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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Editors

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

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

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Contributors

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

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

Contributors

xv

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

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

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Contributors

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

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

Contributors

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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

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

CHEMISTRY AND BIOCHEMISTRY I .

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

1970: 4 million tons. mostly Pangasius species).4 ◾ Handbook of Seafood and Seafood Products Analysis number of species and variability.7 million tons).1 million tons). burden of pollutants. appearance. The major aquaculture (excluding plants) producers (>1 million tons) in 2005 were China (32. 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).6 million tons). Chub mackerel (2. and Norway (2. whereas crustaceans are fourth by quantity at 6. India (3.0 million tons).1 million tons). Blue whiting (2. whereof the major part are cyprinids like carp). 25% is converted into fishmeal and other nonfood products. nutritional status.3 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.9 million tons).6 million tons). 1980: 7 million tons.1 million tons). About 75% of the world’s total seafood supply is used for human consumption. India (2. Aquatic plants that are popular in Southeast Asia are second in quantity at 23. They deteriorate at ambient temperature in a few days.2 million tons). Mollusks (bivalves and cephalopods) are the third most important group both by quantity and by value at 22. including plants) [1]. Another difference compared with land-living animals is the fact that the quality (size. fish is the top group in aquaculture at 47. 40% is consumed as wet fish without any further technological processing or preservation. about 20% is converted into deep frozen products. infestation with parasites. aquaculture is dramatically growing (1960: 2 million tons. Thailand (2. The most important primary product producing countries of marine and inland (freshwater) fisheries in 2005 were China (17. Alaska Pollock (2.8 million tons).4 million tons). and sensory properties. respectively. The world’s aquaculture provided 52 million tons (36%). 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.) of aquatic animals when captured by fishing techniques is—with few exceptions—completely unknown. Most fish was caught in the Pacific Ocean (Northeast and Southeast) followed by Northeast Atlantic Ocean. Skipjack tuna (2. The total world seafood supply for 2007 amounted to 143 million tons. Russia (3. 8% is transformed into cured products.000–35. The fish group alone is represented by 25. Although land-based animals are today tailor made according to industry’s and consumer’s wishes in weight. the United States (4.4 million tons.2% but second by value at 20. 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. body composition. and the captured fish.4 million tons.2%.4%. 91 million tons (64%) of the total supply.3% and 14. However.4%.2 million tons). The top 10 species being caught in huge amounts in 2005 were Anchoveta (10. Vietnam (1. fish and other seafood are highly perishable products when stored without chilling. 1990: 16 million tons. Peru (9.5 million tons).4 million tons).3 million tons). and another 8% into canned products. Largehead hairtail (1. By major groupings. Atlantic herring (2.4% by quantity. and Yellowfin tuna (1. Indonesia (1.4 million tons).4 million tons). Japan (4. etc. Further. Further. The stagnation of captured fish is mainly due to fully exploited or partially overfished stocks.1 million tons).3 million tons). Japanese anchovy (1.1 million tons). and Thailand (1.000 species. . Indonesia (4. 2000: 40 million tons.3 million tons).8 million tons). only some of these 5% have the desired sensory properties and give a good or satisfying fillet yield that catching and processing them can be justified. Chile (4. Chilean jack mackerel (1. state of maturity.

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

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

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

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

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

2006. U. Surrey.K. 456p. 11. and Oehlenschläger. 363p. 15. Control of Fish Quality (3rd edn. 2008... Børresen.. (Ed. Wageningen.. Anon. Careche. VCH.-K.. 2004.. Pommepuy. L. Methods to Determine the Freshness of Fish in Research and Industry. 396p.. Paris.K. J. G.B. et al. State of world aquaculture 2006. (Eds.. Woodhead Publishing Limited. Luten. and Heia. Børresen. 500. K. 134p. T.). Oehlenschläger. Oehlenschläger.R.). 2.. Cambridge. Wageningen. 3. et al. Cambridge. J. Nunes.. 1990.. and Oehlenschläger. in Quality of Fish from Catch to Consumer—Labelling. Børresen.. in Improving Seafood Products for the Consumer. 2009. 10. U. (Eds. Wageningen. 5.. 1998. et al.. Lee.J.).. and Olafsdottir.M.). J.). Knöchel. et al. U. 212p. Olafsdottir. (Eds. (Eds. Connell. Luten. FAO.B. (Eds. Oehlenschläger. 12. in Improving Seafood Products for the Consumer. et al. Luten. T.). Aachen... in Improving Seafood Products for the Consumer. Detecting virus contamination in seafood. Multisensor for fish quality determination. Fishery Products—Quality.K. 2003.. Botta. 1995. 180p. Reducing microbial risk associated with shellfish in European countries. 7. Evaluation of Seafood Freshness Quality. Wageningen Academic Publishers. Fishing News Books. Olafsdottir.. Cambridge. B. J.J.. J. Safety and Authenticity. FAO Fisheries Department. Bosch. A. No. 13. G. 247p. P. 57p. Jacobsen C. Jørgensen. Luten. Rome. V. Martinsdottir. Shaker Verlag GmbH. Bacterial pathogens in seafood.. M. 567p. and Olafsdottir.). Barr. Quality of Fish from Catch to Consumer—Labelling. Wageningen Academic Publishers.. SEQUID: A New Method for Measurement of the Quality of Seafood. et al.. Rehbein. Sæbø. U. G. T. J.. 2008. J. R.. M. K. Verrez-Bagnis. T. Woodhead Publishing Limited. Tejada. J. J. 14. G. Farnham. A study of the attitudes of the European fish sector towards quality monitoring and labeling.. Wageningen Academic Publishers. (Ed.. 86. New York. 227p. 477p. 2003. (Eds.). in Improving Seafood Products for the Consumer. A. 2008.. WileyBlackwell. .K. Woodhead Publishing Limited. Kent.). 194p. Woodhead Publishing Limited.. Monitoring and Traceability. FAO Fisheries Technical Paper. M. E.). M. 2005. Bekaert. (Ed. Seafood Research from Fish to Dish. Technol. (Ed. Monitoring and Traceability. Børresen. J.. Cambridge. Trends Food Sci. International Institute of Refrigeration. Thorkelsson.). U. 8. Dalgaard. 4. J... H. G. 2006. 2008.10 ◾ Handbook of Seafood and Seafood Products Analysis References 1. Mild processing techniques and development of functional marine protein and peptide ingredients.B.. 9. 216p. 6.. R.

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

The Kjel-Foss® instrument mechanizes the entire micro-Kjeldahl procedure while the Kjel-Tec® instrument uses a digestion block together with an apparatus for automated distillation and titration. The factor can be calculated from the amino acid composition of the proteins. The Kjeldahl method was first published in 1883 but has been extensively modified since then. 2.56 [1]. which has been used for more than 75 years.2 Total Content of Proteins The total content of proteins is usually determined by the Kjeldahl or the Dumas method. and trapping of ammonia and titration steps. The Dumas method is quicker and cheaper and easier to perform and is therefore now considered on equal terms with the Kjeldahl method [1]. Many proteins have protein contents that deviate from this. which is distilled and trapped in boric acid.25 is usually used. gelling. The method includes sample digestion. assuming a nitrogen content of 16% in the proteins. It is important that the methods to analyze food proteins are robust [1]. has been shown to be too high for animal proteins. and textural properties. it is difficult to start using other and more correct factors [5]. For animal proteins the value 6. neutralization. Retaining the functional properties through preservation and processing operations is therefore of great importance. distillation. This factor is the amount of nitrogen that contains 1 g of nitrogen. the water-holding capacity and the gelling properties which determine the textural attributes of the products are important quality parameters [3].25. Originally only sulfuric acid was used for digestion of the samples. nitrogen from other nitrogen-containing compounds such as free amino acids. and urea will also contribute to the calculated protein content. For products such as fish mince and surimi. The disadvantage is that the method requires use of hazardous and toxic chemicals. emulsification. The Technicon AutoAnalyzer uses continuous flow analysis [1]. . This method is used as a reference method by many national and international organizations. both different types of raw materials and processed foods.82 are given. The Kjeldahl method determines the nitrogen content as ammonia. When the protein content is calculated based on determination of the nitrogen content. but other chemicals such as potassium sulfate and mercury oxide are also used. For fish. This means that it should be possible to use the method on different types of foods. for instance collagen has a nitrogen content of 18%. The ammonium sulfate is converted into ammonia. nucleotides. The method should also require minimal sample pretreatment to decrease analytical error and reduce costs. and the amount of protein is calculated by multiplication with a Kjeldahl factor. which gives a factor of 5. In addition to the high nutritional value.5]. During digestion the nitrogen in the sample is converted to ammonium sulfate. and the amount of nitrogen is determined by titration [1]. It is also possible to determine the nitrogen content using elemental analysis (C/N analyzers) [4]. The Kjeldahl method has been automated and several instruments for automated analysis are available.12 ◾ Handbook of Seafood and Seafood Products Analysis essential amino acids lysine and methionine. amines. It is also possible to determine the amount of ammonia by different colorimetric methods [1].43 to 5. and that other components in the food such as lipids and pigments should not interfere with the analysis. Tables of conversion factors are given in several papers such as [1. The advantage of this method is that it gives accurate results for all types of samples. Mariotti and coworkers discuss conversion factors in their critical review and conclude that even if a factor of 6. fish proteins also have good functional properties such as water-holding capacity. values from 5.

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

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

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

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

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

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

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

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................................................................ 28 3.3.... 24 3..............2 Proteome Analysis by 2DE .................1 Whole Larval Proteomes........................3.............. 24 3.............2 Basic 2DE Methods Overview .......................................3 The Degradome .............. 34 3............................31 3..............2................2.... 34 Acknowledgments ...........................................................3.......... 22 3.............................. 28 3.......................... 32 3..2..........................................................1 Protein Autolysis and Oxidation during Storage and Processing ........2..............3..............25 3...........................................3 Species Authentication ......................... 32 3............................2..................................... 28 3......................................................25 3..........................................................2.2........Chapter 3 Proteomics Hólmfríður Sveinsdóttir.25 3...........................1 Sample Extraction and Cleanup ....2.............................2......................................................2.................2.3....2 Aquaculture and Antemortem Effects on Quality and Processability ..................................................................31 3.......2 First-Dimension Electrophoresis ........ Ágústa Guðmundsdóttir............................................................ and Oddur Vilhelmsson Contents 3....................... 22 3........................4 Second-Dimension Electrophoresis ............................3.............2.......... 22 3...........................................................................5 Staining ............................2.....2................................ 27 3......2.........................2..................................................6 Analysis ..................................................................35 21 .................................4 Allergen Identification ......35 References ...2 Quality Involution .............................................................................................................2...........2..33 3......................................................................3 Protein Identification by Peptide Mass Fingerprinting ...................................1 Sample Matrix Considerations .....................................1......................1...3 Equilibration .........................1 Development ........................... 27 3...3 Applications of 2DE in Seafood Analysis ...................2.. 22 3.2 Muscle Proteomes ...1..........................................................1 Introduction ....................2....

Like other vertebrates. or with the human immune system after consumption.13 skeletal muscle. then. in the following sections. is the simultaneous separation of hundreds. both on the cellular and tissue-wide levels. the interactions of proteins with one another or with other food components. the “classic” process of two-dimensional (2D) gel polyacrylamide electrophoresis (2DE) followed by protein identification via peptide mass fingerprinting of trypsin digests (Figure 3.12 kidney. is regulated and brought about by proteins. where the bulk of the food matrix is constructed from proteins. 3. Proteome analysis allows us to examine the effects of environmental factors on larval global protein expression. This chapter will therefore focus on 2DE.9–11 heart. along with some of the main improvements that have developed since. for example.4 hold great promise and are deserving of discussion in their own right.1 Whole Larval Proteomes The production of good quality larvae is still a challenge in marine fish hatcheries.2 Proteome Analysis by 2DE 2DE.22 ◾ Handbook of Seafood and Seafood Products Analysis 3. 3. growth depression. giving valuable insight into the composition of the raw materials. 3. is a tool that can be of great value to the food scientist. the cornerstone of most proteomics research. largely because of its high resolution. the proteome varies from tissue to tissue. and mass accuracy. and low survival rate. Proteomics.6–8 liver.1. This is especially true of fish and meat. Several environmental factors can interfere with the protein expression of larvae leading to poor larval quality like malformations. Studies on whole larvae.12 and rectal gland12 have been reported. foodstuffs are in large part made up of proteins.2 surface-enhanced laser desorption/ionization3 or protein arrays. It stands to reason. simplicity. In the following sections. Selection of tissues for protein extraction is therefore an important issue that needs to be considered before a seafood proteomic study is embarked upon. Furthermore. that proteome analysis. we present some issues and challenges related to sample matrices of particular interest to the seafood scientist. based on liquid chromatography tandem mass spectrometry (LC–MS/MS). of proteins on a 2D polyacrylamide slab gel.2. also known as proteomics. While high-throughput. 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. or even thousands. fish possess a number of tissues amenable to 2DE-based proteome analysis. during. quality involution within the product before. .19 intestine.12 brain.1 Sample Matrix Considerations Unlike the genome.1) remains the workhorse of most proteomics work.12. as well as with time and in response to environmental stimuli.12.1 Introduction As with all living matter. the construction of the food matrix. succinctly defined as “the study of the entire proteome or a subset thereof”1 is currently a highly active field possessing a wide spectrum of analytical methods that continue to be developed at a brisk pace. and after processing or storage.2. gel-free methods.14–18 gill.

Only a few proteome analysis studies on fish larvae have been published. Nevertheless. it is excised from the gel. posttranslational modifications and redistribution of specific proteins within cells.6. In many cases this is sufficient for identification purposes. Once a protein of interest has been identified. First. where the majority of the highly abundant proteins were identified as muscle proteins.22 Also.21.22 Three of these publications have focused on the whole larval proteomes in Atlantic cod (Gadus morhua)6.20 all important information for controlling factors influencing the aptitude to continue a normal development until adult stages.7. a protein extract (crude or fractionated) from the tissue of choice is subjected to 2D PAGE. allowing identification of ca. cytoskeletal .6. 85% of the of the selected protein spots.22 and zebrafish (Danio rerio). there are several drawbacks when working with the whole larval proteome. such as the gastrointestinal tract or the central nervous system. subjected to degradation by trypsin (or other suitable protease) and the resulting peptides analyzed by mass spectrometry. These proteins may mask subtle changes in proteins expressed in other tissues or systems.1 An overview over the “classic approach” in proteomics. but if needed.6.7 These studies provided protocols for the production of high-resolution 2D gels.23 This is reflected in our studies on whole cod larval proteome. yielding a peptide mass fingerprint. The axial musculature is the largest tissue in larval fishes as it constitutes approximately 40% of their body mass. like the overwhelming presence of muscle and skin proteins. See text for further details.Proteomics ◾ 23 2D PAGE Trypsin digestion MS fingerprinting MS/MS sequencing Figure 3. peptides can be dissociated into smaller fragment and small partial sequences obtained by MS/MS. Peptide mass mapping using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry was performed only on the cod larval proteins.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.

and biochemistry.3 The Degradome The degradome may be a subproteome of particular interest to the food scientist. preventing identification of holistic alterations in the analyzed proteomes. it may also result in a loss of other proteins. Proteomic analysis on lysosomes has been successfully performed in mammalian (human) systems.30 preparative isoelectrofocusing31 or solubility in the presence of various detergents32 or chaotropes33 have been described.2. whereby proteins are targeted for destruction by the proteasome by covalent . protein turnover is a major regulatory engine of cellular structure. and simply increasing the amount of sample is usually not an option.45. Removal of those proteins may increase detection of other proteins present at low concentrations.29. For example.34–41 3. has profound implications for quality and processability of the fish flesh. Cellular protein turnover involves at least two major systems: the lysosomal system and the ubiquitin–proteasome system. such as actin and tubulin. In addition to having a hand in controlling autolysis determinants. but for other applications low-abundance proteins.5 The remaining option.1.43 The 20S proteasome has been found to have a role in regulating the efficiency with which rainbow trout (Oncorhynchus mykiss) deposit protein. fish skeletal muscle is the main component. No amplification method analogous to PCR exists for proteins. are particularly abundant in the skeletal muscle proteome. A myriad of methods suitable for subsequent 2DE exist for fractionating the proteome into defined subproteomes. in which protein deposition is regulated.2 Muscle Proteomes In most seafood products. allowing a larger sample of the remaining proteins to be analyzed. The fish muscle proteome is therefore likely to be of comparatively high interest to the seafood scientist. are suitable for rigorous investigation using proteomic methods. Fractionation methods for a variety of sample matrices have been reviewed recently. as many textural and other quality factors of muscle foods are related to proteolytic activity in the muscle tissue before. Swamping of low-abundance spots by highly abundant ones may not be a problem for applications relating specifically to structural proteins. such as the ubiquitin–proteasome or the lysosome systems. as it will give rise to overloading artifacts in the gels.2.42. are of keen interest. particularly in muscle tissue. function. then.44 It seems likely that the manner.1. Protein turnover systems. is fractionation of the protein sample in order to weed out the high-abundance proteins. 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. during and after processing. such as those associated with individual organelles or cell compartments28 or by protein biochemical methods such as affi nity chromatography. Structural proteins. Various strategies have been presented for the removal of highly abundant proteins24 or enrichment of lowabundant proteins.24 ◾ Handbook of Seafood and Seafood Products Analysis proteins were prominent among the identified proteins. However.46 An exploitable property of proteasome-mediated protein degradation is the phenomenon of polyubiquitination. An unfractionated 2DE map of the muscle proteome therefore tends to be dominated by comparatively few high-abundance protein spots.25. which include most regulatory proteins and many important metabolic enzymes. rendering analysis of low-abundance proteins difficult or impossible.26 3.

Although a number of refinements have been made to 2DE since O’Farrell’s paper. which proteins are being degraded by the proteasome at a given time or under given conditions.52–57 3.2 Some proteolysis systems.. or fluorescent dyes. may be less directly amenable to proteomic study. most notably the introduction of immobilized pH gradients (IPGs) for IEF.47 By targeting these ubiquitin-labeled proteins.51 the procedure remains essentially as outlined earlier. 0. In the following sections. 3.2. and how they vary with environmental or dietary variables. We have found direct extraction into the gel reswelling buffer (7 M urea.2. 3. For more detailed. such as Coomassie blue. such as that of the matrix metalloproteases.1 Sample Extraction and Cleanup For most applications. The map can be visualized and individual proteins quantified by radiolabeling or by using any of a host of protein dyes and stains. silver stains. Gygi and coworkers have developed methods to study the ubiquitin–proteasome degradome in the yeast Saccharomyces cerevisiae using multidimensional LC–MS/MS.e.” i.58 Thorough homogenization is essential to ensure complete and reproducible extraction of the proteome. which is most conveniently performed using commercial dry IPG gel strips.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 larvae6.3% (w/v) DTT [dithiothreitol]. Ready-made IPG strips are currently available in a variety of linear and . yielding a two-dimensional map (Figure 3. This separates the proteins according to their molecular charge. These strips consist of a dried IPG-containing polyacrylamide gel on a plastic backing.2 Basic 2DE Methods Overview O’Farrell’s original 2DE method first applies a process called isoelectric focusing (IEF). up-to-date protocols.2) rather than the familiar banding pattern observed in one-dimensional (1D) SDS-PAGE.48–50 Monitoring of the expression levels of these regulatory enzymes.2 First-Dimension Electrophoresis The extracted proteins are first separated by IEF. 2 M thiourea. a general protocol is outlined briefly with some notes of special relevance to the seafood scientist. Cleanup of samples using commercial 2D sample cleanup kits may be beneficial for some sample types. sample treatment prior to electrophoresis should be minimal in order to minimize in-sample proteolysis and other sources of experimental artifacts.Proteomics ◾ 25 binding to multiple copies of ubiquitin. where an electric field is applied to a tube gel on which the protein sample and carrier ampholytes have been deposited. it is possible to observe the ubiquitin–proteasome “degradome. Activity of matrix metalloproteases is regulated via a complex network of specific proteases.43. may be more conveniently carried out using transcriptomic methods. The tube gel is then transferred onto a polyacrylamide slab gel and the isoelectrically focused proteins are further separated according to their molecular mass by conventional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).22 and Arctic charr (Salvelinus alpinus) liver. 0. the reader is referred to any of a number of excellent reviews and laboratory manuals.2. 4% (w/v) CHAPS [3-(3-chloramidopropyl)dimethylamino-1-propanesulfonate].2.2.

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

3) and cemented in place using a molten agarose solution. Görg et al. 0.2. The gel is run at a constant current of 25 mA until the bromophenol blue dye front has reached the bottom of the gel. 3. remains the most popular one. avoiding trapping air bubbles. thus reducing vertical streaking. During the equilibration step. and that the strip is pressed gently onto the SDS gel. This is best performed using a dentist’s tool or other appropriate implement. it is applied to the top edge of an SDS-PAGE slab gel (Figure 3. The manufacturer’s instructions should be followed. that the gel side of the IPG strip faces the notched side of the glass plate.8. trace amount of bromophenol blue. 2% SDS. While some reviewers recommend alternative buffer systems. 1% DTT. taking care to put the pressure on the IPG strip’s plastic backing rather than the gel itself.1% SDS) at both electrodes.56 reviewed IEF for 2DE applications.2. This will alkylate thiol groups and prevent their reoxidation during electrophoresis. A typical equilibrationbuffer recipe is as follows: 50 mM Tris–HCl at pH 8.61 using glycine as the trailing ion and the same buffer (25 mM Tris. Figure 3.Proteomics ◾ 27 the equipment used.60 the Laemmli method. A tracking dye for the second electrophoresis step is also normally added at this point.2.59 3. it needs to be equilibrated for 30–45 min in a buffer-containing SDS and a reducing agent such as DTT.5% iodoacetamide and without DTT (otherwise identical buffer) may be required for some applications. 30% glycerol. the SDS–polypeptide complex that affords protein-size-based separation will form and the reducing agent will preserve the reduced state of the proteins.4 Second-Dimension Electrophoresis Once the gel strip has been equilibrated.3 Equilibration Before the isoelectrofocused gel strip can be applied to the second-dimension slab gel. Care must be taken that the (+) end of the strip is on the same side of all slab gels.3 Orientation and placement of an isoelectrofocused IPG strip onto the top of the second-dimension gel. 192 mM glycine. but for most applications gradient gels or gels of about 10% or 12% polyacrylamide are appropriate. Optimal pore size depends on the size of the target proteins. .2. Ready-made gels suitable for analytical 2DE are available commercially. A second equilibration step in the presence of 2. 6 M urea.

2.65–67 3. such as with [35S] methionine. These include radiolabeling. Multivariate analysis has been successfully used by several investigators in recent years.2.2. however.3 Protein Identification by Peptide Mass Fingerprinting Identification of proteins on 2DE gels is most commonly achieved via mass spectrometry of trypsin digests. spot matching between gels tends to be time-consuming and has proved difficult to automate. followed by staining for several days in 0.6 Analysis Although commercial 2DE image analysis software. has improved by leaps and bounds in recent years. such as ImageMaster (Amersham). analysis of the 2DE gel image.28 ◾ Handbook of Seafood and Seafood Products Analysis 3. and followed by destaining for several hours in water. followed by several 30 min washing steps in water.64 These difficulties arise from several sources of variation among individual gels.2. followed by incubation for 1 h in 17% ammonium sulfate/34% methanol/2% ortho-phosphoric acid. and the resulting peptide mixture is analyzed by mass spectrometry. UV-absorbing molecules (such as 2. Pooling samples may also be an option and this depends on the type of experiment. including protein spot definition. Patton published a detailed review of visualization techniques for proteomics. or Progenesis (Nonlinear Dynamics). such as Student’s t-test.1% Coomassie Blue G-250/17% ammonium sulfate/34% methanol/2% ortho-phosphoric acid. and therefore individual variation is a major concern and needs to be accounted for in any statistical treatment of the data. commonly used to assess the significance of observed protein expression differences. A great many alternative visualization methods are available. Briefly.5-dihydroxybenzoic acid) followed by ionization by a laser at the excitation wavelength of the matrix molecules and acceleration of the ionized peptides in an electrostatic field into a flight tube where the time of flight of each peptide is measured and this gives its expected mass. . The most popular mass spectrometry method is MALDI-TOF mass spectrometry. organic. 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.63 In particular. such as the SYPRO or Cy series of dyes. and staining with fluorescent dyes. There are. matching. A typical staining procedure includes fi xing the gel for several hours in 50% ethanol/2% ortho-phosphoric acid. digested with trypsin (or another suitable protease). commercially available colloidal Coomassie staining kits that do not require fi xation or destaining. 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. Also.62 3. gene expression in several tissues varies considerably among the individuals of the same species. and individual protein quantification. 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. remains the bottleneck of 2DE-based proteome analysis and still requires a substantial amount of subjective input by the investigator. such as in difference gel electrophoresis (DIGE). PDQuest (BioRad). such as protein load variability due to varying IPG strip reswelling or protein transfer from strip to slab gel.68 where peptides are suspended in a matrix of small. These multiple sources of variation has led some investigators63–65 to cast doubt on the suitability of univariate tests.

however.4 A trypsin digest mass spectrometry fingerprint of an Atlantic cod larval protein spot. In their work on the rainbow trout liver proteome.50 30 856.63 1258. As can be seen in Table 3.60 Peptides identified as those derived from Atlantic cod β-tubulin Trypsin autolysis peaks 1159.70 1697.35 1621.4) is then used for protein identification by searching against expected peptide masses calculated from data in protein sequence databases.50 20 10 0 741. 2506.4 2212. using the appropriate software. How useful this method is will depend on the length and quality of the available nucleotide sequences.06 1575.00 1196. The resulting spectrum of peptide masses (Figure 3.36 2564.25 Figure 3.9 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.1.8 1652. It is important to realize.96 60 870. 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. 100% agreement was observed between the two methods.Proteomics ◾ 29 842. Several programs are available. 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. which in many cases is more extensive than the protein sequences available. to obtain a tentative identity.69 1659. correlated activity measurements.71 100 90 80  Intensity 1061.54 50 40 1287.expasy.51 1040.56 1974.2 2798.43 . Martin et al.86 1028. identified as b-2 tubulin.07 1131. excluded from the analysis.10 and Vilhelmsson et al.0 1229.801616. this problem is surprisingly acute for species of commercial importance. The open markers indicate mass peaks corresponding to trypsin self-digestion products and were. The solid markers indicate the peaks that were found to correspond to expected b-2 tubulin peptides.61 1272.98 1886. To circumvent this problem. The ExPASy Tools web site (http://www.90 1822. therefore.6 Mass (m/z) 2108. it is possible to take advantage of the available nucleotide sequences.54 70 1960. In those cases where both the protein and nucleotide databases yielded results. many with a web-based open-access interface. such as the National Centre for Biotechnology Information (NCBI) nonredundant protein sequences database.org/tools) contains links to most of the available software for protein identification and several other tools. or transcript abundance).

489 130.006 121. scallops.008 Mollusca (Mollusks) Bivalvia (mussels. incl.) Astacidea (lobsters and crayfishes) Brachyura (short-tailed crabs) 21.046. incl.407 84.122 268 26. incl. etc.533 1.585 1.007 911 303 18.138 36.424 2.344 5.798 81.656 2.203 467. turbot.845 10. sole.287 8.30 ◾ Handbook of Seafood and Seafood Products Analysis Table 3. etc. sea bass. and pollock) Lophiiformes (anglerfishes.245 2.380 898.592 735 179 585 768. incl.864 47.) Gastropoda (incl.933 3. saithe.210 Chondrichthyes (Cartilagenous Fishes) Carcharhiniformes (ground sharks and dogfishes) Lamniformes (mackrel sharks) Rajiformes (skates and rays) 3.1 Families of Some Commercially Important Seafood Species and the Availability of Protein and Nucleotide Sequence Data as of March 27. whelks and abalone) Cephalopoda (squid and octopi) 32.896 3.751 Crustacea (Crustaceans) Caridea (shrimps.680 1.871 32. halibut.237 2.626 138 16.063 3. monkfish) Perciformes (perch-likes.381 45. tuna.353 287 170.442 237 2.208 2. sea bream.782. mackrel. cod.762 726. haddock.845 999. incl. 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.284 2.158 20. and wolffish) Pleuronectiformes (flatfishes. and plaice) Zeiformes (dories) Scorpaeniformes (scorpionfishes.557 . redfish and lumpfishes) 185.086 2.

enhances the specificity of the method even further.88.81 Gygi and Aebersold. which. These variations may.94 Proteome analysis provides valuable information on the variations that occur within the proteome of organisms.72 Today. posttranslational modifications. yielding a second layer of information. In the peptide mass fingerprinting discussed earlier.76 Nyman.77 Damodaran et al. and development.23.85 wheat flour baking quality factors.Proteomics ◾ 31 A more direct.71.. fish physiology.89 A brief discussion of a few emerging areas within fish and seafood proteomics is given as follows. Furthermore. the higher the number of possible combinations. if rather more time-consuming. proteomic investigations on fish and seafood products. as well as in aquaculture. the identified proteins consisted mainly of proteins located in the cytosol. way of obtaining protein identities is by direct sequence comparison.1 Development Fishes go through different developmental stages (embryo. and vastly superior protein spot identification techniques. several short stretches of amino acid sequence will be obtained for each peptide. and nucleus. Until recently. physiology. or redistribution of specific proteins within cells. for example. by Yates. when combined with the peptide and fragment masses obtained. 3. have gained considerable momentum. and behavior of the fish...82 Lin et al. In both these studies.78 Thiede et al. each peptide mass can potentially represent any of a large number of possible amino acid sequence combinations..95 resulting in different expression of proteins.73–75 Mass spectrometry methods in proteomics have been reviewed. The larger the mass (and longer the sequence). for example. improved reproducibility and resolving power of electrophoretic separation techniques. and adult) during their life span that coincide with changes in the morphology.93 This is reflected in the variations of global protein expression and posttranslational modifications of the proteins that may cause alterations in protein function. larva.3.3 Applications of 2DE in Seafood Analysis The two-dimensional electrophoresis has been in use within food science for at least two decades. Correlating this spectrum with the candidate peptides identified in the first round narrows down the number of candidates.84 3.90–92 The morphological and physiological changes that occur during these developmental stages are characterized by differential cellular and organelle functions.83 and Delahunty and Yates. reflect a response to biological perturbations or external stimuli9–11.86 and soybean protein bodies.20 To date few studies on fish development exist in which proteome analysis techniques have been applied.80 Mo and Karger. Early studies focused on relatively small. 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.79 Rappsilber et al. Proteome analyses in developing organisms have shown that many .87 With the lower cost. 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. cytoskeleton. this was accomplished by N-terminal or internal (after proteolysis) sequencing by the Edman degradation of eluted or electroblotted protein spots. the method of choice is tandem mass spectrometry (MS/MS). clearly defined subproteomes and included such applications as the characterization of bovine caseins.

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. 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. Furthermore.98 Different isoforms generated by posttranslational modifications are largely overlooked by studies based on RNA expression.2 Quality Involution Degradation of proteins during chilled storage.3.99.108 3. although degradation of myofibrillar proteins by calpains and cathepsins112. developmental stage specific muscle protein isoforms have gained a special attention.113 . and pI of the protein present in a tissue. be distinguished on 2DE gels.99–107 The developmental changes in the composition of muscle protein isoforms have been tracked by proteome analysis in African catfish (Heterobranchus longifilis). usually have different molecular weight or pI and can. many of which are correlated with specific textural properties in seafood products.94. Thus. In a recent study on the proteome of embryonic zebrafish. This fact further indicates the importance of the proteome approach to understand cellular mechanisms that underlie fish development. where differences are expected to occur in the number. the embryos were deyolked to enrich the pool of embryonic proteins and to minimize ions and lipids found in the yolk prior to 2D gel analysis.99–107 In this context. molecular mass.21.27 is probably due to dechorionation prior to the deyolking of the embryos.7 Despite this undertaking. specific isoforms of myofibrillar proteins. such as curing. whether they be encoded in structural genes or brought about by posttranslational modification.110 Problems of this kind.111 3. and their oxidation during frozen storage.21 and dorada (Brycon moorei).8. can be observed using 2DE or other proteomic methods. are well suited for investigation using 2DE-based proteomics.109. Link et al.3.2. Studies on various proteins have shown that during fish development sequential synthesis of different isoforms appear successively.88.102 common sole (Solea solea).27 published a method to efficiently remove the yolk from large batches of embryos without losing cellular proteins. These interfere with any proteomic application that intends to target the cells of the embryo proper.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. a large number of yolk proteins remained prominently present in the embryonic protein profiles. The success in the removal of yolk proteins by Link et al. several commercially important fish muscle processing techniques. the embryos fall out of their chorions facilitating the removal of the yolk.8.32 ◾ 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. fermentation. For example. By dechorionation.101 These studies demonstrated that the muscle shows the usual sequential synthesis of protein isoforms in the course of development. and production of surimi and conserves occur under conditions conducive to endogenous proteolysis.21. The major obstacle on the use of proteomics in embryonic fish has been the high proportion of yolk proteins. It is also worth noting that protein isoforms other than proteolytic ones. are among persistent quality problems in the seafood industry and have deleterious effects on fish flesh texture. therefore.

Proteomics ◾ 33 and degradation of the extracellular matrix by the matrix metalloproteases and matrix serine proteases114.127. the proteome analysis identified a number of metabolic pathways sensitive to plant protein substitution in rainbow trout feed.111. In the context of this chapter.117. Whatever may be the mechanism.123 found these to comprise several members of the glycolytic and Krebs cycle pathways. For example. flesh softening during storage. where individual physiological characteristics.112. furthermore. such as pathways involved in cellular protein degradation. Olsson et al. the effects on the proteasome are particularly noteworthy.10. etc. The diet was found to have a marked effect on product texture. appear to display seasonal variations.126 in fish fed with the experimental diets. the interplay between these physiological parameters and environmental and dietary variables needs to be understood in detail. as opposed to wild fish catching. To achieve that goal. affect the involution of quality characteristics in the fish product. Kjærsgård et al. the ubiquitin–proteasome pathway has been shown to be downregulated in response to starvation129 and have a role in regulating protein deposition efficiency. We are aware of two recent studies where Atlantic cod muscle proteomes have been compared between farmed and wild fish. Indeed.128 In rainbow trout.2 Aquaculture and Antemortem Effects on Quality and Processability It is well known that an organism’s phenotype.119. this is fast becoming feasible. 3.125 and the liver proteome was analyzed9. in turn. such as proteomics. Furthermore.17 used 2DE. 67. and NADPH metabolism.2. is determined by environmental as well as genetic factors.115 are thought to be among the main culprits. The proteasome is a multisubunit enzyme complex that catalyzes proteolysis via the ATP-dependent ubiquitin–proteasome pathway which.15. Huss noted in his review122 that product quality differences within the same fish species can depend on feeding and rearing conditions. such as those governing gaping tendency. including quality characteristics.123 Both studies indicated that several proteins are differentially expressed in farmed versus wild cod. is thought to be responsible for a large fraction of cellular proteolysis. it is clear.1 .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. in mammals.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. fatty acid breakdown. various quality characteristics of fillet and body were measured124.. and the amount and composition of free amino acids in the fish flesh. that these quality changes are species dependent116. Martinez et al. They found fish muscle proteins to be differentially carbonylated during frozen storage and were able to identify several carbonylated proteins using LC–MS/MS. The practice of rearing fish in aquaculture. differences that can affect postmortem biochemical processes in the product which. 2D-immunoblots and LC–MS/MS to study changes in protein oxidation during frozen storage of rainbow trout.117 and. therefore raises the tantalizing prospect of managing quality characteristics of the fish flesh antemortem.120 and have demonstrated the importance and complexity of proteolysis and oxidative changes in seafood proteins during storage and processing. are optimized. In a recent study on the feasibility of substituting fish meal in rainbow trout diets with protein from plant sources. With the ever increasing resolving power of molecular techniques.3.118 Several 2DE studies have been performed on postmortem changes in seafood flesh14–17.

The identity was further corroborated by cloning and sequencing the relevant cDNA. the proteomes of even closely related fish species are be easily distinguishable by eye from one another on 2D gels1 indicating that diagnostic protein spots may be used to distinguish closely related species.5% of young adults are allergic to shrimp.14 Unlike the genome.143 Indeed.140. blotted the 2D gel onto a PVDF membrane. the proteome varies from tissue to tissue and with environmental conditions. Penaeus monodon. They found the difference to be due to a single T to D amino acid substitution. These authors.143 Lopez and coworkers.141 More recently. From early on. as well as being relevant from a public health standpoint.147 at National Taiwan University. Martinez et al. During the 1960s. performed a 2DE on crude protein extracts from the tiger prawn.4 Allergen Identification Allergenic potential is food safety issue of particular concern to the seafood producer.3 Species Authentication Processed fish products are increasingly common in the market and. particularly for addressing questions on the health status of the fish in question. 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.3. The allergens were then identified by MALDITOF MS of tryptic digests. found that M. The allergen was identified as a protein with close similarity to arginine kinase.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. such as the gadoids or several flat fishes. about 0. Allergic reactions to seafood affect a significant part of the population. Mytilus galloprovincialis.139 These early efforts were reviewed in 1980. proteomics-based species identification methods are likely to develop rapidly and find commercial uses within this field.145 Seafood allergies are caused by an immunoglobulin E-mediated response to particular proteins.142. studying the cause of shrimp allergy in humans.34 ◾ Handbook of Seafood and Seafood Products Analysis 3.135–137 which was soon followed by methods to identify species in processed or cooked products. trossulus could be distinguished from the other two species on foot extract 2D gels by a difference in a tropomyosin spot. the presence of stress-factors or contamination levels at the place of breeding. and probed the membranes with serum from confirmed shrimp allergic patients.134 recently reviewed proteomic and other methods for species authentication in foodstuffs. A final proof was obtained by purifying the protein. 1D electrophoretic techniques were developed to identify the raw flesh of various species. including structural proteins such as tropomyosin.138. this makes the issue of species authentication an area of increasing economic importance.144 Martinez and Jakobsen Friis concluded that the identification of not only the species present. demonstrating that it had arginine kinase . Proteome analysis can therefore potentially yield more information than genomic methods.3. possibly indicating freshness and tissue information in addition to species. 2DE-based methods have been developed to distinguish various closely related species. and postmortem treatment.18. studying three species of European mussels: Mytilus edulis. 3. proteomic methods have been recognized as a potential way of fish species identification. For example. as different fish species have different market values. 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. and Mytilus trossulus. 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.

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.... Fisheries............................. which has revolutionized biology....................................... all fish that would be of use to us..................... some nearly destitute of scales.......................... the flesh of which is unrivalled.6 Concluding Remarks ........2 Genetics and Genomics...........................................1 Introduction The development of high-throughput DNA sequencing methods has opened the era of genomics.............. Twenty Thousand Leagues under the Sea 4........ 43 ..............................................Chapter 4 Seafood Genomics Astrid Böhne................................52 There the nets brought up beautiful specimens of fish: Some with azure fins and tails like gold........................................................................* Delphine Galiana-Arnoux.................. The rise of * Equal contributors...................... Jules Verne.......52 References ........................ as good as bonitos................. 49 4.5 Genomics and Aquaculture ...............* Christina Schultheis.....* Frédéric Brunet....................... and biotechnology over the last decade................4 Genomics................ 43 4........................... with bony jaws.1 Introduction ... medicine........... others............................51 Acknowledgments . and yellow-tinged gills.......................... and Jean-Nicolas Volff Contents 4......................45 4..47 4................. but of exquisite flavour........................................3 Genomic Resources and Genome Projects for Aquatic Species .......................................................... and the Management of Biodiversity ........................ 44 4...............

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

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]). For example. for example. Additional approaches are required to study gene expression (transcriptomics. shotgun sequencing. The relative position of two contigs can also be estimated cytogenetically using double fluorescent in situ hybridization [8]. 4. cloned in a bacterial vector and constituting a so-called genomic library. This provides a physical map respecting the “real” base pair distance between genes and markers. Large-scale expression studies at the transcriptional level are generally performed using microarrays or other methods of high-throughput expression profiling. 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.Seafood Genomics ◾ 45 A clone-by-clone approach can be used as an alternative to. see Ref. a new revolution of large-scale sequencing is ushering in a second era of genomics.org/).g. The overlapping between these clones and their relative arrangement in the genome can be determined through fingerprint analysis (e. with novel methods allowing very rapid and much cheaper sequencing of large amounts of DNA [11–13].. Barcoding is based on a sequence of short standard parts of the genome. 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. Generally. A method called “DNA barcoding” should help to identify species and phylogenetic units. for instance. Bacterial artificial chromosomes (BACs) accepting inserts from several hundreds of kilobases are frequently used as vectors. hereby contributing to species conservation and management of global fish biodiversity (http://www. useful in the case of regions rich in repetitive sequences posing problems to assembly after whole genome shotgun sequencing. Of particular interest are expressed sequence tags (ESTs). These fragments are either integrated in the genome of a host cell line from a different organism in radiation hybrid (RH) mapping [9] or diluted to give aliquots containing approximately one haploid genome equivalent (HAPPY mapping [10]). through the identification of common restriction fragments). can be sequenced either to completion or from their ends.fishbol. [16]). In addition. 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]. 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. obtained through sequencing of complementary DNA (cDNA) libraries. proteomics) and function (functional genomics) as well as interactions with the environment (environmental genomics). which can be very useful to precisely determine the relative position of sequence contigs assembled “in silico” from whole genome shotgun sequencing data.3 Genomic Resources and Genome Projects for Aquatic Species Genetic and genomic resources have been generated for many aquatic species of economical interest. zebrafish and medaka are two complementary fish models to study . for SNP detection and phylogenetic reconstructions. or even better in combination with. ESTs can also be used. Importantly. Such an approach is. 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.

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

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

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

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

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

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

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

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....................................2 Nucleotides and Nucleosides Determination ........3................................................1 31Phosphorous-Nuclear Magnetic Resonance Spectroscopy .. Aleida S..................................61 5.............59 5........3. the adenosine triphosphate (ATP) regeneration that occurs in vivo stops and ATP is degraded until 57 ............... Thus..............................59 5................................3.......2.......... or eviscerated fish) or canned fish...4 Enzymatic Analysis..........................3...........................2.3 Analysis of ATP-Related Compounds ..................... the autolytic process derived from tissue enzymatic activity and lipid oxidations also contributes to fish maturation and subsequent spoilage......2 Chemical Structure of Main Seafood Nucleosides and Nucleotides ....57 5.61 5.............................61 5.....................Chapter 5 Nucleotides and Nucleosides M.............61 5...........65 5......3....2............... objective methods for freshness determination are required and the determination of the biochemical changes occurring in early postmortem in fish constitute a helpful tool............................ 64 References .................. Nevertheless.... Leticia Mora. Sensory methods to evaluate fish quality are subjective and difficult to use in the evaluation of processed (fillets.............................. 60 5..1 Introduction Bacterial growth is the main factor limiting fish commercial life by producing its alteration and unpleasant flavor......2 Capillary Electrophoresis ....................................................3.....................1 Introduction ........................ Concepción Aristoy.............. Hernández-Cázares............... The first autolytic process taking place in fish affects carbohydrates and nucleotides. beheaded........................................ After death.2.....................................................1 Extraction of Nucleotides and Nucleosides ........................ and Fidel Toldrá Contents 5...3 Chromatography.....

whereas AMP remains major in crustaceans. As a result of endogenous enzymes action. IMP degradation to inosine (Ino) and its disappearance have been correlated with lack of freshness in some fish species. . (2006) published a review of the concentration of IMP. IMP is the main nucleotide present in fish species.4 However. which is oxidized to xanthine (Xa) and uric acid in the presence of xanthine oxidase (XO) enzyme.1. 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. 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. ATP molecule is rapidly degraded to adenosine monophosphate (AMP) and afterward to inosine monophosphate (IMP).1 Degradation of ATP in postmortem fish muscle. and either of the two may be used as freshness indicators. although it might be accelerated by the action of different bacteria.2 Howgate et al. Ino and Hx concentrations increased during storage. This process involves a series of reactions commonly represented according to the sequence shown in Figure 5.3 In all cases. This enzyme is mainly generated in muscle from biochemical processes of microorganisms. the use of a single compound as freshness indicator is not always advisable. and Hx in the flesh of some species of fish during chilled storage. Ino.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. Inosine is transformed to hypoxanthine (Hx) by the action of the enzyme nucleoside phosphorylase (NP). which is accumulated in postharvest fish.

K value is defined as the ratio of Ino and Hx to the sum of ATP and related compounds expressed as a percentage.17 5.14 Measurement of ATP-related compounds is also useful for the quality control of retorted fishes.12 However.7–9 Nevertheless. or hypoxanthine is attached to a ribose. also. to which one or two additional phosphate groups are attached through pyrophosphate bonds (∼P) (Figure 5. ATP.2). and it is important to stop this reaction drastically at the sampling time. Nucleotides are o-phosphoric acid esters of the nucleosides. Ino. whereas IMP evokes a fresh meaty taste sensation. ADP and ATP are derived from the AMP. This is achieved by immediately freezing the excised muscle under liquid nitrogen to stop all enzymatic reactions. 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. a high content of Hx is related with the bitter off taste of spoiled fish. The ratio Hx/AMP was considered an adequate alternative to characterize fish freshness due to its constant increment with time. ATP-chain degradation occurs very fast. generally within 1 day of storage in ice after death in all fish species.15. IMP is derived from the inosine in which a phosphate group is attached to the 5-ribose carbon. Some of them are briefly described here. respectively. and thus. These cold conditions must be held along the sample preparation.2. . adenosine diphosphate (ADP). AMP or adenylic acid is derived from the adenosine in which a phosphate group is attached at the 5-ribose carbon. 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.11 and.5 and. for several species. On the other hand. Nucleosides are glycosylamines that are formed when a nucleobase (purine or pyrimidine base) attaches to a ribose or deoxyribose ring. 5. the knowledge of their molecular structure is important.13. a high accumulation of Ino occurs during ATP degradation.18 After this. making K value inadequate as a freshness indicator.Nucleotides and Nucleosides ◾ 59 nucleotide degradation such as the type of spoilage bacteria and mechanical handling of fish. Nucleosides currently analyzed in seafoods are those in which a purine ring. In this way. often designed K ′ value or Ki index. the disappearance of the degradation products differs from one species to another3 as mentioned here. even at refrigeration temperatures.6 This value has been used as one of the freshness indexes to evaluate the quality change of postharvest fish. adenine. In order to achieve this rapid freezing.3 Analysis of ATP-Related Compounds The correct analysis of ATP-related compounds must take into account that early postmortem fish muscle is very sensitive to temperature. nucleotides and nucleosides should be extracted and analyzed.16 Another suggestion to use nucleotide compounds as a measurement of seafood quality is their relation with sensory attributes. consequently. and AMP disappear early postmortem.16. 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. it is advisable to collect small tissue samples and immerse them into liquid nitrogen. This is the main reason for the use of indexes with more than one compound from the ATP-degradation chain. and Hx expressed as percentage. a revised K value.10.2 Chemical Structure of Main Seafood Nucleosides and Nucleotides To a better understanding of the methods of analysis of these compounds. For this reason. forming the adenosine or inosine.

Once the extract is centrifuged (15. although storage at −18°C has been demonstrated to be enough to preserve fish samples and fish extracts for the analysis of IMP. is the following: 5 g or less of muscle tissue are excised and quickly frozen with liquid nitrogen.60 ◾ Handbook of Seafood and Seafood Products Analysis Adenosine nucleoside N N NH2 OH HO P O O OH P O O O P O OH HO O N N OH Ribose Adenine purine base AMP ADP ATP Adenosine nucleotide Figure 5. and the tissue is homogenized with a stomacher-type homogenizer for a few minutes under cold conditions. These fish extracts are used in enzymatic assays with biosensors19. This neutralized extract is kept in an ice bath for 15 min and centrifuged again (15.000 g for 10 min). The supernatant is filtered through a 0.5–6.3. and Hx. 5.2 μm membrane filter and stored under frozen storage at temperatures below −20°C until analysis.000 g for 20 min).22 .000 g for 15 min). cold 0.20 and/or spectrophotometers as well as in capillary electrophoresis (CE)21 or ion chromatography (IC). The neutralized extract must be made up to 5 mL with 20 mM phosphate buffer pH 7. they are neutralized with 2 M sodium hydroxide. 3–5 vol.5 g of fish sample with 10% trichloroacetic acid and.17 Other extraction methods consist in the homogenization of 2. the supernatant is filtered through glass wool and neutralized to pH 6.45 mm membrane.8 and then filtered with a 0.1 Extraction of Nucleotides and Nucleosides A typical extraction procedure for the analysis of fish samples by reversed-phase chromatography. with or without employing an ion-pairing agent.2 Structure of adenosine-derived nucleotides. The frozen tissue is minced.6 M perchloric acid is added.8 by adding solid potassium carbonate or 1 M potassium hydroxide. after centrifugation (27. Ino. avoiding any thawing.

22 and enzymatic assays.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. this technique can present problems in reproducibility. the addition of an ion-pair to the mobile phase greatly improves the separation by increasing the retention time of charged molecules (ATP. including fish extract.26 reversed-phase high-performance liquid chromatography (RP-HPLC) with and without ion pair. pH 11.2. and hypoxanthine would be a potential of 416 V/cm of capillary using 100 mM 3-[cyclohexylamino]-1-propanesulfonic acid (CAPS) buffer.3. Thus. and the K′ or K i index will be usually enough to characterize fish .25 thin-layer chromatography (TLC). Capillary electrophoresis has been used in many nucleotide analysis applications as in the study of nucleotide degradation in fish tissues.24 5. nucleotides will disappear at the rigor mortis state (normally 1 day after catch). which may be adsorbed on capillary walls. ADP. In particular.Nucleotides and Nucleosides ◾ 61 In the development of biosensor analysis. In the analysis of complex biological samples. among other chromatographic techniques. The mode of separation will depend on the analyte of interest.23. because these samples usually contain significant amounts of ions.28 Also in vitro 31P-NMR spectroscopy has been applied to both excised tissue and perchloric acid extracts of fish muscle. ion-exchange HPLC. HPLC has been shown to be the most widely used technique to analyze nucleotides and nucleosides.3. to analyze nucleotides. Thus.2.2.3. in vivo 31P-NMR spectroscopy has been used as a powerful technique to characterize the biochemical changes that occur in live.2 Nucleotides and Nucleosides Determination Several methods have been used to measure nucleotides and nucleosides.3 Chromatography At present.21 Typical conditions to get a good separation of IMP. radioimmunoassay.19 5. RP-HPLC and ion-paired reverse-phase are the methods of choice for this analysis.27 IC. both a microwave oven at 500 V for 5 s and heating at 100°C for 60 min have been used. 5. However. AMP). high-performance capillary electrophoresis (HPCE). 5. Nevertheless. intact fishes after being submitted to physical and chemical stressors such as hypoxia. the reconditioning of the capillary surface is ensured by washing 1 min with 1M NaOH. including nuclear magnetic resonance spectroscopy (NMR).3.2 Capillary Electrophoresis CE is a powerful separation technique that can provide high separation efficiency and high sample throughput with minimal sample volume and buffer consumption. inosine. followed by 2 min of the running buffer used. some authors have described extraction methods that consisted of heated fish sample. In this way.

29 phosphorylated metabolites are also well separated in the chromatogram.29 With buffer pH 7. The column used is an analytical reversed-phase RP-18 column.62 ◾ Handbook of Seafood and Seafood Products Analysis freshness or quality. (4) AMP. 5.3. In Figure 5.3. (3) ADP. and (6) inosine. (5) hypoxanthine. . 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. a simple RP-HPLC with a phosphate buffer as mobile phase will be adequate. (1) IMP. The separation was achieved with an RP C-18 column at 35°C and a gradient between phosphate buffer at pH 7 and acetonitrile. 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 5.17.3 RP-HPLC chromatograms of standards (a) and hake (b) ATP-derived compounds.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.2.3 both chromatograms of standards and hake nucleosides and nucleotides are shown.1 Reversed-Phase HPLC The chromatographic analysis should be performed in a liquid chromatograph equipped with an UV detector (254 nm). Quantitative analysis can be performed by external or internal standard method. which differ mainly in the pH of the mobile phase. There are many approaches to analyze nucleotides and nucleosides by this technique. Then. (2) ATP.

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

respectively. Ino. while the depletion of oxygen is measured by a Clark-type elec- . because no interference of salt in the medium was observed here as was in the case using the HPLC method. electrodes. the analysis may be performed with one or more enzymes.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. oxidizes the hypoxanthine to xanthine and uric acid. and IMP may be determined spectrophotometrically by a sequential addition of XO. 5.4.3. or sensors have a limited shelf life. This option offers some advantages in relation to the free enzyme.39 This procedure was also used to analyze ATP and related compounds in fish sauces with very good results.8 and 30°C–37°C.33.64 ◾ Handbook of Seafood and Seafood Products Analysis 5. inosine. and hypoxanthine will be oxidized to uric acid and H2O2.2. environmental.41 or for the evaluation of chicken32 and beef meat33 aging. the application in the food industry is still restricted36 mainly due to critical stages such as enzyme immobilization or sample preparation for analysis. although these applications used to be achieved with at least one of these enzymes immobilized as described earlier.1 Enzymatic Methods with the Enzyme in Solution The concentration of Hx.3.4 Enzymatic Analysis The use of enzymatic methods to analyze nucleotides in seafood is widespread due to their high specificity. and 5′-nucleotidase (NT) into a reaction phosphate buffer containing the fish extract sample at pH 7.6–7. agricultural. because the denaturalization of the enzymes with time.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. although biosensors have shown its utility in some applications such as clinical.36 Nevertheless. In these conditions. IMP.20. In addition. 5. The depletion of oxygen or the formation of hydrogen peroxide or uric acid may be detected amperometrically.31 Another possibility consisted in monitoring the oxygen consumption after these enzymatic reactions with an amperometric-type sensor (oxymeter).2. Indeed. enzyme-coated strips.4. These assays may be carried out with the enzymes in solution31.2. constituting what is known as enzyme sensors o biosensors. which will be further quantified by measuring the absorbance at 290 nm and by polarimetry. and biotechnology. which is further coupled to a chemical transducer. due to its specificity. In this sensor. and rapid response.20.2 Enzymatic Methods with Immobilized Enzymes In this case. This sensor has been developed mainly for assessing the freshness of fish meat40.32 or immobilized. in which an enzyme or a group of enzymes are immobilized in a membrane or other supports. simplicity. and thus test kits. NP. the use of commercial kits or disposals presents some problems. 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. but they remain immobilized in different supports. which is immobilized in a membrane fi xed in the sensing area of the electrode. These enzymes act by oxidizing the substrates (analyte) while consuming oxygen or producing hydrogen peroxide or uric acid. all the approaches to date need the sample preparation described earlier. XO enzyme.3.35 The most used is the biosensor based on the measure of hypoxanthine.36–38 The most used biosensors for the nucleotide-related compound analysis are electrochemical sensors.

E. Nucleotide degradation in sardine (Sardina pilchardus) stored in different storage condition at 4°C. IMP. Lugo-Sánchez. and Hx amounts.. Fish. 2005. K i. some authors have described this type of biosensor coupled with an oxygen electrode. An immobilized NT was used for the previous conversion of IMP to Ino. Thus. Surette. 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. Sci. 1988. Most recent approaches to determine Hx are based on the incorporation of the XO enzyme in a graphite/Teflon matrix.44 a polyaniline film by electropolimerization.. Matsuyoshi.Nucleotides and Nucleosides ◾ 65 trode at a platinum cathode (−0.A. R.. Y. which can be present in the sample or formed during the enzymatic reaction. M.L. Soc.33 although this relation should be confirmed in each particular system. A similar application was proposed12. an Ag/AgCl reference electrode. Sci.51 The use of multienzymatic biosensors to measure fish freshness has been very helpful for the simultaneous determination of AMP. D. Pacheco-Aguilar. 65: 40–47. Gill.. Özogul. P.56 References 1. E.E. J. Japan). K.E. IMP.. and. Ino.47 On the other hand. Massa.L. M. Food Biochem. Ino was converted to Hx.. Mendes. In the measurement of hypoxanthine. Quinta..23. 2007. T. Food Res. 36: 19–22. et al.. The consumed oxygen produces a current decrease that can be correlated to the concentration of Hx. 1: 13–19. Özogul. Food Sci. 7.53. J. Technol. Int.. In this way. Jpn. different supports have been used for the immobilization of the XO enzyme.. 24: 749–750. Changes in baseline levels of nucleotides during ice storage of fish and crustaceans from the Portuguese coast.18 and a silk fibron membrane in combination with a cellulose acetate membrane42 or a nylon net43 have been used. thus.48 IMP.. M. cellulose triacetate. A new method for estimating the freshness of fish. or H2O2. Postmortem biochemical and functional characteristic of Monterey sardine muscle stored at 0°C. 1959. Robles-Burgueno. Bull. 2000. 41: 341–353. Formed Hx was measured with an amperometric sensor that detected uric acid + hydrogen peroxide in an additive matter. M. and Nguyen52 and afterward it has been commercialized as a Freshness Meter KV-101 (Oriental Electric Co.E. Howgate.54 In fact. Biochemical basis of postmortem nucleotide catabolism in cod (Gadus morhua) and its relationship to spoilage.55. Kuley.. 1 mol of Hx would be converted to 1 mol of uric acid and 2 mol of hydrogen peroxide. Luong and Male20 used a multienzymatic biosensor system to determine the H value as a fish freshness indicator. Postmortem changes in quality indices of ice-stored flounder (Paralichthys patagonicus).45 a nafion-coated platinum disc electrode. P. 212: 141–146. M. 5. specific biosensors to determine AMP. Arai. and then after adding a soluble NP.6 to −0. Most of these supports have been developed with the aim of eliminating interferences due to ascorbic acid. F. Palacios. 29: 570–590.9 V) vs.46 or even in a carbon paste electrode modified with electrodeposited gold nanoparticles. Food Chem. . A review of the kinetics of degradation of inosine monophosphate in some species of fish during chilled storage. 2001. Food Sci. and Hx using a cellulose triacetate membrane have been described.41 preactivated nylon. 6. Eur. J. A.34 to obtain the Ki parameter as a freshness indicator. and H values. Male. Paredi.. 2.49 and Ino50 and a multienzymatic sensor to analyze simultaneously AMP. Comparable results to that of HPLC were reported. Ltd. J.53.. Nunes. 2006. Technol. Ino. This method was patented by Luong. T. The proposed relation 1 Hx for ½ X for each oxygen molecule formed must be taken into account to quantify the Hx. uric acid. R..R. Saito. J.J. Both Hx and X are substrates for the XO action and will be oxidized either simultaneously or sequentially. 4. Fish. Agric. LeBlanc. M. necessary to obtain K. R. 3.

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

.....5......................................... 75 6.......5 Qualitative and Quantitative Analyses of Marine Lipid Classes ......................................... 73 6..............1............1..... 71 6........1.....................2 Marine Lipid Characteristics .............................................1 Lipid Saponification...................3............ 77 6..........3 Column Chromatography............................................. Aubourg Contents 6............3.3..............................................................2........3...........................................................................................................76 6...........76 6........ 78 6...........1 Fatty Acid Methyl Esters Preparation ...76 6..1 Spectrophotometric Assessments of Total Lipid Extract .. 78 6......... 73 6......1 General Aspects of Lipid Compounds ..............................................2 Stereospecific Analysis of Lipid Classes ..............................................2............................... 72 6.........................5....................................................1.................2.....................2......................................................................................... 73 6...................76 6............................2 Quantitative Estimation of Fatty Acid Composition ..............................2 Base-Catalyzed Transesterification ..................1 Introduction .............3 Analysis of Ether Lipids .................. 77 6........4 Lipid Quantification ..................................4.......4 Analysis of Marine Nonsaponifiable Matter . 73 6...............................2 Analysis of Sterols ......5......................................................2..............................................1 Acid-Catalyzed Esterification and Transesterification ...................................................................................................................2..................... 75 6............................ 79 69 ....... 73 6.......................................2 GLC Analysis of FAME....................................................................... 75 6...............1 Qualitative Analysis of Fatty Acid Composition ...1...............................Chapter 6 Lipid Compounds Santiago P..................................... 72 6..............................................4.... 70 6........................2 First Steps in Marine Lipid Analysis ........4..................................................3..1 Isolation of Lipids from Tissues .........3 Lipid Manipulation and Storage .... 70 6.....2 Removal of Nonlipid Contaminants ....... 72 6....................3 Lipid Analysis in Marine Products ......3......3 Marine Fatty Acid Analysis .................. 70 6..................

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

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

72 ◾ Handbook of Seafood and Seafood Products Analysis 6. Although there are limitations to its use and alternatives are frequently suggested. which employs chloroform–methanol (2:1) in a solvent–tissue ratio of 20:1. 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. this method applies a single-phase solubilization of the lipids using chloroform–methanol (1:1) in a solvent–tissue ratio of 4:1.2.2 First Steps in Marine Lipid Analysis 6. it is advisable to include an additional antioxidant at a level of 50–100 mg/L to the solvents.1 Isolation of Lipids from Tissues Ideally. 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.. . In addition. may on occasion be extracted by these when they are in the presence of large amounts of simple lipids such as TG. all solvents can contain contaminants. As an advanced alternative. and as large volumes of solvents may be used to obtain small amounts of lipids. or lyso-phosphatidylglycerols in lipid extracts. 0. the procedure of Bligh and Dyer [9] offers some advantages as it does not use large volumes of solvent. For all extraction methods. peptides.88% KCl) [8]. The second problem is endogenous lipolytic enzymes that can lead to large amounts of unesterified fatty acids.g. phosphatidic acid. When this is not feasible. such as proteins. Two main problems can arise with lipid fraction when employing inconvenient storage conditions. However. 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. amino acids. though more time-consuming. and salts. Its employment has recently been reviewed [10]. most workers in the field appear to accept two basic routines currently in general use. Pure single lipid classes are soluble in a wide variety of organic solvents. Most of the contaminating compounds can be removed from the lipid extract mixtures simply by shaking the combined solvents with one-quarter their total volume of a dilute salt solution (e. which yield essentially quantitative extractions of the major lipid classes when applied to homogenates of whole marine tissue extractions.2. [8]. diacylglycerides. it should not be so polar that TG and other nonpolar simple lipids do not dissolve and are left adhered to the tissues. although endogenous tissue antioxidants can provide some protection. a major driving force being the environmental concern regarding the use of organic solvents. First. marine tissues should be extracted from the living organism as soon as possible after catching or slaughtering [2]. the tissue should be kept frozen (about −60°C or less) as rapidly as possible. A more elegant and complete. The most popular is the method of Folch et al. polar complex lipids. PUFA can autoxidize as a result of endogenous oxidant enzymes. 6. urea.2 Removal of Nonlipid Contaminants Most polar organic solvents used to extract lipids from tissues also extract significant amounts of nonlipid contaminants such as sugars. which do not normally dissolve readily in nonpolar solvents. any such impurities can be troublesome. At the same time. supercritical fluid extraction shows an increasing demand. This type of washing procedure was first developed by Wells and Dittmer [11] and simplified later by Wuthier [12] for large numbers of samples. Where large amounts of tissue have to be extracted.

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

16.8.11.10.9. the double-bond configuration is “cis.10.14.19-Docosapentaenoic 4.15-Octadecatetraenoic 5.12-Octadecadienoic 9.7.14-Eicosatetraenoic 5.19-Docosahexaenoic Linoleic Linolenic Stearidonic Araquidonic EPA DPA or clupanodonic DHA or cervonic In all cases.15-Octadecatrienoic 6.74 ◾ Handbook of Seafood and Seafood Products Analysis Table 6.17-Eicosapentaenoic 7.8.12.13.12.13.16.11.” .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.

2 GLC Analysis of FAME The advent of GLC revolutionized the analysis of the fatty acid components of lipids. However. 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. aldehydes are not liberated from plasmalogens and amide-bound fatty acids are not affected. FFA are not esterified. whereas aldehydes are liberated from plasmalogens under acidic conditions.Lipid Compounds ◾ 75 from amide-bound lipids (sphingolipids) are also transesterified. 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. although potassium methoxide or hydroxide have also been used as catalysts.3. FAME are obtained by heating the reaction mixture in a stoppered tube at 50°C overnight. glasspacked columns were widely employed [18]. . accordingly. In order to guarantee complete solution of nonpolar lipid classes.2.2 Base-Catalyzed Transesterification O-acyl lipids are transesterified very rapidly in anhydrous methanol in the presence of a basic catalyst. a PUFA loss. prepared simply by dissolving fresh clean sodium in dry methanol. This reagent has been applied directly to fish muscle to obtain FAME without previous lipid extraction [17]. the application of wall-coated open tubular (WCOT) columns to the analysis of fatty acids has provided a better knowledge of the complexity of marine fatty acids [19]. The reagent has a limited shelf life unless refrigerated. 6. Later on.1 Qualitative Analysis of Fatty Acid Composition During the previous decades. Initially. Parallel to ECL value employment. The commonest and mildest reagent used for the purpose is anhydrous hydrogen chloride in methanol.1. This is simply prepared by adding acetyl chloride slowly to cooled dry methanol. parameters known as equivalent chain lengths (ECLs) or carbon numbers have considerably been employed.3. so most performances have been carried out for qualitative and quantitative analysis [16]. As with acid-catalyzed procedures. However. The retention times of the unknown acids should be measured under identical operating conditions. Boron trifluoride in methanol is also used as a transmethylation catalyst and in particular as a rapid esterifying reagent for FFA. and the ECL values are read directly from the graph. an additional solvent is necessary to solubilize nonpolar lipids such as cholesterol esters or TG. and the use of old or too concentrated solutions may result in the production of methoxy-substituted acids from unsaturated fatty acids and. a further solvent such as toluene should be employed. under base catalysis. 6.3. A different possibility consists of employing a solution of 1%–2% (v/v) concentrated sulfuric acid in methanol. Transesterification is carried out in the same manner and at much the same rate as with methanolic hydrogen chloride. The commonest reagent used for this purpose has been sodium methoxide in anhydrous methanol.

as these are easily prepared and are widely used in chromatographic analysis.4 Analysis of Marine Nonsaponifiable Matter 6.16]. linearly proportional to the amount (by weight) of material eluting from the columns [15. or oxygenated chains. the resulting FFA have to be transformed into their corresponding FAME for further analysis by an acid-catalyzed method. quantitative results would first be calculated on its basis. Finally. On the other hand. Problems of measuring this area arise mainly when components are not completely separated. If necessary. and polyenoic fatty acids should be analyzed under the same GLC conditions for checking the quantification results. pyridinecontaining derivatives. the areas under the peaks on the GLC traces are. based on the Liebermann–Buchardt reaction. Results can be expressed as weight percentages of the fatty acids present or as molar amounts of each fatty acid. Total sterol content can be determined directly and spectrophotometrically from the lipid extract by using the method of Huang et al. branched. A high proportion of the available data has been obtained for the methyl ester derivatives of fatty acids. sterols can be fractionated and analyzed by means of different . within limits. and there is no way of overcoming this difficulty entirely. 6.4.2 Quantitative Estimation of Fatty Acid Composition With reliable modern gas chromatographs equipped with flame ionization detectors (FID). unsaturated. cyclic. as well as the deacylated residues of ether lipid compound. A known quantity of an internal standard should be added to the lipid sample. calibration factors may have to be calculated for each fatty acid component to correct the areas of the relevant peaks in the mixtures analyzed.3.4. such as picolinyl esters. However. the fatty acids on one side and diethyl-ether-soluble nonsaponifiable materials on the other side are separately recovered for further analysis [2]. Such compounds can be divided into sterols and “ether lipids. In most cases. 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]. 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]. [22].2.1 Lipid Saponification Lipids may be hydrolyzed (saponified) by heating them under reflux with an excess of dilute aqueous ethanolic alkali. have been shown to be suitable for direct mass spectrometric structural analysis of acids containing straight. monoenoic.2 Analysis of Sterols Sterols are biological compounds.76 ◾ Handbook of Seafood and Seafood Products Analysis In recent years. 4. commercially available standard mixtures containing accurately known amounts of methyl esters of saturated.” 6. On the other hand. For a complete analysis. this is specially relevant for PUFA. the basic structure of which includes the cyclopentanophenanthrene ring. nonadecanoic acid is employed and added before the methylation step. 6. the nonsaponifiable layer will contain any long-chain alcohols and sterols originally present in the lipid sample in the esterified form.

v/v) as a solvent system. it can be cleaved by acid-catalyzed transmethylation.24].3 Analysis of Ether Lipids Marine lipids may contain fatty acid residues as the only radicals. which in turn migrate just in front of TG. Adsorption thin-layer chromatography (TLC) on silica gel layers can be used to separate simple alkyl and alkenyl lipids. marine sterols have to be converted into more volatile compounds such as acetate [25] and trimethylsilyl (TMS) [26] derivatives. Cholesterol has been shown to be the most abundant sterol in all marine living beings. although invertebrates have shown a significant presence of other sterols [27]. trifluoroacetate. high-performance liquid chromatography (HPLC) methods can offer a nondestructive alternative. TMS. and combinations of techniques must be used until the required purposes are served. The first type is the major one in marine lipids. The alkyl groups of 1-alkyl-2. Often. although a great interest has been accorded to their isolation because of their medical and cosmetic applications [30]. Chromatographic methods for cholesterol analysis [28] are of relevant importance in foods in relation to human health concerns.32]. batyl.4%) in 2M HCl. In this section. thus. Neutral plasmalogens may be detected by spraying the TLC plates with 2.4. but they suffer from the limitation of the lack of a distinctive chromophore in the analyte. and 18:1. Concerning alkenylglycerols. chimyl. such as acetate.3. The alkyl moieties are usually analyzed in the form of 1-alkylglycerol or as volatile nonpolar derivatives of this compound. and its analysis has already been discussed in Section 6. information on ether lipid composition in marine PL is less abundant. Although the GLC is normally carried out with cholestane as internal standard. 6. or isopropylidene derivatives by GLC. being normally placed as the radical in position 1 and specially abundant in marine invertebrates [5. Further. supercritical fluid chromatography (SFC) has been employed for the glycerol ether analysis of liver oils of shark species [34]. and selachyl alcohols were found to be the most abundant. Methods for separating and quantifying ether-linked glycerides have been reviewed [31.27].Lipid Compounds ◾ 77 chromatographic techniques [23. alkenyl compounds have been directly identified and quantified by GLC together with FAME [35]. Although the vinyl ether linkage is unaffected by basic hydrolysis conditions. 6. The others are often united into a group called “ether lipids. mostly). or they may include alkyl and alkenyl radicals.3-diacyl-sn-glycerols are generally saturated or cis-monoenoic even– numbered components (16:0. neutral plasmalogens tend to migrate ahead of alkyldiacylglycerols.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]. For GLC analysis. different analytical . They can be separated by a double development in a single direction with hexane–diethyl ether (95:5. such compounds tend to be decomposed during GLC analysis and are best reduced by catalytic hydrogenation to alkylglycerols.4-dinitrophenyl-hydrazine (0. which generates dimethyl acetals from the liberated fatty aldehydes. Great attention has been accorded to the assessment of cholesterol oxide formation in marine products [29]. 18:0. no single procedure will achieve the desired analysis. 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]. Unlike fatty acids. whereas no simple spot test is available for the identification of alkyldiacylglycerols. Accordingly.” Such compounds are basically found as PL classes (specially in phosphatidylethanolamine).

5. [39] without previous digestion. these enzymes can be isolated and used in simple incubations in vitro as an aid in structural analyses of lipids. This is due to the great complexity of fatty acids present in these oils and fats. since the presence of double bonds in the proximity of a carbonyl group of fish PUFA reduces the rate of deacylation of glycerides. titrimetric methods were used for many years.3. 6. Compared with the data compiled for plant oils and for fats from land animals. This method can be applied to total lipid extract or to any lipid class after previous isolation [7.2 Stereospecific Analysis of Lipid Classes The determination of fatty acid composition at each location in lipid classes has ever since attracted great attention. traditional methodologies are still employed in cases where such advanced technologies are not available and are reviewed in this section. Procedures that involve spectrophotometric measurement of highly colored copper complexes are now favored. A wide use was found for lipase hydrolysis. then. HPLC.2 [22]. For FFA assessment. The fatty acid composition of each lipid class can be determined by GLC of the methyl ether derivatives. such functional groups are made to react with hydroxamic acid and further complexed with Fe (III).78 ◾ Handbook of Seafood and Seafood Products Analysis approaches will be discussed. Additionally. PL present in the lipid extract are made to react with ammonium molybdate in an organic phase and then measured spectrophotometrically. a method for the quantification of esterified and unesterified total sterols is mentioned in Section 6. where an FFA-cupric acetate-pyridine complex is involved.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. Traditional determination of PL content in lipid extracts has involved the digestion of PL with the release of inorganic phosphate. which is reduced and determined spectrophotometrically [38]. In many cases. preparative TLC on silicic acid impregnated with 5% (w/w) boric acid has been applied to prevent acyl migration during chromatographic separation. giving rise to a tremendous number of species.5. although some interference of polar lipids was found. prepared by esterification or transesterification of the purified lipid class. 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. 6. . in it. this is made to react with ammonium molybdate to form phosphomolybdic acid. the results so far reported for aquatic animals are few. although it turned out not to be accurate enough for marine lipids. this including chromatographic separation and further analysis of fatty acids after previous methylation and transmethylation. In it. However. Ester linkages can be quantified by the method of Vioque and Holman [40]. An alternative and successful method has been proposed by Raheja et al. according to details explained in Section 6. and GLC technologies combined with MS in the last decades has provided quick and useful procedures for the stereospecific lipid analysis. focused on the qualitative and quantitative analyses of marine lipid classes. Finally. Accordingly. A very popular one is that proposed by Lowry and Tinsley [36]. a rapid NIR spectrometry has been applied for the direct FFA determination in fish oil [37].41]. according to the information provided in following sections.4. More recently. the Grignard reagent has widely been employed in the case of marine substrates [42]. The advent of new NMR.

However.41].5 mm thickness [7. or florisil as adsorbents.5.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. MS. which has been used routinely for lipid analysis in the last decades. Separation can be carried out on silicic acid. whereas complex lipids are recovered by elution with methanol [41. and quantification [48. 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.47]. infrared (IR) spectrometry. which include highly automated techniques right from sample application and development to detection and quantification. It combines the separation capabilities of conventional TLC with the quantification power of the FID and has application in the quantitative analysis of all substances separable by conventional TLC.47]. Such techniques would include high-pressure TLC (HPTLC). For preparative purposes. Aminopropyl-bonded phase cartridges have been much used for the isolation of simple and complex lipid fractions. in spite of the relatively higher costs [50].5 High-Performance Liquid Chromatography In recent years. 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. diethyl ether. 6. lipid classes can be detected by any of the nonspecific available reagents and identified by their migration characteristics relative to authentic standards chromatographed simultaneously alongside the samples under investigation.49].5. and acetic (or formic) acid in various proportions.5. 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 tubular TLC (TTLC). HPLC has undoubtedly been the most widely applied separation technique in the analysis of most simple and complex lipid classes [48. In all cases. Such stereospecific studies have widely been focused onto TG [43.44] and PL [45.4 Thin-Layer Chromatography Many text books and reviews report TLC application on lipids for routine separations. The system has been successfully used for marine lipid class analysis [51].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. In addition. being simple lipids eluted in a stepwise sequence with hexane containing increasing proportions of diethyl ether. overpressure TLC (OPTLC). coupling of TLC with other techniques such as HPLC. The improvement and versatility of TLC enable it to be used for several modern applications.52]. Those used most frequently contain hexane. 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. although lengthy conditioning may be necessary before columns can be employed [2.46] classes. The perceived weakness of TLC has been recognized as the quantification aspect. and NMR has increased its analytical power in several applications. acid-washed florisil. identification. HPLC is much more expensive than . 6. although particular care is required to recover the acidic lipids quantitatively. precoated plates are much more convenient than laboratory-made plates. 6. and this has led to the evolution of the TLC/FID Iatroscan system.

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

5. mono. although GLC is conveniently coupled to electron impact ionization (EI) and chemical ionization (CI) sources. After different approaches. a rapid and structure-specific method for the determination of ω3-PUFA in fish lipids was presented.65. A good agreement could be observed between NMR values and those from the GLC analysis. 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. and stearidonic acid. This NMR technique can provide a single signal for each PL class. its intensity should be proportional to the quantity.8 Mass Spectrometry MS has long been used as a powerful tool for the analysis of the molecular weight. Thus. The information-rich nature of MS makes it the most desirable detector for many explanations. thermospray. 13C-NMR was employed for the plasmalogen analysis in fish lipid samples showing a good agreement with the data obtained by GLC [64]. and highly unsaturated fatty acids of lipid extract of Atlantic salmon muscle. ω6.66]. In a first attempt for 13C-NMR application [60]. Among the different food lipids. probably due to their more complicated structure. Recent developments in MS have been very interesting for complex lipid molecules. Signals in the spectra were assigned.95 ppm) with respect to the methyl resonance of all other fatty acids (δ = 0. marine lipids have received lesser attention. the molecules or their fragments can be separated and identified on the basis of their mass-to-charge ratio (m/z).and diene-. empirical formula. ω3. although an increasing importance has been obtained lately for quantitative analysis [20. Finally. the condensed mobile phase used for liquid separations is not readily compatible with high vacuum ionization sources. 2. 13C-NMR spectrometry was successfully used to determine the proportions of saturated. but. The different chemical shifts observed for the methyl resonance of ω3-PUFA (δ = 0. Later on [61]. The first step for any MS method is ionization of the sample molecules in the gas phase. and attention was focused on the identification of specific signals for ω3 fatty acid group and also individually for DHA. soft ionization MS techniques such as fast atom bombardment. 6. the high-resolution NMR spectra of four fish oils were recorded. FFA carbonyl resonances were detected at the lower field of the carbonyl region. so little application is specially available for marine lipids [58].Lipid Compounds ◾ 81 Based on 1H-NMR spectrometry [59]. Application of 31P-NMR has shown to be far shorter than with 1H and 13C. and electrospray have the ability to ionize lipid molecules without causing extensive fragmentation. Quantitative analysis of fatty acid composition and alpha-beta distribution in TG tuna fish was achieved [62]. EPA. and 3 locations) of ω3 fatty acids in depot fat of several fish species was examined by 13C-NMR [63]. according to each corresponding resonance. thus providing a suitable tool for lipolysis analysis. This development paralleled the development of atmospheric pressure chemical ionization (APCI). The positional distribution (1. many of the advances in MS have involved new ionization techniques.86 ppm) provided the possibility of proposing this new analytical tool. results obtained using high-resolution 13C-NMR were in good agreement with those obtained by GLC. . The 31P-NMR application has also shown the possibility of analyzing the ether structures within the glycerol backbone of phosphatidylethanolamine and phosphatidylcholine. Thus. and complete structure of an unknown compound. Some applications concerning the marine lipids’ study will now be mentioned. Following ionization to a negatively or positively charged species (most commonly the later). Over the years. Arpino [67] likened the HPLC-MS union. It could be observed that DHA was concentrated in the 2-location of TG in depot fats.

. and Oehlenschläger.4. An important advantage is that it is compatible with FID. References 1. The mass spectrometer was operated in the EI mode (70 eV). U. Nutritional aspects of fish. Elsevier Science. Börrensen. Integrated Approach to Quality. Handbook of Lipid Research. The Physical Chemistry of Lipids. whereas quantification of TG... 1986. 6.71]. Later on. Analytical SFC has been shown to be particularly applicable to the analysis of higher molecular weight lipid moieties. in Seafood From Producer to Consumer. in a first attempt Baltic herring flesh TG were separated in eight fractions by Ag+-TLC. analytes are eluted from a capillary chromatographic column. which uses a highly compressed gas above its critical temperature and critical pressure.-dimethyloxazoline derivatives [69]. Purification of PUFA (DHA and EPA) ethyl esters from tuna oil was carried out by SFC [74]. 3. J. Lately. an optimization of process parameters was achieved to obtain a maximal production rate.. A. 2.. 4. p. eds. and the four most unsaturated fractions were analyzed by capillary SFC according to their acyl carbon numbers [72]. Concerning marine species analysis. the use of SFC can substantially reduce the dependence on organic solvents in solvent extraction or HPLC analysis. p. J. W.. cholesterol esters. simple classes from marine oils of different species were separated and quantified by capillary SFC [73]. Vol. In addition. the method was based on the use of preparative reversed-phase HPLC followed by subsequent identification by APCI HPLC-MS. Plenum Press. p. 17. and a FID were employed in it.K. D. 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. such as mixed glyceride compositions ranging from 200 to 900 in molecular weight. The liver oils of several shark species were analyzed by SFC [34]. A wide range of cholesterol oxides were identified and quantified. .. Simopoulos. a nonpolar capillary column. the method was capable of direct quantification of squalene and cholesterol. and diacylglycerol ethers required TLC fractionation before SFC analysis. analysis was carried out in conjunction with FAME by means of their dimethyl acetal derivatives resulting from the acid transmethylation of lipid extracts. 89. Small. whereas its critical pressure and critical density are high enough for good solvation of many potential analytes. Lipid Analysis. Oxford.. U.5.K. Carbon dioxide is by far the most commonly used SFC mobile phase because of its low critical temperature. 1982. 589.82 ◾ 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]. New York.9 Supercritical Fluid Chromatography In this advanced technique [10. which has great sensitivity and linearity. 2nd edn. The qualitative and quantitative compositions of 1-O-alk-1-enylglycerolipids of albacore tuna (Thunnus alalunga) were studied along the canning process [35]. and several nonmethylene interrupted fatty acids were singled out. London.. Luten. Christie.. Pergamon Press. thus. carbon dioxide as the mobile phase. T. 1997. Minor fatty acids from mussels (Mytilus galloprovincialis) were enriched by Ag+-TLC and then analyzed by GLC-MS as 2-alkenyl-4.

King. D. V. A. p. 14. Lipid content in herring (Clupea harengus L. 15. 229. Lees. 1957. 1. in Advances in Lipid Methodology—Five. and Wrebiakowski. U.. in Marine Biogenic Lipids. and Gallardo. Purification of lipids from nonlipid contaminants on sephadex bead columns. poultry and fish. One-step conversion of fatty acids into their 2-alkenyl-4. Prost. F.. eds. 1. and Garrido. 2. 11. and Stanley. p. 1977... p. Champaign. 23. Technol. R. Biol. Elsevier Applied Science. 7. W. Lipid Res. 193. IL.... 149. p. 114. p. Christie. and New York.. in Analysis of Oils and Fats. 624. Huang. p. 671.K. and Rossell.. 1405. 27. R. 49. A. Champaign. J. Ackman. 1972. England. J.. Anal. 12.. Eur. and Panunzio. 2003. Lipid Res. M. J. 22.. J.. A simple method for the isolation and purification of total lipids from animal tissue. and Shorland. 1989. Ackman. Bridgwater. 479. U. J. 28. ed. J. Can. Ackman. in Marine Biogenic Lipids. H. 1989. p. in Analysis of Oils and Fats. Adlof. 37. Fats. 1995. ed. NIR and NMR. and Oils. Int.-dimethyloxazoline derivatives directly from total lipids. 5. Medina.. 2006. Chrom. 341. p. 17. in New Trends in Lipid and Lipoprotein Analyses. 113. WCOT (capillary) Gas–liquid chromatography. 23. 1995. R. Distribution and composition of lipids in marine invertebrates. 27. J. E. 301. Hyldig. Packed-column gas chromatography. 1990.Lipid Compounds ◾ 83 4.. The Oily Press. 369... Hammond. S. 108.). and Oils. C.. Seasonal study of the lipid composition in different tissues of the common octopus (Octopus vulgaris)... and Nielsen H. U. Technol. 2. Unters. 673. 1961.. Chem. and Rocha... J. 38. Adv. Boca Raton. Fatty Acids. Lepage. J... J. Pearson. in Handbook of Lipid Research. 341. T. E. A stable reagent for the Liebermann-Buchardt reaction. ed. FL. 6. and Rossell.. J. Fats and Oils. L. 1992... R. Food Sci. Food Sci. Fenton. p. FL. 2. G. Fatty Acids and Glycerides. 9. Wuthier. Patterson. R. A rapid method of total extraction and purification. Influence of biological factors and comparison of different methods of analyses: Solvent extraction.. “Warmed-over” flavor in meat. 20. Chrom.. Wefler. eds. E. 537. Forsch. J. Biochem. Sieiro. Phospholipids.. Gas chromatography-mass spectrometry and tandem mass spectrometry in the analysis of fatty acids. AOCS Press. 911. mollusks and fish. p. Evaluation of soxhlet’s and Bligh and Dyer’s methods in the determination of fat in meat. Kuksis. Chen.. Vol.. Technol. 1963.. 1959. J. Fatmeter... Ackman. Krzynowek. M. 226.K. Lebensm. eds. Sterols and crustaceans.. Boca Raton.. 1. Aubourg. Chromatographic methods in the analysis of cholesterol and related lipids. Vaskowski. 33. J.. 21. 558. Supercritical fluid chromatography (SFC)-Global perspective and applications in lipid technology. G. Bridgwater. J.K. Cholesterol and fatty acids in several species of shrimp. Direct transesterification of all classes of lipids in a one step reaction. and Dyer. A. R. FL... 101.4.. 37. Nielsen. G. Pérez-Martín. 26. B. J...K. ed. J.. and Roy. A. V. 18. 1986.. The use of sephadex for the removal of nonlipid contaminants from lipid extracts. 2003.. and Raftery... Le Quéré. J. 54.. London. 24. Chrom. S. The Oily Press. F.. Biochemistry. 1986. New York. Stability of lipids of frozen albacore (Thunnus alalunga) during steam cooking.. Aubourg. Lipid Analysis... American Oil Chemists’ Society Press. CRC Press.. 1989. Boca Raton. Ackman. Vol. J. 24. and New York. 237.. P. J. 2005. 199.. 16.. 1259. 1978.... 1966.... and Nes. E. Love. 8. .. Nielsen. 191. in Marine Biogenic Lipids. 1994. Folch. CRC Press. Chem. U. Vol. in Physiology and Biochemistry of Sterols. and Dittmer. Food Res. 19. S. W. London. W. Fats. M.. I.. Teshima. R. 497. eds. 13. 103. J. Plenum Press. and Perkins. 7. G. Wells. E. 25. 1989. C... A. Sebedio. R. Hamilton. 10. R. Separation and determination of structure of fatty acids. 1986. 137.. Food Sci. J. J. Bligh.. Elsevier Applied Science. 3rd edn. Chromatographic separation of cholesterol in foods. Hoving. Physiol. ed.. Lipid Sci. IL. Joseph. Z. 1989. Hamilton. R. Kuksis. CRC Press Inc.

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

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

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

7. The secondary oxidation products include both low molecular weight. A simple titration method where the sample is dissolved in chloroform–acetic acid (or isooctane–acetic acid) is often used for fats and oils. making it even more difficult to determine the degree of rancidity. leading to a wide variety of reaction products. and reduced sales. it is both one of the oldest and one of the most used methods. which makes it difficult to find where the components originated. acids. The PV is expressed in milliequivalent of iodine per kilogram of lipid or as millimolar of peroxide per kilogram of lipid [7]. This method requires a sample of 5 g if the PV is below 10 and about 1 g if the PV is higher [3]. The secondary oxidation products can also react further. These include nonradical species such as aldehydes.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. [5] or [6] before analysis. and enzymatic oxidation. Free radicals are formed when hydrogen ions are extracted from the fatty acids. complaints from the consumers. and alcohols. and water.5 meq/kg. 7. resulting in a wide variety of degradation products. volatile compounds and nonvolatile components with a relatively high molecular weight. PV is one of the classical methods for determination of oxidative status. and the liberated iodine is titrated with sodium thiosulfate with starch as an indicator. The sensitivity is about 0. this is oxidized by the hydroperoxides or other components present in the sample.2.88 ◾ Handbook of Seafood and Seafood Products Analysis loss of products.1 Primary Oxidation Products The most common methods to determine primary oxidation products are peroxide value (PV) and conjugated dienes. the molecule is called a core aldehyde. methods that determine the primary oxidation products and methods that measure the secondary oxidation products. photooxidation. Potassium iodide is added. the influence of these compounds has been little studied [3]. Several analytical procedures are available. carbohydrates. The peroxides are easily broken down to alkoxy radicals. The radicals react with oxygen forming peroxy radicals and hydroperoxides. The fatty acids and the lipid oxidation products in foods can also react with other components in the food such as proteins. but this can be improved by determining the endpoint colorimetrically or by . However. and also more complex reaction products such as epoxy and polymeric compounds are formed during the propagation and termination steps [4]. 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]. Autooxidation of lipids takes place when the unsaturated fatty acids are exposed to oxygen and proceeds through an autocatalytic chain reaction [3]. If this contains a terminal carbonyl group. This also makes the determination of the degree of oxidation a challenging task. autoxidation. For determination of PVs in foods. the lipid can be extracted using the methods of Ref. Lipid oxidation can be divided into three types. When the decomposition of a hydroperoxide has resulted in the formation of a low-molecular weight volatile compound. the parent triglyceride is left with a shorter fatty acid. Some of the reaction products from lipid oxidation may also have negative health effects. ketones. Methods to determine the degree of lipid oxidation can be divided into two main groups.

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

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

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

Fluorescence spectroscopy on intact samples has been shown to be a sensitive technique. So far. little has been done to study the fluorescence spectra of the different oxidation products that are formed in foods. Fluorescence spectroscopy has a great potential for on-line or at-line applications.35]. and also cyclic compounds. [28] concluded that fluorescence spectroscopy may be able distinguish between different oxidation products formed but that this would require using the whole spectrum and not only the intensity at the maximum wavelength. Fourier-transform near-infrared (FT-NIR). In a study of different model systems including fish and meat. and a few minutes of oxidation of docosahexaenoate (DHA) resulted in significant changes in the ESR spectra.32. new methods have been developed. the Rancimat test [30].3 Stability Methods Several techniques based on accelerated oxidation are used for evaluation of oxidation. adipose tissue. but it measures the time taken to reach a certain PV. oil stability index (OSI). such as different hydroperoxides. This results in the formation of low molecular weight acids that are flushed out with the air and collected in vessels containing distilled water. obtaining information . and additives may contribute to the spectra.33]. and Oxidograph are techniques for measuring the stability of oils toward oxidation. and the point where it changes most is called the induction time.37]. active oxygen method (AOM). The gas chromatography–mass spectrometry (GC–MS) techniques can be used to determine a wide range of volatile secondary lipid oxidation products [36].92 ◾ Handbook of Seafood and Seafood Products Analysis spectroscopy can be used. The Rancimat. the oil can be heated to 80°C or more while air is bubbled through it. 1H NMR spectra can be used to study specific lipid oxidation products.4 Instrumental Methods Many instrumental methods have been developed for the determination of oxidation parameters in oils and foods. comparable to sensory analysis and gas chromatography. The level of hydroperoxides in fish oil can be determined using a rapid CL method [14]. Lipid oxidation products can produce very weak chemiluminescence (CL). One challenge is that fluorescence spectra can be very complex and that not only the oxidation products but also connective tissue. 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. Free radical assessments by the ESR spin-trapping technique detected the very early stages of lipid oxidation. Veberg et al. It has been shown that sodium hypochlorite-induced decomposition of hydroperoxides gives strong CL [34. 7.2. 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]. these include the oil stability index method [29]. Using solid-phase fluorescence is a relatively new approach. The AOM method is performed in a somewhat similar way. including near-infrared spectroscopy (NIR). The liquid chromatography–mass spectrometry (LC–MS) techniques can also determine nonvolatile products—of special interest are the core aldehydes [3. In Rancimat and OSI instruments. In recent years. but studies on the use of this technique in dried fish were published in 1992 [27]. 7. The change in conductivity is measured. 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. porphyrins. for measuring lipid oxidation [28]. and FT-IR spectroscopy methods [16. aldehydes.2. and oxidative stability measurement by Oxidograph [31] and they are all suitable for analyzing oil systems.

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

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

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

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...................Chapter 8 Volatile Aroma Compounds in Fish Guðrún Ólafsdóttir and Rósa Jónsdóttir Contents 8........ and Cabbage-Like Odors .......... 98 Development of Fish Aroma....................2.........................................108 8...........1 Sweet................ Onion...3 Putrid....................1 Cooked Odor—Boiled Potato and Rancid Odors ......4...................................................4..............103 8.................4..4................. and Malty Odors ....................113 References ..................2 Oxidatively Derived Odors ......................105 8..................3..... 111 8..............1....................4..........................................3 8....................4 Introduction ....1.2.......................................................... 99 Identification of Quality Indicators ........................................................4 Miscellaneous ........................4.......................................1....5 Conclusions ..........................106 8....................................... 98 Fresh Fish Odors .4..........................4...............................112 8...... 111 8..103 8.......................1.....3 Processing Odors .............................................3...........................................................................................4.........................1 8................ and Stale Odors ...............2 Ripening Odor—Salted and Dried Fish Odor......................................... Ammonia-Like.......................2 Dried Fish.....................................105 8......2 Washed Cod Muscle System ..... 100 8................................. Sour........4...105 8...........................1 Smoked Fish Odors ...................................................................................................................................................1 Microbial Spoilage Odors ......................................2 8...........113 97 .............................106 8......4.....................

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

lipoxygenase. salting. malty. caramel.14.5. which have potent green. contributing to green. and mushroom-like odors are unsaturated carbonyl compounds and alcohols with six.5-ocatadien-1-ol. polyphenols Lipids phosholipids/PUFA Proteins sarcoplasmic. Newly caught marine fish contains low levels of volatile compounds and is nearly odorless. 1. and melon-like odors. On the other hand. polyunsaturated fatty acids. were characteristic for freshwater and euryhaline fish. LOX activity on the skin and gills of both freshwater and marine species (rainbow trout. cucumber-. Some components are desirable at low levels. hemoglobin.Volatile Aroma Compounds in Fish ◾ 99 Handling chilling. drying. ammonia-like Oxidized aroma Processed aroma green-like. stale. whereas volatiles generated from fat result in variation in the specific flavor character of different fish species.15]. spoiled. but the mechanism of this activity is not fully elucidated [9]. neutral Spoilage aroma sweet. Josephson et al. amino acids Fresh fish aroma seaweedy. trimethylamineoxide.. popcorn. hydrolases. and cooking Processing smoking. including calpains (neutral calcium-dependent proteases) and cathepsins (lysosomal proteases). The compounds that contribute to the characteristic plant-. TMAO. and processed fish. cucumber-.1 Overview of changes in fish influencing the development of characteristic aroma of fresh. cucumber. and 2. 8.10–13]. LOX proteases. the unsaturated C9 carbonyl compounds such as 2. and hydrolysis Endogenous enzymes i. ascorbic acid. Hb. NPN. peptides Soluble substances.5-octadien-3-ol. putrid. rancid potato.6-nonadienal. myoglobin. phospholipases TMAOase Microbial metabolism Specific spoilage organisms (SSO) Oxidation Prooxidants: metals (Fe. The overall perceived odor is dependent on the level of influential compounds and their odor thresholds along with possible synergistic effects. LOX. nucleotides. freezing. PUFA. nonprotein nitrogen-containing compounds. were responsible for the moderate. and sardines) plays a role in the formation of odorous volatiles. river trout. pleasant aromas of fish [6. mushroom. specific spoilage organisms. or nine carbon atoms [4. boiled potato. activity influences the deterioration of fish muscle. eight. Mb. [5] summarized the occurrences of volatile compounds in freshwater and saltwater species and concluded that the four common compounds found in saltwater species. but . malty.Cu) Hb. Mb Antioxidants: α-tocopherol.3 Fresh Fish Odors The delicate flavor of fish is mostly contributed by volatile compounds and taste active substances in the aqueous phase. 1-octen-3-ol. oxidized. stockfish. sour. NPN. dried fish. melon-. hexanal. metallic. plant-.e. cucumber Figure 8. faint odor of saltwater species. SSO. Soon after harvest.

21–22. where it has been demonstrated by monitoring key volatiles to study changes in different fish products during storage. This has been the approach in our studies. which are present in higher levels and can be quantified. Both single compounds such as TMA and ethanol and multicompound indices based on combination of alcohols. Fatty species develop rancid odors and taste. esters. Rapid methods can then be applied to detect indicators or alternatively classes of compounds if the pattern of the volatile compounds is known and a connection has been verified between the indicator compounds and the compounds that are responsible for the odors and quality changes. and sweet odors. odor.22] and in smoked salmon [23]. and species of the salmonidae family develop earthy. Table 8. in nominal levels the bromophenols appear to contribute to natural sea-. they may contribute to off odors.6-Nonadienal was identified to be the most characteristic compound for the cucumber-like capelin odor [19]. 8. An example is the enzymically derived long-chain alcohols and carbonyls that exhibit characteristic fresh.1 summarizes the occurrence of volatile compounds detected in our studies on cod [22] and haddock fillets [31] and smoked salmon [23]. Another example is iodine-like off flavor in prawns associated with bromophenols originating from the feed chain [17]. (E. and sulfur compounds representing the different changes occurring during storage have been suggested by numerous researchers as indicators for freshness and spoilage [22. but when accumulated in higher levels because of autooxidation. plant-like notes in fresh fish. 2. 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. GC–MS. and identification was based on GC–FID. The main classes of compounds detected during storage are alcohols. Therefore. 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.Z)-2. . and GC–O. including (E)-2-nonenal. which has a very characteristic cucumber odor during spawning. boiled potato-. as seen by the detected odors listed in Table 8. muddy. and these are difficult to detect by analytical techniques. Purge and trap on Tenax and SPME methods were applied for sampling.6-nonadien-1-ol in sweet smelt tissues [20].24–29]. iodine-. Accumulation of certain hydroperoxide isomers coincided with the period of enhancement of characteristic aroma in sweet smelt. lean species typically develop sweet. for example. acids. They were suggested as the possible precursors of nine-carbon volatile compounds. and sulfur compounds. amines. aldehydes. C9 LOX-derived compounds have been found in higher levels in spawning euryhaline and freshwater fish [5]. The volatile pattern changes in mature salmon when migrating from the sea for spawning. However. and marine-like flavors of seafood [18]. and 3. and quality changes can be explained in. a saltwater species. Some of the influential odor compounds that have very low odor thresholds are often present in low levels.1. Studies performed in Japan.6-nonadienal. ketones. and amine-like odors. amines.100 ◾ Handbook of Seafood and Seafood Products Analysis if their concentration increases. cod during storage [21.4 Identification of Quality Indicators Different characteristic odors develop in various fish species during storage.30–36]. it is useful to monitor the overall pattern of volatile compounds and select indicator compounds. The aldehydes contribute most to the spoilage odors because of their low flavor thresholds. they contribute to oxidized and fishy odors in stale fish [16]. on accumulation of hydroperoxides in fish tissues. Environmental conditions and seasonal effects like spawning can influence the odor quality of fish. Seasonal effects have also been reported for capelin. indicate their involvement in the development of fresh fish aroma associated with seasonal variation.

floral Sweet. 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.4-Heptadienal. caramel.1 Volatile Compounds Detected in Cod [22]. flowery — Ketones 2-Butanone 2. fish fillet Sweet.Volatile Aroma Compounds in Fish ◾ 101 Table 8. candy × — — Sweet.3-Butandiol 1-Octen-3-ol 2-Ethyl-1-hexanol 1-Octanol × × × × × × × × × × × × × × × × × × × × × × × × — — — — — — Mushroom — — Aldehydes Acetaldehyde 2-Methyl-propanal 2-Methyl-butanal 3-Methyl-butanal Hexanal cis-4-Heptenal Heptanal 2.E)Nonanal Decanal Undecanal × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × Rancid Boiled potato. Haddock Fillets [31]. (E.3-Butandione × × × × — N/A (continued) . fatty — Fresh. earthy Sweet.

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

the aroma of the fillet is described as sweet and reminiscent of shellfish. mainly amino acids.1). Haddock Fillets [31]. not detected by GC–O. Sweet-milky and vanilla/caramel-like odors are typical in cooked fish. the fish is no longer fit for consumption. sour. mushroom-. During prolonged storage boiled potato odor develops. alcohols. 8.4.1 based on GC–O analysis of cod and smoked salmon represent most of these overall changes. In general when fish is cooked. and when combined with frozen storage odor. 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. sour.34]. meat-like. and 2. and sulfur compounds produced by microbial degradation of fish components. The odor descriptors in Table 8. and temperature conditions during storage [33. The microbially derived alcohols 2-methyl-1-propanol. cabbage Volatiles in boiled cod were analyzed in samples of raw chilled cod fillets [22] by heating corresponding samples at 80°C for 60 min. Sour. and TMA-like smell. cucumber-. cooling. 8. 3-methyl-1-butanol. and sometimes metallic. data not available for haddock. development of spoilage odors. Late spoilage changes. caramel-like. The flavor thresholds . Identification of volatile compounds was based on GC–MS analysis (see Table 8. as well as green plant-. and quantification of the main classes of compounds was based on the sum of the PAR for respective compounds in each class. or geraniumlike odors are characteristic sensory odor descriptors for fresh whole fish. N/A. Seaweedy and marine-like odors. which is mostly affected by handling. 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. and the end of shelf life of cod fillets on day 12 of storage are explained by the presence of TMA.5°C) [22]. dried fish/stockfish.1 Sweet. The loss of freshness of cod fillets and early spoilage changes were related to the formation of ketones.3-butandiol were found in the highest levels on day 12 at sensory rejection. and aldehydes.1. alcohols. —. An example of the spoilage pattern of volatile compounds in chilled fish is illustrated in Figure 8.2. and Malty Odors Ketones.4. acids. 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 freshness notes disappear and the odor of the uncooked fish becomes neutral.1 (continued) Volatile Compounds Detected in Cod [22]. packaging. and malty spoilage odors. contributing to sweet.2) were associated with the development of sweet. sulfur. showing results from a storage study of cod fillets packed in styrofoam boxes during chilled storage (0. After several days of storage. and finally sour and dirty tablecloth odor. and malty odors. esters.1 Microbial Spoilage Odors The spoilage odors in chilled fish vary depending on the dominant microflora in the products.

3-pentanone. that were present in cod fillets throughout . Dimethyl disulfide and dimethyl trisulfide were detected at the end of storage time when samples were spoiled. and 3-methyl-butanal probably originate from degradation of valine and leucine. Volatile compounds as quality indicators in fish during chilled storage: Evaluation of microbial metabolites by an electronic nose. respectively. was characterized by sweet. and. ethyl acetate.. therefore. and 3-methyl-1-butanol as potential indices of refrigerated fish spoilage based on studies of freshwater whitefish. 3-hydroxy-butanone. and they did not contribute to the odor of the fillets as evaluated by GC–O (Table 8. dimethyl disulfide. Lindsay [8] suggested using short-chain alcohols such as ethanol. and fish-fillet-like odors by GC–O in our study. 2-methyl-1-propanol. whereas the formation of branched-chain alcohols and aldehydes such as 2-methyl-1-propanol. caramel. methanethiol. dimethyl trisulfide. G. and the carotenoid-derived 6-methyl-5-heptene-2-one. University of Iceland. TMA. 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. 2005.1) [22].5°C until sensory rejection on day 12.) of alcohols are higher than those of carbonyls. PhD thesis. 3-methyl-1-butanol. The concentration of acetoin was much higher than the lipid derived ketones detected. such as 2-butanone. and butanoic acid ethyl ester were found in the highest amounts and increased with storage. 3-methyl-butanal. piperidine. and acetic acid were identified as spoilage indicators [29].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. In chilled haddock fillets stored in styrofoam boxes. TMA. The formation of acetoin (3-hydroxy-2-butanone) was characteristic for the spoilage of chilled cod fillets packed in styrofoam boxes and was attributed to the growth of Photobacterium phosphoreum [22]. 3-methyl-1-butanol.2 GC–MS analysis of volatile compounds showing changes in the levels (PAR. Levels of acetoin increased earlier than those of TMA. it is more useful to monitor the loss of freshness as an early indicator of spoilage. butanol. 3-methyl-1-butanol. whereas dimethyl sulfide was detected initially and throughout storage [31]. In cultured and wild sea bream stored in ice for 23 days. Ethanol was detected in high levels initially (on days 4 and 7) and then declined. Propanol was suggested as a potential indicator when using modified atmosphere packaging techniques. 1-penten-3-ol. Reykjavík. (Modified from Ólafsdóttir.

2 shows that TMA was detected in high levels on day 12. which may have influenced the overall odor perception leading to the sensory rejection of the fillets. Ketones can influence the overall odor because of their low odor thresholds. and dimethyl disulfide have been suggested as the main cause of putrid spoilage aromas [41]. Additionally.1. Ammonia-Like. decane. ammonia-like odor. 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.2) indicated that they were not important in the spoilage of chilled cod fillets stored in styrofoam boxes. Pseudomonas species have also been found responsible for the formation of volatile sulfides. has been suggested as a freshness indicator along with its precursor TMAO (trimethylamine oxide) [27]. 1-penten-3-ol). and Stale Odors The development of dried fish.39].1. 8. The lipid-derived saturated aldehydes detected on day 12 at sensory rejection also contributed to the overall sweet aroma. alcohols (3-methyl-1-butanol.4. Dimethyl trisulfide has also been associated with spoilage in fish and associated with the growth of Shewanella putrecfaciens [25. ammonia-like. methyl mercaptan. Onion.2 Dried Fish.4. numerous branched chain . The odor of ethyl butanoate. 8.38. fruity off odors [37. and the incorporation of hydrogen sulfide yields dimethyl trisulfide [38].1.Volatile Aroma Compounds in Fish ◾ 105 storage. but no obvious increase occurred until at the end of shelf-life and during continued storage.4. and stale odors by amines during fish spoilage is well known. 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]. contributing to the stale and putrid off odors in fish because of amino acid and lipid degradation [39]. Figure 8. and Cabbage-Like Odors Low levels of sulfur compounds (Figure 8. and undecane) appeared to be similar throughout storage in chilled cod fillets [22]. which forms very early after harvest of fish. The origin of the sulfur compounds is microbial degradation of cysteine and methionine to form hydrogen sulfide and methyl mercaptan. Enzymically produced DMA (dimethylamine). At this point there was an increase in the pH value. which contributed to boiled potato-like odors (Table 8. methyl sulfide. described as sickeningly sweet and nauseous.4 Miscellaneous The concentration of the straight chain alkanes (nonane. 8. TMA is characteristic for the spoilage odors of fish. contributed to the sensory rejection of chilled cod fillets on day 12 and suggested the role of Pseudomonas fragi in the development of sweet.3 Putrid. dried fish.1). In whole fish stored in ice. volatile sulfur compounds such as hydrogen sulfide. TMA is a potent odorant with a characteristic fishy.38]. TMA has been noted for intensifying fishiness by a synergistic action with certain volatile unsaturated aldehydes derived from autoxidation of polyunsaturated fatty acids [40]. The onset of stale odors can be explained by cis-4-heptenal and heptanal. and ketones (2-butanone). Oxidative processes are involved in the formation of dimethyl sulfide from methyl mercaptan and further oxidation of dimethyl disulfide. Additionally. and measurements of volatile amines such as TMA or total volatile bases (TVB-N) have been used in the fish industry as indicators of quality for fish and fish products.

2. such as hexanal.4. Phospholipids are the main membrane-bound lipids.7-decatrienal) should not be overlooked. although their overall levels were lower. heptanal. 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. which are known to be more susceptible to oxidation than triacylglycerols in fat deposits [45]. 3-methyl-butanal.106 ◾ Handbook of Seafood and Seafood Products Analysis alkanes were detected. These oxidation products contributed to the overall characteristic sweet. cis-4-heptenal. and decanal. were detected in the fillets throughout the storage time. ketones.7-decadienal. but the knowledge of the formation of these compounds is obscure. The origin of limonene in fish is most likely related to the diet derived from algae or plant source. 8. The influence of other aroma active compounds present in lower levels such as the unsaturated autoxidatively derived aldehydes (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].3 to demonstrate which odors are most dominating in the aroma profile [48].4-heptadienal. 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]. Similarly. fish-like odors of chilled cod fillets in combination with other carbonyls (3-hydroxy-2-butanone. they are in particular sensitive to oxidation. that contribute to the development of rancid cold store flavors [47]. and overall the alkanes showed an increasing trend with storage time. lipid-derived saturated aldehydes. their impact was greater than alcohols and ketones. Limonene has also been detected in sea bream during storage [29]. A characteristic earthy odor in many species residing in ponds has been associated with piperidine and its reaction products. Aldehydes generally have low odor thresholds.4. Limonene has low odor threshold and a fresh lemon odor was detected by GC–O analysis of cod. which is further enhanced by preprocessing and storage of fish. aromatics. and 6-methyl-5-heptene-2one). However. Several odor active terpene derivatives have been identified in fish. Boiled potato. and terpenes found in wild sea bream compared with those of its cultured counterpart [29].4. and 2.1 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. Various pro and antioxidants influence the stability of the muscle and have been studied in relation to the oxidative stability of phospholipids [46]. 3-pentanone. Piperidine levels have been reported to increase in spawning salmon and contribute to off odors [43]. 8.4-heptadienal and 2. 2-butanone. they are not considered of interest as quality indicators. Piperidine was tentatively identified in chilled cod fillets [22] and has also been suggested as a quality indicator in sea bream [29].and potato-like odors contributed by . since they are not aroma active.4. but the sampling techniques used were not sensitive enough to allow quantification of these compounds. suggesting that it may have an impact on the overall odor of fish fillets [22]. in similar or slightly increasing levels. such as hexanal. These compounds have been associated with rancid and dried fish odors. therefore. the feed may have influenced higher levels of aldehydes. Oxidative processes occurring during storage of fish result in the accumulation of aldehydes. and because of their high unsaturation. and.2.

this aldehyde does not exhibit a fishy-type aroma by itself. (From Jónsdóttir. 2004.4-Heptadienal Rancid Geranium-like 1-Octen-3-ol Mushroom Earthy. Overall earthy. In fresh baked herring (200°C. rancid odors Flowery 2. In fact. green-like. and hexanal were abundant in headspace. sweet. 20 min) 3-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 (---▲---). Principal component analysis (PCA) was performed (Figure 8.3) were fatty. Fatty. but it rather participates in the expression of the overall fishy odor. sweet. R. 2-heptanone. green-like. melon 2-Nonenal Cucumber Fatty Fatty. Taking into account the complexity of the spoilage processes. heptanal. multivariate data analysis is useful to explore the overall trend of the main quality indicators. some confusion exists about the role of cis-4heptenal as the “cold-storage compound” [8]. However. Hexanal. and fish oil notes in the same study. 1-penten-3-ol. and after storage for 3 days the proportions of 4-heptenal. although the level of the compounds may vary and explain the differences in the characteristic odor of these species. 2-methylbutanal. and octatriene increased significantly. and octadienes also increased many-fold during further storage.4) on data from our studies on volatiles in cod [22] during prolonged storage for 17 days and compared with corresponding . flowery. Ideally. and green-like odors were associated with oxidatively derived 3-pentanone. sourish. Unpublished data. Other pronounced odors detected in boiled cod (Figure 8. pop-like Earthy-like odors Figure 8.52].. sweet. The occurrence of cis-4-heptenal has been associated with the “cold storage flavor” of cod [47]. earthy Potato-like Boiled potato cis-4-Heptenal Heptanal ◾ 107 Cucumber. Its odor has been described both as cardboardy. this is not always the trend for dynamic microbial and oxidative changes and the formation of volatiles in fish during storage [22]. Baltic herring has been reported to have a similar development of volatiles. paint-like [50]. quality indicators should demonstrate clear increasing or decreasing levels with storage time. green-like odors Grass Hexanal Heavy Mushroom.) heptanal and cis-4-heptenal were the most potent odors. and rancid odors contributed by 2-nonenal and 2. sour. however. microbial metabolites such as 3-methyl1-butanol and cresol were identified [53]. and after 8 days of storage at 6°C. as well as boiled potato-like [51. The fresh raw salmon odor was characterized as cucumber-like with weak sweet. and the most pronounced attribute was a boiled potato odor [49]. 1-penten-3-ol and hexanal. G.Volatile Aroma Compounds in Fish DMS Sulfur 5 4 3 2 1 Fishy odors Fishy 3-Pentanone 1-Penten-3-ol Flowery 2-Penten-1-ol Flowery Fatty. and fish oil notes were characteristic for fresh cooked salmon.4-heptadienal. and Ólafsdóttir.

The characteristic pattern or trend in volatiles in raw and boiled fish is clearly different. 7.2 0.5-octadien-3-one. 3-methyl-butanal in combination with acetaldehyde. increased with time and were pronounced in the spoiled raw samples (R-D14 and R-D17). boiled and storage days. However. methional.0 Figure 8. On the basis of odor evaluation.Z)-2. 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).E)-2.108 ◾ Handbook of Seafood and Seafood Products Analysis PC2 Bi-plot 1. 19% PC1 1. that is.0 B-D17 1-Penten-3-ol 3-me-butanal Acetic acid 0. samples after heating (see Table 8. hexanal.6-nonadienal. Samples are labeled with R.4-decadienal from PUFA were determined as character impact odorants of boiled cod [54].5 –0. The PCA demonstrates how volatile compounds can explain the variation in quality of samples according to storage time and handling (raw and boiled).4). and dimethyl trisulfide were detected in higher levels in the boiled samples (data not shown). in particular the role of volatile compounds derived from oxidation in heated/boiled samples. 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. as indicated by the arrows (Figure 8. Autoxidatively produced unsaturated carbonyl compounds were the most abundant components in boiled and canned fish. Sulfur compounds dimethyl sulfide. The oxidatively formed compounds. D (4. . and (E.1).4). and 17 days). and oxidatively derived (Z)-1. 3-methyl-butanal was correlated to the boiled stored cod (B-D17) (Figure 8.4 Principal component analysis of raw and boiled cod.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. and 6-methyl-5-hepten-2-one. dimethyl disulfide. Interestingly. 12. The effect of oxidation induced by cooking and formation of oxidation products such as heptenal and nonanal characterizes the (B-D4) sample. In boiled trout.2 Washed Cod Muscle System Rancid odor development during chilled storage of fish has commonly been associated with fatty species.4 –0.2. X-expl: 53%. oxidation of membrane-bound phospholipids in lean species can cause fishy.6 0.4 0.2 Raw and boild c…. methional with a characteristic boiled potato-like odor dominated the odor of the aldehyde fraction of the headspace volatiles. in agreement with earlier studies [54].8 R-D17 –0. decanal.4. 14. (E. especially in trout [15]. 8. raw and B. The malty flavor of 3-methyl butanal was suggested earlier to be mainly responsible for the malty off flavor defect of boiled cod [54]. Only the spoiled raw samples (R-D14 and R-D17) can be correlated with the freshly boiled (B-D4) sample. 10.

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

painty. Active research is ongoing on the application of various natural antioxidants based on polyphenols like flavonoids (i. R.) .110 ◾ 1000 800 ng/g Handbook of Seafood and Seafood Products Analysis Hexanal 30 25 20 ng/g 15 10 5 0 0 1 Blank-II 2 Hb-Char-II 3 Hb-Cod-II 4 0 1 Blank-II 2 Hb-Char-II 3 4 cis-4-Heptenal 600 400 200 0 Hb-Cod-II Figure 8. 2007.5 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 (-♦-. et al. and in TBARS (Figure 8. and Ólafsdóttir. 16. respectively) and raw without hemoglobin (blank).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.. Some promising results have been reported. described as rancid.. -■-. Hb-Char-II..) Thermal treatment of the cod model system significantly enhanced the oxidation of the model on day 1. Blank-II. citric acid. Matis Report 08. caffeic acid) [65] as well as application of tocopherol. 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. and EDTA [66]. G..e.e. 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. -▲-. With permission. where commercially available green tea polyphenols were shown to effectively inhibit the LOX activity of mackerel muscle [67]. HbCod-II). R. J. Studies on LOX inhibitors are of interest in preventing the initiation of oxidation in fish. Aquat. ( Adapted from Jónsdóttir. Food Prod. as measured by rapid increase in rancid odor. 2008. 67. with added hemoglobin (raw and cooked. (From Jónsdóttir.. and dried fish odors.6) as well as more rapid loss of red color (not shown) already on the first day of storage. catechins from tea) and cinnamic acid derivatives (i. 73.

Phenolic derivatives like guaiacol (2-methoxyphenol) and syringol (2. also contribute to the aroma of seafood flavorants [70]. thermal degradation.7) (e. which has a characteristic potato-like odor. aldehydes. Key volatile compounds identified in enzymatically produced seafood flavorants are formed via Maillard reaction and Strecker degradation of amino acids. Guillén et al. Some of these compounds were selected as key spoilage indicators for smoked salmon based on their high levels and contribution to sweet and fruity spoilage off odors in our study on smoked salmon (Figure 8. Volatile compounds like alkyl-pyrazines and sulfur-containing compounds have been found in cooked crustaceans. and 1-octen-3-ol.3. giving the flesh its typical fishy odor [71. and 1-propanol [28. Other oxidatively derived compounds like 1-penten-3-ol. 3-methyl-butanal.4.71.g.4. 2-pentanone. plays important roles in the formation of complicated processing flavors. and 3-methyl-1-butanol) [23].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. like cis-4-heptenal. where groups of phenol pyrolysis were most noticeable in the smoke flavor volatiles. it is clear that their presence contributes to the characteristic fish odor of smoked salmon products. sweet/sour rancid.4-decadienal. and although they contributed less to the odors. These are compounds like methional. the Strecker aldehyde produced from methionine. The typical smoked salmon aroma results from a number of chemicals found in the smoke. Figure 8. Lipid-derived aldehydes play an important role in flavor formation and have been reported to contribute to the characteristic fish-like. such as heptanal and (E. including Strecker degradation. like 3-methyl butanal.72].72]. potato-like odors. 3-methyl-1-butanol. In addition to phenolic compounds.Z)-2. 3-hydroxy-2-butanone. and decanal were among key volatiles. hexanal. hexanal.72]. 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].7) [23]. Microbially produced ketones. 2-methyl-1-butanol. and lipid oxidation. 1-octen-3-ol. nonanal. [74] analyzed headspace components of cod and swordfish. . and 2. contributing to mushroom-like odor. furan-like compounds have been reported to be responsible for the smoked odor in smoked salmon. whereas carbonyl compounds. Additionally. were characteristic in unsmoked fish. Maillard reaction. giving rancid.7 illustrates the main odors that were present in smoked fish samples after 14 days of chilled storage. associated with spoilage off flavors. 8. it was verified that selected key volatile compounds performed better as predictors to explain variation in sensory attributes (smoked.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. The oxidatively derived compounds cis-4-heptenal and heptanal.. Lipid-derived components. and alcohols were abundant in the headspace of cold smoked salmon products during storage. and furans have been found in spray-dried shrimp powder and shrimp hydrolysate [69]. sweet odors of processed seafood like those in smoked salmon [23. 1-penten-3-ol.4-heptanal. ethanol. which is typical for products on the market [23]. 2. gave the most intense odors of smoked salmon and contributed to the fish-like earthy odors and fatty and rancid odors (Figure 8.Volatile Aroma Compounds in Fish ◾ 111 8. and 2-acetyl-1-pyrroline.6-nonadienal. 2.6-nonadienal. giving a popcorn-like odor that can be thermally generated. but it is mostly attributed to the phenols. and off odor and flavor) than traditional chemical and microbial variables. 2-butanone.75].1 Smoked Fish Odors Degradation compounds from Maillard reactions and lipid oxidation are the main compounds contributing to the aroma of smoked salmon [72].

sweet Flowery. 109.2 Ripening Odor—Salted and Dried Fish Odor Numerous volatile compounds have been detected in ripened products like dry cured ham. and free amino acids. smoke 3-Methyl butanal Sweet. 184. sweet Smoke-like 2.7 GC–O evaluation of volatile compounds detected in cold smoked salmon after 14 days of storage at 5°C. most of them generated from chemical or enzymatic oxidation of unsaturated fatty acids and further interactions with proteins. The rancid. 4-Heptadienal Sweet. smoke Mushroom.4-heptadienal and 3. potato-like odors together with cucumber-like odor [82]. Thus. both oxidatively derived compounds. where methional derived from methionine and 2.6-nonadienal from fatty acid oxidation were the main odorants in sugar salted. Methional and (Z)-1.5octadien-3-one were also identified as potent odorants in ripened anchovy [81]. In our study. probably originating from amino acids. 2008. sweet Flowery. ripened roe products [79] Similarly. the highest odor scores were given for boiled potato and rancid. and aldehydes such as acetaldehyde. 2-methylpropanal and 3-methylbutanal were the key. mushroom 2. fruity Flowery. burnt. they suggested that lipid autoxidation during ripening was primarily responsible for aroma development. et al. the desired flavor and texture develop as a consequence of protein and fat degradation. potato-like odor was identified as cis4-heptenal and the boiled potato-like odor. smoke. Similar processes have been reported in ripened seafood products.3. sweet Wood.4. (Modified from Jónsdóttir.and 3-Methyl phenol Guaiacol 4-Methyl-guaiacol Sweet and fruity-like odors Wood. caramel Smoke-house.. [76–78]. geranium Rancid cis-4-Heptenal Heptanal Earthy-like odors 1-Octen-3-ol Figure 8. R. Triqui and Reineccius [80] found that 2. manufacturers of ripened products have observed that some degree of proteolysis is necessary before flavor can develop. However. and rancid-like odors Burnt. Food Chem. During ripening of salted cod. peptides. . Salted cod are traditional products from the North-Atlantic fisheries and are highly regarded as ripened fish products in many countries. where the ripening of salted cod (Gadus morhua) produced by different salting methods was studied. fatty Boiled potato-like Fatty. especially those in the Mediterranean.5-octadien-2-one were associated with the development of the typical flavor obtained after anchovy ripening. earthy.112 ◾ Handbook of Seafood and Seafood Products Analysis Smoked salmon odors Characteristic smoke odor Sweet. highly volatile components of ripened anchovy.) 8.. as heptanal.

to increase trust between buyers and sellers in trade. . were the most intense character impact compounds of salted cod and smoked salmon. FAO Fisheries Technical Paper. amines. esters. Huss. Other key volatile compounds in salted cod are derived form lipid oxidation. such as ketones. 2. A similar set of sensors with selectivity and sensitivity toward the main quality-indicating classes of compounds. could also be responsible for the boiled potato-like odor.5 Conclusions Although aldehydes. and 2-butanone. potato-like odors. and other prooxidants in combination with mild heating treatment are important factors to maintain the delicate flavor and odor of fish products. Therefore. Development of smart sensor technologies like the electronic nose to detect microbial metabolites and oxidation products is of interest to verify the quality of products to facilitate process management. However. New York. Quality and quality changes in fresh fish. Studies on hemoglobin-induced oxidation in the washed cod model system and enhanced oxidation after heating verified the role of the oxidatively derived compounds contributing to off odors in chilled stored and boiled cod. In addition. alcohols. 8. and in retail for consumers as smart sensors imprinted on packaging. References 1. acids. Lipid oxidation during ripening appears to be primarily responsible for desirable aroma development in processed fish. The cucumber-like odor detected is possibly 2.6-nonadienal. Knowledge of the spoilage pattern of volatile compounds is the basis for the development of rapid techniques like smart sensor technologies. Botta. although the compound could not be identified by GC–MS. 193 pp. careful evaluation of the quality of product is needed to ensure acceptable flavor. and sulfur compounds. such as heptanal and (E. VCH Publishers Inc. aldehydes. Evaluation of Seafood Freshness Quality.6-nonadienal. 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. Detection of microbial metabolites originating mainly from soluble aqueous fractions of the muscle can be directly related to the quality of products.. 1995.83]. 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. Rome. and 1-octen-3-ol. No. H. microbial growth can be limited by effective cooling techniques. 1995. J. Volatile compounds as indicators of freshness quality and spoilage can be monitored to determine the quality of fish products.R.H. Proper handling and application of natural antioxidants to control oxidative processes caused by lipoxygenase. FAO. 1–67. their presence at nominal levels gives the characteristic and desirable fishy odor in fresh and processed fish. and new packaging technologies.Z)-2. 348. for example. temperature control. hexanal. proper handling. contributing to mushroom-like odor. according to retention index (RI) of standard and odor evaluation.Volatile Aroma Compounds in Fish ◾ 113 Methional. and myoglobin. exhibiting rancid. cause off odors in fish during storage. derived from methionine and eluting at a similar time as cis-4-heptenal and heptanal. can be used for a variety of fish species that are stored and processed by different techniques. The oxidatively derived compounds cis-4-heptenal and heptanal. 1-penten-3-ol. hemoglobin.

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

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PROCESSING CONTROL II .

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........................................................ and Data Analysis ...................... Measurement Principles..................................................................1............................................................................................................................................................................... 128 9........................................... 122 9..... 130 9....................................1 Determination of Basic Composition........1...........2 Imaging Spectroscopy ...................................4 X-Ray Imaging ......... 128 9................1 Theory and Measurement Principles ...............................................3.3.............................................2 Analysis .....4............................................... 122 9.. 130 9..........................1 Theory............................. 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......................1........................... 128 9................................................................132 9...............................2 Theory...............................5 Summary ............................................ Karsten Heia.............................. 130 9.............................Chapter 9 Basic Composition: Rapid Methodologies Heidi Nilsen................................................... 121 ..........................................4...............2.........................................3....................... 122 9.................................1 Near-Infrared Spectroscopy...... and Margrethe Esaiassen Contents 9.................3 Analysis of Basic Constituents .....3 Analysis of Basic Constituents .......................................2 Analysis of Basic Constituents ...... Measurement Principles.............................................132 9............. 124 9............................1 Determination of Basic Composition..............................132 9..............................133 Acknowledgment ....................................2...... 134 References .....................................131 9. and Analysis ...3 NMR Spectroscopy ......................2 Theory and Measurement Principles ..............

and Data Analysis The electromagnetic range applied in NIR spectroscopy spans from 700 to 2500 nm. In the following section. comprising the frequencies just below those of visible light. which is a prerequisite for a methodology to be applied along a production line. we review some of the most relevant methods for assessing the basic composition of fish and seafood as presented in scientific literature. throughout the years the method has also proven useful for the analysis of seafood and seafood products [8]. The work in food analysis tends to have a focus within the agricultural sector [1]. seafood is considered highly fragile and perishable with a short shelf life and delicate texture. Another aspect to be considered is the increased consumer awareness regarding the quality of their food. 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.122 ◾ Handbook of Seafood and Seafood Products Analysis Compared with the production and distribution of meat from the agricultural sector. and so the need for measurement and documentation of such parameters is both a consumer requirement and also issued by law. In this perspective there is an obvious need for objective methods for evaluating and documenting the basic composition of fish and seafood. 9.1. There are several reasons why NIR as a food analytical tool has caught attention and approval during the last decennia. C−O. 9. Regarding industrialized food production. as well as x-rays. to analysis related to the environment and the petrochemical sector [1]. followed by a presentation of the usage of NIR measurements for the rapid determination of basic constituents in fish and seafood products. hence. The documentation of basic nutritional composition of foods is a legal requirement in many countries. In this chapter. These methods are near-infrared (NIR) spectroscopy. we give a short introduction to the principles of NIR spectroscopy.2 Theory. and hence these issues must be considered during the processing and characterization of the material. The basic principles of the techniques are described as well as a presentation of the use and applicability of quality measures of fish.1. frequently consumers want readily accessible information about nutritional parameters and food quality. In this context seafood is particularly challenging as it comprises a vast number of different species with their own characteristics and qualities. However.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. The measurements are based on light interaction with material. 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]. Measurement Principles. requirements for such a method would preferably be that it is rapid and nondestructive. magnetic resonance. facilitating a rapid response. and additionally the method may be applied with little or no obtrusion to the material sample. C−H. Another benefit is the potential of simultaneous measurements of more parameters.1 9. . imaging techniques. The absorption of light is due to the response of the molecular bonds O−H. pharmaceutical applications. these techniques may be applied in or at a production line. The four methods presented fit with the requirements of speed and nonobtrusiveness.

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

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

in.603947 0. The study concluded that NIR is well suited for nondestructive quality evaluation of salmon fillets. [21.22] also conducted studies documenting the efficiency of applying NIR spectroscopy in different measurement modes to assess fat and water content in salmon. Isaksson et al. 8) Figure 9. whether pre-. PC): (%fettHS. NIR spectroscopy has been used for evaluating the chemical composition of several other fish species as well. water. Unpublished data.937575 0.2 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. An example illustrating the use of NIR spectroscopy for assessing fat content in farmed salmon is given in Figure 9.985681 0. fat. applying minced samples for the spectral readings. and Tuene [24] made use of NIR transmission spectroscopy to assess protein. Silver Spring). and NIR spectroscopy. The fat content of herring has also been assessed by the use of NIR spectroscopy. Spectroscopic readings obtained on the minced samples correlated better with the reference measurements on fat.264047 0. water. In both the works of Vogt et al. In a research article published in 2004. microwave.) Spectral measurements were performed on intact fillets by transflection measurements by use of the fiber optic probe of the instrument NIRS6500 (Perstorp Analytical Inc.. This work also emphasized the impact of the conditional state of the fish when making calibration models. N.. (From Nilsen.Basic Composition: Rapid Methodologies ◾ 125 salmon. [27]. H. 1998. Torry fatmeter. Transmission spectroscopy was also employed for the analysis of fat and dry matter in capelin [25]. (Y–var.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. as well as on minced salmon muscle. and additionally the spectroscopic measurements could be used for origin identification or authentication of the samples. In this work they applied a fiber optic measurement setup.2. Wold et al. Xiccato et al. [28] and Nielsen et al. and Sørensen. . In a recent work by Khodabux et al. [20] conducted a study in which they compared NIR measurements on intact salmon fillet. [26] showed that NIR spectroscopy could be used to estimate lipid. or postspawning. and dry matter in halibut fillet. and protein than those made on intact muscles. [29] one question of interest was the comparison of different methods for measuring fat content. NIR spectroscopy was proven to be a useful tool for the evaluation of basic constituents of different types of tuna. Torrissen.600067 0. and protein content of European sea bass. Measured Y Elements: Slope: Offset: Correlation: RMSEP: SEP: 21 Bias: 24 78 0.K. Nortvedt.

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

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

1 Theory.2. However. Oslo. the spectrograph and the object must move relative to each other. As described in Section 9. the relative motion is accomplished by mounting the imaging spectrograph above a conveyer belt where each captured frame images a line perpendicular to the direction of motion. in general. Most of them are on foods such as fruits. this technique also provides spatial information. 9. As these techniques only use the spectral information. as well as transflection measurements. Between each captured frame. Typically.1 on NIR spectroscopy. the feasibility of the method for the analysis of basic composition of foods. Measurement Principles. and acidity (expressed as pH) [47–49]. this can be illustrated as simultaneously recording information about shape and color.2. Several solutions have also been developed for detection of defects and contaminations on fruits. is a new technique that has been developed during the last decade [43. The realization of a commercial processing analytical tool for the simultaneous analysis of several parameters makes the technology interesting for a broad range of fish and seafood processing industries. or the result from these techniques can be postprocessed to utilize the spatial information [46]. demonstrates the potential of the method in the seafood sector as well. A novel example of this is the development of the QMonitor (QVision AS. Typically. To simplify the concept. It has been shown that NIR hyperspectral imaging techniques are . it uses a two-dimensional sensor. In order to illustrate the potential parameters to be assessed by imaging spectroscopy. In addition to what traditional spectroscopy can facilitate. 9.128 ◾ Handbook of Seafood and Seafood Products Analysis the near-infrared spectra. Depending on the applied sensor technology. Norge). Imaging spectroscopy can be implemented for transmission. and each frame captured provides full spectral information for one line across the object to be imaged. and meat. total soluble solids. For instance. an imaging spectrograph operates in the following way. an analytical tool for industrial quality control of clipfish and salmon fillets. also known as multispectral imaging or hyperspectral imaging.2 Imaging Spectroscopy 9. some examples related to the agricultural sector are referred.2 Analysis of Basic Constituents During the last decade several applications within food-quality inspection have been developed based on imaging spectroscopy. There are still relatively few reports on imaging spectroscopy applied for the analysis of fish and seafood.44]. It has become a widely used technique within fields spanning microscopy to satellite remote sensing. the spectra may be recorded in the visible and near-infrared region. In this way an image of the object is built line by line. For fruits and vegetables more articles report on determination of chemical constituents such as moisture content. 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]. reflection. the hyperspectral data can be preprocessed based on spatial features before applying analytical spectral techniques. Th is means that for each spatial location it is possible to access the full spectral information. improved results can be obtained by combining these techniques with more traditional image processing techniques. The analytical techniques described in that section are also applied to imaging spectroscopy data. vegetables. and Analysis Imaging spectroscopy. this method is an indirect measurement technique.

A recent work on quality assessment of pork has been reported by Qiao et al.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. Norway) has also developed an industrial solution based on multispectral imaging for measuring the fat content in salmon fi llets (see Figure 9. whereas the local fat content varies from approximately 6% up to 43%. . Hence. and cadaver [54. color. measuring the water content in one spot is not necessarily representative for the whole fish.Basic Composition: Rapid Methodologies ◾ 129 useful for automatic online detection of surface defects and contaminations on apples [50–52].3055% Share: 21.3%. Inspection systems based on hyperspectral imaging have been tested for poultry carcass inspection focusing on classification of carcasses into normal. septicemic. and different texture features. [35]. QVision (Oslo. In this publication the importance of including spatial information is illustrated. Fisk: 1 Fettfisk: 18. A thorough review of imaging spectroscopy applications within fruits and vegetables is presented by Nicolai et al. Peeling of shrimps and detection of nematodes were mentioned as possible applications for the future.4 Fat distribution in salmon fillet measured by the multispectral imaging system QMonitor fabricated by QVision (Oslo. [59. pH. [53].60] where several quality parameters were evaluated by imaging spectroscopy. [61]. The first article addressing analysis of fish or seafood by imaging spectroscopy was published in 2000 by Sigernes et al. The parameters included were drip loss. The color bar to the right indicates the correspondence between color and fat content in percentage.4). Since 2000. imaging spectroscopy solutions for detection of contaminants such as fecal and ingesta on poultry carcasses have been studied [56–58]. When drying fish. Regarding the determination of basic chemical composition of fish and seafood. Norway). the moisture content of the fish varies from the thinner parts to the thicker parts of the fish. the main activities within imaging spectroscopy and fish analysis have been focused on online solutions for assessing chemical composition and detection of quality defects in fish products. The mean fat content for this fillet is 18. Further on. there is one recent publication on assessing water content in salted dried cod by Wold et al.55].

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

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

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

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

200. for providing the example picture used in Figure 9. Thyholdt. and Isakson. using near infrared spectroscopy. Pawlinsky. Determination of chemical composition of beef meat by NIRS. et al. T. and Tandberg. Acknowledgment The authors would like to thank Dr Jens Petter Wold. 121–128. Naes. 1986.. J. 8.. H. Blanco. Part of the explanation for this could be the cost level of the equipment in question.. The price of measurement equipment for NIR. Isaksson. NIR. therefore. T. 1997. T. Osborne.. imaging. 70(8). M. K. 2002.4. pp. Thybo. U. K. Nofima Food. 4. NIR spectroscopy: A rapid-response analytical tool. in Fishery Products: Quality. U. Uddin. Harlow.. Eds. However.. 103–111. H. and x-rays are operated at a speed that makes it possible to perform measurements at or in a processing line. Safety and Authenticity. Prediction of sensory texture of cooked potatoes using uniaxial compression. quality seafood products will contribute to retaining the good reputation of fish and seafood in the years to come. and progress in data processing and analytical tools has facilitated usability and ease of interpretation of measurement results. Trends in Analytical Chemistry. NMR. 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. Reykjavik. 33(2).K. . M. P. 1998...G.. England. 240–250. 2. 21(4). and x-rays is considerable and. and Villarroya. however.I. and Williams. nearinfrared spectroscopy and low-field H-1 NMR spectroscopy. 3. Norway) confirms that these techniques may be applied in commercial and industrial high-speed fish processing applications. Near Infrared Spectroscopy in Food Analysis. K. I. et al.. Non-destructive Visible/NIR Spectroscopy for differentiation of fresh and frozenthawed fish. T. Ellis Horwood.. J.. 73(4). not easily applicable for small-scale industries as is often the case in the fish processing industry. 2000.K. Journal of Food Science. A.A. Norway. the finding of a universal measurement tool to meet with this variety is a challenging task. 89–104. C506–C510. et al. Due to the spread and diversity in fish species and sizes as well as the seasonal difference in bodily composition. Lebensmittelwissenschaft und Technologie. Longman Scientific & Technical. VIS/NIR spectroscopy. in Near InfraRed Spectroscopy... Hildrum. 2005. 7. This chapter. Eds. the technological development exemplified by SensorX (Marel hf. Nilsen. References 1. NMR. also makes it clear that although proven useful and promising in laboratory-scale trials. A. In addition. 525–532..J. Prediction of wheat bread-baking functionality in whole kernels. p. Another issue is the substantial variety and heterogeneity of the material to be analyzed. Oslo. Differentiation of frozen and unfrozen beef using near-infrared spectroscopy. and Fearn. and Heia. 6. 2009. hence allowing for measurements to be performed on large-scale quantities. 1992.134 ◾ Handbook of Seafood and Seafood Products Analysis of simplicity in sample preparation. 339–344. De Boever. Oxford. 5. Rehbein. Iceland) and QMonitor (QVision AS. 6. T. Wiley-Blackwell Publishing.K. Introducing and applying these methods to industrial applications and enabling production of well-documented. Journal of Near Infrared Spectroscopy. and Oehlenschläger. B. Journal of the Science of Food and Agriculture.

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

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

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

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

and so on. backscattered electrons. polarizing microscopy. so microanalysis can be carried out by means of x-ray.) before examination in the LM. etched. the sample is frozen in liquid nitrogen and then freeze-dried before being coated and observed.4) and quickly transferred under vacuum to a cold stage fit on a microscope where the frozen sample is fractured. In this way.02–1. and staining the ultrathin sections with heavy metal solutions such as lead citrate or uranyl acetate. showing alternate arrangements of .. in this way. dehydration in a series of ethanol dilutions of increasing concentration.0 mm in diameter. phase contrast or differential interference contrast (Nomarski). and observed. these different signals can be captured by the appropriate detector in each case. When physical fixation is used. infiltration and embedding in resin. and coating with a conducting metal for SEM imaging or with carbon for x-ray.1–2 mm (Figure 10. the sample can be observed with all its constituent water. iodine. There are two ways of preparing samples for SEM: chemical fi xation and physical fi xation (Figure 10. The light microscope (LM) is a very versatile tool that works in different applications such as bright field. cutting ultrathin sections (5–100 nm) in an ultramicrotome. 2008). Once the semithin sections are obtained. red oil. coated..2) are primary fi xation with aldehydes such as glutaraldehyde. At each subdivision there are macroscopic collagenous dividing lines (myocommata). Finally. For this.1). they are mounted in glass slides and stained with different dyes (toluidine blue. The sections are obtained using a microtome after embedding the food in paraffin or resin or using a cryotome after freezing the sample with CO2 or liquid N2. the sample has to be prepared in semithin sections of about 0. The SEM method observes the surface of the sample. image analysis relies heavily on computer technology to obtain quantitative results from microscopy observation. etc.2 Fish Muscle Microstructure Fish muscle consists of myotomes. The muscle cells are short and 0.3). the sample preparation steps are chemical fi xation (with aldehydes and osmium tetroxide. Besides the secondary electrons. In both methods. They are each surrounded by the sarcolemma membrane and by a thin layer of connective tissue (endomysium). In recent years considerable progress has been made in the field of SEM through vitrification techniques. secondary fi xation with osmium tetroxide. the samples need to be prepared first. or fluorescence microscopy. dehydration in a series of ethanol dilutions of increasing concentration. which is contiguous to the myocommata (Ofstad et al. Many of the endomysia are connected to the perymisium. may be generated as a result of the electron beam striking the specimen (Pérez-Munuera et al. critical point drying. sudan. In the former. The fibers are essentially the same as those of terrestrial animals in terms of the arrangement of the thick and thin filaments. as for TEM). 2006). light green. The most useful application for studying seafood structure is bright field microscopy. the sample is frozen in slush nitrogen (Figure 10. ions or molecules can be identified and quantified in situ using specific detectors coupled to the electron microscope. The steps in preparing samples for TEM observation (Figure 10. They are arranged in concentric circles forming subdivisions of striated muscle (Figure 10. so there is no need to section it. Electron microscopy (EM) allows food structures to be studied at higher magnifications than those used in LM. Two types of microscopes use electron beams as their source of illumination: transmission electron microscopes (TEM) and scanning electron microscopes (SEM). In Cryo-SEM. 10.140 ◾ Handbook of Seafood and Seafood Products Analysis provides a detailed description of the protocols often followed to obtain information about seafood microstructure.5). other emanations or signals such as x-rays.

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

2 Preparation of samples for TEM observation. 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 10.142 ◾ Handbook of Seafood and Seafood Products Analysis Food Specimen portions Fixation 2. 90%. Araldite Spurr’s. 50%.5% Glutaraldehyde 2% Os O4 Dehydration Ethanol (30%. 70%. . 100%) Infiltration and embedding in resine Epoxi resin.

50.Microstructure ◾ 143 Food Specimen portions Fixation Physical fixation Chemical fixation Quick freezing in liquid N2 2.5% Glutaraldehyde 2% Os O4 Ethanol (30. 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. Pd. for X-ray) SEM observation Figure 10. 90. . for SEM imaging) (C.3 Preparation of samples for SEM observation. 70.

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. C. vaccuum) Au deposition Coating (Au.) (into Cryo-SEM) (–130°C.4 Preparation of samples for Cryo-SEM observation. . . vaccuum) Cryo-SEM observation Figure 10..

where the Z disks can be distinguished. The fiber is composed of myofibrils in which Z disks are distinguished in the areas where the sarcolemma is broken. the perymisial connective tissue that surrounds the muscle bundles can be seen. The myofibrils are .Microstructure ◾ 145 a b Figure 10.2.6E). b: myocommata. Figure 10. where the fat is viewed as globules on the surface of the fiber. The myofibrils are shown in longitudinal section in this sample (Figure 10. 1990). A and I bands (Pérez-Munuera et al. but the total collagen content is lower. A micrograph cross section of the fibers shows them surrounded by the sarcolemma.6B).7C shows the inside of a muscle fiber with the myofibrils perfectly bundled. it is possible to observe ultrastructural details. At low magnification. a: myotome.5 Schematic representation of fish muscle with myotomes. reveals the myofibrils inside each cell. Examples of different fresh fish tissues observed by several techniques are described here. The layouts of the Z disks that mark the length of the sarcomere are visible. 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.7A shows a herring sample stained with toluidine blue and observed by LM. the fat can be observed covering the fibers in a longitudinal section of herring muscle (Figure 10..1 Herring Figure 10. When the muscle fibers are observed using the Cryo-SEM technique. The typical fish muscle fibers can be seen. Figure 10. Figure 10. which is mainly composed of collagen. 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.7B. the aggregation of solutes produced during the etching of the sample generates the typical eutectic artifact observed in Figure 10. When ultrathin sections of herring muscle tissue are studied by TEM.6D shows the microstructure of herring tissue at a higher magnification. the myofibrils at the cell edges have a less rounded section than the central myofibrils and are arranged like a palisade. obtained by the same technique but observed at a higher magnification. fat globules of different sizes are observed occupying the interfibrillar spaces and myofibrils are distinguished inside the cells.6C). 2008). 10. In a cross section of the sample fi xed in osmium tetroxide (Figure 10.6F. since the water in which the fish live lends support for the body (Lampila. The longitudinal section in 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.

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

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

(2000) used LM to observe the changes that occurred in the salmon during the smoking process and quantified them by image analysis. the greater the shrinkage.10B shows a detail of an intercellular space created by the conjunction of three fibers or cells. Gudmundsson and (A) 100 μm (B) 10 μm Figure 10.1 Smoked Salmon A cross section of a smoked salmon sample obtained using the Cryo-SEM technique is seen in Figure 10. longitudinal filament connecting Z-Z.9 Cytoskeletal structure of hake observed by TEM. The micrograph shows geometrically shaped fibers surrounded by a connective tissue. The data of the average cross-sectional area of muscle fibers showed that the smoking process produces shrinkage of the fibers. With permission. Z.) 10. The fiber shrinkage and the space between the fibers both increased to a greater extent in the muscle from the salmon that were frozen before smoking than in muscle smoked from fresh salmon.10 Smoked salmon. 2005. (A and B) Cryo-SEM. Figure 10. Biochem. Physiol. Z-disk.3 Processed Fish Microstructure 10. . M. B.3. (Reprinted from Pagano.10A. the higher the smoking temperature.. 141. 13. et al. LZ.148 ◾ Handbook of Seafood and Seafood Products Analysis Z LZ IZ DZ 8. Com.R.000 X Figure 10. Sigurgisladottir et al.

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

Such a product is often sold as “crab sticks” or “seafood sticks. Technomic Publishing Co.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.. Lancaster.3.. 2001.3 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. Figure 10. where the presence of salt makes the etching of the sample for observation difficult and masks the underlying structures.13 Schematic representation of (A) squid mantle and (B) arrangement of muscle cells.12A. et al. the paste is shaped and an “artificial fish muscle” is obtained. obtained by SEM. The Chemical and Functional Properties of Food Proteins. PA. With permission..A. The cross section (Figure 10.12B) shows the typical concentric layers of this type of surimi product.” Lean fish meat is minced to a paste. M. Inc. 10. after adding different additives. shows a longitudinal section of a crab stick where the “artificial fibers” can be observed.150 ◾ Handbook of Seafood and Seafood Products Analysis observed by Cryo-SEM. (From Lluch.

Effect of electric field pulses on microstructure of muscle foods and roes.. s. H.14A and B) (Llorca et al. sarcolemma.. . Radial bands (10–15 mm thick) comprise fibers that connect two tunics of connective tissue. Circumferential muscle bands (100–200 mm) comprise fibers running about the entire circumference of the mantle cone. The inner and outer tunics are covered by a visceral lining and outer lining. (C) LM. 225. Muscle fibers are grouped in bands that are arranged orthogonally.Microstructure ◾ 151 –200 μm –10 μm (A) 10 μ (B) 3. Th is gel network structure gives surimi its characteristic elasticity and texture (Sikorski. myofibril. 2001. E. l. and (D) TEM. References Gudmundsson. poultry and fish muscle. 122–128. 807. With permission. L. 10. all the muscle fibers are thin. Technol. This fiber distribution can also be observed by LM in samples stained with toluidine blue (Figure 10.. Muscle Foods. The fibers arranged in circumferential and radial bands were observed by SEM in samples fi xed with glutaraldehyde (Figure 10. 12. Tech. 2007.) ties of surimi.75 μ m s (C) (D) l Figure 10. a central sarcoplasm is shown to be surrounded by myofibrils. respectively. 247–267. et al. (From Llorca. 1.. Regardless of their orientation.14 Squid.4 Squid Microstructure The squid mantle is composed of muscle tissue sandwiched between two tunics of connective tissue (Figure 10. Lampila. Eur.5 mm in diameter (Lluch et al. J. m.. Comparative microstructure of red meat.14D). Food Res. 2007). 2001). Trends Food Sci.. 1990). central sarcoplasm. LM makes it possible to distinguish a peripheral area in blue and a central core in white inside each cell.14C).. 1990.13).. and Hafsteinsson. (A and B) SEM. Each fiber is surrounded by a sarcolemma (Llorca et al. 2001). When TEM is used to study the ultrastructure of fresh squid (Figure 10. M. the intermyofibrillar spaces between these can be observed.E. approximately 3.

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

...............................158 11........................................................3............................158 11.......159 11........3...............................3 Mass Transducers .........................................5 The Application of Electronic Noses for Fish Freshness and Quality Measurement .........1 Introduction .............Chapter 11 Chemical Sensors Corrado Di Natale Contents 11................................................................................................3..........157 11............ is one of the most used method to assess freshness by both consumers and industries [2]..................................157 11...................160 11..............................2 Amperometric Gas Sensors ...........................................157 11...... For these reasons the knowledge of the chemical 153 .............................................................158 11............ for which the human perception of airborne chemicals...............3 Chemical Sensor Technologies .......................3.......................160 11..........................................................................1...........................1 Sensors Based on Conductance Changes ......... called odor... some are of great importance to define overall properties such as freshness or quality [1]...........................164 References ......................3.........................................3.153 11.......4 Electronic Noses ............................................159 11............5 Color Indicators ..........................................................2 Sensor Parameters...154 11...........................4 Field-Effect Transistors .................1 Introduction Among the thousands of molecules composing food complex mixtures.............1 Metal-Oxide Semiconductors .......2 Conducting Polymers and Molecular Aggregates ...................................................3.............6 Conclusions ..............................1..................................................................................................................................................165 11............................... The relationship between chemistry and food properties is particularly interesting in the case of fish and seafood in general.........

do not require any sample treatment. capacitors. Figure 11. a complex structure is necessary. namely a solid-state layer of molecules that can interact with the molecules in the environment. 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. or optical absorbance are among those that can be transduced into an electric signal by suitable transducers. in order to achieve chemical sensors. The interaction with a target molecule (hereafter called analyte) and a solid-state layer is a chemical event that.” The matching between sensitive material and transducer is not univocal: a single sensitive material can be coupled with many different transducers and vice versa. work function. These methods require in some cases complex sample treatments and instrumentation such as gas chromatography or spectrophotometers. As an example. and the development of rapid and reliable chemical analyzers has been pursued since decades. These interactions can be of different nature.2 Sensor Parameters A sensor is an electronic device whose parameters depend on some external quantity of whatever nature [4].154 ◾ Handbook of Seafood and Seafood Products Analysis profile of food is considered of great value. In practice. The device is composed of two parts. whose electrical parameters may depend on the chemical composition of the environment in which they are in contact. Analytical chemistry is naturally based on “separation” approaches: namely. can modify the physical properties of the sensing layer. in the sense that the interaction of human senses with complex mixtures provides a global perception rather than a list of compounds. In the same way there are devices that from the electronic point of view are resistors. 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. as a consequence. These analyzers are chemical sensors. Global perceptions may be enough in many cases to detect freshness or edibility. according to this definition there are resistors whose resistance is a function of external temperature (thermistors) or diodes whose current–voltage relationship is strongly altered once they are illuminated by light (photodiodes). and ultimately they are of paramount importance to determine the acceptance of foodstuff [3]. A sort of combination of natural and analytical approaches has been pursued since decades. These transducers are the second component of a chemical sensor. Differently from analytical instrumentations. it is known that Nature provides living beings chemical senses that. there are many possibilities of assembling a chemical sensor. Chemical analysis of foodstuff is a large part of the analytical chemistry discipline. On the other hand. or even field effect transistors. Electronic properties of materials may hardly be directly influenced by the ambiental chemistry. natural senses are not analytical. The optimal matching between a sensitive layer and transducer is fundamental to achieving a well-performing sensor. and they are sometimes called “basic devices. and a number of methods and protocols for different food are available. 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. The first is a chemically interactive material. Properties such as conductivity. in order to be reliable. and the more utilized are adsorption and reaction phenomena. . mass. 11.1 shows what can be considered as the general structure of a chemical sensor.

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

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

Since the material is a semiconductor.3. It is important to remark that this kind of sensors needs to be operated at high temperature. Paradigmatic.4. which reacts with the bounded oxygen to form carbon dioxide. the number of conductance electrons is limited. At sufficiently high temperature (above 200°C). 11. 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. and a lowering of the potential barrier.1 Metal-Oxide Semiconductors Changes in conductance become appreciable in materials characterized by a limited number of mobile charge carriers. and as a consequence. For instance. The consequence of the exposure to oxygen is a reduction of the surface conductance. a reduction of the surface conductance band depletion. 11. providing the maximum sensitivity.3 Chemical Sensor Technologies In this section the basic principles of the most popular categories of chemical sensors are illustrated. A charge transfer occurs between the material and the adsorbed oxygen atom with the consequence that the conductance band in proximity of the surface becomes depleted. Metal oxide semiconductor chemoresistors have been used several times in fish freshness applications. In practical. dissociative adsorption sites of molecular oxygen are active on the oxide surface. The characteristics of these structures. Metal-oxide semiconductor sensors can be prepared in many different ways. in this regard. These are oxides of transition metals.12]. The amount of depletion and the barrier height are proportional to the number of adsorbed molecules. . The main sensitivity mechanism is related to the role played by oxygen. metal oxide growth in regular shapes such as nanosized belts [9] has shown peculiar properties. the sensitivity to trimethylamine and dimethylamine of aluminum-doped ZnO films was demonstrated [10] as well as the sensitivity to trimethylamine of SnO2 and CuO [11. is the case of carbon monoxide. and then the amount of oxygen molecules that can be adsorbed at the surface is also limited. the most known and studied of which is SnO2 [7]: a wide band gap n-type semiconductor. The most popular materials undergoing a conductance change on interaction with gases are metaloxide semiconductors. although interesting. releasing an electron back to the conductance band.3. 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. This is only one of the many interactions taking place on the surface of metal oxides. The sensitivity can be further modified adding ultrathin amounts of noble catalytic metal atoms on the surface. and the sensitivity of these devices is extended to many different kinds of volatile compounds [8]. Selectivity will be reconsidered in Section 11. semiconductors are subjected to large changes of conductance also in the presence of a modest variation in the number of conductance electrons or holes. have not yet resulted in practical improvements of performances.Chemical Sensors ◾ 157 any reliable information about the chemical composition of the measured sample. Recently.1.1 Sensors Based on Conductance Changes 11. an electrically actuated heater is integrated in the device. in any case. Sensors are here classified according to the physical intermediate quantity. and a surface potential barrier is formed.

Due to the piezoelectric effect. Since crystal resonance is extremely efficient.1.3. More sophisticated mass transducers were proposed by using resonant cantilevers similar to those adopted in atomic force microscopy [19]. These are thin slabs of AT cut quartz oscillating at a frequency between 5 and 50 MHz approximately [17]. aldehydes. The frequency of the mechanical oscillation decreases almost linearly with the mass gravitating onto the quartz surface. and a sensor for SO2 can detect volatile sulfides. aggregates of polypyrrole or polythiophene have a semiconducting character. these sensors were properly used to detect fish freshness [16].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. with broader scopes related to the possibility of developing a novel sort of electronics based on carbon chemistry [13]. the measurement of these mass shifts can allow the evaluation of the amount of adsorbed molecules. If the quartz is connected to an oscillator circuit. conducting polymers sensors can be prepared for different applications. Due these cross-selectivities.3.158 ◾ Handbook of Seafood and Seafood Products Analysis 11. With respect to metal oxides these sensors have two important advantages: they are operated at room temperature and. and food freshness is among them [15].3 Mass Transducers The adsorption of molecules into a sorbent layer produces a change of mass. A typical QMB has a limit of detection around 1 ng. 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. sensors designed for CO are found to be sensitive toward alcohols. their chemical sensitivity can be changed at synthesis level modifying the chemical structure of the monomer [14]. Thanks to this versatility. In spite of the claimed properties. and esters. This property is largely exploited in electronics to build stable oscillators as clock references. most important. Piezoelectric effect can also be exploited in other configurations such as those based on surface acoustic waves. the electric resonance is characterized by a very large quality factor (Q). QMB coated by sensitive layers was used for many applications. The main mechanism is the catalytic reaction occurring on the surface of a noble metal electrode.2 Amperometric Gas Sensors Electrolytic cells based on either solid-state or liquid-ionic conductors are used to detect several kinds of gases. As an example. a sensor for ammonia can detect amines. the electric frequency decreases linearly with the mass. the mechanical resonance of the crystal is coupled with an electric resonance. and their conductance can change after exposure to volatile compounds. 11. an amount that is sufficient in many practical applications.3. The same effect is exploited for chemical sensing adopting particularly shaped crystals such as in quartz microbalances (QMB). these sensors were never demonstrated in practical applications. Although designed for polluting gases. Indeed. the possibility of using these sensors to measure fish freshness was demonstrated with metalloporphyrin coating [18]. Chemical sensors based on conducting polymers may be considered as a lateral result of these studies. For instance. these sensors demonstrated a good sensitivity for compounds relevant for fish freshness. 11. . The measurement of small mass changes is made possible by piezoelectric resonators. A piezoelectric resonator is a piece of piezoelectric crystal properly cut along a well-specified crystalline axis.

combining wavelengths in the optical range. an important gas for fish freshness and quality. and screens. Nonetheless. whose characteristics largely fit the requirements necessary to capture change in optical properties of sensitive layers in many practical applications. H2 molecules dissociate into atomic hydrogen at the palladium surface. also appreciable by eye. allowing a simultaneous evaluation of absorbance and fluorescence of samples. although under constant bias. and hydrogen atoms can diffuse through the palladium film until they reach the oxide surface. FET structures were also modified to accommodate. In particular. and cellular phones all endowed with color screen. was also obtained [21]. 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. as a sensing part. In this way sensitivity to ammonia. This difference can be modulated by a layer of electric dipoles that can reach the metal–oxide interface. . the chemical practice of this approach is badly balanced by the transducer counterpart.Chemical Sensors ◾ 159 11. cameras. known as computer screen photo assisted technique (CSPT). organic molecular layers. On the other hand. where they form an ordered dipoles layer. 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]. the current flowing in the FET changes revealing the chemical interaction. Nonetheless. the colorimetric detection of fish freshness recently received a novel interest. whose sensitivity toward amine was also recently measured [23]. 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]. Indeed. This basic structure was successively modified changing the gate metal and thickness to extend the range of measured gases.3. 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.3. The feasibility of this approach has been demonstrated using as sensitive layer a film of a sodium salt (bromocresol green) [25]. Furthermore. Compared with the use of digital scanners. standard optical instrumentations are usually expensive. and. the visual determination limits the performance and may greatly vary between individuals. This salt exhibits a rather large change in color. in the last decade we have seen rapid growth in performance in fields such as consumer electronics. furthermore. As a result. such as metalloporphyrins [22]. The principle was adequately exploited with a palladium gate FET exposed to hydrogen gas [20]. 11.4 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.5 Color Indicators Although known for several years [24]. The method demonstrated also the possibility to identify a number of different amines [28]. Due to the large diffusion of portable computers. This last technique. 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). is based on the fact that a computer screen can be easily programmed to display millions of colors. 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]. giving rise to a number of low-cost advanced optical equipments such as digital scanners. PDAs.

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

flat. Standard analytical methods for volatile amines and also sensors for some specific amines have been used to inspect fish freshness. In Figure 11. conducting polymer sensors [43]. and sweet conditions are hardly identified. The representation plane is determined as that where the data variance is maximized and then the statistical properties of the dataset are. for example. This result is rather surprising because fish spoilage is in general expected to be a linear and somewhat straightforward process. Minor contributions to the fish headspace come from contamination of the environment (e. let us discuss a simulation of a case study. it is more realistic to consider an array of sensors specific for a single interaction mechanism. Most of these are feasibility studies. The same nonlinearity is observed with electronic noses. In this regard. and optical indicators [46]. In order to understand the potential of electronic noses to detect fish freshness. and acid.6 . let us consider an array specific for each LSER interaction and one compound for family. In addition. In recent years attempts to use electronic nose technology to track the spoilage processes occurring in fishes have been reported in numerous articles. from fish processing. 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. preserved [49]. hydrogen bond basic. Instruments based on different sensor technologies have been used. with a super impression of 6th and 1st days. quartz microbalance sensors [44.4 demonstrate a continuous progress after the 8th day. dipolarity. five different kinds of interactions are considered: dispersion. Sensor data can be conveniently represented by a principal component analysis (PCA) scores plot. PCA is a data analysis method allowing the representation of a multidimensional dataset in a reduced dimensionality space. and fresh. amperometric sensors [42]. and finally from products of lipid oxidation [37]. In gas chromatography. the progress of spoilage is less linear with respect to Figure 11. Analyzing the data with PCA the plot of Figure 11. The number of compounds whose concentrations are only partially correlated makes this application particularly appealing for sensor arrays of partially selective chemical sensors.. as much as possible. MOSFET sensors [41]. with an array of selective sensors it is not possible to distinguish between fresh and flat fishes.3. Since LSER was fruitfully used to model polymer-based chemical sensors [52]. When properly analyzed by pattern recognition methods.3 the time evolution of the major families of volatile compounds found in the headspace of fishes is shown. In LSER. petroleum in sea). and the decrease in others result in a nonlinear problem. the chemical complexity of the problem. Figure 11. but the behavior at the beginning is absolutely nonlinear. a plane. such as metal-oxide chemoresistor sensors [38–40]. the interaction between polymers and volatile compounds is often described by the linear sorption energy relationship (LSER) model [51]. typically according to the freshness or more precisely according to the balance between fresh and spoilage produced compounds. nonetheless. amines become instrumentally appreciable only when spoilage processes take place. The sensitivity of chemical sensors is not immediately related to the molecular family but rather to the interaction mechanism. the accumulation of some compound.5 is obtained. As a result. Results shown in Figure 11.Chemical Sensors ◾ 161 are considered as the typical markers for fish freshness detection.g. showing the ability of the electronic nose to track the different spoilage levels occurring at different storage times. Apparently. Data are extrapolated from an investigation by Strachan and Nicholson [48]. hybrid electronic noses were used combining different sensor technologies such as QMB and amperometric sensors [47]. Nevertheless. polarity. Let us consider the use of an array of sensors absolutely selective for each individual family of compounds mentioned in Figure 11. Each sensor then provides a signal proportional to the concentration of each family.4. in case of fishes. the data produced by a sensor array can classify samples according to some of their global features.45]. An imperfect application of this method was demonstrated with engineered polymer-coated QMB [50].

humans provide a more reliable identification of fish freshness.1 0. 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]. an integration of instruments is necessary. and groupies [59]. in order to measure the quality of fish instrumentally. and olfaction (to smell the gill odor) [54]. Nonetheless. sardines [58].3 Time evolution of the major families of volatile compounds in fish headspace. Results are qualitatively similar to those shown in Figure 11.5. This feature that can be interpreted as a failure of the electronic nose is likely due to an intrinsic nonlinearity of the studied problem. The experiment was related to COD fishes.01 0 5 10 15 Days 20 25 30 35 Figure 11. 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 devices measuring electrical properties) has been illustrated in different applications related to cods [56. with a folding back of the spoilage process in the representation plane.g. image analyzers. olfaction) provides several errors of evaluation. texture meters. . The typical sensorial description is also reported.57]. As a consequence. color meters. tactile (to test the flesh firmness and elasticity). shows the scores plot of a partial least-squares discriminant analysis model related to an array of metalloporphyrin-coated QMBs.. The possibility of developing a multisensor device to measure and/or estimate fish freshness with a combination of instrumental techniques (electronic noses. and original data were previously published [53]. spectroscopic methods. each able to capture different aspects of fishes. and the use of only one sense (e.

5 –2 –2.5 1 PC 2 (15.5 1 0.5 24 PC 2 (19.3 are used.39%) Figure 11. Scores plot 1.4 PCA scores plot of a simulated experiment where sensors selective for the compound family in Figure 11.5 –6 –4 –2 0 2 4 PC 1 (72. . Data show the impossibility of distinguishing the spoilage process in the first 6 days and an abrupt change between 6th and 8th days.93%) 0 –0.5 –1 24 10 12 8 28 2 4 6 1 ◾ 163 22 20 18 16 14 –1.5 –1 –1.Chemical Sensors Scores plot 2 30 1.03%) –1 0 1 2 30 2 28 22 20 10 10 12 14 16 8 1 Figure 11.5 0 –0.76%) 0.5 PCA scores plot of data related to a virtual array of sensors.5 –6 4 6 –5 –4 –3 –2 PC 1 (80. each specific for a single interaction mechanism among those modeled by LSER.

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

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

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

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

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

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

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

1 Ultrasounds—Acoustic Spectroscopy Ultrasonic is a rapidly growing field of research. When these waves pass through foods (or are refracted by them).19]. Ultrasonic velocity in fish tissues. and physical state of foods [20]. and raw meat mixtures can be related to its composition using semiempirical equations [7]. structure. Ultrasound imaging is a versatile. which is finding increasing use in the food industry for the analysis of food products. For fish samples. and visible.3.13] in foodstuffs. which enable a variety of applications [18. determine the velocity of a moving tissue. It is also necessary to work at very low power in order to not cause permanent effects such as heating. Thanks to advancing technology. and dielectric measurements at microwave frequencies can be used to analyze water activity [11] and water content [12. fulfilling the initial premise. Ultrasound is a form of energy generated by sound (really pressure) waves of frequencies that are too high to be detected by human ear. 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. which. It is virtually impossible for . thermal and near-infrared (NIR). 12. well-established. some of their propagation parameters are modified. It is impossible to address all these techniques with precision. and biochemical effects can be observed. the reason is. a number of physical. below are cited some examples of the use of these new technologies in the quality control of foodstuffs. must act in real time and without producing permanent effects on the food. it is almost imperative to resort to elastic (sonic) waves such as ultrasounds or to nonionizing electromagnetic radiation. i. in this chapter. in the case of Doppler-based modes. Nevertheless.172 ◾ Handbook of Seafood and Seafood Products Analysis Given the premise that online control requires a nondestructive method. moreover. chemical. Ultrasound. the modification of these parameters can be measured in real time. 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. exposing their main disadvantages and highlighting the advances in the field of seafood. The main disadvantage of ultrasound is that the energy propagates poorly through a gaseous medium. induces compressions and depressions of the medium particles. such as radio frequency (RF). [21] published a report on how the ultrasonic velocity measurements show potential for analyzing fish composition.e. Normally the modification of any quality parameter is macroscopically correlated to the change in any wave parameter that can be controlled. and a high amount of energy can be imparted. above 16 kHz [17].. NIR measurements are widely used in the food industry to determine the sugar content in fruits [9]. the other techniques that enable online control have been briefly commented on below. microwave. The spectroscopic techniques use the information found in the spectrum that is emitted for the food to predict certain of its qualities. The salt and water content are related to dielectric properties of cod at microwave frequencies [14–16]. Ultrasound attenuation spectroscopy (acoustic spectroscopy) is a method for characterizing properties of fluids and dispersed particles. chicken. Visible (and near UV) transmittance method has been investigated to inspect the internal quality (freshness) of intact chicken egg [8]. Depending on the frequency used and the sound wave amplitude applied. when propagated through a biological structure. and widely used diagnostic tool. The interaction between wave radiation and matter as a function of wavelength or frequency is called spectroscopy. Highfrequency. Suvanich et al. we concentrate on electromagnetic methods at microwave frequencies. impedance measurements (RF) can determine salt and water content in salmon filets [10].

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

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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

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

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

and flavor during spoilage and have been used as quality indicators since the first commercial version of the Torrymeter in 1970 [43]. followed by strengthening of these odors Shellfish.. et al. tallow Flavor Watery. acetic or butyric acids) decomposed grass.2 Torry Score Sheet for Freshness Evaluation of Cooked Cod Fillets Odor Initially weak odor of sweet. and cooking [39. TMA Strong bitterness. rubber. J. however.M. Texture includes the most common characteristics such as hardness.3 Physical Methods 13.194 ◾ Handbook of Seafood and Seafood Products Analysis Table 13. 13. J.40]. springiness. natural odor Wood shavings. Numerous mechanical methods have been used to measure texture. there is little agreement on which is the best method [42]. These changes occurring at microscopic level are related to alterations in appearance. starchy. boiled potato Milk jug odors. seaweed. Sci. hardness is the most important to the consumer.2 The Torrymeter The Torry fish freshness meter “Torrymeter” was developed at Torry Research Station in Aberdeen. 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. 4. vanillin Condensed milk. Dielectric properties of fish are used for determination of freshness.g. trace of “off” flavors Slight bitterness. turnip. sour. “off” flavors. 1953. metallic. flounder. Among textural attributes. A linear relationship was found between Torrymeter readings and sensory attributes for cod. during processing.1 Texture Analysis Texture analyses for seafood are extremely important in research. sour milk. boiled milk. odor. quality control. blue whiting. and chewiness of food. and product development in the seafood industry [38].3. starchy. Dielectric properties of fish skin and muscle alter in a systematic way during spoilage as tissue components degrade. Fish muscle has higher levels of indigenous proteases. Food Agric. Baltic herring. With permission. reminiscent of boiled clothes Lactic acid. boiled meat Loss of odor. Scotland. deciding the commercial value of the meat [41]. Fish muscle may become soft or mushy as a result of autolytic degradation or tough as a result of frozen storage [16]. soapy. . mackerel. which immediately begin to break down the proteins after the harvesting. slight sulfide Score 10 9 8 7 6 5 4 3 Source: Shewan. TMA Lower fatty acids (e. 13. wood sap.. improper handling storage. hake.3.. texture. 283.

and readings from all instruments decrease with storage time. Therefore. and fish-handling procedures. which interfere with the reading of both instruments as they are based on electrical properties of skin. RT-Freshtester reflects dielectrical properties of fish. Inácio et al. 13. Gelman et al. This could be explained by seawater containing ions. However. fast and nondestructive. these instruments need calibration depending on sample preparation. The electric properties of fish can change after death of the fish due to disruption of the cell membranes by autolysis. . The skin of fish could be affected by osmolarity and contact with electrically charged particles [51]. fishing grounds. works only on whole fish and fillets with skin on. [51] also studied the effect of washing with tap and treated seawater on the quality of whole scad (Trachurus trachurus) and found that Torrymeter and RT-Freshmeter readings were significantly (P < 0. measuring the electric properties (resistance. allows automatic grading of 60–70 fish/min. 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]. Fat also has an effect on the dielectric properties of fish and tends to make observed Torrymeter values more variable [47].4 The RT-Freshtester Like Torrymeter and the Intellectron Fischtester VI.3. They are unsuitable for frozen or thawed fish. partly frozen such as superchilled fish. as well as rapid and nondestructive. [50] found strong correlation between the organoleptic and Cosmos results for six species of fish and concluded that application of the “Cosmos” instrument for objective quantitative evaluation of fresh and chilled fish quality by determination of smell intensity appears to be practicable. the “Cosmos” instrument is handheld. 13. iced gilthead sea bream. RT-Freshtester. and farmed Senegalese sole [43–49]. season.3.Methods for Freshness Quality and Deterioration ◾ 195 whole. Mechanical abuse and freezing can affect the readings.3. it could be used for evaluation of fresh and chilled fish in the seafood industry and on fishing vessels. Like other instruments.3 The Intellectron Fischtester VI The basic principles of Torrymeter (the United Kingdom) and the Intellectron Fischtester VI (Germany) are similar. and fish chilled in refrigerated seawater [54]. conductivity. Gelman et al. 13.05) lower in fish washed with seawater than fish washed with tap water or unwashed. However. [50] found that the Torrymeter readings obtained from six species of different origin were poorly correlated with sensory evaluation. portable. and capacitance) of the fish flesh [52]. therefore. The Intellectron Fischtester VI gives reliable information about the days in ice and left of iced stored fish. The loss of skin and muscle integrity and deterioration of the skin caused by bruising during harvesting and packing operations would result in more variable Torrymeter values. The method is based on conduction through skin and.5 The Cosmos The “Cosmos” instrument developed by Japanese is applied for the evaluation of fish quality by determination of smell intensity. The Fischtester readings can be used as an objective criterion for the state of freshness/spoilage together with sensory data across the fish chain.

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

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

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

Among the biogenic amines. and reproducibility.S. depending on the species being examined [10. spermine. postmortem temperature. The formation of biogenic amines results from microbial degradation during the later storage of fish. and arginine leads to putrescine. the disadvantages of using biogenic amines as an index of freshness quality are that their absence does not necessarily indicate a high-quality product [113]. The importance of estimating the concentration of biogenic amines in fish and fish products is related to their impact on human health and food quality. capillary zone electrophoresis (CZE) [128. The QI and the biogenic amine index (BAI) were proposed by Mietz and Karmas [120] and Veciana-Nogues et al.Methods for Freshness Quality and Deterioration ◾ 199 13. Postmortem pH varies from 5.4. tryptamine. Process technology is influenced by rigor development.109. since microbial flora vary seasonally [11].3 pH The pH is also an important parameter to show depletion in tissue and quality of flesh during storage. Consumption of seafood containing high amounts of these amines can have toxicological effects.127]. tyrosine produces tyramine. Since the amines are produced by spoilage bacteria toward the end of shelf life of a fish. [121] for determination of quality of fish. including thin-layer chromatography (TLC) [122. Biogenic amines are generated by microbial decarboxylation of specific free amino acids in fish or shellfish tissue [111].2 Biogenic Amines The concentration of biogenic amines has been reported to be a reliable method of measuring the quality of fish. HPLC [120.5.123]. spermidine. 2-phenylethylamine. tyramine. tryptamine from tryptophan. Among these techniques.129]. Cadaverine is derived from lysine. cadaverine. The most significant biogenic amines produced postmortem in fish and shellfish products are histamine.11. the moment of capture. and use of a biosensor [130–132]. the presence of decarboxylase-positive microorganisms. The others especially putrescine and cadaverine have been reported to enhance the toxicity of histamine [115]. and 2-phenylethylamine is derived from phenylalanine. By means of decarboxylation reactions. the enzyme responsible for its detoxification. cadaverine. Food and Drug Administration [117] and the EU [118]. histamine is potentially hazardous and the causative agent of histaminic intoxication [114].0 to 7. free amino acid content [112]. The formulas used were as follows: QI = (histamine + putrescine + cadaverine)/1 + (spermidine + spermine) BAI = (histamine + putrescine + cadaverine + tyramine) QI is based on the increases in putrescine. The biogenic amine content of fish depends on fish species. their levels are considered as indices of spoilage rather than freshness [112]. and other factors . and stomach contents at death.107. species. respectively. whereas BAI is based on increases in histamine. In addition. Putrescine is also an intermediate of a metabolic pathway that leads to spermidine and spermine [119]. and agmatine. GC [126.124. There are various analytical techniques used to determine the concentration of biogenic amines. putrescine. The hazardous concentrations of histamine are 5 mg/100 g and 20 mg/100 g fish—the legal limit for histamine set by the U. and tyramine. 13.108]. respectively. and the concentration of these increases with storage time [91. putrescine.and diamine oxidase activity [116].125]. and histamine and decreases in spermine and spermidine during storage of fish. reliability.1 depending on season. These problems may be more severe in sensitive consumers who have a reduced mono. and pH [133].4. cadaverine. histidine yields histamine.110]. HPLC is mostly performed because of its sensitivity.

The first one includes direct distillation of fish after adding magnesium oxide. The level of TVB-N in freshly caught fish is generally between 5 and 20 mg N/100 g muscle. Low initial pH is associated with higher stress before slaughtering [13.4. Therefore. was compared with two routine methods. TMA is produced by the decomposition of TMAO due to bacterial spoilage and enzymatic activity [150. 144]. The EC reference method for TVB-N determination. Based on the results obtained from the literature. TVB-N should be used as a chemical check. It was found that there was a good correlation between three methods. Since the activity of enzymes depends on pH. It is well known that determination of TVB-N differs systematically according to the procedures used. However. 13. bacteria act upon TMAO to produce TMA. The European Commission (Council Regulation No. Therefore. produced by spoilage bacteria). the levels of 30–35 mg N/100 g muscle are considered the limit of acceptability for icestored cold-water fish [17. Atlantic cod [143].136–139].59]. The analyses of these indicators are considered unreliable because they reflect later stages of spoilage rather than freshness [140]. turbot [92]. Low pH also promotes oxidation of myoglobin and lipids [134]. and direct distillation methods have been recommended as a rapid routine method. 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. and DMA (produced by autolytic enzymes during frozen storage). TVB-N level correlated with fish quality.151]. such as frozen eel [145]. whereas the second one includes the use of trichloroacetic acid instead of perchloric acid [149]. A relatively low pH may cause a decrease in water binding to the myofibrils. involving preliminary deproteinization with perchloric acid. and European eel [13].4. it could not be regarded as a good indicator of fish freshness and proved to be better as a spoilage index. sardine [12. Low pH is used as an indicator of stress at the time of slaughtering of many animals.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.135]. ammonia (produced by deamination of amino acids and nucleotide catabolites). with the production of lactate.141]. 13. as shown in a variety of fish such as European hake [142]. affecting light scattering and the appearance of fish. total volatile basic nitrogen (TVB-N) primarily includes trimethylamine (TMA. Th is is caused by the depletion of energy reserves. as shown below: Following death of fish. mainly glycogen.200 ◾ Handbook of Seafood and Seafood Products Analysis [134. it affects reactions taking place during storage of fish. which is considered to be the main cause of off odors in fish products [58.4 Total Volatile Basic Nitrogen In seafood. pike perch [146]. the level of TVB-N was not correlated with the time of storage of some fish species. farmed gilthead sea bream [147]. However. and it has been used as an indicator of marine fish spoilage: CH3 CH3 – N=O CH3 TMAO CH3 CH3 – N CH3 TMA . and hake [148].

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

Excess free radicals and peroxides in foods cause destruction of essential fatty acids and vitamins A.K. the type and concentrations of antioxidants.180]. which are the volatile products causing off flavor in products. and pantothenic acid. 3rd edn. The amount of reactive compounds increases gradually.202 ◾ Handbook of Seafood and Seafood Products Analysis Initiation: Initiators (heat. and they break down to aldehydes. 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. B6. The hydroperoxide value is generally shortened to peroxide value (PV). The amount of hydroperoxides can be used as a measure of the extent of oxidation in the early stages. (From Hamilton. lipid oxidation compounds interact with proteins. The peroxide radical can attack another lipid molecule RH. R.C. ketones. resulting in peroxide (ROOH) and new free radical (propagation phase). temperature. Seafood has highly unsaturated lipid content. Peroxides are not stable compounds.182]. and taints [179. 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. Allen. They also destroy pigments. Many factors affect the onset and development of rancidity (oxidative and hydrolytic degradation of lipids). forming stable deterioration products (termination phase) [181. pp. London. in Rancidity in Foods. including the degree of unsaturation of the oil. fish and fish oils are highly susceptible to the development of oxidative rancidity. (Eds. Fish oil contains about 20% of their total fatty acids as long-chain PUFA. U. 1–22.175–177]. and degree of exposure to light [178–180]. Under chilled/frozen conditions. heat) convert RH to free radicals (initiation phase). Peroxides can also react with proteins and result in a decrease in their nutritional value. off flavors. and termination (Figure 13. nutritional losses. pro-oxidants.2 The autoxidation of fatty acids.. C. R. leading to protein denaturation. and free radicals react with oxygen to produce peroxide radicals (ROO). J. and modification of electrophoretic profiles of proteins [172. Initiators (such as light. Free radicals from oxidizing lipids can polymerize with proteins and destroy certain amino acids. There are three steps in autoxidation of unsaturated fatty acids. and alcohols. and Hamilton.J. produce toxins. and then the quantity of radicals and peroxides decreases.). Chapman & Hall.) Off taste and off odor are usually defined as rancidity.C. thiamine. unpleasant odors. oxygen availability. moisture content.. 1994. E. The major chemical indicators for the determination of the extent of oxidative rancidity . and cause off flavor/odors [183]. light.2). propagation.. initiation. consequently.

and lean fish such as blue whiting. 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].188. or enterococci [195].190. Many methods have been employed for the measurements of lipid oxidation in foods as a means of determining the degree of damage [20. European eel. as oxidation proceeds the PV can start to fall.4. During prolonged storage of seafood. and thiobarbituric acid (TBA). Increase in the PV is most useful as an index of the earlier stages of oxidation. which break down to secondary products of oxidation or react with proteins. PV. However. there are some difficulties with common methods when quality has to be assessed. coliforms. reach a peak. Cozzolino et al. [188] and it was found that fluorescence detection of interaction compounds can provide an accurate method to assess quality differences during frozen storage of sardine.186].191]. cod [192. 13. and TBA values may increase. 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. It was indicated that the main requirements for shelf life predictions are to collect information about SSO. 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. [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. spoilage domain such as the range of environmental conditions over which a particular SSO is responsible for spoilage and spoilage level [198]. since they interact with myofibrillar proteins and promote protein aggregation [189].185]. AV.193]. AV and TBA values measure the secondary products of lipid oxidation. 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). and also freshwater fish [194]. TOTOX (2VP + AV).9 Lipid Hydrolysis Hydrolysis leads to hydrolytic rancidity and involves hydrothermal or enzymic (lipase) hydrolysis to FFA and other products. haddock. sardine. FFAs and their oxidation products would have an effect on muscle texture and functionality. horse mackerel [13. Shewanella putrefaciens . causing production of interaction products [187]. PV. 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. Mathematics models have been well established for the growth of spoilage bacteria such as Photobacterium phosphoreum.Methods for Freshness Quality and Deterioration ◾ 203 are anisidine value (AV). since oxidation products are unstable and react with biological amino constituents. PV measures primary products of lipid oxidation. free amino acids. Analysis of these interaction products by fluorescence detection as a quality assessment index for frozen-stored sardine was studied by Aubourg et al. Spoilage of fish and fish products is a result of the production of off odors and flavors mainly caused by bacterial metabolites [197]. It is possible to predict shelf life of seafood based on knowledge of initial numbers and growth of SSO. such as proteins. and phospholipids. peptides.” or numbers of CFU of indicator organisms such as Enterobacteriaceae.5 Microbiological Methods Numbers and types of microbes present in foods are important indicators of safety and quality.

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

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

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

N. Depending on the type of sample and its use. classification based on morphological criteria demands the presence of the morphologic diagnostic characters. for example by chelating divalent cations using EDTA and EGTA. in most breeding programs the fish are indeed selected based on commercially interesting traits such as growth performance.9 resistance to diseases or to stress.1 Sample Preservation and DNA Extraction Methods The sample should be extracted as soon as possible after sampling. Samples fi xed in ethanol must be allowed to dry completely. The basic steps in all DNA extraction methods include the inactivation of nucleases. The DNA .9 Genetic analyses have allowed the differentiation of wild from farmed fish populations in a variety of species. in particular if it has a high enzymatic activity (for example if it contains the hepatopancreas in a crustacean).Analytical Methods to Differentiate Farmed from Wild Seafood ◾ 217 a translucent grayish aspect.).). Th is step is not necessary in samples preserved in ethanol. in contrast to the white opaque color of the wild. and the liver in farmed cod is much bigger than the liver of wild fish.3 Genetic Analysis Doyle et al. and then be rehydrated in water or in the extraction buffer. The DNA is then separated from the contaminating cellular components by salt precipitation. or by the use of Proteinase K) and proteins are removed usually by incubation with Proteinase K. treatment with Chelex. Then. If the sample must be preserved.A Stool DNA Isolation Kit (United Bioinformatica Inc. such as Qiagen. since enzymatic activity also takes place at subzero temperatures. However.Z. give satisfactory results. which are usually absent in many intermediate products as well as in the ready-to-eat dish.11–13 and a loss of rare alleles has usually been observed in the farmed populations.10 and optimal adaptation to different environments. GeneRelease (Bio Venture Inc.11.8. the best method is to freeze it in liquid nitrogen or in a biofreezer. If this policy had been followed. One requisite condition for any genetic analysis is the obtention of good quality DNA suitable for PCR amplification. so that all the ethanol is evaporated. thus limiting its application. Each kit is provided with a detailed description of how to use it. Wizard (Promega).3. For very long periods.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. and then recovered by ethanol or isopropanol precipitation. Many commercial kits. Dynal (Invitrogen). a normal freezer (−20°C) may also be used. the cells are opened (by heat treatment. since diversity would be one of the selected traits in the farmed fish. To extract frozen samples we recommend to start the procedure before the sample is completely thawed. we recommend preservation in 96% ethanol.14–16 Hayes et al. chloroform extraction. Amersham Biosciences (GE Healthcare). which is the most common analysis. 14. sonication. because the enzymes are inactivated by the fi xation. Delays and the use of preservations methods will diminish the quality and the yield of DNA. Nucleospin (Clontech).18 or gel filtration.6 However. and others. it would be relatively difficult to find markers for wild and farmed fish.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. 14. Genomics analyses are dealt with in more detail in Chapter 4 of this handbook. E.

however. salmonids (salmon.2 DNA Markers In recent years. and production method. and the DNA is reconstituted in double-distilled sterile water or in a slightly alkaline buffer (50 mm Tris–EDTA. differences in the protein pattern of liver25 and muscle26. In the future.13. and bass. traits.3. DNA analyses may be performed using chips that permit the determination in one fast step of many characteristics simultaneously. which markers and how many of them are necessary to differentiate a wild from a farmed specimen are completely dependant on the species and the breeding stock and need to be examined on an individual basis. the United States. cod.21 When using the salt extraction method with heavily degraded samples. In these samples the pellet may be practically invisible. SNPs. 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. sample preparation. since some alleles are more frequent in one group than in the other.18 and the salt extraction method. by using a series of SNP and microsatellite polymorphisms by PCR and by oligonucleotide ligation assay (OLA). pH 8. Three more methods that have reputedly produced good quality DNA suitable for amplification. which may be used to identify the strains of the farmed individuals that display an increased frequency of the desired traits. It is worth mentioning. University of the Basque Country. or mitochondrial. Japan. has the potential to be useful to differentiate farmed from wild specimens of a given species. including oysters.23 In principle.17 However. . genomic. protein markers commonly used for genetic analyses have the potential to be used as markers for farmed or wild. cod. which further increases the amount of samples that can be processed. that the Norwegian company GenoMar has patented a method to trace back farmed individual Atlantic salmon. However. China. shrimp. however. breeding stock. Arctic charr).27 tissues between farmed and wild salmon and cod have been reported. whether microsatellites. have started programs to map the whole genome of some species. 14. such as Norway. 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. so the next step.218 ◾ Handbook of Seafood and Seafood Products Analysis pellet is usually washed at least once with 70% ethanol. any marker. Atlantic halibut. and others. several countries. India.19 the Chelex method. including the species.20. catfish. trout. On other occasions. etc. tilapia. An additional advantage is that it is possible to use robots for many of the steps (DNA preparation.15. The outcome of these programs is already producing lists of genetic markers linked to traits of interest. and also for forensic studies.11.14 In addition. from food matrices include the use of hexadecyl–trimethyl ammonium bromide (CTAB). personal communication).).0). must be performed very carefully not to lose the sample.24 The method requires that all parent fish of the brood stock are DNA typed as well as all the individuals under examination. This method has been successfully used by the authors of this paper (unpublished results) and by Bucklin and Kochert22 with whole individuals of Calanus. tilapia. Spain.4 Analysis of Proteins No clear protein marker has been identified to discriminate farmed from wild seafood. the alcohol is allowed to evaporate. 14. which is washing the pellet with 50% ethanol. and sea bass.

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

3 Analysis of Proteins As already mentioned. and. but they will hamper the study of protease activities that may be relevant in some other works. cooking. this may be because some commercial preparations of these enzymes are contaminated with proteases. for a wide screening. In addition to published works. and reducing agents (b-mercaptoethanol. therefore. In our experience.). Afterward. It should be noted that any preservation procedure will alter the protein profile in the sample. there are many methods suitable for protein analyses. Their use should be evaluated for each particular study. due to the great diversity and properties of the proteins contained in the edible tissues of seafood. as well as the different degrees of processing to which the sample may have been submitted (freezing. and fluorescent labeling or staining (of intermediate sensibility and also compatible with MS). usually 8%–20% or 12% PAGE. and digested. we focus on the use of techniques with the potential to identify such markers. Since current studies are still trying to identify markers. detergents (CHAPS. Some authors have claimed that the use of DNase I and RNase in the extraction buffer increases the number of spots in the gels. the strip containing the proteins separated by their pI is loaded on top of the second-dimension SDS-PAGE gel. dithiothreitol (DTT). Triton X-100.4. It is common to use several buffers with increasing concentration of chaotropic agents (urea. one should be very careful when comparing samples preserved and stored under different conditions. 14. SDS).2 Protein Extraction There are many methods for extraction of proteins. but 3–10 are commonly used for wide screenings. Proteomic techniques have a clear advantage in this field. its application is widespread in many fields. Proteomics permits the separation of many proteins (often thousands) from a complex protein mixture in one step. Both first and second-dimension gels can be purchased as precast. etc.4. and there are special protocols for each application. and peptide mass fingerprinting of the digests is then . 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 pH range to choose depends on the sample. usually with trypsin. 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 tryptic fragments are cleaned from contaminants. the gels can be stained by Coomassie Blue (low sensitivity but compatible with mass spectrometry (MS) analysis necessary for subsequent peptide fingerprinting and sequencing). 14. and. the optimal extraction procedure for any given sample must be determined empirically. reduced. However. The first step in proteomic analyses is to extract as many proteins as possible from the sample. destained. alkylated. silver (high sensitivity.220 ◾ Handbook of Seafood and Seafood Products Analysis may be acceptable. ready to use gels from several companies. 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. but not all protocols are compatible with MS). We therefore recommend not to use such enzymes.25–27 BioRad39 and GE Healthcare Amersham have some excellent manuals about protein extraction and analysis. thiourea). The choice of method depends on the protein and the property one wishes to examine. tributylphosphine) to solubilize the widest possible spectrum of proteins. After separation. therefore. depending on the proteins one wishes to examine.

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

5. that is. The most commonly used HR-NMR techniques in wild/farmed classification are 1H NMR and 13C NMR. which leads to less overlapping of signals. including fish and fish products. because it provides multicomponent information and can be applied nondestructively. it is particularly important to exercise care when working with marine lipids: it is recommended to use low temperature (work in ice or in a cold room) and avoid or minimize exposition to air and light in order to prevent lipid hydrolysis.5. NMR spectroscopy can be used to identify functional groups. The most commonly measured nucleus is 1H (the most receptive isotope at natural abundance). 19F.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. 31P. The spectrum is often used to obtain information about the number and type of molecules in a mixture. 2H. it is best to use as low a temperature as possible. 23Na.. 35Cl. both of which are able to detect a range of metabolites in a nontargeted way. 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.g. 10B. oxidation. the reader is directed to several publications46. the ratio n3/n6 was not fully restored.65 NMR gives a fingerprint of the sample analyzed. −80°C. For detailed descriptions of the analysis of fish samples.52 that give detailed descriptions of the procedure. 14. 195Pt). but NMR is applicable to any nucleus possessing spin (e.50.64 may contribute to the difficulty of performing correct classifications as wild/ farmed based only on the FA composition.61 As indicated by Refsgaard et al. The major advantage of 1H NMR spectroscopy compared with 13C NMR is the higher sensitivity and thereby shorter acquisition times per experiment. 17O. 11B. 14. On the other hand.222 ◾ Handbook of Seafood and Seafood Products Analysis for vegetable oils. 29Si. since in a one-dimensional spectrum each peak is produced by those nuclei placed in an identical local chemical environment.2 Lipid Extraction and Gas Chromatography Procedures for lipid extraction are described in another book chapter of this series.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. 14.63. and in particular.1 Sample Preservation Due to the high levels of PUFAs.. and an inert atmosphere. and even after the levels of C20:5n3 and C22:6n3 were restored to the original high levels. and is the preferred tool in lipid analysis when interpretation of . which may be used as a rapid profiling technique. 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. Fresh samples should be kept wrapped in air. If freezing is required. 14N.and light-tight containers and stored at low temperatures. the latter seems to be the most persistent after a dietary switch to fish oil diet. which.52. 13C. HR-NMR has been particularly valuable in the study of marine lipids. 13C NMR has a greater range of chemical shifts.5. 15N. and polymerization.

73 which is of value for authentication purposes.77 Regarding reproducibility issues. even though the signal intensities within each spectrum are not quantitative. It is expected that in the future the use of flow injection systems.0 ppm for 13C NMR. Multivariate methods are frequently applied to study differences among NMR spectra. although this approach has still not been widely used for authentication purposes.70. Normally.76 In some studies. The application of multivariate statistics to NMR spectral data increases the potential of the technique considerably.67 1H NMR has been used to perform quantitative measurements of total n-3 FAs and of the levels of DHA. 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. have allowed the differentiation between wild and farmed salmon74 and cod51 of different origins. the chemical shift scale is referred to the shift of TMS or indirectly to TMS by the peaks from chloroform at 7. will increase the sample throughput significantly. ideally for screening many samples with short acquisition time. the relative intensities for corresponding signals across different spectra are comparable.78 . Both HR 1H and 13C NMR. Typically.. and other intermolecular interactions. or differences in relative concentrations of the samples analyzed. Regions without signals or unwanted signals are removed before multivariate analysis. interactions with metal ions.68. such as instabilities in apparatus. Typically. Both the area/ intensities of peaks and full spectra can be input for multivariate analysis. may lead to erroneous classification. due to the fact that quantitative measurements require a considerable longer experimental time.66. Standardized procedures should be followed to ensure repeatability and comparability.77 It is advisable to check that all spectra have acceptable linewidth and lineshape after the NMR analysis. but it is not necessary for classification purposes. 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.Analytical Methods to Differentiate Farmed from Wild Seafood ◾ 223 spectra is the goal. 1H NMR has also been applied to differentiate between wild and farmed salmon and sea bream of different origins. temperature variations. 99. Tetramethylsilane (TMS) is usually added as a chemical shift and intensity reference. phasing and baseline correction are applied but no zero fi lling.8% CDCl3). in conjunction with chemometrics. although the optimal sample size depends on the instrument.71 13C NMR gives information about FA composition of fish72 and the positional distribution of PUFAs in triacylglycerols and phospholipids. The assignment of spectral resonances gives information about the chemical composition of the samples. a semiquantitative 13C NMR approach has been chosen.75 Small differences in experimental conditions.5–0.71 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). a sample size of 50–100 mg of lipid in 0. Factors that affect the exact chemical shift of NMR signals include the type of solvent used.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. leaving the sample ready for analysis.8 mL solvent is used.75 and it is important that all the samples contain the same volume. hydrogen bondings.66 Potential problems about inconsistencies in ppm values between samples in the data analyses should be solved by manual alignment or data pretreatment methods. inhomogeneities in the applied magnetic field. they are normally converted to ASCII or JCAMP file formats. because it may interfere with the multivariate data analysis. However. pH.28 ppm for 1H NMR and by the triplet of CDCl3 at 77. The most commonly used solvent in the analysis of neutral lipids is deuterated chloroform (i. When full spectra are used. which is easily evaporated.e.

. although many broadleaf plants are also C4).23‰) than the aquaculture specimens.46 were equally successful classifying sea bass using the FA profile. equilibrium reactions also lead to a fractionation of the isotopic forms. 17O (0. depending on their diet and their position in the trophic chain: the higher its position in the trophic chain.224 ◾ Handbook of Seafood and Seafood Products Analysis 14. a kinetic fractionation occurs. N2O and CH4 exhibit wide isotopic variation. and oxygen as three: 16O (99. In contrast. while typical d13C mean values of C3 plants may be −26/−28‰. d13C of individual FA. For example. Some atmospheric gases. such as CO2. exhibit limited variation.037%). they were also able to correctly classify the fish according to their geographic origin.75. because the enzymatic reaction rates on substrates that contain the lighter isotopic forms are faster than in reactions involving the heavier isotopic forms. The isotopic abundances in animal tissues and animal food products are the summation of the feeds ingested throughout all their life. Differences in the 15N/14N ratio also result essentially from diet.204%). Moreover. 171 Atlantic salmon specimens originating from three continents and 15 different geographic regions. and oxygen has been proposed as a method suitable for food authentication.79 Carbon exists as two stable isotopes: 12C (abundance 98. For example. SD ± 0.50 were also able to correctly classify Atlantic salmon according to their geographic origin and production method by using four FA compositions (C16:0. Usually animal products become enriched in the heavier isotope (15N and 13C).52 were able to classify correctly according to the production method. the higher the proportion of the heavier isotope.759%). and O2. C18:1n-9. Samples of muscle tissue of wild salmon were significantly more enriched in nitrogen (d15N: mean = 12.37%). the 13C/12C ratio for both milk fat and cheese protein give information on the type of forage fed to the cows.51. Introducing the percentage of C18:2n6 as a third variable in their model. In addition. Thomas et al.89%) and 13C (1. such as maize. SD ± 0. typically tropical grasses. since the physical properties of molecules containing heavier isotopic forms are different. nitrogen. resulting in a complete separation of the two groups.38‰) but depleted in lipid-corrected carbon (d13C: mean = −20.81 This is because the 13C/12C ratio depends almost exclusively on the photosynthetic mechanism used by the plants for CO2 fi xation. Thus. the abundances of the stable isotopes differ between substrate and product.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.63) and 15N (0.11%). Using the d15N of choline and the d18O of total oil.6 Stable Isotopes The variation in the abundance of the stable isotopes of carbon.80 The natural isotopic abundance largely varies depending on the chemical forms. Aursand et al. nitrogen as two: 14N (99. plus the kinetic fractionations occurring in animal metabolism. the abundance of stable isotopes varies among different compounds. N2. C16:1n-9. 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. C4 plants may have d13C mean values of −12/−14‰. and they reflect both significant isotopic fractionation by microbes and the different biological substrates producing these gases. 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). and 18O (0. 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. Bell et al. Since a molecule containing heavier isotopic forms has stronger chemical bonds.

The research of Sweeting et al. it must not be washed in the laboratory after collection (which may alter the O and H profile of the sample). Enriched samples contain relatively more of the heavier isotopes. thus hydrogen is introduced as H2.6.84 has helped to understand the nitrogen isotopic variations in fishes. the collected tissue samples are dried at a constant temperature of approximately 50°C for 48 h. the ions are finally detected at the detector. The C and O isotopic profiles of fish tissues may be altered if CO is used for stunning or killing. 14. SNIF–NMR can only be applied to the few isotopomers possessing spin. and a detector to measure the different isotopic species. and oxygen isotopes. 14. whereas IRMS gives only an average value of the isotopic forms in the molecule. organic. carbon as CO. and the abundance ratios of the heavy and light isotopic species are then calculated. nitrogen as N2. experimental duration. whereas IRMS can be applied to all except 12 elements. 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.1 were able to differentiate wild. a flight tube with a magnet. The element is converted to a gaseous form to be analyzed by the mass spectrometer.86 To facilitate comparisons between specimens with differing lipid contents. and so on. and stored in glass desiccation vials until analyzed. since these authors assessed the effects of body size. are typically determined with a gas isotope rationing mass spectrometer. the d15N values of heart and liver were also affected by environmental temperature. The ionized gas is then introduced in the flight tube under vacuum or carried by helium. All international standards are .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. ground tissue is used in the simultaneous analysis of stable C and N isotopes. and oxygen as CO2. Usually. The light elements.2 SNIF–NMR and IRMS Two methods are used to assess stable isotopes: SNIF–NMR and IRMS. and d15N of the glycerol choline fraction of flesh phospholipids. and environmental conditions on fish tissue.6. The instrument consists of an ionizing source. The gas is introduced in the mass spectrometer and is ionized by removal of an electron in the ion source.48 NMR techniques have been described previously. probably reflecting the metabolic functions of these tissues and their associated turnover rates. pulverized to a fine powder using a ball mill grinder. Approximately 1 mg of dried.Analytical Methods to Differentiate Farmed from Wild Seafood ◾ 225 d13C and d18O of total muscle oil. for nitrogen 15N:14N). and commercially farmed Atlantic salmon measuring d13C and d15N by IRMS in raw fillets. respectively (for carbon 13C:12C. For example. where R is the heavy:light isotopic ratio of the sample or standard. The assumption that fractionation was independent of body mass was upheld for muscle and heart tissue but not for liver.85. such as carbon. d13C values are normalized for lipid content following techniques developed by McConnaughey and McRoy87 and validated by Kline et al. Interestingly. Molkeltin et al. the paths of isotopic species are deflected by the magnet by an angle that is a direct function of their mass over charge ratio. However.1 Sample Preservation It is very important not to contaminate the sample during handling. nitrogen. as follows: d = (R sample/R standard − 1) × 1000‰.

. it is very important to avoid contaminating the sample during sampling. such as uranium. Carbonate rock from the Pee Dee Belemnite formation89 and nitrogen gas in the atmosphere90 are used as the standards for carbon and nitrogen.5 M nitric acid and rinsed with Milli-Q ultrapure deionized water. cadmium. cadmium and arsenic levels in the muscles of clams from China and Korea were higher than those of clams from Japan. All implements and containers should be cleaned with 0. Thirteen elements were shown to be the most diagnostic. multiple elemental analysis could also be used in this case to identify imported clams from China and Korea. 14. and China. 14. 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. in addition to DNA-based species identification techniques. The sample may be stored in a centrifuge tube at a temperature of −40°C or lower until analyzed. Recently.1 Sample Preservation As for stable isotope analysis. in particular since the analysis may detect contaminants at the pM level. and price.7 Trace Element Fingerprint Sometimes. and vanadium. with the exception that clams from Miyagi had high arsenic content. attributable to manganese and vanadium levels. Rare trace elements taken from the environment. Biochemical analytical techniques using multiple elemental analysis. attributable to cobalt. respectively. and it must be accurately weighed with a microbalance. and analysis. and often the geographic origin of both farmed and wild seafood may be of relevance for its safety. lead.226 ◾ Handbook of Seafood and Seafood Products Analysis set at 0‰ by convention. farmed and wild specimens of the same species have different geographic distributions. Korea. Thus. The . Taiwan. have been used to differentiate the geographical distribution of origin of farmed Japanese eel.95 By using ICP-MS analysis the sensitivity in the determination of rare trace elements can be increased from the nM to pM level. as well as vitamin K and its metabolites. The same research group (Yamashita et al. Multivariate trace elemental analysis is increasingly used as a technique to differentiate the geographic origins of foodstuff. otolith chemistry is used as a recorder of time and environmental conditions. and strontium levels and Factor 3. Therefore. In addition.91 In the case of fish.7. copper. 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 they found distinct patterns for each of the three origins.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. handling. quality. The first step in the analysis is the digestion of the sample: 0. Each sample should be separated from the tissues using ceramic knives and scissors and Teflon-coated tweezers to avoid contamination of metals. multivariate trace elemental analysis is expected to be helpful in determining whether the fish was farmed and its geographic distribution.92–94 Otolith chemistry is useful for identifying the natal origin and assessing the relative contribution of different nursery areas to mixed adult stocks. unpublished data) examined the trace element composition of the muscle and shell of littleneck clams collected in Japan. false labeling problems were encountered in which imported live Japanese eels from Taiwan were illegally sold as being of Japanese origin. were shown to be of relevance to determine the origin of eels. The origins of farmed and wild eel collected from different regions in Japan.

Analytical Methods to Differentiate Farmed from Wild Seafood ◾ 227 digestion may be carried out by placing the tubes in a microwave oven (for example. Afterwards. and stored at room temperature until use. 14.2 ICP-MS Multielement determination of trace elements is usually measured by inductively coupled plasma mass spectroscopy (ICP-MS).8 Other Methods Depending on the species. the samples are diluted to a final volume of 50 mL in Digitube (SCP Science. 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. atomization. a calibration blank and calibration standards are used as surrogate test samples after every 10 analyses. Each solution (5 mL) of the microwave samples is applied to the atomic absorption spectrophotometer. Analysis by chiral chromatography can be used to identify a chiral form (the meso form 3R. much of the astaxanthin used in fish feed nowadays is produced from cultured microalgae or from krill. and the ion information is processed by a data handling system. To initiate the proper operating configuration of the instrument and data system. where energy transfer processes cause desolvation. the use of carotenoids is allowed. Multiwave 3000 Microwave Oven. five internal standards are used: Sc.96 Samples digested as described above are introduced by pneumatic nebulization into a radio frequency plasma. In. the instrument is recalibrated. which suffers from severe memory effects. To verify that the instrument is properly calibrated on a continuous basis. the Norwegian Research .97 using an automatic mercury analyzer (Hiranuma HG-200. Japan). For the determination of mercury. and the last 10 samples are analyzed again.98 making this approach more unreliable than it used to be.3′S) that does not occur naturally and can therefore be used as a marker for farmed salmon. For internal standardization. In the farming of salmon for example. an internal standard mixture is added. Tb. Canada).98 However. there might be specific requirements that may be targeted to identify the production method.7. and Bi. If the measured concentration deviates from the true concentration by more than 10%. Perkin-Elmer). Ions transmitted through the quadrupole are detected by continuous dynode electron multiplier assembly. Y. the total mercury concentration is determined by cold vapor atomic absorption spectrometry. 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. most artificial feeds contain a mixture of canthaxanthin and astaxanthins of different origins (both natural and synthetic). and the resulting digest is a clear liquid with a yellow tint.42. 14. and ionization. but although the diet of wild salmon contains astaxanthin. the mass calibration and resolution are checked using diluted metal solutions as standards.

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

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

..9.. and reduced risk of accidents due to fire.... a water-soluble phase. and their uses in the seafood industry are increasing.. The fractionation of smoke condensates allows obtaining a high diversity of SFs (powders..... Wood sawdust is pyrolyzed in a furnace with low oxygen content...... This kind of smoke flavoring (SFs) appears as an alternative to the smoking process as it is carried out in Europe... are recycled and directed also to ................251 15.................. 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............ a better preservation of the combustible.................. 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. which give rise to new perspectives in the food industry [3].......... the crude smoke condensates are separated in three phases: a water insoluble heavy oil by-products phase..) with a wide range of organoleptic qualities..... hickory.... 15.. and marple... obtained after a settling out time (several days) of the smoke condensates in the settling tank..1...... The gaseous smoke can be cooled down by water or by organic solvents..... However. Simultaneously...... aqueous solutions.................. wood smoke phenolic components are known to be antioxidants..........234 ◾ Handbook of Seafood and Seafood Products Analysis 15................................. By comparison with the smoking technology..... Moreover. SFs are obtained by the condensation of wood smoke and can be further fractionated...... The heavy oil by-products... In United States where 75% of smoked foods are treated by liquid smokes..... beech................... It also allows the reduction of the PAH final concentrations..2 European Regulations on PAH Concentration in Food Treated by SF . oils........ The main woods used for smoke production are oak. Indeed........ The combustible gases are recycled and directed to the furnace................ Indeed....... Today..... that is. mainly hard woods. and provides a higher diversity of smoked food [1]....250 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]... it allows decreasing microorganism activity.1 Introduction Smoking is the oldest food preservation technique.... or concentrated..... purified. liquid smokes are commercialized since the end of the nineteenth century. especially thanks to the industrial benefits brought about by their use.. the use of liquid smokes avoids the release of smokes. the chemical composition of soft woods is responsible for the generation of higher quantities of contaminants as PAH. After condensation...10 Conclusion .... and a water insoluble tar phase.... allows a better control of PAH in the final product. The smoke is filtered to eliminate particules and condensed......... This industrial process leads to more homogenous products smoked with a repeatable intensity and provides an easier cleaning of the smokehouse..... SFs are widely used in the meat industry... between 20% and 30% of European smoked food is treated by liquid smokes.. wood smoke imparts desired organoleptic characteristics such as smoky flavor........ the use of SF allows an easier storage (SF bottles versus wood logs)... A simplified version of SF processes is presented in Figure 15.250 References ..... etc.................. Coupled to salting and drying steps.... However..........

Condensing tower Filter stage two Settling tank Oil exchange system Filter stage one Dryer/blender Further processing Aqueous smokes Recycled combustible gases Smoke oils Patented furnace Recycled heavy oil by-products Smoke powders Wood dust by-products Smoke Flavoring Technology in Seafood Figure 15.1 Diagram of fabrication of SFs. ◾ 235 .

allowing better water solubility. Different SFs (liquid smokes. . Aqueous flavors.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. smoke powders. soluble aqueous flavors. These products have a pH greater than 4 and can also be added to the brine. can be used directly or diluted for applications requiring lower concentrations [4]. or smoke by-products) can be obtained after different steps of filtration. the furnace because they cannot be used for human consumption. Finally.2.2 SFs from primary products.2. Therefore. 15. concentrates of liquid smokes. They are used when intermediate product dispersion is required as in brine. smoke oils. flavors) and also the characteristic aspect and color of smoked food. Concentrates of liquid smokes consist of concentrated versions of aqueous flavors and require lower usage quantities. They are employed to confer smoky organoleptic qualities (tastes. smoke condensates obtained from PTF and PSC are named primary smoke products (PP). This form of SF is especially used for the smoky taste that it confers to the food. They can be employed in sauces or marinades of seafood products. They are especially used when the final water rate in the treated food must be low. The water-soluble phase leads to primary smoke condensates (PSC). However. They are presented in Figure 15. buffered aqueous flavors are partially neutralized or buffered aqueous flavors. a purified extract of the high-density water insoluble tar phase can be used for the production of SFs and is called primary tar fraction (PTF). Soluble aqueous flavors are aqueous flavors that contain an emulsifier such as polysorbates.1 Liquid Smokes Different kinds of liquid smokes are available: aqueous flavors. and buffered aqueous flavors. due to their low pH. separation. or drying/blending of these PP.

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

Drenching/soaking is the opposite of showering technique. According to the final product. vaporized liquid smoke is similar to real wood smoke. Smoke oils are preferred for lipidic emulsions or lipidic sauces. taste. 15. Other devices have been optimized in order to generate a similar physical composition of . This difference constitutes a critical point in the liquid smoking status. especially used for meat products. mainly liquid smoke concentrates on the products in a smokehouse. but it is also employed in the seafood industry. Products are dipped in SFs solution for short periods (from 5 to 60 s).3 Use of Smoke Flavorings There are four techniques to incorporate or deposit SFs in or on seafood products: showering. Direct addition consists in the incorporation of SFs during the fabrication of the food products. The mist obtained is constituted of small droplets with a similar size as in real wood smoke. From a physical point of view. However. an emulsifier must be added in the SFs. Liquid smoke solution is therefore recycled and filtered and the concentration is readjusted. 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. Therefore. especially in the labeling of the smoked products. The mist generated is composed only by small droplets and there is no gaseous phase. which carries a particulate or dispersed phase [8]. In France. atomization of SFs consists in the vaporization of liquid smokes. and this technique appears as an alternative to the smoking process. which can be injected into the product as for the salting step. between 15 and 20 mm. The progress made during the last decades in elucidating the chemical composition of wood smoke gave rise to attempts aiming at producing SF. direct addition.238 ◾ Handbook of Seafood and Seafood Products Analysis The development of synthetic SFs must be also noticed. SFs can be incorporated directly with the ingredients during the formulation or through injection needles when the shape of the product cannot be modified. meat treated by this process is considered as smoked but « smoked by liquid smoke » must appear on the package. Besides. liquid smokes are also employed in the curing brine. Soluble aqueous flavors or buffered aqueous flavors are mainly used. because to guarantee the homogeneity of the SFs during the treatment and to prevent the settling out of smoke condensates in water. the composition of liquid smoke mist is not similar to real wood smoke. Indeed. because the products are immersed in the SFs solution instead of pouring the SFs solution on the products. there are different carriers of SFs. from the granulometry point of view. Smoke powders are preferred when water use is impossible as in dehydrated mixes. composed entirely of synthetic compounds or partly from a liquid smoke base [7]. drenching/soaking. They provide a better water solubility and prevent the heterogeneity of layer formation on the product surface or the product separation during storage. but aqueous liquid smokes are the most used SFs in this technique. Water-based composed SFs such as soluble aqueous flavors or buffered aqueous flavors are commonly used in this technique. However. Diluted SFs fall by gravity through perforated plates on the hung products. wood smoke is composed by a gaseous phase formed by the most volatile compounds. and atomization. the synthetic SFs created are not sufficiently similar to real wood smoke or to SFs. SF is sprayed with air under pressure through special nozzles and forms a wood smoke mist in the cell of smoking. etc. that is. Showering is a technique currently used in North America.) are dependant of the dilution of SFs in water according to proportions varying between 20% and 25% for SFs and 75% and 80% for water. products treated by liquid smoke atomization are considered as flavored and not smoked. The final organoleptic qualities (color. Finally. in numerous European countries. Indeed.

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

it seems difficult to generate the desired organoleptic volatile compounds without PAH contaminants. differences can be observed depending on the molecules. lignin dimers. A slow combustion is reached with weak air velocity. The high moisture allows to reduce the wood combustion efficiency. The combustion is faster when the granulometry of the wood raw material is important [23. The rate of acids is higher for temperature lower than 300°C and decreases after 300°C with the increase in temperature. Therefore. PAH must also be surveyed. a lower temperature is reached and allows increasing the generation of smoke volatile compounds and minimizing PAH formation. and the enolones derivatives [14]. According to the pyrolysis process. and humidity of air constitute key parameters of SF composition. Due to their . the pyrolysis temperature. The air velocity indirectly influences the SF composition by the modification of pyrolysis temperature or smoke temperature [21.18. known as the smoky skeleton of SF.24]. 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 main organoleptically active volatile compounds generated during the pyrolysis process can be sorted in three groups of molecules: the phenolic compounds. the furannic derivatives. whereas a rate between 20% and 30% has been reported as optimal to reduce the emission of particules [11]. and phenolic compounds [10. 15. between 280°C and 320°C for cellulose. the velocity. Therefore. Indeed. A step of filtration is almost obligatory to avoid these contaminants. steps of SF purification through filters or apolar solvent washes are often required to decrease the PAH levels. different groups of compounds are formed. and trimers [12]. because their contents in smoke or in food increase from 400°C to 1000°C. The wood moisture appears as the second important parameter [20]. exothermic reactions of pyrolysis of wood components occur between 200°C and 250°C for hemicellulose. However. The use of hardwood. From 200°C to 600°C. phenol amount is multiplied by two between 450°C and 650°C. whereas syringol quantity is tripled. and 400°C for lignin [17]. Wood granulometry can also influence SF composition.19].240 ◾ Handbook of Seafood and Seafood Products Analysis ethers from lignans. the diversity of settings of pyrolysis parameters can explain the diversity of organoleptic volatile compounds and the diversity of qualities of SF. The manufacturer can choose SF according to the required result on the organoleptic characteristics of the final product. Lower concentrations of oxygenated compounds have been found to be caused by an oxygen depletion during combustion [20]. but they have a weak impact on the smoky flavor of SF and food processed with SF [13]. As the best pyrolysis temperature to obtain the required volatile compounds are between 380°C and 500°C. After the water release (close to 120°C–150°C). is recommended because it burns slower. the quantity of phenolic compounds increases with a maximum close to 500°C and decreases after 500°C.22]. Then. The generation of volatile compounds is dependent on the wood pyrolysis temperature [16]. An optimal moisture is planned in the industry between 17% and 20%. the wood granulometry and moisture. with a lower water rate than that in softwood. because it plays a role in the pyrolysis temperature. A temperature of 450°C–500°C was reported to lead to the best composition for the creation of carbonyls. The rate of carbonyl compounds increases gradually with the temperature from 200°C to 600°C.5 Role of SF Process Parameters in Volatile Compounds Generation Except the wood type that influences the smoke quality strongly [15]. For example. furannic compounds.

The role of syringol is important. taste. Similarly. and alkylguaiacol may also contribute to imparting a smoky flavor to smoked fishes [13. cresols. but it may not be the main contributor to wood smoke flavor. odor. making them odorant at low concentrations. color. and methylsyringol has a pure and characteristic smoky flavor [10]. guaiacol.29].Smoke Flavoring Technology in Seafood ◾ 241 chemical composition.28. They are the major compounds in SF with a wide range of odorant thresholds (Table 15. Some of them have very low odorant thresholds. which gather furannic and enolone derivatives.34].6 Organoleptic Roles of Volatile Compounds of SF 15.6. These observations have been recently corrected [14. Phenolic compounds of medium volatility have been considered as the most important odorant molecules.1 Role of Volatile Compounds of SF in the Odor Even if the concentrations of odorant volatile compounds in SF can be various.2). Many studies have indicated that phenolic compounds present in the vapor phase of smoke may be odor-active compounds [30–32]. two main classes of odor-impact molecules can be defined: phenolic compounds and carbonyl compounds. phenolic compounds are not sufficient to explain the SF role in smoked fish odors. The medium-boiling fraction (91°C–132°C) composed of isoeugenols. and preservation of the product. syringol. 15.1) [14.33. Table 15.1 Odorant Thresholds of Various Phenolic Compounds Phenolic Compounds Phenol o-Cresol m-Cresol p-Cresol Guaiacol 4-Methylguaiacol 4-Ethylguaiacol 4-Vinylguaiacol Vanilline Syringol Eugenol Ethylvanilline Odorant Thresholds in Water (μg/L) 5900 650 680 55 3–21 90 50 3 20–200 1850 6–30 100 References [25] [25] [25] [26] [25] [25] [27] [26] [25] [25] [26] [25] . Phenolic compounds of low-boiling fraction (60°C–90°C) composed mainly of phenol.34] (Table 15. Phenolic compounds are known to constitute the odorant “smoky” skeleton of wood smoke and smoked fish. the diversity of SF causes diverse consequences on the texture. A much more complex mixture of compounds is responsible for the characteristic aroma of smoked fishes [35].

55 ± 39. spicy Spicy. STD MS.53 360.98 20. LRI.97 23. milk Cooked/soup.74 ± 27. ink Marine. green Cooked vegetable. LRI.27 ± 0. LRI MS. green Cooked vegetable. LRI MS.6-Dimethylphenol 1130 .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 2.48 ± 8.34 (24.07 (1. LRI.50) 65. spicy.22 ± 9. LRI. STD MS. spicy/ woody (3) (2) 5 6 (2) 4 4 3 5 6 7 (2) 4 4. STD MS.55 ± 9. fatty Roasty.45 ± 172.27 ± 2.63 ± 13. STD MS.25 6 (5) 6 7 (4) 7 7 6 8 8 8 Chemical. green. roasty Chemical. spicy. potato Cooked potato.24 ± 63. STD MS.Table 15.04 7 4 16.37 ± 3. Animal. mushroom Cooked.53) 8.3-Dimethyl-2cyclopentenone 1052 o-Cresol 1068 p-Cresol 1093 Guaiacol 1110 2. LRI MS. chemical Green. LRI. STD MS. vanilla. STD MS. burnt. wood fire.94 49.12 42. burnt. metallic.75) MS.44 17.18 ± 37. 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. earthy.90 (1. STD Cooked. LRI. STD MS. LRI MS. STD MS. LRI. LRI.62 74. chemical.33 ± 1.64 ± 18.2 Odorant Characteristics and Concentrations of the Most Potent Odorant Volatile Compounds in Salmon Fillets Treated by Liquid Smoke Means of Identificationa Mean ± SDe 124.17 ± 26. milk Smoke. LRI. burnt Smoked. STD MS. LRI.6-Dimethylpyridine 890 2. LRI.

51 ± 18.4Trimethylcyclopenten-1one MS MS.21 ± 7. STD MS. LRI MS.13 6. smoked Candy. green 6 3 11. LRI MS.79 (6.95) 8. vanilla Cooked. spicy.12 4.86 (2. STD MS. spicy. LRI 1160–1180 4-Methylguaiacol 1192 482. LRI.50 18. violet.62 ± 4.85 ± 40.2-Dimethoxybenzene 1147 2. LRI.E)-2. spicy Oily.5-Dimethoxytoluene 1282 4-Ethylguaiacol 1287 Indanone 1307 4-Vinylguaiacol 1330 (E.91 36. STD MS.16 ± 5.82 ± 6. smoked Cooked vegetable. STD MS.17 15. STD Cucumber.35 3-Ethyl-2-hydroxy-2cyclopentenone 1140 1.3.and 2.2.61 ± 22. LRI.15) 86.71 (3.49 ± 6. vanilla.26 ± 1.4. LRI MS.46 Solvent. STD MS. LRI. spicy Spicy. spicy.87 ± 1. green. 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.2. chemical Green. clove Green. LRI MS. green. green. medicinal 7 4 10. burnt Smoke. LRI. milk Burnt. LRI.36 1132 MS Cooked. green. LRI MS.96 ± 10.15 ± 1. clove Sawdust.15) (continued) 2. smoke.3-Trimethoxy-5methylbenzene 243 .3-Dimethoxytoluene 1247 (E)-2-Decenal 1266 3.25 ± 10. fatty.97 2.15 ± 243. spicy 6 5 17.72 44. fatty Burnt rubber.24 ± 1.4-Decadienal 1330 Syringol 1365 Eugenol 1370 Smoke Flavoring Technology in Seafood 4-Propylguaiacol 1382 1400 ◾ 1.25 ± 4.5Dimethylphenol/ (E)-2-nonenal MS. smoke. LRI. LRI. STD MS. green. STD MS. rotten. green Spicy.

81 ± 11.40 ± 3. . roasty. odor intensity.87 ± 2. J. V.. and odor description of an identified compound correspond to the retention time.23 ± 0. LRI.68 MS.. spectrum.77 24. Average intensity of the eight judges is rounded to the nearest whole number. spicy 6 3 Odorant Attributes Given by the Judgesb Number of Judgesc Average Intensityd 244 ◾ Compounds LRI (DB5) (Z)-Isoeugenol 1423 (E)-Isoeugenol 1473 2. green. In micrograms equivalents of dodecane per 100 g of smoked salmon. STD. Food Chem. 55.5 is rounded to 5 and an intensity between 5. LRI MS.Table 15. LRI. it must be considered as an attempt of identification. et al. The odor given corresponds to the odor detected by the judges during olfactometric analysis for its retention time but not surely due to the compound that we try to identify. STD MS.35 (20. When only MS is available for identification. and quantities of odor-active compounds detected by fewer than six judges are indicated in parenthesis. 2007. standard (when the retention time. rotten 7 4 Spicy.3.48) 1.55 ± 8. a b c Handbook of Seafood and Seafood Products Analysis d e Means of identification: MS. Number of judges (out of eight) who have detected an odor. woody (4) (2) Clove. roasty 7 4 Burnt rubber. linear retention index (when the LRI of the identified compound corresponds to the LRI in the literature). and odor description of the injected standard of this compound). chemical 6 4 Smoke. An intensity between 5 and 5. spectrum.39 6. 9 = very strong odor intensity). LRI Animal.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 7.5-Trimethoxytoluene 1527 4-Allylsyringol 1615 8-Heptadecene 1680 Source: Varlet. Note: Frequency of detection. mass spectrum (identified using the mass spectra of the compounds). LRI. 4518.5 and 6 is rounded to 6 (1 = very weak odor. STD MS. LRI MS. Agric. Means of three fillets.

2 Role of Volatile Compounds of SF in the Flavor Phenolic compounds were shown as the major contributors of the smoky flavor [35]. The high-boiling fraction of phenolic compounds (133°C–200°C) was described with an acid and chemical property that was judged of poor quality. and seem to contribute little to overall aroma. the determination of the effects of compounds of SF on the flavor is complex. phenolic compounds are not the only flavor-active compounds. the same compounds responsible for the odor should be involved in the flavor that SF confers to seafood. sometimes cooked. or oils. because they are not the compounds mainly detected in SF and seafood treated by SF odors by sensory analysis [37]. furannic compounds were found to play a role in cold smoke odors of liquid smoke or fishes treated by liquid smoke [14. as the other minor odor-active compounds. they may contribute in mixture to the overall odor. However.34]. The determination of the role of SF components in the final product odor is complex due to the odorant interactions that can occur between the odor-active compounds. Recently. The results of this fractionation are given in Table 15. Furthermore. Furannic compounds were thought to contribute to soften the heavy smoky aromas associated with phenolic compounds [37. 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].3. 4-methylguaiacol. As for the assessment of the odor. 15. The oils used in smoke oils can soften the smoke aroma. whereas syringol and 4-methylguaiacol showed the same but lower effect than guaiacol [40]. it was commonly admitted that syringol derivatives impart a smoky odor and guaiacol derivatives contribute to a smoky flavor. but taste was not investigated as much as odor. They were isolated early from wood smoke and described as grassy. 4-methylguaiacol perception was superior to that of syringol and guaiacol. Enolone derivatives are compounds derived from cyclopentenone. Concerning the bitter taste.38].Smoke Flavoring Technology in Seafood ◾ 245 Carbonyl compounds have also been reported as contributors to the smoky aroma of wood smoke. Two categories of carbonyl compounds can be differentiated: furannic compounds and enolone derivatives. Furfural and homologues exhibit cooked/roasty aromatic notes. A polyfunctional carbonyl subfraction was isolated from wood smoke and possessed a caramellic/burnt sugar aromatic note [36]. Synergic or masking effects are possible and make the final odor complex. However. the physical state of SF can also influence the aroma. and isoeugenol in spicy/sweet flavor [41]. guaiacol derivatives and more generally the phenolic compounds of low-boiling fraction molecules (60°C–90°C) have been shown to cause the odor. if they do not have a strong individual influence. Moreover.6. Then. early works performed on individual phenolic compounds have identified the impact of guaiacol on the smoky flavor. More recently.39]. reactions between liquid smoke compounds and the components of the matrix can occur through Maillard and Strecker reactions. However. . and very little information is available. For several decades. and the odorant contribution of odor-active compounds cannot be the same if the SF is in the form of powders. Sensory analysis performed on standards confirmed the importance of guaiacol and o-cresol in the smoky flavor and dimethylphenol. The taste thresholds of some phenolic compounds were determined [40] and showed a high diversity between the molecules. 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. liquids.

which has been reported in wood smoke and smoked meat [41] but not in smoked fishes until now. The acidic aqueous SF can also increase the coagulation of proteins and act on the texture. histidine. furannic compounds could also have an effect on the flavor [43]. 5. Formaldehyde. phenolic subfraction. These . Other compounds such as enolone derivatives could also play a role in the SF flavor. Indeed. 15. 2. because they are added in the product during its fabrication. they could play a role in the inner texture of the product. which have brown/yellow characteristic color. which can vary from golden yellow to dark brown according to the nature of the wood. 3.3 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.6.6. and the intensity of the process. Formaldehyde was shown to react with the amino group of the N-terminal amino acid residue and the side chains of arginine. the eventual dilutions of SF. distilled at 133°C– 200°C. cysteine.246 ◾ Handbook of Seafood and Seafood Products Analysis Table 15. According to their concentrations found in the different SF.3 Role of Volatile Compounds of SF in the Texture The texture of smoked products is due to coagulation of proteins. Studies on standards have shown that cyclotene was a flavor-active compound [41]. 15. terpene subfraction. In the liquid smoking process. distilled at 67°C–90°C. the product must also be placed in a dry and hot ambient atmosphere for short periods in order to favor color formation. 11 = highest value. SFs under powder or oil forms do not act on the surface texture. After scission and dehydration. 1. smoke condensates are colored mainly due to phenolic compounds. melanoidines could be created by polymerization through aldolic condensations.4 Role of Volatile Compounds of SF in the Aspect and Color The color of seafood treated by SF can derive from physical and chemical reactions. distilled at 91°C–132°C. whole liquid smoke. 4. Maillard and Strecker compounds can also be responsible of the color of the smoked product [45]. the deposition of Maillard compounds leads to a darker color of fish flesh [46]. and lysine residues [44]. can react with proteins. Thus. A brief drying after smoke absorption can cause a higher level of dehydration and lead to higher amounts of Maillard products. Their physical deposition of SF on seafood can confer its color to the product. 6. However. However. 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].

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

Therefore.8 epoxyde OH B[a]P 7.1 Polycyclic Aromatic Hydrocarbons Properties and Toxicology PAHs are well known as being food contaminants and carcinogens [52]. they are considered as environmental pollutants and can contaminate the human feed raw material [53. These compounds have been studied for several years. 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. 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. particularly smoked food [57.3). They are considered as carcinogenic contaminants. . more toxic than light PAHs. PAHs are formed by the incomplete burning of carbon-containing material. PAHs are generated during smoke production by wood pyrolysis.58].248 ◾ Handbook of Seafood and Seafood Products Analysis 15. it is used as the leading substance to illustrate PAH contamination. PAHs can cross the biological membranes and accumulate in tissues.8 15. In SF.3 Benzo[a]pyrene metabolization.8-dihydrodiol-9.8. As they can be absorbed by animals.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. especially benzo[a]pyrene (B[a]P) [59]. which are constituted by less than four benzene rings. hence their toxicity (Figure 15.10-epoxyde Glutathion S OH OH OH O B[a]P 7. they are considered as heavy PAHs. Owing to their lipophilic properties (log Kow between 4 and 7). Hundreds of individual PAHs may be formed and released during the process of incomplete burning of the wood.8-dihydrodiol OH B[a]P glutathion conjugate OH OH B[a]P 7. because their catabolism leads to poly-hydroxy-epoxy-PAH suitable for binding to DNA adducts. Therefore. However. PAHs are considered as carcinogenic contaminants of processed food [56]. home cooking and industrial food processes represent the major source of human contamination [55].54]. PAHs comprise fused aromatic rings made up of carbon and hydrogen atoms: up to four fused benzene rings.

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. that is. but. In the 1980s. 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. Among them. For the separation of the PAHs extracted from SF or seafood treated by SF. it is essential to quantify only the toxictargeted compounds. the analysis step is the most critical point. Other extraction devices have been developed to investigate PAH in smoked food such as accelerated solvent extraction (ASE) [65]. because they do not have the same toxicity.9. and liquid chromatography is coupled to ultraviolet or fluorimetric detector [59–63]. the chromatography must be sufficiently efficient to separate isomers of PAH. In the case of liquid matrices as liquid smoke.S. Gas chromatography is coupled to mass spectrometry [58. solid–liquid extraction can be carried out. In both condensates. even if they were found in weak quantities. For example. PSC and PTF. a liquid–liquid solvent extraction is often used [61–63].1 European Regulations on PAH Found in SF In 2003. or tandem mass spectrometry [70]. the maximum levels of B[a]A and B[a]P were set at 20 and 10 mg/kg of liquid smoke. The nature of the SPE cartridge phase is linked to the extraction method and the biochemical composition of the initial matrix. . respectively [73].69]. purification and/or delipidation steps such as saponification are often applied to reduce the fat matter rate of samples [64]. However.Smoke Flavoring Technology in Seafood ◾ 249 safety avoiding PAH contamination. Thus. these PAHs were considered as toxic at low levels. supercritical fluid extraction (SFE) [66] or solid-phase microextraction (SPME) [67]. respectively [74]. Therefore. In the case of solid seafood treated by liquid smoke. gas chromatography and liquid chromatography are the most used techniques [55]. to our knowledge. This harmonization was necessary to homogenize the legislation about SFs.8. with a weak toxicity but high concentrations in the samples analyzed. and lead to mistakes in the identification. Several devices are therefore developed to optimize the analysis. PAHs are often coextracted with fat matter. Environmental Protection Agency (US-EPA) identified a list of 16 PAHs as the most frequently found [60]. The extraction step must integrate the composition of the matrix. 15. cause chromatographic coelutions. Although all steps are important. and stir bar sorptive extraction (SBSE) [68] but not on liquid smokes or seafood treated by SF. 15.9 Legislative Aspects 15. Parameters of the chromatographic separation and detection must be adjusted to avoid coelutions with interferences from lipids. which can disturb the extraction. Purification was especially performed on an alumina or silica column. but solid-phase extraction (SPE) cartridges are now more frequently employed. in Italy. such as bidimensional chromatography at the gaseous phase (GC/GC) [71] or liquid phase (LC/LC) [72]. a European regulation set the maximum contents of PAH in the primary products (PP) of smoke condensates used for the production of SF. Moreover. The eight heavy PAHs left were shown as being carcinogenic or mutagenic contaminants and gave rise to serious health concern. which combines a separation step and a detection step. eight light PAHs were considered as environmental contaminants.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. flame ionization detector (FID) [60]. the U. Indeed.

the respective legal B[a]P amount is 5 mg/kg. However.03 mg/kg and leads often to noncompliant smoked products. Therefore. The main criticism that can be formulated against SF is the lack of control of the final organoleptic qualities of such processed food.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. atomization of liquid smoke must be lower than 0. This value is very low compared to those authorized in PP. SFs are used in higher quantities than those employed in flavoring processes. for meat industry. excluding bivalve molluscs. and head and thorax meat of lobster and similar large crustaceans [76. whereas food is treated by SF and not directly by PP. Therefore. Moreover.03 mg/kg. In this case. However. All these benefits could help to reconsider the status of atomization of liquid smoke and the maximum PAH contents related. 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. 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]. a European regulation set the maximum content of B[a]P in foodstuffs treated by SF at 0. This value is the result of the necessary harmonization between the national laws of European countries [79]. . Nevertheless. However. the smoking regulations set a maximum B[a]P value of 5 mg/kg of smoked fishery products and smoked crustaceans.9. Indeed.63] which justifies controls and regulations. if it is considered as smoking technique.2 European Regulations on PAH Concentration in Food Treated by SF In 1988. very small amounts.75]. it is paradoxical to apply flavoring regulations to the smoking process. Th is fact can be understood by the use of smoke condensates in flavoring quantities.03 mg/kg. as drenching or showering. it leads to lower PAH contents. according to the origin of SF and industrial manufacturers. As for SF. the 2003 maximum values must be reviewed again. brown meat of crab. Finally.78].69. but it could also initiate an international consideration of labeling of smoked and flavored food. this process can also be considered as a flavoring of the surface of the product. In certain countries such as France. 15. Therefore. Thus. Indeed. leading to less PAH contaminated food by comparison with the traditional smoking techniques [80]. because these values were set for PP and not SF. the PAH contamination was only set for B[a]P [77]. 15. The content of PAH in SFs and in the final product can be better controlled than during traditional smoking. that is. it is necessary to better control the composition of SFs and to improve knowledge about the influence of the pyrolysis parameters (wood nature. Moreover. Indeed. Moreover. that is. atomization of liquid smoke would constitute the smoking technique. the liquid smoking process decreases the emissions of PAH compounds to the environment. important differences in PAH concentrations are noticeable [57. 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. 0. Indeed.10 Conclusion A wide range of SFs and uses of SFs are now available to flavor seafood products. it can lead to problems of labeling. the PAH contamination reached in SF is largely below the values authorized in PP [60. the vaporization of SF in a smokehouse causes a loophole in the legislation. SFs appear as a safe alternative to smoking techniques.

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

2007. Contribution of smoke compounds to sensory. 34. G.. and Burtles. Pol. Agric. 31. ASTM Data Series DS 48A. The development of flavour in potable spirits. J. T. Chem.. 43. 1970. 2002... Fish. Varlet. Biol. CRC Press LLC. 28. 8. 102. 2001..C. Rusz. Contribution of volatiles to rice aroma. . American Society for Testing and Materials.. 137. Daun. 21. Pure Appl. Food Chem. Soc. 2006. PA.. A. M. M. Clifford. B. 1977. Meet. 39.L. J. et al. 1977. Tang. Wasserman. W. Chemical composition and application of smoke flavor. et al. Hamm... et al.. et al. 1999. Feranoli’s Handbook of flavor Ingredients. and Fujimaki... Pure Appl. 53.. Chemical references in sensory analysis of smoke flavourings... Process Biochem.-Technol.E..G.. 18(5). 49.L. 42. 1977. Metz. J.. 85.D. 22. A.D.. Thesis. J. 25. C. 1977.. S. 203. 4126. and Toledo. N. humidity and air flow on smoke penetration into fish muscle.M. 2004. S.. and Vaisey. Food Chem..S. Smoke flavor as related to phenol. Turnbaugh.... Board Can. Agric.J. 1974. 40. Chem. and air velocity.. Proc.. Fiddler.Z. A. Kim.S. Eur. Fazzalari. 29. Kurata. J. Wasserman. J.. R. FL. F 7:1. F. B. wood. Sensory properties of phenolic compounds isolated from curing smoke as influenced by its generation parameters. 2395... 1970. J. Manzanos. 49. Baryłko-Pikielna. Adv. humidity. 1976.. M. bacteriostatic and antioxidative effects in smoked foods.. Guillén.. M. U. Meat Res. University of Sciences of Nantes. 78(4).-Wiss. and Ibargoitia. Compilation of odor and taste threshold values data. and Eyo.. 36. Influence of the moisture content on the composition of the liquid moke produced in the pyrolysis process of Fagus sylvatica L. 1975.. 240. Acta Aliment. 1966. Agric. Caractérisation des composes volatils responsables des qualities odorants du saumon fume (Salmo salar) et evaluation des contaminants du fumage (Hydrocarbures Aromatiques Polycycliques). and Doerr. Flavor effects of different woods on whitefish smoked in a kiln with controlled temperature. et al.. Agric. 33. M. Chem.. W. Effects of the smoking process on odour characteristics of smoked herring (Cuplea harengus) and relationships with phenolic compound content.M.W. Philadelphia.T. 433.. Cardinal. Pure Appl. 1969. A.. M. Food Res. Chan.L.. Analysis of smoke and smoke products. 2004. M.C. Lantz. Boca Raton. Toth... Organoleptic evaluation of three phenols present in wood smoke. 36(5) 1006. M. carbonyl and acid content of bologna. 38. Food Chem. Chem. 32. Sérot. A.. 1667.A. 7(2). Burdock. 49. Food Sci. Guillén. Food Sci. 1980. J. 934. and Potthast. T.A. 1639. Food Chem. Chemical aspects of the smoking of meat and meat products. Biol. and Miler. 201. R. Physical and chemical processes involved in the production and application of smoke. 26.B.. Swan. Effect of smoking processes on the contents of 10 major phenolic compounds in smoked fillets of herring (Cuplea harengus). 49. H. 37. Chem. Lebensm. 40. 96.. Isolation and identification of some components of the lower-boiling fraction of commercial smoke flavourings. 146.252 ◾ Handbook of Seafood and Seafood Products Analysis 20. R. 2002. 31. M. 1988. 47... V. Food Chem. et al. Food Chem.N. 27.G. 111. noncarbonyl neutral and basic fractions of aqueous smoke condensates. Carbohydrate and nitrogenated compounds in liquid smoke flavorings. J. 29. Food Sci. L. 23... and Ibargoitia. Effect of smokehouse temperature. M. 44. Chem. J. June/July. Res. 24.. Bratzler.E.A. 22. Identification of flavour constituents in carbonyl. 6235. 1201. Identification of formaldehyde-induced modifications in proteins: reactions with model peptides. Olsen. 5(3). 38. 1972. 1655. 34... 1978. Radecki. Smoking of foods. Food Chem.. 41. Rev. K.. K. J. Buttery. Agric. and Ling. 1978. 35.. 1984.. K. L. 1005. A “smoke” flavor fraction of a liquid smoke solution. 30. L. 3. 87. Workers. J. Ojeda. 279.J. 27(7).

. 2000.P. Validation (in-house and collaborative) of a method based on liquid chromatography for the quantitation of 15 European-priority polycyclic aromatic hydrocarbons in smoke flavourings: HPLC-method validation for 15 EU priority PAH in smoke condensates.. V.. P. Food Addit. and Partearroyo. 52. J. and Anklam. M. Des techniques ancestrales à leurs réalisations contemporaines: salage. Suñen. 2005. Comparison of two clean-up methodologies for the gas chromatographic/ma ss spectrometric determination of low nanogram/gram levels of polynuclear aromatic hydrocarbons in seafood. 27. 2000. Suñen.. 2007. A.. Agric. Lavoisier. and Turesky. Food Chem. W. Sopelana.. Hooven. Food Chem. 2004.. B. 1996. E. C. J.. Agric. Chromatogr. 126. 48. Changes of benzo(a)pyrene contents in smoked fish during storage. J. Nyman.. Müller. 53. Palme. fumage. La fumaison. Determination of polycyclic aromatic hydrocarbons in smoked meat products and smoke flavourings additives. Contam. 1977. S. 49.J. 56. W. Fleischwirtsch..J. Curing and smoking. and Sikorski. 3. Antibacterial activity of smoke wood condensates against Aeromonas hydrophila. 1991.. M. 489. Technol. Carcinogenic polycyclic aromatic hydrocarbonDNA adducts and mechanism of action. Eur. séchage. Scientific Committee on Food (SCF). R. Stołyhwo. V.. 40. 46..A. 1103.. cold-smoked rainbow trout stored at 4°C.. Contam. 44. Baird...... P. Šimko.M. 63. 1988. and Anklam. 48. and Sérot. J.. 71(1). 208. M. C. Chromatogr.Y. A.D. Mottier. Prost. 2003.387.. 307.Smoke Flavoring Technology in Seafood ◾ 253 45. 1536. Environ. 293. and Partearroyo. Food Chem. C. 2005. P. Agric. 36. Mol. 2244. Guillén.. Determination of polycyclic aromatic hydrocarbons in commercial liquid smoke flavorings of different compositions by gas chromatography–mass spectrometry.. and Mahadevan. J.. B. Food Microbiol.. Polycyclic aromatic hydrocarbons in smoked fish—A critical review. J. M. 51. 61. and Fernandez-Galian. 18. occurrence and mechanisms of formation. P. 47. 49.. Food Res. 59. Food Chem. SCF/ CS/CNTM/PAH/29 final.. 91(2). Z. C. 17(1).H. E. and Aristimuño. T.A. Evaluation of analysis of polycyclic aromatic hydrocarbons in meat products by liquid chromatography. 218. in Technologie de la viande et des produits carnés.A. Šimko. S. 2000. B. P. 45. Fernandez-Galian. Food Res. . Food Addit. Volatile aldehydes in smoked fishes: Analysis methods. Tilgner.. 770. and Listeria monocytogenes at low temperature. L’industrie alimentaire halieutique. 219. 62... 106. Jira. Single-laboratory validation of a gas chromatography–mass spectrometry method for quantitation of 15 European priority polycyclic aromatic hydrocarbons in spiked smoke flavourings... 10(5). Simon.. Varlet. 2002. Guillén. 105.. B. B.J.. Sainclivier. Chen.. 104(2). 2002. Yersinia enterocolitica. 171.. Int. et al. 1985. 2001. 58. R. 61. Simon. Mutagen.. Study of several aspects of a general method for the determination of polycyclic aromatic hydrocarbons in liquid smoke flavourings by gas chromatography-mass spectrometry. Bulletin scientifique et technique de l’Ecole Nationale Supérieure Agronomique Centre de Recherches de Rennes. 111. M.. Activity of smoke wood condensates against Aeromonas hydrophila and Listeria monocytogenes in vacuum-packaged. Palme. 2006. E. 48. 64. 1993. D. P.. marinage. E.... C. W. Opinion of the Scientific Committee on Food on the risks to human health of polycyclic aromatic hydrocarbons in food (expressed on 4 December 2002). Sopelana. and Chiu. R. 1160.. Wang.D. 1629. 57. Food Chem.. 1991. hydrolysats. Parisod.P.E.... L. 876. Quantitative determination of polycyclic aromatic hydrocarbons in barbecued meat sausages by gas chromatography coupled to mass spectrometry.D. 303. Food Chem... The phenomena of quality in the smoke curing process. Paris. Aristimuño. 60. Girard. Food Chem. 54.. 50. Pure Appl.. 55. A GC/MS method for the determination of carcinogenic polycyclic aromatic hydrocarbons (PAH) in smoked meat products and liquid smokes. 2007. Chem.

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

NUTRITIONAL QUALITY III .

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

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

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

Fosslet. the fat content can be calculated theoretically by the formula Fat% = 80 − water % . etc.) Microwave drying The sample is dried and from the water content found.55. 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. fexICA).Table 16. lipid content.1 (continued) Advantages Rapid.22. (SoxTech. processing) [46] Automatic. location of lipids. 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. nondestructive (See under NMR below) [13–15.57] Excellent for determination of fat and water content or even distinguish lipid classes and water properties.

portable (small size). NIR/NIT Transmitted or transflected Near Infrared light (800–1700 nm). location of lipids. rapid. easy. and portable Allows in vivo measurements Nondestructive and rapid Broad range of applications. and nondestructive and allows in vivo measurements (continued) 261 . Low-field NMR See NMR above Composition and Calories NMR mouse ◾ Nondestructive and rapid.8] [46. lipid content. may also provide other nutrient data in the same analysis Some portable instruments are available Allows in vivo measurements Expensive.50.55] Fat meters Determination of water by analyzing the dielectric properties using a microwave strip (calculation of lipids as for the drying method). The NMR mouse is rapid. processing) Needs to be calibrated for the individual species Most suitable for neutral lipid determination See NIR/NIT above [4–6. nondestructive.Nondestructive Methods: No laboratory facilities are required Precision level may be dependent on sample (maturity stages of the fish. requires specific calibrations Traditional low-field instruments require withdrawal of homogeneous samples for analysis (invasive) [14–16.51–52] Relatively inexpensive.22.

since all nitrogen in foods is not in the form of protein.67.1 (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. easy to perform. 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. low sensitivity. independent of physical state of sample Does not give a measure of the true protein.Table 16. Absorbance depends on the type of protein analyzed . 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. safe (no chemical exposure). and environmentally friendly High initial costs.120. Difficult to obtain the accurate protein concentration [53.133] Direct Protein Determination on Soluble Proteins Rapid. trapping of ammonia. 74. high precision and good reproducibility. hazardous. inexpensive to use.133] Kjeldahl Sample digestion followed by neutralization. interference by nonprotein nitrogen compounds. standard method for comparison. distillation. time-consuming.

no addition of reagents required Low sensitivity. Other compounds can interfere. nondestructive. 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.133. High amounts of endogenous proteases may cause errors.175] High sensitivity and easy to perform Standard curve is nonlinear. detergents [133. Unstable reagents are used. technique is less sensitive to protein type: it utilizes absorption involving peptide bonds that are common to all proteins. and reaction products are detected between 500 and 750 nm Rapid. variation of binding capacity for different batches of commercial grade dyes [68. high sensitivity. Absorbance at 540 nm Lowry protein assay Copper(II) ions in alkaline solution react with protein to form complexes. interference by UV-absorbing compounds (nucleic acids and nucleotides). depends on amino acid composition 263 (continued) . easy to perform. color development depends on amino acid composition [67. 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.Biuret method (Alkaline copper reagent test) Negligible interference from materials that absorb at lower wavelengths. The amount of absorption is proportional to the protein present Color formation and binding depend on proteins present. buffers salts.131. which react with the Folinphenol reagent. easy to perform Relatively low sensitivity compared with other UV-visible methods. interference from common laboratory chemicals. interference from ammonia.176.177] Composition and Calories Near-UV absorption Measurement of UV absorption (280 nm) [133] ◾ Rapid.

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

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

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

20 have been successfully used as extraction solvents. sulfosalicylic (SSA). Sample cleanup is necessary to eliminate proteins and polypeptides by means of the deproteinization process. It is usually achieved by homogenization of the ground sample in an appropriate solvent by using a Stomacher. which increases the protein stability in the harsh extracellular environment by conferring proteolytic resistance. and individuals with certain metabolic disease or who suffer from malabsorption syndromes. Several chemical methods include the use of concentrated strong acids such as phosphotungstic (PTA). and so forth. In this chapter.1 N hydrochloric acid solution.22 trichloroacetic (TCA). sulfur-containing amino acids (methionine and cystine/cysteine).01–0. The extraction consists in the separation of the free amino acid fraction from the insoluble portion of the matrix (fish muscle).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. isoleucine. 0. Sample preparation will depend on whether free or total essential amino acids have to be analyzed.21 perchloric (PCA). In some cases.13. cysteine may be essential for infants.12.11 17. The extraction solvent can be hot water. with the additional advantage that proteins are not extracted and. the sample is centrifuged at more than 10. and leucine).16. or (3) its Maillard reaction with sugars yielding characteristic flavors.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. which can be achieved through different chemical or physical procedures.14 6% of perchloric acid. which confers numerous biological functions to this amino acid (precursor to the antioxidant glutathione). then. concentrated strong acid solutions such as 4% of 5-sulfosalicylic acid. the analysis of essential amino acids in seafood is important for the evaluation of both the nutritive value and the sensory quality of seafood.18.1 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.4 Branched-chain essential amino acids (valine. A more detailed description of amino acid methods of analysis may be found in the work of Aristoy and Toldrá. 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 elderly. there is no need for further cleaning up of the sample.23–25 and picric .5–10 Thus. Although classified as nonessential. or diluted phosphate buffers. are described. (2) its ability to cross-link proteins. and aromatic amino acids (phenylalanine and tyrosine) are the most important from this point of view.2. especially of those considered essentials. in rare cases. or by means of a simple stirring in warm solvent.15 or a rich alcohol-containing solution (>75%) such as ethanol16–18 or methanol19.288 ◾ Handbook of Seafood and Seafood Products Analysis compounds through Maillard reactions and Strecker degradations.13 5% of trichloroacetic acid. Polytron. 17. 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. Once homogenized. methods for the analysis of amino acids in seafood.

22.41 The use of microwave technology for the hydrolysis has been assayed by some authors. liquid phase or vapor phase.40.2 Sample Preparation for Total or Hydrolyzed Essential Amino Acid Analysis The total essential amino acid profile is usually requested. Some physical methods consist in centrifugation through cutoff membrane filters (1. the tubes containing the samples are located inside large vessels containing the acid. and so forth.2. but the duration of the treatment is shorter (less than 20 min). Liquid-phase. The most common method used for complete hydrolysis of proteins is acid digestion. In the vapor-phase hydrolysis method. In both cases. etc.35–38 These temperatures in such acidic and oxidative medium may degrade some amino acids.22 Under these conditions. Nitrogen atmosphere and sealed vials are required during the hydrolysis to minimize the degradation. creating an appropriate atmosphere inside the vessels to ensure low amino acid degradation.000. which permits the alternative air evacuating/inert gas purging. 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. Some comparative studies have been published on these deproteinization techniques. Typically. Hydrolysis may be improved by optimizing the temperature and time of incubation41 or with the addition of amino acid oxidation protective compounds.Essential Amino Acids ◾ 289 (PA)26–28 acids or organic solvents such as methanol. whereas free amino acids remain in solution. only the acid vapor comes into contact with the sample. 30. addition of constant boiling hydrochloric acid and additives. which is easily separated by centrifugation. also disposes of an oven to accomplish the hydrolysis. which is easily neutralized by the addition of KOH or potassium bicarbonate. oxygen is removed and substituted by nitrogen or other inert gas.000. When limited amounts of sample are available. thus excluding nonvolatile contaminants. to rend insoluble potassium perchlorate.30 All these methods give a sample solution rich in free amino acids but free of proteins. 10.34 17.18. a system capable of alternative air evacuating/inert gas purging to get a correct deaeration inside is valuable. compatibility with derivatization (pH.39 Some commercial systems are available. samples are treated with constant boiling 6 N hydrochloric acid in an oven at around 110°C for 20–96 h. has also given very good results. and performance under vacuum) is similar to that of a conventional oven. or separation method (interferences in the chromatogram. all of them .). Therefore.29. Proteins must be hydrolyzed into their constituent amino acids before the analysis. A good choice may be the use of 0.42 Sample manipulation (sample evaporation to dryness. The use of organic solvents. The hydrolysis may be accomplished using either liquid-phase or vapor-phase methods. etc. recovery of amino acids. presence of salts. the vapor-phase hydrolysis method is preferred to minimize contaminants coming from aqueous 6 N hydrochloric acid. because it gives information on the nutritional value of fish meat. by mixing two or three volumes of organic solvent with one volume of extract. 5.000 Da) that allow free amino acids through while retaining large compounds.6 N PCA.). resulting in a very simple deproteinization procedure with no interferences. where the hydrochloric acid contacts the sample directly. Upon heating.39. proteins precipitate by denaturation.31–33 with amino acid recoveries around 100% for all them. ethanol. is well suited to hydrolyze large amounts or complex samples. or acetonitrile. Digestion at 145°C for 4 h has also been proposed.000. The presence of appropriate antioxidants/scavengers during hydrolysis can prevent losses of the most labile amino acids. An additional advantage is the easy evaporation to concentrate the sample. Differences among all these chemical and physical methods are caused by several aspects such as differences in the cutoff molecular weight.

The use of alkylating agents to stabilize the previous hydrolysis of cysteine constitutes a valid alternative.60 and books.1% sodium sulfite.51 3-bromopropylamine. making the posterior analysis easier. cysteine sulfinic acid. or pronase. and so on.43–50 improves cysteine (and methionine) recoveries. methionine. Cyst(e)ine is partially oxidized during acid hydrolysis yielding several adducts: cystine.30. has been extensively reported in papers35.52 4-vinyl pyridine53. 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. or BaOH. a compromise of conditions offers the best overall estimation for the largest number of amino acids. The optimization of conditions for hydrolysis based on the study of hydrolysis time and temperature.61. The choice mainly depends on the equipment available or personal preferences.36. A third way to hydrolyze proteins is enzymatic hydrolysis by proteolytic enzymes such as trypsin. 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. with or without the addition of 1% (w/v) thiodiglycol for 18 h at 110°C. protective agents currently used. threonine. amino acids used to be derivatized to allow their separation or to enhance their detection. carboxypeptidase. unless a very selective way of detection is used. presence and concentration of oxidation protective agents.54 or 3.67–69 17.290 ◾ Handbook of Seafood and Seafood Products Analysis essential amino acids. Before or after this separation. Additionally. adequate hydrolysis procedure as the performic acid oxidation before the hydrolysis is a good alternative. 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. When high sensitivity is required.62 An alternative to acid hydrolysis is the alkaline hydrolysis with 4.58. when the analysis of cyst(e)ine would be necessary. serine. Tryptophan is often completely destroyed by hydrochloric acid hydrolysis.2 M of either NaOH. thermolysin. In fact. no single set of conditions will yield the accurate determination of all essential amino acids. chymotrypsin.59. being enough for the requirements of any food industry. cysteine. and tryptophan. papain. acid-to-protein ratio. (2) give a quantitative and reproducible reaction. such as tyrosine.55. because each possible methodology has advantages and drawbacks. In general. 41.38 Alkaline hydrolysis instead of acid hydrolysis is also proposed (see below).56 As can be observed in this section. which is recommended by many authors47. The previous performic acid oxidation of cysteine to cysteic acid. This option is chosen to analyze specific amino acid sequences or single amino acids because of their specific and well-defined activity.3¢-dithiodipropionic acid. The separation of the individual amino acids in a mixture requires very efficient separation. Good recoveries have been achieved by using 3-bromopropionic acid.63–66 for a better tryptophan determination. and cysteic acid making its analysis rather difficult. KOH.3 Seafood Essential Amino Acid Analysis The analysis of individual amino acids needs a previous separation of all others. improve the recovery of nearly all of these amino acids except tryptophan and cysteine. importance of a correct deaeration. although considerable recoveries have been found if no oxygen is present. .36. up to 1% phenol or 0. Derivatization is a usual practice in amino acid analysis. yields acceptable results for the majority of amino acids. such as chromatographic (liquid or gas chromatography (GC)) or capillary electrophoresis (CE) techniques. LiOH. in which methionine is also oxidized to methionine sulfone. Thus. Some additives have been proposed to protect tryptophan against oxidation as is the case of thioglycolic acid.

amino acids were converted into colored ninhydrin derivatives for spectrophotometric (colorimetric) detection.68 Thus. The HPLC techniques to analyze amino acids are cation exchange and reversed-phase (RP) chromatography and are described in Sections 17. because their spectral (high-ultraviolet (UV) absorbing or fluorescence properties) or electrochemical characteristics will affect the sensitivity and selectivity of detection.1.1 and 17. The derivatization reaction can be performed after separation of the amino acids (postcolumn derivatization) or before separating them (precolumn derivatization).3. After separation. and they include derivatives for spectroscopic or for electrochemical detection.1 Cation Exchange Chromatography This methodology is based on the amino acid charge. and those with more than one primary amino group or possessing a guanidyl residue elute at the end of the chromatogram. the more acidic amino acids elute first. 17. and (7) have no interferences due to by-products or excess of reagent. permitting 5–10 pmol sensitivity as standard. The elution involves a stepwise increase in both pH and sodium or lithium ion concentration. 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 classical procedure has been improved with a new polystyrene matrix that offers better resolution power due to smaller particle size. The original method required two separate columns and needed about 4 h to achieve a complete analysis. and better detection systems. (6) have good stability of the derivatization products.3-oxadiazole postcolumn derivatization to obtain highly fluorescent derivatives with enhanced sensitivity. the spectroscopic detection of amino acids requires their previous derivatization to obtain an UV absorbing or fluorescent molecule. Under these conditions. which facilitates a more selective detection. Amino acids. precolumn techniques can be run either offline or online.3. Nevertheless.12 Two types of derivatives are obtained depending on the chosen separation and/or detection technique.3.2. although unidentified.Essential Amino Acids ◾ 291 (3) yield a single derivative of each amino acid.1. or 4-fluoro-7-nitrobenzo-2. The latest generation of Moore and Stein amino acid analyzers also use o-phthaldialdehyde (OPA). tyrosine.70 fluorescamine. 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.1 High-Performance Liquid Chromatographic Methods HPLC is the preferred technique to analyze amino acids. It must be remarked that the use of sufficient amount of reagent is of special importance when dealing with biological samples. in their native form. pellicular packaging. Only three amino acids (phenylalanine. The second type are derivatives that allow gas chromatographic amino acid separation by increasing their volatility and temperature stability. (5) have the possibility of automation. 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). (4) have mild and simple reaction conditions. are always present. absorb at 210 nm and thus cannot be used for spectroscopic detection. as it is a very unspecific detection wavelength. The first type are derivatives that enhance amino acid detection in liquid media.15.1. because reagent-consuming amines. l em = 345 nm). Although postcolumn techniques should be run online for maximum accuracy. recent improvements of the ninhydrin derivatization method71–73 . 17.38.1. Tryptophan also possesses native fluorescence (l ex = 295 nm.3. speed.

the highly complex mobile phase composition. The main drawbacks of this methodology are the high cost of the ion exchange amino acid analyzer and its maintenance. tissues.e.42. Biotronik.70. through a mixing manifold. Hitachi. .76 There are other reports of applying this technique to the amino acid analysis in food and tissues. Amersham Biosciences.74 Nowadays. and tissues). The PTC-amino acids are moderately stable at room temperature for 1 day and much longer when kept under frozen storage. There are many manufacturers (Beckman. the formed molecule improves sensitivity and selectivity at the detection by allowing the spectroscopic (UV or fluorescent) detection of amino acids. feed.77 After separation. which makes it a reference method for amino acid analysis. and an optimized methodology with the advantage of ease of use and reliability. In this way. 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. 17.75. LKB. some difficulties to analyze some essential or sulfur-containing amino acid derivatives). biological fluids. each new methodology must contrast its results with those obtained by cation exchange chromatography (CEC). and the long time of analysis. All PTC-amino acids have similar response factors. Dionex. or the stability of formed derivatives. the derivatizing reagent is pumped into the effluent from the column system. Another disadvantage is the peak broadening produced by the dead volume introduced behind the column. followed by a reaction coil. plants). 66. postcolumn derivatization is not suitable for narrow-bore HPLC. Although this broadening may not affect when using standard-bore columns with flow rates above 1 mL/min.3. which constitutes an advantage. time for sample preparation and amino acids separation. Obviously. Pickering. To choose the most appropriate method some aspects must be taken into account such as the following: the disposable detector (fluorescence or UV). buffer system. which are detectable at UV (254 nm) with detection limits around 5–50 pmol. The most usual derivatizing agents for tissue amino acids are described below. because it requires only a standard equipment that can be shared by different types of analysis. 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. plants. This method has been employed in the classical Moore and Steintype commercial amino acid analyzers. especially in a dry condition. Precolumn amino acid derivatization may be necessary to confer hydrophobicity to the amino acid molecule. the analysis requirements for free or hydrolyzed amino acids or required sensibility. with which many of them have been marketed. biological fluids. 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. but. also. possibility of automation of the derivatization reaction (in the autosampler). Phenylisothiocyanate (PITC): This methodology involves the conversion of primary and secondary amino acids to their phenylthiocarbamyl (PTC) derivatives. Kontron. 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.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. and finally the derivatized amino acids reach an online detector system. making it adequate for partition based on chromatography.1.. (i. etc. The advantage of this method is the accurate results for all known sample types (food.2 Reversed-Phase High-Performance Liquid Chromatography RP-HPLC has been widely used.) who offer integrated commercial systems including the column.

78–80 The chromatographic separation takes around 20 min for hydrolyzed amino acids and 60 min for physiological. The only limitation is the determination of PTC cystine that gives a poor linearity. Moreover. the residual PITC reagent left after evaporation will cause damage to the column package.5).29. This method is available as a commercially prepackaged system named Pico-Tag (Waters Associates. which makes the quantitation of free cystine nonfeasible with this method.1 Reversed-phase HPLC chromatogram of PTC amino acids from hydrolyzed hake muscle. standards.Essential Amino Acids ◾ 293 The methodology is well described in the literature. The reaction time is less than 10 min even though 20 min are recommended for a complete reaction. Both examples applied to the analysis of total amino acids from hake and free amino acids from salmon are shown in Figures 17.1 and 17. Milford. the last one being the elimination of the excess of reagent that may cause some damage to the chromatographic column. respectively.2. It is important to ensure a basic pH to get adequate derivatization recoveries. Massachusetts).29. Sarwar et al.78–80 Sample preparation is quite tedious: it requires a basic medium (pH = 10. because no buffer is used during the reaction. as some columns are more resistant than others.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). which includes the analytical column. and solvents. . which is more critical when amino acids from acid hydrolysis are analyzed. 700 Ala 600 Gly 500 Absorbance at 254 nm (mAU) Glu 400 lS Lys Asp 200 Ser 100 OHpro 300 Arg Thr Leu Pro Tyr Val Met lle Phe His 0 0 2 4 6 8 10 12 14 16 18 Retention time (min) Figure 17. IS. which is achieved by the addition of triethylamine and includes several drying steps.82 The selection of the column is critical to get a good resolved separation especially when the analysis of physiological amino acids is involved. internal standard nor-Leucine.

By-products originating from an excess of reagent absorb at the same wavelength and thus they appear in the chromatogram. Detection limits are in the low picomole range. The high wavelength of absorption makes the baseline chromatogram very stable with a large variety of solvents and gradient systems. taurine. and a reaction time of 1 h at room temperature (in the dark). Ans.2 Reversed-phase HPLC chromatogram of PITC-free amino acids from salmon muscle extract. l em = 510 nm) although UV (l = 250 nm) detection may also be used.83. anserine. However.87 or even 2 min at 100°C. IS. 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. and only needs a basic pH. 15 min at 60°C. The reaction time is around 15 min at 70°C and takes place in a basic medium with an excess of reagent. 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.294 ◾ Handbook of Seafood and Seafood Products Analysis 1400 1200 Glu Ans Absorbance at 254 nm (mAU) 1000 800 Gly 600 Tau βAla His Ala 400 Asp 200 OHpro Ser Lys Pro Thr Arg Tyr Val Met lle lS Leu Trp Phe Orn 0 0 Asn Gln 10 20 30 Retention time (min) 40 50 Figure 17. The dansylated amino acids are stable for 1 day85 or until 7 days when kept at −4°C86 and protected from light. Tau. United States).84 Derivatives are very stable (weeks) and can be formed from both primary and secondary amino acids.33 To overcome this problem and obtain an accurate calibration. The sample derivatization is rather simple. standard amino acid solution should be derivatized under similar conditions. because it is especially affected by the presence of high levels of some chloride salts. presenting a maximum from 448 to 468 nm. Stocchi et al. around 9. the reaction conditions . Nevertheless. Palo Alto California.84 Detection is by absorption in the visible range. Reaction efficiency is highly matrix dependent and variable for different amino acids.58.5. internal standard nor-Leucine.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.

The choice of the mercaptan can affect derivative stability. temperature. In the second option. which is present in excess as it is highly fluorescent and probably interferes into the chromatogram as a huge peak.86. reaction conditions.97. This is relatively easy because the reaction is fast and no heating is necessary. and 3-mercaptopropionic acid are the most frequently used. which includes the addition of ADAM. is of great advantage. 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. several methods have been proposed before derivatization.93 The fluorescence is recorded at 455 or 470 nm after excitation at 230 or 330 nm.88 Even so. 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. cysteine and cystine are quantified together. Tryptophan adducts do not fluoresce and histidine and cyst(e)ine adducts fluoresce weakly. The reaction time is fast (45–90 s) and does not require any heating. reinforcing the poor reproducibility of its results. The major disadvantage is due to the reagent. The addition of detergents like Brij 35 to the derivatization reagent seems to increase the fluorescence response of lysine.89 9-Fluorenylmethyl chloroformate (FMOC): This reagent yields stable derivatives (days) with primary and secondary amines. This excess is hydrolyzed to dansyl sulfonic acid. and fluorescent intensity. this problem is overcome by standardizing the time between sample derivatization and column injection by automation.99 In the case of cysteine. and tyrosine.5) medium.94 into the automatic sample preparation protocol described by Schuster. The derivatization is fast (1–3 min) and is performed at room temperature in alkaline (pH 9. The yield with lysine and cysteine is low and variable.Essential Amino Acids ◾ 295 (pH. Some reports have been published proposing several ways of automation. OPA derivatives can be detected by UV absorption (338 nm) as well. The derivative is fluorescent (l ex = 265 nm. the reaction of the excess of reagent with a very hydrophobic amine as 1-adamantylamine (ADAM) gives a late-eluting noninterfering peak.91 This method is preferred because the addition of ADAM is more easily automatized. many automatic injectors are programmable and able to achieve automatic derivatizations.101 or the formation of the mixed disulfide S-2-carboxyethylthiocysteine (Cys-MPA) from cysteine and cystine. itself or hydrolyzed.94–96 2-mercaptoethanol. using 3. because it guarantees the repeatability of parameters. as well as reaction time.82. have to be optimized very carefully. this methodology reveals excellent linearity for cystine and also cystine-containing short-chain peptides. lysine. ethanethiol. as it is highly fluorescent and then.92.32 . such as FMOC/amino acid ratio. In order to obtain reliable and precise results. chromatographic selectivity. On the contrary. Histidine gives a very poor fluorescence response (10% of the other amino acids). One of the main disadvantages of this procedure is the inability of OPA to react with secondary amines. OPA amino acids are not stable.82 Another problem is the large excess of reagent needed to assure a quantitative reaction.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. and the reagent itself is not fluorescent. An automated precolumn derivatization routine. Nowadays. and excess of reagent) must be carefully fi xed to optimize the product yield and to minimize secondary reactions.3′-dithiodipropionic acid55 and incorporated by Godel et al. this will commonly form multiple derivatives with histidine. l em = 315 nm) and is detected at the femtomole range. The first option was included in the automated AminoTag method90 developed by Varian (Varian Associates Limited). which is not the case with any essential amino acid. 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.

it will be necessary to readjust the chromatographic conditions to get a good separation of all amino acids. as a consequence. even those made by the same manufacturer. 1 min. potential. Milford. Indeed. l em = 395 nm). Only amino acids with aromatic rings or sulfur-containing side chains are sufficiently electrochemically active to be detected by this method.and di-derivatives are the initial adducts from tyrosine. which are separated by RP-HPLC.107. . which is only weakly fluorescent at the amino acid derivatives detection conditions and does not interfere in the chromatogram. conductance. because they are molecules with electroactive functional groups. It means that when transferring a published method to a particular set of samples. United States). Electrochemical detection consists in one electrode or an array of electrodes mounted in a cell with an applied potential difference. The methodology has been marketed as a prepackaged AccQ Tag kit (Waters Corporation.e. The excess of reagent is consumed during the reaction to form aminoquinoline (AMQ). can cause unwanted tailing of peaks (especially for the basic amino acids). detergents. Nowadays. In this case. and UV detection at 254 nm may be used for its analysis. 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. mainly octadecylsilane.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. However. accessible to sample molecules.108 If the choice of the derivative reaction is a challenge. yielding very stable derivatives (1 week at room temperature) with fluorescent properties (l ex = 250 nm. different selectivity may be found among same columns. Some of these derivatives are also susceptible to electrochemical detection. making them very adequate for biochemical research. Both facts facilitate sample preparation.3B shows the same sample but submitted to a performic acid oxidation before the hydrolysis in which the CisH peak appears by 7. and the separation of physiological amino acids is improved. peptides.. and other compounds naturally occurring in biological samples and foods. Cystine and cysteine may be analyzed after their conversion to cysteic acid (CisH) by performic acid oxidation. lipids. the choice of the RP column is not an easy subject because of the great variability of commercially available RP columns. The most used column packaging consists of alkyl-bonded silica particles. is related to the analyte concentration. the addition of a strong cation (i. because both mono. different selectivity. The chromatographic separation of these derivatives has been optimized for the amino acids from hydrolyzed proteins. Sensitivity is in the femtomole range.50).5 min. the optimum pH for the reaction is in a broad range. from 8. the AMQ peak is very large at the beginning of the chromatogram and may interfere with the first eluting peaks (see Bosch et al. UV detection (254 nm) may also be used. Reaction time is short. triethylamine) to the mobile phase can overcome the problem.3A shows the separation of hydrolyzed amino acids from salmon. Due to these variables. The presence of residual uncapped silanol groups on the silica surface. The fluorescence of tryptophan derivative is very poor.2 to 10. Massachusetts. or charge. Figure 17. Only columns manufactured in the same batch are guaranteed to give the same selectivity if the rest of parameters are fi xed. whereas Figure 17. because the resulting CisH is well separated inside the chromatogram. Any electrical measure. columns are more carefully manufactured with these silanol groups blocked or inaccessible by steric impediment avoiding the tailing. In these cases.296 ◾ Handbook of Seafood and Seafood Products Analysis 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC): It reacts with primary and secondary amines from amino acids. OPA/mercaptoethanol or OPA/sulfite104. but 10 min at 55°C would be necessary if a tyrosine monoderivative is required. such as current. Furthermore.105 in addition to fluorescent properties possesses electroactivity (750 mV) and PITC106 has again the advantage of reacting with secondary amines. and proteins.

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

or GC and LC with MS detection. speed. In many cases. and even though a particular pH can significantly . and acidic constituents. The main advantages of these detectors are their high sensitivity and wide linear range. neutral. This methodology has been patented as EZ:faast and commercialized by Phenomenex (Torrance. capable of separating 50 compounds. and the derivatives are stable and ready for GC/FID. nuts.3.113. Gas liquid chromatography (GLC) is not often used for the determination of amino acids from tissues or foods. carnosine. and balenine may complicate the amino acid analysis. United States). physiological amino acids has to be analyzed.3. Reactions consist of two stages: an esterification with an acidified alcohol followed by N-acylation with an acid anhydride in an anhydrous medium. Amino acids constitute a mix of basic.120. and urine matrices but not in tissues in which the presence of natural dipeptides.112 milk. a very highly efficient technique adequate for the amino acid analysis.110 comparing GLC with cation exchange chromatography reported different conclusions when analyzing some hydrolyzed food samples. Described applications are available for the analysis of physiological amino acids in blood. 17. The buffer may be constituted by less than 100 mM concentration of acetate or phosphate. dipeptides. especially. although applications on meat samples are scarcely described. plasma.117–119 where the separation was achieved by using chiral-GC stationary phases. and amines. GLC has been combined with mass spectrometry (MS) for detection and identification.121 The high efficiency. which is universal and the most widely used. in summary.298 ◾ Handbook of Seafood and Seafood Products Analysis with an organic phase constituted by acetonitrile and/or methanol and/or tetrahydrofuran. has been developed.114 In their analysis by GLC. 17. California. A finely adjusted binary (most used) or ternary gradient elution is often necessary when the overall amino acid profile from hydrolyzed and.109. Nevertheless. a very fast GC analysis of physiological amino acids. which are much more sensitive than FID for such compounds.113 and cheese. especially since the capillary columns appeared. Some applications67. and beans111 or other results obtained in honey.3 Capillary Zone Electrophoretic Methods Capillary zone electrophoretic technique is extremely efficient for the separation of charged solutes. and the equipment is very versatile and usually available in any analytical laboratory. and phosphotyrosine. GLC is not very expensive because no solvent is used. Protein removal is not required. and low amount of sample make this technique very interesting when compared with classical electrophoresis and chromatographic techniques. the technique is very efficient and it is worth mentioning the separation of 32 nonprotein amino acids from edible seeds.2 Gas Liquid Chromatographic Methods The extremely high-resolution capacity is the main advantage of GC. especially in the analysis of D isomers. GLC is. anserine. in comparison with liquid chromatographic techniques. whereas thermionic-N-P (NPD) or flame photometric detector (FPD) are selective toward organic compounds containing phosphorous and nitrogen. The detector used is the flame ionization detector (FID). GC/NPD. the amino acids must be converted to volatile and thermostable molecules. The NPD was used by Buser and Erbersdobler115 and FPD by Kataoka et al. phosphothreonine. including amino acids. 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.116 to analyze phosphoserine. The difficulty of separating amino acids by this technique relies on their structure. Recently.

This report includes the optimization of important parameters like the choice of a volatile electrolyte (1 M formic acid) for the electrophoresis. showing that higher efficiency is obtained by the MECC methods with sodium dodecyl sulfate (SDS) as micelle-forming substance.136. methanol. Other additives commonly used in this analysis are organic modifiers (acetonitrile.) and instrumentation (CE. or to allow fluorescence or electrochemical132 detection of amino acids. allowed a rapid development and the onset of these complementary techniques. This technique has also been termed micellar electrokinetic capillary chromatography (MECC or MEKC). and others. CZE shows poor ability for the separation of neutral compounds.138 and OPA139 compared with the separation of OPA-amino acid derivatives by CZE with normal and micellar solutions.118. When sensitivity is the target. to enhance UV detection. etc.4 Mass Spectrometry MS is based on the conversion of components of a sample into rapidly moving gaseous ions. tetrahydrofurane. 19 amino acids were analyzed by CE-ESI-MS in only 17 min with a minimal sample preparation and no matrix interference. With few exceptions. etc.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. nonprotein amino acids. The effect of these additives on the electro-osmotic mobility and electrophoretic mobility of the micelle has been studied.122.144.140 or even urea141 have been assayed. Unfortunately.124 Basic theoretical considerations on this technique125 and its food applications126 are described elsewhere. Good separations have been reported for precapillary derivatized amino acids with dansyl-Cl. the species with different charge can be simultaneously analyzed but with serious doubts in their adequate resolution.133–135 PITC. isobutanol.and l-isomer mixtures. Under the conditions of electro-osmotic flow in CE.127–131 derivatization is used to improve separation.).147 17. although this detector may be used for more complex identifications as in d.131 and thus. Some reviews covering high-sensitivity detection following CE have been published. biomedical or pharmaceutical research. it is likely to cause overlap with the others. which constitutes an important limitation of this technique. and the composition and flow rate of the sheath liquid to obtain the best sensitivity.119 have been reported. A good compatibility between both techniques. Nevertheless.146.113 o-tyrosine in chicken148 or pork149 tissues. although other additives such as dodecyltrimethylammonium bromide. SDS is indeed the most used additive to form micelles in this kind of analysis. Terabe et al. The identification of the 22 protein amino acids may not be a problem.21 chiral amino acids. and so forth.Essential Amino Acids ◾ 299 improve the resolution of one kind. microcolumn liquid chromatography.3. o-tyrosine analysis. . which can be resolved on the basis on their mass-to-charge ratios that are characteristic of each ion and allow its identification. Application in foods such as in the identification of nonprotein amino acids. etc. in particular when capillary columns were available.136 phenylthiohydantoin. the high cost of purchase and maintenance of mass spectrometers has inhibited their more widespread use in the food industry and/or food control.137.139 which is usually enough for food analysis or an LIF (laser-induced fluorescence) detector. Mass spectrometer detectors were first connected to GC equipments. it is relatively easy to analyze low picomol levels of OPA derivatives in micellar solutions by using a conventional fluorometric detector.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.141–143 The CE coupled to electrospray ionization (ESI) MS (CE-ESI-MS) allows direct amino acid analysis without derivatization. reports in the literature of its applications are increasing rapidly.138 Tween 20. compatible with MS.).125.

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

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

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

mainly of plant origin. etc.12 In biological systems various biochemical defense mechanisms. and angiotoxic effects. atherogenic. including enzymatic systems and nonenzymatic antioxidants. The health implications of tissue lipid oxidation are numerous and well documented. The major components of the antioxidant defense system together with their proposed mechanisms of action are presented in Table 18. antioxidants are . Nonradical photooxidation seems to be a minor reaction compared with the 3O2-induced radical chain autoxidation. that is. heat. There is increasing evidence that oxidative stress is implicated in the pathogenesis of many inflammatory and degenerative diseases and conditions. For food systems. because of their high degree of unsaturation.9 Preventive antioxidants hinder ROS formation or scavenge spe• cies responsible for oxidation initiation (O2−.1.7 18. for which the human sensory apparatus has a low threshold.Antioxidants ◾ 311 loss or oxidation product development.10 Lipids deteriorate in seafood products during processing. 1O2. Nevradical oxidation propagators (LOO ertheless. carcinogenic. (2) nonenzymatic and nonradical photooxidation.9 18.8 Studies on the pathological significance of dietary lipid oxidation products have indicated that some lipid oxidation products have cytotoxic. handling. Moreover. antioxidants are molecules that protect macromolecules from being oxidized. preventive antioxidants. 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.3 Marine Lipid Oxidation Compared with other food lipids. and (3) enzymatic oxidation. marine lipids are relatively more susceptible to oxidation.13 Antioxidants counteract oxidation in two different ways: They protect lipids from oxidation initiators. significantly delays or prevents oxidation of that substrate. and they stall the propagation phase. antioxidants often act via more than one mechanism that combines different types of antioxidant activity. mutagenic. Lipid oxidation of omega-3 polyunsaturated fatty acids (PUFA)-rich food products results in the development of particularly unpleasant off flavors. Autoxidation occurs through a three-phase process. are essential for counteracting oxidative stress. and storage. Lipid oxidation is a complex procedure induced by oxygen in the presence of initiators such as light. antioxidants supplied by foods.2 Lipid Peroxidation A very damaging effect of oxidant reactive intermediates is lipid peroxidation. 18. free radicals.3. Three reaction pathways have been proposed: (1) nonenzymatic chain autoxidation. chain-breaking antioxidants.1. This can be especially damaging to lipid-rich cell membranes.2 Antioxidants Antioxidants are defined as any substance that when present at low concentrations compared with those of an oxidizable substrate. Chain-breaking antioxidants intercept •) or participate in halting radical chain propagation. protect the cellular components from oxidative damage. propagation. in which polyunsaturated fatty acids on lipid molecules are attacked and oxidized. and metal ions. initiation. In the first two cases a combination of reactions involving 3O2 and 1O2 occurs. and termination.1.1.11 and unhealthy compounds that reduce their shelf life and nutritive value.).1. In general.1.

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

blue-green algae. thiols. widely distributed in aerobic cells that help in preventing the accumulation of H2O2 within cells. column chromatography and the more sophisticated gas chromatography (GC). and heart.22 whereas in nine Atlantic fish species total SOD values ranged between 157 and 796 U/g fish and Mn-SOD ranged between 45 and 751 U/g. and chromatographic methods. bilirubin.3.16 and Laguerre et al. and uric acid. and crustaceans from the Mediterranean sea.20 Superoxide dismutases (SOD): SODs are metalloproteins.7 U/mg of protein. whereas Fe SOD were purified from red algae. together with traces of phenolic compounds. kidneys. the correlation of instrumental and sensory methods with multivariate data analysis should be followed..17 and Wood et al. At higher levels most of them behave as prooxidants possibly due to their involvement in the initiation reactions. one represented by enzymes and the second represented by low molecular mass compounds.15 Griffiths et al. The available methods for monitoring the antioxidant capacity in biological and food systems in vitro or in vivo were recently reviewed by MacDonald-Wicks et al. Catalase activities ranged between 386 and 1523 mmol/ min/g tissue in several Atlantic fish and was higher in liver. spleen.20 . and algae.2 and 18. tocopherols.1 Antioxidant Enzymes Catalases are metal-containing enzymes. Zn.21 In several species of teleosts.19 concluded that. 18. and high-performance liquid chromatography (HPLC) alone or combined with mass spectroscopy. Kolanowski et al.9 and 9.9 The determination of specific waterand fat-soluble antioxidants is discussed in Sections 18.1 Cu/Zn SOD were purified from marine fish tissues. or Fe in their active site.3 Antioxidants in Seafood and Seafood Products In living organisms. polarographic. voltammetric. thin-layer. and in red muscle compared with that in white.18 In reviewing methods and tests for the assessment of lipid oxidation. such as ascorbic acid. with Mn. oxidative damage to macromolecules is controlled by two types of antioxidant systems. glutathione (GSH).3.Antioxidants ◾ 313 effective at very low concentration levels. cephalopods. carotenoids. 18. which act as primary preventive inhibitors and catalyze the dismutation of superoxide anion • • (O2−) by reducing one O2− to H2O2 and oxidizing another one to O2. coenzyme Q. The available methods have been reviewed by Rajalakshmi and Narasimhan. for quality control of fish oil and fish oil-containing foods.3.3.2 Determ