Seafood and Seafood Products Analysis


Seafood and Seafood Products Analysis
Edited by


Boca Raton London New York

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

CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2010 by Taylor and Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S. Government works Printed in the United States of America on acid-free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number: 978-1-4200-4633-5 (Hardback) This book contains information obtained from authentic and highly regarded sources. Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume responsibility for the validity of all materials or the consequences of their use. The authors and publishers have attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged please write and let us know so we may rectify in any future reprint. Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright.com (http:// www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Library of Congress Cataloging-in-Publication Data Handbook of seafood and seafood products analysis / editors, Leo M.L. Nollet, Fidel Toldrá. p. cm. Includes bibliographical references and index. ISBN 978-1-4200-4633-5 (hardcover : alk. paper) 1. Seafood--Analysis--Handbooks, manuals, etc. I. Nollet, Leo M. L., 1948- II. Toldrá, Fidel. III. Title. TX385.H36 2010 641.3’92--dc22 Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com 2009034833

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

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

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

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

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

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

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

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

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


vi ◾


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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Contents ◾


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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

viii ◾


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

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

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

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

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

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

There are several seafood and seafood products, which represent some of the most important foods in almost all types of societies, including those in developed and developing countries. The intensive production of fish and shellfish has raised some concerns related to the nutritional and sensory qualities of cultured fish in comparison to their wild-catch counterparts. In addition, there are several processing and preservation technologies, from traditional drying or curing to high-pressure processing, and different methods of storage. This increase of variability in products attending the consumers’ demands necessitates the use of adequate analytical methodologies as presented in this book. These analyses will be focused on the chemistry and biochemistry of postmortem seafood; the technological, nutritional, and sensory qualities; as well as the safety aspects related to processing and preservation. This book contains 44 chapters. Part I—Chemistry and Biochemistry (Chapters 1 through 8)—focuses on the analysis of the main chemical and biochemical compounds of seafood. Chapter 1 provides a general introduction to the topics covered in this book. Part II—Processing Control (Chapters 9 through 15)—describes the analysis of technological quality and the use of some nondestructive techniques. Various methods to differentiate between farmed and wild seafood, to check freshness, and to evaluate smoke flavoring are discussed in these chapters. Part III—Nutritional Quality (Chapters 16 through 21)—deals with the analysis of nutrients in muscle foods such as essential amino acids, omega fatty acids, antioxidants, vitamins, minerals, and trace elements. Part IV—Sensory Quality (Chapters 22 through 27)—covers the sensory quality and the main analytical tools to determine the color texture, the flavor and off-flavor, etc. Sensory descriptors and sensory aspects of heat-treated seafood are also discussed. Finally, Part V—Safety (Chapters 28 through 44)—is concerned with safety, especially related to analytical tools, for the detection of pathogens, parasites, viruses, marine toxins, antibiotics, adulterations, and chemical toxic compounds from the environment generated during processing, or intentionally added, that can be found in either cultured or wild-catch seafood. The last chapter also deals with the analysis of genetically modified ingredients in fish feed. This book provides an overview of the analytical tools available for the analysis of seafood, either cultured fish or their wild-catch counterparts, and its derived products. It also provides an extensive description of techniques and methodologies for quality assurance, and describes analytical methodologies for safety control. In summary, this handbook deals with the main types of analytical techniques available worldwide, and the methodologies for the analysis of seafood and seafood products.



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

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



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

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




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

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



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

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



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

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



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

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

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



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

and Thailand (1.1 million tons).3 million tons). nutritional status. Alaska Pollock (2. Chilean jack mackerel (1.4 million tons). body composition.) of aquatic animals when captured by fishing techniques is—with few exceptions—completely unknown. However.4 million tons). and only correct storage of wet fish in melting ice or of certain products at chilled temperatures can prolong the shelf life up to weeks or months. Indonesia (1. and Norway (2.7 million tons). fish and other seafood are highly perishable products when stored without chilling. . Another difference compared with land-living animals is the fact that the quality (size. Chile (4. 40% is consumed as wet fish without any further technological processing or preservation. India (3. only some of these 5% have the desired sensory properties and give a good or satisfying fillet yield that catching and processing them can be justified.2 million tons). mostly Pangasius species).4 million tons).2% but second by value at 20. only a little proportion of this large number of about 5% is present in the world’s oceans in amounts huge enough to allow an economical use (catch and following processing).4 ◾ Handbook of Seafood and Seafood Products Analysis number of species and variability. The most important primary product producing countries of marine and inland (freshwater) fisheries in 2005 were China (17. 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. 8% is transformed into cured products. Chub mackerel (2.6 million tons).8 million tons). They deteriorate at ambient temperature in a few days. burden of pollutants. Mollusks (bivalves and cephalopods) are the third most important group both by quantity and by value at 22.4 million tons). infestation with parasites. 1980: 7 million tons. Thailand (2. state of maturity.1 million tons). 91 million tons (64%) of the total supply. The major aquaculture (excluding plants) producers (>1 million tons) in 2005 were China (32.5 million tons). and the captured fish. Aquatic plants that are popular in Southeast Asia are second in quantity at 23. About 75% of the world’s total seafood supply is used for human consumption.0 million tons).4% by quantity. By major groupings. Although land-based animals are today tailor made according to industry’s and consumer’s wishes in weight. Skipjack tuna (2. whereas crustaceans are fourth by quantity at 6. Blue whiting (2. Japan (4. aquaculture is dramatically growing (1960: 2 million tons.1 million tons). 1970: 4 million tons.3% and 14. etc.4 million tons. Further. The world’s aquaculture provided 52 million tons (36%).000 species. Vietnam (1. 25% is converted into fishmeal and other nonfood products.1 million tons).4 million tons.2 million tons).4%. respectively. Largehead hairtail (1. Peru (9. whereof the major part are cyprinids like carp).2%. 1990: 16 million tons. fish is the top group in aquaculture at 47.6 million tons). 2000: 40 million tons.1 million tons). Further. Most fish was caught in the Pacific Ocean (Northeast and Southeast) followed by Northeast Atlantic Ocean. The stagnation of captured fish is mainly due to fully exploited or partially overfished stocks. Russia (3. Atlantic herring (2. The fish group alone is represented by 25. India (2. and Yellowfin tuna (1.3 million tons). The total world seafood supply for 2007 amounted to 143 million tons. appearance. Indonesia (4. 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.3 million tons). the United States (4.4%. The top 10 species being caught in huge amounts in 2005 were Anchoveta (10.000–35. about 20% is converted into deep frozen products. and another 8% into canned products. and sensory properties.3 million tons).8 million tons). including plants) [1]. Japanese anchovy (1.9 million tons).

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

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

and microbiological methods. After sampling is completed successfully. the whole body may be sampled and analyzed (mussels. The first concerted action in this area was “Evaluation of fish freshness” from 1995 to 1997. in medium-sized specimen. The objective methods are chemical/biochemical methods. Methods that are still in use are among others k-value.Introduction ◾ 7 The number of individuals should be big when a small specimen has to be analyzed. is necessary. a careful selection of individuals that have not been mechanically damaged by the catching technique. it is advisable to concentrate on a muscle part that is simple to identify and can be dissected without destroying the fish completely (examples are muscle below gill cover. or tail end of fillet). shark). it is recommended to store all samples (also solutions) in deep frozen conditions (<−18°C. sprat. and in big fish (tuna. and total volatile basic nitrogen (TVB-N). In addition. which is based on ATP breakdown products. 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).14] that form a very rich source of information about seafood analysis. When sampling for later microbiological analyses. smaller when medium-sized animals are the target. analysis of trimethyl amine. physical methods. The chemical/biochemical methods are mostly traditional methods that were developed earlier than the physical (instrumental) methods and have been mostly applied as methods for freshness/spoilage determinations. it has to be made under strict hygienic conditions to avoid any microbial contamination. These projects brought the scientists together in conferences. When sampling is done onboard a vessel. other species or mud. preferably at −30°C) until analysis. determination of thiobarbituric acid and formaldehyde. tail muscle) must be taken due to intrinsic variations in fillet parts and after homogenization subsamples can be taken. and the second concerted action was “Fish quality labeling and monitoring” (FQLM) from 1998 to 2000. calibrations. Another method that was developed recently is the two-dimensional gel electrophoresis (2DE). snails). head end. precaution must be taken not to contaminate the sample by instruments used during manipulation (scissors. ammonia. knives) or by protective clothes or gloves. dimethyl amine. In small specimens that are consumed totally. The main results of these projects have been published in books [2–4. trimethyl amine oxide. always the whole edible part (fillet. sand. 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 2003.6 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. two books shall be mentioned that have been published earlier but still contain a significant amount of basic knowledge about analytical methods for seafood quality determination [5. and comparative analyses with different instruments. and practical work-ins and allowed on-site measurements. . and so forth. and only a few samples are taken from big individuals. workshops. and analysis of biogenic amines as histamine or cadaverine.6]. 1. The analytical methods used for seafood analyses can be divided into objective methods and sensory methods. While sampling is done.

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

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

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

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

The disadvantage is that the method requires use of hazardous and toxic chemicals. The ammonium sulfate is converted into ammonia. and textural properties. During digestion the nitrogen in the sample is converted to ammonium sulfate. both different types of raw materials and processed foods. values from 5. which is distilled and trapped in boric acid. For animal proteins the value 6.5]. This method is used as a reference method by many national and international organizations.12 ◾ Handbook of Seafood and Seafood Products Analysis essential amino acids lysine and methionine.82 are given. nitrogen from other nitrogen-containing compounds such as free amino acids. The Kjeldahl method was first published in 1883 but has been extensively modified since then. but other chemicals such as potassium sulfate and mercury oxide are also used. nucleotides. Originally only sulfuric acid was used for digestion of the samples. The Kjeldahl method determines the nitrogen content as ammonia. The factor can be calculated from the amino acid composition of the proteins. It is also possible to determine the amount of ammonia by different colorimetric methods [1].43 to 5.25 is usually used. The method includes sample digestion. It is also possible to determine the nitrogen content using elemental analysis (C/N analyzers) [4]. For fish. It is important that the methods to analyze food proteins are robust [1]. gelling. which has been used for more than 75 years. distillation. has been shown to be too high for animal 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. it is difficult to start using other and more correct factors [5]. amines. which gives a factor of 5. and trapping of ammonia and titration steps. When the protein content is calculated based on determination of the nitrogen content. 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. and the amount of nitrogen is determined by titration [1]. Retaining the functional properties through preservation and processing operations is therefore of great importance. and the amount of protein is calculated by multiplication with a Kjeldahl factor. This means that it should be possible to use the method on different types of foods. neutralization. Many proteins have protein contents that deviate from this. for instance collagen has a nitrogen content of 18%. In addition to the high nutritional value.25. assuming a nitrogen content of 16% in the proteins. . and urea will also contribute to the calculated protein content. 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. The method should also require minimal sample pretreatment to decrease analytical error and reduce costs. For products such as fish mince and surimi. and that other components in the food such as lipids and pigments should not interfere with the analysis.2 Total Content of Proteins The total content of proteins is usually determined by the Kjeldahl or the Dumas method. emulsification.56 [1]. The Dumas method is quicker and cheaper and easier to perform and is therefore now considered on equal terms with the Kjeldahl method [1]. The Technicon AutoAnalyzer uses continuous flow analysis [1]. This factor is the amount of nitrogen that contains 1 g of nitrogen. The Kjeldahl method has been automated and several instruments for automated analysis are available. 2. the water-holding capacity and the gelling properties which determine the textural attributes of the products are important quality parameters [3].

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

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

this is often not possible or practical. However. Th is figure varies for different collagen types such as collagen from fish skins from different fish species [24]. There are different dye-binding methods. but detergents. small peptides and free amino acids. The Coomassie Brilliant Blue method is also used for visualizing proteins in electrophoretic gels. The silver staining methods are 100 times more sensitive than the CBBG staining.1). Silver binding is also being used as a method to analyze concentration of soluble proteins [19]. Hydroxylysine is an amino acid that is almost exclusively found in collagen. and denaturing agents such as urea and guanidine hydrochloride cause less interference. an accurate determination requires that the amount of hydroxylysine residues per 100 residues in the collagen is known. while methods such as Biuret and Biorad only determine peptide chains above a certain length. as different amino acids and peptides give different colors in the Lowry method. Finally he compares the Lowry method with other methods to determine protein concentration and concludes that the advantages of the Lowry method are simplicity. for lipids. reducing agents. The amount of collagen can be determined by analysis of the hydroxylysine content by the Neuman and Logan method as modified by Leach [23]. chelators such as EDTA. and one of the most widely used is the Biorad method based on binding of Coomassie Brilliant Blue G (CBBG) [22]. it uses only one reagent instead of two as in the Lowry procedure [1]. The Lowry method determines both proteins. Th is method is faster to perform than the Lowry procedure (5 min development compared to 30–45 min). The method is compatible with a wide range of buffers/substances. sensitivity. Use of bicinchonic acid (BCA) was introduced as an easier way to determine protein.000 1. and acids and alkali cause interference. However. This method is based on the color change taking place when CBBG binds to proteins under acidic conditions. Sensitivity is similar to the Lowry procedure.Peptides and Proteins ◾ 15 have been suggested for the Lowry method. the method is highly protein dependent (Table 2. and precision.000–10.1 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) 500–30.000 10–300 20–140 1–20 1–50 100–300 . However. It would be best if the protein being analyzed could be used as the standard protein. Table 2. the disadvantages are interfering substances and time—compared to some of the dye-binding methods such as the Coomassie Blue methods. and stable reagents and kits are available. however. All the methods discussed above are highly protein dependent and this should be kept in mind when applying these methods for analysis of the protein content. The ability of proteins to bind silver has also been used as a very sensitive method to visualize proteins in gel electrophoresis. buffer salts.

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

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

. wettability). 2. and E. 87: 31–41. Venugopal. D. Time–temperature tolerance and physical-chemical quality tests for frozen Red Hake.K. shape. Non-destructive determination of fat. p. Journal of Food Quality. Kirsten. V.M.. 73: R91–R98. adhesiveness. Iced storage of Atlantic salmon (Salmo salar)—Effects on endogenous enzymes and their impact on muscle proteins and texture. 879–942.L. Journal of Food Science.. 1995. F. 96: 491–495. J.J. 10.. 2006. Journal of Food Science & Technology. O. sulphur and sulphur alone in organic and inorganic materials. 13. 7. and (3) properties related with the protein surface (emulsifying and foaming activities. 25: 289–307.. and quaternary). Hultin. 11. and T. Hultmann. Food Chemistry. . 5: 215–234. 1982. thickening. 48: 177–184. and H. R. References 1. 51: 1173–1179. M.C. W. 6. and gelation). V. It is therefore difficult to compare results from different laboratories. Marcel Dekker: New York. hydrophobicity. 1979. 69: 95–100. net charge. 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.. Connelly. Characteristics of edible muscle tissue. Tome.25 and Jones’ factors. The book edited by Hall [38] gives a good overview of methods to determine protein functionality. solubility. 1992.. L. Shahidi. 463. Converting nitrogen into protein—Beyond 6. salt concentration). amino acid composition and sequence. distribution. J. molecular flexibility/rigidity in response to external environment (pH. Mariotti. hydrogen.R. Venugopal. Fennema. 35: 431–435.A. Relation between protein extractability and free fatty acid production in cod muscle aged in ice. Foegeding.K. and F. or interaction with other food constituents. Critical Reviews in Food Science and Nutrition. T. Ed. 12. A description of the properties of the proteins important for functional properties was given by Damodaran [37]: The physicochemical properties that influence functional behavior of proteins in food include their size. 1996. Food Protein Analysis: Quantitative Eff ects on Processing. Uddin. 4. Journal of the Science of Food and Agriculture. Value added products from underutilised fish species. and consumption [36]. Nondestructive determination of water and protein in surimi by near-infrared spectroscopy. storage. structures (secondary. and P. Functional properties can be divided in several groups. elasticity. Nutritional. New York: Marcel Dekker. Food Research International. Isaksson. Haard.P. 2004. 2008.J. cooking. and R. temperature. E. T. Bock. 2002. Lanier. and biological values are sometimes included in the functional properties. tertiary. (2) properties related with the protein structure and rheological characteristics (viscosity. whippability). aggregation. sensory.. Automatic methods for the simultaneous determination of carbon. Anderson. in Food Chemistry. 32: 1–12. Food Chemistry. Mirand. formation of protein–lipid films. 1995. moisture and protein in salmon fillets by use of near-infrared diff use spectroscopy. hydrophilicity. Licciardello. Ravesi. Methods for processing and utilization of low cost fishes: A critical appraisal.18 ◾ Handbook of Seafood and Seafood Products Analysis processing.F. Rustad. 5. Control of chemical composition and food quality attributes of cultured fish.. et al.E. Owusu-Apenten. Journal of Food Science & Nutrition. 2008. N. Innovative uses of near-infrared spectroscopy in food processing. 1995. 25: 2025–2069. Analytical chemistry. 9. M. 8. et al. It is usual to classify them according to mechanism of action into three main groups: (1) properties related with hydration (absorption of water/oil. 3. et al. pp. Journal of Fisheries Research Board Of Canada.. 1968.

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


.... 22 3......................2............................2 Muscle Proteomes ...........................................2 Quality Involution .................................25 3.......................................2..........2...............................33 3................ 22 3.................................3..........1 Sample Matrix Considerations ....................25 3....................3 Applications of 2DE in Seafood Analysis ..............................2........2....2.............. 24 3..................... and Oddur Vilhelmsson Contents 3.......2 Basic 2DE Methods Overview ...........................2...........................2...........4 Second-Dimension Electrophoresis .............................................................................2...........2................................................. 28 3..................................... 22 3................................................35 References ..............................3........1...............2....................................................................2..........3...........................................................35 21 .1 Development .................2......2......Chapter 3 Proteomics Hólmfríður Sveinsdóttir............................................6 Analysis .. 24 3.....31 3........1 Whole Larval Proteomes.................. 34 3.. 22 3......4 Allergen Identification ......................... 32 3.......... 27 3......................................2.1 Sample Extraction and Cleanup ..............2 Aquaculture and Antemortem Effects on Quality and Processability ...................3 Equilibration ....2 Proteome Analysis by 2DE .....................................3 Protein Identification by Peptide Mass Fingerprinting ...............................................................1 Introduction ...................................3..........................2.............3..1........................5 Staining .......2.....................31 3.........3 Species Authentication .........................1...............2........ 28 3.....2...................3 The Degradome ...........2............................................................................................2 First-Dimension Electrophoresis .......................25 3............................................... 32 3........................ 27 3. Ágústa Guðmundsdóttir...................................................................................................................1 Protein Autolysis and Oxidation during Storage and Processing .............................. 28 3........................................................................................................ 34 Acknowledgments ........3.....................................

2.2. 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.1. where the bulk of the food matrix is constructed from proteins. Proteome analysis allows us to examine the effects of environmental factors on larval global protein expression.14–18 gill. Several environmental factors can interfere with the protein expression of larvae leading to poor larval quality like malformations. the proteome varies from tissue to tissue.12 kidney.12 brain. 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. Selection of tissues for protein extraction is therefore an important issue that needs to be considered before a seafood proteomic study is embarked upon.6–8 liver. the cornerstone of most proteomics research. gel-free methods.13 skeletal muscle. is the simultaneous separation of hundreds.2 Proteome Analysis by 2DE 2DE. is regulated and brought about by proteins. along with some of the main improvements that have developed since. It stands to reason.4 hold great promise and are deserving of discussion in their own right.22 ◾ Handbook of Seafood and Seafood Products Analysis 3. quality involution within the product before. giving valuable insight into the composition of the raw materials.19 intestine. While high-throughput. Studies on whole larvae. largely because of its high resolution. This is especially true of fish and meat. simplicity. or even thousands. Proteomics. in the following sections. fish possess a number of tissues amenable to 2DE-based proteome analysis. the “classic” process of two-dimensional (2D) gel polyacrylamide electrophoresis (2DE) followed by protein identification via peptide mass fingerprinting of trypsin digests (Figure 3. and low survival rate. of proteins on a 2D polyacrylamide slab gel.9–11 heart. both on the cellular and tissue-wide levels. This chapter will therefore focus on 2DE.1 Whole Larval Proteomes The production of good quality larvae is still a challenge in marine fish hatcheries. foodstuffs are in large part made up of proteins. as well as with time and in response to environmental stimuli.1 Introduction As with all living matter. that proteome analysis. based on liquid chromatography tandem mass spectrometry (LC–MS/MS). In the following sections. is a tool that can be of great value to the food scientist. Like other vertebrates. Furthermore. growth depression.12 and rectal gland12 have been reported. . for example. we present some issues and challenges related to sample matrices of particular interest to the seafood scientist. during. the construction of the food matrix.12. also known as proteomics.1) remains the workhorse of most proteomics work. or with the human immune system after consumption. and mass accuracy. then. 3.1 Sample Matrix Considerations Unlike the genome. and after processing or storage. 3. the interactions of proteins with one another or with other food components.2 surface-enhanced laser desorption/ionization3 or protein arrays. 3.12.

1 An overview over the “classic approach” in proteomics. posttranslational modifications and redistribution of specific proteins within cells.6.22 The advantage of working with whole larvae versus distinct tissues is the ease of keeping the sample handling to a minimum in order to avoid loss or modification of the proteins.22 and zebrafish (Danio rerio). These proteins may mask subtle changes in proteins expressed in other tissues or systems. it is excised from the gel. like the overwhelming presence of muscle and skin proteins. Peptide mass mapping using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry was performed only on the cod larval proteins. See text for further details. such as the gastrointestinal tract or the central nervous system. yielding a peptide mass fingerprint.7. First.22 Three of these publications have focused on the whole larval proteomes in Atlantic cod (Gadus morhua)6.Proteomics ◾ 23 2D PAGE Trypsin digestion MS fingerprinting MS/MS sequencing Figure 3. Only a few proteome analysis studies on fish larvae have been published.22 Also. cytoskeletal . allowing identification of ca.21. a protein extract (crude or fractionated) from the tissue of choice is subjected to 2D PAGE. there are several drawbacks when working with the whole larval proteome. Nevertheless. where the majority of the highly abundant proteins were identified as muscle proteins. subjected to degradation by trypsin (or other suitable protease) and the resulting peptides analyzed by mass spectrometry.20 all important information for controlling factors influencing the aptitude to continue a normal development until adult stages.6. 85% of the of the selected protein spots.23 This is reflected in our studies on whole cod larval proteome. In many cases this is sufficient for identification purposes.6.7 These studies provided protocols for the production of high-resolution 2D gels. The axial musculature is the largest tissue in larval fishes as it constitutes approximately 40% of their body mass. peptides can be dissociated into smaller fragment and small partial sequences obtained by MS/MS. but if needed. Once a protein of interest has been identified.

fish skeletal muscle is the main component. function.5 The remaining option.46 An exploitable property of proteasome-mediated protein degradation is the phenomenon of polyubiquitination. then. Protein turnover systems.2. and simply increasing the amount of sample is usually not an option.1. No amplification method analogous to PCR exists for proteins. In addition to having a hand in controlling autolysis determinants. preventing identification of holistic alterations in the analyzed proteomes. protein turnover is a major regulatory engine of cellular structure. Removal of those proteins may increase detection of other proteins present at low concentrations. For example. Fractionation methods for a variety of sample matrices have been reviewed recently. particularly in muscle tissue.43 The 20S proteasome has been found to have a role in regulating the efficiency with which rainbow trout (Oncorhynchus mykiss) deposit protein. rendering analysis of low-abundance proteins difficult or impossible. it may also result in a loss of other proteins. and biochemistry. Proteomic analysis on lysosomes has been successfully performed in mammalian (human) systems. whereby proteins are targeted for destruction by the proteasome by covalent . Cellular protein turnover involves at least two major systems: the lysosomal system and the ubiquitin–proteasome system. has profound implications for quality and processability of the fish flesh.44 It seems likely that the manner. However. An unfractionated 2DE map of the muscle proteome therefore tends to be dominated by comparatively few high-abundance protein spots. but for other applications low-abundance proteins. Various strategies have been presented for the removal of highly abundant proteins24 or enrichment of lowabundant proteins. as many textural and other quality factors of muscle foods are related to proteolytic activity in the muscle tissue before.42. in which protein deposition is regulated. such as the ubiquitin–proteasome or the lysosome systems. A myriad of methods suitable for subsequent 2DE exist for fractionating the proteome into defined subproteomes. as it will give rise to overloading artifacts in the gels.45. The fish muscle proteome is therefore likely to be of comparatively high interest to the seafood scientist.2 Muscle Proteomes In most seafood products. are of keen interest.1.34–41 3. 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. such as actin and tubulin. are suitable for rigorous investigation using proteomic methods. Swamping of low-abundance spots by highly abundant ones may not be a problem for applications relating specifically to structural proteins.24 ◾ Handbook of Seafood and Seafood Products Analysis proteins were prominent among the identified proteins. allowing a larger sample of the remaining proteins to be analyzed.3 The Degradome The degradome may be a subproteome of particular interest to the food scientist. such as those associated with individual organelles or cell compartments28 or by protein biochemical methods such as affi nity chromatography. which include most regulatory proteins and many important metabolic enzymes. Structural proteins.29. are particularly abundant in the skeletal muscle proteome.30 preparative isoelectrofocusing31 or solubility in the presence of various detergents32 or chaotropes33 have been described.2.25. is fractionation of the protein sample in order to weed out the high-abundance proteins. during and after processing.26 3.

and how they vary with environmental or dietary variables. This separates the proteins according to their molecular charge.43. These strips consist of a dried IPG-containing polyacrylamide gel on a plastic backing. most notably the introduction of immobilized pH gradients (IPGs) for IEF. 3.22 and Arctic charr (Salvelinus alpinus) liver. up-to-date protocols. the reader is referred to any of a number of excellent reviews and laboratory manuals. may be less directly amenable to proteomic study.2. such as that of the matrix metalloproteases.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 larvae6.51 the procedure remains essentially as outlined earlier. where an electric field is applied to a tube gel on which the protein sample and carrier ampholytes have been deposited..2. Activity of matrix metalloproteases is regulated via a complex network of specific proteases. such as Coomassie blue. which is most conveniently performed using commercial dry IPG gel strips. Gygi and coworkers have developed methods to study the ubiquitin–proteasome degradome in the yeast Saccharomyces cerevisiae using multidimensional LC–MS/MS. yielding a two-dimensional map (Figure 3.2 Basic 2DE Methods Overview O’Farrell’s original 2DE method first applies a process called isoelectric focusing (IEF). The tube gel is then transferred onto a polyacrylamide slab gel and the isoelectrically focused proteins are further separated according to their molecular mass by conventional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). 0. 2 M thiourea. Although a number of refinements have been made to 2DE since O’Farrell’s paper.52–57 3.2 Some proteolysis systems.1 Sample Extraction and Cleanup For most applications.3% (w/v) DTT [dithiothreitol].2 First-Dimension Electrophoresis The extracted proteins are first separated by IEF. 4% (w/v) CHAPS [3-(3-chloramidopropyl)dimethylamino-1-propanesulfonate]. silver stains.2.e. may be more conveniently carried out using transcriptomic methods. In the following sections. The map can be visualized and individual proteins quantified by radiolabeling or by using any of a host of protein dyes and stains. a general protocol is outlined briefly with some notes of special relevance to the seafood scientist.48–50 Monitoring of the expression levels of these regulatory enzymes.2. it is possible to observe the ubiquitin–proteasome “degradome. We have found direct extraction into the gel reswelling buffer (7 M urea.Proteomics ◾ 25 binding to multiple copies of ubiquitin. which proteins are being degraded by the proteasome at a given time or under given conditions.” i. Cleanup of samples using commercial 2D sample cleanup kits may be beneficial for some sample types.47 By targeting these ubiquitin-labeled proteins. 0. or fluorescent dyes. 3.58 Thorough homogenization is essential to ensure complete and reproducible extraction of the proteome. For more detailed.2) rather than the familiar banding pattern observed in one-dimensional (1D) SDS-PAGE.2. Ready-made IPG strips are currently available in a variety of linear and . sample treatment prior to electrophoresis should be minimal in order to minimize in-sample proteolysis and other sources of experimental artifacts.

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

thus reducing vertical streaking.Proteomics ◾ 27 the equipment used. it is applied to the top edge of an SDS-PAGE slab gel (Figure 3. but for most applications gradient gels or gels of about 10% or 12% polyacrylamide are appropriate. This will alkylate thiol groups and prevent their reoxidation during electrophoresis. A second equilibration step in the presence of 2. A tracking dye for the second electrophoresis step is also normally added at this point. The manufacturer’s instructions should be followed. Ready-made gels suitable for analytical 2DE are available commercially. 192 mM glycine. the SDS–polypeptide complex that affords protein-size-based separation will form and the reducing agent will preserve the reduced state of the proteins.3 Equilibration Before the isoelectrofocused gel strip can be applied to the second-dimension slab gel.2.2. that the gel side of the IPG strip faces the notched side of the glass plate.60 the Laemmli method.61 using glycine as the trailing ion and the same buffer (25 mM Tris. 1% DTT.5% iodoacetamide and without DTT (otherwise identical buffer) may be required for some applications. During the equilibration step.2.59 3. This is best performed using a dentist’s tool or other appropriate implement. taking care to put the pressure on the IPG strip’s plastic backing rather than the gel itself. remains the most popular one.2. 3.1% SDS) at both electrodes. 30% glycerol. it needs to be equilibrated for 30–45 min in a buffer-containing SDS and a reducing agent such as DTT. Optimal pore size depends on the size of the target proteins. While some reviewers recommend alternative buffer systems.3) and cemented in place using a molten agarose solution. A typical equilibrationbuffer recipe is as follows: 50 mM Tris–HCl at pH 8. and that the strip is pressed gently onto the SDS gel. 6 M urea. Care must be taken that the (+) end of the strip is on the same side of all slab gels. 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 Orientation and placement of an isoelectrofocused IPG strip onto the top of the second-dimension gel. trace amount of bromophenol blue.56 reviewed IEF for 2DE applications. Görg et al. 0. . Figure 3. avoiding trapping air bubbles.4 Second-Dimension Electrophoresis Once the gel strip has been equilibrated.8. 2% SDS.

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

In their work on the rainbow trout liver proteome.60 Peptides identified as those derived from Atlantic cod β-tubulin Trypsin autolysis peaks 1159. In those cases where both the protein and nucleotide databases yielded results.50 30 856. or transcript abundance).25 Figure 3.61 1272.43 . 100% agreement was observed between the two methods. excluded from the analysis. identified as b-2 tubulin. As can be seen in Table 3.90 1822. such as the National Centre for Biotechnology Information (NCBI) nonredundant protein sequences database. to obtain a tentative identity. Several programs are available. many with a web-based open-access interface.51 1040. To circumvent this problem. 2506.4 2212.org/tools) contains links to most of the available software for protein identification and several other tools.07 1131. this problem is surprisingly acute for species of commercial importance. The solid markers indicate the peaks that were found to correspond to expected b-2 tubulin peptides.801616.6 Mass (m/z) 2108.69 1659.70 1697.36 2564. using the appropriate software.63 1258.98 1886. it is possible to take advantage of the available nucleotide sequences.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.1.71 100 90 80  Intensity 1061.4 A trypsin digest mass spectrometry fingerprint of an Atlantic cod larval protein spot. The ExPASy Tools web site (http://www.0 1229. How useful this method is will depend on the length and quality of the available nucleotide sequences.06 1575. It is important to realize. therefore. 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. Martin et al.10 and Vilhelmsson et al.56 1974. correlated activity measurements.8 1652.54 70 1960.35 1621.50 20 10 0 741. that an identity obtained in this manner is less reliable than that obtained through protein sequences and should be regarded only as tentative in the absence of corroborating evidence (such as 2D immunoblots.86 1028. The open markers indicate mass peaks corresponding to trypsin self-digestion products and were.96 60 870.4) is then used for protein identification by searching against expected peptide masses calculated from data in protein sequence databases.2 2798.expasy.Proteomics ◾ 29 842. The resulting spectrum of peptide masses (Figure 3.00 1196. however. which in many cases is more extensive than the protein sequences available.54 50 40 1287.

046.864 47. saithe.680 1.158 20. haddock. sea bass.344 5. 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. and pollock) Lophiiformes (anglerfishes.30 ◾ Handbook of Seafood and Seafood Products Analysis Table 3.086 2. cod. turbot. mackrel.592 735 179 585 768.237 2. etc. etc. incl. halibut.210 Chondrichthyes (Cartilagenous Fishes) Carcharhiniformes (ground sharks and dogfishes) Lamniformes (mackrel sharks) Rajiformes (skates and rays) 3.006 121.442 237 2. incl. monkfish) Perciformes (perch-likes.933 3.407 84.656 2.122 268 26. redfish and lumpfishes) 185.245 2.585 1.533 1.557 . incl.380 898. sole.798 81.007 911 303 18. scallops.871 32.1 Families of Some Commercially Important Seafood Species and the Availability of Protein and Nucleotide Sequence Data as of March 27.896 3.) Astacidea (lobsters and crayfishes) Brachyura (short-tailed crabs) 21.751 Crustacea (Crustaceans) Caridea (shrimps.845 10. and plaice) Zeiformes (dories) Scorpaeniformes (scorpionfishes. tuna.762 726.287 8. and wolffish) Pleuronectiformes (flatfishes.008 Mollusca (Mollusks) Bivalvia (mussels. whelks and abalone) Cephalopoda (squid and octopi) 32.208 2.138 36.) Gastropoda (incl.845 999. sea bream.063 3.353 287 170.424 2. incl.626 138 16.489 130.782.381 45. incl.284 2.203 467.

94 Proteome analysis provides valuable information on the variations that occur within the proteome of organisms. the higher the number of possible combinations. clearly defined subproteomes and included such applications as the characterization of bovine caseins. Furthermore. 3. by Yates. posttranslational modifications. Proteome analyses in developing organisms have shown that many .84 3. fish physiology. or redistribution of specific proteins within cells. and vastly superior protein spot identification techniques. Early studies focused on relatively small. larva. 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.20 To date few studies on fish development exist in which proteome analysis techniques have been applied.3 Applications of 2DE in Seafood Analysis The two-dimensional electrophoresis has been in use within food science for at least two decades.72 Today.71. In both these studies. and development. reflect a response to biological perturbations or external stimuli9–11. and adult) during their life span that coincide with changes in the morphology. These variations may. for example. cytoskeleton. the identified proteins consisted mainly of proteins located in the cytosol.. have gained considerable momentum. the method of choice is tandem mass spectrometry (MS/MS).90–92 The morphological and physiological changes that occur during these developmental stages are characterized by differential cellular and organelle functions.93 This is reflected in the variations of global protein expression and posttranslational modifications of the proteins that may cause alterations in protein function. physiology.83 and Delahunty and Yates. each peptide mass can potentially represent any of a large number of possible amino acid sequence combinations.89 A brief discussion of a few emerging areas within fish and seafood proteomics is given as follows.80 Mo and Karger.87 With the lower cost. Until recently. 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. In the peptide mass fingerprinting discussed earlier. proteomic investigations on fish and seafood products. which. yielding a second layer of information.3. several short stretches of amino acid sequence will be obtained for each peptide.81 Gygi and Aebersold. when combined with the peptide and fragment masses obtained..Proteomics ◾ 31 A more direct.23. and nucleus.73–75 Mass spectrometry methods in proteomics have been reviewed. way of obtaining protein identities is by direct sequence comparison. enhances the specificity of the method even further. improved reproducibility and resolving power of electrophoretic separation techniques.79 Rappsilber et al. this was accomplished by N-terminal or internal (after proteolysis) sequencing by the Edman degradation of eluted or electroblotted protein spots.. if rather more time-consuming. Correlating this spectrum with the candidate peptides identified in the first round narrows down the number of candidates. as well as in aquaculture.77 Damodaran et al. and behavior of the fish.1 Development Fishes go through different developmental stages (embryo.78 Thiede et al.82 Lin et al..95 resulting in different expression of proteins.88.86 and soybean protein bodies. for example.85 wheat flour baking quality factors. The larger the mass (and longer the sequence).76 Nyman.

109. whether they be encoded in structural genes or brought about by posttranslational modification. such as curing.21 and dorada (Brycon moorei).3.1 Protein Autolysis and Oxidation during Storage and Processing The specifics of fish muscle protein autolysis during storage and processing still remain in large part to be elucidated.3. Furthermore. a large number of yolk proteins remained prominently present in the embryonic protein profiles. can be observed using 2DE or other proteomic methods. developmental stage specific muscle protein isoforms have gained a special attention. It is also worth noting that protein isoforms other than proteolytic ones. and production of surimi and conserves occur under conditions conducive to endogenous proteolysis.108 3. 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. molecular mass.113 . fermentation. usually have different molecular weight or pI and can.99. This fact further indicates the importance of the proteome approach to understand cellular mechanisms that underlie fish development.88.99–107 In this context.2 Quality Involution Degradation of proteins during chilled storage. several commercially important fish muscle processing techniques.8. The major obstacle on the use of proteomics in embryonic fish has been the high proportion of yolk proteins. In a recent study on the proteome of embryonic zebrafish. The success in the removal of yolk proteins by Link et al. many of which are correlated with specific textural properties in seafood products.7 Despite this undertaking. and pI of the protein present in a tissue. where differences are expected to occur in the number. although degradation of myofibrillar proteins by calpains and cathepsins112.111 3. 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.8.2. Studies on various proteins have shown that during fish development sequential synthesis of different isoforms appear successively. By dechorionation.98 Different isoforms generated by posttranslational modifications are largely overlooked by studies based on RNA expression. For example.99–107 The developmental changes in the composition of muscle protein isoforms have been tracked by proteome analysis in African catfish (Heterobranchus longifilis).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.21. the embryos fall out of their chorions facilitating the removal of the yolk. therefore.101 These studies demonstrated that the muscle shows the usual sequential synthesis of protein isoforms in the course of development. are among persistent quality problems in the seafood industry and have deleterious effects on fish flesh texture. are well suited for investigation using 2DE-based proteomics. be distinguished on 2DE gels.27 is probably due to dechorionation prior to the deyolking of the embryos.21. These interfere with any proteomic application that intends to target the cells of the embryo proper. Thus.94. Link et al. specific isoforms of myofibrillar proteins.27 published a method to efficiently remove the yolk from large batches of embryos without losing cellular proteins. Proteomic techniques have thus been shown to be applicable for investigating cellular and molecular mechanisms involved in the morphological and physiological changes that occur during fish development.102 common sole (Solea solea).110 Problems of this kind. and their oxidation during frozen storage.

In a recent study on the feasibility of substituting fish meal in rainbow trout diets with protein from plant sources. 67.127. With the ever increasing resolving power of molecular techniques. Kjærsgård et al. Huss noted in his review122 that product quality differences within the same fish species can depend on feeding and rearing conditions. and the amount and composition of free amino acids in the fish flesh.2.15. is thought to be responsible for a large fraction of cellular proteolysis.123 found these to comprise several members of the glycolytic and Krebs cycle pathways. in turn. therefore raises the tantalizing prospect of managing quality characteristics of the fish flesh antemortem.Proteomics ◾ 33 and degradation of the extracellular matrix by the matrix metalloproteases and matrix serine proteases114. furthermore. various quality characteristics of fillet and body were measured124.3.1 .10. are optimized. The diet was found to have a marked effect on product texture.125 and the liver proteome was analyzed9. such as proteomics.120 and have demonstrated the importance and complexity of proteolysis and oxidative changes in seafood proteins during storage and processing.117 and.128 In rainbow trout. Whatever may be the mechanism. the proteome analysis identified a number of metabolic pathways sensitive to plant protein substitution in rainbow trout feed. as opposed to wild fish catching. such as those governing gaping tendency. In the context of this chapter.126 in fish fed with the experimental diets.17 used 2DE. The practice of rearing fish in aquaculture. fatty acid breakdown. in mammals. the ubiquitin–proteasome pathway has been shown to be downregulated in response to starvation129 and have a role in regulating protein deposition efficiency. appear to display seasonal variations. 3.44 The results led the authors to speculate that the difference in texture and postmortem amino acid-free pool development are affected by antemortem proteasome activity. flesh softening during storage.118 Several 2DE studies have been performed on postmortem changes in seafood flesh14–17.112. 2D-immunoblots and LC–MS/MS to study changes in protein oxidation during frozen storage of rainbow trout.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. that these quality changes are species dependent116. Martinez et al.117. etc.. They found fish muscle proteins to be differentially carbonylated during frozen storage and were able to identify several carbonylated proteins using LC–MS/MS. is determined by environmental as well as genetic factors. this is fast becoming feasible.119. such as pathways involved in cellular protein degradation. the effects on the proteasome are particularly noteworthy. and NADPH metabolism. Indeed.2 Aquaculture and Antemortem Effects on Quality and Processability It is well known that an organism’s phenotype. including quality characteristics. We are aware of two recent studies where Atlantic cod muscle proteomes have been compared between farmed and wild fish. differences that can affect postmortem biochemical processes in the product which.123 Both studies indicated that several proteins are differentially expressed in farmed versus wild cod. where individual physiological characteristics. For example. Olsson et al. Furthermore.115 are thought to be among the main culprits. the interplay between these physiological parameters and environmental and dietary variables needs to be understood in detail. To achieve that goal. The proteasome is a multisubunit enzyme complex that catalyzes proteolysis via the ATP-dependent ubiquitin–proteasome pathway which.111. affect the involution of quality characteristics in the fish product. it is clear.

and postmortem treatment. the proteome varies from tissue to tissue and with environmental conditions. proteomic methods have been recognized as a potential way of fish species identification. 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. blotted the 2D gel onto a PVDF membrane. Mytilus galloprovincialis. 3. Proteome analysis can therefore potentially yield more information than genomic methods.147 at National Taiwan University.3.146 Proteome analysis can be a valuable tool for the identification and the characterization of allergens as exemplified by the study of Yu et al. The allergen was identified as a protein with close similarity to arginine kinase. as well as being relevant from a public health standpoint. possibly indicating freshness and tissue information in addition to species. 2DE-based methods have been developed to distinguish various closely related species.140.144 Martinez and Jakobsen Friis concluded that the identification of not only the species present. including structural proteins such as tropomyosin. These authors.141 More recently. the presence of stress-factors or contamination levels at the place of breeding. and Mytilus trossulus.4 Allergen Identification Allergenic potential is food safety issue of particular concern to the seafood producer. 1D electrophoretic techniques were developed to identify the raw flesh of various species. The identity was further corroborated by cloning and sequencing the relevant cDNA. During the 1960s.5% of young adults are allergic to shrimp. found that M. The allergens were then identified by MALDITOF MS of tryptic digests. proteomics-based species identification methods are likely to develop rapidly and find commercial uses within this field. 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. performed a 2DE on crude protein extracts from the tiger prawn. Piñeiro and coworkers have found that Cape hake (Merluccius capensis) and European hake (Merluccius merluccius) can be distinguished on 2D gels from other closely related species by the presence of a particular protein spot identified as corresponding to nucleoside diphosphate kinase.135–137 which was soon followed by methods to identify species in processed or cooked products. trossulus could be distinguished from the other two species on foot extract 2D gels by a difference in a tropomyosin spot.134 recently reviewed proteomic and other methods for species authentication in foodstuffs. They found the difference to be due to a single T to D amino acid substitution. From early on.142.3. studying the cause of shrimp allergy in humans. 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.138.34 ◾ Handbook of Seafood and Seafood Products Analysis 3.3 Species Authentication Processed fish products are increasingly common in the market and.18. as different fish species have different market values.14 Unlike the genome.145 Seafood allergies are caused by an immunoglobulin E-mediated response to particular proteins. this makes the issue of species authentication an area of increasing economic importance. particularly for addressing questions on the health status of the fish in question.143 Lopez and coworkers.139 These early efforts were reviewed in 1980. Allergic reactions to seafood affect a significant part of the population. and probed the membranes with serum from confirmed shrimp allergic patients.143 Indeed. demonstrating that it had arginine kinase . such as the gadoids or several flat fishes. studying three species of European mussels: Mytilus edulis. about 0. Penaeus monodon. A final proof was obtained by purifying the protein. For example. Martinez et al.

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

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

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

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

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

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

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

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

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

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

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

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

ATP. whereas IMP evokes a fresh meaty taste sensation.18 After this. making K value inadequate as a freshness indicator. 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.15.14 Measurement of ATP-related compounds is also useful for the quality control of retorted fishes.5 and. consequently. 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 revised K value. even at refrigeration temperatures.17 5. AMP or adenylic acid is derived from the adenosine in which a phosphate group is attached at the 5-ribose carbon. The ratio Hx/AMP was considered an adequate alternative to characterize fish freshness due to its constant increment with time. often designed K ′ value or Ki index. also. On the other hand. for several species. . and thus. a high accumulation of Ino occurs during ATP degradation.10. adenine. 5. This is the main reason for the use of indexes with more than one compound from the ATP-degradation chain. Ino. Some of them are briefly described here. or hypoxanthine is attached to a ribose.13.6 This value has been used as one of the freshness indexes to evaluate the quality change of postharvest fish. a hypoxanthine ratio or H value (Hx/(IMP + Ino + Hx) × 100) was considered as a better indicator of fish freshness in this type of species. to which one or two additional phosphate groups are attached through pyrophosphate bonds (∼P) (Figure 5.12 However. generally within 1 day of storage in ice after death in all fish species. nucleotides and nucleosides should be extracted and analyzed. ATP-chain degradation occurs very fast.16 Another suggestion to use nucleotide compounds as a measurement of seafood quality is their relation with sensory attributes.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. it is advisable to collect small tissue samples and immerse them into liquid nitrogen.Nucleotides and Nucleosides ◾ 59 nucleotide degradation such as the type of spoilage bacteria and mechanical handling of fish.2 Chemical Structure of Main Seafood Nucleosides and Nucleotides To a better understanding of the methods of analysis of these compounds. ADP and ATP are derived from the AMP. forming the adenosine or inosine. For this reason. This is achieved by immediately freezing the excised muscle under liquid nitrogen to stop all enzymatic reactions.7–9 Nevertheless. the disappearance of the degradation products differs from one species to another3 as mentioned here. Nucleosides currently analyzed in seafoods are those in which a purine ring. the knowledge of their molecular structure is important. and it is important to stop this reaction drastically at the sampling time. K value is defined as the ratio of Ino and Hx to the sum of ATP and related compounds expressed as a percentage. respectively.2). IMP is derived from the inosine in which a phosphate group is attached to the 5-ribose carbon. Nucleotides are o-phosphoric acid esters of the nucleosides.16. adenosine diphosphate (ADP).2. and AMP disappear early postmortem. a high content of Hx is related with the bitter off taste of spoiled fish.11 and. In order to achieve this rapid freezing. and Hx expressed as percentage. These cold conditions must be held along the sample preparation. Nucleosides are glycosylamines that are formed when a nucleobase (purine or pyrimidine base) attaches to a ribose or deoxyribose ring. In this way.

and the tissue is homogenized with a stomacher-type homogenizer for a few minutes under cold conditions. This neutralized extract is kept in an ice bath for 15 min and centrifuged again (15.60 ◾ 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 5.6 M perchloric acid is added.000 g for 20 min).22 . The neutralized extract must be made up to 5 mL with 20 mM phosphate buffer pH 7.8 and then filtered with a 0.3. the supernatant is filtered through glass wool and neutralized to pH 6. The supernatant is filtered through a 0. 5.1 Extraction of Nucleotides and Nucleosides A typical extraction procedure for the analysis of fish samples by reversed-phase chromatography.8 by adding solid potassium carbonate or 1 M potassium hydroxide.5 g of fish sample with 10% trichloroacetic acid and. 3–5 vol. These fish extracts are used in enzymatic assays with biosensors19. with or without employing an ion-pairing agent.000 g for 10 min).45 mm membrane.5–6. is the following: 5 g or less of muscle tissue are excised and quickly frozen with liquid nitrogen. after centrifugation (27. and Hx. The frozen tissue is minced.20 and/or spectrophotometers as well as in capillary electrophoresis (CE)21 or ion chromatography (IC).2 Structure of adenosine-derived nucleotides.2 μm membrane filter and stored under frozen storage at temperatures below −20°C until analysis. they are neutralized with 2 M sodium hydroxide.000 g for 15 min). cold 0.17 Other extraction methods consist in the homogenization of 2. Ino. avoiding any thawing. 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.

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. 5. intact fishes after being submitted to physical and chemical stressors such as hypoxia.3. nucleotides will disappear at the rigor mortis state (normally 1 day after catch). and the K′ or K i index will be usually enough to characterize fish . ADP. the addition of an ion-pair to the mobile phase greatly improves the separation by increasing the retention time of charged molecules (ATP. Thus. in vivo 31P-NMR spectroscopy has been used as a powerful technique to characterize the biochemical changes that occur in live.3.24 5.3.23. both a microwave oven at 500 V for 5 s and heating at 100°C for 60 min have been used. 5. In particular. 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. RP-HPLC and ion-paired reverse-phase are the methods of choice for this analysis. among other chromatographic techniques.27 IC. including nuclear magnetic resonance spectroscopy (NMR). radioimmunoassay. However. some authors have described extraction methods that consisted of heated fish sample.21 Typical conditions to get a good separation of IMP.19 5.26 reversed-phase high-performance liquid chromatography (RP-HPLC) with and without ion pair. including fish extract.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. The mode of separation will depend on the analyte of interest. In the analysis of complex biological samples.Nucleotides and Nucleosides ◾ 61 In the development of biosensor analysis. which may be adsorbed on capillary walls. pH 11. and hypoxanthine would be a potential of 416 V/cm of capillary using 100 mM 3-[cyclohexylamino]-1-propanesulfonic acid (CAPS) buffer. ion-exchange HPLC. inosine. Thus.2 Nucleotides and Nucleosides Determination Several methods have been used to measure nucleotides and nucleosides. AMP).2.2. In this way. the reconditioning of the capillary surface is ensured by washing 1 min with 1M NaOH.22 and enzymatic assays. high-performance capillary electrophoresis (HPCE). followed by 2 min of the running buffer used. this technique can present problems in reproducibility. HPLC has been shown to be the most widely used technique to analyze nucleotides and nucleosides.25 thin-layer chromatography (TLC). to analyze nucleotides. Nevertheless. Capillary electrophoresis has been used in many nucleotide analysis applications as in the study of nucleotide degradation in fish tissues.3.3 Chromatography At present.2.

In Figure 5. The column used is an analytical reversed-phase RP-18 column.1 Reversed-Phase HPLC The chromatographic analysis should be performed in a liquid chromatograph equipped with an UV detector (254 nm).2.15 The identification of the chromatographic peaks can be performed by comparing the peak retention times and spectral characteristics (if a diode array detector is available) with those of standards. The separation was achieved with an RP C-18 column at 35°C and a gradient between phosphate buffer at pH 7 and acetonitrile. Quantitative analysis can be performed by external or internal standard method. (1) IMP. a simple RP-HPLC with a phosphate buffer as mobile phase will be adequate. 5.62 ◾ Handbook of Seafood and Seafood Products Analysis freshness or quality. Then.3.17. . (5) hypoxanthine. (2) ATP. and (6) inosine. (4) AMP. There are many approaches to analyze nucleotides and nucleosides by this technique. 1000 (a) 6 800 600 400 Absorbance at 254 nm (mAU) 1 2 3 4 5 200 0 1200 1000 800 600 400 200 0 0 2 4 6 8 10 2 3 6 (b) 1 5 4 Retention time (min) Figure 5. (3) ADP. which differ mainly in the pH of the mobile phase.3 both chromatograms of standards and hake nucleosides and nucleotides are shown.3 RP-HPLC chromatograms of standards (a) and hake (b) ATP-derived compounds. All of them use a phosphate buffer as the mobile phase and a gradient with methanol or acetonitrile should be accomplished to improve the Ino resolution and reduce the chromatogram time.3.29 phosphorylated metabolites are also well separated in the chromatogram.29 With buffer pH 7.

4 shows an ion-paired chromatogram of a 48 h postmortem sardine extract. as well as the resolution. 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. (4) AMP.17. because the ion pair enhances the retention time and separation.Nucleotides and Nucleosides ◾ 63 5.30 This ion-paired technique is especially useful when di. ATP-derived compounds. (5) hypoxanthine.4 Ion-paired HPLC chromatograms of salmon (a) and sardine (b). and the key is to add an ion pair (an ion of charge opposite to that of the analyte molecule). this method is more expensive than the more simple technique previously described. The separation is achieved in a reversed-phase column. (3) ADP. . (1) IMP. due to the ionic nature of the phosphate esters that facilitates strong interactions with the ion-pair reagent at the appropriate pH. Nevertheless. Thus. 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 tri-nucleotides have to be analyzed.2. which is especially useful in separating mixtures of charged and uncharged molecules. (2) ATP. Figure 5.3. and (6) inosine.3. Due to the negative charge of the phosphorylated groups of nucleotides. making it less dependant on the type of column.2 Ion-Pair RP-HPLC The most common technique used for the separation of nucleotides is ion-pair RP-HPLC.

although these applications used to be achieved with at least one of these enzymes immobilized as described earlier. In this sensor. and IMP may be determined spectrophotometrically by a sequential addition of XO.4 Enzymatic Analysis The use of enzymatic methods to analyze nucleotides in seafood is widespread due to their high specificity.3. and rapid response. The depletion of oxygen or the formation of hydrogen peroxide or uric acid may be detected amperometrically.20. oxidizes the hypoxanthine to xanthine and uric acid. NP. although biosensors have shown its utility in some applications such as clinical.31 Another possibility consisted in monitoring the oxygen consumption after these enzymatic reactions with an amperometric-type sensor (oxymeter). simplicity. all the approaches to date need the sample preparation described earlier. due to its specificity. which is immobilized in a membrane fi xed in the sensing area of the electrode. 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. in which an enzyme or a group of enzymes are immobilized in a membrane or other supports. agricultural. This option offers some advantages in relation to the free enzyme.2. which is further coupled to a chemical transducer.36–38 The most used biosensors for the nucleotide-related compound analysis are electrochemical sensors. Ino. and 5′-nucleotidase (NT) into a reaction phosphate buffer containing the fish extract sample at pH 7.2. These assays may be carried out with the enzymes in solution31. In addition. while the depletion of oxygen is measured by a Clark-type elec- . 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.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. environmental. the application in the food industry is still restricted36 mainly due to critical stages such as enzyme immobilization or sample preparation for analysis.41 or for the evaluation of chicken32 and beef meat33 aging. the use of commercial kits or disposals presents some problems.4.3. and biotechnology. but they remain immobilized in different supports. Indeed. and hypoxanthine will be oxidized to uric acid and H2O2. which will be further quantified by measuring the absorbance at 290 nm and by polarimetry.6–7. constituting what is known as enzyme sensors o biosensors.1 Enzymatic Methods with the Enzyme in Solution The concentration of Hx. inosine. IMP. because no interference of salt in the medium was observed here as was in the case using the HPLC method. 5. or sensors have a limited shelf life.35 The most used is the biosensor based on the measure of hypoxanthine.32 or immobilized.8 and 30°C–37°C. electrodes. respectively.2 Enzymatic Methods with Immobilized Enzymes In this case.2. 5.39 This procedure was also used to analyze ATP and related compounds in fish sauces with very good results. This sensor has been developed mainly for assessing the freshness of fish meat40. enzyme-coated strips.33.3.20. These enzymes act by oxidizing the substrates (analyte) while consuming oxygen or producing hydrogen peroxide or uric acid.36 Nevertheless. XO enzyme.64 ◾ Handbook of Seafood and Seafood Products Analysis 5. because the denaturalization of the enzymes with time. and thus test kits. In these conditions.4.

Luong and Male20 used a multienzymatic biosensor system to determine the H value as a fish freshness indicator. Kuley. 2. Nucleotide degradation in sardine (Sardina pilchardus) stored in different storage condition at 4°C.18 and a silk fibron membrane in combination with a cellulose acetate membrane42 or a nylon net43 have been used. Jpn. 36: 19–22. M. 1 mol of Hx would be converted to 1 mol of uric acid and 2 mol of hydrogen peroxide. specific biosensors to determine AMP.Nucleotides and Nucleosides ◾ 65 trode at a platinum cathode (−0. Food Res. Food Biochem. In the measurement of hypoxanthine. 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. Fish. uric acid.L. Bull.48 IMP.45 a nafion-coated platinum disc electrode. J. J. 1959. Comparable results to that of HPLC were reported. Arai.51 The use of multienzymatic biosensors to measure fish freshness has been very helpful for the simultaneous determination of AMP. M. Ino. 2007.. J. LeBlanc. 2001. Postmortem changes in quality indices of ice-stored flounder (Paralichthys patagonicus).56 References 1.44 a polyaniline film by electropolimerization. Most recent approaches to determine Hx are based on the incorporation of the XO enzyme in a graphite/Teflon matrix. The proposed relation 1 Hx for ½ X for each oxygen molecule formed must be taken into account to quantify the Hx. Özogul.55. different supports have been used for the immobilization of the XO enzyme. T.. some authors have described this type of biosensor coupled with an oxygen electrode. Lugo-Sánchez. M. and then after adding a soluble NP.. T. R. Ltd. Japan).9 V) vs.E.. R. P. Y. Int. 41: 341–353. and Hx amounts.. Quinta. Technol.A. 65: 40–47. K i. Most of these supports have been developed with the aim of eliminating interferences due to ascorbic acid. This method was patented by Luong. 1988. necessary to obtain K.34 to obtain the Ki parameter as a freshness indicator. Both Hx and X are substrates for the XO action and will be oxidized either simultaneously or sequentially. 212: 141–146..33 although this relation should be confirmed in each particular system.L. Biochemical basis of postmortem nucleotide catabolism in cod (Gadus morhua) and its relationship to spoilage. F.. Mendes. Özogul. A review of the kinetics of degradation of inosine monophosphate in some species of fish during chilled storage. Food Sci. M.. 2006.6 to −0. Nunes. M.23. 4.53. and H values. Food Chem.. J. an Ag/AgCl reference electrode.. In this way. E.41 preactivated nylon.E. IMP. Ino. 2005. 29: 570–590. Palacios. Changes in baseline levels of nucleotides during ice storage of fish and crustaceans from the Portuguese coast. and Hx using a cellulose triacetate membrane have been described.R. Howgate.49 and Ino50 and a multienzymatic sensor to analyze simultaneously AMP. Postmortem biochemical and functional characteristic of Monterey sardine muscle stored at 0°C.47 On the other hand. thus. Ino was converted to Hx. or H2O2.54 In fact. Male..53. 1: 13–19. . and. Matsuyoshi. Sci. et al. Sci. cellulose triacetate. P. 24: 749–750. J. The consumed oxygen produces a current decrease that can be correlated to the concentration of Hx. 6.46 or even in a carbon paste electrode modified with electrodeposited gold nanoparticles. Eur. An immobilized NT was used for the previous conversion of IMP to Ino. Gill. 3. Formed Hx was measured with an amperometric sensor that detected uric acid + hydrogen peroxide in an additive matter.. Thus. Robles-Burgueno.. IMP.. 7. R.J. K. Agric. A similar application was proposed12. and Nguyen52 and afterward it has been commercialized as a Freshness Meter KV-101 (Oriental Electric Co. M. Pacheco-Aguilar. Soc.E. 2000. Fish. Saito. Massa. A new method for estimating the freshness of fish. A..E. Food Sci. Surette. Paredi. D. 5. Technol. which can be present in the sample or formed during the enzymatic reaction.

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

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

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

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

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

4 Lipid Quantification For most common purposes.2.3. near-infrared (NIR) spectrometry. Conversely. should not exceed about 40°C. Small volumes of solvent can be evaporated by carefully directing a stream of nitrogen onto the surface of the solvent. in general. glass is the best choice.1. Autoxidation of double bonds in marine lipid fatty acids is particularly troublesome. Two basic strategies can be applied [15. According to the special relevance recently acquired by noninvasive technologies.1 Fatty Acid Methyl Esters Preparation Fatty acids are essential components of lipids. Natural tissue antioxidants. Their measurement by gas–liquid chromatography (GLC) is the most commonly used method for lipid analysis. 6.2. since plasticizers are very easily leached out. and Fatmeter measurements is proving to be of increasing interest [14]. it has been shown that lipids can themselves dissolve in some plastics. 6. 6. In it.1). such as tocopherols. Lipids should not be left for any time in the dry state and should be stored in an inert nonalcoholic solvent such as chloroform from which air is excluded by flushing with a stream of nitrogen. since PUFA will oxidize rapidly in air [2].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. leading to selective losses of a proportion of the less polar constituents.16]: acid catalysis and base catalysis. 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. relatively important errors are obtained. Owing to the wide variety of fatty acid compounds in marine lipids (Table 6.3 Marine Fatty Acid Analysis 6. a large diethyl ether volume is employed. Then.3.3 Lipid Manipulation and Storage Wherever possible. afford some protection to lipid extracts. In addition. the Soxhlet method of extraction has been developed [13]. Storage temperature should be −30°C as the highest temperature. and the resulting lipid extract can no more be employed for further analysis. As storage containers. This method proved to be accurate in the case of a high lipid content (low complex lipid content). large volumes of solvents are best removed by means of a rotatory film evaporator at a temperature that. application of nuclear magnetic resonance (NMR). fatty acids . When it is necessary to concentrate lipid extracts. if not. this analysis is more complicated than that with other kinds of living organisms. fatty acid methyl esters (FAME) obtained are usually introduced in the GLC system without previous removal of contaminants. provided water absorption onto the dry extract lipid is avoided. a known aliquot of the purified lipid extract is softly heated and the resulting dry lipid matter weighted. Plastic ware of all kinds (other than that made from TeflonTM) can be specially troublesome and is best avoided. and care must be taken at all steps in the analysis of lipids.Lipid Compounds ◾ 73 6. For fast purposes. lipids should be handled in an atmosphere of nitrogen.

14-Eicosatetraenoic 5.15-Octadecatetraenoic 5. the double-bond configuration is “cis.15-Octadecatrienoic 6.8.19-Docosapentaenoic 4.13.” . 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 Linoleic Linolenic Stearidonic Araquidonic EPA DPA or clupanodonic DHA or cervonic In all cases.10.74 ◾ Handbook of Seafood and Seafood Products Analysis Table 7.11.12-Octadecadienoic

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

4. According to the previous section. and there is no way of overcoming this difficulty entirely.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]. and polyenoic fatty acids should be analyzed under the same GLC conditions for checking the quantification results. pyridinecontaining derivatives. Finally.76 ◾ Handbook of Seafood and Seafood Products Analysis In recent years.” 6. [22].2 Analysis of Sterols Sterols are biological compounds. 6. On the other hand. monoenoic. calibration factors may have to be calculated for each fatty acid component to correct the areas of the relevant peaks in the mixtures analyzed. based on the Liebermann–Buchardt reaction. However. the basic structure of which includes the cyclopentanophenanthrene ring. this is specially relevant for PUFA.3. the fatty acids on one side and diethyl-ether-soluble nonsaponifiable materials on the other side are separately recovered for further analysis [2]. the areas under the peaks on the GLC traces are. as these are easily prepared and are widely used in chromatographic analysis.4. the nonsaponifiable layer will contain any long-chain alcohols and sterols originally present in the lipid sample in the esterified form.16]. On the other hand. unsaturated. 6. A high proportion of the available data has been obtained for the methyl ester derivatives of fatty acids. as well as the deacylated residues of ether lipid compound.4. 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]. For a complete analysis. Problems of measuring this area arise mainly when components are not completely separated. cyclic. Results can be expressed as weight percentages of the fatty acids present or as molar amounts of each fatty acid. nonadecanoic acid is employed and added before the methylation step.2 Quantitative Estimation of Fatty Acid Composition With reliable modern gas chromatographs equipped with flame ionization detectors (FID). such as picolinyl esters.1 Lipid Saponification Lipids may be hydrolyzed (saponified) by heating them under reflux with an excess of dilute aqueous ethanolic alkali.2. within limits. sterols can be fractionated and analyzed by means of different . Such compounds can be divided into sterols and “ether lipids. branched. or oxygenated chains. A known quantity of an internal standard should be added to the lipid sample. linearly proportional to the amount (by weight) of material eluting from the columns [15. Total sterol content can be determined directly and spectrophotometrically from the lipid extract by using the method of Huang et al. commercially available standard mixtures containing accurately known amounts of methyl esters of saturated. the resulting FFA have to be transformed into their corresponding FAME for further analysis by an acid-catalyzed method. have been shown to be suitable for direct mass spectrometric structural analysis of acids containing straight.4 Analysis of Marine Nonsaponifiable Matter 6. quantitative results would first be calculated on its basis. If necessary.

18:0.24]. and combinations of techniques must be used until the required purposes are served. thus. or isopropylidene derivatives by GLC. marine sterols have to be converted into more volatile compounds such as acetate [25] and trimethylsilyl (TMS) [26] derivatives. TMS.27].3 Analysis of Ether Lipids Marine lipids may contain fatty acid residues as the only radicals. 6. The first type is the major one in marine lipids. Adsorption thin-layer chromatography (TLC) on silica gel layers can be used to separate simple alkyl and alkenyl lipids. Although the vinyl ether linkage is unaffected by basic hydrolysis conditions. The alkyl moieties are usually analyzed in the form of 1-alkylglycerol or as volatile nonpolar derivatives of this compound.4-dinitrophenyl-hydrazine (0. In this section. although a great interest has been accorded to their isolation because of their medical and cosmetic applications [30]. supercritical fluid chromatography (SFC) has been employed for the glycerol ether analysis of liver oils of shark species [34]. 6.” Such compounds are basically found as PL classes (specially in phosphatidylethanolamine). They can be separated by a double development in a single direction with hexane–diethyl ether (95:5. alkenyl compounds have been directly identified and quantified by GLC together with FAME [35]. Unlike fatty acids.4. but they suffer from the limitation of the lack of a distinctive chromophore in the analyte. Further. being normally placed as the radical in position 1 and specially abundant in marine invertebrates [5. Great attention has been accorded to the assessment of cholesterol oxide formation in marine products [29]. which in turn migrate just in front of TG.3-diacyl-sn-glycerols are generally saturated or cis-monoenoic even– numbered components (16:0. or they may include alkyl and alkenyl radicals. different analytical . Although the GLC is normally carried out with cholestane as internal standard. neutral plasmalogens tend to migrate ahead of alkyldiacylglycerols. For GLC analysis. mostly). although invertebrates have shown a significant presence of other sterols [27].32]. v/v) as a solvent system. it can be cleaved by acid-catalyzed transmethylation. which generates dimethyl acetals from the liberated fatty aldehydes. Chromatographic methods for cholesterol analysis [28] are of relevant importance in foods in relation to human health concerns. no single procedure will achieve the desired analysis.3.5 Qualitative and Quantitative Analyses of Marine Lipid Classes Lipid samples obtained from extraction of biological material are complex mixtures of individual lipid classes [16]. Concerning alkenylglycerols. information on ether lipid composition in marine PL is less abundant. The determination of double-bond positions in long alkyl chains has been carried out by means of picolinyl and nicotinylidene derivatives by GLC-MS [33]. and its analysis has already been discussed in Section 6. and 18:1. The others are often united into a group called “ether lipids. The alkyl groups of 1-alkyl-2. whereas no simple spot test is available for the identification of alkyldiacylglycerols.Lipid Compounds ◾ 77 chromatographic techniques [23. Accordingly. such as acetate. trifluoroacetate. high-performance liquid chromatography (HPLC) methods can offer a nondestructive alternative. Neutral plasmalogens may be detected by spraying the TLC plates with 2. batyl. Methods for separating and quantifying ether-linked glycerides have been reviewed [31. and selachyl alcohols were found to be the most abundant.4%) in 2M HCl. such compounds tend to be decomposed during GLC analysis and are best reduced by catalytic hydrogenation to alkylglycerols. chimyl. Often. Cholesterol has been shown to be the most abundant sterol in all marine living beings.

5. 6.3. in it. PL present in the lipid extract are made to react with ammonium molybdate in an organic phase and then measured spectrophotometrically. according to details explained in Section 6. Traditional determination of PL content in lipid extracts has involved the digestion of PL with the release of inorganic phosphate. A very popular one is that proposed by Lowry and Tinsley [36]. Procedures that involve spectrophotometric measurement of highly colored copper complexes are now favored.2 Stereospecific Analysis of Lipid Classes The determination of fatty acid composition at each location in lipid classes has ever since attracted great attention. In it. An alternative and successful method has been proposed by Raheja et al. this is made to react with ammonium molybdate to form phosphomolybdic acid. where an FFA-cupric acetate-pyridine complex is involved. which is reduced and determined spectrophotometrically [38]. More recently. according to the information provided in following sections. Ester linkages can be quantified by the method of Vioque and Holman [40]. The fatty acid composition of each lipid class can be determined by GLC of the methyl ether derivatives. the Grignard reagent has widely been employed in the case of marine substrates [42].41]. and GLC technologies combined with MS in the last decades has provided quick and useful procedures for the stereospecific lipid analysis. 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.78 ◾ Handbook of Seafood and Seafood Products Analysis approaches will be discussed. HPLC. This is due to the great complexity of fatty acids present in these oils and fats.4. A wide use was found for lipase hydrolysis. preparative TLC on silicic acid impregnated with 5% (w/w) boric acid has been applied to prevent acyl migration during chromatographic separation.5. traditional methodologies are still employed in cases where such advanced technologies are not available and are reviewed in this section. prepared by esterification or transesterification of the purified lipid class. the results so far reported for aquatic animals are few. Accordingly. titrimetric methods were used for many years. a method for the quantification of esterified and unesterified total sterols is mentioned in Section 6. although it turned out not to be accurate enough for marine lipids. a rapid NIR spectrometry has been applied for the direct FFA determination in fish oil [37]. For FFA assessment. since the presence of double bonds in the proximity of a carbonyl group of fish PUFA reduces the rate of deacylation of glycerides. However. these enzymes can be isolated and used in simple incubations in vitro as an aid in structural analyses of lipids. 6. The advent of new NMR. Compared with the data compiled for plant oils and for fats from land animals. Additionally. this including chromatographic separation and further analysis of fatty acids after previous methylation and transmethylation. focused on the qualitative and quantitative analyses of marine lipid classes. Finally. . giving rise to a tremendous number of species. then.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. such functional groups are made to react with hydroxamic acid and further complexed with Fe (III). [39] without previous digestion. although some interference of polar lipids was found.2 [22]. This method can be applied to total lipid extract or to any lipid class after previous isolation [7. In many cases.

or florisil as adsorbents. being simple lipids eluted in a stepwise sequence with hexane containing increasing proportions of diethyl ether. and quantification [48. in spite of the relatively higher costs [50]. Separation can be carried out on silicic acid.47].5. Those used most frequently contain hexane. although particular care is required to recover the acidic lipids quantitatively. HPLC is much more expensive than .47]. identification. The improvement and versatility of TLC enable it to be used for several modern applications. precoated plates are much more convenient than laboratory-made plates.5 High-Performance Liquid Chromatography In recent years.49]. and NMR has increased its analytical power in several applications. which include highly automated techniques right from sample application and development to detection and quantification. HPLC has undoubtedly been the most widely applied separation technique in the analysis of most simple and complex lipid classes [48. which has been used routinely for lipid analysis in the last decades.44] and PL [45. The perceived weakness of TLC has been recognized as the quantification aspect.5. In addition. 6. Such techniques would include high-pressure TLC (HPTLC).46] classes.5 mm thickness [7.5. However. acid-washed florisil. although lengthy conditioning may be necessary before columns can be employed [2. MS.41]. 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. and this has led to the evolution of the TLC/FID Iatroscan system. In all cases.52]. 6. overpressure TLC (OPTLC). 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. and acetic (or formic) acid in various proportions.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. Aminopropyl-bonded phase cartridges have been much used for the isolation of simple and complex lipid fractions. 6.4 Thin-Layer Chromatography Many text books and reviews report TLC application on lipids for routine separations.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. whereas complex lipids are recovered by elution with methanol [41. For preparative purposes. 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. 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. diethyl ether. Such stereospecific studies have widely been focused onto TG [43. 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. and tubular TLC (TTLC). A variety of solvent systems have been used to separate simple lipids on an analytical or semipreparative scale by single or two-dimensional TLC. coupling of TLC with other techniques such as HPLC. infrared (IR) spectrometry. The system has been successfully used for marine lipid class analysis [51].

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

The 31P-NMR application has also shown the possibility of analyzing the ether structures within the glycerol backbone of phosphatidylethanolamine and phosphatidylcholine. FFA carbonyl resonances were detected at the lower field of the carbonyl region. Finally. the condensed mobile phase used for liquid separations is not readily compatible with high vacuum ionization sources. the high-resolution NMR spectra of four fish oils were recorded. Recent developments in MS have been very interesting for complex lipid molecules. 6.5. and complete structure of an unknown compound. and 3 locations) of ω3 fatty acids in depot fat of several fish species was examined by 13C-NMR [63]. ω6.and diene-. probably due to their more complicated structure. A good agreement could be observed between NMR values and those from the GLC analysis. Over the years. its intensity should be proportional to the quantity. but. empirical formula. Later on [61]. 13C-NMR was employed for the plasmalogen analysis in fish lipid samples showing a good agreement with the data obtained by GLC [64].66]. Following ionization to a negatively or positively charged species (most commonly the later). In a first attempt for 13C-NMR application [60]. although GLC is conveniently coupled to electron impact ionization (EI) and chemical ionization (CI) sources. according to each corresponding resonance. mono. Quantitative analysis of fatty acid composition and alpha-beta distribution in TG tuna fish was achieved [62].65.8 Mass Spectrometry MS has long been used as a powerful tool for the analysis of the molecular weight. and stearidonic acid. Signals in the spectra were assigned. It could be observed that DHA was concentrated in the 2-location of TG in depot fats. and attention was focused on the identification of specific signals for ω3 fatty acid group and also individually for DHA. Thus. EPA. the molecules or their fragments can be separated and identified on the basis of their mass-to-charge ratio (m/z). . This development paralleled the development of atmospheric pressure chemical ionization (APCI). The different chemical shifts observed for the methyl resonance of ω3-PUFA (δ = 0. The information-rich nature of MS makes it the most desirable detector for many explanations. fragmentation of the molecular ion species produced by soft ionization processes can further be achieved in a second mass spectrometer (MS/MS) by collision-induced dissociation. 13C-NMR spectrometry was successfully used to determine the proportions of saturated. The first step for any MS method is ionization of the sample molecules in the gas phase. After different approaches. a rapid and structure-specific method for the determination of ω3-PUFA in fish lipids was presented. and highly unsaturated fatty acids of lipid extract of Atlantic salmon muscle. soft ionization MS techniques such as fast atom bombardment. results obtained using high-resolution 13C-NMR were in good agreement with those obtained by GLC. thus providing a suitable tool for lipolysis analysis. many of the advances in MS have involved new ionization techniques. Some applications concerning the marine lipids’ study will now be mentioned. marine lipids have received lesser attention. although an increasing importance has been obtained lately for quantitative analysis [20. Arpino [67] likened the HPLC-MS union. Among the different food lipids. Thus. and electrospray have the ability to ionize lipid molecules without causing extensive fragmentation.86 ppm) provided the possibility of proposing this new analytical tool. thermospray. so little application is specially available for marine lipids [58]. ω3.Lipid Compounds ◾ 81 Based on 1H-NMR spectrometry [59]. This NMR technique can provide a single signal for each PL class. Application of 31P-NMR has shown to be far shorter than with 1H and 13C. 2.95 ppm) with respect to the methyl resonance of all other fatty acids (δ = 0. The positional distribution (1.

K. analysis was carried out in conjunction with FAME by means of their dimethyl acetal derivatives resulting from the acid transmethylation of lipid extracts.. analytes are eluted from a capillary chromatographic column. Later on. Concerning marine species analysis.. T. 4. the method was capable of direct quantification of squalene and cholesterol. 3. and Oehlenschläger.K. which has great sensitivity and linearity. Elsevier Science. Handbook of Lipid Research. In addition. the use of SFC can substantially reduce the dependence on organic solvents in solvent extraction or HPLC analysis.4. References 1. Small. Christie. thus. the method was based on the use of preparative reversed-phase HPLC followed by subsequent identification by APCI HPLC-MS. whereas quantification of TG. Oxford. 6... J. A.-dimethyloxazoline derivatives [69]. Nutritional aspects of fish. cholesterol esters.. 589. D. Minor fatty acids from mussels (Mytilus galloprovincialis) were enriched by Ag+-TLC and then analyzed by GLC-MS as 2-alkenyl-4. such as mixed glyceride compositions ranging from 200 to 900 in molecular weight.. Lipid Analysis. The liver oils of several shark species were analyzed by SFC [34]. New York. Simopoulos..9 Supercritical Fluid Chromatography In this advanced technique [10. and the four most unsaturated fractions were analyzed by capillary SFC according to their acyl carbon numbers [72]. in a first attempt Baltic herring flesh TG were separated in eight fractions by Ag+-TLC. The qualitative and quantitative compositions of 1-O-alk-1-enylglycerolipids of albacore tuna (Thunnus alalunga) were studied along the canning process [35]. A wide range of cholesterol oxides were identified and quantified. 1997. Börrensen. The Physical Chemistry of Lipids. J. Analytical SFC has been shown to be particularly applicable to the analysis of higher molecular weight lipid moieties. U. Pergamon Press. 17. p. London. 89... and several nonmethylene interrupted fatty acids were singled out. in Seafood From Producer to Consumer. W. An important advantage is that it is compatible with FID. and diacylglycerol ethers required TLC fractionation before SFC analysis. . eds.5. 1982. Carbon dioxide is by far the most commonly used SFC mobile phase because of its low critical temperature. simple classes from marine oils of different species were separated and quantified by capillary SFC [73]. 2.71]. U. Plenum Press. which uses a highly compressed gas above its critical temperature and critical pressure. 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. Integrated Approach to Quality. a nonpolar capillary column. carbon dioxide as the mobile phase. The mass spectrometer was operated in the EI mode (70 eV). an optimization of process parameters was achieved to obtain a maximal production rate. 1986. Lately. 2nd edn.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]. Purification of PUFA (DHA and EPA) ethyl esters from tuna oil was carried out by SFC [74].. p. and a FID were employed in it. Vol. whereas its critical pressure and critical density are high enough for good solvation of many potential analytes. p. Luten.

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

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

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

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

many other components in foods can react with TBA or interfere with the measurements. The TBARS values for different foods with the same level of oxidation (based on flavor scores) can vary significantly [3. hence the name TBARS. the volatiles can be thermally desorbed into a gas chromatograph for separation. Many variations of this test are being used. All the methods are based on the pink color absorbance formed by reaction between TBA and oxidation products of polyunsaturated lipids. In another method. and 2. In the micromethod of Ke and Woyewoda [17]. where the headspace volatiles over the samples are sampled. the TBARS are separated by steam distillation or HPLC to increase selectivity.90 ◾ Handbook of Seafood and Seafood Products Analysis anisidine value (AnV). After sampling. static headspace and solid-phase microextraction (SPME) are the least sensitive. are highly sensitive. which are secondary oxidation products. and the absorbance at 350 nm is determined after 10 min [15]. AnV can also be determined using Fourier transform infrared (FT-IR) [16]. In addition. the colored complex was ascribed to the condensation of two moles of TBA and one mole of malonaldehyde (MDA). Some of the MDA detected in this test is formed during the peroxidation of the lipids. and the absorbance of the solution is read at 530 nm. and chloroform before adding TCA. the lipids are dissolved in a solution of thiobarbituric acid in butanol. and the color is formed by many different secondary oxidation products. and the optical density of the water phase is determined at 538 nm. metal ions. The determination of TBARS (or TBA) is a common method to determine secondary oxidation products. antioxidants.3. the reaction is not specific. but most of it is formed during the decomposition of the lipid peroxides during the acid heating stage. the AnV is a common method.13. Different types of headspace analyses can be used. The TBA test can be standardized using MDA. different methods give different results.4-dienals). but as for the determination of PV. forming a yellow pigment absorbing light at 450 nm. In the AOCS method [13]. This determines the amount of aldehydes (mainly 2-alkenals and 2.18].4-dienals also react with TBA. the sample is incubated at 95°C for 2 h. Of these methods. and identified using different gas sensors. separated. Many factors influence the color in the TBA test—temperature. and antioxidants. TBARS values have been found to correlate with sensory scores within the same materials [19]. alkanals. p-anisidine dissolved in acetic acid is added. In other variations. nitrite. Purge and trap techniques. However. the lipids are boiled for 45 min with a mixture of TBA. The Totox value is still one of the most commonly used oxidation parameters used in commercial laboratories and laboratories in the edible oil industry. where the samples are flushed or purged with nitrogen and the volatiles in the gas flow are trapped on a solid absorber. amino acids. antioxidants. which is formed as a decomposition product from lipid hydroperoxides under the acidic test conditions [3]. in addition H2O2. alkenals. reaction products from browning reactions. There are many published methods to determine TBARS. These methods determine the presence of aldehydes. The mass spectra of the compounds can also be compared with spectra of pure standard compounds and . sucrose and other sugars. sulfite. This value is a combination of the PV and the AV. Originally. and trace metals can influence the result [3. However. The AnV of freshly deodorized oils is caused by core aldehydes.13]. The volatile compounds formed as a result of lipid oxidation can be analyzed using electronic noses/gas-sensor array systems [20]. which is generated by acid hydrolysis of 1. pH. time of heating. Dienals also give a red pigment absorbing at 530 nm. For determination of secondary oxidation products. However. This process is accelerated by metal ions [12]. the oxidation products are extracted in trichloroacetic acid (TCA) before the reaction with TBA.3-tetraethoxypropane [3]. The Totox value is given as 2*PV + AnV. The sample is dissolved in isooctane. and chelating agents may also influence the peroxide decomposition during the assay. nucleic acids.1. Protein.

Analysis of volatiles is discussed by Ólafsdóttir and Jónsdóttir in Chapter 8.1 Excitation and Emission Maxima for Chromophores Formed as a Result of Oxidized Lipids. The advantages of this method are that it is flexible. However. and the results are dependant on the sample material. the data handling is also difficult. forming Schiff bases. reactions between oxidized lipids and proteins/ peptides. Lipid oxidation products can interact with other components in food. This reaction can lead to formation of brown-colored compounds [22. When the samples are turbid or solid or the concentration is high. Aubourg and Medina [26] extracted fish muscle with a 2/2/1. quenching. The fluorescence intensities were divided by the fluorescence intensity of quinine sulfate and the fluorescence shift calculated. quantification of headspace data. such as amino acids. Fluorescence has traditionally been applied to samples in solution. and the concentration is below a certain level. front-face fluorescence Table 7.1. peptides. deoxyribonucleic acid (DNA).23]. When fluorescence measurements are done on samples in solution. The different chromophores formed as a result of oxidized lipids. nucleic acids. 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 . and the amount of sample and sampling conditions can be varied according to the needs. as measured by methods such as PV and TBARS. phospholipids. Small variations in sampling procedures can give large variations in the data. proteins. Hydroperoxides (primary lipid oxidation products) and aldehydes (secondary oxidation products) can react with amino groups in proteins. and so on. and form fluorescent products. or reactions between oxidized lipids and DNA have different excitation and emission maxima as shown in Table 7. They measured the fluorescence intensity both at 393/463 nm and 327/415 nm.8 chloroform/methanol/water mixture and measured fluorescence both in the water and in the organic phase.Lipid Oxidation ◾ 91 identified [21]. destroy this relationship. is complicated. 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. scatter. Fluorescence techniques are highly sensitive and 10–100 times more sensitive for detection of MDA than TBARS [3]. Reactions between lipid oxidation products and other components in seafood or seafood products may lead to underestimation of the degree of lipid oxidation. for example. Instead. The fluorescence shift was found to be a more effective index of changes in fish quality than other commonly used methods. for assessment of lipid oxidation during fish processing [24–26]. especially from solid matrixes. and so on. the measured intensity follows the Beer–Lambert law.

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

In general. 226: 497–509. 2nd ed. 2005. and the use of chemical and instrumental analyses is recommended to support and complement the sensory analysis [3]. 206.. 2006.K. intermediary goods. C. K. Norwegian University of Science and Technology: Trondheim. in Department of Biotechnology.. today it is not possible to use only one method to determine lipid oxidation.. The detection of these low levels is not straightforward with classical lipid oxidation measurement methods. the sensitivity was low (detection levels ∼0. sensory analysis requires relatively large amounts of samples.A. 5. 7. 2000. Sloan Stanley. E. even if there are many different methods that are used to determine lipid oxidation.2. Dietary fat.01 nM). and inflammatory disease. Miayshita. Physiological effects of eicosapentanoic acid (EPA) and docosahexanoic acid (DHA)—A review. Ed.Lipid Oxidation ◾ 93 that cannot usually be obtained by single conventional analytical methods [4]. U. through the nose (nasal) or through the mouth (retronasal). It can also be difficult to compare data from different panels using different vocabularies or data from the same panel analyzed at different times.N. M. Boissonneault.3 Summary Many different methods for the analysis of lipid oxidation exist. Even if sensory methods can give sufficient information. Biol. Falch. for many of these methods the results obtained vary not only with the method used but also with the analytical procedure that is performed.H. Food Rev. Lipids from residual fish raw material. 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]. Int. their use is limited by the cost of employing a trained panel. 2005... 4. and finished products during seafood processing. immunity. However. 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. Chow. 2005. In addition... Marcel Dekker: New York. pp. G.. A simple method for the isolation and purification of total lipids from animal tissues. Frankel. 777–795. 22: 291–306. Folch. so care should be taken in standardizing the procedures.K. 7. Odor threshold values vary both with the chemical structure of the carbonyl compounds and with the food matrix and based on how the sensory detection is performed. A trained panel can be a very valuable tool for detection of early lipid oxidation of foods containing n-3 fatty acids. Lees. and G. 3.5 Sensory Analysis of Rancidity The ultimate measurement of rancid odor and taste is sensory analysis by a trained panel. References 1. The sensitivity could be improved by the use of CryoProbe technology. however. and M.. Chem. E. Narayan. However. the oxidation products from n-3 fatty acids have a lower sensory threshold than those of oxidation products from other fatty acids. in Fatty Acids in Foods and Their Health Implications. The Oily Press: Bridgewater. but for many of these methods calibration and verification are needed before they can be used for routine analysis. J. There is. a rapid development in analytical methods to determine lipid oxidation. . Lipid Oxidation. 2. 1957. Hosakawa. J.. 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. B. However.

J. Halliwell. 106: 279–284. Vogt. 50: 1–7. 1977. pp. Food Lipids.: New York. 1994. 12. K. and I. 1959. O. Sci. LWT-Food Sci. 2003. Pokorny. Gallardo. Endo. 2001. Chemiluminescence of fish oils and its flavour quality. Marcel Dekker Inc. and M. 2002. IL. J.W. Hasegawa. 32: 497–502. Type V collagen in trout (Salmo gairdneri) muscle and its solubility change during chilled storage of muscle. 1998. Agric. Fourier transform infrared spectra data versus peroxide and anisidine values to determine oxidative stability of edible oils.. Oxidative deterioration in dried fi sh model systems assessed by solid sample fluorescence spectrophotometry. 1995. Sci. 2006. 1992. Agric. Rapid assessment of rancidity in complex meat products by front face fluorescence spectroscopy. Comparison of wet-chemical methods for determination of lipid hydroperoxides. Lipids. S. Structural and functional changes in myofibrillar proteins of sea salmon (Pseudopercis semifascata) by interaction with malonaldehyde (RI). and J. Sci.. of Chemistry.P. 2005.94 ◾ Handbook of Seafood and Seafood Products Analysis 6. K. 78: 441–450. A rapid method of total lipid extraction and purification. Method Cd 8-53. D. Int. and A. Method Cd 18–90. in Dept. Food Chem. Undeland. AOCS Official Method Ti 1a-64. Ed.C. 16. D. Dyer. P. Norway. J. Anon. Analysis of early lipid oxidation in foods with n-3 fatty acids. Wold. in Official Methods and Recommended Practices of the American Oil Chemists’ Society. J. Hayahashi. H. Ueda. Biotechnology and Food Science. and W... 160. AOCS. 46: 3662–3666. 10. 14. Food Chem. pp. and M. Wiley-VCH Verlag GmbH: Weinheim.R. 1998. 18. Nielsen. AOCS: Champaign. Food Agric. V. Biol. Ed. La Rivista Italiana Delle Sostanze Grasse. Gutteridge. Ed. AOCS: Champaign. . S. E. Hübschmann. in Handbook of GC/MS-Fundamentals and Applications.A. 21. M. Cabo... Influence of skinning on lipid oxidation in different horizontal layers of herring (Clupea harengus) during frozen storage. 8. N. and C.. and K. Food Agric. Quality assessment of blue whiting (Micrometistius poutassou) during chilled storage by monitoring lipid damages.. Lignert. 7. 67: 930–935. 2002.C. M. Chim. 1991..S. J. AOCS: Champaign. 225–319. 67: 2397–2404. IL. Aubourg. Microdetermination of thiobarbituric acid values in marine lipids by a direct spectrophotometric method with a monophasic reaction system. J. 24. 19. W.. 65: 307–313. 22. Ke. Firestone. Medina. Influence of storage time and temperature on lipid deterioration during cod (Gadus morhua) and haddock (Melanogrammus aeglefinus) frozen storage. 1990.G. 25. 1996. 1979. 10: 35–50. 2002. 1995. et al.P. Aubourg..... 1999. 20. Anal. Basics. Ed... 1999. S.. I. Food Sci. M. J.. Food Chem.M. 13. A. Olsen.. 9.. 15: 129–135. IL. J. 23. 77: 503–510. Nawar. Jacobsen. The measurement and mechanism of lipid peroxidation in biological systems. 27.. J. Firestone. Interaction of oxidised lipids with protein.J. I.. and J. Fluorescence in aldehyde model systems related to lipid oxidation.M. 79: 1943–1948. Physiol. in Official Methods and Recommended Practices of the American Oil Chemists’ Society.D.. M. Technol. Guillen. B. in Food Chemistry. Firestone. and N. Veberg. Woyewoda. 26.. 57: 1123–1126. Chem.P. Stading. Sato. Wold. 17. Tironi. Food Sci.. J. Y. Fennema. Norwegian University of Life Sciences: Ås. 39: 1222–1225. 39: 562–570..J. Sci. J. J. Trends Biochem. S. Tomas. and H. G. Bligh.. Food Res.. 37: 911–917..C. M. J. 27: 389–393. Food Agric. Namiki. Timm-Heinrich. AOCS.. Medina. Lipid damage detection during the frozen storage of an underutilized fish species. Acta.. and J. et al. 11. 1986..D.. E. Agric.-J. Pettersen. Food Sci. D. Aubourg. Effect of ascorbic acid in a model food system. AOCS. Biochem. Fujimoto. 7–212. Can.. 1995. 28. 15.

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....2 Washed Cod Muscle System ................................................................................................................................................ Ammonia-Like................................................105 8.....................................3 8.....................................................1 8.....................................................1 Microbial Spoilage Odors ...................4..1 Sweet.......1 Cooked Odor—Boiled Potato and Rancid Odors ..............................4........................................ 111 8...................................................103 8........4 Miscellaneous .........5 Conclusions .............................................113 References ..........................4..........................3............................................4....................... 98 Fresh Fish Odors .......4.................4...............................3 Putrid..................... and Malty Odors ...........................105 8........................................3.106 8..........4...4............................................... Onion........1 Smoked Fish Odors ...............4.......112 8....................... 111 8.113 97 ..............................1..........1..103 8.........1...........2....2 Ripening Odor—Salted and Dried Fish Odor................................. and Stale Odors ......................108 8................2 Dried Fish...4 Introduction ................4...........................................................3 Processing Odors .........................................................................Chapter 8 Volatile Aroma Compounds in Fish Guðrún Ólafsdóttir and Rósa Jónsdóttir Contents 8....................................... 100 8........................................2 Oxidatively Derived Odors ...1.... 98 Development of Fish Aroma.105 8.....106 8.................................2....2 8....... Sour... and Cabbage-Like Odors .......................4............. 99 Identification of Quality Indicators ................

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

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

in nominal levels the bromophenols appear to contribute to natural sea-. which are present in higher levels and can be quantified. 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 [19.24–29].1 summarizes the occurrence of volatile compounds detected in our studies on cod [22] and haddock fillets [31] and smoked salmon [23]. Accumulation of certain hydroperoxide isomers coincided with the period of enhancement of characteristic aroma in sweet smelt. a saltwater species. including (E)-2-nonenal. and these are difficult to detect by analytical techniques. The volatile pattern changes in mature salmon when migrating from the sea for spawning. acids. (E. and sulfur compounds representing the different changes occurring during storage have been suggested by numerous researchers as indicators for freshness and spoilage [22. The main classes of compounds detected during storage are alcohols. indicate their involvement in the development of fresh fish aroma associated with seasonal variation. and quality changes can be explained in. An example is the enzymically derived long-chain alcohols and carbonyls that exhibit characteristic fresh. it is useful to monitor the overall pattern of volatile compounds and select indicator compounds. Both single compounds such as TMA and ethanol and multicompound indices based on combination of alcohols. 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. Table 8. for example. Purge and trap on Tenax and SPME methods were applied for sampling. and identification was based on GC–FID. 2.22] and in smoked salmon [23]. Fatty species develop rancid odors and taste. This has been the approach in our studies.6-Nonadienal was identified to be the most characteristic compound for the cucumber-like capelin odor [19]. and marine-like flavors of seafood [18]. on accumulation of hydroperoxides in fish tissues. ketones.6-nonadienal. They were suggested as the possible precursors of nine-carbon volatile compounds.Z)-2.6-nonadien-1-ol in sweet smelt tissues [20]. esters. and 3.100 ◾ Handbook of Seafood and Seafood Products Analysis if their concentration increases. they may contribute to off odors.1. The aldehydes contribute most to the spoilage odors because of their low flavor thresholds. lean species typically develop sweet.4 Identification of Quality Indicators Different characteristic odors develop in various fish species during storage. Some of the influential odor compounds that have very low odor thresholds are often present in low levels. amines. but when accumulated in higher levels because of autooxidation. Another example is iodine-like off flavor in prawns associated with bromophenols originating from the feed chain [17]. 8. and sweet odors. boiled potato-. aldehydes. Studies performed in Japan. . Environmental conditions and seasonal effects like spawning can influence the odor quality of fish. and amine-like odors. and GC–O. Therefore. C9 LOX-derived compounds have been found in higher levels in spawning euryhaline and freshwater fish [5]. where it has been demonstrated by monitoring key volatiles to study changes in different fish products during storage. which has a very characteristic cucumber odor during spawning. as seen by the detected odors listed in Table 8. they contribute to oxidized and fishy odors in stale fish [16]. and sulfur compounds. However. muddy. iodine-. cod during storage [21. 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. odor.30–36]. GC–MS. Seasonal effects have also been reported for capelin. and species of the salmonidae family develop earthy. amines.21–22. plant-like notes in fresh fish.

fish fillet Sweet. floral Sweet.3-Butandione × × × × — N/A (continued) . candy × — — Sweet. caramel.Volatile Aroma Compounds in Fish ◾ 101 Table 8. earthy Sweet.E)Nonanal Decanal Undecanal × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × Rancid Boiled potato. (E. caramel. fatty — Fresh. flowery — Ketones 2-Butanone 2. Haddock Fillets [31].4-Heptadienal.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 2.1 Volatile Compounds Detected in Cod [22]. 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 — — — Sweet. 3-methyl. sweet. S-methylester Propanoic acid. ethylester Acetic acid. heavy. dried fish Acid Acetic acid × × × — Esters Ethyl acetate Ethanthiocacid. sour Flowery. ethyl ester 2-Butenoic acid. vomit N/A N/A N/A N/A Sulfur Compounds Methanethiol Dimethyl sulfide × × × × — — . ethyl ester × × × × × × × × × × × × × × × — N/A — N/A N/A Sickenly sweet. Haddock Fillets [31].3-Pentanedione 3-Hexanone 3-Methyl-2-butanone 3-Hydroxy-2-butanone 6-Methyl-5-hepten-2-one × × × × × × × × × × × Raw Cod Boiled Cod Raw Haddock Smoked Salmon × Odor Description (GC–O) — Sweet.1 (continued) Volatile Compounds Detected in Cod [22]. 2-methylpropyl ester Butanoic acid. ethyl ester Propanoicacid-2-methyl. spicy Amine Trimethylamine × × × TMA-like. ethyl ester Butanoic acid.102 ◾ Handbook of Seafood and Seafood Products Analysis Table 8. 2-methyl. ethylester Butanoic acid. ethylester Hexanoic acid. and Smoked Salmon [23] during Chilled Storagea Compound 2-Pentanone 3-Pentanone 2.

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. alcohols. and 2. cooling. Seaweedy and marine-like odors. Haddock Fillets [31]. meat-like. N/A. and TMA-like smell. and quantification of the main classes of compounds was based on the sum of the PAR for respective compounds in each class.1 Microbial Spoilage Odors The spoilage odors in chilled fish vary depending on the dominant microflora in the products. mushroom-. Late spoilage changes.2) were associated with the development of sweet. contributing to sweet. and finally sour and dirty tablecloth odor. and the end of shelf life of cod fillets on day 12 of storage are explained by the presence of TMA. Sweet-milky and vanilla/caramel-like odors are typical in cooked fish. 8. and sulfur compounds produced by microbial degradation of fish components. alcohols. and temperature conditions during storage [33. and when combined with frozen storage odor.1).4. development of spoilage odors.1 (continued) Volatile Compounds Detected in Cod [22].Volatile Aroma Compounds in Fish ◾ 103 Table 8. An example of the spoilage pattern of volatile compounds in chilled fish is illustrated in Figure 8. sulfur. packaging. or geraniumlike odors are characteristic sensory odor descriptors for fresh whole fish. and malty odors. The loss of freshness of cod fillets and early spoilage changes were related to the formation of ketones. The flavor thresholds . and Malty Odors Ketones.4.1. The microbially derived alcohols 2-methyl-1-propanol. and sometimes metallic. the freshness notes disappear and the odor of the uncooked fish becomes neutral. Sour.1 based on GC–O analysis of cod and smoked salmon represent most of these overall changes. not detected by GC–O. 3-methyl-1-butanol. the aroma of the fillet is described as sweet and reminiscent of shellfish. and aldehydes detected on day 4 of storage and their increasing levels on days 7 and 10 (Figure 8.2. showing results from a storage study of cod fillets packed in styrofoam boxes during chilled storage (0. which is mostly affected by handling. the fish is no longer fit for consumption. The odor descriptors in Table 8. dried fish/stockfish. sour.1 Sweet. and Smoked Salmon [23] during Chilled Storagea Compound Dimethyl disulfide Dimethyl trisulfide a Raw Cod × × Boiled Cod × × Raw Haddock × × Smoked Salmon Odor Description (GC–O) Onion like Rotten. After several days of storage. and malty spoilage odors. cucumber-. sour. In general when fish is cooked. and aldehydes. data not available for haddock. mainly amino acids. esters.5°C) [22]. 8. Identification of volatile compounds was based on GC–MS analysis (see Table 8. During prolonged storage boiled potato odor develops. as well as green plant-. —. caramel-like.34].3-butandiol were found in the highest levels on day 12 at sensory rejection. acids. 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.

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

but no obvious increase occurred until at the end of shelf-life and during continued storage. contributed to the sensory rejection of chilled cod fillets on day 12 and suggested the role of Pseudomonas fragi in the development of sweet. whereas DMA may influence the overall fresh flavor of fish in combination with oxidatively formed aldehydes from long-chain fatty acids in fish. Figure 8.2) indicated that they were not important in the spoilage of chilled cod fillets stored in styrofoam boxes. which may have influenced the overall odor perception leading to the sensory rejection of the fillets.4 Miscellaneous The concentration of the straight chain alkanes (nonane. contributing to the stale and putrid off odors in fish because of amino acid and lipid degradation [39]. TMA is characteristic for the spoilage odors of fish.2 shows that TMA was detected in high levels on day 12. methyl mercaptan. and Cabbage-Like Odors Low levels of sulfur compounds (Figure 8.3 Putrid. In whole fish stored in ice. and dimethyl disulfide have been suggested as the main cause of putrid spoilage aromas [41].4.39]. The origin of the sulfur compounds is microbial degradation of cysteine and methionine to form hydrogen sulfide and methyl mercaptan.Volatile Aroma Compounds in Fish ◾ 105 storage. which contributed to boiled potato-like odors (Table 8. decane. and undecane) appeared to be similar throughout storage in chilled cod fillets [22]. has been suggested as a freshness indicator along with its precursor TMAO (trimethylamine oxide) [27]. TMA is a potent odorant with a characteristic fishy. and the incorporation of hydrogen sulfide yields dimethyl trisulfide [38]. Dimethyl trisulfide has also been associated with spoilage in fish and associated with the growth of Shewanella putrecfaciens [25. Additionally. ammonia-like. The odor of ethyl butanoate. Onion. and ketones (2-butanone).38. which forms very early after harvest of fish. 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. alcohols (3-methyl-1-butanol. Ketones can influence the overall odor because of their low odor thresholds.4. 8. methyl sulfide. TMA has been noted for intensifying fishiness by a synergistic action with certain volatile unsaturated aldehydes derived from autoxidation of polyunsaturated fatty acids [40]. Oxidative processes are involved in the formation of dimethyl sulfide from methyl mercaptan and further oxidation of dimethyl disulfide. respectively [41]. ammonia-like odor. Ammonia-Like. 8. The onset of stale odors can be explained by cis-4-heptenal and heptanal.2 Dried Fish. described as sickeningly sweet and nauseous. dried fish. Additionally. and stale odors by amines during fish spoilage is well known. fruity off odors [37. 8. At this point there was an increase in the pH value. numerous branched chain . Pseudomonas species have also been found responsible for the formation of volatile sulfides.1. The lipid-derived saturated aldehydes detected on day 12 at sensory rejection also contributed to the overall sweet aroma. Enzymically produced DMA (dimethylamine).4.1. and Stale Odors The development of dried fish. 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. volatile sulfur compounds such as hydrogen sulfide. 1-penten-3-ol).1.1).38].

and terpenes found in wild sea bream compared with those of its cultured counterpart [29]. Aldehydes generally have low odor thresholds. cis-4-heptenal. and 2. 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]. These oxidation products contributed to the overall characteristic sweet. that contribute to the development of rancid cold store flavors [47]. which is further enhanced by preprocessing and storage of fish. and because of their high unsaturation. and decanal. such as hexanal.2. Various pro and antioxidants influence the stability of the muscle and have been studied in relation to the oxidative stability of phospholipids [46]. were detected in the fillets throughout the storage time. heptanal. aromatics. Piperidine levels have been reported to increase in spawning salmon and contribute to off odors [43]. their impact was greater than alcohols and ketones. suggesting that it may have an impact on the overall odor of fish fillets [22].7-decadienal.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-methyl-butanal.106 ◾ Handbook of Seafood and Seafood Products Analysis alkanes were detected.4. 3-pentanone. 8.3 to demonstrate which odors are most dominating in the aroma profile [48]. Limonene has also been detected in sea bream during storage [29].4.4. Limonene has low odor threshold and a fresh lemon odor was detected by GC–O analysis of cod. in similar or slightly increasing levels. such as hexanal. and 6-methyl-5-heptene-2one). Boiled potato.4.7-decatrienal) should not be overlooked.4-heptadienal and 2. but the knowledge of the formation of these compounds is obscure. although their overall levels were lower. and overall the alkanes showed an increasing trend with storage time. fish-like odors of chilled cod fillets in combination with other carbonyls (3-hydroxy-2-butanone. since they are not aroma active. These compounds have been associated with rancid and dried fish odors. The influence of other aroma active compounds present in lower levels such as the unsaturated autoxidatively derived aldehydes (2. therefore. 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. Phospholipids are the main membrane-bound lipids. The origin of limonene in fish is most likely related to the diet derived from algae or plant source. A characteristic earthy odor in many species residing in ponds has been associated with piperidine and its reaction products. they are not considered of interest as quality indicators. Several odor active terpene derivatives have been identified in fish. Oxidative processes occurring during storage of fish result in the accumulation of aldehydes.4-heptadienal.and potato-like odors contributed by . lipid-derived saturated aldehydes. but the sampling techniques used were not sensitive enough to allow quantification of these compounds. ketones.1 Cooked Odor—Boiled Potato and Rancid Odors Characteristic odors and key volatile compounds in boiled cod stored in closed plastic bags for 22 days compared with fresh boiled cod are shown in Figure 8. 8. Similarly. which are known to be more susceptible to oxidation than triacylglycerols in fat deposits [45]. 2-butanone. 2. the feed may have influenced higher levels of aldehydes. and. However. they are in particular sensitive to oxidation. Piperidine was tentatively identified in chilled cod fillets [22] and has also been suggested as a quality indicator in sea bream [29].

and Ólafsdóttir. (From Jónsdóttir. 2004. G. Principal component analysis (PCA) was performed (Figure 8.4-heptadienal. and after 8 days of storage at 6°C. 2-heptanone. and octatriene increased significantly. earthy Potato-like Boiled potato cis-4-Heptenal Heptanal ◾ 107 Cucumber. and rancid odors contributed by 2-nonenal and 2. Its odor has been described both as cardboardy. and after storage for 3 days the proportions of 4-heptenal. R. and hexanal were abundant in headspace. green-like. although the level of the compounds may vary and explain the differences in the characteristic odor of these species. In fact. sweet. flowery. 1-penten-3-ol and hexanal. 1-penten-3-ol. multivariate data analysis is useful to explore the overall trend of the main quality indicators. and the most pronounced attribute was a boiled potato odor [49]. pop-like Earthy-like odors Figure 8. sweet.) heptanal and cis-4-heptenal were the most potent odors. 2-methylbutanal.4) on data from our studies on volatiles in cod [22] during prolonged storage for 17 days and compared with corresponding . but it rather participates in the expression of the overall fishy odor.3) were fatty.3 Odor profile (GC–O analysis) of boiled cod stored in plastic bags (-♦-) after 22 days of refrigerated storage (3°C) compared with freshly boiled cod (---▲---). sweet. paint-like [50]. Unpublished data. 20 min) 3-methylbutanal. microbial metabolites such as 3-methyl1-butanol and cresol were identified [53]. In fresh baked herring (200°C. The occurrence of cis-4-heptenal has been associated with the “cold storage flavor” of cod [47]. some confusion exists about the role of cis-4heptenal as the “cold-storage compound” [8].. Other pronounced odors detected in boiled cod (Figure 8. however. The fresh raw salmon odor was characterized as cucumber-like with weak sweet. sourish. green-like. sour. as well as boiled potato-like [51. this aldehyde does not exhibit a fishy-type aroma by itself. Baltic herring has been reported to have a similar development of volatiles.4-Heptadienal Rancid Geranium-like 1-Octen-3-ol Mushroom Earthy.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 green-like odors were associated with oxidatively derived 3-pentanone. melon 2-Nonenal Cucumber Fatty Fatty. Hexanal. Ideally. Fatty. Overall earthy. green-like odors Grass Hexanal Heavy Mushroom. rancid odors Flowery 2. and fish oil notes in the same study. heptanal. this is not always the trend for dynamic microbial and oxidative changes and the formation of volatiles in fish during storage [22].52]. Taking into account the complexity of the spoilage processes. However. and octadienes also increased many-fold during further storage. and fish oil notes were characteristic for fresh cooked salmon. quality indicators should demonstrate clear increasing or decreasing levels with storage time.

4.Z)-2. decanal. and (E.5 Undecanal Ethanol 3-me-1-butanol B-D10 0 R-D4 R-D12 R-D7 R-D10 Ethylbutanoate B-D4 Heptanal Nonanal Acetaldehyde Ethylacetate 2-Butanone 3-HO-2-Butanone 2-me-1-propanol TMA Decanal R-D14 6-me-5-h-2-one Hexanal 0 0. and dimethyl trisulfide were detected in higher levels in the boiled samples (data not shown). dimethyl disulfide. However. Interestingly. that is. Autoxidatively produced unsaturated carbonyl compounds were the most abundant components in boiled and canned fish.4 0.5 –0.6-nonadienal. In boiled trout.6 0.E)-2. and 6-methyl-5-hepten-2-one. and oxidatively derived (Z)-1. 12.4). . The oxidatively formed compounds. (E.8 R-D17 –0. The malty flavor of 3-methyl butanal was suggested earlier to be mainly responsible for the malty off flavor defect of boiled cod [54]. boiled and storage days. 14. The PCA demonstrates how volatile compounds can explain the variation in quality of samples according to storage time and handling (raw and boiled). hexanal. 3-methyl-butanal was correlated to the boiled stored cod (B-D17) (Figure 8. samples after heating (see Table 8. raw and B. as indicated by the arrows (Figure 8.5-octadien-3-one.0 B-D17 1-Penten-3-ol 3-me-butanal Acetic acid 0.108 ◾ Handbook of Seafood and Seafood Products Analysis PC2 Bi-plot 1. methional with a characteristic boiled potato-like odor dominated the odor of the aldehyde fraction of the headspace volatiles. Samples are labeled with R. 8.1).2 0.4 Principal component analysis of raw and boiled cod. 10. D (4. methional. oxidation of membrane-bound phospholipids in lean species can cause fishy. 3-methyl-butanal in combination with acetaldehyde. and 17 days). increased with time and were pronounced in the spoiled raw samples (R-D14 and R-D17).0 Figure 8.4-decadienal from PUFA were determined as character impact odorants of boiled cod [54]. The characteristic pattern or trend in volatiles in raw and boiled fish is clearly different. in agreement with earlier studies [54].4 –0.2 Washed Cod Muscle System Rancid odor development during chilled storage of fish has commonly been associated with fatty species. 7. On the basis of odor evaluation. 19% PC1 1. X-expl: 53%. Sulfur compounds dimethyl sulfide.4).2 Raw and boild c…. 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). 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. Only the spoiled raw samples (R-D14 and R-D17) can be correlated with the freshly boiled (B-D4) sample. especially in trout [15].2. in particular the role of volatile compounds derived from oxidation in heated/boiled samples. The effect of oxidation induced by cooking and formation of oxidation products such as heptenal and nonanal characterizes the (B-D4) sample.

. ascorbic acid. These odors were also detected in cod fillets during chilled storage (Table 8. and spicy and flowery notes exhibited by 6-methyl-5-hepten-2-one. grass odor contributed by hexanal. which is not practical for rapid determination of oxidation. earthy. Consequently. interaction with other food components.61]. 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. physical. floral. rancid fish oil like. including hemoglobin from blood [7. and glutathione peroxidase) and aqueous prooxidants in fish muscle. lipid oxidation of muscle phospholipids may be induced by several catalysts. pH) [55. cucumber-like. and a similar trend was observed in the development of cis-4-heptenal (Figure 8. green. Odor development in lean fish studied by hemoglobininduced oxidation in washed cod muscle system showed that sweet. 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.. 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. To accurately evaluate the potential of antioxidants in foods. These compounds can be used as indicator compounds for oxidation. . Sohn et al. in agreement with TBARS and changes in color [62]. and rancid odors dominated the aroma profile [62]. The role of antioxidants (a-tocopherol.56]. Similarly. The prooxidative effect of hemoglobin was evident by the formation of hexanal in high levels. On the other hand. it is possible to detect the most volatile oxidation products like propanal and hexanal by rapid.1). In lean fish such as cod. Washed cod muscle system has been widely used to study oxidation and the influence of prooxidative and antioxidative factors [59. including blood components like inorganic metals iron (Fe) and copper (Cu). TBARS (thiobarbituric reactive substances). and lemon-like odors were explained by 2.58].Volatile Aroma Compounds in Fish ◾ 109 rancid. the concentration and composition of volatile oxidation products analyzed by GC were compared with TBARS measurements.4-heptadienal [62]. static headspace sampling methods. fatty. rancid. and color. sweet. The most potent odors detected in the model system were malty. Furthermore.g.3-pentandione.g. Preconcentration techniques are necessary for the analysis of unsaturated aldehydes. and instrumental color changes. we found in our studies on the washed cod muscle system that hexanal could be used as indicator for rancid odor development. mushroom odor caused by 1-octen-3-ol. free radical scavenging and chelation) but also on factors such as physical location. but the compounds were detected in much lower levels [22]. rancid. and environmental conditions expected in food products.5) as well as 2. [63].4heptadienal that contributed to rancid odor caused by oxidation. The added hemoglobin was very effective as a prooxidant. and caramel-like odors contributed by 3-methylbutanal. has been studied to understand better the mechanisms of oxidation in the muscle [57. sensory assessments. and the overall odor was an intense dried fish. [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. 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. soapy. 2. To monitor the development of rancidity. This is because the activity of antioxidants in food systems depends not only on the chemical reactivity of the antioxidant (e. and environmental conditions (e. as demonstrated by Boyd et al. it is necessary to apply models that take into account the chemical. painty.59]. and 1-penten 3-ol. dried fish-like off odors as discussed before. potato-like odor caused by cis-4-heptenal and heptanal.

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

plays important roles in the formation of complicated processing flavors. Additionally. hexanal. 3-methyl-1-butanol.7) (e.Volatile Aroma Compounds in Fish ◾ 111 8. The typical smoked salmon aroma results from a number of chemicals found in the smoke. [74] analyzed headspace components of cod and swordfish. and although they contributed less to the odors. Lipid-derived components. In addition to phenolic compounds. The oxidatively derived compounds cis-4-heptenal and heptanal.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) [23.72]. and alcohols were abundant in the headspace of cold smoked salmon products during storage. and decanal were among key volatiles. where groups of phenol pyrolysis were most noticeable in the smoke flavor volatiles. and 3-methyl-1-butanol) [23]. giving a popcorn-like odor that can be thermally generated. Guillén et al. and furans have been found in spray-dried shrimp powder and shrimp hydrolysate [69]. including Strecker degradation. and lipid oxidation. ethanol. associated with spoilage off flavors. sweet odors of processed seafood like those in smoked salmon [23.Z)-2. Lipid-derived aldehydes play an important role in flavor formation and have been reported to contribute to the characteristic fish-like. nonanal. 2. and 2-acetyl-1-pyrroline. 1-penten-3-ol. and 2. furan-like compounds have been reported to be responsible for the smoked odor in smoked salmon. potato-like odors. 2-butanone. 2-methyl-1-butanol. Phenolic derivatives like guaiacol (2-methoxyphenol) and syringol (2.71. Other oxidatively derived compounds like 1-penten-3-ol.. 1-octen-3-ol. Microbially produced ketones. 8.6-nonadienal.g. like 3-methyl butanal. sweet/sour rancid. also contribute to the aroma of seafood flavorants [70]. aldehydes.4. such as heptanal and (E.72]. 3-methyl-butanal. contributing to mushroom-like odor.72].7) [23]. Some of these compounds were selected as key spoilage indicators for smoked salmon based on their high levels and contribution to sweet and fruity spoilage off odors in our study on smoked salmon (Figure 8.75]. giving rancid.4.1 Smoked Fish Odors Degradation compounds from Maillard reactions and lipid oxidation are the main compounds contributing to the aroma of smoked salmon [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]. which has a characteristic potato-like odor. These are compounds like methional.4-decadienal.4-heptanal. and off odor and flavor) than traditional chemical and microbial variables. Key volatile compounds identified in enzymatically produced seafood flavorants are formed via Maillard reaction and Strecker degradation of amino acids. Figure 8.3 Processing Odors Flavor development in processed seafood is a result of complex proteolytic and lipolytic reactions induced by different processing parameters like enzymes and temperature. and 1-propanol [28. gave the most intense odors of smoked salmon and contributed to the fish-like earthy odors and fatty and rancid odors (Figure 8. were characteristic in unsmoked fish. which is typical for products on the market [23]. but it is mostly attributed to the phenols. the Strecker aldehyde produced from methionine. thermal degradation. like cis-4-heptenal. and 1-octen-3-ol.7 illustrates the main odors that were present in smoked fish samples after 14 days of chilled storage. Maillard reaction.3. it was verified that selected key volatile compounds performed better as predictors to explain variation in sensory attributes (smoked. it is clear that their presence contributes to the characteristic fish odor of smoked salmon products. 2. giving the flesh its typical fishy odor [71. Volatile compounds like alkyl-pyrazines and sulfur-containing compounds have been found in cooked crustaceans. whereas carbonyl compounds. 2-pentanone. hexanal. .6-nonadienal. 3-hydroxy-2-butanone.

most of them generated from chemical or enzymatic oxidation of unsaturated fatty acids and further interactions with proteins. 109. potato-like odor was identified as cis4-heptenal and the boiled potato-like odor.112 ◾ Handbook of Seafood and Seafood Products Analysis Smoked salmon odors Characteristic smoke odor Sweet.4-heptadienal and 3. 4-Heptadienal Sweet.. smoke 3-Methyl butanal Sweet. Food Chem. However. 2008. the highest odor scores were given for boiled potato and rancid. probably originating from amino acids. 2-methylpropanal and 3-methylbutanal were the key.2 Ripening Odor—Salted and Dried Fish Odor Numerous volatile compounds have been detected in ripened products like dry cured ham. 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. . During ripening of salted cod. Similar processes have been reported in ripened seafood products. and free amino acids. where methional derived from methionine and 2. the desired flavor and texture develop as a consequence of protein and fat degradation. burnt.3. they suggested that lipid autoxidation during ripening was primarily responsible for aroma development. caramel Smoke-house. sweet Smoke-like 2. smoke Mushroom. especially those in the Mediterranean. geranium Rancid cis-4-Heptenal Heptanal Earthy-like odors 1-Octen-3-ol Figure 8.5-octadien-2-one were associated with the development of the typical flavor obtained after anchovy ripening.7 GC–O evaluation of volatile compounds detected in cold smoked salmon after 14 days of storage at 5°C. mushroom 2. peptides. In our study.and 3-Methyl phenol Guaiacol 4-Methyl-guaiacol Sweet and fruity-like odors Wood. sweet Flowery. both oxidatively derived compounds. and rancid-like odors Burnt. sweet Flowery. earthy. [76–78]. highly volatile components of ripened anchovy. and aldehydes such as acetaldehyde. fatty Boiled potato-like Fatty. sweet Wood. Thus. The rancid. smoke. fruity Flowery. manufacturers of ripened products have observed that some degree of proteolysis is necessary before flavor can develop.5octadien-3-one were also identified as potent odorants in ripened anchovy [81].6-nonadienal from fatty acid oxidation were the main odorants in sugar salted. (Modified from Jónsdóttir. as heptanal. ripened roe products [79] Similarly.4. et al. potato-like odors together with cucumber-like odor [82]. Methional and (Z)-1. R. 184.) 8.. Triqui and Reineccius [80] found that 2.

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

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

and Data Analysis The electromagnetic range applied in NIR spectroscopy spans from 700 to 2500 nm. and so the need for measurement and documentation of such parameters is both a consumer requirement and also issued by law. The work in food analysis tends to have a focus within the agricultural sector [1].1.2 Theory. There are several reasons why NIR as a food analytical tool has caught attention and approval during the last decennia. requirements for such a method would preferably be that it is rapid and nondestructive. The absorption of light is due to the response of the molecular bonds O−H. frequently consumers want readily accessible information about nutritional parameters and food quality.1.122 ◾ Handbook of Seafood and Seafood Products Analysis Compared with the production and distribution of meat from the agricultural sector. Measurement Principles. imaging techniques. In this perspective there is an obvious need for objective methods for evaluating and documenting the basic composition of fish and seafood. as well as x-rays. hence. . we review some of the most relevant methods for assessing the basic composition of fish and seafood as presented in scientific literature.1 9.1 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. followed by a presentation of the usage of NIR measurements for the rapid determination of basic constituents in fish and seafood products. The measurements are based on light interaction with material. 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. In the following section. However. 9. The basic principles of the techniques are described as well as a presentation of the use and applicability of quality measures of fish. and additionally the method may be applied with little or no obtrusion to the material sample. comprising the frequencies just below those of visible light. In this context seafood is particularly challenging as it comprises a vast number of different species with their own characteristics and qualities. Regarding industrialized food production. C−H. magnetic resonance. The four methods presented fit with the requirements of speed and nonobtrusiveness. 9. and hence these issues must be considered during the processing and characterization of the material. to analysis related to the environment and the petrochemical sector [1]. pharmaceutical applications. seafood is considered highly fragile and perishable with a short shelf life and delicate texture. The documentation of basic nutritional composition of foods is a legal requirement in many countries. which is a prerequisite for a methodology to be applied along a production line. In this chapter. throughout the years the method has also proven useful for the analysis of seafood and seafood products [8]. During the last 30 years the use of NIR spectroscopy has gained increased importance in the evaluation of a number of different food quality parameters [2–7]. These methods are near-infrared (NIR) spectroscopy. these techniques may be applied in or at a production line. C−O. we give a short introduction to the principles of NIR spectroscopy. Another benefit is the potential of simultaneous measurements of more parameters. facilitating a rapid response. Another aspect to be considered is the increased consumer awareness regarding the quality of their food.

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

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

937575 0. (From Nilsen. or postspawning.22] also conducted studies documenting the efficiency of applying NIR spectroscopy in different measurement modes to assess fat and water content in salmon. Silver Spring). Transmission spectroscopy was also employed for the analysis of fat and dry matter in capelin [25]. microwave. In a research article published in 2004.. The study concluded that NIR is well suited for nondestructive quality evaluation of salmon fillets. and additionally the spectroscopic measurements could be used for origin identification or authentication of the samples. NIR spectroscopy has been used for evaluating the chemical composition of several other fish species as well. whether pre-. Torry fatmeter. Unpublished data. fat. H. In this work they applied a fiber optic measurement setup. 1998.Basic Composition: Rapid Methodologies ◾ 125 salmon. water. N. In a recent work by Khodabux et al. water. PC): (%fettHS. Xiccato et al. 8) Figure 9. and dry matter in halibut fillet. An example illustrating the use of NIR spectroscopy for assessing fat content in farmed salmon is given in Figure 9.985681 0.603947 0. Isaksson et al.K. NIR spectroscopy was proven to be a useful tool for the evaluation of basic constituents of different types of tuna.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 Hsfett1.600067 0. [26] showed that NIR spectroscopy could be used to estimate lipid. Measured Y Elements: Slope: Offset: Correlation: RMSEP: SEP: 21 Bias: 24 78 0. This work also emphasized the impact of the conditional state of the fish when making calibration models. (Y–var.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. In both the works of Vogt et al. [20] conducted a study in which they compared NIR measurements on intact salmon fillet. as well as on minced salmon muscle. The fat content of herring has also been assessed by the use of NIR spectroscopy. Nortvedt. in. and Sørensen. [29] one question of interest was the comparison of different methods for measuring fat content. [28] and Nielsen et al. [27]. [21.2. . and protein content of European sea bass.264047 0. Torrissen. Wold et al. and protein than those made on intact muscles.) Spectral measurements were performed on intact fillets by transflection measurements by use of the fiber optic probe of the instrument NIRS6500 (Perstorp Analytical Inc.. applying minced samples for the spectral readings. Spectroscopic readings obtained on the minced samples correlated better with the reference measurements on fat. and NIR spectroscopy. and Tuene [24] made use of NIR transmission spectroscopy to assess protein.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

They are arranged in concentric circles forming subdivisions of striated muscle (Figure 10. etc. 2008).1–2 mm (Figure 10. backscattered electrons. At each subdivision there are macroscopic collagenous dividing lines (myocommata).2) are primary fi xation with aldehydes such as glutaraldehyde. polarizing microscopy. In recent years considerable progress has been made in the field of SEM through vitrification techniques. sudan. phase contrast or differential interference contrast (Nomarski).5). Finally.) before examination in the LM. For this. coated. the sample can be observed with all its constituent water. In Cryo-SEM. Many of the endomysia are connected to the perymisium.1). The fibers are essentially the same as those of terrestrial animals in terms of the arrangement of the thick and thin filaments.140 ◾ Handbook of Seafood and Seafood Products Analysis provides a detailed description of the protocols often followed to obtain information about seafood microstructure. 2006). In the former. the sample is frozen in slush nitrogen (Figure 10. In this way. so microanalysis can be carried out by means of x-ray. infiltration and embedding in resin. and so on. The steps in preparing samples for TEM observation (Figure 10. and coating with a conducting metal for SEM imaging or with carbon for x-ray.4) and quickly transferred under vacuum to a cold stage fit on a microscope where the frozen sample is fractured. When physical fixation is used. They are each surrounded by the sarcolemma membrane and by a thin layer of connective tissue (endomysium). the sample preparation steps are chemical fi xation (with aldehydes and osmium tetroxide. Besides the secondary electrons. critical point drying. In both methods. 10. There are two ways of preparing samples for SEM: chemical fi xation and physical fi xation (Figure 10. the sample is frozen in liquid nitrogen and then freeze-dried before being coated and observed. so there is no need to section it. other emanations or signals such as x-rays. iodine. and staining the ultrathin sections with heavy metal solutions such as lead citrate or uranyl acetate. the samples need to be prepared first. in this way. Electron microscopy (EM) allows food structures to be studied at higher magnifications than those used in LM. which is contiguous to the myocommata (Ofstad et al. they are mounted in glass slides and stained with different dyes (toluidine blue. Once the semithin sections are obtained. these different signals can be captured by the appropriate detector in each case.02–1. as for TEM). The most useful application for studying seafood structure is bright field microscopy. etched. and observed. may be generated as a result of the electron beam striking the specimen (Pérez-Munuera et al. ions or molecules can be identified and quantified in situ using specific detectors coupled to the electron microscope.2 Fish Muscle Microstructure Fish muscle consists of myotomes... dehydration in a series of ethanol dilutions of increasing concentration. image analysis relies heavily on computer technology to obtain quantitative results from microscopy observation.3). The muscle cells are short and 0.0 mm in diameter. Two types of microscopes use electron beams as their source of illumination: transmission electron microscopes (TEM) and scanning electron microscopes (SEM). showing alternate arrangements of . The SEM method observes the surface of the sample. or fluorescence microscopy. dehydration in a series of ethanol dilutions of increasing concentration. light green. The light microscope (LM) is a very versatile tool that works in different applications such as bright field. the sample has to be prepared in semithin sections of about 0. 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. secondary fi xation with osmium tetroxide. cutting ultrathin sections (5–100 nm) in an ultramicrotome. red oil.

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

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

Pd. 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. 70. . for X-ray) SEM observation Figure 10.5% Glutaraldehyde 2% Os O4 Ethanol (30.Microstructure ◾ 143 Food Specimen portions Fixation Physical fixation Chemical fixation Quick freezing in liquid N2 2. 50.3 Preparation of samples for SEM observation. for SEM imaging) (C. 90.

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

2.7A shows a herring sample stained with toluidine blue and observed by LM. the perymisial connective tissue that surrounds the muscle bundles can be seen. Fixing in osmium tetroxide shows the distribution of fat in the herring tissue. but the total collagen content is lower. In a cross section of the sample fi xed in osmium tetroxide (Figure 10.7C shows the inside of a muscle fiber with the myofibrils perfectly bundled.6C).6A shows a cross section of herring tissue fi xed with glutaraldehyde and observed by SEM. surrounded by the sarcolemma and by the endomysial connective tissue. since the water in which the fish live lends support for the body (Lampila.6F.6B). At low magnification. Figure 10.5 Schematic representation of fish muscle with myotomes. The longitudinal section in Figure 10. it is possible to observe ultrastructural details. The myofibrils are shown in longitudinal section in this sample (Figure 10. which is mainly composed of collagen.6D shows the microstructure of herring tissue at a higher magnification. A and I bands (Pérez-Munuera et al. a: myotome.Microstructure ◾ 145 a b Figure 10. with the endomysial connective tissue keeping the muscle fibers firmly attached to one another. where the fat is viewed as globules on the surface of the fiber. 1990). The typical fish muscle fibers can be seen. 2008). obtained by the same technique but observed at a higher magnification.7B. Examples of different fresh fish tissues observed by several techniques are described here. A micrograph cross section of the fibers shows them surrounded by the sarcolemma. When ultrathin sections of herring muscle tissue are studied by TEM. the myofibrils at the cell edges have a less rounded section than the central myofibrils and are arranged like a palisade.6E). The separation that can be observed between the muscle cells is usually attributed to the effect produced by chemical fixation and dehydration during preparation for SEM. the fat can be observed covering the fibers in a longitudinal section of herring muscle (Figure 10.. reveals the myofibrils inside each cell. When the muscle fibers are observed using the Cryo-SEM technique. fat globules of different sizes are observed occupying the interfibrillar spaces and myofibrils are distinguished inside the cells. The layouts of the Z disks that mark the length of the sarcomere are visible. Figure 10. The myofibrils are . Figure 10. b: myocommata. The fiber is composed of myofibrils in which Z disks are distinguished in the areas where the sarcolemma is broken.1 Herring Figure 10. where the Z disks can be distinguished. 10. the aggregation of solutes produced during the etching of the sample generates the typical eutectic artifact observed in Figure 10.

TEM and SEM studies demonstrated an extensive network of filaments connecting Z structures that were regularly spaced and connected by sets of longitudinal.9). connective tissue. The main difference is their size: hake fibers are thicker than herring ones. which are the components of the cytoskeletal network that links the myofibrils to one another and to the sarcolemma.. (A–E) SEM. a. The cytoskeletal ultrastructure of hake was studied by Pagano et al. Hake fibers surrounded by connective tissue can be observed in Figure 10. connected to each other at the Z disk level by the costameres. Z. the A and I bands. 10. along with the M and Z lines. can be seen. The TEM technique allows images to be obtained at higher magnification and with better resolution than other microscopy techniques (Figure 10. 2007). f. The structural elements that constitute the sarcomere. fat globule.2. . (2005) after depleting the thick and thin filaments with a potassium iodide treatment.8. and roughly parallel filaments (Figure 10.7D). (F) Cryo-SEM. eutectic artifact.6 Herring tissue. pork meat (raw ham) (Larrea et al. for example. Z disk.2 Hake The observation of hake muscle by SEM after fi xing with glutaraldehyde allows distinguishing that the fibers of hake muscle tissue are very similar to those of herrings. The same structure has also been observed in different meat products.146 ◾ Handbook of Seafood and Seafood Products Analysis Z (A) 300 μm (B) 6 μm I (C) f 40 μm f (D) a 10 μm f (E) 60 μm (F) c 30 μm Figure 10. continuous. c.

(C and D) TEM. I. Z.7 Herring tissue. c. M line. Z disks.Microstructure ◾ 147 p m m 30 μm (A) c M 10 μm (B) m m A Z (C) (D) Figure 10. A. I band. m. M. . (A and B) LM. P. A band. myofibrils in a “palisade” ringing the edge. 100 μm Figure 10.8 Hake tissue observed by SEM. costameres. perymisial connective tissue.

141. Com.9 Cytoskeletal structure of hake observed by TEM. The fiber shrinkage and the space between the fibers both increased to a greater extent in the muscle from the salmon that were frozen before smoking than in muscle smoked from fresh salmon. the greater the shrinkage. Biochem. (2000) used LM to observe the changes that occurred in the salmon during the smoking process and quantified them by image analysis. The micrograph shows geometrically shaped fibers surrounded by a connective tissue.10 Smoked salmon. The data of the average cross-sectional area of muscle fibers showed that the smoking process produces shrinkage of the fibers. Z-disk.. longitudinal filament connecting Z-Z.10B shows a detail of an intercellular space created by the conjunction of three fibers or cells. Sigurgisladottir et al.3. B. M. With permission.1 Smoked Salmon A cross section of a smoked salmon sample obtained using the Cryo-SEM technique is seen in Figure 10.148 ◾ Handbook of Seafood and Seafood Products Analysis Z LZ IZ DZ 8.) 10. 2005. Figure 10. . LZ. Z.10A.000 X Figure 10. Gudmundsson and (A) 100 μm (B) 10 μm Figure 10.3 Processed Fish Microstructure 10. (A and B) Cryo-SEM.R. et al. 13. Physiol. (Reprinted from Pagano. the higher the smoking temperature.

3.11A) 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) (B) (C) Figure 10. Figure 10. (B) cross section. (A) SEM. . (B) Cryo-SEM. (A) longitudinal section.2 Salted Cod The presence of salt deposits in the cod tissue can be observed by SEM (Figure 10. and gaps were formed in the tissue structure.11 Salted cod.12 Seafood stick (surimi) observed by SEM. A combination of PEF and high pressure had a more detrimental effect on smoked salmon microstructure than PEF treatment alone. 10. where two fibers can be observed completely covered by salt deposits. these treatments decreased the cell size compared with fresh salmon. and (C) protein network.11B shows a cross section of salted cod tissue (A) 100 μm (B) 300 μm Figure 10.Microstructure ◾ 149 Hafsteinsson (2001) studied the effect of pulsed electric fields (PEF) and a combination of PEF and high pressure on smoked salmon microstructure. Figure 10.

M. 2001..” Lean fish meat is minced to a paste. the paste is shaped and an “artificial fish muscle” is obtained.. obtained by SEM.12A. The cross section (Figure 10.12B) shows the typical concentric layers of this type of surimi product. The formation of a new network with the myofibrillar protein (Figure 10.A.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. Such a product is often sold as “crab sticks” or “seafood sticks. PA. Lancaster.3. where the presence of salt makes the etching of the sample for observation difficult and masks the underlying structures. after adding different additives.. 10. (From Lluch. Figure 10.150 ◾ Handbook of Seafood and Seafood Products Analysis observed by Cryo-SEM. et al. With permission.3 Surimi One of the most common surimi products on the market is artificial crab muscle. shows a longitudinal section of a crab stick where the “artificial fibers” can be observed. Technomic Publishing Co. Inc. The Chemical and Functional Properties of Food Proteins.) .13 Schematic representation of (A) squid mantle and (B) arrangement of muscle cells.

When TEM is used to study the ultrastructure of fresh squid (Figure 10. (A and B) SEM. The inner and outer tunics are covered by a visceral lining and outer lining. 1990. a central sarcoplasm is shown to be surrounded by myofibrils. Eur. J. Muscle Foods.E.4 Squid Microstructure The squid mantle is composed of muscle tissue sandwiched between two tunics of connective tissue (Figure 10. (From Llorca.. 2001).. Each fiber is surrounded by a sarcolemma (Llorca et al. Muscle fibers are grouped in bands that are arranged orthogonally. m. Comparative microstructure of red meat. Lampila. This fiber distribution can also be observed by LM in samples stained with toluidine blue (Figure 10. Trends Food Sci. Th is gel network structure gives surimi its characteristic elasticity and texture (Sikorski. 122–128.Microstructure ◾ 151 –200 μm –10 μm (A) 10 μ (B) 3. the intermyofibrillar spaces between these can be observed. and (D) TEM. With permission. . Regardless of their orientation. 807. 2007). central sarcoplasm. Tech. H.14C). l. and Hafsteinsson.13).75 μ m s (C) (D) l Figure 10.) ties of surimi. 2001). Food Res.. s. Technol. L.. all the muscle fibers are thin. sarcolemma. Effect of electric field pulses on microstructure of muscle foods and roes. The fibers arranged in circumferential and radial bands were observed by SEM in samples fi xed with glutaraldehyde (Figure 10. et al. 247–267. 1990). approximately 3. (C) LM.. 225. 2001. 2007. 10. LM makes it possible to distinguish a peripheral area in blue and a central core in white inside each cell. E. M.. 1. Circumferential muscle bands (100–200 mm) comprise fibers running about the entire circumference of the mantle cone..14A and B) (Llorca et al. 12. Radial bands (10–15 mm thick) comprise fibers that connect two tunics of connective tissue. References Gudmundsson. respectively.5 mm in diameter (Lluch et al. poultry and fish muscle..14 Squid.14D). myofibril.

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

3 Mass Transducers ..................................6 Conclusions ............................3....................1 Sensors Based on Conductance Changes ..................................154 11...............................1................... some are of great importance to define overall properties such as freshness or quality [1]...................3......................... is one of the most used method to assess freshness by both consumers and industries [2]...............................2 Conducting Polymers and Molecular Aggregates ............................4 Electronic Noses ..................164 References ...160 11..............2 Amperometric Gas Sensors ..............................2 Sensor Parameters.............................................................165 11........................1 Introduction .........4 Field-Effect Transistors ............................................................ For these reasons the knowledge of the chemical 153 .....................1 Metal-Oxide Semiconductors ........................................3 Chemical Sensor Technologies ........................................................... for which the human perception of airborne chemicals......................160 11.............5 The Application of Electronic Noses for Fish Freshness and Quality Measurement ..........158 11................................157 11........................................................ called odor..................................159 11..........153 11...........................................................................3.......1 Introduction Among the thousands of molecules composing food complex mixtures..............................................158 11..................157 11....................159 11......................3.....Chapter 11 Chemical Sensors Corrado Di Natale Contents 11......158 11..................................................................... The relationship between chemistry and food properties is particularly interesting in the case of fish and seafood in general.................157 11.................3.......................................3.3...........................................................1.........5 Color Indicators .....................................................................................

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

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

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

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

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

H2 molecules dissociate into atomic hydrogen at the palladium surface. allowing a simultaneous evaluation of absorbance and fluorescence of samples. 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). Indeed. Furthermore. In this way sensitivity to ammonia. such as metalloporphyrins [22]. Nonetheless. furthermore.3. organic molecular layers. The feasibility of this approach has been demonstrated using as sensitive layer a film of a sodium salt (bromocresol green) [25]. Due to the large diffusion of portable computers. giving rise to a number of low-cost advanced optical equipments such as digital scanners. the current flowing in the FET changes revealing the chemical interaction. This difference can be modulated by a layer of electric dipoles that can reach the metal–oxide interface. The principle was adequately exploited with a palladium gate FET exposed to hydrogen gas [20]. and cellular phones all endowed with color screen. where they form an ordered dipoles layer. 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. whose sensitivity toward amine was also recently measured [23]. the colorimetric detection of fish freshness recently received a novel interest. is based on the fact that a computer screen can be easily programmed to display millions of colors. This salt exhibits a rather large change in color. and hydrogen atoms can diffuse through the palladium film until they reach the oxide surface. was also obtained [21]. In particular. the chemical practice of this approach is badly balanced by the transducer counterpart. also appreciable by eye. FET structures were also modified to accommodate. known as computer screen photo assisted technique (CSPT). On the other hand. 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]. cameras. This basic structure was successively modified changing the gate metal and thickness to extend the range of measured gases. although under constant bias. 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]. whose characteristics largely fit the requirements necessary to capture change in optical properties of sensitive layers in many practical applications.4 Field-Effect Transistors Most of the properties of field-effect transistors (FET) depend on the difference between the work function of electrons in the metal gate and in the semiconductor. . The method demonstrated also the possibility to identify a number of different amines [28]. as a sensing part. and screens.3. and. This last technique. 11. As a result. an important gas for fish freshness and quality.5 Color Indicators Although known for several years [24].Chemical Sensors ◾ 159 11. Nonetheless. PDAs. the visual determination limits the performance and may greatly vary between individuals. standard optical instrumentations are usually expensive. in the last decade we have seen rapid growth in performance in fields such as consumer electronics. combining wavelengths in the optical range. Compared with the use of digital scanners. Lundström and Filippini proved that it is possible to assemble a sort of spectrophotometer using the computer screen monitor as a programmable source and a web camera as detector [29]. 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.

and aromatic. 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]. and each molecule is sensed by many receptors [33]. Since the 1980s. After this discovery. and such systems were soon dubbed as “electronic noses. Their concentration and the presence of other compounds are rather typical of each species. Investigations about olfaction receptors show that Nature strategies for odor recognition are completely different from those of analytical chemistry. The concentrations of these chemicals are directly correlated to the degree of spoilage. the application of the CSPT concept may be foreseen as greatly extending the analytical capacity worldwide. observation of Nature offered a useful suggestion about the use of such devices. The physiology of olfaction has made considerable advances. CSPT has demonstrated its utility in particular to classify airborne chemicals reading absorbance and fluorescence changes in chemical dyes such as metalloporphyrins [30]. Recent studies are also beginning to unveil the signal pathways leading from the generation of olfactory neuron signal to the conscious identification of odors [32]. models of receptor mechanisms explaining the sensitivity to volatile compounds are now available. whereas CSPT arrangement gives the possibility of evaluating at the same time both the effects. 11. N-cyclic. and acid compounds. bromophenols. and the genes expressed by olfactive receptors are known [31].5 The Application of Electronic Noses for Fish Freshness and Quality Measurement The composition of fish headspace is a source of information about its freshness. 11. After this conjecture. To this point of view. Receptors were found to be rather unselective. amines. Among these compounds amines . Standard optochemical sensors are based either on absorbance or on fluorescence. the possibility of developing artificial olfaction systems became possible.4 Electronic Noses As discussed above. allowing for electronic nose application oriented optimizations. the lack of selectivity of many chemical sensors was considered as one of the main problems limiting their diff usion for practical applications. Odor classification properties of artificial systems were tested on several different fields proving that electronic noses could be in principle used to replace human olfaction in practical applications such as food quality and medical diagnosis [35]. sulfur compounds. each receptor senses several kinds of molecules. On the other side. 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. it was proposed that arrays of nonselective chemical sensors may show properties similar to those of natural olfaction [34]. and N-cyclic compounds. Nonetheless. almost all sensor technologies were used to build such systems. organic synthetic receptors offer an unlimited number of possibilities to assemble molecules endowed with differentiated sensing features.” This denomination is currently given to any array of unselective chemical sensor coupled with some multicomponent classifier. and an even more extended computation capabilities.160 ◾ Handbook of Seafood and Seafood Products Analysis camera. The features of electronic noses are fundamentally dependent on the sensing properties of the artificial receptors. The most important chemicals involved in the fresh fish odor are long-chain alcohols and carbonyls.

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

The possibility of developing a multisensor device to measure and/or estimate fish freshness with a combination of instrumental techniques (electronic noses. an integration of instruments is necessary.3 Time evolution of the major families of volatile compounds in fish headspace.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. This feature that can be interpreted as a failure of the electronic nose is likely due to an intrinsic nonlinearity of the studied problem. Nonetheless. sardines [58].5.57].g. and original data were previously published [53]. and the use of only one sense (e. with a folding back of the spoilage process in the representation plane. The experiment was related to COD fishes. olfaction) provides several errors of evaluation. humans provide a more reliable identification of fish freshness. . tactile (to test the flesh firmness and elasticity). and groupies [59].01 0 5 10 15 Days 20 25 30 35 Figure 11. in order to measure the quality of fish instrumentally.. and olfaction (to smell the gill odor) [54]. As a consequence. each able to capture different aspects of fishes. spectroscopic methods. Results are qualitatively similar to those shown in Figure 11. texture meters. 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]. shows the scores plot of a partial least-squares discriminant analysis model related to an array of metalloporphyrin-coated QMBs. color meters.1 0. The typical sensorial description is also reported. It is important to consider that sensorial methods of freshness appraisal involve the use of sight (to evaluate the skin appearance and the color and the global aspect of eyes). image analyzers. and devices measuring electrical properties) has been illustrated in different applications related to cods [56.

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 –6 4 6 –5 –4 –3 –2 PC 1 (80.5 0 –0.3 are used.5 –6 –4 –2 0 2 4 PC 1 (72.5 –2 –2.5 PCA scores plot of data related to a virtual array of sensors. each specific for a single interaction mechanism among those modeled by LSER.Chemical Sensors Scores plot 2 30 1. .5 1 0.76%) 0. Scores plot 1.5 –1 24 10 12 8 28 2 4 6 1 ◾ 163 22 20 18 16 14 –1.4 PCA scores plot of a simulated experiment where sensors selective for the compound family in Figure 11.39%) Figure 11.5 24 PC 2 (19.03%) –1 0 1 2 30 2 28 22 20 10 10 12 14 16 8 1 Figure 11.5 1 PC 2 (15.5 –1 –1.

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

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

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


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

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

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

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

[28] applied NIR spectroscopy to assess the end point temperature (EPT) of heated fish and shellfish meats.5 and 20 micrometers. The region of the electromagnetic spectrum under consideration in Raman spectroscopy is similar to that in MIR. the energy at defined frequencies can be partially absorbed.000 and 400 cm−1 (2. the usefulness of visible spectroscopy/near infrared spectroscopy (VS/NIRS) has been researched for many quality aspects [23–25]. NIR spectra of foods comprise broad bands arising from overlapping absorptions corresponding mainly to overtones and combinations of vibration modes involving C–H. 12.3. the freshness of cod was estimated by Heia et al. thus. All these techniques have been gradually implemented as monitoring systems in food processing [31]. which is also called thermal infrared (TIR) refers to electromagnetic waves with a wavelength of between 3. This technique measures the reflectance of light from the product in the visible and NIR wavelength range. but it involves a scattering process. Thermal infrared imagers translate the energy transmitted in the infrared wavelength into data that can be processed . 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. ultrasound transducers must have airless contact with the sample during examinations [22]. This complicates the noncontact measurements. the main disadvantage of this method is that only the surface of the sample is examined.2 Visible Spectroscopy In recent years. When radiation with energy corresponding to the MIR range interacts with a molecule. In the fish sector. Marquardt and Wold [34] concluded that Raman spectroscopy might be a useful tool for rapid and nondestructive analysis of fish quality. 12. Other information should be used in conjunction with visible spectra in determining the specific properties of interest. The most popular IR spectroscopy is the NIR one. Mid-infrared (MIR) and Raman spectroscopy have high structural selectivity and contain more of the type of information needed in structural elucidation studies. but it is measured in terms of tenths of a millimeter [32] and is dependent on less-precise reference methods [27]. [33] applied MIR spectroscopy combined with chemometric tools to determine whether fish has been frozen–thawed. and it is able to provide thermal information. For example. MIR spectroscopy concerns the region of the spectrum lying between 4. and N–H chemical bonds [27]. O–H. Most industrial processes require the measurement of temperature. NIR technology has been widely developed as an analytical tool.000 nm).500–25.3. 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. This makes it very feasible for measurements to be made in organic and biological systems.3 IR Spectroscopy In the recent years. NIR spectroscopy is based on the absorption of electromagnetic radiation at wavelengths in the range 780–2500 nm. A rapid. Karoui et al. but it is not the only one. Focusing on fish products.Physical Sensors and Techniques ◾ 173 ultrasound to pass through air. Uddin et al. The far IR. the visible spectrum is a function of the entire structure of the compound rather than specific bonds. but their use is limited by their low penetration in the product (it depends on the wave length. [26] using the visible wavelengths only. a multispectral imaging NIR transflectance system was developed for online determination of moisture content in dried salted codfish [29].


Handbook of Seafood and Seafood Products Analysis

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


RF Spectroscopy—Impedance Spectroscopy

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

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

Physical Sensors and Techniques


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

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


Handbook of Seafood and Seafood Products Analysis

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


Overview of Microwave Theory

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

E1, A1, θ1

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

E2, A2, θ2

, θ3 E 3, A 3

Product characterization

E1, A1, θ1

, θ5 E 5, A 5

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

E4, A4, θ4 Processes control (or monitoring)

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

Physical Sensors and Techniques


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


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

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

ε˝ i + – + + – MW + – + – dw

+ + – – + –

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

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

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


Handbook of Seafood and Seafood Products Analysis

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

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

Physical Sensors and Techniques
ε˝ 4


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

+ –

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

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


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

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


Determination of Moisture Content

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

Physical Sensors and Techniques


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

Tea leaves

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


Handbook of Seafood and Seafood Products Analysis

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


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

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

Physical Sensors and Techniques


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

0.2 GHz 0.3 GHz

0.9 GHz 1.8 GHz 2.45 GHz

10 GHz

ε˝ t



1E + 08

1E + 09 Frequency

1E + 10

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

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


Handbook of Seafood and Seafood Products Analysis



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

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

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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).


Handbook of Seafood and Seafood Products Analysis

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

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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).


Handbook of Seafood and Seafood Products Analysis

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

Chapter 13

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


Handbook of Seafood and Seafood Products Analysis

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



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


Sensory Methods

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

Methods for Freshness Quality and Deterioration


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


The European Union Freshness Grading (EU or EC Scheme)

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

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

QDA provides a detailed description of all flavor characteristics in a qualitative and quantitative way. However. sole. The advantages of QIM are that it requires short training. brill. the words for describing the odor and flavor of the fish can be categorized into two groups. positive and negative sensory parameters based on whether fish are fresh fish or fish at the end of the storage period [37].3 The Torry Scheme In contrast to the QIM. Objective sensory methods are essential for quality control and estimation of shelf life of seafood. medium fat. This method is considered to be a relatively fast. pollock. Rapid PC-based QIM is also available on the Internet at http://www.2. and flavor. instrumental methods are also needed to satisfy the need for quality measurements in fish industry. The Torry Scheme. a higher score can be given for a single parameter [27]. nondestructive method based on direct observation of sensory parameters of fish and can also be specific for species. Sebastes mentella marinus. The method can also be used for texture. It has been widely used in its original or modified forms.1) [34]. and is nondestructive and can be used as a tool in production planning and quality warranty work [27]. Solea vulgaris.5 may be used as the limit for consumption [21].dfu. plaice. Therefore. panelists evaluate the odor and flavor of cooked fillets. Hyldig [29] indicated that the QIM is expected to become the leading reference method for the assessment of fresh fish within the European community.2.dk/QIMRS/qim_0202. The average score of 5. expensive. herring (Clupea harengus) (Table 13. and fat fish species. is rapid and easy to perform. sensory methods are time consuming. Objective terms should be used rather than subjective terms. odor. 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]. and not always practical for large-scale commercial purposes. Pleuronectes platessa. farmed Atlantic salmon (Salmo salar) [31]. redfish shrimp. In QDA. During spoilage. Recently developed QIM schemes were presented for raw gilthead sea bream (Sparus aurata) [30]. QIM Rating system software was developed for cod.2). QIM Eurofish published a manual [21] containing QIM schemes for 12 fish species and information about how to use the QIM schemes (QIM-Eurofish 2004). and Scopthalmus maximus. saithe. trained personnel required. Pollachius virens. Melanogrammus aelefigus. . fresh cod (Gadus morhua) [32]. the Torry Scheme was developed at the Torry Research Station for use with expert and trained judges.min. herring. The most comprehensive scoring scheme to assess fish is the Torry Scheme [36]. In addition. Pandalus borealis.htm.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. In this scheme. 13.192 ◾ Handbook of Seafood and Seafood Products Analysis quality expressed as a demerit score and storage life on ice. Descriptive words should be carefully selected. is a descriptive 10-point scale and has been developed for lean. haddock. the QIM is suitable for early stage of storage of fish where other instrumental methods are not accurate [28]. often referred to as the Torry scale. and redfish by the Danish Institute for Fisheries Research. and the panelists trained should agree with the terms. common octopus (Octopus vulgaris) [33]. which makes it possible to predict remaining storage life on ice. 13. The scores are given from 10 (very fresh) to 3 (spoiled) (Table 13. and turbot (Scopthalmus rhombus. respectively) [21].

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. 37–59.1 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... S. Int. seaweedy. pp. Nordic Industrial Fund (in Danish). 1992. With permission. D. 2004..Methods for Freshness Quality and Deterioration Table 13. Quality Standards for Fish: Final Report Phase II. matte. 37. Food Res. developed by Nielsen. . G. and Hyldig. 975. brown Odor Fresh.

texture. odor.3..1 Texture Analysis Texture analyses for seafood are extremely important in research. With permission. vanillin Condensed milk. Sci. sour. and chewiness of food. improper handling storage. hardness is the most important to the consumer. soapy. and product development in the seafood industry [38]. wood sap. starchy. . boiled milk. boiled potato Milk jug odors. acetic or butyric acids) decomposed grass. Dielectric properties of fish are used for determination of freshness. seaweed. These changes occurring at microscopic level are related to alterations in appearance. during processing. Scotland. Numerous mechanical methods have been used to measure texture. 4.2 The Torrymeter The Torry fish freshness meter “Torrymeter” was developed at Torry Research Station in Aberdeen. which immediately begin to break down the proteins after the harvesting. tallow Flavor Watery. however. and cooking [39. Dielectric properties of fish skin and muscle alter in a systematic way during spoilage as tissue components degrade. Among textural attributes. TMA Strong bitterness. followed by strengthening of these odors Shellfish. Fish muscle has higher levels of indigenous proteases. Baltic herring.2 Torry Score Sheet for Freshness Evaluation of Cooked Cod Fillets Odor Initially weak odor of sweet.. J. rubber.. blue whiting. TMA Lower fatty acids (e. A linear relationship was found between Torrymeter readings and sensory attributes for cod. turnip. and flavor during spoilage and have been used as quality indicators since the first commercial version of the Torrymeter in 1970 [43]. quality control. mackerel. sour milk. natural odor Wood shavings.M. springiness. 13.3. 283.3 Physical Methods 13. metallic. Texture includes the most common characteristics such as hardness. deciding the commercial value of the meat [41]. slight sulfide Score 10 9 8 7 6 5 4 3 Source: Shewan. J. flounder. et al. “off” flavors.40]. 1953. Food Agric.g. starchy. hake. 13. trace of “off” flavors Slight bitterness. reminiscent of boiled clothes Lactic acid. Fish muscle may become soft or mushy as a result of autolytic degradation or tough as a result of frozen storage [16]. boiled meat Loss of odor. 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. there is little agreement on which is the best method [42].194 ◾ Handbook of Seafood and Seafood Products Analysis Table 13.

4 The RT-Freshtester Like Torrymeter and the Intellectron Fischtester VI. it could be used for evaluation of fresh and chilled fish in the seafood industry and on fishing vessels. The skin of fish could be affected by osmolarity and contact with electrically charged particles [51]. measuring the electric properties (resistance.3. Therefore.Methods for Freshness Quality and Deterioration ◾ 195 whole. these instruments need calibration depending on sample preparation. The method is based on conduction through skin and. Inácio et al. It has also reported that there is a linear correlation between the instrument readings obtained on the day of harvest/catch and the date of spoilage [53]. iced gilthead sea bream. The Intellectron Fischtester VI gives reliable information about the days in ice and left of iced stored fish. portable. and capacitance) of the fish flesh [52]. They are unsuitable for frozen or thawed fish. therefore. The Fischtester readings can be used as an objective criterion for the state of freshness/spoilage together with sensory data across the fish chain. fast and nondestructive.3. . 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. [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. season. [50] found strong correlation between the organoleptic and Cosmos results for six species of fish and concluded that application of the “Cosmos” instrument for objective quantitative evaluation of fresh and chilled fish quality by determination of smell intensity appears to be practicable. Mechanical abuse and freezing can affect the readings. Like other instruments. However. The electric properties of fish can change after death of the fish due to disruption of the cell membranes by autolysis. 13. fishing grounds. 13.05) lower in fish washed with seawater than fish washed with tap water or unwashed. and fish chilled in refrigerated seawater [54]. However. and fish-handling procedures.3. Gelman et al. 13. and readings from all instruments decrease with storage time.5 The Cosmos The “Cosmos” instrument developed by Japanese is applied for the evaluation of fish quality by determination of smell intensity. conductivity. [50] found that the Torrymeter readings obtained from six species of different origin were poorly correlated with sensory evaluation. which interfere with the reading of both instruments as they are based on electrical properties of skin. partly frozen such as superchilled fish. RT-Freshtester. RT-Freshtester reflects dielectrical properties of fish. works only on whole fish and fillets with skin on. This could be explained by seawater containing ions. allows automatic grading of 60–70 fish/min. and farmed Senegalese sole [43–49].3 The Intellectron Fischtester VI The basic principles of Torrymeter (the United Kingdom) and the Intellectron Fischtester VI (Germany) are similar. Fat also has an effect on the dielectric properties of fish and tends to make observed Torrymeter values more variable [47]. the “Cosmos” instrument is handheld. Gelman et al. as well as rapid and nondestructive.

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

13. For the first time. Currently. and the chemical compound that is determined should increase or decrease as microbial spoilage or autolysis progresses [16]. Traceability is becoming a method of providing safer food supplies and of connecting producers and consumers. This alternative method could be cost effective and definitely more reliable. The traceability system can also be used for the determination of fish freshness. The most used procedures for the objective measurements of seafood quality are given in the next sections.1 shown. which is the main energy source for metabolic activity. 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]. and it has been indicated that this spectroscopic technique is useful in assessing the freshness and quality of sardine during iced storage [84]. The sequences of nucleotide catabolism proceed as shown in Figure 13. 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].1. 13. sensitive. degradation of ATP proceeds according to the sequence .1 ATP and Its Breakdown Products Rigor mortis occurs in postmortem muscle tissue and is associated with stiffness of muscle or flesh. recording the product temperature from the moment of catch. The oxidation Adenosine triphosphate (ATP) Adenosine diphosphate (ADP) Adenosine monophosphate (AMP) Inosine monophosphate (IMP) Inosine (Ino) Hypoxanthine (Hx) Xanthine (Xa) Uric acid (Uric) Figure 13.4 Chemical and Biochemical Methods Chemical and biochemical methods for the evaluation of seafood quality are more reliable and accurate. The initial stage of the reaction catalyzed by endogenous enzymes takes place quickly. These objective methods should correlate with sensory quality.4. and requires a small amount of sample. This process results from breakdown of adenosine triphosphate (ATP). Nucleotide breakdown reflects both action of autolytic enzymes and bacterial action [16]. this technique has been applied to sardine muscle during iced storage. since they eliminate personal opinions on the product quality. In postmortem fish muscle. cheap. leading to accumulation of adenosine diphosphate (ADP) and inosine monophosphate (IMP). It has been indicated that there is a correlation between nucleotide catabolism and loss of freshness.Methods for Freshness Quality and Deterioration ◾ 197 its use is simple.

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

The others especially putrescine and cadaverine have been reported to enhance the toxicity of histamine [115]. HPLC [120. histidine yields histamine. and use of a biosensor [130–132]. capillary zone electrophoresis (CZE) [128. 13. Since the amines are produced by spoilage bacteria toward the end of shelf life of a fish. and 2-phenylethylamine is derived from phenylalanine. Food and Drug Administration [117] and the EU [118]. cadaverine.108]. Cadaverine is derived from lysine. including thin-layer chromatography (TLC) [122.110]. Biogenic amines are generated by microbial decarboxylation of specific free amino acids in fish or shellfish tissue [111]. Among the biogenic amines.129]. and stomach contents at death. The biogenic amine content of fish depends on fish species. tyrosine produces tyramine. 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]. species. 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. The most significant biogenic amines produced postmortem in fish and shellfish products are histamine.124. There are various analytical techniques used to determine the concentration of biogenic amines. reliability. free amino acid content [112].S. By means of decarboxylation reactions. whereas BAI is based on increases in histamine. spermine. In addition. 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.123].107. tryptamine.0 to 7. their levels are considered as indices of spoilage rather than freshness [112]. the moment of capture. respectively. tyramine.109. since microbial flora vary seasonally [11]. depending on the species being examined [10. and histamine and decreases in spermine and spermidine during storage of fish.125]. putrescine. and the concentration of these increases with storage time [91. Consumption of seafood containing high amounts of these amines can have toxicological effects.3 pH The pH is also an important parameter to show depletion in tissue and quality of flesh during storage.127]. respectively.1 depending on season. Process technology is influenced by rigor development. and tyramine. cadaverine. histamine is potentially hazardous and the causative agent of histaminic intoxication [114]. postmortem temperature. GC [126. Among these techniques. putrescine. and reproducibility. HPLC is mostly performed because of its sensitivity.4. 2-phenylethylamine. and other factors . and agmatine. These problems may be more severe in sensitive consumers who have a reduced mono.11. spermidine. Postmortem pH varies from 5. the enzyme responsible for its detoxification.Methods for Freshness Quality and Deterioration ◾ 199 13. [121] for determination of quality of fish. The hazardous concentrations of histamine are 5 mg/100 g and 20 mg/100 g fish—the legal limit for histamine set by the U.5.and diamine oxidase activity [116]. and arginine leads to putrescine.2 Biogenic Amines The concentration of biogenic amines has been reported to be a reliable method of measuring the quality of fish. tryptamine from tryptophan. The formation of biogenic amines results from microbial degradation during the later storage of fish. Putrescine is also an intermediate of a metabolic pathway that leads to spermidine and spermine [119]. cadaverine. the presence of decarboxylase-positive microorganisms.4. The QI and the biogenic amine index (BAI) were proposed by Mietz and Karmas [120] and Veciana-Nogues et al. and pH [133].

200 ◾ Handbook of Seafood and Seafood Products Analysis [134. farmed gilthead sea bream [147].135]. the levels of 30–35 mg N/100 g muscle are considered the limit of acceptability for icestored cold-water fish [17. affecting light scattering and the appearance of fish. pike perch [146]. which is considered to be the main cause of off odors in fish products [58.151]. The EC reference method for TVB-N determination. Th is is caused by the depletion of energy reserves. bacteria act upon TMAO to produce TMA. TMA is produced by the decomposition of TMAO due to bacterial spoilage and enzymatic activity [150. The analyses of these indicators are considered unreliable because they reflect later stages of spoilage rather than freshness [140]. total volatile basic nitrogen (TVB-N) primarily includes trimethylamine (TMA. Low pH is used as an indicator of stress at the time of slaughtering of many animals. produced by spoilage bacteria). Low initial pH is associated with higher stress before slaughtering [13. and direct distillation methods have been recommended as a rapid routine method. Based on the results obtained from the literature. the level of TVB-N was not correlated with the time of storage of some fish species. The European Commission (Council Regulation No. TVB-N should be used as a chemical check. Therefore. It is well known that determination of TVB-N differs systematically according to the procedures used. and DMA (produced by autolytic enzymes during frozen storage). ammonia (produced by deamination of amino acids and nucleotide catabolites). turbot [92].136–139]. Therefore. sardine [12. 144]. and hake [148]. whereas the second one includes the use of trichloroacetic acid instead of perchloric acid [149]. it could not be regarded as a good indicator of fish freshness and proved to be better as a spoilage index. as shown in a variety of fish such as European hake [142]. 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 it has been used as an indicator of marine fish spoilage: CH3 CH3 – N=O CH3 TMAO CH3 CH3 – N CH3 TMA .141]. However. it affects reactions taking place during storage of fish. 13.4.4 Total Volatile Basic Nitrogen In seafood. It was found that there was a good correlation between three methods. A relatively low pH may cause a decrease in water binding to the myofibrils.5 Trimethylamine The one type of spoilage caused by microorganisms often detected as a fishy odor is due to the decomposition of trimethylamine oxide (TMAO) via the enzyme TMAOase demethylase. The first one includes direct distillation of fish after adding magnesium oxide. TVB-N level correlated with fish quality.59]. such as frozen eel [145]. Atlantic cod [143]. involving preliminary deproteinization with perchloric acid. However. was compared with two routine methods. as shown below: Following death of fish.4. and European eel [13]. Since the activity of enzymes depends on pH. 13. The level of TVB-N in freshly caught fish is generally between 5 and 20 mg N/100 g muscle. with the production of lactate. Low pH also promotes oxidation of myoglobin and lipids [134]. mainly glycogen.

164]. The formation of these products may cause severe quality changes or spoilage during prolonged frozen storage.150]. fish size. Conway microdiff usion and titration [159]. Several assays have been described for the determination of TMAOase activity in fish muscle [151. 13. the storage temperature. fish contain TMAO. when bacterial growth is inhibited. A close relationship has been found between lipid damage and quality of the final product [173]. a capillary electrophoresis method [165]. enzymatic and nonenzymatic lipid oxidation occurs. which results in a dry and firm texture of the fish muscle [174]. .5 mg TMA/100 g in fresh cod. its usefulness depends on time of year.6 Dimethylamine As mentioned earlier. During chilled or frozen storage of fish. biosensor using flavin-containing monooxygenase type-3 [168].4. and low-molecular weight compounds. terminal amino groups. but values increase during spoilage. especially in gadoid fish. lipid oxidation is the limiting factor in fatty fish species. The amount of DMA produced depends on species (except gadoid species. Seawater fish have 1–100 mg TMAO in every 100 g muscular tissue. location of catching. The TMAO content of seafood varies with species. However. flowinjection-gas diff usion method [167]. The formaldehyde content of frozen seafood is generally used as a spoilage index. However. this reaction is replaced by a slow conversion by an enzyme to DMA and formaldehyde [16. causing denaturation and cross-linking of proteins [171]. type of storage and processing. but it can react with a number of chemical compounds such as amino acid residues. 13. Fresh fish has a very low amount of TMA with values less than 1. including steam distillation [158].4. DMA can be used as a spoilage index during frozen storage of some species such as frozen hake [170]. 13.157]. The limiting factor of frozen storage in lean fish species is denaturation of proteins. age. and time. The fish is considered stale when the rate of TMA production is higher than 30 mg/100 g cod [155]. and environmental factors [152]. stage of spoilage.4. whereas fish can be stored in a frozen state for several months without severe changes in quality. DMA. HPLC method [162]. but it appears after 3 or 4 days. TMA is not produced in a significant amount during the early stages of chilled storage of fish. which is converted to TMA by bacteria in iced fish. This reduces the solubility of myofibrillar proteins [172]. Fresh fish has a limited shelf life and is prone to deterioration.7 Formaldehyde The formaldehyde content in seafood products is generally considered as nontoxic. time of year. colorimetric method [160]. other species do not develop adequate amounts of DMA).Methods for Freshness Quality and Deterioration ◾ 201 TMAO appears to be part of the system used for osmoregulation. after which the rate of production of TMA parallels the bacterial proliferation pattern [154]. photometry [161].156. and solid-state sensors based on bromocresol green [169]. TMA can be used as a spoilage indicator and not as an index of freshness. and methods employed for analysis. semiconducting metal–oxide array [166].8 Lipid Oxidation Indicators During processing and storage. resulting in rancidity. whereas freshwater fish generally contain only 5–20 mg% [153]. Many analytical methods have been developed for the measurements of TMA. or TVB-N contents. GC method [163.

(Eds.2 The autoxidation of fatty acids. which are the volatile products causing off flavor in products. temperature. consequently. Chapman & Hall. Many factors affect the onset and development of rancidity (oxidative and hydrolytic degradation of lipids). and Hamilton. London. Excess free radicals and peroxides in foods cause destruction of essential fatty acids and vitamins A. There are three steps in autoxidation of unsaturated fatty acids. 1994. ketones. The hydroperoxide value is generally shortened to peroxide value (PV). forming stable deterioration products (termination phase) [181. 1–22.202 ◾ Handbook of Seafood and Seafood Products Analysis Initiation: Initiators (heat. U. Under chilled/frozen conditions. nutritional losses. the type and concentrations of antioxidants. and free radicals react with oxygen to produce peroxide radicals (ROO).C.180]. moisture content. 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. The peroxide radical can attack another lipid molecule RH. and modification of electrophoretic profiles of proteins [172.C. Free radicals from oxidizing lipids can polymerize with proteins and destroy certain amino acids. (From Hamilton.K.182].2). Initiators (such as light. Seafood has highly unsaturated lipid content. R.) Off taste and off odor are usually defined as rancidity. heat) convert RH to free radicals (initiation phase). off flavors. and pantothenic acid. light. and degree of exposure to light [178–180]. oxygen availability. J.).. and alcohols. lipid oxidation compounds interact with proteins. produce toxins. including the degree of unsaturation of the oil. and then the quantity of radicals and peroxides decreases. They also destroy pigments. initiation. propagation. Fish oil contains about 20% of their total fatty acids as long-chain PUFA. in Rancidity in Foods. C. leading to protein denaturation. B6. pp. and termination (Figure 13. The major chemical indicators for the determination of the extent of oxidative rancidity . Allen. resulting in peroxide (ROOH) and new free radical (propagation phase). E. pro-oxidants. The amount of reactive compounds increases gradually. unpleasant odors. fish and fish oils are highly susceptible to the development of oxidative rancidity. R. and taints [179. and cause off flavor/odors [183]. 3rd edn. Several chemical and physical techniques applied alone or together have been used to determine the degree of oxidation and hydrolytic degradation of lipids in edible oils. and they break down to aldehydes. thiamine.. The amount of hydroperoxides can be used as a measure of the extent of oxidation in the early stages.. Peroxides are not stable compounds. Peroxides can also react with proteins and result in a decrease in their nutritional value.175–177].J.

Many methods have been employed for the measurements of lipid oxidation in foods as a means of determining the degree of damage [20. and decline [184. cod [192. 13.9 Lipid Hydrolysis Hydrolysis leads to hydrolytic rancidity and involves hydrothermal or enzymic (lipase) hydrolysis to FFA and other products. horse mackerel [13. spoilage domain such as the range of environmental conditions over which a particular SSO is responsible for spoilage and spoilage level [198]. 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). sardine. 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.4.190. 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. PV. haddock.193]. and lean fish such as blue whiting. and TBA values may increase. free amino acids.191].186].5 Microbiological Methods Numbers and types of microbes present in foods are important indicators of safety and quality. It is possible to predict shelf life of seafood based on knowledge of initial numbers and growth of SSO.185]. such as proteins.Methods for Freshness Quality and Deterioration ◾ 203 are anisidine value (AV). Mathematics models have been well established for the growth of spoilage bacteria such as Photobacterium phosphoreum. causing production of interaction products [187]. Shewanella putrefaciens . which break down to secondary products of oxidation or react with proteins. peptides. PV. Increase in the PV is most useful as an index of the earlier stages of oxidation. However. as oxidation proceeds the PV can start to fall. Cozzolino et al. European eel. coliforms.” or numbers of CFU of indicator organisms such as Enterobacteriaceae. FFAs and their oxidation products would have an effect on muscle texture and functionality. AV and TBA values measure the secondary products of lipid oxidation. [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.188. AV. since they interact with myofibrillar proteins and promote protein aggregation [189]. Spoilage of fish and fish products is a result of the production of off odors and flavors mainly caused by bacterial metabolites [197]. or enterococci [195]. Microbial assessments have been carried out to monitor the numbers of various groups of microorganisms during the production process as part of food safety objectives and also hazard analysis critical control point (HACCP) systems [196]. 13. since oxidation products are unstable and react with biological amino constituents. PV measures primary products of lipid oxidation. and thiobarbituric acid (TBA). TOTOX (2VP + AV). During prolonged storage of seafood. and phospholipids. reach a peak. Analysis of these interaction products by fluorescence detection as a quality assessment index for frozen-stored sardine was studied by Aubourg et al. It was indicated that the main requirements for shelf life predictions are to collect information about SSO. and also freshwater fish [194]. [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. there are some difficulties with common methods when quality has to be assessed. 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.

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

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

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

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

which is the most common analysis. chloroform extraction.3. If the sample must be preserved. GeneRelease (Bio Venture Inc. E. since enzymatic activity also takes place at subzero temperatures. we recommend preservation in 96% ethanol.11. and then be rehydrated in water or in the extraction buffer. the cells are opened (by heat treatment. For very long periods. One requisite condition for any genetic analysis is the obtention of good quality DNA suitable for PCR amplification. 14.7 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. give satisfactory results. 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. Samples fi xed in ethanol must be allowed to dry completely.18 or gel filtration. Each kit is provided with a detailed description of how to use it.9 resistance to diseases or to stress.Z. Genomics analyses are dealt with in more detail in Chapter 4 of this handbook.6 However. in particular if it has a high enzymatic activity (for example if it contains the hepatopancreas in a crustacean). Th is step is not necessary in samples preserved in ethanol. in contrast to the white opaque color of the wild.).3 Genetic Analysis Doyle et al.N. and others.Analytical Methods to Differentiate Farmed from Wild Seafood ◾ 217 a translucent grayish aspect. If this policy had been followed. treatment with Chelex. Dynal (Invitrogen). classification based on morphological criteria demands the presence of the morphologic diagnostic characters. The basic steps in all DNA extraction methods include the inactivation of nucleases.1 Sample Preservation and DNA Extraction Methods The sample should be extracted as soon as possible after sampling.11–13 and a loss of rare alleles has usually been observed in the farmed populations. and then recovered by ethanol or isopropanol precipitation. However. in most breeding programs the fish are indeed selected based on commercially interesting traits such as growth performance.8. it would be relatively difficult to find markers for wild and farmed fish. thus limiting its application. Then. sonication. a normal freezer (−20°C) may also be used.9 Genetic analyses have allowed the differentiation of wild from farmed fish populations in a variety of species. The DNA .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. so that all the ethanol is evaporated. for example by chelating divalent cations using EDTA and EGTA. Amersham Biosciences (GE Healthcare). Depending on the type of sample and its use. To extract frozen samples we recommend to start the procedure before the sample is completely thawed. which are usually absent in many intermediate products as well as in the ready-to-eat dish.14–16 Hayes et al. Nucleospin (Clontech). and the liver in farmed cod is much bigger than the liver of wild fish.).A Stool DNA Isolation Kit (United Bioinformatica Inc. such as Qiagen. 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. Many commercial kits. since diversity would be one of the selected traits in the farmed fish. the best method is to freeze it in liquid nitrogen or in a biofreezer. Wizard (Promega). 14.10 and optimal adaptation to different environments.

3. In the future. since some alleles are more frequent in one group than in the other. the alcohol is allowed to evaporate.15. tilapia. so the next step. that the Norwegian company GenoMar has patented a method to trace back farmed individual Atlantic salmon.24 The method requires that all parent fish of the brood stock are DNA typed as well as all the individuals under examination. . etc. China. tilapia.20. differences in the protein pattern of liver25 and muscle26. and sea bass. however. including the species.19 the Chelex method.0). whether microsatellites.27 tissues between farmed and wild salmon and cod have been reported. traits.23 In principle. In these samples the pellet may be practically invisible. However. such as Norway. shrimp. Three more methods that have reputedly produced good quality DNA suitable for amplification.4 Analysis of Proteins No clear protein marker has been identified to discriminate farmed from wild seafood. India. which is washing the pellet with 50% ethanol. personal communication).21 When using the salt extraction method with heavily degraded samples. by using a series of SNP and microsatellite polymorphisms by PCR and by oligonucleotide ligation assay (OLA). and production method.13. salmonids (salmon. 14. An additional advantage is that it is possible to use robots for many of the steps (DNA preparation. or mitochondrial. which may be used to identify the strains of the farmed individuals that display an increased frequency of the desired traits. 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. sample preparation. which further increases the amount of samples that can be processed. any marker. have started programs to map the whole genome of some species. breeding stock. 14. It is worth mentioning. pH 8. This method has been successfully used by the authors of this paper (unpublished results) and by Bucklin and Kochert22 with whole individuals of Calanus. and also for forensic studies. On other occasions. SNPs. cod. Atlantic halibut. including oysters.218 ◾ Handbook of Seafood and Seafood Products Analysis pellet is usually washed at least once with 70% ethanol. 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.18 and the salt extraction method.14 In addition. 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. and others.17 However. from food matrices include the use of hexadecyl–trimethyl ammonium bromide (CTAB). Arctic charr). and the DNA is reconstituted in double-distilled sterile water or in a slightly alkaline buffer (50 mm Tris–EDTA.). genomic. University of the Basque Country. protein markers commonly used for genetic analyses have the potential to be used as markers for farmed or wild. must be performed very carefully not to lose the sample. cod. has the potential to be useful to differentiate farmed from wild specimens of a given species. The outcome of these programs is already producing lists of genetic markers linked to traits of interest. the United States. trout. DNA analyses may be performed using chips that permit the determination in one fast step of many characteristics simultaneously. Japan.11. catfish. and bass. several countries.2 DNA Markers In recent years. Spain. however.

When this is not possible.4. several enzymes. have prompted the development of feed formulations based on vegetable oils and proteins. Fe. that is. −20°C .32 Unfortunately. and proteolysis) of the proteins in the sample should be chosen. attributed to the fish’s increased requirement for energy metabolism. which would be a natural diet. Zn. which is reflected in the composition of their organs. and this may induce stress in the farmed animals. The authors noted a downregulation of some structural proteins in fish-fed soy proteins. with no preservation at all. Soft texture.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. lectins. antigenic proteins. it has been shown that components in fish feeds may contain very high levels of metals (Cu.27 also registered the altered expression of five enzymes implicated in the glycolytic pathway and citric acid cycle in farmed cod. Thus.1 Sample Preservation The optimal case would be when the extraction of proteins can take place on the sample immediately after the experimental treatment. several enzymes involved in anabolic metabolism were downregulated in fish fed the diet rich in soybean meal. including heat shock proteins. Proteins and proteomic analyses are dealt with in more detail in a different chapter of this handbook.37 Martinez et al. Using proteomic analysis. and structural and FA-binding proteins. 14. and they require high levels of dietary protein (30%–60%). and feed diets based on plant protein require supplementation with synthetic amino acids. and the proteasome.26 examined the protein expression in skeletal muscle of farmed and wild cod by high-resolution twodimensional electrophoresis and found differences between the two.. Although feeds and breeding conditions need to be developed and optimized for each species. Some enzymatic systems that may be responsible for the muscle softening are metalloproteases and collagenases. Johnston et al. Interestingly. a preservation procedure that minimizes the modifications (denaturation. usually considered negative. which were attributed to increased proteolytic activity in the muscle of the farmed compared with the wild cod. neutral calcium-activated calpains.25–27 In addition. the amino acid profiles of plant proteins do not meet the essential amino acid requirements of fish. it is common to apply directly to new species those conditions that have proven successful for other species. loss of functional groups. and then modify them depending on the results. Optimal methods include fast freezing and frozen storage using temperatures as low as possible.33 Moreover. For short periods of time. 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.36 Martin et al. which they use as their preferred energy source. neither feeds nor breeding conditions may be optimal for farming. is more common in stressed and in farmed than in wild fish. and Ca)34 and that vegetable meals may contain antinutritional factors (protease inhibitors. Olsson et al. these authors identified 33 differentially expressed proteins. lysosomal cathepsins.33.38 found that the reason for the softening in this species did not seem to be the faster growth of the farmed fish. Thus. 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. aggregation. Texture is an important quality attribute of the fish flesh.)29 that may have adverse effects on fish. However. and they hypothesized that the greater concentration of insoluble collagen present in wild salmon may contribute to their firmer texture. indicating increased emphases on catabolism relative to anabolism in the fish fed this diet.Analytical Methods to Differentiate Farmed from Wild Seafood ◾ 219 Industrial fish farming is a relatively new activity compared with farming of land animals. Mg.35. etc.28–31 The source of protein in teleost fish is very important. In addition. the depletion of the wild stocks of pelagic fish and the high price of feeds based on fish meal and oil.

The choice of method depends on the protein and the property one wishes to examine. etc. The use of protease inhibitors should always be considered: use of some inhibitors and cocktails may help to preserve the sample during the extraction procedure. and digested. its application is widespread in many fields. alkylated. usually 8%–20% or 12% PAGE. The optimal pH range to choose depends on the sample. there are many methods suitable for protein analyses. In our experience. but not all protocols are compatible with MS). Both first and second-dimension gels can be purchased as precast. silver (high sensitivity. therefore. 14. tributylphosphine) to solubilize the widest possible spectrum of proteins. ready to use gels from several companies. Proteomic techniques have a clear advantage in this field. dithiothreitol (DTT). We therefore recommend not to use such enzymes. It should be noted that any preservation procedure will alter the protein profile in the sample. After separation. Triton X-100.4. but 3–10 are commonly used for wide screenings. The first step in proteomic analyses is to extract as many proteins as possible from the sample. 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. Since current studies are still trying to identify markers. 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. we focus on the use of techniques with the potential to identify such markers. as well as the different degrees of processing to which the sample may have been submitted (freezing. Afterward. thiourea).25–27 BioRad39 and GE Healthcare Amersham have some excellent manuals about protein extraction and analysis.). the strip containing the proteins separated by their pI is loaded on top of the second-dimension SDS-PAGE gel. It is common to use several buffers with increasing concentration of chaotropic agents (urea. usually with trypsin. detergents (CHAPS. reduced. due to the great diversity and properties of the proteins contained in the edible tissues of seafood. However. Their use should be evaluated for each particular study. destained.4. and. one should be very careful when comparing samples preserved and stored under different conditions. 14.3 Analysis of Proteins As already mentioned. but they will hamper the study of protease activities that may be relevant in some other works. and. and reducing agents (b-mercaptoethanol. depending on the proteins one wishes to examine. and peptide mass fingerprinting of the digests is then . SDS). cooking. and there are special protocols for each application.2 Protein Extraction There are many methods for extraction of proteins. this may be because some commercial preparations of these enzymes are contaminated with proteases.220 ◾ Handbook of Seafood and Seafood Products Analysis may be acceptable. the gels can be stained by Coomassie Blue (low sensitivity but compatible with mass spectrometry (MS) analysis necessary for subsequent peptide fingerprinting and sequencing). The tryptic fragments are cleaned from contaminants. Proteomics permits the separation of many proteins (often thousands) from a complex protein mixture in one step. the optimal extraction procedure for any given sample must be determined empirically. In addition to published works. for a wide screening. therefore. Some authors have claimed that the use of DNase I and RNase in the extraction buffer increases the number of spots in the gels. and fluorescent labeling or staining (of intermediate sensibility and also compatible with MS).

and sardines than in capelin.42. there are more FAs present in detectable amounts. In Atlantic salmon for example. the total amount of TGs alone may be used as a criterion to differentiate farmed from wild fish. however.44 and this FA fingerprint has often been successfully used49–52 as a diagnostic to identify the production method. has often given correct classification of farmed and wild specimens.25 and reviewed by Granvogl et al. NCBI) using suitable software (MASCOT.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.).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. As in vegetable oils.55–58 Specific FAs are selectively retained or used. Development of protein chips will facilitate the simultaneous screening of many targets and samples. and it is seldom that only one of them makes more than 25% of the total.5 Analysis of the Lipid Content The analysis of the triglyceride (TG) fraction. etc. The FA profile of vegetable oils (such as corn.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. cottonseed. whereas C18:1n9. This is probably the most time consuming step of the whole procedure. rapeseed. C16:0 and C18:1n9 are relatively abundant in all fish oils. The FA composition of fish oils is more complex.). the FA profile of TGs reflects that of the feed. lateral flow strip tests permit in situ easy and fast screening of seafood samples. 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. olive. but C22:1 (several isomers) is relatively more abundant in Coho salmon. herring. Proteomic analysis is a complicated procedure necessary to identify the biological markers. palm. However. The proteins are afterward identified by searching in databases (National Centre for Biotechnology Information. The whole procedure is described in detail by Martin et al. etc.44 Frequently. ProteinLynx Global SERVER). and often a single FA may account for about 50% of the total FA content in these oils. in control laboratories. it has been shown that there was selective deposition and retention of C22:6n3. the final identification and assignment of the spots in the gels must be performed visually by trained personnel. which uses MS data to identify proteins from primary sequence databases. Once the diagnostic proteins are identified. so that the concentrations in flesh were higher than in the diet. which FA is more abundant is species dependant. C22:1n11. and C18:3n3 were selectively metabolized. 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. that is. spot cutters. due to frequent errors in the automated spot identification procedure (because of imperfect spot separation and identification caused by overlapping of spots.60 The FAs C18:1n9 and 18:2n6 may act as markers . and ELISA format will permit the routine analyses of many samples. sand eel. and sand eel and C22:6n3 is more abundant (over 10%) in Atlantic and Coho salmon. C18:2n6. capelin. soybean. identification of diagnostic spots. or menhaden. For example.Analytical Methods to Differentiate Farmed from Wild Seafood ◾ 221 usually performed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS.53. 14.45–48 In addition. linseed. because farmed fish usually have a much higher content than wild ones. very different staining intensities. in particular when combined with stable isotope composition (see the following paragraph). for example. herring.

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

Regions without signals or unwanted signals are removed before multivariate analysis.66. Tetramethylsilane (TMS) is usually added as a chemical shift and intensity reference. which is easily evaporated. inhomogeneities in the applied magnetic field.67 1H NMR has been used to perform quantitative measurements of total n-3 FAs and of the levels of DHA. a sample size of 50–100 mg of lipid in 0. When full spectra are used. in conjunction with chemometrics.75 Small differences in experimental conditions. or differences in relative concentrations of the samples analyzed. pH.68. have allowed the differentiation between wild and farmed salmon74 and cod51 of different origins.77 Regarding reproducibility issues. may lead to erroneous classification. but it is not necessary for classification purposes.78 . the chemical shift scale is referred to the shift of TMS or indirectly to TMS by the peaks from chloroform at 7.28 ppm for 1H NMR and by the triplet of CDCl3 at 77. due to the fact that quantitative measurements require a considerable longer experimental time. interactions with metal ions.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. phasing and baseline correction are applied but no zero fi lling.66 Potential problems about inconsistencies in ppm values between samples in the data analyses should be solved by manual alignment or data pretreatment methods. ideally for screening many samples with short acquisition time. temperature variations. It is expected that in the future the use of flow injection systems.71 13C NMR gives information about FA composition of fish72 and the positional distribution of PUFAs in triacylglycerols and phospholipids. even though the signal intensities within each spectrum are not quantitative.76 In some studies. and other intermolecular interactions. The assignment of spectral resonances gives information about the chemical composition of the samples. a semiquantitative 13C NMR approach has been chosen.70. However. although the optimal sample size depends on the instrument.5–0. 13C NMR and 1H NMR spectra are fi rst obtained by Fourier transformation of the resulting free-induction decay (FID) function after applying a prospective line-broadening function. the whole procedure from sample preparation to analysis by a data exploration technique can be affected by factors unrelated to the sample characteristic of interest.e.8% CDCl3). Factors that affect the exact chemical shift of NMR signals include the type of solvent used. although this approach has still not been widely used for authentication purposes.77 It is advisable to check that all spectra have acceptable linewidth and lineshape after the NMR analysis.Analytical Methods to Differentiate Farmed from Wild Seafood ◾ 223 spectra is the goal.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). leaving the sample ready for analysis.0 ppm for 13C NMR. they are normally converted to ASCII or JCAMP file formats..73 which is of value for authentication purposes. Both HR 1H and 13C NMR. because it may interfere with the multivariate data analysis. Typically. hydrogen bondings. Typically. The most commonly used solvent in the analysis of neutral lipids is deuterated chloroform (i. The application of multivariate statistics to NMR spectral data increases the potential of the technique considerably. Normally. 99. Both the area/ intensities of peaks and full spectra can be input for multivariate analysis. will increase the sample throughput significantly.8 mL solvent is used. Multivariate methods are frequently applied to study differences among NMR spectra. 1H NMR has also been applied to differentiate between wild and farmed salmon and sea bream of different origins. the relative intensities for corresponding signals across different spectra are comparable. such as instabilities in apparatus. Standardized procedures should be followed to ensure repeatability and comparability.75 and it is important that all the samples contain the same volume.

the abundance of stable isotopes varies among different compounds. SD ± 0. C18:1n-9.51.82 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. N2O and CH4 exhibit wide isotopic variation.89%) and 13C (1.759%).63) and 15N (0. because the enzymatic reaction rates on substrates that contain the lighter isotopic forms are faster than in reactions involving the heavier isotopic forms. For example. Some atmospheric gases. resulting in a complete separation of the two groups. the higher the proportion of the heavier isotope. Usually animal products become enriched in the heavier isotope (15N and 13C). C16:1n-9.11%). and they reflect both significant isotopic fractionation by microbes and the different biological substrates producing these gases. they were also able to correctly classify the fish according to their geographic origin. Introducing the percentage of C18:2n6 as a third variable in their model.46 were equally successful classifying sea bass using the FA profile. Aursand et al. 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. Thomas et al. nitrogen. 17O (0. and oxygen has been proposed as a method suitable for food authentication. Bell et al. since the physical properties of molecules containing heavier isotopic forms are different.224 ◾ Handbook of Seafood and Seafood Products Analysis 14. while typical d13C mean values of C3 plants may be −26/−28‰. depending on their diet and their position in the trophic chain: the higher its position in the trophic chain. plus the kinetic fractionations occurring in animal metabolism. N2. 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). exhibit limited variation. 171 Atlantic salmon specimens originating from three continents and 15 different geographic regions.6 Stable Isotopes The variation in the abundance of the stable isotopes of carbon. and 18O (0. and O2. and oxygen as three: 16O (99. In contrast. The isotopic abundances in animal tissues and animal food products are the summation of the feeds ingested throughout all their life. such as maize. such as CO2. although many broadleaf plants are also C4). In addition. d13C of individual FA. nitrogen as two: 14N (99. SD ± 0. typically tropical grasses.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 13C/12C ratio for both milk fat and cheese protein give information on the type of forage fed to the cows. . equilibrium reactions also lead to a fractionation of the isotopic forms. Differences in the 15N/14N ratio also result essentially from diet. For example.50 were also able to correctly classify Atlantic salmon according to their geographic origin and production method by using four FA compositions (C16:0.79 Carbon exists as two stable isotopes: 12C (abundance 98.204%). 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 CO2.38‰) but depleted in lipid-corrected carbon (d13C: mean = −20.37%). Moreover. Samples of muscle tissue of wild salmon were significantly more enriched in nitrogen (d15N: mean = 12. Using the d15N of choline and the d18O of total oil. Since a molecule containing heavier isotopic forms has stronger chemical bonds.52 were able to classify correctly according to the production method.80 The natural isotopic abundance largely varies depending on the chemical forms.23‰) than the aquaculture specimens. the abundances of the stable isotopes differ between substrate and product.037%). a kinetic fractionation occurs.75. C4 plants may have d13C mean values of −12/−14‰.

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

lead. handling. multiple elemental analysis could also be used in this case to identify imported clams from China and Korea. attributable to cobalt. Multivariate analysis showed that differences in elemental composition in the muscle between Japanese and imported clams were mainly due to two factors: Factor 1. Multivariate trace elemental analysis is increasingly used as a technique to differentiate the geographic origins of foodstuff. and they found distinct patterns for each of the three origins. The sample may be stored in a centrifuge tube at a temperature of −40°C or lower until analyzed. in particular since the analysis may detect contaminants at the pM level. cadmium. All implements and containers should be cleaned with 0.. it is very important to avoid contaminating the sample during sampling. otolith chemistry is used as a recorder of time and environmental conditions. such as uranium.1 Sample Preservation As for stable isotope analysis. quality. multivariate trace elemental analysis is expected to be helpful in determining whether the fish was farmed and its geographic distribution. farmed and wild specimens of the same species have different geographic distributions. 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. Each sample should be separated from the tissues using ceramic knives and scissors and Teflon-coated tweezers to avoid contamination of metals. were shown to be of relevance to determine the origin of eels. copper. Korea. and China.95 By using ICP-MS analysis the sensitivity in the determination of rare trace elements can be increased from the nM to pM level. and vanadium. have been used to differentiate the geographical distribution of origin of farmed Japanese eel. and it must be accurately weighed with a microbalance. and price. and analysis. Taiwan. 14. The . Thus. unpublished data) examined the trace element composition of the muscle and shell of littleneck clams collected in Japan. Recently. 14. and strontium levels and Factor 3. The first step in the analysis is the digestion of the sample: 0. as well as vitamin K and its metabolites.92–94 Otolith chemistry is useful for identifying the natal origin and assessing the relative contribution of different nursery areas to mixed adult stocks. false labeling problems were encountered in which imported live Japanese eels from Taiwan were illegally sold as being of Japanese origin. respectively. The origins of farmed and wild eel collected from different regions in Japan. Rare trace elements taken from the environment. Therefore. Thirteen elements were shown to be the most diagnostic. The same research group (Yamashita et al. cadmium and arsenic levels in the muscles of clams from China and Korea were higher than those of clams from Japan.5 M nitric acid and rinsed with Milli-Q ultrapure deionized water. Biochemical analytical techniques using multiple elemental analysis.1–1 g of tissue samples are placed into 50 mL Teflon tubes and 8–16 sample volumes of a mixture of concentrated trace-metal-grade nitric acid/hydroperoxide mixture (5:3) is added.7 Trace Element Fingerprint Sometimes. in addition to DNA-based species identification techniques. and often the geographic origin of both farmed and wild seafood may be of relevance for its safety.91 In the case of fish.226 ◾ Handbook of Seafood and Seafood Products Analysis set at 0‰ by convention.7. attributable to manganese and vanadium levels. Carbonate rock from the Pee Dee Belemnite formation89 and nitrogen gas in the atmosphere90 are used as the standards for carbon and nitrogen. with the exception that clams from Miyagi had high arsenic content. In addition.

but although the diet of wild salmon contains astaxanthin.98 making this approach more unreliable than it used to be. Canada). which suffers from severe memory effects. where energy transfer processes cause desolvation. the samples are diluted to a final volume of 50 mL in Digitube (SCP Science. an internal standard mixture is added. Ions transmitted through the quadrupole are detected by continuous dynode electron multiplier assembly. Afterwards. Each solution (5 mL) of the microwave samples is applied to the atomic absorption spectrophotometer. 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. a calibration blank and calibration standards are used as surrogate test samples after every 10 analyses. 14.97 using an automatic mercury analyzer (Hiranuma HG-200. and stored at room temperature until use. the total mercury concentration is determined by cold vapor atomic absorption spectrometry. five internal standards are used: Sc. there might be specific requirements that may be targeted to identify the production method. 14. In the farming of salmon for example. For internal standardization. Tb. the mass calibration and resolution are checked using diluted metal solutions as standards. Y. To verify that the instrument is properly calibrated on a continuous basis. the Norwegian Research . To initiate the proper operating configuration of the instrument and data system. Analysis by chiral chromatography can be used to identify a chiral form (the meso form 3R. Japan). In. If the measured concentration deviates from the true concentration by more than 10%. and the resulting digest is a clear liquid with a yellow tint.8 Other Methods Depending on the species. and ionization.3′S) that does not occur naturally and can therefore be used as a marker for farmed salmon.7. and Bi. For the determination of mercury. and the ion information is processed by a data handling system. much of the astaxanthin used in fish feed nowadays is produced from cultured microalgae or from krill. the use of carotenoids is allowed.98 However. atomization. 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.2 ICP-MS Multielement determination of trace elements is usually measured by inductively coupled plasma mass spectroscopy (ICP-MS).96 Samples digested as described above are introduced by pneumatic nebulization into a radio frequency plasma. the instrument is recalibrated. and the last 10 samples are analyzed again.42.Analytical Methods to Differentiate Farmed from Wild Seafood ◾ 227 digestion may be carried out by placing the tubes in a microwave oven (for example. most artificial feeds contain a mixture of canthaxanthin and astaxanthins of different origins (both natural and synthetic). Multiwave 3000 Microwave Oven. Perkin-Elmer).

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

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

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

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

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

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

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

Wood granulometry can also influence SF composition. An optimal moisture is planned in the industry between 17% and 20%. whereas a rate between 20% and 30% has been reported as optimal to reduce the emission of particules [11]. Due to their . The generation of volatile compounds is dependent on the wood pyrolysis temperature [16]. Then. differences can be observed depending on the molecules. furannic compounds. The high moisture allows to reduce the wood combustion efficiency.18. lignin dimers. the diversity of settings of pyrolysis parameters can explain the diversity of organoleptic volatile compounds and the diversity of qualities of SF.22]. and 400°C for lignin [17]. the velocity. For example. known as the smoky skeleton of SF. phenol amount is multiplied by two between 450°C and 650°C. and phenolic compounds [10.19]. and the enolones derivatives [14]. between 280°C and 320°C for cellulose. the furannic derivatives. steps of SF purification through filters or apolar solvent washes are often required to decrease the PAH levels. According to the pyrolysis process. and trimers [12]. exothermic reactions of pyrolysis of wood components occur between 200°C and 250°C for hemicellulose. Lower concentrations of oxygenated compounds have been found to be caused by an oxygen depletion during combustion [20]. The combustion is faster when the granulometry of the wood raw material is important [23. whereas syringol quantity is tripled.240 ◾ Handbook of Seafood and Seafood Products Analysis ethers from lignans. a lower temperature is reached and allows increasing the generation of smoke volatile compounds and minimizing PAH formation. the quantity of phenolic compounds increases with a maximum close to 500°C and decreases after 500°C. The manufacturer can choose SF according to the required result on the organoleptic characteristics of the final product. The use of hardwood. Indeed. it seems difficult to generate the desired organoleptic volatile compounds without PAH contaminants. The wood moisture appears as the second important parameter [20]. the pyrolysis temperature. The rate of acids is higher for temperature lower than 300°C and decreases after 300°C with the increase in temperature. However. Therefore. A slow combustion is reached with weak air velocity.24]. the wood granulometry and moisture. because it plays a role in the pyrolysis temperature. Therefore. The air velocity indirectly influences the SF composition by the modification of pyrolysis temperature or smoke temperature [21. A temperature of 450°C–500°C was reported to lead to the best composition for the creation of carbonyls. From 200°C to 600°C. As the best pyrolysis temperature to obtain the required volatile compounds are between 380°C and 500°C.5 Role of SF Process Parameters in Volatile Compounds Generation Except the wood type that influences the smoke quality strongly [15]. 15. the main organoleptically active volatile compounds generated during the pyrolysis process can be sorted in three groups of molecules: the phenolic compounds. but they have a weak impact on the smoky flavor of SF and food processed with SF [13]. 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. because their contents in smoke or in food increase from 400°C to 1000°C. A step of filtration is almost obligatory to avoid these contaminants. PAH must also be surveyed. After the water release (close to 120°C–150°C). with a lower water rate than that in softwood. different groups of compounds are formed. is recommended because it burns slower. and humidity of air constitute key parameters of SF composition. The rate of carbonyl compounds increases gradually with the temperature from 200°C to 600°C.

34] (Table 15. color. Table 15. They are the major compounds in SF with a wide range of odorant thresholds (Table 15.33. 15.1) [14.2). taste. cresols. Many studies have indicated that phenolic compounds present in the vapor phase of smoke may be odor-active compounds [30–32].Smoke Flavoring Technology in Seafood ◾ 241 chemical composition. A much more complex mixture of compounds is responsible for the characteristic aroma of smoked fishes [35]. Some of them have very low odorant thresholds. but it may not be the main contributor to wood smoke flavor. odor. and alkylguaiacol may also contribute to imparting a smoky flavor to smoked fishes [13.6.28. The medium-boiling fraction (91°C–132°C) composed of isoeugenols.1 Role of Volatile Compounds of SF in the Odor Even if the concentrations of odorant volatile compounds in SF can be various. which gather furannic and enolone derivatives. These observations have been recently corrected [14. 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. and methylsyringol has a pure and characteristic smoky flavor [10].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] . and preservation of the product.29]. The role of syringol is important. Phenolic compounds of low-boiling fraction (60°C–90°C) composed mainly of phenol.6 Organoleptic Roles of Volatile Compounds of SF 15. phenolic compounds are not sufficient to explain the SF role in smoked fish odors.34]. two main classes of odor-impact molecules can be defined: phenolic compounds and carbonyl compounds. syringol. making them odorant at low concentrations. Phenolic compounds of medium volatility have been considered as the most important odorant molecules. guaiacol. Similarly.

18 ± 37. spicy Spicy. LRI MS. STD Cooked. wood fire. STD MS. vanilla. ink Marine.22 ± 9.3-Dimethyl-2cyclopentenone 1052 o-Cresol 1068 p-Cresol 1093 Guaiacol 1110 2.55 ± 39.64 ± 18. STD MS. 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 2.45 ± 172.4-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 2.53 360. STD MS.25 6 (5) 6 7 (4) 7 7 6 8 8 8 Chemical.33 ± 1. mushroom Cooked.07 (1. STD MS. green Cooked vegetable.62 74. LRI. LRI.6-Dimethylphenol 1130 . burnt Smoked.37 ± 3.Table 15. STD MS.53) 8. metallic. STD MS. chemical Green. milk Smoke. burnt. LRI. LRI. LRI. LRI MS.48 ± 8.50) 65. STD MS. LRI MS.12 42.94 49. LRI. LRI. green.34 (24. LRI.55 ± 9. potato Cooked potato. chemical. LRI. spicy.44 17. Animal. earthy. burnt.98 20.24 ± 63.75) MS. STD MS. fatty Roasty.27 ± 2.27 ± 0. LRI. spicy/ woody (3) (2) 5 6 (2) 4 4 3 5 6 7 (2) 4 4.6-Dimethylpyridine 890 2.04 7 4 16. LRI MS.90 (1. milk Cooked/soup. spicy.63 ± 13.17 ± 26. STD MS.74 ± 27.97 23. LRI. green Cooked vegetable.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. roasty Chemical. STD MS.

3. clove Green. chemical Green. spicy Oily.4.4-Decadienal 1330 Syringol 1365 Eugenol 1370 Smoke Flavoring Technology in Seafood 4-Propylguaiacol 1382 1400 ◾ 1. green. smoked Cooked vegetable. medicinal 7 4 10.5-Dimethoxytoluene 1282 4-Ethylguaiacol 1287 Indanone 1307 4-Vinylguaiacol 1330 (E.13 6. STD MS.72 44.21 ± 7. LRI.15) (continued) 2. green Spicy. LRI. LRI MS. LRI MS. vanilla Cooked. vanilla.25 ± 10. smoked Candy.85 ± 40.16 ± 5.4Trimethylcyclopenten-1one MS MS.50 18. LRI MS. LRI.51 ± 18. green 6 3 11. 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. milk Burnt.62 ± 4. spicy 6 5 17.86 (2.2-Dimethoxybenzene 1147 2. LRI. green.49 ± 6. violet. smoke.E)-2. spicy.35 3-Ethyl-2-hydroxy-2cyclopentenone 1140 1. STD Cucumber. LRI MS.82 ± 6.15) 86. LRI.5Dimethylphenol/ (E)-2-nonenal MS.26 ± 1.17 15. rotten. green. smoke.61 ± 22.97 2. spicy Spicy.3-Dimethoxytoluene 1247 (E)-2-Decenal 1266 3.12 4.3-Trimethoxy-5methylbenzene 243 .95) 8. fatty. green. LRI MS.87 ± 1. STD MS.96 ± 10.71 (3.and 2.15 ± 243. STD MS.36 1132 MS Cooked. fatty Burnt rubber.91 36. burnt Smoke. spicy. STD MS.25 ± 4. STD MS. LRI.24 ± 1.2.15 ± 1. LRI. spicy.2.79 (6. clove Sawdust. LRI 1160–1180 4-Methylguaiacol 1192 482. green.46 Solvent. STD MS. STD MS. LRI.

LRI MS. green. Average intensity of the eight judges is rounded to the nearest whole number. and odor description of an identified compound correspond to the retention time. Food Chem. spectrum.48) 1.40 ± 3. 55. spectrum. STD MS..81 ± 11.39 6. LRI. roasty. LRI. and quantities of odor-active compounds detected by fewer than six judges are indicated in parenthesis. J.5-Trimethoxytoluene 1527 4-Allylsyringol 1615 8-Heptadecene 1680 Source: Varlet. Note: Frequency of detection. V.5 is rounded to 5 and an intensity between 5. .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. standard (when the retention time. STD.68 MS. spicy 6 3 Odorant Attributes Given by the Judgesb Number of Judgesc Average Intensityd 244 ◾ Compounds LRI (DB5) (Z)-Isoeugenol 1423 (E)-Isoeugenol 1473 2. a b c Handbook of Seafood and Seafood Products Analysis d e Means of identification: MS. chemical 6 4 Smoke. LRI MS. woody (4) (2) Clove. linear retention index (when the LRI of the identified compound corresponds to the LRI in the literature). 9 = very strong odor intensity). 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. An intensity between 5 and 5.23 ± 0.35 (20.. LRI Animal. LRI. Means of three fillets.87 ± 2. When only MS is available for identification. rotten 7 4 Spicy. Number of judges (out of eight) who have detected an odor. In micrograms equivalents of dodecane per 100 g of smoked salmon. roasty 7 4 Burnt rubber. Agric.Table 15. 4518. it must be considered as an attempt of identification.77 24.55 ± 8. and odor description of the injected standard of this compound).5 and 6 is rounded to 6 (1 = very weak odor. 2007. mass spectrum (identified using the mass spectra of the compounds). et al.3. odor intensity.

However. or oils. Enolone derivatives are compounds derived from cyclopentenone. 4-methylguaiacol. More recently. it was commonly admitted that syringol derivatives impart a smoky odor and guaiacol derivatives contribute to a smoky flavor. 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. the determination of the effects of compounds of SF on the flavor is complex. whereas syringol and 4-methylguaiacol showed the same but lower effect than guaiacol [40]. 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. A polyfunctional carbonyl subfraction was isolated from wood smoke and possessed a caramellic/burnt sugar aromatic note [36]. furannic compounds were found to play a role in cold smoke odors of liquid smoke or fishes treated by liquid smoke [14.2 Role of Volatile Compounds of SF in the Flavor Phenolic compounds were shown as the major contributors of the smoky flavor [35]. The fractionation of a commercial liquid smoke preparation evaluated by a sensory panel concluded that the phenol fraction was essential but not complete from a sensory standpoint [42]. and seem to contribute little to overall aroma. The taste thresholds of some phenolic compounds were determined [40] and showed a high diversity between the molecules. and the odorant contribution of odor-active compounds cannot be the same if the SF is in the form of powders. Furfural and homologues exhibit cooked/roasty aromatic notes. sometimes cooked.Smoke Flavoring Technology in Seafood ◾ 245 Carbonyl compounds have also been reported as contributors to the smoky aroma of wood smoke. the same compounds responsible for the odor should be involved in the flavor that SF confers to seafood.3.6.34].39]. they may contribute in mixture to the overall odor. reactions between liquid smoke compounds and the components of the matrix can occur through Maillard and Strecker reactions. Synergic or masking effects are possible and make the final odor complex.38]. . 4-methylguaiacol perception was superior to that of syringol and guaiacol. Furthermore. early works performed on individual phenolic compounds have identified the impact of guaiacol on the smoky flavor. 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. Two categories of carbonyl compounds can be differentiated: furannic compounds and enolone derivatives. Then. However. as the other minor odor-active compounds. Concerning the bitter taste. phenolic compounds are not the only flavor-active compounds. and isoeugenol in spicy/sweet flavor [41]. Moreover. The results of this fractionation are given in Table 15. The oils used in smoke oils can soften the smoke aroma. They were isolated early from wood smoke and described as grassy. but taste was not investigated as much as odor. Recently. because they are not the compounds mainly detected in SF and seafood treated by SF odors by sensory analysis [37]. and very little information is available. For several decades. 15. liquids. Sensory analysis performed on standards confirmed the importance of guaiacol and o-cresol in the smoky flavor and dimethylphenol. As for the assessment of the odor. Furannic compounds were thought to contribute to soften the heavy smoky aromas associated with phenolic compounds [37. However. the physical state of SF can also influence the aroma. if they do not have a strong individual influence. guaiacol derivatives and more generally the phenolic compounds of low-boiling fraction molecules (60°C–90°C) have been shown to cause the odor.

because they are added in the product during its fabrication. smoke condensates are colored mainly due to phenolic compounds. the eventual dilutions of SF. 5. distilled at 67°C–90°C. However. phenolic subfraction. The acidic aqueous SF can also increase the coagulation of proteins and act on the texture. can react with proteins. histidine. 15. furannic compounds could also have an effect on the flavor [43]. which has been reported in wood smoke and smoked meat [41] but not in smoked fishes until now. However. they could play a role in the inner texture of the product. distilled at 133°C– 200°C. and the intensity of the process. the product must also be placed in a dry and hot ambient atmosphere for short periods in order to favor color formation. whole liquid smoke. terpene subfraction. In the liquid smoking process. Studies on standards have shown that cyclotene was a flavor-active compound [41]. 6. 1. Thus. which can vary from golden yellow to dark brown according to the nature of the wood. Maillard and Strecker compounds can also be responsible of the color of the smoked product [45].3 Role of Volatile Compounds of SF in the Texture The texture of smoked products is due to coagulation of proteins. the deposition of Maillard compounds leads to a darker color of fish flesh [46]. 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]. These . Formaldehyde. A brief drying after smoke absorption can cause a higher level of dehydration and lead to higher amounts of Maillard products. Formaldehyde was shown to react with the amino group of the N-terminal amino acid residue and the side chains of arginine. Their physical deposition of SF on seafood can confer its color to the product. After scission and dehydration. 15. Other compounds such as enolone derivatives could also play a role in the SF flavor. cysteine. According to their concentrations found in the different SF. 4.6. melanoidines could be created by polymerization through aldolic condensations. 3.6.4 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.246 ◾ Handbook of Seafood and Seafood Products Analysis Table 15. 2. which have brown/yellow characteristic color. SFs under powder or oil forms do not act on the surface texture. Indeed. distilled at 91°C–132°C.3 Sensory Taste Intensities of Liquid Smoke Fractions Fractionb Taste Property a 1 6 3 1 1 2 7 1 1 2 3 3 2 3 3 4 11 0 0 0 5 4 6 1 0 6 10 1 0 0 Smoke taste intensity Tarry taste intensity Chemical taste intensity Acidulous taste intensity a b Intensity scale: 0 = below threshold. and lysine residues [44]. 11 = highest value.

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

PAHs are formed by the incomplete burning of carbon-containing material. especially benzo[a]pyrene (B[a]P) [59]. As they can be absorbed by animals. 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 7.8-dihydrodiol-9.8 15.8. PAHs are considered as carcinogenic contaminants of processed food [56].8 epoxyde OH B[a]P 7. PAHs are generated during smoke production by wood pyrolysis. Owing to their lipophilic properties (log Kow between 4 and 7). it is used as the leading substance to illustrate PAH contamination. . 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. These compounds have been studied for several years. particularly smoked food [57.58]. However. In SF. 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. they are considered as heavy PAHs. They are considered as carcinogenic contaminants. more toxic than light PAHs.3). Therefore. home cooking and industrial food processes represent the major source of human contamination [55]. they are considered as environmental pollutants and can contaminate the human feed raw material [53.8-catechol O OH S O COOH O O OH OH OH OH OH O B[a]P sulfo-conjugate B[a]P glucuronide Detoxification products Figure 15. PAHs comprise fused aromatic rings made up of carbon and hydrogen atoms: up to four fused benzene rings. because their catabolism leads to poly-hydroxy-epoxy-PAH suitable for binding to DNA adducts.3 Benzo[a]pyrene metabolization.54]. hence their toxicity (Figure 15.10-epoxyde Glutathion S OH OH OH O B[a]P 7. Therefore. which are constituted by less than four benzene rings.248 ◾ Handbook of Seafood and Seafood Products Analysis 15.1 Polycyclic Aromatic Hydrocarbons Properties and Toxicology PAHs are well known as being food contaminants and carcinogens [52]. PAHs can cross the biological membranes and accumulate in tissues.

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

whereas food is treated by SF and not directly by PP. 0. the PAH contamination reached in SF is largely below the values authorized in PP [60. This value is very low compared to those authorized in PP. it is necessary to better control the composition of SFs and to improve knowledge about the influence of the pyrolysis parameters (wood nature. this process can also be considered as a flavoring of the surface of the product. SFs appear as a safe alternative to smoking techniques. important differences in PAH concentrations are noticeable [57. SFs are used in higher quantities than those employed in flavoring processes. Finally. Indeed.69. 15.9. Moreover.75]. The high maximum values authorized in PP do not seem well adjusted with the weak final PAH contamination of SF. Indeed.250 ◾ Handbook of Seafood and Seafood Products Analysis However. As for SF. the PAH contamination was only set for B[a]P [77]. However. the liquid smoking process decreases the emissions of PAH compounds to the environment. 15. it is legitimate to wonder if the exclusive monitoring of the B[a]A and B[a]P in PP is adequate to illustrate the PAH contamination of SF. atomization of liquid smoke must be lower than 0. Indeed. it can lead to problems of labeling. Therefore. Thus. leading to less PAH contaminated food by comparison with the traditional smoking techniques [80]. Th is fact can be understood by the use of smoke condensates in flavoring quantities. All these benefits could help to reconsider the status of atomization of liquid smoke and the maximum PAH contents related. the toxicity of other heavy PAHs was recently demonstrated and the monitoring of these PAHs was recommended by a European regulation published in 2005 [76].2 European Regulations on PAH Concentration in Food Treated by SF In 1988. as drenching or showering. The main criticism that can be formulated against SF is the lack of control of the final organoleptic qualities of such processed food.78]. the smoking regulations set a maximum B[a]P value of 5 mg/kg of smoked fishery products and smoked crustaceans. . Therefore. and head and thorax meat of lobster and similar large crustaceans [76. Moreover. However. the 2003 maximum values must be reviewed again. it is paradoxical to apply flavoring regulations to the smoking process. if it is considered as smoking technique. because these values were set for PP and not SF.10 Conclusion A wide range of SFs and uses of SFs are now available to flavor seafood products. but it could also initiate an international consideration of labeling of smoked and flavored food. In this case. a European regulation set the maximum content of B[a]P in foodstuffs treated by SF at 0. the vaporization of SF in a smokehouse causes a loophole in the legislation. This value is the result of the necessary harmonization between the national laws of European countries [79].03 mg/kg and leads often to noncompliant smoked products. atomization of liquid smoke would constitute the smoking technique. The content of PAH in SFs and in the final product can be better controlled than during traditional smoking.03 mg/kg. excluding bivalve molluscs. Nevertheless. it leads to lower PAH contents. that is. In certain countries such as France. according to the origin of SF and industrial manufacturers. Moreover. the respective legal B[a]P amount is 5 mg/kg.63] which justifies controls and regulations.03 mg/kg. However. atomization of liquid smoke in a smokehouse is considered as a smoking process but the maximum level of PAH must not overcome that of flavoring legislation. Therefore. for meat industry. brown meat of crab. that is. very small amounts. Indeed.

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

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

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

making it possible to measure over the whole NIR spectrum and not only on a small number of selected filters. Diff use transmittance measurements are usually carried out in the 800–1100 nm region of the spectrum.258 ◾ Handbook of Seafood and Seafood Products Analysis 16. The development of NIR in food analysis started with the development of analysis of cereal grains and oilseeds in Canada [2]. the range 1100–2500 nm. and minerals [1]. and sizes. which involves concentrated sulfuric acid and heavy metal catalysts.org). to ensure obtaining data on the exact proximate composition. geographical locations. but the available detectors cover a smaller range.7 deals with the different methods to determine and calculate calories in fish and shellfish. The end result is a calibration equation from which the constituent of interest is calculated from a linear combination of spectral data.5 m per year and a saving for the environment by replacing the Kjeldahl system. stages of maturity. As well as increased efficiency of the Canadian wheat segregation program. Proximate data on different fish species are collected in databases such as the FishBase (www. the silicon detector covers the range 400–1100 nm. In fish meat these constituents make up about 98% of the total mass. However. Nearinfrared spectroscopy (NIR) is the most common method for such analysis and is therefore comprehensively presented in this chapter. During the 1980s monochromator instruments were developed.2 Nondestructive Analysis of Total Proximate Composition Analysis of each nutrient separately is time-consuming and requires a diverse set of equipments. and then one can perform a linear regression on the principal components.fishbase. Methods for simultaneous determination of the major components are therefore valuable. transmitted. The first instruments on the market were filter instruments measuring in reflectance mode. The NIR spectrum is defined between the wavelength 800 and 2500 nm. an indium gallium arsenic covers the range 800–1700 nm. making it difficult to use this spectral range before the development of multivariate calibration technology. 600. the chemical composition of fish generally varies due to seasons. by the chemical-free NIR method. and so on. or reflected depending on the interaction with NIR wavelength and physical status of the sample as transparent or nontransparent. 16. When Williams was running the program for the Canadian Grain Commission. vitamins. NIR has been found to be a reliable. analysis should be performed on the specific samples.1 Introduction The proximate composition in most fish and shellfish is primarily water. 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. proteins.000 Kjeldahl analyses were conducted per year and incidentally producing 47 ton of caustic waste in the process. and a lead sulfide. and lipids. or whole grain. however. Section 16.1 and further discussed in the text below. The spectral data will be reduced by principal component analysis. in this spectral region the spectrum of the transmitted light is very compact and no single peaks are visible. such as the introduction of partial-least squares (PLS) by Martens in 1982 [3]. Therefore. cheese. and easy to perform nondestructive analysis for simultaneous determination of the major components in fish. rapid. and the other minor constituents include carbohydrates. where the weak absorptions enable useful data to be obtained using sample thickness of 1–2 cm of samples such as meat. the adoption of NIR testing resulted in a total cost saving of CAN$ 2. The NIR radiation interacting with a sample may be absorbed. .

precise. fully automated. Nondestructive and can be used on live fish. expensive Few articles on fish composition Need more research Composition and Calories [21. Calibrations need to be made against reference methods. and can be performed online [17–20.8] For reflectance instruments (surface analysis) some drawbacks such as interference by starch and lipids.172–174] (continued) ◾ 259 . physiological and physical states can affect values of conductivity. or transmission of nearinfrared light (850–1700 nm) Ultrasound Measurement of ultrasonic velocity Rapid. can be used on live fish Samples are placed in an electromagnetic field and electric conductivity is measured Specific for different species. can be nonsensitive. nondestructive.173] Total body electrical conductivity (TOBEC) May obtain data on water. and protein.133] NIR/NIT Reflection. Different calibrations for different species and organs. displacement of reflectance spectrum by moisture content. water. The equipments are relatively expensive. transflection.Table 16. and disturbance by particle size in samples Ultrasonic properties of tissue depend on composition and temperature [11. and protein content noninvasively. [4–6.1 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. nondestructive. lipids. Calibrations require skilled personnel.

etc. Fosslet.Table 16. the fat content can be calculated theoretically by the formula Fat% = 80 − water % .55. fexICA).1 (continued) Advantages Rapid.) Microwave drying The sample is dried and from the water content found. location of lipids. 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.57] Excellent for determination of fat and water content or even distinguish lipid classes and water properties. 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.22. (SoxTech. processing) [46] Automatic. nondestructive (See under NMR below) [13–15. lipid content.

easy.51–52] Relatively inexpensive. and nondestructive and allows in vivo measurements (continued) 261 . rapid.22. The NMR mouse is rapid. lipid content. may also provide other nutrient data in the same analysis Some portable instruments are available Allows in vivo measurements Expensive. and portable Allows in vivo measurements Nondestructive and rapid Broad range of applications.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) [14–16.50.55] Fat meters Determination of water by analyzing the dielectric properties using a microwave strip (calculation of lipids as for the drying method). portable (small size). nondestructive. processing) Needs to be calibrated for the individual species Most suitable for neutral lipid determination See NIR/NIT above [4–6. location of lipids. Low-field NMR See NMR above Composition and Calories NMR mouse ◾ Nondestructive and rapid.8] [46. NIR/NIT Transmitted or transflected Near Infrared light (800–1700 nm).

since all nitrogen in foods is not in the form of protein. hazardous.67. standard method for comparison. trapping of ammonia. inexpensive to use and sensitive to low concentrations of proteins Limited to soluble proteins Most samples must undergo steps of sample preparation before they can be analyzed. and titration with acid Handbook of Seafood and Seafood Products Analysis Dumas combustion method High temperature combustion and detection of N by thermal conductivity detector [53. easy to perform. interference by nonprotein nitrogen compounds.120. distillation. safe (no chemical exposure). low sensitivity.1 (continued) Advantages Drawbacks Selected References Overview of the Most Common Methods for Analysis of Proximate Composition in Fish and Fish Products 262 ◾ Methods Principle Protein Determination Proteins Total Nitrogen Determination Widely used internationally. Absorbance depends on the type of protein analyzed . 74.Table 16.133] Kjeldahl Sample digestion followed by neutralization. inexpensive to use. Difficult to obtain the accurate protein concentration [53. and environmentally friendly High initial costs. independent of physical state of sample Does not give a measure of the true protein.133] Direct Protein Determination on Soluble Proteins Rapid. potentially toxic chemicals are used Rapid. time-consuming. high precision and good reproducibility.

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

option to quantify free amino acids. Derivatization agents: OPA: no derivatization with secondary amino acids. no addition of reagents required. chromatographic separation.1 (continued) Advantages Rapid. derivatization.97. low interference from nucleic acids and nucleotides Interference by oxygen and UV-absorbing compounds (buffer. high sensitivity. influence by lipids and sample particle size.133] Faster than ion exchange chromatography. quantifies amino acids. 108. sensitive Most methods do not include all amino acids.178] Drawbacks Selected References Overview of the Most Common Methods for Analysis of Proximate Composition in Fish and Fish Products 264 ◾ Methods Principle Far-UV absorption Measurement of UV absorption Highperformance chromatography (HPLC) Hydrolysis. 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) . FMOC: less soluble.Table 16. might interfere [90–92. hydrolysis destroys some of the amino acids. low dependency of signal response on amino acid composition. salts) [132.96. multicomponent analysis See NIR/NIT above Strong interference by water. and detection of amino acids with UV absorbance. value for net protein. nondestructive. fluorescence Handbook of Seafood and Seafood Products Analysis Nondestructive Determination Rapid. nondestructive.

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


Handbook of Seafood and Seafood Products Analysis

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

Composition and Calories




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

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


Handbook of Seafood and Seafood Products Analysis

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

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

Composition and Calories


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

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



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

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Methods for Protein Determination

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


Determination of Total Nitrogen

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


Direct Methods for Soluble Protein Determination

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

Composition and Calories


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

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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].

Composition and Calories


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

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

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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].



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

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

Composition and Calories


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

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


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

In some cases. the analysis of essential amino acids in seafood is important for the evaluation of both the nutritive value and the sensory quality of seafood.20 have been successfully used as extraction solvents. or by means of a simple stirring in warm solvent. the elderly. the sample is centrifuged at more than 10.2. The extraction solvent can be hot water. (2) its ability to cross-link proteins.18.22 trichloroacetic (TCA).23–25 and picric .13. concentrated strong acid solutions such as 4% of 5-sulfosalicylic acid. Free amino acids initiate important changes at early postmortem and during storage and can be very useful as quality indices of processing and storage. The extraction consists in the separation of the free amino acid fraction from the insoluble portion of the matrix (fish muscle). methods for the analysis of amino acids in seafood. which confers numerous biological functions to this amino acid (precursor to the antioxidant glutathione).16.15 or a rich alcohol-containing solution (>75%) such as ethanol16–18 or methanol19. especially of those considered essentials. or diluted phosphate buffers. Sample cleanup is necessary to eliminate proteins and polypeptides by means of the deproteinization process.11 17.1 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. 0.21 perchloric (PCA). cysteine may be essential for infants. It is usually achieved by homogenization of the ground sample in an appropriate solvent by using a Stomacher.1 N hydrochloric acid solution. and individuals with certain metabolic disease or who suffer from malabsorption syndromes. are described. which can be achieved through different chemical or physical procedures. and leucine). Sample preparation will depend on whether free or total essential amino acids have to be analyzed. In this chapter. then.288 ◾ Handbook of Seafood and Seafood Products Analysis compounds through Maillard reactions and Strecker degradations. Polytron. sulfosalicylic (SSA). or (3) its Maillard reaction with sugars yielding characteristic flavors. Although classified as nonessential. Once homogenized. Several chemical methods include the use of concentrated strong acids such as phosphotungstic (PTA). isoleucine. which increases the protein stability in the harsh extracellular environment by conferring proteolytic resistance.14 6% of perchloric acid.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.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. in rare cases. 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.01–0. there is no need for further cleaning up of the sample.4 Branched-chain essential amino acids (valine. 17.13 5% of trichloroacetic acid. with the additional advantage that proteins are not extracted and. and aromatic amino acids (phenylalanine and tyrosine) are the most important from this point of view.5–10 Thus. A more detailed description of amino acid methods of analysis may be found in the work of Aristoy and Toldrá. and so forth. sulfur-containing amino acids (methionine and cystine/cysteine).12.

because it gives information on the nutritional value of fish meat. which is easily neutralized by the addition of KOH or potassium bicarbonate. In the vapor-phase hydrolysis method. 10. or separation method (interferences in the chromatogram. Upon heating. or acetonitrile. resulting in a very simple deproteinization procedure with no interferences.41 The use of microwave technology for the hydrolysis has been assayed by some authors. The most common method used for complete hydrolysis of proteins is acid digestion.2.). When limited amounts of sample are available. Nitrogen atmosphere and sealed vials are required during the hydrolysis to minimize the degradation. In both cases. Some comparative studies have been published on these deproteinization techniques. Proteins must be hydrolyzed into their constituent amino acids before the analysis. thus excluding nonvolatile contaminants. A good choice may be the use of 0. creating an appropriate atmosphere inside the vessels to ensure low amino acid degradation. 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. whereas free amino acids remain in solution.35–38 These temperatures in such acidic and oxidative medium may degrade some amino acids. addition of constant boiling hydrochloric acid and additives. 5.22 Under these conditions. only the acid vapor comes into contact with the sample. presence of salts. 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. liquid phase or vapor phase. ethanol.000. Differences among all these chemical and physical methods are caused by several aspects such as differences in the cutoff molecular weight. etc. recovery of amino acids. to rend insoluble potassium perchlorate. oxygen is removed and substituted by nitrogen or other inert gas. which is easily separated by centrifugation. proteins precipitate by denaturation.000 Da) that allow free amino acids through while retaining large compounds. Some physical methods consist in centrifugation through cutoff membrane filters (1. The presence of appropriate antioxidants/scavengers during hydrolysis can prevent losses of the most labile amino acids.39 Some commercial systems are available.6 N PCA. and so forth.29. etc.39. but the duration of the treatment is shorter (less than 20 min). where the hydrochloric acid contacts the sample directly. The use of organic solvents. Liquid-phase. Typically. also disposes of an oven to accomplish the hydrolysis. An additional advantage is the easy evaporation to concentrate the sample. which permits the alternative air evacuating/inert gas purging. by mixing two or three volumes of organic solvent with one volume of extract.40.31–33 with amino acid recoveries around 100% for all them.000.Essential Amino Acids ◾ 289 (PA)26–28 acids or organic solvents such as methanol. all of them . compatibility with derivatization (pH.2 Sample Preparation for Total or Hydrolyzed Essential Amino Acid Analysis The total essential amino acid profile is usually requested.22. the vapor-phase hydrolysis method is preferred to minimize contaminants coming from aqueous 6 N hydrochloric acid. and performance under vacuum) is similar to that of a conventional oven.42 Sample manipulation (sample evaporation to dryness. the tubes containing the samples are located inside large vessels containing the acid.).000.18. Digestion at 145°C for 4 h has also been proposed.30 All these methods give a sample solution rich in free amino acids but free of proteins.34 17. Therefore. The hydrolysis may be accomplished using either liquid-phase or vapor-phase methods. 30. Hydrolysis may be improved by optimizing the temperature and time of incubation41 or with the addition of amino acid oxidation protective compounds. a system capable of alternative air evacuating/inert gas purging to get a correct deaeration inside is valuable. is well suited to hydrolyze large amounts or complex samples.

30.56 As can be observed in this section.3 Seafood Essential Amino Acid Analysis The analysis of individual amino acids needs a previous separation of all others. 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. and tryptophan. Thus. or pronase. KOH. The choice mainly depends on the equipment available or personal preferences. Tryptophan is often completely destroyed by hydrochloric acid hydrolysis. cysteine. cysteine sulfinic acid. The use of alkylating agents to stabilize the previous hydrolysis of cysteine constitutes a valid alternative. because each possible methodology has advantages and drawbacks.36. when the analysis of cyst(e)ine would be necessary. carboxypeptidase. chymotrypsin. Some additives have been proposed to protect tryptophan against oxidation as is the case of thioglycolic acid. no single set of conditions will yield the accurate determination of all essential amino acids.67–69 17.60 and books. Before or after this separation. or BaOH. and so on.43–50 improves cysteine (and methionine) recoveries. . importance of a correct deaeration.2 M of either NaOH. methionine. 41. unless a very selective way of detection is used. has been extensively reported in papers35. LiOH. yields acceptable results for the majority of amino acids. The separation of the individual amino acids in a mixture requires very efficient separation. making the posterior analysis easier. In general.290 ◾ Handbook of Seafood and Seafood Products Analysis essential amino acids. adequate hydrolysis procedure as the performic acid oxidation before the hydrolysis is a good alternative. A third way to hydrolyze proteins is enzymatic hydrolysis by proteolytic enzymes such as trypsin. although considerable recoveries have been found if no oxygen is present. The previous performic acid oxidation of cysteine to cysteic acid. in which methionine is also oxidized to methionine sulfone. Good recoveries have been achieved by using 3-bromopropionic acid.38 Alkaline hydrolysis instead of acid hydrolysis is also proposed (see below). acid-to-protein ratio. (2) give a quantitative and reproducible reaction.3¢-dithiodipropionic acid. Additionally.55. In fact. 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.62 An alternative to acid hydrolysis is the alkaline hydrolysis with 4.61. improve the recovery of nearly all of these amino acids except tryptophan and cysteine. Cyst(e)ine is partially oxidized during acid hydrolysis yielding several adducts: cystine. with or without the addition of 1% (w/v) thiodiglycol for 18 h at 110°C. presence and concentration of oxidation protective agents. 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. such as chromatographic (liquid or gas chromatography (GC)) or capillary electrophoresis (CE) techniques. a compromise of conditions offers the best overall estimation for the largest number of amino acids. When high sensitivity is required. serine. papain.36. being enough for the requirements of any food industry. threonine. such as tyrosine.1% sodium sulfite. up to 1% phenol or 0. thermolysin. protective agents currently used. amino acids used to be derivatized to allow their separation or to enhance their detection. which is recommended by many authors47. This option is chosen to analyze specific amino acid sequences or single amino acids because of their specific and well-defined activity. and cysteic acid making its analysis rather difficult.54 or 3. Derivatization is a usual practice in amino acid analysis.52 4-vinyl pyridine53. The optimization of conditions for hydrolysis based on the study of hydrolysis time and temperature.51 3-bromopropylamine.63–66 for a better tryptophan determination.59.58.

or 4-fluoro-7-nitrobenzo-2. The HPLC techniques to analyze amino acids are cation exchange and reversed-phase (RP) chromatography and are described in Sections 17.1. The original method required two separate columns and needed about 4 h to achieve a complete analysis. absorb at 210 nm and thus cannot be used for spectroscopic detection. in their native form.3. as it is a very unspecific detection wavelength.68 Thus.1 Cation Exchange Chromatography This methodology is based on the amino acid charge. and they include derivatives for spectroscopic or for electrochemical detection.12 Two types of derivatives are obtained depending on the chosen separation and/or detection technique. l em = 345 nm). The first type are derivatives that enhance amino acid detection in liquid media. It must be remarked that the use of sufficient amount of reagent is of special importance when dealing with biological samples. the more acidic amino acids elute first. permitting 5–10 pmol sensitivity as standard.1. Nevertheless.3. and those with more than one primary amino group or possessing a guanidyl residue elute at the end of the chromatogram. and better detection systems. The second type are derivatives that allow gas chromatographic amino acid separation by increasing their volatility and temperature stability. After separation.Essential Amino Acids ◾ 291 (3) yield a single derivative of each amino acid. Only three amino acids (phenylalanine. speed.3-oxadiazole postcolumn derivatization to obtain highly fluorescent derivatives with enhanced sensitivity. tyrosine. The elution involves a stepwise increase in both pH and sodium or lithium ion concentration.1 High-Performance Liquid Chromatographic Methods HPLC is the preferred technique to analyze amino acids. Amino acids. the spectroscopic detection of amino acids requires their previous derivatization to obtain an UV absorbing or fluorescent molecule. which facilitates a more selective detection. recent improvements of the ninhydrin derivatization method71–73 . (6) have good stability of the derivatization products.38.1 and 17. The latest generation of Moore and Stein amino acid analyzers also use o-phthaldialdehyde (OPA). and (7) have no interferences due to by-products or excess of reagent.15. (5) have the possibility of automation. The classical procedure has been improved with a new polystyrene matrix that offers better resolution power due to smaller particle size. are always present. because reagent-consuming amines. pellicular packaging.3. amino acids were converted into colored ninhydrin derivatives for spectrophotometric (colorimetric) detection. although unidentified. 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). because their spectral (high-ultraviolet (UV) absorbing or fluorescence properties) or electrochemical characteristics will affect the sensitivity and selectivity of detection. 17. (4) have mild and simple reaction conditions. Under these conditions.1. Although postcolumn techniques should be run online for maximum accuracy. 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 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.1.2. The derivatization reaction can be performed after separation of the amino acids (postcolumn derivatization) or before separating them (precolumn derivatization). Tryptophan also possesses native fluorescence (l ex = 295 nm. precolumn techniques can be run either offline or online.3.70 fluorescamine. 17.

biological fluids. which constitutes an advantage. plants). some difficulties to analyze some essential or sulfur-containing amino acid derivatives). especially in a dry condition. The most usual derivatizing agents for tissue amino acids are described below.) who offer integrated commercial systems including the column. (i.76 There are other reports of applying this technique to the amino acid analysis in food and tissues. and the long time of analysis. Phenylisothiocyanate (PITC): This methodology involves the conversion of primary and secondary amino acids to their phenylthiocarbamyl (PTC) derivatives. time for sample preparation and amino acids separation. Hitachi. which are detectable at UV (254 nm) with detection limits around 5–50 pmol. followed by a reaction coil. 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. Amersham Biosciences. To choose the most appropriate method some aspects must be taken into account such as the following: the disposable detector (fluorescence or UV). There are many manufacturers (Beckman. because it requires only a standard equipment that can be shared by different types of analysis. The main drawbacks of this methodology are the high cost of the ion exchange amino acid analyzer and its maintenance. Obviously. etc. and an optimized methodology with the advantage of ease of use and reliability. LKB. 17.75.2 Reversed-Phase High-Performance Liquid Chromatography RP-HPLC has been widely used. The PTC-amino acids are moderately stable at room temperature for 1 day and much longer when kept under frozen storage. possibility of automation of the derivatization reaction (in the autosampler).e. postcolumn derivatization is not suitable for narrow-bore HPLC. Dionex.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. Pickering. 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. The advantage of this method is the accurate results for all known sample types (food.77 After separation. making it adequate for partition based on chromatography. or the stability of formed derivatives. with which many of them have been marketed. feed. biological fluids. buffer system.74 Nowadays. through a mixing manifold. and finally the derivatized amino acids reach an online detector system. the formed molecule improves sensitivity and selectivity at the detection by allowing the spectroscopic (UV or fluorescent) detection of amino acids. the highly complex mobile phase composition.1. the analysis requirements for free or hydrolyzed amino acids or required sensibility. tissues. Kontron. and tissues). but. This method has been employed in the classical Moore and Steintype commercial amino acid analyzers. 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. Another disadvantage is the peak broadening produced by the dead volume introduced behind the column. 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.70.42. Precolumn amino acid derivatization may be necessary to confer hydrophobicity to the amino acid molecule. 66. Biotronik. also. .. Although this broadening may not affect when using standard-bore columns with flow rates above 1 mL/min. each new methodology must contrast its results with those obtained by cation exchange chromatography (CEC). plants.3. the derivatizing reagent is pumped into the effluent from the column system. which makes it a reference method for amino acid analysis. All PTC-amino acids have similar response factors.

which is achieved by the addition of triethylamine and includes several drying steps. 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 17.29.78–80 Sample preparation is quite tedious: it requires a basic medium (pH = 10.78–80 The chromatographic separation takes around 20 min for hydrolyzed amino acids and 60 min for physiological. which makes the quantitation of free cystine nonfeasible with this method. Sarwar et al.2. because no buffer is used during the reaction.5). The only limitation is the determination of PTC cystine that gives a poor linearity. and solvents. which includes the analytical column.1 Reversed-phase HPLC chromatogram of PTC amino acids from hydrolyzed hake muscle.Essential Amino Acids ◾ 293 The methodology is well described in the literature. This method is available as a commercially prepackaged system named Pico-Tag (Waters Associates.1 and 17. standards. The reaction time is less than 10 min even though 20 min are recommended for a complete reaction. internal standard nor-Leucine. the residual PITC reagent left after evaporation will cause damage to the column package.29. the last one being the elimination of the excess of reagent that may cause some damage to the chromatographic column. Both examples applied to the analysis of total amino acids from hake and free amino acids from salmon are shown in Figures 17. Moreover.82 The selection of the column is critical to get a good resolved separation especially when the analysis of physiological amino acids is involved. as some columns are more resistant than others. IS. which is more critical when amino acids from acid hydrolysis are analyzed. Massachusetts). Milford. It is important to ensure a basic pH to get adequate derivatization recoveries.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). respectively. .

IS. Detection limits are in the low picomole range. The dansylated amino acids are stable for 1 day85 or until 7 days when kept at −4°C86 and protected from light. United States). The high wavelength of absorption makes the baseline chromatogram very stable with a large variety of solvents and gradient systems.2 Reversed-phase HPLC chromatogram of PITC-free amino acids from salmon muscle extract. presenting a maximum from 448 to 468 nm. Ans. Palo Alto California.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 17.84 Detection is by absorption in the visible range. 15 min at 60°C. By-products originating from an excess of reagent absorb at the same wavelength and thus they appear in the chromatogram.84 Derivatives are very stable (weeks) and can be formed from both primary and secondary amino acids. Nevertheless. standard amino acid solution should be derivatized under similar conditions. anserine. However. Stocchi et al. internal standard nor-Leucine. and only needs a basic pH. around 9.83. l em = 510 nm) although UV (l = 250 nm) detection may also be used. 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. because it is especially affected by the presence of high levels of some chloride salts. The reaction time is around 15 min at 70°C and takes place in a basic medium with an excess of reagent. The sample derivatization is rather simple. Commercial System Gold/Dabsylation Kit™ uses this technique (Beckman Instruments.33 To overcome this problem and obtain an accurate calibration.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.58. Tau.87 or even 2 min at 100°C. Reaction efficiency is highly matrix dependent and variable for different amino acids. and a reaction time of 1 h at room temperature (in the dark). 4-Dimethyl-aminoazobenzene-4′-sulfonyl chloride (Dabsyl-Cl): This reagent was first described in 1975 for use in amino acid analysis. taurine.5. the reaction conditions .

and excess of reagent) must be carefully fi xed to optimize the product yield and to minimize secondary reactions. several methods have been proposed before derivatization. Tryptophan adducts do not fluoresce and histidine and cyst(e)ine adducts fluoresce weakly.32 . 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. which is not the case with any essential amino acid.86. The choice of the mercaptan can affect derivative stability. Nowadays.92. itself or hydrolyzed.82 Another problem is the large excess of reagent needed to assure a quantitative reaction. and fluorescent intensity. chromatographic selectivity. which is present in excess as it is highly fluorescent and probably interferes into the chromatogram as a huge peak.97. which includes the addition of ADAM. The reaction time is fast (45–90 s) and does not require any heating. OPA derivatives can be detected by UV absorption (338 nm) as well.89 9-Fluorenylmethyl chloroformate (FMOC): This reagent yields stable derivatives (days) with primary and secondary amines. as it is highly fluorescent and then. and the reagent itself is not fluorescent. On the contrary. The first option was included in the automated AminoTag method90 developed by Varian (Varian Associates Limited).5) medium.88 Even so. this problem is overcome by standardizing the time between sample derivatization and column injection by automation. Histidine gives a very poor fluorescence response (10% of the other amino acids).Essential Amino Acids ◾ 295 (pH.91 This method is preferred because the addition of ADAM is more easily automatized. Another proposal102 consists of a slight modification in the OPA derivatization method by using 2-aminoethanol as a nucleophilic agent and altering the order of the addition of reagents in the automated derivatization procedure. The major disadvantage is due to the reagent.32 In these methods.93 The fluorescence is recorded at 455 or 470 nm after excitation at 230 or 330 nm. The yield with lysine and cysteine is low and variable. reinforcing the poor reproducibility of its results. the reaction of the excess of reagent with a very hydrophobic amine as 1-adamantylamine (ADAM) gives a late-eluting noninterfering peak. temperature. The derivative is fluorescent (l ex = 265 nm.98 and some of them have been patented and commercially marketed (AutoTag OPA from Waters Associates).101 or the formation of the mixed disulfide S-2-carboxyethylthiocysteine (Cys-MPA) from cysteine and cystine. cysteine and cystine are quantified together. such as FMOC/amino acid ratio. many automatic injectors are programmable and able to achieve automatic derivatizations. The addition of detergents like Brij 35 to the derivatization reagent seems to increase the fluorescence response of lysine. this will commonly form multiple derivatives with histidine. One of the main disadvantages of this procedure is the inability of OPA to react with secondary amines.82.99 In the case of cysteine. lysine. because it guarantees the repeatability of parameters.94–96 2-mercaptoethanol. is of great advantage. In order to obtain reliable and precise results. ethanethiol. OPA amino acids are not stable.3′-dithiodipropionic acid55 and incorporated by Godel et al. Some reports have been published proposing several ways of automation. 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. and 3-mercaptopropionic acid are the most frequently used. reaction conditions. An automated precolumn derivatization routine. and tyrosine. In the second option. The derivatization is fast (1–3 min) and is performed at room temperature in alkaline (pH 9. This excess is hydrolyzed to dansyl sulfonic acid. as well as reaction time.94 into the automatic sample preparation protocol described by Schuster. have to be optimized very carefully. this methodology reveals excellent linearity for cystine and also cystine-containing short-chain peptides. l em = 315 nm) and is detected at the femtomole range. using 3. This is relatively easy because the reaction is fast and no heating is necessary. the excess may interfere in the chromatogram and for this reason it must be extracted (with pentane or diethyl ether) or converted into noninterfering adduct before injection.

the AMQ peak is very large at the beginning of the chromatogram and may interfere with the first eluting peaks (see Bosch et al.108 If the choice of the derivative reaction is a challenge. The chromatographic separation of these derivatives has been optimized for the amino acids from hydrolyzed proteins. different selectivity may be found among same columns. . or charge. Indeed. because both mono.105 in addition to fluorescent properties possesses electroactivity (750 mV) and PITC106 has again the advantage of reacting with secondary amines. Any electrical measure. potential. different selectivity. In these cases. The excess of reagent is consumed during the reaction to form aminoquinoline (AMQ). and UV detection at 254 nm may be used for its analysis. columns are more carefully manufactured with these silanol groups blocked or inaccessible by steric impediment avoiding the tailing. lipids.3A shows the separation of hydrolyzed amino acids from salmon. Due to these variables.and di-derivatives are the initial adducts from tyrosine. the optimum pH for the reaction is in a broad range. is related to the analyte concentration. 1 min.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. Milford.296 ◾ Handbook of Seafood and Seafood Products Analysis 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC): It reacts with primary and secondary amines from amino acids. Electrochemical detection consists in one electrode or an array of electrodes mounted in a cell with an applied potential difference. OPA/mercaptoethanol or OPA/sulfite104.5 min. Furthermore. Only columns manufactured in the same batch are guaranteed to give the same selectivity if the rest of parameters are fi xed. peptides.50). Some of these derivatives are also susceptible to electrochemical detection.2 to 10. because the resulting CisH is well separated inside the chromatogram. In this case. The most used column packaging consists of alkyl-bonded silica particles. detergents. conductance. The methodology has been marketed as a prepackaged AccQ Tag kit (Waters Corporation. Reaction time is short. yielding very stable derivatives (1 week at room temperature) with fluorescent properties (l ex = 250 nm. which is only weakly fluorescent at the amino acid derivatives detection conditions and does not interfere in the chromatogram. and other compounds naturally occurring in biological samples and foods. which are separated by RP-HPLC. The fluorescence of tryptophan derivative is very poor. United States). because they are molecules with electroactive functional groups.107. even those made by the same manufacturer. and proteins.. It means that when transferring a published method to a particular set of samples. Only amino acids with aromatic rings or sulfur-containing side chains are sufficiently electrochemically active to be detected by this method. and the separation of physiological amino acids is improved. Both facts facilitate sample preparation.e. the choice of the RP column is not an easy subject because of the great variability of commercially available RP columns. Cystine and cysteine may be analyzed after their conversion to cysteic acid (CisH) by performic acid oxidation. from 8.3B shows the same sample but submitted to a performic acid oxidation before the hydrolysis in which the CisH peak appears by 7. as a consequence. accessible to sample molecules. it will be necessary to readjust the chromatographic conditions to get a good separation of all amino acids. Massachusetts. Sensitivity is in the femtomole range. l em = 395 nm). but 10 min at 55°C would be necessary if a tyrosine monoderivative is required. mainly octadecylsilane. Nowadays. such as current. The presence of residual uncapped silanol groups on the silica surface. the addition of a strong cation (i. UV detection (254 nm) may also be used. However. can cause unwanted tailing of peaks (especially for the basic amino acids). Figure 17. whereas Figure 17. making them very adequate for biochemical research. triethylamine) to the mobile phase can overcome the problem. 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.

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

especially in the analysis of D isomers. and amines.3. which is universal and the most widely used. This methodology has been patented as EZ:faast and commercialized by Phenomenex (Torrance. which are much more sensitive than FID for such compounds. Recently. especially since the capillary columns appeared. dipeptides. United States). and balenine may complicate the amino acid analysis. in summary. the amino acids must be converted to volatile and thermostable molecules. A finely adjusted binary (most used) or ternary gradient elution is often necessary when the overall amino acid profile from hydrolyzed and. a very highly efficient technique adequate for the amino acid analysis. a very fast GC analysis of physiological amino acids. carnosine. although applications on meat samples are scarcely described. The buffer may be constituted by less than 100 mM concentration of acetate or phosphate.3 Capillary Zone Electrophoretic Methods Capillary zone electrophoretic technique is extremely efficient for the separation of charged solutes. 17.2 Gas Liquid Chromatographic Methods The extremely high-resolution capacity is the main advantage of GC. including amino acids. Some applications67.112 milk. in comparison with liquid chromatographic techniques. Gas liquid chromatography (GLC) is not often used for the determination of amino acids from tissues or foods.113 and cheese.117–119 where the separation was achieved by using chiral-GC stationary phases. especially. and the derivatives are stable and ready for GC/FID. and urine matrices but not in tissues in which the presence of natural dipeptides. Described applications are available for the analysis of physiological amino acids in blood.3. Nevertheless. the technique is very efficient and it is worth mentioning the separation of 32 nonprotein amino acids from edible seeds.120.114 In their analysis by GLC. GLC is. The detector used is the flame ionization detector (FID). or GC and LC with MS detection. Protein removal is not required. physiological amino acids has to be analyzed.113. capable of separating 50 compounds. plasma. In many cases. The main advantages of these detectors are their high sensitivity and wide linear range. and the equipment is very versatile and usually available in any analytical laboratory. Amino acids constitute a mix of basic. California. GLC has been combined with mass spectrometry (MS) for detection and identification.121 The high efficiency. GC/NPD. The difficulty of separating amino acids by this technique relies on their structure.116 to analyze phosphoserine. whereas thermionic-N-P (NPD) or flame photometric detector (FPD) are selective toward organic compounds containing phosphorous and nitrogen. anserine. and beans111 or other results obtained in honey. Reactions consist of two stages: an esterification with an acidified alcohol followed by N-acylation with an acid anhydride in an anhydrous medium. and phosphotyrosine.298 ◾ Handbook of Seafood and Seafood Products Analysis with an organic phase constituted by acetonitrile and/or methanol and/or tetrahydrofuran. The NPD was used by Buser and Erbersdobler115 and FPD by Kataoka et al.109. and low amount of sample make this technique very interesting when compared with classical electrophoresis and chromatographic techniques. has been developed. GLC is not very expensive because no solvent is used. phosphothreonine. nuts. speed. 17. The method yields a full amino acid profile (33 amino acids) in 15 min including a 7 min extraction-derivatization step plus 8 min for the gas chromatographic separation. and even though a particular pH can significantly .110 comparing GLC with cation exchange chromatography reported different conclusions when analyzing some hydrolyzed food samples. and acidic constituents. neutral.

nonprotein amino acids. A good compatibility between both techniques. showing that higher efficiency is obtained by the MECC methods with sodium dodecyl sulfate (SDS) as micelle-forming substance.118. and the composition and flow rate of the sheath liquid to obtain the best sensitivity. microcolumn liquid chromatography. etc.136 phenylthiohydantoin.146. it is likely to cause overlap with the others.124 Basic theoretical considerations on this technique125 and its food applications126 are described elsewhere.140 or even urea141 have been assayed. to enhance UV detection. 19 amino acids were analyzed by CE-ESI-MS in only 17 min with a minimal sample preparation and no matrix interference. allowed a rapid development and the onset of these complementary techniques. Some reviews covering high-sensitivity detection following CE have been published. Unfortunately.119 have been reported.). and others.and l-isomer mixtures. Terabe et al. Under the conditions of electro-osmotic flow in CE. or to allow fluorescence or electrochemical132 detection of amino acids. The identification of the 22 protein amino acids may not be a problem.138 and OPA139 compared with the separation of OPA-amino acid derivatives by CZE with normal and micellar solutions.133–135 PITC. isobutanol. the species with different charge can be simultaneously analyzed but with serious doubts in their adequate resolution.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. 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. This report includes the optimization of important parameters like the choice of a volatile electrolyte (1 M formic acid) for the electrophoresis. When sensitivity is the target.139 which is usually enough for food analysis or an LIF (laser-induced fluorescence) detector.Essential Amino Acids ◾ 299 improve the resolution of one kind. which can be resolved on the basis on their mass-to-charge ratios that are characteristic of each ion and allow its identification. reports in the literature of its applications are increasing rapidly.4 Mass Spectrometry MS is based on the conversion of components of a sample into rapidly moving gaseous ions. and so forth. With few exceptions. although other additives such as dodecyltrimethylammonium bromide. the high cost of purchase and maintenance of mass spectrometers has inhibited their more widespread use in the food industry and/or food control. SDS is indeed the most used additive to form micelles in this kind of analysis.136. which constitutes an important limitation of this technique.137. This technique has also been termed micellar electrokinetic capillary chromatography (MECC or MEKC).) and instrumentation (CE.127–131 derivatization is used to improve separation. in particular when capillary columns were available.138 Tween 20. Other additives commonly used in this analysis are organic modifiers (acetonitrile. o-tyrosine analysis. The effect of these additives on the electro-osmotic mobility and electrophoretic mobility of the micelle has been studied. . although this detector may be used for more complex identifications as in d.113 o-tyrosine in chicken148 or pork149 tissues. methanol.125.144. Nevertheless. CZE shows poor ability for the separation of neutral compounds. it is relatively easy to analyze low picomol levels of OPA derivatives in micellar solutions by using a conventional fluorometric detector. Mass spectrometer detectors were first connected to GC equipments. compatible with MS. tetrahydrofurane.3.141–143 The CE coupled to electrospray ionization (ESI) MS (CE-ESI-MS) allows direct amino acid analysis without derivatization.122. etc.147 17. Good separations have been reported for precapillary derivatized amino acids with dansyl-Cl.21 chiral amino acids.131 and thus.). Application in foods such as in the identification of nonprotein amino acids. biomedical or pharmaceutical research.

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

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..............310 18......................314 18..............................317 18...316 18.......................313 18....318 309 .......................1......................................3 Occurrence of Ascorbic Acid in Marine Organisms .......................................... 315 18..............1...................317 18..................3..........3 Occurrence of Carotenoids ...............................3.............................................................Chapter 18 Antioxidants Nick Kalogeropoulos and Antonia Chiou Contents 18.........1 Ascorbic Acid Functions ........................3..........................................................4...................... 315 18.................3..........311 18........311 18..3 Antioxidants in Seafood and Seafood Products ...314 18................................................1 Antioxidant Enzymes ......2 Carotenoid Determination .........2..........1.....................3...1......2.......3..............3............................313 18.....310 18.......................3...........................................1 Antioxidant and Other Functions of Carotenoids ...............1...........................318 18.................3 Vitamin E .......3.3........................2.4................317 18..2 Lipid Peroxidation..3........................2 Ascorbic Acid Analysis .........2 Antioxidants..................313 18.......................................................3..316 18...........................1 Oxidation and Its Implications..........................................4.2 Vitamin E Determination ..........................3.................................3 Marine Lipid Oxidation .........311 18...............................................................................................3..........................1...............1.............................................2 Determination of Antioxidants and Antioxidant Capacity in Biological and Food Systems ................310 18..............2 Ascorbic Acid ...........................................................................................................4 Carotenoids .....................1......................3.............................................................1 Oxidative Stress and Its Implications ............1 Vitamin E as an Antioxidant.......3......................................1 Introduction .........314 18.......3 Occurrence of Vitamin E ..........

... that is.....319 18......2 Natural Antioxidants .... Aging........... Under conditions of oxidative stress..3............... if ROS are not immediately intercepted by antioxidants........4...............319 18...........5 Ubiquinone .........319 18.1 Introduction Antioxidants evolved together with the emergence of photosynthesis by cyanobacteria......3............ polyphenols............................ Until subsequent free radicals are deactivated................ and hydroxyl hydrogen peroxide (H2O2)................. where antioxidants are mainly produced by photosynthetic organisms and are consequently transported through the trophic web...................1.........................2 Determination of Ubiquinone ......... 320 18......... as a defense against oxygen toxicity........... being exposed to the oxygen they produce....................6 Other Endogenous Antioxidants ........................319 18.... peroxyl (ROO •)... 320 18..... 18........... since oxygen is the final electron acceptor in the electron flow system that produces energy...................................................................................... alkoxyl (RO •)......................1 Oxidative Stress and Its Implications Oxidative stress occurs when the prooxidant–antioxidant balance becomes too favorable to the prooxidants.............. Although the initial attack causes the neutralization of the free radical......... they may oxidize several cell components............. This is also followed in the marine environment......... such as peroxynitrite (ONOO−)..3.........1 Synthetic Antioxidants .4..3............... more than 2 billion years ago..........1.............................310 ◾ Handbook of Seafood and Seafood Products Analysis 18....321 References ..................... •)............5..... nucleic acids.4 and environmental stress5..1 Indeed cyanobacteria and the laterevolved green plants.........6 have been reported to cause oxidative stress to fish or bivalves....................... are rich in antioxidants such as vitamins C and E......... generation of free radicals occurs..... resulting in an increased antioxidant activity and 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. and carotenoids.............. Humans and most animals cannot synthesize the majority of these antioxidants and depend on the dietary intake from plant consumption......... ROS production in organisms is related to both the basal metabolism and the influence of environmental factors2........1.... 18. which include among others superoxide anion radical (O2−).............4 Added Antioxidants .... 320 18......3 Occurrence of Ubiquinone......... electrically charged compounds that seek out and capture electrons from other compounds in order to neutralize themselves.......... and proteins may be damaged by reactive oxidants. Common free radicals in biological systems are the so• called reactive oxygen species (ROS). When the electron flow becomes uncoupled (transfer of unpaired single electrons)............................. lipids.................................. thousands of free radical reactions may occur within a few seconds............. and the so-called reactive nitrogen species (RNS)... another free radical is generated in the process.3 pollution. nitric oxide (NO (OH•) radicals............3.. resulting in a chain reaction......5...5.....1 Function of Ubiquinone .......321 18.

free radicals. that is. significantly delays or prevents oxidation of that substrate. Three reaction pathways have been proposed: (1) nonenzymatic chain autoxidation. The major components of the antioxidant defense system together with their proposed mechanisms of action are presented in Table 18. There is increasing evidence that oxidative stress is implicated in the pathogenesis of many inflammatory and degenerative diseases and conditions. and storage. carcinogenic.3 Marine Lipid Oxidation Compared with other food lipids.7 18. and metal ions. This can be especially damaging to lipid-rich cell membranes. The health implications of tissue lipid oxidation are numerous and well documented.9 Preventive antioxidants hinder ROS formation or scavenge spe• cies responsible for oxidation initiation (O2−. because of their high degree of unsaturation. Lipid oxidation of omega-3 polyunsaturated fatty acids (PUFA)-rich food products results in the development of particularly unpleasant off flavors.1. in which polyunsaturated fatty acids on lipid molecules are attacked and oxidized. antioxidants are molecules that protect macromolecules from being oxidized. propagation. protect the cellular components from oxidative damage. Lipid oxidation is a complex procedure induced by oxygen in the presence of initiators such as light. handling.8 Studies on the pathological significance of dietary lipid oxidation products have indicated that some lipid oxidation products have cytotoxic. Nonradical photooxidation seems to be a minor reaction compared with the 3O2-induced radical chain autoxidation. Moreover.13 Antioxidants counteract oxidation in two different ways: They protect lipids from oxidation initiators. etc. and angiotoxic effects.2 Lipid Peroxidation A very damaging effect of oxidant reactive intermediates is lipid peroxidation. 18. 1O2. preventive antioxidants.2 Antioxidants Antioxidants are defined as any substance that when present at low concentrations compared with those of an oxidizable substrate. In general. atherogenic.11 and unhealthy compounds that reduce their shelf life and nutritive value. mainly of plant origin. antioxidants are . 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. For food systems.1.Antioxidants ◾ 311 loss or oxidation product development.3. Chain-breaking antioxidants intercept •) or participate in halting radical chain propagation. mutagenic. and they stall the propagation phase. and termination. antioxidants supplied by foods. and (3) enzymatic oxidation. (2) nonenzymatic and nonradical photooxidation.12 In biological systems various biochemical defense mechanisms. including enzymatic systems and nonenzymatic antioxidants.1. are essential for counteracting oxidative stress.1.9 18.). antioxidants often act via more than one mechanism that combines different types of antioxidant activity.1. heat. chain-breaking antioxidants.1.10 Lipids deteriorate in seafood products during processing. for which the human sensory apparatus has a low threshold. initiation. marine lipids are relatively more susceptible to oxidation. Autoxidation occurs through a three-phase process. Nevradical oxidation propagators (LOO ertheless. In the first two cases a combination of reactions involving 3O2 and 1O2 occurs.

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

and algae.3. Kolanowski et al. one represented by enzymes and the second represented by low molecular mass compounds. oxidative damage to macromolecules is controlled by two types of antioxidant systems. thiols. voltammetric. 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. 18. cephalopods. the latter including paper. spleen.2 and 18.3 Antioxidants in Seafood and Seafood Products In living organisms. bilirubin. At higher levels most of them behave as prooxidants possibly due to their involvement in the initiation reactions. 18. widely distributed in aerobic cells that help in preventing the accumulation of H2O2 within cells.1 Antioxidant Enzymes Catalases are metal-containing enzymes. or Fe in their active site.17 and Wood et al. and uric acid. glutathione (GSH). together with traces of phenolic compounds. 18.9 The determination of specific waterand fat-soluble antioxidants is discussed in Sections 18.Antioxidants ◾ 313 effective at very low concentration levels. coenzyme Q. and chromatographic methods. The available methods have been reviewed by Rajalakshmi and Narasimhan. Cu. Zn.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.16 and Laguerre et al. thin-layer. blue-green algae.7 U/mg of protein.1 Cu/Zn SOD were purified from marine fish tissues.2 Determination of Antioxidants and Antioxidant Capacity in Biological and Food Systems Analytical techniques developed for some of the common endogenous or exogenous antioxidants range from qualitative detection by color-developing reactions to semiquantitative and quantitative determinations by means of spectroscopic.20 .18 In reviewing methods and tests for the assessment of lipid oxidation. and in red muscle compared with that in white.9 and 9. carotenoids. Catalase activities ranged between 386 and 1523 mmol/ min/g tissue in several Atlantic fish and was higher in liver. such as ascorbic acid. whereas Fe SOD were purified from red algae..19 concluded that. tocopherols. for quality control of fish oil and fish oil-containing foods. and high-performance liquid chromatography (HPLC) alone or combined with mass spectroscopy.21 In several species of teleosts. These antioxidants act in a concerted way to protect sensitive molecules such as the unsaturated fatty acids from oxidation. and heart.3.15 Griffiths et al. and crustaceans from the Mediterranean sea. with Mn. polarographic. column chromatography and the more sophisticated gas chromatography (GC).20 Superoxide dismutases (SOD): SODs are metalloproteins. T