addition.3 the time evolution of the major families of volatile compounds found in the headspace of fishes is shown. and optical indicators [46].6 .3. let us consider an array specific for each LSER interaction and one compound for family. Each sensor then provides a signal proportional to the concentration of each family. Apparently. In recent years attempts to use electronic nose technology to track the spoilage processes occurring in fishes have been reported in numerous articles. and the decrease in others result in a nonlinear problem. In order to understand the potential of electronic noses to detect fish freshness. Let us consider the use of an array of sensors absolutely selective for each individual family of compounds mentioned in Figure 11. and fresh. and finally from products of lipid oxidation [37]. In this regard. hydrogen bond basic. the interaction between polymers and volatile compounds is often described by the linear sorption energy relationship (LSER) model [51]. the progress of spoilage is less linear with respect to Figure 11. conducting polymer sensors [43]. as much as possible. The same nonlinearity is observed with electronic noses. Figure 11. An imperfect application of this method was demonstrated with engineered polymer-coated QMB [50]. preserved [49]. Most of these are feasibility studies. for example.

in order to measure the quality of fish instrumentally. each able to capture different aspects of fishes.g. color meters. humans provide a more reliable identification of fish freshness.01 0 5 10 15 Days 20 25 30 35 Figure 11. Nonetheless. The typical sensorial description is also reported. texture meters.. with a folding back of the spoilage process in the representation plane. shows the scores plot of a partial least-squares discriminant analysis model related to an array of metalloporphyrin-coated QMBs. This feature that can be interpreted as a failure of the electronic nose is likely due to an intrinsic nonlinearity of the studied problem.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. 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). As a consequence. . olfaction) provides several errors of evaluation. and devices measuring electrical properties) has been illustrated in different applications related to cods [56. sardines [58].5. Results are qualitatively similar to those shown in Figure 11. and original data were previously published [53]. tactile (to test the flesh firmness and elasticity).1 0. and groupies [59]. an integration of instruments is necessary.3 Time evolution of the major families of volatile compounds in fish headspace. 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].57]. and olfaction (to smell the gill odor) [54]. The experiment was related to COD fishes. The possibility of developing a multisensor device to measure and/or estimate fish freshness with a combination of instrumental techniques (electronic noses. image analyzers. spectroscopic methods. and the use of only one sense (e.

3 are used.5 –2 –2.5 1 PC 2 (15.5 1 0.4 PCA scores plot of a simulated experiment where sensors selective for the compound family in Figure 11.5 –1 24 10 12 8 28 2 4 6 1 ◾ 163 22 20 18 16 14 –1.03%) –1 0 1 2 30 2 28 22 20 10 10 12 14 16 8 1 Figure 11.5 24 PC 2 (19. each specific for a single interaction mechanism among those modeled by LSER. .76%) 0.39%) Figure 11.5 –6 –4 –2 0 2 4 PC 1 (72.5 –1 –1. Scores plot 1. Data show the impossibility of distinguishing the spoilage process in the first 6 days and an abrupt change between 6th and 8th days.Chemical Sensors Scores plot 2 30 1.5 –6 4 6 –5 –4 –3 –2 PC 1 (80.5 0 –0.93%) 0 –0.5 PCA scores plot of data related to a virtual array of sensors.

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

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

. A. 352. Neurobiol. Enokihara. 10–14. 44. Lundstrom. Optical sensing looks to new field. Sens. 2001. et al. Friedrich. Rapid gas sensor measurements to predict the freshness of capelin (Mallotus villosus). Proposed modification of dyer’s method for trimethylamine determination in cod fish. Chim. Receptor cell responses to odorants: Similiarities and differences among odorants. Liston (eds. Ólafsdóttir. 292. 2001. R. Microbiological.. 31. 2007. 11. 1975. I. et al. Gauglitz. Molecular recognition and discrimination of amines with a colorimetric array.. K. Olafsdottir. 1969. Curr. 29.. 2000. et al. 1997. 102. 2005. 55–69. 43. Commun.. Andersson. Angew. D. Acoustic Wave Sensors.. Cell. Kluwer. Chem. A novel multigene family may encode odorant receptors: A molecular basis for odor recognition. 1997. The application of metalloporphyrins as coating material for quartz microbalance based chemical sensors. W. L. . Filippini. and Weimar. Recent dynamics in olfactory population coding. Paquit. and Fleurence. 17. Nature. 839. Actuators B. 1984. Tozawa. Computer screen as a programmable light source for visible absorption characterization of (bio)chemical assays. et al.X. and electronic nose evaluations of yellowfin tuna under various storage conditions. 22. 32. 37. Lett. Opin. Barsan. J. G.. M. and Axel. Appl. 240. F.). Persaud. Academic Press. Monitoring of fish freshness using tin oxide sensors. 20. 2008. Development of a smart packaging for the monitoring of fish spoilage.. Elsevier.H. 16. Sens. Soc. 30.M. Lindsay. 45. E. A. Electrical detection of amine ligation to a metalloporphyrin via a hybrid SOIMOSFET. Analysis of discrimination mechanisms in the mammalian olfactory system using a model nose. 34. Agric.. 26. D. 18. J. Ólafsson. and Olafsdottir. Chemical sensing with familiar devices. Sicard.. Gardner. 35. A hydrogen sensitive MOS field effect transistor. 28. Measurement of volatile aroma constituents as a means for following sensory deterioration of fresh fish and fishery products. (eds.. 26. M.. Prot. D. Kramer. Svensson. Winquist. 64.. J. Lett..N. A colorimetric sensor array for odour visualization. 1986. J. 55. K. 2226. 325.W. 2027. Development of a ChemFET sensor with molecular films of porphyrins as sensitive layers.. J. et al. Buck. L.. Trends Anal. 24.166 ◾ Handbook of Seafood and Seafood Products Analysis 15. and Lundström. 27. Phys. and Holley. N. 299. and Stopfer. K. 2002. Takulapalli. Ed. 705. Brunink. Appl.. CA. 77. H. in Seafood Quality Determination Symposium. 33. Martinsdóttir. Filippini. Dordrecht. in Methods to Determine the Freshness of Fish in Research and Industry. G. and Jónsson. 25. F. 53. Battiston. G. Int.. Electronic nose: Current status and future trends. et al. S. 1982. Food. 77. 1996. A chemical sensor based on a microfabricated cantilever array with simultaneous resonance-frequency and bending readout. P.. K. 406. and Dodds. R. et al. and Amano. Rakow et al. 2001. Chem. Evaluation of fish freshness using volatile compounds: Classification of volatile compounds in fish. D. 1997. R. Am. 108. San Diego.. Du. Amsterdam. J. 175. G.. 257. Acta.). E. Angew. Technical Conference on Fish Inspection and Quality Control. and Suslick. Anal.W. 2003. B. et al. 2006. Chem. International Institute of Refrigeration. 466. 19. U. Nantes. 710. 23. N. sensory. 4458. Brain Res. et al.. Nature. Chem. Bartlett. 705. Phys. Chem. Röck. Halifax (Canada). Josephson. Rev. 2006. et al. 25. Talanta. 122..S. I. G. Chem. the Netherlands. 108. 567.. p. Food Chem. 65. 130. 1983. Ed. in Sensors and Sensory Systems for an Electronic Nose. 3800. D. 36. 21. R. 468. November 12–14. the Netherlands. 2654. 1991. 283. Int. 45. 2008. Modified palladium metal-oxide semiconductor structure with increased ammonia gas sensitivity. Rakow. Actuators B. F. 1992. Ballantine. et al. 38. 15–25 July. November 1986. G. Proceedings of the Final meeting of the Concerted Action “Evaluation of Fish Freshness” AIR3 CT94 2283. 748.

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

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

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

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

a number of physical. chemical. thermal and near-infrared (NIR). Ultrasound imaging is a versatile. when propagated through a biological structure. below are cited some examples of the use of these new technologies in the quality control of foodstuffs. the reason is. we concentrate on electromagnetic methods at microwave frequencies. and a high amount of energy can be imparted. Nevertheless. Ultrasound attenuation spectroscopy (acoustic spectroscopy) is a method for characterizing properties of fluids and dispersed particles. in the case of Doppler-based modes. and biochemical effects can be observed. chicken. Ultrasound. above 16 kHz [17]. Thanks to advancing technology. some of their propagation parameters are modified. Ultrasound is a form of energy generated by sound (really pressure) waves of frequencies that are too high to be detected by human ear. 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. the other techniques that enable online control have been briefly commented on below. which. exposing their main disadvantages and highlighting the advances in the field of seafood. 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.172 ◾ Handbook of Seafood and Seafood Products Analysis Given the premise that online control requires a nondestructive method. The main disadvantage of ultrasound is that the energy propagates poorly through a gaseous medium. For fish samples. NIR measurements are widely used in the food industry to determine the sugar content in fruits [9]. Normally the modification of any quality parameter is macroscopically correlated to the change in any wave parameter that can be controlled. moreover. The interaction between wave radiation and matter as a function of wavelength or frequency is called spectroscopy. fulfilling the initial premise.19].13] in foodstuffs. Suvanich et al. and physical state of foods [20]. The salt and water content are related to dielectric properties of cod at microwave frequencies [14–16]. which enable a variety of applications [18. which is finding increasing use in the food industry for the analysis of food products. in this chapter. and widely used diagnostic tool. microwave.e. It is virtually impossible for . It is also necessary to work at very low power in order to not cause permanent effects such as heating.3. must act in real time and without producing permanent effects on the food. It is impossible to address all these techniques with precision. well-established. and raw meat mixtures can be related to its composition using semiempirical equations [7]. The spectroscopic techniques use the information found in the spectrum that is emitted for the food to predict certain of its qualities. Ultrasonic velocity in fish tissues. Visible (and near UV) transmittance method has been investigated to inspect the internal quality (freshness) of intact chicken egg [8]. such as radio frequency (RF).. and dielectric measurements at microwave frequencies can be used to analyze water activity [11] and water content [12. [21] published a report on how the ultrasonic velocity measurements show potential for analyzing fish composition. impedance measurements (RF) can determine salt and water content in salmon filets [10]. 12. i. determine the velocity of a moving tissue.1 Ultrasounds—Acoustic Spectroscopy Ultrasonic is a rapidly growing field of research. and visible. it is almost imperative to resort to elastic (sonic) waves such as ultrasounds or to nonionizing electromagnetic radiation. the modification of these parameters can be measured in real time. Highfrequency. induces compressions and depressions of the medium particles. structure. When these waves pass through foods (or are refracted by them). Depending on the frequency used and the sound wave amplitude applied.

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

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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

redfish shrimp.3 The Torry Scheme In contrast to the QIM. Melanogrammus aelefigus. Descriptive words should be carefully selected. and the panelists trained should agree with the terms.2).dk/QIMRS/qim_0202. QIM Rating system software was developed for cod. and is nondestructive and can be used as a tool in production planning and quality warranty work [27].1) [34]. The method can also be used for texture. herring. Rapid PC-based QIM is also available on the Internet at http://www. common octopus (Octopus vulgaris) [33]. sensory methods are time consuming. brill. and flavor. In addition. respectively) [21]. medium fat. fresh cod (Gadus morhua) [32].2. 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).192 ◾ Handbook of Seafood and Seafood Products Analysis quality expressed as a demerit score and storage life on ice. Objective sensory methods are essential for quality control and estimation of shelf life of seafood. The most comprehensive scoring scheme to assess fish is the Torry Scheme [36].2.4 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. is rapid and easy to perform. sole.min. However. In QDA. is a descriptive 10-point scale and has been developed for lean. Sebastes mentella marinus. 13. which makes it possible to predict remaining storage life on ice. Therefore. and Scopthalmus maximus. pollock. The advantages of QIM are that it requires short training.dfu. Pollachius virens. panelists evaluate the odor and flavor of cooked fillets. trained personnel required. positive and negative sensory parameters based on whether fish are fresh fish or fish at the end of the storage period [37]. saithe. The average score of 5. 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]. QDA provides a detailed description of all flavor characteristics in a qualitative and quantitative way. and not always practical for large-scale commercial purposes. This method is considered to be a relatively fast. Pandalus borealis. farmed Atlantic salmon (Salmo salar) [31]. 13. often referred to as the Torry scale. Recently developed QIM schemes were presented for raw gilthead sea bream (Sparus aurata) [30].htm. odor. The Torry Scheme. and redfish by the Danish Institute for Fisheries Research. . a higher score can be given for a single parameter [27]. In this scheme. The scores are given from 10 (very fresh) to 3 (spoiled) (Table 13. the QIM is suitable for early stage of storage of fish where other instrumental methods are not accurate [28]. It has been widely used in its original or modified forms. and fat fish species. nondestructive method based on direct observation of sensory parameters of fish and can also be specific for species. plaice. Solea vulgaris. the words for describing the odor and flavor of the fish can be categorized into two groups. haddock. Hyldig [29] indicated that the QIM is expected to become the leading reference method for the assessment of fresh fish within the European community.5 may be used as the limit for consumption [21]. the Torry Scheme was developed at the Torry Research Station for use with expert and trained judges. During spoilage. herring (Clupea harengus) (Table 13. and turbot (Scopthalmus rhombus. Objective terms should be used rather than subjective terms. Pleuronectes platessa. expensive. instrumental methods are also needed to satisfy the need for quality measurements in fish industry.

With permission. Quality Standards for Fish: Final Report Phase II. 37. D.Methods for Freshness Quality and Deterioration Table 13. 975. . pp. 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. G. 2004. seaweedy. 1992.1 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. matte. Int. S.. developed by Nielsen.. Food Res. Nordic Industrial Fund (in Danish).. 37–59. brown Odor Fresh. and Hyldig.

With permission. 283. TMA Strong bitterness. metallic. followed by strengthening of these odors Shellfish. flounder. J. there is little agreement on which is the best method [42]. wood sap.194 ◾ Handbook of Seafood and Seafood Products Analysis Table 13. springiness. slight sulfide Score 10 9 8 7 6 5 4 3 Source: Shewan. vanillin Condensed milk. Dielectric properties of fish skin and muscle alter in a systematic way during spoilage as tissue components degrade. starchy. and cooking [39. TMA Lower fatty acids (e. boiled potato Milk jug odors. and product development in the seafood industry [38]. deciding the commercial value of the meat [41]. trace of “off” flavors Slight bitterness. acetic or butyric acids) decomposed grass.40]. mackerel.3. sour milk.M.. soapy. sour. and flavor during spoilage and have been used as quality indicators since the first commercial version of the Torrymeter in 1970 [43].3 Physical Methods 13. texture. seaweed. natural odor Wood shavings.3.2 The Torrymeter The Torry fish freshness meter “Torrymeter” was developed at Torry Research Station in Aberdeen. boiled milk. improper handling storage. 13. Numerous mechanical methods have been used to measure texture. during processing..1 Texture Analysis Texture analyses for seafood are extremely important in research.2 Torry Score Sheet for Freshness Evaluation of Cooked Cod Fillets Odor Initially weak odor of sweet. starchy. Fish muscle has higher levels of indigenous proteases. Dielectric properties of fish are used for determination of freshness. Baltic herring. and chewiness of food. rubber. et al. hake.. Sci. J. quality control. hardness is the most important to the consumer. 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. odor. Scotland. turnip. Fish muscle may become soft or mushy as a result of autolytic degradation or tough as a result of frozen storage [16]. These changes occurring at microscopic level are related to alterations in appearance. Texture includes the most common characteristics such as hardness. Food Agric. reminiscent of boiled clothes Lactic acid. blue whiting. “off” flavors. tallow Flavor Watery. however. 1953. 4. boiled meat Loss of odor.g. Among textural attributes. A linear relationship was found between Torrymeter readings and sensory attributes for cod. 13. which immediately begin to break down the proteins after the harvesting. .

However. and fish-handling procedures. conductivity. 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. as well as rapid and nondestructive. The electric properties of fish can change after death of the fish due to disruption of the cell membranes by autolysis. Therefore. Inácio et al. and capacitance) of the fish flesh [52]. They are unsuitable for frozen or thawed fish. The skin of fish could be affected by osmolarity and contact with electrically charged particles [51]. The method is based on conduction through skin and. RT-Freshtester reflects dielectrical properties of fish. Like other instruments. fast and nondestructive. 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]. iced gilthead sea bream.3. [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. [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. 13.05) lower in fish washed with seawater than fish washed with tap water or unwashed. measuring the electric properties (resistance. Gelman et al. season. The Intellectron Fischtester VI gives reliable information about the days in ice and left of iced stored fish. fishing grounds. Gelman et al. works only on whole fish and fillets with skin on. therefore. and readings from all instruments decrease with storage time. these instruments need calibration depending on sample preparation. portable. 13.5 The Cosmos The “Cosmos” instrument developed by Japanese is applied for the evaluation of fish quality by determination of smell intensity. . Fat also has an effect on the dielectric properties of fish and tends to make observed Torrymeter values more variable [47]. This could be explained by seawater containing ions. and fish chilled in refrigerated seawater [54].3. the “Cosmos” instrument is handheld.3 The Intellectron Fischtester VI The basic principles of Torrymeter (the United Kingdom) and the Intellectron Fischtester VI (Germany) are similar. Mechanical abuse and freezing can affect the readings.Methods for Freshness Quality and Deterioration ◾ 195 whole.4 The RT-Freshtester Like Torrymeter and the Intellectron Fischtester VI. partly frozen such as superchilled fish. allows automatic grading of 60–70 fish/min. However. 13. [50] found that the Torrymeter readings obtained from six species of different origin were poorly correlated with sensory evaluation. and farmed Senegalese sole [43–49]. The Fischtester readings can be used as an objective criterion for the state of freshness/spoilage together with sensory data across the fish chain. which interfere with the reading of both instruments as they are based on electrical properties of skin. RT-Freshtester.

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

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. These objective methods should correlate with sensory quality. 13. Nucleotide breakdown reflects both action of autolytic enzymes and bacterial action [16]. and it has been indicated that this spectroscopic technique is useful in assessing the freshness and quality of sardine during iced storage [84]. which is the main energy source for metabolic activity. For the first time.1 shown. and the chemical compound that is determined should increase or decrease as microbial spoilage or autolysis progresses [16].1 ATP and Its Breakdown Products Rigor mortis occurs in postmortem muscle tissue and is associated with stiffness of muscle or flesh. recording the product temperature from the moment of catch.4. The sequences of nucleotide catabolism proceed as shown in Figure 13. This alternative method could be cost effective and definitely more reliable. The traceability system can also be used for the determination of fish freshness. 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. Traceability is becoming a method of providing safer food supplies and of connecting producers and consumers. degradation of ATP proceeds according to the sequence . the most used method to evaluate fish freshness is to combine several measurements obtained from different methods and correlate the findings with sensory analysis [59]. cheap. This process results from breakdown of adenosine triphosphate (ATP). since they eliminate personal opinions on the product quality. The most used procedures for the objective measurements of seafood quality are given in the next sections. this technique has been applied to sardine muscle during iced storage. 13.1. sensitive.Methods for Freshness Quality and Deterioration ◾ 197 its use is simple. 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]. and requires a small amount of sample. In postmortem fish muscle. leading to accumulation of adenosine diphosphate (ADP) and inosine monophosphate (IMP).4 Chemical and Biochemical Methods Chemical and biochemical methods for the evaluation of seafood quality are more reliable and accurate. Currently.

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

1 depending on season. 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]. These problems may be more severe in sensitive consumers who have a reduced mono.4.125]. The formation of biogenic amines results from microbial degradation during the later storage of fish. HPLC [120. and use of a biosensor [130–132]. Cadaverine is derived from lysine. Postmortem pH varies from 5. their levels are considered as indices of spoilage rather than freshness [112]. and stomach contents at death.107.S. 2-phenylethylamine. By means of decarboxylation reactions. In addition. capillary zone electrophoresis (CZE) [128. and reproducibility. species. depending on the species being examined [10. 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. The QI and the biogenic amine index (BAI) were proposed by Mietz and Karmas [120] and Veciana-Nogues et al.127]. 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. tryptamine. and agmatine. cadaverine. Consumption of seafood containing high amounts of these amines can have toxicological effects. 13.129]. The biogenic amine content of fish depends on fish species. The most significant biogenic amines produced postmortem in fish and shellfish products are histamine.108]. the presence of decarboxylase-positive microorganisms. [121] for determination of quality of fish. Since the amines are produced by spoilage bacteria toward the end of shelf life of a fish. since microbial flora vary seasonally [11].Methods for Freshness Quality and Deterioration ◾ 199 13. tyrosine produces tyramine.123]. and tyramine.110]. histamine is potentially hazardous and the causative agent of histaminic intoxication [114]. tryptamine from tryptophan. putrescine. There are various analytical techniques used to determine the concentration of biogenic amines. and arginine leads to putrescine. reliability. spermine. free amino acid content [112]. and other factors . HPLC is mostly performed because of its sensitivity.and diamine oxidase activity [116]. histidine yields histamine. cadaverine.124. the moment of capture.11.109. 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. putrescine. whereas BAI is based on increases in histamine. Among these techniques.2 Biogenic Amines The concentration of biogenic amines has been reported to be a reliable method of measuring the quality of fish. The others especially putrescine and cadaverine have been reported to enhance the toxicity of histamine [115]. tyramine. including thin-layer chromatography (TLC) [122. GC [126. cadaverine. and the concentration of these increases with storage time [91. respectively. postmortem temperature. Among the biogenic amines.5. spermidine. and histamine and decreases in spermine and spermidine during storage of fish. and pH [133]. Putrescine is also an intermediate of a metabolic pathway that leads to spermidine and spermine [119]. and 2-phenylethylamine is derived from phenylalanine.3 pH The pH is also an important parameter to show depletion in tissue and quality of flesh during storage.0 to 7. respectively. Food and Drug Administration [117] and the EU [118]. Process technology is influenced by rigor development. the enzyme responsible for its detoxification. Biogenic amines are generated by microbial decarboxylation of specific free amino acids in fish or shellfish tissue [111].4.

A relatively low pH may cause a decrease in water binding to the myofibrils. The EC reference method for TVB-N determination. total volatile basic nitrogen (TVB-N) primarily includes trimethylamine (TMA.4. involving preliminary deproteinization with perchloric acid. Th is is caused by the depletion of energy reserves. The European Commission (Council Regulation No. turbot [92]. pike perch [146]. mainly glycogen. Since the activity of enzymes depends on pH.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 is well known that determination of TVB-N differs systematically according to the procedures used. However. as shown in a variety of fish such as European hake [142]. 13. Therefore.200 ◾ Handbook of Seafood and Seafood Products Analysis [134. the levels of 30–35 mg N/100 g muscle are considered the limit of acceptability for icestored cold-water fish [17.141]. Atlantic cod [143].151]. which is considered to be the main cause of off odors in fish products [58. ammonia (produced by deamination of amino acids and nucleotide catabolites). as shown below: Following death of fish. 144]. and European eel [13]. The level of TVB-N in freshly caught fish is generally between 5 and 20 mg N/100 g muscle. it affects reactions taking place during storage of fish.4 Total Volatile Basic Nitrogen In seafood. TVB-N level correlated with fish quality. with the production of lactate. and it has been used as an indicator of marine fish spoilage: CH3 CH3 – N=O CH3 TMAO CH3 CH3 – N CH3 TMA .59]. sardine [12. farmed gilthead sea bream [147]. whereas the second one includes the use of trichloroacetic acid instead of perchloric acid [149]. However. TMA is produced by the decomposition of TMAO due to bacterial spoilage and enzymatic activity [150. was compared with two routine methods. it could not be regarded as a good indicator of fish freshness and proved to be better as a spoilage index. It was found that there was a good correlation between three methods. Low pH also promotes oxidation of myoglobin and lipids [134]. Low pH is used as an indicator of stress at the time of slaughtering of many animals. 13. and hake [148]. The analyses of these indicators are considered unreliable because they reflect later stages of spoilage rather than freshness [140]. Therefore.135]. such as frozen eel [145]. affecting light scattering and the appearance of fish. and direct distillation methods have been recommended as a rapid routine method. produced by spoilage bacteria). The first one includes direct distillation of fish after adding magnesium oxide.4. bacteria act upon TMAO to produce TMA. and DMA (produced by autolytic enzymes during frozen storage).136–139]. TVB-N should be used as a chemical check. 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. Low initial pH is associated with higher stress before slaughtering [13. Based on the results obtained from the literature.

13. photometry [161]. but it can react with a number of chemical compounds such as amino acid residues. when bacterial growth is inhibited.4. 13. The limiting factor of frozen storage in lean fish species is denaturation of proteins. whereas freshwater fish generally contain only 5–20 mg% [153]. whereas fish can be stored in a frozen state for several months without severe changes in quality. The TMAO content of seafood varies with species. HPLC method [162]. flowinjection-gas diff usion method [167]. The fish is considered stale when the rate of TMA production is higher than 30 mg/100 g cod [155]. However. DMA can be used as a spoilage index during frozen storage of some species such as frozen hake [170]. semiconducting metal–oxide array [166]. or TVB-N contents. lipid oxidation is the limiting factor in fatty fish species. A close relationship has been found between lipid damage and quality of the final product [173]. time of year. TMA is not produced in a significant amount during the early stages of chilled storage of fish. and time. and low-molecular weight compounds.150].157]. This reduces the solubility of myofibrillar proteins [172]. age. after which the rate of production of TMA parallels the bacterial proliferation pattern [154]. The amount of DMA produced depends on species (except gadoid species. Seawater fish have 1–100 mg TMAO in every 100 g muscular tissue.6 Dimethylamine As mentioned earlier. Conway microdiff usion and titration [159]. terminal amino groups. GC method [163. and environmental factors [152]. which results in a dry and firm texture of the fish muscle [174]. location of catching. However. resulting in rancidity. a capillary electrophoresis method [165]. causing denaturation and cross-linking of proteins [171]. other species do not develop adequate amounts of DMA). Fresh fish has a limited shelf life and is prone to deterioration. including steam distillation [158]. . The formaldehyde content of frozen seafood is generally used as a spoilage index. stage of spoilage. this reaction is replaced by a slow conversion by an enzyme to DMA and formaldehyde [16. colorimetric method [160]. The formation of these products may cause severe quality changes or spoilage during prolonged frozen storage. fish contain TMAO. Fresh fish has a very low amount of TMA with values less than 1. TMA can be used as a spoilage indicator and not as an index of freshness. During chilled or frozen storage of fish. but it appears after 3 or 4 days.156. its usefulness depends on time of year.7 Formaldehyde The formaldehyde content in seafood products is generally considered as nontoxic.5 mg TMA/100 g in fresh cod.4. 164].8 Lipid Oxidation Indicators During processing and storage. type of storage and processing. enzymatic and nonenzymatic lipid oxidation occurs. biosensor using flavin-containing monooxygenase type-3 [168]. DMA. which is converted to TMA by bacteria in iced fish. Many analytical methods have been developed for the measurements of TMA. but values increase during spoilage. especially in gadoid fish. the storage temperature. Several assays have been described for the determination of TMAOase activity in fish muscle [151. and solid-state sensors based on bromocresol green [169].4. 13. and methods employed for analysis.Methods for Freshness Quality and Deterioration ◾ 201 TMAO appears to be part of the system used for osmoregulation. fish size.

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

185]. peptides. 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. sardine.Methods for Freshness Quality and Deterioration ◾ 203 are anisidine value (AV). which break down to secondary products of oxidation or react with proteins. coliforms. However. 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]. PV. haddock. and decline [184. or enterococci [195]. Cozzolino et al.191].4. free amino acids.” or numbers of CFU of indicator organisms such as Enterobacteriaceae.188. and TBA values may increase. reach a peak. as oxidation proceeds the PV can start to fall. European eel. Many methods have been employed for the measurements of lipid oxidation in foods as a means of determining the degree of damage [20. It was indicated that the main requirements for shelf life predictions are to collect information about SSO.9 Lipid Hydrolysis Hydrolysis leads to hydrolytic rancidity and involves hydrothermal or enzymic (lipase) hydrolysis to FFA and other products. and phospholipids.193]. PV measures primary products of lipid oxidation. 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). It is possible to predict shelf life of seafood based on knowledge of initial numbers and growth of SSO. FFAs and their oxidation products would have an effect on muscle texture and functionality. [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. there are some difficulties with common methods when quality has to be assessed. Mathematics models have been well established for the growth of spoilage bacteria such as Photobacterium phosphoreum.186]. During prolonged storage of seafood. 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. such as proteins. spoilage domain such as the range of environmental conditions over which a particular SSO is responsible for spoilage and spoilage level [198]. and thiobarbituric acid (TBA). cod [192. Shewanella putrefaciens .5 Microbiological Methods Numbers and types of microbes present in foods are important indicators of safety and quality. AV and TBA values measure the secondary products of lipid oxidation. 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. 13.190. PV. since oxidation products are unstable and react with biological amino constituents. [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. AV. 13. and lean fish such as blue whiting. TOTOX (2VP + AV). horse mackerel [13. since they interact with myofibrillar proteins and promote protein aggregation [189]. causing production of interaction products [187]. and also freshwater fish [194]. Spoilage of fish and fish products is a result of the production of off odors and flavors mainly caused by bacterial metabolites [197]. 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.

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

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

............. salar parr.... 226 14....... JAS Law......... and these are seldom present in farmed seafood......2 Also in an earlier study...7.............................. the most prominent differences are the higher condition factor....................... and the United States (The Federal Food................ In small Salmo salar...................... For example......... The production method is also part of the information essential to fulfill the traceability of a product and.................... Correct information about the production method of seafood is also important. including morphological examination.. and Cosmetic Act and The Fair Packaging and Labeling).................. shape of the head. 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.....................................5 The flesh of farmed cod sometimes presents .... 227 14............... 227 References .. analytical methods should be made available to confirm it... but wild specimens may contain parasites harmful to humans. 227 Acknowledgments ... and caudal peduncles could be used for a total correct classification of wild.. and length of the pectoral fin....8 Other Methods . therefore...... fins...... the set of technologies to apply are basically the same as those described here...... as well as examination of the stable isotope and trace element profiles.......................................... farmed.............................2 ICP-MS ............................ commercially farmed specimens may contain residues of veterinary drugs whose presence is unlikely in wild seafood........................ 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..........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..............7 Trace Element Fingerprint.....1 Sample Preservation ......................... In particular... and sea-ranched S.............. 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.....................................7........... of 1999). Although in this work no special mention is made to organic farming... 226 14..... and smaller head4 as well as backbone malformations in farmed specimens.. 228 14...... larger liver.............. Several methods have been successfully applied to differentiate farmed from wild seafood...................... 100% discrimination between farmed (AquaGen strain) and wild parr was achieved by examining the body form.................... genetic analyses.. Drug...........1 14...3 it was shown that the morphology of the head........... The later study showed that the environmentally induced phenotypic divergence increased with age and with the numbers of generations under domestication....... Standards for organic farming are still under development in many countries....... size of the eyes and mouth..... In cod..................2 Morphological Examination There are few publications and no official guidelines for the morphological differentiation of farmed and wild aquatic organisms................216 ◾ Handbook of Seafood and Seafood Products Analysis 14.. analysis of the protein and lipid contents...............

To extract frozen samples we recommend to start the procedure before the sample is completely thawed. Delays and the use of preservations methods will diminish the quality and the yield of DNA. or by the use of Proteinase K) and proteins are removed usually by incubation with Proteinase K. Many commercial kits.8. Each kit is provided with a detailed description of how to use it.11–13 and a loss of rare alleles has usually been observed in the farmed populations.11. the cells are opened (by heat treatment. give satisfactory results. in most breeding programs the fish are indeed selected based on commercially interesting traits such as growth performance. in particular if it has a high enzymatic activity (for example if it contains the hepatopancreas in a crustacean). sonication.Z. which is the most common analysis. and then be rehydrated in water or in the extraction buffer.6 However. so that all the ethanol is evaporated. Dynal (Invitrogen). 14. GeneRelease (Bio Venture Inc.18 or gel filtration. Depending on the type of sample and its use. Nucleospin (Clontech).10 and optimal adaptation to different environments. Th is step is not necessary in samples preserved in ethanol. because the enzymes are inactivated by the fi xation. a normal freezer (−20°C) may also be used. thus limiting its application. Amersham Biosciences (GE Healthcare). The basic steps in all DNA extraction methods include the inactivation of nucleases. One requisite condition for any genetic analysis is the obtention of good quality DNA suitable for PCR amplification. and others.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. Then.1 Sample Preservation and DNA Extraction Methods The sample should be extracted as soon as possible after sampling. it would be relatively difficult to find markers for wild and farmed fish.). If this policy had been followed. Samples fi xed in ethanol must be allowed to dry completely. For very long periods. and then recovered by ethanol or isopropanol precipitation. The DNA .14–16 Hayes et al.3 Genetic Analysis Doyle et al.Analytical Methods to Differentiate Farmed from Wild Seafood ◾ 217 a translucent grayish aspect. If the sample must be preserved. However. Wizard (Promega). chloroform extraction. in contrast to the white opaque color of the wild. we recommend preservation in 96% ethanol. which are usually absent in many intermediate products as well as in the ready-to-eat dish.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.9 Genetic analyses have allowed the differentiation of wild from farmed fish populations in a variety of species. Genomics analyses are dealt with in more detail in Chapter 4 of this handbook. treatment with Chelex. since enzymatic activity also takes place at subzero temperatures. E. such as Qiagen. the best method is to freeze it in liquid nitrogen or in a biofreezer. 14. for example by chelating divalent cations using EDTA and EGTA. The DNA is then separated from the contaminating cellular components by salt precipitation.9 resistance to diseases or to stress.A Stool DNA Isolation Kit (United Bioinformatica Inc.3. and the liver in farmed cod is much bigger than the liver of wild fish.N.). since diversity would be one of the selected traits in the farmed fish. classification based on morphological criteria demands the presence of the morphologic diagnostic characters.

23 In principle.2 DNA Markers In recent years.3. Spain. 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. 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. The outcome of these programs is already producing lists of genetic markers linked to traits of interest. Atlantic halibut.14 In addition.20. University of the Basque Country. SNPs. and sea bass.0). It is worth mentioning. An additional advantage is that it is possible to use robots for many of the steps (DNA preparation. and bass. Arctic charr). 14. protein markers commonly used for genetic analyses have the potential to be used as markers for farmed or wild.218 ◾ Handbook of Seafood and Seafood Products Analysis pellet is usually washed at least once with 70% ethanol. have started programs to map the whole genome of some species. personal communication). which may be used to identify the strains of the farmed individuals that display an increased frequency of the desired traits. tilapia. or mitochondrial. and others. DNA analyses may be performed using chips that permit the determination in one fast step of many characteristics simultaneously. any marker. catfish. cod. sample preparation. and also for forensic studies.). However. This method has been successfully used by the authors of this paper (unpublished results) and by Bucklin and Kochert22 with whole individuals of Calanus. tilapia. whether microsatellites. so the next step. Three more methods that have reputedly produced good quality DNA suitable for amplification. genomic.17 However.4 Analysis of Proteins No clear protein marker has been identified to discriminate farmed from wild seafood. 14. traits. from food matrices include the use of hexadecyl–trimethyl ammonium bromide (CTAB). must be performed very carefully not to lose the sample. such as Norway. however.19 the Chelex method. cod. which further increases the amount of samples that can be processed. since some alleles are more frequent in one group than in the other. which is washing the pellet with 50% ethanol. India.24 The method requires that all parent fish of the brood stock are DNA typed as well as all the individuals under examination. and production method. salmonids (salmon. On other occasions. pH 8. China. the United States.13.21 When using the salt extraction method with heavily degraded samples. and the DNA is reconstituted in double-distilled sterile water or in a slightly alkaline buffer (50 mm Tris–EDTA. differences in the protein pattern of liver25 and muscle26. 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. however. etc. trout. shrimp. by using a series of SNP and microsatellite polymorphisms by PCR and by oligonucleotide ligation assay (OLA).11. several countries. .15.18 and the salt extraction method. that the Norwegian company GenoMar has patented a method to trace back farmed individual Atlantic salmon. breeding stock. including oysters. the alcohol is allowed to evaporate. In these samples the pellet may be practically invisible. including the species. Japan. In the future. has the potential to be useful to differentiate farmed from wild specimens of a given species.27 tissues between farmed and wild salmon and cod have been reported.

neither feeds nor breeding conditions may be optimal for farming. In addition.28–31 The source of protein in teleost fish is very important.4.38 found that the reason for the softening in this species did not seem to be the faster growth of the farmed fish. it has been shown that components in fish feeds may contain very high levels of metals (Cu. and they hypothesized that the greater concentration of insoluble collagen present in wild salmon may contribute to their firmer texture. −20°C . which they use as their preferred energy source. which were attributed to increased proteolytic activity in the muscle of the farmed compared with the wild cod. and feed diets based on plant protein require supplementation with synthetic amino acids.36 Martin et al. neutral calcium-activated calpains.37 Martinez et al. several enzymes involved in anabolic metabolism were downregulated in fish fed the diet rich in soybean meal. lectins. including heat shock proteins. and this may induce stress in the farmed animals. Using proteomic analysis. 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. and they require high levels of dietary protein (30%–60%). loss of functional groups. antigenic proteins. it is common to apply directly to new species those conditions that have proven successful for other species.Analytical Methods to Differentiate Farmed from Wild Seafood ◾ 219 Industrial fish farming is a relatively new activity compared with farming of land animals. However. Some enzymatic systems that may be responsible for the muscle softening are metalloproteases and collagenases.26 examined the protein expression in skeletal muscle of farmed and wild cod by high-resolution twodimensional electrophoresis and found differences between the two. Olsson et al. and Ca)34 and that vegetable meals may contain antinutritional factors (protease inhibitors. which would be a natural diet. and then modify them depending on the results.27 also registered the altered expression of five enzymes implicated in the glycolytic pathway and citric acid cycle in farmed cod. have prompted the development of feed formulations based on vegetable oils and proteins. and the proteasome. 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.1 Sample Preservation The optimal case would be when the extraction of proteins can take place on the sample immediately after the experimental treatment. Although feeds and breeding conditions need to be developed and optimized for each species. lysosomal cathepsins. etc. with no preservation at all. Thus.)29 that may have adverse effects on fish.33. Fe. and proteolysis) of the proteins in the sample should be chosen. When this is not possible. several enzymes. the depletion of the wild stocks of pelagic fish and the high price of feeds based on fish meal and oil. a preservation procedure that minimizes the modifications (denaturation. For short periods of time. that is. Thus. indicating increased emphases on catabolism relative to anabolism in the fish fed this diet. 14. aggregation.33 Moreover. the amino acid profiles of plant proteins do not meet the essential amino acid requirements of fish. Optimal methods include fast freezing and frozen storage using temperatures as low as possible. Zn.32 Unfortunately. The authors noted a downregulation of some structural proteins in fish-fed soy proteins. attributed to the fish’s increased requirement for energy metabolism.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. is more common in stressed and in farmed than in wild fish.35. Proteins and proteomic analyses are dealt with in more detail in a different chapter of this handbook.25–27 In addition. usually considered negative. these authors identified 33 differentially expressed proteins. Interestingly. Johnston et al.. Mg. which is reflected in the composition of their organs. Soft texture. Texture is an important quality attribute of the fish flesh. and structural and FA-binding proteins.

we focus on the use of techniques with the potential to identify such markers. The first step in proteomic analyses is to extract as many proteins as possible from the sample. therefore. the gels can be stained by Coomassie Blue (low sensitivity but compatible with mass spectrometry (MS) analysis necessary for subsequent peptide fingerprinting and sequencing). Triton X-100. It should be noted that any preservation procedure will alter the protein profile in the sample. etc. due to the great diversity and properties of the proteins contained in the edible tissues of seafood. In our experience. 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 is common to use several buffers with increasing concentration of chaotropic agents (urea. destained. but not all protocols are compatible with MS). depending on the proteins one wishes to examine. In addition to published works. alkylated. thiourea). However. 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. and there are special protocols for each application. one should be very careful when comparing samples preserved and stored under different conditions. this may be because some commercial preparations of these enzymes are contaminated with proteases. Proteomic techniques have a clear advantage in this field. and. usually 8%–20% or 12% PAGE.4. for a wide screening. We therefore recommend not to use such enzymes. and reducing agents (b-mercaptoethanol. cooking. the optimal extraction procedure for any given sample must be determined empirically. but 3–10 are commonly used for wide screenings. usually with trypsin. and peptide mass fingerprinting of the digests is then .4. there are many methods suitable for protein analyses. therefore. The choice of method depends on the protein and the property one wishes to examine. but they will hamper the study of protease activities that may be relevant in some other works. and fluorescent labeling or staining (of intermediate sensibility and also compatible with MS).3 Analysis of Proteins As already mentioned. reduced. Proteomics permits the separation of many proteins (often thousands) from a complex protein mixture in one step.220 ◾ Handbook of Seafood and Seafood Products Analysis may be acceptable. The tryptic fragments are cleaned from contaminants. Both first and second-dimension gels can be purchased as precast. Afterward. 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.2 Protein Extraction There are many methods for extraction of proteins.). Their use should be evaluated for each particular study. 14. SDS). tributylphosphine) to solubilize the widest possible spectrum of proteins. ready to use gels from several companies. detergents (CHAPS. its application is widespread in many fields. the strip containing the proteins separated by their pI is loaded on top of the second-dimension SDS-PAGE gel. 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 digested. dithiothreitol (DTT). After separation. 14. The optimal pH range to choose depends on the sample. Since current studies are still trying to identify markers.25–27 BioRad39 and GE Healthcare Amersham have some excellent manuals about protein extraction and analysis. and. silver (high sensitivity. as well as the different degrees of processing to which the sample may have been submitted (freezing.

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

it is best to use as low a temperature as possible. and even after the levels of C20:5n3 and C22:6n3 were restored to the original high levels. because it provides multicomponent information and can be applied nondestructively.63. 29Si. 31P.g.and light-tight containers and stored at low temperatures.2 Lipid Extraction and Gas Chromatography Procedures for lipid extraction are described in another book chapter of this series. −80°C. the reader is directed to several publications46.52 that give detailed descriptions of the procedure. NMR spectroscopy can be used to identify functional groups. 195Pt).5. The most commonly measured nucleus is 1H (the most receptive isotope at natural abundance). 14. The major advantage of 1H NMR spectroscopy compared with 13C NMR is the higher sensitivity and thereby shorter acquisition times per experiment. and polymerization... which leads to less overlapping of signals. and in particular. including fish and fish products.5. which. 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. The spectrum is often used to obtain information about the number and type of molecules in a mixture. HR-NMR has been particularly valuable in the study of marine lipids. 2H. 14.222 ◾ Handbook of Seafood and Seafood Products Analysis for vegetable oils. that is. 17O. 23Na.1 Sample Preservation Due to the high levels of PUFAs. which may be used as a rapid profiling technique. On the other hand.52. the ratio n3/n6 was not fully restored. 13C NMR has a greater range of chemical shifts.64 may contribute to the difficulty of performing correct classifications as wild/ farmed based only on the FA composition. If freezing is required. the latter seems to be the most persistent after a dietary switch to fish oil diet. 35Cl. and is the preferred tool in lipid analysis when interpretation of . 11B.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. 14. 14N. Fresh samples should be kept wrapped in air. and an inert atmosphere. The most commonly used HR-NMR techniques in wild/farmed classification are 1H NMR and 13C NMR.50. 10B. both of which are able to detect a range of metabolites in a nontargeted way.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. but NMR is applicable to any nucleus possessing spin (e. 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.5. 15N. 19F.61 As indicated by Refsgaard et al. since in a one-dimensional spectrum each peak is produced by those nuclei placed in an identical local chemical environment. 13C. oxidation.65 NMR gives a fingerprint of the sample analyzed. For detailed descriptions of the analysis of fish samples. 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.

have allowed the differentiation between wild and farmed salmon74 and cod51 of different origins. a semiquantitative 13C NMR approach has been chosen. The most commonly used solvent in the analysis of neutral lipids is deuterated chloroform (i. Factors that affect the exact chemical shift of NMR signals include the type of solvent used.71 13C NMR gives information about FA composition of fish72 and the positional distribution of PUFAs in triacylglycerols and phospholipids. they are normally converted to ASCII or JCAMP file formats.. The assignment of spectral resonances gives information about the chemical composition of the samples.78 . which is easily evaporated.8% CDCl3). The application of multivariate statistics to NMR spectral data increases the potential of the technique considerably. the relative intensities for corresponding signals across different spectra are comparable. leaving the sample ready for analysis. Typically. even though the signal intensities within each spectrum are not quantitative.66.77 It is advisable to check that all spectra have acceptable linewidth and lineshape after the NMR analysis.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). 1H NMR has also been applied to differentiate between wild and farmed salmon and sea bream of different origins.75 and it is important that all the samples contain the same volume. Standardized procedures should be followed to ensure repeatability and comparability. the chemical shift scale is referred to the shift of TMS or indirectly to TMS by the peaks from chloroform at 7. temperature variations.28 ppm for 1H NMR and by the triplet of CDCl3 at 77. in conjunction with chemometrics. because it may interfere with the multivariate data analysis. and other intermolecular interactions. will increase the sample throughput significantly.e. Tetramethylsilane (TMS) is usually added as a chemical shift and intensity reference. However.67 1H NMR has been used to perform quantitative measurements of total n-3 FAs and of the levels of DHA.70.8 mL solvent is used. phasing and baseline correction are applied but no zero fi lling.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. Normally.76 In some studies. although this approach has still not been widely used for authentication purposes.5–0. It is expected that in the future the use of flow injection systems. Regions without signals or unwanted signals are removed before multivariate analysis. due to the fact that quantitative measurements require a considerable longer experimental time. interactions with metal ions. inhomogeneities in the applied magnetic field. Both HR 1H and 13C NMR. When full spectra are used. pH. a sample size of 50–100 mg of lipid in 0.77 Regarding reproducibility issues.66 Potential problems about inconsistencies in ppm values between samples in the data analyses should be solved by manual alignment or data pretreatment methods.0 ppm for 13C NMR. Typically. may lead to erroneous classification. Multivariate methods are frequently applied to study differences among NMR spectra. such as instabilities in apparatus. Both the area/ intensities of peaks and full spectra can be input for multivariate analysis. hydrogen bondings. or differences in relative concentrations of the samples analyzed.75 Small differences in experimental conditions.Analytical Methods to Differentiate Farmed from Wild Seafood ◾ 223 spectra is the goal. 99. ideally for screening many samples with short acquisition time. although the optimal sample size depends on the instrument. 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.73 which is of value for authentication purposes. 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. but it is not necessary for classification purposes.68.

because the enzymatic reaction rates on substrates that contain the lighter isotopic forms are faster than in reactions involving the heavier isotopic forms. SD ± 0. typically tropical grasses. nitrogen as two: 14N (99. such as CO2. Thus. the abundances of the stable isotopes differ between substrate and product.89%) and 13C (1. Introducing the percentage of C18:2n6 as a third variable in their model. SD ± 0. Samples of muscle tissue of wild salmon were significantly more enriched in nitrogen (d15N: mean = 12.37%). resulting in a complete separation of the two groups. and 18O (0.224 ◾ Handbook of Seafood and Seafood Products Analysis 14. Using the d15N of choline and the d18O of total oil. Since a molecule containing heavier isotopic forms has stronger chemical bonds. since the physical properties of molecules containing heavier isotopic forms are different. For example. 17O (0. exhibit limited variation. C18:1n-9. they were also able to correctly classify the fish according to their geographic origin.75.23‰) than the aquaculture specimens.037%). Bell et al. Aursand et al. In addition. and oxygen has been proposed as a method suitable for food authentication. Thomas et al. For example. the abundance of stable isotopes varies among different compounds.50 were also able to correctly classify Atlantic salmon according to their geographic origin and production method by using four FA compositions (C16:0. and O2.81 This is because the 13C/12C ratio depends almost exclusively on the photosynthetic mechanism used by the plants for CO2 fi xation.759%).38‰) but depleted in lipid-corrected carbon (d13C: mean = −20. plus the kinetic fractionations occurring in animal metabolism. 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.79 Carbon exists as two stable isotopes: 12C (abundance 98. N2. and oxygen as three: 16O (99. . equilibrium reactions also lead to a fractionation of the isotopic forms.82 Dempson and Power83 examined the potential of using stable isotopes of carbon and nitrogen 13C and d15N) by isotope ratio mass spectrometry (IRMS) to identify escaped farmed Atlantic (d salmon. The isotopic abundances in animal tissues and animal food products are the summation of the feeds ingested throughout all their life.52 were able to classify correctly according to the production method. 171 Atlantic salmon specimens originating from three continents and 15 different geographic regions. while typical d13C mean values of C3 plants may be −26/−28‰. although many broadleaf plants are also C4).46 were equally successful classifying sea bass using the FA profile. Usually animal products become enriched in the heavier isotope (15N and 13C).63) and 15N (0.51.80 The natural isotopic abundance largely varies depending on the chemical forms.204%). a kinetic fractionation occurs. C4 plants may have d13C mean values of −12/−14‰. d13C of individual FA. and they reflect both significant isotopic fractionation by microbes and the different biological substrates producing these gases. Some atmospheric gases. such as maize. depending on their diet and their position in the trophic chain: the higher its position in the trophic chain.6 Stable Isotopes The variation in the abundance of the stable isotopes of carbon. nitrogen. N2O and CH4 exhibit wide isotopic variation. Moreover. 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. C16:1n-9.11%). the higher the proportion of the heavier isotope. Differences in the 15N/14N ratio also result essentially from diet. the 13C/12C ratio for both milk fat and cheese protein give information on the type of forage fed to the cows. In contrast. 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).

2 SNIF–NMR and IRMS Two methods are used to assess stable isotopes: SNIF–NMR and IRMS.1 were able to differentiate wild. and so on. pulverized to a fine powder using a ball mill grinder.1 Sample Preservation It is very important not to contaminate the sample during handling. experimental duration. and a detector to measure the different isotopic species. are typically determined with a gas isotope rationing mass spectrometer. such as carbon.Analytical Methods to Differentiate Farmed from Wild Seafood ◾ 225 d13C and d18O of total muscle oil. The light elements.6. The gas is introduced in the mass spectrometer and is ionized by removal of an electron in the ion source. nitrogen as N2. and the abundance ratios of the heavy and light isotopic species are then calculated. carbon as CO. The assumption that fractionation was independent of body mass was upheld for muscle and heart tissue but not for liver. Approximately 1 mg of dried. respectively (for carbon 13C:12C. 14. nitrogen. All international standards are . 14.86 To facilitate comparisons between specimens with differing lipid contents. The ionized gas is then introduced in the flight tube under vacuum or carried by helium. The instrument consists of an ionizing source. the d15N values of heart and liver were also affected by environmental temperature. d13C values are normalized for lipid content following techniques developed by McConnaughey and McRoy87 and validated by Kline et al. The element is converted to a gaseous form to be analyzed by the mass spectrometer. and environmental conditions on fish tissue. Molkeltin et al. since these authors assessed the effects of body size. For example.48 NMR techniques have been described previously. and commercially farmed Atlantic salmon measuring d13C and d15N by IRMS in raw fillets. The C and O isotopic profiles of fish tissues may be altered if CO is used for stunning or killing. Interestingly. thus hydrogen is introduced as H2. 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.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. a flight tube with a magnet. ground tissue is used in the simultaneous analysis of stable C and N isotopes. whereas IRMS can be applied to all except 12 elements. and oxygen as CO2. where R is the heavy:light isotopic ratio of the sample or standard. SNIF–NMR can only be applied to the few isotopomers possessing spin. organic. Enriched samples contain relatively more of the heavier isotopes.85. and stored in glass desiccation vials until analyzed. and d15N of the glycerol choline fraction of flesh phospholipids. for nitrogen 15N:14N). the collected tissue samples are dried at a constant temperature of approximately 50°C for 48 h. probably reflecting the metabolic functions of these tissues and their associated turnover rates. and oxygen isotopes. the ions are finally detected at the detector. it must not be washed in the laboratory after collection (which may alter the O and H profile of the sample). However. whereas IRMS gives only an average value of the isotopic forms in the molecule. as follows: d = (R sample/R standard − 1) × 1000‰. the paths of isotopic species are deflected by the magnet by an angle that is a direct function of their mass over charge ratio.84 has helped to understand the nitrogen isotopic variations in fishes. Usually. The research of Sweeting et al.6.

Biochemical analytical techniques using multiple elemental analysis.1–1 g of tissue samples are placed into 50 mL Teflon tubes and 8–16 sample volumes of a mixture of concentrated trace-metal-grade nitric acid/hydroperoxide mixture (5:3) is added. in particular since the analysis may detect contaminants at the pM level. All implements and containers should be cleaned with 0. The same research group (Yamashita et al. lead. Taiwan. and China. Therefore. Multivariate trace elemental analysis is increasingly used as a technique to differentiate the geographic origins of foodstuff. farmed and wild specimens of the same species have different geographic distributions. Thus. handling. multiple elemental analysis could also be used in this case to identify imported clams from China and Korea. were shown to be of relevance to determine the origin of eels. cadmium. The . 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. Multivariate analysis showed that differences in elemental composition in the muscle between Japanese and imported clams were mainly due to two factors: Factor 1.91 In the case of fish.1 Sample Preservation As for stable isotope analysis.226 ◾ Handbook of Seafood and Seafood Products Analysis set at 0‰ by convention. it is very important to avoid contaminating the sample during sampling. cadmium and arsenic levels in the muscles of clams from China and Korea were higher than those of clams from Japan.95 By using ICP-MS analysis the sensitivity in the determination of rare trace elements can be increased from the nM to pM level. false labeling problems were encountered in which imported live Japanese eels from Taiwan were illegally sold as being of Japanese origin. 14. copper. and strontium levels and Factor 3. with the exception that clams from Miyagi had high arsenic content. multivariate trace elemental analysis is expected to be helpful in determining whether the fish was farmed and its geographic distribution. as well as vitamin K and its metabolites. Korea. The first step in the analysis is the digestion of the sample: 0. such as uranium. 14.7.92–94 Otolith chemistry is useful for identifying the natal origin and assessing the relative contribution of different nursery areas to mixed adult stocks. Rare trace elements taken from the environment.7 Trace Element Fingerprint Sometimes. 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. unpublished data) examined the trace element composition of the muscle and shell of littleneck clams collected in Japan. and it must be accurately weighed with a microbalance. Carbonate rock from the Pee Dee Belemnite formation89 and nitrogen gas in the atmosphere90 are used as the standards for carbon and nitrogen. Thirteen elements were shown to be the most diagnostic. quality.5 M nitric acid and rinsed with Milli-Q ultrapure deionized water. otolith chemistry is used as a recorder of time and environmental conditions.. respectively. in addition to DNA-based species identification techniques. and vanadium. and analysis. attributable to manganese and vanadium levels. The sample may be stored in a centrifuge tube at a temperature of −40°C or lower until analyzed. Each sample should be separated from the tissues using ceramic knives and scissors and Teflon-coated tweezers to avoid contamination of metals. attributable to cobalt. and they found distinct patterns for each of the three origins. and price. have been used to differentiate the geographical distribution of origin of farmed Japanese eel. Recently. In addition.

8 Other Methods Depending on the species. Canada). Japan). where energy transfer processes cause desolvation. 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-2004-506359.98 making this approach more unreliable than it used to be. and the resulting digest is a clear liquid with a yellow tint.97 using an automatic mercury analyzer (Hiranuma HG-200. but although the diet of wild salmon contains astaxanthin. and ionization. and the ion information is processed by a data handling system. For internal standardization. If the measured concentration deviates from the true concentration by more than 10%. Each solution (5 mL) of the microwave samples is applied to the atomic absorption spectrophotometer. the samples are diluted to a final volume of 50 mL in Digitube (SCP Science. Y. most artificial feeds contain a mixture of canthaxanthin and astaxanthins of different origins (both natural and synthetic). 14.2 ICP-MS Multielement determination of trace elements is usually measured by inductively coupled plasma mass spectroscopy (ICP-MS). To verify that the instrument is properly calibrated on a continuous basis. and stored at room temperature until use. an internal standard mixture is added. Afterwards. the mass calibration and resolution are checked using diluted metal solutions as standards. which suffers from severe memory effects.3′S) that does not occur naturally and can therefore be used as a marker for farmed salmon. In the farming of salmon for example. Ions transmitted through the quadrupole are detected by continuous dynode electron multiplier assembly. Tb. there might be specific requirements that may be targeted to identify the production method. the Norwegian Research . Multiwave 3000 Microwave Oven.7. To initiate the proper operating configuration of the instrument and data system. Analysis by chiral chromatography can be used to identify a chiral form (the meso form 3R. and the last 10 samples are analyzed again.Analytical Methods to Differentiate Farmed from Wild Seafood ◾ 227 digestion may be carried out by placing the tubes in a microwave oven (for example. much of the astaxanthin used in fish feed nowadays is produced from cultured microalgae or from krill. 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. In.98 However. atomization. the instrument is recalibrated. Perkin-Elmer). the total mercury concentration is determined by cold vapor atomic absorption spectrometry. five internal standards are used: Sc. the use of carotenoids is allowed.42.96 Samples digested as described above are introduced by pneumatic nebulization into a radio frequency plasma. and Bi. For the determination of mercury. a calibration blank and calibration standards are used as surrogate test samples after every 10 analyses. 14.

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..2..........................3 Smoke Powders ............................... 236 15...................8 Polycyclic Aromatic Hydrocarbons........ 239 15.............................. 237 15.......................................................9 Legislative Aspects...........249 15........2 Extraction and Analysis Methods of PAH in SF and Seafood Treated by SF ... 248 15......5 Role of SF Process Parameters in Volatile Compounds Generation ..........1 Properties and Toxicology ...................................................................... 246 15...................6............................................................. 245 15......2..... 241 15..............1 European Regulations on PAH Found in SF ...........2 Smoke Flavoring Process .......... 240 15........................................1 Role of Volatile Compounds of SF in the Odor ......................................................................1 Introduction .....6................................2 Role of Volatile Compounds of SF in the Flavor ..........6........1 Liquid Smokes.249 15.............................Chapter 15 Smoke Flavoring Technology in Seafood Vincent Varlet...........6..............................................7 Role of Volatile Compounds of SF in Preservation ......4 Smoke By-Products ......................241 15...............249 233 ............................................................................................................................. 246 15.........4 Chemical Composition of Liquid Smokes .............9.........4 Role of Volatile Compounds of SF in the Aspect and Color .......... 248 15................................................. 234 15........................................................2 Smoke Oils .. Thierry Serot..............................................................3 Role of Volatile Compounds of SF in the Texture ......................6 Organoleptic Roles of Volatile Compounds of SF ............................. 238 15............................. 234 15...............................247 15.................................... 237 15..................................................................................... 237 15.....................................................3 Use of Smoke Flavorings .........................................................8.2............8.. and Carole Prost Contents 15................................................................................................2....................

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

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 15.1 Diagram of fabrication of SFs. ◾ 235 .

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

whereas liquid smokes are used for the characteristic smoky odor and color of smoked products. SFs for herring.2 Smoke Oils Smoke oils are made by blending liquid smokes with vegetable oils.2. Smoke by-products constitute more complex SFs. 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. or fish oils. 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. 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. Therefore. Indeed.2. hundreds of smoke by-products are available. the liquid smokes and smoke powders can be added to salt or in brine but not smoke oils. smoke powders are mainly used to confer smoky tastes to the final product. salmon. because smoke oils are especially employed in food preparations such as emulsions. . 15. We must distinguish dry salting and wet salting. in seafood industry. Therefore. Dry salting (or dry curing if nitrited salts are used) is made with dry salt deposited directly on food.4 Smoke By-Products Smoke by-products are constituted by smoke extracts and smoke distillates [6]. Today. 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 distillates are obtained by the fractionating distillation of PP. and so forth. the organoleptic qualities can vary in a high range changing the food matrix. 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. Nitrited salts. these SFs are not used much in seafood industry. These molecules can increase the generation of carcinogenic nitrosamines. 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. These smoke powders can be added to salt used for salting steps or to dehydrated sauces or soups elaborated from seafood products. are present in the market. As smoke oils. The distillation is commonly performed with steam water at atmospheric pressure.2. However. smoke oils can be only used in preparations such as taramas. consequent to the reaction between amino acids and nitrite. Smoke powders can also be rehydrated and used in brine as liquid smokes. Consequently.Smoke Flavoring Technology in Seafood ◾ 237 15.3 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]. Consequently. fish sauces. They are less acidic than aqueous flavors and allow to exhibit more complex smoky tastes. 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. smoke manufacturers can control their products and can create smoke by-products whose uses are recommended for a kind of fish. 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. most frequently in 90:10 (v/v) proportions. Therefore. 15. generally forbidden in seafood industry according to the countries.

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

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

The combustion is faster when the granulometry of the wood raw material is important [23. The high moisture allows to reduce the wood combustion efficiency. and 400°C for lignin [17]. but they have a weak impact on the smoky flavor of SF and food processed with SF [13]. lignin dimers. steps of SF purification through filters or apolar solvent washes are often required to decrease the PAH levels. the quantity of phenolic compounds increases with a maximum close to 500°C and decreases after 500°C. The generation of volatile compounds is dependent on the wood pyrolysis temperature [16]. known as the smoky skeleton of SF. Therefore. and trimers [12]. furannic compounds. For example. it seems difficult to generate the desired organoleptic volatile compounds without PAH contaminants. phenol amount is multiplied by two between 450°C and 650°C. The air velocity indirectly influences the SF composition by the modification of pyrolysis temperature or smoke temperature [21.19]. PAH must also be surveyed. with a lower water rate than that in softwood. differences can be observed depending on the molecules. 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]. and phenolic compounds [10. 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. The rate of carbonyl compounds increases gradually with the temperature from 200°C to 600°C. Wood granulometry can also influence SF composition. A temperature of 450°C–500°C was reported to lead to the best composition for the creation of carbonyls. and humidity of air constitute key parameters of SF composition. the diversity of settings of pyrolysis parameters can explain the diversity of organoleptic volatile compounds and the diversity of qualities of SF. After the water release (close to 120°C–150°C). The wood moisture appears as the second important parameter [20]. is recommended because it burns slower. a lower temperature is reached and allows increasing the generation of smoke volatile compounds and minimizing PAH formation. between 280°C and 320°C for cellulose. Then. exothermic reactions of pyrolysis of wood components occur between 200°C and 250°C for hemicellulose. As the best pyrolysis temperature to obtain the required volatile compounds are between 380°C and 500°C.24]. the wood granulometry and moisture. different groups of compounds are formed. whereas syringol quantity is tripled. the main organoleptically active volatile compounds generated during the pyrolysis process can be sorted in three groups of molecules: the phenolic compounds.240 ◾ Handbook of Seafood and Seafood Products Analysis ethers from lignans. An optimal moisture is planned in the industry between 17% and 20%. The manufacturer can choose SF according to the required result on the organoleptic characteristics of the final product. A step of filtration is almost obligatory to avoid these contaminants. According to the pyrolysis process. the pyrolysis temperature. The use of hardwood. However. the furannic derivatives. Therefore. A slow combustion is reached with weak air velocity. because their contents in smoke or in food increase from 400°C to 1000°C. Due to their . the velocity. The rate of acids is higher for temperature lower than 300°C and decreases after 300°C with the increase in temperature. Lower concentrations of oxygenated compounds have been found to be caused by an oxygen depletion during combustion [20].22].18. Indeed. and the enolones derivatives [14].5 Role of SF Process Parameters in Volatile Compounds Generation Except the wood type that influences the smoke quality strongly [15]. because it plays a role in the pyrolysis temperature. 15.

15. the diversity of SF causes diverse consequences on the texture. A much more complex mixture of compounds is responsible for the characteristic aroma of smoked fishes [35]. two main classes of odor-impact molecules can be defined: phenolic compounds and carbonyl compounds. Phenolic compounds of low-boiling fraction (60°C–90°C) composed mainly of phenol. odor. The role of syringol is important. These observations have been recently corrected [14. syringol. Phenolic compounds are known to constitute the odorant “smoky” skeleton of wood smoke and smoked fish.33. Phenolic compounds of medium volatility have been considered as the most important odorant molecules.1 Role of Volatile Compounds of SF in the Odor Even if the concentrations of odorant volatile compounds in SF can be various. Many studies have indicated that phenolic compounds present in the vapor phase of smoke may be odor-active compounds [30–32]. taste.2).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] . Some of them have very low odorant thresholds.34] (Table 15. phenolic compounds are not sufficient to explain the SF role in smoked fish odors.29]. guaiacol. Table 15. cresols. Similarly. They are the major compounds in SF with a wide range of odorant thresholds (Table 15.Smoke Flavoring Technology in Seafood ◾ 241 chemical composition. and preservation of the product. and alkylguaiacol may also contribute to imparting a smoky flavor to smoked fishes [13. making them odorant at low concentrations. and methylsyringol has a pure and characteristic smoky flavor [10]. but it may not be the main contributor to wood smoke flavor.34].1) [14.6 Organoleptic Roles of Volatile Compounds of SF 15.6. which gather furannic and enolone derivatives.28. color. The medium-boiling fraction (91°C–132°C) composed of isoeugenols.

burnt.12 42. earthy. LRI MS.18 ± 37. spicy Spicy. LRI.24 ± 63.53) 8. fatty Roasty.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. STD MS. LRI.27 ± 0. vanilla. LRI.50) 65. STD MS.98 20. LRI MS.94 49. STD MS.27 ± 2.97 23.25 6 (5) 6 7 (4) 7 7 6 8 8 8 Chemical.33 ± 1. potato Cooked potato. wood fire.Table 15. STD MS. spicy/ woody (3) (2) 5 6 (2) 4 4 3 5 6 7 (2) 4 4. LRI. metallic. burnt. milk Smoke.45 ± 172. STD Cooked.74 ± 27. roasty Chemical.48 ± 8. 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 2.63 ± 13. ink Marine. LRI.90 (1.55 ± 9. LRI.3-Dimethyl-2cyclopentenone 1052 o-Cresol 1068 p-Cresol 1093 Guaiacol 1110 2. burnt Smoked. green Cooked vegetable.17 ± 26.62 74.34 (24.4-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. milk Cooked/soup.22 ± 9.53 360. mushroom Cooked.04 7 4 16.6-Dimethylphenol 1130 . STD MS. STD MS.07 (1. STD MS.55 ± 39. LRI.6-Dimethylpyridine 890 2. STD MS. STD MS. chemical. Animal. LRI.37 ± 3. spicy.75) MS. LRI MS. LRI MS. chemical Green. spicy. LRI.64 ± 18. STD MS. green Cooked vegetable.44 17. green. LRI. LRI.

STD MS.5-Dimethoxytoluene 1282 4-Ethylguaiacol 1287 Indanone 1307 4-Vinylguaiacol 1330 (E.49 ± 6. burnt Smoke.36 1132 MS Cooked.24 ± 1.97 2.12 4.and 2. clove Sawdust. smoke. LRI MS.21 ± 7. smoked Candy.51 ± 18. LRI. STD MS. green. STD Cucumber. clove Green. STD MS.26 ± 1. fatty Burnt rubber. violet.35 3-Ethyl-2-hydroxy-2cyclopentenone 1140 1.96 ± 10.72 44. medicinal 7 4 10. green. spicy. vanilla.4Trimethylcyclopenten-1one MS MS.16 ± 5. LRI. LRI. rotten.71 (3.50 18. STD MS.15 ± 1. spicy.3-Trimethoxy-5methylbenzene 243 .5Dimethylphenol/ (E)-2-nonenal MS.4-Decadienal 1330 Syringol 1365 Eugenol 1370 Smoke Flavoring Technology in Seafood 4-Propylguaiacol 1382 1400 ◾ 1. LRI. LRI. spicy Oily.2.E)-2. STD MS. green. STD MS. LRI MS. green. milk Burnt. green 6 3 11.15) (continued) 2.61 ± 22. green.25 ± 10. LRI. LRI MS. LRI.86 (2.87 ± 1. LRI 1160–1180 4-Methylguaiacol 1192 482. LRI MS.95) 8. chemical Green. LRI MS. smoked Cooked vegetable.46 Solvent.17 15. vanilla Cooked.2-Dimethoxybenzene 1147 2.62 ± 4. smoke.2. STD MS.91 36.79 (6.13 6.4. green Spicy. spicy Spicy. LRI.25 ± 4.15) 86. spicy 6 5 17.85 ± 40. fatty.15 ± 243. 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.82 ± 6.3.3-Dimethoxytoluene 1247 (E)-2-Decenal 1266 3. spicy.

and quantities of odor-active compounds detected by fewer than six judges are indicated in parenthesis. STD MS. When only MS is available for identification. roasty 7 4 Burnt rubber.23 ± 0.55 ± 8.35 (20.48) 1.3. Agric. LRI. 55. V. spectrum. LRI MS. and odor description of an identified compound correspond to the retention time. LRI Animal. LRI.40 ± 3.68 MS. and odor description of the injected standard of this compound). STD. LRI MS.5 is rounded to 5 and an intensity between 5.. STD MS.. roasty. spectrum.39 6. a b c Handbook of Seafood and Seafood Products Analysis d e Means of identification: MS. standard (when the retention time. Means of three fillets. et al. chemical 6 4 Smoke.81 ± 11. mass spectrum (identified using the mass spectra of the compounds). odor intensity. 4518.77 24. it must be considered as an attempt of identification.5-Trimethoxytoluene 1527 4-Allylsyringol 1615 8-Heptadecene 1680 Source: Varlet.5 and 6 is rounded to 6 (1 = very weak odor. LRI. Average intensity of the eight judges is rounded to the nearest whole number.87 ± 2. Note: Frequency of detection. woody (4) (2) Clove. An intensity between 5 and 5. J. 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. green. 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. rotten 7 4 Spicy. linear retention index (when the LRI of the identified compound corresponds to the LRI in the literature). 9 = very strong odor intensity). Food Chem. 2007. .Table 15. Number of judges (out of eight) who have detected an odor.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. In micrograms equivalents of dodecane per 100 g of smoked salmon.

early works performed on individual phenolic compounds have identified the impact of guaiacol on the smoky flavor. . the same compounds responsible for the odor should be involved in the flavor that SF confers to seafood. More recently. Moreover.3. they may contribute in mixture to the overall odor. if they do not have a strong individual influence. the determination of the effects of compounds of SF on the flavor is complex. However. guaiacol derivatives and more generally the phenolic compounds of low-boiling fraction molecules (60°C–90°C) have been shown to cause the odor.38]. as the other minor odor-active compounds. They were isolated early from wood smoke and described as grassy. As for the assessment of the odor. 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. However. Furthermore. whereas syringol and 4-methylguaiacol showed the same but lower effect than guaiacol [40]. Recently. Two categories of carbonyl compounds can be differentiated: furannic compounds and enolone derivatives. or oils. 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. Concerning the bitter taste. Then. 15. phenolic compounds are not the only flavor-active compounds. Synergic or masking effects are possible and make the final odor complex. it was commonly admitted that syringol derivatives impart a smoky odor and guaiacol derivatives contribute to a smoky flavor. Furannic compounds were thought to contribute to soften the heavy smoky aromas associated with phenolic compounds [37. liquids. reactions between liquid smoke compounds and the components of the matrix can occur through Maillard and Strecker reactions. For several decades. Enolone derivatives are compounds derived from cyclopentenone. and the odorant contribution of odor-active compounds cannot be the same if the SF is in the form of powders. sometimes cooked.2 Role of Volatile Compounds of SF in the Flavor Phenolic compounds were shown as the major contributors of the smoky flavor [35]. The oils used in smoke oils can soften the smoke aroma. 4-methylguaiacol perception was superior to that of syringol and guaiacol. 4-methylguaiacol. the physical state of SF can also influence the aroma. but taste was not investigated as much as odor. The fractionation of a commercial liquid smoke preparation evaluated by a sensory panel concluded that the phenol fraction was essential but not complete from a sensory standpoint [42]. and seem to contribute little to overall aroma. because they are not the compounds mainly detected in SF and seafood treated by SF odors by sensory analysis [37].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]. The taste thresholds of some phenolic compounds were determined [40] and showed a high diversity between the molecules. 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. furannic compounds were found to play a role in cold smoke odors of liquid smoke or fishes treated by liquid smoke [14.34]. The results of this fractionation are given in Table 15. and isoeugenol in spicy/sweet flavor [41]. Furfural and homologues exhibit cooked/roasty aromatic notes.6. However. Sensory analysis performed on standards confirmed the importance of guaiacol and o-cresol in the smoky flavor and dimethylphenol.39]. and very little information is available.

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

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

they are considered as heavy PAHs.3). . Therefore. These compounds have been studied for several years. Owing to their lipophilic properties (log Kow between 4 and 7). more toxic than light PAHs. which are constituted by less than four benzene rings. Therefore.248 ◾ Handbook of Seafood and Seafood Products Analysis 15. Hundreds of individual PAHs may be formed and released during the process of incomplete burning of the wood. In SF. they are considered as environmental pollutants and can contaminate the human feed raw material [53. However.8-dihydrodiol-9. particularly smoked food [57. hence their toxicity (Figure 15.8.10-epoxyde Glutathion S OH OH OH O B[a]P 7. They are considered as carcinogenic contaminants. 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. because their catabolism leads to poly-hydroxy-epoxy-PAH suitable for binding to DNA adducts.3 Benzo[a]pyrene metabolization.1 Polycyclic Aromatic Hydrocarbons Properties and Toxicology PAHs are well known as being food contaminants and carcinogens [52].8-dihydrodiol OH B[a]P glutathion conjugate OH OH B[a]P 7. home cooking and industrial food processes represent the major source of human contamination [55].54]. PAHs are considered as carcinogenic contaminants of processed food [56]. As they can be absorbed by animals.58]. 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. especially benzo[a]pyrene (B[a]P) [59]. PAHs can cross the biological membranes and accumulate in tissues.8 15. PAHs are formed by the incomplete burning of carbon-containing material. PAHs comprise fused aromatic rings made up of carbon and hydrogen atoms: up to four fused benzene rings.8 epoxyde OH B[a]P 7. PAHs are generated during smoke production by wood pyrolysis.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. it is used as the leading substance to illustrate PAH contamination.

Therefore. . Apolar solvents or mixes of apolar and semipolar solvents are used to extract the maximum of PAHs. solid–liquid extraction can be carried out. that is. Moreover. the maximum levels of B[a]A and B[a]P were set at 20 and 10 mg/kg of liquid smoke. Several devices are therefore developed to optimize the analysis. they have not been applied to SF or seafood treated by SF. or tandem mass spectrometry [70]. Although all steps are important. to our knowledge.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 was especially performed on an alumina or silica column. gas chromatography and liquid chromatography are the most used techniques [55]. However. which combines a separation step and a detection step.S. eight light PAHs were considered as environmental contaminants. This harmonization was necessary to homogenize the legislation about SFs.1 European Regulations on PAH Found in SF In 2003. and liquid chromatography is coupled to ultraviolet or fluorimetric detector [59–63]. the analysis step is the most critical point. In the case of liquid matrices as liquid smoke. Among them. in Italy. 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. such as bidimensional chromatography at the gaseous phase (GC/GC) [71] or liquid phase (LC/LC) [72]. The nature of the SPE cartridge phase is linked to the extraction method and the biochemical composition of the initial matrix. Gas chromatography is coupled to mass spectrometry [58. these PAHs were considered as toxic at low levels. cause chromatographic coelutions. because they do not have the same toxicity. with a weak toxicity but high concentrations in the samples analyzed.9 Legislative Aspects 15.69]. 15. Environmental Protection Agency (US-EPA) identified a list of 16 PAHs as the most frequently found [60]. 15. even if they were found in weak quantities.Smoke Flavoring Technology in Seafood ◾ 249 safety avoiding PAH contamination. which can disturb the extraction. The eight heavy PAHs left were shown as being carcinogenic or mutagenic contaminants and gave rise to serious health concern. In the 1980s. a European regulation set the maximum contents of PAH in the primary products (PP) of smoke condensates used for the production of SF. Indeed. but solid-phase extraction (SPE) cartridges are now more frequently employed. The extraction step must integrate the composition of the matrix. For example. respectively [73]. For the separation of the PAHs extracted from SF or seafood treated by SF. In both condensates. respectively [74]. In the case of solid seafood treated by liquid smoke. Other extraction devices have been developed to investigate PAH in smoked food such as accelerated solvent extraction (ASE) [65].9. the U. PSC and PTF. Thus. supercritical fluid extraction (SFE) [66] or solid-phase microextraction (SPME) [67]. a liquid–liquid solvent extraction is often used [61–63]. PAHs are often coextracted with fat matter.8. Parameters of the chromatographic separation and detection must be adjusted to avoid coelutions with interferences from lipids. and stir bar sorptive extraction (SBSE) [68] but not on liquid smokes or seafood treated by SF. purification and/or delipidation steps such as saponification are often applied to reduce the fat matter rate of samples [64]. flame ionization detector (FID) [60]. the chromatography must be sufficiently efficient to separate isomers of PAH. and lead to mistakes in the identification. it is essential to quantify only the toxictargeted compounds. but.

but it could also initiate an international consideration of labeling of smoked and flavored food. Moreover. this process can also be considered as a flavoring of the surface of the product. . Indeed. 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. and head and thorax meat of lobster and similar large crustaceans [76. This value is the result of the necessary harmonization between the national laws of European countries [79]. the vaporization of SF in a smokehouse causes a loophole in the legislation. 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]. The main criticism that can be formulated against SF is the lack of control of the final organoleptic qualities of such processed food. Therefore. whereas food is treated by SF and not directly by PP. the respective legal B[a]P amount is 5 mg/kg. 15.75]. if it is considered as smoking technique. it leads to lower PAH contents. In certain countries such as France. important differences in PAH concentrations are noticeable [57. a European regulation set the maximum content of B[a]P in foodstuffs treated by SF at 0. This value is very low compared to those authorized in PP.10 Conclusion A wide range of SFs and uses of SFs are now available to flavor seafood products. that is. Indeed. leading to less PAH contaminated food by comparison with the traditional smoking techniques [80]. that is.03 mg/kg.69. as drenching or showering. SFs appear as a safe alternative to smoking techniques. However. Thus. Indeed. Therefore. atomization of liquid smoke would constitute the smoking technique. SFs are used in higher quantities than those employed in flavoring processes. 15. because these values were set for PP and not SF. very small amounts. All these benefits could help to reconsider the status of atomization of liquid smoke and the maximum PAH contents related. the liquid smoking process decreases the emissions of PAH compounds to the environment. However. it is paradoxical to apply flavoring regulations to the smoking process. As for SF.2 European Regulations on PAH Concentration in Food Treated by SF In 1988. brown meat of crab. Finally. Th is fact can be understood by the use of smoke condensates in flavoring quantities. 0. Moreover. Moreover. the 2003 maximum values must be reviewed again. However.03 mg/kg. excluding bivalve molluscs. The content of PAH in SFs and in the final product can be better controlled than during traditional smoking. it can lead to problems of labeling.78]. 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.9.250 ◾ Handbook of Seafood and Seafood Products Analysis However. Indeed. the PAH contamination was only set for B[a]P [77]. the PAH contamination reached in SF is largely below the values authorized in PP [60. Nevertheless. it is necessary to better control the composition of SFs and to improve knowledge about the influence of the pyrolysis parameters (wood nature. the smoking regulations set a maximum B[a]P value of 5 mg/kg of smoked fishery products and smoked crustaceans. atomization of liquid smoke must be lower than 0. In this case.63] which justifies controls and regulations. according to the origin of SF and industrial manufacturers. The high maximum values authorized in PP do not seem well adjusted with the weak final PAH contamination of SF.03 mg/kg and leads often to noncompliant smoked products. for meat industry. Therefore.

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

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

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

1473. J.. J. et al. 309. T.. Contam... Union. Determination of polycyclic aromatic hydrocarbons in water by solid-phase microextraction–gas chromatography–mass spectrometry.. Polycyclic aromatic hydrocarbons from wood pyrolysis in charcoal production furnaces. 1062. Food Chem. 76.. P. A. 1999. Determination of polycyclic aromatic hydrocarbons in vegetable oils using solidphase microextraction—Comprehensive two-dimensional gas chromatography coupled with time-offlight mass spectrometry. EC 2065/2003. 77. G. Eur. L. Lebensm... 284.254 ◾ Handbook of Seafood and Seafood Products Analysis 65. Food Chem... Technol. 1. 71..-Technol. Off. Toxicol.. 66.L. Union. 897. Readman. Chromatogr.. 2004. Pimenta. Use of supercritical fluid extraction-high performance chromatography in the determination of polynuclear aromatic hydrocarbons from smoked and broiled fish.J. 364: 5. et al. T. Ré-Poppi. Italian Law Decree. et al. Contam.. 716. and Santiago-Silva. R. 1. L. 47. 38. Analytical methods for polycyclic aromatic hydrocarbons (PAHs) in food and the environment needed for new food legislation in the European Union. and Tapanainen. Evaluation of acute toxicity and genotoxicity of liquid products from pyrolysis of Eucalyptus grandis wood. 78. G. E. Moret. Res. Hattula. L. 2006. 74. 2001. U. Chim. 1161. W. Varlet et al. J. 79. 47. 2007. N. Relat. M. Union. 72. 2006. Eur. L 34: 43. 68. Allegato III. Järvenpää. Wenzl. Chromatogr..-Wiss. 69. Determination of PAH profiles by GC-MS/MS in salmon muscle processed according to four different smoking techniques. J. J. . D. J. J.S. 19(9). 523(2). A. Chem. Anal.M. 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.. Assessment of polycyclic aromatic hydrocarbon content of smoked fish by means of a fast HPLC/HPLC method. 101. 73.. J. Union. Popp. et al. Trends Anal. Huopalahti.. 2003. et al. P. 1367.. 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. 259. 2007. EC 2005/108. 184. Liq.. 744. 521. 2000. Off. and Dean. 75. 25(7). EC 88/388. Conte. 2006. L. et al.. J. 2005. Commission Regulation of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs. 1999. 153. Environ. 24(7). Chromatogr. 34.. S. J. 70. Accelerated solvent extraction and gas chromatography/mass spectrometry for determination of polycyclic aromatic hydrocarbons in smoked food samples. 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. A. A. Agric. 1–2. Purcaro.. Decreto Legislativo N°107 del 25/01/1992. Use of liquid smoke flavouring as an alternative to traditional flue gas smoking of rainbow trout fillets (Oncorhyncus mykiss). and Zhou... Environ. Eur. Wang. J. 169.. Determination of polycyclic aromatic hydrocarbons in wastewater by off-line coupling of solid-phase microextraction with column liquid chromatography. Commission Recommendation of 4 February 2005 on the further investigation into the levels of polycyclic aromatic hydrocarbons in certain foods.. Eur. King. Agric. Food Addit. Acta. EC 1881/2006. Off. 1996. 80. 1988. Dos Santos Barbosa. 67. 304. Off. 2003. Arch.

NUTRITIONAL QUALITY III .

.

............4..................................................270 16....................................................3 Lipids .........3......... 273 16.....267 16....276 257 ..................................................... 269 16................2 Methods for Determination of Total Lipids .................................................................4 Comparison of Methods .......................7 Calories ..274 16.................................................. and Rasa Slizyte Contents 16.....................................................3......3 Nondestructive Methods ..............................................................................................................275 16..................4 Proteins ................270 16..........................7...267 16..... 258 16...................2 Nondestructive Analysis of Total Proximate Composition..........................................Chapter 16 Composition and Calories Eva Falch......................3........................................... 258 16.........................................................................274 16......................................6 Determination of Water Content .........................................................270 16............... 269 16....3 Food Composition Tables and Databases ................................................2 Indirect Measurements of Energy.........1 Direct Measurement of Energy .....................4 Direct Methods for Soluble Protein Determination ..........................4...................... 269 16...........5 Nondestructive Analysis of Proteins .................4.................. Christel Solberg..............................................4............................................................274 16.7...............276 References ..................................................................4........................................................7.................1 Introduction ...........................................................................................................1 Nutritional Aspects ......2 Methods for Protein Determination .................3.................................................267 16..1 Nutritional Aspects ..... Ingrid Overrein..................... 268 16..................5 Determination of Carbohydrate Content ................................................................. 273 16......3 Determination of Total Nitrogen .......................................

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

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

55. etc. processing) [46] Automatic.) Microwave drying The sample is dried and from the water content found. location of lipids.Table 16.57] Excellent for determination of fat and water content or even distinguish lipid classes and water properties.22. lipid content. Fosslet. 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. 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.1 (continued) Advantages Rapid. the fat content can be calculated theoretically by the formula Fat% = 80 − water % . nondestructive (See under NMR below) [13–15. (SoxTech.

8] [46. and nondestructive and allows in vivo measurements (continued) 261 . requires specific calibrations Traditional low-field instruments require withdrawal of homogeneous samples for analysis (invasive) [14–16. lipid content. portable (small size). location of lipids. and portable Allows in vivo measurements Nondestructive and rapid Broad range of applications.55] Fat meters Determination of water by analyzing the dielectric properties using a microwave strip (calculation of lipids as for the drying method). easy. may also provide other nutrient data in the same analysis Some portable instruments are available Allows in vivo measurements Expensive. nondestructive.51–52] Relatively inexpensive.50. processing) Needs to be calibrated for the individual species Most suitable for neutral lipid determination See NIR/NIT above [4–6.22. NIR/NIT Transmitted or transflected Near Infrared light (800–1700 nm). The NMR mouse is rapid.Nondestructive Methods: No laboratory facilities are required Precision level may be dependent on sample (maturity stages of the fish. Low-field NMR See NMR above Composition and Calories NMR mouse ◾ Nondestructive and rapid. rapid.

independent of physical state of sample Does not give a measure of the true protein. low sensitivity. 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 [53.133] Kjeldahl Sample digestion followed by neutralization. Difficult to obtain the accurate protein concentration [53. safe (no chemical exposure). distillation. and environmentally friendly High initial costs. inexpensive to use.67. interference by nonprotein nitrogen compounds.120. easy to perform.133] Direct Protein Determination on Soluble Proteins Rapid. Absorbance depends on the type of protein analyzed . hazardous. high precision and good reproducibility.Table 16.1 (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. potentially toxic chemicals are used Rapid. time-consuming. 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. trapping of ammonia. 74. since all nitrogen in foods is not in the form of protein. standard method for comparison.

Unstable reagents are used.177] Composition and Calories Near-UV absorption Measurement of UV absorption (280 nm) [133] ◾ Rapid.175] High sensitivity and easy to perform Standard curve is nonlinear.133.176.133] A violet-purplish color is produced when copper(II) ions interact with peptide bonds under alkaline conditions. easy to perform Relatively low sensitivity compared with other UV-visible methods. High amounts of endogenous proteases may cause errors. no addition of reagents required Low sensitivity. technique is less sensitive to protein type: it utilizes absorption involving peptide bonds that are common to all proteins. 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.131. The amount of absorption is proportional to the protein present Color formation and binding depend on proteins present. Absorbance at 540 nm Lowry protein assay Copper(II) ions in alkaline solution react with protein to form complexes. Other compounds can interfere. variation of binding capacity for different batches of commercial grade dyes [68. and reaction products are detected between 500 and 750 nm Rapid. color development depends on amino acid composition [67. interference from common laboratory chemicals. high sensitivity. easy to perform. which react with the Folinphenol reagent. buffers salts. interference from ammonia. depends on amino acid composition 263 (continued) .Biuret method (Alkaline copper reagent test) Negligible interference from materials that absorb at lower wavelengths. nondestructive. protein-dye complex adsorbs on glass surface. detergents [133. interference by UV-absorbing compounds (nucleic acids and nucleotides).

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) . quantifies amino acids. nondestructive.133] Faster than ion exchange chromatography. low interference from nucleic acids and nucleotides Interference by oxygen and UV-absorbing compounds (buffer. influence by lipids and sample particle size. FMOC: less soluble. might interfere [90–92. derivatization. sensitive Most methods do not include all amino acids. fluorescence Handbook of Seafood and Seafood Products Analysis Nondestructive Determination Rapid. nondestructive. option to quantify free amino acids. high sensitivity. value for net protein.Table 16. salts) [132. hydrolysis destroys some of the amino acids.1 (continued) Advantages Rapid. no addition of reagents required. Derivatization agents: OPA: no derivatization with secondary amino acids.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.96. multicomponent analysis See NIR/NIT above Strong interference by water. low dependency of signal response on amino acid composition. chromatographic separation. 108.97. and detection of amino acids with UV absorbance.

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. Vacuum drying: may be difficult to keep uniform temperature distribution in the oven Air or vacuum drying The sample is dried until constant weight (e. Infrared drying Microwave drying Drying by irradiation Dean and Stark method Volumetric analysis of water after boiling in toluene See NIR/NIT above Possible to distinguish between free and bounded water NIR/NIT See NIR/NIT above NMR See NMR above Composition and Calories ◾ 265 .Water Determination Simple to use and inexpensive equipments required [151] Shorter analysis time compared with air and vacuum drying.g.178] [151] Long analysis time. Air drying (101°C) may lead to thermal damage.. 12 or 24 h) and water evaporated is determined.

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

Several chemical methods include the use of concentrated strong acids such as phosphotungstic (PTA). which confers numerous biological functions to this amino acid (precursor to the antioxidant glutathione). The extraction solvent can be hot water. In this chapter. with the additional advantage that proteins are not extracted and.13 5% of trichloroacetic acid. which can be achieved through different chemical or physical procedures. Although classified as nonessential.18. and individuals with certain metabolic disease or who suffer from malabsorption syndromes.16. 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. isoleucine. in rare cases.11 17. or (3) its Maillard reaction with sugars yielding characteristic flavors.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. sulfosalicylic (SSA). sulfur-containing amino acids (methionine and cystine/cysteine). cysteine may be essential for infants.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. In some cases. The extraction consists in the separation of the free amino acid fraction from the insoluble portion of the matrix (fish muscle). then. methods for the analysis of amino acids in seafood. Polytron. the elderly. A more detailed description of amino acid methods of analysis may be found in the work of Aristoy and Toldrá.5–10 Thus.21 perchloric (PCA). or by means of a simple stirring in warm solvent. especially of those considered essentials.20 have been successfully used as extraction solvents.4 Branched-chain essential amino acids (valine.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.2. (2) its ability to cross-link proteins. the analysis of essential amino acids in seafood is important for the evaluation of both the nutritive value and the sensory quality of seafood.1 N hydrochloric acid solution.22 trichloroacetic (TCA). Free amino acids initiate important changes at early postmortem and during storage and can be very useful as quality indices of processing and storage. are described.01–0.12. It is usually achieved by homogenization of the ground sample in an appropriate solvent by using a Stomacher. Sample preparation will depend on whether free or total essential amino acids have to be analyzed. 17. Once homogenized. there is no need for further cleaning up of the sample.14 6% of perchloric acid. and leucine). which increases the protein stability in the harsh extracellular environment by conferring proteolytic resistance. and aromatic amino acids (phenylalanine and tyrosine) are the most important from this point of view.15 or a rich alcohol-containing solution (>75%) such as ethanol16–18 or methanol19.13. concentrated strong acid solutions such as 4% of 5-sulfosalicylic acid. Sample cleanup is necessary to eliminate proteins and polypeptides by means of the deproteinization process. and so forth.288 ◾ Handbook of Seafood and Seafood Products Analysis compounds through Maillard reactions and Strecker degradations.23–25 and picric . 0. or diluted phosphate buffers. the sample is centrifuged at more than 10.

41 The use of microwave technology for the hydrolysis has been assayed by some authors.40. Proteins must be hydrolyzed into their constituent amino acids before the analysis. all of them . 30.6 N PCA. samples are treated with constant boiling 6 N hydrochloric acid in an oven at around 110°C for 20–96 h. Liquid-phase. An additional advantage is the easy evaporation to concentrate the sample. ethanol. In both cases. Nitrogen atmosphere and sealed vials are required during the hydrolysis to minimize the degradation. which is easily neutralized by the addition of KOH or potassium bicarbonate. only the acid vapor comes into contact with the sample.000. 5. where the hydrochloric acid contacts the sample directly.22 Under these conditions. The hydrolysis may be accomplished using either liquid-phase or vapor-phase methods. Some physical methods consist in centrifugation through cutoff membrane filters (1. Typically. presence of salts. 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.).39 Some commercial systems are available. and performance under vacuum) is similar to that of a conventional oven.000. Differences among all these chemical and physical methods are caused by several aspects such as differences in the cutoff molecular weight.42 Sample manipulation (sample evaporation to dryness. and so forth. or separation method (interferences in the chromatogram. When limited amounts of sample are available. which is easily separated by centrifugation. Digestion at 145°C for 4 h has also been proposed.22. to rend insoluble potassium perchlorate. etc. thus excluding nonvolatile contaminants.). a system capable of alternative air evacuating/inert gas purging to get a correct deaeration inside is valuable. has also given very good results. addition of constant boiling hydrochloric acid and additives. also disposes of an oven to accomplish the hydrolysis. Hydrolysis may be improved by optimizing the temperature and time of incubation41 or with the addition of amino acid oxidation protective compounds. Upon heating. The use of organic solvents. is well suited to hydrolyze large amounts or complex samples. proteins precipitate by denaturation.30 All these methods give a sample solution rich in free amino acids but free of proteins. or acetonitrile.2 Sample Preparation for Total or Hydrolyzed Essential Amino Acid Analysis The total essential amino acid profile is usually requested. recovery of amino acids. but the duration of the treatment is shorter (less than 20 min). by mixing two or three volumes of organic solvent with one volume of extract.31–33 with amino acid recoveries around 100% for all them. compatibility with derivatization (pH.39. resulting in a very simple deproteinization procedure with no interferences.000 Da) that allow free amino acids through while retaining large compounds. Some comparative studies have been published on these deproteinization techniques. The most common method used for complete hydrolysis of proteins is acid digestion. the tubes containing the samples are located inside large vessels containing the acid.000. whereas free amino acids remain in solution. which permits the alternative air evacuating/inert gas purging.34 17. creating an appropriate atmosphere inside the vessels to ensure low amino acid degradation. The presence of appropriate antioxidants/scavengers during hydrolysis can prevent losses of the most labile amino acids. 10.Essential Amino Acids ◾ 289 (PA)26–28 acids or organic solvents such as methanol. Therefore. liquid phase or vapor phase. In the vapor-phase hydrolysis method. the vapor-phase hydrolysis method is preferred to minimize contaminants coming from aqueous 6 N hydrochloric acid. because it gives information on the nutritional value of fish meat. oxygen is removed and substituted by nitrogen or other inert gas. A good choice may be the use of 0.29.35–38 These temperatures in such acidic and oxidative medium may degrade some amino acids. etc.2.18.

2 M of either NaOH. The optimization of conditions for hydrolysis based on the study of hydrolysis time and temperature. papain. being enough for the requirements of any food industry. 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.63–66 for a better tryptophan determination. chymotrypsin.290 ◾ Handbook of Seafood and Seafood Products Analysis essential amino acids. This option is chosen to analyze specific amino acid sequences or single amino acids because of their specific and well-defined activity. threonine. The previous performic acid oxidation of cysteine to cysteic acid.55.51 3-bromopropylamine. no single set of conditions will yield the accurate determination of all essential amino acids. Cyst(e)ine is partially oxidized during acid hydrolysis yielding several adducts: cystine. importance of a correct deaeration. up to 1% phenol or 0. although considerable recoveries have been found if no oxygen is present.58. acid-to-protein ratio.3¢-dithiodipropionic acid.60 and books. has been extensively reported in papers35. adequate hydrolysis procedure as the performic acid oxidation before the hydrolysis is a good alternative. with or without the addition of 1% (w/v) thiodiglycol for 18 h at 110°C. Good recoveries have been achieved by using 3-bromopropionic acid.43–50 improves cysteine (and methionine) recoveries. when the analysis of cyst(e)ine would be necessary. 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.36. methionine. (2) give a quantitative and reproducible reaction. Derivatization is a usual practice in amino acid analysis. in which methionine is also oxidized to methionine sulfone. The choice mainly depends on the equipment available or personal preferences. a compromise of conditions offers the best overall estimation for the largest number of amino acids. such as tyrosine. The use of alkylating agents to stabilize the previous hydrolysis of cysteine constitutes a valid alternative.54 or 3. cysteine. such as chromatographic (liquid or gas chromatography (GC)) or capillary electrophoresis (CE) techniques. LiOH.59. 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.36.1% sodium sulfite. or pronase. or BaOH. When high sensitivity is required. yields acceptable results for the majority of amino acids. Before or after this separation. cysteine sulfinic acid. KOH. Additionally. Tryptophan is often completely destroyed by hydrochloric acid hydrolysis. thermolysin. 41.3 Seafood Essential Amino Acid Analysis The analysis of individual amino acids needs a previous separation of all others. Some additives have been proposed to protect tryptophan against oxidation as is the case of thioglycolic acid.61. presence and concentration of oxidation protective agents. Thus. improve the recovery of nearly all of these amino acids except tryptophan and cysteine. making the posterior analysis easier. serine. which is recommended by many authors47. In fact.62 An alternative to acid hydrolysis is the alkaline hydrolysis with 4. unless a very selective way of detection is used. and tryptophan. and cysteic acid making its analysis rather difficult. carboxypeptidase. A third way to hydrolyze proteins is enzymatic hydrolysis by proteolytic enzymes such as trypsin.56 As can be observed in this section. . because each possible methodology has advantages and drawbacks. amino acids used to be derivatized to allow their separation or to enhance their detection. The separation of the individual amino acids in a mixture requires very efficient separation. In general.38 Alkaline hydrolysis instead of acid hydrolysis is also proposed (see below).30.52 4-vinyl pyridine53. protective agents currently used. and so on.67–69 17.

Nevertheless.3-oxadiazole postcolumn derivatization to obtain highly fluorescent derivatives with enhanced sensitivity. which facilitates a more selective detection. the spectroscopic detection of amino acids requires their previous derivatization to obtain an UV absorbing or fluorescent molecule. Only three amino acids (phenylalanine.68 Thus. are always present. and they include derivatives for spectroscopic or for electrochemical detection. tyrosine. After separation. The classical procedure has been improved with a new polystyrene matrix that offers better resolution power due to smaller particle size.1 High-Performance Liquid Chromatographic Methods HPLC is the preferred technique to analyze amino acids. pellicular packaging.70 fluorescamine. The latest generation of Moore and Stein amino acid analyzers also use o-phthaldialdehyde (OPA).3.1 and 17.1. amino acids were converted into colored ninhydrin derivatives for spectrophotometric (colorimetric) detection. and better detection systems. 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).12 Two types of derivatives are obtained depending on the chosen separation and/or detection technique.15. The HPLC techniques to analyze amino acids are cation exchange and reversed-phase (RP) chromatography and are described in Sections 17. The original method required two separate columns and needed about 4 h to achieve a complete analysis. Although postcolumn techniques should be run online for maximum accuracy.3.1. (4) have mild and simple reaction conditions.1.1. The derivatization reaction can be performed after separation of the amino acids (postcolumn derivatization) or before separating them (precolumn derivatization). although unidentified. Under these conditions. 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. speed. the more acidic amino acids elute first. or 4-fluoro-7-nitrobenzo-2. l em = 345 nm). recent improvements of the ninhydrin derivatization method71–73 . The first type are derivatives that enhance amino acid detection in liquid media. 17. 17. because their spectral (high-ultraviolet (UV) absorbing or fluorescence properties) or electrochemical characteristics will affect the sensitivity and selectivity of detection. It must be remarked that the use of sufficient amount of reagent is of special importance when dealing with biological samples.3. in their native form. The second type are derivatives that allow gas chromatographic amino acid separation by increasing their volatility and temperature stability.Essential Amino Acids ◾ 291 (3) yield a single derivative of each amino acid. because reagent-consuming amines.3. permitting 5–10 pmol sensitivity as standard. absorb at 210 nm and thus cannot be used for spectroscopic detection. precolumn techniques can be run either offline or online. Tryptophan also possesses native fluorescence (l ex = 295 nm. and (7) have no interferences due to by-products or excess of reagent. as it is a very unspecific detection wavelength. (5) have the possibility of automation. 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. Amino acids. The elution involves a stepwise increase in both pH and sodium or lithium ion concentration.1 Cation Exchange Chromatography This methodology is based on the amino acid charge. and those with more than one primary amino group or possessing a guanidyl residue elute at the end of the chromatogram.38. (6) have good stability of the derivatization products.2.

some difficulties to analyze some essential or sulfur-containing amino acid derivatives). plants).70. also. through a mixing manifold. feed. and the long time of analysis.e.76 There are other reports of applying this technique to the amino acid analysis in food and tissues.1. buffer system. 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. and an optimized methodology with the advantage of ease of use and reliability. The most usual derivatizing agents for tissue amino acids are described below. biological fluids. the formed molecule improves sensitivity and selectivity at the detection by allowing the spectroscopic (UV or fluorescent) detection of amino acids. (i. the derivatizing reagent is pumped into the effluent from the column system.3. or the stability of formed derivatives. LKB. Amersham Biosciences.. 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. 66.75.292 ◾ 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. which makes it a reference method for amino acid analysis. etc. 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.77 After separation.74 Nowadays. followed by a reaction coil. 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. In this way. biological fluids. which constitutes an advantage. All PTC-amino acids have similar response factors. The main drawbacks of this methodology are the high cost of the ion exchange amino acid analyzer and its maintenance. Kontron. 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. Pickering. Hitachi. but. Another disadvantage is the peak broadening produced by the dead volume introduced behind the column. which are detectable at UV (254 nm) with detection limits around 5–50 pmol. The advantage of this method is the accurate results for all known sample types (food. This method has been employed in the classical Moore and Steintype commercial amino acid analyzers. The PTC-amino acids are moderately stable at room temperature for 1 day and much longer when kept under frozen storage. and tissues). Precolumn amino acid derivatization may be necessary to confer hydrophobicity to the amino acid molecule. Biotronik.42. and finally the derivatized amino acids reach an online detector system. . tissues. Obviously. Dionex. plants. time for sample preparation and amino acids separation. There are many manufacturers (Beckman.) who offer integrated commercial systems including the column. To choose the most appropriate method some aspects must be taken into account such as the following: the disposable detector (fluorescence or UV). Phenylisothiocyanate (PITC): This methodology involves the conversion of primary and secondary amino acids to their phenylthiocarbamyl (PTC) derivatives. making it adequate for partition based on chromatography. the highly complex mobile phase composition. Although this broadening may not affect when using standard-bore columns with flow rates above 1 mL/min.2 Reversed-Phase High-Performance Liquid Chromatography RP-HPLC has been widely used. especially in a dry condition. with which many of them have been marketed. 17. each new methodology must contrast its results with those obtained by cation exchange chromatography (CEC). possibility of automation of the derivatization reaction (in the autosampler). the analysis requirements for free or hydrolyzed amino acids or required sensibility.

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

15 min at 60°C. taurine.5. and a reaction time of 1 h at room temperature (in the dark). anserine.58.84 Detection is by absorption in the visible range. IS. internal standard nor-Leucine. standard amino acid solution should be derivatized under similar conditions. 4-Dimethyl-aminoazobenzene-4′-sulfonyl chloride (Dabsyl-Cl): This reagent was first described in 1975 for use in amino acid analysis. Ans. presenting a maximum from 448 to 468 nm. Nevertheless.84 Derivatives are very stable (weeks) and can be formed from both primary and secondary amino acids.2 Reversed-phase HPLC chromatogram of PITC-free amino acids from salmon muscle extract. the reaction conditions .58 obtained a good separation of 35 dabsyl-amino acids and by-products in a 15 cm C18 column packed with 3 mm particle size.33 To overcome this problem and obtain an accurate calibration. 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. around 9. United States). l em = 510 nm) although UV (l = 250 nm) detection may also be used. Tau. and only needs a basic pH. Detection limits are in the low picomole range. Palo Alto California. because it is especially affected by the presence of high levels of some chloride salts. The high wavelength of absorption makes the baseline chromatogram very stable with a large variety of solvents and gradient systems. Commercial System Gold/Dabsylation Kit™ uses this technique (Beckman Instruments.87 or even 2 min at 100°C. However. Reaction efficiency is highly matrix dependent and variable for different amino acids.83. By-products originating from an excess of reagent absorb at the same wavelength and thus they appear in the chromatogram. The dansylated amino acids are stable for 1 day85 or until 7 days when kept at −4°C86 and protected from light.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. Stocchi et al. The sample derivatization is rather simple.

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

It means that when transferring a published method to a particular set of samples. as a consequence. The fluorescence of tryptophan derivative is very poor. Due to these variables.2 to 10. l em = 395 nm).296 ◾ Handbook of Seafood and Seafood Products Analysis 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC): It reacts with primary and secondary amines from amino acids. conductance. UV detection (254 nm) may also be used. In this case. In these cases. Only columns manufactured in the same batch are guaranteed to give the same selectivity if the rest of parameters are fi xed. detergents. even those made by the same manufacturer. different selectivity may be found among same columns. because they are molecules with electroactive functional groups. Indeed. from 8.5 min. and the separation of physiological amino acids is improved. Furthermore. the choice of the RP column is not an easy subject because of the great variability of commercially available RP columns. Electrochemical detection consists in one electrode or an array of electrodes mounted in a cell with an applied potential difference. it will be necessary to readjust the chromatographic conditions to get a good separation of all amino acids.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.. because both mono. and UV detection at 254 nm may be used for its analysis. yielding very stable derivatives (1 week at room temperature) with fluorescent properties (l ex = 250 nm. Figure 17. Sensitivity is in the femtomole range.and di-derivatives are the initial adducts from tyrosine. potential. is related to the analyte concentration. The chromatographic separation of these derivatives has been optimized for the amino acids from hydrolyzed proteins. which is only weakly fluorescent at the amino acid derivatives detection conditions and does not interfere in the chromatogram. columns are more carefully manufactured with these silanol groups blocked or inaccessible by steric impediment avoiding the tailing. Reaction time is short. which are separated by RP-HPLC. Only amino acids with aromatic rings or sulfur-containing side chains are sufficiently electrochemically active to be detected by this method. United States). peptides. the optimum pH for the reaction is in a broad range. The most used column packaging consists of alkyl-bonded silica particles. . can cause unwanted tailing of peaks (especially for the basic amino acids).3B shows the same sample but submitted to a performic acid oxidation before the hydrolysis in which the CisH peak appears by 7. However.e. Milford. The excess of reagent is consumed during the reaction to form aminoquinoline (AMQ). the addition of a strong cation (i. and other compounds naturally occurring in biological samples and foods. The presence of residual uncapped silanol groups on the silica surface. Massachusetts. such as current. Some of these derivatives are also susceptible to electrochemical detection. the AMQ peak is very large at the beginning of the chromatogram and may interfere with the first eluting peaks (see Bosch et al. but 10 min at 55°C would be necessary if a tyrosine monoderivative is required. Cystine and cysteine may be analyzed after their conversion to cysteic acid (CisH) by performic acid oxidation. Both facts facilitate sample preparation.108 If the choice of the derivative reaction is a challenge.3A shows the separation of hydrolyzed amino acids from salmon. making them very adequate for biochemical research. whereas Figure 17.50). lipids.105 in addition to fluorescent properties possesses electroactivity (750 mV) and PITC106 has again the advantage of reacting with secondary amines. or charge. accessible to sample molecules. Nowadays. The methodology has been marketed as a prepackaged AccQ Tag kit (Waters Corporation. 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. and proteins. 1 min. Any electrical measure.107. different selectivity. because the resulting CisH is well separated inside the chromatogram. OPA/mercaptoethanol or OPA/sulfite104. triethylamine) to the mobile phase can overcome the problem. mainly octadecylsilane.

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

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

nonprotein amino acids.119 have been reported. reports in the literature of its applications are increasing rapidly. 19 amino acids were analyzed by CE-ESI-MS in only 17 min with a minimal sample preparation and no matrix interference. microcolumn liquid chromatography.140 or even urea141 have been assayed. The effect of these additives on the electro-osmotic mobility and electrophoretic mobility of the micelle has been studied.4 Mass Spectrometry MS is based on the conversion of components of a sample into rapidly moving gaseous ions. With few exceptions.127–131 derivatization is used to improve separation.and l-isomer mixtures.146. compatible with MS.124 Basic theoretical considerations on this technique125 and its food applications126 are described elsewhere.118.125. although this detector may be used for more complex identifications as in d.144. methanol. although other additives such as dodecyltrimethylammonium bromide. . etc. it is likely to cause overlap with the others. Mass spectrometer detectors were first connected to GC equipments. Other additives commonly used in this analysis are organic modifiers (acetonitrile. A good compatibility between both techniques. tetrahydrofurane.137. biomedical or pharmaceutical research.). which can be resolved on the basis on their mass-to-charge ratios that are characteristic of each ion and allow its identification. the species with different charge can be simultaneously analyzed but with serious doubts in their adequate resolution.136 phenylthiohydantoin.138 Tween 20. which constitutes an important limitation of this technique. o-tyrosine analysis. Application in foods such as in the identification of nonprotein amino acids. Good separations have been reported for precapillary derivatized amino acids with dansyl-Cl. isobutanol. Nevertheless. or to allow fluorescence or electrochemical132 detection of amino acids. allowed a rapid development and the onset of these complementary techniques.).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. in particular when capillary columns were available. The identification of the 22 protein amino acids may not be a problem.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.113 o-tyrosine in chicken148 or pork149 tissues.122. This technique has also been termed micellar electrokinetic capillary chromatography (MECC or MEKC). and so forth.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. etc. the high cost of purchase and maintenance of mass spectrometers has inhibited their more widespread use in the food industry and/or food control.) and instrumentation (CE.131 and thus. it is relatively easy to analyze low picomol levels of OPA derivatives in micellar solutions by using a conventional fluorometric detector. Under the conditions of electro-osmotic flow in CE. Some reviews covering high-sensitivity detection following CE have been published. This report includes the optimization of important parameters like the choice of a volatile electrolyte (1 M formic acid) for the electrophoresis. and others.133–135 PITC.141–143 The CE coupled to electrospray ionization (ESI) MS (CE-ESI-MS) allows direct amino acid analysis without derivatization.136. showing that higher efficiency is obtained by the MECC methods with sodium dodecyl sulfate (SDS) as micelle-forming substance. Terabe et al.Essential Amino Acids ◾ 299 improve the resolution of one kind. to enhance UV detection. CZE shows poor ability for the separation of neutral compounds. SDS is indeed the most used additive to form micelles in this kind of analysis.147 17. Unfortunately.21 chiral amino acids. etc.138 and OPA139 compared with the separation of OPA-amino acid derivatives by CZE with normal and micellar solutions. When sensitivity is the target.

atmospheric pressure chemical ionization (APCI).. In general. Adv. which means a minor sample manipulation. once again. K. but tedious and time-consuming sample derivatization is required. 2. and the technique is widespread although it is still expensive. and many applications can be found in other matrices like cheese or meat. Konosu.151. G.. Protein digestibility: In vitro methods of assessment.4 Conclusions To obtain the total essential amino acids profile of a given seafood. 1981. S. 1991.. acid hydrolysis with HCl 6 N (110°C for 22 h or 145°C for 4 h) with an oxidation protective agent. Yamaguchi.2. such as phenol. small peptides. the convenience of purchasing commercially available prepackaged kits should be considered. high mobile-phase flow rate vs. Food Sci. were compared by Kwon and Moini150 in relation to sensitivity. 479–483. H. The requirements in resolution are not so exigent as those for physiological amino acids. atmospheric pressure microwave-induced plasma ionization (AP-MIPI). Food Nutr. offering the additional advantage of analyzing the amino acids without derivatization. RP-HPLC methods with precolumn OPA or PITC derivatization are very convenient methods to use. and ESI. 185–236. due to its high specificity. Fisheries Sci. Therefore. 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. Res.L. Swaisgood. The best results were obtained by using AP-MIPI in conjunction with a dual oscillating capillary nebulizer. In general. which may be consulted. is enough for the majority of purposes. the first decision is the choice of the hydrolysis method. References 1. 62. Three types of ionization modes. a complete resolution of the whole peaks is really difficult. Any separation strategy may give good results.E. and. 17. H. may appear in the chromatogram. the analytical technique for a determined sample must be carefully chosen based on the literature. Catignani. Since many peaks corresponding to protein and nonprotein amino acids. the most important factors to take into account are the resolution power and selectivity. and so on. 821–824.2. because fewer peaks appear in the chromatogram. R. Particular hydrolysis problems related with certain amino acids are described in Section 17. T. However.. One of the main requirements for samples to be analyzed by MS is that analytes. J.300 ◾ Handbook of Seafood and Seafood Products Analysis MS has also been used as a spectroscopic detector after HPLC or CZE.152 A very careful control of the derivatization reactions and chromatographic conditions are necessary for a consistent and reproducible analysis. Nevertheless. amino acids in this case. and taking care of avoiding the presence of oxygen with vacuum and nitrogen purging. Sensory analysis of taste-active components in the extract of boiled snow crab meat. The highest resolution is obtained by GC with the capillary column technique. nucleosides. 46. Yamanaka. 1996. Shimada. nowadays these difficulties have been overcome with the development of new interfaces. vacuum). cation exchange and postcolumn derivatization or RP-HPLC precolumn derivatization techniques are the preferred methods. Post-mortem biochemical changes in the muscle of Japanese spiny lobster during storage. . and. reduced problems related with matrix interferences or poor resolution between peaks. 35. When amino acids from seafood proteins have to be analyzed. The majority of published reports in which seafood amino acids are analyzed have used the cation exchange method. 3. Hayashi. The convenience of purchasing commercially available kits must be evaluated.

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1.....311 18..........................................................................3....................3....1...........3...........3....2 Vitamin E Determination ..........2.............318 18.....................1 Vitamin E as an Antioxidant.....................3..3 Vitamin E .................................................................................3.....4........................1.....................................3............................................................................318 309 .......................3.......... 315 18.......................................1..................................1 Oxidation and Its Implications..................................310 18.................................................................................311 18...................................314 18....3......................................316 18..1 Antioxidant Enzymes .....3 Occurrence of Ascorbic Acid in Marine Organisms ......2 Lipid Peroxidation.................... 315 18....Chapter 18 Antioxidants Nick Kalogeropoulos and Antonia Chiou Contents 18...................................2 Antioxidants.....2 Ascorbic Acid Analysis .................................................................3...310 18.....3 Marine Lipid Oxidation ...............3 Occurrence of Vitamin E ...................1 Ascorbic Acid Functions ..3.........................317 18............1 Introduction ..............1 Antioxidant and Other Functions of Carotenoids ....3 Antioxidants in Seafood and Seafood Products ...............................................4 Carotenoids ...........................317 18..2........................311 18..................................310 18.......................4.................313 18......................................3........1 Oxidative Stress and Its Implications ......2 Determination of Antioxidants and Antioxidant Capacity in Biological and Food Systems ........2.........................................................................313 18.............................316 18.........................313 18......4................................3.1........1............2 Carotenoid Determination ........................3.......................317 18.........................2 Ascorbic Acid ..........314 18......314 18.............3 Occurrence of Carotenoids .............3.................................1...................................1..........................3.....

.1 Oxidation and Its Implications Oxidation is the transfer of electrons from one atom to another and represents an essential part of aerobic life.............................. being exposed to the oxygen they produce.....3..........2 Determination of Ubiquinone .........................5....... electrically charged compounds that seek out and capture electrons from other compounds in order to neutralize themselves................. they may oxidize several cell components.................................. •)..... 18..............1 Indeed cyanobacteria and the laterevolved green plants........... Common free radicals in biological systems are the so• called reactive oxygen species (ROS)................... Aging...... and carotenoids... generation of free radicals occurs. alkoxyl (RO •)........................ such as peroxynitrite (ONOO−)................ 320 18.................. This is also followed in the marine environment....... resulting in a chain reaction.......... polyphenols.3 pollution................ Although the initial attack causes the neutralization of the free radical.................. Humans and most animals cannot synthesize the majority of these antioxidants and depend on the dietary intake from plant consumption.. 320 18....... and the so-called reactive nitrogen species (RNS)........... more than 2 billion years ago.. 18.2 Natural Antioxidants ..4 Added Antioxidants ....319 18......... nitric oxide (NO (OH•) radicals..1 Function of Ubiquinone .1.... another free radical is generated in the process......3......... When the electron flow becomes uncoupled (transfer of unpaired single electrons)...............3........5 Ubiquinone .....321 18..........319 18..319 18............ and proteins may be damaged by reactive oxidants......6 Other Endogenous Antioxidants .........3........310 ◾ Handbook of Seafood and Seafood Products Analysis 18.............. ROS production in organisms is related to both the basal metabolism and the influence of environmental factors2........................... which include among others superoxide anion radical (O2−)..... 320 18....4....... are rich in antioxidants such as vitamins C and E........... Until subsequent free radicals are deactivated........3.... resulting in an increased antioxidant activity and antioxidants ...............................321 References ...............4.....1 Introduction Antioxidants evolved together with the emergence of photosynthesis by cyanobacteria. since oxygen is the final electron acceptor in the electron flow system that produces energy..............1..... lipids......319 18............ where antioxidants are mainly produced by photosynthetic organisms and are consequently transported through the trophic web.5..........................1 Oxidative Stress and Its Implications Oxidative stress occurs when the prooxidant–antioxidant balance becomes too favorable to the prooxidants.6 have been reported to cause oxidative stress to fish or bivalves........ nucleic acids.....1.................. thousands of free radical reactions may occur within a few seconds....4 and environmental stress5....................... and hydroxyl hydrogen peroxide (H2O2)................................ if ROS are not immediately intercepted by antioxidants......... as a defense against oxygen toxicity.1 Synthetic Antioxidants ..............3 Occurrence of Ubiquinone...................................... that is........ peroxyl (ROO •)..................... Under conditions of oxidative stress............5..

Lipid oxidation of omega-3 polyunsaturated fatty acids (PUFA)-rich food products results in the development of particularly unpleasant off flavors. Lipid oxidation is a complex procedure induced by oxygen in the presence of initiators such as light. free radicals. For food systems. that is. carcinogenic. antioxidants are molecules that protect macromolecules from being oxidized. propagation. Autoxidation occurs through a three-phase process. Nonradical photooxidation seems to be a minor reaction compared with the 3O2-induced radical chain autoxidation.3 Marine Lipid Oxidation Compared with other food lipids.12 In biological systems various biochemical defense mechanisms. Chain-breaking antioxidants intercept •) or participate in halting radical chain propagation. significantly delays or prevents oxidation of that substrate. atherogenic. Nevradical oxidation propagators (LOO ertheless. preventive antioxidants.11 and unhealthy compounds that reduce their shelf life and nutritive value.13 Antioxidants counteract oxidation in two different ways: They protect lipids from oxidation initiators. 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.1. in which polyunsaturated fatty acids on lipid molecules are attacked and oxidized. and (3) enzymatic oxidation. heat. and they stall the propagation phase. This can be especially damaging to lipid-rich cell membranes. chain-breaking antioxidants. and storage. for which the human sensory apparatus has a low threshold.1. Moreover. The health implications of tissue lipid oxidation are numerous and well documented.1. are essential for counteracting oxidative stress. There is increasing evidence that oxidative stress is implicated in the pathogenesis of many inflammatory and degenerative diseases and conditions. mutagenic. and termination.10 Lipids deteriorate in seafood products during processing. and angiotoxic effects. because of their high degree of unsaturation.1. antioxidants are .). including enzymatic systems and nonenzymatic antioxidants.1. and metal ions.2 Lipid Peroxidation A very damaging effect of oxidant reactive intermediates is lipid peroxidation. In the first two cases a combination of reactions involving 3O2 and 1O2 occurs. mainly of plant origin.9 Preventive antioxidants hinder ROS formation or scavenge spe• cies responsible for oxidation initiation (O2−.7 18. The major components of the antioxidant defense system together with their proposed mechanisms of action are presented in Table 18.3. (2) nonenzymatic and nonradical photooxidation. handling. initiation. protect the cellular components from oxidative damage.2 Antioxidants Antioxidants are defined as any substance that when present at low concentrations compared with those of an oxidizable substrate. 1O2.Antioxidants ◾ 311 loss or oxidation product development.1. 18. etc. marine lipids are relatively more susceptible to oxidation. Three reaction pathways have been proposed: (1) nonenzymatic chain autoxidation. In general. antioxidants often act via more than one mechanism that combines different types of antioxidant activity. antioxidants supplied by foods.8 Studies on the pathological significance of dietary lipid oxidation products have indicated that some lipid oxidation products have cytotoxic.9 18.

scavenges peroxyradicals. EDTA. carnosine. ROS detoxification (hydroperoxides). sex hormones melatonin.K. 1O2 quencher Compounds with proven antioxidant activity in vitro. citric acid Polyphenols (exogenous) Chain-breaking antioxidant. chain-breaking antioxidants Synergistic to vitamin E Scavenges NO2 Transient metal chelators. 275. melanins (endogenous) Source: Adapted from Willcox. Crit.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. synergistic to vitamin E Transient metal chelators Transient metal chelators (the ones with o-diphenolic structure).. but uncertain in vivo Vitamin E (exogenous) Bilirubin. Cu) Transient metal chelators (chelates Cu) Transient metal chelators (chelates Fe) Transient metal chelators (chelates Fe) Transient metal chelators (chelates Cd. et al. Rev. . Food Sci. Zn. 2004. 2-oxo acids. 44..312 ◾ Handbook of Seafood and Seafood Products Analysis Table 18. chain-breaking antioxidants. J. Cu) Low Molecular Mass Ascorbic acid (exogenous) Carotenoids (exogenous) Coenzyme Q (endogenous) Urate (endogenous) Phospholipids (endogenous) Polyphosphates. anserine. regenerate oxidized vitamin E Chain-breaking antioxidant. lipoic acid. regenerates oxidized vitamin E 1 O2 quenchers.

Antioxidants ◾ 313 effective at very low concentration levels. 18.1 Antioxidant Enzymes Catalases are metal-containing enzymes. widely distributed in aerobic cells that help in preventing the accumulation of H2O2 within cells. whereas Fe SOD were purified from red algae. and chromatographic methods. Zn. column chromatography and the more sophisticated gas chromatography (GC). thin-layer. one represented by enzymes and the second represented by low molecular mass compounds. or Fe in their active site. and crustaceans from the Mediterranean sea. kidneys. The available methods for monitoring the antioxidant capacity in biological and food systems in vitro or in vivo were recently reviewed by MacDonald-Wicks et al. the latter including paper.1 Cu/Zn SOD were purified from marine fish tissues. bilirubin. Cu.15 Griffiths et al. carotenoids.9 and 9. The available methods have been reviewed by Rajalakshmi and Narasimhan.16 and Laguerre et al.9 The determination of specific waterand fat-soluble antioxidants is discussed in Sections 18. Catalase activities ranged between 386 and 1523 mmol/ min/g tissue in several Atlantic fish and was higher in liver. oxidative damage to macromolecules is controlled by two types of antioxidant systems.3.7 U/mg of protein.. such as ascorbic acid. polarographic.20 Superoxide dismutases (SOD): SODs are metalloproteins. 18. 18.3.3 Antioxidants in Seafood and Seafood Products In living organisms. together with traces of phenolic compounds. 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. and heart.19 concluded that. blue-green algae.21 In several species of teleosts. and uric acid. thiols.3. the correlation of instrumental and sensory methods with multivariate data analysis should be followed.17 and Wood et al. Cu/Zn-SOD activities ranged between 1.3. voltammetric. glutathione (GSH). Kolanowski et al. cephalopods. tocopherols.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.22 whereas in nine Atlantic fish species total SOD values ranged between 157 and 796 U/g fish and Mn-SOD ranged between 45 and 751 U/g.18 In reviewing methods and tests for the assessment of lipid oxidation. spleen.2 and 18. and in red muscle compared with that in white. and high-performance liquid chromatography (HPLC) alone or combined with mass spectroscopy. for quality control of fish oil and fish oil-containing foods. These antioxidants act in a concerted way to protect sensitive molecules such as the unsaturated fatty acids from oxidation.20 . and algae. At higher levels most of them behave as prooxidants possibly due to