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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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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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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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

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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

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

CHEMISTRY AND BIOCHEMISTRY I .

.

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

8 million tons). The total world seafood supply for 2007 amounted to 143 million tons. Russia (3.2%. 2000: 40 million tons.000–35. and Norway (2. fish and other seafood are highly perishable products when stored without chilling. the United States (4.2 million tons). body composition. 25% is converted into fishmeal and other nonfood products.3 million tons). Atlantic herring (2. Thailand (2. 1980: 7 million tons. However. 8% is transformed into cured products. The fish group alone is represented by 25. respectively.4%.4 million tons). Mollusks (bivalves and cephalopods) are the third most important group both by quantity and by value at 22. The top 10 species being caught in huge amounts in 2005 were Anchoveta (10. The stagnation of captured fish is mainly due to fully exploited or partially overfished stocks.4 million tons. 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. They deteriorate at ambient temperature in a few days. Japan (4. and the captured fish. burden of pollutants.4 million tons.9 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.7 million tons). 91 million tons (64%) of the total supply. about 20% is converted into deep frozen products.6 million tons). fish is the top group in aquaculture at 47. whereof the major part are cyprinids like carp). Indonesia (1. Further.2 million tons). state of maturity.4 ◾ Handbook of Seafood and Seafood Products Analysis number of species and variability.) of aquatic animals when captured by fishing techniques is—with few exceptions—completely unknown. Skipjack tuna (2. Chilean jack mackerel (1. Blue whiting (2.1 million tons).1 million tons). in the case of captured seafood we have to accept what we find in the trawl despite modern advanced technology of sonar and echo sounders.1 million tons). 1990: 16 million tons. India (2. etc. Although land-based animals are today tailor made according to industry’s and consumer’s wishes in weight.0 million tons). Chub mackerel (2. The major aquaculture (excluding plants) producers (>1 million tons) in 2005 were China (32.3 million tons).3 million tons). The most important primary product producing countries of marine and inland (freshwater) fisheries in 2005 were China (17. mostly Pangasius species).4 million tons).4 million tons). and Thailand (1. nutritional status.3% and 14. 40% is consumed as wet fish without any further technological processing or preservation. and sensory properties.1 million tons). Further.4 million tons).3 million tons).6 million tons). Alaska Pollock (2. Largehead hairtail (1. Chile (4.8 million tons). Most fish was caught in the Pacific Ocean (Northeast and Southeast) followed by Northeast Atlantic Ocean. Indonesia (4. Vietnam (1.4%.1 million tons). Peru (9.5 million tons). only a little proportion of this large number of about 5% is present in the world’s oceans in amounts huge enough to allow an economical use (catch and following processing). appearance. Aquatic plants that are popular in Southeast Asia are second in quantity at 23. 1970: 4 million tons. whereas crustaceans are fourth by quantity at 6. India (3. and Yellowfin tuna (1. Japanese anchovy (1. including plants) [1]. aquaculture is dramatically growing (1960: 2 million tons.4% by quantity.000 species. infestation with parasites. 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. . The world’s aquaculture provided 52 million tons (36%). and another 8% into canned products.2% but second by value at 20. About 75% of the world’s total seafood supply is used for human consumption. Another difference compared with land-living animals is the fact that the quality (size. By major groupings.

which continues until a state of spoilage is reached. leading to several diseases such as diarrhetic shellfi sh poisoning (DSP). Not only weight and length are varying with age but also other factors such as proximate composition.g. After catch and harvest. eellike fishes.2 Variability of Aquatic Animals The variability of aquatic animals can be described and explained in many different ways. crustaceans. fat. which are at the end of the marine food web. we have different groups such as bony and cartilaginous fishes. and so forth. Mackerel is a typical pelagic swarm fish occurring in big schools. Based on taxonomic criteria. these are all very rough classifications. and mollusks.Introduction ◾ 5 1. in one haul specimen of 5% fat and 35% fat are present. 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. and before analyzing fish. or according to their occurrence in the ocean’s water column into pelagic fish. flat fishes. a certain degree of variability is found. A drastic example illustrating the variability in fish is the Atlantic mackerel. season. which are subject to variations based on state of maturity. medium fatty fish species (>1% to <10% fat). Besides this more general aspect. and protein are not even distributed in the edible part and also trace element concentrations vary from head to tail or back to belly. cestodes) that can be harmful to humans when they enter live and intact into the human body. pollution of water. Toxins from dinoflagellates can accumulate in bivalve mollusks. demersal fish. We can also group them according to their fat content into three groups: lean fish species (<1% fat). composition. fishing area. With all these variations in the raw seafood material before the analysis of any components. When captured during the spawning season. which are very different from each other in appearance. some groups offer special problems to which a lot of attention has to be given: aquatic animals may contain parasites (e. Predatory fish species such as sharks. When concentrating on fish as the major group contributing to the world’s fish supply.3 Special Problems with Aquatic Animals The main problem with aquatic animals is the fact that from the moment that they are caught or harvested. In addition. not only spoilage and freshness parameters are changing due to metabolic (autolytic) and microbiological processes but also the microbial flora is changing. paralytic shellfish poisoning (PSP). However. This can lead to extreme problems not only in processing but also in analysis. bottom fish. a careful consideration has to be made if the variation is important and if it is worth or essential knowing (leading to analysis of individuals) or if a more general impression about the target component is sufficient (pooled samples). and nutritive properties. nematodes. Also within the fish body. This means that each fish can be different and unique. and trace element content. and the spawned fish can exhibit fillet fat contents of down to 5%. decisions must be made where the results should be used and how detailed an analysis must be. and ground fish. since parallel with fat content. The prespawning fish can have a fat content in fillet up to 35%. 1. and so on. and fatty fish species (>10% fat). a change in properties starts. other parameters such as organic pollutant concentrations vary. neurotoxic shellfish poisoning (NSP). and .. mineral. can accumulate mercury during their long life span to quantities that exceed legal limits. Components like water. we arrange them in order according to their shape into round fish.

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

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

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

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

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

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

The ammonium sulfate is converted into ammonia. Retaining the functional properties through preservation and processing operations is therefore of great importance. and trapping of ammonia and titration steps. The method includes sample digestion. In addition to the high nutritional value. 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. has been shown to be too high for animal proteins. When the protein content is calculated based on determination of the nitrogen content. The disadvantage is that the method requires use of hazardous and toxic chemicals. for instance collagen has a nitrogen content of 18%. values from 5.5]. . distillation. and that other components in the food such as lipids and pigments should not interfere with the analysis. the water-holding capacity and the gelling properties which determine the textural attributes of the products are important quality parameters [3].82 are given. and textural properties. This factor is the amount of nitrogen that contains 1 g of nitrogen. For products such as fish mince and surimi. and the amount of protein is calculated by multiplication with a Kjeldahl factor. It is important that the methods to analyze food proteins are robust [1]. For fish. which is distilled and trapped in boric acid. It is also possible to determine the nitrogen content using elemental analysis (C/N analyzers) [4].25 is usually used.56 [1]. This means that it should be possible to use the method on different types of foods.12 ◾ Handbook of Seafood and Seafood Products Analysis essential amino acids lysine and methionine. 2. Originally only sulfuric acid was used for digestion of the samples.25. both different types of raw materials and processed foods. For animal proteins the value 6. Tables of conversion factors are given in several papers such as [1. The Kjeldahl method has been automated and several instruments for automated analysis are available. The Kjeldahl method was first published in 1883 but has been extensively modified since then. and urea will also contribute to the calculated protein content. Many proteins have protein contents that deviate from this. The method should also require minimal sample pretreatment to decrease analytical error and reduce costs. The Technicon AutoAnalyzer uses continuous flow analysis [1]. it is difficult to start using other and more correct factors [5]. amines. fish proteins also have good functional properties such as water-holding capacity. The advantage of this method is that it gives accurate results for all types of samples. nitrogen from other nitrogen-containing compounds such as free amino acids. During digestion the nitrogen in the sample is converted to ammonium sulfate. assuming a nitrogen content of 16% in the proteins. neutralization. nucleotides. Mariotti and coworkers discuss conversion factors in their critical review and conclude that even if a factor of 6.2 Total Content of Proteins The total content of proteins is usually determined by the Kjeldahl or the Dumas method. It is also possible to determine the amount of ammonia by different colorimetric methods [1]. This method is used as a reference method by many national and international organizations. and the amount of nitrogen is determined by titration [1]. gelling. which has been used for more than 75 years. emulsification. The Dumas method is quicker and cheaper and easier to perform and is therefore now considered on equal terms with the Kjeldahl method [1]. but other chemicals such as potassium sulfate and mercury oxide are also used.43 to 5. The Kjeldahl method determines the nitrogen content as ammonia. which gives a factor of 5. The factor can be calculated from the amino acid composition of the proteins.

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

The extraction in NaOH was repeated five times and the supernatants were pooled for the analysis of alkali-soluble collagen content.4 Analysis of Soluble Proteins There are many indirect colorimetric methods to determine protein content. However. Since all proteins absorb UV/visible light to varying degrees. After extraction. The purple complex is relatively stable and has an absorption maximum at 540–560 nm. [18]. the presence of nonprotein UV-absorbing groups such as nucleic acids and nucleotides which absorb strongly at 260 nm further complicate matters. The protein concentration can then be calculated from the Lambert–Beer law: A = ε cl where A is the absorption at a given wavelength c is the molar protein concentration l is the path length for the light (cm) e is the molar absorption or extinction coefficient (M−1 cm−1) The molar absorptivity can be determined by dry weight estimation of a purified protein. one of the simplest methods is to determine absorbance in the far UV range. Reducing agents and sucrose as well as several common buffers interfere with the Lowry method. which is a modification of the method described by Sato et al. but the method is simple and inexpensive. 2. the method therefore has protein-to-protein variations. The review also discusses many of the modifications that . The Lowry method [20] is based on a Biuret-type reaction between protein and copper(II) ions under alkaline conditions. Measurement of UV absorption at 280 nm is a simple and popular method to determine protein concentration. Absorption at 280 nm is mainly due to tryptophan and tyrosine residues with smaller contributions from phenylalanine and the sulfur-containing amino acids. The reactions are highly pH dependent. The precipitate was stirred with 0.1 M NaOH and centrifuged.5 M acetic acid for 2 days at room temperature and centrifuged as above. and a few of them will be treated here. The product becomes reduced to molybdenum/tungsten blue and can be measured at 750 nm. the complexes react with the Folin-phenol reagent a mixture of phosphotungstic acid and phosphomolybdic acid in phenol. This was the acid-soluble collagen. Light scattering because of large particles or aggregates can also lead to errors. In addition. by absorbance at 205 nm or from knowledge of amino acid composition [19]. Methods exist to correct for the influence of light scattering and nucleic acids/ nucleotides [19]. Peterson have reviewed the Lowry method [21] and listed interfering substances. giving upper tolerable limits for a long range of these as well as some methods for coping with the effect of these substances. The sensitivity can be increased by measuring absorbance at 310 nm or by increasing the time for the Biuret reaction.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]. some of these methods reduce the speed and simplicity of the method [19]. A standard curve is needed. Samples were homogenized in 0. The Biuret method is based on the formation of complexes between copper salts and peptide bonds under alkaline conditions. However mg quantities of protein are generally required. the concentration of the soluble proteins can be analyzed with a wide variety of methods. The method is not very sensitive. measuring concentrations between 1 and 10 mg/mL.

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

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

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

Journal of Food Quality. 1992. D.18 ◾ Handbook of Seafood and Seafood Products Analysis processing.25 and Jones’ factors. Automatic methods for the simultaneous determination of carbon. Mirand. 2002. formation of protein–lipid films. Venugopal. It is therefore difficult to compare results from different laboratories. Characteristics of edible muscle tissue. Non-destructive determination of fat. Journal of Food Science & Technology.M. Mariotti. and H. Food Chemistry. V. 12. 879–942. Nutritional. L. N. and P. 1979. 1968..A..K.. 87: 31–41. whippability). 25: 2025–2069. Food Protein Analysis: Quantitative Eff ects on Processing. Iced storage of Atlantic salmon (Salmo salar)—Effects on endogenous enzymes and their impact on muscle proteins and texture. Marcel Dekker: New York. et al. 5: 215–234.F. net charge. elasticity. Functional properties can be divided in several groups. temperature. Journal of Fisheries Research Board Of Canada. thickening. 2006. Bock. Tome.. and gelation).E. Licciardello. 35: 431–435. 13. References 1. amino acid composition and sequence. 1995. T. distribution. 73: R91–R98. 1996.C. 463. aggregation.R. salt concentration). et al. and R. Ed. 9. Converting nitrogen into protein—Beyond 6. Hultmann. Fennema. sensory. J. structures (secondary. 5.. 1995.P. Journal of the Science of Food and Agriculture. hydrophobicity. cooking. adhesiveness. and consumption [36]. solubility.K. Nondestructive determination of water and protein in surimi by near-infrared spectroscopy. tertiary. Food Research International. pp. Relation between protein extractability and free fatty acid production in cod muscle aged in ice. Innovative uses of near-infrared spectroscopy in food processing. 48: 177–184. 3. Venugopal. F. New York: Marcel Dekker. wettability). Uddin. (2) properties related with the protein structure and rheological characteristics (viscosity. 2008. Foegeding. et al. and quaternary). . Rustad. 69: 95–100. and biological values are sometimes included in the functional properties. M. 2004.. Ravesi. Connelly. Anderson. Haard. 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. 2. Analytical chemistry. Isaksson. in Food Chemistry. 1982. Critical Reviews in Food Science and Nutrition. Lanier. It is usual to classify them according to mechanism of action into three main groups: (1) properties related with hydration (absorption of water/oil. O. storage. 25: 289–307. E. 96: 491–495. Methods for processing and utilization of low cost fishes: A critical appraisal. and (3) properties related with the protein surface (emulsifying and foaming activities. 10. Journal of Food Science. 4.J. M. Shahidi. molecular flexibility/rigidity in response to external environment (pH.. 6.L. Control of chemical composition and food quality attributes of cultured fish. hydrogen. 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. Food Chemistry.. moisture and protein in salmon fillets by use of near-infrared diff use spectroscopy. A description of the properties of the proteins important for functional properties was given by Damodaran [37]: The physicochemical properties that influence functional behavior of proteins in food include their size. 2008. J. 1995. Owusu-Apenten. and E. hydrophilicity. and F. 7. and T. V. sulphur and sulphur alone in organic and inorganic materials.J. 8. 51: 1173–1179. R. T. Kirsten. 32: 1–12. W. Hultin. or interaction with other food constituents. 11... Journal of Food Science & Nutrition. shape. p. Value added products from underutilised fish species.

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

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

and after processing or storage.2 Proteome Analysis by 2DE 2DE. along with some of the main improvements that have developed since. that proteome analysis. or even thousands. the proteome varies from tissue to tissue.14–18 gill. for example.12. the interactions of proteins with one another or with other food components. and mass accuracy.2. This chapter will therefore focus on 2DE.1. is a tool that can be of great value to the food scientist.22 ◾ Handbook of Seafood and Seafood Products Analysis 3. growth depression.19 intestine. 3. largely because of its high resolution. also known as proteomics. in the following sections.1 Sample Matrix Considerations Unlike the genome. of proteins on a 2D polyacrylamide slab gel. simplicity. based on liquid chromatography tandem mass spectrometry (LC–MS/MS). the construction of the food matrix. and low survival rate. 3. quality involution within the product before. In the following sections. Studies on whole larvae.12 and rectal gland12 have been reported. fish possess a number of tissues amenable to 2DE-based proteome analysis.1 Whole Larval Proteomes The production of good quality larvae is still a challenge in marine fish hatcheries. or with the human immune system after consumption. Like other vertebrates.2 surface-enhanced laser desorption/ionization3 or protein arrays. foodstuffs are in large part made up of proteins. 3.13 skeletal muscle. during.12 kidney. we present some issues and challenges related to sample matrices of particular interest to the seafood scientist. gel-free methods. . Selection of tissues for protein extraction is therefore an important issue that needs to be considered before a seafood proteomic study is embarked upon.1) remains the workhorse of most proteomics work. This is especially true of fish and meat.9–11 heart. both on the cellular and tissue-wide levels. giving valuable insight into the composition of the raw materials.6–8 liver. the “classic” process of two-dimensional (2D) gel polyacrylamide electrophoresis (2DE) followed by protein identification via peptide mass fingerprinting of trypsin digests (Figure 3. It stands to reason.12.2. Several environmental factors can interfere with the protein expression of larvae leading to poor larval quality like malformations. where the bulk of the food matrix is constructed from proteins.4 hold great promise and are deserving of discussion in their own right.12 brain. While high-throughput. is the simultaneous separation of hundreds. Proteomics. is regulated and brought about by proteins. then.1 Introduction As with all living matter. the cornerstone of most proteomics research. 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. Proteome analysis allows us to examine the effects of environmental factors on larval global protein expression. as well as with time and in response to environmental stimuli. 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. Furthermore.

22 Also. yielding a peptide mass fingerprint.7 These studies provided protocols for the production of high-resolution 2D gels.22 and zebrafish (Danio rerio). In many cases this is sufficient for identification purposes. See text for further details. allowing identification of ca. there are several drawbacks when working with the whole larval proteome. The axial musculature is the largest tissue in larval fishes as it constitutes approximately 40% of their body mass.7. 85% of the of the selected protein spots. cytoskeletal . First. where the majority of the highly abundant proteins were identified as muscle proteins. Peptide mass mapping using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry was performed only on the cod larval proteins.23 This is reflected in our studies on whole cod larval proteome. Once a protein of interest has been identified.6. such as the gastrointestinal tract or the central nervous system. but if needed. Only a few proteome analysis studies on fish larvae have been published. posttranslational modifications and redistribution of specific proteins within cells.20 all important information for controlling factors influencing the aptitude to continue a normal development until adult stages.22 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. These proteins may mask subtle changes in proteins expressed in other tissues or systems.6. it is excised from the gel.21.1 An overview over the “classic approach” in proteomics. Nevertheless.6.Proteomics ◾ 23 2D PAGE Trypsin digestion MS fingerprinting MS/MS sequencing Figure 3. a protein extract (crude or fractionated) from the tissue of choice is subjected to 2D PAGE. like the overwhelming presence of muscle and skin proteins.22 Three of these publications have focused on the whole larval proteomes in Atlantic cod (Gadus morhua)6. subjected to degradation by trypsin (or other suitable protease) and the resulting peptides analyzed by mass spectrometry. peptides can be dissociated into smaller fragment and small partial sequences obtained by MS/MS.

during and after processing.42.34–41 3.43 The 20S proteasome has been found to have a role in regulating the efficiency with which rainbow trout (Oncorhynchus mykiss) deposit protein. No amplification method analogous to PCR exists for proteins.3 The Degradome The degradome may be a subproteome of particular interest to the food scientist. allowing a larger sample of the remaining proteins to be analyzed. and simply increasing the amount of sample is usually not an option. protein turnover is a major regulatory engine of cellular structure.30 preparative isoelectrofocusing31 or solubility in the presence of various detergents32 or chaotropes33 have been described. For example.46 An exploitable property of proteasome-mediated protein degradation is the phenomenon of polyubiquitination. such as those associated with individual organelles or cell compartments28 or by protein biochemical methods such as affi nity chromatography. However. but for other applications low-abundance proteins.29. In addition to having a hand in controlling autolysis determinants.45. and biochemistry. then.44 It seems likely that the manner. has profound implications for quality and processability of the fish flesh. Swamping of low-abundance spots by highly abundant ones may not be a problem for applications relating specifically to structural proteins. Proteomic analysis on lysosomes has been successfully performed in mammalian (human) systems. which include most regulatory proteins and many important metabolic enzymes. An unfractionated 2DE map of the muscle proteome therefore tends to be dominated by comparatively few high-abundance protein spots. Cellular protein turnover involves at least two major systems: the lysosomal system and the ubiquitin–proteasome system. whereby proteins are targeted for destruction by the proteasome by covalent . such as actin and tubulin. A myriad of methods suitable for subsequent 2DE exist for fractionating the proteome into defined subproteomes.24 ◾ Handbook of Seafood and Seafood Products Analysis proteins were prominent among the identified proteins. Various strategies have been presented for the removal of highly abundant proteins24 or enrichment of lowabundant proteins.2 Muscle Proteomes In most seafood products.1. Removal of those proteins may increase detection of other proteins present at low concentrations. as many textural and other quality factors of muscle foods are related to proteolytic activity in the muscle tissue before.26 3. The fish muscle proteome is therefore likely to be of comparatively high interest to the seafood scientist.2.2. is fractionation of the protein sample in order to weed out the high-abundance proteins. it may also result in a loss of other proteins.5 The remaining option. are particularly abundant in the skeletal muscle proteome.25. are of keen interest. are suitable for rigorous investigation using proteomic methods. function. such as the ubiquitin–proteasome or the lysosome systems. Structural proteins. preventing identification of holistic alterations in the analyzed proteomes. in which protein deposition is regulated. 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. Fractionation methods for a variety of sample matrices have been reviewed recently. Protein turnover systems. particularly in muscle tissue. as it will give rise to overloading artifacts in the gels. fish skeletal muscle is the main component. rendering analysis of low-abundance proteins difficult or impossible.1.

This separates the proteins according to their molecular charge.48–50 Monitoring of the expression levels of these regulatory enzymes. 4% (w/v) CHAPS [3-(3-chloramidopropyl)dimethylamino-1-propanesulfonate]. 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).e.2.2. sample treatment prior to electrophoresis should be minimal in order to minimize in-sample proteolysis and other sources of experimental artifacts. Ready-made IPG strips are currently available in a variety of linear and .” i.2. Although a number of refinements have been made to 2DE since O’Farrell’s paper. The map can be visualized and individual proteins quantified by radiolabeling or by using any of a host of protein dyes and stains. or fluorescent dyes.2 First-Dimension Electrophoresis The extracted proteins are first separated by IEF.22 and Arctic charr (Salvelinus alpinus) liver. which is most conveniently performed using commercial dry IPG gel strips. In the following sections. 0. We have found direct extraction into the gel reswelling buffer (7 M urea. may be less directly amenable to proteomic study.2 Basic 2DE Methods Overview O’Farrell’s original 2DE method first applies a process called isoelectric focusing (IEF). most notably the introduction of immobilized pH gradients (IPGs) for IEF. Activity of matrix metalloproteases is regulated via a complex network of specific proteases. Cleanup of samples using commercial 2D sample cleanup kits may be beneficial for some sample types. 2 M thiourea.1 Sample Extraction and Cleanup For most applications. which proteins are being degraded by the proteasome at a given time or under given conditions.2.2) rather than the familiar banding pattern observed in one-dimensional (1D) SDS-PAGE.47 By targeting these ubiquitin-labeled proteins.58 Thorough homogenization is essential to ensure complete and reproducible extraction of the proteome. yielding a two-dimensional map (Figure 3.Proteomics ◾ 25 binding to multiple copies of ubiquitin.2 Some proteolysis systems. up-to-date protocols. 0. where an electric field is applied to a tube gel on which the protein sample and carrier ampholytes have been deposited.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. and how they vary with environmental or dietary variables. the reader is referred to any of a number of excellent reviews and laboratory manuals.52–57 3.3% (w/v) DTT [dithiothreitol]. 3. such as that of the matrix metalloproteases. For more detailed. a general protocol is outlined briefly with some notes of special relevance to the seafood scientist.43. such as Coomassie blue.51 the procedure remains essentially as outlined earlier. Gygi and coworkers have developed methods to study the ubiquitin–proteasome degradome in the yeast Saccharomyces cerevisiae using multidimensional LC–MS/MS.. silver stains. it is possible to observe the ubiquitin–proteasome “degradome. 3. may be more conveniently carried out using transcriptomic methods. These strips consist of a dried IPG-containing polyacrylamide gel on a plastic backing.2.

IEF is normally performed for several hours at high voltage and low current. morhua) larval proteins with pI between 4 and 7 and molecular mass about 10–100 kDa.500 V.26 ◾ Handbook of Seafood and Seafood Products Analysis MW (kDa) 60 42 30 22 17 4 5 pl 6 7 Figure 3. If the protein sample is to be applied during the reswelling process. 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. Typically.000 Vh. but also on .2.. usually totaling about 10.2 A 2DE protein map of whole Atlantic cod (G.g. the starting voltage is about 150 V. Isoelectrofocusing was by pH 4–7 IPG strip and the second dimension was in a 12% polyacrylamide slab gel. the dried gel needs to be reswelled to its original volume. Optimal conditions for reswelling are normally provided by the IPG strip manufacturer.1. extraction directly into the reswelling buffer is recommended. sigmoidal pH ranges. The proteins are separated according to their pI in the horizontal dimension and according to their mass in the vertical dimension.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. which is then increased stepwise to about 3. pH 3–10) are commonly used for whole-proteome analysis of tissue samples.2. A recipe for a typical reswelling buffer is presented in Section 3. Reswelling is normally performed overnight at 4°C. The appropriate IEF protocol will depend not only on the sample and IPG strip. Application of a low voltage current may speed up the reswelling process. although this will depend on the IPG gradient and the length of the strip. Before electrophoresis. This method is thus suitable for most 2DE applications and has all but completely replaced the older and less reproducible method of IEF by carrier ampholytes in tube gels. Broad-range linear strips (e.

2.3) and cemented in place using a molten agarose solution. but for most applications gradient gels or gels of about 10% or 12% polyacrylamide are appropriate. During the equilibration step. While some reviewers recommend alternative buffer systems.4 Second-Dimension Electrophoresis Once the gel strip has been equilibrated. This is best performed using a dentist’s tool or other appropriate implement.60 the Laemmli method. A tracking dye for the second electrophoresis step is also normally added at this point. A typical equilibrationbuffer recipe is as follows: 50 mM Tris–HCl at pH 8. Care must be taken that the (+) end of the strip is on the same side of all slab gels. 2% SDS.2.3 Orientation and placement of an isoelectrofocused IPG strip onto the top of the second-dimension gel.1% SDS) at both electrodes. Görg et al.2.8.56 reviewed IEF for 2DE applications. The manufacturer’s instructions should be followed.Proteomics ◾ 27 the equipment used. 192 mM glycine. .3 Equilibration Before the isoelectrofocused gel strip can be applied to the second-dimension slab gel.2. Figure 3. taking care to put the pressure on the IPG strip’s plastic backing rather than the gel itself. that the gel side of the IPG strip faces the notched side of the glass plate.61 using glycine as the trailing ion and the same buffer (25 mM Tris. 3. 0. 30% glycerol.59 3. 1% DTT. trace amount of bromophenol blue. 6 M urea. A second equilibration step in the presence of 2. thus reducing vertical streaking. the SDS–polypeptide complex that affords protein-size-based separation will form and the reducing agent will preserve the reduced state of the proteins. Optimal pore size depends on the size of the target proteins. and that the strip is pressed gently onto the SDS gel. The gel is run at a constant current of 25 mA until the bromophenol blue dye front has reached the bottom of the gel. it is applied to the top edge of an SDS-PAGE slab gel (Figure 3. Ready-made gels suitable for analytical 2DE are available commercially.5% iodoacetamide and without DTT (otherwise identical buffer) may be required for some applications. This will alkylate thiol groups and prevent their reoxidation during electrophoresis. it needs to be equilibrated for 30–45 min in a buffer-containing SDS and a reducing agent such as DTT. avoiding trapping air bubbles. remains the most popular one.

such as the SYPRO or Cy series of dyes. Patton published a detailed review of visualization techniques for proteomics. organic. and therefore individual variation is a major concern and needs to be accounted for in any statistical treatment of the data.63 In particular.1% Coomassie Blue G-250/17% ammonium sulfate/34% methanol/2% ortho-phosphoric acid.64 These difficulties arise from several sources of variation among individual gels. These multiple sources of variation has led some investigators63–65 to cast doubt on the suitability of univariate tests. and individual protein quantification. or Progenesis (Nonlinear Dynamics). 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. followed by incubation for 1 h in 17% ammonium sulfate/34% methanol/2% ortho-phosphoric acid. A typical staining procedure includes fi xing the gel for several hours in 50% ethanol/2% ortho-phosphoric acid. These include radiolabeling. such as in difference gel electrophoresis (DIGE). A great many alternative visualization methods are available.6 Analysis Although commercial 2DE image analysis software. Pooling samples may also be an option and this depends on the type of experiment.2. and followed by destaining for several hours in water. the spot of interest is excised from the gel.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.2. matching. 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.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. Briefly.3 Protein Identification by Peptide Mass Fingerprinting Identification of proteins on 2DE gels is most commonly achieved via mass spectrometry of trypsin digests. such as Student’s t-test.62 3. UV-absorbing molecules (such as 2.2. digested with trypsin (or another suitable protease). followed by several 30 min washing steps in water. Multivariate analysis has been successfully used by several investigators in recent years. and the resulting peptide mixture is analyzed by mass spectrometry.68 where peptides are suspended in a matrix of small. commonly used to assess the significance of observed protein expression differences. and staining with fluorescent dyes. 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. such as ImageMaster (Amersham). analysis of the 2DE gel image. Also.2. There are. has improved by leaps and bounds in recent years. PDQuest (BioRad). commercially available colloidal Coomassie staining kits that do not require fi xation or destaining. . The most popular mass spectrometry method is MALDI-TOF mass spectrometry. such as protein load variability due to varying IPG strip reswelling or protein transfer from strip to slab gel.28 ◾ Handbook of Seafood and Seafood Products Analysis 3.65–67 3.2. however. followed by staining for several days in 0. gene expression in several tissues varies considerably among the individuals of the same species. such as with [35S] methionine. including protein spot definition.

this problem is surprisingly acute for species of commercial importance.70 1697. 100% agreement was observed between the two methods.4) is then used for protein identification by searching against expected peptide masses calculated from data in protein sequence databases.8 1652.50 30 856. As can be seen in Table 3. The solid markers indicate the peaks that were found to correspond to expected b-2 tubulin peptides.86 1028. How useful this method is will depend on the length and quality of the available nucleotide sequences. Several programs are available. using the appropriate software. correlated activity measurements. In their work on the rainbow trout liver proteome. It is important to realize.90 1822.63 1258. to obtain a tentative identity.61 1272.10 and Vilhelmsson et al.org/tools) contains links to most of the available software for protein identification and several other tools.25 Figure 3. which in many cases is more extensive than the protein sequences available.4 2212.0 1229. excluded from the analysis.69 1659. 2506.4 A trypsin digest mass spectrometry fingerprint of an Atlantic cod larval protein spot. such as the National Centre for Biotechnology Information (NCBI) nonredundant protein sequences database.54 50 40 1287.54 70 1960. 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. 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.56 1974.06 1575.Proteomics ◾ 29 842. therefore.60 Peptides identified as those derived from Atlantic cod β-tubulin Trypsin autolysis peaks 1159.98 1886.expasy.43 . many with a web-based open-access interface.50 20 10 0 741.6 Mass (m/z) 2108.9 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.07 1131. identified as b-2 tubulin. however.801616. The open markers indicate mass peaks corresponding to trypsin self-digestion products and were. Martin et al. To circumvent this problem.00 1196. The ExPASy Tools web site (http://www.51 1040.96 60 870. it is possible to take advantage of the available nucleotide sequences.2 2798.1. The resulting spectrum of peptide masses (Figure 3.35 1621. In those cases where both the protein and nucleotide databases yielded results.36 2564.71 100 90 80  Intensity 1061. or transcript abundance).

) Astacidea (lobsters and crayfishes) Brachyura (short-tailed crabs) 21. haddock.237 2. incl. halibut.896 3.864 47.158 20. and plaice) Zeiformes (dories) Scorpaeniformes (scorpionfishes. incl.1 Families of Some Commercially Important Seafood Species and the Availability of Protein and Nucleotide Sequence Data as of March 27.046. and pollock) Lophiiformes (anglerfishes.208 2.557 .284 2.407 84.086 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.138 36.933 3.30 ◾ Handbook of Seafood and Seafood Products Analysis Table 3.424 2.845 999. turbot. incl. whelks and abalone) Cephalopoda (squid and octopi) 32.626 138 16. incl. cod. redfish and lumpfishes) 185.871 32.533 1.008 Mollusca (Mollusks) Bivalvia (mussels.122 268 26. etc. mackrel.751 Crustacea (Crustaceans) Caridea (shrimps.) Gastropoda (incl.203 467. etc.063 3. sole. sea bass.585 1. incl.006 121. saithe.489 130.245 2. sea bream.287 8. and wolffish) Pleuronectiformes (flatfishes.007 911 303 18. monkfish) Perciformes (perch-likes.845 10.353 287 170.798 81.680 1. tuna.344 5.442 237 2.592 735 179 585 768.762 726.782.380 898.210 Chondrichthyes (Cartilagenous Fishes) Carcharhiniformes (ground sharks and dogfishes) Lamniformes (mackrel sharks) Rajiformes (skates and rays) 3. scallops.656 2.381 45.

3 Applications of 2DE in Seafood Analysis The two-dimensional electrophoresis has been in use within food science for at least two decades. In the peptide mass fingerprinting discussed earlier. Proteome analyses in developing organisms have shown that many .Proteomics ◾ 31 A more direct. and adult) during their life span that coincide with changes in the morphology. clearly defined subproteomes and included such applications as the characterization of bovine caseins. yielding a second layer of information.73–75 Mass spectrometry methods in proteomics have been reviewed. proteomic investigations on fish and seafood products. larva. cytoskeleton.79 Rappsilber et al. each peptide mass can potentially represent any of a large number of possible amino acid sequence combinations.83 and Delahunty and Yates.1 Development Fishes go through different developmental stages (embryo.80 Mo and Karger. way of obtaining protein identities is by direct sequence comparison. and behavior of the fish. 3.77 Damodaran et al.20 To date few studies on fish development exist in which proteome analysis techniques have been applied.. 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. fish physiology. Furthermore.85 wheat flour baking quality factors.86 and soybean protein bodies.81 Gygi and Aebersold. which.23.71.. Until recently. several short stretches of amino acid sequence will be obtained for each peptide. the method of choice is tandem mass spectrometry (MS/MS). improved reproducibility and resolving power of electrophoretic separation techniques. reflect a response to biological perturbations or external stimuli9–11. as well as in aquaculture.90–92 The morphological and physiological changes that occur during these developmental stages are characterized by differential cellular and organelle functions. the identified proteins consisted mainly of proteins located in the cytosol.3.76 Nyman. if rather more time-consuming. Early studies focused on relatively small.84 3.. for example. and nucleus. have gained considerable momentum. and vastly superior protein spot identification techniques. In both these studies. for example. this was accomplished by N-terminal or internal (after proteolysis) sequencing by the Edman degradation of eluted or electroblotted protein spots.78 Thiede et al. physiology.88. 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. when combined with the peptide and fragment masses obtained.89 A brief discussion of a few emerging areas within fish and seafood proteomics is given as follows.94 Proteome analysis provides valuable information on the variations that occur within the proteome of organisms.95 resulting in different expression of proteins.72 Today. the higher the number of possible combinations. or redistribution of specific proteins within cells. The larger the mass (and longer the sequence). Correlating this spectrum with the candidate peptides identified in the first round narrows down the number of candidates. by Yates. posttranslational modifications. and development. These variations may.. enhances the specificity of the method even further.87 With the lower cost.93 This is reflected in the variations of global protein expression and posttranslational modifications of the proteins that may cause alterations in protein function.82 Lin et al.

8. and their oxidation during frozen storage. whether they be encoded in structural genes or brought about by posttranslational modification. can be observed using 2DE or other proteomic methods. Studies on various proteins have shown that during fish development sequential synthesis of different isoforms appear successively. therefore. molecular mass.98 Different isoforms generated by posttranslational modifications are largely overlooked by studies based on RNA expression.99–107 The developmental changes in the composition of muscle protein isoforms have been tracked by proteome analysis in African catfish (Heterobranchus longifilis). and pI of the protein present in a tissue. 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.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.102 common sole (Solea solea).7 Despite this undertaking. developmental stage specific muscle protein isoforms have gained a special attention.2. It is also worth noting that protein isoforms other than proteolytic ones. This fact further indicates the importance of the proteome approach to understand cellular mechanisms that underlie fish development. The major obstacle on the use of proteomics in embryonic fish has been the high proportion of yolk proteins.27 published a method to efficiently remove the yolk from large batches of embryos without losing cellular proteins.113 .108 3. are among persistent quality problems in the seafood industry and have deleterious effects on fish flesh texture. many of which are correlated with specific textural properties in seafood products. By dechorionation. where differences are expected to occur in the number. a large number of yolk proteins remained prominently present in the embryonic protein profiles.8.109.2 Quality Involution Degradation of proteins during chilled storage. 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.1 Protein Autolysis and Oxidation during Storage and Processing The specifics of fish muscle protein autolysis during storage and processing still remain in large part to be elucidated.110 Problems of this kind.111 3. The success in the removal of yolk proteins by Link et al. be distinguished on 2DE gels. These interfere with any proteomic application that intends to target the cells of the embryo proper.21. and production of surimi and conserves occur under conditions conducive to endogenous proteolysis. Link et al. several commercially important fish muscle processing techniques.27 is probably due to dechorionation prior to the deyolking of the embryos. fermentation. are well suited for investigation using 2DE-based proteomics. the embryos fall out of their chorions facilitating the removal of the yolk.99. 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.101 These studies demonstrated that the muscle shows the usual sequential synthesis of protein isoforms in the course of development. In a recent study on the proteome of embryonic zebrafish.21 and dorada (Brycon moorei). Thus. For example.3.3. Furthermore.94.99–107 In this context. usually have different molecular weight or pI and can.88. such as curing.21. specific isoforms of myofibrillar proteins. although degradation of myofibrillar proteins by calpains and cathepsins112.

123 Both studies indicated that several proteins are differentially expressed in farmed versus wild cod. in turn. Kjærsgård et al. where individual physiological characteristics.17 used 2DE. Huss noted in his review122 that product quality differences within the same fish species can depend on feeding and rearing conditions.1 . various quality characteristics of fillet and body were measured124. is thought to be responsible for a large fraction of cellular proteolysis. The proteasome is a multisubunit enzyme complex that catalyzes proteolysis via the ATP-dependent ubiquitin–proteasome pathway which. In a recent study on the feasibility of substituting fish meal in rainbow trout diets with protein from plant sources.128 In rainbow trout. appear to display seasonal variations. Indeed.117. etc. Olsson et al. the interplay between these physiological parameters and environmental and dietary variables needs to be understood in detail. Whatever may be the mechanism.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. We are aware of two recent studies where Atlantic cod muscle proteomes have been compared between farmed and wild fish.120 and have demonstrated the importance and complexity of proteolysis and oxidative changes in seafood proteins during storage and processing. this is fast becoming feasible. furthermore. Furthermore. such as pathways involved in cellular protein degradation.3.111. Martinez et al.10. the effects on the proteasome are particularly noteworthy.123 found these to comprise several members of the glycolytic and Krebs cycle pathways. 3.127. the ubiquitin–proteasome pathway has been shown to be downregulated in response to starvation129 and have a role in regulating protein deposition efficiency. differences that can affect postmortem biochemical processes in the product which. In the context of this chapter.. therefore raises the tantalizing prospect of managing quality characteristics of the fish flesh antemortem.126 in fish fed with the experimental diets. 2D-immunoblots and LC–MS/MS to study changes in protein oxidation during frozen storage of rainbow trout. They found fish muscle proteins to be differentially carbonylated during frozen storage and were able to identify several carbonylated proteins using LC–MS/MS. flesh softening during storage.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.125 and the liver proteome was analyzed9. such as proteomics.118 Several 2DE studies have been performed on postmortem changes in seafood flesh14–17. it is clear. in mammals.Proteomics ◾ 33 and degradation of the extracellular matrix by the matrix metalloproteases and matrix serine proteases114. are optimized.119. The diet was found to have a marked effect on product texture. including quality characteristics.115 are thought to be among the main culprits. For example.117 and.2 Aquaculture and Antemortem Effects on Quality and Processability It is well known that an organism’s phenotype. With the ever increasing resolving power of molecular techniques. 67. and the amount and composition of free amino acids in the fish flesh.15. and NADPH metabolism.112. such as those governing gaping tendency. as opposed to wild fish catching. is determined by environmental as well as genetic factors. To achieve that goal. fatty acid breakdown. the proteome analysis identified a number of metabolic pathways sensitive to plant protein substitution in rainbow trout feed. affect the involution of quality characteristics in the fish product.2. that these quality changes are species dependent116. The practice of rearing fish in aquaculture.

proteomic methods have been recognized as a potential way of fish species identification.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.138.3. about 0.135–137 which was soon followed by methods to identify species in processed or cooked products. particularly for addressing questions on the health status of the fish in question. 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. For example.14 Unlike the genome.147 at National Taiwan University.18. Martinez et al. 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. and probed the membranes with serum from confirmed shrimp allergic patients.142. possibly indicating freshness and tissue information in addition to species. as different fish species have different market values. the presence of stress-factors or contamination levels at the place of breeding.145 Seafood allergies are caused by an immunoglobulin E-mediated response to particular proteins.144 Martinez and Jakobsen Friis concluded that the identification of not only the species present. 1D electrophoretic techniques were developed to identify the raw flesh of various species.34 ◾ Handbook of Seafood and Seafood Products Analysis 3. Proteome analysis can therefore potentially yield more information than genomic methods. as well as being relevant from a public health standpoint. blotted the 2D gel onto a PVDF membrane. 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.143 Indeed. studying three species of European mussels: Mytilus edulis.5% of young adults are allergic to shrimp.3. During the 1960s. 3. Penaeus monodon. studying the cause of shrimp allergy in humans.134 recently reviewed proteomic and other methods for species authentication in foodstuffs. and Mytilus trossulus. including structural proteins such as tropomyosin. The identity was further corroborated by cloning and sequencing the relevant cDNA. trossulus could be distinguished from the other two species on foot extract 2D gels by a difference in a tropomyosin spot. The allergen was identified as a protein with close similarity to arginine kinase. 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. found that M. the proteome varies from tissue to tissue and with environmental conditions. demonstrating that it had arginine kinase .139 These early efforts were reviewed in 1980.4 Allergen Identification Allergenic potential is food safety issue of particular concern to the seafood producer. this makes the issue of species authentication an area of increasing economic importance. 2DE-based methods have been developed to distinguish various closely related species.143 Lopez and coworkers. Mytilus galloprovincialis. and postmortem treatment.3 Species Authentication Processed fish products are increasingly common in the market and. From early on. such as the gadoids or several flat fishes. They found the difference to be due to a single T to D amino acid substitution. performed a 2DE on crude protein extracts from the tiger prawn.140. Allergic reactions to seafood affect a significant part of the population.141 More recently. The allergens were then identified by MALDITOF MS of tryptic digests. proteomics-based species identification methods are likely to develop rapidly and find commercial uses within this field. A final proof was obtained by purifying the protein. These authors.

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

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

3 Genomic Resources and Genome Projects for Aquatic Species Genetic and genomic resources have been generated for many aquatic species of economical interest.. Of particular interest are expressed sequence tags (ESTs). can be sequenced either to completion or from their ends. through the identification of common restriction fragments). Importantly. A method called “DNA barcoding” should help to identify species and phylogenetic units. 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. 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. Additional approaches are required to study gene expression (transcriptomics. ESTs can also be used. cloned in a bacterial vector and constituting a so-called genomic library. 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]). which can be very useful to precisely determine the relative position of sequence contigs assembled “in silico” from whole genome shotgun sequencing data. or even better in combination with.g. obtained through sequencing of complementary DNA (cDNA) libraries. Generally. 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]. The overlapping between these clones and their relative arrangement in the genome can be determined through fingerprint analysis (e. for SNP detection and phylogenetic reconstructions. In addition. a new revolution of large-scale sequencing is ushering in a second era of genomics. for instance. Barcoding is based on a sequence of short standard parts of the genome. proteomics) and function (functional genomics) as well as interactions with the environment (environmental genomics). Bacterial artificial chromosomes (BACs) accepting inserts from several hundreds of kilobases are frequently used as vectors. [16]). for example. zebrafish and medaka are two complementary fish models to study . useful in the case of regions rich in repetitive sequences posing problems to assembly after whole genome shotgun sequencing.fishbol. Such an approach is. The relative position of two contigs can also be estimated cytogenetically using double fluorescent in situ hybridization [8]. 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. 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. Parts of the genome.Seafood Genomics ◾ 45 A clone-by-clone approach can be used as an alternative to. For example. Large-scale expression studies at the transcriptional level are generally performed using microarrays or other methods of high-throughput expression profiling. 4. hereby contributing to species conservation and management of global fish biodiversity (http://www. This provides a physical map respecting the “real” base pair distance between genes and markers. see Ref. shotgun sequencing.org/). Sequence data can be used among others to identify similarities and differences between species and study genome evolution (comparative genomics [14]) or to infer reliable phylogenetic relationships between organisms (molecular phylogenetics and phylogenomics [15]).

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

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

Populations of North-East Arctic cod and Norwegian coastal cod have been analyzed. For several species. Genomics and transcriptomics can allow assessing the genetic and functional consequences of interbreeding between farmed and wild fish. brown trout.62]. with possible detection of DNA sequences promoting evolution in their genomes [17]. species-rich groups such as the East African cichlids [64] might be preserved with priority since their evolution potential might predispose them to serve as progenitors of future biodiversity [52]. Different types of markers have been used for the estimation of natural population and the determination of conservation genetic parameters in salmonids [54] and to estimate quantitative genetic parameters under wild conditions [55]. This approach has already been used to identify adaptive differences between natural populations in several species. for example. For example. Population genomics is a form of population genetics extending the analysis of genetic variation in natural populations to the scale of the genome itself. it has been. microsatellite data indicated marked genetic changes in declining North Sea cod [57]. conservation efforts could focus on the preservation of genetic diversity allowing biota to adapt to new conditions. Molecular markers can be used to monitor the efficiency of programs aiming to supplement declining wild populations through individuals reared in captivity.48 ◾ Handbook of Seafood and Seafood Products Analysis RAPD markers. 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. SNPs. for which large annual escapees of farmed Atlantic salmon enhance the risk of extinction of wild populations. and others (for review. phylogenetics and phylogenomics are of major importance for the recognition of endangered taxa from the systematic point of view. sufficient genetic data might be available to provide at least basic information on genetic structure and genetic units for biologically sustainable use [56]. In contrast. Finally. and pike.59]. 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]. Genetic monitoring of diversity using polymorphic markers allows monitoring population size and diversity over time. thereby identifying loci potentially influenced by natural selection [53]. multiple SNPs have been generated for Atlantic cod. taxa can also be considered as conservation units. heralding a new era in the analysis of adaptive evolution and functional variation [58. including the European flounder and the brown trout [61. The effects of stress factors contributing to species collapse and . including Atlantic herring. observed that reintroduced steelhead trout presented reduced reproductive capabilities caused by genetic effects of domestication [66]. This type of study has been performed on Atlantic salmon. Atlantic salmon. Through pedigree reconstruction with microsatellite markers. see Ref. [1]). 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. For example. European eel. resulting in potentially detrimental effects on survival of these populations [67]. Genome-wide gene expression profiling can also be used to detect variations in gene expression within and among natural populations [60]. With the development of much faster and cheaper high-throughput sequencing methods. Beside populations. this field will certainly be of major importance in the future of fisheries management and biodiversity conservation. the available population genetic information is insufficient for most other species. with the discovery of new groupings and the determination of divergence times and molecular clocks [63]. turbot. Accordingly. with poorly represented phylogenetic groups receiving high conservation priorities [52].

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

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

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

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

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....59 5........Chapter 5 Nucleotides and Nucleosides M.................59 5....................61 5.3...............3 Chromatography.........3................................. Aleida S............................................... Sensory methods to evaluate fish quality are subjective and difficult to use in the evaluation of processed (fillets....................................................2..................... Leticia Mora.1 Introduction Bacterial growth is the main factor limiting fish commercial life by producing its alteration and unpleasant flavor..............1 31Phosphorous-Nuclear Magnetic Resonance Spectroscopy ............. Concepción Aristoy.. 64 References ...................................................................1 Introduction ..........................................................................2....................2 Nucleotides and Nucleosides Determination ...........................................3.61 5................ and Fidel Toldrá Contents 5......................4 Enzymatic Analysis..........3................. Hernández-Cázares........ the adenosine triphosphate (ATP) regeneration that occurs in vivo stops and ATP is degraded until 57 ...................... objective methods for freshness determination are required and the determination of the biochemical changes occurring in early postmortem in fish constitute a helpful tool.3 Analysis of ATP-Related Compounds ......................... or eviscerated fish) or canned fish...61 5................... beheaded...........61 5................................ the autolytic process derived from tissue enzymatic activity and lipid oxidations also contributes to fish maturation and subsequent spoilage. The first autolytic process taking place in fish affects carbohydrates and nucleotides............................65 5...2...2 Capillary Electrophoresis ......57 5...2.3.................... Thus.....3...................... Nevertheless.... After death.. 60 5.......................1 Extraction of Nucleotides and Nucleosides ..2 Chemical Structure of Main Seafood Nucleosides and Nucleotides ...

IMP degradation to inosine (Ino) and its disappearance have been correlated with lack of freshness in some fish species. Inosine is transformed to hypoxanthine (Hx) by the action of the enzyme nucleoside phosphorylase (NP). and either of the two may be used as freshness indicators. 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.3 In all cases. .1 Degradation of ATP in postmortem fish muscle. This process involves a series of reactions commonly represented according to the sequence shown in Figure 5. As a result of endogenous enzymes action. Ino. 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. (2006) published a review of the concentration of IMP. Ino and Hx concentrations increased during storage.1 The following IMP dephosphorylation to obtain inosine is mainly autolytic and occurs at a slower rate during the first stage of cold storage.58 ◾ Handbook of Seafood and Seafood Products Analysis rigor mortis is reached. This enzyme is mainly generated in muscle from biochemical processes of microorganisms. although it might be accelerated by the action of different bacteria.4 However. 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.2 Howgate et al. IMP is the main nucleotide present in fish species.1. which is accumulated in postharvest fish. ATP molecule is rapidly degraded to adenosine monophosphate (AMP) and afterward to inosine monophosphate (IMP). the use of a single compound as freshness indicator is not always advisable. whereas AMP remains major in crustaceans.

Some of them are briefly described here. 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. For this reason.12 However. to which one or two additional phosphate groups are attached through pyrophosphate bonds (∼P) (Figure 5. adenosine diphosphate (ADP). These cold conditions must be held along the sample preparation. Nucleosides are glycosylamines that are formed when a nucleobase (purine or pyrimidine base) attaches to a ribose or deoxyribose ring.16 Another suggestion to use nucleotide compounds as a measurement of seafood quality is their relation with sensory attributes. This is achieved by immediately freezing the excised muscle under liquid nitrogen to stop all enzymatic reactions.2. forming the adenosine or inosine. often designed K ′ value or Ki index. a revised K value. Nucleosides currently analyzed in seafoods are those in which a purine ring. generally within 1 day of storage in ice after death in all fish species. and it is important to stop this reaction drastically at the sampling time. it is advisable to collect small tissue samples and immerse them into liquid nitrogen. consequently.11 and. ATP-chain degradation occurs very fast. ATP. for several species.6 This value has been used as one of the freshness indexes to evaluate the quality change of postharvest fish. and thus. 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. This is the main reason for the use of indexes with more than one compound from the ATP-degradation chain. even at refrigeration temperatures. IMP is derived from the inosine in which a phosphate group is attached to the 5-ribose carbon.17 5. a high content of Hx is related with the bitter off taste of spoiled fish. adenine. ADP and ATP are derived from the AMP. and AMP disappear early postmortem. the knowledge of their molecular structure is important. Ino. or hypoxanthine is attached to a ribose.14 Measurement of ATP-related compounds is also useful for the quality control of retorted fishes.2).16.15. In order to achieve this rapid freezing.13. nucleotides and nucleosides should be extracted and analyzed.2 Chemical Structure of Main Seafood Nucleosides and Nucleotides To a better understanding of the methods of analysis of these compounds. 5.3 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. whereas IMP evokes a fresh meaty taste sensation. and Hx expressed as percentage. In this way.18 After this.7–9 Nevertheless. The ratio Hx/AMP was considered an adequate alternative to characterize fish freshness due to its constant increment with time. On the other hand. respectively. . K value is defined as the ratio of Ino and Hx to the sum of ATP and related compounds expressed as a percentage.10. a high accumulation of Ino occurs during ATP degradation.5 and. making K value inadequate as a freshness indicator. also. 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. AMP or adenylic acid is derived from the adenosine in which a phosphate group is attached at the 5-ribose carbon. Nucleotides are o-phosphoric acid esters of the nucleosides.Nucleotides and Nucleosides ◾ 59 nucleotide degradation such as the type of spoilage bacteria and mechanical handling of fish. the disappearance of the degradation products differs from one species to another3 as mentioned here.

000 g for 10 min).3. is the following: 5 g or less of muscle tissue are excised and quickly frozen with liquid nitrogen.8 and then filtered with a 0.2 μm membrane filter and stored under frozen storage at temperatures below −20°C until analysis.6 M perchloric acid is added.17 Other extraction methods consist in the homogenization of 2. avoiding any thawing. 5.5 g of fish sample with 10% trichloroacetic acid and.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 5.20 and/or spectrophotometers as well as in capillary electrophoresis (CE)21 or ion chromatography (IC). and the tissue is homogenized with a stomacher-type homogenizer for a few minutes under cold conditions. they are neutralized with 2 M sodium hydroxide.8 by adding solid potassium carbonate or 1 M potassium hydroxide. This neutralized extract is kept in an ice bath for 15 min and centrifuged again (15. Ino.2 Structure of adenosine-derived nucleotides.1 Extraction of Nucleotides and Nucleosides A typical extraction procedure for the analysis of fish samples by reversed-phase chromatography. after centrifugation (27.000 g for 20 min). with or without employing an ion-pairing agent. 3–5 vol.5–6. cold 0.45 mm membrane. Once the extract is centrifuged (15. These fish extracts are used in enzymatic assays with biosensors19.000 g for 15 min). The frozen tissue is minced. The neutralized extract must be made up to 5 mL with 20 mM phosphate buffer pH 7. The supernatant is filtered through a 0.22 . although storage at −18°C has been demonstrated to be enough to preserve fish samples and fish extracts for the analysis of IMP. and Hx. the supernatant is filtered through glass wool and neutralized to pH 6.

in vivo 31P-NMR spectroscopy has been used as a powerful technique to characterize the biochemical changes that occur in live. to analyze nucleotides.23.27 IC. 5. the addition of an ion-pair to the mobile phase greatly improves the separation by increasing the retention time of charged molecules (ATP. the reconditioning of the capillary surface is ensured by washing 1 min with 1M NaOH. some authors have described extraction methods that consisted of heated fish sample. 5. both a microwave oven at 500 V for 5 s and heating at 100°C for 60 min have been used. Thus. Capillary electrophoresis has been used in many nucleotide analysis applications as in the study of nucleotide degradation in fish tissues. among other chromatographic techniques. In this way. AMP). The mode of separation will depend on the analyte of interest.3.25 thin-layer chromatography (TLC).3. Thus. high-performance capillary electrophoresis (HPCE). ADP. and the K′ or K i index will be usually enough to characterize fish . followed by 2 min of the running buffer used.3.22 and enzymatic assays.24 5. ion-exchange HPLC.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. RP-HPLC and ion-paired reverse-phase are the methods of choice for this analysis.3.19 5.21 Typical conditions to get a good separation of IMP. nucleotides will disappear at the rigor mortis state (normally 1 day after catch). HPLC has been shown to be the most widely used technique to analyze nucleotides and nucleosides.28 Also in vitro 31P-NMR spectroscopy has been applied to both excised tissue and perchloric acid extracts of fish muscle.2.2 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. which may be adsorbed on capillary walls. including fish extract.Nucleotides and Nucleosides ◾ 61 In the development of biosensor analysis.2. Nevertheless. However. because these samples usually contain significant amounts of ions. In particular.2 Nucleotides and Nucleosides Determination Several methods have been used to measure nucleotides and nucleosides.2. pH 11. this technique can present problems in reproducibility. and hypoxanthine would be a potential of 416 V/cm of capillary using 100 mM 3-[cyclohexylamino]-1-propanesulfonic acid (CAPS) buffer.1 31 31 Phosphorous-Nuclear Magnetic Resonance Spectroscopy The phosphorous nuclear magnetic resonance spectroscopy (31P-NMR) technique makes it possible to perform multiple determinations of high-energy phosphates in vivo in the same muscle sample. inosine.3 Chromatography At present. including nuclear magnetic resonance spectroscopy (NMR). radioimmunoassay. In the analysis of complex biological samples.

(2) ATP.3 both chromatograms of standards and hake nucleosides and nucleotides are shown. (4) AMP. .17. (5) hypoxanthine. The separation was achieved with an RP C-18 column at 35°C and a gradient between phosphate buffer at pH 7 and acetonitrile.2.1 Reversed-Phase HPLC The chromatographic analysis should be performed in a liquid chromatograph equipped with an UV detector (254 nm). 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. 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. In Figure 5.29 phosphorylated metabolites are also well separated in the chromatogram. (3) ADP.3 RP-HPLC chromatograms of standards (a) and hake (b) ATP-derived compounds. There are many approaches to analyze nucleotides and nucleosides by this technique.15 The identification of the chromatographic peaks can be performed by comparing the peak retention times and spectral characteristics (if a diode array detector is available) with those of standards. The column used is an analytical reversed-phase RP-18 column. which differ mainly in the pH of the mobile phase.29 With buffer pH 7. and (6) inosine.3.3. (1) IMP.62 ◾ Handbook of Seafood and Seafood Products Analysis freshness or quality. a simple RP-HPLC with a phosphate buffer as mobile phase will be adequate. Quantitative analysis can be performed by external or internal standard method. 5. Then.

30 This ion-paired technique is especially useful when di. the ion pair should be a positive ion with a hydrophobic rest to improve the affinity with the stationary phase. Figure 5.17. Thus. ATP-derived compounds. either tetrabutylammonium hydrogen sulfate or phosphate is the ion pair most used.3. The separation is achieved in a reversed-phase column.2.4 Ion-paired HPLC chromatograms of salmon (a) and sardine (b). and (6) inosine. Nevertheless.and tri-nucleotides have to be analyzed.2 Ion-Pair RP-HPLC The most common technique used for the separation of nucleotides is ion-pair RP-HPLC. which is especially useful in separating mixtures of charged and uncharged molecules. (5) hypoxanthine. this method is more expensive than the more simple technique previously described. 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. because the ion pair enhances the retention time and separation.Nucleotides and Nucleosides ◾ 63 5. (4) AMP. . (1) IMP. making it less dependant on the type of column. (2) ATP.4 shows an ion-paired chromatogram of a 48 h postmortem sardine extract. and the key is to add an ion pair (an ion of charge opposite to that of the analyte molecule).3. as well as the resolution. Due to the negative charge of the phosphorylated groups of nucleotides. due to the ionic nature of the phosphate esters that facilitates strong interactions with the ion-pair reagent at the appropriate pH. (3) ADP.

2.2.36 Nevertheless.36–38 The most used biosensors for the nucleotide-related compound analysis are electrochemical sensors. 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). because the denaturalization of the enzymes with time.3. and 5′-nucleotidase (NT) into a reaction phosphate buffer containing the fish extract sample at pH 7. although these applications used to be achieved with at least one of these enzymes immobilized as described earlier.32 or immobilized. and thus test kits. The depletion of oxygen or the formation of hydrogen peroxide or uric acid may be detected amperometrically. and rapid response.33.3.20.2 Enzymatic Methods with Immobilized Enzymes In this case.4. and IMP may be determined spectrophotometrically by a sequential addition of XO. and hypoxanthine will be oxidized to uric acid and H2O2. but they remain immobilized in different supports. although biosensors have shown its utility in some applications such as clinical. the analysis may be performed with one or more enzymes. XO enzyme. which is immobilized in a membrane fi xed in the sensing area of the electrode.8 and 30°C–37°C.2. simplicity.41 or for the evaluation of chicken32 and beef meat33 aging. 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. or sensors have a limited shelf life. In this sensor. all the approaches to date need the sample preparation described earlier. the use of commercial kits or disposals presents some problems. IMP. In these conditions. 5. because no interference of salt in the medium was observed here as was in the case using the HPLC method. electrodes. inosine. in which an enzyme or a group of enzymes are immobilized in a membrane or other supports. while the depletion of oxygen is measured by a Clark-type elec- .1 Enzymatic Methods with the Enzyme in Solution The concentration of Hx.20. which is further coupled to a chemical transducer. environmental.6–7.35 The most used is the biosensor based on the measure of hypoxanthine. These enzymes act by oxidizing the substrates (analyte) while consuming oxygen or producing hydrogen peroxide or uric acid. constituting what is known as enzyme sensors o biosensors.64 ◾ Handbook of Seafood and Seafood Products Analysis 5.4. This sensor has been developed mainly for assessing the freshness of fish meat40.35 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. which will be further quantified by measuring the absorbance at 290 nm and by polarimetry. due to its specificity. enzyme-coated strips. Indeed. the application in the food industry is still restricted36 mainly due to critical stages such as enzyme immobilization or sample preparation for analysis. Ino. NP.39 This procedure was also used to analyze ATP and related compounds in fish sauces with very good results.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. oxidizes the hypoxanthine to xanthine and uric acid. and biotechnology.4 Enzymatic Analysis The use of enzymatic methods to analyze nucleotides in seafood is widespread due to their high specificity. agricultural. In addition.3. These assays may be carried out with the enzymes in solution31. respectively. 5.

51 The use of multienzymatic biosensors to measure fish freshness has been very helpful for the simultaneous determination of AMP. Male. Ino was converted to Hx. thus. Howgate...R. which can be present in the sample or formed during the enzymatic reaction. T. Food Chem. Lugo-Sánchez. Thus. A similar application was proposed12. Ino. A new method for estimating the freshness of fish.. The proposed relation 1 Hx for ½ X for each oxygen molecule formed must be taken into account to quantify the Hx. J. different supports have been used for the immobilization of the XO enzyme. and then after adding a soluble NP. Most recent approaches to determine Hx are based on the incorporation of the XO enzyme in a graphite/Teflon matrix. An immobilized NT was used for the previous conversion of IMP to Ino. 2006. J. In this way. 2007. 65: 40–47. Özogul. and H values. or H2O2. Sci. Fish. Sci.44 a polyaniline film by electropolimerization. E.23. 2000. 1 mol of Hx would be converted to 1 mol of uric acid and 2 mol of hydrogen peroxide. 36: 19–22. Soc. LeBlanc. Biochemical basis of postmortem nucleotide catabolism in cod (Gadus morhua) and its relationship to spoilage. 29: 570–590. 2001. M. R. P. Nucleotide degradation in sardine (Sardina pilchardus) stored in different storage condition at 4°C..6 to −0. uric acid. F. R. Surette. In the measurement of hypoxanthine. Kuley.34 to obtain the Ki parameter as a freshness indicator. 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. Pacheco-Aguilar. Japan). Fish.46 or even in a carbon paste electrode modified with electrodeposited gold nanoparticles. 1988. Robles-Burgueno. 2. et al.J. 1959.33 although this relation should be confirmed in each particular system. M. Saito..18 and a silk fibron membrane in combination with a cellulose acetate membrane42 or a nylon net43 have been used.9 V) vs.48 IMP. 212: 141–146.53. 41: 341–353. K i. Arai. necessary to obtain K. Comparable results to that of HPLC were reported. Nunes. R. 1: 13–19. Paredi.. specific biosensors to determine AMP. Technol. Food Sci. 4. D. K. Massa. Technol. M.56 References 1. Postmortem biochemical and functional characteristic of Monterey sardine muscle stored at 0°C. Formed Hx was measured with an amperometric sensor that detected uric acid + hydrogen peroxide in an additive matter.E.. Agric.E.. and Hx using a cellulose triacetate membrane have been described. Y. some authors have described this type of biosensor coupled with an oxygen electrode. Postmortem changes in quality indices of ice-stored flounder (Paralichthys patagonicus). J. Özogul. P. Palacios.45 a nafion-coated platinum disc electrode. 3. A review of the kinetics of degradation of inosine monophosphate in some species of fish during chilled storage. 2005. Food Sci. Luong and Male20 used a multienzymatic biosensor system to determine the H value as a fish freshness indicator.A. 24: 749–750. Most of these supports have been developed with the aim of eliminating interferences due to ascorbic acid.. Quinta. 6. T.L. ..Nucleotides and Nucleosides ◾ 65 trode at a platinum cathode (−0. Changes in baseline levels of nucleotides during ice storage of fish and crustaceans from the Portuguese coast. Gill.41 preactivated nylon. Matsuyoshi. an Ag/AgCl reference electrode. Bull. 5. Mendes. Ltd. Both Hx and X are substrates for the XO action and will be oxidized either simultaneously or sequentially.. This method was patented by Luong.. Int.. M.E.53. J. and Nguyen52 and afterward it has been commercialized as a Freshness Meter KV-101 (Oriental Electric Co. M. Eur.47 On the other hand. IMP..49 and Ino50 and a multienzymatic sensor to analyze simultaneously AMP.55. and Hx amounts.L. J. Food Res. cellulose triacetate.54 In fact. Ino..E. Food Biochem. The consumed oxygen produces a current decrease that can be correlated to the concentration of Hx. Jpn. IMP. M. A. and. 7.

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

.......1..2 Analysis of Sterols ............................................1 Introduction .2 First Steps in Marine Lipid Analysis ........ 72 6............4.. 73 6................. 70 6...............3 Lipid Analysis in Marine Products ................. 77 6....................................2 Quantitative Estimation of Fatty Acid Composition ...............Chapter 6 Lipid Compounds Santiago P..............................................5 Qualitative and Quantitative Analyses of Marine Lipid Classes ...............3......................3..........1 Acid-Catalyzed Esterification and Transesterification .................... Aubourg Contents 6....3 Column Chromatography...................................................2 Marine Lipid Characteristics .....................1 Lipid Saponification............................................................................................................................................................4 Lipid Quantification .......................2 Stereospecific Analysis of Lipid Classes ................3.............1 Spectrophotometric Assessments of Total Lipid Extract ...........................................3 Analysis of Ether Lipids .5........3 Marine Fatty Acid Analysis ................. 70 6..............2........... 78 6.................................................... 72 6..........4......3.............................. 75 6....2.5........................................... 75 6........3............76 6................ 73 6... 73 6.......1 Fatty Acid Methyl Esters Preparation ....... 73 6..................2 Removal of Nonlipid Contaminants .................................................... 70 6.....1 Qualitative Analysis of Fatty Acid Composition ......................................... 79 69 ...................................................................1............1.........................76 6.......................3 Lipid Manipulation and Storage .................................... 77 6.......5.........1..1 Isolation of Lipids from Tissues ........................................2................ 72 6.......................2.......76 6.....................2.........4.....................................2 Base-Catalyzed Transesterification ................................................ 75 6............................................ 71 6...................................4 Analysis of Marine Nonsaponifiable Matter ..................................................................1........................76 6..............................................3...................... 78 6................2 GLC Analysis of FAME...............................................................................2......................1 General Aspects of Lipid Compounds ........................................ 73 6...

82 6.........5... indeed.... Such diverse compounds as hydrocarbons.. and lipopolysaccharides would be included.... phospholipids (PL)............................. lipid is usually the second largest biochemical constituent after protein..8 Mass Spectrometry ...... in it... a widely accepted division has been difficult... Because of their structural and functional variety.4 Thin-Layer Chromatography ........6 Silver Ion Chromatography .... however..... differ from the other sources in that they contain a wider range of fatty acids........... gangliosides..... 82 References ...... and sphingolipids) would yield three or more types of products per mol........................................................... 80 6. longer-chain fatty acids.....81 6.......... “simple lipids” (fatty acid and alcohol components) would be those that yield on hydrolysis at most two types of different products per mol........ including cardiovascular ones [3].. and alcohols....... an inverse ..... ethers.............2 Marine Lipid Characteristics In many marine organisms........ Seafood lipids are known to provide high contents of important components for the human diet.....1................ Most textbooks describe lipids as a group of naturally occurring compounds....... carotenoids...... fatty alcohols (wax esters).......... Related to exogenous effects.. and a larger proportion of highly unsaturated fatty acids...1 Introduction 6....5..... 79 6....................9 Supercritical Fluid Chromatography ...... Marine lipids.... chloroform......5.7 Nuclear Magnetic Resonance (NMR) Spectrometry ................. such as hexane............................... 80 6........ glycoglycerolipids......... 6.... toluene...................5......................5 High-Performance Liquid Chromatography ... DHA) [4].. An alternative division into two broad classes has been shown to be convenient for lipid analysts [2]......70 ◾ Handbook of Seafood and Seafood Products Analysis 6...... A simple physicochemical classification that empirically groups lipid molecules according to the hydrophilic–lipophilic balance has been proposed [1]................. such as nutritional lipid-soluble vitamins (namely A and D) and essential and ω3 polyunsaturated fatty acids (PUFA) that have shown a positive role in preventing certain human diseases................. 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).. 79 6.....1.. and amines (PL).5.......................5....... sterols (sterol esters)................ Different attempts have been carried out to define what is meant by the term lipid. Marine species have shown large variations in lipid content and composition as a result of endogenous and exogenous effects [5–7]................... soaps...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.... although no satisfactory or widely accepted definition exists............... the catching season has been shown to play a key role regarding temperature and feeding availability. triacylglycerols (TG)............... particularly C20:5ω3 (eicosapentaenoic acid....... which have in common a ready solubility in organic solvents...... whereas “complex lipids” (glycerophospholipids....... steroids...... EPA) and C22:6ω3 (docosahexaenoic acid.... phosphoric acid..

With respect to endogenous effects. . and instrumentation available. 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. differences according to the type of muscle and its location. A basic protocol procedure is exposed in Figure 6. 6.1.1. sex.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. content variations have specially been observed in fish locations to be employed as lipid depots.Lipid Compounds ◾ 71 relationship between unsaturated fatty acid content and environmental temperature has been confirmed for many marine fish.1 Basic steps to be carried out for the lipid analysis of marine products. The approach to the analysis of lipids in a given marine sample will depend on the amount of material in the sample. lipid matter has been described to exhibit a heterogeneous distribution throughout the body of marine species. Thus. age. but mainly the amount of information required. In all cases. and sexual maturation have been pointed out. probably affected by physiological and anatomical factors. 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 equipment.

6.g.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. The second problem is endogenous lipolytic enzymes that can lead to large amounts of unesterified fatty acids.. although endogenous tissue antioxidants can provide some protection. and salts. . peptides. PUFA can autoxidize as a result of endogenous oxidant enzymes. may on occasion be extracted by these when they are in the presence of large amounts of simple lipids such as TG. When this is not feasible. For all extraction methods. any such impurities can be troublesome. Where large amounts of tissue have to be extracted. which employs chloroform–methanol (2:1) in a solvent–tissue ratio of 20:1. phosphatidic acid. Two main problems can arise with lipid fraction when employing inconvenient storage conditions. Its employment has recently been reviewed [10]. [8]. 0.72 ◾ Handbook of Seafood and Seafood Products Analysis 6. amino acids.2 First Steps in Marine Lipid Analysis 6. At the same time.88% KCl) [8]. the tissue should be kept frozen (about −60°C or less) as rapidly as possible. polar complex lipids. 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. However. which do not normally dissolve readily in nonpolar solvents. such as proteins. the procedure of Bligh and Dyer [9] offers some advantages as it does not use large volumes of solvent. As an advanced alternative.2. urea. and as large volumes of solvents may be used to obtain small amounts of lipids. Pure single lipid classes are soluble in a wide variety of organic solvents. though more time-consuming. 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. marine tissues should be extracted from the living organism as soon as possible after catching or slaughtering [2]. it should not be so polar that TG and other nonpolar simple lipids do not dissolve and are left adhered to the tissues.1 Isolation of Lipids from Tissues Ideally. First. a major driving force being the environmental concern regarding the use of organic solvents. 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. most workers in the field appear to accept two basic routines currently in general use.2. or lyso-phosphatidylglycerols in lipid extracts. In addition. 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. This type of washing procedure was first developed by Wells and Dittmer [11] and simplified later by Wuthier [12] for large numbers of samples. which yield essentially quantitative extractions of the major lipid classes when applied to homogenates of whole marine tissue extractions. all solvents can contain contaminants. Although there are limitations to its use and alternatives are frequently suggested. The most popular is the method of Folch et al. supercritical fluid extraction shows an increasing demand. diacylglycerides. A more elegant and complete. this method applies a single-phase solubilization of the lipids using chloroform–methanol (1:1) in a solvent–tissue ratio of 4:1. it is advisable to include an additional antioxidant at a level of 50–100 mg/L to the solvents.

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

” .7. the double-bond configuration is “cis.15-Octadecatetraenoic 5.14.8.16.12.10.11.14-Eicosatetraenoic 5.13.19-Docosapentaenoic 4.9.8.16.12-Octadecadienoic 9.15-Octadecatrienoic 6.74 ◾ Handbook of Seafood and Seafood Products Analysis Table 6.1 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 9.10.11.13.19-Docosahexaenoic Linoleic Linolenic Stearidonic Araquidonic EPA DPA or clupanodonic DHA or cervonic In all cases.12.17-Eicosapentaenoic 7.

6. ECL values can be calculated from an equation similar to that for Kovats’ indices or found by reference to the straight line obtained by plotting the logarithms of the retention times of a homologous series of straight-chain saturated FAME against the number of carbon atoms in the aliphatic chain of each acid. 6. FAME are obtained by heating the reaction mixture in a stoppered tube at 50°C overnight. although potassium methoxide or hydroxide have also been used as catalysts. aldehydes are not liberated from plasmalogens and amide-bound fatty acids are not affected. 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]. The commonest reagent used for this purpose has been sodium methoxide in anhydrous methanol. a PUFA loss.3. However. a further solvent such as toluene should be employed. prepared simply by dissolving fresh clean sodium in dry methanol. 6.1. .3. This reagent has been applied directly to fish muscle to obtain FAME without previous lipid extraction [17]. The commonest and mildest reagent used for the purpose is anhydrous hydrogen chloride in methanol. This is simply prepared by adding acetyl chloride slowly to cooled dry methanol. FFA are not esterified. Later on. and the ECL values are read directly from the graph.2.2 Base-Catalyzed Transesterification O-acyl lipids are transesterified very rapidly in anhydrous methanol in the presence of a basic catalyst. glasspacked columns were widely employed [18].2 GLC Analysis of FAME The advent of GLC revolutionized the analysis of the fatty acid components of lipids. As with acid-catalyzed procedures. 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. Initially. The reagent has a limited shelf life unless refrigerated. parameters known as equivalent chain lengths (ECLs) or carbon numbers have considerably been employed. The retention times of the unknown acids should be measured under identical operating conditions.Lipid Compounds ◾ 75 from amide-bound lipids (sphingolipids) are also transesterified. an additional solvent is necessary to solubilize nonpolar lipids such as cholesterol esters or TG. A different possibility consists of employing a solution of 1%–2% (v/v) concentrated sulfuric acid in methanol.1 Qualitative Analysis of Fatty Acid Composition During the previous decades. under base catalysis. and the use of old or too concentrated solutions may result in the production of methoxy-substituted acids from unsaturated fatty acids and.3. However. whereas aldehydes are liberated from plasmalogens under acidic conditions. Boron trifluoride in methanol is also used as a transmethylation catalyst and in particular as a rapid esterifying reagent for FFA. Transesterification is carried out in the same manner and at much the same rate as with methanolic hydrogen chloride. so most performances have been carried out for qualitative and quantitative analysis [16]. accordingly. In order to guarantee complete solution of nonpolar lipid classes. Parallel to ECL value employment.

the fatty acids on one side and diethyl-ether-soluble nonsaponifiable materials on the other side are separately recovered for further analysis [2]. as these are easily prepared and are widely used in chromatographic analysis.76 ◾ Handbook of Seafood and Seafood Products Analysis In recent years. If necessary. the resulting FFA have to be transformed into their corresponding FAME for further analysis by an acid-catalyzed method. On the other hand.” 6. or oxygenated chains. For a complete analysis. based on the Liebermann–Buchardt reaction. calibration factors may have to be calculated for each fatty acid component to correct the areas of the relevant peaks in the mixtures analyzed.16]. On the other hand.2. 4. 6. Results can be expressed as weight percentages of the fatty acids present or as molar amounts of each fatty acid. sterols can be fractionated and analyzed by means of different . quantitative results would first be calculated on its basis. have been shown to be suitable for direct mass spectrometric structural analysis of acids containing straight. as well as the deacylated residues of ether lipid compound. commercially available standard mixtures containing accurately known amounts of methyl esters of saturated. Problems of measuring this area arise mainly when components are not completely separated. Finally. nonadecanoic acid is employed and added before the methylation step.2 Quantitative Estimation of Fatty Acid Composition With reliable modern gas chromatographs equipped with flame ionization detectors (FID). branched. A high proportion of the available data has been obtained for the methyl ester derivatives of fatty acids. [22]. this is specially relevant for PUFA. Such compounds can be divided into sterols and “ether lipids.1 Lipid Saponification Lipids may be hydrolyzed (saponified) by heating them under reflux with an excess of dilute aqueous ethanolic alkali. and polyenoic fatty acids should be analyzed under the same GLC conditions for checking the quantification results. within limits. A known quantity of an internal standard should be added to the lipid sample. However. and there is no way of overcoming this difficulty entirely. According to the previous section.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]. unsaturated. 6. linearly proportional to the amount (by weight) of material eluting from the columns [15. monoenoic.4. the basic structure of which includes the cyclopentanophenanthrene ring. the nonsaponifiable layer will contain any long-chain alcohols and sterols originally present in the lipid sample in the esterified form. the areas under the peaks on the GLC traces are. such as picolinyl esters. Total sterol content can be determined directly and spectrophotometrically from the lipid extract by using the method of Huang et al.4.3. 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]. In most cases. cyclic. pyridinecontaining derivatives.2 Analysis of Sterols Sterols are biological compounds.4 Analysis of Marine Nonsaponifiable Matter 6.

although a great interest has been accorded to their isolation because of their medical and cosmetic applications [30]. Concerning alkenylglycerols. 18:0. Accordingly. batyl. alkenyl compounds have been directly identified and quantified by GLC together with FAME [35].5 Qualitative and Quantitative Analyses of Marine Lipid Classes Lipid samples obtained from extraction of biological material are complex mixtures of individual lipid classes [16]. 6. or they may include alkyl and alkenyl radicals. high-performance liquid chromatography (HPLC) methods can offer a nondestructive alternative. Further. The alkyl moieties are usually analyzed in the form of 1-alkylglycerol or as volatile nonpolar derivatives of this compound.Lipid Compounds ◾ 77 chromatographic techniques [23.24]. Although the GLC is normally carried out with cholestane as internal standard.27]. The first type is the major one in marine lipids. such as acetate. and its analysis has already been discussed in Section 6. The others are often united into a group called “ether lipids. marine sterols have to be converted into more volatile compounds such as acetate [25] and trimethylsilyl (TMS) [26] derivatives. For GLC analysis.3-diacyl-sn-glycerols are generally saturated or cis-monoenoic even– numbered components (16:0. neutral plasmalogens tend to migrate ahead of alkyldiacylglycerols. In this section. Adsorption thin-layer chromatography (TLC) on silica gel layers can be used to separate simple alkyl and alkenyl lipids.32]. information on ether lipid composition in marine PL is less abundant. The alkyl groups of 1-alkyl-2. Methods for separating and quantifying ether-linked glycerides have been reviewed [31. or isopropylidene derivatives by GLC. trifluoroacetate.4%) in 2M HCl. thus. and selachyl alcohols were found to be the most abundant. They can be separated by a double development in a single direction with hexane–diethyl ether (95:5. which generates dimethyl acetals from the liberated fatty aldehydes. Although the vinyl ether linkage is unaffected by basic hydrolysis conditions. but they suffer from the limitation of the lack of a distinctive chromophore in the analyte. 6. whereas no simple spot test is available for the identification of alkyldiacylglycerols. Great attention has been accorded to the assessment of cholesterol oxide formation in marine products [29]. and 18:1. being normally placed as the radical in position 1 and specially abundant in marine invertebrates [5. v/v) as a solvent system.3. it can be cleaved by acid-catalyzed transmethylation. Often.” Such compounds are basically found as PL classes (specially in phosphatidylethanolamine). no single procedure will achieve the desired analysis. such compounds tend to be decomposed during GLC analysis and are best reduced by catalytic hydrogenation to alkylglycerols. Unlike fatty acids.3 Analysis of Ether Lipids Marine lipids may contain fatty acid residues as the only radicals.4. 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]. Cholesterol has been shown to be the most abundant sterol in all marine living beings. Chromatographic methods for cholesterol analysis [28] are of relevant importance in foods in relation to human health concerns.4-dinitrophenyl-hydrazine (0. and combinations of techniques must be used until the required purposes are served. although invertebrates have shown a significant presence of other sterols [27]. Neutral plasmalogens may be detected by spraying the TLC plates with 2. chimyl. which in turn migrate just in front of TG. supercritical fluid chromatography (SFC) has been employed for the glycerol ether analysis of liver oils of shark species [34]. TMS. mostly). different analytical .

PL present in the lipid extract are made to react with ammonium molybdate in an organic phase and then measured spectrophotometrically. Compared with the data compiled for plant oils and for fats from land animals.2 Stereospecific Analysis of Lipid Classes The determination of fatty acid composition at each location in lipid classes has ever since attracted great attention. The advent of new NMR.2 [22]. However. A very popular one is that proposed by Lowry and Tinsley [36]. Accordingly. and GLC technologies combined with MS in the last decades has provided quick and useful procedures for the stereospecific lipid analysis. titrimetric methods were used for many years. Finally. which is reduced and determined spectrophotometrically [38]. 6. 6. For FFA assessment. 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. HPLC. then. a method for the quantification of esterified and unesterified total sterols is mentioned in Section 6. In many cases. according to the information provided in following sections. traditional methodologies are still employed in cases where such advanced technologies are not available and are reviewed in this section. . [39] without previous digestion. the Grignard reagent has widely been employed in the case of marine substrates [42].5. Ester linkages can be quantified by the method of Vioque and Holman [40]. although it turned out not to be accurate enough for marine lipids. preparative TLC on silicic acid impregnated with 5% (w/w) boric acid has been applied to prevent acyl migration during chromatographic separation. in it. A wide use was found for lipase hydrolysis. according to details explained in Section 6. although some interference of polar lipids was found. these enzymes can be isolated and used in simple incubations in vitro as an aid in structural analyses of lipids. Additionally. This is due to the great complexity of fatty acids present in these oils and fats.4. This method can be applied to total lipid extract or to any lipid class after previous isolation [7. Traditional determination of PL content in lipid extracts has involved the digestion of PL with the release of inorganic phosphate.78 ◾ Handbook of Seafood and Seafood Products Analysis approaches will be discussed. focused on the qualitative and quantitative analyses of marine lipid classes. where an FFA-cupric acetate-pyridine complex is involved. the results so far reported for aquatic animals are few. The fatty acid composition of each lipid class can be determined by GLC of the methyl ether derivatives. a rapid NIR spectrometry has been applied for the direct FFA determination in fish oil [37].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. giving rise to a tremendous number of species.5.41]. such functional groups are made to react with hydroxamic acid and further complexed with Fe (III). this including chromatographic separation and further analysis of fatty acids after previous methylation and transmethylation. In it. this is made to react with ammonium molybdate to form phosphomolybdic acid. prepared by esterification or transesterification of the purified lipid class. An alternative and successful method has been proposed by Raheja et al.3. 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. Procedures that involve spectrophotometric measurement of highly colored copper complexes are now favored.

5 mm thickness [7. 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. and quantification [48. In addition. 6. HPLC has undoubtedly been the most widely applied separation technique in the analysis of most simple and complex lipid classes [48.52]. in spite of the relatively higher costs [50].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. precoated plates are much more convenient than laboratory-made plates.41]. diethyl ether.47]. It combines the separation capabilities of conventional TLC with the quantification power of the FID and has application in the quantitative analysis of all substances separable by conventional TLC.5.4 Thin-Layer Chromatography Many text books and reviews report TLC application on lipids for routine separations. whereas complex lipids are recovered by elution with methanol [41. Such techniques would include high-pressure TLC (HPTLC).44] and PL [45. For preparative purposes. Separation can be carried out on silicic acid. 6. HPLC is much more expensive than . acid-washed florisil. In all cases. A variety of solvent systems have been used to separate simple lipids on an analytical or semipreparative scale by single or two-dimensional TLC. and acetic (or formic) acid in various proportions. identification. which include highly automated techniques right from sample application and development to detection and quantification. being simple lipids eluted in a stepwise sequence with hexane containing increasing proportions of diethyl ether. 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. infrared (IR) spectrometry. and tubular TLC (TTLC).47]. The improvement and versatility of TLC enable it to be used for several modern applications. The perceived weakness of TLC has been recognized as the quantification aspect. overpressure TLC (OPTLC). The system has been successfully used for marine lipid class analysis [51]. MS. Such stereospecific studies have widely been focused onto TG [43. although lengthy conditioning may be necessary before columns can be employed [2. although particular care is required to recover the acidic lipids quantitatively. or florisil as adsorbents.5. which has been used routinely for lipid analysis in the last decades. Those used most frequently contain hexane.5 High-Performance Liquid Chromatography In recent years. and NMR has increased its analytical power in several applications.46] classes. However. 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. 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. and this has led to the evolution of the TLC/FID Iatroscan system. coupling of TLC with other techniques such as HPLC. Aminopropyl-bonded phase cartridges have been much used for the isolation of simple and complex lipid fractions.49]. 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.

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

probably due to their more complicated structure. 6.65. although GLC is conveniently coupled to electron impact ionization (EI) and chemical ionization (CI) sources. This NMR technique can provide a single signal for each PL class. mono. The 31P-NMR application has also shown the possibility of analyzing the ether structures within the glycerol backbone of phosphatidylethanolamine and phosphatidylcholine. This development paralleled the development of atmospheric pressure chemical ionization (APCI). a rapid and structure-specific method for the determination of ω3-PUFA in fish lipids was presented. empirical formula.Lipid Compounds ◾ 81 Based on 1H-NMR spectrometry [59]. FFA carbonyl resonances were detected at the lower field of the carbonyl region. Thus. thus providing a suitable tool for lipolysis analysis.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. many of the advances in MS have involved new ionization techniques. 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]. A good agreement could be observed between NMR values and those from the GLC analysis.86 ppm) provided the possibility of proposing this new analytical tool. but. It could be observed that DHA was concentrated in the 2-location of TG in depot fats. and complete structure of an unknown compound. and 3 locations) of ω3 fatty acids in depot fat of several fish species was examined by 13C-NMR [63]. Finally. After different approaches. Later on [61]. Following ionization to a negatively or positively charged species (most commonly the later). and attention was focused on the identification of specific signals for ω3 fatty acid group and also individually for DHA.66]. according to each corresponding resonance. The different chemical shifts observed for the methyl resonance of ω3-PUFA (δ = 0. results obtained using high-resolution 13C-NMR were in good agreement with those obtained by GLC. Thus. Application of 31P-NMR has shown to be far shorter than with 1H and 13C. the high-resolution NMR spectra of four fish oils were recorded. Quantitative analysis of fatty acid composition and alpha-beta distribution in TG tuna fish was achieved [62]. the condensed mobile phase used for liquid separations is not readily compatible with high vacuum ionization sources. marine lipids have received lesser attention. 2. the molecules or their fragments can be separated and identified on the basis of their mass-to-charge ratio (m/z). thermospray. so little application is specially available for marine lipids [58]. Arpino [67] likened the HPLC-MS union. soft ionization MS techniques such as fast atom bombardment. although an increasing importance has been obtained lately for quantitative analysis [20. and electrospray have the ability to ionize lipid molecules without causing extensive fragmentation.8 Mass Spectrometry MS has long been used as a powerful tool for the analysis of the molecular weight. .and diene-. Over the years. its intensity should be proportional to the quantity. In a first attempt for 13C-NMR application [60]. The information-rich nature of MS makes it the most desirable detector for many explanations. Among the different food lipids. and stearidonic acid. ω3.5. EPA. and highly unsaturated fatty acids of lipid extract of Atlantic salmon muscle. Some applications concerning the marine lipids’ study will now be mentioned. The first step for any MS method is ionization of the sample molecules in the gas phase. 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. ω6. Recent developments in MS have been very interesting for complex lipid molecules.

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

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

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

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

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

The mass spectra of the compounds can also be compared with spectra of pure standard compounds and . metal ions. hence the name TBARS. and chelating agents may also influence the peroxide decomposition during the assay. and the absorbance at 350 nm is determined after 10 min [15]. The AnV of freshly deodorized oils is caused by core aldehydes.4-dienals also react with TBA. In other variations.13]. and antioxidants.3. These methods determine the presence of aldehydes. and 2. However. which is formed as a decomposition product from lipid hydroperoxides under the acidic test conditions [3]. the oxidation products are extracted in trichloroacetic acid (TCA) before the reaction with TBA. different methods give different results. Different types of headspace analyses can be used. forming a yellow pigment absorbing light at 450 nm.1. For determination of secondary oxidation products. the volatiles can be thermally desorbed into a gas chromatograph for separation. the sample is incubated at 95°C for 2 h. sulfite. the TBARS are separated by steam distillation or HPLC to increase selectivity. sucrose and other sugars. The Totox value is given as 2*PV + AnV. antioxidants. The determination of TBARS (or TBA) is a common method to determine secondary oxidation products.4-dienals). This value is a combination of the PV and the AV. There are many published methods to determine TBARS. separated. Of these methods. nucleic acids. AnV can also be determined using Fourier transform infrared (FT-IR) [16]. The TBARS values for different foods with the same level of oxidation (based on flavor scores) can vary significantly [3. Protein. where the samples are flushed or purged with nitrogen and the volatiles in the gas flow are trapped on a solid absorber. which is generated by acid hydrolysis of 1. In addition. which are secondary oxidation products. Many factors influence the color in the TBA test—temperature. The Totox value is still one of the most commonly used oxidation parameters used in commercial laboratories and laboratories in the edible oil industry. reaction products from browning reactions. and the optical density of the water phase is determined at 538 nm. However. pH. where the headspace volatiles over the samples are sampled. After sampling. and the absorbance of the solution is read at 530 nm. TBARS values have been found to correlate with sensory scores within the same materials [19]. This process is accelerated by metal ions [12]. and identified using different gas sensors. the AnV is a common method. and the color is formed by many different secondary oxidation products. The volatile compounds formed as a result of lipid oxidation can be analyzed using electronic noses/gas-sensor array systems [20]. All the methods are based on the pink color absorbance formed by reaction between TBA and oxidation products of polyunsaturated lipids.3-tetraethoxypropane [3]. alkanals. In the micromethod of Ke and Woyewoda [17]. Some of the MDA detected in this test is formed during the peroxidation of the lipids. Originally. and trace metals can influence the result [3. but most of it is formed during the decomposition of the lipid peroxides during the acid heating stage.90 ◾ Handbook of Seafood and Seafood Products Analysis anisidine value (AnV). In another method. amino acids. time of heating.18]. Many variations of this test are being used. Purge and trap techniques. but as for the determination of PV. alkenals. nitrite. In the AOCS method [13]. and chloroform before adding TCA. the lipids are dissolved in a solution of thiobarbituric acid in butanol. many other components in foods can react with TBA or interfere with the measurements. the lipids are boiled for 45 min with a mixture of TBA. are highly sensitive. The sample is dissolved in isooctane. This determines the amount of aldehydes (mainly 2-alkenals and 2.13. However. the reaction is not specific. in addition H2O2. static headspace and solid-phase microextraction (SPME) are the least sensitive. The TBA test can be standardized using MDA. antioxidants. p-anisidine dissolved in acetic acid is added. the colored complex was ascribed to the condensation of two moles of TBA and one mole of malonaldehyde (MDA). Dienals also give a red pigment absorbing at 530 nm.

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

In Rancimat and OSI instruments. Fourier-transform near-infrared (FT-NIR). 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.37]. The AOM method is performed in a somewhat similar way. Veberg et al. and additives may contribute to the spectra. The Rancimat. these include the oil stability index method [29]. and FT-IR spectroscopy methods [16. Fluorescence spectroscopy on intact samples has been shown to be a sensitive technique. the Rancimat test [30]. and also cyclic compounds. The change in conductivity is measured. for measuring lipid oxidation [28]. the oil can be heated to 80°C or more while air is bubbled through it.2. and a few minutes of oxidation of docosahexaenoate (DHA) resulted in significant changes in the ESR spectra. including near-infrared spectroscopy (NIR). One challenge is that fluorescence spectra can be very complex and that not only the oxidation products but also connective tissue. and Oxidograph are techniques for measuring the stability of oils toward oxidation.92 ◾ Handbook of Seafood and Seafood Products Analysis spectroscopy can be used. comparable to sensory analysis and gas chromatography. The level of hydroperoxides in fish oil can be determined using a rapid CL method [14]. In recent years. So far.33]. Lipid oxidation products can produce very weak chemiluminescence (CL). porphyrins. new methods have been developed. Fluorescence spectroscopy has a great potential for on-line or at-line applications. such as different hydroperoxides. 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].32. and the point where it changes most is called the induction time. 7. 1H NMR spectra can be used to study specific lipid oxidation products. Free radical assessments by the ESR spin-trapping technique detected the very early stages of lipid oxidation. It has been shown that sodium hypochlorite-induced decomposition of hydroperoxides gives strong CL [34. In a study of different model systems including fish and meat. Using solid-phase fluorescence is a relatively new approach. but studies on the use of this technique in dried fish were published in 1992 [27]. This results in the formation of low molecular weight acids that are flushed out with the air and collected in vessels containing distilled water. oil stability index (OSI). active oxygen method (AOM).4 Instrumental Methods Many instrumental methods have been developed for the determination of oxidation parameters in oils and foods. obtaining information . adipose tissue.35]. The gas chromatography–mass spectrometry (GC–MS) techniques can be used to determine a wide range of volatile secondary lipid oxidation products [36].2. and oxidative stability measurement by Oxidograph [31] and they are all suitable for analyzing oil systems. little has been done to study the fluorescence spectra of the different oxidation products that are formed in foods. but it measures the time taken to reach a certain PV. The liquid chromatography–mass spectrometry (LC–MS) techniques can also determine nonvolatile products—of special interest are the core aldehydes [3.3 Stability Methods Several techniques based on accelerated oxidation are used for evaluation of oxidation. 7. 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. aldehydes. [28] concluded that fluorescence spectroscopy may be able distinguish between different oxidation products formed but that this would require using the whole spectrum and not only the intensity at the maximum wavelength.

. intermediary goods. References 1. Narayan. The Oily Press: Bridgewater. Biol. 2006.. pp.. 5. the sensitivity was low (detection levels ∼0.K. J. 1957. The detection of these low levels is not straightforward with classical lipid oxidation measurement methods. sensory analysis requires relatively large amounts of samples. A simple method for the isolation and purification of total lipids from animal tissues.. A trained panel can be a very valuable tool for detection of early lipid oxidation of foods containing n-3 fatty acids. Int. There is. in Fatty Acids in Foods and Their Health Implications. Frankel. today it is not possible to use only one method to determine lipid oxidation. U. E. 2000. M. Dietary fat. 4. However.01 nM). Ed. 2. so care should be taken in standardizing the procedures. B. Sloan Stanley. and inflammatory disease. Lees. Lipid Oxidation. Chow. even if there are many different methods that are used to determine lipid oxidation. 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.. 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. 226: 497–509. Falch. Miayshita.. E. 2nd ed. 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. . Lipids from residual fish raw material. Marcel Dekker: New York.5 Sensory Analysis of Rancidity The ultimate measurement of rancid odor and taste is sensory analysis by a trained panel.. and the use of chemical and instrumental analyses is recommended to support and complement the sensory analysis [3]. 7. Norwegian University of Science and Technology: Trondheim. Physiological effects of eicosapentanoic acid (EPA) and docosahexanoic acid (DHA)—A review. their use is limited by the cost of employing a trained panel. and M..H.A. In general.N. In addition. It can also be difficult to compare data from different panels using different vocabularies or data from the same panel analyzed at different times. C.K. in Department of Biotechnology. 2005. However. 3. However. immunity. and finished products during seafood processing. 2005. K.Lipid Oxidation ◾ 93 that cannot usually be obtained by single conventional analytical methods [4]. 7. a rapid development in analytical methods to determine lipid oxidation. but for many of these methods calibration and verification are needed before they can be used for routine analysis. Food Rev. 777–795. however. J. 22: 291–306. 206. the oxidation products from n-3 fatty acids have a lower sensory threshold than those of oxidation products from other fatty acids. for many of these methods the results obtained vary not only with the method used but also with the analytical procedure that is performed.. Folch. Chem. Even if sensory methods can give sufficient information. Hosakawa. through the nose (nasal) or through the mouth (retronasal).. 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]. 2005.2. The sensitivity could be improved by the use of CryoProbe technology. Boissonneault.3 Summary Many different methods for the analysis of lipid oxidation exist. and G. G.

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

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

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..........1.............2 8............................. and Cabbage-Like Odors ....4......1...........................4.......4......................108 8.................................................4...........4........2 Washed Cod Muscle System .....................................2 Oxidatively Derived Odors .......105 8.......................................103 8....113 97 ...... 111 8......... 98 Fresh Fish Odors ..4.......... Onion. and Stale Odors .........3...................4..........Chapter 8 Volatile Aroma Compounds in Fish Guðrún Ólafsdóttir and Rósa Jónsdóttir Contents 8........................2...106 8.................105 8.......................................103 8.2 Dried Fish........4 Introduction ...............................2...............................................................................3.............................................................105 8..............................................................................................1 Sweet.........1 Smoked Fish Odors ................................1 8...............................................4........106 8.....................................................3 Processing Odors ......................................2 Ripening Odor—Salted and Dried Fish Odor..................3 Putrid......................................1.................................... 99 Identification of Quality Indicators ...............4 Miscellaneous .................................................... 100 8....................................................................4..............5 Conclusions ... 98 Development of Fish Aroma...........1 Cooked Odor—Boiled Potato and Rancid Odors ....... 111 8......................................................................................................1 Microbial Spoilage Odors ...112 8.............................................. Ammonia-Like....................................4. Sour...............113 References ....3 8................4.........1............ and Malty Odors .................

oxidative processes causing odors and texture changes become noticeable during extended storage and limit the shelf life. anserine). the changes are dominated by autolytic activity. The understanding of odor development by chemical. However. or hydrolyzed products and as ingredients in functional foods. Consequently.1 Introduction Health and wellness are the main drivers in new product development. formation of taste. Fish being a valuable source of polyunsaturated fatty acids (PUFA) and other nutrients is a prominent candidate as the healthy choice for consumers. 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. Endogenous enzyme . processed. They are composed of the various nonprotein nitrogenous components (NPN). and glutamic acid are known to contribute to taste together with the degradation components of the nucleotides such as inosine. leading to the formation of secondary oxidation products and off flavors [8].e. Initially. the proliferation of the specific spoilage organisms (SSO) results in the development of volatile compounds. amino acids. cooked. followed by oxidation processes. lowering of pH and endogenous enzyme activity.2 Development of Fish Aroma An overview of changes during handling and processing influencing the development of aroma in fish is generalized in Figure 8.98 ◾ Handbook of Seafood and Seafood Products Analysis 8. including degradation of nucleotides. resulting in undesirable texture changes in fish. Finally. Enhanced oxidation during cooking resulting in off odor development is of concern and an obstacle for application of fish in convenience food. 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. contributing to spoilage changes and thus influencing the freshness and quality of the end product of chilled fish [1–3]. As a result. extension in shelf life of fresh chilled fish has been achieved. valine. 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]. including small peptides such as carnosine and anserine. Research over the years has led to improved chilling and packaging technologies aimed at reducing microbial growth. 8. and nucleotides. Some of these compounds influence the taste of fish-like peptides (i. Volatile compounds play an important role in the odor quality characteristics and consumer acceptance of fish. Proteolysis plays a critical role in postmortem changes. biochemical. alanine.. and the individual amino acids glycine. A prerequisite for increased consumption of fish products is their availability on the market as fresh and high-quality products of delicate flavor. TMAO. Other prooxidants like hemeproteins (hemoglobin and myoglobin) are also involved in the initiation of the oxidative processes in fish muscle [7]. 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. The pool of components that are degraded and cause off flavors because of microbial growth are mainly soluble substances in the muscle. guanidine compounds like creatine. active inosine. and accumulation of hypoxanthine (Hx). Degradation of soluble muscle constituents such as sarcoplasmic proteins and microbial metabolism contributes to changes in the aroma profile of fish during storage. studies on application of natural antioxidants are of prime interest to underpin further utilization of fish in innovative product development as fresh.1.

dried fish. LOX activity on the skin and gills of both freshwater and marine species (rainbow trout. LOX. Josephson et al. Mb Antioxidants: α-tocopherol. were characteristic for freshwater and euryhaline fish. On the other hand.5. including calpains (neutral calcium-dependent proteases) and cathepsins (lysosomal proteases). The overall perceived odor is dependent on the level of influential compounds and their odor thresholds along with possible synergistic effects. popcorn. trimethylamineoxide. which have potent green. peptides Soluble substances. whereas volatiles generated from fat result in variation in the specific flavor character of different fish species. 1. TMAO.5-octadien-3-ol. hemoglobin. faint odor of saltwater species. lipoxygenase. plant-.1 Overview of changes in fish influencing the development of characteristic aroma of fresh. metallic. 8. 1-octen-3-ol. LOX proteases.3 Fresh Fish Odors The delicate flavor of fish is mostly contributed by volatile compounds and taste active substances in the aqueous phase. and melon-like odors. hexanal.10–13]. ascorbic acid. neutral Spoilage aroma sweet. drying. but the mechanism of this activity is not fully elucidated [9]. malty. and sardines) plays a role in the formation of odorous volatiles. cucumber Figure 8. melon-. and cooking Processing smoking. cucumber-. Newly caught marine fish contains low levels of volatile compounds and is nearly odorless. the unsaturated C9 carbonyl compounds such as 2. or nine carbon atoms [4. boiled potato. polyphenols Lipids phosholipids/PUFA Proteins sarcoplasmic. Soon after harvest. Mb. eight.14. SSO. activity influences the deterioration of fish muscle. Some components are desirable at low levels. The compounds that contribute to the characteristic plant-. hydrolases.5-ocatadien-1-ol.Volatile Aroma Compounds in Fish ◾ 99 Handling chilling. myoglobin.e. rancid potato. stockfish. oxidized. sour. and 2. specific spoilage organisms. and mushroom-like odors are unsaturated carbonyl compounds and alcohols with six. caramel. pleasant aromas of fish [6.15]. nucleotides. cucumber-. but . amino acids Fresh fish aroma seaweedy. freezing. putrid. contributing to green.Cu) Hb.6-nonadienal. and processed fish. stale. were responsible for the moderate. mushroom. spoiled. Hb. polyunsaturated fatty acids. and hydrolysis Endogenous enzymes i.. malty. PUFA. nonprotein nitrogen-containing compounds. [5] summarized the occurrences of volatile compounds in freshwater and saltwater species and concluded that the four common compounds found in saltwater species. NPN. salting. cucumber. phospholipases TMAOase Microbial metabolism Specific spoilage organisms (SSO) Oxidation Prooxidants: metals (Fe. ammonia-like Oxidized aroma Processed aroma green-like. NPN. river trout.

(E. they may contribute to off odors. 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. cod during storage [21. for example. a saltwater species. The volatile pattern changes in mature salmon when migrating from the sea for spawning. and species of the salmonidae family develop earthy.4 Identification of Quality Indicators Different characteristic odors develop in various fish species during storage. aldehydes. Another example is iodine-like off flavor in prawns associated with bromophenols originating from the feed chain [17]. amines.6-nonadien-1-ol in sweet smelt tissues [20]. on accumulation of hydroperoxides in fish tissues. plant-like notes in fresh fish. . indicate their involvement in the development of fresh fish aroma associated with seasonal variation. Both single compounds such as TMA and ethanol and multicompound indices based on combination of alcohols. The aldehydes contribute most to the spoilage odors because of their low flavor thresholds. boiled potato-. and identification was based on GC–FID. and these are difficult to detect by analytical techniques. 2.100 ◾ Handbook of Seafood and Seafood Products Analysis if their concentration increases.6-Nonadienal was identified to be the most characteristic compound for the cucumber-like capelin odor [19]. Accumulation of certain hydroperoxide isomers coincided with the period of enhancement of characteristic aroma in sweet smelt. and GC–O. Therefore. and quality changes can be explained in. C9 LOX-derived compounds have been found in higher levels in spawning euryhaline and freshwater fish [5]. where it has been demonstrated by monitoring key volatiles to study changes in different fish products during storage. they contribute to oxidized and fishy odors in stale fish [16]. and 3.22] and in smoked salmon [23]. acids. GC–MS. it is useful to monitor the overall pattern of volatile compounds and select indicator compounds. However. which are present in higher levels and can be quantified.1. Table 8. An example is the enzymically derived long-chain alcohols and carbonyls that exhibit characteristic fresh. including (E)-2-nonenal. esters. Environmental conditions and seasonal effects like spawning can influence the odor quality of fish.30–36]. but when accumulated in higher levels because of autooxidation. as seen by the detected odors listed in Table 8. The main classes of compounds detected during storage are alcohols. Studies performed in Japan. They were suggested as the possible precursors of nine-carbon volatile compounds. and sweet odors.24–29]. and amine-like odors. Fatty species develop rancid odors and taste. muddy. and sulfur compounds representing the different changes occurring during storage have been suggested by numerous researchers as indicators for freshness and spoilage [22. 8. 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. odor. amines. ketones. iodine-.21–22. and sulfur compounds.Z)-2.6-nonadienal. Purge and trap on Tenax and SPME methods were applied for sampling. lean species typically develop sweet.1 summarizes the occurrence of volatile compounds detected in our studies on cod [22] and haddock fillets [31] and smoked salmon [23]. in nominal levels the bromophenols appear to contribute to natural sea-. which has a very characteristic cucumber odor during spawning. Volatile compounds formed by microbial metabolism and oxidation contributing to these odors have been identified by gas chromatography methods and suggested as indicators of quality. and marine-like flavors of seafood [18]. This has been the approach in our studies. Seasonal effects have also been reported for capelin. Some of the influential odor compounds that have very low odor thresholds are often present in low levels.

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. caramel. Haddock Fillets [31]. earthy Sweet. floral Sweet. fatty — Fresh. fish fillet Sweet.Volatile Aroma Compounds in Fish ◾ 101 Table 8.1 Volatile Compounds Detected in Cod [22]. flowery — Ketones 2-Butanone 2.3-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 2.3-Butandione × × × × — N/A (continued) .E)Nonanal Decanal Undecanal × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × Rancid Boiled potato. (E.4-Heptadienal. candy × — — Sweet.

ethylester Butanoic acid. dried fish Acid Acetic acid × × × — Esters Ethyl acetate Ethanthiocacid. ethyl ester 2-Butenoic acid. ethyl ester Butanoic acid. Haddock Fillets [31]. ethyl ester × × × × × × × × × × × × × × × — N/A — N/A N/A Sickenly sweet. 3-methyl. sour Flowery. spicy Amine Trimethylamine × × × TMA-like. ethylester Hexanoic acid. 2-methylpropyl ester Butanoic acid. S-methylester Propanoic acid. heavy. vomit N/A N/A N/A N/A Sulfur Compounds Methanethiol Dimethyl sulfide × × × × — — . sweet. ethylester Acetic acid. and Smoked Salmon [23] during Chilled Storagea Compound 2-Pentanone 3-Pentanone 2.102 ◾ Handbook of Seafood and Seafood Products Analysis Table 8. 2-methyl. caramel — — — Sweet. ethyl ester Propanoicacid-2-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.1 (continued) Volatile Compounds Detected in Cod [22].

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. An example of the spoilage pattern of volatile compounds in chilled fish is illustrated in Figure 8.4. The flavor thresholds . The odor descriptors in Table 8. and finally sour and dirty tablecloth odor. Sweet-milky and vanilla/caramel-like odors are typical in cooked fish. cucumber-. and quantification of the main classes of compounds was based on the sum of the PAR for respective compounds in each class. mushroom-.1. After several days of storage. 8. and aldehydes detected on day 4 of storage and their increasing levels on days 7 and 10 (Figure 8.Volatile Aroma Compounds in Fish ◾ 103 Table 8.3-butandiol were found in the highest levels on day 12 at sensory rejection. —.1 Microbial Spoilage Odors The spoilage odors in chilled fish vary depending on the dominant microflora in the products. Sour. as well as green plant-. esters. contributing to sweet. 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. The loss of freshness of cod fillets and early spoilage changes were related to the formation of ketones. N/A. and aldehydes. or geraniumlike odors are characteristic sensory odor descriptors for fresh whole fish.1 Sweet. and sometimes metallic. packaging. 3-methyl-1-butanol. mainly amino acids. acids.2) were associated with the development of sweet. The microbially derived alcohols 2-methyl-1-propanol. dried fish/stockfish. In general when fish is cooked. Late spoilage changes.5°C) [22]. sulfur. the fish is no longer fit for consumption. and malty spoilage odors. and when combined with frozen storage odor. 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. Haddock Fillets [31]. 8. and malty odors.1 (continued) Volatile Compounds Detected in Cod [22]. Seaweedy and marine-like odors. the freshness notes disappear and the odor of the uncooked fish becomes neutral. and TMA-like smell.34]. During prolonged storage boiled potato odor develops. Identification of volatile compounds was based on GC–MS analysis (see Table 8. and temperature conditions during storage [33. meat-like. data not available for haddock. alcohols. which is mostly affected by handling. sour. and the end of shelf life of cod fillets on day 12 of storage are explained by the presence of TMA. caramel-like. cooling. not detected by GC–O. and 2. development of spoilage odors. alcohols.1 based on GC–O analysis of cod and smoked salmon represent most of these overall changes.4. and sulfur compounds produced by microbial degradation of fish components. sour.2. the aroma of the fillet is described as sweet and reminiscent of shellfish. and Malty Odors Ketones. showing results from a storage study of cod fillets packed in styrofoam boxes during chilled storage (0.1).

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

described as sickeningly sweet and nauseous. Oxidative processes are involved in the formation of dimethyl sulfide from methyl mercaptan and further oxidation of dimethyl disulfide. In whole fish stored in ice.2 shows that TMA was detected in high levels on day 12. Pseudomonas species have also been found responsible for the formation of volatile sulfides. The onset of stale odors can be explained by cis-4-heptenal and heptanal. but no obvious increase occurred until at the end of shelf-life and during continued storage.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. The origin of the sulfur compounds is microbial degradation of cysteine and methionine to form hydrogen sulfide and methyl mercaptan. TMA is characteristic for the spoilage odors of fish.39].4. contributing to the stale and putrid off odors in fish because of amino acid and lipid degradation [39]. numerous branched chain . fruity off odors [37.Volatile Aroma Compounds in Fish ◾ 105 storage. and the incorporation of hydrogen sulfide yields dimethyl trisulfide [38]. ammonia-like. and Cabbage-Like Odors Low levels of sulfur compounds (Figure 8. methyl mercaptan. and Stale Odors The development of dried fish. At this point there was an increase in the pH value. The lipid-derived saturated aldehydes detected on day 12 at sensory rejection also contributed to the overall sweet aroma. and dimethyl disulfide have been suggested as the main cause of putrid spoilage aromas [41]. which forms very early after harvest of fish.2) indicated that they were not important in the spoilage of chilled cod fillets stored in styrofoam boxes.4 Miscellaneous The concentration of the straight chain alkanes (nonane. and stale odors by amines during fish spoilage is well known. TMA is a potent odorant with a characteristic fishy. Ketones can influence the overall odor because of their low odor thresholds. has been suggested as a freshness indicator along with its precursor TMAO (trimethylamine oxide) [27].2 Dried Fish. decane. and undecane) appeared to be similar throughout storage in chilled cod fillets [22].1). Ammonia-Like. and ketones (2-butanone). 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. The odor of ethyl butanoate. which may have influenced the overall odor perception leading to the sensory rejection of the fillets. 8. 1-penten-3-ol). Enzymically produced DMA (dimethylamine). methyl sulfide. dried fish. ammonia-like odor. Additionally. whereas DMA may influence the overall fresh flavor of fish in combination with oxidatively formed aldehydes from long-chain fatty acids in fish. respectively [41].38].1. 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. 8.38.1.1. volatile sulfur compounds such as hydrogen sulfide. Dimethyl trisulfide has also been associated with spoilage in fish and associated with the growth of Shewanella putrecfaciens [25. Figure 8. which contributed to boiled potato-like odors (Table 8. 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]. alcohols (3-methyl-1-butanol.3 Putrid. 8. Onion.4.

they are not considered of interest as quality indicators. Limonene has also been detected in sea bream during storage [29]. which is further enhanced by preprocessing and storage of fish. Several odor active terpene derivatives have been identified in fish.4-heptadienal and 2.2 Oxidatively Derived Odors Initiation of lipid oxidation in fish is generally associated with the polyunsaturated fatty acids in phospholipids of muscle cell membranes [44]. Oxidative processes occurring during storage of fish result in the accumulation of aldehydes. Various pro and antioxidants influence the stability of the muscle and have been studied in relation to the oxidative stability of phospholipids [46]. suggesting that it may have an impact on the overall odor of fish fillets [22]. but the sampling techniques used were not sensitive enough to allow quantification of these compounds.4. Limonene has low odor threshold and a fresh lemon odor was detected by GC–O analysis of cod. 3-methyl-butanal. in similar or slightly increasing levels. 3-pentanone. 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]. 2-butanone. 2. heptanal. and 6-methyl-5-heptene-2one). and overall the alkanes showed an increasing trend with storage time. and 2. Piperidine levels have been reported to increase in spawning salmon and contribute to off odors [43].4. However. Boiled potato. and decanal. These compounds have been associated with rancid and dried fish odors. 8. therefore. and. but the knowledge of the formation of these compounds is obscure.7-decadienal. although their overall levels were lower. 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]. their impact was greater than alcohols and ketones.3 to demonstrate which odors are most dominating in the aroma profile [48]. and terpenes found in wild sea bream compared with those of its cultured counterpart [29]. such as hexanal. Aldehydes generally have low odor thresholds.2. were detected in the fillets throughout the storage time. A characteristic earthy odor in many species residing in ponds has been associated with piperidine and its reaction products. Phospholipids are the main membrane-bound lipids. The influence of other aroma active compounds present in lower levels such as the unsaturated autoxidatively derived aldehydes (2. they are in particular sensitive to oxidation.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. aromatics. 8. the feed may have influenced higher levels of aldehydes.7-decatrienal) should not be overlooked.4-heptadienal. ketones. such as hexanal. since they are not aroma active.4. Piperidine was tentatively identified in chilled cod fillets [22] and has also been suggested as a quality indicator in sea bream [29]. and because of their high unsaturation.4. 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.106 ◾ Handbook of Seafood and Seafood Products Analysis alkanes were detected. which are known to be more susceptible to oxidation than triacylglycerols in fat deposits [45]. Similarly. fish-like odors of chilled cod fillets in combination with other carbonyls (3-hydroxy-2-butanone.and potato-like odors contributed by . These oxidation products contributed to the overall characteristic sweet. cis-4-heptenal. lipid-derived saturated aldehydes.

Hexanal. and octatriene increased significantly. flowery. and fish oil notes in the same study. The fresh raw salmon odor was characterized as cucumber-like with weak sweet. green-like odors Grass Hexanal Heavy Mushroom.4-Heptadienal Rancid Geranium-like 1-Octen-3-ol Mushroom Earthy. 1-penten-3-ol. pop-like Earthy-like odors Figure 8. and Ólafsdóttir. sweet. and octadienes also increased many-fold during further storage. multivariate data analysis is useful to explore the overall trend of the main quality indicators. green-like. this aldehyde does not exhibit a fishy-type aroma by itself. and fish oil notes were characteristic for fresh cooked salmon.4-heptadienal. and green-like odors were associated with oxidatively derived 3-pentanone.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. However. Ideally. Taking into account the complexity of the spoilage processes. and rancid odors contributed by 2-nonenal and 2. some confusion exists about the role of cis-4heptenal as the “cold-storage compound” [8]. microbial metabolites such as 3-methyl1-butanol and cresol were identified [53]. sourish. 2004.4) on data from our studies on volatiles in cod [22] during prolonged storage for 17 days and compared with corresponding . heptanal. Overall earthy. 1-penten-3-ol and hexanal. The occurrence of cis-4-heptenal has been associated with the “cold storage flavor” of cod [47]. however.. green-like. G. In fact. but it rather participates in the expression of the overall fishy odor.3) were fatty. sour. and after storage for 3 days the proportions of 4-heptenal. 2-methylbutanal.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 (---▲---).52]. earthy Potato-like Boiled potato cis-4-Heptenal Heptanal ◾ 107 Cucumber. although the level of the compounds may vary and explain the differences in the characteristic odor of these species. quality indicators should demonstrate clear increasing or decreasing levels with storage time. this is not always the trend for dynamic microbial and oxidative changes and the formation of volatiles in fish during storage [22]. 20 min) 3-methylbutanal. 2-heptanone. sweet. and the most pronounced attribute was a boiled potato odor [49]. Unpublished data. melon 2-Nonenal Cucumber Fatty Fatty. and after 8 days of storage at 6°C. (From Jónsdóttir. and hexanal were abundant in headspace. Fatty. Its odor has been described both as cardboardy.) heptanal and cis-4-heptenal were the most potent odors. In fresh baked herring (200°C. Other pronounced odors detected in boiled cod (Figure 8. Baltic herring has been reported to have a similar development of volatiles. as well as boiled potato-like [51. rancid odors Flowery 2. R. Principal component analysis (PCA) was performed (Figure 8. paint-like [50]. sweet.

X-expl: 53%. and 6-methyl-5-hepten-2-one. 3-methyl-butanal in combination with acetaldehyde.4 Principal component analysis of raw and boiled cod. methional with a characteristic boiled potato-like odor dominated the odor of the aldehyde fraction of the headspace volatiles. Autoxidatively produced unsaturated carbonyl compounds were the most abundant components in boiled and canned fish. 10. oxidation of membrane-bound phospholipids in lean species can cause fishy. that is. 14.5 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 0.0 B-D17 1-Penten-3-ol 3-me-butanal Acetic acid 0. The effect of oxidation induced by cooking and formation of oxidation products such as heptenal and nonanal characterizes the (B-D4) sample.Z)-2. 3-methyl-butanal was correlated to the boiled stored cod (B-D17) (Figure 8. boiled and storage days. On the basis of odor evaluation.2 0.5-octadien-3-one. Only the spoiled raw samples (R-D14 and R-D17) can be correlated with the freshly boiled (B-D4) sample. The PCA demonstrates how volatile compounds can explain the variation in quality of samples according to storage time and handling (raw and boiled). The characteristic pattern or trend in volatiles in raw and boiled fish is clearly different. samples after heating (see Table 8. increased with time and were pronounced in the spoiled raw samples (R-D14 and R-D17). and (E.2 Washed Cod Muscle System Rancid odor development during chilled storage of fish has commonly been associated with fatty species. 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).0 Figure 8. 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. decanal.108 ◾ Handbook of Seafood and Seafood Products Analysis PC2 Bi-plot 1. However.4). raw and B. hexanal. 12.6 0. Sulfur compounds dimethyl sulfide. methional.E)-2. .2. in agreement with earlier studies [54].5 –0.4. in particular the role of volatile compounds derived from oxidation in heated/boiled samples.4-decadienal from PUFA were determined as character impact odorants of boiled cod [54]. Samples are labeled with R. as indicated by the arrows (Figure 8.8 R-D17 –0. 8. and 17 days). D (4. The malty flavor of 3-methyl butanal was suggested earlier to be mainly responsible for the malty off flavor defect of boiled cod [54]. dimethyl disulfide.4 –0. and dimethyl trisulfide were detected in higher levels in the boiled samples (data not shown). Interestingly.1).6-nonadienal. and oxidatively derived (Z)-1. especially in trout [15]. In boiled trout. (E. 19% PC1 1.4). The oxidatively formed compounds.4 0. 7.2 Raw and boild c….

it is necessary to apply models that take into account the chemical. These odors were also detected in cod fillets during chilled storage (Table 8.58].. green. the concentration and composition of volatile oxidation products analyzed by GC were compared with TBARS measurements. 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. mushroom odor caused by 1-octen-3-ol. including blood components like inorganic metals iron (Fe) and copper (Cu). The prooxidative effect of hemoglobin was evident by the formation of hexanal in high levels. in agreement with TBARS and changes in color [62]. In lean fish such as cod.4heptadienal that contributed to rancid odor caused by oxidation. TBARS (thiobarbituric reactive substances). and lemon-like odors were explained by 2. fatty. cucumber-like. Furthermore.59]. 2. sweet. 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. earthy. ..1). potato-like odor caused by cis-4-heptenal and heptanal. and a similar trend was observed in the development of cis-4-heptenal (Figure 8. ascorbic acid.5) as well as 2. Consequently. 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. 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. 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. lipid oxidation of muscle phospholipids may be induced by several catalysts. pH) [55.61]. The most potent odors detected in the model system were malty.g. rancid. and the overall odor was an intense dried fish. and instrumental color changes. To monitor the development of rancidity. and caramel-like odors contributed by 3-methylbutanal. and rancid odors dominated the aroma profile [62]. [63]. 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.g. and glutathione peroxidase) and aqueous prooxidants in fish muscle. The added hemoglobin was very effective as a prooxidant. free radical scavenging and chelation) but also on factors such as physical location. and color. painty. floral. Washed cod muscle system has been widely used to study oxidation and the influence of prooxidative and antioxidative factors [59. and environmental conditions (e. Sohn et al. grass odor contributed by hexanal. and environmental conditions expected in food products. but the compounds were detected in much lower levels [22]. [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. These compounds can be used as indicator compounds for oxidation. rancid fish oil like. including hemoglobin from blood [7. physical. as demonstrated by Boyd et al. which is not practical for rapid determination of oxidation. it is possible to detect the most volatile oxidation products like propanal and hexanal by rapid.56]. rancid. and 1-penten 3-ol. sensory assessments. has been studied to understand better the mechanisms of oxidation in the muscle [57.4-heptadienal [62]. interaction with other food components.3-pentandione. Preconcentration techniques are necessary for the analysis of unsaturated aldehydes. On the other hand. and spicy and flowery notes exhibited by 6-methyl-5-hepten-2-one. To accurately evaluate the potential of antioxidants in foods.Volatile Aroma Compounds in Fish ◾ 109 rancid. Similarly. static headspace sampling methods. soapy. dried fish-like off odors as discussed before.

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

75]. Additionally. 2-pentanone.7 illustrates the main odors that were present in smoked fish samples after 14 days of chilled storage. [74] analyzed headspace components of cod and swordfish.6-nonadienal. 3-methyl-butanal. 2-methyl-1-butanol. giving the flesh its typical fishy odor [71.Z)-2. 8. and 2-acetyl-1-pyrroline. where groups of phenol pyrolysis were most noticeable in the smoke flavor volatiles.72]. but it is mostly attributed to the phenols. Other oxidatively derived compounds like 1-penten-3-ol. Key volatile compounds identified in enzymatically produced seafood flavorants are formed via Maillard reaction and Strecker degradation of amino acids.Volatile Aroma Compounds in Fish ◾ 111 8. 3-hydroxy-2-butanone.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. and 1-octen-3-ol. Lipid-derived components. Lipid-derived aldehydes play an important role in flavor formation and have been reported to contribute to the characteristic fish-like.7) (e.4-heptanal. sweet odors of processed seafood like those in smoked salmon [23.6-nonadienal. also contribute to the aroma of seafood flavorants [70]. and 2.4.7) [23]. nonanal. thermal degradation. Figure 8. Phenolic derivatives like guaiacol (2-methoxyphenol) and syringol (2. and although they contributed less to the odors. The typical smoked salmon aroma results from a number of chemicals found in the smoke.72]. 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. it is clear that their presence contributes to the characteristic fish odor of smoked salmon products. like 3-methyl butanal. and lipid oxidation. 1-octen-3-ol. associated with spoilage off flavors. giving a popcorn-like odor that can be thermally generated. contributing to mushroom-like odor. and decanal were among key volatiles.71. 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]. In addition to phenolic compounds. hexanal. which has a characteristic potato-like odor. furan-like compounds have been reported to be responsible for the smoked odor in smoked salmon. giving rancid. plays important roles in the formation of complicated processing flavors. which is typical for products on the market [23].3. and furans have been found in spray-dried shrimp powder and shrimp hydrolysate [69].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. hexanal. whereas carbonyl compounds.. and off odor and flavor) than traditional chemical and microbial variables. The oxidatively derived compounds cis-4-heptenal and heptanal. gave the most intense odors of smoked salmon and contributed to the fish-like earthy odors and fatty and rancid odors (Figure 8.4. 3-methyl-1-butanol. were characteristic in unsmoked fish. 2. it was verified that selected key volatile compounds performed better as predictors to explain variation in sensory attributes (smoked. and 1-propanol [28.g. ethanol.4-decadienal. These are compounds like methional. Guillén et al. the Strecker aldehyde produced from methionine. Volatile compounds like alkyl-pyrazines and sulfur-containing compounds have been found in cooked crustaceans. including Strecker degradation. and alcohols were abundant in the headspace of cold smoked salmon products during storage. Microbially produced ketones. aldehydes. 2-butanone. Maillard reaction. 2. 1-penten-3-ol.72]. such as heptanal and (E. . like cis-4-heptenal. sweet/sour rancid. potato-like odors. 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].

fatty Boiled potato-like Fatty.2 Ripening Odor—Salted and Dried Fish Odor Numerous volatile compounds have been detected in ripened products like dry cured ham. Similar processes have been reported in ripened seafood products. especially those in the Mediterranean.6-nonadienal from fatty acid oxidation were the main odorants in sugar salted.5-octadien-2-one were associated with the development of the typical flavor obtained after anchovy ripening.4-heptadienal and 3. and rancid-like odors Burnt.7 GC–O evaluation of volatile compounds detected in cold smoked salmon after 14 days of storage at 5°C. geranium Rancid cis-4-Heptenal Heptanal Earthy-like odors 1-Octen-3-ol Figure 8. Salted cod are traditional products from the North-Atlantic fisheries and are highly regarded as ripened fish products in many countries. both oxidatively derived compounds. and aldehydes such as acetaldehyde. R. During ripening of salted cod. In our study. the highest odor scores were given for boiled potato and rancid. smoke Mushroom. 109. 2-methylpropanal and 3-methylbutanal were the key. 4-Heptadienal Sweet. probably originating from amino acids. peptides. (Modified from Jónsdóttir. caramel Smoke-house.4. as heptanal. However.112 ◾ Handbook of Seafood and Seafood Products Analysis Smoked salmon odors Characteristic smoke odor Sweet. sweet Flowery. most of them generated from chemical or enzymatic oxidation of unsaturated fatty acids and further interactions with proteins. mushroom 2.and 3-Methyl phenol Guaiacol 4-Methyl-guaiacol Sweet and fruity-like odors Wood.. smoke 3-Methyl butanal Sweet. The rancid. . highly volatile components of ripened anchovy. the desired flavor and texture develop as a consequence of protein and fat degradation. smoke. burnt. Food Chem. Triqui and Reineccius [80] found that 2. fruity Flowery. sweet Smoke-like 2. et al.) 8. potato-like odors together with cucumber-like odor [82]. where the ripening of salted cod (Gadus morhua) produced by different salting methods was studied. sweet Flowery. 2008. earthy. potato-like odor was identified as cis4-heptenal and the boiled potato-like odor. ripened roe products [79] Similarly. Thus. where methional derived from methionine and 2. sweet Wood. manufacturers of ripened products have observed that some degree of proteolysis is necessary before flavor can develop. and free amino acids. [76–78]. Methional and (Z)-1. 184.3.5octadien-3-one were also identified as potent odorants in ripened anchovy [81].. they suggested that lipid autoxidation during ripening was primarily responsible for aroma development.

1995.6-nonadienal. The cucumber-like odor detected is possibly 2. potato-like odors. Botta. such as heptanal and (E. Quality and quality changes in fresh fish. 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. New York.R. Therefore. according to retention index (RI) of standard and odor evaluation. proper handling.Z)-2. and sulfur compounds.6-nonadienal. and new packaging technologies. were the most intense character impact compounds of salted cod and smoked salmon. could also be responsible for the boiled potato-like odor. . H. No. Huss. to increase trust between buyers and sellers in trade. Rome. Detection of microbial metabolites originating mainly from soluble aqueous fractions of the muscle can be directly related to the quality of products. alcohols. and other prooxidants in combination with mild heating treatment are important factors to maintain the delicate flavor and odor of fish products. 348. careful evaluation of the quality of product is needed to ensure acceptable flavor. FAO Fisheries Technical Paper. Knowledge of the spoilage pattern of volatile compounds is the basis for the development of rapid techniques like smart sensor technologies. In addition. 2. such as ketones. 1-penten-3-ol. temperature control. J.Volatile Aroma Compounds in Fish ◾ 113 Methional. 1–67. and myoglobin. However. esters. cause off odors in fish during storage. Evaluation of Seafood Freshness Quality.5 Conclusions Although aldehydes. exhibiting rancid. FAO. hexanal. their presence at nominal levels gives the characteristic and desirable fishy odor in fresh and processed fish. derived from methionine and eluting at a similar time as cis-4-heptenal and heptanal. acids. 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. Other key volatile compounds in salted cod are derived form lipid oxidation. and in retail for consumers as smart sensors imprinted on packaging. contributing to mushroom-like odor. aldehydes. 1995. 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. hemoglobin. and 2-butanone. microbial growth can be limited by effective cooling techniques. Proper handling and application of natural antioxidants to control oxidative processes caused by lipoxygenase. Lipid oxidation during ripening appears to be primarily responsible for desirable aroma development in processed fish. References 1. although the compound could not be identified by GC–MS. 193 pp. 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. Volatile compounds as indicators of freshness quality and spoilage can be monitored to determine the quality of fish products. A similar set of sensors with selectivity and sensitivity toward the main quality-indicating classes of compounds.83]. The oxidatively derived compounds cis-4-heptenal and heptanal.. can be used for a variety of fish species that are stored and processed by different techniques.H. amines. 8. VCH Publishers Inc. and 1-octen-3-ol. for example.

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

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

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

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

and additionally the spectroscopic measurements could be used for origin identification or authentication of the samples. (From Nilsen. [20] conducted a study in which they compared NIR measurements on intact salmon fillet. 1998. An example illustrating the use of NIR spectroscopy for assessing fat content in farmed salmon is given in Figure 9. water. Torry fatmeter. NIR spectroscopy has been used for evaluating the chemical composition of several other fish species as well. [26] showed that NIR spectroscopy could be used to estimate lipid. In this work they applied a fiber optic measurement setup. and protein than those made on intact muscles.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. N. Isaksson et al. Measured Y Elements: Slope: Offset: Correlation: RMSEP: SEP: 21 Bias: 24 78 0.937575 0. as well as on minced salmon muscle. [29] one question of interest was the comparison of different methods for measuring fat content. Wold et al.2. Unpublished data. Spectroscopic readings obtained on the minced samples correlated better with the reference measurements on fat. whether pre-.Basic Composition: Rapid Methodologies ◾ 125 salmon. [28] and Nielsen et al. In both the works of Vogt et al. microwave. applying minced samples for the spectral readings.K.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.985681 0. H..) 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 NIR spectroscopy. The study concluded that NIR is well suited for nondestructive quality evaluation of salmon fillets. and protein content of European sea bass.600067 0. PC): (%fettHS. The fat content of herring has also been assessed by the use of NIR spectroscopy.603947 0. (Y–var. This work also emphasized the impact of the conditional state of the fish when making calibration models. fat. and Sørensen. Transmission spectroscopy was also employed for the analysis of fat and dry matter in capelin [25]. . in. [27]. Xiccato et al. 8) Figure 9. In a research article published in 2004.22] also conducted studies documenting the efficiency of applying NIR spectroscopy in different measurement modes to assess fat and water content in salmon. and dry matter in halibut fillet. and Tuene [24] made use of NIR transmission spectroscopy to assess protein. Nortvedt. [21. or postspawning. water. Silver Spring).. NIR spectroscopy was proven to be a useful tool for the evaluation of basic constituents of different types of tuna.264047 0. Torrissen. In a recent work by Khodabux et al.

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

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

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

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

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

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

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

Iceland). 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.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. This instrument can detect bones and bone fragments down to a diameter of 0. Marel developed an X-ray-based bone detection unit (SensorX) that was commercially available on the market in 2003 [87]. 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. 9. A similar work has been carried out by Hancz et al. instrumental means capable of objective and rapid determination of basic composition are also available.Basic Composition: Rapid Methodologies ◾ 133 Figure 9.5 Detection of pin bones in fish fillets by x-ray imaging using the SensorX instrumentation (Marel. Iceland). authors recommended CT scanning as an online technique for carcass evaluation.5 for an example). [86] showing good results predicting fat content of common carp based on CT scanning. Throughout development all presented techniques have met the requirements .

1986.. Osborne. NIR.. 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. 3. pp. Oslo. Part of the explanation for this could be the cost level of the equipment in question. K.K. nearinfrared spectroscopy and low-field H-1 NMR spectroscopy. 6.I.. Safety and Authenticity. Nilsen. Another issue is the substantial variety and heterogeneity of the material to be analyzed. imaging. VIS/NIR spectroscopy. H. 7. et al. Determination of chemical composition of beef meat by NIRS. T. in Fishery Products: Quality. and x-rays is considerable and. T. for providing the example picture used in Figure 9. 73(4). 339–344. This chapter. Lebensmittelwissenschaft und Technologie. C506–C510.. 200. and Heia. in Near InfraRed Spectroscopy. U.. Eds. In addition. therefore. Thybo. B. and Isakson. Introducing and applying these methods to industrial applications and enabling production of well-documented. 2005. 2000.A. 8. Thyholdt. M. 21(4).G. NMR. Rehbein. Norway. Blanco. 103–111. Ellis Horwood. 70(8). Hildrum. A. 240–250. Iceland) and QMonitor (QVision AS. Differentiation of frozen and unfrozen beef using near-infrared spectroscopy.K. 2009. however. 89–104. H. and Fearn. . J. NIR spectroscopy: A rapid-response analytical tool..4. the technological development exemplified by SensorX (Marel hf. Journal of Food Science. Due to the spread and diversity in fish species and sizes as well as the seasonal difference in bodily composition. T. I. using near infrared spectroscopy. Near Infrared Spectroscopy in Food Analysis. Eds. K. Trends in Analytical Chemistry. References 1. et al. et al. Journal of the Science of Food and Agriculture. 6.134 ◾ Handbook of Seafood and Seafood Products Analysis of simplicity in sample preparation.. Journal of Near Infrared Spectroscopy. However. J. 1992. Uddin. U. the finding of a universal measurement tool to meet with this variety is a challenging task. Pawlinsky. and progress in data processing and analytical tools has facilitated usability and ease of interpretation of measurement results.. Prediction of sensory texture of cooked potatoes using uniaxial compression. P.. K.. 1998. and Oehlenschläger. 1997.J. Nofima Food. 2. Prediction of wheat bread-baking functionality in whole kernels. and Villarroya.. and Tandberg. England. also makes it clear that although proven useful and promising in laboratory-scale trials. Norway) confirms that these techniques may be applied in commercial and industrial high-speed fish processing applications. Harlow. and x-rays are operated at a speed that makes it possible to perform measurements at or in a processing line. quality seafood products will contribute to retaining the good reputation of fish and seafood in the years to come. Acknowledgment The authors would like to thank Dr Jens Petter Wold. 2002. Isaksson. A.. Oxford. T. The price of measurement equipment for NIR. 33(2). and Williams. Reykjavik. p. Naes. 525–532. Longman Scientific & Technical. NMR.. 4.. Wiley-Blackwell Publishing. De Boever.. Non-destructive Visible/NIR Spectroscopy for differentiation of fresh and frozenthawed fish. 5. not easily applicable for small-scale industries as is often the case in the fish processing industry. M. hence allowing for measurements to be performed on large-scale quantities. T.K. 121–128.

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

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

The SEM method observes the surface of the sample.. etc. critical point drying.3). Finally. and staining the ultrathin sections with heavy metal solutions such as lead citrate or uranyl acetate. sudan. as for TEM). At each subdivision there are macroscopic collagenous dividing lines (myocommata). the sample can be observed with all its constituent water. the sample is frozen in liquid nitrogen and then freeze-dried before being coated and observed.1). In Cryo-SEM. infiltration and embedding in resin.2) are primary fi xation with aldehydes such as glutaraldehyde.1–2 mm (Figure 10. light green. The fibers are essentially the same as those of terrestrial animals in terms of the arrangement of the thick and thin filaments. they are mounted in glass slides and stained with different dyes (toluidine blue. secondary fi xation with osmium tetroxide. There are two ways of preparing samples for SEM: chemical fi xation and physical fi xation (Figure 10. backscattered electrons. Many of the endomysia are connected to the perymisium. and so on..) before examination in the LM. 2006). these different signals can be captured by the appropriate detector in each case. They are arranged in concentric circles forming subdivisions of striated muscle (Figure 10. Two types of microscopes use electron beams as their source of illumination: transmission electron microscopes (TEM) and scanning electron microscopes (SEM).5). the sample is frozen in slush nitrogen (Figure 10. red oil. the sample has to be prepared in semithin sections of about 0. The light microscope (LM) is a very versatile tool that works in different applications such as bright field. and observed.0 mm in diameter. 10. polarizing microscopy. other emanations or signals such as x-rays.140 ◾ Handbook of Seafood and Seafood Products Analysis provides a detailed description of the protocols often followed to obtain information about seafood microstructure. dehydration in a series of ethanol dilutions of increasing concentration. ions or molecules can be identified and quantified in situ using specific detectors coupled to the electron microscope. so microanalysis can be carried out by means of x-ray. the samples need to be prepared first. The steps in preparing samples for TEM observation (Figure 10. so there is no need to section it. phase contrast or differential interference contrast (Nomarski). Electron microscopy (EM) allows food structures to be studied at higher magnifications than those used in LM. The muscle cells are short and 0. etched. They are each surrounded by the sarcolemma membrane and by a thin layer of connective tissue (endomysium).02–1.2 Fish Muscle Microstructure Fish muscle consists of myotomes. 2008). In recent years considerable progress has been made in the field of SEM through vitrification techniques. 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. When physical fixation is used. showing alternate arrangements of . Once the semithin sections are obtained. In the former. which is contiguous to the myocommata (Ofstad et al. In both methods. coated. the sample preparation steps are chemical fi xation (with aldehydes and osmium tetroxide. The most useful application for studying seafood structure is bright field microscopy. iodine. cutting ultrathin sections (5–100 nm) in an ultramicrotome. may be generated as a result of the electron beam striking the specimen (Pérez-Munuera et al. in this way. 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. For this. In this way.4) and quickly transferred under vacuum to a cold stage fit on a microscope where the frozen sample is fractured. image analysis relies heavily on computer technology to obtain quantitative results from microscopy observation. Besides the secondary electrons. or fluorescence microscopy.

1% red oil.. LM observation Figure 10. . liquid N2) Preparation for slicing Cold knife CO2 Knife Slice Semithin sections (0.1 Preparation of samples for LM observation..11–2 μm) Cold stub Microtome Cryotome Mounting in glass slides Staining specimen 1% Toluidine blue 1% Lugol.Microstructure ◾ 141 Food Specimen portions Embedding in paraffin or resin Freezing (CO2. .

50%. Araldite Spurr’s. .142 ◾ Handbook of Seafood and Seafood Products Analysis Food Specimen portions Fixation 2.2 Preparation of samples for TEM observation. 70%.5% Glutaraldehyde 2% Os O4 Dehydration Ethanol (30%. 100%) Infiltration and embedding in resine Epoxi resin. 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. 90%.

Pd.Microstructure ◾ 143 Food Specimen portions Fixation Physical fixation Chemical fixation Quick freezing in liquid N2 2. for SEM imaging) (C. . 70. 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.3 Preparation of samples for SEM observation. 50.5% Glutaraldehyde 2% Os O4 Ethanol (30. for X-ray) SEM observation Figure 10. 90.

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

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

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

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

9 Cytoskeletal structure of hake observed by TEM. Sigurgisladottir et al. Physiol.000 X Figure 10. (A and B) Cryo-SEM. 2005.R. (Reprinted from Pagano. B. With permission. longitudinal filament connecting Z-Z. .10B shows a detail of an intercellular space created by the conjunction of three fibers or cells. The micrograph shows geometrically shaped fibers surrounded by a connective tissue.10A. Figure 10.3.1 Smoked Salmon A cross section of a smoked salmon sample obtained using the Cryo-SEM technique is seen in Figure 10. Z-disk. 13.. (2000) used LM to observe the changes that occurred in the salmon during the smoking process and quantified them by image analysis. the higher the smoking temperature. Com.) 10. Z. Biochem. et al. LZ. M. The fiber shrinkage and the space between the fibers both increased to a greater extent in the muscle from the salmon that were frozen before smoking than in muscle smoked from fresh salmon. The data of the average cross-sectional area of muscle fibers showed that the smoking process produces shrinkage of the fibers.148 ◾ Handbook of Seafood and Seafood Products Analysis Z LZ IZ DZ 8.10 Smoked salmon.3 Processed Fish Microstructure 10. the greater the shrinkage. 141. Gudmundsson and (A) 100 μm (B) 10 μm Figure 10.

Figure 10. (A) (B) (C) Figure 10.12 Seafood stick (surimi) observed by SEM. (B) Cryo-SEM. these treatments decreased the cell size compared with fresh salmon. 10.3.11B shows a cross section of salted cod tissue (A) 100 μm (B) 300 μm Figure 10. where two fibers can be observed completely covered by salt deposits. Figure 10.Microstructure ◾ 149 Hafsteinsson (2001) studied the effect of pulsed electric fields (PEF) and a combination of PEF and high pressure on smoked salmon microstructure. (B) cross section.11A shows a longitudinal section of salted cod. (A) longitudinal section.11A) in samples that have been obtained using physical fi xing (freeze-drying) instead of chemical fi xing.2 Salted Cod The presence of salt deposits in the cod tissue can be observed by SEM (Figure 10. and (C) protein network. A combination of PEF and high pressure had a more detrimental effect on smoked salmon microstructure than PEF treatment alone. . (A) SEM. and gaps were formed in the tissue structure.11 Salted cod.

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

13). Tech. 2007. and (D) TEM. s.) ties of surimi. 10. poultry and fish muscle. a central sarcoplasm is shown to be surrounded by myofibrils. Effect of electric field pulses on microstructure of muscle foods and roes. Food Res. myofibril. et al. (C) LM. all the muscle fibers are thin. J. 1990.. and Hafsteinsson. References Gudmundsson. LM makes it possible to distinguish a peripheral area in blue and a central core in white inside each cell. 1990). Muscle Foods. Each fiber is surrounded by a sarcolemma (Llorca et al.14 Squid.. the intermyofibrillar spaces between these can be observed.Microstructure ◾ 151 –200 μm –10 μm (A) 10 μ (B) 3. (From Llorca.4 Squid Microstructure The squid mantle is composed of muscle tissue sandwiched between two tunics of connective tissue (Figure 10. Comparative microstructure of red meat. Muscle fibers are grouped in bands that are arranged orthogonally. Regardless of their orientation. Trends Food Sci. 2001. H. E. respectively. Th is gel network structure gives surimi its characteristic elasticity and texture (Sikorski.5 mm in diameter (Lluch et al. . 247–267. 12. Radial bands (10–15 mm thick) comprise fibers that connect two tunics of connective tissue. (A and B) SEM.14D). Circumferential muscle bands (100–200 mm) comprise fibers running about the entire circumference of the mantle cone. 1. 225. This fiber distribution can also be observed by LM in samples stained with toluidine blue (Figure 10. m. 2001). 122–128. L. M. The inner and outer tunics are covered by a visceral lining and outer lining. Eur. 807. sarcolemma. central sarcoplasm..14C). Lampila.E. Technol. 2001). l. With permission.. approximately 3.75 μ m s (C) (D) l Figure 10. The fibers arranged in circumferential and radial bands were observed by SEM in samples fi xed with glutaraldehyde (Figure 10... 2007). When TEM is used to study the ultrastructure of fresh squid (Figure 10...14A and B) (Llorca et al.

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

..5 The Application of Electronic Noses for Fish Freshness and Quality Measurement .........................................3........... some are of great importance to define overall properties such as freshness or quality [1]......................................................165 11....6 Conclusions .......1 Introduction Among the thousands of molecules composing food complex mixtures..............158 11.......................153 11....157 11...............3.. For these reasons the knowledge of the chemical 153 .......1 Sensors Based on Conductance Changes ...................................... called odor...............159 11.2 Amperometric Gas Sensors ...................... The relationship between chemistry and food properties is particularly interesting in the case of fish and seafood in general... for which the human perception of airborne chemicals.......................................................................................................................................................154 11.......................................................158 11....4 Field-Effect Transistors ................................................................2 Sensor Parameters...................158 11...............................3.4 Electronic Noses ............................. is one of the most used method to assess freshness by both consumers and industries [2]..............5 Color Indicators ........157 11............................................................3.............................................................................................................................157 11.............................................2 Conducting Polymers and Molecular Aggregates ............3 Chemical Sensor Technologies .............1 Introduction ......................1.Chapter 11 Chemical Sensors Corrado Di Natale Contents 11...............................160 11............3....................3.....................................................................1.............164 References ............................................3 Mass Transducers ..................................................1 Metal-Oxide Semiconductors .........3..........................................160 11........................................159 11.................................

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

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

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

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

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

known as computer screen photo assisted technique (CSPT). and screens. and. This last technique. Due to the large diffusion of portable computers. as a sensing part. 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]. H2 molecules dissociate into atomic hydrogen at the palladium surface. cameras.Chemical Sensors ◾ 159 11. The method demonstrated also the possibility to identify a number of different amines [28]. the chemical practice of this approach is badly balanced by the transducer counterpart. Nonetheless. The principle was adequately exploited with a palladium gate FET exposed to hydrogen gas [20]. organic molecular layers. . and cellular phones all endowed with color screen. also appreciable by eye. and hydrogen atoms can diffuse through the palladium film until they reach the oxide surface. PDAs. such as metalloporphyrins [22]. 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. giving rise to a number of low-cost advanced optical equipments such as digital scanners. the colorimetric detection of fish freshness recently received a novel interest. whose sensitivity toward amine was also recently measured [23]. the current flowing in the FET changes revealing the chemical interaction. Compared with the use of digital scanners. Nonetheless. This salt exhibits a rather large change in color.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. The feasibility of this approach has been demonstrated using as sensitive layer a film of a sodium salt (bromocresol green) [25]. 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]. 11. where they form an ordered dipoles layer. was also obtained [21]. In particular. On the other hand. standard optical instrumentations are usually expensive. This difference can be modulated by a layer of electric dipoles that can reach the metal–oxide interface. In this way sensitivity to ammonia. furthermore. although under constant bias.3. is based on the fact that a computer screen can be easily programmed to display millions of colors. an important gas for fish freshness and quality.3. whose characteristics largely fit the requirements necessary to capture change in optical properties of sensitive layers in many practical applications. combining wavelengths in the optical range. As a result. Lundström and Filippini proved that it is possible to assemble a sort of spectrophotometer using the computer screen monitor as a programmable source and a web camera as detector [29]. the use of pH indicators is limited by the fact that mainly amines are considered (limiting the detection not to freshness but rather to spoilage). This basic structure was successively modified changing the gate metal and thickness to extend the range of measured gases. 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. allowing a simultaneous evaluation of absorbance and fluorescence of samples. in the last decade we have seen rapid growth in performance in fields such as consumer electronics. the visual determination limits the performance and may greatly vary between individuals.5 Color Indicators Although known for several years [24]. Furthermore. Indeed. FET structures were also modified to accommodate.

it was proposed that arrays of nonselective chemical sensors may show properties similar to those of natural olfaction [34]. and such systems were soon dubbed as “electronic noses. Nonetheless. observation of Nature offered a useful suggestion about the use of such devices. To this point of view. and each molecule is sensed by many receptors [33]. Among these compounds amines . 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].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. microbial spoilage produces short-chain alcohols and carbonyls. Standard optochemical sensors are based either on absorbance or on fluorescence. The physiology of olfaction has made considerable advances. the possibility of developing artificial olfaction systems became possible. Their concentration and the presence of other compounds are rather typical of each species. and acid compounds. and the genes expressed by olfactive receptors are known [31]. 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]. 11. Receptors were found to be rather unselective. The most important chemicals involved in the fresh fish odor are long-chain alcohols and carbonyls. bromophenols. sulfur compounds. and aromatic. 11.4 Electronic Noses As discussed above.160 ◾ Handbook of Seafood and Seafood Products Analysis camera. amines. After this conjecture. each receptor senses several kinds of molecules. and an even more extended computation capabilities. models of receptor mechanisms explaining the sensitivity to volatile compounds are now available. almost all sensor technologies were used to build such systems. 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. CSPT has demonstrated its utility in particular to classify airborne chemicals reading absorbance and fluorescence changes in chemical dyes such as metalloporphyrins [30]. allowing for electronic nose application oriented optimizations.” This denomination is currently given to any array of unselective chemical sensor coupled with some multicomponent classifier. whereas CSPT arrangement gives the possibility of evaluating at the same time both the effects. The concentrations of these chemicals are directly correlated to the degree of spoilage. the lack of selectivity of many chemical sensors was considered as one of the main problems limiting their diff usion for practical applications. Investigations about olfaction receptors show that Nature strategies for odor recognition are completely different from those of analytical chemistry. On the other side. N-cyclic. and N-cyclic compounds. Since the 1980s. 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]. organic synthetic receptors offer an unlimited number of possibilities to assemble molecules endowed with differentiated sensing features. After this discovery. 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.

in case of fishes. Data are extrapolated from an investigation by Strachan and Nicholson [48]. In order to understand the potential of electronic noses to detect fish freshness.3 the time evolution of the major families of volatile compounds found in the headspace of fishes is shown. The representation plane is determined as that where the data variance is maximized and then the statistical properties of the dataset are. Instruments based on different sensor technologies have been used. Analyzing the data with PCA the plot of Figure 11. As a result. from fish processing. In LSER. Results shown in Figure 11. In this regard.4 demonstrate a continuous progress after the 8th day. amperometric sensors [42]. nonetheless. and fresh.g. Sensor data can be conveniently represented by a principal component analysis (PCA) scores plot..5 is obtained. the interaction between polymers and volatile compounds is often described by the linear sorption energy relationship (LSER) model [51]. Figure 11. and acid. dipolarity. such as metal-oxide chemoresistor sensors [38–40]. 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. the data produced by a sensor array can classify samples according to some of their global features. but the behavior at the beginning is absolutely nonlinear. typically according to the freshness or more precisely according to the balance between fresh and spoilage produced compounds. amines become instrumentally appreciable only when spoilage processes take place. quartz microbalance sensors [44. as much as possible. 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. hydrogen bond basic. it is more realistic to consider an array of sensors specific for a single interaction mechanism. In Figure 11.3. Each sensor then provides a signal proportional to the concentration of each family. and the decrease in others result in a nonlinear problem. This result is rather surprising because fish spoilage is in general expected to be a linear and somewhat straightforward process. let us consider an array specific for each LSER interaction and one compound for family. Most of these are feasibility studies. In addition. a plane. with an array of selective sensors it is not possible to distinguish between fresh and flat fishes. Standard analytical methods for volatile amines and also sensors for some specific amines have been used to inspect fish freshness. petroleum in sea). Nevertheless. Minor contributions to the fish headspace come from contamination of the environment (e. showing the ability of the electronic nose to track the different spoilage levels occurring at different storage times. hybrid electronic noses were used combining different sensor technologies such as QMB and amperometric sensors [47]. the progress of spoilage is less linear with respect to Figure 11.6 . The sensitivity of chemical sensors is not immediately related to the molecular family but rather to the interaction mechanism. Apparently. and optical indicators [46].4. and finally from products of lipid oxidation [37]. and sweet conditions are hardly identified. with a super impression of 6th and 1st days. let us discuss a simulation of a case study. In recent years attempts to use electronic nose technology to track the spoilage processes occurring in fishes have been reported in numerous articles. flat. An imperfect application of this method was demonstrated with engineered polymer-coated QMB [50].45]. Since LSER was fruitfully used to model polymer-based chemical sensors [52]. The same nonlinearity is observed with electronic noses. the chemical complexity of the problem. for example. MOSFET sensors [41]. In gas chromatography. PCA is a data analysis method allowing the representation of a multidimensional dataset in a reduced dimensionality space. preserved [49]. conducting polymer sensors [43]. polarity. When properly analyzed by pattern recognition methods. the accumulation of some compound.Chemical Sensors ◾ 161 are considered as the typical markers for fish freshness detection. Let us consider the use of an array of sensors absolutely selective for each individual family of compounds mentioned in Figure 11.

tactile (to test the flesh firmness and elasticity). sardines [58]. image analyzers. Results are qualitatively similar to those shown in Figure 11. and the use of only one sense (e.57]. 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]. The typical sensorial description is also reported. Nonetheless. humans provide a more reliable identification of fish freshness. shows the scores plot of a partial least-squares discriminant analysis model related to an array of metalloporphyrin-coated QMBs. texture meters. As a consequence. The experiment was related to COD fishes. each able to capture different aspects of fishes. in order to measure the quality of fish instrumentally.3 Time evolution of the major families of volatile compounds in fish headspace. olfaction) provides several errors of evaluation. an integration of instruments is necessary. 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).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. and groupies [59].01 0 5 10 15 Days 20 25 30 35 Figure 11. The possibility of developing a multisensor device to measure and/or estimate fish freshness with a combination of instrumental techniques (electronic noses. color meters. spectroscopic methods. with a folding back of the spoilage process in the representation plane. . and olfaction (to smell the gill odor) [54]. This feature that can be interpreted as a failure of the electronic nose is likely due to an intrinsic nonlinearity of the studied problem.1 0. and devices measuring electrical properties) has been illustrated in different applications related to cods [56.5. and original data were previously published [53]..g.

5 –2 –2.5 –6 4 6 –5 –4 –3 –2 PC 1 (80.39%) Figure 11.5 –6 –4 –2 0 2 4 PC 1 (72.5 PCA scores plot of data related to a virtual array of sensors. . Data show the impossibility of distinguishing the spoilage process in the first 6 days and an abrupt change between 6th and 8th days.4 PCA scores plot of a simulated experiment where sensors selective for the compound family in Figure 11.5 1 0.5 –1 –1. each specific for a single interaction mechanism among those modeled by LSER.5 0 –0. Scores plot 1.93%) 0 –0.03%) –1 0 1 2 30 2 28 22 20 10 10 12 14 16 8 1 Figure 11.5 1 PC 2 (15.3 are used.5 24 PC 2 (19.5 –1 24 10 12 8 28 2 4 6 1 ◾ 163 22 20 18 16 14 –1.76%) 0.Chemical Sensors Scores plot 2 30 1.

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

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

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

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

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

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

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

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

For example.000 nm). The most popular IR spectroscopy is the NIR one. Marquardt and Wold [34] concluded that Raman spectroscopy might be a useful tool for rapid and nondestructive analysis of fish quality. Focusing on fish products. but it is measured in terms of tenths of a millimeter [32] and is dependent on less-precise reference methods [27]. and N–H chemical bonds [27]. the energy at defined frequencies can be partially absorbed. the usefulness of visible spectroscopy/near infrared spectroscopy (VS/NIRS) has been researched for many quality aspects [23–25]. All these techniques have been gradually implemented as monitoring systems in food processing [31]. 12. This technique measures the reflectance of light from the product in the visible and NIR wavelength range. but it involves a scattering process. [28] applied NIR spectroscopy to assess the end point temperature (EPT) of heated fish and shellfish meats. O–H. thus. 12. a multispectral imaging NIR transflectance system was developed for online determination of moisture content in dried salted codfish [29]. [33] applied MIR spectroscopy combined with chemometric tools to determine whether fish has been frozen–thawed. and it is able to provide thermal information. which is also called thermal infrared (TIR) refers to electromagnetic waves with a wavelength of between 3. Uddin et al. 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.Physical Sensors and Techniques ◾ 173 ultrasound to pass through air.000 and 400 cm−1 (2.3. NIR spectroscopy is based on the absorption of electromagnetic radiation at wavelengths in the range 780–2500 nm.5 and 20 micrometers. 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. ultrasound transducers must have airless contact with the sample during examinations [22]. Mid-infrared (MIR) and Raman spectroscopy have high structural selectivity and contain more of the type of information needed in structural elucidation studies. the visible spectrum is a function of the entire structure of the compound rather than specific bonds. Other information should be used in conjunction with visible spectra in determining the specific properties of interest. NIR technology has been widely developed as an analytical tool.3 IR Spectroscopy In the recent years. This makes it very feasible for measurements to be made in organic and biological systems. Thermal infrared imagers translate the energy transmitted in the infrared wavelength into data that can be processed . The far IR. In the fish sector. This complicates the noncontact measurements. but it is not the only one.3. the main disadvantage of this method is that only the surface of the sample is examined.500–25. MIR spectroscopy concerns the region of the spectrum lying between 4. Karoui et al. The region of the electromagnetic spectrum under consideration in Raman spectroscopy is similar to that in MIR.2 Visible Spectroscopy In recent years. When radiation with energy corresponding to the MIR range interacts with a molecule. but their use is limited by their low penetration in the product (it depends on the wave length. Most industrial processes require the measurement of temperature. the freshness of cod was estimated by Heia et al. A rapid. [26] using the visible wavelengths only. NIR spectra of foods comprise broad bands arising from overlapping absorptions corresponding mainly to overtones and combinations of vibration modes involving C–H.

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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

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

Int. G. Quality Standards for Fish: Final Report Phase II. 2004. 1992. developed by Nielsen. matte. Nordic Industrial Fund (in Danish). 37.Methods for Freshness Quality and Deterioration Table 13. seaweedy. D. and Hyldig. With permission.. brown Odor Fresh.. Food Res. 37–59. S.. 975.1 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. 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. .

boiled meat Loss of odor. . hardness is the most important to the consumer. however. “off” flavors. starchy. Scotland. J. J. flounder. hake. boiled potato Milk jug odors. and product development in the seafood industry [38]. With permission. wood sap. Dielectric properties of fish are used for determination of freshness. during processing. 13. TMA Lower fatty acids (e. slight sulfide Score 10 9 8 7 6 5 4 3 Source: Shewan.g. A linear relationship was found between Torrymeter readings and sensory attributes for cod. 1953. These changes occurring at microscopic level are related to alterations in appearance. blue whiting. 4. trace of “off” flavors Slight bitterness..194 ◾ Handbook of Seafood and Seafood Products Analysis Table 13. Food Agric. sour milk.3 Physical Methods 13.2 Torry Score Sheet for Freshness Evaluation of Cooked Cod Fillets Odor Initially weak odor of sweet. Numerous mechanical methods have been used to measure texture. Among textural attributes. rubber. improper handling storage. Fish muscle may become soft or mushy as a result of autolytic degradation or tough as a result of frozen storage [16]. sour. et al. natural odor Wood shavings. metallic.3. quality control.. 13. soapy.2 The Torrymeter The Torry fish freshness meter “Torrymeter” was developed at Torry Research Station in Aberdeen. and flavor during spoilage and have been used as quality indicators since the first commercial version of the Torrymeter in 1970 [43]. boiled milk. and chewiness of food.1 Texture Analysis Texture analyses for seafood are extremely important in research. and cooking [39. followed by strengthening of these odors Shellfish. texture. odor. 283. reminiscent of boiled clothes Lactic acid. tallow Flavor Watery. springiness. Sci. seaweed. Dielectric properties of fish skin and muscle alter in a systematic way during spoilage as tissue components degrade.3.40]. which immediately begin to break down the proteins after the harvesting. turnip.M. Fish muscle has higher levels of indigenous proteases.. 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. Baltic herring. acetic or butyric acids) decomposed grass. starchy. there is little agreement on which is the best method [42]. TMA Strong bitterness. mackerel. deciding the commercial value of the meat [41]. vanillin Condensed milk. Texture includes the most common characteristics such as hardness.

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

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

For the first time.Methods for Freshness Quality and Deterioration ◾ 197 its use is simple. since they eliminate personal opinions on the product quality. The traceability system can also be used for the determination of fish freshness.4.4 Chemical and Biochemical Methods Chemical and biochemical methods for the evaluation of seafood quality are more reliable and accurate. 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]. The most used procedures for the objective measurements of seafood quality are given in the next sections. This process results from breakdown of adenosine triphosphate (ATP). recording the product temperature from the moment of catch. which is the main energy source for metabolic activity. It has been indicated that there is a correlation between nucleotide catabolism and loss of freshness. this technique has been applied to sardine muscle during iced storage. 13. and requires a small amount of sample. cheap.1. In postmortem fish muscle. Currently. This alternative method could be cost effective and definitely more reliable. These objective methods should correlate with sensory quality. 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].1 shown. leading to accumulation of adenosine diphosphate (ADP) and inosine monophosphate (IMP). The sequences of nucleotide catabolism proceed as shown in Figure 13. degradation of ATP proceeds according to the sequence . 13.1 ATP and Its Breakdown Products Rigor mortis occurs in postmortem muscle tissue and is associated with stiffness of muscle or flesh. Nucleotide breakdown reflects both action of autolytic enzymes and bacterial action [16]. sensitive. and the chemical compound that is determined should increase or decrease as microbial spoilage or autolysis progresses [16]. and it has been indicated that this spectroscopic technique is useful in assessing the freshness and quality of sardine during iced storage [84]. Traceability is becoming a method of providing safer food supplies and of connecting producers and consumers. The initial stage of the reaction catalyzed by endogenous enzymes takes place quickly. 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 results showed that measuring the concentration of single nucleotide degradation product to determine freshness quality of seafood is not appropriate. indicating its superiority to Ki value [101].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]. [97] proposed the Ki value. season. although it was observed to decrease during the first 2 or 3 days of iced storage. [102]. stress during capture. body location (dark or white muscle). before its subsequent increase. P. With some species. The K. The IMP is associated with fresh fish flavor. Luong et al. and Gill et al. Karube et al. H values have been described by Luong et al. [101] as an index of freshness quality. Therefore. [100] was found to be superior to Ki value for iced Atlantic cod.13. However. in some species ATP. which excludes ATP. Shahidi et al. The K value proposed by Saito et al. and sea bream [104]. Since adenosine nucleotides are almost converted to IMP within 24 h postmortem [96].95]. Ki. whereas inosine and Hx reflect poor quality [87]. it is difficult to obtain meaningful G and P values since fatty fish deteriorate due to rancidity [103]. Karube et al. and storage conditions [105. The K value includes intermediate breakdown products. Burns et al. ADP. [97]. However. G. the Ki value has been shown to increase very rapidly and then remain constant even though freshness quality continues to decrease greatly [98. [93]. ADP. The H value of iced Pacific cod was observed to increase steadily. 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. Gill et al. P value has been described by Shahidi et al. handling.9. and Fr values are calculated by the procedures described by Saito et al. In addition. [100]. respectively. H. and AMP remain even after 2 weeks [97].106]. but measuring the concentration of ATP and its degradation products can be useful in determining freshness quality [20]. and it varies within species of fish [94. Several methods have been proposed for the analysis of single or a combination of nucleotide catabolites. The rate of nucleotide degradation varies with species. the K value can be superior to the other values. and adenosine monophosphate (AMP). [102] also proposed Fr value for yellow fin tuna. [101].99].88–92]. Determination of G and P values are useful with lean fish. European eel [13]. but the high-performance liquid chromatography (HPLC) method is the most reliable among them. [103]. It was reported that K and related values increased linearly (except Fr value) with storage time in turbot [91]. The concentrations of ATP and its breakdown products have been used as indicators of freshness in many fish species [8. [93] is a biochemical index for fish quality assessment based on nucleotide degradation. [103].

Methods for Freshness Quality and Deterioration ◾ 199 13. their levels are considered as indices of spoilage rather than freshness [112]. spermine. and agmatine.5. since microbial flora vary seasonally [11].and diamine oxidase activity [116]. 13. Since the amines are produced by spoilage bacteria toward the end of shelf life of a fish. and pH [133]. and tyramine. respectively. The most significant biogenic amines produced postmortem in fish and shellfish products are histamine. In addition. including thin-layer chromatography (TLC) [122. and 2-phenylethylamine is derived from phenylalanine. cadaverine. the enzyme responsible for its detoxification. species. There are various analytical techniques used to determine the concentration of biogenic amines. tyrosine produces tyramine.4. HPLC [120.0 to 7.108]. and histamine and decreases in spermine and spermidine during storage of fish. and reproducibility. 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.4. free amino acid content [112]. HPLC is mostly performed because of its sensitivity. Process technology is influenced by rigor development. and use of a biosensor [130–132]. These problems may be more severe in sensitive consumers who have a reduced mono. 2-phenylethylamine. tryptamine. and other factors . 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.129]. reliability. tyramine. By means of decarboxylation reactions.2 Biogenic Amines The concentration of biogenic amines has been reported to be a reliable method of measuring the quality of fish. putrescine. capillary zone electrophoresis (CZE) [128. respectively. putrescine. the presence of decarboxylase-positive microorganisms. [121] for determination of quality of fish. tryptamine from tryptophan. The biogenic amine content of fish depends on fish species.123].109. cadaverine. Putrescine is also an intermediate of a metabolic pathway that leads to spermidine and spermine [119]. Postmortem pH varies from 5. whereas BAI is based on increases in histamine.124. and stomach contents at death. The QI and the biogenic amine index (BAI) were proposed by Mietz and Karmas [120] and Veciana-Nogues et al.110]. histamine is potentially hazardous and the causative agent of histaminic intoxication [114].125]. GC [126.1 depending on season. Among the biogenic amines. The others especially putrescine and cadaverine have been reported to enhance the toxicity of histamine [115].127]. the moment of capture.S. 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. Cadaverine is derived from lysine. Among these techniques.11. cadaverine. Consumption of seafood containing high amounts of these amines can have toxicological effects. 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].3 pH The pH is also an important parameter to show depletion in tissue and quality of flesh during storage.107. depending on the species being examined [10. Biogenic amines are generated by microbial decarboxylation of specific free amino acids in fish or shellfish tissue [111]. spermidine. The formation of biogenic amines results from microbial degradation during the later storage of fish. Food and Drug Administration [117] and the EU [118]. postmortem temperature. and arginine leads to putrescine. histidine yields histamine. and the concentration of these increases with storage time [91.

total volatile basic nitrogen (TVB-N) primarily includes trimethylamine (TMA. Th is is caused by the depletion of energy reserves. However. and direct distillation methods have been recommended as a rapid routine method. However. Low pH is used as an indicator of stress at the time of slaughtering of many animals. The level of TVB-N in freshly caught fish is generally between 5 and 20 mg N/100 g muscle. A relatively low pH may cause a decrease in water binding to the myofibrils. and it has been used as an indicator of marine fish spoilage: CH3 CH3 – N=O CH3 TMAO CH3 CH3 – N CH3 TMA .4 Total Volatile Basic Nitrogen In seafood. It was found that there was a good correlation between three methods. such as frozen eel [145]. The European Commission (Council Regulation No. Therefore. Low initial pH is associated with higher stress before slaughtering [13. TVB-N level correlated with fish quality. mainly glycogen. 13. the levels of 30–35 mg N/100 g muscle are considered the limit of acceptability for icestored cold-water fish [17. it could not be regarded as a good indicator of fish freshness and proved to be better as a spoilage index. as shown in a variety of fish such as European hake [142]. was compared with two routine methods. TMA is produced by the decomposition of TMAO due to bacterial spoilage and enzymatic activity [150. ammonia (produced by deamination of amino acids and nucleotide catabolites).141]. The EC reference method for TVB-N determination. farmed gilthead sea bream [147]. The analyses of these indicators are considered unreliable because they reflect later stages of spoilage rather than freshness [140]. Atlantic cod [143]. it affects reactions taking place during storage of fish. affecting light scattering and the appearance of fish. Since the activity of enzymes depends on pH.4. 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 pH also promotes oxidation of myoglobin and lipids [134]. and DMA (produced by autolytic enzymes during frozen storage).136–139]. whereas the second one includes the use of trichloroacetic acid instead of perchloric acid [149]. Therefore. bacteria act upon TMAO to produce TMA.200 ◾ Handbook of Seafood and Seafood Products Analysis [134. 13. as shown below: Following death of fish. which is considered to be the main cause of off odors in fish products [58.5 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. sardine [12. It is well known that determination of TVB-N differs systematically according to the procedures used.4. produced by spoilage bacteria). Based on the results obtained from the literature.59]. pike perch [146].135].151]. TVB-N should be used as a chemical check. and European eel [13]. turbot [92]. with the production of lactate. the level of TVB-N was not correlated with the time of storage of some fish species. 144]. involving preliminary deproteinization with perchloric acid. The first one includes direct distillation of fish after adding magnesium oxide. and hake [148].

Fresh fish has a very low amount of TMA with values less than 1.4. The amount of DMA produced depends on species (except gadoid species. but it appears after 3 or 4 days. TMA can be used as a spoilage indicator and not as an index of freshness. whereas freshwater fish generally contain only 5–20 mg% [153].6 Dimethylamine As mentioned earlier. Several assays have been described for the determination of TMAOase activity in fish muscle [151. DMA can be used as a spoilage index during frozen storage of some species such as frozen hake [170].7 Formaldehyde The formaldehyde content in seafood products is generally considered as nontoxic. especially in gadoid fish. TMA is not produced in a significant amount during the early stages of chilled storage of fish. resulting in rancidity. The formaldehyde content of frozen seafood is generally used as a spoilage index. which results in a dry and firm texture of the fish muscle [174]. The limiting factor of frozen storage in lean fish species is denaturation of proteins.4. age. this reaction is replaced by a slow conversion by an enzyme to DMA and formaldehyde [16. time of year. the storage temperature. The TMAO content of seafood varies with species. including steam distillation [158]. stage of spoilage. after which the rate of production of TMA parallels the bacterial proliferation pattern [154]. The fish is considered stale when the rate of TMA production is higher than 30 mg/100 g cod [155]. A close relationship has been found between lipid damage and quality of the final product [173]. colorimetric method [160]. 13. The formation of these products may cause severe quality changes or spoilage during prolonged frozen storage. 13. whereas fish can be stored in a frozen state for several months without severe changes in quality.150]. GC method [163. DMA. location of catching. Fresh fish has a limited shelf life and is prone to deterioration. Seawater fish have 1–100 mg TMAO in every 100 g muscular tissue. This reduces the solubility of myofibrillar proteins [172]. semiconducting metal–oxide array [166]. and solid-state sensors based on bromocresol green [169]. other species do not develop adequate amounts of DMA).156. which is converted to TMA by bacteria in iced fish. and environmental factors [152]. fish contain TMAO. and methods employed for analysis. but it can react with a number of chemical compounds such as amino acid residues. biosensor using flavin-containing monooxygenase type-3 [168]. fish size. terminal amino groups. its usefulness depends on time of year. However.157]. and low-molecular weight compounds. Many analytical methods have been developed for the measurements of TMA. when bacterial growth is inhibited.4. or TVB-N contents. Conway microdiff usion and titration [159]. lipid oxidation is the limiting factor in fatty fish species.5 mg TMA/100 g in fresh cod. However. During chilled or frozen storage of fish. 164]. but values increase during spoilage. a capillary electrophoresis method [165]. 13.Methods for Freshness Quality and Deterioration ◾ 201 TMAO appears to be part of the system used for osmoregulation.8 Lipid Oxidation Indicators During processing and storage. HPLC method [162]. type of storage and processing. photometry [161]. flowinjection-gas diff usion method [167]. . and time. enzymatic and nonenzymatic lipid oxidation occurs. causing denaturation and cross-linking of proteins [171].

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

peptides. AV. as oxidation proceeds the PV can start to fall. Shewanella putrefaciens . which break down to secondary products of oxidation or react with proteins. coliforms. 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. or enterococci [195]. 13. TOTOX (2VP + AV). PV. It was indicated that the main requirements for shelf life predictions are to collect information about SSO.188. 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.186]. and lean fish such as blue whiting. It is possible to predict shelf life of seafood based on knowledge of initial numbers and growth of SSO. Mathematics models have been well established for the growth of spoilage bacteria such as Photobacterium phosphoreum. cod [192. [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. Increase in the PV is most useful as an index of the earlier stages of oxidation. causing production of interaction products [187].” or numbers of CFU of indicator organisms such as Enterobacteriaceae.4. and also freshwater fish [194].185].9 Lipid Hydrolysis Hydrolysis leads to hydrolytic rancidity and involves hydrothermal or enzymic (lipase) hydrolysis to FFA and other products. and decline [184. 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).193].Methods for Freshness Quality and Deterioration ◾ 203 are anisidine value (AV). Analysis of these interaction products by fluorescence detection as a quality assessment index for frozen-stored sardine was studied by Aubourg et al. haddock. However. Spoilage of fish and fish products is a result of the production of off odors and flavors mainly caused by bacterial metabolites [197]. spoilage domain such as the range of environmental conditions over which a particular SSO is responsible for spoilage and spoilage level [198]. such as proteins. since they interact with myofibrillar proteins and promote protein aggregation [189]. FFAs and their oxidation products would have an effect on muscle texture and functionality.190. and thiobarbituric acid (TBA). Many methods have been employed for the measurements of lipid oxidation in foods as a means of determining the degree of damage [20.191]. and phospholipids. sardine. Cozzolino et al. PV measures primary products of lipid oxidation. since oxidation products are unstable and react with biological amino constituents.5 Microbiological Methods Numbers and types of microbes present in foods are important indicators of safety and quality. PV. AV and TBA values measure the secondary products of lipid oxidation. [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. During prolonged storage of seafood. free amino acids. 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. reach a peak. and TBA values may increase. European eel. horse mackerel [13. there are some difficulties with common methods when quality has to be assessed. 13. 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].

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

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

........ farmed........................................................7 Trace Element Fingerprint................................. Correct information about the production method of seafood is also important.................. Drug................................................ shape of the head.......1 14.. 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.................. Standards for organic farming are still under development in many countries... The later study showed that the environmentally induced phenotypic divergence increased with age and with the numbers of generations under domestication............8 Other Methods .... and Cosmetic Act and The Fair Packaging and Labeling)............... Several methods have been successfully applied to differentiate farmed from wild seafood.............2 ICP-MS .. size of the eyes and mouth. 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...... larger liver..........216 ◾ Handbook of Seafood and Seafood Products Analysis 14........... In small Salmo salar.............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. of 1999)........ For example... stable isotope analyses combined with fatty acid (FA) profiles have proven particularly useful when tested................................. 228 14......... and sea-ranched S......... and smaller head4 as well as backbone malformations in farmed specimens........2 Also in an earlier study........... salar parr.. The production method is also part of the information essential to fulfill the traceability of a product and... 227 References .. 100% discrimination between farmed (AquaGen strain) and wild parr was achieved by examining the body form........ 226 14............2 Morphological Examination There are few publications and no official guidelines for the morphological differentiation of farmed and wild aquatic organisms.......... as well as examination of the stable isotope and trace element profiles............. but wild specimens may contain parasites harmful to humans..... and these are seldom present in farmed seafood. and the United States (The Federal Food................ therefore.... 227 14.7...........7....... analytical methods should be made available to confirm it..................... analysis of the protein and lipid contents. and length of the pectoral fin.3 it was shown that the morphology of the head...... In particular........................... JAS Law.......... 226 14.. commercially farmed specimens may contain residues of veterinary drugs whose presence is unlikely in wild seafood....... genetic analyses...... fins....................... 227 Acknowledgments ....................... Although in this work no special mention is made to organic farming.......... because farmed and wild organisms carry different hazards and are therefore submitted to different regulations and analytical controls... In cod........ and caudal peduncles could be used for a total correct classification of wild....................... the set of technologies to apply are basically the same as those described here..... including morphological examination................ the most prominent differences are the higher condition factor....1 Sample Preservation .5 The flesh of farmed cod sometimes presents .....................................

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

such as Norway. Arctic charr). and others.14 In addition. tilapia. breeding stock. and bass. etc. must be performed very carefully not to lose the sample. .4 Analysis of Proteins No clear protein marker has been identified to discriminate farmed from wild seafood. has the potential to be useful to differentiate farmed from wild specimens of a given species.21 When using the salt extraction method with heavily degraded samples. 14. Spain. protein markers commonly used for genetic analyses have the potential to be used as markers for farmed or wild. 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. An additional advantage is that it is possible to use robots for many of the steps (DNA preparation. since some alleles are more frequent in one group than in the other. by using a series of SNP and microsatellite polymorphisms by PCR and by oligonucleotide ligation assay (OLA). any marker. DNA analyses may be performed using chips that permit the determination in one fast step of many characteristics simultaneously. or mitochondrial. however. SNPs. whether microsatellites. It is worth mentioning.20. and production method. and also for forensic studies. This method has been successfully used by the authors of this paper (unpublished results) and by Bucklin and Kochert22 with whole individuals of Calanus. the alcohol is allowed to evaporate. China. pH 8. that the Norwegian company GenoMar has patented a method to trace back farmed individual Atlantic salmon.23 In principle.13.27 tissues between farmed and wild salmon and cod have been reported. The outcome of these programs is already producing lists of genetic markers linked to traits of interest. cod.218 ◾ Handbook of Seafood and Seafood Products Analysis pellet is usually washed at least once with 70% ethanol. salmonids (salmon.).2 DNA Markers In recent years.18 and the salt extraction method.17 However. the United States. In these samples the pellet may be practically invisible. cod. In the future. 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. including oysters. several countries. have started programs to map the whole genome of some species.3. Three more methods that have reputedly produced good quality DNA suitable for amplification. On other occasions. we have found it helpful to leave the tubes after the first precipitation of DNA with isopropanol in a freezer at −20°C or at −80°C for a few hours before centrifugation (Marian Martinez de Pancorbo. However. traits. 14. personal communication).19 the Chelex method. Atlantic halibut. which may be used to identify the strains of the farmed individuals that display an increased frequency of the desired traits. differences in the protein pattern of liver25 and muscle26. including the species.11. however. so the next step. which is washing the pellet with 50% ethanol. genomic. sample preparation.24 The method requires that all parent fish of the brood stock are DNA typed as well as all the individuals under examination. shrimp. and the DNA is reconstituted in double-distilled sterile water or in a slightly alkaline buffer (50 mm Tris–EDTA.15. Japan. and sea bass. from food matrices include the use of hexadecyl–trimethyl ammonium bromide (CTAB). catfish. which further increases the amount of samples that can be processed.0). tilapia. trout. India. University of the Basque Country.

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

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

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. in particular when combined with stable isotope composition (see the following paragraph). The FA composition of fish oils is more complex. herring. etc. and ELISA format will permit the routine analyses of many samples. but C22:1 (several isomers) is relatively more abundant in Coho salmon. soybean. linseed. etc. so that the concentrations in flesh were higher than in the diet. The FA profile of vegetable oils (such as corn.42. sand eel. ProteinLynx Global SERVER). the total amount of TGs alone may be used as a criterion to differentiate farmed from wild fish. which uses MS data to identify proteins from primary sequence databases. which FA is more abundant is species dependant. or menhaden. 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. has often given correct classification of farmed and wild specimens. C18:2n6. and C18:3n3 were selectively metabolized.53.25 and reviewed by Granvogl et al. rapeseed. it has been shown that there was selective deposition and retention of C22:6n3.45–48 In addition. and sand eel and C22:6n3 is more abundant (over 10%) in Atlantic and Coho salmon. and it is seldom that only one of them makes more than 25% of the total. and sardines than in capelin. that is.44 Frequently. and often a single FA may account for about 50% of the total FA content in these oils. the final identification and assignment of the spots in the gels must be performed visually by trained personnel. olive.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. in control laboratories. identification of diagnostic spots. very different staining intensities. spot cutters.). palm. In Atlantic salmon for example. 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. however. Development of protein chips will facilitate the simultaneous screening of many targets and samples. for example. due to frequent errors in the automated spot identification procedure (because of imperfect spot separation and identification caused by overlapping of spots.44 and this FA fingerprint has often been successfully used49–52 as a diagnostic to identify the production method.Analytical Methods to Differentiate Farmed from Wild Seafood ◾ 221 usually performed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS. herring. This is probably the most time consuming step of the whole procedure.). C16:0 and C18:1n9 are relatively abundant in all fish oils. lateral flow strip tests permit in situ easy and fast screening of seafood samples. there are more FAs present in detectable amounts. The proteins are afterward identified by searching in databases (National Centre for Biotechnology Information. because farmed fish usually have a much higher content than wild ones.55–58 Specific FAs are selectively retained or used. For example. capelin. Proteomic analysis is a complicated procedure necessary to identify the biological markers. 14.60 The FAs C18:1n9 and 18:2n6 may act as markers . However. whereas C18:1n9. cottonseed. the FA profile of TGs reflects that of the feed. As in vegetable oils. NCBI) using suitable software (MASCOT.5 Analysis of the Lipid Content The analysis of the triglyceride (TG) fraction.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. Once the diagnostic proteins are identified. C22:1n11. The whole procedure is described in detail by Martin et al.

oxidation.222 ◾ Handbook of Seafood and Seafood Products Analysis for vegetable oils. 31P. because it provides multicomponent information and can be applied nondestructively. which. and polymerization.64 may contribute to the difficulty of performing correct classifications as wild/ farmed based only on the FA composition.5. 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.63. since in a one-dimensional spectrum each peak is produced by those nuclei placed in an identical local chemical environment. 35Cl. 15N. The spectrum is often used to obtain information about the number and type of molecules in a mixture.. both of which are able to detect a range of metabolites in a nontargeted way. 2H. NMR spectroscopy can be used to identify functional groups.1 Sample Preservation Due to the high levels of PUFAs. For detailed descriptions of the analysis of fish samples.and light-tight containers and stored at low temperatures. 14. −80°C. The most commonly used HR-NMR techniques in wild/farmed classification are 1H NMR and 13C NMR. 10B. On the other hand.50. which may be used as a rapid profiling technique. HR-NMR has been particularly valuable in the study of marine lipids.52 that give detailed descriptions of the procedure. 13C. 14. 195Pt). including fish and fish products. 17O. and is the preferred tool in lipid analysis when interpretation of .61 As indicated by Refsgaard et al. the ratio n3/n6 was not fully restored.65 NMR gives a fingerprint of the sample analyzed. 23Na. The major advantage of 1H NMR spectroscopy compared with 13C NMR is the higher sensitivity and thereby shorter acquisition times per experiment.5.52. it is best to use as low a temperature as possible.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.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.. that is. which leads to less overlapping of signals. 29Si.g. 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. the reader is directed to several publications46. Fresh samples should be kept wrapped in air. 19F. and in particular. but NMR is applicable to any nucleus possessing spin (e. 11B. 13C NMR has a greater range of chemical shifts. The most commonly measured nucleus is 1H (the most receptive isotope at natural abundance). 14. 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. and an inert atmosphere. 14N. and even after the levels of C20:5n3 and C22:6n3 were restored to the original high levels.2 Lipid Extraction and Gas Chromatography Procedures for lipid extraction are described in another book chapter of this series. the latter seems to be the most persistent after a dietary switch to fish oil diet. If freezing is required.5.

.75 and it is important that all the samples contain the same volume. 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. Standardized procedures should be followed to ensure repeatability and comparability. a semiquantitative 13C NMR approach has been chosen. due to the fact that quantitative measurements require a considerable longer experimental time. ideally for screening many samples with short acquisition time. 99.71 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). although this approach has still not been widely used for authentication purposes. in conjunction with chemometrics. 1H NMR has also been applied to differentiate between wild and farmed salmon and sea bream of different origins.66 Potential problems about inconsistencies in ppm values between samples in the data analyses should be solved by manual alignment or data pretreatment methods. but it is not necessary for classification purposes.e. leaving the sample ready for analysis. inhomogeneities in the applied magnetic field. will increase the sample throughput significantly.66. Typically. When full spectra are used. pH.75 Small differences in experimental conditions. the relative intensities for corresponding signals across different spectra are comparable. a sample size of 50–100 mg of lipid in 0. hydrogen bondings.70. or differences in relative concentrations of the samples analyzed. may lead to erroneous classification.77 It is advisable to check that all spectra have acceptable linewidth and lineshape after the NMR analysis. However. It is expected that in the future the use of flow injection systems.8 mL solvent is used.76 In some studies. Factors that affect the exact chemical shift of NMR signals include the type of solvent used. Normally.Analytical Methods to Differentiate Farmed from Wild Seafood ◾ 223 spectra is the goal.77 Regarding reproducibility issues. Typically. temperature variations.5–0.71 13C NMR gives information about FA composition of fish72 and the positional distribution of PUFAs in triacylglycerols and phospholipids.78 .0 ppm for 13C NMR. which is easily evaporated. 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. Regions without signals or unwanted signals are removed before multivariate analysis. The assignment of spectral resonances gives information about the chemical composition of the samples. Both HR 1H and 13C NMR. Both the area/ intensities of peaks and full spectra can be input for multivariate analysis.68. although the optimal sample size depends on the instrument. even though the signal intensities within each spectrum are not quantitative. have allowed the differentiation between wild and farmed salmon74 and cod51 of different origins.67 1H NMR has been used to perform quantitative measurements of total n-3 FAs and of the levels of DHA. The most commonly used solvent in the analysis of neutral lipids is deuterated chloroform (i.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. and other intermolecular interactions. they are normally converted to ASCII or JCAMP file formats. interactions with metal ions. the chemical shift scale is referred to the shift of TMS or indirectly to TMS by the peaks from chloroform at 7. such as instabilities in apparatus.28 ppm for 1H NMR and by the triplet of CDCl3 at 77.8% CDCl3). Tetramethylsilane (TMS) is usually added as a chemical shift and intensity reference. phasing and baseline correction are applied but no zero fi lling. because it may interfere with the multivariate data analysis. Multivariate methods are frequently applied to study differences among NMR spectra. The application of multivariate statistics to NMR spectral data increases the potential of the technique considerably.

such as maize. and O2. plus the kinetic fractionations occurring in animal metabolism. the higher the proportion of the heavier isotope. equilibrium reactions also lead to a fractionation of the isotopic forms. the 13C/12C ratio for both milk fat and cheese protein give information on the type of forage fed to the cows. SD ± 0. Some atmospheric gases.11%). since the physical properties of molecules containing heavier isotopic forms are different.6 Stable Isotopes The variation in the abundance of the stable isotopes of carbon. and they reflect both significant isotopic fractionation by microbes and the different biological substrates producing these gases. and 18O (0. .52 were able to classify correctly according to the production method. Bell et al. In contrast. Using the d15N of choline and the d18O of total oil.037%). Aursand et al.63) and 15N (0. For example. Introducing the percentage of C18:2n6 as a third variable in their model. Samples of muscle tissue of wild salmon were significantly more enriched in nitrogen (d15N: mean = 12. a kinetic fractionation occurs. C4 plants may have d13C mean values of −12/−14‰. N2.51. 17O (0. For example. 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.75. SD ± 0. and oxygen has been proposed as a method suitable for food authentication.224 ◾ Handbook of Seafood and Seafood Products Analysis 14. C16:1n-9. The isotopic abundances in animal tissues and animal food products are the summation of the feeds ingested throughout all their life. while typical d13C mean values of C3 plants may be −26/−28‰. they were also able to correctly classify the fish according to their geographic origin. typically tropical grasses.204%). the abundance of stable isotopes varies among different compounds.37%).759%).81 This is because the 13C/12C ratio depends almost exclusively on the photosynthetic mechanism used by the plants for CO2 fi xation. In addition. and oxygen as three: 16O (99. although many broadleaf plants are also C4). the abundances of the stable isotopes differ between substrate and product. such as CO2. 171 Atlantic salmon specimens originating from three continents and 15 different geographic regions. depending on their diet and their position in the trophic chain: the higher its position in the trophic chain. Moreover. nitrogen as two: 14N (99.23‰) than the aquaculture specimens.38‰) but depleted in lipid-corrected carbon (d13C: mean = −20. d13C of individual FA. Differences in the 15N/14N ratio also result essentially from diet. because the enzymatic reaction rates on substrates that contain the lighter isotopic forms are faster than in reactions involving the heavier isotopic forms.80 The natural isotopic abundance largely varies depending on the chemical forms.50 were also able to correctly classify Atlantic salmon according to their geographic origin and production method by using four FA compositions (C16:0. nitrogen.79 Carbon exists as two stable isotopes: 12C (abundance 98. Thomas et al. Usually animal products become enriched in the heavier isotope (15N and 13C). Thus. N2O and CH4 exhibit wide isotopic variation. 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 CO2.89%) and 13C (1.82 Dempson and Power83 examined the potential of using stable isotopes of carbon and nitrogen 13C and d15N) by isotope ratio mass spectrometry (IRMS) to identify escaped farmed Atlantic (d salmon. and 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). C18:1n-9. resulting in a complete separation of the two groups.46 were equally successful classifying sea bass using the FA profile. exhibit limited variation. Since a molecule containing heavier isotopic forms has stronger chemical bonds.

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

Taiwan. Biochemical analytical techniques using multiple elemental analysis. The sample may be stored in a centrifuge tube at a temperature of −40°C or lower until analyzed. false labeling problems were encountered in which imported live Japanese eels from Taiwan were illegally sold as being of Japanese origin. cadmium and arsenic levels in the muscles of clams from China and Korea were higher than those of clams from Japan. handling.5 M nitric acid and rinsed with Milli-Q ultrapure deionized water. All implements and containers should be cleaned with 0.91 In the case of fish. in particular since the analysis may detect contaminants at the pM level. The same research group (Yamashita et al. Each sample should be separated from the tissues using ceramic knives and scissors and Teflon-coated tweezers to avoid contamination of metals. copper. with the exception that clams from Miyagi had high arsenic content. and China. farmed and wild specimens of the same species have different geographic distributions.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. Rare trace elements taken from the environment. lead. Carbonate rock from the Pee Dee Belemnite formation89 and nitrogen gas in the atmosphere90 are used as the standards for carbon and nitrogen. Recently. Therefore. have been used to differentiate the geographical distribution of origin of farmed Japanese eel. cadmium. respectively.92–94 Otolith chemistry is useful for identifying the natal origin and assessing the relative contribution of different nursery areas to mixed adult stocks. 14. The origins of farmed and wild eel collected from different regions in Japan. such as uranium. Multivariate analysis showed that differences in elemental composition in the muscle between Japanese and imported clams were mainly due to two factors: Factor 1. and it must be accurately weighed with a microbalance.1 Sample Preservation As for stable isotope analysis. The . attributable to manganese and vanadium levels. Thirteen elements were shown to be the most diagnostic. and often the geographic origin of both farmed and wild seafood may be of relevance for its safety. otolith chemistry is used as a recorder of time and environmental conditions.95 By using ICP-MS analysis the sensitivity in the determination of rare trace elements can be increased from the nM to pM level. multivariate trace elemental analysis is expected to be helpful in determining whether the fish was farmed and its geographic distribution. and they found distinct patterns for each of the three origins. were shown to be of relevance to determine the origin of eels. Korea.7. In addition. 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. quality. multiple elemental analysis could also be used in this case to identify imported clams from China and Korea. Multivariate trace elemental analysis is increasingly used as a technique to differentiate the geographic origins of foodstuff.. Thus. and analysis. as well as vitamin K and its metabolites. and vanadium. 14.226 ◾ Handbook of Seafood and Seafood Products Analysis set at 0‰ by convention. and price. attributable to cobalt. it is very important to avoid contaminating the sample during sampling. in addition to DNA-based species identification techniques. and strontium levels and Factor 3. The first step in the analysis is the digestion of the sample: 0. unpublished data) examined the trace element composition of the muscle and shell of littleneck clams collected in Japan.7 Trace Element Fingerprint Sometimes.

a calibration blank and calibration standards are used as surrogate test samples after every 10 analyses. the total mercury concentration is determined by cold vapor atomic absorption spectrometry. the mass calibration and resolution are checked using diluted metal solutions as standards. For the determination of mercury. most artificial feeds contain a mixture of canthaxanthin and astaxanthins of different origins (both natural and synthetic). In. 14. If the measured concentration deviates from the true concentration by more than 10%. 14. Tb. Analysis by chiral chromatography can be used to identify a chiral form (the meso form 3R.97 using an automatic mercury analyzer (Hiranuma HG-200. To initiate the proper operating configuration of the instrument and data system. 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. the instrument is recalibrated. Canada). much of the astaxanthin used in fish feed nowadays is produced from cultured microalgae or from krill.96 Samples digested as described above are introduced by pneumatic nebulization into a radio frequency plasma. and the resulting digest is a clear liquid with a yellow tint. Multiwave 3000 Microwave Oven. where energy transfer processes cause desolvation.7. To verify that the instrument is properly calibrated on a continuous basis. and stored at room temperature until use. Each solution (5 mL) of the microwave samples is applied to the atomic absorption spectrophotometer. the Norwegian Research . Perkin-Elmer). Afterwards. and the last 10 samples are analyzed again. and ionization. In the farming of salmon for example. an internal standard mixture is added.8 Other Methods Depending on the species. the use of carotenoids is allowed. and Bi. five internal standards are used: Sc. atomization.98 making this approach more unreliable than it used to be. For internal standardization. the samples are diluted to a final volume of 50 mL in Digitube (SCP Science.2 ICP-MS Multielement determination of trace elements is usually measured by inductively coupled plasma mass spectroscopy (ICP-MS). Japan).3′S) that does not occur naturally and can therefore be used as a marker for farmed salmon.98 However. 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. Y. and the ion information is processed by a data handling system. Ions transmitted through the quadrupole are detected by continuous dynode electron multiplier assembly. but although the diet of wild salmon contains astaxanthin. which suffers from severe memory effects.Analytical Methods to Differentiate Farmed from Wild Seafood ◾ 227 digestion may be carried out by placing the tubes in a microwave oven (for example. there might be specific requirements that may be targeted to identify the production method.42.

20. 343. 15.M. 69. J. 2007. B. 261.F. Gadus morhua L. 137. G... and Nakamura. 15. 1995.. Digestive enzyme activities in starved pre-slaughter farmed and wild-captured.J.. 2005. D. et al. J. M. Aquaculture.P. Aquaculture. Fevolden. Aquaculture. 7. 2003. 48. 209. Genetic improvement of fanned tilapias: Composition and genetic parameters of a synthetic base population of Oreochromis niloticus for selective breeding. References 1. Dahle. 2005. 3. Microsatellite DNA variation in wild populations and farmed strains of turbot from Ireland and Norway: A preliminary study. J. and Gjerde. 4.R. Blood vessel melanosis: A physiological detoxification mechanism in Atlantic cod (Gadus morhua). 701. 1994. 4692. Ø. 148. S.Ø. 2004.K. 10. Genetic characteristics of broodstock collected from four Norwegian coastal cod (Gadus morhua) populations.. Coughlan. Aquaculture. S. Aljanabi.. Chapela. and Fjalestad.. 240. and Martinez. C. 52. Fish Biol. Skaala. 67.. Paralichthys olivaceus (Temminck and Schlegel) using microsatellite markers. Atlantic cod (Gadus morhua). 2007.. 6. A.W.. S. Sci.D. 51. Kang. Gildberg. Sci. Chromatogr. Dykes. I. Molkentin. Sonesson.. 9.. M. Phenotypic divergence of sea-ranched. 118. 1988. et al. Ø.. 131. Fleming. 1466. Can. J. 21. J. Genetic differences between five major domesticated strains of Atlantic salmon and wild salmon. 16... 25. Eur. Doyle. Friars. 1. et al. Aquaculture. 37. and Kjønes. Aquaculture. H. Aquat.. 2006.. 556. Evaluation of three methods for effective extraction of DNA from human hair. 12. H. J.W. et al. Ø. 67. et al. A combined salt and confinement stress enhances mortality in rainbow trout (Oncorhynchus mykiss) selected for high stress responsiveness. Roed.A.. 13. Fish Biol. . 16. 2007.. and Midling K. A simple salting put procedure fore extracting DNA from human nucleated cells. Sci. 137... J. Microsatellite analysis in domesticated and wild Atlantic salmon (Salmo salar L. O.K.H. Food Res.A. 2006. Fish Biol. Forestry and Fisheries of Japan. G. J. Int.. 238. Bailey. and by grants from the Fisheries Research Agency and the Ministry of Agriculture. 250. 1988. 535. 5. 1991. 2004. Comparison of DNA extraction methods from muscle of canned tuna for species identification. 2006. 2006. ICES J. et al. J. 216. NAR. K. 1215. Suenaga. 273. Selective diversification of aquaculture stocks: A proposal for economically sustainable genetic stock conservation. Aquacult. Universal and rapid salt-extraction of high quality genomic DNA for PCR-based techniques. Mar.K. 1997. A. Technol. and Polesky. Fish.. Inter. Aquat. 2. Jonsson. M. et al. Applications of selection for multiple traits in cagereared Atlantic salmon (Salmo salar).. 2006. 916. et al. B. Solem.. Genetic relationship between broodstocks of olive flounder. R. 19. 70. Res. NAR. Miller. K. farmed and wild salmon. 18. with particular reference to growth under farmed condition. E.). Pampoulie.E. K.. A.H. Fish.. I. 1211. Can. J. A review of the physiological and nutritional energetics of cod. 17. 14.. Aquacult.J. 1998. Berg. et al. B. Jobling. A... Aquaculture.and intra-population morphological differences between wild and farmed Atlantic salmon juveniles. Hayes.. Taggart. M. Cooper. 2808.228 ◾ Handbook of Seafood and Seafood Products Analysis Council. Skaala.): Allelic diversity and identification of individuals.E... Identification of organically farmed Atlantic salmon by analysis of stable isotopes and fatty acids.. F. Aquaculture. 11. and Gunnes.M. SINTEF Fisheries and Aquaculture..B. B. 70. 8. 2005. Evaluation of three strategies using DNA markers for traceability in aquaculture species. 63. Eknath. 1. J. and O’Flynn.. Genetic comparison of experimental farmed strains and wild Icelandic populations of Atlantic cod (Gadus morhua L. 820. 213. and Gross. Food Control. 224. 43. 18.

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Aursand. 58. 49. Agric. Australia.230 ◾ Handbook of Seafood and Seafood Products Analysis 42.. 5934.. Are modifications in tissue fatty acid profiles following a change in diet the results of dilution? Test of a simple dilution model. Grigorakis. 989. Froyland. U.. Aquaculture. 52. Mooney.... Seasonal variations in chemical and sensory characteristics of farmed and wild Atlantic halibut (Hippoglossus hippoglossus). in Fishery Products: Quality. 2002.. H.S. et al. 2002.. London. Gunstone. J. I. Polyunsaturated fatty acid composition of the salmon (Salmo salar L. G. I. Aquaculture. Chapman & Hall. Safety and Authenticity. Nutritional Value of Australian Seafood II. M... J. et al. 2008. High-resolution C-13 nuclear magnetic resonance spectroscopy pattern recognition of fish oil capsules. 2003. 218.. 47. Food Chem. J.. and Martin. D. Aquacult. Food Chem. F. 515.B. et al.. Aursand. muscle lipid composition. 55. 541. and Padley. J. Biochim... J. et al. 1996. ISBN 1-876-996-07-2. J. Rapeseed oil as an alternative to marine fish oil in diets of post-smolt Atlantic salmon (Salmo salar): Changes in flesh fatty acid composition and effectiveness of subsequent fish oil “wash out. J. J. 289. G.. Food Chem. 77. 56. Tasmania. 19.B. Martinez.T. 50. Bell. K.. Bell.. 44. 46. Quality. et al.G. 2003. and Elliot. I. Torstensen.. p. I. Harwood. Biophys. and Rehbein. 2000. 175. Martinez. CSIRO Marine Research and FRDC. 2nd ed. and Axelson.. Compositional and organoleptic quality of farmed and wild gilthead sea bream (Sparus aurata) and sea bass (Dicentrarchus labrax) and factors affecting it: A review. heart histology and standard oxygen consumption of Atlantic salmon (Salmo salar) raised at two temperatures. 207. JAOCS.. Nutr. F. 2009.B.G. Olsson. Aquaculture. J. 217. 105. Replacing dietary fish oil with increasing levels of rapeseed oil and olive oil—Effects on Atlantic salmon (Salmo salar L. JAOCS. 10.. 51. I. and Park. 388.) pineal organ: Modification by diet and effect on prostaglandin production.. 54.. P.J. 198pp.G.. Nutr. J. feed utilization. Gunstone. Eds. 123. B. Influence of dietary blends of menhaden oil and canola oil on growth. 52 p. Wageningen Academic Publishers. Standal. 45. 631. 55.” Aquaculture. Ø.) tissue and lipoprotein lipid composition and lipogenic enzyme activities. 1998. 85.E. et al.. tissue fatty acid composition.3. I.L..B. M.G. and thyroidal status of Atlantic salmon (Salmo salar) in sea water. Authenticity assessment based on other principles: Analysis of lipids. 60. 1994..D. Biochem.. 2004.. 191..B. 2007. Discrimination of cod liver oil according to wild/farmed and geographical origins by GC and 13C NMR. the Netherlands. M. J. et al. R. 48. 43. et al. F. 2006. J. Jobling. 2007... Hobart. Standal.D. Determination of origin of Atlantic salmon (Salmo salar): The use of multiprobe and multielement isotopic analyses in combination with fatty acid composition to assess wild or farmed origin.D. 59. 2007.. p. 232. Differentiation of cultured and wild sturgeon based on fatty acid composition.K. 55. Blackwell Publishing. and Lie. stable isotopes and trace elements. B.. 1299. et al. Grisdale-Helland. B. 60. Replacement of fish oil with rapeseed oil in diets of Atlantic salmon (Salmo salar) affects tissue lipid compositions and hepatocyte fatty acid metabolism. N. 2004.. 2001. 272. Martinez.. Safety and Processing of Wild and Farmed Fish. Oehlenschläger.E. 55.8. B. 1535.. Discrimination of wild and cultured European sea bass (Dicentrarchus labrax) using chemical and isotopic analyses. 311. 551. Aquaculture. 38. Influence of high contents of dietary soybean oil on growth. 61. L.. SEAFOODplus report 6. F. The Lipid Handbook. . ISBN 978-87-7075-001-1. Mabon. Nichols.. 1995. Characterization of farmed and wild salmon (Salmo salar). 131. Agric. Agric.. Chen. F. 659.. Acta. 2008.. Bell.. 56. Eds. Food Sci..G. et al. M. Luten. Revision of analytical methodologies to verify the production method of fish. Bell. in Seafood from Fish to Dish. 2006. Dosanjh. Fish Physiol. Eds... Evaluation of the profile of lipids as a tool to discriminate wild from farmed salmon.. Thomas. 57. 53. Henderson.J.D..

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

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

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

They are used when intermediate product dispersion is required as in brine. They are especially used when the final water rate in the treated food must be low.2.236 ◾ 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. or smoke by-products) can be obtained after different steps of filtration. This form of SF is especially used for the smoky taste that it confers to the food. or drying/blending of these PP. Aqueous flavors. They are employed to confer smoky organoleptic qualities (tastes.2 SFs from primary products. These products have a pH greater than 4 and can also be added to the brine.2. due to their low pH.1 Liquid Smokes Different kinds of liquid smokes are available: aqueous flavors. Different SFs (liquid smokes. Therefore. concentrates of liquid smokes. Concentrates of liquid smokes consist of concentrated versions of aqueous flavors and require lower usage quantities. . They are presented in Figure 15. 15. smoke oils. The water-soluble phase leads to primary smoke condensates (PSC). buffered aqueous flavors are partially neutralized or buffered aqueous flavors. the furnace because they cannot be used for human consumption. flavors) and also the characteristic aspect and color of smoked food. and buffered aqueous flavors. However. separation. smoke powders. Soluble aqueous flavors are aqueous flavors that contain an emulsifier such as polysorbates. allowing better water solubility. a purified extract of the high-density water insoluble tar phase can be used for the production of SFs and is called primary tar fraction (PTF). Finally. can be used directly or diluted for applications requiring lower concentrations [4]. smoke condensates obtained from PTF and PSC are named primary smoke products (PP). soluble 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. smoke powders are mainly used to confer smoky tastes to the final product. 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. hundreds of smoke by-products are available. Consequently. most frequently in 90:10 (v/v) proportions. Smoke distillates are obtained by the fractionating distillation of PP. 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. smoke oils can be only used in preparations such as taramas. As seafood emulsions are not very common. Dry salting (or dry curing if nitrited salts are used) is made with dry salt deposited directly on food. smoke manufacturers can control their products and can create smoke by-products whose uses are recommended for a kind of fish. consequent to the reaction between amino acids and nitrite. They are less acidic than aqueous flavors and allow to exhibit more complex smoky tastes. these SFs are not used much in seafood industry. Wet salting (or curing if nitrited salts are used) is made with brines spread on food or in which the food is dipped. These molecules can increase the generation of carcinogenic nitrosamines. Today. 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. salmon. Therefore. These smoke powders can be added to salt used for salting steps or to dehydrated sauces or soups elaborated from seafood products.4 Smoke By-Products Smoke by-products are constituted by smoke extracts and smoke distillates [6].2. Indeed. However. SFs for herring. in seafood industry. . The distillation is commonly performed with steam water at atmospheric pressure. Smoke powders can also be rehydrated and used in brine as liquid smokes. Consequently.2 Smoke Oils Smoke oils are made by blending liquid smokes with vegetable oils. are present in the market. Therefore. As smoke oils. because smoke oils are especially employed in food preparations such as emulsions. generally forbidden in seafood industry according to the countries.Smoke Flavoring Technology in Seafood ◾ 237 15. or fish oils. their uses are really characteristic of a product and cannot be employed for a wide variety of food due to their typical organoleptic qualities. and so forth. 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. the organoleptic qualities can vary in a high range changing the food matrix. Smoke by-products constitute more complex SFs. 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. the liquid smokes and smoke powders can be added to salt or in brine but not smoke oils. Therefore. A reaction between the phenolic compounds of SFs and these nitrited salts or powders can lead to a nitrosation and to nitrophenols.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]. 15. We must distinguish dry salting and wet salting. 15. 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.2.2. fish sauces. Nitrited salts.

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

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

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

phenolic compounds are not sufficient to explain the SF role in smoked fish odors. taste. and methylsyringol has a pure and characteristic smoky flavor [10]. which gather furannic and enolone derivatives. Phenolic compounds of medium volatility have been considered as the most important odorant molecules.1) [14. the diversity of SF causes diverse consequences on the texture. Table 15. The medium-boiling fraction (91°C–132°C) composed of isoeugenols. Similarly.6 Organoleptic Roles of Volatile Compounds of SF 15. Many studies have indicated that phenolic compounds present in the vapor phase of smoke may be odor-active compounds [30–32].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] . They are the major compounds in SF with a wide range of odorant thresholds (Table 15.6.34] (Table 15. These observations have been recently corrected [14. The role of syringol is important. Some of them have very low odorant thresholds. color.29]. syringol. A much more complex mixture of compounds is responsible for the characteristic aroma of smoked fishes [35]. and preservation of the product. and alkylguaiacol may also contribute to imparting a smoky flavor to smoked fishes [13.1 Role of Volatile Compounds of SF in the Odor Even if the concentrations of odorant volatile compounds in SF can be various.33. guaiacol.2). odor. Phenolic compounds of low-boiling fraction (60°C–90°C) composed mainly of phenol.Smoke Flavoring Technology in Seafood ◾ 241 chemical composition. Phenolic compounds are known to constitute the odorant “smoky” skeleton of wood smoke and smoked fish.28. two main classes of odor-impact molecules can be defined: phenolic compounds and carbonyl compounds. making them odorant at low concentrations.34]. but it may not be the main contributor to wood smoke flavor. 15. cresols.

55 ± 9. STD MS.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. LRI. LRI MS. Animal.3-Dimethyl-2cyclopentenone 1052 o-Cresol 1068 p-Cresol 1093 Guaiacol 1110 2. LRI. LRI. LRI. wood fire. STD MS.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 2.04 7 4 16. earthy. milk Cooked/soup. LRI. LRI. STD MS.90 (1.25 6 (5) 6 7 (4) 7 7 6 8 8 8 Chemical. green Cooked vegetable.53) 8. STD Cooked.64 ± 18. roasty Chemical. metallic.34 (24. STD MS. burnt Smoked. mushroom Cooked. ink Marine. green Cooked vegetable.45 ± 172. fatty Roasty. green.27 ± 2.44 17. vanilla.75) MS. LRI MS.97 23.24 ± 63. chemical Green. STD MS.12 42.6-Dimethylpyridine 890 2.48 ± 8.27 ± 0. LRI MS. chemical. STD MS.94 49. STD MS. spicy/ woody (3) (2) 5 6 (2) 4 4 3 5 6 7 (2) 4 4.37 ± 3. LRI. milk Smoke. spicy. LRI MS.74 ± 27. STD MS. LRI.17 ± 26. STD MS.50) 65. LRI.33 ± 1.53 360. 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. LRI.07 (1.6-Dimethylphenol 1130 . burnt. spicy Spicy.22 ± 9.63 ± 13. LRI. spicy. burnt.55 ± 39.Table 15.98 20. potato Cooked potato.18 ± 37.62 74. STD MS.

LRI.25 ± 10.3-Dimethoxytoluene 1247 (E)-2-Decenal 1266 3. green.17 15. STD MS.87 ± 1.16 ± 5.24 ± 1. violet.82 ± 6. LRI. clove Green. LRI MS.26 ± 1. STD MS.51 ± 18. medicinal 7 4 10.21 ± 7. 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. smoke.15) (continued) 2. LRI. STD MS.15 ± 1. LRI.35 3-Ethyl-2-hydroxy-2cyclopentenone 1140 1. LRI MS.3.4Trimethylcyclopenten-1one MS MS. LRI.13 6.79 (6.85 ± 40.4. rotten. clove Sawdust. chemical Green.12 4. LRI.50 18. green. vanilla. LRI MS. milk Burnt. spicy. green Spicy. STD Cucumber. fatty. smoked Candy. spicy. spicy Oily. fatty Burnt rubber. LRI.3-Trimethoxy-5methylbenzene 243 . LRI 1160–1180 4-Methylguaiacol 1192 482.2.97 2. spicy 6 5 17.4-Decadienal 1330 Syringol 1365 Eugenol 1370 Smoke Flavoring Technology in Seafood 4-Propylguaiacol 1382 1400 ◾ 1. green.2-Dimethoxybenzene 1147 2. spicy Spicy. STD MS.61 ± 22. LRI MS.15 ± 243. STD MS. smoked Cooked vegetable.95) 8.5Dimethylphenol/ (E)-2-nonenal MS. burnt Smoke. spicy. green.5-Dimethoxytoluene 1282 4-Ethylguaiacol 1287 Indanone 1307 4-Vinylguaiacol 1330 (E.46 Solvent.36 1132 MS Cooked. LRI MS. vanilla Cooked. LRI. STD MS. STD MS. green.25 ± 4.72 44.86 (2.96 ± 10.62 ± 4. green 6 3 11.2. smoke.E)-2.91 36.15) 86.71 (3.49 ± 6.and 2.

5-Trimethoxytoluene 1527 4-Allylsyringol 1615 8-Heptadecene 1680 Source: Varlet. Average intensity of the eight judges is rounded to the nearest whole number.3.87 ± 2. chemical 6 4 Smoke.48) 1. When only MS is available for identification. 9 = very strong odor intensity).55 ± 8. odor intensity. STD MS.Table 15. spectrum. and odor description of an identified compound correspond to the retention time.35 (20. An intensity between 5 and 5. spectrum. LRI.23 ± 0.39 6. green.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 7.40 ± 3. and quantities of odor-active compounds detected by fewer than six judges are indicated in parenthesis. 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. LRI MS. LRI. LRI. 2007. rotten 7 4 Spicy. . Note: Frequency of detection. LRI Animal.68 MS.77 24. Means of three fillets. and odor description of the injected standard of this compound). mass spectrum (identified using the mass spectra of the compounds). roasty 7 4 Burnt rubber. et al. roasty. In micrograms equivalents of dodecane per 100 g of smoked salmon. 55. standard (when the retention time. linear retention index (when the LRI of the identified compound corresponds to the LRI in the literature). STD MS.5 is rounded to 5 and an intensity between 5. LRI MS. Agric. 4518. a b c Handbook of Seafood and Seafood Products Analysis d e Means of identification: MS.81 ± 11. Number of judges (out of eight) who have detected an odor. J. STD. 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. V. woody (4) (2) Clove.. Food Chem.5 and 6 is rounded to 6 (1 = very weak odor. it must be considered as an attempt of identification..

39]. and seem to contribute little to overall aroma. The taste thresholds of some phenolic compounds were determined [40] and showed a high diversity between the molecules. whereas syringol and 4-methylguaiacol showed the same but lower effect than guaiacol [40]. 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. Concerning the bitter taste. sometimes cooked. 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]. Enolone derivatives are compounds derived from cyclopentenone. However. early works performed on individual phenolic compounds have identified the impact of guaiacol on the smoky flavor. and the odorant contribution of odor-active compounds cannot be the same if the SF is in the form of powders. it was commonly admitted that syringol derivatives impart a smoky odor and guaiacol derivatives contribute to a smoky flavor. Synergic or masking effects are possible and make the final odor complex. furannic compounds were found to play a role in cold smoke odors of liquid smoke or fishes treated by liquid smoke [14. reactions between liquid smoke compounds and the components of the matrix can occur through Maillard and Strecker reactions. For several decades.34]. and very little information is available. Two categories of carbonyl compounds can be differentiated: furannic compounds and enolone derivatives. Sensory analysis performed on standards confirmed the importance of guaiacol and o-cresol in the smoky flavor and dimethylphenol. Moreover. A polyfunctional carbonyl subfraction was isolated from wood smoke and possessed a caramellic/burnt sugar aromatic note [36]. Furfural and homologues exhibit cooked/roasty aromatic notes. More recently. but taste was not investigated as much as odor. and isoeugenol in spicy/sweet flavor [41]. Furannic compounds were thought to contribute to soften the heavy smoky aromas associated with phenolic compounds [37. if they do not have a strong individual influence. 4-methylguaiacol. 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. As for the assessment of the odor.6. 15. the physical state of SF can also influence the aroma. Furthermore.3. they may contribute in mixture to the overall odor. or oils.2 Role of Volatile Compounds of SF in the Flavor Phenolic compounds were shown as the major contributors of the smoky flavor [35]. 4-methylguaiacol perception was superior to that of syringol and guaiacol.38]. guaiacol derivatives and more generally the phenolic compounds of low-boiling fraction molecules (60°C–90°C) have been shown to cause the odor. The results of this fractionation are given in Table 15. The oils used in smoke oils can soften the smoke aroma. They were isolated early from wood smoke and described as grassy. as the other minor odor-active compounds. liquids. the same compounds responsible for the odor should be involved in the flavor that SF confers to seafood.Smoke Flavoring Technology in Seafood ◾ 245 Carbonyl compounds have also been reported as contributors to the smoky aroma of wood smoke. the determination of the effects of compounds of SF on the flavor is complex. Then. 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. Recently. . However. phenolic compounds are not the only flavor-active compounds. However. because they are not the compounds mainly detected in SF and seafood treated by SF odors by sensory analysis [37].

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

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

Owing to their lipophilic properties (log Kow between 4 and 7). which are constituted by less than four benzene rings. particularly smoked food [57.10-epoxyde Glutathion S OH OH OH O B[a]P 7. . These compounds have been studied for several years.8.248 ◾ Handbook of Seafood and Seafood Products Analysis 15. 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.8 15.3). In SF.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. because their catabolism leads to poly-hydroxy-epoxy-PAH suitable for binding to DNA adducts. PAHs can cross the biological membranes and accumulate in tissues. more toxic than light PAHs. However. especially benzo[a]pyrene (B[a]P) [59]. home cooking and industrial food processes represent the major source of human contamination [55]. Therefore. They are considered as carcinogenic contaminants.3 Benzo[a]pyrene metabolization.58].8-dihydrodiol-9.8-dihydrodiol OH B[a]P glutathion conjugate OH OH B[a]P 7. PAHs are generated during smoke production by wood pyrolysis. As they can be absorbed by animals.8 epoxyde OH B[a]P 7. it is used as the leading substance to illustrate PAH contamination. they are considered as environmental pollutants and can contaminate the human feed raw material [53. they are considered as heavy PAHs. PAHs are considered as carcinogenic contaminants of processed food [56]. 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. 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.1 Polycyclic Aromatic Hydrocarbons Properties and Toxicology PAHs are well known as being food contaminants and carcinogens [52]. Therefore. Hundreds of individual PAHs may be formed and released during the process of incomplete burning of the wood.54]. hence their toxicity (Figure 15.

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. Thus. cause chromatographic coelutions. In the case of liquid matrices as liquid smoke. gas chromatography and liquid chromatography are the most used techniques [55].S. 15.2 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. that is. However. In the case of solid seafood treated by liquid smoke. 15. Although all steps are important. solid–liquid extraction can be carried out. because they do not have the same toxicity. it is essential to quantify only the toxictargeted compounds. Several devices are therefore developed to optimize the analysis. PSC and PTF. Purification was especially performed on an alumina or silica column.69]. respectively [74]. the analysis step is the most critical point. Gas chromatography is coupled to mass spectrometry [58. Apolar solvents or mixes of apolar and semipolar solvents are used to extract the maximum of PAHs. they have not been applied to SF or seafood treated by SF. For example. a European regulation set the maximum contents of PAH in the primary products (PP) of smoke condensates used for the production of SF.Smoke Flavoring Technology in Seafood ◾ 249 safety avoiding PAH contamination. The extraction step must integrate the composition of the matrix. respectively [73]. purification and/or delipidation steps such as saponification are often applied to reduce the fat matter rate of samples [64]. even if they were found in weak quantities. The eight heavy PAHs left were shown as being carcinogenic or mutagenic contaminants and gave rise to serious health concern. and lead to mistakes in the identification. which combines a separation step and a detection step. the maximum levels of B[a]A and B[a]P were set at 20 and 10 mg/kg of liquid smoke. flame ionization detector (FID) [60]. Indeed. Moreover.9. supercritical fluid extraction (SFE) [66] or solid-phase microextraction (SPME) [67]. which can disturb the extraction.9 Legislative Aspects 15. Parameters of the chromatographic separation and detection must be adjusted to avoid coelutions with interferences from lipids. Among them. PAHs are often coextracted with fat matter. in Italy. The nature of the SPE cartridge phase is linked to the extraction method and the biochemical composition of the initial matrix. or tandem mass spectrometry [70]. Therefore. For the separation of the PAHs extracted from SF or seafood treated by SF. a liquid–liquid solvent extraction is often used [61–63]. In both condensates. the chromatography must be sufficiently efficient to separate isomers of PAH.1 European Regulations on PAH Found in SF In 2003. the U. and liquid chromatography is coupled to ultraviolet or fluorimetric detector [59–63]. . but solid-phase extraction (SPE) cartridges are now more frequently employed. such as bidimensional chromatography at the gaseous phase (GC/GC) [71] or liquid phase (LC/LC) [72]. and stir bar sorptive extraction (SBSE) [68] but not on liquid smokes or seafood treated by SF. but. Environmental Protection Agency (US-EPA) identified a list of 16 PAHs as the most frequently found [60]. to our knowledge. these PAHs were considered as toxic at low levels. with a weak toxicity but high concentrations in the samples analyzed. eight light PAHs were considered as environmental contaminants. In the 1980s.8. Other extraction devices have been developed to investigate PAH in smoked food such as accelerated solvent extraction (ASE) [65]. This harmonization was necessary to homogenize the legislation about SFs.

Finally. The content of PAH in SFs and in the final product can be better controlled than during traditional smoking. it is necessary to better control the composition of SFs and to improve knowledge about the influence of the pyrolysis parameters (wood nature. SFs are used in higher quantities than those employed in flavoring processes. if it is considered as smoking technique. the respective legal B[a]P amount is 5 mg/kg. but it could also initiate an international consideration of labeling of smoked and flavored food. This value is the result of the necessary harmonization between the national laws of European countries [79]. 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. Indeed. Moreover. brown meat of crab.78]. it is paradoxical to apply flavoring regulations to the smoking process. it can lead to problems of labeling.03 mg/kg. . a European regulation set the maximum content of B[a]P in foodstuffs treated by SF at 0. Indeed. the PAH contamination reached in SF is largely below the values authorized in PP [60. important differences in PAH concentrations are noticeable [57. leading to less PAH contaminated food by comparison with the traditional smoking techniques [80]. that is.03 mg/kg and leads often to noncompliant smoked products. because these values were set for PP and not SF. the PAH contamination was only set for B[a]P [77]. excluding bivalve molluscs. atomization of liquid smoke must be lower than 0. 0. the liquid smoking process decreases the emissions of PAH compounds to the environment. This value is very low compared to those authorized in PP. However. Therefore. atomization of liquid smoke would constitute the smoking technique. In this case. the 2003 maximum values must be reviewed again. that is. Indeed. for meat industry. Moreover. very small amounts. Therefore. However.63] which justifies controls and regulations. according to the origin of SF and industrial manufacturers. it leads to lower PAH contents. 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. In certain countries such as France. as drenching or showering. the vaporization of SF in a smokehouse causes a loophole in the legislation. Th is fact can be understood by the use of smoke condensates in flavoring quantities. Thus.10 Conclusion A wide range of SFs and uses of SFs are now available to flavor seafood products. 15.9. the smoking regulations set a maximum B[a]P value of 5 mg/kg of smoked fishery products and smoked crustaceans. The main criticism that can be formulated against SF is the lack of control of the final organoleptic qualities of such processed food.03 mg/kg. Nevertheless. Therefore. Moreover.75].250 ◾ Handbook of Seafood and Seafood Products Analysis However. The high maximum values authorized in PP do not seem well adjusted with the weak final PAH contamination of SF. this process can also be considered as a flavoring of the surface of the product. whereas food is treated by SF and not directly by PP. 15. All these benefits could help to reconsider the status of atomization of liquid smoke and the maximum PAH contents related. 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].69. and head and thorax meat of lobster and similar large crustaceans [76. SFs appear as a safe alternative to smoking techniques.2 European Regulations on PAH Concentration in Food Treated by SF In 1988. However. As for SF.

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

T. Wasserman. Food Chem. and Ling. 1978.. Compilation of odor and taste threshold values data.S. M... Identification of formaldehyde-induced modifications in proteins: reactions with model peptides. 36. Caractérisation des composes volatils responsables des qualities odorants du saumon fume (Salmo salar) et evaluation des contaminants du fumage (Hydrocarbures Aromatiques Polycycliques). Olsen. M.L. 1977. 2001. F. 2007. Guillén. 1966. Physical and chemical processes involved in the production and application of smoke.. Biol. Lebensm. W. Chem.E. et al. 146. Meat Res.-Technol. 49. Carbohydrate and nitrogenated compounds in liquid smoke flavorings. and Eyo. M. 35. 1972. Chem. 240. U.. Organoleptic evaluation of three phenols present in wood smoke. Wasserman.. J. W. 27.. Pure Appl. 29. A.M. J. and Potthast. J.C. Agric. 85. Swan. Food Chem. M. 1980. 1969. A. 26.. 2395.J. A. Kim. Baryłko-Pikielna. Radecki. Fazzalari.L. Chan. Varlet. Fish. J. Agric. 1976.. N.. Contribution of smoke compounds to sensory. Effects of the smoking process on odour characteristics of smoked herring (Cuplea harengus) and relationships with phenolic compound content. M. Process Biochem. 1639. Influence of the moisture content on the composition of the liquid moke produced in the pyrolysis process of Fagus sylvatica L.L. and Fujimaki. Chem. 6235.. 24. Smoke flavor as related to phenol. Metz. Chem. 1005.D. Clifford. Food Chem.. 43. FL. and air velocity. 34. A. Isolation and identification of some components of the lower-boiling fraction of commercial smoke flavourings. Thesis.J. Food Chem. Pure Appl.C. Rev. Philadelphia. Toth.. Sensory properties of phenolic compounds isolated from curing smoke as influenced by its generation parameters. Hamm. J. 1975. Contribution of volatiles to rice aroma.. Acta Aliment.G. Board Can. M.T. J. Flavor effects of different woods on whitefish smoked in a kiln with controlled temperature. M.-Wiss. 3.. Res. Chemical references in sensory analysis of smoke flavourings.... Food Sci. et al. Agric. wood. 279. ASTM Data Series DS 48A. and Miler. and Ibargoitia. 203. 934. 1977. Guillén. Manzanos. 1667. . 1974. 1977. 1999.G.. 21.. L. 49. 39. A “smoke” flavor fraction of a liquid smoke solution. L. Smoking of foods. Food Chem. 47. Chem. June/July. V.W. The development of flavour in potable spirits. Sérot. 31. Food Chem. J. Ojeda. Analysis of smoke and smoke products. Workers. 42. Food Chem. Effect of smoking processes on the contents of 10 major phenolic compounds in smoked fillets of herring (Cuplea harengus)... Agric. Lantz. 44.. 1970. Chem. 38. carbonyl and acid content of bologna. Soc. J. 201. Bratzler. C. Burdock. American Society for Testing and Materials. Daun. 49. A. K. bacteriostatic and antioxidative effects in smoked foods. 1655. 1988. 53.252 ◾ Handbook of Seafood and Seafood Products Analysis 20. 1977. Chemical composition and application of smoke flavor.N. 22... Agric. et al. Chemical aspects of the smoking of meat and meat products. T. 8. Pol. 38. 41. R. 30. 49. 1984. R. 37. Tang. and Toledo. Rusz. H.. B.. 2006... S. University of Sciences of Nantes. 137. F 7:1. CRC Press LLC. 31.. humidity and air flow on smoke penetration into fish muscle...Z. humidity. Biol. 33. 40. Cardinal.A. and Doerr. J. K. J. et al. Feranoli’s Handbook of flavor Ingredients. 111. 96. 2002. Pure Appl. and Burtles..D. 102... M.S. 1201.A.. Meet. 2004. Identification of flavour constituents in carbonyl. Eur..M.. 32... Food Res. Buttery.. 40. 5(3)... 34. 433. B. 23. 27(7). Adv. 28. 18(5)... 4126. 1978. 7(2).. noncarbonyl neutral and basic fractions of aqueous smoke condensates. PA. Food Sci... J. 87. and Ibargoitia.. L.. G. Food Sci. Turnbaugh.. 25. Proc.B. 78(4). J. S. M.A. R. 29. Effect of smokehouse temperature. K.. Fiddler. and Vaisey. M. et al. 36(5) 1006. Boca Raton. Kurata. 2002.. et al..E. 1970.. 22.. 2004.

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.. P. Arch. 47. Wenzl.. J. Readman. A. 66. Evaluation of acute toxicity and genotoxicity of liquid products from pyrolysis of Eucalyptus grandis wood. Allegato III. 1367. Off. 69. A. 2000.. Off. 47. Ré-Poppi. J. Off. EC 1881/2006. Moret. J. 1.. Environ. Union. and Santiago-Silva. 2004. L. M. T.. Italian Law Decree. T. Eur. Determination of polycyclic aromatic hydrocarbons in water by solid-phase microextraction–gas chromatography–mass spectrometry.. and Dean. Varlet et al. Decreto Legislativo N°107 del 25/01/1992. A. 25(7). 73. Agric.. D. Dos Santos Barbosa. 744. 2005. 259.. Acta. Chromatogr.-Wiss.M. Environ. 1. 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. Purcaro. Contam. Union. et al. 2007. 75. U. 716. N. Eur. 24(7). 70. 309. 169. J.. 68. Res. 34. 153. 897. EC 2065/2003.. 2001. Food Chem. 71. 72. 184. 79. Huopalahti. et al. 364: 5. Commission Regulation of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs. Use of liquid smoke flavouring as an alternative to traditional flue gas smoking of rainbow trout fillets (Oncorhyncus mykiss). J.. 304. . 2007. 77.. 80. Off. 1999. and Tapanainen.254 ◾ Handbook of Seafood and Seafood Products Analysis 65. EC 88/388. Food Addit. Liq. Union. 101. J. J. 19(9). Food Chem. Chim. Accelerated solvent extraction and gas chromatography/mass spectrometry for determination of polycyclic aromatic hydrocarbons in smoked food samples. Determination of polycyclic aromatic hydrocarbons in vegetable oils using solidphase microextraction—Comprehensive two-dimensional gas chromatography coupled with time-offlight mass spectrometry. L. Determination of polycyclic aromatic hydrocarbons in wastewater by off-line coupling of solid-phase microextraction with column liquid chromatography..-Technol. et al. Use of supercritical fluid extraction-high performance chromatography in the determination of polynuclear aromatic hydrocarbons from smoked and broiled fish.. Polycyclic aromatic hydrocarbons from wood pyrolysis in charcoal production furnaces.. Popp. R.. L. Chromatogr. Assessment of polycyclic aromatic hydrocarbon content of smoked fish by means of a fast HPLC/HPLC method. Determination of PAH profiles by GC-MS/MS in salmon muscle processed according to four different smoking techniques. 1–2. 78. 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. P. G. J. Contam. 1999. Eur... 521. Järvenpää. J.. 1988. Union. Agric. 2003. 1062... Lebensm. et al. Anal.. Wang. J. King. 1473.S. 1161. Conte. A. 2003. Pimenta. L. J.. 284.. et al. Analytical methods for polycyclic aromatic hydrocarbons (PAHs) in food and the environment needed for new food legislation in the European Union. E. Commission Recommendation of 4 February 2005 on the further investigation into the levels of polycyclic aromatic hydrocarbons in certain foods. 2006. W. 2006. 76. L 34: 43. 523(2).. 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. EC 2005/108. et al. G. and Zhou.J. Eur. 74. 67. 38. Trends Anal. 2006. 1996. S. Hattula. Relat. Toxicol. Technol. Chromatogr.L.. Chem.

NUTRITIONAL QUALITY III .

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

7 deals with the different methods to determine and calculate calories in fish and shellfish. the adoption of NIR testing resulted in a total cost saving of CAN$ 2.258 ◾ Handbook of Seafood and Seafood Products Analysis 16. the chemical composition of fish generally varies due to seasons. but the available detectors cover a smaller range. The development of NIR in food analysis started with the development of analysis of cereal grains and oilseeds in Canada [2]. analysis should be performed on the specific samples.org). 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. However. Section 16. which involves concentrated sulfuric acid and heavy metal catalysts. and lipids. and the other minor constituents include carbohydrates. making it difficult to use this spectral range before the development of multivariate calibration technology. rapid. the range 1100–2500 nm. The NIR spectrum is defined between the wavelength 800 and 2500 nm. Nearinfrared spectroscopy (NIR) is the most common method for such analysis and is therefore comprehensively presented in this chapter. Diff use transmittance measurements are usually carried out in the 800–1100 nm region of the spectrum. and sizes. The NIR radiation interacting with a sample may be absorbed. 16. such as the introduction of partial-least squares (PLS) by Martens in 1982 [3]. geographical locations.000 Kjeldahl analyses were conducted per year and incidentally producing 47 ton of caustic waste in the process. The first instruments on the market were filter instruments measuring in reflectance mode. The spectral data will be reduced by principal component analysis. Therefore. and a lead sulfide. where the weak absorptions enable useful data to be obtained using sample thickness of 1–2 cm of samples such as meat. The end result is a calibration equation from which the constituent of interest is calculated from a linear combination of spectral data. When Williams was running the program for the Canadian Grain Commission. and then one can perform a linear regression on the principal components. and minerals [1].fishbase. Proximate data on different fish species are collected in databases such as the FishBase (www. stages of maturity. In fish meat these constituents make up about 98% of the total mass. As well as increased efficiency of the Canadian wheat segregation program. to ensure obtaining data on the exact proximate composition. in this spectral region the spectrum of the transmitted light is very compact and no single peaks are visible. and easy to perform nondestructive analysis for simultaneous determination of the major components in fish. . NIR has been found to be a reliable. and so on.5 m per year and a saving for the environment by replacing the Kjeldahl system. the silicon detector covers the range 400–1100 nm.2 Nondestructive Analysis of Total Proximate Composition Analysis of each nutrient separately is time-consuming and requires a diverse set of equipments. an indium gallium arsenic covers the range 800–1700 nm. making it possible to measure over the whole NIR spectrum and not only on a small number of selected filters. During the 1980s monochromator instruments were developed. by the chemical-free NIR method. cheese. Methods for simultaneous determination of the major components are therefore valuable. or whole grain. transmitted. however. 600. proteins. vitamins. 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.1 and further discussed in the text below.

nondestructive.133] NIR/NIT Reflection. precise. The equipments are relatively expensive. and protein content noninvasively. transflection. Calibrations require skilled personnel.172–174] (continued) ◾ 259 . Different calibrations for different species and organs. expensive Few articles on fish composition Need more research Composition and Calories [21.1 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. can be nonsensitive. Nondestructive and can be used on live fish. and can be performed online [17–20.8] For reflectance instruments (surface analysis) some drawbacks such as interference by starch and lipids. can be used on live fish Samples are placed in an electromagnetic field and electric conductivity is measured Specific for different species. or transmission of nearinfrared light (850–1700 nm) Ultrasound Measurement of ultrasonic velocity Rapid. displacement of reflectance spectrum by moisture content. and protein.173] Total body electrical conductivity (TOBEC) May obtain data on water. physiological and physical states can affect values of conductivity. Calibrations need to be made against reference methods. nondestructive. [4–6.Table 16. fully automated. lipids. and disturbance by particle size in samples Ultrasonic properties of tissue depend on composition and temperature [11. water.

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. (SoxTech. Fosslet. location of lipids. fexICA). Need of sample specific calibrations Weaknesses due to quantification of proteins without combining with destructive methods Drawbacks Selected References Overview of the Most Common Methods for Analysis of Proximate Composition in Fish and Fish Products 260 ◾ Methods Principle Nuclear magnetic resonance (NMR) Nuclei of atoms in a sample provide spectra when the sample is exposed to a magnetic field Total Lipid Determination Chemical Extractions: Provides high total lipid yield Time-consuming Use of health hazard chemicals Destructive technique Requires well-trained laboratory personnel May discriminate structured fat (such as phospholipids) Requires laboratory facilities Physical and chemical changes might occur during examination Precision level may be dependent on sample (maturity stages of the fish.55.) Microwave drying The sample is dried and from the water content found. etc.57] Excellent for determination of fat and water content or even distinguish lipid classes and water properties. lipid content.Table 16. nondestructive (See under NMR below) [13–15. the fat content can be calculated theoretically by the formula Fat% = 80 − water % . processing) [46] Automatic.22.1 (continued) Advantages Rapid.

NIR/NIT Transmitted or transflected Near Infrared light (800–1700 nm). nondestructive.22. location of lipids. and nondestructive and allows in vivo measurements (continued) 261 . The NMR mouse is rapid. and portable Allows in vivo measurements Nondestructive and rapid Broad range of applications. Low-field NMR See NMR above Composition and Calories NMR mouse ◾ Nondestructive and rapid. requires specific calibrations Traditional low-field instruments require withdrawal of homogeneous samples for analysis (invasive) [14–16. rapid.8] [46. may also provide other nutrient data in the same analysis Some portable instruments are available Allows in vivo measurements Expensive. easy.50.51–52] Relatively inexpensive. lipid content. processing) Needs to be calibrated for the individual species Most suitable for neutral lipid determination See NIR/NIT above [4–6.Nondestructive Methods: No laboratory facilities are required Precision level may be dependent on sample (maturity stages of the fish.55] Fat meters Determination of water by analyzing the dielectric properties using a microwave strip (calculation of lipids as for the drying method). portable (small size).

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

buffers salts. The amount of absorption is proportional to the protein present Color formation and binding depend on proteins present. nondestructive.176. detergents [133. no addition of reagents required Low sensitivity. interference from ammonia. high sensitivity. High amounts of endogenous proteases may cause errors. easy to perform Relatively low sensitivity compared with other UV-visible methods. Unstable reagents are used. which react with the Folinphenol reagent.177] Composition and Calories Near-UV absorption Measurement of UV absorption (280 nm) [133] ◾ Rapid. depends on amino acid composition 263 (continued) . and reaction products are detected between 500 and 750 nm Rapid. interference from common laboratory chemicals.Biuret method (Alkaline copper reagent test) Negligible interference from materials that absorb at lower wavelengths. protein-dye complex adsorbs on glass surface.133] A violet-purplish color is produced when copper(II) ions interact with peptide bonds under alkaline conditions. Absorbance at 540 nm Lowry protein assay Copper(II) ions in alkaline solution react with protein to form complexes. easy to perform. 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. interference by UV-absorbing compounds (nucleic acids and nucleotides). technique is less sensitive to protein type: it utilizes absorption involving peptide bonds that are common to all proteins. variation of binding capacity for different batches of commercial grade dyes [68. Other compounds can interfere.133. color development depends on amino acid composition [67.175] High sensitivity and easy to perform Standard curve is nonlinear.

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

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

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

2. sulfosalicylic (SSA). and leucine).21 perchloric (PCA). (2) its ability to cross-link proteins. cysteine may be essential for infants. which increases the protein stability in the harsh extracellular environment by conferring proteolytic resistance. Once homogenized. In this chapter. and aromatic amino acids (phenylalanine and tyrosine) are the most important from this point of view. Free amino acids initiate important changes at early postmortem and during storage and can be very useful as quality indices of processing and storage. The extraction consists in the separation of the free amino acid fraction from the insoluble portion of the matrix (fish muscle). the analysis of essential amino acids in seafood is important for the evaluation of both the nutritive value and the sensory quality of seafood. It is usually achieved by homogenization of the ground sample in an appropriate solvent by using a Stomacher.16. or diluted phosphate buffers.22 trichloroacetic (TCA). 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.18.1 N hydrochloric acid solution. which confers numerous biological functions to this amino acid (precursor to the antioxidant glutathione).20 have been successfully used as extraction solvents.12. then. in rare cases.01–0.2 Sample Preparation for the Analysis of Seafood Essential Amino Acids Free or total essential amino acids are analyzed from the whole amino acid profile. Polytron. especially of those considered essentials.5–10 Thus. 0. and so forth.288 ◾ Handbook of Seafood and Seafood Products Analysis compounds through Maillard reactions and Strecker degradations. Sample preparation will depend on whether free or total essential amino acids have to be analyzed.4 Branched-chain essential amino acids (valine. the sample is centrifuged at more than 10. there is no need for further cleaning up of the sample.13. sulfur-containing amino acids (methionine and cystine/cysteine).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. Several chemical methods include the use of concentrated strong acids such as phosphotungstic (PTA).13 5% of trichloroacetic acid.11 17. Sample cleanup is necessary to eliminate proteins and polypeptides by means of the deproteinization process. methods for the analysis of amino acids in seafood. isoleucine. Although classified as nonessential. the elderly. or (3) its Maillard reaction with sugars yielding characteristic flavors. concentrated strong acid solutions such as 4% of 5-sulfosalicylic acid. with the additional advantage that proteins are not extracted and. The extraction solvent can be hot water.15 or a rich alcohol-containing solution (>75%) such as ethanol16–18 or methanol19. A more detailed description of amino acid methods of analysis may be found in the work of Aristoy and Toldrá.23–25 and picric . are described. which can be achieved through different chemical or physical procedures. 17.14 6% of perchloric acid. and individuals with certain metabolic disease or who suffer from malabsorption syndromes. or by means of a simple stirring in warm solvent.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.

resulting in a very simple deproteinization procedure with no interferences.31–33 with amino acid recoveries around 100% for all them. which is easily neutralized by the addition of KOH or potassium bicarbonate.40. proteins precipitate by denaturation. Nitrogen atmosphere and sealed vials are required during the hydrolysis to minimize the degradation. etc. Differences among all these chemical and physical methods are caused by several aspects such as differences in the cutoff molecular weight.6 N PCA. oxygen is removed and substituted by nitrogen or other inert gas.000. or acetonitrile.39. Proteins must be hydrolyzed into their constituent amino acids before the analysis. whereas free amino acids remain in solution. Some comparative studies have been published on these deproteinization techniques. When limited amounts of sample are available.). 5. creating an appropriate atmosphere inside the vessels to ensure low amino acid degradation.Essential Amino Acids ◾ 289 (PA)26–28 acids or organic solvents such as methanol. recovery of amino acids. which is easily separated by centrifugation. by mixing two or three volumes of organic solvent with one volume of extract.000. addition of constant boiling hydrochloric acid and additives.2 Sample Preparation for Total or Hydrolyzed Essential Amino Acid Analysis The total essential amino acid profile is usually requested.30 All these methods give a sample solution rich in free amino acids but free of proteins.42 Sample manipulation (sample evaporation to dryness. Liquid-phase. compatibility with derivatization (pH.22 Under these conditions.). has also given very good results. to rend insoluble potassium perchlorate. The presence of appropriate antioxidants/scavengers during hydrolysis can prevent losses of the most labile amino acids.41 The use of microwave technology for the hydrolysis has been assayed by some authors. Therefore. ethanol. and so forth. 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. all of them . presence of salts. A good choice may be the use of 0. The hydrolysis may be accomplished using either liquid-phase or vapor-phase methods. where the hydrochloric acid contacts the sample directly. etc.22.34 17.18. only the acid vapor comes into contact with the sample. In the vapor-phase hydrolysis method. 30.35–38 These temperatures in such acidic and oxidative medium may degrade some amino acids.2. The use of organic solvents.000. and performance under vacuum) is similar to that of a conventional oven. also disposes of an oven to accomplish the hydrolysis.29. which permits the alternative air evacuating/inert gas purging. a system capable of alternative air evacuating/inert gas purging to get a correct deaeration inside is valuable. samples are treated with constant boiling 6 N hydrochloric acid in an oven at around 110°C for 20–96 h. In both cases. thus excluding nonvolatile contaminants. Upon heating.000 Da) that allow free amino acids through while retaining large compounds. because it gives information on the nutritional value of fish meat. the tubes containing the samples are located inside large vessels containing the acid.39 Some commercial systems are available. Hydrolysis may be improved by optimizing the temperature and time of incubation41 or with the addition of amino acid oxidation protective compounds. liquid phase or vapor phase. Digestion at 145°C for 4 h has also been proposed. 10. Some physical methods consist in centrifugation through cutoff membrane filters (1. An additional advantage is the easy evaporation to concentrate the sample. the vapor-phase hydrolysis method is preferred to minimize contaminants coming from aqueous 6 N hydrochloric acid. or separation method (interferences in the chromatogram. but the duration of the treatment is shorter (less than 20 min). is well suited to hydrolyze large amounts or complex samples. The most common method used for complete hydrolysis of proteins is acid digestion. Typically.

KOH.3¢-dithiodipropionic acid.58. no single set of conditions will yield the accurate determination of all 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. .38 Alkaline hydrolysis instead of acid hydrolysis is also proposed (see below). making the posterior analysis easier.55. has been extensively reported in papers35.56 As can be observed in this section. LiOH.290 ◾ Handbook of Seafood and Seafood Products Analysis essential amino acids. 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. improve the recovery of nearly all of these amino acids except tryptophan and cysteine. or pronase. importance of a correct deaeration.43–50 improves cysteine (and methionine) recoveries. such as chromatographic (liquid or gas chromatography (GC)) or capillary electrophoresis (CE) techniques. chymotrypsin. 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. Some additives have been proposed to protect tryptophan against oxidation as is the case of thioglycolic acid. acid-to-protein ratio. and cysteic acid making its analysis rather difficult. When high sensitivity is required. and so on. being enough for the requirements of any food industry.62 An alternative to acid hydrolysis is the alkaline hydrolysis with 4. yields acceptable results for the majority of amino acids. threonine.61. in which methionine is also oxidized to methionine sulfone.2 M of either NaOH. Thus. Before or after this separation. serine. and tryptophan.67–69 17. carboxypeptidase.51 3-bromopropylamine.3 Seafood Essential Amino Acid Analysis The analysis of individual amino acids needs a previous separation of all others. The previous performic acid oxidation of cysteine to cysteic acid. unless a very selective way of detection is used. although considerable recoveries have been found if no oxygen is present. when the analysis of cyst(e)ine would be necessary. methionine. a compromise of conditions offers the best overall estimation for the largest number of amino acids. up to 1% phenol or 0.60 and books. 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. 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. Derivatization is a usual practice in amino acid analysis. thermolysin. presence and concentration of oxidation protective agents.59. In general. The choice mainly depends on the equipment available or personal preferences. cysteine. The separation of the individual amino acids in a mixture requires very efficient separation. protective agents currently used.52 4-vinyl pyridine53. The use of alkylating agents to stabilize the previous hydrolysis of cysteine constitutes a valid alternative.30. 41.36. Cyst(e)ine is partially oxidized during acid hydrolysis yielding several adducts: cystine. Additionally.54 or 3.63–66 for a better tryptophan determination. Good recoveries have been achieved by using 3-bromopropionic acid. which is recommended by many authors47. cysteine sulfinic acid. Tryptophan is often completely destroyed by hydrochloric acid hydrolysis. amino acids used to be derivatized to allow their separation or to enhance their detection. such as tyrosine. The optimization of conditions for hydrolysis based on the study of hydrolysis time and temperature. In fact.36. because each possible methodology has advantages and drawbacks.1% sodium sulfite. or BaOH. papain. A third way to hydrolyze proteins is enzymatic hydrolysis by proteolytic enzymes such as trypsin. (2) give a quantitative and reproducible reaction.

The elution involves a stepwise increase in both pH and sodium or lithium ion concentration. are always present.12 Two types of derivatives are obtained depending on the chosen separation and/or detection technique. The classical procedure has been improved with a new polystyrene matrix that offers better resolution power due to smaller particle size. or 4-fluoro-7-nitrobenzo-2.3. Amino acids. although unidentified.1.1 High-Performance Liquid Chromatographic Methods HPLC is the preferred technique to analyze amino acids. pellicular packaging.2. (5) have the possibility of automation. in their native form. 17.3.3-oxadiazole postcolumn derivatization to obtain highly fluorescent derivatives with enhanced sensitivity. Under these conditions.38. (4) have mild and simple reaction conditions. which facilitates a more selective detection. After separation.1. the spectroscopic detection of amino acids requires their previous derivatization to obtain an UV absorbing or fluorescent molecule. because reagent-consuming amines. and tryptophan) have a chromophore moiety that confers a suitable maximum absorbance for more specific UV detection (280 nm for tyrosine and tryptophan and 254 nm for phenylalanine). The second type are derivatives that allow gas chromatographic amino acid separation by increasing their volatility and temperature stability.15.1. permitting 5–10 pmol sensitivity as standard. The original method required two separate columns and needed about 4 h to achieve a complete analysis.1 and 17.3. 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. The HPLC techniques to analyze amino acids are cation exchange and reversed-phase (RP) chromatography and are described in Sections 17.3.1. and those with more than one primary amino group or possessing a guanidyl residue elute at the end of the chromatogram.68 Thus.Essential Amino Acids ◾ 291 (3) yield a single derivative of each amino acid. The derivatization reaction can be performed after separation of the amino acids (postcolumn derivatization) or before separating them (precolumn derivatization). recent improvements of the ninhydrin derivatization method71–73 . Although postcolumn techniques should be run online for maximum accuracy. tyrosine. The latest generation of Moore and Stein amino acid analyzers also use o-phthaldialdehyde (OPA). 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.70 fluorescamine.1 Cation Exchange Chromatography This methodology is based on the amino acid charge. and they include derivatives for spectroscopic or for electrochemical detection. because their spectral (high-ultraviolet (UV) absorbing or fluorescence properties) or electrochemical characteristics will affect the sensitivity and selectivity of detection. and better detection systems. Tryptophan also possesses native fluorescence (l ex = 295 nm. absorb at 210 nm and thus cannot be used for spectroscopic detection. the more acidic amino acids elute first. l em = 345 nm). and (7) have no interferences due to by-products or excess of reagent. It must be remarked that the use of sufficient amount of reagent is of special importance when dealing with biological samples. amino acids were converted into colored ninhydrin derivatives for spectrophotometric (colorimetric) detection. as it is a very unspecific detection wavelength. The first type are derivatives that enhance amino acid detection in liquid media. 17. Only three amino acids (phenylalanine. precolumn techniques can be run either offline or online. (6) have good stability of the derivatization products. speed. Nevertheless.

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

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

58. The sample derivatization is rather simple. around 9. 1-Dimethylamino-naphthalene-5-sulfonyl chloride (Dansyl-Cl): Dansyl-Cl reacts with both primary and secondary amines to give a highly fluorescent derivative (l ex = 350.2 Reversed-phase HPLC chromatogram of PITC-free amino acids from salmon muscle extract. and only needs a basic pH.84 Detection is by absorption in the visible range.294 ◾ Handbook of Seafood and Seafood Products Analysis 1400 1200 Glu Ans Absorbance at 254 nm (mAU) 1000 800 Gly 600 Tau βAla His Ala 400 Asp 200 OHpro Ser Lys Pro Thr Arg Tyr Val Met lle lS Leu Trp Phe Orn 0 0 Asn Gln 10 20 30 Retention time (min) 40 50 Figure 17. Reaction efficiency is highly matrix dependent and variable for different amino acids. standard amino acid solution should be derivatized under similar conditions. The reaction time is around 15 min at 70°C and takes place in a basic medium with an excess of reagent. IS. anserine. The high wavelength of absorption makes the baseline chromatogram very stable with a large variety of solvents and gradient systems. 15 min at 60°C. and a reaction time of 1 h at room temperature (in the dark). Stocchi et al. Ans.5. United States). the reaction conditions . However. because it is especially affected by the presence of high levels of some chloride salts. Nevertheless. Tau.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.84 Derivatives are very stable (weeks) and can be formed from both primary and secondary amino acids.83. Detection limits are in the low picomole range. The dansylated amino acids are stable for 1 day85 or until 7 days when kept at −4°C86 and protected from light. internal standard nor-Leucine. Palo Alto California. By-products originating from an excess of reagent absorb at the same wavelength and thus they appear in the chromatogram. presenting a maximum from 448 to 468 nm.33 To overcome this problem and obtain an accurate calibration. l em = 510 nm) although UV (l = 250 nm) detection may also be used. taurine. 4-Dimethyl-aminoazobenzene-4′-sulfonyl chloride (Dabsyl-Cl): This reagent was first described in 1975 for use in amino acid analysis. Commercial System Gold/Dabsylation Kit™ uses this technique (Beckman Instruments.87 or even 2 min at 100°C.

as it is highly fluorescent and then. The yield with lysine and cysteine is low and variable.88 Even so. the reaction of the excess of reagent with a very hydrophobic amine as 1-adamantylamine (ADAM) gives a late-eluting noninterfering peak. The addition of detergents like Brij 35 to the derivatization reagent seems to increase the fluorescence response of lysine. reaction conditions. In order to obtain reliable and precise results. cysteine and cystine are quantified together. using 3. Nowadays. The reaction time is fast (45–90 s) and does not require any heating. and fluorescent intensity. this methodology reveals excellent linearity for cystine and also cystine-containing short-chain peptides.93 The fluorescence is recorded at 455 or 470 nm after excitation at 230 or 330 nm.Essential Amino Acids ◾ 295 (pH. The derivative is fluorescent (l ex = 265 nm. and tyrosine. An automated precolumn derivatization routine. temperature. 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. OPA derivatives can be detected by UV absorption (338 nm) as well.5) medium. which includes the addition of ADAM.91 This method is preferred because the addition of ADAM is more easily automatized. because it guarantees the repeatability of parameters. many automatic injectors are programmable and able to achieve automatic derivatizations. This excess is hydrolyzed to dansyl sulfonic acid. ethanethiol. lysine. The derivatization is fast (1–3 min) and is performed at room temperature in alkaline (pH 9. which is present in excess as it is highly fluorescent and probably interferes into the chromatogram as a huge peak. In the second option. These methods include the conversion of cysteine and cystine to cysteic acid by oxidation with performic acid or carboxymethylation of the sulfhydryl residues with iodoacetic100.32 In these methods. Histidine gives a very poor fluorescence response (10% of the other amino acids). and excess of reagent) must be carefully fi xed to optimize the product yield and to minimize secondary reactions. is of great advantage.94–96 2-mercaptoethanol. chromatographic selectivity. such as FMOC/amino acid ratio.32 . itself or hydrolyzed. Some reports have been published proposing several ways of automation.89 9-Fluorenylmethyl chloroformate (FMOC): This reagent yields stable derivatives (days) with primary and secondary amines. The first option was included in the automated AminoTag method90 developed by Varian (Varian Associates Limited). 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. The choice of the mercaptan can affect derivative stability. as well as reaction time.97. several methods have been proposed before derivatization.82 Another problem is the large excess of reagent needed to assure a quantitative reaction. l em = 315 nm) and is detected at the femtomole range. On the contrary. and the reagent itself is not fluorescent. One of the main disadvantages of this procedure is the inability of OPA to react with secondary amines. have to be optimized very carefully.86.3′-dithiodipropionic acid55 and incorporated by Godel et al.101 or the formation of the mixed disulfide S-2-carboxyethylthiocysteine (Cys-MPA) from cysteine and cystine.82.99 In the case of cysteine. this problem is overcome by standardizing the time between sample derivatization and column injection by automation. this will commonly form multiple derivatives with histidine.92. reinforcing the poor reproducibility of its results. Tryptophan adducts do not fluoresce and histidine and cyst(e)ine adducts fluoresce weakly. This is relatively easy because the reaction is fast and no heating is necessary.94 into the automatic sample preparation protocol described by Schuster. and 3-mercaptopropionic acid are the most frequently used. OPA amino acids are not stable. which is not the case with any essential amino acid.98 and some of them have been patented and commercially marketed (AutoTag OPA from Waters Associates). 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.

it will be necessary to readjust the chromatographic conditions to get a good separation of all amino acids. The chromatographic separation of these derivatives has been optimized for the amino acids from hydrolyzed proteins. Furthermore. Only columns manufactured in the same batch are guaranteed to give the same selectivity if the rest of parameters are fi xed. Massachusetts. Only amino acids with aromatic rings or sulfur-containing side chains are sufficiently electrochemically active to be detected by this method. OPA/mercaptoethanol or OPA/sulfite104. Any electrical measure. The presence of residual uncapped silanol groups on the silica surface. Milford. such as current. or charge. Cystine and cysteine may be analyzed after their conversion to cysteic acid (CisH) by performic acid oxidation. lipids.3A shows the separation of hydrolyzed amino acids from salmon. the choice of the RP column is not an easy subject because of the great variability of commercially available RP columns.50). Due to these variables. mainly octadecylsilane.e. the AMQ peak is very large at the beginning of the chromatogram and may interfere with the first eluting peaks (see Bosch et al. and the separation of physiological amino acids is improved. which is only weakly fluorescent at the amino acid derivatives detection conditions and does not interfere in the chromatogram. Some of these derivatives are also susceptible to electrochemical detection. 1 min. yielding very stable derivatives (1 week at room temperature) with fluorescent properties (l ex = 250 nm. triethylamine) to the mobile phase can overcome the problem. columns are more carefully manufactured with these silanol groups blocked or inaccessible by steric impediment avoiding the tailing. . from 8.108 If the choice of the derivative reaction is a challenge.3B shows the same sample but submitted to a performic acid oxidation before the hydrolysis in which the CisH peak appears by 7. The excess of reagent is consumed during the reaction to form aminoquinoline (AMQ). and UV detection at 254 nm may be used for its analysis. It means that when transferring a published method to a particular set of samples. different selectivity. is related to the analyte concentration. conductance. 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. Figure 17. The fluorescence of tryptophan derivative is very poor.296 ◾ Handbook of Seafood and Seafood Products Analysis 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC): It reacts with primary and secondary amines from amino acids. United States). detergents. accessible to sample molecules.2 to 10. and other compounds naturally occurring in biological samples and foods. potential. because the resulting CisH is well separated inside the chromatogram. different selectivity may be found among same columns. Nowadays. The methodology has been marketed as a prepackaged AccQ Tag kit (Waters Corporation. but 10 min at 55°C would be necessary if a tyrosine monoderivative is required. l em = 395 nm). the optimum pH for the reaction is in a broad range. Both facts facilitate sample preparation.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. Electrochemical detection consists in one electrode or an array of electrodes mounted in a cell with an applied potential difference. even those made by the same manufacturer. However. can cause unwanted tailing of peaks (especially for the basic amino acids).105 in addition to fluorescent properties possesses electroactivity (750 mV) and PITC106 has again the advantage of reacting with secondary amines.and di-derivatives are the initial adducts from tyrosine. In these cases. the addition of a strong cation (i. because they are molecules with electroactive functional groups.. Indeed. Reaction time is short.107. because both mono.5 min. The most used column packaging consists of alkyl-bonded silica particles. which are separated by RP-HPLC. UV detection (254 nm) may also be used. In this case. whereas Figure 17. Sensitivity is in the femtomole range. and proteins. making them very adequate for biochemical research. as a consequence. peptides.

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

which are much more sensitive than FID for such compounds.113 and cheese.3 Capillary Zone Electrophoretic Methods Capillary zone electrophoretic technique is extremely efficient for the separation of charged solutes.113. and beans111 or other results obtained in honey.110 comparing GLC with cation exchange chromatography reported different conclusions when analyzing some hydrolyzed food samples. and urine matrices but not in tissues in which the presence of natural dipeptides. 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. especially in the analysis of D isomers. a very fast GC analysis of physiological amino acids.298 ◾ Handbook of Seafood and Seafood Products Analysis with an organic phase constituted by acetonitrile and/or methanol and/or tetrahydrofuran.120.3. and even though a particular pH can significantly . phosphothreonine. neutral. and the derivatives are stable and ready for GC/FID. has been developed. and the equipment is very versatile and usually available in any analytical laboratory. capable of separating 50 compounds. The detector used is the flame ionization detector (FID). dipeptides.112 milk. Recently. Nevertheless. Reactions consist of two stages: an esterification with an acidified alcohol followed by N-acylation with an acid anhydride in an anhydrous medium. The buffer may be constituted by less than 100 mM concentration of acetate or phosphate. physiological amino acids has to be analyzed. GLC has been combined with mass spectrometry (MS) for detection and identification. a very highly efficient technique adequate for the amino acid analysis. and phosphotyrosine. in summary. Gas liquid chromatography (GLC) is not often used for the determination of amino acids from tissues or foods. including amino acids. GLC is.121 The high efficiency.117–119 where the separation was achieved by using chiral-GC stationary phases. whereas thermionic-N-P (NPD) or flame photometric detector (FPD) are selective toward organic compounds containing phosphorous and nitrogen. In many cases. Protein removal is not required.116 to analyze phosphoserine. the technique is very efficient and it is worth mentioning the separation of 32 nonprotein amino acids from edible seeds. Described applications are available for the analysis of physiological amino acids in blood. A finely adjusted binary (most used) or ternary gradient elution is often necessary when the overall amino acid profile from hydrolyzed and. especially since the capillary columns appeared. and balenine may complicate the amino acid analysis. and acidic constituents. Amino acids constitute a mix of basic. GC/NPD. speed. The main advantages of these detectors are their high sensitivity and wide linear range. 17. This methodology has been patented as EZ:faast and commercialized by Phenomenex (Torrance. The difficulty of separating amino acids by this technique relies on their structure. plasma.2 Gas Liquid Chromatographic Methods The extremely high-resolution capacity is the main advantage of GC. which is universal and the most widely used. especially.3. anserine.114 In their analysis by GLC. nuts. although applications on meat samples are scarcely described.109. GLC is not very expensive because no solvent is used. the amino acids must be converted to volatile and thermostable molecules. in comparison with liquid chromatographic techniques. and amines. The NPD was used by Buser and Erbersdobler115 and FPD by Kataoka et al. Some applications67. 17. carnosine. California. and low amount of sample make this technique very interesting when compared with classical electrophoresis and chromatographic techniques. United States). or GC and LC with MS detection.

138 Tween 20.118.146. microcolumn liquid chromatography.131 and thus. showing that higher efficiency is obtained by the MECC methods with sodium dodecyl sulfate (SDS) as micelle-forming substance.125. which can be resolved on the basis on their mass-to-charge ratios that are characteristic of each ion and allow its identification. it is likely to cause overlap with the others. SDS is indeed the most used additive to form micelles in this kind of analysis.139 which is usually enough for food analysis or an LIF (laser-induced fluorescence) detector. isobutanol.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.136.123 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. Mass spectrometer detectors were first connected to GC equipments. 19 amino acids were analyzed by CE-ESI-MS in only 17 min with a minimal sample preparation and no matrix interference.136 phenylthiohydantoin. .21 chiral amino acids.141–143 The CE coupled to electrospray ionization (ESI) MS (CE-ESI-MS) allows direct amino acid analysis without derivatization.138 and OPA139 compared with the separation of OPA-amino acid derivatives by CZE with normal and micellar solutions. Some reviews covering high-sensitivity detection following CE have been published. This technique has also been termed micellar electrokinetic capillary chromatography (MECC or MEKC).). nonprotein amino acids.3. and others. methanol. which constitutes an important limitation of this technique.147 17.Essential Amino Acids ◾ 299 improve the resolution of one kind.122. compatible with MS. etc. in particular when capillary columns were available. Under the conditions of electro-osmotic flow in CE. Application in foods such as in the identification of nonprotein amino acids.133–135 PITC. Unfortunately. the species with different charge can be simultaneously analyzed but with serious doubts in their adequate resolution. biomedical or pharmaceutical research. although this detector may be used for more complex identifications as in d. The effect of these additives on the electro-osmotic mobility and electrophoretic mobility of the micelle has been studied. CZE shows poor ability for the separation of neutral compounds.124 Basic theoretical considerations on this technique125 and its food applications126 are described elsewhere. and so forth.127–131 derivatization is used to improve separation. When sensitivity is the target. o-tyrosine analysis. With few exceptions. Nevertheless. tetrahydrofurane.137. although other additives such as dodecyltrimethylammonium bromide. Good separations have been reported for precapillary derivatized amino acids with dansyl-Cl. it is relatively easy to analyze low picomol levels of OPA derivatives in micellar solutions by using a conventional fluorometric detector.140 or even urea141 have been assayed. etc. Other additives commonly used in this analysis are organic modifiers (acetonitrile. Terabe et al. and the composition and flow rate of the sheath liquid to obtain the best sensitivity.113 o-tyrosine in chicken148 or pork149 tissues.144. This report includes the optimization of important parameters like the choice of a volatile electrolyte (1 M formic acid) for the electrophoresis. reports in the literature of its applications are increasing rapidly.and l-isomer mixtures. The identification of the 22 protein amino acids may not be a problem. or to allow fluorescence or electrochemical132 detection of amino acids.) and instrumentation (CE.4 Mass Spectrometry MS is based on the conversion of components of a sample into rapidly moving gaseous ions.).119 have been reported. to enhance UV detection. etc. A good compatibility between both techniques. the high cost of purchase and maintenance of mass spectrometers has inhibited their more widespread use in the food industry and/or food control. allowed a rapid development and the onset of these complementary techniques.

T. and the technique is widespread although it is still expensive. RP-HPLC methods with precolumn OPA or PITC derivatization are very convenient methods to use. Food Sci. because fewer peaks appear in the chromatogram. Particular hydrolysis problems related with certain amino acids are described in Section 17. Fisheries Sci. offering the additional advantage of analyzing the amino acids without derivatization. nowadays these difficulties have been overcome with the development of new interfaces.2. Shimada. the analytical technique for a determined sample must be carefully chosen based on the literature. K. and ESI. S. and many applications can be found in other matrices like cheese or meat. H. and so on. G. . The highest resolution is obtained by GC with the capillary column technique. Hayashi. 1981. is enough for the majority of purposes. cation exchange and postcolumn derivatization or RP-HPLC precolumn derivatization techniques are the preferred methods. Yamanaka. small peptides. The connection of HPLC and MS detector is much more problematic than with GLC because of the incompatibility between both techniques (solvents from chromatography. and taking care of avoiding the presence of oxygen with vacuum and nitrogen purging. Adv. may appear in the chromatogram. once again. 185–236. Three types of ionization modes. which means a minor sample manipulation. When amino acids from seafood proteins have to be analyzed. the first decision is the choice of the hydrolysis method. 35. One of the main requirements for samples to be analyzed by MS is that analytes. Therefore. reduced problems related with matrix interferences or poor resolution between peaks.152 A very careful control of the derivatization reactions and chromatographic conditions are necessary for a consistent and reproducible analysis. In general. due to its high specificity. Yamaguchi. acid hydrolysis with HCl 6 N (110°C for 22 h or 145°C for 4 h) with an oxidation protective agent. 17. 2.4 Conclusions To obtain the total essential amino acids profile of a given seafood. The majority of published reports in which seafood amino acids are analyzed have used the cation exchange method.. but tedious and time-consuming sample derivatization is required. Nevertheless. were compared by Kwon and Moini150 in relation to sensitivity. nucleosides. the most important factors to take into account are the resolution power and selectivity.. Swaisgood. atmospheric pressure microwave-induced plasma ionization (AP-MIPI). Catignani. and.2. The best results were obtained by using AP-MIPI in conjunction with a dual oscillating capillary nebulizer. Since many peaks corresponding to protein and nonprotein amino acids. References 1. 821–824.300 ◾ Handbook of Seafood and Seafood Products Analysis MS has also been used as a spectroscopic detector after HPLC or CZE. which may be consulted. R. 479–483. However. the convenience of purchasing commercially available prepackaged kits should be considered. a complete resolution of the whole peaks is really difficult.151.. Any separation strategy may give good results. The convenience of purchasing commercially available kits must be evaluated. Sensory analysis of taste-active components in the extract of boiled snow crab meat. H. 3. 1991. J. 46.. 1996. Protein digestibility: In vitro methods of assessment. must be ionized. The requirements in resolution are not so exigent as those for physiological amino acids. Post-mortem biochemical changes in the muscle of Japanese spiny lobster during storage. Konosu. Food Nutr. Res. 62.E. In general.L. atmospheric pressure chemical ionization (APCI). high mobile-phase flow rate vs. such as phenol. vacuum). and. amino acids in this case.

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.2.........310 18.......................4...311 18..2 Ascorbic Acid Analysis ....3 Occurrence of Ascorbic Acid in Marine Organisms .........................3...........................................318 18...............................................................1................3 Vitamin E .......................1 Introduction ...................................3................3 Marine Lipid Oxidation ..........314 18........3..313 18..........................................1 Oxidative Stress and Its Implications ..3...........316 18..............314 18...........................316 18....................................310 18..............1....318 309 ...........................3..................................1................................1..............................2...Chapter 18 Antioxidants Nick Kalogeropoulos and Antonia Chiou Contents 18.................................................................3............1 Oxidation and Its Implications...........................314 18.....................1.........1 Ascorbic Acid Functions ........................4......................2 Vitamin E Determination ......3......................3.........1 Antioxidant and Other Functions of Carotenoids ......3......2 Determination of Antioxidants and Antioxidant Capacity in Biological and Food Systems .................................................................3......................317 18.313 18....1..3 Occurrence of Vitamin E .....................................................311 18....1 Antioxidant Enzymes ..........................................................4 Carotenoids .....3 Occurrence of Carotenoids .317 18..........2 Antioxidants.........1 Vitamin E as an Antioxidant.............. 315 18................2..................................................................................................................317 18..............310 18.........................3................ 315 18...3.....................................3............................1..............313 18................................................................3.......................311 18.........2 Ascorbic Acid .........4............................................................................................................................2 Carotenoid Determination ..................3 Antioxidants in Seafood and Seafood Products ....2 Lipid Peroxidation.................3........3...1.........

.............1 Synthetic Antioxidants ........................ since oxygen is the final electron acceptor in the electron flow system that produces energy........... generation of free radicals occurs........... Common free radicals in biological systems are the so• called reactive oxygen species (ROS). more than 2 billion years ago............................... resulting in an increased antioxidant activity and antioxidants . nucleic acids.........3........3............. Until subsequent free radicals are deactivated.....................4... 320 18................... Although the initial attack causes the neutralization of the free radical. where antioxidants are mainly produced by photosynthetic organisms and are consequently transported through the trophic web....... Humans and most animals cannot synthesize the majority of these antioxidants and depend on the dietary intake from plant consumption................................ lipids..... 320 18.. Aging.........................321 18..... 18........5...... and carotenoids.1. 18........ and hydroxyl hydrogen peroxide (H2O2).....3.1 Oxidative Stress and Its Implications Oxidative stress occurs when the prooxidant–antioxidant balance becomes too favorable to the prooxidants....2 Determination of Ubiquinone .....1 Indeed cyanobacteria and the laterevolved green plants.......319 18.......3 Occurrence of Ubiquinone.............................. that is........................ 320 18........319 18... such as peroxynitrite (ONOO−)......... Under conditions of oxidative stress........................319 18................6 have been reported to cause oxidative stress to fish or bivalves......4 and environmental stress5.............. peroxyl (ROO •)..2 Natural Antioxidants ......310 ◾ Handbook of Seafood and Seafood Products Analysis 18.........................4 Added Antioxidants ..321 References ... being exposed to the oxygen they produce......3...... as a defense against oxygen toxicity......... thousands of free radical reactions may occur within a few seconds..6 Other Endogenous Antioxidants ........3.... polyphenols........ electrically charged compounds that seek out and capture electrons from other compounds in order to neutralize themselves........... This is also followed in the marine environment....1 Introduction Antioxidants evolved together with the emergence of photosynthesis by cyanobacteria.................. and the so-called reactive nitrogen species (RNS)............................4... ROS production in organisms is related to both the basal metabolism and the influence of environmental factors2.. they may oxidize several cell components........... When the electron flow becomes uncoupled (transfer of unpaired single electrons)..... alkoxyl (RO •)....... resulting in a chain reaction...............1...5 Ubiquinone ........................ and proteins may be damaged by reactive oxidants......... nitric oxide (NO (OH•) radicals.................. which include among others superoxide anion radical (O2−)........1.......5........................319 18............... if ROS are not immediately intercepted by antioxidants......................... are rich in antioxidants such as vitamins C and E...............3 pollution.......1 Function of Ubiquinone ..........1 Oxidation and Its Implications Oxidation is the transfer of electrons from one atom to another and represents an essential part of aerobic life.... •)........ another free radical is generated in the process....................5..............

Three reaction pathways have been proposed: (1) nonenzymatic chain autoxidation. This can be especially damaging to lipid-rich cell membranes.13 Antioxidants counteract oxidation in two different ways: They protect lipids from oxidation initiators. chain-breaking antioxidants.12 In biological systems various biochemical defense mechanisms. mainly of plant origin. because of their high degree of unsaturation. are essential for counteracting oxidative stress.2 Antioxidants Antioxidants are defined as any substance that when present at low concentrations compared with those of an oxidizable substrate. etc. including enzymatic systems and nonenzymatic antioxidants. The health implications of tissue lipid oxidation are numerous and well documented.11 and unhealthy compounds that reduce their shelf life and nutritive value. Autoxidation occurs through a three-phase process. atherogenic. and metal ions. free radicals.9 18. Nevradical oxidation propagators (LOO ertheless. initiation. antioxidants supplied by foods.Antioxidants ◾ 311 loss or oxidation product development. heat.). mutagenic. Lipid oxidation is a complex procedure induced by oxygen in the presence of initiators such as light. 1O2. protect the cellular components from oxidative damage. 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. propagation. and (3) enzymatic oxidation. carcinogenic. for which the human sensory apparatus has a low threshold.8 Studies on the pathological significance of dietary lipid oxidation products have indicated that some lipid oxidation products have cytotoxic.1.9 Preventive antioxidants hinder ROS formation or scavenge spe• cies responsible for oxidation initiation (O2−. For food systems.1. antioxidants often act via more than one mechanism that combines different types of antioxidant activity. In the first two cases a combination of reactions involving 3O2 and 1O2 occurs. that is.10 Lipids deteriorate in seafood products during processing. preventive antioxidants. marine lipids are relatively more susceptible to oxidation. and angiotoxic effects. Nonradical photooxidation seems to be a minor reaction compared with the 3O2-induced radical chain autoxidation. There is increasing evidence that oxidative stress is implicated in the pathogenesis of many inflammatory and degenerative diseases and conditions. and termination. in which polyunsaturated fatty acids on lipid molecules are attacked and oxidized. (2) nonenzymatic and nonradical photooxidation.2 Lipid Peroxidation A very damaging effect of oxidant reactive intermediates is lipid peroxidation. and they stall the propagation phase. antioxidants are .7 18. handling.1. The major components of the antioxidant defense system together with their proposed mechanisms of action are presented in Table 18. Moreover. significantly delays or prevents oxidation of that substrate. Lipid oxidation of omega-3 polyunsaturated fatty acids (PUFA)-rich food products results in the development of particularly unpleasant off flavors.1.1.1.3 Marine Lipid Oxidation Compared with other food lipids. Chain-breaking antioxidants intercept •) or participate in halting radical chain propagation. 18.3. In general. and storage. antioxidants are molecules that protect macromolecules from being oxidized.

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

3 Antioxidants in Seafood and Seafood Products In living organisms. oxidative damage to macromolecules is controlled by two types of antioxidant systems. blue-green algae. 18. for quality control of fish oil and fish oil-containing foods. and high-performance liquid chromatography (HPLC) alone or combined with mass spectroscopy. or Fe in their active site. thiols. The available methods have been reviewed by Rajalakshmi and Narasimhan.19 concluded that.1 Cu/Zn SOD were purified from marine fish tissues. kidneys. the correlation of instrumental and sensory methods with multivariate data analysis should be followed. At higher levels most of them behave as prooxidants possibly due to their involvement in the initiation reactions. widely distributed in aerobic cells that help in preventing the accumulation of H2O2 within cells.3.3. and uric acid. Zn.1 Antioxidant Enzymes Catalases are metal-containing enzymes. Kolanowski et al. coenzyme Q.20 . carotenoids. glutathione (GSH). polarographic.3.18 In reviewing methods and tests for the assessment of lipid oxidation.. and crustaceans from the Mediterranean sea. and in red muscle compared with that in white. one represented by enzymes and the second represented by low molecular mass compounds. cephalopods. Catalase activities ranged between 386 and 1523 mmol/ min/g tissue in several Atlantic fish and was higher in liver.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.17 and Wood et al. spleen. the latter including paper.Antioxidants ◾ 313 effective at very low concentration levels. and algae. bilirubin. 18. column chromatography and the more sophisticated gas chromatography (GC). whereas Fe SOD were purified from red algae.21 In several species of teleosts. These antioxidants act in a concerted way to protect sensitive molecules such as the unsaturated fatty acids from oxidation.2 and 18.16 and Laguerre et al. voltammetric. and heart. and chromatographic methods. Cu.22 whereas in nine