Fishery Products: Quality, Safety and Authenticity

FISHERY PRODUCTS Quality, safety and authenticity

Edited by Hartmut Rehbein Jörg Oehlenschläger

A John Wiley & Sons, Ltd., Publication

FISHERY PRODUCTS

FISHERY PRODUCTS Quality, safety and authenticity

Edited by Hartmut Rehbein Jörg Oehlenschläger

A John Wiley & Sons, Ltd., Publication

This edition first published 2009 © 2009 Blackwell Publishing Ltd Blackwell Publishing was acquired by John Wiley & Sons in February 2007. Blackwell’s publishing programme has been merged with Wiley’s global Scientific, Technical, and Medical business to form Wiley-Blackwell. Registered office John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, United Kingdom Editorial offices 9600 Garsington Road, Oxford, OX4 2DQ, United Kingdom 2121 State Avenue, Ames, Iowa 50014-8300, USA For details of our global editorial offices, for customer services and for information about how to apply for permission to reuse the copyright material in this book please see our website at www.wiley.com/wileyblackwell. The right of the author to be identified as the author of this work has been asserted in accordance with the Copyright, Designs and Patents Act 1988. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, except as permitted by the UK Copyright, Designs and Patents Act 1988, without the prior permission of the publisher. Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic books. Designations used by companies to distinguish their products are often claimed as trademarks. All brand names and product names used in this book are trade names, service marks, trademarks or registered trademarks of their respective owners. The publisher is not associated with any product or vendor mentioned in this book. This publication is designed to provide accurate and authoritative information in regard to the subject matter covered. It is sold on the understanding that the publisher is not engaged in rendering professional services. If professional advice or other expert assistance is required, the services of a competent professional should be sought. Library of Congress Cataloging-in-Publication Data Fishery products : quality, safety and authenticity / edited by Hartmut Rehbein, Jörg Oehlenschläger. p. cm. Includes bibliographical references and index. ISBN 978-1-4051-4162-8 (hardback: alk. paper) 1. Fishery products–Quality control. 2. Fishery processing–Quality control. I. Rehbein, Hartmut. II. Oehlenschläger, Jörg. SH335.5.Q35F58 2009 664′.94–dc22 2008039852 A catalogue record for this book is available from the British Library. Set in 10 on 12 pt Times by SNP Best-set Typesetter Ltd., Hong Kong Printed in Singapore 1

2009

Contents

List of contributors Preface Introduction

Chapter 1

Basic facts and figures Jörg Oehlenschläger and Hartmut Rehbein 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9

Chapter 2

Chapter 3

xi xiii xv

Introduction World fishery production Categories of fish species Fish muscle Nutritional composition Vitamins Minerals Post mortem changes in fish muscle References and further reading

1 1 1 3 4 4 10 15 15 17

Traditional methods Peter Howgate

19

2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 2.10 2.11

19 20 23 29 30 31 32 33 33 34 35

Introduction TVB-N Methylamines Volatile acids Volatile reducing substances Indole Proteolysis and amino acids pH Refractive index of eye fluids Discussion and summary References

Biogenic amines Rogério Mendes

42

3.1 3.2 3.3 3.4

42 44 47 49

Introduction Factors affecting amine decarboxylase activity Safety aspects Quality assessment

v

vi

Contents

3.5 3.6 3.7 Chapter 4

Chapter 5

Regulatory issues Methods of biogenic amine quantification References

54 55 59

ATP-derived products and K-value determination Margarita Tejada

68

4.1 4.2 4.3 4.4 4.5

68 69 79 81 81

VIS/NIR spectroscopy Heidi Anita Nilsen and Karsten Heia 5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.9

Chapter 6

Introduction Analytical principles and measurements Constituents: assessment of chemical composition Freshness and storage time Authentication Safety Other quality parameters Summary and future perspectives References

89 89 89 92 96 98 98 99 100 101

Electronic nose and electronic tongue Corrado Di Natale and Gudrun Ólafsdóttir

105

6.1 6.2 6.3

105 106

6.4 6.5 6.6 6.7 6.8 6.9 Chapter 7

In vivo role of nucleotides Post mortem changes Methodology for evaluating the K-value or related compounds Conclusions References

Introduction to the electronic nose and olfaction Application of the electronic nose and electronic tongue Colorimetric techniques, optical equipment and consumer electronics Classification of fish odours Quality indicators in fish during chilled storage: gas chromatography analysis of volatile compounds Application of the electronic nose for evaluation of fish freshness Combined electronic noses for estimating fish freshness Conclusions and future outlook References

108 109 111 114 116 119 120

Colour measurement Reinhard Schubring

127

7.1 7.2 7.3

127 128 130

Introduction Instrumentation Novel methods of colour evaluation

Contents

7.4 7.5 7.6 Chapter 8

Chapter 10

Chapter 11

131 159 159

Differential scanning calorimetry Reinhard Schubring

173

8.1 8.2 8.3 8.4

173 174 178

8.5 8.6 Chapter 9

Colour measurement on fish and fishery products Summary References

vii

Introduction Principle of function of the instruments First applications of DSC on fish muscle and other seafood Recent applications of DSC for investigating quality and safety Summary References

181 204 204

Instrumental texture measurement Mercedes Careche and Marta Barroso

214

9.1 9.2 9.3 9.4 9.5

214 216 229 231 231

Introduction Instrumental texture Texture measurement for quality classification or prediction Conclusions References

Image processing Michael Kroeger

240

10.1 10.2 10.3 10.4 10.5 10.6 10.7 10.8 10.9 10.10

240 241 243 244 244 245 246 246 249 249

Introduction Quality characteristics from images Spectral signature of images Elastic properties from images Analysis of image data Results and discussion Freshness determination from images Firmness information from images Conclusions References

Nuclear magnetic resonance Marit Aursand, Emil Veliyulin, Inger B. Standal, Eva Falch, Ida G. Aursand and Ulf Erikson

252

11.1 11.2 11.3 11.4 11.5 11.6

252 253 257 259 265 266

Introduction Magnetic resonance imaging Low-field NMR High-resolution NMR The future of NMR in seafood References

viii

Contents

Chapter 12 Time domain spectroscopy Michael Kent and Frank Daschner 12.1 12.2 12.3 12.4 12.5 Chapter 13

Chapter 14

Introduction Measurement system Time domain reflectometry measurements Conclusions References

273 275 278 283 285

Measuring electrical properties Michael Kent and Jörg Oehlenschläger

286

13.1 13.2 13.3 13.4 13.5 13.6

Introduction Fischtester Torrymeter Use of the Fischtester Summary References

286 286 287 294 296 297

Two-dimensional gel electrophoresis Flemming Jessen

301

14.1 14.2 14.3 14.4 14.5

301 302 305 310 312

Introduction Two-dimensional gel electrophoresis (2DE) 2DE applications in seafood science 2DE-based seafood science in the future References

Chapter 15 Microbiological methods Ulrike Lyhs 15.1 15.2 15.3 15.4 15.5 Chapter 16

273

Microorganisms in fish and fish products General aspects of microbiological methods Most probable number method Molecular methods References

318 318 320 336 336 338

Protein-based methods Hartmut Rehbein

349

16.1 16.2 16.3 16.4 16.5 16.6 16.7 16.8

349 349 351 356 357 357 359 359

Introduction Fish muscle proteins Electrophoretic methods for fish species identification High-performance liquid chromatography Immunological methods and detection of allergenic proteins Determination of heating temperature Differentiation of fresh and frozen/thawed fish fillets References

Contents

Chapter 17

Chapter 18

DNA-based methods Hartmut Rehbein

363

17.1 17.2 17.3 17.4 17.5 17.6

363 364 366 368 379 380 388

18.1 18.2

388

Chapter 21

Introduction Species and breeding stock identification by lipid analysis Verification of the production method Identification of the geographic origin Future prospects References

389 394 398 403 404

Sensory evaluation of seafood: general principles and guidelines Emilia Martinsdóttir, Rian Schelvis, Grethe Hyldig and Kolbrun Sveinsdóttir

411

19.1 19.2

411

19.3 Chapter 20

Introduction DNA in fishery products Genes used for species identification Methods Conclusions and outlook References

Other principles: analysis of lipids, stable isotopes and trace elements Iciar Martinez

18.3 18.4 18.5 18.6 Chapter 19

ix

General principles for sensory analysis Application of sensory evaluation to fish and other seafood References

417 422

Sensory evaluation of seafood: methods Emilia Martinsdóttir, Rian Schelvis, Grethe Hyldig and Kolbrun Sveinsdóttir

425

20.1 20.2 20.3 20.4 20.5 20.6 20.7

Introduction Difference tests Grading schemes Quality index method Descriptive sensory analysis Consumer tests (hedonic) References

425 425 427 430 438 440 440

Data handling by multivariate data analysis Bo M. Jørgensen

444

21.1 21.2 21.3 21.4

444 444 446 447

Introduction What is multivariate data analysis? Arrangement of data for bi-linear modelling The outcome of bi-linear modelling

x

Contents

21.5 21.6 21.7 Chapter 22

451 453 453

Traceability as a tool Erling P. Larsen and Begoña Pérez Villarreal

458

22.1 22.2 22.3

458 460

22.4 22.5 22.6 Index

Validation and prediction Real examples and further reading References

Introduction Traceability from older times to the present Traceability research in the seafood sector and other EU-funded food traceability projects Validation of traceability data Traceability in a global perspective References

465 466 468 470 472

List of Contributors

Ida G. Aursand, SINTEF Fisheries and Aquaculture, N-7465 Trondheim, Norway; and Department of Biotechnology, NTNU, N-7491, Trondheim, Norway Marit Aursand, SINTEF Fisheries and Aquaculture, N-7465 Trondheim, Norway Marta Barroso, Instituto del Frío CSIC, c/José Antonio Novais 10, 28040 Madrid, Spain Mercedes Careche, Instituto del Frío CSIC, c/José Antonio Novais 10, 28040 Madrid, Spain Frank Daschner, Technische Fakultät der Christian-Albrecht-Universität, Institut für Elektrotechnik und Informationstechnik, Kaiserstrasse 2, D-24143 Kiel, Germany Corrado Di Natale, Department of Electronic Engineering, University of Rome ‘Tor Vergata’, Via del Politecnico 1; 00 133 Roma, Italy Ulf Erikson, SINTEF Fisheries and Aquaculture, N-7465 Trondheim, Norway Eva Falch, SINTEF Fisheries and Aquaculture, N-7465 Trondheim, Norway Karsten Heia, Nofima, Marine, N-9291 Tromsø, Norway Peter Howgate, 26 Lavender Row, Stedham, Midhurst, West Sussex GU29 0NS, UK Grethe Hyldig, DTU Aqua, National Institute of Aquatic Resources, Technical University of Denmark, Søltofts Plads, Bygning 221, DK-2800 Kongens Lyngby, Denmark Flemming Jessen, DTU Aqua, National Institute of Aquatic Resources, Technical University of Denmark, Søltofts Plads, Building 221, DK-2800 Kongens Lyngby, Denmark Bo M. Jørgensen, DTU Aqua, National Institute of Aquatic Resources, Technical University of Denmark, Søltofts Plads, Building 221, DK-2800 Kongens Lyngby, Denmark Michael Kent, The White House, Greystone, Carmyllie, by Arbroath, Angus DD11 2RJ, UK Michael Kroeger, technet GmbH, Pestalozzistrasse 8, D-70563 Stuttgart, Germany Erling P. Larsen, DTU Aqua, National Institute of Aquatic Resources, Technical University of Denmark, Søltofts Plads, DTU, Bygning 221, DK-2800 Kongens Lyngby, Denmark Ulrike Lyhs, Ruralia-Institute, Seinäjoki Unit, University of Helsinki, Kampusranta 9C, 60320 Seinäjoki, Finland Iciar Martinez, SINTEF Fisheries and Aquaculture Ltd, 7465 Trondheim, Norway xi

xii

List of Contributors

Emilia Martinsdóttir, Matís (Food research, Innovation and safety), Skulagata 4, IS-101 Reykjavík, Iceland Rogério Mendes, Department of Technological Innovation and Upgrading of Fishery Products, INRB/IPIMAR, Av. De Brasilia, 1449-006 Lisboa, Portugal Heidi Anita Nilsen, NOFIMA, Marine, N-9291 Tromsø, Norway Jörg Oehlenschläger, Max Rubner Institute, Federal Research Institute for Nutrition and Food, Unit for Seafood Quality, Palmaille 9, D-22767 Hamburg, Germany Gudrun Ólafsdóttir, Department of Food Science and Nutrition, Faculty of Science, University of Iceland, Hjardarhagi 2-6, 107 Reykjavík, Iceland; and Syni Laboratory Service, Lyngháls 3, 110 Reykjavík, Iceland Begoña Pérez Villarreal, Food Research Division, Txatxarramendi Ugartea z/g, 48395 Sukarrieta (Bizkaia), Spain Hartmut Rehbein, Max Rubner Institute, Federal Research Institute for Nutrition and Food, Unit for Seafood Quality, Palmaille 9, D-22767 Hamburg, Germany Rian Schelvis, Wageningen IMARES, P.O. Box 68, NL-1970 AB IJmuiden, The Netherlands Reinhard Schubring, Max Rubner Institute, Federal Research Institute for Nutrition and Food, Unit for Seafood Quality, Palmaille 9, D-22767 Hamburg, Germany Inger B. Standal, SINTEF Fisheries and Aquaculture, N-7465 Trondheim, Norway; and Department of Biotechnology, NTNU, N-7491, Trondheim, Norway Kolbrun Sveinsdóttir, Matís (Food research, Innovation and safety), Skulagata 4, IS-101 Reykjavík, Iceland Margarita Tejada, Instituto del Frío (CSIC), José Antonio Novais, 10, 28040 Madrid, Spain Emil Veliyulin, SINTEF Fisheries and Aquaculture, N-7465 Trondheim, Norway

Preface

The contribution of fisheries and aquaculture to the human food supply has increased very significantly in recent decades. What is remarkable for this part of the food sector is the large share of fish that enters international trade, with some 37% of all fish caught and cultured being traded across national borders. So it can be argued that fish and fishery products are in the forefront of globalization, as products from all corners of the world can be found on the international market. There are many interesting facets to how this came about, in particular how well developing countries have adapted to the strict trading regimes of the modern marketplace for fish and fishery products. As food retailers consolidate in ever-larger units, the competition for customers intensifies. This has direct effects through the whole supply chain, not least primary producers. Besides, large retailers now have so much reputation at stake that they spend large sums of money to minimize the risk of ‘food scandals’ ever being attributable to the products they sell. This translates into ever more and stricter food safety and quality criteria with which all the actors in the food chain have to comply. This is one of the reasons for a rise in private standards of various sorts that are stricter than the standards set by governments. This rise in private standards is seen by many as a potential new form of protectionism. The objective of the World Trade Organization (WTO) is to facilitate free trade between nations to ‘improve the welfare of the peoples of the Member Countries’. The WTO Agreements, particularly the Sanitary and Phytosanitary Agreement (SPS) and the Technical Barriers to Trade Agreement (TBT), were set as the framework within which technical standards would be operated. In 1995 it was decided that the food standards of the Codex Alimentarius would be the standards used to resolve safety and quality questions in international trade disputes. Free trade is a very important issue on the international agenda. The international system created through the WTO is meant to create a ‘level playing field’ so that all can participate in international trade and to allow ‘trade to flow smoothly, freely, fairly and predictably.’ Thus, the importance of food standards to ascertain if they comply with agreed minimum criteria. The SPS Agreement stipulates that food standards should be based on sound science and be risk based. There is also a call for harmonization of standards and equivalence of different national standards relating to food safety management systems as long as they adhere to the same level of protection. That is a brief description of the framework, but all food standards are linked to specific methods by which compliance with them is measured. This book deals with the methods commonly used to measure the quality of fish and fishery products. Going through it is truly a story attesting to the great progress that has been made in this area in recent decades. It is interesting to see how the science has moved forward to increasing automation and online, non-destructive methods to ascertain characteristics of the products. It is also interesting to see how sensory evaluation, which not so long ago was considered subjective and thus unscientific, has been turned into an objective scientific tool in its own right. Competition in the food market makes it imperative for retailers not only to present products that are safe to eat and taste good, but also nutritionally balanced. Increasingly they xiii

xiv

Preface

also have to comply with environmental criteria such as not originating from a fish stock that is overfished or from vessels fishing illegally. In such a competitive environment, it is tempting for producers to cut corners. Water can be added to increase weight, expensive fish species can be substituted by cheaper ones or chemicals can be added to remove smells, to name only a few examples. At the same time, any wrongdoing or fraud can inflict huge damage in lost reputation for the producing countries and retailers. Therefore, objective analytical methods to verify and check compliance are of the utmost importance at all levels in today’s food chains. I therefore congratulate the authors for this important contribution to a seafood sector that aspires to deliver safe, tasty and wholesome food to the demanding modern consumer. The seafood sector has been in the forefront of globalization and this book will contribute to ensuring that quality and safety standards will be implemented by processors both in developed and developing countries. Grimur Valdimarsson Director Fish Products and Industry Division Fisheries and Aquaculture Department Food and Agriculture Organization of the United Nations Rome Italy

Introduction

When we started our careers as analytical chemists and biochemists in a seafood-dedicated institution more than 30 years ago, there were only a few analytical methods known to analyse safety and quality of seafood and which were used as well in research as in industry. The important methods were few: electrical devices as the Fischtester and Torrymeter; total volatile basic nitrogen (TVBN) for the determination of spoilage (often wrongly referred to as a freshness determination method); colony-forming units (cfu) in microbiology; and some methods for the determination of proximate composition, additives and non-desirable components. Textbooks, especially about analytical methods applied in seafood research and seafood-related industry, were missing; and monographs in this field were rare. Worthy of mention are the books written by Ludorff and Meyer (1973, in German), Connell (1995, 4th edition) and Botta (1995). More recently (2003), a book was edited by Luten, Oehlenschläger and Olafsdottir containing some chapters with information about instrumental and sensory measurement techniques. During the past 30 years the situation has changed dramatically. The changes were mainly initiated by two major causes. Firstly, there was a rapid development in the field of analytical instrumental methods, both in further development or application of existing and novel methods. Secondly, development in the research framework programmes of the European Commission enabled scientists not only to travel to congresses to meet and talk to their peers but also to conduct integrated research projects together. The latter has been very effective in the past 15–20 years. The results of these research projects and Concerted Actions led to a jump forward in seafood-related instrumental techniques in Europe. We ourselves have participated in several research projects, of which a few that were very productive and future-orientated are worth mentioning. The Concerted Actions ‘Evaluation of fish freshness’ and ‘Fish quality labelling and monitoring’, and the Research Projects ‘Multi-sensor techniques for monitoring the quality of fish (MUSTEC)’, ‘Seafood quality identification (SEQUID)’, and ‘Identification of species in processed seafood products using DNA-based diagnostic techniques’. Many of the authors in this book have also been partners in such research projects and have been selected to be contributors based on their skills and experience provided there. Unfortunately, many results obtained in the research projects have been hidden in confidential reports and never published. One of us (J.O.) has been chairman of the ‘WEFTA (Western European Fish Technologists’ Association) Working Group on Analytical Methods in Fish and Fishery Products’ since 1988. During this time,we increasingly desired to collect and publish the knowledge about modern analytical methods from all the different disciplines in a comprehensive book to make it available in a convenient form for the reader. From the first idea to realisation took many years, but the results are now in your hands. To our knowledge, this is the first book that concentrates on instrumental methods used in the seafood world. It is not a textbook about analytical methods but a guidebook for applied methodologies in the seafood area. It was our aim to present chapters that could be used by a wide range of interested readers, from students or beginners in the field who want xv

xvi

Introduction

to get a first overlook about the topic and who can be guided further by the numerous references, to advanced readers who will certainly find some new information that cannot be obtained elsewhere in such a concentrated and condensed form. The book contains chapters about traditional, instrumental, microbiological, sensory and authenticity methods. Finally, it has two chapters about multivariate data analysis and traceability. The book does not include chapters about determination of organic and inorganic pollutants for two reasons: (1) there are excellent books already available on analytical chemistry; (2) the importance of the topic and the multitude of analytical methods and pollutants would need a separate book. The chapters have been written by scientists who are all intensively working in their respective areas and who are highly specialised. Nevertheless, it is our hope that the chapters and the whole book are easily readable and understandable. We are very grateful to all authors who have contributed to this book; we thank them deeply for their patience and willingness to consider our wishes for changes, amendments and additions to the chapters during the preparation of the book. We especially thank Dr Ute Ostermeyer (Max Rubner-Institute, Hamburg, Germany), who has considerably contributed to the section about vitamins in the introductory chapter. We further thank the anonymous reviewers who have read the chapters and given valuable advice. May this book be a reference for seafood-related analytical techniques for years to come! Hartmut Rehbein Jörg Oehlenschläger

Chapter 1

Basic facts and figures Jörg Oehlenschläger and Hartmut Rehbein

1.1

Introduction

With more than 30,000 known species, fish form the biggest group in the animal kingdom that is used for the production of animal-based foods. Only about 700 of these species are commercially fished and used for food production. Further, some 100 crustacean and 100 molluscan species (for example mussels, snails and cephalopods) are used as food for humans. The amount captured worldwide is registered annually by the Food and Agriculture Organization of the United Nations (FAO). Fish and other seafood are very important in covering a part of the protein demand for humans. In 2000, food fish contributed 15.9% to the human diet on a worldwide basis (fish as a percentage of total animal protein intake). There are, however, great differences between continents and countries. In low-income, food-deficient countries (LIFDC) fish contributes 20.6%, in Asia 23.3%, in China 21.1%, whereas in South America the contribution amounts only to 5.7%, in North and Central America to 7.1% and in Europe to 10.3%. The average contribution in developed countries is 12% whereas it is 18.8% in developing countries (FAO).

1.2

World fishery production

World fishery production has been developing rapidly since 1950 (Table 1.1). In 1948 only 22 million metric tonnes of fish were captured, whereas in 2004 the world production amounted to more than 140 million tonnes. The dramatic increase of captured fish from 1950 to 1975 was followed by a somewhat more moderate increase between 1975 and 1990, and stagnation since then. Today, most fish stocks are fully exploited and a few are even overexploited. The growth in world fishery production in the past 10–15 years is based on a steadily growing aquaculture. The proportion of species farmed by aquaculture of the total world fishery production amounts today to more than 40%. The countries contributing most to total world fishery production of 140,457 million tonnes in 2004 are listed in Table 1.2. In Table 1.3 the major captured fish species are listed, and in Table 1.4 the major species farmed by aquaculture. Of this world fishery production of 140,457 million tonnes, 105,632 million tonnes (75.2%) were used for human consumption. Of these 75%, 39% were marketed fresh, 19% frozen, 8% cured and 9% canned. 1

2

Fishery Products: Quality, safety and authenticity

Table 1.1 Development of world fish production (catch and aquaculture) since 1900. Year Million tonnes

1900 4

1948 22

1958 40

1968 67

1978 73

1988 99

1995 117

1998 118

2004 140

Table 1.2 World fishery production: top 10 countries in 2004. Country China Peru India Indonesia Chile USA Japan Thailand Norway Vietnam

Capture

Aquaculture

Total (tonnes)

16,892,793 9,613,180 3,615,724 4,811,320 4,935,376 4,959,826 4,401,341 2,845,088 2,522,225 1,879,488

30,614,968 22,199 2,472,335 1,045,051 674,979 606,549 776,421 1,172,866 637,993 1,198,617

47,507,761 9,635,379 6,088,059 5,856,371 5,610,355 5,566,375 5,177,762 4,017,954 3,160,218 3,078,105

Table 1.3 World fishery production in 2004: fish species captured (greater than 1 million metric tons). Species Peruvian anchovy Alaska pollock Blue whiting Skipjack tuna Atlantic herring Chub mackerel Japanese anchovy Chilean jack mackerel Largehead hairtail Yellowfin tuna European pilchard

Taxonomic name

Amount captured (tonnes)

Engraulis ringens Theragra chalcogramma Micromesistius poutassou Katsuwonus pelamis Clupea harengus Scomber japonicus Engraulis japonicus Trachurus murphyi Trichiurus lepturus Thunnus albacares Sardina pilchardus

10,679,338 2,691,939 2,427,862 2,092,356 2,019,933 2,017,276 1,795,844 1,778,777 1,587,452 1,384,358 1,062,432

Table 1.4 World aquaculture production of fish, crustaceans and molluscs in 2004 (greater than 1 million metric tonnes). Species Pacific oyster Silver carp Grass carp Carp Japanese carpet shell Bighead carp Crucian carp Nile tilapia Whiteleg shrimp Atlantic salmon Japanese scallop

Taxonomic name

Quantity (tonnes)

Crassostrea gigas Hypophthalmichthys molitrix Ctenopharyngodon idellus Cyprinus carpio Ruditapes philippinarum Hypophthalmichthys nobilis Carassius carassius Oreochromis niloticus Penaeus vannamei Salmo salar Patinopecten yessoensis

4,429,337 3,979,292 3,876,868 3,387,918 2,860,152 2,101,688 1,949,758 1,495,744 1,386,382 1,244,637 1,126,159

Basic facts and figures

3

Part of the world fishery catch is processed into fish meal, which is used as a fertiliser or as animal (mainly fish) feed. For this, some target fish species such as sand eel and anchoveta (so-called ‘industry fish’) are caught. The fish oil recovered during the fish meal process is to some extent also used for human nutrition. The stagnation of the world fish catch has led to an intensive discussion about better use and management of the resources. Also, quality aspects that have been neglected for many years are back on the agenda (careful handling of the the catch, prolonged shelf life of ice- and frozen-stored fish, optimisation of yield in fish processing machines, etc.).

1.3 Categories of fish species Fish species can be divided into categories, for example according to their habitat as marine and freshwater species. Some species such as European eel and most salmon can live in both Table 1.5 Categories of marine and freshwater fish species according to their total fat content in edible tissue (fillet).

Category

Total fat content (%)

Common names

Taxonomic names

<1

Cod, saithe, haddock, whiting, ling, cusk, grenadier, blue whiting, blue ling, pike, perch, pikeperch, monkfish or angler, lemon sole, Alaska pollack

Species with low fat content

>1–5

White halibut, wolffish, plaice, hake, dab, grey mullet, red mullet, redfish, whitch, sole, turbot, brill, trout, tench, whitefish

Medium fatty fish species

>5–10

Redfish, sardine, swordfish, Bream, catfish, albacore, dogfish, tuna, conger, salmon

>10

Sprat, black halibut, mackerel, herring, eel

Gadus morhua, Pollachius pollachius, Pollachius virens, Melanogrammus aeglefinus, Molva molva, Brosme brosme, Coryphaenoides rupestris, Macrourus berglax, Micromesistius poutassou, Molva dypterygia, Esox lucius, Perca fluviatilis, Stizostedion lucioperca, Lophius piscatorius, Microstomus kitt, Theragra chalcogramma Hippoglossus hippoglossus, Anarhichas lupus, Anarhichas minor, Pleuronectes platessa, Merluccius merluccius, Limanda limanda, Mugil cephalus, Mullus surmuletus, Sebastes marinus, Sebastes mentella, Glyptocephalus cynoglossus, Solea solea, Psetta maxima, Scophtalmus rhombus, Trutta trutta, Tinca tinca, Coregonus sp. Sebastes marinus, Sebastes mentella, Sardina pilchardus, Xiphias gladius, Abramis brama, Silurus glanis, Squalus acanthias, Thunnus thynnus, Conger conger, Salmo salar Sprattus sprattus, Hippoglossoides platessoides, Scomber scombrus, Clupea harengus, Anguilla anguilla

Lean fish species

Fatty fish species

4

Fishery Products: Quality, safety and authenticity

environments. Typical representatives of freshwater fish are: carp, pike, perch, pikeperch, tench; examples of marine fish are: cod, saithe, redfish, mackerel and herring. Often, their anatomical shape is also used for categorisation: roundfish such as saithe, cod and hake; flat fish species such as plaice, dab and flounder; and snake-shaped fish such as eel, lamprey and moray. Also, the categories groundfish and swarmfish are used. Groundfish are those species that search for their prey close to the bottom of the sea (cod, flat fish); swarm fish are those that gather in big schools, mainly the small, pelagic, fatty species such as herring, sprat and sardine. Most species belong to the group of bony fish (Osteichthyes), which means that they have a fully developed bony skeleton. Some fish species have so-called real bones which are not attached to the backbone or to other bones but are located free in the muscle tissue. These bones are formed by hardened connective tissue. Sharks, rays and chimaeras belong to the group of cartilagenous fish (Chondrichthyes). They have no bones, but a cartilaginous tissue which is enforced by calcium carbonate. Fish species can also be divided into four classes (Table 1.5) based on their nutritional properties, for example their fat content, which can vary in some species depending on their state of maturity from 1% to 30%.

1.4

Fish muscle

Fish flesh, fish muscle or fish fillet is the name for the body musculature of fish reaching from head to tail: this muscle forms the major part of the edible portion of fish. This side muscle consists of segments (myomers) lying between connective tissue layers (myocommata). The muscle fibres within the myomers are longitudinally orientated. The proportion of fish flesh to total body weight varies between 40% and 65%, depending of species, shape, age and the physiological status of the fish. Fish with more elliptical cross sections (tuna, herring and salmon) exhibit a much higher proportion of the edible part than flatfish species or species with very big heads such as monkfish. Fish flesh consists of light and dark musculature. Both types can be differentiated by chemical composition, physiological importance and nutritional value. Most species have more light than dark muscle. Herring and mackerel have approximately equal amounts of light and dark muscle. The dark muscle occurs just below the skin in the area of the lateral line, and continues as a wedge shape to the backbone. The light musculature is used for rapid, sudden movements and obtains energy mainly from anaerobic glycolysis. For continuous swimming, fish use their dark musculature. This type of muscle is therefore well developed in pelagic species (herring, mackerel, tuna), well supplied with blood and rich in myoglobin. The metabolism of dark muscle is aerobic; energy is provided by lipids and carbohydrates.

1.5

Nutritional composition

The nutritional composition of fish (Tables 1.6 and 1.7) is comparable to that of warmblooded animals; in relation to essential elements such as selenium and iodine, it is superior.

Table 1.6 Compositional data of edible part (fillet) of marine and freshwater fish species. Data are average values calculated or estimated and rounded from several food composition tables as well as from our analyses and can be subject to great variations depending on intrinsic fish parameters such as state of maturity, sex, age, season, nutritional status, etc. Fish species

Components

22 : 6 (n − 3) (g/100 g) Cholesterol (mg/100 g)

Sea bass

Bluefish

Burbot

Common carp

Channel catfish

Cod

Pacific cod

Cusk

Eel

Engraulis encrasicolus

Morone saxiatilis

Pomatomus saltatrix

Lota lota

Cyprinus carpio

Ictalurus punctatus

Gadus morhua

Gadus macrocephalus

Brosme brosme

Anguilla anguilla

73 20 5 1.4 131 548 147 41 174 383 104 1.7 0.2 0.07 37

79 18 2 1 97 406 15 40 198 256 69 0.4 0.03 0.12 375

71 20 4 1 124 519 7 33 227 327 60 0.08 0.05 0.2 375

67 18 6 1.5 127 531 41 29 415 333 49 1.5 0.06 0.04 13

80 16 3 1 95 397 14 23 209 358 43 0.5 0.03 0.03 13

81 18 0.4 1.2 82 343 7 24 174 403 71 0.4 0.03 0.01 37

76 19 0.7 1.3 87 364 10 31 204 392 31 0.3 0.02 0.02 37

68 18 12 1.4 184 770 20 20 216 272 51 1.6 0.02 0.04 7

0.08

Total PUFA 0.28 41

79 19 1 1.2 90 377 50 32 200 404 97 0.8 0.2 0.7 136

0.538

0.169

0.252

0.07

0.238

0.13

81 18 0.5 1.2 82 343 16 32 203 413 54 0.5 0.03 0.02 33 187 0.064

0.911 30

0.585 80

0.519 59

0.096 60

0.114 66

0.234 58

0.12 39

0.135 37

0.084 0.063 51

Basic facts and figures

Moisture (g/100 g) Raw protein (g/100 g) Total lipids (g/100 g) Ash (g/100 g) Energy (kcal/100 g) Energy (kJ/100 g) Calcium (mg/100 g) Magnesium (mg/100 g) Phosphorus (mg/100 g) Potassium (mg/100 g) Sodium (mg/100 g) Zinc (mg/100 g) Copper (mg/100 g) Manganese (mg/100 g) Selenium (μg/100 g) Iodine (μg/100 g) 20 : 5 (n − 3) (g/100 g)

Anchovy

5

6

Continued Fish species

Components Moisture (g/100 g) Raw protein (g/100 g) Total lipids (g/100 g) Ash (g/100 g) Energy (kcal/100 g) Energy (kJ/100 g) Calcium (mg/100 g) Magnesium (mg/100 g) Phosphorus (mg/100 g) Potassium (mg/100 g) Sodium (mg/100 g) Zinc (mg/100 g) Copper (mg/100 g) Manganese (mg/100 g) Selenium (μg/100 g) Iodine (μg/100 g) 20 : 5 (n − 3) (g/100 g) 22 : 6 (n − 3) (g/100 g) Cholesterol (mg/100 g)

Haddock

White halibut

Greenland halibut

Herring

Ling

Mackerel

Monkfish

Mullet

Redfish

Melanogrammus aeglefinus

Hippoglossus hippoglossus

Rheinhardtius hippoglossoides

Clupea harengus

Molva molva

Scomber scombrus

Lophius piscatorius

Mugil cephalus

Sebastes marinus

78 19 0.7 1.2 87 364 33 39 188 311 68 0.3 0.06 0.03 30 186 0.059 0.126

78 21 2.3 1.4 110 460 47 83 222 450 54 0.4 0.03 0.02 37 22 0.071 0.292

70 14 14 1 186 778 3 26 164 268 80 0.4 0.03 0.01 37 74 0.526 0.393

72 18 9 1.5 158 661 57 32 236 327 90 1 0.09 0.04 36 41 0.709 0.862

80 19 0.6 1.4 87 364 34 63 198 379 135 0.3 0.1 0.03 36 175 Total PUFA 0.22 31

64 19 14 1.4 205 858 12 76 217 314 90 0.6 0.07 0.02 44 109 0.898 1.401

83 15 1.5 1.2 76 318 8 21 200 400 18 0.3 0.03 0.02 36 27 Total PUFA 0.61 33

77 19 3.8 1.2 117 490 41 29 221 357 65 0.5 0.05 0.02 36

79 19 1.6 1.2 94 393 107 30 216 273 75 0.5 0.03 0.02 43 70 0.08 0.211

36

45

42

31

33

0.217 0.108 49

42

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Table 1.6

Fish species

Components

22 : 6 (n − 3) (g/100 g) Cholesterol (mg/100 g)

Saithe

Alaska Pollack

Orange roughy

Bluefin tuna

Skipjack

Yellowfin tuna

Turbot

Wolffish

Esox lucius

Pollachius virens

Theragra chalcogramma

Hoplostetus atlanticus

Thunnus thynnus

Euthynnus pelamis

Thunnus albacares

Psetta maxima

Anarhichas lupus

79 19 0.7 1.2 88 368 57 31 220 259 39 0.67 0.05 0.2 12

78 19 1 1.4 92 385 60 67 221 356 86 0.37 0.05 0.02 36 121 0.071

82 17 0.8 1.2 81 339 5 57 376 326 99 0.44 0.04 0.02 22 46 0.15

76 15 0.7 0.9 69 289 30 30 200 300 63 0.75 0.1 0.02 36

68 23 5 1.2 144 602 8 50 254 252 39 0.6 0.09 0.02 36

71 22 1 1.3 103 431 29 34 222 407 37 0.82 0.09 0.02 36

71 23 15 1.3 168 452 16 50 191 444 37 0.52 0.06 0.02 36

77 16 3 2 95 397 18 51 129 238 150 0.22 0.04 0.02 36 180 Total PUFA 0.88

80 18 2 1.2 96 402 6 30 200 300 85 0.78 0.03 0.02 36

0.033 0.074 39

0.35 31

0.283

0.22 71

0.890 20

38

0.071 0.185 47

0.037 0.181 45

39

0.307 0.316 43 Continued

Basic facts and figures

Moisture (g/100 g) Raw protein (g/100 g) Total lipids (g/100 g) Ash (g/100 g) Energy (kcal/100 g) Energy (kJ/100 g) Calcium (mg/100 g) Magnesium (mg/100 g) Phosphorus (mg/100 g) Potassium (mg/100 g) Sodium (mg/100 g) Zinc (mg/100 g) Copper (mg/100 g) Manganese (mg/100 g) Selenium (μg/100 g) Iodine (μg/100 g) 20 : 5 (n − 3) (g/100 g)

Pike

7

8

Continued Fish species

Components Moisture (g/100 g) Raw protein (g/100 g) Total lipids (g/100 g) Ash (g/100 g) Energy (kcal/100 g) Energy (kJ/100 g) Calcium (mg/100 g) Magnesium (mg/100 g) Phosphorus (mg/100 g) Potassium (mg/100 g) Sodium (mg/100 g) Zinc (mg/100 g) Copper (mg/100 g) Manganese (mg/100 g) Selenium (μg/100 g) Iodine (μg/100 g) 20 : 5 (n − 3) (g/100 g) 22 : 6 (n − 3) (g/100 g) Cholesterol (mg/100 g)

Salmon

Chinook

Keta

Coho

Swordfish

Salmo salar

Oncorhynchus tschawytscha

Oncorhynchus keta

Oncorhynchus kisutch

Xiphias gladius

75 20 4 1.2 120 502 11 22 283 429 50 0.5 0.06 0.02 35

73 22 6 1.2 146 611 36 31 262 423 46 0.4 0.05 0.01 36

76 20 4 1.5 121 506 4 27 263 288 90 1.2 0.1 0.02 48

69 20 6 2.5 142 594 12 29 200 490 44 0.6 0.3 0.02 37 45 0.32 1.12 26

73 20 10 1.4 180 753 22 95 289 394 47 0.4 0.04 0.02 36 0.788 0.567 66

0.233 0.394 74

0.429 0.656 45

0.108 0.531 39

Fishery Products: Quality, safety and authenticity

Table 1.6

Table 1.7 Compositional data of edible part (fillet) of marine crustacean and molluscan shellfish species. Data are average values calculated or estimated and rounded from several food composition tables as well as from our analyses and can be subject to great variations depending on intrinsic fish parameters such as state of maturity, sex, age, season, nutritional status, etc. Crustacean and molluscan shellfish species

Components

22 : 6 (n − 3) (g/100 g) Cholesterol (mg/100 g)

American oyster

Common octopus

Blue mussle

American lobster

Snow crab

Dungeness crab

Blue crab

King crab

Crassostrea gigas

Crassostrea virginica

Octopus vulgaris

Mytilus edulis

Homarus americanus

Chionoectes opilio

Cancer magister

Callinectes sapidus

Paralithodes camtschatica

81 12 2 1.6 86 360 26 4 34 197 320 286 1.6 0.1 3.4 45 99 0.188

77 19 1 2.2 90 377 48 0.3 27 144 275 296 3 1.7 0.06 41

81 19 1 2 90 377 26 2.5 49 133 173 539 2.8 0.6 0.03 35

79 17 1 1.7 86 360 46 0.4 45 182 354 295 4.3 0.7 0.08 37

79 18 1 1.8 87 364 89 0.7 34 229 329 293 3.5 0.7 0.2 37

80 18 0.6 1.8 84 351 46 0.6 49 219 204 836 6 0.9 0.04 36

0.268

80 15 1 1.6 82 343 53 5.3 30 186 350 230 1.7 0.4 0.03 45 25 0.076

0.292 53

0.081 48

0.253 28

82 9.5 2 1.2 81 339 8 5 22 162 168 106 17 1.6 0.6 77 0.438 0.25 50

85 7 2 1.4 68 285 45 6.7 47 135 156 211 91 4.5 0.4 64

Total PUFA 0.15 95

0.259

0.219

0.17

0.113 55

0.088 59

0.15 78

Total PUFA 0.13 42

Basic facts and figures

Moisture (g/100 g) Raw protein (g/100 g) Total lipids (g/100 g) Ash (g/100 g) Energy (kcal/100 g) Energy (kJ/100 g) Calcium (mg/100 g) Iron (mg/100 g) Magnesium (mg/100 g) Phosphorus (mg/100 g) Potassium (mg/100 g) Sodium (mg/100 g) Zinc (mg/100 g) Copper (mg/100 g) Manganese (mg/100 g) Selenium (μg/100 g) Iodine (μg/100 g) 20 : 5 (n − 3) (g/100 g)

Pacific oyster

9

10

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The protein of fish muscle is rich in essential amino acids, has a high biological value and can be digested easily. The amount of connective tissue is low (1–2%) compared with warm-blooded animals (10–13%). The content of non-protein nitrogen (NPN) components in fish flesh is high. The main components are creatine (200–700 mg/100 g), trimethylamine oxide (100–1000 mg/100 g), adenosine nucleotides (200–400 mg/100 g), free amino acids and dipeptides. Chondrichthyes contain high amounts of urea. The average sum of NPN amounts to 420 mg/100 g and contributes to 15% of raw protein content (nitrogen content × 6.25). The fat content of fish varies greatly in quantity and fatty acid composition. The protein content is almost constant. The fat content is mainly dependent on biological state of maturity, but also on nutritional status, age, catching ground and season. The fat is not homogeneously distributed in the body. In lean fish species, it is located in the liver as an energy reservoir; in fatty species, it is deposited in the muscle tissue, as a subcutaneous layer under the skin or in the intestines. In many fatty fish species, a linear correlation exists between the fat and water content of muscle tissue. Lean fish species have a higher proportion of polar lipids (phosphatidylcholine and phosphatidylethanolamine) than fatty fish species, in which the fat consists mainly of neutral lipids (triacylglycerols). The polar lipids are mainly located in the lipid bilayer of the cell membranes, whereas the neutral lipids are located in the fat cells of the energy reservoirs (liver, muscle). The cholesterol content of fish muscle is generally low (35 mg/100 g). Fish lipids differ from those of terrestrial animals mainly in their high content of longchain, highly unsaturated fatty acids of the n–3 series (eicosapentaenoic acid, 20 : 5 and docosahexaenoic acid, 22 : 6), often referred to as polyunsaturated fatty acids (PUFAs). The content of these PUFAs in fatty fish species can be high: dogfish 3 g/100 g, herring 2.3 g/100 g, mackerel 4.6 g/100 g, salmon 2.3 g/100 g and tuna 2.1 g/100 g. The highly unsaturated character of these fatty acids is the reason why they are susceptible to lipid oxidation and oxidative degradation. Fatty fish species therefore have a tendency to exhibit rancid tastes and odours after limited storage time.

1.6

Vitamins

The vitamin contents in fish are species specific. They can vary considerably within one species with age, size, sex, season, diet, state of health and geographic location. In fish farmed by aquaculture, the contents of vitamins reflect the composition of the corresponding components in the fish feed. Therefore, the vitamin content of wild and farmed fish can be different. From different food composition tables, the mean vitamin contents in raw muscle are summarised in Table 1.8a, b for several marine fish and in Table 1.9a, b for freshwater fish.

1.6.1

Fat-soluble vitamins

The liver of fish is a rich source of fat-soluble vitamins (A, D, E and K). In fish flesh, dark muscle contains more fat-soluble vitamins than white muscle because of its higher fat content.

Table 1.8a Fat-soluble vitamins in marine fish species (μg/100 g edible portion). Species Anchovy Anglerfish Atlantic halibut Catfish (wolffish) Cod Dab Flounder Greenland halibut Haddock Hake Herring (Atlantic) Herring (Baltic Sea) Horse mackerel Ling Mackerel Mullet Plaice Redfish (ocean perch) Saithe

n.d., No data.

Vitamin A

Vitamin D

Vitamin E

2.3 0.1–1.5 1.6–10.4 2.0–2.5 0.3–0.7 1.0–1.6 0.7–3.2 9.8–14.1 0.2–1.0 0.4–2.5 14.0–17.8 9.2 3.9–5.6 0.6–2.8 3–30 4.2–4.3 1.4–1.9 0.9–3.6 0.2–0.9 0.3–0.9 4.5–5.2 1.26 1.7–1.8 1.1–7.1 10.5–17.6 4.0–4.4 15.1–15.5 1.1–4.9 10.0 1.7–3.0 0.2–0.5 0.6–0.7 1.1

19 8–80 <2–33 18–27 2–11 14 9–10 5–50 2–17 15 6–38 9.7 12–40 2–10 51–214 45–47 0–16 13–14 2–20 2–11 19–20 3.0 11 <2–4 100–150 20–36 450–464 370 370 1–12 2–10 3–15 1

20 1–2 5–18 0.5–1.8 1.2–1.5 1.5 0.8 11–15 1 1 11–27 7.8 0.5 1–3.4 1–13 3 3.0–9.0 2.3 1–2.2 0.7–0.8 8.5–11

500 500–1000 700–1000 1100–2150 449–1100 400 360–1000 850–2200 390–500 600 600–1800 2000 700 300 600–1550 1000 600–800 900–1300 200–700 360–600 465

1 8 18.7–20 2 4.5–5.0 2.9–5.0 5.4 1.7–2 1 0.2 0.01

100 600–800 1200–2500 1000 1171 1200 1200 600 100–300 200–230 800

Vitamin K1

n.d.

0.02–0.05 0.01 0.2 0.03 0.01 0.04 0.08

5.0 0.6 0.05 0.04

1.0

n.d. 0.03

11

Turbot Tusk Whiting Witch

Engraulis encrasicolus Lophius piscatorius Hippoglossus hippoglossus Anarhichas lupus Gadus morhua Limanda limanda Platichthys flesus Rheinhardtius hippoglossoides Melanogrammus aeglefinus Merluccius merluccius Clupea harengus Clupea harengus membras Trachurus trachurus Molva molva Scomber scombrus Mugilidae spp. Pleuronectes platessa Sebastes marinus Pollachius pollachius Pollachius virens Sardina pilchardus Raja spp. Osmerus eperlanus Solea solea / Solea vulgaris Sprattus sprattus Xiphias gladius Thunnus spp. Thunnus thynnus Thunnus alalunga Psetta maxima Brosme brosme Merlangius merlangus Glyptocephalus cynoglossus

Fat (g/100 g)

Basic facts and figures

Sardine (pilchard) Skate (ray) Smelt Sole Sprat Swordfish Tuna

Taxonomic name

Anchovy Anglerfish Atlantic halibut Catfish (wolffish) Cod Dab Flounder Greenland halibut Haddock Hake Herring Horse mackerel Ling Mackerel Mullet Plaice Redfish Saithe: P. pollachius P. virens Sardine (pilchard) Skate (ray) Smelt Sole Sprat Swordfish Tuna Thunnus thynnus Thunnus alalunga Turbot Tusk Whiting Witch

Thiamin B1

Riboflavin B2

Niacin B3

Vitamin B6

10–70 24 40–78 70–200 50–56 100–103 150–220 46–65 50–70 90–100 40–60 140–180 50–70 100–140 60–65 150–259 50–110 50–170 47–100 10–20 20–30 50–100 60–72 40–80 36–50 160–176 163–241 50–160 20–50 30–50 40–100 89

120–270 60 45–80 60–80 46–110 80–84 200–210 70–80 100–170 80–200 200–300 140–421 80 314–360 138–150 90–220 50–90 100–350 185–200 250–300 10–400 120–140 50–100 150–260 80–82 160–166 80–251 110–160 80–150 60–150 50–200 79

3100–20,000 1683 4000–6000 2200–2400 2000–2300 2300 3000–3500 1300–1500 3100–4000 1600–2900 3756–4500 3400–8320 2300–2500 7500–9400 3800–4033 3500–4000 1200–2500 1290–4000 3270–3500 7400–12933 2000–2500 1450–1900 3000–3500 4167–5000 7600–7947 8500–8519 8500–8640 1200–8500 1100–6100 2300–2800 2000–6100 3424

143–180 165 354–500 300–357 200–230 190–257 250 430–600 120–500 230–880 346–500 310–700 260–304 426–800 424 220–347 230–372 100–400 287–500 270–960 750 120–183 259–314 200–230 330 460–612 450–1000 440–460 152–300 300–387 240 249

Pantothenic acid B5

Biotin B7

773 225 250–400 570–600 200–256 860 680–1000 250–300 200–300 190 940–1000 350–500 300–320 460–1000 300 767–800 360 140–300 380–400 660–758

7 2.0 3.1–5 2.0 2.2 1.2 1.2 2.0 2.4–5.0 4.3 4.5–10 2 4.2–6.4 1.5–7 3 1.2–90 1–11 3.2–7 7–7.2 8.4

4–12.3 11 2.6–12 1 8.0–12 5.0 5–15 12 0.8–13 14 5–10 1 11 1–8 14 11–14 13 10 10 4

640–920 300–322 600–1000 412 660–739 500–1054 420–660 250–1000 120–300 190 325

30 1.2–4.4 6 1.5 1.5 1.5–5 1.5–3 3.2 0.1 4.3 5

5–37 10 5 2 15 15 15 8–16 2 14 11

Folic acid B9

Vitamin B12

Vit. C

0.6–3.3 2.0 0.8–1.1 2–2.2 0.6–1.2 1.5 1.0–3.0 1.0 0.7–2 2.1 8.5–14 4.4–12.7 0.5–0.6 8.4–12 2.2 1.5–10 1–3.8 1–3.5 3.2–4 0.1–17 1 1.8–3.4 0.8–1.0 7 0.6–1.8 4.3–4.5 3.4–9.4 1.7–4.3 0.8–1.7 0.3–1 2.1 1.1

500 1000 1 1000 2000 1000 857–1000 1000 2000 1583 500–667 1000 2000 0–800 600 1500–1633 800–829 1500 1000 400 3000 2000 200 850 1000 2000–2600 5000 Trace 3000 1500 1

Fishery Products: Quality, safety and authenticity

Species

12

Table 1.8b Water-soluble vitamins in marine fish species (μg/100 g edible portion).

Basic facts and figures

13

Table 1.9a Fat-soluble vitamin content in edible part of freshwater fish species (μg/100 g edible portion). Species

Taxonomic name

Brook trout

Salvelinus fontinalis Cyprinus carpio Anguilla anguilla Perca fluviatilis Esox lucius Stizostedion lucioperca Coregonus spp. Salmo salar Tinca tinca Salmo trutta Oncorhynchus mykiss

Carp Eel Perch Pike Pike-perch Pollan Salmon Tench Trout Rainbow trout

Fat (g/100 g) 2.1

Vitamin A 10

Vitamin D 1

Vitamin E

Vitamin K1

100

4.8–4.9 24.5–32.5 0.80–1.3 0.9 0.7

44 600–1800 6.5–7 14–15 1

0.5 20–30 0.2–0.8 2 0.2–0.7

500 2800–8000 1200–1500 850–910 1350–1450

3.0–3.2 7–23 0.7 2.7–3.3 3.3–10.2

21–22 15–41 1 12–32 10–18

1 8–30 0.2 2.1 13.0–32.9

2678–2700 600–4000 100 650–1700 1600–2700

0.2 0.7–2.8

0.08

0.07

The flesh of fatty fish still contains moderate amounts of vitamin A, but lean fish contain only trace amounts. Fish and fish products are commonly regarded as the most important natural food sources of vitamin D. Vitamin D contents differ greatly between species. In general, the higher the fat content of the fish meat, the higher is the vitamin D content. Vitamin E functions as a natural antioxidant to prevent lipids from becoming rancid. Fish flesh is only a low to modest source of vitamin E. Relatively few values for the vitamin K content of fish are available. The highest concentrations are found in muscle of marine and freshwater fish with high fat content, and in the liver. Lean fish contain only very small amounts of vitamin K. Fish muscle, compared with some green vegetables, represents only a minor source of vitamin K.

1.6.2

Water-soluble vitamins

Most fish species cannot synthesise vitamin C (ascorbic acid). The average vitamin C content of fish ranges from 1 to 5 mg per 100 g. These small amounts are without any nutritional significance for humans. The natural thiamin (vitamin B1) content of most fish and fishery products is relatively low. A special problem with some fresh- and seawater fish species is the occurrence of thiaminases. These enzymes cleave the thiamin molecule. They occur especially in the viscera of fish. For example, carp, mackerel and mussels have high thiaminase activity. The enzymes can act during food storage, but they are inactivated by heat, so cooking and smoking destroy them. All plant and animal cells contain riboflavin (vitamin B2), but there are few rich sources. Fish is a modest source. Only the dark muscle, the roe and the liver of several fish species contain higher concentrations of riboflavin.

14

Species Brook trout Carp Eel Perch Pike Pike-perch Pollan Salmon Tench Trout Rainbow trout

Thiamin B1

Riboflavin B2

Nicotinamid B3

Pyridoxin B6

Pantothenic acid B5

100 68–110 150–180 70–75 81–90 160 88 170–230 75 84–100 90–100

70 52–80 40–320 70–120 55–70 250 108 100–170 180 76–140 80–210

4000 1863–3000 2600–3800 1700–4000 1600–1700 2300 3168 6000–8200 4000 3400–3500 3666–5500

980 70–156 244–300 230 140–150 240 367 811–980 290 539 258–700

1000 150–592 140–200 190 240 170 658 1000–2000 670 1700–1950 970–2000

Biotin B7 3.0 8.5 5.1 4.3 2 2.1 10 5–7.4 4.3 4.5 4.5

Folic acid B9

B12

C

26 22–70 12–14 14 6–13 10 9–23 3.4–26 21 9.4–16 8–22

3.0 1.5–3.2 1.0–4.4 1.0 2 1.6 3.2 2.9–6.2 2.1 5 3–5

3000 1000–1086 1000–1800 2000 3725 1000 900 1000 1000 1100 3400

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Table 1.9b Water-soluble vitamin content in edible part of freshwater fish species (μg/100 g edible portion).

Basic facts and figures

15

Seafood is rich in niacin. Lean fish contain lower levels than fatty fish like mackerel, salmon and tuna. Although niacin is equally spread throughout the fish body, liver, roe and sperm can contain higher amounts. With the exception of salmon and trout, fish generally contain only small amounts of pantothenic acid. In fish, the highest concentrations are found in the ovaries and the lowest in the flesh. Fish and shellfish are very good sources of vitamin B6. Mackerel, herring, tuna, sardine and salmon are especially rich in this vitamin. A 200 g portion of fish fillet contains on average approximately 30–60% of the daily requirement of a human. Fish liver contains particularly high concentrations. Fish organs like liver and kidneys contain more folic acid than the flesh. Fish are poor sources compared with plant products. These small amounts are without any nutritional significance for humans. Biotin is found only in small amounts in most fish. One fish meal (200 g) covers 10–20% of the daily requirement of a human. Vitamin B12 originates from synthesis by microorganisms. It is only found in animal foods. Seafood is an important food source. In particular, organs like liver and heart are very rich sources. Dark-fleshed fish like herring and mackerel contain more vitamin B12 than whitefleshed fish like cod and flatfish. The dark muscle of fish usually contains higher levels of the water-soluble vitamins than the white meat. In general, fish meat is a rich source of vitamin D and B12. Many species like Atlantic halibut, herring, mackerel, swordfish, tuna, trout and salmon also contain considerable amounts of niacin and vitamin B6. The higher the fat content of the fish meat, the higher is the content of fat-soluble and some water-soluble vitamins.

1.7

Minerals

There are numerous minerals in fish. The two most important, found predominantly in marine fish species, are the essential elements selenium and iodine. Fish is the only major natural source for these elements. The regular intake of these elements through food is of great importance because the population in many European countries is not sufficiently supplied with these elements. Insufficient iodine can lead to goitre and other diseases. Also, lack of selenium leads to numerous diseases. The iodine content in marine fish varies, according to species, between 50 and 800 μg/100 g. Fish skin is extremely rich in iodine. Freshwater species contain only 5 μg iodine/100 g on average. The selenium concentration in marine fish is 0.35–0.60 mg/kg. The daily requirement of these essential elements can be met by eating a 200 g portion of marine fish.

1.8

Post mortem changes in fish muscle

Directly after death of the fish, a series of biochemical reactions starts, which is of paramount importance for the quality and shelf life of products. These reactions depend on several different factors: the type of fish species, the physiological condition of the fish, as well as

16

Fishery Products: Quality, safety and authenticity

environmental influences (for example water temperature, salinity) the living fish has been exposed to, may influence quality loss and spoilage. In addition, catching and harvesting methods, killing procedures and the performance of slaughtering have a great effect on the biochemical reactions related to disintegration of the fish fillet by gaping for example, or to the so-called ‘tuna burn’, which is a term describing pale and soft flesh of tuna. Details of post mortem metabolism are discussed in most of the following chapters of this book, as changes in the metabolome (for example in nucleotide concentrations) can be used to follow quality loss and spoilage of fish. This introductory chapter only gives an overview of the important biochemical reactions related to quality changes. Even during the catching process, the concentration of ammonium ions in the fillet increases and glycogen stores are reduced. When the fish has been killed, anaerobic glycolysis continues, increasing the concentration of L-lactate in the fillet with a concomitant decrease in the pH value. The concentration of creatine phosphate and adenosine triphosphate (ATP) decreases, and the onset of rigor mortis starts when the concentration of ATP is no longer sufficient to remove the connection between thick (myosin) and thin (actin) filaments of the myofibrils. Freezing of fish or fish fillet directly after catch more or less halts most of the enzymatic reactions, depending on the temperature of the frozen fish. However, during later thawing, chilled storage or further processing of the fish, glycolysis, proteolysis, lipolysis and other enzymatic reactions continue and may result in quality losses. Decrease of water-binding capacity and texture deterioration are examples of the effects of ‘thaw rigor’. The start, extent and length of rigor mortis vary considerably between different fish species. Rigor mortis may last for several days before the fish flesh becomes tender due to the action of endogenous proteases, without, however, reaching the same elasticity as before rigor. Several proteolytic systems, consisting of enzymes and inhibitors, are involved in the degradation of the structural proteins of fish muscle: these are the acid cathepsins located in lysosomes, alkaline proteinases, proteosomes (multicatalytic proteinase complexes), calpains, aminopeptidases, collagenases and elastases. Enzymes bound in cell organelles or located in the cell membranes are gradually released during storage of fish in melting ice or at higher temperatures in the refrigerator. Fish muscle lysosomes contain a multitude of hydrolytic enzymes apart from cathepsins, which may influence metabolite changes of fresh fish. Mitochondrial enzymes are involved in ATP degradation and the increase of calcium ions (Ca2+) in the sarcoplasma. The heavy destruction of cell organelles during freezing and thawing has been used to develop methods of distinguishing fresh from thawed fish (see Chapter 16). Storage of fish being in rigor at elevated temperatures (for example 17 °C for Atlantic cod, Gadus morhua) results in heavy shortening of the muscle. The connective tissue becomes partly denatured, and the connections between the myocommata and the muscle fibres are removed. The result is ‘gaping’, which is characterised by fissures and cracks in the fillet. As the muscle tissue of living marine fish is sterile, the quality of fish stored in melting ice is initially mainly influenced by autolytic reactions. ATP is degraded in several steps into hypoxanthine and ribose or ribose phosphate. Formation of hypoxanthine can be used as a freshness indicator, as discussed in detail in Chapter 4. Gadoid fishes, like cod, hake (Merluccius spp.) or Alaska pollack (Theragra chalcogramma), contain the enzyme trimethylamine oxide demethylase (TMAOase), which catalyses the cleavage of trimethylamine oxide (TMAO) into dimethylamine (DMA) and

Basic facts and figures

17

formaldehyde (FA). Gadoid fish produce DMA and FA during processing (for example filleting) or in the first days of storage in melting ice. Later TMAOase activity is inhibited by oxygen, and the reaction ends after a few days. Later (±10 days after catch), bacterial reduction of TMAO to trimethylamine (TMA) starts, a process that dominates during further storage of the fish or fillet. TMA is responsible for the fishy flavour of spoiled marine fish. TMA and ammonia are the main components of the ‘total volatile basic nitrogen’ (TVBN) fraction of chilled stored fish. The TVBN fraction is obtained by steam distillation of alkalised fish muscle or fish muscle extract. Fish exceeding certain limits of the TVBN value is considered unsuitable for human consumption when sensory assessment leads to the same conclusion. Ammonia is the main component of the TVBN fraction in the first phase of storage lasting 1 to 2 weeks, depending on species and storage conditions, whereas the rise of TMA is correlated to spoilage of the fish (see Chapter 2). Directly after the catch, the muscle tissue of healthy marine fish is free from bacteria, but not the gills, skin and intestines. The bacteria penetrate into the fillet mainly through the gills and body cavity during storage and processing, accompanied by changes in the composition of the bacterial flora. Gram-negative psychrotrophic rods (Shewanella spp., Pseudomonas spp., Vibrio spp. and Aeromonas spp.) are important spoilage bacteria (see Chapter 15). Bacteria are also responsible for the formation of biogenic amines from precursor amino acids in spoiling fish by decarboxylation. Histamine is produced from histidine, cadaverine from lysine, putrescine from ornithine, tryptamine from tryptophane, tyramine from tyrosine and agmatine from arginine. Dark-fleshed fish species, like scombroids (for example tuna, mackerel) or some clupeids (for example anchovy), which contain high concentrations of histidine, are especially important histamine formers if stored at higher temperatures (see Chapter 3). Fish lipids are not stable during storage at any temperature. Lipolysis and lipid oxidation may occur in chilled or frozen fish, leading to unpleasant flavours and tastes caused by carbonyl compounds and short-chain carbonic acids. Binding of free fatty acids to fish muscle proteins may result in texture deterioration. Speed and extent of lipolysis and of oxidation of unsaturated fatty acids are higher in dark muscle than in white muscle, like many other biochemical reactions in the fillet post mortem. The activity of enzymes (for example lysosomal enzymes, TMAOase) and other proteins like myoglobin, as well as the concentration of metal ions (for example Fe2+/3+), relevant to spoilage is higher in red than in white muscle. During frozen storage, lipid degradation by lipolysis (lipases, phospholipases) is not completely stopped but continues at a lower rate, leading to increased concentrations of free fatty acids, which can be used as quality indicators.

1.9 References and further reading Anon (2001) What’s so healthy about seafood – a guide for seafood marketers. Fisheries Research and Development Corporation, Australia, 37 pp. Danish Food Composition Databank www.foodcomp.dk. Delbarre-Ladrat, C., Cheret, R., Taylor, R. and Verrez-Bagnis, V. (2006) Trends in postmortem aging in fish: understanding of proteolysis and disorganization of the myofibrillar structure. Critical Reviews in Food Science and Nutrition 46: 409–421.

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Fraser, O. and Sumar, S. (1998a) Compositional changes in fish – an introduction. Nutrition & Food Science 5: 275–279. Fraser, O. and Sumar, S. (1998b) Compositional changes in fish (part II) – microbiological induced deterioration. Nutrition & Food Science 6: 325–329. Gordon, D.T. and Martin, R.E. (1982) In: R.E. Martin, G.F. Flick, C.E. Hebard and D.R. Ward (Eds.): Chemistry and Biochemistry of Marine Food Products, AVI Publishing Company, Westport, pp. 429–445. Huss, H.H. (1994) Assurance of seafood quality. FAO Fisheries Technical Paper 334, FAO, Rome, 169 pp. Huss, H.H., Ababouch, L. and Gram, L. (2004) Assessment and management of seafood safety and quality. FAO Fisheries Technical Paper 444, FAO, Rome, 230 pp. Lall, S.P. and Parazo, M.P. (1995) Vitamins in fish and shellfish. In: A. Ruiter (Ed.) Fish and Fishery Products, CAB International, New York, pp. 157–186. Nettleton, J. (Ed.) (1985) Seafood Nutrition: Facts, Issues and Marketing of Nutrition in Fish and Shellfish, Osprey Books, Huntington, pp. 44–54. Oehlenschläger, J. (2006) Cholesterol content in seafood, data from the last decade: A review. In J.B. Luten, C. Jacobsen, K. Bekaert, A. Saeboe and J. Ochlenschläger (Eds.): Seafood research from fish to dish, Quality, safety and processing of wild and farmed fish, Wageningen Academic Publishers, 41–57. Ostermeyer, U. and Schmidt, T. (2001) Determination of vitamin K in the edible part of fish by highperformance liquid chromatography. European Food Research and Technology 212: 518–528. Ostermeyer, U. and Schmidt, T. (2006) Vitamin D and provitamin D in fish. European Food Research and Technology 222: 403–413. Souci, S.W., Fachmann, W. and Kraut, H. (2008) Food Composition and Nutrition Tables, 7th edn. Medpharm Scientific Publishers, Stuttgart. Valfre, F., Caprino, F. and Turchini, G.M. (2003) The health benefit of seafood. Veterinary Research Communications 27 (Suppl. 1): 507–512.

Chapter 2

Traditional methods Peter Howgate

2.1

Introduction

The systematic study of the spoilage of fish and of its measurement began in the early years of the last century. Anderson (1908) described in detail the changes in sensory attributes of spoiling fish and reported some microbiological measurements, but did not measure any chemical changes. Clark and Almy (1917) discussed various chemical tests that might be used to monitor spoilage of fish and evaluated some of them with model solutions, but did not apply any of them to samples of fish. The first reference to the use of a chemical test for evaluation of the quality of a fishery product seems to be Weber and Wilson (1919), who reported using ammonia content and what they called ‘total volatile nitrogen’ content as indices of freshness of canned sardines from as early as 1913. Tillmans and Otto (1924) evaluated several chemical tests based on nitrogen-containing compounds for their potential to measure the spoilage of fish, but concentrated on what they called ‘ammoniak’, ammonia, though their ‘ammoniak’ included methylamines and was equivalent to Weber and Wilson’s ‘total volatile nitrogen’. This test, now more commonly referred to, in English anyway, as total volatile bases (TVB) has been by far the most commonly used chemical index of spoilage for a wide range of species of fish up to the present time, and will form a large part of this review. There was not much development of procedures for measuring freshness after that (Boury and Schvinte 1935; Reay 1935) until the burst of activity in research in fish processing technology in the years just after the Second World War when improved methods for analysis of amines were developed, and new principles for measurement of freshness based on analysis of nucleotides were introduced. Farber (1965) is a review with a comprehensive bibliography of methods used for measuring freshness up to the early 1960s, and Oehlenschläger (1997a) is a review of literature on volatile amines as measures of spoilage. The purpose of this chapter is to summarise and review the chemical tests introduced in early studies of spoilage of fish up to about the middle of the last century, and which can now be considered as ‘traditional’ methods, and to consider to what extent, if at all, they can still be considered as effective measures of quality in present day inspection and quality assurance of fishery products. The word ‘fish’ in the following discussion should be construed to include vertebrate and invertebrate species unless otherwise specified. 19

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Fishery Products: Quality, safety and authenticity

The procedures discussed here do not directly measure the safety of fishery products, though in some circumstances spoilage of a product might be associated with a hazard to the health of a consumer.

2.2 2.2.1

TVB-N Measurement of TVB

One of the reasons why TVB has been so popular as a measure of spoilage of fishery products, far more so than other traditional methods, is undoubtedly the simplicity of the analytical procedure, and the low level of resources, laboratory and human, required for the measurement. The principle is quite straightforward: a suspension of fish muscle or an extract of fish muscle is made alkaline and the free bases are distilled, usually at boiling point at atmospheric pressure, collected, and estimated using standardised acid or alkali. The measurements can be performed by relatively inexperienced analysts using standard laboratory glassware or equipment. TVB contains ammonia, trimethylamine (TMA) and dimethylamine (DMA), and can be estimated by measuring the concentrations of the individual components and summing them, although this detracts from the simplicity and low costs of the traditional procedures. Because the amines comprising TVB contain one atom of nitrogen per molecule, it is conventional to express TVB, and constituent amines and ammonia, on a nitrogen basis, e.g. TVB nitrogen (TVBN). Although the principles of the analytical procedure can be simply stated, and though the method has been in use for nearly a century now, it has proved very difficult to develop an accepted standardised practical procedure for its measurement. The problem resides in the fact that fish muscle and extracts of muscle have nitrogencontaining substances other than those contributing to the intrinsic TVB in the sample. These substances can decompose during the analysis, depending on the conditions, producing ammonia, which contributes to the measured TVB content. Low alkalinity and low temperature during distillation results in no or negligible decomposition, but increases the time for its analysis or makes the procedure more complex, whereas high temperature and strong alkalinity favours decomposition and overestimation of TVB content, but allows for simpler equipment and fast analysis. The literature on TVB determination in fish is replete with reports of new or revised procedures for its measurement and with comparisons of procedures, but still without resolving the problem. Formation of decomposition ammonia during distillation at room temperature or a little above is negligible. Two of the early papers cited above, Clark and Almy (1917) and Weber and Wilson (1919), used aeration at room temperature as their procedure. Stansby et al. (1944) included aeration in their comparison of methods for determination of TVB. In this method, a suspension of a small amount of sample is made alkaline and the liberated bases purged from the suspension by a current of air to be trapped in standard acid and measured. Apart from the complexity of the apparatus needed, the procedure requires 4–5 hours for the aeration step, and the method has not been adopted for routine use for measuring TVB. Distillation at low temperature at atmospheric pressure can be effected by microdiffusion in the Conway cell (Conway 1950). The first explicit account of the use of the microdiffusion procedure for TVB measurement seems to be Stansby et al. (1944), although Beatty

Traditional methods

21

and Gibbons (1937) had earlier used the procedure to measure TMA. The time for complete recovery of TVB in the Conway cell is 2 hours at room temperature or one hour at 37 °C, but these times are acceptable in practice. The difficulties of the procedure are in the care necessary for accurate and precise results. The central well contains only 1.0 ml of absorbing acid and the back titration is usually less than that, so the analyst must be experienced in accurately titrating small volumes of solution using a microburette. Other aspects of the procedure require scrupulous attention to detail. Spinelli (1964) and Shewan et al. (1971) have discussed the precautions required for accurate results. The advantages of the Conway method for determination of TVB are that the result gives the true intrinsic content of TVB in the sample because there is no concomitant decomposition of nitrogenous substances under the conditions of measurement, and the apparatus required for the analysis is cheap. In the circumstances, it is therefore perhaps not surprising that the method is popular; a survey performed by this reviewer of a 100 or so reports on TVB contents in spoiling fish revealed that the Conway procedure was the most frequently used method for its measurement. Distillation at around 50 °C under reduced pressure using low pH alkalisers such as magnesium oxide, MgO, does not result in appreciable formation of decomposition ammonia (Tillmans and Otto 1924; Boury and Schvinte 1935; Hjorth-Hansen and Bakken 1947; Tomiyama and Harada 1952; Tomiyama et al. 1956; Pearson and Muslemuddin 1968) at the expense of complexity of the apparatus required and the long time taken to dismantle and reassemble the apparatus between analyses. Distillation at reduced pressure does not appear to have been used often as a method for determining TVB other than in the laboratories describing the procedures. Distillation of the bases is much faster when performed at higher temperatures, for example at boiling point at atmospheric pressure. Apart from speed, an advantage of this approach is that distillation can be performed in apparatus used in the distillation step of the determination of nitrogen by the Kjeldahl method, equipment that is readily available in food laboratories. There are broadly two approaches to the distillation: direct distillation of an aqueous suspension of fish muscle in macro-scale Kjeldahl distillation apparatus; and steam distillation of a protein-free extract of fish muscle using semi-micro apparatus. The first approach is described in detail in Lücke and Geidel (1935) and is usually referred to as the Lücke and Geidel (L&G) method (not always spelled correctly), though it is based on a procedure previously described in Tillmans and Mildner (1916). In the L&G procedure, 10 g of a blended sample of fish muscle is suspended in 300 ml of water, made alkaline with 1–2 g of MgO, and distilled. The rate of heating is adjusted so that the suspension reaches boiling point in 10 minutes and the distillation is performed for 25 minutes. There is no commonly accepted procedure for determination of TVB using protein-free extracts, and many variations of the basic procedure have been described. The common precipitants are 7.5% trichloroacetic acid and 0.6 M perchloric acid, although ratios of sample weight to precipitant volume used range widely. It is convenient to use semi-micro stills such as the Markham still or the Hopkins apparatus that empty the contents of the distilling tube when the distillation is completed. Typically the distillation times in such apparatus is about 10 minutes. Since the 1970s, rapid distillation units have been developed for the Kjeldahl analysis which can also be used for determination of TVB. The first account of their use for this purpose seems to be that of Billon et al. (1979), but since then distillation units from different suppliers have been cited in several papers on studies of TVB in fish. Distillation times in these units are of the order of 5–10 minutes.

22

2.2.2

Fishery Products: Quality, safety and authenticity

Formation of decomposition ammonia during measurement of TVB

Although distillation at high temperature compared with distillation at low temperature has the advantage of speed, it does mean there is appreciable formation of decomposition ammonia and the TVB value obtained is higher than the true, intrinsic, value. Proposed procedures then attempt to optimise full recovery of the TVB while minimising formation of decomposition ammonia. Several factors must be considered. The distilling mix must be alkaline enough to liberate the bases in their un-ionised form, but increasing alkalinity promotes decomposition. The acid dissociation constant (pKa) values of ammonia, TMA and DMA at 25 °C are 9.25, 9.81 and 10.73 respectively, and the pH of a suspension of MgO, the alkaliser used in the L&G procedure, in water is around 10.5 at room temperature. At this temperature approximately 93, 80 and 32% of the ammonia, TMA and DMA are in the un-ionised form. It is not necessary for a base to be all in the free base form for all of it to distil over – the system is in equilibrium and as undissociated base is removed, dissociated base become undissociated – but the distillation rate is slowed when an appreciable portion of the base is in the dissociated form. However, pKa is temperature dependent, and in the case of ammonia becomes lower as temperature rises (Emerson et al. 1975). Assuming that this condition applies to the methylamines means that higher proportions of the bases than quoted above are in the un-ionised form at the distillation temperature. Another factor that affects rate of distillation is the volatilities of the bases, a property measured by the Henry’s law constant for the base. Egnér and Johansson (1938) made a study of the thermodynamics of the distillation of ammonia and showed that the proportion of a substance distilled over by both direct distillation or by steam distillation is related to the proportion of water in the distilling mix distilled over and to the value of the Henry’s law coefficient. Hjorth-Hansen and Bakken (1947) measured the time course of distillation of ammonia and methylamines in the L&G procedure and showed that the observed time course of distillation of the bases agreed with the Egnér and Johansson’s (1938) expression. Their investigations showed that TMA is the most volatile of the bases, followed by ammonia then DMA. From Egnér and Johansson’s (1938) equations, it can be calculated that more than 99% of the bases are distilled over by either direct or steam distillation when 30% of the water has distilled over in the case of direct distillation, or 30% of the original volume of aliquot in the distilling unit has distilled over in the case of steam distillation. Although there is a large literature on procedures for determination of TVB by distillation, there is very little on details of recovery of the bases and on rates of formation of decomposition ammonia. Pearson and Muslemuddin (1968, 1969a,b) measured the cumulative TVB content during distillation by the L&G procedure up to and beyond the specified 25 minutes distillation time for three species of fish and four freshnesses within each. They showed that after 25 minutes the TVB content increased linearly with time, suggesting that by this time all of the methylamines had been distilled followed by ammonia formed by decomposition processes. The average rate in this time over all samples was 0.34 mg N/100 g/min, and the 25 minutes distillation time specified in the L&G procedure would be expected to result in an overestimate of the true TVB content by around 8–9 mg N/100 g. Egnér and Johansson’s (1938) equation shows that it is the proportion of the initial volume distilled over that matters.If the sample is suspended in 200 ml of water rather than the 300 ml specified, the

Traditional methods

23

distillation time can be reduced to 17 minutes and the overestimate reduced to around 6 mg N/100 g. The steam distillation glassware used in the semi-micro Kjeldahl nitrogen determination is not amenable to solid samples, but Antonacopoulos (1960) describes an apparatus for steam distillation of solid foodstuffs that has been extensively used for determination TVB in fish. The minced fish sample is washed into the distilling tube with a small amount of water, MgO added, and the mix distilled at 10 ml/min for 12 minutes, giving a distillation volume of 120 ml. The amount of decomposition ammonia under these conditions would be around 3.5 mg N/100 g. If the volume in the distilling tube of the apparatus is as much as 50 ml – it is likely to be less than this – the required volume of distillate for recovery of the bases using Egnér and Johansson’s (1938) equation is 15 ml, an amount that could be achieved in less than 2 minutes, resulting in decomposition ammonia equivalent to 0.7 mg N/100 ml. Amide groups in the muscle proteins are the main source of decomposition ammonia in the distillation of muscle tissue. This source is eliminated by the use of protein-free extracts. The distillation is conveniently performed in a semi-micro Kjeldahl distillation apparatus with a solution of alkali, usually sodium hydroxide, as the alkalising agent. Although the free amide content of fish muscle is low and its decomposition would contribute only negligible amounts to TVB by this method, there are other nitrogenous components present in the extract that are decomposed by the strong alkali to produce ammonia. Little is known of the nature and amounts of these compounds, but the rate of formation of decomposition ammonia is about the same as that by the L&G procedure, depending on species, strength of alkali and possibly the staleness of the fish (Rehbein and Oehlenschläger 1988; Oehlenschläger 1988). Distillation of protein-free extracts at a pH below 10.0, measured at room temperature (Oehlenschläger, 1988) or with MgO (Pearson and Muslemuddin 1969a), does not result in formation of decomposition ammonia. It is clear from the examples discussed above that the value of TVB obtained in an analysis is dependent on the procedure used, and that variations in analytical conditions will result in variations in the result. Although it might not be difficult to standardise procedures within a laboratory, it does not appear easy to do so between laboratories judging from reports of collaborative trials (Vyncke et al. 1987; Antonacopoulos and Vyncke 1989). The error due to variation in the amounts of decomposition ammonia in the analytical procedure would be eliminated if a procedure that did not result in decomposition were used. The Conway procedure fulfils this requirement, but has some drawbacks. Alternatively, distillation of a protein-free extract with a buffer below a pH of 10.0 or with MgO could be used. There are some practical difficulties using solid MgO with semi-micro nitrogen distillation apparatuses, but not with the rapid distillation units increasingly being used in food laboratories. The reviewer is aware of only one report of this approach to measuring TVB (Hjartarson et al. 1998), but he believes it is worthy of further evaluation.

2.3 2.3.1

Methylamines Determination of TMA and DMA

It has been known from the early days of measuring TVB in fish that the TMA is a component of the mixture of bases (for example Weber and Wilson (1919)). However, in the early

24

Fishery Products: Quality, safety and authenticity

decades of the century, TMA was determined by a tedious analytical procedure based on separate measurement of ammonia and selective decomposition of the amines with nitrous acid (Weber and Wilson 1918; Okoloff 1932). The procedures are also described in the reviews by Hjorth-Hansen and Bakken (1947) and by Boury and Schvinte (1935). A much simpler method of analysis was introduced by Beatty and Gibbons (1937) using the Conway cell, in which formaldehyde is added to the sample and saturated potassium carbonate is used as the alkaliser. Under these conditions the formaldehyde complexes the ammonia and dimethylamine to leave to TMA to distil over into the central well. The method has the same advantages and disadvantages as the Conway method for determination of TVB, and it has largely been displaced by the picrate method developed by Dyer (1945). In the picrate procedure, an aliquot of a trichloroacetic extract of fish muscle is taken, formaldehyde added, and the mixture made alkaline. The free bases are extracted into toluene and reacted with picric acid to give a yellow-coloured picrate salt that can be measured in a photometer. In the original Dyer (1945) method, saturated potassium carbonate was used as the alkaliser, but later studies showed that potassium hydroxide was more efficient in suppressing interference from dimethylamine and in giving higher recoveries of TMA (Hashimoto and Okaichi 1957; Shewan et al. 1971; Tozawa et al. 1971; Keay and Hardy 1972; Murray and Gibson 1972; Bullard and Collins 1980). The equipment required for the picrate method is quite cheap and the procedure can be performed by any competent laboratory assistant, which makes it suitable for use by laboratories in the fish processing industry as well as in research. The most expensive item is a photometer, but a reasonably accurate estimate of optical density can be achieved by comparing the test sample against a suitable range of standards. Amines were among the first groups of compounds to be separated by gas–liquid chromatography (GLC) when the technique was invented in the early 1950s.The first application to fishery products seems to be Hughes (1959). Since then, many analytical procedures have been published and a variety of columns and column packings have been described for effecting the separation. The advantages of GLC procedures are that they are specific for individual amines; the disadvantages are the cost of the equipment and the need for analysts trained in its use. Another approach to the determination of TMA is to use the property of sequestration of ammonia and lower methylamines with formaldehyde, as in the Conway method referred to above. TMA can be determined by adding formaldehyde solution to the neutralised distillate from a TVB determination (the bases must be distilled into standard acid, not boric acid), and back-titrating the released acid (Analytical Methods Committee 1979). The difference between the original TVB titration and the formol titration is equivalent to the TMA content. This procedure is a convenient extension to the normal TVB determination, but because the TMA content is based on the difference between two large values, the titrations must be performed carefully to obtain accurate and precise results. Malle and Tao (1987) describe a method for determination of TMA by adding formaldehyde to a TCA extract before making it alkaline with NaOH and distilling as for TVB measurement. Stansby et al. (1944) also allude to using this method, but give no details or results, and Norris and Benoit (1945) used it in their procedure for measuring TMAO. Malle and Tao (1987) claim that the formaldehyde suppresses distillation of ammonia and DMA, but an analysis of the data tabulated in the paper shows that their procedure overestimates TMA content by about 20% compared with the Conway method. Although the Malle and Tao (1987) procedure has the merit of

Traditional methods

25

simplicity, it should be used with caution because of the likelihood of overestimation of TMA content. Analytical methods for TMA determination based on other principles have been described – the difference between using potassium carbonate and potassium hydroxide in the picrate method (Castell et al. 1974), specific ion electrode (Chang et al. 1976), high-performance liquid chromatography (HPLC) (Gill and Thompson 1984), enzymic (Wong and Gill 1987), pH-sensitive test strip (Wong et al. 1988), gas sensor (Ohashi 1991), flow injection (Sadok et al. 1996) – but these methods seem more suited to use in research laboratories and need further evaluation before possible use in commercial quality assurance. DMA is usually measured as the dithiocarbamate by a procedure first applied to fish by Dyer and Mounsey (1945) and later modified to replace benzene as the solvent by chloroform (Mackie and Thomson 1974), or by GLC.

2.3.2

Formation of ammonia and methylamines in spoiling fish

The many published studies of TVB in spoiling fish are consistent in reporting that the bases comprise predominately ammonia and TMA with, in some species, a small contribution of DMA. Extensive microbiological investigations have shown that the TMA is formed by reduction of trimethylamine oxide (TMAO) by some species in the bacteriological flora of spoiling fish (Barrett and Kwan 1985; Gram and Huss 1996; Gram and Dalgaard 2002). DMA is formed by enzymatic splitting of TMAO into DMA and formaldehyde. The process is active only in some species of fish, mainly gadoids (Hebard et al. 1982). Ammonia is present shortly after death in all species of fish from the deamination of adenosine monophosphate to inosine monophosphate, and more is formed in later stages of spoilage by the action of proteolytic bacteria. A comprehensive summary of the typical changes of TMA, DMA and ammonia in ice-stored Atlantic cod (Gadus morhua) is given in Oehlenschläger (1997b). The figures in the paper show an initial ammonia concentration of about 8 mg N/100 g, remaining at this value until about 15 days of storage when it increases approximately exponentially to 14 mg N/100 g of muscle at 23 days. The DMA concentration is shown increasing linearly from zero at the start of storage to 3 mg N/100 g at 23 days of storage. The initial concentration of TMA is 0.3 mg N/100 g, remaining at this value until 10 days in ice when it increases exponentially with storage time to 44 mg N/100 g at 23 days. The TVB content is initially 13 mg N/100 g and rises slightly until 8 days in ice when it then increases exponentially to 63 mg N/100 g at 23 days of storage. The increase in TVB during storage is predominately due to the increase in TMA. When the TVB concentrations reported in the Oehlenschläger (1997b) study are corrected for the increases in ammonia and DMA during storage, the slope of the functional relation between corrected TVB and TMA is not significantly different from 1.0. This result, and values of TVB and TMA concentrations from other similar studies, show that TVB content can be accurately predicted, at least in vertebrate fish, from the expression TVB = TMA + c where c is a constant. When TVB is determined without decomposition of nitrogenous substance, c is the intrinsic concentration of ammonia, which differs among species, but typically is in the range 6–10 mg N/100 g of muscle tissue. In the Oehlenschläger (1997b) study, c = 15 mg N/100 g which, after correcting for the intrinsic ammonia concentration, gives the contribution of decomposition ammonia as 7 mg N/100 g

26

Fishery Products: Quality, safety and authenticity

Concentration in muscle (mg N/100 g)

60 50 TMA DMA Ammonia TVB

40 30 20 10 0 0

5

10 15 Storage time at 0°C (days)

20

Figure 2.1 Changes in concentrations of ammonia, methylamines and TVB in muscle tissue of typical demersal marine fish stored at 0 °C. The lines are based on results from several sources of data and should be considered representative of demersal marine species such as gadoids rather than indicating any particular set of results. The TVB line is representative of results obtained by distillation of a protein-free extract with strong alkali.

(the TVB was determined by steam distillation of a perchloric acid extract). The changes in concentrations of ammonia, methylamines and TVB that can be expected in typical demersal marine fish are shown in Figure 2.1. In a closed system, the increase in TMA content is matched by a stoichiometrically equivalent loss of TMAO (Beatty 1938) and the sum of TMAO and TMA, on a nitrogen basis is constant during storage. However, fish stowed in boxes of ice is not a closed system, and TMAO and its decomposition products can be lost by leaching. This process is apparent in the data of Figure 7 of Oehlenschläger (1997b), which shows the TMAO concentrations decreasing by about 3.5 mg N/100 g/day over the first 12 days of storage then by 6 mg N/100 g/day overall for the rest of the storage period. The first period of loss of TMAO can be attributed to leaching, the second to the combination of leaching and of reduction to TMA. The sum of TMAO and TMA concentrations shows a trend for the values after 12 days of storage to continue the rate of change before that time. The rate of loss of the sum over the total storage time is equivalent to 2.45%/day cumulative. Although there are very many publications reporting TVB and TMA concentrations in spoiling vertebrate fish, very few also include measurement of TMAO concentrations and it is not possible to estimate the size of any leaching as has been done here for the Oehlenschläger (1997b) data. Howgate (2006) reviewed the kinetics of the degradation of inosine monophosphate in iced fish and found that in about half of the 53 data sets studied the effect of leaching was significant in the mathematical model used to evaluate the kinetics. There is no doubt that leaching can, and does, occur during typical storage of fish in ice – many of the published figures or data sets in reports of spoiling fish show a decrease in TVB concentrations in the first few days of storage – but it is rarely commented on in the reports or taken into account when discussing quantitative changes during storage.

Traditional methods

27

The formation of TMA during spoilage requires that the muscle should contain sufficient amounts of TMAO for TMA to be formed. Typically, muscle tissue of fish from the marine environment contains adequate amounts (Hebard et al. 1982) and consequently TMA is almost always found in spoiling marine fish. Where the fish contains large amounts of TMAO, the maximum TMA formed is dependent on the growth of relevant microorganisms rather than availability of substrate. In this case, TMA concentrations can exceed 50 mg TMA nitrogen (TMAN)/100 g of muscle tissue when very spoiled. Some species of marine fish, for example many pelagic species, have only low concentrations of TMAO in the muscle tissue, and TMA concentrations in very spoiled fish may be less than 20 mg TMAN/100 g. Muscle tissue of fishery products from freshwaters typically have no, or only low, concentrations of TMAO, although there are many exceptions (Hebard et al. 1982; Anthoni et al. 1990). The production of ammonia only at late stages of spoilage is true for non-elasmobranch vertebrate fish; in elasmobranchs, large amounts of ammonia are formed by enzymatic degradation of the urea which is a feature of the biochemistry of this group of fish. In addition, crustaceans at least among the invertebrates have high intrinsic concentrations of ammonia in the muscle tissue as well as enzymes that produce even more (Yeh et al. 1978). It has already been mentioned that DMA is produced in the muscle of some species of fish by enzymatic splitting of TMAO and that low concentrations of the amine are formed in chill-stored fish of these species. However, the amount produced is very variable within and among species (Hebard et al. 1982). Almost invariably studies of TMA formation in stored fish show an initial dwell during which TMA, and TVB, contents do not increase significantly followed by an exponential increase in concentration. The exponential growth phase can be linearised by converting values to their logarithms. For example, Hoogland (1958) used the TMA index (TMAI), defined as TMAI = log10 (TMA + 1) in the freshness grading of fish. The logarithmic transformation also equalises the standard deviations (which increase with increasing values of TMA) around the line. The constant 1 is added to the TMA concentration to avoid negative values of the index. A similar index can be used for TVB concentrations (Shewan and Ehrenberg 1957). A plot of TMAI against storage time shows a linear increase after the dwell period – around 7 days or so in well-iced demersal species – until advanced spoilage.

2.3.3

Sources of variance in measured TMA and TVB contents

TMA measured in protein-free extracts is obtained by blending a sample with a proteinprecipitating solution and the TMA content determined in an aliquot of the filtrate. The amount in the aliquot is scaled up to concentrations expressed as milligrams of TMA nitrogen/100 g of sample, but procedures differ as to how the scaling factor is derived. One approach assumes the bases are distributed in the whole mass of extract mixture, that is the sum of sample weight and extractant volume, and the concentration is scaled from the aliquot to this amount. The other assumes the bases are distributed in the aqueous phase of the mix,

28

Fishery Products: Quality, safety and authenticity

that is the sum of the volume of extractant and the volume of water in the sample, and the concentration is scaled to this amount from the aliquot. For example, a common extraction procedure is to blend 100 g of sample with 200 ml of extractant. In the first case above, the TMA content of the aliquot would be scaled by 300/v, where v is volume of aliquot, whereas in the second case the scaling fraction would be 280/v assuming the water content of the sample is 80%. The first procedure will overestimate the TMA content by 7% compared with the second. The same consideration applies to those analytical methods for TVB content that are based on distillation of extracts. Boury and Schvinte (1935) appear to be the first to point out explicitly that the amines are distributed in the water phase. They drew attention to the need to take the water content of the muscle into account when calculating TVB content using extracts. Unfortunately, very few of the publications on TVB or TMA in fish explicitly state the basis of calculating concentrations, or they cite references in which the calculation is not stated. Dyer (1959) provides a detailed description of the picrate procedure, and there the calculation scales up from the aliquot by the total mass of extractant plus sample. On the other hand, the procedures recommended by the Analytical Methods Committee (1979) for TVB and for TMA scale by the volume of the total aqueous phase. Some authors refer to a ‘Codex’ method for determination of TVB, which was presented to the Codex Committee on Fish and Fishery Products in 1968 for consideration as a recommended procedure (though the proposal was not adopted by the Committee and the methodology does not appear in lists of Codex Alimentarius documents). The experimental procedure is described in detail in Vyncke et al. (1987), but the paper does not describe the calculation. Nor do any other publications that cite the ‘Codex’ procedure, but the author is informed that it is based on the total mass. The European Commission (1995) has issued a recommended procedure for determination of TVB based on rapid distillation of a perchloric acid extract. The calculation step scales the TVB from the aliquot by the total mass of the extraction mixture rather than the volume of the water phase, but in this case the bias is only 2% because the ratio of sample to extractant is 1 : 9. Other examples of papers in which the authors report their calculation of concentration and have used the total mass of extraction mix in scaling up the aliquot include Billon et al. (1979), Malle and Tao (1987), Antonacopoulos and Vyncke (1989), Civera et al. (1993); examples in which the volume of water phase is used include Lundstrom and Racicot (1983) and Gill and Thompson (1984). It seems surprising, and disappointing, to this reviewer that after several decades of using protein-free extracts for the measurement of TMA and of TVB there is not agreement on how concentrations in the muscle tissue should be calculated. The opinion of this reviewer is that the amines should be considered as being distributed in just the aqueous phase, and the correct scaling factor to use is the total water content of the mix. There is a large bibliography of studies of TVB and TMA concentrations in fish muscle and the effects of storage on them. A noticeable feature of the results is the high variance associated with the measurements. Part of this variance is due to variations in procedures, even when nominally standard methods are used, on fixed samples, as evidenced by the rather poor outcomes of collaborative trials (Vyncke et al. 1987; Antonacopoulos and Vyncke 1989; Ellis et al. 2000). TVB is the sum of the concentrations of ammonia and methylamines, and variances in concentrations of each of the components contribute additively to the overall variance. The intrinsic initial ammonia concentrations vary within a species (Beatty and Gibbons 1937) and so do the compounds that decompose during analysis to ammonia.

Traditional methods

29

The increase in TMA during spoilage, which accounts almost entirely for the increase in TVB, is the result of microbiological activity of some species of microorganisms in the bacterial flora on fish. Initial loads of these responsible organisms, and growth of them, would be expected to vary with biological factors such as season, fishing grounds and species, post-rigor pH, all of which will add to the variance of TMA and TVB content. Although these sources of variation are often referred to in writings about TMA and TVB, there are almost no quantitative data on the size of the variances. The most detailed study of biological factors on formation of TMA in iced fish is reported in Burt et al. (1975). In this study, 14 batches of cod from different fishing grounds in the North Sea and caught at various time of the year were carefully stored in boxes in ice and sampled at intervals up to 20 days of storage. Spoilage was measured by various sensory and non-sensory methods including TMA, but not TVB. It was found that regressions of TMAI on storage time after the dwell period of seven days for the various batches were significantly different from each other. The original paper should be consulted for details of sources of variance, but as a summary, the storage time predicted from the mean TMA concentration of sample of six fish from a box using the overall regression of TMAI on storage time for all grounds and seasons had a prediction error of ±4 days (95% confidence limits). This error in predicting storage time from TMAI is valid only for the set of samples and for the sampling protocol from which the prediction equation was derived, and might not be correct in other situations. However, it gives a feel for the error of prediction of storage time from TMA content; the error would be greater in the case of TVB contents. The effects of leaching already discussed above have also been commented on (Shewan and Liston 1956; Oehlenschläger 1997b).However, other than Karnop (1976) and LapaGuimarães et al. (2005), rates of leaching have not been reported. As discussed above, the rate of leaching can be appreciable even under good storage practice and could differ quite markedly among conditions of storage: storage of iced fish under ambient temperature close to 0 °C would result in very low rates of leaching, if any, whereas storage at high ambient temperature would result in high rates of melting of ice and consequently high rates of leaching. Storage in slush ice or refrigerated water systems could lead to high rates of leaching. It is very common for reported data on TVB concentrations to show a decrease over the first few days of storage, and this dip can be, and has been, attributed to leaching.

2.4

Volatile acids

The determination of total volatile acids (TVA) can be considered the obverse of TVB. A suspension of muscle tissue or an extract of it is made acidic, distilled, and the acidic substances recovered quantitatively measured. TVA as a measure of spoilage is not discussed in early reviews of the subject though Farber (1965) cites papers by Tillmans and colleagues in 1927 and by Boury in 1934 and 1935. Most information on TVA and spoilage comes from a series of papers by Hillig and co-workers between 1938 and 1960 (see Hillig et al. (1960) for a summary), and a few other papers since then (Sigurdsson 1947; Lobben and Lee 1968; Quaranta and Curzio 1983; Hollingworth et al. 1990, 1991). TVA can be determined using ordinary laboratory glassware (Clark and Hillig 1938; Friedemann 1938) as well as more specialised apparatus (Tomiyama et al. 1956; Antonacopoulos 1960). In principle, the analytical procedures are similar to those for

30

Fishery Products: Quality, safety and authenticity

measuring TVB, but there are some special aspects to take into account. The partitioning of the lower molecular mass acids is very much in favour of the water phase; that is, the Henry’s law constant of the ratio of concentration in the vapour phase to concentration in the water phase is very low, much lower than for the volatile amines, which means that a much higher proportion of the distilling mix has to be distilled over to achieve almost complete recovery, approaching 100% compared with 30% in the case of the amines (Clark and Hillig 1938). Clark and Hillig (1938) showed that rates of distillation of the individual volatile acids are not the same, the rates increasing with molecular mass. This property was used by Hillig and Knudsen (1942) to estimate separately the concentrations of formic and acetic acids. Partitioning of the acids into the vapour phase is increased by including a high concentration of salts in the distilling mix (Friedemann 1938) and seems to be the preferred procedure if just the TVA is required (Sigurdsson 1947). The other consideration is that the distilling mix should not contain carbon dioxide, a condition that can be achieved by using freshly distilled water for making the extracts and by allowing the acidified mix to stand for a short time to allow any carbon dioxide to diffuse away before distillation. The sodium hydroxide used to titrate the acids should also be carbonate-free. Wekell et al. (1987) have described a flow injection analysis (FIA) procedure for TVA in fish, and Conway (1950) describes a procedure for measuring volatile acids using the Conway cell, though it does not seem to have been used to determine TVA in fish. There are no reports of rapid distillation units being used in the measurement of TVA, but there is no reason why they cannot be so used provided that high concentrations of salts such as magnesium or sodium sulphates are present in the distilling mix. TVA as an index of spoilage has an advantage over TVB in that volatile acids are not produced as an artefact of the analytical procedure. This aspect will remove one of the sources of variation that occurs in the TVB method. There are not many reports of changes in TVA during storage in ice (Hillig et al. (1960) and Reppond et al. (1979) are examples), but they show that TVA content of fresh muscle tissue is low and increases rapidly after a few days in ice. Lobben and Lee (1968) studied aspects of the bacteriology of the formation of volatile acids in fish muscle. They showed that the acids are not formed in sterile muscle and that only some of the species of the spoilage flora produce them. Like TVB content, TVA content does not predict storage times at early times of storage and is a predictor of storage times only after the product starts to spoil. There are not many data available to evaluate the accuracy and precision of estimates of storage time from TVA, but given the simplicity of the analytical procedure perhaps TVA should be re-evaluated as an index of spoilage. It might be especially useful in the case of species, such as freshwater fish, that have little or no TMAO in the tissue. TVA content of fish muscle is not increased or decreased in the canning process and has been used to indicate the freshness of the raw material used in the process, and hence the quality grade of the canned product.

2.5

Volatile reducing substances

This test lies between TVB and TVA in that it measures substances that are volatile from fish muscle at its natural neutral, or near neutral, pH. The sample, usually a press juice, is distilled by aeration at room temperature and the volatiles are collected in alkaline potassium permanganate, which is reduced by the volatiles, or at least a fraction of the volatiles.

Traditional methods

31

The excess potassium permanganate is back-titrated to give the amount of reducible substances, hence the description, VRS. The method was first described by Lang et al. (1944) who used a somewhat complex train of four wash bottles to clean the purging air, a distillation flask and two absorbers. A simpler, recirculating, apparatus has been described by Farber and Ferro (1956). The complete apparatus cannot be assembled completely from standard laboratory glassware and some components have to be specially fabricated. The analysis time from receipt of sample to result is about an hour, though of course more than one aeration unit can be used to increase throughput. Most of the information on VRS in fish and its use as an index of quality is contained in a series of papers by Farber and his colleagues between 1944 and 1961 – summarised in Farber and Lerke (1958 1960) – and in a few other papers. The Farber and Lerke (1958, 1960) summaries have only a few examples of changes in VRS during storage, and only some of those relate to trials of fish held in ice. The changes during storage are similar to those seen in TVB, TMA and TVA contents, a dwell of a few days followed by an approximately exponential increase in concentration. Indeed, examination of the data shows good linear associations between VRS and TVB/TMA within data sets. The method does not estimate specific chemicals, but those such as alcohols, carbonyls and sulphides, which are known to be present in spoiling fish as a product of bacterial action and are readily oxidised by cold alkaline permanganate solution. There is insufficient information in reports of VRS in fish to determine sources and sizes of variation in VRS, or to estimate the accuracy and precision of estimates of storage times. Farber and Lerke (1958, 1960) suggest that VRS can be used to differentiate between ‘passable’ and ‘not passable’ fish as judged on sensory criteria, and presence or absence of spoilage odours.

2.6

Indole

Indole was included in Clark and Almy’s (1917) list of possible indicators of spoilage in fish and seems to have been first measured in fishery products by Clough et al. (1925). Indole content, along with TVB, is one of the traditional tests for spoilage still being used as a measure of quality at the present time. It can be determined colorimetrically by the pink colour formed with p-dimethylaminobenzaldehyde (Erlich’s reagent), but the indole has first to be recovered from the product matrix by steam distillation (Clough et al. 1925; Boury and Schvinte 1935) or by extraction into solvent (Cheuk and Finne 1981; Snellings et al., 2003). Ponder (1978) has described a fluorimetric method which is not quite as sensitive as the colorimetric procedure, and which seems to have been little used in studies of spoilage of fishery products. Indole can be determined by GLC (Chambers 1982) and by HPLC (Chambers et al. 1981; Schulz 1986). Indole is formed in spoiling foods by bacterial action on tryptophan, which is split into indole, pyruvic acid and ammonia. A range of bacterial species can produce indole in foods, and they are all mesophilic in nature (Oehlenschläger and Luten, 2005). Vertebrate fish contain free tryptophan in their muscles, and potentially indole can be formed in this group of fishery products during spoilage, but almost all the studies on indole formation are concerned with spoilage of invertebrate species, predominately shrimp. Clough et al. (1925) reported that indole was formed in salmon (species not stated) during storage at ambient

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Fishery Products: Quality, safety and authenticity

temperature, but the paper does not give any data, and Schulz (1986) measured indole in herring and turbot. It is clear from all the studies on indole formation in fishery products that it is not produced in any significant amounts when the product is well chilled, but is formed during storage at higher temperatures, above about 10 °C (Oehlenschläger and Luten 2005). This means that the presence of high concentrations of indole in shrimp is indicative of temperature abuse and extensive spoilage, but low concentrations do not mean that the product is not spoiled; it could have spoiled by long-term storage in ice, for example, without any appreciable increase in indole concentration. According to chemical data sheets, indole is not toxic in the amounts that can occur even in very spoiled material, but high levels point to temperature abuse and therefore possibly the presence of agents injurious to health. Otherwise, there is a general consensus in reports on indole production in shrimp that it is not effective as an index of spoilage as such. Oehlenschläger and Luten (2005) is a comprehensive review of indole in shrimp and its use as an index of quality and in official inspection of shrimp.

2.7

Proteolysis and amino acids

Clark and Lamy’s (1917) list includes measurement of what they called ‘amino-acid nitrogen’ as a possible measure of spoilage, and tested the Sørensen procedure with model mixtures of amino acids and amines as surrogates for actual fish preparations. The principle of this approach to the measurement of spoilage is that as spoilage progresses the activity of hydrolytic bacteria on muscle proteins releases peptides and amino acids, which can be measured to provide an index of spoilage. The traditional procedures for measuring amino acids in the early 20th century were the Sørensen method – titration after addition of formaldehyde – and the more cumbersome van Slyke method – manometric determination of carbon dioxide after reacting the amino acids with ninhydrin. The latter procedure is more specific for amino acids, but the former measures any α-amino nitrogen including that in peptides. Total amino-acid content can also be measured colorimetrically with ninhydrin or by reacting with copper, and individual amino acids by HPLC. Although there is a large literature on hydrolysis of fish proteins, there is only a little in the context of spoilage and measurement of spoilage. Tillmans and Otto (1924) and Boury and Schvinte (1935) present a small amount of data obtained using Sørensen’s, method, but neither group considered the results were useful measures of spoilage. Sigurdsson (1947), in his comparison of procedures for measuring spoilage of herring, included measurement of amino acids by the copper method, but found the increase during storage was small and considered that free amino-acid content was not suitable as an index of spoilage. Though there is no doubt that some spoilage organisms on fish are hydrolytic, it is unlikely in principle that measurement of amino acids, or α-amino nitrogen generally, would be an effective index of spoilage. The intrinsic free amino-acid content of fish flesh varies markedly among and within species (Mackie and Ritchie 1974), and any attempt to specify values of hydrolytic products as criteria of quality would be subject to very high uncertainty. Tyrosine content has been suggested as an index of hydrolysis (Farber 1965), but this is only one of the amino acids released by hydrolysis and its selection probably has more to do with its ease of analysis by a colorimetric reaction than with its utility.

Traditional methods

2.8

33

pH

Throughout the long history of studies of spoilage of fish, it has been common to measure the pH of the muscle tissue. The natural pH of live fish is just above 7.0, typically about 7.3, but this falls markedly after death as the fish goes through rigor mortis and glycogen is converted to lactic acid. In most species, the post mortem pH is between 6.0 and 6.8, but in some species, for example tunas, it is below 6.0 because of high initial concentrations of glycogen. Within a species, the glycogen content in a fish at death depends on biological factors such as nutritional status at the time, and the amount of activity, which depletes glycogen content, just before death. The results of pH measurements during spoilage invariably show that after the resolution of rigor mortis the pH increases, usually after a dwell of a few days depending on the conditions of storage. However, the great variability of intrinsic pH between species, effects of biological conditions and harvesting procedures, and between fish within a batch, preclude it being an effective measure of spoilage.

2.9 Refractive index of eye fluids This method is more properly a physico-chemical, rather than a chemical, procedure as it measures a chemical reaction by a physical technique. The principle is that the refractive index (RI) of fluid, the vitreous humour, recovered from the interior of the eye is measured and related to the storage time of the fish. The method was first used for measuring freshness of fish by Proctor et al. (1959), who showed that the RI increased approximately linearly during storage in ice. (These authors do not discuss why they considered the RI of eye fluid to be a promising method, and they do not cite any references to previous studies in fish or any other animal. However, the forensic literature refers to measurement of inorganic salts, especially potassium, in aqueous humour being used to estimate time of death, and perhaps they were aware of this.) Subsequently, a few other studies using the procedure have confirmed this increase in the RI of eye fluids during storage (Kietzmann et al. 1964; Abdalla et al. 1989; Yapar and Yetim 1998; Gökolu and Yerlikaya 2004). The RI is measured in an Abbé refractometer, and variations in the procedure are in the preparation of the sample. Proctor et al. (1959) sampled the aqueous humour by cutting the eye and allowing the liquid to drain, centrifuging the liquid and filtering it to remove any gel before measurement, but other workers have measured the RI of the humour directly (Yapar and Yetim 1998; Gökoglu and Yerlikaya 2004). There have been no conclusive studies of the nature of the changes that are being measured by RI, though Proctor et al. (1959) established that bacterial growth in the eye was not responsible. A likely cause is salts such as potassium, which are substantially present in the living fish within the cells lining the eye, but not in the extracellular fluid, leaching into the vitreous humour as the ion pump system across the cell membrane degrades on the death of the fish. Measurement of RI of eye fluids is a simple and cheap procedure and is probably worth pursuing as an index of storage time. It has the considerable advantage of increasing from soon after the death of the fish, within a day or so judging from the experimental data, and in principle could discriminate qualities of fish before spoilage starts. Measuring RI of the eye fluids is not the same as measuring changes in the opacity of the lens during iced storage (Love 1954).

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2.10

Fishery Products: Quality, safety and authenticity

Discussion and summary

One of the objectives of this review is to consider whether traditional, non-sensory methods of assessing quality have any use in current practices for quality assurance of fishery products. However, before examining this point, it would be useful to discuss briefly what is meant by quality in the context of the situations where the methods might be applied. There are various ways in which quality can be defined (Bremner 2002), but for the following discussion the concept of the aggregate of properties that influence a consumer’s liking or disliking of a product will be used. Leaving aside aspects such as price and convenience of purchase and preparation, the properties of interest to the consumer are the product’s sensory properties of which flavour and odour of the cooked product are the most relevant to this discussion. (Appearance and texture of fish are affected by length of storage, but they probably affect overall hedonic response less than do odour and flavour.) The literature on sensory evaluation of fishery products has descriptions of changes in these properties for several species of fish, often in tabular form (see Shewan et al. (1953) for a typical example). Whatever the species, two main phases of changes during storage can be recognised. The odour and flavour of the cooked fish when freshly harvested is variously described in terms such as sweet, creamy, meaty, marine, depending on the species, and these characteristic odours and flavours decrease in intensity without changing much in character during the first few days of storage in ice – faster of course at higher temperatures. Changes in this phase are attributed to the effects of enzyme activity, especially those concerned with degradation of nucleotides. After a few days in ice the effects of bacterial spoilage start to become apparent and the odours and flavours change in character, becoming progressively more unpleasant and ultimately offensive enough that the product is considered unfit for consumption (Shewan 1977). A common feature of all the traditional methods discussed above, with the exception of refractive index of eye fluids, is that they monitor effects of bacterial action on the flesh of the fish; that is, they monitor changes during the spoilage phase of deterioration. This feature has been realised from the earliest days of the introduction and development of the methods. Many authors of original papers and of reviews comment that the methods do not measure changes in earlier stages of storage and are only adequate to indicate that spoilage has occurred. The methods were developed in Europe and North America at a time when those regions had distant-water fishing fleets which were at sea for long times and perhaps used less than optimum storage practices. Consequently, an appreciable proportion, perhaps a major portion, of the catch was spoiling or was even in a state of being unfit for consumption on discharge at the ports. The traditional methods described above could have had some application at that time in measuring or grading quality, but even so many commentators doubted if they were very effective. The situation now with regard to fish supplies and fish processing and marketing is markedly different from that in the first seven decades or so of the last century. Changes in circumstances relating to capture of fish – introduction of freezing at sea, imposition of Exclusive Economic Zones, unfavourable economics of distant water fishing based on icing of the catch – led to the demise of distant-water fleets based on icing the catch, and most fish landed in the chilled state are now caught by near-shore and middle-water fleets. This has resulted in a marked reduction in the amount of spoiling fish being landed and has almost eliminated from the market fish that are unfit for consumption

Traditional methods

35

due to advanced spoilage. Additionally, over the past couple of decades, aquacultured products, which have the potential of offering very fresh products to the consumer, have made a significant contribution to supplies. Consequently, the requirement to measure the freshness of fish that might be in the spoilage phase of deterioration is now much reduced compared with much of the previous century. Along with these changes in the supply situation have been changes in the marketing of fish. At the time the traditional methods were being developed and promoted, fish was sold to consumers by small-scale fishmongers who had little opportunity to exert control over the quality of their supplies unless they were in a position to buy at port markets. Nowadays, in countries with advanced economies, and increasingly in countries with developing economies, much fish, in common with other foods, is sold by multiple retailers, supermarkets. These companies are very conscious of the requirement to ensure that their customers are pleased with the products the store sells and set standards of quality for the products they take in. The standards for fishery products will require that they are at least not spoiled and, better still, should retain some of their intrinsic flavours. They also require, and check, that suppliers of fishery products for their stores apply effective quality assurance procedures in their processing plants to meet these quality standards. Multiple retailers often market fish as pre-packaged products and indicate the shelf life of the product by suitable labelling, for example ‘use by’ or ‘best before’ dates. Usually food legislation requires such labelling, and the legislation might indicate what properties should determine the end of shelf life. For example, the relevant European Union (EU) legislation defines the ‘best before’ date as the ‘date until which the foodstuff retains its specific properties when properly stored’. If having intrinsic fresh fish flavours can be considered as a specific property, then the EU Directive requires the product, by its ‘best before’ date, should have the characteristics of the fresh product and be free of spoilage characteristics. None of the traditional methods discussed above other than perhaps refractive index of eye fluid would be able to discriminate among qualities within the ‘best before’ range, nor reliably detect samples that were just beyond it. The emphasis in current practices for quality assurance of fishery products, as it is for foods generally, is for control over the quality of raw materials and control of processing, storage and distribution systems. There is little scope in this approach for chemical tests for spoilage of any kind except perhaps in the checking of stable starting materials such as frozen fish. Certainly, the traditional methods would not be effective in this situation. Chemical tests might have a role in cases of complaints or rejection of material by consumers or intermediaries in the supply chain. If sensory evaluation suggests that rejection can be attributed to spoilage then perhaps measurement of TMA would be the best of the traditional methods for providing additional information. Overall it must be concluded that the traditional methods of measuring quality have no, or at best, a very limited place in current practices of quality assurance of fishery products.

2.11

References

Abdalla, M.A., Hassan, I.M., Shalaby, A.R. and Naguib, K. (1989) Physicochemical and bacteriological changes occurring during storage of sardine fish. Grasas y Aceites 40: 389–398.

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Analytical Methods Committee. (1979) Recommended general methods for the examination of fish and fish products. Analyst 104: 434–450. Anderson, A.G. (1908) On the decomposition of fish. 26th Annual Report of the Fishery Board for Scotland 1907, Part III, Scientific Reports. HMSO, London, pp. 13–39. Anthoni, U., Børrensen, T., Chrostophersen, C., Gram, L. and Nielsen, P.H. (1990) Is trimethylamine oxide a reliable indicator for the marine origin of fish? Comparative Biochemistry and Physiology 97B: 569–571. Antonacopoulos, N. (1960) Improved apparatus for quantitative distillation of steam volatile substances. (Verbesserte Apparatur zur quantitativen Destillation wasserdampfflüchter Stoffe.) Zeitschrift für Lebensmittel -Untersuchung und -Forschung 113: 113–116. (In German) Antonacopoulos, N. and Vyncke, W. (1989) Determination of volatile basic nitrogen: a third collaborative study by The West European Fish Technologists’ Association (WEFTA) Zeitschrift für Lebensmittel-Untersuchung und -Forschung 189: 309–316. Barrett, E.L. and Kwan, H.S. (1985) Bacterial reduction of trimethylamine oxide. Annual Review of Microbiology 39: 131–149. Beatty, S.A. (1938) Studies in fish spoilage. II. The origin of trimethylamine during the spoilage of cod muscle press juice. Journal of the Fisheries Research Board of Canada 4: 63–68. Beatty, S.A. and Gibbons, N.E. (1937) The measurement of spoilage in fish. Journal of the Biological Board of Canada 3: 77–91. Billon, J., Ollieuz, N. and Tao, S.H. (1979) Study of a new method of dosage of total volatile basic nitrogen for the qualitative evaluation of fishery products. (Etude d’une nouvell méthode de dosage de l’azote basique volatil total (ABVT) pour l’évaluation qualitative des produits de la pêche.) Revue Technique Veterinaire de l’Alimentation 149: 13–17. (In French) Boury, M. and Schvinte, J. (1935) Spoilage of fish. (L’altération du poisson.) Revue des Traveaux de l’Office des Peches Maritimes 8: 282–333. (In French) Bremner, H.A. (2002) Understanding the concepts of quality and freshness in fish. In: H.A. Bremner (Ed.) Safety and Quality Issues in Fish Processing Woodhead Publishing Ltd, Cambridge, UK, pp. 163–172. Bullard, F.A. and Collins, J. (1980) An improved method to analyse trimethylamine in fish and the interference of ammonia and dimethylamine. Fishery Bulletin 78: 465–473. Burt, J.R., Gibson, D.M., Jason, A.C. and Sanders, H.R. (1975) Comparison of methods of freshness assessment of wet fish. Part II. Instrumental and chemical assessment of boxed experimental fish. Journal of Food Technology 11: 73–89. Castell, C.H., Smith, B. and Dyer, W.J. (1974) Simultaneous measurements of trimethylamine and dimethylamine in fish and their use for estimating quality of frozen stored gadoid fillets. Journal of the Fisheries Research Board of Canada 31: 383–389. Chambers, T.L. (1982) Modification of the AOAC gas-liquid chromatographic method for indole in shrimp: collaborative study. Journal of the Association of Official Analytical Chemists 65: 842–845. Chambers, T.L., Staruszkiewicz, W.F., Bohm, G., Bond, J.F., Carr, R., Edge, D.J., Everett, R.L., Illuminati, J.C., La Rose, J., McMurtrey, K., Miller, G., Panaro, K.W. and Smith, B.H. (1981) High pressure liquid chromatographic method for indole in shrimp: development of method and collaborative study. Journal of the Association of Official Analytical Chemists 64: 592– 602. Chang, G.W., Chang, W.L. and Lew, K.B.K. (1976) Trimethylamine-specific electrode for fish quality control. Journal of Food Science 41: 723–724. Cheuk, W.L. and Finne, G. (1981) Modified colorimetric method for determining indole in shrimp. Journal of the Association of Official Analytical Chemists 64: 783–785. Civera, T., Turi, R.M., Bisio, E., Parisi, E. and Fazio, G. (1993) Sensory and chemical assessment of marine teleosteans. Sciences des Aliments 13: 109–117.

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Clark, E.D. and Almy, L.H. (1917) Preliminary studies on chemical methods of detecting deterioration in fish flesh. Journal of the Association of Official Agricultural Chemists 2: 231–236. Clark, E.P. and Hillig, F. (1938) Concerning the Dyer method for the identification and determination of fatty acids. Journal of the Association of Official Agricultural Chemists 21: 684–688. Clough, R.M., Shostrom, O.E. and Clark, E.D. (1925) Notes on the presence of indol in sea food and other food products. University of Washington Publications in Fisheries 1: 101–108. Conway, E.J. (1950) Microdiffusion Analysis and Volumetric Error, 3rd edn. Crosby Lockwood and Son Ltd, London. Dyer, W.J. (1945) Amines in fish muscle: I. Colorimetric determination of trimethylamine as the picrate salt. Journal of the Fisheries Research Board of Canada 6: 351–358. Dyer, W.J. (1959) Report on trimethylamine in fish. Journal of the Association of Official Analytical Chemists 42: 292–294. Dyer, W.J. and Mounsey, Y.A. (1945) Amines in fish muscle: II Development of trimethylamine and other amines. Journal of the Fisheries Research Board of Canada 6: 359–367. Egnér, H. and Johansson, V.J. (1938) Distillation of ammonia. Annals of the Agricultural College of Sweden 5: 113–130. Ellis, P.C., Pivarnik, L.F. and Thuam, M. (2000) Determination of volatile bases in seafood using the ammonia on selective electrode: collaborative study. Journal of AOAC International 83: 933–943. Emerson, K., Russo, R.C., Lund, R.C. and Thurson, R.V. (1975) Aqueous ammonia equilibrium calculations: effects of pH and temperature. Journal of the Fisheries Research Board of Canada 32: 2379–2383. European Commission (2005) Commission Regulation No. 2074/2005 of 5 December 2005 laying down implementing measures for certain products under Regulation (EC) No 853/2004 of the European Parliament and of the Council and for the organisation of official controls under Regulation (EC) No 854/2004 of the European Parliament and of the Council and Regulation (EC) No 882/2004 of the European Parliament and of the Council, derogating from Regulation (EC) No 852/2004 of the European Parliament and of the Council and amending Regulations (EC) No 853/2004 and (EC) No 854/2004. Official Journal of the European Communities L338: 27–59. Farber, L. (1965) Freshness tests. In: G. Borgstrom (Ed.) Fish as Food. vol IV. Processing: Part 2, chapter 2. Academic Press, New York and London, pp. 65–126. Farber, L. and Ferro, M. (1956) Volatile reducing substances (VRS) and volatile nitrogen compounds in relation to spoilage of canned fish. Food Technology 10: 303–304. Farber, L. and Lerke, P.A. (1958) A review of the value of volatile reducing substances substances for the chemical assessment of freshness of fish and fish products. Food Technology 12: 677–680. Farber, L. and Lerke, P.A. (1960) The objective assessment of raw fish quality. In: E. Hess and G.N. Subba Rao (Eds), Chilling of Fish. Fish processing technologists meeting, Rotterdam, The Netherlands, 25–29 June 1956. Ministry of Agriculture, Fisheries and Food, The Hague, pp. 221–233. Friedemann, T. (1938) The identification and quantitative determination of volatile alcohols and acids. Journal of Biological Chemistry 123: 161–184. Gill, T.A. and Thompson, J.W. (1984) Rapid, automated analysis of amines in seafood by ionmoderated partition HPLC. Journal of Food Science 49: 603–606. Gökoglu, N. and Yerlikaya, P. (2004) Use of eye fluid refractive index in sardine (Sardina pilchardus) as a freshness indicator. European Food Research and Technology 218: 295–297. Gram, L. and Dalgaard, P. (2002) Fish spoilage bacteria B problems and solutions. Current Opinion in Biotechnology 13: 262–266. Gram, L. and Huss, H.H. (1996) Microbiological spoilage of fish and fish products. International Journal of Food Microbiology 33: 121–137. Hashimoto, Y. and Okaichi, T. (1957) On the determination of trimethylamine and trimethylamine oxide. A modification of the Dyer method. Bulletin of the Japanese Society of Scientific Fisheries 23: 269–272. (In Japanese)

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Hebard, C.E., Flick, G.J. and Martin, R.E. (1982) Occurrence and significance of trimethylamine oxide its derivatives in fish and shellfish. In: R.E. Martin, G.J. Flick, C.E. Hebard and D.R. Ward (Eds) Chemistry and Biochemistry of Marine Food Products, chapter 12. Avi Publishing Co., Westport, CO, pp. 149–304. Hillig, F. and Knudsen, L.F. (1942) Determination of volatile fatty acids. Journal of the Association of Official Agricultural Chemists 25: 176–195. Hillig, F., Shelton, L.R., Loughtey, J.H. and Fitzgerals, B.F. (1960) Chemical indices of decomposition in ocean perch. Journal of the Association of Official Analytical Chemists 43: 433– 438. Hjartarson, S.V., Einarsson, H., Magnússon, H. and Arnason, S.V. (1998) Bulk storage of cod. Comparison of ice, CSW and CO2 – Saturated CSW storage. Icelandic Fisheries Latoratories Report 7–98. Hjorth-Hansen, S. and Bakken, K. (1947) Investigations on analytical methods for estimation of ammonia and methylamines in fish. (Undersøkelser over analysemetoder for ammoniak og metylaminer i fisk.) Reports from the Norwegian Fisheries Research Laboratory 1, No. 6. (Fiskeridirektoratets Skrifter. Serie Undersøkelser ved Statens Fiskeriforsøksstatsjon 1, No. 6) (In Norwegian) Hollingworth, T.A., Kaysner, C.A., Colburn, K.G., Sullivan, J.J., Abeyta, C., Walker, K.D, Torkelson, J.D., Throm, H.R. and Wekell, M.M. (1991) Chemical and microbiological analysis of vacuumpacked, pasteurized flaked imitation crabmeat. Journal of Food Science 56: 164–167. Hollingworth, T.A., Wekell, M.M., Sullivan, J.J., Torkelson, J.D. and Throm, H.R. (1990) Chemical indicators of decomposition for raw surimi and flaked artificial crab. Journal of Food Science 55: 349–352. Hoogland, P.L. (1958) Grading fish for quality. Part 2. Statistical analysis of results of experiments regarding grades and trimethylamine values. Journal of the Fisheries Research Board of Canada 15: 717–728. Howgate, P. (2006) A review of the kinetics of degradation of inosine monophosphate in some species of fish during chilled storage. International Journal of Food Science and Technology 41: 341–353. Hughes, R.B. (1959) Chemical studies on the herring (Clupea harengus) I. Trimethylamine oxide and volatile amines in fresh, spoiling and cooked herring flesh. Journal of the Science of Food and Agriculture 10: 431–436. Karnop, G. (1976) The local distribution of volatile bases (TVB-N) in the tissue of whole fish during iced storage. (Die lokale Verteilung flüchtiger Basen (TVB-N) im Gewebe von Ganzfischen während der Eislagerung.) Archiv für Fischereiwissenschaft 27: 159–169. (In German) Keay, J.N. and Hardy, R. (1972) The separation of aliphatic amines in dilute aqueous solution by gas chromatography and application of this technique to the quantitative analysis of tri- and dimethylamine in fish. Journal of the Science of Food and Agriculture 23: 9–19. Kietzmann, H.J., Wegner, K., Priebe, U. and Rakow, D. (1964) Freshness test of fish using measurement of refractive index of eye fluid and of mussel fluid of fish. (Frischetest durch refraktometrische Messungen der Augenflüssigkeit und des Muskelwassers bei Fischen.) Zentralblatt der Veterinärmedezin B 11: 511–560. Lang, O.W., Farber, L., Beck, C. and Yerman, F. (1944) Determination of spoilage in protein foodstuffs, with particular reference to fish. Industrial and Engineering Chemistry 16: 490–494. Lapa-Guimarães, J., de Felício, P.E. and Guzmán, E.S.C. (2005) Chemical and microbial analyses of squid muscle (Loligo plei) during storage in ice. Food Chemistry 91: 477–483. Lobben, J.C. and Lee, J.S. (1968) Roles of microorganisms in the deterioration of rockfish. Applied Microbiology 16: 1320–1325. Love, R.M. (1954) Post-mortem changes in the lenses of fish eyes: assessment of storage time and fish quality. Journal of the Science of Food and Agriculture 5: 566–572.

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39

Lücke, F. and Geidel, W. (1935) Determination of volatile basic nitrogen in fish as a measure of their freshness. (Bestimmung des flüchtigen basischen Stickstoffs in Fischen als Maßstab fur ihren Frischezustand) Zeitschrift für Untersuchung der Lebensmittel, 70: 441–458. (In German) Lundstrom, R.C. and Racicot, L.D. (1983) Gas chromatographic determination of dimethylamine and trimethylamine in seafoods. Journal of the Association of Official Analytical Chemists 66: 1158–1163. Mackie, I.M. and Ritchie, A.H. (1974) Free amino acids of fish flesh. Proceedings of the IVth International Congress of Food Science and Technology, Instituto de Agroquímica y Tecnología de Alimentos, Madrid, 23–27 September 1974, pp. 29–38. Mackie, I.M. and Thomson, B.W. (1974) Decomposition of trimethylamine oxide during iced and frozen-storage of whole and comminuted tissue of fish. In: Proceedings of the IVth International Congress of Food Science and Technology, Instituto de Agroquímica y Tecnología de Alimentos, Valencia, Spain, pp. 243–250. Malle, P. and Tao, S.H. (1987) Rapid, quantitative determination of trimethylamine using steam distillation. Journal of Food Protection 50: 756–760. Murray, C.K. and Gibson, D.M. (1972) An investigation of the method of determining trimethylamine in fish muscle extracts by the formation of its picrate salt. Part II. Journal of Food Technology 7: 47–51. Norris, E.R. and Benoit, G.J (1945) Studies on trimethylamine oxide I. Occurrence of trimethylamine oxide in marine organisms. Journal of Biological Chemistry 158: 433–438. Oehlenschläger, J. (1988) The determination of volatile basic nitrogen (TVB-N) Effect of pH on the deamination of acid extracts of sea fish during steam distillation after alkilisation. (Zur bestimmung des flüchtigen Basenstickstoffe (TVB-N) Einfluss des pH-Wertes auf die Desaminierung bei der Wasserdampfdestillation von sauren Extrakten aus Seefischen nach Alkalisierung.) Informationen für die Fischwirtschaft 35: 31–34. (In German) Oehlenschläger, J. (1997a) Volatile amines as freshness/spoilage indicators. A literature review. In: J.B. Luten, T. Børrensen and J. Oehlenschläger (Eds) Seafood from Producer to Consumer, Integrated Approach to Quality. Elsevier, Amsterdam, pp. 571–584. Oehlenschläger, J. (1997b) Suitability of ammonia-N, dimethylamine-N, trimeyhylamine-N, trimethylamine oxide-N and total volatile basic nitrogen as freshness indicators in seafoods. In: G. Ólafsdóttir, J. Luten, P. Dalgaard, M. Careche, V. Verrez-Bagnis, E. Martinsdottir and E. Heia (Eds) 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, Nantes, November 12–14 1997. International Institute of Refrigeration, Paris, pp. 92–99. Oehlenschläger, J. and Luten, J.B. (2005) Review: indole as a quality indicator in shrimps and prawns. Archiv für Lebensmittelhygiene 56: 52–57. Ohashi, E., Takao, Y., Fujita, T., Shimizu, Y. and Egashira, M. (1991) Semiconductive trimethylamine gas sensor for detecting fish freshness. Journal of Food Science 56: 1275–1278, 1286. Okoloff, F. (1932) Determination of ammonia, trimethylamine and other amines in foods. (Bestimmung von Ammoniak, Trimethylamin und anderen Aminen in Nahrungsmitteln.) Zeitschrift für Untersuchung der Lebensmittel 63: 129–154. (In German) Pearson, D. and Muslemuddin, M. (1968) The accurate determination of total volatile nitrogen in meat and fish, Part I. Techniques and application to beef and salmon. Journal of the Association of Public Analysts 6: 117–123. Pearson, D. and Muslemuddin, M. (1969a) The accurate determination of total volatile nitrogen in meat and fish. Part II application to white teleostean fish. Journal of the Association of Public Analysts 7: 50–54. Pearson, D. and Muslemuddin, M. (1969b) The accurate determination of total volatile nitrogen in meat and fish. Part III. Application to elasmobranch fish. Journal of the Association of Public Analysts 7: 73–82.

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Ponder, C. (1978) Decomposition in foods (chemical methods) Fluorometric determination of indole in shrimp. Journal of the Association of Official Analytical Chemists 61: 1089–1091. Proctor, B.E., Nickerson, J.T.R., Fazzina, T.L., Ronsivalli, L., Smith, R.K. and Stern, J. (1959) Rapid determination of the quality of whole eviscerated haddock. Food Technology 31: 224– 228. Quaranta, H.O. and Curzio, O.A. (1983) Total volatile acids content as a quality index of irradiated hake (Merluccius merluccius hubbsii) fillets. Food Chemistry 11: 27–30. Reay, G.A. (1935) Some observations on methods of estimating the degree of preservation of white fish. Chemistry and Industry 54: 145–148. Rehbein, H. and Oehlenschläger, J. (1988) Determination of volatile basic nitrogen (TVB-N) Deamination of pure substances occurring in fish muscle during steam distillation after mild alkalization. (Desaminierung von im Fischmuskel vorkommenden Reinsubstanzen bei Wasserdampfdestillation nach milder Alkalisierung.) Informationen für die Fischwirtschaft 35: 136–139. (In German) Reppond, K.D., Bullard, F.A. and Collins, J. (1979) Walleye pollock, Theragra chalcogramma: physical, chemical, and sensory changes when held in ice and in carbon dioxide modified refrigerated seawater. Fishery Bulletin 77: 481–488. Sadok, S., Uglow, R.F. and Haswell, S.J. (1996) Determination of trimethylamine in fish by flow injection analysis. Analytica Chimica Acta 321: 69–74. Schulz, H. (1986) Determination of indole and skatole in seafood using high performance liquid chromatography (HPLC) (Bestimmung von Indol und Skatol in Meerestieren durch HochleistungsFlüssigchromatographie (HPLC).) Zeitschrift für Lebensmittel- Untersuchung und -Forschung 183: 331–334. (In German) Shewan, J.M. (1977) The bacteriology of fresh and spoiling fish and the biochemical changes induced by bacterial action. In: P. Sutcliffe and J. Disney (Eds) Proceedings of the Conference on the handling, processing and marketing of tropical fish, London 5–9 July 1976. Tropical Products Institute, London, pp. 51–56. Shewan, J.M. and Ehrenberg, A.C.S. (1957) Volatile bases as quality indices of iced North Sea cod. Journal of the Science of Food and Agriculture 8: 227–231. Shewan, J.M. and Liston, J. (1956) Recent work on the use of total volatile bases and trimethylamine contents and tetrazolium salt reduction for assessing the quality of iced fish. In: E. Hess and G.N. Subba Rao (Eds), Chilling of Fish. Fish processing technologists meeting, Rotterdam, The Netherlands, 25–29 June 1956. Ministry of Agriculture, Fisheries and Food, The Hague, pp. 204–214. Shewan, J.M., Gibson, D.M. and Murray, C.K. (1971) The estimation of trimethylamine in fish muscle. In: R. Kreuzer (Ed.) Fish Inspection and Quality Control. Fishing News (Books) Limited, London, pp. 183–186. Shewan, J.M., MacIntosh, R.G., Tucker, C.G. and Ehrenberg, A.S.C. (1953) The development of a numerical scoring system for the sensory assessment of the spoilage of wet white fish stored in ice. Journal of the Science of Food and Agriculture 4: 283–298. Sigurdsson, G.J. (1947) Comparison of chemical tests of the quality of fish. Analytical Chemistry 19: 892–902. Snellings, S.L., Takenaka, N.E., Kim-Hayes, Y. and Miller, D.W. (2003) Rapid colorimetric method to detect indole in shrimp with gas chromatography mass spectrometry confirmation. Journal of Food Science 68: 1548–1553. Spinelli, J. (1964) Evaluation of the micro-diffusion method for the determination of tertiary volatile base in marine products. Fishery Industrial Research 2: 17–19. Stansby, M.A., Harrison, R.W., Dassow, J. and Sater, M. (1944) Determining volatile bases in fish – comparison of precision of certain methods. Industrial and Engineering Chemistry, Analytical Edition 16: 593–596.

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Tillmans, J. and Mildner, H. (1916) The detection of the onset of spoilage of flesh. (Über den Nachweiss beginnender Fleischfäulnis.) Zeitschrift für Untersuchungs der Nahrung und Genussmittel 32: 65–75. (In German) Tillmans, J. and Otto, R. (1924) The detection of the onset of spoilage of fish. (Über den Nachweis der beginnenden Fischfäulnis.) Zeitschrift für Untersuchungs der Nahrungs- und Genussmittel 47: 25–37. (In German) Tomiyama, T. and Harada, Y. (1952) Studies on the method for testing the spoilage of food – IV. A rapid vacuum-distilling method for the determination of volatile base. Bulletin of the Japanese Society of Scientific Fisheries 18: 112–116. Tomiyama, T., de Costa, A.A. and Stern, J.A. (1956) A rapid vacuum distillation procedure for the determination of volatile acids and volatile bases in fresh fish. Food Technology 10: 614–617. Tozawa, H., Enokihara, K. and Amano, K. (1971) Proposed modification of Dyer’s method for trimethylamine determination in cod fish. In: R. Kreuzer (Ed.) Fish Inspection and Quality Control Fishing News (Books) Limited, London, pp. 187–190. Vyncke, W., Luten, J., Brünner, K. and Moermans, R. (1987) Determination of total volatile bases in fish: a collaborative study by the West European Fish Technologists’ Association (WEFTA) Zeitschrift für Lebensmittel-Untersuchung und -Forschung 184: 110–114. Weber, F.C. and Wilson, J.B. (1918) A method for the separation and quantitative determination of the lower alkylamines in the presence of ammonia. Journal of Biological Chemistry 15: 385–410. Weber, F.C. and Wilson, J.B. (1919) The formation of ammonia and amines in canned sardines during storage. Journal of Industrial and Engineering Chemistry 11: 121–126. Wekell, M.M., Hollingworth, T.A. and Sullivan, J.J. (1987) Application of flow injection analysis (FIA) to the determination of seafood quality. In: D.E. Kramer and J. Liston (Eds) Seafood Quality Determination. Elsevier, Amsterdam, pp. 17–26. Wong, K. and Gill, A. (1987) Enzymatic determination of trimethylamine and its relationship to fish quality. Journal of Food Science 52: 1–3, 6. Wong, K., Bartlett, F. and Gill, T.A. (1988) A diagnostic test strip for the semiquantitative determination of trimethylamine in fish. Journal of Food Science 53: 1653–1655. Yapar, A. and Yetim, H. (1998) Determination of anchovy freshness by refractive index of eye fluid. Food Research International 31: 693–695. Yeh, C.P.S., Nickelson, R. and Finne, G. (1978) Ammonia-producing enzymes in white shrimp tails. Journal of Food Science 43: 1400–1401, 1404.

Chapter 3

Biogenic amines Rogério Mendes

3.1

Introduction

By definition, biogenic amines are basic nitrogenous compounds formed mainly by decarboxylation of amino acids or by amination and transamination of aldehydes and ketones (Askar and Treptow 1986; Maijala et al. 1993). Biogenic amines are organic bases of low molecular mass and according to the chemical structure can either be aliphatic (putrescine, cadaverine, spermine, spermidine), aromatic (tyramine, β-phenylethylamine) or heterocyclic (histamine, tryptamine). These compounds are synthesised by microbial, vegetable and animal metabolism (Brink et al. 1990). Polyamines such as putrescine, spermidine, spermine and cadaverine are fundamental components of living cells and play an important role in the regulation of nucleic acid function and protein synthesis, and likely also in the stabilisation of membranes (Bardoz et al. 1993; Maijala et al. 1993; Halász et al. 1994). Initially it was thought that fish autolysis was the main reason for formation of biogenic amines. However, deeper studies showed that production of biogenic amines reported by some researchers was caused by a previous bacterial contamination or a deficient sterilisation process (Arnold and Brown 1978). Histamine, cadaverine and putrescine are diamines that may be produced post mortem from the decarboxylation of specific amino acids in fish and shellfish tissue (Santos 1996). Biogenic amines present in fish are almost totally the result of the action of exogenous enzymes released by the various microorganisms associated with the seafood products (Frank et al. 1981). Confirming this fact is the inhibition of histamine formation when antibiotics such as penicillin and tetracycline are used. Histamine, putrescine, cadaverine, tyramine, tryptamine, β-phenylethylamine, spermine and spermidine are considered generally the most relevant biogenic amines in foods (Shalaby 1996). β-phenylethylamine, spermine and spermidine are, however, not end products of bacterial decomposition in fishery products. Figure 3.1 presents the biogenic amines and their chemical precursors. Endogenous decarboxylase enzymes naturally occurring in fish or shellfish tissue may also contribute to the production of biogenic amines. This pathway is, however, insignificant compared with exogenous production (Wendakoon and Sakaguchi 1992). Studies by Wendakon et al. (1990) show the existence of a correlation between the formation of biogenic amines and the decrease of free amino acids. At 20 °C the rate of decrease in histidine, 42

Biogenic amines N

(CH2)2 NH2 HO

Tyramine

(CH2)2 NH2

N H

HO

Tyrosine

N H

Tryptamine

(1)

CH2 CH COOH

N

CH2

CH2 CH2 NH2

HO

N (1) H Histamine

(3)

43

CH2 CH COOH

NH2

NH2

Histidine

N H Tryptophan

CH COOH NH2

COOH H2N CH (CH2)4 NH2

Protein

H2N

NH2

NH2

COOH

O

COOH

(CH2)5

Cadaverine (2)

Lysine

H2N CH (CH2)3 NH C NH

H2N CH (CH2)2 C NH2 Glutamine

Arginine

COOH

H

H2N CH (CH2)3 NH2 Ornithine

H2N

(CH2)4 N

NH2 C NH

Agmatine

H2N (CH2)4 NH2 Putrescine (2) H H2N

(CH2)3

N

H (CH2)4

N

(CH2)3 NH2

Spermine H H2N

(CH2)3 N (CH2)4 NH2 Spermidine

Figure 3.1 Metabolic pathways of biogenic amine formation in fish: (1) heterocyclic amine; (2) aliphatic amine; (3) aromatic amine. (Adapted from Halász et al. 1994.)

arginine and lysine was proportional to the raise in the correspondent biogenic amines. Free amino acids are in general present in high quantities in the muscle of living fish and may increase even further post mortem. The high activity of proteolytic enzymes present in the viscera is responsible for the rapid development of the autolytic process (Gildberg 1978; Aksnes 1988) and the high content of free amino acids in fishery products.

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Softening of tissues as a result of post mortem autolytical changes has also an important role in the release of amino acids to the muscular tissue, thus allowing an easier penetration of bacteria and favourable conditions for the action of the respective decarboxylase (Brink et al. 1990). Formation of biogenic amines by microorganisms involves several prerequisites, namely: availability of free amino acids, though not always leading to amine production (Marklinder and Lönner 1992) presence of microorganisms as producers of decarboxylase enzymes (Brink et al. 1990; Huis in’t Veld et al. 1990) existence of conditions for bacterial development, decarboxylase synthesis and decarboxylation activity (Brink et al. 1990).

3.2

Factors affecting amine decarboxylase activity

Taking into account that amines result from enzymatic activity of the food or bacteria with active decarboxylase, control or inhibition of such processes and prevention of bacterial growth are key factors fundamental for limiting the amine content in food. It is well known that temperature has a marked effect on the formation of histamine by bacteria present in fish. However, optimal conditions for bacterial growth are different from those necessary for optimal histidine decarboxylase synthesis (Arnold et al. 1980; Eitenmiller et al. 1982; Spreekens 1987). According to Eintenmiller et al. (1982) and Okuzumi et al. (1984), the maximum of histidine decarboxylase activity is attained when the bacterial culture is incubated at room temperature (about 24 °C) whereas the higher bacterial growth is visible at 37 °C for mesophiles and 20 °C for psychrophiles. In relation to the specific enzyme activity, several authors (Eintenmiller et al. 1982; Olley and Baranowski 1985; Abell and O’Leary 1985) reported 37 °C as the optimal temperature for enzyme activity, though the enzyme showed some activity at temperatures lower than 10 °C (Olley and Baranowski 1985). Maximum histamine production is therefore the result of a compromise between the production of histidine decarboxylase by bacteria and optimal temperature activity of amino-acid decarboxylase activity. In what concerns thermal stability, histidine decarboxylase is sensitive to temperatures above room temperatures. However, though the inactivation of the enzyme occurs at moderate temperatures (40 °C) it is not sufficiently quick to prevent the production of significant amounts of histamine in tuna at the beginning of the steam pre-cooking phase (Olley and Baranowski 1985). According to Frank and Yoshinaga (1984), production of histamine is negligible above 50 °C and below 0 °C. Arnold et al. (1980) reported that histamine development in Morganella morganii and Pseudomonas vulgaris cultures was optimal at 30 °C and considerably slower at 7 °C. Similar results were obtained by Eitenmiller et al. (1981) and Behling and Taylor (1982) with mesophile bacterial producers of histamine. Lower temperature limits for significant histamine production in tuna fish infusion broth for some decarboxylase bacteria are 7 °C for Klebsiella pneumoniae, 15 °C for Morganella morgani and 30 °C for Hafnia alvei, Citrobacter freundi and Escherichia coli (Behing and Taylor 1982). M. morganii and K. pneumoniae are, however, capable of growth at 4 °C, being in this case the histamine production negligible for the first and moderate for the second

Biogenic amines

45

bacterial species (Frank and Yoshinaga 1984). Some bacterial cultures, though not developing at low temperature, remain viable and also produce histamine in small quantities (Ratkowsky et al. 1982; Taylor and Speckard 1983; Olley and Baranowski 1985; Wei et al. 1990). Clostridium perfrigens, for instance, does not growth at low temperature though is able to produce histamine at 4 °C (Frank and Yoshinaga 1984). Therefore if a sufficiently high number of bacteria are produced before refrigeration, histidine decarboxylase may have an important role in production of histamine at low temperatures. Similarly, psychrophile bacteria may have an important action in the accumulation of histamine in refrigerated fish, as these may grow and produce histamine at low temperatures during long incubation periods (Okuzumi et al. 1981; Ryser et al. 1984; Spreekens 1987). A new bacterium that can grow and produce toxic concentrations of histamine at temperatures as low as 2 °C, called Morganella psychrotolerans, was reported by Emborg et al. (2006). With Photobacterium phosphoreum, M. psychrotolerans belongs to the psychrotolerant and strongly histamine-producing bacteria. Several well-known bacteria, such as M. morganii, are able to form toxic concentrations of histamine in seafood when products are kept above 7–10 °C. Chilling seafood to 2–5 °C eliminates histamine production by mesophilic bacteria. However, psychrotolerant bacteria can produce toxic concentrations of histamine in seafood at 2–5 °C. In the case of M. psychrotolerans and for fresh and lean tuna loins, a modified atmosphere packaging was shown to reduce histamine formation significantly compared with vaccum packaging (Emborg et al. 2005). For sliced and vaccumpacked cold-smoked tuna, the addition of salt was suggested to limit histamine formation by M. psychrotolerans. Histamine accumulation in refrigerated fish can therefore be the result of: bacteria such as K. pneumoniae that grow and decarboxylate histidine at low temperatures (Frank and Yoshinaga 1984) bacteria such as C. perfrigens that grow at moderate temperature but produce enzymes, which decarboxylate histidine at low temperatures (Frank and Yoshinaga 1984) psychrophile bacteria such as the ones from ‘Group N’ that grow and decarboxylate histidine at temperatures around 0 °C (Okuzumi et al. 1981; Okuzumi and Awano 1983) and like M. psychrotolerans that grow at 2 °C. The effect of temperature in the production of histamine has been discussed by several authors (Edmunds and Eintenmiller 1975; Arnold and Brown 1978; Salguero and Mackie 1979) but a significant inconsistency exists in what concerns the optimal temperature range for production. The main reason for the differences observed appears to be related to the difference between the fish species studied, differences in the contaminating microflora as well as differences in fish handling before incubation (Frank et al. 1981). According to Salguero and Mackie (1979), low concentrations of histamine (0.07– 1.24 mg/100 g) in fresh mackerel (Scomber scombrus) were observed during storage at 0 °C. A slight increase was observed after 14 days at this temperature, attaining 1.86 mg/100 g after 18 days of storage at 0 °C. This value is, however, considerably lower than the maximum allowed (20 mg/100 g). Studies by other authors (Hardy and Smith 1976; Ritchie and Mackie 1980; Wendakoon et al. 1990) agree that production of histamine at 0 °C or at lower temperatures is negligible, even when advanced states of decomposition are attained.

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Some researchers suggest 10 °C as the minimal temperature necessary for histamine production and do not refer any significant amounts of this amine at lower temperatures (Edmund and Eintemiller 1975; Hardy and Smith 1976; Salguero and Mackie 1979). After one month of incubation at 10 °C, no histamine was formed by Pseudomonas morganis, Pseudomonas vulgaris or Hafnia strains (Halász et al. 1994). Klausen and Lund (1986) report similar data and refer the amine content in mackerel and herring as being temperature dependent and at 10 °C 2–20 times higher than at 2 °C. However, Ababouch et al. (1991) indicate that low storage temperatures are not sufficient to inhibit the formation of toxic amines such as histamine. Taylor (1986) states that fish kept for a reduced period (1 day) at a temperature of 20 °C may produce histamine during the storage at lower temperatures as a result of the activity of histamine decarboxylase produced by bacteria in the initial period at higher temperature. Santos et al. (1986a) observed also that storage temperature did not significantly delay the start of the amine increase. Furthermore, several other studies refer the production of histamine at temperatures around 4–5 °C (Arnold et al. 1980; Baldrati et al. 1980; Frank and Yoshinaga 1984; Okuzumi et al. 1984; Yamanaka et al. 1984; Morii et al. 1986; Veciana-Nogués et al. 1990; Wei et al. 1990). Yamanaka et al. (1984) observed, for example, a gradual increase in the histamine content of several fish species kept at 5 °C, which reached 100–600 mg/100 g after 8 days of storage. Emborg et al. (2005) also showed that psychrotolerant bacteria, like Morganella morganii and Photobacterium phophoreum, characterised by growing well at 0–2 °C but not at 35–37 °C, were able at about 2 °C to form toxic histamine concentrations in chilled vacuum-packaged tuna steaks. The authors further add that these bacteria may produce histamine in appropriately chilled fresh food and that vacuum-packaged tuna steaks that comply with existing chilling requirements may have contributed to the substantial histamine fish poisoning problems observed in Europe and North America during the past decade. Dalgaard et al. (2006) also reported that biogenic amines, for example histamine above 1000 parts per million (ppm), can be formed in fresh garfish (Belone belone) at 0–5 °C. This was primarily due to the growth and activity of P. phosphoreum, and only when aerobic plate counts were about 107 cfu/g or above was histamine observed in chilled fish. Amounts of histamine produced thereafter strongly depended on storage temperature and previous freezing of fish. However, the formation of detectable amounts of histamine and other amines mainly increased at and after the time of sensory product rejection. Noteworthy was the marked delay of histamine formation in thawed garfish compared with fresh fish. In fact, freezing inactivated P. phosphoreum, extended shelf life and markedly reduced histamine formation in thawed modified atmosphere packaged garfish during chilled storage. Existent data therefore show the importance of understanding and limiting the growth and activity of P. phosphoreum and other psychrotolerant histamine-producing bacteria in chilled foods, because even if chilled they may represent a histamine fish-poisoning risk. Furthermore, the existence of such heat-labile bacteria responsible for high concentrations of histamine suggests that the often-applied practice of evaluating the capacity of microorganisms for histamine production by experiments performed above 30 °C is not optimal (Taylor and Woychik 1982). Lack of agreement also exists in relation to the optimal temperature for bacterial production of amines. In some studies, optimal temperature for histamine production is reported between 15 and 20 °C (Baldrati et al. 1980; Yamanaka et al. 1984), whereas in

Biogenic amines

47

others the range reported is 30–38 °C (Frank et al. 1981, 1983; Frank and Yoshinaga 1984; Pan 1985). However, most studies refer the maximum histamine production in storage temperatures of 20–25 °C (Kimata 1961; Edmunds and Eintenmiller 1975; Hardy and Smith 1976; Salguero and Mackie 1979; Eintenmiller et al. 1981; Okuzumi et al. 1984; Wei et al. 1990).

3.3

Safety aspects

Biogenic amines such as histamine, tyramine and putrescine play an important role in many critical functions in man and animals. However, consumption of food containing high amounts of these amines may have deleterious effect on human health. The most important food-borne intoxication caused by biogenic amines results from histamine action. Secondary amines such as putrescine and cadaverine can also react with nitrite to form heterocyclic carcinogenic nitrosamines, nitrosopyrolidine and nitrosopiperidine (Huis in’t Veld et al. 1990). Histamine and other biogenic amines are toxic because of their interaction with receptors H1 and H2 on cellular membranes (Santos 1996; Lehane and Olley 2000). Histamine in particular has been implicated as the causative agent in several food poisoning incidents occurring after heating fish. An incident of food-borne poisoning causing illness in three victims due to ingestion of canned mackerel occurred in December 2001, in Taipei Prefecture, northern Taiwan, and was reported by Tsai et al. (2005). Analysis of biogenic amines showed that the leftovers of the victims’ canned mackerel contained 153.9 mg/100 g of histamine. The three other mackerel cans of the same brand and lot number as the suspected canned sample had histamine greater than the hazard action level of 50 mg/100 g. The contents of the other biogenic amines in all four canned samples were found to be less than 10 mg/100 g. On account of the high histamine content and the presence of other biogenic amines in the suspected canned mackerel, the food poisoning reported was strongly suspected as being due to the histamine intoxification. Histamine causes changes in the cardiovascular system, particularly dilation of peripheral blood vessels and urticaria, hypotension, flushing and headache (Stratton et al. 1991). Contraction of intestinal smooth muscle induced by histamine and mediated by the H1 receptors may be responsible for abdominal cramps, diarrhoea and vomiting (Taylor 1986). Histamine also regulates the gastric acid secretion through receptors H2 located on the parietal cells (Soll and Vollin 1977). Nevertheless, it is not known if this process is responsible for some of the symptoms observed during histamine food poisoning (Taylor 1986). These symptoms are generally similar to those found in immunoglobulin-E-mediated food allergies (Taylor et al. 1989) and as in these, there is also individual susceptibility to biogenic amines. Clinical symptoms of histamine food poisoning are more intense when medication that inhibits enzymes that normally detoxify histamine in the intestine is used. To treat these symptoms, antihistamines are used effectively. Histamine is the most toxic amine detected in food (Brink et al. 1990; Huis in’t Veld et al. 1990) and although there is clear evidence implicating it as the main cause of histamine food poisoning, a direct dose–response relation is lacking. In fact, spoiled fish with high amounts of histamine have been found to be more toxic than the same amount of pure histamine taken orally. Furthermore, several authors (Clifford et al. 1989, 1991a,b; Ijomah et al. 1991a,b) also suggest that the action of external histamine in opposition to endogenous

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histamine is reduced, and no clear evidence exists in relation to the potentiation effect of other external amines. Based on the concentration of histamine found in products involved in histamine food poisoning and despite all uncertainties reported, it is clear that the toxicological effects depend on histamine intake concentration. Levels above 500–1000 mg/kg are considered potentially dangerous to human health (Brink et al. 1990). Less in known about the presence of other amines and their toxic doses. For tyramine and β-phenylethylamine, threshold values around 100–800 mg/kg and 30 mg/kg have been suggested, respectively (Brink et al. 1990). A survey of scombrotoxic fish poisoning in Britain (Bartholomew et al. 1987) suggests that most incidents were clearly basic histamine poisoning, but that other toxins, likely other biogenic amines that acted as histamine potentiators or toxic in their own right, may have been involved when suspect fish presented low histamine levels or when the symptoms were not typical. The intestinal tract of mammals has, in general, relatively efficient detoxification mechanisms capable of processing normal dietary consumption of histamine (Huis in’t Veld et al. 1990). When exogenous amines are absorbed from food in normal dietary intakes, they are quickly detoxified under the action of amine oxidases or by conjugation. However, when allergenic individuals are considered, when monoamine oxidase inhibitors are used or when levels of biogenic amines that are too high are ingested, the detoxification process is altered and amines start to accumulate in the body. Monoamine oxidase (MAO) and diamine oxidase (DAO) are enzymes activated by the presence of mono or diamines (Wendakoon and Sakaguchi 1993) and play an important role in this defensive mechanism. Whenever their action is affected by one or more substances known as potentiators, the detoxification process is inhibited. Potentiators may be food-borne putrefactive amines or pharmacological agents (Stratton et al. 1991). Putrescine and cadaverine are known to inhibit histamine-detoxifying enzymes. Tyramine (inhibits MAO), tryptamine (inhibits DAO) and β-phenylethylamine (inhibits DAO and histamine N-methyltransferase) are suggested as having a similar role (Stratton et al. 1991). In favour of the hypothesis about the inhibition of histamine detoxification by histamine potentiators, several scientists have reported that histamine is enhanced by some other component or components in toxic fish (Bjeldanes et al. 1978; Taylor and Lieber 1979; Chu and Bjeldanes 1981; Lyons et al. 1983; Taylor 1986; Stratton et al. 1991). These potentiators would lower the threshold level of histamine needed to produce an adverse reaction. Taylor (1986) reported that mild histamine reactions were produced by doses of pure histamine several times higher than the doses producing severe symptoms when consumed in spoiled fish. However, variability of histamine level in spoiled fish did not allow an accurate estimation of the toxic threshold although a limit of 50 mg/kg of flesh was adopted (US Food and Drug Administration: FDA 2001). Supporting the above thesis, it is also reported that biogenic amines such as putrescine and cadaverine occur in considerable amounts in toxic fish (Arnold and Brown 1978) and at low levels in non-toxic fish (Mietz and Karmas 1977). In laboratory studies with animals, these amines potentiate the biological activity of histamine when higher ratios relative to histamine than those usually present in toxic fish are used. When comparing the spoilage of mackerel and herring at low temperatures, Klausen and Lund (1986) found that the concentrations of cadaverine in mackerel may exceed those of histamine by two to five times, which, according to the authors, could explain the reason why mackerel is often associated with histamine fish poisoning.

Biogenic amines

3.4

49

Quality assessment

Although a considerable amount of research has been done over the years, the reasons for biogenic amine determination in foods remain today unchanged and continue to be twofold. The first is the potential toxicity whereas the second is the possibility of using them as markers of food quality. It is well established that although low amounts of biogenic amines are used in many physiological processes (Bardocz et al. 1995; Eliassen et al. 2002), the absorption of large quantities may induce health problems (Joosten 1988). For these reasons research is still ongoing in this field. Being the result of bacterial action, the presence of histamine should, according to Taylor and Summer (1986), be an indicator of fish quality/deterioration. Histamine may also induce lipid oxidation and therefore rancidification during processing and storage of tuna (Uchida et al. 1990). However, the production of histamine cannot be detected by simple methods like, for instance, change in appearance or development of odours. Fish having high histamine content may display normal appearance and odour (Arnold and Brown 1978). Although several methods are described in the literature, there is not a unique and objective method of quality assessment of fish (Gill 1990). Published methods are essentially based on autolytic changes, bacterial development and lipid oxidation (Sorensen 1992). Among these, the most common are: measurements of volatile basic nitrogen compounds such as trimethylamine (TMA), dimethylamine (DMA), ammonia and other volatile basic compounds associated with fish deterioration; the degradation of nucleotides, for example measurement of a final ATP decomposition product like hypoxanthine (Gill 1990); measurement of fish muscle conductivity (Sorensen 1992); lipid oxidation measurement by the peroxide index or the quantification of malonaldehyde with the thiobarbituric acid method (Sorensen 1992); and the detection and quantification of microbial contamination (Connell 1990). Nevertheless, and despite the development of various objective methods of fish quality evaluation, the main method used in fish inspection is still sensorial analysis. The production of biogenic amines may not, however, result in changes of sensory characteristics (Wurziger and Dickhaut 1978). On account of the high rate of histamine production that characterises some bacterial species, fish contaminated for a short period may be sensorically acceptable but present high levels of histamine (Middlebrooks et al. 1998). To determine the usefulness of histamine as a quality indicator, studies were performed for comparison with other chemical quality indices. Several authors (Khayat 1977; Yamanaka et al. 1980; Nagayama et al. 1985; Putro and Saleth 1985; Veciana-Nogués 1990) reported the existence of a correlation between histamine development in fish and other quality indices such as volatile acids, hypoxanthine and total volatile basic nitrogen compounds (TVBN). However, high histamine levels were also determined in fish that could be considered fresh on account of the low TVBN and bacterial count levels (Yamanaka et al. 1982). Kimata (1961) compared the production of histamine and ammonia in chub mackerel as a function of the storage temperature. Production of histamine at 35 °C was almost negligible compared with ammonia, whereas at lower temperatures (17 °C) the production of histamine was higher than ammonia. Histamine may also be produced in the initial phase of degradation before there are noticeable changes in pH and TVBN (Yamanaka et al. 1984, 1986; Vidal-Carou et al. 1990c). Yamanaka et al. (1984) detected large quantities of histamine

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Fishery Products: Quality, safety and authenticity

during storage of sardine, chub mackerel and saury pike at 5 and 20 °C, though TVBN levels were lower than 20 mg/100 g. The use of efficient and rapid methods of histamine detection is therefore highly important because, as suggested by Arnold and Brown (1978), there is not a direct relation between changes in fish freshness and the histamine level. Despite the conflicting results between biogenic amines and other chemical quality indices, the assessment of amine content in food has received considerable attention for many years for the possibility of using it as an index of quality. Considering that histamine alone is not always useful as an indicator of fish quality, Mietz and Karmas (1977) proposed a chemical quality index to establish the extent of decomposition in fresh tuna before canning. The relationship of five amines (histamine, putrescine, cadaverine, spermine and spermidine) in canned fish was quantified and calculated on a part per million basis in order to be used as an index of tuna decomposition: Index:

Histamine (ppm) + Putrescine (ppm) + Cadaverine (ppm) 1 + Spermine (ppm) + Spermidine (ppm)

Studies of samples collected and decomposed in controlled conditions have shown that the chemical index scores generated with the above formula compared favourably with the sensorial analysis and therefore with the decomposition changes in the samples. This method measures several different compounds produced during different decomposition processes and could be used as a chemical indicator of decomposition of tuna provided biogenic amine determinations are easier to perform for routine analysis. In general, the use of more than a single biogenic amine is advised to overcome the limitation of possible variability in the concentration of one amine, and has been considered as a more appropriate quality indicator. Along with the index of Mietz and Karmas, other examples are the sum of cadaverine and putrescine (Stede and Stockemer 1981), the index described by Veciana-Nogués et al. (1997) for tuna which considers the sum of putrescine, cadaverine, histamine and tyramine, and cadaverine alone as an index of decomposition of salmonoid fishes (Yamanaka et al. 1989). Although not mentioned as a possible scombroid toxin, Baranowski (1985) suggested that urocanic acid could be a useful alternative to histamine as a spoilage index in fish species rich in endogenous histidine. Urocanic acid is an imidazole compound and a histidine metabolite of spoiling fish (Mackie and Salguero 1977; Baranowski 1985). This acid has recently been suggested as the cause of histamine production in ‘in vivo’ experiments with mice (Hart et al. 1997), and which induces the release of histamine by degradation of mast cells in human skin cultures (Wille et al. 1999) in a process similar to that occurring in the gastrointestinal tract. To follow biogenic amines as quality indices, the content of histamine, cadaverine, putrescine, tyramine, agmatine, spermidine, tryptamine, spermine and trimethylamine were studied in parallel with the development of microbial populations and sensory evaluation during the storage of gilthead seabream (Sparus aurata) at three temperatures, 0, 8 and 15 °C (Koutsoumanis et al. 1999). Pseudomonas spp. and H2S-producing bacteria were the most abundant microorganisms while Enterobacteriaceae and lactic acid bacteria were also present in the fish microflora. Of the biogenic amines, putrescine and cadaverine were detected when Pseudomonas spp. exceeded 106–107 cfu/g and reached the highest levels in samples stored at 15 °C after 120 h. Histamine was produced only at 15 °C and reached levels higher than

Biogenic amines

51

50 ppm at 48 h. Agmatine, tryptamine, tyramine and trimethylamine were absent, regardless of the storage temperature. On account of the changes, putrescine and cadaverine were the only amines considered useful as an index of freshness. The production of cadaverine and putrescine by microorganisms may be considered as a natural process because the covalent linking of cadaverine and putrescine to the peptidoglycan is fundamental for regular microbial development (Suzuki et al. 1988). Despite the importance of the production of these two amines, their role and significance in food safety and biogenic amine poisoning is not yet clear. Quality assessment by biogenic amines was also studied by Mendes (1999), who reported changes in histamine, cadaverine, putrescine and agmatine content in sardine (Sardina pilchardus), Atlantic horse mackerel (Trachurus trachurus), chub mackerel (Scomber japonicus) and Atlantic mackerel (Scomber scombrus) during storage at room temperature (20–23 °C) and in ice (2–3 °C). The aerobic colony counts were initially high, 105–106 cfu/g at day 0 and reached a maximum level within 48–55 h in fish stored at 22–23 °C, but only after a prolonged period (10–16 days) in fish at 2–3 °C. Histamine, as well as other amine formation, varied greatly with fish species and storage conditions. Levels of histamine, cadaverine and putrescine increased gradually in all species as decomposition progressed, regardless of storage temperatures, and attained maximum limits for human consumption after 24 h of storage at room temperature. In contrast, amine production in iced fish was considerably reduced and histamine concentration increased slowly until day 7, after which a considerable increase was observed, nevertheless generally below 100 mg/kg. The evaluation of the changes in fish quality showed that no correlation existed between the content of histamine and other amines and the degree of fish decomposition. Thus, the use of histamine or other amines as a freshness index of the studied fish species was not considered appropriate. The proposal that decomposition protects consumers from the action of hazardous biogenic amines therefore seems doubtful. Going against the above conclusions, a report from Kaneko (2000) suggests that odours of decomposition are reliable indicators of histamine risk and that sensory evaluation is an effective tool in the hazard assessment critical control point (HACCP) plans used in the Hawaiian fisheries. From the 583 mixed pelagic fish analysed, 119 showed signs of decomposition and were therefore rejected. From these, only 14 fish exceeded the histamine defect action level of 50 mg/100 g, but it is worth mentioning that they had first been rejected because of odours of decomposition. In this study none of the inspected fish that passed the sensory evaluation exceeded the defect action level. Within the same objective of quality assessment, other fish like fresh pink salmon (Oncorhyncus gorbuscha) fillets and whole Pacific herring (Clupea harengus pallasi) were also stored for 2 weeks at 10 °C to determine if significant amounts of histamine developed before spoilage (Crapo and Himelbloom 1999). Salmon developed moderate spoilage odours by day 4 and intense ones by day 7. In herring, detectable spoilage started also on day 4 and became significant on day 6. Aerobic bacterial counts changed from initial values of 102–103 cfu/g to 107–108 cfu/g at the end of the 2 weeks of storage. Salmon did not show signs of detectable histamine during the storage period, whereas herring presented levels around 55 ppm at day 14. Because toxic levels of histamine were not attained before the onset of visible decomposition, it is therefore adequate to use the spoilage level as a safety indicator against histamine poisoning when 10 °C is used as the storage temperature for salmon and herring.

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Fishery Products: Quality, safety and authenticity

Although the detection of histamine in fish muscle is a clear sign that decomposition has taken place, its occurrence is highly variable. From the above, it is safe to assume that its production is a function of several factors such as: (1) the species of fish and the individual fish, (2) the part of the body of the fish sampled, (3) time and temperatures of storage and (4) types and numbers of bacteria present in the fish (Rawles et al. 1996). Quality assessment using biogenic amines has also been done for freshwater species. Krizet et al. (2004) followed quality changes of vacuum-packed and non-vacuum-packed flesh of carp (Cyprinus carpio) during storage at different temperatures (3 and 15 °C). Chemical, sensory and microbial qualities were measured throughout the storage time to evaluate the effects of both temperature and packaging type. Seven biogenic amines (putrescine, cadaverine, spermidine, spermine, histamine, tyramine and tryptamine) were determined. Low temperature had a dominant effect, resulting in low biogenic amine content and better quality of samples. Biogenic amine formation was reported to be more related to the activity of mesophilic than psychrotrophic bacteria. The content of these amines in the stored carp flesh was shown not to represent any health hazard for individuals, because the content of the most problematic amines (histamine and tyramine) was low. Putrescine and cadaverine showed notable correlation with both sensory levels and total microbial counts within all storage trials and therefore were considered good quality indicators. The biogenic amine content of other freshwater species like rainbow trout, whole and filleted, was also monitored during ice storage (18 days) and related to the respective microbial and sensorial changes (Chytiri et al. 2004). Eight amines were determined (putrescine, cadaverine, tyramine, spermidine, tryptamine, β-phenylethylamine, spermine and histamine). In all cases, concentration was higher in fillet than whole trout samples. Pseudomonas spp., H2S-producing bacteria and to a lesser extent Enterobacteriaceae counts remained below 106 cfu/g throughout the entire storage period, accounting for the low production of histamine. A putrescine value of 13–14 mg/kg and a spermine value of approximately 7 mg/ kg obtained after 12 and 9 days, respectively, was proposed as a freshness indicator and, therefore, as the upper limit for spoilage initiation of fresh rainbow trout based on sensorial and microbiological data (total viable counts of 106–107 cfu/g). For other amines, both tyramine and spermine were also reported as being useful as freshness indicators, preferably for whole trout. Because tryptamine, β-phenylethylamine, histamine and cadaverine were only produced during the later stages of storage, they were not considered suitable as freshness indicators. Mietz and Karmas (1978) also found that histamine content varied extensively with the fish species and did not recommend using its determination for quality assessment of fish flesh. The same opinion was confirmed by Mendes (1999), who examined histamine formation on sardine and mackerel. No correlation was found between histamine production and organoleptic quality. However, different results were obtained by Ozogul et al. (2004) when performing the quality assessment of sardine (Sardina pilchardus) stored in modified atmosphere packaging, vacuum packaging and in air. The rate of increase of histamine during storage of samples at 4 °C was fairly linear with storage time in all three cases. At the time of sensorial rejection, after 12 days in modified atmosphere packaging, 9 days in vacuum packaging and 3 days in air, the content of histamine was similar and ranged between 7.5 and 9 mg/100 g. Similar results were reported by Pacheco-Aguilar et al. (2000), who found the histamine content of Monterey sardine (Sardinops sagax caerulea) stored in air at 4 °C to be less than 10 mg/100 g at the last day of sensory acceptability. El Marrackchi et al.

Biogenic amines

53

(1990) reported that the amount of histamine in sardine (S. pilchardus) flesh at the time of rejection (12 days) in ice was 16.2 mg/100 g. At the time of rejection, the content of histamine in herring and mackerel stored in vacuum packaging at 2 °C were 42 and 43 ppm, respectively (Klausen and Lund 1986). Histamine levels in salmon steaks during refrigeration storage (2 °C) were reported to be at the time of rejection, 12.23 ppm, 33.24 ppm and 58.69 ppm when stored in air, CO2/air (20/80) and CO2/air (40/60), respectively (Hoz et al. 2000). The suitability of biogenic amines, considered either individually or in combination, as chemical markers of freshness was also followed in the overall evaluation of hake hygienic quality during ice storage (Baixas-Nogueras et al. 2005). The earlier and higher cadaverine production compared with the other biogenic amines, and its association with the development of the specific spoilage organism Shewanella putrefaciens, suggested that this amine is the specific spoilage biogenic amine in hake stored in ice. On the contrary, agmatine does not seem to be appropriate to evaluate hake freshness, because it did not present a regular pattern of change throughout ice storage. In this study, histamine levels were relatively low (less than 3.5 μg/g) and always lower than those described in pelagic fish. Nevertheless detectable amounts of histamine appeared before the rejection point established by sensory analysis. Despite the absence of toxicological significance, the detection of histamine even at such low levels was suggested as qualitatively meaningful from the hygienic point of view, because it is the result of a noticeable decrease in hake freshness. Taking into account that higher histamine amounts are produced during storage at abusive temperatures (BaixasNogueras et al. 2001), the detection of histamine in hake may help to identify a possible failure in the chilling chain. At the point of rejection (8–10 days) by sensory and microbial limiting values, biogenic amines in hake stored in ice ranged between 8 and 16 mg/g for cadaverine, 3 and 5 mg/g for putrescine and histamine, and 4 and 6 mg/g for tyramine. Though the concentration of histamine in hake at the time of rejection was lower than the reported for tuna by Veciana-Nogues et al. (1997), the levels of the other amines, namely putrescine, cadaverine and tyramine, were similar or slightly higher. Because hake showed considerable biogenic amine accumulation during storage, the use of amines was considered appropriate as indicators for freshness or quality assessment. The limit of 50 mg/g reported for the biogenic amine index (putrescine + cadaverine + histamine + tyramine) of tuna was, however, not observed in hake, even after both sensory and microbial rejection, suggesting that this limit should be specifically determined for hake. Considering that fish spoilage is highly variable and depends on several factors, such as handling procedures and spoilage flora, Baixas-Nogueras et al. (2005) proposed a preliminary biogenic amine index in the range 15–20 mg/g as a limit for acceptability of hake. In a similar work with the same species, Ruiz-Capillas and Moral (2001) also reported that cadaverine presented the highest increase during ice storage and with agmatine occurred before spoilage, therefore indicating that these amines may indicate the freshness of hake stored in ice. Safety concerns and quality loss because of biogenic amine formation during storage were also followed in fish from tropical and sub-tropical countries. Guizami et al. (2005) studied the effect of storage temperature on histamine production and freshness of yellowfin tuna (Thunnus albacares). During storage at 0 and 8 °C, yellowfin tuna were rejected by sensory analysis before histamine reached the toxic level proposed by the US FDA (5 mg/100 g). For histamine formation in yellowfin tuna stored at 8 and 20 °C, the loss of shelf life correlated with the loss of the safety. Nevertheless, tuna stored at 0 °C remained safe during

54

Fishery Products: Quality, safety and authenticity

the storage period (17 days) whereas their shelf life was limited to 12 days. Preservation of yellowfin tuna safety at 0 °C was probably the result of histamine decomposition at this temperature. Histamine content is known to depend not only on histamine-forming bacteria but also on the presence of histamine-decomposing bacteria within the flora (Sato et al. 1994). A similar finding was observed by Mazorra-Manzano et al. (2000), who found that black skipjack maintained its safety for more than 21 days at 0 °C, whereas the K value as the freshness parameter reached 57.8% in 14 days, thus indicating the end of shelf life. Freezing of fish can significantly reduce bacterial counts. However, it will not inhibit the activity of decarboxylase enzymes produced before freezing. Staruszkiewicz et al. (2004) studied the effect of onboard and dockside handling on the formation of biogenic amines in mahimahi (Coryphena hippurus), skipjack tuna (Katsuwonus pelamis) and yellowfin tuna (Thunnus albacares), and reported that histidine decarboxylase activity was retained in some frozen samples of the fish. This behaviour could result in further increases in histamine during thawing. Knowledge of the time–temperature history of frozen fish is therefore very important because outbreaks of histamine poisoning can be caused by the consumption of thawed fish containing biogenic amines if the fish was previously stored at an inappropriate temperature (Flick et al. 2001).

3.5

Regulatory issues

On account of the recurrence of histamine poisoning as a global problem and because of the importance of the international trade of the problematic fish species, many countries established maximal limits or guidelines on levels of traded fish. The European Union demands that nine samples must be analysed from each batch of fishery products from fish species associated with a high amount of histamine, particularly fish species of the following families: Scombridae, Clupeidae, Engraulidae, Coryphaenidae, Pomatomidae and Scombresosidae (Official Journal of the European Union 2005). The histamine content in the samples must fulfil the following requirements to make samples acceptable for human consumption: the mean content must not exceed 10 mg/100 g two samples may have a value of more than 10 mg/100 g but less than 20 mg/ 100 g no sample may show a value exceeding 20 mg/100 g. In fishery products that have undergone enzymatic maturation treatment in brine, manufactured from fish species of the above-mentioned families, the histamine limit values are increased to 20 and 40 mg/100 g, respectively. In other countries such as Australia, New Zealand and Brazil, the level of histamine in a composite sample of fish or fish products must not exceed 10 mg/100 g. In what concerns the US FDA, guidelines have been established for tuna, mahi mahi and related fish species that specify 50 mg/100 g as the toxicity level and 5 mg/100 g as the defect action level because histamine is not uniformly distributed in a decomposed fish. Thus, if 5 mg/100 g is detected in one section of the fish, there is a possibility that other sections may exceed 50 mg/100 g (FDA 2001). In fact, histamine concentrations can vary considerably between fish from a single catch and even between different portions of a tuna

Biogenic amines

55

fish (Lerke et al. 1978; Frank et al. 1981). Samples from the dorsal muscle of fresh yellowfin tuna were reported by Lerke et al. (1978) as having 52 ± 15 mg/kg of histamine, whereas samples from the bell cavity of the same fish stored under the same conditions showed 4400 ± 2700 mg/kg of histamine. During a chilli-marination experiment using tuna implicated in a histamine fish poisoning outbreak in Denmark, Emborg et al. (2005) also referred also to the differences in histamine concentrations between the prepared (chilli-marinated and grilled, 7100–9100 mg/kg) and the unprepared (70–236 mg/kg) tuna samples, probably reflecting differences between the loins of the tuna rather than the effect of product preparation. This study also confirmed that evaluation of low concentrations in test samples is relevant to ensure absence of toxic histamine concentrations in a batch of tuna, and further support the low concentration of histamine allowed in US and EU regulations compared with the toxicity levels of histamine (<50 mg/kg to 13,280 mg/kg) reported to cause histamine fish poisoning (Bartholomew et al. 1987; Brett 2000; Kan et al. 2000). It is clear that variable and differing data are frequently reported and that the fish species affect the production of biogenic amines. All the reported results and the conflicting evidence present a real problem if biogenic amines are to be used in quality and safety assessment and therefore as legal safety indices, thus suggesting that further studies are required in this area.

3.6 Methods of biogenic amine quantification Different methods of assaying the biogenic amine content in fish and fish products have been reported. One of the first amines to be detected in fish was histamine. The method of detection was based on the fact that it causes contraction of guinea-pig ileum (Geiger 1944). In 1957, Sager and Horwitz compared this method with a colorimetric method of histamine determination. Results with the biological method were consistently higher than those obtained by the chemical method, and the extraction technique accounted for the difference. Once the extraction procedure was modified, the values obtained were coincident and the method using the contraction of guinea pig ileum became the biological method of election of the AOAC (Official Method 954.04), because it allowed a rapid screening of histamine in many samples. Chronologically, the second official method for detecting and quantifying histamine in seafood (AOAC Official Method 957.07) begins with a complex chemical extraction using methanol, benzaldehyde, a sodium hydroxide solution and a benzene-n-butanol mixture, after which the extracted sample is passed through a crude cotton acid succinate column. The eluted fraction is collected and quantified by reading at 475 nm in a spectrophotometer (AOAC 1990). In the search for a simple and fast method of histamine detection, other biological methods that relied on the sensitivity to histamine of different biological systems, like protozoans, fish, insects, bull sperm, cellular cultures and microorganisms were also developed (De Waart et al. 1972). Blonz and Olcott (1977) suggested, for example, the use of a small crustacean, Daphnia artemia, because of its high mortality (90–100%) when exposed to tuna extracts responsible for histamine fish poisoning outbreaks. Biological methods have several disadvantages, however, mainly the need for many animals, the possibility of individual variation in the response to histamine and issues related

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with animal welfare. Because of this, since the time of the first methods, many diverse chemical analytical procedures have been published for histamine in raw and canned fish. One of the most popular methods is the colorimetric determination of histamine. This technique involves extraction processes and chromatographic purification of histamine by the use of carboxylic cation exchangers coupled with reaction with a diazonium salt/2,4dinitroflurobenzene and detection at 495 nm (Code and McIntire 1956; Sager and Horwitz 1957; Hardy and Smith 1976; Kawabata et al. 1960). The reaction between the purified histamine and copper and a dye to form a red complex that could be easily quantified was suggested by Bateman et al. (1994). A purple compound upon oxidation of histamine when DAO, horseradish peroxidase and leuco-crystal violet were used was also reported by Lerke et al. (1983) for histamine determination. Rodriguez-Jerez et al. (1994) further developed this enzymatic test and suggested the use of a detection wavelength of 580 nm and an incubation time of 15 min for the reaction. Nevertheless, because of the lower sensitivity and specificity of the colorimetric methods used for histamine determination, other techniques were developed. Some of the more relevant are listed in Table 3.1. Most of the methods listed were, in general, developed to determine histamine and other biogenic amines in research applications. Rogers and Staruszkiewicz (2000) summarised the known limitations of these techniques for more widespread and routine use by industry. Methods involving chromatography of histamine derivatives use expensive instrumentation such as HPLC or gas chromatography. Furthermore, although HPLC with either pre-column or post-column derivatisation can be automated and used for the simultaneous analysis of several amines, pre-column derivatisation involves liquid/liquid extractions which are a limiting factor in quantification (Hui and Taylor 1978; Mietz and Karmas 1978). On the other hand, in HPLC with post-column methods the pumps have been considered a frequent source of variation, especially when buffers are in the mobile phase (Veciana-Nogues et al. 1995). In HPLC with on-column fluorescence, derivatisation effort for the isolation of the analyte is considerably reduced but some background problems are still present (Saito et al. 1992). Thin-layer chromatography is inexpensive and allows many analyses on one plate, but is only semi-quantitative (Lieber and Taylor 1978). The method with an oxygen-sensorbased electrode uses a trichloroacetic extraction and an enzyme that is not commercially available (Ohashi et al. 1994). Other enzyme-based screening tests are unsuitable for routine analysis because they use several perchloric acid extractions and a 2-hour incubation period (Lerke et al. 1983; Lopez and Sabater 1993). The solid-phase dipstick method is rapid but only allows the determination of histamine in a very narrow range (Hall et al. 1995). In flow injection analysis, no pre-treatment of the extract is necessary. However, meticulous selection of the concentration of the reagents and careful control of four pumps is needed to maintain specificity for the histamine derivative (Hungerford et al. 1990). The disadvantages of the laboratory-based methods therefore mean that there is a need for a simple, rapid test for histamine that can be performed easily in a commercial environment. To overcome this problem, several rapid testing methods have been produced, which are advertised as being easy to use, rapid and capable of giving accurate results at low cost. Table 3.2 summarises some of the commercially available rapid tests for histamine analysis. The kits are classified as either qualitative or quantitative in the range 1–500 ppm. Most are based on an enzyme-linked immunosorbent assay (ELISA) that measures the direct competition between the histamine to be assayed and the enzyme-labelled histamine conjugate. Some other tests are based on a chemical colorimetric analysis.

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Table 3.1 Methods used in the analysis of histamine in fish products. Type of fishery product Canned fish

Fish

Methodology High-performance liquid chromatography (HPLC) Fluorimetric method HPLC-pre column method Amine oxidase-based flow biosensor Capillary zone electrophoresis (CE) Colorimetric method with imidazole reacting p-phenyldiazonium sulfonate DAO-based amperometric biosensor Electrochemical biosensor Enzyme sensor array Enzyme–based screening test

Seafood

Tuna

Flow injection determination with a histamine dehydrogenase-based sensor Fluorimetric method HPLC-post column method Ion chromatography-integrated pulsed amperometric detection Monoclonal anti-body based ELISA Oxygen-sensor based method Thin-layer chromatographic method Automated kinetics-enhanced flow injection method Biological method Chemical method Flow injection method Fluorimetric method HPLC-oncolumn fluorescence derivatisation method Ion-exchange chromatographic method Modification of AOAC method Cooper chelation method Solid phase-dipstick method Thin-layer chromatographic method

Reference Yen and Hsieh 1991 Lerke and Bell 1976 Mietz and Karmas 1977; Hui and Taylor 1983 Frebort et al. 2000 Mopper and Sciacchitano 1994; Trenerry et al. 1998 Patange et al. 2005 Male et al. 1996 Draisci et al. 1998b Lange and Whittmann 2002 Lerke et al. 1983; Lopez-Sabater et al. 1993 Takagi and Shikata 2004 Taylor et al. 1978 Veciana-Nogues et al. 1995 Draisci et al. 1998a Serrar et al. 1995 Ohashi et al. 1994 Shultz et al. 1976 Hungerford et al. 2001 AOAC 1995a AOAC 1995b Hungerford et al. 1990 AOAC 1995c Saito et al. 1992 Simon-Sarkadi and Kovács 2002 Rogers and Staruszkiewicz 1997 Bateman et al. 1994 Hall et al. 1995 Lieber and Taylor 1978

Some of the rapid tests referred in Table 3.2, namely Histamarine Enzyme Immunoassay kit, K1-HTM, K3-HTM, ALERT® kit and the VERATOX Histamine kit, were evaluated by Rogers and Staruszkiewicz (2000). The histamine content determined by each test kit was compared with the histamine concentration using AOAC method 977.13 (fluorimetric method). The results showed that all the kits were acceptable for use as screening test for histamine and were able to distinguish between products that contained less than 50 ppm and those that contained more than 50 ppm. The same authors suggest that both the ALERT® and VERATOX Histamine kits have several practical advantages over the other test kits, especially for easier extractions, in having all the required reagents and plastic ware, all

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Table 3.2 Rapid methods used in the analysis of histamine in fish products. Histamine test kit HISTAMARINE Enzyme Immunoassay kit (AOAC approved) K1-HTM (quantitative test) K3-HTM (qualitative test) HISTAQUANT (quantitative test) HISTAMETER (qualitative test) ALERT® kit VERATOX Histamine kit RIDASCREEN RIDAQUICK Histamine TRANSIA Tube Histamine kit (semi-quantitative) HisQuick TM Histamine Rapid Test HISTAMINE Food HISTAMINE ELISA kit

Company Immunotech (Colter Corp., OPA Locka, FL, USA) Immuno-Diagnostic Reagents (Vista, CA, USA) Biomedix (CA, USA) Neogen Corporation (lansing, MI, USA) R-Biopharm, Inc. (Marshall, MI, USA) GENE-TRACK Systems (Hopkinton, MA, USA) BioSource Europe S.A. (Nivelles, Belgium) Immuno Biological Laboratories (Hamburg, Germany)

volumes to be measured being 100 μl (requiring only one pipette), the results being easily read visually or with a plate reader, and having significantly reduced incubation times compared with the other ELISA-based tests. Despite these rapid methods, most frequently used analyses of biogenic amines in fish involve chromatography of histamine and biogenic amine derivatives using HPLC or gas chromatography (Redmond and Tseng 1979; Henion et al. 1981; Hayman et al. 1985; Suzuki et al. 1990; Yen and Hsieh 1991; Jeyashakila et al. 2001; Ozogul et al. 2002). However, a considerably fewer reports have been published on the simultaneous determination of multiple biogenic amines. The AOAC procedure (Method 977.13 2002) is the official method for analysing histamine in foods and has been used routinely in the USA as a basis for taking regulatory action on fish containing histamine (Staruszkiewicz et al. 1977). The method involves extraction of histamine with hot methanol and ion exchange chromatography before derivatisation with o-phthaldehyde. The fluorescence of the formed compound is measured. A modification, changing the analyte extraction solvent from 75 to 100% methanol, was further approved in a second collaborative study to allow other biogenic amines, such as cadaverine and putrescine, to be determined from the same extracts (Rogers and Staruszkiewicz 1997). In the European Union, a liquid chromatographic method described by Malle et al. (1996) is used as reference for histamine analysis (EU 2005). This method allows the quantitative determination of putrefaction amines: putrescine, cadaverine, histamine, spermidine and spermine. These amines are extracted from fish by grinding with perchloric acid at −20 °C. The amines react with dansyl chloride at alkaline pH, are dried under a stream of nitrogen, placed in an automatic injector at −20 °C, separated in a C18 reversed-phase column at 25 °C and quantified in an ultraviolet detector. For future trends in biogenic amine analysis, the most promising technology for a rapid test appears to be an electrode (Hibi and Senda 2000; Zeng et al. 2000; Niculescu et al. 2001; Iwaki et al. 2002; Lange and Whittmann 2002; Lili et al. 2002) capable of detecting

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59

histamine and other diamines rather than HPLC or capillary zone electrophoresis. These recent publications on histamine electrodes suggest the technology has improved from the first stages and that a stable histamine electrode may be available soon.

3.7

References

Ababouch, L., Afilal, M.E. and Benabdeljelil, H. (1991) Quantitative changes in bacteria, amino acids and biogenic amines in sardine (Sardina pilchardus) stored at ambient temperature (25–28 °C) and in ice. International Journal of Food Science & Technology 26: 297–306. Abell, L.M. and O’Leary, M.H. (1988) Isotope effect studies of the pyridoxal 5′-phosphate dependent histidine decarboxylase from Morganella morganni. Biochemistry 27: 5927–5933. Aksnes, A. (1988) Location of enzymes responsible for autolysis in bulk-stored capelin (Mallotus villosus). Journal of the Science of Food and Agriculture 44: 263–271. AOAC (1995a) Histamine in seafood: biological method. Sec. 35.1.30, Method 954.04. In: P.A. Cunniff (Ed.) Official Methods of Analysis of AOAC INTERNATIONAL (16th edn). AOAC International, Gaithersburg, MD, pp. 14–15. AOAC (1995b) Histamine in seafood: chemical method. Sec. 35.5.31, Method 957.07. In: P.A. Cunniff (Ed.) Official Methods of Analysis of AOAC INTERNATIONAL (16th edn). AOAC International, Gaithersburg, MD, pp. 15–16. AOAC (1995c) Histamine in seafood: fluorometric method. Sec. 35.1.32, Method 977.13. In P.A. Cunniff (Ed.) Official Methods of Analysis of AOAC INTERNATIONAL (16th edn). AOAC International, Gaithersburg, MD, pp. 16–17. Arnold, S.H. and Brown, W.D. (1978) Histamine toxicity from fish products. Advances in Food Research 34: 113–154. Arnold, S.H., Price, R.J. and Brown, W.D. (1980). Histamine formation by bacteria isolated from skipjack tuna, Katsuwonus pelamis. Bulletin of the Japanese Society of Scientific Fisheries 46(8): 991–995. Askar, A. and Treptow, H. (1986) Biogene Amine in Lebensmitteln. Vorkommen, Bedeutung und Bestimmung. Eugen Ulmer GmbH and Co, Stuttgart, Germany. Baixas-Nogueras, S., Bover-Cid, S., Vidal-Carou, M.C. and Veciana-Nogues, M.T. (2001) Volatile and nonvolatile amines in Mediterranean hake as a function of their storage temperature. Journal of Food Science 66: 83–88. Baixas-Nogueras, S., Bover-Cid, S., Veciana-Nogues, M.T., Marine-Font, A. and Vidal-Carou, M.C. (2005) Biogenic amine index for freshness evaluation in iced mediterranean hake (Merluccius merluccius). Journal of Food Protection 68(11): 2433–2438. Baldrati, G., Fornari, M.B., Spotti, E. and Incerti, I. (1980) Effect of temperature on histamine formation in fish rich in histidine. Industria Conserve 55: 114–122. Bao, L., Sun, D., Tachikawa, H. and Davidson, V.L. (2002) Improved sensitivity of a histamine sensor using an engineered methylamine dehydrogenase. Analytical Chemistry 74(5): 1144–1148. Baranowski, J.D. (1985) Low-temperature production of urocanic acid by spoilage bacteria isolated from mahimahi (Coryphaena hippurus). Applied and Environmental Microbiology 50(2): 546–547. Bardóz, S., Grant, G., Brown, D.S., Ralph, A. and Pusztai, A. (1993) Polyamines in food – implications for growth and health. Journal of Nutritional Biochemistry 4: 66–71. Bardocz, S., Duguid, T.J., Brown, D.S., Grant, G., Pusztai, A., White, A. and Ralph, A. (1995) The importance of dietary polyamines in cell regeneration and growth. British Journal of Nutrition 73: 819–828.

60

Fishery Products: Quality, safety and authenticity

Bartholomew, B.A., Berry, P.R., Rodhouse, J.C. and Gilbert, R.J. (1987) Scombrotoxic fish poisoning in Britain: feature of over 250 suspected incidents from 1976 to 1986. Epidemiology and Infection 99: 775–782. Bateman, R.C., Eldrige, D.B., Wade, S., McCoy Messer, J., Jester, E.L.E. and Mowdy, D.E. (1994) Copper chelation assay for histamine in tuna. Journal of Food Science 59(3): 517–518, 543. Behling, A.R. and Taylor, S.L. (1982) Bacterial histamine production as a function of temperature and time of incubation. Journal of Food Science 47: 1311–1314, 1317. Bjeldanes, L.F., Schutz, D.E. and Morris, M.M. (1978) On the aetiology of scombroid poisoning: cadaverine potentiation of histamine toxicity in the guinea pig. Food and Cosmetics Toxicology 16(2): 157–159. Blonz, E.R. and Olcott, H.S. (1977) Daphnia magna as a bioassay system for histamine in tuna extracts. Bulletin of the Japanese Society of Scientific Fisheries 44: 517–519. Brett, M. (2000) Scombrotoxic fish poisoning. CDR Weekly 10: 171. Brink, B. ten, Damink, C., Joosten, H.M.L.J. and Huis in’t Veld, J.H.J. (1990) Occurrence and formation of biologically active amines in foods. International Journal of Food Microbiology 11: 73–84. Chu, C.H. and Bjeldanes, L.F. (1981) Effect of diamines, polyamines and tuna fish extracts on the binding of histamine to mucin in vitro. Journal of Food Science 47: 79–88. Chytiri, S., Paleologos, E., Savvaidis, I. and Kontominas, M.G. (2004) Relation of biogenic amines with microbial and sensory changes of whole and filleted freshwater rainbow trout (Onchorynchus mykiss) stored on ice. Journal of Food Protection 67(5): 960–965. Clifford, M.N., Walker, R., Wright, J., Hardy, R. and Murray, C.K. (1989) Studies with volunteers on the role of histamine in suspected scombrotoxicosis. Journal of the Science of Food and Agriculture 47: 365–375. Clifford, M.N., Walker, R., Ijomah, P., Wright, J., Murray, C.K. and Hardy, R. (1991a) Is there a role for amines other than histamine in the aetiology of scombrotoxicosis? Food Additives and Contaminants 8: 641–652. Clifford, M.N., Walker, R., Wright, J., Ijomah, P., Hardy, R., Murray, C.K. and Rainsford, K.D. (1991b) Evidence of histamine being the causative toxin in scombroid-fish poisoning. New England Journal of Medicine 325: 515–516. Code, C.F. and McIntire, F.C. (1956) Methods in Biochemical Analysis III. Wiley, New York, p. 49. Connell, J.J. (1990) Control of Fish Quality (3rd edn). Fishing News Books, Blackwell Scientific Publications, Cambridge. Crapo, C. and Himelbloom, B. (1999) Spoilage and histamine in whole Pacific herring (Clupea harengus pallasi) and pink salmon (Oncorhynchus gorbuscha) fillets. Journal of Food Safety 19: 45–55. Dalgaard, P., Madsen, H.L., Samieian, N. and Emborg, J. (2006) Biogenic amines formation and microbial spoilage in chilled garfish (Belone belone belone) – effect of modified atmosphere packaging and previous frozen storage. Journal of Applied Microbiology 101: 80–95. De Waart, J., van Aken, F. and Pouw, H. (1972) Detection of orally toxic microbial metabolites in foods with bioassay systems. Zentrablatt für Bakteriologie [Orig. A]. 222(1): 96–114. Draisci, R., Volpe, G., Lucentini, L., Cecilia, A., Federico, R. and Palleschi, G. (1998a) Determination of biogenic amines with an electrochemical biosensor and its application to salted anchovies. Food Chemistry 62(2): 225–232. Draisci, R., Giannetti, L., Boria, P., Lucentini, L., Palleschi, L. and Cavalli, S. (1998b) Improved ion chromatography-integrated pulsed amperometric detection method for the evaluation of biogenic amines in food or vegetable or animal origin and in fermented foods. Journal of Chromatography A 798(1–2): 109–116. Edmunds, W.J. and Eitenmiller, R.R. (1975). Effect of storage time and temperature on histamine content and histidine decarboxylase activity of aquatic species. Journal of Food Science 40: 515–519.

Biogenic amines

61

Eitenmiller, R.R., Orr, J.H. and Wallis, W.W. (1982). Histamine formation in fish: microbial and biochemical conditions. In: R.E. Martin, G.J. Flick, C.E. Bebard and D.R. Ward (Eds) Chemistry and Biochemistry of Marine Food Products. Avi, Westport, CT, pp. 39–50. Eitenmiller, R.R., Wallis, J.W., Orr, J.H. and Phillips, R.D. (1981) Production of histidine decarboxylase and histamine by Proteus morganii. Journal of Food Protection 44(11): 815– 820. Eliassen, K.A., Reistad, R., Risoen, U. and Ronning, H.F. (2002) Dietary polyamines. Food Chemistry 78: 273–280. Emborg, J., Dalgaard, P. and Ahrens, P. (2006) Morganella psychrotolerans sp. nov., a histamineproducing bacterium isolated from various seafoods. International Journal of Systematic and Evolutionary Microbiology 56: 2473–2479. Emborg, J., Laursen, B.G. and Dalgaard, P. (2005) Significant histamine formation in tuna (Thunnus albacares) at 2 °C – effect of vacuum- and modified atmosphere-packaging on psychrotolerant bacteria. International Journal of Food Microbiology 101(3): 263–279. EU (2005) Commission Regulation (EC) No 2073/2005. of 15 November 2005 on microbiological criteria for foodstuff. Official Journal of the European Union L338/1 of 22.12.2005. FDA (US Food and Drug Administration) (2001) Fish and Fishery Products Hazards and Controls Guide (3rd edn). FDA, Center for Food Safety and Applied Nutrition, Office of Seafood, Washington DC, USA. www.cfsan.fda.gov/~comm/haccpsea.html. Flick, G.J., Oria, M.P. and Douglas, L. (2001) Potential hazards in cold-smoked fish: biogenic amines. Journal of Food Science 66(Suppl.): S1088–S1099. Frank, H.A. and Yoshinaga, D.H. (1984) Histamine formation in tuna. In: Seafood Toxins. Symposium series no. 262. American Chemical Society, Washington D.C. 19, pp. 443–451. Frank, H.A., Yoshinaga, D.H. and Wu, I.P. (1983) Nomograph for estimating histamine formation in skipjack tuna at elevated temperatures. Marine Fisheries Review 45: 40–44. Frank, H.A., Yoshinaga, D.H. and Nip, W.K. (1981) Histamine formation and honeycombing during decomposition of skipjack tuna, Katsuwonus pelamis, at elevated temperatures. Marine Fisheries Review 43(10): 9–14. Frebort, I., Skoupa, L. and Pec, P. (2000) Amine oxidase-based flow biosensor for the assessment of fish freshness. Food Control 11: 13–18. Geiger, E. (1944) Histamine content of unprocessed and canned fish. A tentative method for quantitative determination of spoilage. Food Research 9(4): 293–297. Gildberg, A. (1978) Proteolytic activity and the frequency of burst bellies in capelin. Journal of Food Technology 13: 409–416. Gill, T.A. (1990) Objective analysis of seafood quality. Food Reviews International 6: 681–714. Guizani, N., Moza, A.A., Ismail, M.A., Mothershaw, A. and Mohammad, S.R. (2005) The effect of storage temperature on histamine production and the freshness of yellowfin tuna (Thunnus albacares). Food Research International 38(29): 215–222. Halász, A., Barath, A., Simon-Sarkadi, L. and Holzapfel, W. (1994) Biogenic amines and their production by microorganisms in food. Trends in Food Science & Technology 5: 42–48. Hall, M., Eldridge, D.B., Saunders, R.D., Fairclough, D.L. and Bateman, R.C. Jr. (1995) A rapid dipstick test for histamine in tuna. Food Biotechnology 9: 39–57. Hardy, R. and Smith, J.G.M. (1976) The storage of mackerel (Scomber scombrus). Development of histamine and rancidity. Journal of the Science of Food and Agriculture 27: 595–599. Hart, P.H., Jaksic, A., Swift, G., Norval, M., El-Ghorr, A.A. and Findlay-Jones, J.J. (1997) Histamine involvement in UVB- and cis-urocanic acid-induced systemic suppression of contact hypersensitivity responses. Immunology 91(4): 601–608. Hayman, A.R., Gray, D.O. and Evans, S.V. (1985) New high-performance liquid chromatographic system for the separation of biogenic amines as their Dns derivatives. Journal of Chromatography 325: 462–466.

62

Fishery Products: Quality, safety and authenticity

Henion, J.D., Nosanchuk, J.S. and Bilder, B.M. (1981) Capillary gas chromatographic–mass spectrometric determination of histamine in tuna fish causing scombroid poisoning. Journal of Chromatography 213: 475–480. Hibi, T. and Senda, M. (2000) Enzymatic assay of histamine by amperometric detection of H2O2 with a peroxidase-based sensor. Bioscience, Biotechnology, and Biochemistry 64(9): 1963–1966. Hoz, L., Lopez-Galvez, D.E., Fernandez, M., Hierro, E. and Ordonez, J.A. (2000) Use of carbon dioxide enriched atmospheres in the refrigerated storage (2 °C) of salmon (Salmo salar) steaks. European Food Research and Technology 210: 179–188. Hui, J.Y. and Taylor, S.L. (1983) High pressure liquid chromatographic determination of putrefactive amines in foods. Journal of AOAC INTERNATIONAL 66(4): 853–857. Huis in’t Veld, J.H.J., Hose, H., Schaafsma, G.J., Silla, H. and Smith, J.E. (1990) Health aspects of food biotechnology. In: P. Zeuthen, J.C. Cheftel, C. Ericksson, T.R. Gormley, P. Link and K. Paulus (Eds). Processing and Quality of Foods, vol. 2. Food Biotechnology: Avenues to healthy and nutritious products. Elsevier Applied Science, London and New York, pp. 2.73–2.97. Hungerford, J.M., Hollingworth, T.A. and Wekell, M.M. (2001) Automated kinetics-enhanced flow-injection method for histamine in regulatory laboratories: rapid screening and suitability requirements. Analytica Chimica Acta 438(1–2): 123–129. Hungerford, J.M., Walker, K.D., Wekell, M.M., LaRose, J.E. and Throm, H.R. (1990) Selective determination of histamine by flow injection analysis. Analytical Chemistry 62(18): 1971–1976. Ijomah, P., Clifford, M.N., Walker, R., Wright, J., Hardy, R. and Murray, C.K. (1991a) Further volunteer studies on scombrotoxicosis. In: J.R. Burt, R. Hardy and K.J. Whittle (Eds). Pelagic Fish: The Resource and its Exploitation, Fishing News Books, Oxford, pp. 194–199. Ijomah, P., Clifford, M.N., Walker, R., Wright, J., Hardy, R. and Murray, C.K. (1991b) The importance of endogenous histamine relative to dietary histamine in the aetiology of scombrotoxicosis. Food Additives and Contaminants 8: 531–542. Iwaki, S., Ogasawara, M., Kurita, R., Niwa, O., Tanizawa, K., Ohashi, Y. and Maeyama, K. (2002) Real-time monitoring of histamine released from rat basophilic leukemia (RBL-2H3) cells with a histamine microsensor using recombinant histamine oxidase. Analytical Biochemistry 304(2): 236–243. Jeyashakila, R., Vasundhara, T.S. and Kumudavally, K.V. (2001) A comparison of TLC-densitometry and HPLC method for the determination of biogenic amines in fish and fishery products. Food Chemistry 75: 255–259. Joosten, H.M.L.J. (1988) The biogenic amine contents of Dutch cheese and their toxicological significance. Netherlands Milk and Dairy Journal 42: 25–42. Kan, K., Ushiyama, H., Shindo, T., Uehara, S. and Yasuda, K. (2000) Outbreak of histamine poisoning due to ingestion of fish (Lepidocybium flavobrunneum). Journal of the Food Hygiene Society of Japan 41: 116–121. Kaneko, J. (PacMar, Inc., Honolulu, Hawaii) (2000) Development of a HACCP-based strategy for the control of histamine for the fresh tuna industry [A report by PacMar, Inc. pursuant to National Oceanographic and Atmospheric Administration]. Honolulu (Hawaii): PacMar. 2000 Jul 31. NOAA Award No. NA86FD0067. 48 p. Kawabata, T., Uchida, Y. and Akano, T. (1960) A simple and rapid method for the determination of histamine in fish flesh. Bulletin of the Japanese Society of Scientific Fisheries 26: 1183– 1191. Khayat, A. (1977) Hydrogen sulfide production by heating tuna meat. Journal of Food Science 42: 601–609. Kimata, M. (1961) The histamine problem. In: G. Borgstrom (ed.) Fish as Food, vol. 1, Academic Press, NY, pp. 329–352. Klausen, N.K. and Lund, E. (1986) Formation of biogenic amines in herring and mackerel. Zeitschrift für Lebensmittel-Untersuchung und -Forschung 182(6): 459–463.

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Koutsoumanis, K., Lampropoulou, K. and Nychas, G.-J.E. (1999) Biogenic amines and sensory changes associated with the microbial flora of Mediterranean Gilt-head sea bream (Sparus aurata) stored aerobically at 0, 8, and 15 °C. Journal of Food Protection 62(4): 398–402. Krizek, M., Vacha, F., Vorlova, L., Lukasova, J. and Cupakova, S. (2004) Biogenic amines in vacuumpacked and non-vacuum-packed flesh of carp (Cyprinus carpio) stored at different temperatures. Food Chemistry 88(2): 185–191. Lange, J. and Wittmann, C. (2002) Enzyme sensor array for the determination of biogenic amines in food samples. Analytical and Bioanalytical Chemistry 372(2): 276–283. Lehane, L. and Olley, J. (2000) Histamine fish poisoning revisited. International Journal of Food Microbiology 58: 1–37. Lerke, P.A. and Bell, L.D. (1976) A rapid fluorometric method for the determination of histamine in canned tuna. Journal of Food Science 41: 1282–1284. Lerke, P.A., Porcuna, M.N. and Chin, H.B. (1983) Screening test for histamine in fish. Journal of Food Science 48: 155–157. Lerke, P.A., Werner, S.B., Taylor, S.L. and Guthertz, L.S. (1978) Scombroid poisoning. Western Journal of Medicine 129: 381–386. Lieber, E.R. and Taylor, S.L. (1978) Thin-layer chromatographic screening methods for histamine in tuna fish. Journal of Chromatography 153: 143–152. Lopez-Sabater, E.I., Rodriguez-Jerez, J.J., Roig-Sagues, A.X. and Mora-Ventura, M.T. (1993) Determination of histamine using an enzymic method. Food Additives & Contaminants 10: 593–602. Lyons, D.E., Beery, J.T., Lyons, S.A. and Taylor, S.L. (1983) Cadaverine and aminoguanidine potentiate the uptake of histamine in vitro in perfused intestinal segments of rats. Toxicology and Applied Pharmacology 70: 445–458. Mackie, I.M. and Salguero, F.J. (1977) Histidine metabolism in fish. Urocanic acid in mackerel (Scomber scombrus). Journal of the Science of Food and Agriculture 28: 935–940. Maijala, R.L., Eerola, S.H., Aho, M.A. and Hirn, J.A. (1993) The effect of GDL-induced pH decrease on the formation of biogenic amines in meat. Journal of Food Protection 50: 125–129. Male, K.B., Bouverette, P., Loung, J.H.T. and Gibbs, B.F. (1996) Amperometric biosensor for total histamine, putrescine and cadaverine using diamine oxidase. Journal of Food Science 61(5): 1012–1016. Malle, P., Valle, M. and Bouquelet, S. (1996) Assay of biogenic amines involved in fish decomposition. Journal of AOAC INTERNATIONAL 79(1): 43–49. Marklinder, I. and Lönner, C. (1992) Fermentation properties of intestinal strains of Lactobacillus, of a sour dough and of a yoghurt starter culture in an oat-based nutritive solution. Food Microbiology 9: 197–205. Marrakchi, A.E., Bennour, M., Bouchriti, N., Hamama, A. and Tagafatit, H. (1990) Sensory, chemical, and microbiological assessments of Moroccan sardines (Sardina pilchardus) stored in ice. Journal of Food Protection 53: 600–605. Mazorra-Manzano, M., Pacheco-Aguilar, R., Diaz-Rojaz, E. and Lugo-Sanchez, M. (2000) Postmortem changes in black skipjack muscle during storage in ice. Journal of Food Science 65(5): 774–779. Mendes, R. (1999) Changes in biogenic amines of major Portuguese bluefish species during storage at different temperatures. Journal of Food Biochemistry 23: 33–43. Mietz, J.L. and Karmas, E. (1977) Chemical quality index of canned tuna as determined by highpressure liquid chromatography. Journal of Food Science 42: 155–158. Mietz, J.L. and Karmas, E. (1978) Polyamine and histamine content of rockfish, salmon, lobster and shrimp as an indicator of decomposition. Journal of AOAC INTERNATIONAL 61: 139–145. Mopper, B. and Sciacchitano, C.J. (1994) Capillary zone electrophoretic determination of histamine in fish. Journal of AOAC INTERNATIONAL 77(4): 881–884.

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Morii, H., Cann, D.C., Taylor, L.Y. and Murray, C.K. (1986) Formation of histamine by luminous bacteria isolated from scombroid fish. Bulletin of the Japanese Society of Scientific Fisheries 52: 2135–2141. Nagayama, T., Tamura, Y., Maki, T., Kan, K., Naoi, Y. and Nishima, T. (1985) Non-volatile amine formation and decomposition in abusively stored fishes and shellfish. Journal of Hygienic Chemistry 31: 362–370. Niculescu, M., Nistor, C., Ruzgas, T., Frébort, I., Sebela, M., Pec, P. and Csöregi, E. (2001) Detection of histamine and other biogenic amines using biosensors based on amine oxidase. Inflammation Research 50(Suppl. 2): S146–S148. (Proceedings of the XXIXth Annual Meeting of EHRS.) OAC (1990) Histamine in seafood: fluorometric method. Official Method of Analysis – AOAC, vol. 2, Food Composition; Additives; Natural Contaminants (15th edn). AOAC, Arlington. 1298 pp. (pp. 876–877). Official Journal of the European Union (2005) Commission Regulation (EC) N° 2073/2005 of 15 November 2005 on microbiological criteria for foodstuffs. Official Journal of the European Union L 338/1–26. Ohashi, M., Nomura, F., Suzuki, M., Otsuka, M., Adachi, O. and Arakawa, N. (1994) Oxygensensor-based simple assay of histamine in fish using purified amine oxidase. Journal of Food Science 59(3): 19–22. Okuzumi, M. and Awano, M. (1983) Seasonal variations in numbers of psychrophilic and halophilic histamine-forming bacteria (N-group bacteria) in seawater and on marine fishes. Bulletin of the Japanese Society of Scientific Fisheries 49: 1285–1291. Okuzumi, M., Okuda, S. and Awano, M. (1981) Isolation of psychrophilic and halophilic histamineforming bacteria from Scomber japonicus. Bulletin of the Japanese Society of Scientific Fisheries 47: 1591–1598. Okuzumi, M., Okuda, S. and Awano, M. (1984) Effects of temperature, pH value and NaCl concentration on histamine formation of N-group bacteria (psychrophilic and halophilic histamine-forming bacteria). Bulletin of the Japanese Society of Scientific Fisheries 50: 1757–1762. Olley, J. and Baranowski, J. (1985) V. Temperature effects on histamine formation. In: Histamine Formation in Marine Products: Production by Bacteria, Measurement and Prediction of Formation, FAO Fisheries Technical Paper, No. 252. Food and Agriculture Organization of the United Nations, Rome, pp. 14–17. Özogul, F., Polat, A. and Özogul, Y. (2004) The effects of modified atmosphere packaging and vacuum packaging on chemical, sensory and microbiological changes of sardines (Sardina pilchardus). Food Chemistry 85(1): 49–57. Ozogul, F., Taylor, K.D.A., Quantick, P. and Ozogul, Y. (2002) Biogenic amine formation in Atlantic herring (Clupea harengus) stored under modified atmosphere packaging using a rapid HPLC method. International Journal of Food Science & Technology 37: 515–522. Pacheco-Aguilar, R., Lugo-Sanchez, M.E. and Robles-Burgueno, M.R. (2000) Post-mortem biochemical and functional characteristic of Monterey sardine muscle stored at 0 °C. Journal of Food Science 65(1): 40–47. Pan, B.S. (1985) VIII.2. Histamine formation in canning processes. In: Histamine Formation in Marine Products: Production by Bacteria, Measurement and Prediction of Formation, FAO Fisheries Technical Paper, No. 252. Food and Agriculture Organization of the United Nations, Rome, pp. 41–44. Patange, S.B., Mukundan, M.K. and Ashok Kumar, K. (2005) A simple and rapid method for colorimetric determination of histamine in fish flesh. Food Control 16(5): 465–472. Putro, S. and Saleh, M. (1985) Effect of delayed icing. (a) Skipjack tuna. In: Histamine Formation in Marine Products: Production by Bacteria, Measurement and Prediction of Formation, FAO Fisheries Technical Paper, No. 252. Food and Agriculture Organization of the United Nations, Rome, pp. 30–32.

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Ratkowsky, D.A., Olley, J. McMeekin, T.A. and Ball, A. (1982) Relationship between temperature and growth rate of bacterial cultures. Journal of Bacteriology 149: 1–5. Rawles, D.D., Flick, G.J. and Martin, R.E. (1996) Biogenic amines in fish and shellfish. Advances in Food Nutrition Research 39: 329–364. Redmond, J.W. and Tseng, A. (1979) High-pressure liquid chromatographic determination of putrescine, cadaverine, spermidine and spermine. Journal of Chromatography 170: 479– 481. Ritchie, A.H. and Mackie, I.M. (1980) The formation of diamines and polyamines during storage of mackerel (Scomber scombrus). In: J. Connell (ed), Advances in Fish Science and Technology. Fishing News Books Ltd, Farnham, Surrey, UK, pp. 489–494. Rodriguez-Jerez, J.J., Grassi, M.A. and Civera, T. (1994) A modification of Lerke enzymatic test for histamine quantification. Journal of Food Protection 57(11): 1019–1021. Rogers, P.L. and Staruszkiewicz, W. (1997) Gas chromatographic method for putrescine and cadaverine in canned tuna and mahi mahi and fluorometric method for histamine (minor modification of AOAC Official Method 977.13): collaborative study. Journal of AOAC INTERNATIONAL 80(3): 591–602. Rogers, P. and Staruszkiewicz, W. (2000) Histamine test kit comparison. Aquatic Food Production Technology 9(2): 5–17. Ruiz Capillas, C. and Moral, A. (2001) Production of biogenic amines and their potential use as quality control indices for hake (Merluccius merluccius, L.) stored in ice. Journal of Food Science 66(7): 1030–1032. Ryser, E.T., Marth, E.H. and Taylor, S.L. (1984). Histamine production by psychrotrophic pseudomonads isolated form tuna fish. Journal of Food Protection 47: 8–80. Sager, O.S. and Horwitz, W. (1957) A chemical method for the determination of histamine in canned tuna fish. Journal of AOAC 40: 892. Saito, K., Horie, M., Nose, N., Nakagomi, K. and Nakazawa, H. (1992) Determination of polyamines in foods by liquid chromatography with on-column fluorescence derivatization. Analytical Science 8: 675–680. Salguero, F.J. and Mackie, I.M. (1979) Histidine metabolism in mackerel (Scomber scombrus). Studies on histidine decarboxylase activity and histamine formation during storage of flesh and liver under sterile and non-sterile conditions. Journal of Food Technology 14: 131–139. Santos, S.M.H. (1996) Biogenic amines: their importance in foods. International Journal of Food Microbiology 29: 213–231. Santos, C., Marine, A. and Rivas, J.C. (1986) Changes of tyramine during storage and spoilage of anchovies. Journal of Food Science 51: 512–513, 515. Sato, T., Fujii, T., Masuda, T. and Okuzumi, M. (1994) Changes in numbers of histamine – metabolic bacteria and histamine content during storage of common mackerel. Fisheries Science 60(3): 299–302. Serrar, D., Brebant, R., Bruneau, S. and Denoyel, G.A. (1995) The development of a monoclonal antibody-based ELISA for the determination of histamine in food: application to fishery products and comparison with the HPLC assay. Food Chemistry 54: 85–91. Shalaby, A.R. (1996) Significance of biogenic amines to food safety and human health. Food Research International 29(7): 675–690. Shultz, D.E., Chang, G.W. and Bjeldanes, L.F. (1976) Rapid thin layer chromatographic method for the determination of histamine in fish products. Journal of AOAC INTERNATIONAL 59(6): 1224–1225. Simon-Sarkadi, L. and Kovács, Á. (2002) Biogenic amine determination in food by different techniques. G.I.T. Laboratory Journal 6(1): 11–13. Soll, A.H. and Wollin, A. (1977) The effects of histamine postglanding E, and secretin on cyclic AMP in separated canine fundic mucosal cells. Gastroenterology 72: 1166.

66

Fishery Products: Quality, safety and authenticity

Sorensen, N.K. (1992) Physical and instrumental methods for assessing seafood quality. In: H.H. Huss, M. Jakobsen and J.C. Liston, J.C. (Eds) Quality Assurance in the Fish Industry. Elsevier Science Publishers B.V., Amsterdam, pp. 309–318. Spreekens, K. (1987) Histamine production by the psychrophilic flora. In: D.E. Kramer and J. Liston (eds) Seafood Quality Determination Elsevier, Amsterdam, pp. 309–318. Staruszkiewicz, W., Waldron, E. and Bond, J. (1977). Fluorometric determination of histamine in tuna. Journal of AOAC INTERNATIONAL 60: 1125–1130. Staruszkiewicz, W.F., Barnett, J.D., Rogers, P.L., Benner, R.A. Jr., Wong, L.L. and Cook, J. (2004) Effects of on-board and dockside handling on the formation of biogenic amines in mahimahi (Coryphaena hippurus), skipjack tuna (Katsuwonus pelamis), and yellowfin tuna (Thunnus albacares). Journal of Food Protection, 67(1): 134–141. Stede, M. and Stockemer, J. (1981). Biogene Amine in Seefischen. Lebensmittel-Wissenschaft und -Technologie 19: 283–287. Stratton, J.E., Hutkins, R.W. and Taylor, S.L. (1991) Biogenic amines in cheese and other fermented foods, a review. Journal of Food Protection 54: 460–470. Suzuki, S., Matsui, Y. and Takama, K. (1988) Profiles of polyamine composition in putrefactive Pseudomonas type III/IV. Microbios Letters 38: 105–109. Suzuki, S., Kobayashi, K., Noda, J., Suzuki, T. and Takama, K. (1990) Simultaneous determination of biogenic amines by reverse-phase high-performance liquid chromatography. Journal of Chromatography 508: 225–228. Takagi, K. and Shikata, S. (2004) Flow injection determination of histamine with a histamine dehydrogenase-based electrode. Analytica Chimica Acta 505: 189–193. Taylor, S.L. (1986) Histamine food poisoning: toxicology and clinical aspects. Critical Reviews in Toxicology 17(2): 91–128 Taylor, S.L. and Lieber, E.R. (1979) In vivo inhibition of rat intestinal histamine-metabolizing enzymes. Food and Cosmetics Toxicology 17: 237–240. Taylor, S.L. and Speckhard, M.W. (1983) Isolation of histamine-producing bacteria from frozen tuna. Marine Fisheries Review 45: 35–39. Taylor, S.L. and Summer, S.S. (1986) Determination of histamine, putrescine, and cadaverine. In: D.E. Kramer and J. Liston (eds) Seafood Quality Determination. Elsevier, Amsterdam, pp. 235–245. Taylor, S.L. and Woychik, N.A. (1982). Simple medium for assessing quantative production of histamine by Enterobacteriaceae. Journal of Food Protection 45: 747–751. Taylor, S.L., Lieber, E.R. and Leatherwood, M. (1978) A simplified method for histamine analysis of foods. Journal of Food Science 43: 247–250. Taylor, S.L., Stratton, J.E. and Nordlee, J.A. (1989) Histamine poisoning (scombroid fish poisoning): an allergy-like intoxication. Clinical Toxicology 27(4 & 5): 225–240. Trenerry, V.C., Marshall, P.A. and Windahl, K. (1998) Method optimisation: determination of histamine in fish by capillary zone electrophoresis. Journal of Capillary Electrophoresis 5(1–2): 27–32. Tsai, Y.H., Kung, H.F., Lee, T.M., Chen, H.C., Chou, S.S., Wei, C.I. and Hwang, D.F. (2006) Determination of histamine in canned mackerel implicated in a food borne poisoning. Food Control 16: 579–585. Uchida, K., Haraguchi, K., Mitsui, M. and Kawakishi, S. (1990) Stimulatory effect of histamine on the peroxidation of linoleic acid. Journal of Agriculture and Food Chemistry 38: 1491– 1493. Veciana-Nogues, M.T., Vidal-Carou, M.C. and Marine-Font, A. (1990) Histamine and tyramine during storage and spoilage of anchovies, Engraulis encrasicholus: relationships with other fish spoilage indicators. Journal of Food Science 55: 1192–1193, 1195. Veciana-Nogues, M.T., Hernandez-Jover, T., Marine-Font, A. and Vidal-Carou, M.C. (1995) Liquid chromatographic method for determination of biogenic amines in fish and fish products. Journal of AOAC INTERNATIONAL 78: 1045–1050.

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Veciana-Nogues, M.T., Marine-Font, A. and Vidal-Carou, M.C. (1997) Biogenic amines as hygienicquality indicators of tuna: relationships with microbial counts, ATP related compounds, volatile amines and organoleptic changes. Journal of Agricultural and Food Chemistry 45: 2036–2041. Vidal-Carou, M.C., Izquierdo-Pulido, M.L., Martin-Morro, M.C. and Marine-Font, A. (1990) Histamine and tyramine in meat products: relationship with meat spoilage. Food Chemistry 37: 239–249. Wei, C.I., Chen, C.M., Koburger, J.A., Otwell, W.S. and Marshall, M.R. (1990). Bacterial growth and histamine production on vacuum packaged tuna. Journal of Food Science 55: 59–63. Wendakoon, C.N. and Sakaguchi, M. (1992) Effects of spices on growth and biogenic amine formation by bacteria in fish muscle. In: H.H. Huss, M. Jakobsen, and J. Liston (Eds) Quality Assurance in the Fish Industry: Proceedings of an International Conference; August 26–30, 1991, Copenhagen, Denmark. Elsevier, Amsterdam, pp. 305–313 (Development in Food Science Series No. 30). Wendakoon, C.N. and Sakaguchi, M. (1993) Combined effect of sodium chloride and clove on growth and biogenic amine formation of Enterobacter aerogenes in mackerel muscle extract. Journal of Food Protection 56: 410–413. Wendakoon, C.N., Murata, M. and Sakaguchi, M. (1990) Comparison of non-volatile amine formation between the dark and white muscles of mackerel during storage. Nippon Suisan Gakkaishi 56(5): 809–818. Wille, J.J., Kydonieus, A.F. and Murphy, G.F. (1999) cis-Urocanic acid induces mast cell degranulation and release of preformed TNF-alpha: a possible mechanism linking UVB and cis-urocanic acid to immunosuppression of contact hypersensitivity. Skin Pharmacology and Applied Skin Physiology 12(1–2): 18–27. Wurziger, J. and Dickhaut, G. (1978) Estimation of histamine in fish and fish products in the light of the food regulations. Die Fleischwirtschaft 6: 963–964. Yamanaka, H., Shiomi, K., Naito, M. and Kikuchi, T. (1980) Histamine content in the canned red meat fish. Bulletin of the Japanese Society of Scientific Fisheries 46: 905–907. Yamanaka, H., Shiomi, K., Kikuchi, T. and Okuzumi, M. (1984) Changes in histamine contents in red meat fish during storage at different temperatures. Bulletin of the Japanese Society of Scientific Fisheries 50: 695–701. Yamanaka, H., Shimakura, K., Shiomi, K. and Kikuchi, T. (1986) Changes in non-volatile amine contents of the meats of sardine and saury pike during storage. Bulletin of the Japanese Society of Scientific Fisheries 52: 127–130. Yamanaka, H., Shiomi, K. and Kikuchi, T. (1989) Cadaverine as a potential index for decomposition of salmonoid fishes. Journal of the Food Hygiene Society of Japan 30: 170–174. Yen, G. and Hsieh, C. (1991) Simultaneous analysis of biogenic amines in canned fish by HPLC. Journal of Food Science 56(1): 158–160. Zeng, K., Tachikawa, H., Zhu, Z. and Davidson, V.L. (2000) Amperometric detection of histamine with a methylamine dehydrogenase polypyrole-based sensor. Analytical Chemistry 72(10): 2211–2215.

Chapter 4

ATP-derived products and K-value determination Margarita Tejada

4.1

In vivo role of nucleotides

Nucleotides are the 5′-phosphate esters of nucleosides. The most important nucleotide in all living organisms is adenosine 5′-triphosphate (ATP), which consists of the nucleoside adenosine linked to three phosphate groups. ATP functions as the universal carrier of energy, transferring energy from chemical bonds to endergonic reactions within the cell. The energy is stored in the covalent bonds between phosphates, which are considered the energy source for biological systems. The key chemical reaction for bioenergetics is the inter-conversion of ATP and ADP (ADP + Pi + energy ↔ ATP). The energy is used in the building up of cell components, muscle contraction, transmission of nerve impulses and many other functions. ATP has been termed the cell’s energy currency. The energy source for ATP generation in fish muscle is different according to the type of muscle. In aerobic conditions, glycogen is the source of energy in the light (ordinary) muscle, whereas the dark muscle may also use lipids. According to Love (1970), the relatively small change in the glycogen and lactic acid content of dark muscle compared with ordinary muscle in fish under stress by forced swimming is probably because the energy for dark muscle contraction is derived mostly from lipids. A major difference is, further, that the dark muscle contains many more mitochondria than light muscle, thus enabling the dark muscle to operate an extensive aerobic energy metabolism. In anaerobic conditions the light muscle, mostly generating energy by anaerobic glycolysis, accumulates lactic acid which has to be transported to the liver to be further metabolized. The different metabolic patterns found in the two muscle types makes the dark muscle suitable for continuous muscle movements whereas the light muscle is excellently fitted for strong, short muscle movements. A review of the physiological role of both types of muscle in fish was made by Love (1970). The role of nucleotides in muscle contraction has been studied for decades. ATP is required for both contraction and relaxation of muscle, supplying the energy required for the sliding of the myosin filaments for contraction and for the separation of myosin and actin during the relaxation. Both the sarcoplasmic reticulum (SR) and the major myofibrillar protein, myosin, have ATPase activity, which varies according to myosin isoforms fast (white muscle) and slow (red muscle) myosin, and the fish species (Rowlerson et al. 1985). The sliding 68

Level of toughness, K-value, VBN and total viable cell counts

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Autolysis Spoilage Death of fish ↓ Anaerobic glycolysis ↓ Formation of lactic acid Feedback inhibition ↓ pH 7.0 → pH 5.0 (muscle) ↓ Activation of ATPase ↓ Degradation of ATP (→ ADP → AMP→ IMP → Inosine→ Hypoxantine → Uric acid ↓ histamine degradation of protein → polypeptide → amino acid Rigor mortis NH3 TMAO → TMA → DMA + formaldehyde rapid increase of bacteria

K-value

for sashimi

Total viable cell counts

VBN

for cooking

rigor mortis

Death

1

2

3

Storage days at 5°C Figure 4.1

Post mortem changes in fish meat (From Hamada-Sato et al. 2005.)

of the filaments is the result of interactions between the myosin cross-bridges and the actin filaments. The cross-bridges reversibly bind to actin and produce a mechanical impulse, which results in force transmission along the filaments, which in turn results in force production and shortening. Both Ca2+ and Mg2+ play an important role in the splitting of ATP and the contraction–relaxation cycle (Leadbeater and Perry 1963). Muscle contraction and relaxation are controlled by the concentration of calcium ions within the muscle cell. When Mg2+ ions are present, ATP splits actomyosin into actin and myosin. To relax the muscle, Ca2+ATPase of the SR pumps calcium ions from the cytoplasm into the SR against a concentration gradient, thereby causing the relaxation of muscle cells. A sudden inflow of Ca2+ is the trigger for muscle contraction. After the contraction, the Ca2+ is rapidly pumped back into the SR, so the muscle can contract again.

4.2

Post mortem changes

The regulatory functions operating in vivo cease post mortem. Nevertheless the reactions in fish muscle continue leading at the spoilage of the fish. Post mortem changes in fish are summarized in Figure 4.1.

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Rigor mortis

After death, the oxygen supply to the fish tissues ceases and anaerobic metabolism is established. Nevertheless the two main anaerobic sources of ATP, creatine phosphate and anaerobic glycolysis, fail rapidly, and the energy resources in the muscle are soon depleted. When the intracellular level of ATP reaches its minimum (1–2 μmol/g tissue according to Iwamoto et al. (1988)) myosin and actin are interconnected irreversibly to form inextensible actomyosin. The muscles become stiff as a result of the accumulation of Ca2+ ions inside the cell due to the failure of the calcium pump of the SR. The stiffening of skeletal muscle that appears shortly after death is known as rigor mortis. This process is also accompanied by a decrease of the pH of the muscle to a minimum due to the accumulation of lactic acid formed as end-product of glycolysis. At this pH, the charge of muscle proteins is close to their isoelectric point. In fish with high amounts of red muscle the final pH is lower than in fish with a high amount of white muscle, but is seldom as low as that observed for post mortem mammalian muscle (Huss 1995). Fish generally exhibit rigor mortis starting from about 1–6 h after death although the onset, intensity and resolution of rigor depend on several factors such as species, size, stress, killing method, catching conditions, handling ante- and post mortem and temperature, among others (Stroud 1981; Erikson 2001). Rigor starts in different muscles according to the depletion of ATP and progressively extends to the whole fish. Usually rigor starts at the tail, and the muscles harden gradually along the body towards the head until the whole fish is quite stiff (Stroud 1981). However, in salmon the onset of the rigor starts in the neck region (Berg et al. 1997). ATP is rapidly depleted at high temperatures, producing a strong rigor tension over the whole fish. This is important mainly when the muscle is not supported by the spine, where a rapid shortening may appear with an increase of hardness and stiffness (Figure 4.2). Handling of fish once rigor has started is not recommended because the yield of fillets is poorer and the handling may cause gaping. The delay in the onset and resolution of rigor mortis extend the shelf life of the fish. After the maximum stiffness of the muscle is obtained (completion of the rigor) the softening of the muscle starts (resolution of rigor mortis). Autolytic enzymes are activated due to the pH drop and the increase of calcium ions in the sarcoplasm. Both calpains, activated by calcium, and cathepsins, released from the lysosomes and activated by the pH drop post mortem, degrade the structural proteins, causing an increase in tenderness of the muscle (‘aging effect’). It is accepted that the resolution of rigor mortis produces the softening of the fish muscle and determines the texture in the early steps of tenderization of the muscle. Proteolysis degrades bonds inside the cytoskeleton including proteins building the myofibrillar structure (titin, nebulin, M- and Z-line proteins) proteins connecting myofibrils (desmin and skelemin), and proteins in the external cytoskeleton, i.e. proteins connecting myofibrils with sarcolemma through costamers and proteins that anchor muscle fibres in surrounding membrane. Proteolysis also involves degradation of the connective tissue, probably because of the supporting action of lysosomal glycosidases. So far, there is no evidence that the lengthening of the sarcomere that is observed often during meat storage is caused by degradation of actomyosin. The mechanism of softening of the muscle after rigor continues to be under discussion (Pospiech et al. 2003).

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Figure 4.2 Shortening of fillets due to time of processing. Left: fillet cut from the frame bone prerigor; right: fillet cut from the frame bone post-rigor. Copyright: Torbjørn Tobiassen, Fiskeriforskning, Norway.

Thaw rigor and cold shortening are severe types of rigor mortis. Cold shortening is the intense shrinkage of the muscle at temperatures above freezing due to the release of Ca2+ from the SR while ATP is still present. Thaw rigor is a rapid and intense contraction of fish muscle frozen pre-rigor before ATP and glycogen stores have been depleted; when the muscle is thawed at high temperature, preserved ATP and glycogen are rapidly metabolized (Jones 1965, 1969). In mammalians, the two terms are very well defined and cold shortening is applied to the shortening that occurs when the post-slaughter meat is immediately chilled. However, in fish muscle the term cold shortening is sometimes applied to the shrinkage observed when muscle that was frozen pre-rigor is thawed. The shortening of fish muscle is accompanied by a severe drip loss and is mainly observed in fillets, where no mechanical forces due to the insertion to the spine may counteract the strong contraction. Large differences in the shortening and drip loss of fish fillets frozen pre-rigor have been reported between lean and fat species, ranging from 3 to 40% with less shrinkage in the fatty species (Slinde et al. 2001). The intense contraction is due to an increase in the calcium ions inside the cells leaking from the SR or to the rupture of the SR membranes during freezing and thawing when ATP levels in the muscle are high (Ma et al. 1992; Erikson 2001; Slinde et al. 2001).

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ATP degradation takes place during the freezing process, and ATP can be depleted in the frozen muscle depending on the temperature and time of storage. Even if ATP is degraded during frozen storage, the fish might still contain a depot of glycogen that allows synthesis of ATP during the thawing. This has been observed in small samples as well as in whole fish during thawing at 0 °C. It has been demonstrated that synthesis of ATP through glycolysis also occurs during freezing. Nevertheless, in frozen fish the enzymatic reactions in relation to glycolysis and ATP breakdown act independently of each other (Cappeln et al. 1999; Cappeln and Jensen 2001).

4.2.2

Nucleotide degradation

Nucleotide degradation in post mortem fish muscle has been examined by different authors for decades and is considered one of the earliest indices to asses fish freshness (Saito and Arai 1957a,b: Saito et al. 1959a,b; Arai and Saito 1961; Kassemsarn et al. 1963; Ehira and Uchiyama 1987, among others). After death, the ATP is degraded in the fish muscle by endogenous enzymes causing the successive formation of adenosine-5′-diphosphate (ADP), adenosine-5′-monophosphate (AMP), inosine-5′-monophosphate (IMP), inosine (Ino or HxR) and hypoxanthine (Hx) that degrades to xanthine (X) and uric acid (U). The degradation of ATP up to IMP is very fast, but as degradation of IMP is relatively slow, IMP is predominantly accumulated in fish muscle. This reaction is believed to be autolytic (Jones 1965; Hiltz et al. 1971; Surette et al. 1988). In marine invertebrates, AMP has been identified in several species as the major nucleotide present from ATP degradation. Accumulation of AMP instead of IMP has been measured in several molluscs and crustaceans during ice storage (Arai and Saito 1961; Groninger and Brandt 1969; Mendes et al. 2001). The conversion of AMP to Ino was proposed to be by adenosine (Ado) rather than IMP (Saito et al. 1958a,b; Arai 1960; Arai and Saito 1961; Hiltz and Dyer 1970, Yoneda et al. 2002). Both nucleotides have been detected in Dungeness crab (Cancer magister) muscle (Groninger and Brand 1969) and kuruma prawn (Penaeus japonicus) (Shirai and Kikuchi 2002). Accumulation post mortem of IMP in different amounts has been reported in bivalves, scallops, squid muscle and Norway lobster (Sakamoto et al. 1973; Nakamura et al. 1976; Suwetja et al. 1989; Sagedhal et al. 1997; Massa et al. 2003; Gornik et al. 2006). It is widely accepted that nucleotides influence taste and flavour of fish and mollusc meat (Matsumoto and Yamanaka 1991). IMP contributes to the pleasant, sweet taste and fresh flavour of the meat and has been recognized for decades as a flavour enhancer (‘umami’ taste) (Kuninaka et al. 1964) which is preserved even when the muscles are cooked (Dingle and Hines 1971). AMP also contributes to the flavour-enhancing effect in prawns (Fuke et al. 1994; Shirai and Kikuchi 2002). However, IMP plays only minor role in the flavour of Dungeness crab and king crab, even when IMP is added to the meat before cooking (Groninger and Brandt 1969). In contrast, the degradation of IMP to Ino and Hx has been associated with the detection of bitter flavours and the progressive loss of desirable flavour (Kazeniac 1961; Kassemsarn et al. 1963; Jones and Murray 1964); nevertheless Spinelli (1965) did not detect any changes in bitterness associated with an increase of Ino and Hx. Different fish stocks might differ in activities of IMP-degrading enzymes. The season of harvesting and rearing practice in farmed fish might vary initial concentrations of ATP, and

ATP-derived products and K-value determination

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hence of IMP. Genetic fish selection for high initial ATP concentration, or development of cultural practices that increase initial ATP concentration, could result in products with high IMP concentrations at the time of consumption, which, given a link between IMP concentration and pleasant flavours, could enhance the desirability of the product (Howgate 2006).

4.2.3

Enzymes that degrade ATP and related compounds

The enzymes that intervene in the degradation of ATP in fish muscle have been studied since the early work of Tarr (1966) and Ehira (1976). The pathway of ATP breakdown in fish muscle and related enzymes is shown in Figure 4.3. Hx is further degraded by xanthine oxidase to xanthine(X) and uric acid (U). In marine invertebrates, the conversion of AMP to Ino via Ado rather than IMP was attributed to a lower level of AMP deaminase activity than in fish (Fujisawa and Yoshino 1985). The initial levels and rate of breakdown of ATP and related compounds are subjected to large inter- and intra-species differences (Figure 4.4). In the degradation process, ATP, ADP and AMP are rapidly degraded to IMP, resulting in accumulation of inosine and hypoxanthine. Many factors alter the rate of degradation and the accumulation of ATP derivatives during storage of fish. The degradation of ATP to IMP has generally been attributed to muscle enzymes whereas the degradation of IMP to Ino and Hx has been attributed additionally to the growth of bacteria (Surette et al. 1988). However, in studies performed in fish muscle maintained in aseptic and non-aseptic conditions during ice storage, the degradation of ATP and related compounds has been found to be nearly identical (Uchiyama and Ehira 1974; Ehira and Uchiyama 1987). Nucleotide degradation correlates well with the shelf life of a wide range of fish species (Jones and Murray 1962, 1964; Spinelli et al. 1964). In tropical species, the shelf life of the fish was related to the rate of IMP breakdown rather than to bacterial counts (Bremner et al. 1988). Large differences between individual fish of the same catch and between dark and white muscle have also been reported (Hattula 1997). These differences have been associated with differences in the pH of both types of muscle (Dingle and Hines 1971; Obatake et al. 1988). One of the main factors affecting the degradation of ATP derivatives among fish species is the rate of breakdown of IMP due to different activity of 5′-nucleotidase (Marseno et al. 1992). The rate-limiting step in nucleotide degradation in fish muscle is the conversion of IMP to Ino and ultimate formation of Hx, xanthine and uric acid (Jones and Murray 1964). The concentration of IMP in fish during chilled storage decreases as a first-order reaction (Howgate 2005). In crustaceans the rate-limiting step differs according to the species: in king crab muscle the rate-limiting step is the conversion of AMP to IMP, whereas in Dungeness crab muscle it is the conversion of AMP to IMP and of IMP to Ino (Groninger and Brandt 1969). Differences are also found in the same species reared in seawater or in freshwater. In trout reared in seawater the data fit a mathematical model assuming that only endogenous enzymes act in a sequence of consecutive first-order reactions. In trout reared in freshwater, the mathematical model assuming Ino was additionally converted to Hx by bacterial action better fitted the data (Howgate 2005). The kinetics of degradation of IMP in several species of fish during chilled storage has been recently reviewed (Howgate 2006). The formation and the accumulation in the muscle of Ino and Hx differs among fish species (Ehira and Uchiyama 1969, 1973; Ehira 1976; Mendes et al. 2001; Tejada et al. 2006a,b).

74

O

O

HO-P-O-P-O-P-OCH2

NH2 C N N C CH HC C N N

ip O

O

NH2 C N N C CH HC C N N

ip

H2O3POCH2 O

(myokinase) (ATPase) HO-P-O-P-OCH2 O OH OH OH OH OH H H H H H H OH OH OH OH adenosine–5′-diphosphate (ADP) adenosine–5′-triphosphate (ATP) O

OH C N N C CH HC C N N H2O3POCH2 O

(phosphatase) HOCH

2

H H H

H OH OH

inosine–5′-monophosphate (IMP)

O

H

OH C N N C CH HC C N N H hypoxanthine (6–hydroxypurine) (Hx)

( nucleoside hydrolase )

H nucleoside OH OH phosphorylase inosine (HxR)

(

H OH OH

adenosine–5′-monophosphate (AMP)

H H H

(deaminase)

H H

OH C N N C CH HC C N N

ip

NH2

)

H H C O C H H CHOH H HO C C H OH OH H C O D-ribose C H H CHOPO H 3 2 H HO C C OH OH D-ribose–1–phosphate

Figure 4.3 Pathway of ATP breakdown in fish muscle and related enzymes. (Adapted from Ehira, 1976.)

Fishery Products: Quality, safety and authenticity

O

NH2 C N N C CH HC C N N

ATP-derived products and K-value determination Gilthead seabream

Seabass

75

Senegalese Sole

Total micromol/g sample

10 9

Hx

8

INO

7 6

IMP

5

AMP

4

ADP

3

ATP

2 1 0 1

9

22

1

9

22

7

14

28

Days of storage Figure 4.4 Nucleotides and derivatives extracted from post-rigor muscle during ice storage of farmed fish.

Ehira (1976) divided fish species in Ino-intermediate- or Hx-forming species according to the molar ratio of Ino : Hx. The loss of ATP-derived products by leaching when unprotected fish is stored in ice also modifies the accumulation of Ino or Hx in the muscle (Howgate 2006). For this reason, using Hx content without using other indices of freshness may be misleading. The degradation of nucleic acids as possible precursors of mononucleotides during ice storage of fish muscle was studied by Ehira (1976). Both RNA and DNA decreased little during the ice storage of muscle, as they seemed to be protected or masked by proteins against the attack of nucleases (Tomlinson and Creelman 1960).

4.2.4

K-value

The selection of an index for estimating the freshness of fish by measuring ATP breakdown products during storage has to be done, taking into account the major final products formed. Indices based on the detection of Hx or Ino used as freshness indicators for fish may not be appropriated to evaluate fish freshness in several species owing to big differences in the final ATP-related products formed and all the factors that intervene in ATP degradation. To avoid this problem, the extent of breakdown of ATP and related products is expressed as the K-value, defined as the percentage of the amount of Ino and Hx to the total amount of ATP-related compounds: K -value (%) = [(Ino + Hx ) (ATP + ADP + AMP + Ino + Hx )] × 100 (Saito et all. 1959c). The K-value is one of the indices that has an early and linear response with time during fish stored at temperatures above freezing and is used extensively as a commercial index in Japan for estimating fish freshness (Ehira and Uchiyama 1987). Because ATP is rapidly degraded

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to IMP after death in most fish species and remains very low during storage, other indices that do not require determination of ATP, ADP or AMP have been proposed to evaluate fish freshness. Indices relating degradation products of ATP are: K1 -value (%) = [(Ino + Hx ) (IMP + Ino + Hx )] × 100 (Karube et al. 1984) G-value (%) = [(Ino + Hx ) (AMP + IMP + Ino)] × 100 (Burns et al. 1985); Fr -value (%) = [(IMP ) (IMP + Ino + Hx )] × 100 (Gill et al. 1987) H-value (%) = [(Hx ) (IMP + Ino + Hx )] × 100 (Luong et al. 1992); P -value (%) = [(Ino + Hx) (AMP + IMP + Ino + Hx )] × 100 (Shahidi et al. 1994) Higher K, K1, H, G and P values correspond to a decrease in the freshness of the fish. The usefulness of these freshness indices depends on the fish species being examined. Different indices have been found to correlate better with freshness in farmed or wild fish (Alasalvar et al. 2002). In general, all these values reduce variability compared with former methods such as determination of hypoxanthine or free ribose concentration, and are better correlated with the freshness of the fish. K- and K1-values are used as quality criteria to determine the consumption limit for raw, chilled fish. Large differences in the rate of increase of the K-value have been observed among fish species. Nevertheless, the values tend to be quite homogeneous for the same species, irrespective of the total amount of ATP derivatives measured in different lots (Huidobro 2001; Tejada et al. 2006a,b). Maximum K-values at the rejection of the fish have been reported in several species ranging from: 80% in European whitefish (Coregonus wartmanni) (Hattula et al. 1993); 70–80% in Atlantic salmon (Salmo salar) (Erikson et al. 1997); 60–70% in farmed seabass (Dicentrarchus labrax) (Tejada et al. 2006a), farmed turbot (Psetta maxima) (Rodriguez et al. 2006) and wild turbot (Scophthalmus maximus) (Özogul et al. 2006); to 30–45% in hoki (Macruronus novaezelandiae) (Ryder et al. 1993), farmed gilthead seabream (Sparus aurata) (Huidobro et al. 2001; Tejada et al. 2006a), and Senegalese sole (Solea senegalensis) (Tejada et al. 2006b). A K- or K1-value of 20% has been defined by Japanese researchers as the limit for raw fish (‘sashimi’ grade) consumption. The limit for consuming fish as sashimi slightly differs among species and data are still needed to clarify the relation between temperature background and the K1 value (Hamada-Sato et al. 2005). The time in ice reported in several species to reach this value ranges between 1 day for Pacific cod (Gadus morhua macrocephalus) and 14 days for sharp-toothed eel (Muraenesox cinereus) and flying fish (Cypselurus opisthopus hiraii) (Ehira 1976). In farmed fish, the period ranged between 3 and 4 days in seabass, 7 and 8 days in sea bream, and 8 and 10 days in Senegalese sole, depending on the batch studied (Huidobro et al. 2001; Tejada et al. 2006a). Differences had been found in reaching this value depending on the method of storage. K-values of 20% were measured in farmed turbot stored in slurry ice at 10 days of storage; however, in the fish stored in flake ice the K-value increased faster (Piñeiro et al. 2005). Degradation of nucleotides and K-value have been also used as freshness indicators for raw material used to make fish gels (‘kamaboko’ type gels) (Onibala et al. 1997). However, other indices such as ATPase activity of myosin, actomyosin or myofibrils are better related

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77

to the gel-forming ability of the fish muscle and surimi, and are generally used for measuring the native myosin content and changes in fish muscle and surimi during storage (Kawashima 1973a,b,c; Kato 1979; Careche and Tejada 1991; Koseki et al. 2005).

4.2.5

Other factors that affect the degradation of ATP-related compounds

Differences in the degradation of ATP and related compounds have been associated with the type of muscle fibre in species with different proportions of red and white muscle. A higher activity of 5′-nucleotidase has been found in red than in pink or white muscle, and subsequently a lower amount of IMP and a rapid increase of Ino and Hx are measured in the red muscle (Saito et al. 1959a; Dingle and Hines 1971; Kanoh et al. 1986; Obatake et al. 1988). This is observed as a faster rate of increase of the K-value in red fibres, and slower and intermediate rates in white and pink muscle fibres, respectively (Yada et al. 2000). The interposition of pink fibres into dorsal ordinary muscle considerably accelerates the increase of the K-value in various fish species (Obatake et al. 1988; Yada et al. 2000, 2001a,b). Inter- and intraspecific differences in the degradation of nucleotides due to habitat temperature have been found. In the latter case, the rate of increase of the K-value measured at 32 °C [ΔK32 (%/h)] in cultured carp acclimated at 10 °C was higher than in carp acclimated at 30 °C (Tsuchimoto et al. 1988a,b). The acceleration of the K-value has been associated with the higher interposition of pink fibres in ordinary muscle in fish living at lower temperatures (Yada et al. 2001a). Seasonal differences in nucleotide degradation during storage have been reported in several fish species. In farmed seabass and gilthead seabream, higher values and rates of K-value increase have been found during early spoilage in summer fish (Grigorakis et al. 2003, 2004). Differences in the total amount of ATP and breakdown products have been also found when these species were harvested at different seasons and years, although the rate of increase of the K-value during storage was in the same range in most of the lots. Other rearing factors may modify the initial amount of ATP and the reaction rates (Huidobro et al. 2001; Tejada et al. 2006a). Initial levels of ATP in fish muscle also depend on the struggle of the fish during catching or harvesting. It has long been known that capture, confinement and handling cause physiological stress in fish with significant reductions in high-energy fuels such as creatine phosphate, ATP and muscle glycogen (Gallauger and Farrell 1999; Farrell et al. 2001; Pottinger 2001). Rapid killing of the fish has been shown to produce slower changes in energy-related compounds than in those fish that struggled (Mochizuki and Sato 1994, 1996). Rapid killing by brain spiking has been widely used as a killing procedure. In contrast, slow-acting slaughter methods such as death in air or in ice, or processes causing greater muscle activity such as carbon dioxide stunning, have detrimental effects (Kestin et al. 1995). Treatments that depress the stress during the handling and harvesting of fish slow down the degradation of ATP (Robb 2001). In unstressed (anaesthetized) Atlantic salmon, a high concentration of ATP delays the onset of the rigor mortis compared with stressed fish, although the influence in the K-value was only observed during the first 2 days post mortem (Erikson et al. 1997). In post-rigor gilthead seabream, no significant differences were found in relation to the method of slaughter in the evolution of the K-value during ice storage (Huidobro et al. 2001).

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Accumulation of IMP with storage time has been observed mainly in white muscle of struggled fish (Obatake et al. 1988). The degradation of nucleotides and derivatives is accelerated when the fish muscle loses its integrity. A faster formation of Ino and Hx in cod fillets than in gutted cod has been reported. Handling pre- and post-mortem and subsequent mechanical damage to muscle tissue accelerate the rate of IMP, Ino and Hx accumulation and degradation. This is associated with enzyme decompartmentalization caused when the integrity of the muscle is lost, rendering the substrates more accessible (Surette et al. 1988). Different results in the evolution of the K-value with gutting of the fish have been observed. Higher K-values for gutted lots have been found in several fish species at the end of storage (Scott et al. 1986; Hattula et al. 1993). However, no differences during the commercial life of gutted and ungutted farmed gilthead seabream were observed (Huidobro et al. 2001). Different results for the effect of gutting on the shelf life of ice-stored fish have been linked to possible damage of the fish during gutting. Bleeding of the fish slows down the degradation of ATP, delaying rigor development to some extent and lowering the K-values (Chiba et al. 1991; Mochizuki et al. 1998; Erikson et al. 1999). Nevertheless, the stress caused to the fish at bleeding may mask the beneficial effect. The degradation of ATP-related compounds during storage is often accelerated at higher temperatures of storage (Oka 1989; Ponce de Leon et al. 1994; Hattula 1997). The decrease in ATP and the progress of rigor mortis in plaice muscle were slower at storage temperatures ranging from 5 to 15 °C than at 0 and 20 °C, although the slower decrease of ATP at 15 °C did not correspond with the freshness of the fish (Iwamoto et al. 1985). This effect was not observed in post-rigor plaice, where the K-value increased at higher storage temperature (Iwamoto et al. 1987). Technological or food processing treatments given to the fish may alter the rate of nucleotide degradation. No changes in the K-values during freezing and frozen storage have been observed, nor in the rates of IMP degradation in frozen-thawed fish compared with unfrozen fish (Kemp and Spinelli 1969; Raja and Moorjani 1971). The increase in the K-value reported in frozen stored fish has been attributed to incorrect handling of the fish during thawing or during the treatment of the sample before extraction. Changes in nucleotide degradation differ when fish is hot or cold smoked. Hot smoking accelerates the degradation of nucleotides and increases the K-value; however, no nucleotide breakdown occurs to a marked extent during subsequent storage of the smoked fish. This has been attributed to the denaturation of the nucleotide-degrading enzymes. On the contrary, cold smoking does not modify the K-value; however, the K-value increases during storage, as in raw fish (Hattula and Kiesvaara 1996). The rate of increase of the K-value is reduced by salt content but the activity differs according to the concentration and type of salt used. Differences among fish species have been attributed to a different inhibiting activity of the NaCl, KCl and CaCl2 salts on the 5′-nucleotidase (Oba and Niwa 1992, 1993). Changes in the K-value during storage of fish under modified atmosphere packaging (MAP) differ according to the species. Storage of sardine and herring under MAP conditions decreased the production of ATP derivatives and the rate of increase of the K-value; also, a shelf life extension of the MAP-stored fish, longer than for fish stored under vacuum or air, was obtained (Özogul et al. 2000, 2004). However, in sole fillets no effect of the atmosphere

ATP-derived products and K-value determination

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on the K-value has been found (López Gálvez et al. 1998). The K-value of MAP-stored fillets of catfish at different temperatures did not correspond with the spoilage detected by sensory assessment (Reddy et al. 1997). Changes in the K-value have been closely related to sensory evaluation in farmed fish species harvested in different years and seasons, and is considered to be one of the best chemical indices for calibrating equipment or techniques for measuring fish freshness (Hamada-Sato et al. 2005; Raatikainen et al. 2005; Tejada et al. 2006a,b).

4.3 Methodology for evaluating the K-value or related compounds Inosine and/or hypoxanthine accumulate during storage of fish regardless of the species, although the ATP degradation rate may differ. Hypoxanthine was measured in the early methodology and was associated with fish freshness, but because of the different rate of producing and accumulating inosine or hypoxanthine, the early methodology to determine hypoxanthine in fish muscle was based on chromatographic methods (Jones and Murray 1964), immobilization of enzymes and coupling of the reaction with indicator dyes or enzyme electrodes (Jans et al. 1976; Ehira and Uchiyama 1969) was considered not suitable for many fish species (Ehira 1976; Luong and Male 1992). In the original K-value method, Ino and Hx were eluted together from ATP and other related compounds from the acid extracts of the muscle (Saito et al. 1959c). The initial method has been improved to get a better separation of the nucleotides and derivatives and/or to speed up the determination. Reviews of the methodology have been made by Ehira and Uchiyama (1987) and Gill (1992). The most common methodology used to evaluate ATP and its breakdown products as the K-value or other related indices described previously are based on extraction of the nucleotides and derivatives in acid (perchloric or trichloroacetic acid), neutralization of the extracts and further separation by high-performance liquid chromatography (HPLC). One of the most common techniques used is that proposed by Ryder (1985). The HPLC methods are usually used to contrast other techniques. Enzymatic methods that involve the analysis of individual nucleotide catabolites or a combination of several compounds are also used. The most common enzymatic methods use immobilized enzymes and electrochemical devices for detecting either oxygen consumed by hypoxanthine oxidation or H2O2 produced in the oxidation of hypoxanthine to xanthine and to uric acid by means of xanthine oxidase. Colorimetric methods have been also developed using enzymes immobilized in paper strips coupled to the colour change of an indicator dye when the reaction takes place. The most advantageous feature of the enzyme sensors is their selectivity, because measurements are usually made on mixtures of various compounds using multifunctional enzyme sensors, which allow components in a sample to be determined without prior separation. Multielectrode enzyme sensors that combine immobilized enzymes with electrodes have been used to calculate the K1-value assembling four enzyme sensors for AMP, IMP, HxR and Hx in a flow cell. No prior separation is needed and the compounds are determined from oxygen consumption (Watanabe et al. 1984). A hypoxanthine biosensor using immobilized xanthine oxidase and a polarographic electrode was developed by Muchaldani et al. (1990). The [Hx + HxR] concentration in the tissue extract was measured by nucleoside phosphorylase and xanthine oxidase immobilized on a commercially available pre-activated nylon

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membrane and attached to the tip of a polarographic electrode. For determination of [IMP + HxR + Hx], IMP was first converted to HxR by nucleotidase immobilized on the wall of a polystyrene tube. The polarographic electrode detected hydrogen peroxide and uric acid released during the enzymatic reaction. The K-value for each sample can be determined in approximately 10 min. To monitor the H-value, a biosensor used for determination of Hx was developed by Luong and Male (1992). The system consisted of xanthine oxidase immobilized on a membrane, soluble nucleoside phosphorylase and nucleotidase immobilized on a polystyrene tube. Hydrogen peroxide and uric acid released from hypoxanthine are detected by the amperometric probe. Nucleotidase was immobilized on the wall of a polystyrene tube pre-coated with polyethylenimine and used for conversion of IMP to HxR. After injection of soluble nucleoside phosphorylase (NP) and phosphate to the detection chamber, the resulting HxR was introduced for determination of [Hx + HxR + IMP]. For repeated assays, guanosine was used to prevent interfering signals due to residual soluble nucleoside phosphorylase during the course of Hx measurement. Another system based on double multi-enzyme reactor electrodes has been proposed by Okuma and Watanabe (2002). It consists of a pair of enzyme reactors with an oxygen electrode positioned close to the respective reactor. One enzyme reactor (I) is packed with nucleoside phosphorylase and xanthine oxidase immobilized simultaneously on chitosan beads (immobilized enzyme A). The other enzyme reactor (II) is packed with immobilized enzyme A and immobilized enzyme B (co-immobilized alkaline phosphatase and adenosine deaminase). The total amount of IMP, Ino and Hx is determined from the output of the electrode corresponding to the amount of oxygen consumed by Hx oxidation. One assay requires about 5 min, including the time for sample preparation. Capillary electrophoresis has also been used to measure the K-value (Nguyen et al. 1990; Luong et al. 1992). Systems based on chemiluminescence (CL)–flow injection analysis (FIA) using the chemiluminescence of luminol have been applied to the development of fish freshness sensors. Chemiluminescence of luminol has been widely accepted as a sensitive method for determination of H2O2 produced in the oxidation of hypoxanthine to xanthine and to uric acid by means of xanthine oxidase (Hayashi et al. 1996). The CL–FIA system used a photodiode as a detector, luminol and peroxidase from Artheromyces ramosus with detection limits of 3 and 10 nM for H2O2 or hypoxanthine, respectively. The time for a single K1 measurement was less than 1 min (Nakamura and Karube 2003). Recently, a quality evaluation sensor for determining the quality of sashimi has been developed by Watanabe et al. (2005). The technique correlates the sensory and K1-values of sashimi with the degree of colour change of thiazole blue (MTT: 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyl-tetrazolium bromide) due to the redox reaction of MTT accompanying the oxidation of hypoxanthine by xanthine oxidase in the sensor. The freshness of chilled or frozen stored fish to be consumed as sashimi is determined from the colour change of the sensor and is expressed as remaining days of validity (RDV) as sashimi (Hamada-Sato et al. 2004). The technique is non-destructive because it is not necessary to test the fish itself since the changes of fish meat agree with the changes of the colour tone of the enzyme reaction in the sensor stored with the fish. Commercial methods for a rapid measurement of fish freshness have been developed. Standard methods to be used by the industry need to be sensitive and rapid, and the devices need to be compact, portable and easy to use. A big effort has been made by companies and researchers to build devices for the fish industry. Enzyme sensor systems have been

ATP-derived products and K-value determination

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developed and commercialized by several companies to monitor the K-, K1- or H-values.

4.4

Conclusions

The measurement of the K- or other related values based on ATP degradation is considered one of the best techniques for evaluating freshness in fish stored at temperatures above freezing. The increase in the K-value is detected very early, the maximum values and the rate of increase remain quite stable for each fish species, and it has been found to be closely related to sensory evaluation in many fish species. The K-value is considered one of the best chemical indices for calibrating equipment or techniques for measuring fish freshness.

Acknowledgements Thanks are due to Professor Dr Ken Ichi Arai, former professor at Hokkaido University, and Professor Dr Noboru Kato and Dr Satomi Koseki, from the School of Marine Science and Technology, Tokai University, Shizuoka, Japan, for their help and advice in the writing of this chapter.

4.5

References

Alasalvar, C., Taylor, K.D.A. and Shahidi, F. (2002) Comparative quality assessment of cultured and wild seabream (Sparus aurata) stored in ice. Journal of Agricultural and Food Chemistry 50: 2039–2045. Arai, K. (1960) Acid soluble nucleotides in muscle marine invertebrate. effect of storing temperature in marine invertebrate. Effect of storing temperature upon the content of muscular nucleotide of some seashells. Bulletin of the Faculty of Fisheries Hokkaido University 11: 67. Arai, K.I. and Saito, T. (1961) Changes in adenine nucleotides in the muscle of some marine invertebrates. Nature 192: 451–452. Berg, T., Erikson, U. and Nordtvedt, T.S. (1997) Rigor mortis assessment of Atlantic salmon (Salmo salar) and effects of stress. Journal of Food Science 62: 439–446. Bremner, H.A., Olley, J., Statham, J.A. and Vail, A.M.A. (1988) Nucleotide catabolism: influence on the storage life of tropical species of fish from the North West Shelf of Australia. Journal of Food Science 53: 6–11. Burns, G.B., Kee, P.J. and Irvine, B.B. (1985) Objective procedure for fish freshness evaluation based on nucleotide changes using a HPLC system. Canadian Technical Report of Fisheries and Aquatic Sciences No. 1373. Cappeln, G. and Jessen, F. (2001) Glycolysis and ATP degradation in cod (Gadus morhua) at subzero temperatures in relation to thaw rigor. Lebensmittel-Wissenschaft Und -Technologie 34: 81–88. Cappeln, G., Nielsen, J. and Jessen, F. (1999) Synthesis and degradation of adenosine triphosphate in cod (Gadus morhua) at subzero temperatures. Journal of the Science of Food and Agriculture 79: 1099–1104.

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Careche, M. and Tejada, M. (1991) Interaction between triolein and natural hake actomyosin during frozen storage at −18 °C. Journal of Food Biochemistry 15: 449–462. Chiba, A., Hamaguchi, M., Kosaka, M., Tokuno, T., Asai, T. and Chichibu, S. (1991) Quality evaluation of fish meat by 31phosphorus-nuclear magnetic resonance. Journal of Food Science 56(3): 660–664. Dingle, J.R. and Hines, J.A. (1971) Degradation of inosine 5′-monophosphate in the skeletal muscle of several North Atlantic fishes. Journal of the Fisheries Research Board of Canada 28(8): 1125–1131. Ehira, S. (1976) A biochemical study on the freshness of fish. Bulletin of the Tokai Regional Fisheries Research Laboratory 88: 1–132. Ehira, S. and Uchiyama, H. (1969) Rapid estimation of freshness of fish by nucleoside phosphorylase and xantine oxidase. Bulletin of the Japanese Society of Scientific Fisheries 35(11): 1080– 1085. Ehira, S. and Uchiyama, H. (1973) Formation of inosine and hypoxanthine in fish muscle during ice storage. Bulletin of the Tokai Regional Fisheries Research Laboratory 75: 63–73. Ehira, S. and Uchiyama, H. (1987) Determination of fish freshness using the K-value and comments on some other biochemical changes in relation to freshness. In: D.E. Kramer and J. Liston (Eds) Seafood Quality Determination. Elsevier Science Publishers B.V., Amsterdam, pp. 185–207. Erikson, U. (2001) Potential effects of preslaughter fasting, handling and transport. In: S.C. Kestin and P.D. Warriss (Eds) Farmed Fish Quality. Fishing News Books, London, pp. 202–219. Erikson, U., Bayer, A.R. and Sigholt, T. (1997) Muscle high-energy phosphates and stress affect K-values during ice storage of Atlantic salmon (Salmo salar). Journal of Food Science 62(1): 43–47. Erikson, U., Sigholt, T., Rustad, T., Einarsdottir, I.E. and Jørgensen, L. (1999) Contribution of bleeding to total handling stress during slaughter of Atlantic salmon. Aquaculture International 7: 101–115. Farrell, A.P., Gallaugher, P.E. and Routledge, R. (2001) Rapid recovery of exhausted adult coho salmon after commercial capture by troll fishing. Canadian Journal of Fisheries and Aquatic Science 58: 2319–2324. Fujisawa, K. and Yoshino, M. (1985) Distribution of AMP deaminase and adenosine desaminase in muscle of mammals, fish, molluscans and crustaceans. Journal of the Japanese Society of Nutrition and Food Science 38: 322. Fuke, S., Watanabe, K. and Konosu, S. (1994) Enhancing effect of nucleotides on sweetness of heated prawn muscle. In: K. Kurihara, N. Suzuki and H. Ogawa (Eds) Olfaction and Taste XI. SpringerVerlag, Tokyo, pp. 357–360. Gallaugher, P. and Farrell, A.P. (1999) Physiological indicators of stress of capture and mortality risk in commercial non-retention salmon fisheries. Report. Fisheries and Oceans Canada. Available at http://www.google.es/search?hl=es&q=ATP+fish+capture&meta. Accessed June 15, 2006. Gill, T.A. (1992) Biochemical and chemical indices of seafood quality. In: H.H. Huss, M. Jakobsen, and J.C. Liston, J.C. (Eds) Quality Assurance in the Fish Industry, Developments in Food Science. Elsevier Science Publishers B.V., Amsterdam, pp. 377–388. Gill, T.A., Thompson, J.W., Gould, S. and Sherwood, D. (1987) Characterization of quality deterioration of yellowfin tuna. Journal of Food Science 52: 580–583. Gornik, A., Albalat, A., Neil, D.M. and Coombs, G.H. (2006) Post mortem changes in Norway lobster (Nephrops norvegicus) – possible reasons for the absence of the rigor-mortis state and the relative short shelf life. TAFT 2006. 2nd Joint Trans–Atlantic Fisheries technology conference. Québec, pp. 51. Grigorakis, K., Taylor, K.D.A. and Alexis, M.N. (2003) Seasonal patterns of spoilage of ice-stored cultured gilthead sea bream (Sparus aurata). Food Chemistry 81: 263–268.

ATP-derived products and K-value determination

83

Grigorakis, K., Alexis, M., Gialamas, I. and Nikolopoulou, D. (2004) Sensory, microbiological, and chemical spoilage of cultured common sea bass (Dicentrarchus labrax) stored in ice: a seasonal differentiation. European Food Research and Technology 219: 584–587. Groniger, H.S. and Brandt, K.R. (1969) Degradation of adenine nucleotides in the muscle of Dungeness crab (Cancer magister) and king crab (Paralithodes camtschatica) during storage and cooking. Journal of Milk and Food Technology 32(1): 1–3. Hamada-Sato, N., Okuma, H. and Watanabe, E. (2004) Theoretical consideration on estimation of the remaining days of validity (RDV) for fresh fish based on K value concept. Nippon Shokuhin Kagaku Kogaku Kaishi 51(9): 495–504. Hamada-Sato, N., Usui, K., Kobayashi, T., Imada, C. and Watanabe, E. (2005) Quality assurance of raw fish based on HACCP concept. Food Control 16: 301–307. Hattula, T. (1997) Adenosine triphosphate breakdown products as a freshness indicator of some fish species and fish products. Technical Research Centre of Finland. VTT Publications No. 297, pp. 48. Hattula, T. and Kiesvaara, M. (1996) Breakdown products of adenosine triphosphate in heated fishery products as an indicator of raw material freshness and of storage quality. Lebensmittel-Wissenschaft Und -Technologie 29: 135–139. Hattula, T., Kiesvaara, M. and Moran, M. (1993) Freshness evaluation in European whitefish (Coregonus wartmanni) during chill storage. Journal of Food Science 58(6): 1212–1215, 1236. Hayashi, K., Sasaki, S., Ikebukuro, K. and Karube, I. (1996) Highly sensitive chemiluminescence flow injection analysis system using microbial peroxidase and a photodiode detector. Analytica Chimica Acta 329: 127–l 34. Hiltz, D.F. and Dyer, W.J. (1970) Principal acid-soluble nucleotides in adductor muscle of the scallop (Placopecten magellanicus) and their degradation during postmortem storage in ice. Journal of the Fisheries Research Board of Canada 27: 83–92. Hiltz, D.F., Dyer, W.J., Nowland, S. and Dingle, J.R. (1971) Variations of biochemical quality indices by biological and technological factors. In: R. Kreuzer (Ed.) Fish Inspection and Quality Control. Fishing News Books, London, pp. 91–195. Howgate, P. (2005) Kinetics of degradation of adenosine triphosphate in chill-stored rainbow trout (Oncorhynchus mykiss). International Journal of Food Science and Technology 40: 579–588. Howgate, P. (2006) A review of the kinetics of degradation of inosine monophosphate in some species of fish during chilled storage. International Journal of Food Science and Technology 41: 341–353. Huidobro, A., Pastor, A. and Tejada, M. (2001) Adenosine triphosphate and derivatives as freshness indicators of gilthead sea bream (Sparus aurata). Food Science and Technology International 7(1): 23–30. Huss, H.H. (1995) Quality and quality changes in fresh fish. FAO Fisheries Technical Paper No. 348. 195 pp. Available at http://www.fao.org/docrep/. Iwamoto, M., Ioka, H., Saito, M. and Yamanaka, H. (1985) Relation between rigor mortis of sea bream and storage temperatures. Nippon Suisan Gakkaishi 51: 443–446. Iwamoto, M., Yamanaka, H., Watabe, S. and Hashimoto, K. (1987) Effect of storage temperature on rigor mortis and ATP degradation in plaice Paralichtys olivaceus muscle. Journal of Food Science 52: 1514–1517. Iwamoto, M., Yamanaka, H., Abe, H., Ushio, H., Watabe, S. and Hashimoto, K. (1988) ATP and creatine phosphate breakdown in spiked plaice muscle during storage and activities of some enzymes involved. Food Science 53: 1662–1665. Janhs, A.C., Howe, J.L., Coduri, R.J. and Rand, A.G. (1976) A rapid visual enzyme test to asses fish freshness. Food Technology 30: 37–30. Jones, N.R. (1965) Problems associated with freezing very fresh fish. In: Proceedings of Meetings on Fish Technology. OECD, Paris, pp. 31–55.

84

Fishery Products: Quality, safety and authenticity

Jones, N.R. (1969) Fish as a raw material for freezing: Factors influencing the quality of products frozen at sea. In: R. Kreuzer (Ed.) Fish Inspection and Quality Control. Fishing News Books, London, pp. 31–39. Jones, N.R. and Murray, J. (1962) Degradation of adenine- and hypoxantine nucleotide in the muscle of trawled cod (Gadus callarias). Journal of the Science of Food and Agriculture 13: 475–480. Jones, N.R. and Murray, J. (1964) Rapid measures of nucleotide dephosphorylation in iced fish muscle. their value as indices of freshness and of inosine 5′-monophosphate concentration. Journal of the Science of Food and Agriculture 15: 763–774. Kanoh, S., Suzuki, T., Maeyama, K., Takewa, T., Watabe, S. and Hashimoto, K. (1986) Comparative studies on ordinary and dark muscles of tuna fish. Nippon Suisan Gakkaishi 52(19): 1807–1816. Karube, I., Matsuoka, H., Suzuki, S., Watanabe, E. and Toyama, K. (1984) Determination of fish freshness with an enzyme sensor system. Journal of Agricultural and Food Chemistry 32: 314–319. Kassemsarn, B.O., Sanz Perez, B., Murray, J. and Jones, N.R. (1963) Nucleotide degradation in the muscle of iced haddock (Gadus aeglefinus) lemon sole (Pleuronectes microcephalus) and plaice (Pleuronectes platessa). Journal of Food Science 28: 28–37. Kato, N., Nozaki, H., Komatsu, K. and Arai, K. (1979) A new method for evaluation of the quality of frozen surimi from Alaska pollack. Relationship between myofibrillar ATPase activity and kamaboko forming ability of frozen surimi. Nippon Suisan Gakkaishi 45: 1027–1032. Kawashima, K., Arai, K. and Saito, T. (1973a) Studies on muscular proteins of fish-IX. An attempt on quantitative determination of actomyosin in frozen surimi from Alaska-pollack. Nippon Suisan Gakkaishi 39: 207–214. Kawashima, K., Arai, K. and Saito, T. (1973b) Studies on muscular proteins of fish-X. The amount of actomyosin in frozen ‘surimi’ from Alaska-pollack. Nippon Suisan Gakkaishi 39: 525–532. Kawashima, K., Arai, K. and Saito, T. (1973c) Studies on muscular proteins of fish-XIII. Relationship between the amount of actomyosin in frozen surimi and the quality of kamaboko from the same material in Alaska-pollack. Nippon Suisan Gakkaishi 39: 1201–1209. Kazeniac, S.J. (1961) Chicken flavour. In: Proceedings of the Flavor Chemistry Symposium, p. 37. Campbell Soup Co., Camden, NJ. Kemp, B. and Spinelli, J. (1969) Comparative rates of IMP degradation in unfrozen and frozen-andthawed (slacked) fish. Journal of Food Science 34; 133–135. Kestin, S., Wotton, S. and Adams, S. (1995) The effect of CO2, concussion or electrical stunning of rainbow trout (Oncorhynchus mykiss) on fish welfare. In: Quality in Aquaculture, Special Publication No. 23, European Aquaculture Society, Ghent, Belgium. pp. 380–381. Koseki, S., Ootake, R., Katoh, N. and Konno, K. (2005) Quality evaluation of frozen surimi by using pH stat for ATPase assay. Fisheries Science 71: 388–396. Kuninaka, A., Kibi, M. and Kin’Ichiro, K. (1964) Development of flavor nucleotides. Food Technology 18(3): 287–293. Leadbeater, L. and Perry, S.V. (1963) The effect of actin on the magnesium-activated adenosine triphosphatase of heavy meromyosin. Biochemical Journal 87: 233–238. López-Galvez, D.E., de la Hoz, L., Blanco, M. and Ordoñez, J.A. (1998) Refrigerated storage (2 °C) of sole (Solea solea) fillets under CO2-enriched atmospheres. Journal of Agricultural and Food Chemistry 46: 1143–1149. Love, R.M. (1970) The Chemical Biology of Fishes. Academic Press, London, pp. 547. Luong, J.H.T. and Male, K.B. (1992) Development of a new biosensor system for the determination of the hypoxanthine ratio, an indicator of fish freshness. Enzyme and Microbial Technology 14: 125–130. Luong, J.H.T., Male, K.B., Masson, C. and Nguyen, A.L. (1992) Hypoxanthine ratio determination in fish extract using capillary electrophoresis and immobilized enzymes. Journal of Food Science 57: 77–81.

ATP-derived products and K-value determination

85

Ma, L.B., Yamanaka, H., Ushio, H. and Watabe, S. (1992) Studies on the mechanism of thaw rigor in carp. Nippon Suisan Gakkaishi 58: 1535–1540. Marseno, S.W., Hori, K. and Miyazawa, K. (1992) Distribution of 5′ nucleotidase in muscle of some marine fishes. Comparative Biochemistry & Physiology 102B: 247–253. Massa, A.E., Paredi, M.E. and Crupkin, M. (2003) A chemical assessment of freshness in stored adductor muscle from scallops. Brazilian Journal of Chemical Engineering 20(2): 147–152. Matsumoto, M. and Yamanaka, H. (1991) –Post mortem biochemical changes in the muscle of kuruma prawn during storage and evaluation of the freshness. Nippon Suisan Gakkaishi 56: 1145–1149. Mendes, R., Quinta, R. and Nunes, M.L. (2001) Changes in baseline levels of nucleotides during ice storage of fish and crustaceans from the Portuguese coast. European Food Research Technology 212: 141–146. Mochizuki, S. and Sato, A. (1994) Effect of various killing procedures and storage temperatures on post mortem changes in the muscle of horse mackerel. Nippon Suisan Gakkaishi 60: 125–130. Mochizuki, S. and Sato, A. (1996) Effect of various killing procedures on post mortem changes in the muscle of chub mackerel and round scad. Nippon Suisan Gakkaishi 62: 453–457. Mochizuki, S., Norita, Y. and Maeno, K. (1998) Effects of bleeding on post mortem changes in the muscle of horse mackerel. Nippon Suisan Gakkaishi 64: 276–279. Mulchandani, A., Male, K.B. and Luong, J.H.T. (1990) Development of a biosensor for assaying postmortem nucleotide degradation in fish tissues. Biotechnology and Bioengineering 35(7): 739–745. Nakamura, H. and Karube, I. (2003) Current research activity in biosensors. Analytical and Bioanalytical Chemistry 377: 446–468. Nakamura, K., Fuji, Y. and Ishikawa, S. (1976) Postmortem changes in the amounts of glycogen and atp in adductor muscle of scallop. Bulletin of the Tokai Regional Fisheries Research Laboratory 84: 21–29. Nguyen, A.L., Luong, J.H.T. and Masson, C. (1990) Determination of nucleotides in fish tissues using capillary electrophoresis. Analytical Chemistry 62: 2490–2493. Oba, K. and Niwa, E. (1992) Suppressive effects of salts on degradation of ATP-related compound in fish flesh during cold storage. Bulletin of the Japanese Society for Food Science and Technology. 39: 1007–1010. Oba, K. and Niwa, E. (1993) The mode of inhibition by salts to two enzymes involved in IMP degradation in fish flesh. Sippon Shokuhin Kogyo Gakkaishi 40: 583–588. Obatake, A., Doi, T. and Ono, T. (1988) Post mortem degradation of inosinic acid and related enzyme activity in the dark muscle of fish. Nippon Suisan Gakkaishi 54: 283–288. Oka, H. (1989) Packaging for freshness and the prevention of discoloration of fish fillets. Packaging Technology and Science 2: 201–213. Okuma, H. and Watanabe, E. (2002) Flow system for fish freshness determination based on double multi-enzyme reactor electrodes. Biosensors and Bioelectronics 17: 367–372. Onibala, H., Takayama, T., Shindo, J., Hayashi, S. and Miki, H. (1997) Influence of freshness on occurrence of setting and disintegrating in heat-induced gels from tilapia. Fisheries Science 63(2): 276–280. Özogul, F., Taylor, K.D.A., Quantick, P.C. and Özogul, Y. (2000) A rapid HPLC-determination of ATP-related compounds and its application to herring stored under modified atmosphere. International Journal of Food Science 35(6): 549–554. Özogul, F., Polat, A. and Özogul, Y. (2004) The effects of modifed atmosphere packaging and vacuum packaging on chemical, sensory and microbiological changes of sardines (Sardina pilchardus). Food Chemistry 85: 49–57. Özogul, Y., Özogul, F., Kuley, E., Özkutuk, A.S., Gökbulut, C. and Köse, S. (2006). Biochemical, sensory and microbiological attributes of wild turbot (Scophthalmus maximus), from the Black Sea, during chilled storage. Food Chemistry 99: 752–758.

86

Fishery Products: Quality, safety and authenticity

Piñeiro, C., Bautista, R., Rodríguez, O., Losada, V., Barros-Velaázquez, J. and Aubourg, S.P. (2005) Quality retention during the chilled distribution of farmed turbot (Psetta maxima): effect of a primary slurry ice treatment. International Journal of Food Science & Technology 40: 817–824. Ponce de Leon, S., Inoue, N. and Shinano, H. (1994) Shelf life of sardine and bacterial flora in brine containing acetic acid during immersed storage. Fisheries Science 60: 429–433. Pospiech, E., Grzes´, B., ´Lyczyn´ski, A., Borzuta, K., Szalata, M. and Mikol´ajczak, B. (2003) Muscle proteins and their changes in the process of meat tenderization. Animal Science Papers and Reports 21(Suppl. 1): 133–151. Pottinger, T.G. (2001) Effects of husbandry stress on flesh quality indicators in fish. In: S.C. Kestin and P.D. Warriss (Eds) Farmed Fish Quality. Fishing News Books, Oxford, pp. 145–160. Raatikainen, O., Reinikainen, V., Minkkinen, P., Ritvanen, T., Muje, P., Pursiainen, J., Hiltunen, T., Hyvönen, P., von Wright, A. and Reinikainen, S.P. (2005) Multivariate modelling of fish freshness index based on ion mobility spectrometry measurements. Analytica Chimica Acta 544: 128–134. Raja, K.C.M. and Moorjan, M.N. (1971) Review: nucleotides – a study of their degradation in fish under various conditions and its impact on flavour. Journal of Food Technology 3(1): 3–6. Reddy, N.R., Roman, M.G., Villanueva, M., Solomon, H.M., Kautter, D.A. and Rhodehamel, E.J. (1997) Shelf life and Clostridium botulinum toxin development during storage and modified atmosphere-packaged fresh catfish fillets. Journal of Food Science 62: 878–884. Robb, D.H.F. (2001) The relationship between killing methods and quality. In: S.C. Kestin and P.D. Warriss (Eds) Farmed Fish Quality. Fishing News Books, Oxford, pp. 220–233. Rodriguez, O., Barros-Velazquez, J.M., Piñeiro, C., Gallardo, J.M. and Aubourg, S.P. (2006) Effects of storage in slurry ice on the microbial, chemical and sensory quality and on the shelf life of farmed turbot (Psetta maxima). Food Chemistry 95(2): 270–278. Rowlerson, A., Scapolo, P.A., Mascarello, F., Carpene, E. and Veggetti, A. (1985) Comparative study of myosins present in the lateral muscle of some fish: species variations in myosin isoforms and their distribution in red, pink and white muscle. Journal of Muscle Research and Cell Motility 6(5): 601–640. Ryder, J.M. (1985) Determination of adenosine triphosphate and its breakdown products in fish muscle by high performance liquid chromatography. Journal of Agricultural and Food Chemistry 33: 678–680. Ryder, J.M., Fletcher, G.C., Stec, M.G. and Seelye, R.J. (1993) Sensory, microbiological and chemical changes in hoki stored in ice. International Journal of Food Science & Technology 28: 169–180. Sagedhal, A., Busalmen, J.P., Roldan, H., Paredi, M.E. and Crupkin, M. (1997) Post-mortem changes in adenosine triphosphate and related compounds in mantle of squid (Ilex argentinus) at different stages of sexual maturation. Journal of Aquatic Food Product Technology 64: 43–56. Saito, T. and Arai, K. (1957a) Slow freezing of carp muscle and inosinic acid formation. Nature 179: 820–821. Saito, T. and Arai, K. (1957b) Studies on the organic phosphates in muscle of aquatic Animals V. Changes in muscular nucleotides of carp during freezing storage. Nippon Suisan Gakkaishi 23(5): 265–269. Saito, T., Arai, K. and Tanaka, T. (1958a) Studies on the organic phosphates in muscle of aquatic Animals VI. Effect of storing temperature upon the content of muscular nucleotides of squid. Bulletin of the Faculty of Fisheries Hokkaido University 9(2): 121–126. Saito, T., Arai, K. and Tanaka, T. (1958b) Changes in adenine nucleotide of squid muscle. Nature 181: 1127–1128. Saito, T., Arai, K.I. and Yajima, T. (1959a) Studies on the organic phosphates in muscle of aquatic animals. VII. Changes in purine nucleotides of red lateral muscle os fish. Nippon Suisan Gakkaishi 25(7): 573–575. Saito, T., Arai, K.I. and Yajima, T. (1959b) Changes in purine nucleotides of red lateral muscle of rainbow trout. Nature 184: 1415.

ATP-derived products and K-value determination

87

Saito, T., Arai, K. and Matsuyoshi, M. (1959c) A new method for estimating the freshness of fish. Bulletin of the Japanese Society of Scientific Fisheries 24(9): 749–750. Sakamoto, M., Fujii, Y. and Nakamura, K. (1973) Changes of the components in adductor muscle of scallop during storage. Bulletin of the Tokai Regional Fisheries Research Laboratory 75: 107–118. Scott, D.N., Fletcher, G.C., Hogg, M.G. and Ryder, J.M. (1986) Comparison of whole with headed and gutted orange roughy stored in ice: sensory, microbiology and chemical assessment. Journal of Food Science 51: 79–83, 86. Shahidi, F., Chong, X. and Dunajski, E. (1994) Freshness quality of harp seal (Phoca groenlandica) meat. Journal of Agricultural and Food Chemistry 42: 868–872. Shirai, T. and Kikuchi, Y. (2002) Extractive components of kuruma prawns Penaeus japonicus. Fisheries Science 68(II): 1386–1389. Slinde, E., Roth, B. and Torrisen, O.J. (2001) Cold shortening and drip loss in Atlantic Salmon (Salmo salar), Rainbow trout (Oncorhyncus mykiss), Halibut (Hippoglossus hipoglossus), cod (Gadus morhua), Haddock (Melanogrammus aeglefinus) and saithe (Pollachius virens) In: S.C. Kestin and P.D. Warriss (Eds) Farmed Fish Quality. Fishing News Books, Oxford, pp. 407–408. Spinelli, J. (1965) Effect of hypoxanthine on the flavour of fish and stored low-dose irradiated petrale sole fillets. Journal of Food Science 30: 1063–1067. Spinelli, J., Eklund, M. and Miyauchi, D. (1964) Measurement of hypoxantine in fish as a method of assessing freshness. Journal of Food Science 29: 719–714. Stroud, G.D. (1981) Rigor in fish. The effect on quality. Torry Advisory Note 36, Torry Research Station, Ministry of Technology (UK). Available at: http://www.fao.org/wairdocs/tan/x5914e/ x5914e00.htm. Surette, M.E., Gill, T.A. and LeBlanc, P.J.J. (1988) Biochemical basis of postmortem nucleotide catabolism in cod (Gadus morhua) and its relationship to spoilage. Journal of Agricultural and Food Chemistry 36: 19–22. Suwetja, I.K., Hori, K., Miyazawa, K. and Ito, K. (1989) Changes in content of ATP-related compounds, homarine, and trigonelline in marine invertebrates during ice storage. Nippon Suisan Gakkaishi 55: 559–566. Tarr, H.L.A. (1966) Post-mortem changes in glycogen, nucleotides, sugar phosphates, and sugars in fish muscles. A review. Journal of Food Science 31(6): 846–854. Tejada, M., Huidobro, A. and Fouad Mohamed, G. (2006a) Evaluation of two quality indices related to ice storage and sensory analysis in farmed gilthead seabream and seabass. Food Science and Technology International 12(3): 261–268. Tejada, M., De las Heras, C. and Kent, M. (2006b) Changes in the quality indices during ice storage of farmed Senegalese sole (Solea senegalensis). European Food Research and Technology DOI 10.1007/s00217-006-0407-9. Tomlinson, N. and Creelman, V.M. (1960) On the source of free ribose formed post mortem in the muscle of lingcod (Ophiodon elongatus). Journal of the Fisheries Research Board of Canada 17(5): 603–606. Tsuchimoto, M., Misima, T., Utsugi, T., Kitajima, S., Yada, S., Senta, T. and Yasuda, M. (1988a) Resolution characteristics of ATP-related compounds in fishes from several waters and the effect of habitat temperatures on the characters. Nippon Suisan Gakkaishi 54: 683–689. Tsuchimoto, M., Tanaka, N., Uesugi, T., Misima, T., Tachibana, T., Yada, S., Senta, T. and Yasuda, M. (1988b) The influence of rearing water temperature on the relative thermostability of myofibrillar Ca2+-ATPase and on the lowering speed of freshness in carp. Nippon Suisan Gakkaishi 54: 117–122. Uchiyama, H. and Ehira, S. (1974) Relation between freshness and acid-soluble nucleotides in aseptic cod and yellowtail muscles during ice storage. Bulletin of the Tokai Regional Fisheries Research Laboratory 78: 23–31.

88

Fishery Products: Quality, safety and authenticity

Watanabe, E., Tokimatsu, S., Toyama, K., Karube, I., Matsuoka, H. and Suzuki, S. (1984) Simultaneous determination of hypoxanthine, inosine, inosine-5′-phosphate and adenosine-5′-phosphate with a multielectrode enzyme sensor. Analytica Chimica Acta 164: 139–146. Watanabe, E., Tamada, Y. and Hamada-Sato, N. (2005) Development of quality evaluation sensor for fish freshness control based on KI value. Biosensors and Bioelectronics 21(3): 534–538. Yada, O., Tsuchimoto, M., Wang, Q., Apablaza, P.A.G., Jabarsyah, A. and Tachibana, K. (2000) Differences of muscle fiber type and temporal change of K-value among parts toward depth of dorsal muscle in carp (cultured). Fisheries Science 66: 147–152. Yada, O., Tsuchimoto, M., Jabarsyah, A., Wang, Q., Apablaza, P.A.G. and Tachibana, K. (2001a) Influence of interposition of pink muscle fiber into dorsal ordinary muscle on increasing rate of K-value in various fish species. Fisheries Science 67: 675–681. Yada, O., Tsuchimoto, M., Wang, Q., Apablaza, P.A.G., Jabarsyah, A. and Tachibana, K. (2001b) Influence of interposition of pink muscle fiber into dorsal ordinary muscle on 5′-IMP degrading activity in various fish species. Fisheries Science 67: 948–955. Yoneda, C., Kasamatsu, C., Hatae, K. and Watabe, S. (2002) Changes in taste and textural properties of the foot of the Japanese cockle (Fulvia mutica) by cooking and during storage. Fisheries Science 68: 1138–1144.

Chapter 5

VIS/NIR spectroscopy Heidi Anita Nilsen and Karsten Heia

5.1

Introduction

In the case of fish products – which are considered particularly fragile and perishable and often have a relatively short shelf life – the characteristic of the quality measuring method is of importance. It should be fast both on performance and on response. The former implies no or little sample preparation, and the latter means that the principle of measurement as well as evaluation of result should be made within minutes or preferably seconds. Another issue is the price: for a method to become commercially viable to several users, the price per unit should not be too high. Measurements based on light spectroscopy have the potential of meeting the above requirements, and particularly near infrared spectroscopy (NIR) has proven to be useful in a variety of food analysis applications (Osborne and Fearn 1986; Pasquini 2003). The broadbanded NIR spectra contain information on several physical and chemical parameters, and so it may give answers to several quality issues simultaneously, as illustrated in Figure 5.1. Over the past two decades there has also been an increasing activity on the application of NIR spectroscopy for assessment of quality parameters related to fish. This review will focus on the use of visible and near infrared (VIS/NIR) spectroscopy and its use for quality and safety determination of fish and fish-based products for human consumption. Initially, a brief introduction to VIS/NIR spectroscopic techniques and the corresponding analytical tools will be given. The following features will be applications of VIS/NIR spectroscopy as reported in the literature. The quality characteristics that are assessed by VIS/NIR spectroscopy will be categorised in five main themes: constituents, freshness, authentication, safety and other quality parameters.

5.2 Analytical principles and measurements 5.2.1

VIS/NIR spectroscopy

The operating principle of the measurement technique is based on sending light onto the sample and then measuring the light coming from the sample at different wavelengths. 89

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Figure 5.1 Spectroscopy may reveal several product aspects simultaneously, for instance fat, water and protein content, as well as freshness and detection of nematodes. Artwork by Oddvar Dahl, Nofima Marine, Norway.

The wavelength region to be discussed ranges from 400 to 2500 nm. This covers the visible (400–700 nm) and near infrared (700–2500 nm) regions. VIS/NIR spectroscopy refers to experimental set-ups where the sample is illuminated with broadbanded VIS/NIR light and the emitted VIS/NIR light spectrum from the sample is recorded. When the light is sent through the sample we talk about a transmission or a transmittance measurement, and 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. When the illumination and detector unit is located on the same side of the sample, the measurement is referred to as a reflection or a reflectance measurement. In this case, the amount of light entering the detector unit is dependent on the illumination pattern and geometry, and the reflectivity of the sample. In early works, these were the two most used measurement configurations. A transflection/transflectance measurement, also called diffuse reflectance, refers to the situation when the sample is illuminated and measured on the same side, but not on the same location. In this case, the amount of light entering the detector unit depends on the illumination, and the scattering and absorption properties of the sample. The three modes of measurement are shown in Figure 5.2. The physical and chemical material characteristics responsible for the interaction with light are beyond the scope of this chapter, but can be found in Osborne and Fearn (1986) and Pasquini (2003). In traditional VIS/NIR spectroscopy, the sample measurement results in a spectrum in the visible and/or near infrared region. In the past decade, a new technique referred to as imaging spectroscopy has been developed (Herrala and Okkonen 1996; Hyvarinen et al. 1998). In addition to the spectral information, this technique also gives the spatial information of the sample. In other words, at each location at the sample a full spectrum is recorded.

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Figure 5.2 The three measurement modes for VIS/NIR spectroscopy applied to fish products. From the left: reflection, transmission and transflection.

Multivariate Analysis

Model Spectra (measured)

Response (measured)

Prediction

Model New spectra (measured)

Response (predicted)

Figure 5.3 Multivariate analysis of VIS/NIR spectra. The first step is to record spectra and build a model between the spectra and a reference measurement. The reference can, for instance, be chemically measured fat content. Secondly, spectra from new samples are recorded and the model is used to predict the wanted parameter.

Imaging spectroscopy can be used for transmission, reflection as well as transflection measurements.

5.2.2

Data analysis

Spectroscopy applied to fish or fish products is not a direct technique, as illustrated in Figure 5.3. This means that further analysis of the recorded spectrum is needed. A common methodology to use is multivariate analysis, also referred to as chemometrics. These techniques

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model the relation between the recorded spectral information and the information searched for. The complex nature of the VIS/NIR spectral region seldom allows the use of single wavelengths for qualitative or quantitative purposes. Typically, a model based on a higher number of wavelengths is required to extract useful information from the spectroscopic data. It is not in the scope of this chapter to cover all details on multivariate analysis, but among the most used techniques are principal component analysis (PCA), partial least square (PLS) regression and soft independent modelling of class analogies (SIMCA) (Martens and Næs 1989). The most frequently used techniques typically allow samples with similar properties to be grouped to establish classification models (qualitative analysis) or to make models for determining some property of the unknown sample (quantitative analysis). The multivariate analysis can be applied directly on the spectroscopic measurement, or some pre-treatment of spectra can be performed. This can be normalisation (Griffiths 1995), derivatives (McLure 1993, 1994), multiplicative scatter correction (Geladi et al. 1985), standard normal variate (Barnes et al. 1989), de-trending (Barnes et al. 1989) or a combination of these treatments.

5.3

Constituents: assessment of chemical composition

To a great extent, work on light spectroscopy and fish quality parameters requires determination of chemical composition. Along with the development of spectroscopic instrumentation and versatility in measurement modes, it has been a development in sample preparation procedures; as instrumentation facilities increased, sample preparation has become simpler and in some cases redundant. Of the earliest reports are the work by Mathias et al. (1987) and Gjerde and Martens (1987). In the former, it was shown how NIR reflectance spectroscopy can be applied for the determination of lipid and protein in rainbow trout and Arctic charr, and in the latter NIR reflectance spectroscopy was used to determine protein, fat and water content in rainbow trout. Before NIR measurements, the fish muscle was ground and freeze-dried (Gjerde and Martens 1987) or freeze-dried and pulverised (Mathias et al. 1987). Also, in the work of Darwish et al. (1989), the procedure for sample preparation of fish material is quite cumbersome. By using mid-infrared transmission spectroscopy they analysed cod, mackerel and tuna samples for fat, protein and water content. For the two former constituents, the fish samples were minced and processed to a milk-like emulsion before NIR measurements. For the determination of moisture, the minced fish muscle was exposed to a methanol extraction procedure before NIR measurements. In this work good correlation between chemical reference measurements and spectroscopic data were obtained, and the conclusion was that mid-infrared transmission spectroscopy could serve as a rapid means of proximate analysis of fish products for quality control. In the following years, reports showed that preparation of fish samples for measurements of chemical composition by use of NIR could be significant. Rasco and co-workers (1991) described how reflectance spectroscopy in the 900–1800 nm region could be applied to estimate moisture, protein and crude lipid content of fresh and frozen trout muscle without grounding, drying or extraction of the muscle sample. In 1992, Sollid and Solberg reported how the fat content of salmon could be estimated by transmission spectroscopy in the 850– 1050 nm region. Samples were prepared by homogenising the muscle, and the spectroscopic

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method gave an error of prediction of less than 0.5%. Solberg (1992) also demonstrated that NIR transmission spectra could be used to discriminate between seasonal variation in cod as well as determining moisture, protein and pH value of cod. The development of methods towards more simplified sample procedures made NIR spectroscopy a more convenient method. Another issue to be resolved was the matter of intrusion of the measurement material. Lee et al. (1992) were the first to introduce shortwavelength NIR (700–1100 nm) as a non-invasive technique for quality determination in fish. Measurement fibres were placed directly onto the fish, with no removal of scales or skin. It proved it possible to determine the crude lipid content in small-sized intact rainbow trout. Although the standard error of prediction of this method was quite high, it was argued that the method would be interesting for analysis of many samples, for instance as a useful tool in grading programmes. In the mid-1990s, there were more reports on non-destructive quality determination of salmon by use of NIR spectroscopy. Isaksson et al. (1995) compared measurements made by fibre-optic diffuse NIR spectroscopy and traditional reflectance NIR spectroscopy. The spectroscopic readings were made to determine fat, moisture and protein in salmon fillets. They found higher prediction errors by applying intact muscle then when using homogenised muscle for measurements. Downey (1996) also applied a fibre-optic probe for the analysis of fat and moisture in farmed salmon. Spectra were recorded through skin and scales at several measurement locations along the dorsal and ventral site of the intact fish. Here it was pointed out that the trade-off of the reduced accuracy when measuring through skin and scales would be the efficiency of the method as well as the advantage of not having to disrupt the samples. An example of a fibre-optic measurement probe is shown in Figure 5.4. Identifying the most suitable measurement location for NIR fat determination was one important issue in the work of Wold and co-workers in 1996. For correlation with chemically measured fat content of the whole fish, the best correlation and lowest error of prediction was obtained by selecting a measurement location in the dorsal region just behind the dorsal fin. They also demonstrated that a subset of nine wavelengths would provide sufficient prediction of the average fat content in the fish. One year later, Wold and Isaksson (1997) presented the use of a transmission probe for the assessment of fat and moisture in whole Atlantic salmon. The method was found viable for screening purposes, classifying salmon into five groups ranging from very low in fat to very high in fat. Solberg et al. (2003) reported on fat determination in live salmon by the use of noninvasive NIR techniques. Two different types of NIR instrumentation were applied. One system used a fibre-optic probe and one instrument measured reflectance. Both systems proved efficient in determining the crude fat content of live, anaesthetised salmon. Analysis was performed within 3 seconds by the reflection system, whereas the fibre-optic system was seven times slower. Salmon and trout seem to have been the most applied species when exploring the feasibility of NIR spectroscopy to determine chemical constituents. One likely reason for this is the commercial impact of these species. The ability to use NIR in registration of nutritional quality has, however, also been reported for other fish species. Nortvedt et al. (1998) used NIR transmission in the range 850–1150 nm to assess fat, protein and dry matter in Atlantic halibut fillet. Measurements were done on minced fillet samples. The best correlation was obtained between crude fat and transmission measurements.

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Figure 5.4 A fibre-optic measurement probe guides the light onto the sample. After the light has interacted with the sample, it is guided back to the spectrograph. Photograph by Frank Gregersen, Nofima Marine, Norway.

Analysis of chemical constituents in capelin by NIR was demonstrated by Solberg and Fredriksen (2003). Transmission measurements were applied on minced capelin muscle to estimate fat and dry matter. Shimamoto et al. (2003) performed non-destructive NIR analysis of frozen skipjack. The fat content of the skipjack was determined both by a portable NIR instrument as well as by a desktop instrument. Both devices provided results with relatively low error of prediction. Fat analysis made by the desktop instrument gave a prediction error of 1.58% whereas the portable instrument provided the result with an error of 1.06%, hence in this study the portable instrument proved to be more accurate than the desktop version. In two recent reports (Vogt et al. 2002; Nielsen et al. 2005) NIR spectroscopy was compared with other measurement methods to find the most convenient method for fat determination in herring. Fatmeter measurements, microwave drying, a modified chemical extraction method, NMR spectroscopy and NIR spectroscopy were all compared with traditional chemical extraction methods as references. In both studies, NIR spectroscopy proved

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to be the most useful method for speed, simplicity of performance and accuracy in the reference method. The spectroscopic readings were also considered most relevant for sorting large quantities of herring in commercial production. NIR spectroscopy has not only been applied for the analysis of raw, frozen or thawed fish. There are several reports on analyses of processed fish and fish products as well. Cured and smoked products have been investigated by NIR spectroscopy. Huang and colleagues (2001) demonstrated how shortwave NIR spectroscopy was useful in predicting the salt and moisture content of cured salmon roe. The sensitivity of the method was said to be limited, but it was still considered a convenient way of non-destructive quality determination. Likewise, salt and moisture contents were determined non-destructively in cold smoked Atlantic salmon (Huang et al. 2002). In two further works, Huang and co-workers performed NIR analysis on cured Atlantic salmon (Huang et al. 2003) as well as on commercial king salmon and chum salmon (Lin et al. 2003). In these studies, measurements were performed by use of NIR spectroscopy in the range 600–1100 nm. Measurements were made by a fibre-optic measurement probe, locating the probe onto the sample material and leaving no disruption. It was possible to predict the salt and/or water content of the samples with acceptable correlation and error levels. The selected wavelength range is interesting in terms of commercial instrumentation for analytical purposes. Instruments with components in this wavelength range tend to be less expensive than instruments with sensitivity in at higher wavelengths. Another application related to salt and curing was suggested by Svensson et al. (2004). NIR reflectance measurements in the range 1000–2500 nm were applied to assess the protein content of brine from barrel-salted herring. The protein content of the brine could be used as an indicator of the progress of ripening process of the salted herring, and so the NIR recordings were an indirect means of determining the quality of the salted product. The latest application for measurements of constituents in salt processed fish is the work by Wold et al. (2006). They introduced near-infrared imaging as a means of representative sampling in the determination of moisture content in salted saithe. Using NIR in combination with imaging of the whole coalfish, it was possible to obtain information on the water distribution in the samples. They also demonstrated that the application of remote transflectance is an attractive method for non-contact measurements of heterogeneous fish samples. In 2004, a study on NIR for quality evaluation of the Greek dish taramosalata was published (Adamopoulos and Goula 2004). Taramosalata is a traditional dish made from dried and salted roe of grey mullet, cod or tuna. In the aforementioned study, reflectance spectroscopy was used to assess moisture, fat and protein of taramosalata, and the authors recommended this method to replace traditional and time-consuming chemical methods in product analyses. An additional refined fish product analysed by NIR spectroscopy is surimi. Uddin et al. (2006) demonstrated how protein and water content of surimi could be determined by non-destructive NIR spectroscopy. Correlations between NIR and chemical measurements were high and prediction errors low, making NIR spectroscopy a potential method for fast and non-destructive analyses of surimi. NIR spectroscopy has also been applied for the analysis of fish oils. Zhang and Lee (1997) used NIR to assess free fatty acids (FFA) in fish oil. Prediction of FFA in mackerel fat could be related to change in mackerel quality, and so this measurement was a means of assessing mackerel quality. Two manuscripts from 2005 give a further illustration of the usefulness of NIR spectroscopy for fish oil analysis. Endo et al. (2005) showed that NIR spectroscopy could be applied to determine iodine and saponification values of the oil. The oils analysed

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in this work were from orange roughy, sardine, tuna, cod and squid. NIR spectroscopy proved to be speedier and require less sample preparation than the titration reference methods. Cozzolino et al. (2005) attempted to correlate NIR measurements of fish oils with free fatty acids, moisture, peroxide value (PV) and anisidine value (AV). The analyses proved successful for the two former parameters, whereas the results for AV and PV showed poor accuracy. The results, however, showed that NIR spectroscopy could be applied to express fish oil degradation.

5.4

Freshness and storage time

One important quality parameter of fish is freshness, and most consumers consider freshness a positive attribute. Documenting fish freshness is not, however, a straightforward task. Deterioration of fresh caught fish is due to chemical and microbiological processes. Many of the traditional chemical or microbiological methods measuring freshness are sensitive in the latter phase of degradation and hence not very useful in terms of freshness grading of the just-caught product. Ólafsdóttir et al. (1997) suggested that light spectroscopy might be a tool to aid in determining fish freshness. Shortly after, NIR spectroscopy was, for the first time, reported as a method for determining fish freshness by Sigernes et al. (1998). In this study, both transmission and transflectance measurements were used, the latter a fibre-optic probe. The measurements were performed on few samples, but the results indicated that NIR correlated well with storage time in ice, and storage time could be predicted with an error of less than 30 hours (see Figure 5.5). Nilsen and colleagues (2002) maintained the work on freshness assessment of ice-stored cod and included freshness assessment of salmon by NIR spectroscopy. Here, the applied spectral range was from 400 to 1100 nm. The measurements were made on intact fillets with a fibre-optic transflection probe. This work concluded that the fillet area best suited for

20

Measured Y

15

Elements: Slope: Offset: Correlation: RMSEP: SEP: Bias:

50 0.979500 0.097524 0.954631 1.152834 1.164135 0.030366

10

5 0 0 5 –5 RESULT1, (Y-var, PC): (Y,5)

10

15

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Predicted Y Figure 5.5 Prediction plot for cod freshness (days in ice) based on VIS/NIR transflectance spectroscopy. The root-mean-square error of prediction is 1.15 days. From Sigernes et al. (1998).

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making these measurements was the upper range of the loin. It was also found that prediction of cod freshness was best made by use of the visible region of the spectrum, whereas for salmon the NIR range gave the better prediction. Storage time in ice for cod was predicted with an error of 1.04 days, and for salmon the prediction error was slightly higher: 1.20 days. Bøknæs and co-workers (2002) made a study on thawed-chilled cod fillets packed in modified atmosphere, stored at 2 °C. The aim of the work was to evaluate the potential of NIR spectroscopy to assess freshness of thawed-chilled fillets as well as frozen storage temperature and frozen storage period. The potential of NIR to obtain information of water holding capacity, drip loss and content of dimethylamine was also evaluated. NIR measurements were made in reflectance mode on minced samples. The spectroscopic measurements correlated rather well with the storage time of the chilled fillets, which was predicted with an error of 3.4 days. The NIR measurements could also be correlated with drip loss. For the other parameters investigated, there was found to be no correlation with NIR measurements. Both Bøknæs et al. (2002) and Nilsen et al. (2002) stated that NIR spectroscopy was a promising method for freshness determination; however, development of a method for freshness assessment based on NIR spectroscopy should be based on studies including variation such as season, origin, species and size. Heia et al. (2003) conducted freshness measurements on ice-stored cod as well as on chilled-thawed hake. They applied a handheld transmission spectrometer operating in the range from 500 to 650 nm on skinned fillets. The storage time of cod was predicted with an error of 23 hours by this instrumentation. The trial on chilled hake was made on relatively few samples, and as found by Bøknæs et al. (2002) also, this study showed good correlation between chilled storage period and spectroscopic measurements. Interestingly, Heia et al. (2003) also found VIS spectroscopy useful for predicting storage time of frozen hake. The error value of prediction was 1.6 months. Pursuing the idea of freshness models based on VIS/NIR spectroscopy Nilsen and Esaiassen (2005) presented a study where the sensory score of ice-stored cod was predicted by use of transflection measurements in the visible region. The sensory tool applied was the quality index method (QIM) (Olafsdottir et al. 1997; Martinsdottir et al. 2003). It was shown that the QIM score of cod could be predicted from visible spectroscopy with an error value of 2.6 QI scores. The cod applied in this work were obtained from both a gill net and a longline catch, and it was shown that one prediction model including both types of sample was sufficient to predict QIM score. The article shows that the spectroscopic method complies with freshness as described by a sensory tool, which may be considered more informative than addressed as storage time in ice. The most recent report on fish freshness determination by NIR spectroscopy was made by Lin and co-workers (2006). They found that shortwave NIR spectroscopy could be applied to detect and monitor microbiological spoilage in rainbow trout. They operated a transflection probe in the wavelength range 600–1100 nm and used minced fillet as well as intact fillets in the study. The minced samples were stored at 21 °C and measured at 0, 2, 4, 6, 8, 10, 12 and 24 hours, respectively; the intact fillet samples were stored at 4 °C and sampled daily for a period of 8 days. Microbial load was measured as total viable count. The best results were obtained by transflection measurements on intact fillet, giving an error of prediction of 0.53 log cfu/g. This result is most interesting in terms of documenting freshness and degree of spoilage in a rapid and non-intrusive way.

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5.5

Authentication

A food-quality issue with increasing focus in the past decade is food authenticity and adulteration. Along with these issues came the need for methods to verify or authenticate that the product fulfils claimed properties in terms of origin or processing. The review of Reid et al. (2006) gave an updated overview of the analytical tools available for authentication, among them NIR spectroscopy. Until now, there have been just a few papers discussing NIR spectroscopy as a possible tool for the authentication of fish. Xiccato et al. (2004) performed a study where the aim was to predict the origin of European sea bass as well as the chemical composition by NIR reflectance spectroscopy. The origin of samples was diversified according to different rearing systems: extensive ponds, semi-intensive ponds, intensive concrete tanks and intensive sea-cages. Analysis was performed on intact fillet, minced muscle and freeze-dried minced muscle. The latter sample preparation proved to be the most suitable for correct classification of rearing system based on the NIR data. The authors concluded that NIR spectroscopy was a promising measurement method both for chemical characterisation of sea bass and authentication. In the work of Uddin and Okazaki (2004), it was pointed out that the fresh versus frozen fish may be subject to adulteration. The main concern is presentation of frozen/thawed fish as fresh, and in this respect giving incorrect information to the buyer of the product. Their study showed the potential of NIR reflectance spectroscopy to differentiate between fresh and thawed fish. Dried drip juice extracts from thawed and fresh horse mackerel was analysed by reflectance spectroscopy in the 1100–2500 nm range. The spectral readings from fresh and thawed samples enabled 100% correct classification according to the temperature history of the samples. It has also been shown that differentiation between fresh and thawed fish can be made by non-destructive NIR measurements (Uddin et al. 2005b). A fibre-optic measurement probe was applied on the skin of fresh and frozen/thawed red sea bream. In this work, all samples were correctly classified according to fresh or thawed origin. It was argued that the reason for this was due to changes in texture as a function of freezing and thawing. Hence NIR spectroscopy provided a feasible easy-to-use tool to differente between fresh and frozen-thawed fish.

5.6

Safety

There are just a few published papers addressing NIR spectroscopy for safety determination in fish for human consumption. One application is the possibility to determine the heating temperature of the food product. Thermal processing is one important hurdle to provide food safety, and so verification of the appropriate temperature treatment is an important issue in food safety. Uddin et al. (2002, 2005a, 2006b) have demonstrated the usefulness of NIR spectroscopy for fish analysis in this area. In their work from 2002, it was shown that NIR reflectance spectroscopy correlated strongly with the end-point temperature of five different fish species: blue marlin, skipjack, red sea bream, kuruma prawn and scallop. In more recent studies, they demonstrated that the technique could be applied to kamaboko gels and other fish-meat gels (Uddin et al. 2005a, 2006a).

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Figure 5.6 Outline of nematode detection. Left image shows a digital image of the front part of a cod fillet with five nematodes. Here the nematodes are indicated with needle pins. In the right image, the classification result with detected nematodes (encircled) is shown. In this example, four out of five nematodes were detected.

Combining VIS/NIR spectroscopy with imaging has given new possibilities for fish quality determination. The technique enables detection and documentation of quality parameters not always easily spotted or recognised upon visual inspection of the fish. One such matter is the detection of parasites in fish fillets. Presence of parasites in fish for human consumption is considered a safety issue if the fish is not exposed to appropriate temperature treatment sufficient to kill them (Audicana et al. 2002). To eliminate this issue, the parasites should therefore be removed from the fish. Parasites in fish fillets have proved to be a challenge for instrument detection at industrial speed (Wold et al. 2001). Wold and co-workers (2001) presented a method of detection based on multispectral imaging in the VIS and NIR range. With this method it was possible to identify parasites on the fillet surface and down to 6 mm into the muscle, and the technology was suggested viable for online implementation. Another approach to this issue has been demonstrated by Heia et al. (2006). They applied imaging spectroscopy in the range 350–950 nm to detect parasites in cod fillets. Neither method complied with the speed requirements of industrial production, but rapid development in instrumentation and computational speed makes these results interesting for the fillet-processing industry. Figure 5.6 shows an example of parasite detection. Based on the analytical results, it will be possible to remove the parasites or the infected regions of the fillet.

5.7 Other quality parameters It has long been known that NIR spectra reflect physical parameters such as texture or particle size in powders (Osborne and Fearn 1986). Also, in fish quality analysis, there are examples of NIR applied to determine physical quality aspects. Bechmann and Jørgensen

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(1998) demonstrated the use of NIR reflectance spectroscopy to assess quality parameters in whole frozen-thawed cod. It was found that spectral recordings on the skin of the cod correlated reasonably well with the water-holding capacity. Another example of NIR used to determine physical quality properties was given by Isaksson et al. (2001). This work showed how NIR spectra could be used to predict the muscle texture of salmon in terms of Kramer shear-force measurements. The NIR measurements were made non-destructively by a fibre-optic probe, and the authors recommended such measurements for rough classification of salmon before sale or processing. A recent study by Nilsen et al. (2006) presented results where NIR spectroscopy was used to identify quality flaws in stockfish (gutted and outdoor dried cod). Some of the defects caused textural changes in the fish, and could thus be identified by NIR spectroscopy. Lin et al. (2003) demonstrated the usefulness of visible and short-wavelength NIR for the non-destructive detection of bruises in Pacific pink salmon. Spectral measurements were made on the skin side of the fish using a fibre-optic probe. Consecutively, the fish was filleted and imaged by a digital camera. The images were then analysed to identify the location of bruises, and this information was applied as reference for the spectral recordings. The authors concluded that the method could be relevant in the aquatic industry, but further studies would be necessary to evaluate the feasibility of the technique for other species. NIR assessment of other physical parameters such as density, refractive index and brix was demonstrated by Ritthiruangdej et al. (2005). The analysis was made on Thai fish sauce. In addition to the physical parameters, an estimate of total nitrogen content and pH was made. The study included an evaluation of which wavelength regions were the most useful for predicting the different parameters. In another application, NIR was used to predict sensory quality criteria for five different fish species. Warm et al. (2001) made a study where NIR measurements on whole fish and fish fillets were correlated with sensory attributes of the cooked fish. NIR predictions for appearance and texture were better than for odour and taste. It was suggested that NIR measurements could be applied as a rapid supplementary tool in monitoring of sensory parameters in fish.

5.8

Summary and future perspectives

As seen from the above, the main activity within VIS/NIR spectroscopy and fish quality determination has been in assessment of chemical constituents. It is interesting to see that within this group of highly heterogeneous set of sample materials, the spectroscopic measurements apply very well in identifying several different quality issues. The spectral recordings comprise information about both chemical and physical attributes to be revealed upon data analysis. However, development of the spectroscopic methods relies on the validity and accuracy of the methods used as reference, so future development also requires development in methods used for analytical reference. Another feature that makes the spectroscopic measurement so attractive is the speed and development of tools that, to a great extent, have enabled non-intrusive and non-destructive sampling. The fact that the sample material can be further processed after VIS/NIR measurements makes the technique a viable tool for at- or on-line analysis. Until now, there have not been very many applications on safety and authentication issues. Seen in view of the simplicity of VIS/NIR as an analytical technique,

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as well as the need for rapid documentation within the field of food safety and authentication, we believe published results on these issues will increase. With this review we did not intend to give the details of all the reported work. Nor did we give a complete report of all the work done within the field of light spectroscopy and quality assessment of fish. However, we did mean to give an overview of the possibilities of VIS/NIR spectroscopy for fish quality analysis. Hopefully, this review may also trigger some ideas for new applications as well as the future realisation of this technology.

5.9

References

Adamopoulos, K.G. and Goula, A.M. (2004) Application of near-infrared reflectance spectroscopy in the determination of major components in taramosalata. Journal of Food Engineering 63: 199–207. Audicana, M.T., Ansotegui, I.J., de Corres, L.F. and Kennedy, M.W. (2002) Anisakis simplex: dangerous – dead and alive? Trends in Parasitology 18(1): 20–25. Barnes, R.J., Dhanoa, M.S. and Lister, S.J. (1989) Standard normal variate transformation and de-trending of near-infrared diffuse reflectance spectra. Applied Spectroscopy 43(5): 772–777. Bechmann, I.B. and Jørgensen, B.M. (1998) Rapid assessment of quality parameters for frozen cod using near infrared spectroscopy. Lebensmittel-Wissenschaft Und -Technologie 31: 648–652. Bøknæs, N., Jensen, K.N., Andersen, C.M. and Martens, H. (2002) Freshness assessment of thawed and chilled cod fillets packed in modified atmosphere using near-infrared spectroscopy. Lebensmittel-Wissenschaft Und -Technologie 35(7): 648–652. Cozzolino, D., Murray, I., Chree, A. and Scaife, J.R. (2005) Multivariate determination of free fatty acids and moisture in fish oils by partial least-squares regression and near-infrared spectroscopy. Lebensmittel-Wissenschaft Und -Technologie 38: 821–828. Darwish, G.S., van de Voort, F.R. and Smith, J.P. (1989) Proximate analysis of fish tissue by midinfrared transmission spectroscopy. Canadian Journal of Fisheries and Aquatic Sciences 46: 644–649. Downey, G. (1996) Non-invasive and non-destructive percutaneous analysis of farmed salmon flesh by near infra-red spectroscopy. Food Chemistry 55(3): 305–311. Dufour, É., Frencia, J.P. and Kane, E. (2003) Development of a rapid method based on front-face fluorescence spectroscopy for the monitoring of fish freshness. Food Research International 36: 415–423. Endo, Y., Tagiri-Endo, M. and Kimura, K. (2005) Rapid determination of iodine value and saponification value of fish oils by near-infrared spectroscopy. Journal of Food Science 70(2): C127–C131. Geladi, P., MacDougall, D. and Martens, H. (1985) Linearization and scatter-correction for near-infrared reflectance spectra of meat. Applied Spectroscopy 39(3): 491–500. Gjerde, B. and Martens, H. (1987) Predicting carcass composition of rainbow trout by near-infrared reflectance spectroscopy. Zeitschrift für Tierzuchtung und Zuchtungsbiologie 104(1–2): 137–148. Griffiths, P.R. (1995) Practical consequences of math pre-treatment of near infrared reflectance data: log(1/R) vs F(R). Journal of Near Infrared Spectroscopy 3: 60–62. Heia, K., Esaiassen, M., Nilsen, H. and Sigernes, F. (2003) Visible spectroscopy – evaluation of storage time of ice stored cod and frozen hake. In: J.B. Luten, J. Oehlenschläger and G. Ólafsdóttir (Eds.) Quality of Fish from Catch to Consumer. Labelling, Monitoring and Traceability. Wageningen Academic Publishers, The Netherlands, pp. 201–209. Heia, K., Sivertsen, A.H., Stormo, S.K., Elvevoll, E., Wold, J.P. and Nilsen, H. (2007) Detection of nematodes in cod (Gadus morhua) fillets by imaging spectroscopy. Journal of Food Science 72(1): E11–E15.

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Herrala, E. and Okkonen, J. (1996) Imaging spectrograph and camera solutions for industrial applications. International Journal of Pattern Recognition and Artificial Intelligence 10: 43–54. Huang, Y., Rogers, T.M., Wenz, M.A., Cavinato, A.G., Mayes, D.M., Bledsoe, G.E. and Rasco, B.A. (2001) Detection of sodium chloride in cured salmon roe by SW-NIR spectroscopy. Journal of Agricultural Food Chemistry 49: 4161–4167. Huang, Y., Cavinato, A.G., Mayes, D.M., Bledsoe, G.E. and Rasco, B.A. (2002) Nondestructive prediction of moisture and sodium chloride in cold smoked Atlantic salmon (Salmo salar). Journal of Food Science 67(7): 2543–2547. Huang, Y., Cavinato, A.G., Mayes, D.M., Kangas, L.J., Bledsoe, G.E. and Rasco, B.A. (2003) Nondestructive determination of moisture and sodium chloride in cured Atlantic salmon (Salmo salar) (teijin) using short-wavelength near-infrared spectroscopy (SW-NIR). Journal of Food Science 68(2): 482–486. Hyvarinen, T.S., Herrala, E. and Dall’Ava, A. (1998) Direct sight imaging spectrograph: a unique add–in component brings spectral imaging to industrial applications. In: G.M. WilliamsJr (Ed.) Digital Solid State Cameras: Designs and Applications (Proc. SPIE 3302) pp. 165–175. Isaksson, T., Tøgersen, G., Iversen, A. and Hildrum, K.I. (1995) Non-destructive determination of fat, moisture and protein in salmon fillets by use of near-infrared diffuse spectroscopy. Journal of the Science of Food and Agriculture 69: 95–100. Isaksson, T., Swensen, L.P., Taylor, R.G., Fjæra, S.O. and Skjervold, P.O. (2001) Non-destructive texture analysis of farmed Atlantic salmon using visual/near-infrared reflectance spectroscopy. Journal of the Science of Food and Agriculture 82: 53–60. Lee, M.H., Cavinato, D. and Rasco, B.A. (1992) Noninvasive short-wavelength near-infrared spectroscopic method to estimate the crude lipid content in the muscle of intact rainbow trout. Journal of Agricultural and Food Chemistry 40: 2176–2181. Lin, M., Cavinato, A.G., Huang, Y. and Rasco, B.A. (2003) Predicting sodium chloride content in commercial king (Oncorhynchus tshawytscha) and chum (O. keta) hot smoked salmon fillet portions by short-wavelength near-infrared (SW-NIR) spectroscopy. Food Research International 36: 761–766. Lin, M., Cavinato, A.G., Mayes, D.M., Smiley, S., Huang, Y., Al-Holy, M. and Rasco, B.A. (2003) Bruise detection in Pacific pink salmon (Oncorhynchus gorbuscha) by visible and short-wavelength near-infrared (SW-NIR) spectroscopy (600–1100 nm). Agricultural and Food Chemistry 51: 6404–6408. Lin, M., Mousavi, M., Al-Holy, M., Cavinato, A.G. and Rasco, B.A. (2006) Rapid near infrared spectroscopic method for the detection of spoilage in rainbow trout (Oncorhynchus mykiss) fillet. Journal of Food Science 71(1): s18–s23. Martens, H. and Næs, T. (1989) Mulitvariate Calibration. John Wiley & Sons Ltd. pp. 419. Martinsdóttir, E., Luten, J.B., Schelvis-Smit, R. and Hyldig, G. (2003) Developments of QIM – past and future. In: J.B. Luten, J. Oehlenschläger and G. Ólafsdóttir (Eds.) Quality of Fish from Catch to Consumer. Labelling, Monitoring and Traceability. Wageningen Academic Publishers, The Netherlands, pp. 265–272. Mathias, J.A., Williams, P.C. and Sobering, D.C. (1987) The determination of lipid and protein in freshwater fish using near-infrared reflectance spectroscopy. Aquaculture 61: 303–311. McLure, W.F. (1993) More on derivatives: part 1. Segments, gaps and ‘ghosts’. NIR News 4(6): 12–12. McLure, W.F. (1994) More on derivatives: part 2. Band shiftings and noise. NIR News 5(1): 12–14. Nielsen, D., Hyldig, G., Nielsen, J. and Nielsen, H.H. (2005) Lipid content in herring (Clupea harengus L.) – influence of biological factors and comparison of different methods of analysis: solvent extraction, Fatmeter, NIR and NMR. Lebensmittel-Wissenschaft Und -Technologie 38: 537–548. Nilsen, H. and Esaiassen, M. (2005) Predicting sensory score of cod (Gadus morhua) from visible spectroscopy. Lebensmittel-Wissenschaft Und -Technologie 38: 95–99.

VIS/NIR spectroscopy

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Nilsen, H., Sivertsen, A., Joensen, S., Bjørkevoll, I. and Heia, K. (2006) Instrumental quality control of stockfish. In: J.B. Luten, C. Jacobsen, K. Bekaert, A. Sæbø and J. Oehlenschläger (Eds) Seafood Research from Fish to Dish. Quality, Safety And Processing of Wild and Farmed Fish. Wageningen Academic Publishers, The Netherlands, pp. 509–517. Nilsen, H., Esaiassen, M., Heia, K., & Sigernes, F. (2002) Visible / near-infrared spectroscopy – a new tool for the evaluation of fish freshness? Journal of Food Science 67(5): 1821– 1826. Nortvedt, R., Torrisen, O.J. and Tuene, S. (1998) Application of near-infrared transmittance spectroscopy in the determination of fat, protein and dry matter in Atlantic halibut fillet. Chemometrics and Intelligent Laboratory Systems 42: 199–207. Ólafsdóttir, G., Martinsdóttir, E., Oehlenschläger, J., Dalgaard, P., Jensen, B., Undeland, I., Mackie, I.M., Henehan, G., Nielsen, J. and Nilsen, H. (1997) Methods to evaluate fish freshness in research and industry. Trends in Food Science and Technology 8(8): 258–265. Osborne, B.G. and Fearn, T. (1986) Near Infrared Spectroscopy in Food Analysis. Longman Scientific & Technical, Harlow, Essex, UK, p. 200. Pasquini, C. (2003) Near infrared spectroscopy: fundamentals, practical aspects and analytical applications. Journal of the Brazilian Chemical Society 14(2): 198–219. Rasco, B.A., Miller, C.E. and King, T.L. (1991) Utilization of NIR spectroscopy to estimate the proximate composition of trout muscle with minimal sample pretreatment. Journal of Agricultural and Food Chemistry 39(1): 67–72. Reid, L.M., O’Donnell, C.P. and Downey, G. (2006) Recent technological advances for the determination of food authenticity. Trends in Food Science & Technology 17: 344–353. Ritthiruangdej, P., Kasemsumran, S., Suwonsichon, T., Haruthaithanasan, V., Thanapase, W. and Ozaki, Y. (2005) Determination of total nitrogen content, pH, density, refractive index and brix in Thai fish sauces and their classification by near-infrared spectroscopy with searching combination moving window partial least squares. Analyst 130: 1439–1445. Shimamoto, J., Hiratsuka, S., Hasegawa, K., Sato, M. and Kawano, S. (2003) Rapid non-destructive determination of fat content in frozen skipjack using a portable near infrared spectrophotometer. Fisheries Science 69: 856–860. Sigernes, F., Esaiassen, M., Heia, K., Wold, J.P., Eilertsen, G. and Sørensen, N.K. (1998) Assessment of fish (cod) freshness by VIS/NIR spectroscopy. In: G. Olafsdóttir, J. Luten, P. Dalgaard, M. Careche, V. Verrez-Bagnis, E. Martinsdóttir and K. Heia (Eds) Methods to Evaluate Fish Freshness in Research and Industry. International Institute of Refrigeration (IIR), Paris, pp. 369–375. Solberg, C. (1992) Near infrared spectroscopy of fish samples. In K. Hildrum, T. Isaksson, T. Naes and A. Tandberg (Eds) Near Infra-Red Spectroscopy. Bridging the Gap between Data Analysis and NIR Applications, Ellis Horwood Series in Analytical Chemistry. Ellis Horwood Limited, Chichester, UK, pp. 223–227. Solberg, C. and Fredriksen, G. (2001) Analysis of fat and dry matter in capelin by near infrared transmission spectroscopy. Journal of Near Infrared Spectroscopy 9: 221–228. Solberg, C., Saugen, E., Swenson, L.P., Brun, L. and Isaksson, T. (2003) Determination of fat in live farmed Atlantic salmon using non-invasive NIR techniques. Journal of the Science of Food and Agriculture 83: 692–696. Sollid, H. and Solberg, C. (1992) Salmon fat content estimation by near infrared transmission spectroscopy. Journal of Food Science 57(3): 792–793. Svensson, V.T., Nielsen, H.H. and Bro, R. (2004) Determination of the protein content in brine from salted herring using near-infrared spectroscopy. Lebensmittel-Wissenschaft Und -Technologie 37: 803–809. Uddin, M. and Okazaki, E. (2004) Classification of fresh and frozen-thawed fish by near-infrared spectroscopy. Journal of Food Science 69(8): C665–C668.

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Uddin, M., Ishizaki, S., Okazaki, E. and Tanaka, M. (2002) Near-infrared reflectance spectroscopy for determining end-point temperature of heated fish and shellfish meats. Journal of the Science of Food and Agriculture 82: 286–292. Uddin, M., Okazaki, E., Ahmad, M.U., Fukuda, Y. and Tanaka, M. (2005a) Noninvasive NIR spectroscopy to verify endpoint temperature of kamaboko gel. Lebensmittel-Wissenschaft Und -Technologie 38: 809–814. Uddin, M., Okazaki, E., Turza, S., Yumiko, Y., Tanaka, M. and Fukuda, Y. (2005b) Non-destructive visible/NIR spectroscopy for differentiation of fresh and frozen-thawed fish. Journal of Food Science 70(8): C506–C510. Uddin, M., Okazaki, E., Fukushima, H., Turza, S., Yumiko, Y. and Fukuda, Y. (2006a) Nondestructive determination of water and protein in surimi by near-infrared spectroscopy. Food Chemistry 96: 491–495. Uddin, M., Okazaki, E., Ahmad, M.U., Fukuda, Y. and Tanaka, M. (2006b) NIR spectroscopy: a nondestructive fast technique to verify heat treatment of fish-meat gel. Food Control 17: 660–664. Vogt, A., Gormley, T.R., Downey, G. and Somers, J. (2002) A comparision of selected rapid methods for fat measurement in fresh herring (Clupea harengus). Journal of Food Composition and Analysis 15: 205–215. Warm, K., Martens, H., Nielsen, J. and Martens, M. (2001) Sensory quality criteria for five fish species predicted from near-infrared (NIR) reflectance measurements. Journal of Food Quality 24(5): 389–403. Wold, J.P. and Isaksson, T. (1997) Non-destructive determination of fat and moisture in whole Atlantic salmon by near-infrared diffuse spectroscopy. Journal of Food Science 62(4): 734–736. Wold, J.P., Jakobsen, T. and Krane, L. (1996) Atlantic salmon average fat content estimated by nearinfrared transmittance spectroscopy. Journal of Food Science 61(1): 74–77. Wold, J.P., Westad, F. and Heia, K. (2001) Detection of parasites in cod fillets by using SIMCA classification in multispectral images in the visible and NIR region. Applied Spectroscopy 55(8): 1025–1034. Wold, J.P., Johansen, I.R., Haugholt, K.H., Tschudi, J., Thielemann, J., Segtnan, V.H., Narum, B. and Wold, E. (2006) Non-contact transflectance near infrared imaging for representative on-line sampling of dried salted coalfish (bacalao). Journal of Near Infrared Spectroscopy 14: 59–66. Xiccato, G., Trocino, A., Tulli, F. and Tibaldi, E. (2004) Prediction of chemical composition and origin identification of European sea bass (Dicentrarchus labrax L.) by near infrared reflectance spectroscopy (NIRS). Food Chemistry 86: 275–281. Zhang, H.Z. and Lee, T.C. (1997) Rapid near-infrared spectroscopic method for the determination of free fatty acid in fish and its application in fish quality assessment. Journal of Agricultural Food Chemistry 45: 3515–3521.

Chapter 6

Electronic nose and electronic tongue Corrado Di Natale and Gudrun Ólafsdóttir

6.1 Introduction on electronic nose and olfaction The analysis of odours, namely the investigation of gaseous samples composed of volatile compounds, is a typical subject of analytical chemistry where several methods are available to separate mixtures in individual compounds (for example gas chromatography). To analyse a gaseous mixture without separation, a set of selective sensors can be applied where each sensor senses only one of the many molecular species. Actually, many solid-state sensors developed since the 1970s are intrinsically non-selective, making them not useful for the goal in analytical chemistry of simultaneously measuring the concentration of many different compounds. The non-selectivity of chemical sensors was considered one of the main problems limiting their practical application. The role of solid-state chemical sensors changes when compared with the properties of natural olfaction. In the past two decades, our understanding of the physiology of olfaction has made considerable advances, models of receptors mechanisms explaining the sensitivity to volatile compounds are now available and the genes expressed by olfactive receptors are known (Buck and Axel 1991). Recent studies have also indicated how to unveil the signal pathways leading from the generation of olfactory neuron signals to the conscious identification of odours (Friedrich and Stopfer 2001). Nonetheless, olfaction remains a mysterious sense because of its strong connection with unconscious perception, which corresponds with an unusual scarcity of semantic expressions limiting the communication of olfactive experiences. Compared with other senses (vision, hearing, and touch), for which several technological devices exist, attempts to endow artificial systems with odour recognition features have been thwarted for a long time. The principle of sample separation has been considered very differently from natural olfaction, where the odour interacts at once with the globality of receptors. Nonetheless, recent studies have shown that both the mucus and the shape of the turbinates have a property of separating airborne chemicals according to their chemical and physical properties (Stitzel et al. 2003). Physiological investigations about olfaction receptors have shown that Nature’s strategy for odour recognition is rather different from the approach of analytical chemistry. Receptors were found to be rather unselective: each receptor senses several kinds of molecule and each molecule is sensed by many receptors (Sicard and Holley 1984). After this discovery, Persaud proposed that arrays of non-selective chemical sensors may show properties similar 105

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to that of natural olfaction (Persaud and Dodds 1982). After Persaud’s conjecture, artificial olfaction systems were developed which were soon dubbed ‘electronic noses’. This denomination is currently given to any array of unselective chemical sensors coupled with some multicomponent classifier. Since the 1980s, almost all sensor technologies have been used to build such systems. Odour classification properties of artificial systems were tested on several different fields, proving that electronic noses could in principle be used to replace human olfaction in practical applications such as food quality and medical diagnosis (Pearce et al. 2003). The same principle of the electronic nose was also applied to sensors working in environments for the classification of liquids (Hayashi et al. 1990; Di Natale et al. 1996b): by analogy with electronic noses, arrays of sensors working in liquids were called ‘electronic tongues’. Apart from a few attempts to use optical sensors (Lavigne et al. 1998) and quartz microbalances (Hauptmann et al. 2000), most of the electronic tongues so far reported have been based either on potentiometric (Legin et al. 1999) or amperometric sensors (Winquist et al. 1997), both radicated in electrochemistry. The sense of taste in mammalians is organised similarly to olfaction. Taste is perceived by non-specific taste buds, situated on the papillae of the tongue. Conventionally, overall taste is correlated with a combination of four basic tastes: sweetness, sourness, bitterness and saltiness, in addition to taste sensations like umami, metallic and astringency, and spicy and cooling effects. Because taste and odour are often perceived simultaneously, the term ‘flavour’ is widely used to describe their combination, especially when speaking about food. The relationship between taste (flavour) and chemical composition is often not known precisely, especially for sweet substances. Another interesting and highly controversial issue is the interaction between different tastes (Stewart and Amerine 1973). In most cases, a desensitising effect or threshold increase takes place when two substances eliciting different tastes are present simultaneously. A further effect is the decrease in sensitivity threshold when substances present at non-perceptible concentrations can be felt, if a contrasting taste substance is applied to the tongue. Perception thresholds of the human tongue to most taste substances are much higher than those for olfaction, with exception of alkaloids, such as quinine. However, differential taste and odour thresholds are comparable. Thus, the mammalian sense of taste function is similar to olfaction but is less developed, possibly because it is less related to the survival of living beings. It has to be noted that the physiology of the taste is completely different to that of olfaction. As an example, taste receptors in mammals are not neurons but cells that generate action potentials and release neurotransmitters in response to taste cues. The activity is revealed and transmitted by neurons innervating taste buds (Scott 2005). The other aspect of taste is the impression obtained when food enters the mouth. The basic taste is then merged with the information from the olfactory receptor cells, when aroma from the food enters the nasal cavities through the inner passage. This merged sensory experience is referred to as the descriptive taste by sensory panels.

6.2

Application of the electronic nose and electronic tongue

More than 10,000 odorous compounds are known to exist in nature, but only a few of these are likely to be important in solving any discrimination task by an electronic nose. The

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electronic nose will not necessarily detect the most odorous compounds, but it may give a robust pattern based on a response to volatile compounds that is indicative of the quality changes. The application of electronic noses and tongues as tools to monitor freshness or quality of various foods was proposed when these instruments were first developed, and gained increasing interest over the years. Since the early 1990s several commercial instruments have been launched on the market for this purpose, but there were some drawbacks to their immediate use in industry. The main reason was that the instruments were not fully developed, and problems with their sensitivities to humidity and environmental conditions led to misinterpretation of their performance. Active research in this area has since focused on better characterising better the overall technique. Improvements in the selectivity, sensitivity and reproducibility of the gas sensors have been the key issues. Identification of other important factors not directly related to the development of the sensor technologies have been brought into focus. These include sampling techniques and data analysis, which are essential parts of the overall technique (Mielle 1996; Haugen and Kvaal 1998; Gardner and Bartlett 1999; Mielle and Marquis 1999; Haugen 2001). The key principles involved in the standard electronic nose concept are: the transfer of the total headspace of a sample to a sensor array that detects the presence of volatile compounds in the headspace; a pattern of signals is provided that are dependent on the sensors’ selectivity and sensitivity; and the characteristics of the volatile compounds in the headspace (Gardner and Bartlett 1999). Feature extraction and pre-processing of the data are essential steps before applying the pattern recognition techniques that are required to interpret the sensors’ signals characterising the samples. The qualitative discriminatory power of the electronic nose has a resemblance to the subjective discrimination of odours by the human nose. The common approach in multipurpose commercial electronic noses is to characterise odours by generating patterns with a sensor array using numerous sensors with partly overlapping selectivity, as discussed before. Another approach is the use of highly selective sensors for specific indicator compounds. For both approaches, a basic understanding of the composition and chemistry of the volatile compounds being measured is a key factor to ensure meaningful evaluation of the sensor responses. For quantitative analysis, a few sensors from the array can give adequate information, given that the selectivities of the sensors cover the different classes of compounds relevant for the particular application. It is unlikely that a universal electronic nose, able to solve all odour detection problems, will become a commercial reality, particularly because creating sensor diversity within an instrument is expensive and most instruments are dedicated to certain applications. Numerous electronic nose instruments based on different types of sensor, sampling system and data analysis procedure have been developed. Active research in recent years on different types of sensor has resulted in commercial gas sensors with dedicated approaches for a wide range of applications, from quality control of various food products to medical diagnosis (Craven et al. 1996; Pearce et al. 2003). Electronic noses have been suggested for various applications related to quality evaluation of different foods, like monitoring freshness and the onset of spoilage or bioprocesses. Many of these applications are based on detecting volatile compounds produced by the growth of fungi, moulds or microbes, or changes occurring in food because of oxidation (Olafsson et al. 1992; Jonsson et al. 1997; Namdev et al. 1998; Schnürer et al. 1999; McEntegart et al. 2000; Keshri et al. 2002; Boothe and Arnold 2002; Olsson et al. 2002). Other applications for electronic noses for food have been suggested, for example quality

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assessment and monitoring of ripening in cheese (Trihaas and Nielsen 2005; Trihaas et al. 2005) and in various other fields like medical applications, the pharmaceutical industry, cosmetics, environmental monitoring, flavour and fragrances, and monitoring tainting in packaging materials. The range of application of electronic tongues depends largely on the characteristics of the sensors. The most important feature of the electronic tongue compared with the electronic nose is that the measured quantity is the sample itself, not its volatile part. Electrochemical sensors require samples in the form of aqueous solutions, but the use of other sensor technologies could in principle remove this limitation. Nonetheless, until now almost all samples analysed by electronic tongues have been electrolytic solutions,and the capability of electronic tongues to classify samples of food and beverages according to their quality and to identify different stages of industrial processes or depuration of water has been abundantly demonstrated in recent years (Toko 2000; Vlasov et al. 2005). In principle, living beings are embedded in an electrolytic solution. Nonetheless, electrochemical measurement conditions are found only over short dimensions, for instance where individual cell properties are measured but the overall properties of the whole sample are not accessible. Therefore, at the scale necessary to define the quality of fish or meat, the sample may be considered a solid. The applicability of the electronic tongue to the analysis of muscle food was demonstrated on fish (Legin et al. 2002) and pork liver (Legin et al. 2001). In both cases, an array of potentiometric sensors was used. In the case of fish, the measurements were made in a homogenate prepared by stirring chopped fish with distilled water. It was found that the system was capable of distinguishing between a sample of freshwater fish and two samples of marine fish. The measurements were performed on fish samples that had been stored in a freezer and at room temperature. The electronic tongue could detect and monitor fish spoilage.

6.3

Colorimetric techniques, optical equipment and consumer electronics

Although known for several years (Tozawa et al. 1971), the colorimetric detection of fish freshness has recently received renewed interest. In particular, the importance of amines as spoilage markers led to the consideration of their reducing role and the possibility of detecting them with functional layers sensitive to pH changes. Paquit et al. (2006) recently demonstrated the feasibility of this approach using a film of a sodium salt (bromocresol green) as the sensitive layer. This salt exhibits a rather large change in colour that is also appreciable by eye. Nonetheless, this method is rather limited because only amines are considered (so limiting the detection not to freshness but rather to spoilage). Furthermore, visual determinations have limited performance and may greatly vary between individuals. Chemical sensing based on optically sensitive layers is a captivating strategy because of the strong influence of target chemicals on the absorption and fluorescence spectra of chosen indicators. Nonetheless, the chemical practice of this approach is weakened by the transducer counterpart. Indeed, traditional optical instrumentation of high quality is usually bulky and expensive. On the other hand, in the past decade we have seen rapid growth in performance in fields such as consumer electronics, giving rise to several low-cost advanced optical technologies

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such as digital scanners, cameras and screens, whose characteristics largely fit the requirements necessary to capture the change in optical properties of sensitive layers in many practical applications. 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 colour changes in chemical dyes due to the adsorption of volatile compounds (Rakow and Suslick 2000). Furthermore, the group of Lundström and Filippini proved that it is possible to assemble a type of spectrophotometer using a computer screen monitor as a programmable source and a webcam as a detector (Filippini et al. 2003). This last technique, known as computer screen photo-assisted technique (CSPT) is based on the fact that a computer screen can be easily programmed to display millions of colours, combining wavelengths in the optical range. Compared with the use of digital scanners, to probe the sample with a variable combination of wavelengths instead of using the white light of scanners, makes possibile an optical fingerprint measurement allowing a simultaneous evaluation of absorbance and fluorescence of samples. Owing to the large diffusion of portable computers, personal digital assistants (PDAs), and cellular phones, all endowed with colour screen, camera and even more extended computation capabilities, the application of the CSPT concept may be foreseen as greatly extending analytical capacity worldwide. CSPT has demonstrated its use particularly in classifying airborne chemicals, reading absorbance and fluorescence changes in chemical dyes such as metalloporphyrins (Filippini et al. 2006). Standard opto-chemical sensors are based either on absorbance or on fluorescence, whereas CSPT gives the possibility of evaluating both effects at the same time. A first application of a CSPT-based electronic nose formed by an array of metalloporphyrins for measuring fish freshness has been recently demonstrated with the spoilage in icestored thawed fish and fresh anchovies (Alimelli et al. 2007).

6.4

Classification of fish odours

The odour of fresh fish is one of the most important quality parameters determining whether fish is acceptable for direct human consumption. The concept of classifying compounds based on their structural and odour characteristics is the basis for the interpretation of electronic nose measurements. It is therefore of interest to analyse the composition of the headspace of different fish products during storage and to identify quality indicators present in high enough concentrations so that they can be detected by electronic nose sensors. The information on the identity and quantity of volatile compounds present in the headspace during storage of fish is essential when selecting sensors in an array for quality monitoring. Moreover, it is important to know the sensitivity of the sensors towards key compounds in the sample for indicating the quality. The gas sensor array can then be designed based on the selectivity and sensitivity of the sensors to the quality-indicating compounds. Descriptions of characteristic odours and odour changes caused mainly by microbial growth in chilled fish during storage are well documented. Sensory schemes based on these attributes have been developed to evaluate spoilage changes and the shelf life of fish. The composition of volatile compounds in fish contributing to the characteristic odours can be determined and related to the quality (Ólafsdóttir and Fleurence 1998). Volatile compounds that are most important to monitoring freshness and spoilage of chilled fish can be divided

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Fish odour

Fresh fish odour

Microbial spoilage odour

Oxidised odour

planty, cucumber and mushroom-like odours

sweet, fruity, ammoniacal, sulphur, and putrid odours

cod liver-oil paint-like

C6–C9 alcohols and carbonyl compounds i.e. 1,5-octadien-3-ol, 2,6-nonadienal

i.e. ethanol, 3-methyl-1butanol, ethylacetate, ammonia, TMA, hydrogen sulphide methyl mercaptan

i.e. hexanal 2,4-heptadienal 2,4,7-decatrienal

Figure 6.1 Odour of fish and volatile compounds contributing to the characteristic odour of fresh, spoiled and oxidised fish. (From Ólafsdóttir et al. 1997.)

into three groups based on their origin, as illustrated in Figure 6.1. These groups are fresh fish odours, microbial spoilage odours and oxidised odours.

6.4.1

Fresh fish odours and oxidised odours

Fish odours are complex, and each species has a characteristic aroma. Concentration of influential compounds and their odour thresholds are important factors. Some compounds are desirable at low levels (parts per billion), like enzymically derived unsaturated carbonyl compounds and alcohols with six, eight or nine carbon atoms that have low odour thresholds and exhibit characteristic fresh-, plant-, cucumber-, melon- and mushroom-like odours in both freshwater and marine species (Josephson et al. 1983, 1984, 1986; Hirano et al. 1992; Milo and Grosch 1993). These compounds are derived from polyunsaturated fatty acids that are susceptible to auto-oxidation during prolonged storage. When accumulated in high levels they contribute to oxidised, rancid and fishy odours (Josephson 1991). In general, speciesrelated odour compounds in fresh fish are present in very low levels (micrograms per kilogram) in the headspace, and it is unlikely that electronic nose techniques will detect these compounds in the total headspace of fish during storage, although lower molecular mass compounds derived from autoxidation like propanal and hexanal may be detected.

6.4.2

Microbial spoilage odours

Microbial degradation of fish components, mainly amino acids and non-protein nitrogenous substances like trimethylamine oxide (TMAO), results in the formation of spoilage odours of fish. Ammonia, dimethylamine (DMA), trimethylamine (TMA), ethanol (which spoilage odour has ethanol?), hydrogen sulphide, methyl mercaptan and sulphides are typical spoilage compounds that exhibit odours such as fishy, stale, rotten and putrid, and are present in the headspace above fish during spoilage at the micrograms per kilogram level.

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120

Peak area ratio (PAR)

100 80

alcohols

60

ketones

40

acetic acid

aldehydes TMA esters

20 0 0

2

4 6 8 10 12 14 Days of storage

Figure 6.2 GC–MS analysis of volatile compounds showing changes in the levels (PAR: peak area ratio) of the main classes of compound contributing to spoilage in cod fillets packed in Styrofoam boxes during storage at 0.5 °C until sensory rejection on day 12. (Adapted from Ólafsdóttir et al. 2005)

The spoilage pattern in chilled fish is characterised by the specific spoilage organisms present. Proliferation of the microflora depends on initial handling and storage conditions (temperature and packaging). As a consequence, the combination and amount of microbial metabolites in the headspace of chilled products varies depending on which bacterium is the dominating specific spoilage organism (Ólafsdóttir 2005). The concentration of microbially formed compounds increases with time as the fish spoils, and it can be used as index of spoilage (Lindsay et al. 1986; Ólafsdóttir 2003, 2005). Single compounds like TMA and a combination of compounds, mainly alcohols, amines and sulphur, have both been suggested as indicators for freshness and spoilage (Lerke and Huck 1977; Human and Khayat 1981; Hebard et al. 1982; Ahmed and Matches 1983; Kelleher and Zall 1983; Josephson et al. 1986; Lindsay et al. 1986; Oehlenschläger 1992; Jörgensen et al. 2000; Alasalvar et al. 2005).

6.5 Quality indicators in fish during chilled storage: gas chromatography analysis of volatile compounds To demonstrate the dynamic evolution of volatile compounds during storage of fish, an example is given from a storage study of cod fillets (Ólafsdóttir et al. 2005). Volatile compounds were monitored by gas chromatography to screen potential quality indicators during chilled storage. Identification of volatile compounds based on gas chromatography–mass spectrometry (GC–MS) and quantification of the main classes of compounds based on the sum of the peak area ratio (PAR) for respective compounds in each class were done for cod fillets packed in Styrofoam boxes during chilled storage (0.5 °C). When the fillets were rejected by sensory analysis on day 12 of storage, ketones were detected at the highest level, followed by amines (TMA), alcohols, aldehydes and acids, and a low level of esters (Figure 6.2). Selective

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sensors for detecting these classes of compound were suggested for monitoring quality changes to cod fillets during storage. The detection of sulphur compounds would be useful only when the products are spoiled.

6.5.1

Loss of freshness formation of stale odours: alcohols and carbonyls

The initial increase in alcohol levels is caused mainly by the production of ethanol. Its formation has been related to the utilisation of carbohydrate sources, whereas the formation of branched-chain alcohols and aldehydes like 2-methyl-1-propanol, 3-methyl-1-butanol and 3-methyl-butanal probably originates from degradation of valine and leucine, respectively. The flavour thresholds of the alcohols are high compared with the carbonyls, and they do not contribute to the odour of the fillets as evaluated by gas chromatography olfactometry (GC-O) (Table 6.1). The branched chain aldehyde 3-methyl butanal is characterised by a malty flavour and caramel-like odours (Ólafsdóttir et al. 2005). The malty flavour of 3methyl butanal was suggested by Milo and Grosch (1996) as being mainly responsible for the malty off-flavour defect of boiled cod. The corresponding alcohols 3-methyl-1-butanol and 2-methyl-1-propanol, which exhibit alcoholic and fruity odours, were found to increase during storage of cod fillets (Ólafsdóttir et al. 2005). Levels of ketones, mainly acetoin, increased earlier than TMA in chilled cod fillets packed in Styrofoam boxes. The concentration of acetoin was much higher than the other lipidderived ketones detected, like 2-butanone, 3-pentanone and the carotenoid-derived 6methyl-5-heptene-2-one, that were present in cod fillets throughout storage, but no obvious increase occurred until at the end of shelf life and during continued storage. Ketones can influence the overall odour because of their typical odours and their low odour thresholds (Table 6.1). Lipid-derived aldehydes, like hexanal, nonanal and decanal, were detected in similar levels throughout the storage time and contributed to the overall sweet odours of cod fillets in combination with other carbonyls (3-hydroxy-2-butanone, acetaldehyde, 2-butanone, 3pentanone and 6-methyl-5-heptene-2-one). Aldehydes have generally low odour thresholds and therefore their odour impact was greater than the alcohols and the ketones although their overall levels were less (Table 6.1). The formation of alcohols and carbonyls contributes to loss of freshness and onset of stale spoilage odours. Pre-concentration techniques are necessary for the analysis of the unsaturated aldehydes, which is not practical for rapid determination of oxidation. On the other hand, it is possible to detect the most volatile oxidation products like propanal and hexanal by rapid, static headspace sampling methods Boyd et al. (1992). 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.

6.5.2

Spoilage odours: amines, acids, esters and sulphur compounds

The development of amines during fish spoilage is well known, and measurements of the very volatile amines such as TMA or total volatile basic nitrogen (TVBN) have been used

Table 6.1 Quality indicators in cod fillets: main classes of quality-indicating compounds, PAR on day 12 at sensory rejection and odour analysis of compounds identified in cod fillets packed in Styrofoam boxes during storage at 0.5°C. (From: Ólafsdóttir 2005.) Quality indicators Class Alcohols

Aldehydes

Acids Amines Esters Sulphur compounds

a c

15%

3%

33%

4% 27% <1%

Compounds ethanol 2-methyl -1-propanol 1-penten-3-ol 3-methyl-1-butanol 2-methyl-1-butanol 2,3-butandiol 2-ethyl-1-hexanol acetaldehyde 3-methyl-butanal hexanal heptanal octanal nonanal decanal undecanal 2-butanone 3-pentanone 3-hydroxy-2-butanone 6-methyl-5-hepten-2-one acetic acid TMA ethyl acetate ethyl butanoate dimethyl sulphide dimethyl disulphide dimethyl trisulphide

PAR Day 12 – 35.0 ± 2.8 1.2 ± 0.0 8.5 ± 1.0 – 4.0 ± 4.4 1.8 ± 0.2 0.8 ± 0.5 1.3 ± 0.2 2.0 ± 0.1 0.7 ± 0.2 3.7 ± 0.6 2.4 ± 0.3 0.4 ± 0.1 – 13.6 ± 6.2 95.3 ± 5.6 1.0 ± 0.1 14.2 ± 13.0 91.6 ± 28.5 0.6 – – – –

Odour scoreb

Odour descriptionc

1.5–3.0

sweet, caramel, fish fillet – earthy, boiled potato – – fresh, floral sweet, candy – sweet, caramel sweet, sour spicy, flowery – TMA-like, dried fish – sickenly sweet, vomit – onion-like rotten, sulphur, cabbage

2.0–3.0

1.5 1.5 1.5–2.0 1.5–2.0 1.5 3.0 2.3 1.5–2.5 2.5

PAR: peak area ratio based on comparison with an internal standard (GC–MS analysis); b Range of odour score in samples during storage, increasing with time (GC-O); based on GC-O analysis (two panelists)

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Ketones

%PARa Day 12

113

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in the fish industry as indicators of quality. Development of ammonia-like and ‘fishy’ off-flavours has been related to specific fish spoilage organisms that can reduce TMAO to TMA. Enzymically produced DMA (dimethylamine), which forms very early after harvest of fish, has been suggested as a freshness indicator along with its precursor TMAO (trimethylamine oxide) (Oehlenschläger 1992). TMA and TVBN values are only useful for advanced spoilage because they only begin to increase at later stages of storage (Oehlenschläger 1998; Baixas-Nogueras et al. 2003). Odour of saltwater species has often been related to the odour of TMA; however, the combination of TMA and other amines, acids and sulphur compounds (such as ammonia, DMA, acetic, formic and propionic acids, hydrogen sulphide and methyl mercaptan) also contribute to saltwater fish spoilage odour rather than TMA alone. The presence of ethyl acetate and low levels of sulphur compounds contributed to the sensory rejection. The volatile sulphur compounds hydrogen sulphide, methyl mercaptan, methyl sulphide and dimethyl disulphide have been suggested as the main cause of putrid spoilage aromas in fish (Herbert et al. 1971). The origin of the sulphur compounds is from microbial degradation of cysteine and methionine to form hydrogen sulphide and methyl mercaptan, respectively (Herbert et al. 1975). Dimethyl trisulphide has also been associated with spoilage in fish (Miller et al. 1973a,b; Lindsay et al. 1986). Milo and Grosch (1996) evaluated the headspace of boiled cod by GC-O and found that dimethyl trisulphide was the most potent odorant contributing to off-odours in cod formed when the raw material was inappropriately stored. During advanced spoilage of fish, the concentration of microbially produced sulphur compounds increases and they tend to dominate the putrid spoilage aroma, although the amines, acids and aromatics in addition give the complete putrid spoilage aromas of fish. Acetic acid was detected in increasing concentrations in cod fillets during chilled storage (Ólafsdóttir et al. 2005). Although alcohols, aldehydes, ketones, acids, sulphur compounds, esters and acids are primarily of interest as spoilage indicators of fish, other classes of compound may also have an impact when measuring the total headspace with electronic noses. Phenol and phenethanol have been found as the major high boiling volatile compounds in haddock during storage (Chen et al. 1974) derived from phenylalanine. Various benzene derivatives have been reported as a part of the total volatiles in chilled fish (Alasalvar et al. 2005). The concentration of the straight chain alkanes (nonane, decane and undecane) appeared to be similar throughout storage in chilled cod fillets. Additionally, numerous branched chain alkanes were detected. The alkanes will not influence the responses of the electrochemical sensors of the electronic nose and are not considered of interest as quality indicators because they are not aroma active.

6.6

Application of the electronic nose for evaluation of fish freshness

In recent years, attempts to use electronic nose technology to track the spoilage processes occurring in fish have been reported in numerous papers. Most of these are feasibility studies, showing the ability of the electronic nose to discriminate between different spoilage levels or storage times of samples. Instruments based on different sensor technologies have been used like metal-oxide chemoresistor sensors (Egashira et al. 1990; Ohashi et al. 1991; Ólafsson et al. 1992; Ólafsdóttir et al. 2005b; Haugen et al. 2006), MOSFET sensors

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(Haugen and Undeland 2003), amperometric sensors (Schweizer-Berberich et al. 1994; Ólafsdóttir et al. 1997b,c; 1998, 2000, 2002; Jónsdóttir et al. 2004), conducting polymer sensors (Luzuriaga and Balaban 1999a,b; Newman et al. 1999; Du et al. 2001, 2002) and quartz microbalance sensors (Di Natale et al. 1996a, 2001; Zhao et al. 2002). Classical chemical methods for the analysis of TVBN and TMA are commonly used for freshness determination of fish, along with sensory and microbial analysis. For accurate evaluation of quality, no single measurement can encompass all the complex changes occurring during spoilage of fish. One means of overcoming this is by developing an instrument that measures a set of attributes that together can give a better estimation of freshness or quality than with one technique alone (Ólafsdóttir et al. 1997a). The possibility of developing a multi-sensor device to measure and/or estimate fish freshness with a combination of instrumental techniques (electronic noses, spectroscopic methods, texture-meters, image analysers, colour meters and devices measuring electrical properties) was investigated in a multinational research project funded by the European Union called ‘Development of Multi-Sensor Techniques for Monitoring the Quality of Fish’ (Di Natale 2003; Ólafsdóttir et al. 2004). In fact, the electronic nose technique by itself is a multisensor approach to evaluate volatile compounds that are developed by different microflora and biochemical and oxidative processes that influence the spoilage odour in fish. Selected standard compounds that are representative of the main classes of compounds causing spoilage odour have been analysed by the electronic nose FreshSense (Ólafsdóttir et al. 1998, 2002). The results showed that the electrochemical gas sensors (Dräger, Germany: CO, SO2; City Technology, UK: NH3) in the FreshSense had different selectivity and sensitivity towards the compounds selected from these classes. The CO sensor was sensitive towards alcohols, aldehydes and esters, the NH3 sensor detected amines like TMA and ammonia and the SO2 sensor detected volatile sulphides. Therefore, the main classes of spoilage indicator compounds present in the headspace can be estimated based on the individual sensor responses. The FreshSense instrument developed in Iceland has been used for freshness monitoring of various fish species like haddock, capelin, redfish and cod that were handled and stored under different conditions. A similar trend in the responses of the electronic nose and the development of volatile compounds was observed for different fish species (Ólafsdóttir et al. 1997a,b, 1998, 2000, 2002, 2005b, 2006a,b; DiNatale et al. 2001). Many studies have stressed the importance of comparing the electronic nose responses to traditional analysis of volatile compounds by gas chromatography. An example of the dynamic evolution of volatile compounds during storage of cod fillets is illustrated in Figure 6.2, showing that ketones and TMA were most abundant. Comparison of the development of the key indicator compounds (acetoin and TMA) analysed by gas chromatography, the FreshSense electronic nose sensors (CO and NH3) and chemical measurement of TVBN is shown in Figure 6.3. The CO sensor was useful for detecting incipient spoilage because response increased significantly between days 7 and 10 and was found to increase rapidly in parallel with the increase in the pH value after day 10 of storage (Figure 6.3). The change in the pH reflecting the autolytic and the microbial degradation processes was not continuous with time. The increasing response of the electronic nose’s CO sensor was explained by the increasing level of alcohols during storage like ethanol, 2-methyl-1-propanol, 3-methyl-1butanol and 2,3-butandiol, in addition to the presence of aldehydes and the formation of esters at the end of the shelf life (Table 6.1). The early detection of alcohols by the CO sensor is of importance for monitoring incipient changes in quality during storage.

Fishery Products: Quality, safety and authenticity 450

7.0 TMA TVB-N CO sensor NH3 sensor acetoin pH

400

PAR–TVB-N sensors

350 300

6.9 6.8 6.7

250 6.6

pH

116

200 6.5 150 6.4

100

6.3

50

6.2

0 0

2

4

6 8 10 12 Days from catch

14 16

Figure 6.3 Response of the electronic nose CO and NH3 sensor GC–MS analysis of TMA and acetoin (PAR), TVB-N and pH in chilled cod fillets packed in Styrofoam boxes during storage at 0.5 °C (From Ólafsdóttir 2005.)

6.7

Combined electronic noses for estimating fish freshness

One of the most interesting applications of electronic noses is their capability of estimating the human perception of airborne chemicals: namely the odour. Odour plays an important role in any sensory analysis scheme applied to fish. In particular, in this case the estimation of the odour score in the frame of the quality index method (QIM) (Luten and Martinsdottir 1998) is illustrated. To provide the reader with data from different sensor technologies, in this example the use of two electronic noses is given. The contemporaneous use in electronic noses of sensors based on different principles and technologies has been proposed as a methodology to improve class separation in many applications. The simplest way to fuse data from different instruments consists of joining together the data matrices of each instrument. In this way, a sort of supra-matrix is formed with several columns equal to the sum of variables of the instruments. This strategy was adopted, for instance, to integrate the data of two electronic noses in the measurement of fish freshness (Di Natale et al. 2001). Herewith a more sophisticated methodology of data fusion based on the outer product is used (Ólafsdóttir et al. 2003). This technique has been introduced in analytical chemistry to study the correlation between different spectral techniques applied to the same samples (Barros et al. 1997). The outer product of two vectors X and Y is a mathematical operation, also known as Cartesian product, consisting in the set of all possible ordered pairs whose first component is a member of X and whose second component is a member of Y. In practice, because electronic nose data are arranged as vectors, the outer product combines the signals of the sensors of one electronic nose with the signals of the sensors of another. Then, for each measurement, a matrix is obtained (Burdick 1995). Given a set of measurements,

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3 QIM mean smell attributes

Acetic acid, sulphuric, very sour (QIM=3)

Yeast, bread, beer, sour milk (QIM=2)

2

1 Neutral, grassy, musty (QIM=1)

Fresh, seaweedy, metallic (QIM=0)

0 0

2

4

6

8 10 12 Storage days

14

16

18

Figure 6.4 QIM mean scores for odour attributes versus the days of storage at 0 °C and description of odours for QIM scores. (From Ólafsdóttir et al. 2003.)

a three-dimensional data structure is generated. Different chemometric techniques are available to analyse tri-dimensional data structures and may be used both for qualitative (classification) and quantitative analysis (Gurden et al. 2001). In this example, the FreshSense electronic nose introduced in the previous section is complemented by the LibraNose. The latter is an electronic nose designed and fabricated at the University of Rome Tor Vergata based on an array of eight thickness shear mode resonators coated with various kinds of metalloporphyrin. These molecules are investigated as receptors of artificial sensor systems (Di Natale et al. 2006). Eight samples per storage day were measured in a total of 72 fishes. The measurements were performed on fillets. For each fish the right side fillet was measured, and the other side reserved for experiments not described here. Fillets were prepared about 1 hour before the analysis and were held constantly on an ice-bed until measured. During the experiment, the bone side of the right fillets was measured for each fish. Here the performance of each electronic nose and that of the joined data sets are evaluated to estimate the smell attribute of fishes as reported by the panel. In QIM, cod smell is divided into three classes corresponding to three different stages of freshness. In the experiment described here, each fish was checked by seven assessors, and the mean of their single evaluation were considered as the final score. Figure 6.4 shows the behaviour of the mean smell attribute with the number of storage days at 0 °C and the odour descriptors for each score. Although a monotonic behaviour with storage time is found, the plot shows three distinct slopes corresponding to three different batches of fish. This shows the well-known dependence of the spoilage processes from the initial conditions. Figures 6.5 and 6.6 show the scatter plot related to the performance of partial least squares models aimed at retrieving the QIM smell attribute from LibraNose and FreshSense data respectively. The data fusion of the sets produced by the two instruments was performed by the analysis of the outer product. Figure 6.7 shows estimated versus true QIM smell attribute evaluated

118

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Measured Y(:,1)

2.5

2

15

17

11771 7

15 1 5 111555

111111 1 1 1

17 17

1 51 5 1 1 11 1

1.5 9 9

1

7 4 4 4

44

99 7 77

3333 33

3

22

2

7

77

9

9

4 4

9

7

0.5

0

22

2

2 2

1 11 11 1 1

0

0.5

1

1.5 2 Predicted Y(:,1)

2.5

3

Figure 6.5 LibraNose performance is shown as a scatter plot of predicted versus measured QIM smell attributes. Estimations were evaluated by a partial least squares model. Numbers indicate storage time in days (From Ólafsdóttir et al. 2003.)

3.5 3 17

Measured Y(:,1)

2.5

1 71 7

2

1 7 1 71 7

1 15 51 1 55 1 51 5 1 5

17

17

15

1 1 1 11111 1 1111

1.5 9

9 9 99

1

9

77777 4 4 4444 3

99

77

33333 3

0.5 2 22222 2

0 0

1 1111 1 1

0.5

1

1.5 2 Predicted Y(:,1)

2.5

3

3.5

Figure 6.6 Scatter plot of FreshSense predicted versus measured QIM smell attributes. Estimations were evaluated by a partial least squares model. Numbers indicate storage time in days (From Ólafsdóttir et al. 2003.)

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3 1 7 1177 1 7

2.5

Measured Y(:,1)

2

17

1 71 71 7

1 5 1 51 51151 5155 1 5 1 11 11 1 1 1

1 1 1 11 1

1.5 9

1

99

9

7 7 77 77 44 4 4 4 3

3

3 33

9 9 9 444

9

7

3

0.5 2 2 222

0 –0.5 –0.5

1

1 11

0

1

2 22

1

0.5

1 1.5 Predicted Y(:,1)

2

2.5

3

Figure 6.7 Scatter plot from a partial least squares model of the unfolded outer product matrices. A net improvement respect to Figures 6.5 and 6.6 is observed. Numbers indicate storage time in days. (From Ólafsdóttir et al. 2003.)

by a partial least squares model of the unfolded outer product matrices. The improvement compared with Figures 6.5 and 6.6 is self-evident. The increase in performance can be numerically expressed through the root mean square errors calculated in calibration and validation (RMSEC and RMSECV, respectively) value. The combined electronic noses achieve 0.27 and 0.30 errors of calibration and validation. The validation errors achieved by each electronic nose were 0.57 and 0.45 for LibraNose and FreshSense, respectively. It is worth mentioning that the whole scale for odour attribution in the QIM goes from 0 to 4. In conclusion, the use of different sensor technologies increases the overall sensitivity, augmenting the information collected from the sample. In particular, in this example the possibility of retrieving the odour index of a fish from instruments has been shown. This feature, although not yet implemented in practice, is of great importance because it allows the possibility of providing with an instrument the same evaluation as a trained human panel (Di Natale 2003; Ólafsdóttir et al. 2004; Macagnano et al. 2005).

6.8 Conclusions and future outlook In the past 20 years, a growing number of researchers have been involved in the design, fabrication and application of electronic noses. The capabilities of this approach to discriminate samples, in different fields, according to their composition have been demonstrated in hundreds of diverse applications and published in several scientific journals. However, this technology is still not completely ready to produce practical equipment for end users. The reasons for this apparent failure can be found in a non-balanced development of the technology,

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which neglected in some cases very important topics about sampling of the headspace and conditioning of the sample. In practice, companies developing electronic noses have concentrated their efforts in building general-purpose equipment that may be useful in laboratories but they have not developed specific applications aimed at solving specific problems. Nonetheless, each step of the food chain has particular needs that an electronic nose approach can, in principle, satisfy. As examples, at producer level the increment of quality and yield, at processor level the screening of quality of incoming products or raw materials to optimise processing and to sort processed food, and finally at consumer level the control of quality and safety both on the market and at home. All these applications require instrumentation that is able to work on-site, avoiding environmental disturbances. As an example, food processing sites are usually highly contaminated from the point of view of odour. On the other hand, there are certainly applications, interesting at the industrial level, where existing electronic noses can be specialised, in terms of sampling and data presentations, to fulfil user requirements. For these developments, strong co-operation between electronic nose producers and end-users is necessary in order to optimise practical solutions. At this level, it is important to obtain correct and careful analysis of user needs and expectations, and to educate the users in order to disseminate the intrinsic novelty carried by the artificial olfaction machines. Finally, it is important to consider that the currently available electronic noses are still based on an oversimplified olfaction model that takes into consideration very little of the complexity of natural olfaction. The next generation of electronic noses is being devised, which include more biological similitudes such as sample separation in some media resembling the function of nasal mucus, or introducing novel data analysis based on biological paradigms. Furthermore, the introduction of transducers based on consumer electronics may greatly help the process of capillary diffusion of electronic noses, simply embedding the sensors into yet existing and used technology, such as computers or cellular phones.

6.9

References

Ahmed, A. and Matches, J.R. (1983) Alcohol production by fish spoilage bacteria. Journal of Food Protection 46: 1055–1059. Alasalvar, C., Taylor, K.D.A. and Shahidi, F. (2005) Comparison of volatiles of cultured and wild sea bream (Sparus aurata) during storage in ice by dynamic headspace analysis gas chromatography mass spectrometry. Journal of Agricultural and Food Chemistry 53: 2616–2622. Alimelli, A., Pennazza, G., Santonico, M., Paolesse, R., Filippini, D., D’Amico, A., Lundström, I. and Di Natale, C. (2007) Fish freshness detection by a computer screen photoassisted based gas sensor array. Analytica Chimica Acta 582(2): 320–328. Baixas-Nogueras, S., Bover-Cid, S., Veciana-Nogues, M.T. and Vidal-Carou, M.C. (2003) Suitability of volatile amines as freshness indexes for iced Mediterranean hake. Journal of Food Science 68: 1607–1610. Barros, A.S., Safar, M., Devaux, M.F., Bertrand, D. and Rutledge, D.N. (1997) Relations between mid-infrared and near-infrared spectra detected by analysis of variance of an intervariable data matrix. Applied Spectroscopy 51: 1384. Boothe, A.A.J. and Arnold, J.W. (2002) Electronic nose analysis of volatile compounds from poultry meat samples, fresh and after refrigerated storage. Journal of the Science of Food and Agriculture 82(3): 315–322.

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Boyd, L.C., King, M.F. and Sheldon, B. (1992) A rapid method for determining the oxidation of n−3 fatty acids. Journal of the American Oil Chemists’ Society 69(4): 325–330. Buck, L. and Axel, R. (1991) A novel multigene family may encode odourant receptors: a molecular basis for odour recognition. Cell 65: 175–187. Burdick, D.S. (1995) An introduction to tensor products with applications to multiway data analysis. Chemometrics and Intelligent Laboratory Systems 28: 229. Chen, T.C., Nawar, W.W. and Levin, R.E. (1974) Identification of major high-boiling volatile compounds produced during refrigerated storage of haddock fillets. Applied Microbiology 28(4): 679–680. Craven, M.A., Gardner, J.W. and Bartlett, P.N. (1996) Electronic noses development and future prospects. Trends in Analytical Chemistry 15(9): 486–493. Di Natale, C. (2003) Data fusion in MUSTEC: towards the definition of an artificial quality index. In: J.B. Luten, J. Oehlenschläger and G. Ólafsdóttir (Eds) Quality of Fish from Catch to Consumer: Labeling, Monitoring and Traceability. Wageningen Academic Publishers, The Netherlands, pp. 273–280. Di Natale, C., Brunink, J.A.J., Bungaro, F., Davide, F., d’Amico, A., Paolesse, R., Boschi, T., Faccio, M. and Ferri, G. (1996a) Recognition of fish storage time by a metalloporphyrin-coated QMB sensor array. Measurement Science and Technology 7: 1103–1114. Di Natale, C., Davide, F., Brunink, J., D’Amico, A., Vlasov, Yu., Legin, A. and Rudnitskaya, A. (1996b) Multicomponent analysis of heavy metal cations and inorganic anions in liquids by a non-selective chalcogenide glass sensor array. Sensors and Actuators B 34: 539–542. Di Natale, C., Ólafsdóttir, G., Einarsson, S., Mantini, A., Martinelli, E., Paolesse, R., Falconi, C. and D’Amico, A. (2001) Comparison and integration of different electronic noses for the evaluation of freshness of cod fish fillets. Sensors and Actuators B 77: 572–578. Di Natale, C., Paolesse, R. and D’Amico, A. (2006) Metalloporphyrins based artificial olfactory receptors. Sensors and Actuators B 121(1): 238–246. Du, W.X., Kim, J., Huang, T.S., Marshall, M.R. and Wei, C.I. (2001) Microbiological, sensory, and electronic nose evaluations of yellowfin tuna under various storage conditions. Journal of Food Protection 64(12): 2027–2036. Du, W.X., Lin, C.M., Huang, T.S., Kim, J., Marshall, M.R. and Wei, C.L. (2002) Potential application of the electronic nose for quality assessment of salmon fillets under various storage conditions. Journal of Food Science 67(1): 307–313. Egashira, M., Shimizu, Y. and Takao, Y. (1990) Trimethylamine sensor based on semiconductive metaloxides for detection of fish freshness. Sensors and Actuators B– 1(1–6): 108–112. Filippini, D., Svensson, S. and Lundström, I. (2003) Computer screen as a programmable light source for visible absorption characterization of (bio)chemical assays. Chemical Communications 2003: 240–241. Filippini, D., Alimelli, A., Di Natale, C., Paolesse, R., D’Amico, A. and Lundström, I. (2006) Chemical sensing with familiar devices. Angewandte Chemie International Edition 45: 3800–3803. Friedrich, R.W. and Stopfer, M. (2001) Recent dynamics in olfactory population coding. Current Opinion in Neurobiology 11468–11474. Gardner, J.W. and Bartlett, P.N. (1999) Electronic Noses. Principles and Applications. Oxford, Oxford University Press. Gurden, S.P., Westerhuis, J.A., Bro, R. and Smilde, A.K. (2001) A comparison of multiway regression and scaling methods. Chemometrics and Intelligent Laboratory Systems 59: 121. Haugen, J.E. (2001) Electronic noses in food analysis. In: R.L. Rouseff and K.R. Cadwallader (Eds) Headspace Analysis of Food and Flavors: Theory and Practice. Kluwer Academic/Plenium Publishers, New York, pp. 43–57. Haugen, J.E. and Kvaal, K. (1998) Electronic nose and artificial neural network. Meat Science 49: 273–286.

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Haugen, J.E. and Undeland, I. (2003) Lipid oxidation in herring fillets (Clupea harengus) during ice storage measured by a commercial hybrid gas-sensor array system. Journal of Agricultural and Food Chemistry 51(3): 752–759. Haugen, J.E., Chanie, E., Westad, F., Jonsdottir, R., Bazzo, S., Labreche, S., Marcq, P., Lundby, F. and Ólafsdóttir, G. (2006) Rapid control of smoked Atlantic salmon quality by electronic nose: correlation with classical evaluation methods. Sensors and Actuators B 116: 72–77. Hautpmann, P., Auge, J., Borngraber, R. and Schroder, J. (2000) Application of novel sensor electronics for quartz resonators in artificial tongue. In: Proceedings of the 2000 IEEE/EIA International Frequency Control Symposium, Kansas City (USA), pp. 100–105. Hayashi, H., Yamanaka, M., Toko, K. and Yamafuji, K. (1990) Multichannel taste sensor using lipid membranes. Sensors and Actuators B 2: 205–213. Hebard, C.E., Flick, G.J. and Martin, R.E. (1982) Occurrence and significance of trimethylamine oxide and its derivatives in fish and shellfish. In: R.E. Martin (Ed.) Chemistry and Biochemistry of Marine Food Products. AVI Publishing Co., Inc., Westport, pp. 149–304. Herbert, R.A., Hendrie, M.S., Gibson, D.M. and Shewan, J.M. (1971) Bacteria active in the spoilage of certain seafoods. Journal of Applied Bacteriology 34: 41–50. Herbert, R.A., Ellis, J.R. and Shewan, J.M. (1975) Isolation and identification of the volatile sulphides produced during chill-storage of North Sea Cod (Gadus morhua). Journal of the Science of Food and Agriculture 26: 1195–1202. Hirano, T., Zhang, C.-H., Morishita, A., Suzuki, T. and Shirai, T. (1992) Identification of volatile compounds in ayu fish and its feeds. Nippon Suisan Gakkaishi 58(3): 547–557. Human, J. and Khayat, A. (1981) Quality evaluation of raw tuna by gas chromatography and sensory methods. Journal of Food Science 46: 868–873. Jónsdóttir, R., Ólafsdóttir, G., Martinsdottir, E. and Stefansson, G. (2004) Flavor characterization of ripened cod roe by gas chromatography, sensory analysis and electronic nose. Journal of Agricultural and Food Chemistry 52: 6250–6256. Jonsson, A., Winquist, F., Schnürer, J., Sundgren, H. and Lundström, I. (1997) Electronic nose for microbial quality classification of grains. International Journal of Food Microbiology 35: 187–193. Jørgensen, L.V., Dalgaard, P. and Huss, H.H. (2000) Multiple compound quality index for cold-smoked salmon (Salmo salar) developed by multivariate regression of biogenic amines and pH. Journal of Agricultural and Food Chemistry 48: 2448–2453. Josephson, D.B. (1991) Seafood. In: H. Maarse (Ed.) Volatile Compounds in Foods and Beverages. Marcel Dekker, New York, pp. 179–202. Josephson, D.B., Lindsay, R.C. and Stuiber, D.C. (1983) Identification of compounds characterizing the aroma of fresh whitefish (Coregonus clupeaformis). Journal of Agricultural and Food Chemistry 31: 326–330. Josephson, D.B., Lindsay, R.C. and Stuiber, D.C. (1984) Variations in the occurrences of enzymically derived volatile aroma compounds in salt- and freshwater fish. Journal of Agricultural and Food Chemistry 32: 1344–1347. Josephson, D.B., Lindsay, R.C. and Ólafsdóttir, G. (1986) Measurement of volatile aroma constituents as a means for following sensory deterioration of fresh fish and fishery products. In: D.E. Kramer and J. Liston (Eds) Proceedings of an International Symposium on Quality Determinations sponsored by the University of Alaska Sea Grant Program, Anchorage, Alaska, U.S.A. Elsevier Science Publishers BV, Amsterdam, pp. 27–47. Kelleher, S.D. and Zall, R.R. (1983) Ethanol accumulation in muscle tissue as a chemical indicator of fish spoilage. Journal of Food Biochemistry 7: 87–91. Keshri, G., Voysey, P. and Magan, N. (2002) Early detection of spoilage moulds in Keshri bread using volatile production patterns and quantitative enzyme assays. Journal of Applied Microbiology 92: 165–172.

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Lavigne, J., Savoy, S., Clevenger, M., Ritchie, J., McDoniel, B., Yoo, S., Anslyn, E., McDevitt, J., Shear, J. and Neikirk, D. (1998) Solution-based analysis of multiple analytes by a sensor array: toward the development of an electronic tongue. Journal of the American Chemical Society 120: 6429–6430. Legin, A., Rudnitskaya, A., Vlasov, Y., Di Natale, C., Mazzone, E. and D’Amico, A. (1999) Application of electronic tongue for quantitative analysis of mineral water and wine. Electroanalysis 11: 814–820. Legin, A., Rudnitskaya, A., Seleznev, B. and Vlasov, Yu. (2002) Recognition of liquid and flesh food using an ‘electronic tongue’. International Journal of Food Science and Technology 37: 375. Legin, A., Rudnitskaya, A., Seleznev, B., Vlasov, Yu. and Velikzhanin, V. (2001) Electronic tongue for recognition of flesh food. In: J. Stetter and W. Penrose (Eds), Artificial chemical sensing (ISOEN 2001), The Electrochemical Society Inc., Pennington, NJ. Lerke, P. and Huck, R.W. (1977) Objective determination of canned tuna quality: identification of ethanol as a potentially useful index. Journal of Food Science 42(3): 755–758. Lindsay, R.C., Josephson, D.B. and Ólafsdóttir, G. (1986) Chemical and biochemical indices for assessing the quality of fish packaged in controlled atmospheres. In D.E. Kramer and J. Liston (Eds) Proceedings of an International Symposium on Quality Determinations sponsored by the University of Alaska Sea Grant Program, Anchorage, Alaska, U.S.A. Elsevier Science Publishers BV, Amsterdam, pp. 221–234. Luten, J.B. and Martinsdóttir, E. (1998) QIM: a European tool for fish freshness evaluation in the fishery chain. In: G. Ólafsdóttir, J. Luten, P. Dalgaard, M. Careche, V. Verrez-Bagnis, E. Martinsdóttir and K. Heia (Eds) Methods to Determine the Freshness of Fish in Research and Industry. Proceedings of the Final Meeting of the concerted action ‘Evaluation of Fish Freshness’ AIR3 CT94 2283. International Institute of Refrigeration, Paris, pp. 287–296. Luzuriaga, D.A. and Balaban, M.O. (1999a) Evaluation of odour decomposition in raw and cooked shrimp: correlation of electronic nose readings, odour sensory evaluation and ammonia levels. In: W.J. Hurst (Ed.) Electronic Nose and Sensor Array Based System, Design and Applications. Proceedings of the 5th International Symposium on Olfaction and the Electronic Nose. Technomic Publ. Co., Inc., USA, pp. 177–184. Luzuriaga, D.A. and Balaban, M.O. (1999b) Electronic nose odour evaluation of salmon fillets stored at different temperatures In: W.J. Hurst (Ed.) Electronic Nose and Sensor Array Based System, Design and Applications. Proceedings of the 5th International Symposium on Olfaction and the Electronic Nose. Technomic Publ. Co., Inc., USA, pp. 162–169. Macagnano, A., Careche, M., Herrero, A., Paolesse, R., Martinelli, E., Pennazza, G., Carmona, P., D’Amico, A. and Di Natale, C. (2005) A model to predict fish quality from instrumental features. Sensors and Actuators B 111: 293–298. McEntegart, C.M., Penrose, W.R., Strathmann, S. and Stetter, J.R. (2000) Detection and discrimination of coliform bacteria with gas sensor arrays. Sensors and Actuators B 70: 170–176. Mielle, P. (1996) Electronic noses: towards the objective instrumental characterisation of food aroma. Trends in Food Science & Technology 7: 432–438. Mielle, P. and Marquis, F. (1999) An alternative way to improve the sensitivity of electronic olfactometers. Sensors and Actuators B 58: 526–535. Miller III, A., Scanlan, R.A., Lee, J.S. and Libbey, L.M. (1973a) Identification of the volatile compounds produced in sterile fish muscle (Sebastes melanops) by Pseudomonas fragi. Applied Microbiology 25(6): 952–955. Miller III, A., Scanlan, R.A., Lee, J.S. and Libbey, L.M. (1973b) Volatile compounds produced by Pseudomonas putrefaciens, Pseudomonas fluorescens and an Achromobacter species. Applied Microbiology 26: 18–21. Milo, C. and Grosch, W. (1993) Changes in the odourants of boiled trout (Salmo fario) as affected by the storage of the raw material. Journal of Agricultural and Food Chemistry 41: 2076–2081.

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Milo, C. and Grosch, W. (1996) Changes in the odourants of boiled salmon and cod as affected by the storage of the raw material. Journal of Agricultural and Food Chemistry 44: 2366–2371. Namdev, P.K., Alroy, Y. and Singh, V. (1998) Sniffing out trouble: use of an electronic nose in bioprocesses. Biotechnology Progress 14: 75–78. Newman, D.J., Luzuriaga, D.A. and Balaban, M.O. (1999) Odour and microbiological evaluation of raw tuna: – correlation of sensory and electronic nose data. In: W.J. Hurst (Ed.) Electronic Nose and Sensor Array Based System, Design and Applications. Proceedings of the 5th International Symposium on Olfaction and the Electronic Nose. Technomic Publ. Co., Inc., USA, pp. pp. 170–176. Oehlenschläger, J. and Sörensen, N.K. (1998) Criteria of seafood freshness and quality aspects. In: G. Ólafsdóttir, J. Luten, P. Dalgaard, M. Careche, V. Verrez-Bagnis, E. Martinsdóttir and K. Heia (Eds) Methods to Determine the Freshness of Fish in Research and Industry. Proceedings of the Final Meeting of the concerted action ‘Evaluation of Fish Freshness’ AIR3 CT94 2283. International Institute of Refrigeration, Paris, pp. 30–35. Oehlenschläger, J. (1992) Evaluation of some well established and some underrated indices for the determination of freshness and/or spoilage of ice stored wet fish. In: H.H. Huss, M. Jakobsen and J. Liston (Eds) Quality Assurance in the Fish Industry. Elsevier Science Publisher, Amsterdam, pp. 339–350. Ohashi, E., Takao, Y., Fujita, T., Shimizu, Y. and Egashira, M. (1991) Semiconductive trimethylamine gas sensor for detecting fish freshness. Journal of Food Science 56(5): 1275–1278. Ólafsdóttir, G. (2003) Developing rapid olfaction arrays for determining fish quality. In: I.E. Tothill (Ed.) Rapid and On-Line Instrumentation for Food Quality Assurance. Woodhead Publishing Ltd., Cambridge, UK, pp. 339–360. Ólafsdóttir, G. (2005) Volatile compounds as quality indicators in fish during chilled storage: evaluation of microbial metabolites by an electronic nose. PhD thesis, Faculty of Science, University of Iceland, Reykjavík, 291 pp. ISBN 9979-70-052-1. Ólafsdóttir, G. and Fleurence, J. (1998) Evaluation of fish freshness using volatile compounds – classification of volatile compounds in fish. In: G. Ólafsdóttir, J. Luten, P. Dalgaard, M. Careche, V. Verrez-Bagnis, E. Martinsdóttir and K. Heia (Eds) Methods to Determine the Freshness of Fish in Research and Industry. Proceedings of the Final Meeting of the concerted action ‘Evaluation of Fish Freshness’ AIR3 CT94 2283. International Institute of Refrigeration, Paris, pp. 55–69. Ólafsdóttir, G., Martinsdóttir, E., Oehlenschläger, J., Dalgaard, P., Jensen, B., Undeland, I., Mackie, I.M., Henehan, G., Nielsen, J. and Nilsen, H. (1997a) Methods to evaluate fish freshness in research and industry. Trends in Food Science & Technology 8: 258–265. Ólafsdóttir, G., Martinsdóttir, E. and Jónsson, E.H. (1997b) Rapid gas sensor measurements to predict the freshness of capelin (Mallotus villosus). Journal of Agricultural and Food Chemistry 45(7): 2654–2659. Ólafsdóttir, G., Martinsdóttir, E. and Jónsson, E.H. (1997c) Gas sensor and GC measurements of volatile compounds in capelin (Mallotus villosus). In: J.B. Luten, T. Börresen and J. Oehlenschläger (Eds) Seafood from Producer to Consumer, Integrated Approach to Quality. Elsevier, Amsterdam, pp. 507–520. Ólafsdóttir, G., Högnadóttir, Á. and Martinsdóttir, E. (1998) Application of gas sensors to evaluate freshness and spoilage of various seafoods. In: G. Ólafsdóttir, J. Luten, P. Dalgaard, M. Careche, V. Verrez-Bagnis, E. Martinsdóttir and K. Heia (Eds) Methods to Determine the Freshness of Fish in Research and Industry. Proceedings of the Final Meeting of the concerted action ‘Evaluation of Fish Freshness’ AIR3 CT94 2283. International Institute of Refrigeration, Paris, pp. 100– 109. Ólafsdóttir, G., Högnadóttir, Á., Martinsdóttir, E. and Jónsdóttir, H. (2000) Application of an electronic nose to predict total volatile bases in capelin (Mallotus villosus) for fishmeal production. Journal of Agricultural and Food Chemistry 48(6): 2353–2359.

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Ólafsdóttir, G., Li, X., Lauzon, H.L. and Jonsdottir, R. (2002) Precision and application of electronic nose measurements for freshness monitoring of redfish (Sebastes marinus) stored in ice and modified atmosphere bulk storage. Journal of Aquatic Food Product Technology 11(3/4): 229–249. Ólafsdóttir, G., Di Natale, C. and Macagnano, A. (2003) Measurements of quality of fish by electronic noses. In: J.B. Luten, J. Oehlenschläger and G. Ólafsdóttir (Eds) Quality of Fish from Catch to Consumer: Labelling, Monitoring and Traceability. Wageningen Academic Publishers, Wageningen, The Netherlands, pp. 225–234. Ólafsdóttir, G., Nesvadba, P., Di Natale, C., Careche, M., Oehlenschläger, J., Tryggvadóttir, S.V., Schubring, R., Kroeger, M., Heia, K., Esaiassen, M., Macagnano, A. and Jørgensen, B.M. (2004) Multisensor for fish quality determination. Trends in Food Science & Technology 15: 86–93. Ólafsdóttir, G., Jonsdottir, R., Lauzon, H.L., Luten, J. and Kristbergsson, K. (2005a) Characterization of volatile compounds in chilled cod (Gadus morhua) fillets by gas chromatography and detection of quality indicators by an electronic nose. Journal of Agricultural Food Chemistry 53(26): 10140– 10147. Ólafsdóttir, G., Chanie, E., Westad, F., Jonsdottir, R., Thalman, C., Bazzo, S., Labreche, S., Marcq, P., Lundby, F. and Haugen, J.E. (2005b) Prediction of microbial and sensory quality of cold smoked Atlantic salmon (Salmo salar) by electronic nose. Journal of Food Science 70(5): 563–574. Ólafsdóttir, G., Lauzon, H., Martinsdottir, E. and Kristbergsson, K. (2006a) Influence of storage temperature on microbial spoilage characteristics of haddock fillets (Melanogrammus aeglefinus) evaluated by multivariate quality prediction. International Journal of Food Microbiology 111: 112–125. Ólafsdóttir, G., Lauzon, H., Martinsdottir, E., Oehlenschläger, J. and Kristbergsson, K. (2006b) Evaluation of shelf-life of superchilled cod (Gadus morhua) fillets and influence of temperature fluctuations on microbial and chemical quality indicators. Journal of Food Science 71(2): 97–109. Ólafsson, R., Martinsdóttir, E., Ólafsdóttir, G., Sigfússon, T.I. and Gardner, J.W. (1992) Monitoring of fish freshness using tin oxide sensors. In: J.W. Gardner and P.N. Bartlett (Eds) Sensors and Sensory Systems for an Electronic Nose. Kluwer, Dordrecht, The Netherlands, pp. 257–272. Olsson, J., Börjesson, T., Lundstedt, T. and Schnürer, J. (2002) Detection and quantification of ochratoxin A and deoxyinvalenol in barley grains by GC-MS and electronic nose. International Journal of Food Microbiology 72: 203–214. Pacquit, A., Lau, K.T., McLaughlin, H., Frisby, J., Quilty, B. and Diamond, D. (2006) Development of a volatile amine sensor for the monitoring of fish spoilage. Talanta 69: 515–520. Pearce, T.C., Schiffman, S.S., Nagle, H.T., Gardner, J.W. (Eds) (2003) Handbook of Machine Olfaction: Electronic Nose Technology. Wiley-VCH, Weinheim. Persaud, K. and Dodds, G. (1982) Analysis of discrimination mechanisms on the mammalian olfactory system using a mode nose. Nature 299: 352–353. Rakow, N. and Suslick, K. (2000) A colorimetric sensor array for odour visualization, Nature 406: 710–712. Schnürer, J., Olsson, J. and Börjesson, T. (1999) Fungal volatiles as indicators of food and feeds spoilage. Fungal Genetics and Biology 27: 209–217. Schweizer-Berberich, P.M., Vaihinger, S. and Göpel, W. (1994) Characterisation of food freshness with sensor arrays. Sensors and Actuators 18–19: 282–290. Scott, K. (2005) Taste recognition: food for thought. Neurons 48: 455–464. Sicard, G. and Holley, A. (1984) Receptor cell responses to odourants: similiarities and differences among odourants. Brain Research 292: 283–291. Stewart, G.F. and Amerine, M.A. (1973) Introduction to Food Science and Technology. Academic Press, New York. Stitzel, S., Stein, D. and Walt, D. (2003) Enhancing vapor sensor discrimination by mimicking a canine nasal cavity flow environment. Journal of the American Chemical Society 125: 3684–3685. Toko, K. (2000) Taste sensor. Sensors and Actuators B 64: 205–215.

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Tozawa, H., Enokihara, K. and Amano, K. (1971). Proposed modification of Dyer’s method for trimethylamine determination in cod fish. In: Fish Inspection and Quality Control. London: Fishing News Books, pp. 187–190. Trihaas, J. and Nielsen, P.V. (2005) Electronic nose technology in quality assessment: monitoring the ripening process of Danish blue cheese. Journal of Food Science 70(1): E44–E49. Trihaas, J., van den Tempel, T. and Nielsen, P.V. (2005) Electronic nose technology in quality assessment: predicting volatile composition of Danish blue cheese during ripening. Journal of Food Science 70(6): E392–E400. Vlasov, Y., Legin, A., Rudnitskaya, A., Di Natale, C. and D’Amico, A. (2005) Nonspecific sensor arrays (‘electronic tongue’) for chemical analysis of liquids (IUPAC technical report). Pure and Applied Chemistry 77: 1965–1983. Winquist, F., Wide, P. and Lundstrom, I. (1997) An electronic tongue based on voltammetry. Analytica Chimica Acta 357: 21. Zhao, C.Z., Pan, Y.Z., Ma, L.Z., Tang, Z.N., Zhao, G.L. and Wang, L.D. (2002) Assay of fish freshness using trimethylamine vapor probe based on a sensitive membrane on piezoelectric quartz crystal. Sensors and Actuators B 81(2–3): 218–222.

Chapter 7

Colour measurement Reinhard Schubring

7.1

Introduction

We perceive the world by our five senses, vision, hearing, touch, taste and smell, of which vision is usually the first used in detecting events and objects. The process of seeing comprises many cooperating activities: first, detection by our eyes, then interpreted by our brain; recognition of movement and location of objects; relationships of objects to their surroundings; the intensity and quality of the light and the colour appearance of objects or events in the visual scene (MacDougall 2002). Scientific understanding of the processes involved in determining colour has been elucidated in the past two to three centuries. The experiments in mixing colours performed during this period clearly demonstrated that people with normal colour vision must have at least three retinal pigments in their eyes, detecting the short-, mid- and long wavelengths of the visible spectrum. The first truly functional system for measuring colour as specified by the Commission Internationale de l’Eclairage (CIE) was the so-called CIE 1931 2° visual field system of colour measurement. Since then, many improvements have been incorporated into the system to make it nearly visually uniform, and this research continues (MacDougall 2002). With the development of the computer, complex colour measurements and calculations are now routinely used in research and the food industry for studies of food functionality, standardisation of product ingredients and process control. Three interacting factors are required to measure the colour appearance of any object. These are an understanding of the human visual process, the effect of light on objects in their environment and the nature of the materials observed (MacDougall 2002). The CIE system of colour measurement transforms the reflection or transmission spectrum of the object into a three-dimensional colour space using the spectral power distribution of the illuminant and the colour matching functions of standard observers. The original 1931 CIE Y, x, y system of colour measurement is not visually uniform. Constant hue and chroma are distorted and equal visual distances increase several-fold from purple–red to green. Near uniform colour spaces of practical importance are the Hunter and CIELAB spaces. The CIE L* a* b*, also known as CIELAB, has generally replaced the Hunter space for industrial application. The coordinates L* a* b* serve to define the locations of any colour in the uniform colour space. Colour terms can be divided into the subjective and the objective. The subjective terms, i.e. the psychosensorial, are brightness, lightness, hue, saturation, chroma and colourfulness. Colourfulness is that 127

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aspect of visual sensation according to which an area appears to exhibit more or less chromatic colour. Although hue is easily understood as that attribute described by colour names red, green, purple, etc., the difference between saturation and chroma is less easily comprehended. Saturation is colourfulness judged in proportion to its brightness, whereas chroma is colourfulness relative to the brightness of its surroundings. A similar difference exists between lightness and brightness. Lightness is relative brightness. Lightness is unaffected by illumination level because it is the proportion of the light reflected, whereas the sensation of brightness increases with the level of illumination. The objective terms, i.e. the psychophysical, are related to the stimulus and are evaluated from spectral power distribution, the reflectance or transmittance of the object and observer response. They provide the basis for the psychometric qualities that correspond most closely to those perceived (MacDougall 2002). For CIELAB space, the terms are lightness L*, hue h* = tan−1 (a*/b*) and chroma C* = (a*2 + b*2)1/2. CIELAB total colour difference can be expressed either as the coordinates of colour space or as the correlates of lightness, chroma and hue. ΔE* = [( ΔL*2 ) + ( Δa*2 ) + ( Δb *2 )]

12

or ΔE* = [( ΔL*2 ) + ( ΔC *2 ) + ( ΔH *2 )]

12

Colour is the perception that results from the detection of light after it has interacted with an object. The perceived colour of an object is affected by three entities: the physical and chemical composition of the object, the spectral composition of the light source illuminating the object, and the spectral sensitivity of the viewer’s eye. Because everyone is sensitive to the colour of foods, appetite is stimulated or dampened in almost direct relation to the observer’s reaction to colour. The colour we see clearly indicates the flavour we will taste (Downham and Collins 2000). For food products, the consumers often assess the initial quality of the product by its colour and appearance. In food processing and cooking, colour serves as a cue for the doneness of foods and is correlated with changes of aroma and flavour. For example, colour or lightness, such as the lightness of canned tuna, is important for identity and quality grading. In general, colour and appearance affect the consumer’s perceptions of other sensory modalities (Lawless and Heymann 1998). The appearance of fish and meat products is an essential factor according to which consumers judge their acceptance (Clydesdale 1991). However, the colour of food is not stable, because it changes with decreasing freshness. The appearance of a newly landed fish is unforgettable, in that the interplay of the subtle shades of beautiful colours makes it a joy to behold and irresistible as an item of food. Just a few hours after death, though, it begins to look less obviously attractive, and its now ‘ordinary’ colours are much more familiar to most of the public (Love 1988).

7.2

Instrumentation

Photoelectric colour measuring instruments can be divided into two classes, trichromatic colorimeters and spectrophotometers. Colorimeters are tristimulus (three-filtered) devices that make use of red, green and blue filters that emulate the response of the human eye to light and colour. In some quality control applications, these tools represent the lowest-cost answer.

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Figure 7.1 Konica Minolta CR-300 Chroma Meter with DP-301 Data Processor.

The more modern tristimulus instruments are linked to computers with automatic compensation and the provision of several colour spaces. A colorimeter uses a light source to light the specimen being measured. The light reflected off the object then passes through the glass filters to simulate the standard observer’s functions for a particular illuminant. A photodetector beyond each filter then detects the amount of light passing through the filters. These signals are then displayed as X, Y and Z values. The Chroma Meter CR-300 (Figure 7.1) offers an 8 mm diameter measuring area. Its measuring head also uses diffuse illumination/0° viewing geometry (specular component included) to provide measurements of a wide variety of surfaces, which correlate well with colour as seen under diffuse lighting conditions. A pulsed xenon arc (PXA) lamp inside a mixing chamber provides diffuse, uniform lighting over the 8 mm diameter specimen area. Only the light reflected perpendicular to the specimen surface is collected by the optical-fibre cable for colour analysis. A white standard CR-A43 is used as illuminant condition D65 and for calibration purposes . Use of the computer software ChromaControl C (release 2.04) allows easy calculation of the hue and chroma. The most accurate instrument for measuring colour is the spectrophotometer. It uses a light source to illuminate the specimen being measured. The light reflected by the object then passes to a grating, which breaks it into the spectrum. The spectrum falls onto a diode array, which measures the amount of light at each wavelength. This spectral data is then sent to the processor where it is multiplied with data-table values for the selected CIE illuminant and the 2° or 10° standard observer functions to the X, Y and Z values. These are further transformed to the CIELAB values L*, a*, b* by the following equations: L* = 116 (Y Yn )

13

− 16

13 13 a* = 500 ⎡⎣( X X n ) − (Y Yn ) ⎤⎦

b* = 200 ⎡⎣− (Y Yn )

13

− ( Z Z n ) ⎤⎦ 13

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Light guide

Light source LCD display

Lens system

Microprocessor

L* 62.1

a* 49.8

b* 31.2

Sample Grating

REF

Measurement/ reference receiver system

M

Prog

ON/C

Keyboard

Figure 7.2 Dr Lange pen-type colorimeter.

Colour measurements on fish samples were performed with the spectral colour meter Spectro-pen® (Dr Lange, Düsseldorf, Germany). This is a colorimeter (Figure 7.2) operating on the spectral method described in DIN 5033 using the 45/0° circular viewing geometry, i.e. the sample is illuminated with polychromatic light encircling it at an angle of 45°, with the optical unit observing the reflected light from a horizontal angle (0°) towards the sample surface. Spectro-pen® is a genuine grating colorimeter measuring the visible spectral range (400–700 nm) at intervals of 10 nm. A 10° standard observer and a D65 illuminant were used (light source: polychromatic with tungsten lamp). The PC software ‘spectral – QC’ allows state-of-the-art data processing. Before measuring each lot, the colorimeter is calibrated against a white standard (LZM 224). The precision of predicting colour parameters from one instrument to another varies with food commodity. The type of sample seems to have a larger effect on the precision of the measurement than the size of the instrument’s measuring area. The variation from one instrument to another was found to systematic and can be described by linear regression. The regression can be used to compare colour values expected from one instrument with those obtained from another. The precision of prediction will increase with increased homogeneity of the food samples (Baardseth et al. 1988). When instrumental colour measurements of raw salmon flesh were performed by one tristimulus filter colorimeter and two spectrophotometers, it was found that the instruments gave different values in absolute terms, depending on sampling conditions. The highest correlation between astaxanthin content and instrumental colour reading was obtained for the a* and chroma values of the spectrophotometers when sampling on 1 cm thick cutlets on white background (Stien et al. 2006a).

7.3

Novel methods of colour evaluation

Image features, i.e. colour, size, shape and texture, have been extensively applied in the food industry for quality evaluation and inspection of a wide variety of food. Colour features are effective tools for indicating reconstruction of components of food product during processing.

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Three types of colour space, hardware orientated, human-orientated and instrumental, are generally used for the extraction of colour features. Hardware-orientated spaces are preferable for observing small changes in the colour of food products during processing. With the remarkable development in computer hardware, it might become realistic to use whole image data as input features, which might be an exceptional indicator of food quality (Zheng et al. 2006). Very recently, an automated image analysis method has been presented that was able to describe quality properties, such as area of the cutlet, dorsal fat depot, red muscle, fat percentage and colour from many scanned images of rainbow trout cutlets. It is also possible to produce images of cutlets of adequate quality for image analysis, using a simple flatbed scanner (Stien et al. 2006a). No elaborate lighting regime is necessary (Stien et al. 2006b). An image acquisition system was presented recently that allows digital images in L*a*b* colour units for each pixel of the digital red, green, blue (RGB) image. Five models were built that are able to measure colour in L*a*b* units and simultaneously measure the colour of each pixel on the target surface. This is not the case with conventional colorimeters. The best results were achieved with the quadratic and neural network model (León et al. 2006). An easier method has been proposed that uses a combination of digital camera, computer and graphic software to analyse the surface colour of food products (Yam and Papadakis 2004). Measuring colour, particularly in the L*a*b* space, provides a better statistical discrimination between the groups of fish studied than sensory analysis. In fact, although in agreement with the results of the panel, the colorimetric method can distinguish all of the groups in terms of the mean colour and the heterogeneity of colour. The very high degree of precision of these results provides an understanding of the effect of the drying processes on the colour of the different samples (Louka et al. 2004). However, it became obvious that the determination of only one quality attribute was not sufficient. Correct classification based on experimental variables measured by discriminant function analysis was poor for colour data alone, acceptable for electronic nose data alone, and excellent with these data combined (Korel et al. 2001). Combining the data from the various sensors improves the estimate of the freshness of fish (Olafsdottir et al. 2004). To demonstrate this, colour, texture and electronic nose measurements were selected, and their calibrated outputs combined to construct a so-called artificial quality index (AQI) (Di Natale 2003). It was reported that machine vision is able to differentiate and quantify colour distributions in fish samples with uneven colour. In the case of fresh tuna, the hue values offered less variability and more monotonous changes with storage time. Colour of fresh tuna exposed to 4% CO for 48 hours stayed unchanged with refrigerated storage time. The colour of control samples did change substantially and turned brown (Balaban et al. 2005).

7.4 Colour measurement on fish and fishery products In contrast to warm-blooded meat, reports on colour measurements taken on fish and/or fishery products appear lacking in books dealing with the colour of foods (Kress-Rogers 1993; Hutchings 1994, 1999, 2003; Pearson and Dutson 1994; Kress-Rogers and Brimelow 2001; MacDougall 2002). Colour measurement on fresh meat (MacDougall 2002) is explained as a typical example for muscle foods, modelling colour stability is discussed on fresh beef (Jakobsen and Bertelsen 2002). It is therefore the aim of this chapter to give an overview

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on the importance and application of colour measurement on fish and fishery products. The following main areas of application of colour measurements can be considered: Aquaculture Fish mince, surimi and surimi-based products Processing effect on colour of fish and fishery products Refrigerated and frozen storage Thermal processing (heating and smoking) High-pressure processing

7.4.1

Aquaculture

In monitoring colour changes that occurred during maturation of farmed Atlantic salmon (Salmo salar), it was found that colour of the fillet of maturing fish was significantly less vivid than in the immature fish for the first time in September. The pigmentation of maturing fish thereafter decreased until a very pale colour was registered in November. Maturing or mature males and females did not differ significantly in fillet colour. In this case, colour was scored using a scale from 1 to 8, with top score for the reddest fillet (Aksnes et al. 1986). The same scale supported by a specially constructed ‘colour fan’ was used for assessment of flesh colour on the wall of the body cavity of gutted Chinook salmon (Oncorhynchus tshawytscha) to investigate whether genetic factors determine the extent to which the dietary carotenoid pigment is deposited in the fish flesh. Results demonstrated that Chinook salmon fed the same diet containing astaxanthin may show wide variations between strain as well as among families. No correlation was found between the degree of pigmentation and the lipid concentration of the flesh (McCallum et al. 1987). Colour standards from the Natural Colour System (NCS) were selected by a sensory panel to match colour of raw flesh of astaxanthin-fed Atlantic salmon. NCS chromaticity and hue were both found to be significantly correlated with carotenoid concentration and with instrumentally obtained CIE a*, b* and h* values (Skrede et al. 1990). Investigations of changes in the muscle of chum salmon (Oncorhynchus keta) during spawning migration revealed that visual grades were significantly correlated, among others, with muscle colour. Hunter L values increased while a, b and a/L decreased with increasing maturity (Reid et al. 1993). However, it was found that the Colour Card provided a better prediction of the astaxanthin concentration at higher astaxanthin levels than the chromameter, whereas a good correlation was found between the Colour Card score and the astaxanthin concentration in the flesh (Christiansen et al. 1995). In farmed rainbow trout (Oncorhynchus mykiss), it was found that muscle carotinoid content increased markedly with increased carotinoid in the diet, and that female muscles were more coloured than male ones. Increased pigmentations caused increased C* and reduced h* and L* in the corresponding muscle samples. Carotinoid concentrations in the muscle were well correlated with h*, C* and L* (Choubert et al. 1992). Already after 5 weeks of feeding on pigmented diets (astaxanthin and canthaxanthin), the effect of carotinoid pigmentation on colour of Arctic char (Salvelinus alpinus) flesh was noticeable. Deposition of carotinoids in the flesh resulted in a decrease in L and an increase in a and b. The major changes of colour parameters were achieved during the first few weeks of feeding. The skin

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colour was also significantly affected by the deposition of carotenoids. However, no correlations were found between colour parameters of skin and flesh from the same fish (Shahidi et al. 1993). Dietary algal (Haematococcus pluvialis) incorporation that contained a total amount of 2% of carotenoids (based on dry algae) increased the pigmentation of the rainbow trout muscle and caused an increase in chroma and a reduction in hue and lightness after 4 weeks of feeding (Choubert and Heinrich 1993). Genetic variation in flesh colour of the coho salmon (Oncorhynchus kisutch) was observed in degree of flesh pigmentation. Chilliwack River coho salmon had the deepest red coloured flesh (a* = 4.34), and Robertson Creek salmon had the lightest flesh colouration (a* = 2.72). Heritability of flesh colour over all populations was 0.19 ± 0.12 (Withler and Beacham 1994). The colour characteristics and deposition of canthaxanthin in cultured Arctic char, stocked at 40, 50 and 75 kg/m3, were studied. L, a and b values of fillets, as such, or in the homogenised tissues, and skin of fish, varied significantly among density groups. For belly skin, the numerical values of colour parameters decreased with increasing stocking density. However, no correlation existed between the total carotenoid contents of fish skin and their L, a and b values. On the other hand, the L values of fillets and homogenised tissues were inversely correlated with their carotenoid contents, whereas their a and b values were directly correlated with carotenoid contents (Metusalach et al. 1997). When Arctic char was reared at different temperatures (8° compared with 12 °C), fish maintained at the lower temperature had significantly higher pigmentation compared with those grown at the higher temperature (Olsen and Mortensen 1997). Increasing carotenoid concentrations led to increased a*, b* and C*, and decreased L* and h*. Redness was the colour parameter most highly correlated with carotenoid concentration in the Arctic char fillets. For a given a* value, b* was higher in sexually mature than in immature Arctic char (Hatlen et al. 1998). Redness of raw flesh of Arctic char, independent of strain, was higher in fish reared at 10 °C than those reared at 15 °C. The fillet from one strain reared at 10 °C had stronger b* and C* than fish of the same strain reared at 15 °C and than the other strain, regardless of the rearing temperature (Ginés et al. 2004b). Colour measurements in pan-size rainbow trout that were fed an astaxanthin-containing diet indicated that colour levels induced by astaxanthin were under genetic control, the largest familial differences being obtained from lightness measurements. L* was found to be positively correlated with h* and with C* (Blanc and Choubert 1993). The effect of the location at which measurements were taken was highly significant on lightness, chroma and hue. Data from tail part of the fish muscle were higher than those from the head. The middle part always exhibited the lowest data (Choubert et al. 1997). In rainbow trout, colorimetry measurements demonstrated large variations in fillet colour irrespective of dietary treatment. Values of a* ranged from 5 to 26, b* from 7 to 31, L* from 35 to 63, whereas C* and h* ranged from 8 to 40 and 34 to 72, respectively. In all treatments, L* was significantly correlated with flesh lipid content; however, only a*, b* and C* values of fish fed low fat diet were significantly correlated with flesh lipid content. At each sampling, the tail regions within all treatments had significantly higher concentrations of astaxanthin and a* than the dorsal and belly fillet regions, although among treatments, there were no significant differences in carotenoid content (Nickell and Bromage 1998). A comparative study of flesh pigmentation efficiency of oil-extracted astaxanthin from langostilla (Pleuroncodes planipes), a red yeast (Phaffia rhodozyma) and synthetic astaxanthin (Carophyll pink) on rainbow trout was conducted. Pigmentation efficiencies of the diet

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supplemented with the langostilla carotenoid (a* = 18.6) and with the yeast astaxanthin (a* = 16.8) were similar after six weeks, whereas the efficiency of synthetic astaxanthin (a* = 21.0) was higher (Coral et al. 1998). Rainbow trout were fed diets supplemented with canthaxanthin, oleoresin paprika and αtocopherol. L* and h* decreased gradually until the end of the feeding period in all treatment groups. Similarly, a* increased during the entire trial. In contrast, b* increased up to four months and then started to decline in all treatment groups (Akhtar et al. 1999). The effects of two red chilli oleoresins and synthetic astaxanthin on colour, carotenoid concentration and carotenoid retention rate in their fresh and minced fillets were compared. The variations of the average values of the colour parameters measured in the fresh muscle of rainbow trout can be attributed to three main factors: (a) a non-uniform distribution of carotenoid in the flesh; (b) the use of few discrete measurement points on the fillet surface; and (c) the incident beam reflectance. In the minced fillet, the colour parameters were higher because the material was more homogeneously distributed, with no discrete points occurring, resulting in higher precision. The increment in the average values of a* and b* and a decrement of L* in the muscle of salmonids with increasing total dietary carotenoid concentration and feeding trial time is well documented when using synthetic pigments (Ingle de la Mora et al. 2006). To investigate astaxanthin isomer ratios in flesh of fish fed different carotenoid sources, three groups of rainbow trout were fed, for 60 days, diets containing astaxanthin from synthetic source, Haematococcus pluvialis algae meal and Phaffia rhodozyma red yeast. Colour values measured in different sites of fillet of rainbow trout fed with different pigment sources showed no significant differences. Similarly, different sources of pigment (natural or synthetic) produced colour values of fresh fillet with no relevant or significant differences (Moretti et al. 2006). Decreases in L* values during the 6 weeks of feeding were more marked in trout fed fish oil and synthetic astaxanthin than in trout fed fish oil and microalgae. Feeding either source of astaxanthin with olive oil resulted in intermediate L* values. Hue was unaffected by diet, but C*, a* and b* were higher for synthetic astaxanthin-fed fish than microalgae-fed fish (Choubert et al. 2006). No significant differences in L, a or b values were found between fillets from the different diets containing 0, 500, 1000 or 3000 mg/kg pure genistein. Therefore, it can be concluded that genistein does not influence fillet colour of rainbow trout. There was also no effect of time of harvest on the colour of the fillets (D’Souza et al. 2005). The effect of feeding diets containing 0%, 20%, and 40% soybean meal (SBM) for 24 weeks on colour of the rainbow trout fillets was evaluated. The fillets from trout fed the 40% SBM diet had significantly higher L* values than trout fed the 0% SBM and 20% SBM diets. The a* values significantly decreased over time for fillets from all three diet groups, and no significant differences in b* were found (D’Souza et al. 2006). The effect of dietary lipid level on flesh colour was found to be pronounced in Atlantic salmon because a* and b* were higher in fish fed high fat diets than those fed medium fat diet, suggesting that intestinal astaxanthin absorption was facilitated by higher dietary fat content (Bjerkeng et al. 1997). The suitability of soybean/corn lecithin and poultry fat as partial replacements for menhaden oil in feeds for rainbow trout was investigated. Significant differences in b* values were observed in raw and cooked fillets that had been stored raw under the different time/temperature conditions. Raw L15 fillets (15% lecithin) were statistically higher in b* values than all of the other treatments under all storage conditions. There was also a trend for L15 fillets that were stored raw under all tested time/temperature

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conditions and then cooked to have higher b* values than the other treatments. Under all storage conditions tested, there were no significant differences in L* values among all treatments of raw and cooked fillets, or in a* values of cooked fillets (Liu et al. 2004). When the pigmentation of Atlantic salmon fed astaxanthin in all meals or in alternating meals was investigated, a high rectilinear relationship was found between total carotenoid concentration and a* (r = 0.87). The b* values exhibited larger variation (r = 0.76), whereas L* was poorly correlated (r = 0.34) to the carotenoid concentration in the flesh. It is concluded that measurements made directly on the fillets yield fairly accurate estimates for astaxanthin concentration and the variation for fish within this concentration range (Wathne et al. 1998). Muscle composition and quality characteristics of fillets were investigated in Atlantic salmon starved at low water temperatures for 0, 3, 7, 14, 30, 58 or 86 days before slaughter. Although astaxanthin concentration was not significantly affected by starvation time, instrumental colour analyses of raw fillets revealed changes in L* and b*. At the start of the experiment, increased L*, a* and b* values were found from 4 to 12 days ice-storage, whereas this was not shown at the end of the experiment. It is suggested that starvation is a rather weak tool for changing fillet quality in Atlantic salmon (Einen and Thomassen 1998). Atlantic salmon were given feeds where the pigment source was astaxanthin only, canthaxanthin only or an astaxanthin/canthaxanthin mix. Results of cross-section assessment for a* and Roche SalmoFan scores also showed an increase in colour with increasing proportions of canthaxanthin in the feed. The data reported clearly indicate that Atlantic salmon of this size deposit canthaxanthin more efficiently than they do astaxanthin (Buttle et al. 2001). The relative pigmentation efficacy of astaxanthin and canthaxanthin in diets for the Atlantic salmon raised in freshwater was assessed. Flesh carotenoid levels generally responded concomitantly with applied dietary dose, directly affecting flesh coloration a* and b* values and SalmoFan score (Baker et al. 2002). Estimates of phenotypic and genetic parameters for flesh colour traits in farmed Atlantic salmon based on a multiple trait animal model were investigated. Three different methods of colour measurement of Atlantic salmon flesh were used in this study: subjective scoring (SalmoFan), colorimetric measurement and carotenoid content as measured by near infrared spectroscopy. It may be that fillet colour is influenced by factors other than pigment retained in the flesh. Genetic correlations between subjective visual scores (two scorers) and colorimetric measurements were all high, with the a* value of the Minolta camera being the most highly correlated with subjectively assessed colour (Norris and Cunningham 2004). A low oil pre-harvest feed (50% crude protein, 18% crude fat) as a means of manipulating the quality of Atlantic salmon growing between 2.5 and 4.5 kg in the spring/summer was tested. There were no significant differences in perceived flesh colour (Roche SalmoFan) at harvest, and using the low oil, pre-harvest feed did not significantly improve instrumentally measured colour (Morris et al. 2005). The absorption efficiency, deposition, retention and possible interaction of two xanthophylls given in diets to the Atlantic salmon were evaluated. Colour values a*, b* and L* were taken at 138 days of feeding. a* readings were stable at around 25, b* at 48 and L* at 27 without any significant difference between the groups. This indicated that lutein did not influence the salmon colour (Olsen and Baker 2006). Rainbow trout and Atlantic salmon were submitted for various slaughter and bleeding procedures to see what effect these would have on blood drainage of the muscles. Results showed that the bleeding method (bled live

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by a gill cut or percussive killed and bled by gutting) is of less importance than the timing. No significant difference in blood spotting was observed between fish that were bled live by a gill cut or percussive killed and bled by gutting. The visual appearance of the fillet was influenced by number and size of the bloodstains. Colour measurements with Hunter L, a, b did not reveal this (Roth et al. 2005b). The effects on fish welfare of commercial and experimental slaughtering techniques were evaluated. Three methods were selected (bleeding in ice slurry, whole-body electrical treatment and percussion), and their influence on flesh colour was studied over a 9 day period. Fish killed by electricity was redder and darker, as indicated by higher a* values and lower L* values respectively. No differences in b* were observed among samples. It therefore appeared that the bleeding process was not as extensive in fish subjected to electricity as it was with the other two methods. Percussion followed by bleeding produced a flesh colour similar to that obtained after bleeding only (Morzel et al. 2003). Diploid and triploid Atlantic salmon were reared for 32 months in seawater and compared for colour. Triploid fish had darker (lower L value) and redder (higher a value) fillet colour than the diploid fish (Bjørnevik et al. 2004). Colour was affected by season, as fillets were paler and redder in the autumn and winter samplings, whereas yellowness was unaffected of harvesting time. During ice storage, fillet colour became lighter and redder, whereas yellowness changed in the fattier fillets upon ice storage (Espe et al. 2004b). The electrically stimulated fish were even more red than both the stressed and rested fish with significant higher a* and C*, but there was little or no difference in colour between the groups measured as L* and h* (Roth et al. 2006). Colour of salmon flesh from different locations on pre- and post-rigor fillets were evaluated. It was found that pre-rigor fillet cuts improved colour characteristics (lower L*, higher a* and b* values) compared with post-rigor fillet cuts. The lower loins had higher colour scores after storage, but these colour differences were more pronounced in the pre-rigor group. Comparison of anterior and caudal loins showed significant differences for colour. The effects on colour of fillets taken pre-rigor compared with post-rigor were more pronounced than the effects of duration of storage (Skjervold et al. 2001). The potential for increasing carbohydrate inclusion levels above those currently used in salmon feeds and thereby substituting a large proportion of fish oil in the feed was investigated for the colour attributes of fish grown on such diets. Flesh colour (a*, b* and C*) was affected throughout the trial by dietary energy content, but significant carbohydrate × energy content interactions occurred in September. Despite this, a*, b* and C* increased throughout the trial, and in May showed a tendency to be strongest in the high-energy-fed fish. The h* value fluctuated throughout the trial irrespective of dietary treatment and was affected by significant carbohydrate × energy content interactions in September. However, by May all fish had lower h* readings as a result of the high-energy feeds, regardless of dietary carbohydrate source (Young et al. 2006). To investigate concerns about the effects of high-energy diets on Atlantic salmon, a trial was undertaken using large fish fed extremely high- or medium-energy diets. Flesh colour appeared strongest in the tail, then dorsal and finally the belly region. In October, diet and feeding intensity had no effect on instrumentally measured L*, a* , b* or C*. However, the value of h* of the flesh of salmon maintained on the high-energy regime was significantly redder than all others. In December, no significant differences were seen in h*, but C* was strongest in the fastest growing fish. It was summarised that, in practical terms, the flesh

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colour of these large salmon was not affected by either growth rate or the use of very high-energy feeds (Young et al. 2005). Farmed Atlantic salmon were slaughtered under either high- or low-stress conditions and analysed for colour. There were no significant differences in L* value. The values of a* and b* were higher under high-stress conditions. The post-thawing analyses of vacuum-packed cutlets frozen for 12 months also showed that salmon slaughtered under high stress were more intensely coloured (Kiessling et al. 2004). There was a significant effect of season on colour attributes measured on flesh of Atlantic salmon. From January to June there was a slight increase of a and C values, but by October all fish had become significantly more red as a, b and C had elevated, and L and h had decreased (Roth et al. 2005a). Growth performance and white muscle cellularity were investigated in Atlantic cod (Gadus morhua) to determine if colour is affected by exercise. The colour of the cod fillet was white (high L value), faint green (−a value) and faint yellow (+b value). There was no correlation between colour values and fibre diameter, but a positive correlation between a and shear force was found (Bjørnevik et al. 2003). Effects of pre-slaughter stress and storage temperature on colour in pre-rigor filleted farmed cod fillets were investigated using three colorimetric instruments. Higher L values were registered as the rigor process progressed, signifying a less transparent (more opaque) fillet. The L values at 20 °C rose to a higher level than at 4 °C. There were also differences in L between unstressed and stressed fish. This was especially apparent for the fish stored at 4 °C, where all the spectrophotometers registered significant higher L values in fillets from the stressed fish than in unstressed fish at the last sampling. Differences between treatments were also found for the h and C values. It was interesting to note that the three instruments’ colour readings differed, both in absolute value and in how the values changed during the rigor process. The main difference was not between the Minolta tristimulus colorimeter and the two spectrophotometers, but between the Hunterlab Miniscan/XE instrument and the two other colorimetric instruments. This can be explained by the differences in output colour model (Hunter L, a, b versus CIE L*, a*, b*). Instrumental differences as the Hunterlab’s large sampling area (25 mm diameter) compared with the two other instruments (Minolta: 8 mm diameter and X-Rite: 6.35 mm diameter) may also play a role (Stien et al. 2005). Colour development was studied during storage of pre-rigor fillets of farmed Atlantic cod. For eight weeks before slaughtering, the fish were fed diets containing either 100% fish oil (M-group) or 60% fish oil and 40% soybean oil (S-group). The L* value increased during the first 4–6 h storage. From 8 to 16 h, the L* value was stable or slightly decreasing. The L* value was consistently higher of the M-group (Mørkøre 2006). The effects of pre-rigor filleting on fillet quality of wild and farmed cod were studied. Quality parameters investigated included among others lightness of the fillet. The time of filleting did not affect L* of the cut muscle surface measured on day 10 after slaughter. When the relationship between L* and the muscle pH 10 days after slaughter is shown as a dot plot, a negative relationship at pH lower than approximately 6.4 appears (Kristoffersen et al. 2006). Immature gilthead sea bream (Sparus aurata) with a mean initial weight of 25.6 g were reared over 11 months to market size under different photoperiods of both 16 hours of light (16L : 8D) and permanent light (24L : 0D) and a control. Of the skin colour measurement variables, only L* differed significantly between treatments. Fish reared under 24L:0D conditions displayed the highest values (85.2) relative to the other two treatments (82.5 and 80.1 for the16L:8D and the control treatments, respectively) (Ginés et al. 2004a).

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The red porgy, Pagrus pagrus, is a fairly new and highly appreciated species in aquaculture. A problem encountered in rearing red porgy is a darkening of the body after capture of wild fish and during farming. The red–silver colour of the body changes into an overall dark grey. Several environmental conditions were evaluated for their potency to influence the skin colour. Background colour was the main factor in controlling skin pigmentation. A light background colour restored the lightness value of the skin up to levels found in wild red porgy (L* = 70). The background effect was enhanced by applying blue illumination. The light intensity had no clear effect on the body colour, but a high density had a negative effect on L* (van der Salm et al. 2004). The effect of diet supplementation with two carotinoid sources on skin colour was evaluated. Only astaxanthin from shrimp shell meal was able to give red porgy skin an overall reddish coloration. Shrimp shell meal diets enhanced reddish h* and C* values. However, L* of red porgy skin was not influenced (Kalinowski et al. 2005). In a comparable experiment it was found that only astaxanthin, but not β-carotene and lycopene, had a significant effect on skin hue, promoting a reddish coloration of the dorsal skin area and a ventral hue similar to wild red porgy (Chatzifotis et al. 2005). An appropriate method for comparisons of the skin chromaticity parameters in wild and farmed red skin Sparidae (Pagrus pagrus, Pagrus caeruleostictus and Dentex gibbosus) was developed and the effect of storage time on skin colour of farmed Pagrus pagrus was investigated. A new index, named the Entire Colour Index (ECI), was developed to express h* and C* which, as combined variables, cannot be considered separately. ECI was calculated as ECIi = C* cos (hi − hmean). In all species there was a remarkable dorsoventral gradient in mean L* and h*, with the ventral area being statistically significant brighter than the dorsal one. ECI value was species specific but did not show any statistically significant dorsoventral gradient, with the exception of P. pagrus. Storage time affected L* and h* only in the dorsal skin area. However, the effect of storage on ice was better reflected in the mean ECI value, which showed a marked decrease from day 3 to day 7 in both the dorsal and ventral skin area. It is concluded that the results provide data for a non-subjective determination of skin colour pattern and show that ECI offers a good index of the actual colour in a meaningful and objective way (Pavlidis et al. 2006). The L* value for wild pikeperch (Sander lucioperca) tissue was very similar to that of cultivated pikeperch. The tissue of both types of fish also had similar values of a*, whereas b* for wild pikeperch was significantly lower than that for cultivated specimens (Jankowska et al. 2003). Colour measurements taken on wild and aquacultured meagre (Sciena umbra) did not show any significant difference in colour parameters measured (Cakli et al. 2006).

7.4.2

Fish mince, surimi and surimi-based products

The second major area in which colour measurements are frequently used is characterised by the comminution of fish muscle and steps to restructure the comminuted muscle to fish muscle analogues mainly by heating. Gel formation necessary to achieve the goal is dependent on the functionality of muscle proteins and supported by addition of substances that are able to support gel formation. Most of these technological steps include characteristic colour changes of fish muscle. Therefore, it is no wonder that colour measurements have been applied at an early stage. In the three relevant textbooks dealing with surimi

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(Lanier and Lee 1992; Park 2000, 2005) colour measurements on surimi gels are highlighted or treated in special chapters (Park 2005). Whiteness, as an index for the general appearance of a surimi gel, has been introduced and can be calculated as: Whiteness = L * − 3b* or whiteness = 100 − ⎡⎣(100 − L*) + a*2 + b*2 ⎤⎦ 2

0.5

Colour changes observed in frozen-stored minced fish flesh were not considered seriously detrimental to quality. However, some colour changes could be measured. For example, deboned flesh of Alaska pollock (Theragra chalcogramma) became less dark, whereas that of shortspine thornyhead (Sebastolobus alascanus), turbot (Atherestres stomias) and dogfish (Squalus acanthias) revealed a gradual shift in the direction of yellowness (Nakayama and Yamamoto 1977). A colour measuring system for minced fish blocks was developed that was based on a reflectance spectrophotometer and Munsell neutral value standards (King and Ryan 1977). Hunter L, a, b values were used to order fish mince colour according to species or processing parameters, and mince colour differences ΔE were used to illustrate colour differences between samples. It was concluded that irrespective of the inherent difficulties of controlling the homogeneity of the prepared minces, colour matching of fish mince provides a good basis for assessing the practical limits of grading for colour (Young and Whittle 1985). Six fish species were manually processed into mince and surimi by using three washing steps. The colour of all washed minces rated better than those that were unwashed and fresh. The relatively higher L values of washed minces indicated that some of the dark pigments were removed during washing, whereas the lower a values indicated that most of the haem pigments were leached out (Eid et al. 1991). Lightness was observed to increase with increasing levels of H2O2 up to 2% in the presence of 1% sodium tripolyphosphate in fillets treated by immersion. The peroxide treatment was more effective in removing colour from the mince than from the fillets (Brown et al. 1993). Soaking in H2O2 of fish mince derived from cod and haddock flaps reduced the superficial blood discoloration of flaps and improved the colour of recovered mince after separation particularly by decreasing a and b values (Himonedes et al. 1999). Colour values of fresh minces prepared from rock sole (Lepidopsetta bilineata) and Alaska pollock did not show any significant differences. After 1 and 6 months of frozen storage both minces darkened slightly and the a* and b* values changed little (Crapo and Himelbloom 1993). When minces produced from Alaska pollock frames were compared against commercially produced fillet mince, it became obvious that frame minces were much darker, redder and yellower. Darkness increased remarkable with increasing frozen storage. Therefore frame minces would be only usable in a product blended with commercial mince (Crapo and Himelbloom 1994). Various anionic and neutral hydrocolloids were added to minced fillets of whiting, and the colour changes during frozen storage at −18 °C for three months observed. L generally decreased slightly. This was accompanied by increases in a and b (da Ponte et al. 1985). Several dairy ingredients, selected on the basis of their potential to act as cryoprotectants, have been chosen as improvers of the quality of frozen fish mince. As examples of fish with different fat and moisture contents cod, haddock, salmon and spent salmon were chosen for mincing. The results for L and h showed that the treatment effects on each of the four fish types were significant (Anese and Gormley 1996).

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Minced sardine (Sardinella aurita) flesh washed with different solutions (sodium bicarbonate and water) was characterised for colour. L* of washed sardine mince increased markedly with 0.5% NaHCO3 solution from 30.9 for unwashed mince to 49.5, whereas in all treatments a* decreased. On the other hand, b* increased in water-washed mince (Barrero and Bello 2000). Mechanical deboning of fish body resulted in mince, and its colour was significantly influenced by pre-processing of the fish. All minces except skin-on fillet mince are significantly different in colour compared with skin-off fillets (ΔE 5–15). The L* values decreased when body parts other than ordinary muscles were used for deboning. Both the a* and b* values were much higher in minces prepared from belly flaps, headed and gutted, backbones and tails (Schubring 1999a). Colour values of channel catfish frame mince were determined during storage at −20, 0 and 5 °C. Hunter L values of catfish frame mince increased after washing. L values of mince did not change during storage at 5, 0 and −20 °C. Hunter a values of catfish frame mince decreased after washing, but did not change during storage at 5, 0, and −20 °C. Washing also reduced b values of catfish frame mince. Hunter b values of catfish frame mince remained unchanged during storage at 5, 0, and −20 °C (Suvanich et al. 2000). Surimi was commercially produced from arrowtooth flounder using a continuous wash decanter process and a conventional surimi process. Colour of both surimi types was comparable, indicating the possibility of producing a commercial grade arrowtooth surimi (Babbitt et al. 1993). Conventional and counter-current leaching processes of surimi manufacture were also compared for the functionality of the surimi produced. Gels made from surimi processed by either method had equivalent colour values (Green and Lanier 1994). L* and b* taken on surimi produced from Pacific whiting (Merluccius productus) did not change during refrigerated storage for up to 5 days but decreased at day 7. However, a* did not change throughout storage. After freezing, surimi samples showed a slight change in L*, a* and b* and remained stable during 2 months of frozen storage (Pipatsattayanuwong et al. 1995). Northern squawfish (Ptychocheilus oregonensis), a freshwater fish, was tested for surimi processing. Significant differences were found in whiteness due to pre-processing freshness of the fish. This change was due primarily to an increase in b* in surimi as the fish was kept longer in ice (Lin and Morrissey 1995). When surimi was prepared with channel catfish mince recovered from fillet frame, significant differences in colour values L, a, b were found between gels prepared with washed and unwashed surimi only after the first wash. There were no additional changes in colour values after two or three washes (Kim et al. 1996). Pacific whiting frame minces were obtained by different processing methods (mechanical deboning and water jet deboning). The lightness of the surimi gels ranged from 70.8 to 75.7. After 6 months of frozen storage the water jet deboning showed significantly lower L* than the mechanical deboning gels. No significant difference in b was found among samples during frozen storage (Wendel et al. 2002). Gels prepared from pollock surimi and arrowtooth flounder (Atherestres stomias) surimi with or without protease inhibitors and 3 : 1 blends of these types of surimi were evaluated for colour. L* of cooked gels without inhibitors was lowest for pollock and highest for arrowtooth flounder. The range in a* was small, being between −1.6 and −3.4, indicating more greenish hue. The b* value for arrowtooth surimi was highest indicating more yellow hue (Reppond et al. 1993). Kamaboko was made through setting and heating procedures, and then colour values of these products were compared. Results obtained showed that the

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addition of 1% sorbitol resulted in kamaboko of high lightness, whereas addition of H2O2, ascorbic acid, sodium erythorbate and cystine improved the lightness of kamaboko. In contrast, calcium carbonate decreased the lightness of the product (Chen et al. 1999). Also, glucono-δ-lactone was found to be an effective whiteness improver for milkfish kamaboko (Chen et al. 1998). Bicarbonate salts added to both pollock and whiting surimi containing 0.3% of a phosphate blend impacted the colour. There was a tendency for the pollock surimi to become less yellow when bicarbonate salts were added (Bledsoe et al. 2000). Several non-muscle proteins were added as gelation aid to horse mackerel surimi. The L* value of kamaboko increased within 0.5 h after cooking and then decreased slightly. The greenness decreased whereas b* increased gradually. Addition of liquid egg white to kamaboko increased its L* but reduced greenness and b*. Reduction of L* was observed when the other proteins were added (Chen 2000). The colour of tropical tilapia surimi gels was compared with Alaska pollock and Pacific whiting gels. Pollock gels were generally whiter than tilapia or whiting gels, which were almost equivalent. In general, whiter gels were obtained for all species when fish proteins were treated at high temperature setting (≥40 °C) followed by 90 °C cooking (Klesk et al. 2000). The effect of chitin and chitosan with different degrees of deacetylation and concentrations on functional properties of surimi prepared from barred garfish (Hemiramphus far) were investigated. Suwari gels were obtained by incubating the sol at 25 °C for 3 h, kamaboko gels were prepared by heating at 90 °C for 20 min. For kamaboko gels, higher L and b values but a lower a value were found compared with suwari. In general, no large differences in colour between the control and gels containing chitosans were observed. No differences in L were observed between the samples with different concentrations of added chitosans. Higher a and b values were obtained in samples with different concentrations of added chitosan compared with the control. When additional CaCl2 was added, the value of L of the kamaboko gels increased compared with the control, whereas no changes in a and b were observed in all samples (Benjakul et al. 2000). Several hydrocolloids (locust bean gum, guar gum, xanthan gum, iota-carrageenan, kappacarrageenan, carboxymethylcellulose and alginate) were added in different concentrations (0.5, 1.0, 2.0, 3.0 and 4.0%) to determine their behaviour as additives in washed blue whiting (Micromesistius poutassou) muscle mince. Gel colour was virtually unaffected by the presence of different hydrocolloid concentrations in the formulation Pérez-Mateos and Montero 2000). To improve the colour of surimi made from dark-fleshed fish species, the effects of water, oil starch, CaCO3 and TiO2 were investigated. Raising the moisture or oil content of the threadfin and hairtail surimis significantly increased the L* and decreased the b* values of the gels. Potato starch decreased the b* value, but not the L* value. Calcium carbonate and titanium dioxide increased the L* value, but not the b* value. Adding water, oil or titanium dioxide was considered an effective way to whiten the colour of surimi gels (Hsu and Chiang 2002). For cod mince, 1 g TiO2/kg was considered the most acceptable level of whiteness compared with the colour of cooked cod fillet (Meacock et al. 1997). Optimum blends of different grades of surimi determined by nonlinear programming can offer a way of using low-grade surimi. The texture and colour properties of surimi gels consisting of pollock surimi (L = 86.9, b = 5.5), golden threadfin-bream surimi (L = 87.2, b = 7.6) and low-grade hairtail surimi (L = 79.6, b = 11.8) in various ratios were determined based on a mixture design. Surimi gels were produced by heating at 90 °C for 20 min with the addition of 2% NaCl. The texture and colour properties of blended surimi from various grades can

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be represented as nonlinear functions. Therefore, nonlinear programming was found to be appropriate for determining the optimum formulation for surimi products blended from various grades of surimi. About 3.3–18.8% of hairtail surimi could be used when blending with high-grade surimi to produce surimi seafood (Hsu and Chiang 2001). The colour of freeze-dried surimi powder from three marine fish species of Malaysian water were checked. Threadfin bream (Nemipterus japonicus) surimi powder showed the greatest L, followed by purple-spotted bigeye (Priacanthus tayenus) and lizardfish (Saurida tumbil), respectively. Similar results were found for a and b. The high b values indicate the surimi powders were yellowish to light brown in colour (Huda et al. 2001). To develop a new processing technique for producing paste-like fish products using lacticacid bacterial fermentation, the colour of the fermented product was evaluated. After 48 h fermentation at 37 °C, L, b and whiteness of samples with starters were significantly higher than those before fermentation and those without starter. However, the value of a of the samples with lactic-acid bacteria was significantly lower than that before fermentation and without starter (Yin et al. 2002). Proteins from herring (Clupea harengus) light muscle were extracted using acidic or alkaline solubilisation. The acid- and alkali-produced proteins made gels with equal colour. L values were 64 and 65, a values 8 and 8.1, and b values −2.4 for both gels, respectively (Undeland et al. 2002). A process based on solubilisation at pH 2.7 gives high yields of herring muscle proteins with good functionality. The stability of these protein isolates during ice storage was followed in terms of colour (a*/b* values). There was a clear loss in a* in the herring mince during acid processing, and a* decreased further during subsequent storage. Yellowness went from 7.1 in the original mince to 0.2–0.7 after processing. No significant changes took place in b* values during ice storage (Undeland et al. 2005). Six different washing treatments at acidic and alkaline pH areas for kamaboko production from sardine (Sardina pilchardus) were applied and their effect on colour of the final product evaluated. Washing had a beneficial effect on the colour of the protein concentrates resulting in higher L* values and whiteness index. Further improvements in the degree of L* and whiteness index were achieved during heating. The values of a* of the protein concentrates were in all cases negative and differentiated between treatments reaching a minimum during alkaline washing at pH 11.5, indicating high greenness (−a*). Modification of the pH during washing showed a significant effect on the b* values of the kamaboko. A marked increase in the b* values of the heat-induced gels was observed (Karayannakidis et al. 2007). Washing increased whiteness of small-scale mud carp (Cirrhiana microlepis) gel by removing sarcoplasmic proteins, including myoglobin, and to some extent blood residues containing haemoglobin. Setting temperature had no effect on the colour values of mince gel. When the whiteness is required, extensive washing is necessary to improve such an attribute (Yongsawatdigul et al. 2006). The whiteness of surimi gel from bigeye snapper (Priacanthus tayenus) added with various phosphate compounds at different levels (0–0.5% w/w) and heated under various conditions was studied. Whiteness of all surimi gels increased as the phosphate concentrations increased. Among all the gels tested, suwari gels had the lowest whiteness compared with the others (kamaboko and directly heated gel). Directly heated gel exhibited similar whiteness to kamaboko gel. Whiteness of kamaboko gel from bigeye snapper surimi added with polyphosphate in the presence of CaCl2 increased with increasing CaCl2 concentrations, regardless of polyphosphate concentration (Julavittayanukul et al. 2006).

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Colour changes during the production of low-salt restructured fish products from Mexican flounder (Cyclopsetta chittendeni) using microbial transglutaminase or whey protein concentrate as binders showed a decrease in L* whereas a* and b* increased compared with the control. L* varied from 70.9 to 78.4, a* varied from −0.9 to 0.7 and b* varied from 10.9 to 13.9 (Ramírez et al. 2006). The effect of amidated low methoxyl pectin levels on the colour of Mexican flounder mince was studied. The addition of pectin significantly increased the L* and b* values of the mince gels, whereas a* increased only slightly (Uresti et al. 2003). The effect of quercetin on the colour characteristics of fish gels, fortified with omega3 fatty acids was studied and evidenced that the addition of quercitin produced important changes in colour. L* is reduced from 71 to 65 and b* is almost doubled compared with gels containing only fish oil (7.3 versus 18.3) (Pérez-Mateos et al. 2005). Heat-induced gelling abilities of surimis prepared by pH shifting (isoelectric precipitation following acid or alkaline solubilisation) were compared with that of conventionally washed surimi. Conventionally washed surimi gels exhibited a higher whiteness than did acid or alkaline surimi gels, owing to the higher L* and lower b* and a* values. Higher whiteness was obtained in alkaline gels than in acid gels (Pérez-Mateos and Lanier 2007). Extraction and recovery of fish muscle proteins with the pH-shift process was investigated for Atlantic croaker and compared with a laboratory-scale surimi process. Although isolate pastes had higher L* than surimi pastes, surimi gels had higher L* than isolate gels. The acid-aided gels had higher levels of b* than the other treatments (Kristinsson and Liang 2006). Proteins of Cape hake (Merluccius capensis) were recovered by solubilisation of washed and unwashed mince at pH 2 and pH 12 followed by precipitation at pH 5.5. The values of L* of all recovered proteins were higher than the mince, as well as the whiteness. Also, higher values of b* were measured in the recovered proteins, with the exception of that from unwashed mince at pH 12. However, the values of a* of all recovered proteins and mince were quite similar (Batista et al. 2006). Effect of beef plasma protein and egg white on proteolysis and gelling properties of lizardfish was investigated. Addition of beef plasma protein resulted in a lower whiteness, whereas no changes in whiteness were observed with gels added with egg white. No marked differences in whiteness among samples added with beef plasma protein or egg white prepared under different heating conditions were observed (Benjakul et al. 2004). The higher whiteness was found in gels from washed mince, compared with those of unwashed mince. Gels from sardine generally showed a higher whiteness than those from mackerel. For both directly heated gels and kamaboko gels from sardine, those prepared from water washed mince showed a greater whiteness than mince washed in NaCl solution. However, no difference in whiteness between gels of water-washed mince and mince washed in NaCl solution was found in mackerel gels (Chaijan et al. 2004). Colours of Alaska pollock and Pacific whiting surimi gels were evaluated and related to compositional and physical conditions during preparation and measurements. Water addition increased L* and decreased b* of gels with all physical conditions treated. Pollock surimi was whiter than whiting surimi owing to an enzyme inhibitor in the whiting sample. Smaller size showed higher L* and b*. Room temperature setting before 90 °C slightly increased L* and b*. Gels measured at 25 °C appeared lighter than those measured at 5 °C. Room light condition at measurement did not affect L*, a* and b*. Freeze–thaw abuse darkened the colours of surimi gels. Gels had a significant reduction of L* after nine cycles. The value of a* was less affected and independent of moisture levels and other physical conditions during freeze–thaw (Park 1995).

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The colour properties of cooked surimi seafood gels fortified with omega-3 fatty acids (fish oil concentrate, menhaden oil and purified marine oil) were monitored during chilled storage for 2 months. Only minor changes in colour occurred. Initially, the control gel (no oil added) was less opaque and significantly lower in whiteness index than those fortified with oils. The increased whiteness can be attributed to the increase in L* caused by the dispersion of light resulting from the presence of emulsified oil droplets. During chilled storage, there was a tendency toward increased whiteness with increased desired level (Pérez-Mateos et al. 2004). Higher L value and whiteness were observed on samples washed with alkaline phosphate or bicarbonate buffers. The L value and whiteness of surimi samples increased with the duration of ozonation and decreasing pH. In addition, the a value decreased as the duration of ozonation increased and with the increase of pH up to 7.0. The L value and whiteness were highest in samples with 30 min ozonation at pH 3.0 and 4.0 (Jiang et al. 1998). Colour tests as affected by moisture content were evaluated for surimi from pollock, Pacific herring, arrowtooth flounder, and Pacific whiting. The L* and whiteness increased with higher moisture content, but changes in a* and b* values were not consistent for surimi from different species (Reppond and Babbitt 1997). To improve the characteristics of fish muscle, Monascus purpureus or Monascus species were inoculated in 2.1% rice powder broth containing 30% minced mackerel tissue to produce a fermented fish product. The Hunter a values of mackerel mince with M. purpureus or Monascus species was significantly higher than that without starter and increased rapidly during the fermentation time of 10 days. The increase in Hunter a was due to the secretion of pigments by Monascus during fermentation. A decrease in Hunter L value was observed on the samples with starters during fermentation. This might have been due to the accumulation of red pigments (Yin et al. 2005). Intermediate moisture fish patties were formulated from rockfish mince with additives. The mixture was dehydrated and vacuum packaged. The colour of the patties were characterised by Hunter values (L = 54.7, a = 2.3, b = 16.8) that not change significantly during storage for two months at 38 °C or −20 °C (Destura and Haard 1999).

7.4.3

Processing effect on colour of fish and fishery products

Refrigerated and frozen storage The refrigerated seawater (RSW)-stored ocean perch (Sebastes marinus) generally kept their skin colour better than the iced fish. The values for a were generally higher for the RSWstored than for the iced fish. Of the two treatments with RSW, that with CO2 tended to preserve higher a values. L and b did not appear to change appreciably from the original values of about 50 and 12, respectively (Longard and Regier 1974). Thornyhead rockfish (Sebastolobus alascanus) were treated with mixed tocopherols, alone or with ascorbic acid (TCAA), butylated hydroxytoluene and sodium etythorbate to preserve red skin colour during frozen storage. Although all treatments significantly improved colour retention compared with the control, at 4 month the TCAA-treated fish had significantly higher red colour scores than any other treatment group. However, there were no significant differences in measured redness of rockfish among the tocopherol without ascorbic acid, butylated hydroxytoluene

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and erythorbate groups. The control rockfish, on the other hand, had lost almost all red colour after four months (Wasson et al. 1991). The effectiveness of natural antioxidant extracted from shrimp waste on colour stability of red species of rockfish was evaluated. After 8 days refrigerated storage, samples treated with either crude extract or sodium erythorbate apparently maintained more skin redness than controls in which a* values decreased from initial values of 19.5 ± 1.8 to 10.9 ± 1.2. The effectiveness of antioxidant extracts from shrimp waste was lower than sodium erythorbate, a commercial antioxidant, which has been commercially used to stabilise the red skin colour (Li et al. 1998). The effect of modified atmosphere packaging (MAP) on colour of commercial Pacific red snapper (Sebastes entomelas, S. flavidus or S. goodei) during storage at 2 °C was investigated. L values decreased in all samples over time. The a values were significantly lower in each treatment after seven days, and no differences in b were observed between treatments. ΔE values were significantly higher in air than in the gas tri-mix at day 7. ΔE values were significantly higher in the gas tri-mix than in air at day 21. It was concluded that there was a significant colour change in air at the first stage and later in the gas tri-mix during storage (Gerdes and Santos Valdez 1991). When colour changes during iced storage of redfish (Sebastes marinus) onboard were monitored, it became obvious that L* increased whereby head side was generally brighter than tail side of fillet. In b*, the same tendencies could be noticed, whereas a* decreased with storage and increased in the direction from head to tail. Immediately (within 1–3 hours) after the fish has been caught in demersal species, for example cod, haddock and redfish, L* in fillets increased along the body axis from head to tail, whereas in pelagic fish like mackerel and herring it decreased in the same direction. Values of a* appeared to increase in fillets in both demersal and pelagic fish, whereas b* increased in demersal and decreased in pelagic species along the body axis from head to tail (Schubring 1999b). Colour measured onboard a research vessel on the skin of iced-stored whole fish showed, when using sardine as well as horse mackerel, no significant differences between specimens stored in flake ice and those stored in ice slurry. Also, when measurements were taken on muscle homogenates or later ashore on frozen thawed samples, colour values were not significantly different between flake ice and ice slurry samples (Schubring and Meyer 2006a, b). Fresh gutted cod stored in ice underwent significant changes in the colour values measured ventrally (L*) and dorsally (a* and b*) on skin. Lightness and redness showed a good linear relationship, with both the quality index method (QIM) value and the sensory value for appearance of the skin. The regression factors were highest for L* versus QIM and appearance of the skin. However, when measurements were taken on thawed fish during subsequent storage in ice or on thawed fish after prolonged frozen storage, colour changes were less pronounced and the correlation with both QIM and appearance of the skin was weak (Schubring 2003). Coloration of the squid Illex illecebrosus was investigated by evaluating the surface colour as well as relating post mortem colour decline to quality characteristics. Using five different storage methods (held at the same temperature), it was concluded that the rate of colour decline was dependent on the method of storage but the decline in quality was not. Even though colour cannot always be used as a quality determinant, the retention of colour is necessary if this product is to be exported in the frozen, round state. Squid stored in aqueous environments showed more rapid colour loss than those stored in contact icing or non-contact

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icing (Hincks and Stanley 1985). It was found that squid skin colour changes to white during ice-cold storage, and it was suggested that such a skin colour change is most likely accelerated by chilling and hypoxia during storage. The avoidance of direct touch with ice causing rapid chilling and the supply of fresh air for aerobic energy metabolism in squid skin would be efficient in preventing fresh squid from becoming an inappropriate commercial value loss by fading skin colour (Okada et al. 2004). Colour measurements during iced storage of ray (Raja clavata) revealed a slight reduction in L, whereas a and b hardly underwent any variation. However, measurements taken on cooked muscle showed higher L and b values, which did not change very much during storage. The parameter that best defined the colour changes that occurred on the surface of iced stored ray wings was L (Pastoriza and Sampedro 1994). Dip treatment in solutions at 4 °C in 2% acetic, citric, hydrochloric, lactic, malic or tartratic acid of catfish fillets caused lighter and yellower colour of fillets than untreated controls, with malic acid producing the least bleaching (Bal’a and Marshall 1998). The effects of sodium acetate, sodium lactate and propyl gallate were determined on colour of filmoverwrapped or vacuum-skin packaged catfish fillets. Products were stored at 4 °C up to 12 days. No significant differences on L, a or b values of catfish fillets in the same packaging method were observed, regardless of treatment (Zhuang et al. 1996). Mackerel fillets were stored under CO2 MAP at −2 °C for 21 days. A significant increase was found in the L and b values at 14 days for raw mackerel fillets, indicating that fillets were becoming lighter from CO2 bleaching of colour pigments and more yellow. In addition, the increase in b was supported by related sensory findings. The a values for raw CO2 MAP mackerel fillets showed significant changes between 7 and 21 days’ storage, indicating a general increase in a. However, after cooking, no significant differences were found in L, a and b for the CO2 MAP mackerel fillets (Hong et al. 1996). The effects on colour attributes achieved by washing black tilapia (Oreochromis mossambicus) flesh with a banana-leaf ash solution were investigated. There were highly significant differences in L when treated fillets were compared with untreated fillets. L values increased with the increase in supernatant strength and washing time. Values of a decreased significantly for samples washed with supernatant. All treated samples were not significantly different among each other in b but they were significantly different from the control. However, cooking the fillets reduced the difference, making it not significant (Mohsin et al. 1999). Catfish fillets containing 2-methylisoborneol were vacuum-tumbled in 0, 0.5 and 2% citric acid solutions, or left untreated. Water alone increased lightness of fish from an L* value of 60.6 to 65.8. Fish tumbled in 2% acid showed significantly greater L* values, followed by the 0.5% acid-treated, water-treated fish and then the untreated samples. Fish tumbled with water were significantly lower in a* and b* than the other treatment groups. The overall colour difference (from the control) increased with increased acid concentration (Forrester et al. 2002). The colour stability of muscle of rainbow trout fed ketocarotenoids stored at 4 °C under vacuum-packaging conditions was studied. The decrease in carotenoid concentrations was not reflected by colour parameters. No significant differences were noted for C* and h* with increasing storage. Colour stability of rainbow trout fillets was studied during a 4 week storage at 4 °C under controlled and modified atmospheres under 100% air, 60 : 40 N2 : CO2 mix and 60 : 40 air : CO2 mix. Fillets from fish fed high-fat diets showed higher C* and higher a* and b* colour parameters than those from fish fed low-fat diets. Storage time increased L* and h* in controlled atmospheres but only L* under modified atmospheres. After storage

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at 4 °C, values of L* of fish fillets stored under modified atmospheres were lower than those stored under controlled atmospheres. Carotenoid source resulted in differences in C* and h* of fish fillet stored under controlled and modified atmospheres. Dietary lipid levels resulted in differences in chroma under controlled atmospheres. Under controlled atmospheres the lower differences between stored-initial values was for N2/CO2 and the higher for air. Under modified atmospheres, air/CO2 and N2/CO2 gave similar results for L*, C* and h*. Experiments demonstrated that colour parameters of fish fillets reacted differently according to gas mixture and storage time (Choubert and Baccaunaud et al. 2006). Flesh portions of king salmon were stored in air packs (0 °C) for 22 days and colour changes evaluated. Colour of the frame sides had significantly higher a* and b* values and lower L* values than the skin sides. L* did not change with time. The a* and b* values did not vary with harvest method, but a* varied with time, with the skin side showing a linear decrease with time (Fletcher et al. 2003). Quality changes of farmed halibut (Hippoglossus hippoglossus) stored on ice for 26 days were evaluated. Fish were fed diets that differed only in the source of lipid. The L* value of the cutlets increased significantly during the first 6 days of storage, and the same was true for the b* value. The negative a* value showed that the cutlets had a greenish colour, and this value decreased significantly between day 4 and day 6. These early colour changes are probably related to muscle structure alterations during rigor and the rigor resolution. The colour parameters did not change significantly after day 6, which agrees with the results from the sensory evaluation of whiteness (Guillerm-Regost et al. 2006). However, significant differences for L* between days 5 and 11 were observed (Gobantes et al. 1998). Channel catfish (Ictalurus punctatus) fillet strips were packaged under aerobic, 25% CO2 and 80% CO2 environments, with the remainder being air, and stored at 2 and 8 °C for 4 weeks. Hunter L values increased in CO2-packaged samples, whereas Hunter a values decreased (Silva and White 1994). The shelf life of MAP and vacuum-packaged fillets of Atlantic halibut after dietary supplementation with α-tocopheryl acetate was evaluated. After slaughter, fillet portions were stored for 11 days at 2 °C in MAP and vacuum packed at −20 °C for 6 weeks. Although colour of fillets of all treatments significantly deteriorated in storage, discoloration was not very obvious. A significant increase in L was observed in chilled MAP fillets. This was also evident, to a lesser extent although significant, in vacuum-packed frozen fillets of Atlantic halibut. The significant decrease in h in chilled MAP and vacuum-packed frozen storage was accompanied by a similar decrease in C. Regression analysis of colour parameters, measured during 11 days of chilled MAP, showed that h and C were best related to Hunter b values. With increasing b, h dropped below 180 and C increased (Ruff et al. 2003). The effect of MAP on quality changes of gutted farmed bass when stored at 3 °C was investigated for up to 9 days. Gutted farmed bass was packed with six different atmospheres (0% O2/ 70% CO2 (A); 20% O2/70% CO2 (B); 30% O2/60% CO2 (C); 40% O2/60% CO2 (D); 30% O2/50% CO2 (E); 21% O2/0% CO2 (F)). During storage time, colorimetric parameters related to the bass abdomen and stomach did not change, whereas those related to the flesh did and their values at different storage times showed some changes. Only a* and b* were affected by the storage time, whereas L* was independent on time. The atmosphere composition had a significant effect on all coordinates. In particular, the parameter a* decreased during storage when bass was packed by using C, D and E, whereas it did not change when bass was packed by A and B. The same was observed in the samples packed by using air as

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initial gas composition. Parameter b* increased during storage in the case of the atmosphere B, C and E, but decreased or remained constant in the other cases (Torrieri et al. 2006). Colour changes in European sea bass fillets packed under 40% CO2 : 60% N2 (MAP) and air (AIR) or prepared from the whole ungutted fish stored in ice (ROUND) were compared. MAP and ROUND fillets had higher L* values at the beginning and at the end of the storage period compared with the AIR fillets; ROUND fillets in turn had higher a* and b* than MAP and AIR fillets, respectively, on the sixth and the second day after packaging, whereas MAP and AIR fillets did not differ between them. No difference emerged for whiteness among the differently preserved fillets (Poli et al. 2006). To investigate the reducing ability of met-myoglobin (metMb) reductase, browned tuna meat was immersed with/without metMb reductase for 120 min at 4 °C. Changes in metMb reductase activity, metMb, Hunter’s a value and colour for both the control and treated samples suggested that immersion in metMb reductase could extend the colour stability of tuna meat, and that the enzymatic reduction of metMb by its reductase occurred during refrigerated storage (Chiou et al. 2001). Hunter values of the various derivates from bluefin tuna Mb were as follows, for L, a, b, respectively (Ochiai et al. 1988): deoxyMb (magenta) 46.4, 43.0, 12.2; oxyMb (bright red) 49.7, 47.6, 26.4; carboxyMb (deep pink) 49.9, 55.5, 21.7; metMb (brown) 57.5, 15.5, 27.7; cyanmetMb (orange) 45.2, 32.3, 26.4. The effect of fasting on colour of ordinary muscles in full-cycle cultured Pacific bluefin tuna (Thunnus orientalis) during chilled storage was evaluated. The colour values (L*, a* and b*) of dorsal ordinary muscles of post-fasting group (post-FG) tuna were lower than for pre-fasting group (pre-FG) tuna throughout the storage period. However, the changes in the values of L* and a* of ventral ordinary muscles of pre- and post-FG tuna throughout the storage period were similar despite showing differences, and the changes in the b* value of ventral ordinary muscle of post-FG tuna were higher than for pre-FG tuna (Nakamura et al. 2006). The redness index (a*/b* ratio) of iced-stored sardine and mackerel muscles decreased when the storage time increased. The redness index of washed mince was lower than that of unwashed mince. This result suggested that some pigments, especially myoglobin and haemoglobin, were removed, leading to the lowered redness. The redness index of washed mince, either with NaCl solution or distilled water, from both species, decreased during the first 6 days of iced storage (Chaijan et al. 2005). Changes in colour measured on yellowtail (Seriola quinqueradiata) muscles during ice storage revealed that the a* value was significantly higher for the dark muscle than the ordinary muscle at 0 days of storage and subsequently decreased significantly during 2 days of ice storage. The b* value tended to increase in both ordinary and dark muscles throughout the storage time. The L* value changed only slightly during 2 days of ice storage. Changes in colour tones of the ordinary and dark muscles during 4 days of ice storage were different for different fish species investigated. No discoloration of the ordinary muscle of Japanese butterfish (Hyperoglyphe japonica) occurred. The colour of ordinary muscle in the Japanese Spanish mackerel (Scomberomorus niphonius) changed slightly from white to pink, and in both Pacific saury (Cololabis saira) and chub mackerel (Scomber japonicus) from light pink to dark brown. A drastic colour change from reddish brown to dark brown in the dark muscles of Japanese butterfish, Pacific saury, Japanese Spanish mackerel and chub mackerel was seen after 4 days of ice storage (Sohn et al. 2005). The Hunter a value decreased from 18.7 to 13.2 during 120 h storage at 4 °C of tuna meat. Progressive browning was also observed during 120 h storage. The Hunter a of both wrapped and unwrapped samples decreased, whereas L of both samples

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significantly increased during storage at 4 °C and was higher in unwrapped tuna than in wrapped samples after 120 h storage (Pong et al. 2000). A process to enhance shelf life of the freshwater fish rohu (Labeo rohita) on ice using combination treatment of coating the fish steaks with gel dispersion from the same fish and low-dose gamma irradiation has been described. Dispersion treatment either alone or in combination with irradiation, however, had some adverse effect on the colour of the product. Although irradiation alone did not bleach the colour, irradiation in combination with dispersion coating resulted in bleaching of the pink colour of the flesh, as observed by higher (>50) Hunter L values. However, bleaching of pigments was prevented by incorporation of antioxidants, namely, butylated hydroxyanisole (BHA) or ascorbic acid at 0.5% (w/v) in the dispersion. Alternatively, vacuum packaging also prevented bleaching of the colour (Panchavarnam et al. 2003). Around 35 years ago, the first instrumental measurements of colour accompanied frozen storage of fish of different initial freshness to indicate changes in quality. A so-called colour ratio (CR) was determined. Although for frozen stored cod a linear relationship was found between CR and storage time dependent on storage temperature, this method did not work well with haddock (Connell and Howgate 1968, 1969). The effects of brine-freezing and plate-freezing at sea on colour were determined at intervals during subsequent frozen storage at −30 °C. It was found that the Hunter a/b ratio for Chinook salmon showed a significant interaction with freezing method and storage time (Botta et al. 1973). Weakfish (Cynoscion regalis) were harvested seasonally for a 12 month period to determine the chemical composition and frozen storage (−18 °C) stability of fillets. One pound (≈0.454 kg) blocks were prepared, frozen and evaluated after 0, 3, 6 and 12 months’ storage. The colour values (L, a, b) for all product forms did not change significantly between months of harvest or during storage (Waters 1983). Frozen storage (3 and 6 months at −20 and −80 °C) of vacuum-packed fillets of rainbow trout resulted in increased L*, a* and b*, and decreased h* values. Colour characteristics from different parts of the fillet differed significantly (No and Storebakken 1991). During frozen storage and after thawing of yellowtail dark muscle the b/a ratio was used as an indicator of browning progress. When the value of b/a exceeds approximately 0.5, browning is only slight; when the ratio exceeds 0.8, browning becomes non-merchantable. It was found that browning of muscle after thawing occurred faster than the discoloration of unfrozen meat during ice storage. Thawed fish lost its commercial value in a short time (Hiraoka et al. 2004). The same ratio b/a was also used when packaging with nitrogen gas was investigated for preventing discoloration of red meat fish (Oka 1989). Rainbow trout fillets cut from fish fed diets containing synthetic astaxanthin or synthetic canthaxanthin had significantly higher a* values after 90 days’ frozen storage than fresh fish, fish frozen for 180 days or fish frozen for 90 days thawed and refrozen for 90 days. No significant differences in a* were found as a result of storage temperature (−18, −28 or −80 °C) (Scott et al. 1994). The cryoprotective effectiveness of sucrose/sorbitol and sodium lactate on colour in frozen rainbow trout fillets, with or without phosphate and MgCl2, was evaluated. For fresh fillets soaked in solutions of cryoprotectants, significantly higher values of L* were observed compared with water-soaked fillets. Values of L* decreased significantly after frozen storage of the non-soaked treatment. After frozen storage, fillets soaked in cryoprotectant solution had lower values of L* than those soaked in water, but no difference was observed between

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non-soaked treatments before and after frozen storage. Fresh fillets soaked in cryoprotectants had lower values of a* and b* than the water control. Frozen storage decreased a*, but did not affect b*. Sucrose/sorbitol resulted in the lowest b* in fresh and frozen samples. Phosphate decreased L* in fresh and frozen samples. MgCl2 did not affect muscle colour for fresh and frozen fillets (Jittinandana et al. 2003). The effects of dietary oil source and frozen storage on flesh colour of Atlantic salmon were evaluated. L* and b* values of fish fed capelin oil were significantly lower than the others, and a* values of fish fed Peruvian fish oil were significantly different from those of fish fed rapeseed oil and soybean oil in raw fillets. Important increases of L*, a*, b* or C* values were observed during frozen storage. Significant interactions between dietary treatment and frozen storage were observed on a*, b* and C* values. Strong correlations were observed between L*, a*, b* or C* values and storage time (Regost et al. 2004). Colour changes of wild salmon and farmed rainbow trout from aquaculture, both packed in transparent vacuum-skin packaging, were followed during storage at −17 °C surface temperature for 6 months in an illuminated freezer cabinet. After three weeks of storage a sudden increase of L was observed caused by a pronounced dehydration of the surface. A subsequent slow decrease in a was most significant for rainbow trout (Andersen et al. 1990). Double-frozen blocks of fillets, produced by thawing frozen gutted fish and refreezing it after filleting and skinning, have become, apart from at-sea frozen blocks, increasingly common in international trade, double freezing being a practice used for decades. For saithe, cod and haddock, fillet colour was found to be affected by both the rigor state and double freezing. The tendencies, however, are not always clear. Irrespective of the rigor state, double freezing mainly caused a significant increase in both L* and b*. However, some exceptions hindered the use of colour changes for discriminating single from double frozen fillets (Schubring 1999b, 2000, 2001a, b, 2002a). Surprisingly, when redfish was used, in almost all samples the colour difference was weak between single-frozen and double-frozen portions of fillets (Schubring 2005b). In frozen-stored herring, there was a large variation in colour within each fillet and between fillets in the same group. During freezing, the amount of yellow colour increased substantially and the amount of red colour increased in the posterior end of the fillets, causing a visible change in appearance. Thereafter, only minor changes occurred until week 10 but, in week 30, the fillets had become less red, causing a less fresh and greyer appearance. Compared with the within-fillet and within-group variations, there were only minor effects of ascorbic acid treatment on colour. It is suggested that shelf life of frozen herring fillets should be set at between 9 and 14 weeks at −30 °C. Treatment with ascorbic acid will not alleviate the discoloration of frozen herring fillets (Hamre et al. 2003a). Seasonal development of colour of fillets from Norwegian spring-spawning herring was investigated. The fillets were lighter, less red and more yellow at the anterior than the posterior end. Further, they tended to become darker and redder with progress of the season. At the anterior end, the fillets also became more yellow with time. The development towards darker and redder fillets was correlated with the decreases in lipid and dry matter contents, whereas the development of yellowness was coupled to dry matter content, only. It was concluded that the colour deterioration in the fillets was not due to lipid oxidation (Hamre et al. 2003b). The effect of long-term frozen storage up to 13 months at three different storage temperatures (−10 or −14, −20 and −28 or −30 °C) on colour of individually packed fish fillets were

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assessed. Only during storage at −10 and −14 °C and somewhat lower at −20 °C a noticeable increase in L* could be observed whereas L* during storage at −28 or −30 °C did not change. A comparable behaviour was found in b* (Schubring 2004, 2005a). Freeze-chilling performs equally to chilling and gives a shelf life of about three days at 4 °C for pre-packaged raw haddock and mackerel fillets. Freeze-chilling, freezing or chilling had no effect on lightness, and the L values were the same as those of the fresh fillets. Fresh mackerel fillets, however, had higher L/b values than the freeze-chilled, frozen or chilled samples, which did not differ among each other were still highly acceptable visually (Fagan et al. 2002). MAP was combined with freeze-chilling to extend the shelf life of raw whiting, mackerel and salmon fillets/portions. MAP had no effect on Hunter L or a values. Hunter b values showed air-packed whiting and salmon portions to be less yellow than those in 100% CO2 (Fagan et al. 2004). Freeze-chilling involves freezing and frozen storage followed by thawing and chilled storage. The effects of the four treatments on the colour of the raw samples (whiting, mackerel and salmon) were small in practical terms (Fagan et al. 2003). The effectiveness of sucrose/sorbitol, trehalose, trehalose/sorbitol, and sodium lactate as cryoprotectants in stabilising the quality of restructured trout products were compared during frozen storage. After 6 months of storage, control and sodium-lactate-treated products were lighter than carbohydrate-treated products. A difference in a* among cryoprotectant treatments was not detected after 6 months of frozen storage. After 6 months of storage, carbohydrate-treated products were less yellow than control and sodium-lactate-treated products. After 6 months of storage, L* of cooked products were not different, cooked product containing sodium lactate was least red, and control and sodium lactate products were more yellow than trehalose/sorbitol products. Prolonged storage to 6 months did not affect L* and a* of control and carbohydrate-containing products (Jittinandana et al. 2005a). After each freeze–thaw cycle, raw products from control and sodium lactate treatments were lighter than raw products from carbohydrate treatments. As the number of freeze–thaw cycles increased, the a* value of cryoprotectant-treated raw samples did not change. After each freeze–thaw cycle, raw products containing carbohydrates were less yellow than control product. In general, carbohydrate-treated products were less red and less yellow, and this response corresponded to lower L* values compared with raw, control and sodium lactate products. Cryoprotectant treatment effects on colour attributes were due to differences in moisture content caused by cryoprotectant addition to the formulation (Jittinandana et al. 2005b). The changes in colour during frozen storage were also slowed down by DE 18 maltodextrin or the combination of sucrose and sorbitol. This was shown by measurements of the Hunter parameters. The values for b were significantly lower and the values for a significantly higher in the treated samples than in the control during most of the period of storage at −10 and −20 °C. Slight differences between treated samples and control were also found for L, which occasionally became significant at −10 °C (Rodríguez-Herrera et al. 2006). Using under-utilised fish, fish burgers from two different formulas were developed and the colour was evaluated during storage at −20 °C for 3 months. L values did not show significant changes in both formulas. Furthermore, no significant changes in a and b were observed during frozen storage (Al-Bulushi et al. 2005). The colour of brown shrimp (Crangon crangon) is influenced by freezing/thawing as well as frozen storage (15 weeks). Both L* and a* values decreased significantly with increasing storage time. However, a

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decrease in b* is caused by freezing followed by an increase in b* during frozen storage to about the same level as for the non-frozen shrimps (Schubring 2002b). Thermal processing (heating and smoking) Already in 1968 a paper had been been published on instrumental colour measurement taken on radiation-sterilised pre-fried cod and halibut patties. The breaded pre-fried products were irradiated at 4.5 Mrad, stored for 12 months at 22 °C and evaluated by colour reflectance measurement. Pre-fried patties became lighter in colour after irradiation. The halibut patties were lighter than the cod patties initially, after irradiation and after storage (Sinnhuber et al. 1968). Research on colorimetry of salmon has been concerned mainly with the colour of the canned product. An overview on early activities in this respect has been given (Francis and Clydesdale 1971). Pre- and post mortem handling of fish (skipjack tuna) did not influence significantly the reflectance values of the canned fish. Differences between samples must therefore be ascribed to individual variation (Little 1972). When the effect of species and formulation on colour characteristics of catfish, ocean perch and Alaska pollock during thermal processing was investigated it was shown that changes in colour values during canning were different between species. L values increased for catfish whereas it decreased for ocean perch and Alaska pollock after thermal processing. The values of a and b increased during canning for all species investigated. The formulation of canned fish products had no effect on colour (Paredes and Baker 1988). Most changes in Hunter colour difference values due to pre-canning tripolyphosphate treatments of thermally processed fresh and frozen mullet were insignificant except for b values. The L and a values were not significantly different between treatments or between fish processed fresh or processed after frozen storage, whereas b values increased slightly due to treatments and when using frozen fish for canning (English et al. 1988). Blue crab meat became darker with increasing heating process; crab harvest location had significant effect on L values of the flesh; and meat that is located in the bottom of a can was darker than that in the top (Requena et al. 1999). Muscle tissue of Pacific chum salmon was heated in water (60–100 °C) for 0–40 min. Increase in processing temperature or time increased L*, but decreased a* and b* of muscle (Bhattacharya et al. 1994). Cooking increased the L value and decreased a and b values for all aquacultured fish fillets analysed (pacu, rainbow trout, hybrid striped bass, catfish and tilapia). The highest amount of change in L was observed in catfish and the least change in hybrid striped bass (Pullela et al. 2000). Some undesirable distinction accompanying the canning of red-fleshed fish, shellfish and crustaceans such as browning, greening, blue discoloration and blackening are described (Ledward 1992). The colour of raw, baked and cold smoked flesh from cultured and wild salmon was compared. The value of a* of raw, baked and smoked salmon increased with increasing carotenoid concentration of the raw flesh, whereas L* decreased. The b* value was not significantly influenced by carotenoid concentration. Baking and smoking caused a* to decrease, whereas L* increased (Skreede and Storebakken 1986a). Colour of wild, astaxanthin pigmented, and farmed, canthaxanthin pigmented, Atlantic salmon was instrumentally evaluated. No significant differences in colour between wild and farmed salmon were found. The values of a* and h* in raw and baked salmon flesh and the a* in smoked salmon were correlated to the pigment concentration in raw salmon. The values of a* and h* in processed salmon were predictable from the a* and h* of raw flesh (Skreede and Storebakken 1986b). Instrumental colour of raw baked

Colour measurement

153

and smoked rainbow trout flesh was related to the carotenoid concentration of the raw flesh. Highly significant relationships between sensory perceived colour and instrumental assessed colour were found (Skrede et al. 1989). The colour and colour stability of smoked fillets during chill storage were not related to the duration of frozen storage before the smoking of fillets. Smoked fillets from fish fed the lowest level of astaxanthin (40 mg/kg) had significantly higher L and lower a and b values than smoked products from fish fed 100 mg astaxanthin/kg. Smoked products from fish fed 32% fat had significantly higher L and lower a and b values than the smoked fillets produced from fish fed the diet with the lowest fat level (27%). Only small changes in colour were found during chill storage (Jensen et al. 1998). Seven treatments were applied on fresh or frozen raw material (ocean ranched salmon and farmed salmon) combining dry or brine salting with cold smoking at 20 or 30 °C. Electrostatic smoking was tested on dry-salted salmon fillets. Before processing, ocean-ranched fish never fed pigments had lower L*, a* and b* values than the two farmed fish groups. There was no significant difference in colour parameters between the two reared fish groups. Smoking led to a reduction of a* values, an increase of b* values regardless of the raw material, and a reduction of L* values (mainly for farmed fish). Ocean-ranched salmon showed the lowest a* and b* values. Smoking temperature, however, did not affect a* and b* in the same way. With a 30 °C smoking temperature, b* values were greater than a* values, whereas with smoking at 20 °C, a* values were higher. Regardless of the raw material, b* values for raw and smoked fillets were higher when fish had been frozen. Despite the low smoking intensity for samples treated by the electrostatic technique, L* was low compared with other samples. No colour difference between samples was observed relative to the salting technique (Cardinal et al. 2001). Colour changes were studied in the muscle of smoked rainbow trout fed diet supplemented with canthaxanthin in combination with three different lipid levels. The cold smoked fish were packed under three different packaging conditions. Smoke-curing lead to a decrease of L* and an increase of h* more marked in fish fed the diet with high lipid level. The use of MAP for the packaging of fillets maintained the colour of the flesh compared with packaging under vacuum or under air (Quinones et al. 2003). The effects of salting method (injection salting compared with dry salting), smoking temperature (20 °C compared with 30 °C) and storage (chilled storage compared with no storage) on surface coloration of cold smoked Atlantic salmon fillets was investigated. Both salting method and smoking temperature had significant effects on changes in colorimetric characteristics. The changes in b*, h* and ΔE* were considerably higher than changes in a*, L* and C*. There was a significantly higher increase in b* and C* and higher h* and ΔE* of dry salted than injection-salted fillets. Changes in L* and a* were not influenced significantly by salting method. The drop in a* was significantly higher when fillets were smoked at 20 °C than at 30 °C, whereas the increase in C* was higher when fillets were smoked at 20 °C than at 30 °C. Changes in L*, b*, and h* were not affected significantly by smoking temperature, but the ΔE* was higher when fillets were smoked at 30 °C than at 20 °C. No interaction effect between salting method and smoking temperature was observed in colour parameter changes (Birkeland et al. 2004). Packages of cold smoked Atlantic salmon were sampled in a French hypermarket. Sliced salmon were originally from fish grown in Norway, Scotland and Ireland. There was a significant effect of country of origin on instrumentally measured colour, with the Irish having higher values (a*, 15.0; b*, 18.0; C*, 23.5 and h*, 50.3) than the Norwegian (a*, 12.0;

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b*, 12.4; C*, 17.3 and h*, 45.7), with the values of the Scottish fish in-between (a*, 13.4; b*, 14.9; C*, 20.0 and h*, 48.0). The appearance of the fish beside the shelf life was the most important criterion when choosing a package of cold smoked salmon in the shop (Espe et al. 2004a; Rørå et al. 2004). When exposed to light changes in colour parameters at the surface of sliced smoked rainbow trout fillets occurred. Significant changes were noted for L* and h*: storage time effect was more pronounced than light effect, L* increased with time and the slices became paler. Nevertheless, the pattern changes in Δh* was different for fillets stored in the light and those stored in the dark (Choubert et al. 2005). On smoked Norwegian salmon slides the influence of a freeze/thaw cycle was evaluated. Already after a short frozen storage of 8 hours a significant colour difference in comparison to the unfrozen sample was observable. This was manifested by an increase in lightness as well as in redness and yellowness as result of the freeze/thaw cycle (Schubring and Oehlenschläger 1996). The instrumentally measured values for a*, b*, h* and C* increased significantly with increasing fat content but were not significantly correlated to the estimated area of fat deposits. Significant reductions in all colour parameters were observed from raw to smoked fillets. There was a lack of agreement between the colour measurements and sensory evaluation of colour in relation to fat content (Rørå et al. 1998). Effects of cold-smoking temperature (range 21.5–29.9 °C) and dietary oil sources (pure Peruvian fish oil or pure soybean oil supplements) on colour characteristics were determined in Atlantic salmon. No significant effects of temperature were observed for L*, a*, h*, C*. Only b* values exhibited significant correlations with temperature. Dietary oil source only had significant effects on a* and C* values during storage of the cold-smoked fillets. Atlantic salmon fed diets with the fish oil were slightly redder than salmon fed soybean oil. Furthermore, the fish oil group had significantly higher C* than the soybean oil group (Rørå et al. 2005). Studying the effects of sodium tripolyphosphate in brine on smoke adsorption and colour of cold smoked mullet (Mugil cephalus) it was observed that Hunter L, reflecting smoke adsorption, was lightest for the control fillets, followed by the 5% salt treatment, and darkest for 5 and 10% sodium tripolyphosphate both with 5% salt (Antoine et al. 2000). A new fish-smoking process was applied using a combination of liquid smoke and steaming at pressures up to 1 bar above atmospheric. All instrumental colour variables were affected by the steam pressure conditions. L* reduced due to increasing pressure and to drying process before processing. All fillets processed at 2 bar steam pressure showed higher b* values than those processed at 1 and 1.5 bar. The increase of pressure conditions seemed to produce fillets with a reverse trend in a*; however no significant differences were detected. These results indicated that increasing pressure conditions led to more yellow and less red final products (Siskos et al. 2005). When steam blanching (5 min), water blanching (1 min) and microwave heating (5 W/g, 2 min) were applied to de-salted cod it was found that thermally treated cod were lighter and slightly yellower. Heat-induced changes in cod were a little smaller than in microwaveheated cod (Fernandez-Segovia et al. 2003). The colour of heavily salted cod as influenced by adjusted levels of calcium, magnesium and pH in the salt was investigated. The pH value of the muscle correlated negatively with the lightness of the cured cod muscle. Calcium and magnesium ions increased the lightness of the cured fillet (Lauritzsen et al. 2004). Colour analysis of skinless catfish fillets obtained from steam-treated catfish revealed no colour differences (L, a, b and whiteness) between steam-treated and control fillets (Bal’a et al.

Colour measurement

155

78 68

L*

a*

b*

CIELAB values

58 48 38 28 18 8 –2 untreated

30

35 40 60 Heating temperature (°C)

65

70

Figure 7.3 Changes of CIELAB values taken on ordinary muscle of rainbow trout as affected by elevated temperature.

2000). The combined effects of sous vide cooking and exudate from the fish (into the sauce) caused a loss of L, a and b in the fish/sauce packs. Sauce colour lightened on sous vide cooking, as indicated by a rise in L/b mean values from 1.8 to 2.0 (Fagan and Gormley 2005). To assess the influence of heating the fish muscle (rainbow trout) on colour, the fresh fish were cut transversally to the backbone into cutlets approximately 2 cm thick. They were packed in cooking bags and heated in a water bath at given temperatures in the range 30– 70 °C until core temperature was equal that of the water bath. Temperature was recorded during heating. Cutlets were left for at least 5 min at final temperature and subsequently cooled down using ice-cold water. Among the colour values, L* was most influenced by heating. Redness did not change markedly whereas b* increased slightly at higher temperatures (Figure 7.3). Increase in L* appeared almost linear up to 60 °C without further changes due to subsequent heating, possibly caused by the almost complete denaturation of muscle proteins at this temperature. As can be seen in Figure 7.4, not only the fish muscle but also muscle of shrimps (Parapenaeus longirostris) underwent colour changes when heated. The patterns are almost comparable to that observed on fish muscle (Figure 7.3). The colour parameters, L*, a* and b* were determined on rainbow trout muscle with a portable spectrophotometer before irradiation (3 h post mortem) and after irradiation (60Co source, a dose of 3 kGy and a dose rate of 3.33 kGy h-1) (5 h post mortem). A significant difference in L* at the time before and after exposure was found. The value of a* was identical, and b* decreased (Dvorák et al. 2005). Instrumental colour measurements on terrestrial snails (spiced-butter preparation) indicated strong differences in L* between Helix aspersa and the others (H. pomatia, H. lucorum and Achatina fulica). Furthermore, H. aspersa differed in a* from the other snail species. Differences in b*, C* and h* were less meaningful. These results were in good agreement with the sensory panel’s, where almost no differences in chroma could be detected among the different snail products. Colour differences, ΔE*, between H. aspersa and the others were very strong (Schubring and Meyer 2002).

156

Fishery Products: Quality, safety and authenticity 65 60 L*

CIE values

55 50 45

b*

6 5 4 3 2 1

none

a*

30

35 40 50 55 Thermal treatment (°C)

60

70

Figure 7.4 Changes of CIELAB values taken on deepwater rose shrimp as affected by elevated temperature.

High-pressure processing Several pelagic and demersal species from different fishing grounds were repeatedly pressure-treated using the same conditions. The results were quite comparable, especially for sardine and pollack (Pollachius pollachius). The influence of storage time post mortem was obviously marginal because colour differences measured on black sea bream (Spondyliosoma cantharus) pre-rigor and post-rigor were almost the same. Colour changes measured on demersal fish caused by pressure treatment resulted from a marked increase in L* and from decreases in both a* and b*. From the species investigated, the flesh of red gunard (Chelidonichthys cuculus) seemed to be most sensitive to colour changes caused by pressure treatment. The flying squid (Todarodes sagittatus) was also used for a long-term pressurisation experiment. The prolongation of holding time from 60 to 900 min did not result in any further changes of ΔE* (Schubring et al. 2005). High-pressure treatments (>150–200 MPa, 5 min) resulted in a cooked appearance of pollack (Pollachius virens), mackerel (Scomber scombrus), tuna (Thunnus thynnus), cod (Gadus morhua), salmon trout (Salmon trutta), carp (Cyprinus carpio), plaice (Pleuronectes platessa) and anglerfish (Lophius piscatorius). Only octopus (Octopus vulgaris) retained a raw appearance until 400–800 MPa (Matser et al. 2000). When bluefish (Pomatomus saltatrix) pastes were processed into gels by heating (60 °C, 60 min) or by pressure treatment (370 MPa, 30 min) with addition of bovine serum extracts containing α2-macroglobulin, L* increased whereas a* and b* decreased. Heat-induced gels had higher L* and b* values but lower a* values than their counterparts formulated by pressure. During iced storage of the gels for 21 days, significant changes, particularly in L* and a*, could be observed (Sareevoravitkul et al. 1996). When high pressure (100–300 MPa) was applied (for 0–30 min) to fresh seafood to control enzyme-related texture, it became obvious that certain time–pressure combination treatments of the fish muscle resulted in significant changes of colour. Colour became lighter with increasing pressure, presenting an increasingly cooked appearance to the flesh. L values showed progressive increase with both the amount of pressure applied and the duration of

Colour measurement

157

pressure application. The b values showed a similar trend whereas a values were reduced with amount of pressure and holding time. ΔE values showed that there was no significant difference in colour between sample treatments up to 200 MPa for 10 min. Beyond this limit the changes were quite significant (Ashie and Simpson 1996). Changes in colour of turbot (Scophthalmus maximus) fillets during frozen storage at −20 °C were followed for pressure-shift and air-blast freezing. Pressure-shift freezing resulted in overall increase in L* and b* values and decrease in a*. Frozen storage did not particularly modify colour parameters of pressure-shift freezing fillets, which were stable during storage period. It was noticed that air-blast freezing did not give a cooked aspect after thawing to the turbot fillets. During the storage of air-blast freezing fillets, the decrease in L* was confirmed after 15 days of storage. However, from 30 days of storage, no significant difference appeared with the control samples. The b* values increased significantly along the storage period (Chevalier et al. 2000). One of the most obvious quality changes caused by high-pressure thawing was in colour. Colour differences measured on both high-pressure and conventionally thawed raw or cooked samples (redfish, cod, rainbow trout, whiting, haddock and salmon) indicate a strong influence of high-pressure treatment. Especially in raw fillets, significant colour changes (verified by very strong colour differences, ΔE*) can be seen, mainly caused by a strong increase in L*. Smaller but also uniform changes were monitored for both a* (decrease) and b* (increase). After heat treatment, the influence of high pressure on colour was obviously much smaller. ΔE* between cooked fillets previously thawed, either by high-pressure treatment or conventionally, varied from negligible to significant (Schubring et al. 2003). Comparable results were recently reported for another muscle food, pressure-assisted thawed pork. There were no differences in colour at ≤100 MPa; however, both L* and b* increased significantly from 150 MPa whereas a* decreased (Park et al. 2006). Atlantic salmon samples were frozen by conventional air freezing, plate freezing and liquid nitrogen freezing, and subjected to different thawing treatments: water immersion thawing (4 and 20 °C) and high-pressure thawing at 100, 150 and 200 MPa with water (containing 2 g oil/100 g) as pressure medium at 20 °C. The high-pressure thawing product at 100 MPa had a value of L* similar to that of control and water immersion thawing samples (0.1 MPa). Only slight discoloration in high-pressure thawing samples at 150 MPa was recognised with the naked eye, but the value of L* was found to be significantly higher than that of control and high-pressure thawing samples at 100 MPa. High-pressure thawing samples at 200 MPa looked pale white as in cooked fish, and resulted in a very high L*. a* and b* varied among different thawing treatments, but the difference was much less than observed with L*. The maximum values of a* and b* appeared at 150 MPa rather than at 200 MPa. Freezing process had little impact on overall colour change in fish samples (Zhu et al. 2004). Carp (Cyprinus carpio) muscles were exposed to high pressures of 50, 100, 300, 400 and 500 MPa for 10 min at room temperature. Carp muscles lost their transparency and showed increased L values parallel with an increase of pressurisation. Only slight colour differences were observed at 100 MPa; however, these differences were appreciable at 300 MPa and remarkable at 500 MPa. Colour differences in heated samples were even greater (Yoshioka and Yamamoto-Munoz et al. 1998). Raw carp fillets were vacuum packed and pressurised at 100, 140, 180 and 200 MPa at 4 °C for 15 and 20 min, and then monitored for changes in colour. The value of L* remained essentially unchanged at 100 MPa regardless of processing time. However, at 140 MPa and above, L* increased with pressure. Similarly, for a given pressure level, L* increased as processing time increased. The values of a* increased as

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pressure increased. The values of b* increased with pressure and with holding times at pressure levels of 140 MPa and above. The value of ΔE* also reflected the progressive colour changes with increasing pressure and pressure-holding times (Sequeira-Munoz et al. 2006). The effect of high-pressure treatment up to 500 MPa for 5 min was studied on physical characteristics of sea bass fillets after 0, 7 and 14 days of refrigerated storage. The storage of non-pressurised fillets led to an increase in L* for a storage time of 7 days, then L* decreased for 14 days of storage. The values of a* and b* values remained almost constant during refrigerated storage. Whatever pressure level, application of pressure on fillet induces an increase in L* and a slight change of h* (Chéret et al. 2005). Fish proteins solubilised with 2.5% salt and 4, 8 and 12% sucrose, sorbitol, trehalose or 1 : 1 mixtures of sucrose– sorbitol and trehalose–sorbitol were subjected to 600 MPa for 5 min. In raw pressuretreated samples, L* and a* ranged from 66.9 to 73.9 and from −2.72 to −1.53, respectively. Sugars and sorbitol at the concentrations tested induced small or no changes in L*, a* and b* colour attributes. The small a* and b* values for all samples indicate that fish paste remained in the greyish colour zone and thus changes in C* and h* values would have low perceptibility (Uresti et al. 2005a). Cod sausages with added chitosan (1.5%) from shrimp shells were obtained by highpressure treatment (350 MPa/7 °C/15 min). With high-pressure treatment, the value of L* increased from about 32 to 34 units in the batters to about 40 units in the pressurised sausage; however, b* underwent a slight decrease, with values ranging from approximately 4.5 in the batter without any added chitosan to 5 in the batters with it. No significant differences in L* were found between the sausages containing chitosan and the control sausages at any time during the storage period. Conversely, b* increased with the addition of chitosan, especially when the chitosan was incorporated in soluble form (López-Caballero et al. 2005). The effect of high-pressure treatments at 400 and 600 MPa for 1 and 5 min on colour of heat-induced fish gels obtained from arrowtooth flounder fish-paste was evaluated. Three thermal treatments of pressure treated and control samples were evaluated: 90 °C for 15 min (kamaboko); 40 °C for 30 min plus 90 °C for 15 min (setting); 60 °C for 30 min plus 90 °C for 15 min (modori). L* values ranged from 76.6 to 81.7, a* varied from −1.37 to −0.69, b* varied from 6.0 to 10.9, C* from 6.1 to 10.9 and h* from 94.8 to 101.2. Modori samples showed the lowest L* values. Changes in a* and b* were observed in the same samples. Hue values were not modified by pressure treatment. The low values of C* indicate that all samples remained in the greyish zone. C* values were slightly higher in samples incubated at 60 °C, suggesting that the modori phenomenon modifies this colour attribute (Uresti et al. 2005b). Restructured fish products from arrowtooth flounder were obtained by hydrostatic pressure processing at 400 and 600 MPa with 0–5 min pressure-holding time. Raw and cooked (90 °C, 15 min) pressure-treated gels were characterised by changes in colour. When the pressure holding time was zero, no differences were observed between L* values for raw fish paste and samples treated at 400 or 600 MPa. At both pressure levels, the L* parameter increased at 1 min pressure-holding time, but no further increases were detected when it was further increased to 5 min. The increase in L* was larger at 600 MPa. The value of a* increased slightly in pressure-treated samples. Raw fish paste had a higher b* value than pressure-treated samples, indicating a decrease in b*. The L* value increased from 69.8 in the non-pressured fish paste to 80.5 in the cooked non-pressured sample. The value of a* decreased from −0.7 in the fish paste to −0.9 to −1.2 in pressured products, but there were no differences between cooked pressured and cooked non-pressured gels. The value of b*

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increased from 4.7 in the fish paste to 8.8 in the cooked non-pressured gels. This parameter was significantly higher (p < 0.05) in cooked pressure treated samples (400 and 600 MPa) than in the fish paste, but lower than for the cooked non-pressured sample. The increase in L* and a* values was lower than in cooked samples of pressured and non-pressured fish paste, and in the former b* decreased while increasing in the latter (Uresti et al. 2004). Alaska pollock and Pacific whiting surimi gels containing potato starch and/or egg white were induced with 400 and 650 MPa for 10 min at 20 °C and compared with heat-induced gels (90 °C, 40 min). Pressure treatment improved whiteness of surimi gels compared with heat-treated surimi gels, whereas additives did not. The whiteness of Alaska pollock surimi gels varied from 67.2% to 76.7%, whereas the whiteness of Pacific whiting surimi gels varied from 62.3% to 76.9%. At 400 MPa, whiteness values were 10% higher than heated gels. However, at 650 MPa, whiteness increased 8% (Tabilo-Munizaga and Barbosa-Cánovas 2004). Minced albacore muscle was treated with high hydrostatic pressure at 275 and 310 MPa for 2, 4 and 6 min and the effects on shelf life at 4 and −20 °C were studied. All changes in L*, a* and b* resulted in a lighter and whiter product as pressure/holding time were incremented. L* increased with pressure applied, giving a lighter product, a* decreased and b* increased, giving a light yellow/greyish hue, cooked appearance as the higher hue values showed. Storage temperature affected the whiteness of minced pressurised tuna, as both temperatures showed a tendency to increase, always showing higher values than control. On the contrary, hue value always showed a decrease toward the end of both experiments owing to the increase in a* (Ramirez-Suarez and Morrissey 2006).

7.5

Summary

The overview given here indicates the importance of colour measurement in the evaluation of quality and safety in fish processing. One of the advantages is that the measuring technique is not expensive and can therefore be applied widely.

7.6

References

Akhtar, P., Gray, J.I., Cooper, T.H., Garling, D.L. and Booren, A.M. (1999) Dietary pigmentation and deposition of alpha-tocopherol and carotenoids in rainbow trout muscle and liver tissue. Journal of Food Science 64: 234–239. Aksnes, A., Gjerde, B. and Roald, S.O. (1986) Biological, chemical and organoleptic changes during maturation of farmed Atlantic salmon, Salmo salar. Aquaculture 53: 7–20. Al-Bulushi, I.M., Kasapis, S., Al-Oufi, H. and Al-Mamari, S. (2005) Evaluating the quality and storage stability of fish burgers during frozen storage. Fisheries Science 71: 648–654. Andersen, H.J., Bertelsen, G., Christophersen, A.G., Ohlen, A. and Skibsted, L.H. (1990) Development of rancidity in salmonoid steaks during retail display. A comparison of practical storage life of wild salmon and farmed rainbow trout. Zeitschrift für Lebensmittel-Untersuchung und -Forschung 191: 119–122. Anese, M. and Gormley, R. (1996) Effects of dairy ingredients on some chemical, physico-chemical and functional properties of minced fish during freezing and frozen storage. LebensmittelWissenschaft und -Technologie 29: 151–157.

160

Fishery Products: Quality, safety and authenticity

Antoine, F.R., Marshall, M.R., Sims, C.A., O’Keefe, S.F. and Wei, C.I. (2000) Phosphate pretreatment on smoke adsorption of cold smoked mullet (Mugil cephalus). Journal of Aquatic Food Product Technology 9: 69–81. Ashie, I.N.A. and Simpson, B.K. (1996) Application of high hydrostatic pressure to control enzyme related fresh seafood texture deterioration. Food Research International 29: 569–575. Baardseth, P., Skrede, G., Naes, T., Thomassen, M.S., Iversen, A. and Kaaber, L. (1988) A comparison of CIE (1976) L*a*b* values obtained from two different instruments on several food commodities. Journal of Food Science 53: 1737–1742. Babbitt, J.K., Reppond, K.D., Kamath, G., Hardy, A.C., Pook, C.J., Kokubu, N. and Berntsen, S. (1993) Evaluation of processes for producing arrowtooth flounder surimi. Journal of Aquatic Food Product Technology 2: 89–95. Baker, R.T.M., Pfeiffer, A.-M., Schöner, F.-J. and Smith-Lemmon, L. (2002) Pigmenting efficacy of astaxanthin and canthaxanthin in fresh-water reared Atlantic salmon, Salmo salar. Animal Feed Science Technology 99: 97–106. Bal’a, M.F.A. and Marshall, D.L. (1998) Organic acid dipping of catfish fillets: effect on color, microbial load, and Listeria monocytogenes. Journal of Food Protection 61: 1470–1474, 1998. Bal’a, M.F.A., Podalak, R. and Marshall, D.L. (2000) Microbial and color quality of fillets obtained from steam-pasteurized deheaded and eviscerated whole catfish. Food Microbiology 17: 625–631. Balaban, M.O., Kristinsson, H.G. and Otwell, W.S. (2005) Evaluation of color parameters in a machine vision analysis of carbon monoxide-treated fish. Part I: fresh tuna. Journal of Aquatic Food Product Technology 14: 5–24. Barrero, M. and Bello, R.E. (2000) Characterisation of sardine minced flesh (Sardinella aurita) washed with different solutions. Journal of Aquatic Food Product Technology 9: 105–114. Batista, I., Pires, C., Nelhas, R. and Godinho, V. (2006) Acid and alkaline-aided protein recovery from Cape hake by-products. In: J.B. Luten, C. Jacobsen, K. Bekaert, A. Sæbø and J. Oehlenschläger (Eds) Seafood Research from Fish to Dish. Quality, Safety and Processing of Wild and Farmed Fish. Wageningen Academic Publishers, Wageningen, pp. 427–437. Benjakul, S., Visessanguan, W., Tanaka, M., Ishizaki, S., Suthidham, R. and Sungpech, O. (2000) Effect of chitin and chitosan on gelling properties of surimi from barred garfish (Hemiramphurus far). Journal of the Science of Food and Agriculture 81: 102–108. Benjakul, S., Visessanguan, W., Tueksuban, J. and Tanaka, M. (2004) Effect of some protein additives on proteolysis and gel-forming ability of lizardfish (Saurida tumbil). Food Hydrocolloids 18: 395–401. Bhattacharya, S., Choudhury, G.S. and Studebaker, S. (1994) Color changes during thermal processing of Pacific chum salmon. Journal of Aquatic Food Product Technology 3: 39–48. Birkeland, S., Haarstad, I. and Bjerkeng, B. (2004) Effects of salt-curing procedure and smoking temperature on astaxanthin stability in smoked salmon. Journal of Food Science 69: FEP198–FEP203. Bjerkeng, B., Refstie, S., Fjalestad, K.T., Storebakken, T., Rodbotten, M. and Roem, A.J. (1997) Quality parameters of the flesh of Atlantic salmon (Salmo salar) as affected by dietary fat content and full-fat soybean meal as a partial substitute for fish meal in the diet. Aquaculture 157: 297–309. Bjørnevik, M., Karlsen, Ø., Johnston, I.A. and Kiessling, A. (2003) Effect of sustained exercise on white muscle structure and flesh quality in farmed cod (Gadus morhua L.). Aquaculture Research 34: 55–64. Bjørnevik, M., Espe, M., Beattie, C., Nortvedt, R. and Kiessling, A. (2004) Temporal variation in muscle fibre area, gaping, texture, colour and collagen in triploid and diploid Atlantic salmon (Salmo salar L). Journal of the Science of Food and Agriculture 84: 530–540. Blanc, J.M. and Choubert, G. (1993) Genetic variation of flesh color in pan-size rainbow trout fed astaxanthin. Journal of Applied Aquaculture 2: 115–123.

Colour measurement

161

Bledsoe, G.E., Rasco, B.A. and Piggott, G.M. (2000) The affect of bicarbonate salt addition on the gel forming properties of Alaska pollock (Theragra chalcogramma) and Pacific whiting (Merluccius productus) surimi. Journal of Aquatic Food Product Technology 9: 31–45. Botta, J.R., Richards, J.F. and Tomlinson, N. (1973) Flesh pH, color, thaw drip, and mineral concentration of Pacific halibut (Hippoglossus stenolepis) and chinook salmon (Concorhynchus tshawytscha) frozen at sea. Journal of the Fisheries Research Board of Canada 30: 71–77. Brown, P., Rasco, B.A. and Borhan, M. (1993) Color removal from the dark muscle of Alaskan pollock (Theragra chalcogramma) fillets and minces using peroxide. Journal of Aquatic Food Product Technology 2: 125–134. Buttle, L.G., Crampton, V.O. and Williams, P.D. (2001) The effect of feed pigment type on flesh pigment deposition and colour in farmed Atlantic salmon, Salmo salar L. Aquaculture Research 32: 103–111. Cakli, S., Dincer, T., Cadun, A., Saka S. and Firat, K. (2006) Seasonal variation of proximate and fatty acid class composition of wild and cultured Brown Meagre (Sciena umbra), a new species for aquaculture. In: J.B. Luten, C. Jacobsen, K. Bekaert, A. Sæbø and J. Oehlenschläger (Eds) Seafood Research from Fish to Dish. Quality, Safety and Processing of Wild and Farmed Fish. Wageningen Academic Publishers, Wageningen, pp. 469–476. Cardinal, M., Knockaert, C., Torrissen, O., Sigurgisladottir, S., Mørkøre, T., Thomassen, M. and Vallet, J.L. (2001) Relation of smoking parameters to the yield, colour and sensory quality of smoked salmon (Salmo salar). Food Research International 34: 537–550. Chaijan, M., Benjakul, S., Visessanguan, W. and Faustman, C. (2004) Characteristics and gel properties of muscles from sardine (Sardinella gibbosa) and mackerel (Rastrelliger kanagurta) caught in Thailand. Food Research International 37: 1021–1030. Chaijan, M., Benjakul, S., Visessanguan, W. and Faustman, C. (2005) Changes of pigments and color in sardine (Sardinella gibbosa) and mackerel (Rastrelliger kanagurta) muscle during iced storage. Food Chemistry 93: 607–617. Chatzifotis, S., Pavlidis, M., Jimeno, C.D., Vardanis, G., Sterioti, A. and Divanach, P. (2005) The effect of different carotenoid sources on skin coloration of cultured red porgy (Pagrus pagrus). Aquaculture Research 36: 1517–1525. Chen, H.H. (2000) Effect of non-muscle protein on the thermogelation of horse mackerel surimi and the resultant cooking tolerance of kamaboko. Fisheries Science 66: 783–788. Chen, W.-L., Chow, C.-J. and Ochiai, Y. (1998) Effects of acid and alkaline reagents on the color and gel-forming ability of milkfish kamaboko. Fisheries Science 64: 160–163. Chen, W.-L., Chow, C.-J. and Ochiai, Y. (1999) Effects of some food additives on the gel-forming ability and colour of milkfish meat paste. Fisheries Science 65: 777–783. Chéret, R., Chapleau, N., Delbarre-Ladrat, C., Verrez-Bagnis, V. and de Lamballerie, M. (2005) Effects of high pressure on texture and microstructure of sea bass (Dicentrarchus labrax L.) fillets. Journal of Food Science 70: E477–E483. Chevalier, D., Sequeira-Munoz, A., Le Bail, A., Simpson, B.K. and Ghoul, M. (2000) Effect of pressure shift freezing, air-blast freezing and storage on some biochemical and physical properties of turbot (Scophthalmus maximus). Lebensmittel-Wissenschaft und -Technologie 33: 570–577. Chiou, T.-K., Pong, C.-Y., Nieh, F.-P. and Jiang, S.-T. (2001) Effect of met-myoglobin reductase on the color stability of blue fin tuna during refrigerated storage. Fisheries Science 67: 694–702. Choubert, G. and Baccaunaud, M. (2006) Colour changes of fillets of rainbow trout (Oncorhynchus mykiss W.) fed astaxanthin or canthaxanthin during storage under controlled or modified atmosphere. Lebensmittel-Wissenschaft und -Technologie 39: 1203–1213. Choubert, G. and Heinrich, O. (1993) Carotenoid pigments of the green alga Haematococcus pluvialis: assay on rainbow trout, Oncorhynchus mykiss, pigmentation in comparison with synthetic astaxanthin and canthaxanthin. Aquaculture 112: 217–226.

162

Fishery Products: Quality, safety and authenticity

Choubert, G., Blanc, J.-M. and Courvalin, C. (1992) Muscle carotenoid content and colour of farmed rainbow trout fed astaxanthin or canthaxanthin as affected by coking and smoke-curing procedures. International Journal of Food Science and Technology 27: 277–284. Choubert, G., Blanc J.-M. and Valle, F. (1997) Colour measurement, using the CIELCH colour space, of muscle of rainbow trout, Oncorhynchus mykiss (Walbaum), fed astaxanthin: effects of family, ploidy, sex, and location of reading. Aquaculture Research 28: 15–22. Choubert, G., Dentella, E., Atgié, C. and Baccaunaud, M. (2005) Effect of light on colour stability of sliced smoked rainbow trout Oncorhynchus mykiss fed astaxanthin. Food Research International 38: 949–952. Choubert, G., Mendes-Pinto, M.M. and Morais, R. (2006) Pigmenting efficacy of astaxanthin fed to rainbow trout Oncorhynchus mykiss: effect of dietary astaxanthin and lipid sources. Aquaculture 257: 429–436. Christiansen, R., Struksnaes, G., Estermann, R. and Torrissen, O.J. (1995) Assessment of flesh colour in Atlantic salmon, Salmo salar L. Aquaculture Research 26: 311–321. Clydesdale, F. M. (1991) Color perception and food quality. Journal of Food Quality 14: 61–74. Connell, J.J. and Howgate, P.F. (1968) Sensory and objective measurements of quality of frozen stored cod of different initial freshnesses. Journal of the Science of Food and Agriculture 19: 342–354. Connell, J.J. and Howgate, P.F. (1969) Sensory and objective measurements of the quality of frozen stored haddock of different initial freshness. Journal of the Science of Food and Agriculture 20: 469–476. Coral, G., Huberman, A., de la Lanza, G. and Monroy-Ruiz, J. (1998) Muscle pigmentation of rainbow trout (Oncorhynchus mykiss) fed on oil-extracted pigment from langostilla (Pleuroncodes planipes) compared with two commercial sources of astaxanthin. Journal of Aquatic Food Product Technology 7: 31–45. Crapo, C. and Himelbloom, B. (1993) Quality and stability of mince from undersized rock sole (Lepidopsetta bilineata). Journal of Aquatic Food Product Technology 2: 47–54. Crapo, C. and Himelbloom, B. (1994) Quality of mince from Alaska pollock (Theragra chalcogramma) frames. Journal of Aquatic Food Product Technology 3: 7–17. Da Ponte, D.J.B., Roozen, J.P. and Pilnik, W. (1985) Effects of additions on the stability of frozen stored minced fillets of whiting. II. Various anionic and neutral hydrocolloids. Journal of Food Quality 8: 183–198. Destura, F.I. and Haard, N.F. (1999) Development of intermediate moisture fish patties from minced rockfish meat (Sebastes sp.). Journal of Aquatic Food Product Technology 8: 77–94. Di Natale, C. (2003) Data Fusion in MUSTEC: Towards the definition of an Artificial Quality Index. In: J.B. Luten, J. Oehlenschläger and G. Olafsdottir (Eds) Quality of Fish from Catch to Consumer: Labelling, Monitoring and Traceability. Wageningen: Wageningen Academic Publishers, pp. 273–282. Downham, A. and Collins, P. (2000) Colouring our foods in the last and next millennium. International Journal of Food Science Technology 35: 5–22. D’Souza, N., Skonberg, D. I., Camire, M.E., Guthrie, K.E., Malison, J. and Lima, L. (2005) Influence of dietary genistein levels on tissue genistein deposition and on the physical, chemical, and sensory quality of rainbow trout, Oncorhynchus mykiss. Journal of Agricultural and Food Chemistry 53: 3631–3636. D’Souza, N., Skonberg, D.I., Stone, D.A.J. and Brown, P.B. (2006) Effect of soybean meal-based diets on the product quality of rainbow trout fillets. Journal of Food Science 71: S337–S342. Dvorák, P., Kratochvíl, B. and Grolichová, M. (2005) Changes of colour and pH in fish musculature after ionising radiation exposure. European Food Research Technology 220: 309–311. Eid, N., Dashti, B. and Sawaya, W. (1991) Sub-tropical fish by-catch for surimi processing. Lebensmittel-Wissenschaft und -Technologie 24: 103–106.

Colour measurement

163

Einen, O. and Thomassen, M.S. (1998) Starvation prior to slaughter in Atlantic salmon (Salmo salar) II. in raw and cooked fillets. Aquaculture 169: 37–53. English, P.M., Gerdes, D.L., Finerty, M.W. and Grodner, R.M. (1988) Effects of tripolyphosphate dips on the quality of thermally processed mullet (Mugil cephalus). Journal of Food Science 53: 1319–1321. Espe, M., Kiessling, A., Lunestad, B.-T., Torrissen, O.J. and Rørå, A.M.B. (2004a) Quality of cold smoked salmon collected in one French hypermarket during a period of 1 year. LebensmittelWissenschaft und -Technologie 37: 627–638. Espe, M., Ruohonen, K., Bjørnevik, M., Frøyland, L., Nortvedt, R. and Kiessling, A. (2004b) Interactions between ice storage time, collagen composition, gaping and textural properties in farmed salmon muscle harvested at different times of the year. Aquaculture 240: 489–504. Fagan, J.D. and Gormley, T.R. (2005) Effect of sous vide cooking, with freezing, on selected quality parameters of seven fish species in a range of sauces. European Food Research Technology 220: 299–304. Fagan, J.D., Gormley, T.R. and Uí Mhuircheartaigh, M.M. (2002) Freeze-chill technology for raw whiting and mackerel fillets. Farm & Food 12: 14–17. Fagan, J.D., Gormley, T.R. and Mhuircheartaigh, M.U. (2003) Effect of freeze-chilling, in comparison with fresh, chilling and freezing, on some quality parameters of raw whiting, mackerel and salmon portions. Lebensmittel-Wissenschaft und -Technologie 36: 647–655. Fagan, J.D., Gormley, T.R. and Uí Mhuircheartaigh, M.M. (2004) Effect of modified atmosphere packaging with freeze-chilling on some quality parameters of raw whiting, mackerel and salmon portions. Innovative Food Science and Emerging Technologies 5: 205–214. Fernandez-Segovia, I., Camacho, M.M., Martinez-Navarrete, N., Escriche, I. and Chiralt, A. (2003) Structure and color changes due to thermal treatments in desalted cod. Journal of Food Processing and Preservation 27: 465–474. Fletcher, G.C., Corrigan, V.K., Summers, G., Leonard, M.J., Jerrett, A.R. and Black, S.E. (2003) Spoilage of rested harvested King salmon (Oncorhynchus tshawytscha). Journal of Food Science 68: 2810–2816. Forrester, P.N., Prinyawiwatkul, W., Godber, J.S. and Plhak, L.C. (2002) Treatment of catfish fillets with citric acid causes reduction of 2-methylisoborneol, but not musty flavour. Journal of Food Science 67: 2615–2618. Francis, F.J. and Clydesdale, F.M. (1971) Colour measurements of foods: XXX salmon. Food Product Development 5: 33–36, 38, 46. Gerdes, D.L. and Santos Valdez, C. (1991) Modified atmosphere packaging of commercial Pacific red snapper (Sebastes entomelas, Sebastes flavidus, or Sebastes goodei). Lebensmittel-Wissenschaft und -Technologie 24: 256–258. Ginés, R., Afonso, J.M., Argüello, A., Zamorano, M.J. and López, J.L. (2004a) The effects of long-day photoperiod on growth, body composition and skin colour in immature gilthead sea bream (Sparus aurata L.). Aquaculture Research 35: 1207–1212. Ginés, R., Valdimarsdottir, T., Sveinsdottir, K. and Thorarensen, H. (2004b) Effects of rearing temperature and strain on sensory characteristics, texture, colour and fat of Arctic charr (Salvelinus alpinus). Food Quality and Preference 15: 177–185. Gobantes, I., Choubert, G. and Gomez, R. (1998) Quality of pigmented (astaxanthin and canthaxanthin) rainbow trout (Oncorhynchus mykiss) fillets stored under vacuum packaging during chilled storage. Journal of Agricultural and Food Chemistry 46: 4358–4362. Green, D.P. and Lanier, T.C. (1999) Comparison of conventional and countercurrent leaching processes of surimi manufacture. Journal of Aquatic Food Product Technology 8: 45–58. Guillerm-Regost, C., Haugen, T., Nortvedt, R., Carlehög, M., Lunestad, B. T., Kiessling, A. and Rørå, A.M.B. (2006) Quality characterization of farmed Atlantic halibut during ice storage. Journal of Food Science 71: S83–S90.

164

Fishery Products: Quality, safety and authenticity

Hamre, K., Lie, O. and Sandnes, K. (2003a) Development of lipid oxidation and flesh colour in frozen stored fillets of Norwegian spring-spawning herring (Clupea harengus L.). Effects of treatment with ascorbic acid. Food Chemistry 82: 447–453. Hamre, K., Lie, O. and Sandnes, K. (2003b) Seasonal development of nutrient composition, lipid oxidation and colour of fillets from Norwegian spring-spawning herring (Clupea harengus L.). Food Chemistry 82: 441–446. Hatlen, B., Jobling, M. and Bjerkeng, B. (1998) Relationships between carotenoid concentration and colour of fillets of Arctic charr, Salvelinus alpinus (L.), fed astaxanthin. Aquaculture Research 29: 191–202. Himonides, A.T., Taylor, K.D.A. and Knowles, M.J. (1999) The improved whitening of cod and haddock flaps using hydrogen peroxide. Journal of the Science of Food and Agriculture 79: 845–850. Hincks, M.J. and Stanley, D.W. (1985) Colour measurement of the squid Illex illecebrosus and its relationship to quality and chromatophore ultrastructure. Canadian Institute of Food Science and Technology Journal 18: 233–241. Hiraoka, Y., Ohsaka, E., Narita, K., Yamabe, K. and Seki, N. (2004) Preventive method of color deterioration of yellowtail dark muscle during frozen storage and post thawing. Fisheries Science 70: 1130–1136. Hong, L.C., Leblanc, E.L., Hawrysh, Z.J. and Hardin, R.T. (1996) Quality of Atlantic mackerel (Scomber scombrus L.) fillets during modified atmosphere storage. Journal of Food Science 61: 646–651. Hsu, C.-K. and Chiang, B.-H. (2001) Optimum blends of different grades of surimi determined by non-linear programming. Fisheries Science 67: 500–505. Hsu, C.-K. and Chiang, B.-H. (2002) Effects of water, oil, starch, calcium carbonate and titanium dioxide on the colour and texture of threadfin and hairtail surimi gels. International Journal of Food Science Technology 37: 387–393. Huda, N., Abdullah, A. and Babji, A.S. (2001) Functional properties of surimi powder from three Malaysian marine fish. International Journal of Food Science Technology 36: 401–406. Hutchings, J.B. (1994) Food Colour and Appearance. Blackie Academic and Professional, Glasgow, 513 pp. Hutchings, J.B. (1999) Food Color and Appearance. Aspen Publ. Inc., Gaithersburg, 610 pp. Hutchings, J.B. (2003) Expectations and the food industry: the impact of color and appearance. Kluwer Academic/Plenum Publishers, Dordrecht, The Netherlands, 198 pp. Ingle de la Mora, G., Arredondo-Figueroa, J.L., Ponce-Palafox, J.T., Barriga-Soca, I.D.L.A. and Vernon-Carter, J.E. (2006) Comparison of red chilli (Capsicum annuum) oleoresin and astaxanthin on rainbow trout (Oncorhynchus mykiss) fillet pigmentation. Aquaculture 258: 487–495. Jakobsen, M. and Bertelsen, G. (2002) Modelling colour stabillity in meat. In: D.B. MacDougall (Ed.) Colour in Food: Improving Quality. Woodhead Publishing Ltd., Abington, Cambridge, pp. 233–247. Jankowska, B., Zakes, Z., Zmijewski, T. and Szczepkowski, M. (2003) A comparison of selected quality features of the tissue and slaughter yield of wild and cultivated pikeperch Sander lucioperca (L.). European Food Research Technology 217: 401–405. Jensen, C., Birk, E., Jokumsen, A., Skibsted, L.H. and Bertelsen, G. (1998) Effect of dietary levels of fat, α-tocopherol and astaxanthin on colour and lipid oxidation during storage of frozen rainbow trout (Oncorhynchus mykiss) and during chill storage of smoked trout. Zeitschrift für LebensmittelUntersuchung und -Forschung A 207: 189–196. Jiang, S.-T., Ho, M.-L., Jiang, S.-H., Lo, L. and Chen, H.-C. (1998) Color and quality of mackerel surimi as affected by alkaline washing and ozonation. Journal of Food Science 63: 652–655. Jittinandana, S., Kenney, P.B. and Slider, S.D. (2005a) Cryoprotectants affect physical properties of restructured trout during frozen storage. Journal of Food Science 70: C35–C42.

Colour measurement

165

Jittinandana, S., Kenney, P.B. and Slider, S.D. (2005b) Cryoprotectants preserve quality of restructured trout products following freeze thaw cycling. Journal of Muscle Foods 16: 354–378. Jittinandana, S., Kenney, P.B., Slider, S.D. and Kiser, R.A. (2003) Cryoprotection of frozen trout fillets for smoked trout production. Journal of Aquatic Food Product Technology 12: 3–21. Julavittayanukul, O., Benjakul, S. and Visessanguan, W. (2006) Effect of phosphate compounds on gel-forming ability of surimi from bigeye snapper (Priacanthus tayenus). Food Hydrocolloids 20: 1153–1163. Kalinowski, C.T., Robaina, L.E., Fernández-Palacios, H., Schuchardt, D. and Izquierdo, M. S. (2005) Effect of different carotenoid sources and their dietary levels on red porgy (Pagrus pagrus) growth and skin colour. Aquaculture 244: 223–231. Karayannakidis, P.D., Zotos, A., Petridis, D. and Taylor, K.D.A. (2007) The effect of initial wash at acidic and alkaline pHs on the properties of protein concentrate (kamaboko) products from sardine (Sardina pilchardus) samples. Journal of Food Engineering 78: 775–783. Kiessling, A., Espe, M., Ruohonen, K. and Mørkøre, T. (2004) Texture, gaping and colour of fresh and frozen Atlantic salmon flesh as affected by pre-slaughter iso-eugenol or CO2 anaesthesia. Aquaculture 236: 645–657. Kim, J.M., Liu, C.H., Eun, J.B., Park, J.W., Oshimi, R., Hayashi, K., Ott, B., Aramaki, T., Sekine, M., Horikita, Y., Fujimoto, K., Aikawa, T., Welch, L. and Long, R. (1996) Surimi from fillet frames of channel catfish. Journal of Food Science 61: 428–431. King, F.J. and Ryan, J.J. (1977) Development of a color measuring system for minced fish blocks. Marine Fisheries Review 39: 18–23. Klesk, K., Yongsawatdigul, J., Park, J.W., Viratchakul, S. and Virulhakul, P. (2000) Gel forming ability of tropical tilapia surimi as compared with Alaska pollock and Pacific whiting surimi. Journal of Aquatic Food Product Technology 9: 91–104. Kress-Rogers, E. (1993) Instrumentation and Sensors for the Food Industry. Butterworth Heinemann, Oxford, 780 pp. Kress-Rogers, E. and Brimelow, C.J.B. (2001) Instrumentation and Sensors for the Food Industry. Woodhead Publishing Ltd., Abington, Cambridge, 836 pp. Kristinsson, H.G. and Liang, Y. (2006) Effect of pH-shift processing and surimi processing on Atlantic croaker (Micropogonias undulates) muscle proteins. Journal of Food Science 71: C304–C312. Kristoffersen, S., Tobiassen, T., Esaiassen, M., Olsson, G.B., Godvik, L.A., Seppola, M.A. and Olsen, R.L. (2006) Effects of pre-rigor filleting on quality aspects of Atlantic cod (Gadus morhua L.). Aquaculture Research 37: 1556–1564. Korel, F., Luzuriaga, D.A. and Balaban, M.Ö. (2001) Objective quality assessment of raw tilapia (Oreochromis niloticus) fillets using electronic nose and machine vision. Journal of Food Science 66: 1018–1024. Lanier, T.C. and Lee, C.M. (1992) Surimi Technology. Marcel Dekker Inc., New York, pp. 528. Lauritzsen, K., Akse, L., Gundersen, B. and Olsen, R.L. (2004) Effects of calcium, magnesium and pH during salt curing of cod (Gadus morhua L.). Journal of the Science of Food and Agriculture 84: 683–692. Lawless, H.T. and Heymann, H. (1998) Sensory Evaluation of Food. Principles and Practices. Chapman & Hall, New York, pp. 379–405. Ledward, D.A. (1992) Colour of raw and cooked meat. In: D.E. Johnston, M.K. Knight, D.A. Ledward (Eds) The Chemistry of Muscle-Based Foods. The Royal Society of Chemistry, Cambridge, pp. 128–145. León, K., Mery, D., Pedreschi, F. and León, J. (2006) Color measurement in L*a*b* units from RGB digital images. Food Research International 39: 1084–1091. Li, S.J., Seymour, T.A., King, A.J. and Morrissey, M.T. (1998) Color stability and lipid oxidation of rockfish as affected by antioxidant from shrimp shell waste. Journal of Food Science 63: 438–441.

166

Fishery Products: Quality, safety and authenticity

Lin, D. and Morrissey, M.T. (1995) Northern squawfish (Ptychocheilus oregonensis) for surimi production. Journal of Food Science 60: 1245–1247. Little, A.C. (1972) Effect of pre- and post-mortem handling on reflectance characteristics of canned skipjack tuna. Journal of Food Science 37: 502. Liu, K.K.M., Barrows, F.T., Hardy, R.W. and Dong, F.M. (2004) Body composition, growth performance, and product quality of rainbow trout (Oncorhynchus mykiss) fed diets containing poultry fat, soybean/corn lecithin, or menhaden oil. Aquaculture 238: 309–328. Longard, A.A. and Regier, L.W. (1974) Color and some composition changes in ocean perch (Sebastes marinus) held in refrigerated sea water with and without carbon dioxide. Journal of the Fisheries Research Board of Canada 31: 456–460. López-Caballero, M.E., Gómez-Guillén, M.C., Pérez-Mateos, M. and Montero, P. (2005) A functional chitosan-enriched fish sausage treated by high pressure. Journal of Food Science 70: M166–M171. Louka, N., Juhel, F., Fazilleau, V. and Loonis, P. (2004) A novel colorimetry analysis used to compare different drying fish processes. Food Control 15: 327–334. Love, R.M. (1988) The Food Fishes. Their Intrinsic Variation and Practical Implications. Farrand Press, London. MacDougall, D.B. (2002) Colour in Food: Improving Quality. Woodhead Publishing Ltd., Abington, Cambridge, 378 pp. Matser, A.M., Stegeman, D., Kals, J. and Bartels, P.V. (2000) Effects of high pressure on colour and texture of fish. High Pressure Research 19: 109–115. McCallum, I.M., Cheng, K.M. and March, B.E. (1987) Carotenoid pigmentation in two strains of chinook salmon (Oncorhynchus tshawytscha) and their crosses. Aquaculture 67: 291– 300. Meacock, G., Taylor, K.D.A., Knowles, M.J. and Himonides, A. (1997) The improved whitening of minced cod flesh using dispersed titanium dioxide. Journal of the Science of Food and Agriculture 73: 221–225. Metusalach, B., Brown, J.A. and Shahidi, F. (1997) Effects of stocking density on colour characteristics and deposition of carotenoids in cultured Arctic charr (Salvelinus alpinus). Food Chemistry 59: 107–114. Mohsin, M., Bakar, J. and Selamat, J. (1999) The effects on colour, texture and sensory attributes achieved by washing black tilapia flesh with a banana leaf ash solution. International Journal of Food Science Technology 34: 359–363. Mørkøre, T. (2006) Relevance of dietary oil source for contraction and quality of pre-rigor filleted Atlantic cod, Gadus morhua. Aquaculture 251: 56–65. Moretti, V.M., Mentasti, T., Bellagamba, F., Luzzana, U., Caprino, F., Turchini, G.M., Giani, I. and Valfrè, F. (2006) Determination of astaxanthin stereoisomers and colour attributes in flesh of rainbow trout (Oncorhynchus mykiss) as a tool to distinguish the dietary pigmentation source. Food Additives & Contaminants 23: 1056–1063. Morris, P.C., Beattie, C., Elder, B., Finlay, J., Gallimore, P., Jewison, W., Lee, D., Mackenzie, K., McKinney, R., Sinnott, R., Smart, A. and Weir, M. (2005) Application of a low oil pre-harvest diet to manipulate the composition and quality of Atlantic salmon, Salmo salar L. Aquaculture 244: 187–201. Morzel, M., Sohier, D. and van de Vis, H. (2003) Evaluation of slaughtering methods for turbot with respect to animal welfare and flesh quality. Journal of the Science of Food and Agriculture 83: 19–28. Nakayama, T. and Yamamoto, M. (1977) Physical, chemical and sensory evaluations of frozen-stored deboned (minced) fish flesh. Journal of Food Science 42: 900–905. Nakamura, Y.-N., Ando, M., Seoka, M., Kawasaki, K.-I., Sawada, Y., Miyashita, S., Okada, T., Kumai, H. and Tsukamasa, Y. (2006) Effect of fasting on physical/chemical properties of ordinary muscles

Colour measurement

167

in full-cycle cultured Pacific bluefin tuna Thunnus orientalis during chilled storage. Fisheries Science 72: 1079–1085. Nickell, D.C. and Bromage, N.R. (1998) The effect of dietary lipid level on variation of flesh pigmentation in rainbow trout (Oncorhynchus mykiss). Aquaculture 161: 237–251. No, H.K. and Storebakken, T. (1991) Color stability of rainbow trout fillets during frozen storage. Journal of Food Science 56: 969–972, 984. Norris, A.T. and Cunningham, E.P. (2004) Estimates of phenotypic and genetic parameters for flesh colour traits in farmed Atlantic salmon based on multiple trait animal model. Livestock Production Science 89: 209–222. Ochiai, Y., Chow, C.-J., Watabe, S. and Hashimoto, K. (1988) Evaluation of tuna meat discoloration by Hunter color difference scale. Nippon Suisan Gakkaishi 54: 649–653. Oka, H. (1989) Packaging for freshness and the prevention of discoloration of fish fillets. Packaging Technology and Science 2: 201–213. Okada, T., Ushio, H. and Ohshima, T. (2004) Skin color changes of squids Todarodes pacificus and Loligo bleekeri during chilled storage. Journal of Food Science 69: S414–S417. Olafsdottir, G., Nesvadba, P., Di Natale, C., Careche, M., Oehlenschläger, J., Tryggvadottir, S.V., Schubring, R., Kroeger, M., Heia, K., Esaiassen, M., Macagnano, A. and Jørgensen, B.M. (2004) Multisensor for fish quality determination. Trends in Food Science and Technology 15: 86–93, 2004. Olsen, R.E. and Baker, R.T.M. (2006) Lutein does not influence flesh astaxanthin pigmentation in the Atlantic salmon (Salmo salar L.). Aquaculture 258: 558–564. Olsen, R.E. and Mortensen, A. (1997) The influence of dietary astaxanthin and temperature on flesh colour in Arctic charr Salvelinus alpinus L. Aquaculture Research 28: 51–58. Panchavarnam, S., Basu, S., Manisha, K., Warrier, S.B. and Venugopal, V. (2003) Preparation and use of freshwater fish, rohu (Labeo rohita) protein dispersion in shelf-life extension of the fish steaks. Lebensmittel-Wissenschaft und -Technologie 36: 433–439. Paredes, M.D.C. and Baker, R.C. (1988) Physical, chemical and sensory changes during thermal processing of three species of canned fish. Journal of Food Processing and Preservation 12: 71–81. Park, J.W. (1995) Surimi gel colors as affected by moisture content and physical conditions. Journal of Food Science 60: 15–18. Park, J.W. (2000) Surimi and Surimi Seafood. Marcel Dekker Inc., New York, 500 pp. Park, J.W. (2005) Surimi and Surimi Seafood, 2nd edition. Marcel Dekker Inc., New York, 923 pp. Park, S.H., Ryu, H.S., Hong, G.P. and Min, S.G. (2006) Physical properties of frozen pork thawed by high pressure assisted thawing process. Food Science and Technology International 12: 347–352. Pastoriza, L. and Sampedro, G. (1994) Influence of ice storage on ray (Raja clavata) wing muscle. Journal of the Science of Food and Agriculture 64: 9–18. Pavlidis, M., Papandroulakis, N. and Divanach, P. (2006) A method for the comparison of chromaticity parameters in fish skin: Preliminary results for coloration pattern of red skin Sparidae. Aquaculture 258: 211–219. Pearson, A.M. and Dutson, T.R. (1994) Quality Attributes and Their Measurement in Meat, Poultry And Fish Products. Blackie Academic and Professional, Glasgow, 505 pp. Pérez-Mateos, M. and Lanier, T.C. (2007) Comparison of Atlantic menhaden gels from surimi processed by acid or alkaline solubilization. Food Chemistry 101: 1223–1229. Pérez-Mateos, M. and Montero, P. (2000) Contribution of hydrocolloids to gelling properties of blue whiting muscle. European Food Research Technology 210: 383–390. Pérez-Mateos, M., Boyd, L. and Lanier, T. (2004) Stability of omega-3 fatty acids in fortified surimi seafoods during chilled storage. Journal of Agricultural and Food Chemistry 52: 7944–7949. Pérez-Mateos. M., Bravo, L., Goya, L., Gomez-Guillen, C. and Montero, P. (2005) Quercetin properties as a functional ingredient in omega-3 enriched fish gels fed to rats. Journal of the Science of Food and Agriculture 85: 1651–1659.

168

Fishery Products: Quality, safety and authenticity

Pipatsattayanuwong, S., Park, J.W. and Morrissey, M.T. (1995) Functional properties and shelf life of fresh surimi from Pacific whiting. Journal of Food Science 60: 1241–1244. Poli, B.M., Messini, A., Parisi, G., Scappini, F., Vigiani, V., Giorni, G. and Vincenzini, M. (2006) Sensory, physical, chemical and microbiological changes in European sea bass (Dicentrarchus labrax) fillets packed under modified atmosphere/air or prepared from whole fish stored in ice. International Journal of Food Science and Technology 41: 444–454. Pong, C.-Y., Chiou, T.-K., Ho, M.-L. and Jiang, S.-T. (2000) Effect of polyethylene package on the metmyoglobin reductase activity and color of tuna muscle during low temperature storage. Fisheries Science 66: 384–389. Pullela, S.V., Fernandes, C.F., Flick, G.J., Libey, G.S., Smith, S.A. and Coale, C.W. (2000) Quality comparison of aquacultured pacu (Piaractus mesopotamicus) fillets with other aquacultured fish fillets using subjective and objective sensorial traits. Journal of Aquatic Food Product Technology 9: 65–76. Quinones, A., Choubert, G. and Gomez, R. (2003) Colour stability of smoked muscle rainbow trout: packaging effect. Sciences des Aliments 23: 132–137. Ramírez, J.A., Del Ángel, A., Velazquez, G. and Vázquez, M. (2006) Production of low-salt restructured fish products from Mexican flounder (Cyclopsetta chittendeni) using microbial transglutaminase or whey protein concentrate as binders. European Food Research Technology 223: 341–345. Ramirez-Suarez, J.C. and Morrissey, M.T. (2006) Effect of high pressure processing (HPP) on shelf life of albacore tuna (Thunnus alalunga) minced muscle. Innovative Food Science and Emerging Technologies 7: 19–27. Reid, R.A., Durance, T.D., Walker, D.C. and Reid, P.E. (1993) Structural and chemical changes in the muscle of chum salmon (Oncorhynchus keta) during spawning migration. Food Research International 26: 1–9. Regost, C., Jakobsen, J.V. and Rørå, A.M.B. (2004) Flesh quality of raw and smoked fillets of Atlantic salmon as influenced by dietary oil sources and frozen storage. Food Research International 37: 259–271. Reppond, K.D. and Babbitt, J.K. (1997) Gel properties of surimi from various fish species as affected by moisture content. Journal of Food Science 62: 33–36. Reppond, K.D., Wasson, D.H. and Babbitt, J.K. (1993) Properties of gels produced from blends of arrowtooth flounder and Alaska pollock surimi. Journal of Aquatic Food Product Technology 2: 83–98. Requena, D.D., Hale, S.A., Green, D.P., McClure, W.F. and Farkas, B.E. (1999) Detection of discoloration in thermally processed blue crab meat. Journal of the Science of Food and Agriculture 79: 786–791. Rodríguez-Herrera, J.J., Bernardez, M., Sampedro, G., Cabo, M.L. and Pastoriza, L. (2006) Possible role for cryostabilizers in preventing protein and lipid alterations in frozen-stored minced muscle of Atlantic mackerel. Journal of Agricultural Food Chemistry 54: 3324–3333. Rørå, A.M.B., Birkeland, S., Hultmann, L., Rustad, T., Skåra, T. and Bjerkeng, B. (2005) Quality characteristics of farmed Atlantic salmon (Salmo salar) fed diets high in soybean or fish oil as affected by cold-smoking temperature. LWT – Food Science and Technology 38: 201–211. Rørå, A. M. B., Kvale, A., Mørkøre, T., Rorvik, K.-A., Steien, S. H. and Thomassen, M. S. (1998) Process yield, colour and sensory quality of smoked Atlantic salmon (Salmo salar) in relation to raw material characteristics. Food Research International 31: 601–609. Rørå, A. M. B., Monfort, M. C. and Espe, M. (2004) Effects of country of origin on consumer preference of smoked salmon collected in a French hypermarket. Journal of Aquatic Food Product Technology 13: 69–85. Roth, B., Johansen, S. J. S., Suontama, J., Kiessling, A., Leknes, O., Guldberg, B. and Handeland, S. (2005a) Seasonal variation in flesh quality, comparison between large and small Atlantic salmon (Salmo salar) transferred into seawater as 0+ or 1+ smolts. Aquaculture 250: 830–840.

Colour measurement

169

Roth, B., Slinde, E. and Arildsen, J. (2006) Pre or post mortem muscle activity in Atlantic salmon (Salmo salar). The effect on rigor mortis and the physical properties of flesh. Aquaculture 257: 504–510. Roth, B., Torrissen, O.J. and Slinde, E. (2005b) The effect of slaughtering procedures on blood spotting in rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar). Aquaculture 250: 796–803. Ruff, N., Fitzgerald, R.D., Cross, T.F. and Kerry, J.P. (2003) Shelf-life evaluation of modified atmosphere and vacuum package fillets of Atlantic halibut (Hippoglossus hippoglossus), following dietary alpha-tocopheryl acetate supplementation. Journal of Aquatic Food Product Technology 12: 23–37. Sareevoravitkul, R., Simpson, B.K. and Ramashwamy, H. (1996) Effects of crude α2-macroglobulin on properties of bluefish (Pomatomus saltatrix) gels prepared by high hydrostatic pressure and heat treatment. Journal of Food Biochemistry 20: 49–63. Schubring, R. (1999a) Determination of fish freshness by instrumental colour measurement. Fleischwirtschaft International 79: 26–29. Schubring, R. (1999b) Einfluß des Doppelgefrierens auf Qualitätsmerkmale des Filets von Seelachs (Pollachius virens) während der TK-Lagerung in Abhängigkeit vom Rigor-Stadium. Deutsche Lebensmittel-Rundschau 95: 61–71. Schubring, R. (2000) Influence of double freezing on quality attributes of lean fish fillet during frozen storage as affected by rigor states. In: Advances in the Refrigeration Systems, Food Technologies and Cold chain. Proceedings of the Conference of Comm. B2 & C2 with D1 & D2/3, Sofia, Bulgaria 1998/6. IIR/IIF, Paris. pp. 504–513. Schubring, R. (2001a) Double freezing of fillets and minces prepared from saithe and haddock: influence on selected sensory and physical attributes. In: A. Gudjonsson, and O. Niclasen (Eds) Proceedings of the 30th WEFTA Plenary Meeting, 19–22 June, Torshavn, Faroe Islands. Anales Societatis Scientarum Faeroensis (Suppl.) XXVIII: 169–179. Schubring, R. (2001b) Double freezing of saithe fillets. Influence on sensory and physical attributes. Nahrung/Food 45: 280–285. Schubring, R. (2002a) Double freezing of cod fillets: influence on sensory, physical and chemical attributes of battered and breaded fillet portions. Nahrung/Food 46: 227–232. Schubring, R. (2002b) Influence of freezing/thawing and frozen storage on the texture and colour of brown shrimp (Crangon crangon). Archiv fur Lebensmittelhygiene 53: 34–36. Schubring, R. (2003) Colour measurement on skin during storage of wet and frozen fish. In: J.B. Luten, J. Oehlenschläger and G. Olafsdottir, (Eds) Quality of Fish from Catch to Consumer: Labelling, Monitoring & Traceability. Wageningen Academic Publishers, Waganingen, pp. 251–263. Schubring, R. (2004) Instrumental colour, texture, water holding and DSC measurements on frozen cod fillets (Gadus morhua) during long term storage at different temperatures. Deutsche Lebensmittel-Rundschau 100: 247–254. Schubring, R. (2005a) Changes in texture, water holding capacity, colour, and thermal stability of frozen cod (Gadus morhua) fillets: effect of frozen storage temperature. Deutsche LebensmittelRundschau 101: 484–493. Schubring, R. (2005b) Quality effects of double freezing on seafood. In: J. Ryder and L. Ababouch (Eds) Fifth World Fish Inspection and Quality Control Congress. The Hague, Netherlands, 20–22 October 2003. FAO Fisheries Proceedings, Rome, pp. 123–130. Schubring, R. and Meyer, C. (2002) Quality factors of terrestrial snail products as affected by the species. Journal of Food Science 67: 3148–3151. Schubring, R. and Meyer, C. (2006a) Ice storage of fish, new aspects: comparison between flake ice and stream ice – part I: sardine (Sardina pilchardus). Deutsche Lebensmittel-Rundschau 102: 405–415.

170

Fishery Products: Quality, safety and authenticity

Schubring, R. and Meyer, C. (2006b) Iced storage of fish, new aspects: comparison between flake ice and stream ice – Part II: horse mackerel (Trachurus trachurus). Deutsche Lebensmittel-Rundschau 102: 508–517. Schubring, R. and Oehlenschläger, J. (1996) Bewertung der Farbe von frischen und tiefgefrorenen Räucherlachsseiten mittels objektiver Farbmessung. Informationen fur die Fischwirtschaft 43: 81–84. Schubring, R., Meyer, C., Schlüter, O., Boguslawski, S. and Knorr, D. (2003) Impact of high pressure assisted thawing on the quality of fillets from various fish species. Innovative Food Science Emerging Technology 4: 257–267. Schubring, R., Schlüter, O., Boguslawski, S., Meyer, C. and Knorr, D. (2005) Off shore high pressure treatment of freshly caught fish. In: P. Fito and F. Toldra (Eds) Innovations in Traditional Foods. Elsevier, London, pp. 1213–1216. Scott, T. M., Ranco, B.A. and Hardy, R.W. (1994) Stability of krill meal, astaxanthin, and canthaxanthin color in cultured rainbow trout (Oncorhynchus mykiss) fillets during frozen storage and cooking. Journal of Aquatic Food Product Technology 3: 53–63. Sequeira-Munoz, A., Chevalier, D., LeBail, A., Ramaswamy, H.S. and Simpson, B.K. (2006) Physicochemical changes induced in carp (Cyprinus carpio) fillets by high pressure processing at low temperature. Innovative Food Science and Emerging Technologies 7: 13–18. Shahidi, F., Synowiecki, J. and Penney, R.W. (1993) Pigmentation of Arctic char (Salvelinus alpinus) by dietary carotenoids. Journal of Aquatic Food Product Technology 2: 99–115. Silva, J.L. and White, T.D. (1994) Bacteriological and colour changes in modified atmospherepackaged channel catfish. Journal of Food Protection 57: 715–719. Sinnhuber, R.O., Landers, M.K., Yu, T.C., Simon, M. and Heiligman, F. (1968) Radiation sterilization of pre-fried cod and halibut patties. Food Technology 22: 74–76. Siskos, I., Zotos, A. and Taylor, K.D.A. (2005) The effect of drying, pressure and processing time on the quality of liquid-smoked trout (Salmo gairdnerii) fillets. Journal of the Science of Food and Agriculture 85: 2054–2060. Skjervold, P.O., Fjæra, S.O., Østby, P.B., Isaksson, T., Einen, O. and Taylor, R. (2001) Properties of salmon flesh from different locations on pre- and post rigor fillets. Aquaculture 201: 91–106. Skrede, G. and Storebakken, T. (1986a) Characteristics of color in raw, baked and smoked wild and pre-reared Atlantic salmon. Journal of Food Science 51: 804–808. Skrede, G. and Storebakken, T. (1986b) Instrumental colour analysis of farmed and wild Atlantic salmon when raw, baked and smoked. Aquaculture 53: 279–286. Skrede, G., Storebakken, T. and Naes, T. (1989) Color evaluation in raw, baked and smoked flesh of rainbow trout (Oncorhynchus mykiss) fed astaxanthin or canthaxanthin. Journal of Food Science 55: 1574–1578. Skrede, G., Risvik, E., Huber, M., Enersen, G. and Bluemlein, L. (1990) Developing a color card for raw flesh of astaxanthin-fed salmon. Journal of Food Science 55: 361–363. Sohn, J.-H., Taki, Y., Ushio, H., Kohata, T., Shioya, I. and Ohshima, T. (2005) Lipid oxidations in ordinary and dark muscles of fish: influences on rancid off-odor development and color darkening of yellowtail flesh during ice storage. Journal of Food Science 70: S490–S496. Stien, L.H., Hirmas, E., Bjørnevik, M., Karlsen, Ø., Nortvedt, R., Rørå, A.M.B., Sunde, J. and Kiessling, A. (2005) The effects of stress and storage temperature on the colour and texture of prerigor filleted farmed cod (Gadus morhua L.). Aquaculture Research 36: 1197–1206. Stien, L.H., Amundsen, A.H., Mørkøre, T., Økland, S.N. and Nortvedt, R. (2006a) Instrumental colour analysis of Atlantic salmon (Salmo salar L.) muscle. In: J.B. Luten, C. Jacobsen, K. Bekaert, A. Sæbø and J. Oehlenschläger (Eds) Seafood Research from Fish to Dish. Quality, Safety and Processing of Wild and Farmed Fish. Wageningen Academic Publishers, Wageningen, pp. 525–539. Stien, L.H., Manne, F., Ruohonene, K., Kause, A., Rungruangsak-Torrissen, K. and Kiessling, A. (2006b) Automated image analysis as a tool to quantify the colour and composition of rainbow trout (Oncorhynchus mykiss W.) cutlets. Aquaculture 261: 695–705, 2006.

Colour measurement

171

Suvanich, V., Marshall, D.L. and Jahncke, M.L. (2000) Microbiological and color quality changes of channel catfish frame mince during chilled and frozen storage. Journal of Food Science 65: 151–154. Tabilo-Munizaga, G. and Barbosa-Cánovas, G.V. (2004) Color and textural parameters of pressurized and heat-treated surimi gels as affected by potato starch and egg white. Food Research International 37: 767–775. Torrieri, E., Cavella, S., Villani, F. and Masi, P. (2006) Influence of modified atmosphere packaging on the chilled shelf life of gutted farmed bass (Dicentrarchus labrax). Journal of Food Engineering 77: 1078–1086. Undeland, I., Kelleher, S.D. and Hultin, H.O. (2002) Recovery of functional proteins from herring (Clupea harengus) light muscle by an acid or alkaline solubilization process. Journal of Agricultural and Food Chemistry 50: 7371–7379. Undeland, I., Hall, G., Wendin, K., Gangby, I. and Rutgersson, A. (2005) Preventing lipid oxidation during recovery of functional proteins from herring (Clupea harengus) fillets by an acid solubilization process. Journal of Agricultural and Food Chemistry 53: 5625–5634. Uresti, R.M., López-Arias, N., Ramírez, J.A. and Vázquez, M. (2003) Effect of amidated low methoxyl pectin on the mechanical properties and colour attributes of fish mince. Food Technology and Biotechnology 41: 131–136. Uresti, R.M., Velazquez, G., Ramírez, J.A., Vázquez, M. and Torres, J.A. (2004) Effect of highpressure treatments on mechanical and functional properties of restructured products from arrowtooth flounder (Atheresthes stomias). Journal of the Science of Food and Agriculture 84: 1741–1749. Uresti, R.M., Velazquez, G., Vázquez, M., Ramírez, J.A. and Torres, J.A. (2005a) Effect of sugars and polyols on the functional and mechanical properties of pressure-treated arrowtooth flounder (Atheresthes stomias) proteins. Food Hydrocolloids 19: 964–973. Uresti, R.M., Velazquez, G., Vázquez, M., Ramírez, J.A. and Torres, J.A. (2005b) Restructured products from arrowtooth flounder (Atheresthes stomias) using high-pressure treatments. European Food Research Technology 220: 113–119. van der Salm, A.L., Martinez, M., Flik, G. and Wendelaar Bonga, S.E. (2004) Effects of husbandry conditions on the skin colour and stress response of red porgy, Pagrus pagrus. Aquaculture 241: 371–386. Wasson, D.H., Reppond, K.D. and Kandianis, T.M. (1991) Antioxidants to preserve rockfish color. Journal of Food Science 56: 1564–1566. Waters, M.E. (1983) Chemical composition and frozen storage stability of weakfish, Cynoscion regalis. Marine Fisheries Review 45: 27–33. Wathne, E., Bjerkeng, B., Storebakken, T., Vassvik, V. and Odland, A.B. (1998) Pigmentation of Atlantic salmon (Salmo salar) fed astaxanthin in all meals or in alternating meals. Aquaculture 159: 217–231. Wendel, A., Park, J.W. and Kristbergsson, K. (2002) Recovered meat from Pacific whiting frame. Journal of Aquatic Food Product Technology 11: 5–18. Withler, R.E. and Beacham, T.D. (1994) Genetic variation in body weight and flesh colour of the coho salmon (Oncorhynchus kisutch) in British Columbia. Aquaculture 119: 135–148. Yam, K.L. and Papadakis, S.E. (2004) A simple digital imaging method for measuring and analyzing color of food surfaces. Journal of Food Engineering 61: 137–142. Yin, L.-J., Lin, H.-Y. and Jiang S.-T. (2002) New technology for producing paste-like fish products using lactic acid bacteria fermentation. Journal of Food Science 67: 3114–3118. Yin, L.-J., Lu, M.-C., Pan, C.-L. and Jiang S.-T.S. (2005) Effect of monascus fermentation on the characteristics of mackerel mince. Journal of Food Science 70: S66–S72. Yongsawatdigul, J., Piyadhammaviboon, P. and Singchan, K. (2006) Gel-forming ability of small scale mud carp (Cirrhiana microlepis) unwashed and washed mince as related to endogenous proteinases and transglutaminase activities. European Food Research Technology 223: 769–774.

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Yoshioka, K. and Yamamoto, T. (1998) Changes of ultrastructure and the physical properties of carp muscle by high pressure. Fisheries Science 64: 89–94. Young, A., Morris, P. C., Huntingford, F.A. and Sinnott, R. (2005) The effects of diet, feeding regime and catch-up growth on flesh quality attributes of large (1 + sea winter) Atlantic salmon, Salmo salar. Aquaculture 248: 59–73. Young, A., Morris, P.C., Huntingford, F.A. and Sinnott, R. (2006) Replacing fish oil with pre-extruded carbohydrate in diets for Atlantic salmon, Salmo salar, during their entire marine grow-out phase: effects on growth, composition and colour. Aquaculture 253: 531–546. Young, K.W. and Whittle, K.J. (1985) Colour measurement of fish minces using Hunter L, a, b values. Journal of the Science of Food and Agriculture 36: 383–392. Zheng, C., Sun, D.W. and Zheng, L. (2006) Recent developments and applications of image features for food quality evaluation and inspection – a review. Trends in Food Science and Technology 17: 642–655. Zhu, S., Ramaswamy, H.S. and Simpson, B.K. (2004) Effect of high-pressure versus conventional thawing on color, drip loss and texture of Atlantic salmon frozen by different methods. Lebensmittel-Wissenschaft und -Technologie 37: 291–299. Zhuang, R.Y., Huang, Y.W. and Beuchat, L.R. (1996) Quality changes during refrigerated storage of packaged shrimp and catfish fillets treated with sodium acetate, sodium lactate or propyl gallate. Journal of Food Science 61: 241–244.

Chapter 8

Differential scanning calorimetry Reinhard Schubring

8.1

Introduction

Differential scanning calorimetry (DSC) is a well-established measuring method that is used on a large scale in different areas of research, development, and quality inspection and testing. Thermal effects can be quickly identified and the relevant temperature and the characteristic caloric values determined using substance quantities in the mgmilligram range. Measurement values obtained by DSC allow heat capacity, heat of transition kinetic data, purity and glass transition to be determined. DSC curves serve to identify substances (Höhne et al. 1996). When a material undergoes a change in physical state such as melting or transition from one crystalline form to another, or when a material reacts chemically, heat is either absorbed or liberated. According to Lund (1983), DSC offers a tremendous potential for studying physicochemical changes that occur in foods. During the 1980s, its use in food research became apparent. DSC is characterised by its usefulness for analysing phase changes. Reactions that can lead to such phase changes are crystallisation of water (melting and freezing), evaporation of water and certain chemical reactions, for example protein denaturation. In DSC the temperature of the sample and reference are maintained the same and amount of heat required to achieve this is recorded. The DSC curve is a plot of heat flow against temperature. Consequently, the enthalpy change involved in the reaction can be determined from the area under the curve of heat-flow versus time. Material of known enthalpy can be used to calibrate the equipment. For samples containing water, evaporation is prevented using sealed containers. For examining frozen water in foods, results that are more reproducible are obtained while thawing frozen products than during cooling and freezing of products, because the variable phenomenon of super-cooling occurs during freezing. For proteins, the temperature corresponding to the maximum peak height is often used as an indication of the denaturation temperature, and the width of the peak as a measure of the complexity of the denaturation reaction. Using these assumptions, it is possible to observe how the denaturation temperature is affected by the changes in pH or the presence of other components, and the variation in enthalpy with denaturation temperature. As a protein becomes more denatured, the size of the peak should decrease. Phase changes in these both main components of the fish flesh (water and protein) are subject of DSC investigation in this special field. DSC has emerged 173

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as a technique of choice for the study of thermal transitions of food. The conversion of a protein from a native to a denatured state by heat is a cooperative phenomenon and is accompanied by a significant uptake of heat, seen as an endothermic peak in the DSC curve. For proteins, the thermally induced process detectable by DSC is the structural melting or unfolding of the molecule, thermal denaturation of proteins being attributed to the rupture of intermolecular hydrogen bonds, the temperatures at which the bonds rupture being a measure of the thermal stability of proteins. Their determination under controlled conditions can provide direct comparison of the thermal stability of the different proteins. The enthalpy value, which is correlated with the net content of the ordered secondary structure of a protein, is actually a net value obtained through the combination of endothermic reactions and exothermic processes, including protein aggregation and the break-up of hydrophobic interactions. A successful approach to the study of the native conformation of proteins is to subject the protein to physical and chemical stresses, followed by a determination of the effect of these stresses on its thermal denaturation. There are numerous papers published dealing with the application of DSC in food in general (Wright 1982; Biliarderis 1983; Lund 1983; Harwalkar and Ma 1990; Ma and Harwalkar 1991; Raemy and Lambelet 1991; Schiraldi et al. 1999). Furthermore, papers dealing with biochemical, biophysical and biological application of DSC should also be mentioned (Sturtevant 1987; Shnyrov et al. 1997; Collett and Brown 1998; Levitzky et al. 1998; Lorinczy 2004). Special attention has been devoted to the component of foods that influences decisively their functional properties – the proteins (Privalov 1989; Brandts and Lin 1990; Stanley and Yada 1992; Smith 1994; Freire 1995; Carey and Surewicz 1996; Boye et al. 1997; Cooper 1999). Papers can be recommended to those who are interested in the general problem of DSC application in foods and food-related fields. However, muscle foods, and particularly fish, are mostly not the subjects of the papers mentioned above. Therefore, this chapter deals particularly with the application of DSC in fishery research. Basically, this means fish processing and processing steps that are of major influence on fish quality and, furthermore, changes in quality that accompany the deterioration of fish and fishery products.

8.2

Principle of function of the instruments

Two types of differential scanning calorimeters must be distinguished (Höhne et al. 1996): the heat flux differential scanning calorimeter; the power compensation differential scanning calorimeter. Both types of calorimeter use a differential method of measurement, which is defined as follows: a method of measurement in which the measurand is compared with a quantity of the same kind, of known value, only slightly different from the value of the measurand, and in which the difference between the two values is measured. A characteristic feature of all DSC measuring systems is the twin-type design and the direct in-difference connection of the two measuring systems, which are of the same kind. It is the decisive advantage of the differential principle that, in first approximation, disturbances such as temperature variations in the environment of the measuring system and

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the like affect the two measuring systems in the same way and are compensated for when the difference between the individual signals is formed. The differential signal can strongly be amplified. It is the essential characteristic of each differential scanning calorimeter. Another characteristic is the dynamic mode of operation. The differential scanning calorimeter can be heated or cooled at a preset heating or cooling rate (isothermal mode is possible as well). Another type of differential scanning calorimeter, the highly sensitive, precise differential scanning calorimeters, should only be briefly mentioned here. They are used to measure thermodynamic data on biopolymer solutions. Detailed information can be found in earlier reviews (Privalov and Plotnikov 1989; Privalov and Potekhin 1986; Benoist 1990) as well as from the website www.calorimertysciences.com, which gives information on the latest development of a nano-scale differential scanning calorimeter that only needs 2 μg of protein in solution to provide quality data yielding accurate values of thermodynamic parameters.

8.2.1

The heat flux differential scanning calorimeter

The heat flux differential scanning calorimeter belongs to the class of heat-exchanging calorimeters (Höhne et al. 1996). In the heat flux calorimeters, a defined exchange of the heat to be measured with the environment takes place through a thermal resistance. The measurement signal is the temperature difference. The most important fundamental types are: the disk-type measuring system with solid sample support (disk) that allows high heating rates; its time constants and the sample volume are small, but it has a high sensitivity per unit volume; the cylinder-type measuring system with integrated sample cavities that allows only low heating rates; its time constant and the sample volume are large, but it has a low sensitivity per sample volume. The characteristic feature of this measuring system is that the main heat flow from the furnace to the sample passes symmetrically through a disk of good thermal conductivity. The samples are positioned on this disk symmetrically in the centre. The temperature sensors are integrated into the disk or fixed on its surface. Each sensor covers more or less the area supporting the respective area (crucible, pan) so that calibration can be done independently of the sample position inside the container. Metals, quartz glass or ceramics are used as disk materials. The type and design of temperature sensors differ (thermocouples, resistance thermometers). When the furnace is heated, heat flows through the disk to the samples. When the arrangement is ideally symmetrical, equally high heat flow rates flow into sample and reference sample. The differential temperature signal, ΔT, is then zero. If this steady-state equilibrium is disturbed by a sample transition, a differential signal is generated that is proportional to the difference between the heat flow rates to the sample and to the reference sample. For my own measurements, a heat flux differential scanning calorimeter with a cylinder-type measuring system was used. In this, a block-type cylindrical furnace is provided with two cylindrical cavities, each containing a cylindrical fixed sample container that is connected with the furnace or directly with the other container by several thermocouples

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Figure 8.1 Heat flux differential scanning calorimeter MicroDSC VII, SETARAM.

(thermopiles), which are the characteristic features of this type of measuring system. The heat flux differential scanning calorimeter used (Figure 8.1) was a Tian-Calvet type microcalorimeter (MicroDSC VII, SETARAM, Caluire, France), in which thermal effects are measured by two fluxmeters (one on the measurement side and one on the reference side), each of which measures the thermal power exchanged at each moment between the experimental vessel and the calorimetric unit. The Tian-Calvet fluxmetric transducer envelopes the sample, making it possible to measure almost all the exchanges between the vessel and unit. This capability gives this device a clear metrological advantage in terms of both the quantity of measurements and their sensitivity (capacity to measure very weak effects). Typical measuring conditions used in my investigations were as follows: sample weight, 300–700 mg (weighed accurately to ±0.1 mg); heating range, 25–95 °C, scanning rate, 0.3 or 0.5 K/min. An example of a differential scanning calorimeter curve obtained on ordinary muscle of untreated rainbow trout when using this instrument is shown in Figure 8.2. Two main peaks are to be seen, the first one at lower temperature comprising myosin and the second one at higher temperature actin. In between both, two smaller transitions can possibly be detected comprising sarcoplasmic protein and connective tissue.

8.2.2

The power compensation DSC

The power compensation differential scanning calorimeter, like the PerkinElmer DSC-7 (Figure 8.3), belongs to the class of heat-compensating calorimeters (Höhne et al. 1996). The heat to be measured is compensated by electrical energy, by increasing or decreasing an adjustable Joule’s heat. The measuring system consists of two microfurnaces of the same type made of a platinum–iridium alloy, each of which contains a temperature sensor (platinum resistance thermometer) and a heating resistor (made of platinum wire). The microfurnace is about 9 mm in diameter, approximately 6 mm in height and has a mass of

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↑ Endo Heat flow (mW)

I 0.01

IV II

22

30

40

III

50 60 70 Temperature (°C)

80

90 95

Heat flow (mW)

↓ Endo

0.25 II

III IV

I 25 30

35 40

45 50 55

60

Temperature (°C) Figure 8.2 DSC curve of ordinary muscle of untreated rainbow trout obtained by using the Perkin Elmer DSC-7 (above) and the SETARAM Micro DSC VII (below) showing peaks of different protein fraction (I, myosin; II, III, sarcoplasmic proteins and connective tissue; IV, actin); higher sensitivity of the MicroDSC VII allows a good presentation of the complex myosin transition.

Figure 8.3 Power compensation differential scanning calorimeter: PerkinElmer DSC 7.

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approximately 2 g. Both microfurnaces – separated from each other (thermally decoupled) – are positioned in an aluminium block of constant temperature. During heating-up, the same heating power is supplied to both microfurnaces by a control circuit in order to change their mean temperature in accordance with the preset heating rate. If there is ideal thermal symmetry, the temperature of both microfurnaces is always the same. When an asymmetry occurs, for example as a result of a sample reaction, a temperature difference results between the microfurnace accommodating the sample and the microfurnace containing the reference sample. The temperature difference is both the measurement signal and the input signal of a second control circuit. This second circuit tries to compensate the reaction heat flow rate by increasing or decreasing an additional heating power. The compensating heating power is proportional to remaining temperature difference. Typical measuring conditions used in my investigations were: sample weight, 15 ± 3 mg (weighed accurately to ±0.1 mg); heating range, 25–95 °C; scanning rate, 10 K/min; 60 μl stainless steel pans (LVC 0319–0218); reference, empty sealed pan; calibration substances, naphthalene and indium. Measurements were performed at least in triplicate; average curves were used as result. An example of a differential scanning calorimeter curve obtained on ordinary muscle of untreated rainbow trout when using this instrument is shown in Figure 8.2.

8.3

First applications of DSC on fish muscle and other seafood

One of the first papers applying DSC to investigate the effect of heat treatment on proteins of marine origin (sperm-whale myoglobin) observed the influence of various water contents on protein transition (Hägerdal and Marten 1976). According to the results obtained, at water contents below 30% only a certain fraction of the heat-treated protein sample underwent irreversible transitions whereas the rest remained native or underwent reversible transition. This fraction of the protein sample increased linearly with the amount of water present during heat treatment. Above 30% water content, the entire protein sample underwent irreversible transition. It was found that the heating rate influenced both the transition temperature and transition enthalpy. Almost at the same time, enthalpy changes associated with the denaturation of acid-soluble and insoluble collagens prepared from sheep, cod, halibut and pike skin were determined by DSC (Menashi et al. 1976). It was found that the enthalpy change associated with the soluble collagens decreased with decreasing imino-acid content (from 1420 cal/mol for sheep to 736 cal/mol for cod) whereas the value for insoluble collagen was approximately constant at 1360 cal/mol. It was further confirmed that the denaturation temperature of collagens increases with increasing imino-acid content, with the exception of halibut collagen. For this anomalous behaviour, no explanation was offered. Imino-acid contents and denaturation temperatures of various collagens including numerous fish skins are given by Burjanadze (1992). A short time later, DSC was used to follow the denaturation of fish muscle proteins occurring during frozen storage (Rodger et al. 1980). The main result from the DSC studies was that changes in protein solubility were not mirrored by similar changes in the thermal stability. DSC curves of samples whose solubility were widely different did not show great differences between each other. At the same time in Japan, the gel-forming ability of fish muscle proteins was measured by DSC (Akahane et al. 1981). DSC curves of the gels

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obtained were characterised by a single deep hollow with a minimum around 105 °C (R105 values), which represents the evaporation of water. When gels were prepared with different ratios of fish muscle protein and egg white, the R105 values illustrated good correlations with the jelly strength. Kamaboko added with different kinds of natural high polymer was subjected to DSC. From the DSC curves obtained, the R105 values represented the amount of captured water in the total water calculated (Niwa et al. 1988). The R105 values of the kamaboko were increased by the addition of each of the cost-effective polymers (agar, κ-carrageenan, corn starch, egg albumin, and soybean protein and wheat gluten). The heat stability of the proteins present in different fish species was compared using DSC and by measuring the solubility of the proteins in weak and strong salt solutions after heat treatment at selected temperatures from 25 to 100 °C. It was found that the stability of both the collagens of the connective tissue and myosin of the myofibrillar proteins varied between species, the more stable proteins being those from the fish found in the waters of higher ambient temperature (Hastings et al. 1985; Poulter et al. 1985). The stability of the sarcoplasmic proteins also exhibited some species dependence, but actin present in the species compared was of similar thermal stability. DSC curves indicated that during frozen storage as well as dehydration some changes in the nature of the myosin molecule occurred. For the influence of pH changes in cod muscle on the thermal stability, it was found that the highest transition temperatures for both myosin and actin occurred at pH 5.0 and 5.5 respectively, near the isoelectric point of the myofibrillar protein (Hastings and Rodger 1985). During frozen storage of cod muscle at pH 6.5, after a reduction in size of the major myosin peak at about 4 weeks no further significant changes were observed. A comparison of cod and snapper revealed that under aggregating conditions of low ionic strength (0.06 mol) and pH 6.0, snapper myosin did have a 10 K greater thermal stability than cod myosin. However, when dissolved at high ionic strength and/or pH 8.0, the two proteins had rather similar thermal stabilities, with the lowest myosin transition close to 310 K in both cases (Davis et al. 1988). Hake (Merluccius hubbsi) muscle free of connective tissue showed two transitions (Tmax 46.5 and 75.3 °C) and ΔH of 4.27 cal/g. The sarcoplasmic proteins contributed to both denaturation peaks. The exudative sarcoplasmic fraction showed three transitions (Tmax 45.2, 59.0 and 75.5 °C) and ΔH of 3.92 cal/g (Beas et al. 1990). The area under the DSC curve corresponding to myosin denaturation was smaller in myofibrillar extracts from prespawning than from post-spawning hake, whereas the areas corresponding to denaturation of actin were similar. It was concluded that proteins of fish in better biological condition (post-spawning) denature more rapidly and completely (Beas et al. 1991). To obtain basic knowledge for processing of fish meat products, like kamaboko, myofibrillar proteins were isolated and submitted to DSC (Akahane et al. 1985). Myosin preparations gave broadly dispersed endothermic regions in each of which a peak was found at 62 °C for rabbit, 50 °C for carp and 40 °C for scallop. Actin showed a peak at 82 °C (rabbit), 75 °C (carp) and 77 °C (scallop). The order of the denaturation temperature generally found during the investigation, namely ‘rabbit, carp and scallop’ appeared to reflect the habitat temperature of these animals. The variation in myofibrillar protein thermal stability was compared by using DSC for various fish species (Howell et al. 1991). Tropical freshwater and marine fish were compared with fish inhabiting cold waters. Onset temperature of myofibrillar protein denaturation occurred up to 11 °C higher for tropical species (43.5 °C, catfish), than for cod (32.6 °C) at pH 7.0 and low ionic strength. As pH (6.0–8.0) and ionic strength (0.05–1.0)

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were increased, thermal denaturation temperatures of myosins from tropical, but not coldwater, species decreased. Enthalpies of myofibrillar denaturation decreased for all species with increasing pH and ionic strength (Howell et al. 1991). When the thermal behaviour of tilapia muscle proteins was investigated by DSC at various stages in the processing of surimi, a shift in the thermal transition of actin to lower temperature was observed and the denaturation enthalpies for both actin and myosin decreased with further processing. Salt addition also induced shifts in denaturation transition to lower temperatures and decreased enthalpies of denaturation (Park and Lanier 1989). The effect of cyclic freezing and thawing on the DSC curves was found to be marginal, whereas other factors, such as salt addition, had a greater influence (Kim et al. 1986). The addition of sugar or salt to leached fish muscle induced a stabilisation or destabilisation of the myofibrillar proteins, respectively. Evidence for this is most pronounced in the shift of transition temperature and is supported by kinetic analysis of the enthalpic changes that occurred during denaturation. Net enthalpic changes, which are the result of both denaturation (endothermic) and aggregation (exothermic) of the proteins, increased in an endothermic direction as heating rate was increased (Park and Lanier 1990). During frozen storage of fish muscle, alterations in fish myofibrillar proteins have been largely accepted as the principal cause of loss of protein functional properties. Identifying cryoprotective substances by DSC was the aim of numerous early published papers (Park and Lanier 1987; Sych et al. 1990a, b, 1991a, b). According to the results, palatinit and polydextrose provided similar cryoprotection to the myofibrillar proteins of cod surimi (Sych et al. 1990a). The greatest stabilising effect to cod surimi proteins was obtained from carbohydrate/polymer treatments at the 8% level. In these investigations, the correlation between certain DSC parameters and salt-extractable proteins was found to be high (Sych et al. 1990b). It was found that addition of lactilol (6–8%) was as effective as 8% sucrose/sorbitol, the additive currently used in the surimi industry. From the results it was concluded further that some DSC parameters such as ΔH-myosin and Tmax myosin appeared to be effective indices for estimating the quantity of functional protein and stability of various protein fractions during frozen storage of cod surimi (Sych et al. 1991a, b). DSC studies revealed that phosphate addition induced stabilisation of myosin, and carbohydrate addition generally induced a one-step denaturation of myosin with higher initial temperature of denaturation. No differences due to phosphate type were observed for any quality parameter of freeze- or heat-induced protein denaturation (Park and Lanier 1987). There has been much research into additives that support the gel-forming ability of surimi using DSC (Wu et al. 1985; Mochizuki et al. 1987; Chung and Lee 1991). Three transitions due to protein denaturation and one transition due to starch gelatinisation during thermal treatment were identified by DSC, whereby thermal transitions of starch and fish protein seemed to proceed independently in mixture systems (Wu et al. 1985). Almost the same was found for mixtures consisting of frozen surimi of Alaska pollack and lard. Thermal transition of surimi, namely gelation, was irreversible; whereas that of lard was reversible and both transitions proceed independently in the mixture (Mochizuki et al. 1987). According to Chung and Lee (1991), the compression-expressible moisture and the DSC-bound water could be used to evaluate gel integrity and cohesiveness. The amount of DSC-measured bound water of the different starch-incorporated surimi gels correlated positively with compression force (r = 0.94) and penetration force (r = 0.99), but inversely with expressible moisture (r = −0.99). In DSC measurements on various fish species, it was found that the

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easy-setting meat and the easy-disintegrating meat required a small quantity of heat to change the structure (Iso et al. 1991). Also, invertebrates such as squid were subjected to DSC. Stanley and Hultin (1984) found the DSC pattern of frozen squid muscle comparable to that of rabbit muscle, and identified actin as the muscle protein most impervious to proteolytic degradation. DSC of autolysed comminutes showed no changes in the characteristic thermal transitions relative to the control sample (Rodger et al. 1984). This overview of early applications of DSC in the investigation of quality and safety connected with fish processing should be completed with an interesting use of DSC to determine the state and content of water in normal fish lenses. Compared with other animals, it was found that fish lenses had both the lowest contents of bound water and total water, whereas avian lenses had the highest amounts of bound water and total water content (Lundgren et al. 1986).

8.4 Recent applications of DSC for investigating quality and safety 8.4.1

Thermal stability of muscle proteins

Fish myosin, the major structural muscle protein, has been the main focus of research. It comprises, together with titin, the thick filaments of the sarcomere. The protein has a molecular mass of about 500 kilodaltons (kDa) and plays a major role in the functionality of the muscle. Myosin is composed of two heavy (about 200 kDa each) and four light (about 20 kDa each) chains. The amino (N)-terminal halves of each heavy chain fold into two globular heads, and the carboxy (C)-terminal ends fold into distinct α-helices that wrap around one another to form a long, fibrous, coiled-coil tail. Myosin constitutes 50–58% of the myofibrillar proteins of fish. From a technological point of view, the structural proteins, namely myosin and the regulatory proteins like actin, tropomyosin and troponin, which are present in the thin filaments, determine the functionality of muscle as food, such as oilemulsification capacity, gel-forming ability, whippability and chewability, among others (Venugopal 2006). Differences of thermal stability of the overall structure, which included an α-helical structure of rod, among fish were elucidated (Ogawa et al. 1993). A total of 10 species were used. DSC curves of most fish myosins showed three peaks, ranging from low to high temperature. In contrast, myosins from horse mackerel and rabbit showed only one major peak. It was concluded that the denaturation process differed from species to species; some fish myosins gave triple peaks, others double peaks. It was also concluded that the thermal denaturation recognised in the endothermic peaks of myosin arises from breaking α-helical structures. The thermal stabilities of the proteins of a range of fish muscles of different habitat temperatures were determined before and after frozen storage at −20 °C (Davies et al. 1994). In whole muscle, a clear relationship was observed between habitat temperature and thermal denaturation of myosin, which persisted when isolated myosins were analysed under conditions close to physiological pH and ionic strength. The ionic environment of the myosin molecules in the whole tissue will, however, not be the same as in the myosin solution. After frozen storage, the myosin transitions in red snapper, a warm-water species, were markedly changed in whole muscle but not in isolated myosin, suggesting the post mortem

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development is an interaction with other muscle proteins or metabolites, salts and others. In contrast, in cod, a cold-water species, changes in myosin transitions were very similar, both in whole muscle and isolated myosin. From the results obtained, it was concluded that: (1) actin and its associated proteins may undergo some modification during prolonged frozen storage, but the effect is minimal compared with myosin; (2) there appears to be a correlation between the extent of ‘freeze-denaturation’ of the muscle (predominantly the myosin and the habitat temperature of the fish; (3) the rate of unfolding of myosin in frozen muscle appears to be similar for both red snapper and cod and appears to continue, for cod, for up to 54 weeks’ frozen storage; and (4) on cooking fresh muscle, the different domains of the myosin molecules denature and aggregate in a different way to that observed during freeze denaturation (Davies et al. 1994). The thermal stability of myosin of carp was compared with that of rabbit by DSC. A structural change of myosin began at 40 °C for rabbit and at 30 °C for carp. Additionally, rabbit myosin unfolded in a single peaked transition whereas carp myosin unfolded in double-peaked transition (Ogawa et al. 1994). In studying the thermal unfolding of myosin rods from carp acclimated to 10 and 30 °C, differences in thermal stability reflecting structural properties were clearly demonstrated (Watabe et al. 1998). Rod of carp acclimated to 10 °C showed three distinct endotherms at 32.9, 33.4 and 44.1 °C, respectively, whereas the transition temperatures from the 30 °C-acclimated carp rod were 34.5, 39.7 and 46.7 °C, respectively. A low thermostability with cold-acclimated carp was found to prevail over most parts of the myosin molecule. Tropomyosin, one of the striated muscle regulatory proteins, forms a family of highly conserved actin-binding proteins (Huang and Ochiai 2005). After isolating tropomyosins from fast skeletal muscle of various fish species, differential scanning microcalorimetry (microDSC) was used to measure their thermal stability. The major endothermic peaks appeared at around 40 °C for all the tropomyosins examined. However, Atlantic salmon showed the lowest transition temperature (37.7 °C), whereas pufferfish showed the highest (46.6 °C). The clear differences in thermostability observed among the tropomyosins from different species led to a suggestion that tropomyosins from all the species modified their structures in response to environmental changes. When Alaska pollock and white croaker myosin were compared with rabbit myosin using microDSC, all of the DSC curves were different from each other, but all showed three major endothermic peaks. Alaska pollock myosin exhibited Tmax at 32.7, 44.1 and 50.4 °C, whereas white croaker myosin exhibited Tmax at 34.8, 44.1 and 58.4 °C, suggesting that the latter is thermally more stable than the former. Rabbit myosin showed Tmax at 45.4, 50.9 and 54.6 °C. According to circular dichroism investigations of these myosins upon heating, it appeared that a close relationship of endothermic peaks in DSC with unfolding of α-helical structure existed (Fukushima et al. 2003a). Light meromyosins (LMMs) of white croaker and walleye pollack were prepared in an expression system using Escherichia coli and determined for their thermodynamic properties by using microDSC. White croaker LMM formed dimer by heating at 80 °C and showed only a single peak at 32.1 °C of temperature transition in DSC. On the other hand, walleye pollack LMM hardly formed polymer and showed four peaks at 27.7, 30.5, 35.8 and 43.9 °C. When Cys525 of white croaker LMM was replaced by alanine, this point-mutated LMM showed no change in its DSC profile but formed no dimer upon heating, suggesting a possible role of Cys525 in dimer formation. On the other hand, walleye pollack LMM, where Cys491 was substituted by alanine, changed its DSC profile, showing four peaks at 27.9, 29.1, 38.4 and 43.9 °C. Results suggest that cysteine residue(s)

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participates in thermal gel formation of LMM when it locates in a suitable position of the sequence (Fukushima et al. 2003b). The thermodynamic properties of myosin and its C-terminal fragment, LMM, from walleye pollack were analysed by using microDSC. Recombinant walleye pollack LMM expressed in Escherichia coli was also subjected to DSC for reference. The two proteins prepared from frozen surimi showed three endothermic peaks, the transition temperatures of which were quite similar, although overall DSC patterns differed considerably from one another. Recombinant LMM gave four endothermic peaks at 27.4, 30.8, 36.5 and 43.4 °C in DSC. The peak at 27.4 °C could not be observed in walleye pollack LMM prepared from frozen surimi (Togashi et al. 2002). Investigating the thermal properties of Japanese stingfish (Sebates inermis), transition temperatures of each protein as determined by DSC were as follows: myosin, 40.9 °C; actin, 61.1 °C; heavy meromyosin, 40.9 and 59.3 °C; and light meromyosin, 62.2 °C. When myosin was digested with trypsin, subfragment-1, the head portion of myosin had a transition temperature similar to myosin. The portion corresponding to subfragment-2, the central region of myosin, corresponded to the high temperature-peak (Nagai et al. 1999). Changes in the conformation of catfish (Ictalurus punctatus) myosin due to (1) anions, (2) acid pH, and (3) salt addition were determined, among others, by microDSC. Acid-treated myosin subjected to heating from 5 to 80 °C showed two sharp endothermic peaks at around 38 and 44 °C. The different acid treatments changed the thermal sensitivity of myosin toward denaturation. The ΔH of acid-treated myosin with salt added before unfolding was significantly higher than myosin with salt added after refolding. This indicates that the addition of salt to myosin before acid treatment provides greater stability against denaturation than the addition of salt after acid treatment. DSC studies indicated major thermal transitions, occurring between 35 and 50 °C. ΔH for both orders of salt addition decreased in the following order: control >Cl− >PO43− treatment. Myosin treated with Cl− and PO43− showed lower ΔH values on heating than the control, which is likely due to denaturation and unfolding of myosin by acid treatments. Among the two anions, PO43− treatment showed lower ΔH than Cl− treatment, indicative of greater denaturation of myosin by PO43− treatment (Raghavan and Kristinsson 2007). Richards et al. (2007) compared thermal stability of haemoglobin from tilapia (Oreochromis niloticus) with that from trout (Onchorhynchus mykiss) by microDSC. Tilapia haemoglobin had a single peak at 61 °C, whereas trout haemoglobin had one peak at 52 °C and another at 60 °C. Scans were conducted only at pH 7.4, as reliable results could not be obtained at pH 6.3 owing to extensive aggregation.

8.4.2

DSC measurements taken on invertebrates

The Argentine group of Crupkin specialises in this field. During the past 10 years, numerous papers have been published dealing with DSC measurements on mussels (Aulacomya ater ater), squid (Illex argentinus) and scallop (Zygochlamys patagonica). Whole adductor muscle of mussel free of connective tissue showed two transitions (Tmax 50.5 and 72.5 °C) and ΔH of 2.5 cal/g. Sarcoplasmic proteins contributed to both denaturation peaks. The DSC curves of actomyosin were similar to those of whole muscle. Two endothermic peaks (36 and 50.5 °C) were observed in the DSC curve of myosin. DSC curves corresponding to

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actomyosin, paramyosin and sarcoplasmic proteins indicated that myosin and paramyosin contribute mainly to the first transition and that actin is responsible for most of the second transition in whole muscle. Thermal stability of whole muscle decreased with increasing pH and ionic strength. Total denaturation enthalpy significantly decreased with an increase of the ionic strength (Paredi et al. 1994). Post mortem thermal behaviour of the striated adductor muscles of mussel stored at 2–4 °C was characterised as follows. The exothermal peak present in pre rigor muscles from other species, such as fish or mammalian muscles, was not evident in DSC curves of either fresh or cold-stored adductor muscles. In the first 8 h of storage, the greatest increases in both total ΔH and ΔH related to the first endothermal transition were observed. It was suggested that onset of rigor mortis in the mussel can be determined by enthalpy measurements (Paredi et al. 1995). Mussels (Perna canicula), which were thermally treated during processing to facilitate an easier removal of shells, were investigated on their degree of doneness by DSC. DSC curves of differently treated samples revealed by the number of peaks identified as well as by their ΔH that trade samples in question were only blanched and not completely well cooked (Rehbein and Schubring 1996). The DSC curves of whole muscle of female squid showed four endothermic transitions, with Tmax equal to 45.9, 56.8, 67.2 and 79.2 °C, respectively. DSC curves of whole muscle of male squid showed three transitions, with Tmax equal to 47.9, 56.8, and 79.2 °C, respectively. Tmax of 67.2 °C, present only in female squid muscle, was related to sarcoplasmic proteins. Myosin and paramyosin contributed to the first transition, connective tissues to the second transition, and actin to the last transition. No major differences were observed in Tmax values that were related to the sex and sexual maturation stage of specimens. The lowest ΔH was found in muscle from immature females. Independent of sex and sexual maturation stage of specimens, no major changes were observed in either Tmax or ΔH during frozen storage of squid (Paredi et al. 1996). DSC curves of both striated and smooth muscles free of connective tissue derived from scallop showed two transitions, Tmax of 53.2 and 79 °C, and Tmax of 52.7 and 78 °C, respectively. These results indicate that the different paramyosin content of the muscles did not influence the thermal stability of their proteins. DSC curves of myofibrils and actomyosin were similar to those corresponding to respective whole muscles. Myosin from striated muscles showed a cooperative single peak, with Tmax equal to 48.8 °C. Similar Tmax values were observed in DSC curves of myosin from smooth muscle. As pH and ionic strength increased, thermal stability of whole muscle decreased. Smooth muscles were more affected than striated muscles. The pH increment significantly affected ΔH of whole smooth muscles. ΔH significantly decreased when ionic strength increased to 0.5 in both types of muscle (Paredi et al. 1998). DSC curves of both striated and smooth whole muscles of scallop showed two transitions, Tmax 55.0, 79.2 °C and Tmax 54.7, 78.7 °C, respectively. The pH increase (5.0 to 8.0) significantly decreased ΔH of whole striated muscles. Significant decreases in ΔH were also observed in DSC curves of smooth muscles at pH 8.0. ΔH significantly decreased when ionic strength increased from 0.05 to 0.5 in both types of muscle. Striated muscles were affected more than smooth muscles by changes in the chemical environment (Paredi et al. 2002). Three major endothermic peaks at 50, 57 and 74 °C were found in the DSC curve of the mantle of cuttlefish (Sepia esculenta). The first and second peaks mainly corresponded with the denaturation of myosin and collagen. The third peak was that of actin, which was almost native up to 63 °C, whereas the other proteins had

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been completely denatured below 63 °C (Mochizuki et al. 1995a, b). Comparable results were reported for Sepia pharaonis (Thanonkaew et al. 2006). They found three endothermic transitions, with Tmax values of 49.8, 59.8 and 74.7 °C in the head portion, and three in the mantle portion with Tmax values of 50.3, 60.3 and 78.8 °C, corresponding to the thermal denaturation of myosin and paramyosin, connective tissues and actin, respectively. It was concluded that the thermal stability of an individual portion varied, depending on cuttlefish portions. In frozen and thawed freshwater prawns, the onset and peak melting temperatures corresponding to myosin denaturation, as well as ΔH of prawn muscle, decreased after freezing– thawing treatments (Srinivasan et al. 1997a). There were no significant differences in the thermal properties of prawns with changes in the rate of freezing. However, the thermal properties were influenced by the rate of thawing. Rapid thawing resulted in a lower thermal stability of prawn proteins compared with slow or moderately fast thawing methods. Keeping prawn shells intact or not intact during freezing–thawing did not alter the thermal properties of the prawn proteins. Fresh prawns were subjected to five freeze–thaw cycles (−29 °C to 22 °C). ΔH decreased from 16.6 J/g (fresh) to 13.5 J/g after one freeze–thaw cycle with minor changes thereafter (Srinivasan et al. 1997b). The influence of protein content (25, 35 and 40%) in the feed given to blue shrimp on their thermal stability was investigated (Rivas-Vega et al. 2001). DSC curves showed three transition peaks, with Tmax values of 52, 72 and 86 °C. An influence of feed was not obvious. When fish (blue marlin, saury and skipjack) and shellfish (scallop and prawn) meat were compared for their thermal properties, three main endothermic peaks were displayed in the DSC curves (Uddin et al. 2001). Values of Tmax for myosin and sarcoplasmic proteins were approximately similar in both fish and shellfish meats, whereas that for actin was significantly higher in shellfish compared with fish meat. From our recently performed DSC measurements on deepwater rose shrimps (Parapenaeus longirostris) caught in Turkey, it became obvious that frozen/thawed muscle of shrimp exhibited three peaks at 33.4, 50.4 and 57.9 °C, respectively. Pre-heating of samples to different temperatures in the range 30–70 °C caused the following changes in the DSC curves: at 40 °C the first peak disappeared, at 50 °C the second peak disappeared additionally, and at 70 °C no peak was detectable. However, in the range 50–65 °C, an additional peak at 73 °C became visible. A comparison of DSC curves taken from deepwater rose shrimp with those of black tiger shrimps (Penaeus monodon) farmed in Vietnam and white shrimps (P. vannamei) farmed in Ecuador revealed that the values of Tmax of the first peak were different, at 40 and 45 °C, respectively. This can be seen as an indication that species are possibly more responsible than environmental temperature for thermal stability of muscle proteins of shrimp. Thermal stability of muscle proteins from black tiger shrimp and from white shrimp were compared by DSC (Sriket et al. 2007). Two major peaks were obtained, corresponding to myosin and actin peaks. The two shrimps had similar Tmax and ΔH values of the first peak, suggesting that myosins of both shrimp muscles had similar temperatures and energies required for denaturation. Tmax of the second peak, representing actin of black tiger shrimp, was lower than that of white shrimp. However, no differences in ΔH of actin were found between the two species. This result revealed that the actin of white shrimp meat was more likely to resist thermal denaturation than that of black tiger shrimp meat. In shark muscle during heating, the following changes measured by DSC were assumed (Chen 1995): (1) changes in conformation of myosin molecules including solubilisation and

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denaturation of myosin tails might occur at a low temperature (30–43 °C), corresponding to the low endothermic peaks; (2) at higher temperatures (47–57 °C), the head portion of the myosin molecule might project from the filament to enable more interactions among head portions; and (3) further interactions between myosin and actin could then form the actomyosin complex. In DSC analysis of small hammerhead shark (Sphyrna levini) muscle during cold storage at 5 °C, an exothermic peak within 25–45 °C and a low-temperature endothermic peak (LTEP) around 35 °C were observed after 8 and 56 hours, respectively (Chen et al. 1996a). The disappearance of LTEP accompanied the increase in the peak area at 61 °C, which was considered attributable to the denaturation of actomyosin complex. The thermal stability of thresher shark (Alopias pelagicus) muscle decreased during frozen storage at −18 °C (Chen et al. 1996b). The exothermic peak between 30 and 40 °C observed for the unfrozen muscle and the LTEP disappeared in the DSC pattern of muscle stored for 150 days. The longer the fish chunks were stored, the sooner the LTEP or endothermic peak around 50 °C disappeared and the endothermic peak at around 62 °C appeared. During frozen storage of silvertip shark (Carcharhinus albimarginatus), it was observed that the exothermic peak at around 45 °C visible for both fresh and 7-day frozenstored muscle disappeared in the muscle stored longer than 14 days. Tmax of the endothermic peak of around 57 °C shifted to a lower temperature, and ΔH of muscle diminished with storage time. This revealed that the thermal stability of shark muscle decreased for the duration of storage time (Chen 1996). The DSC pattern of different shark meat (catsharks, Scyliorhinus spp., smoothhounds, Mustelus spp., liveroil sharks, Galeorhinus spp.) showed two main endothermic peaks and additionally a smaller one at lower temperatures. Skin was characterised by a pronounced peak that can be attributed to collagen. Additionally, DSC measurements were taken on previously heat-treated shark meat and skin (smoothhounds). Peaks disappeared gradually in the DSC curves taken on meat with increasing temperature. In skin samples the collagen peak at around 48 °C disappeared completely after heating to 45–50 °C, whereby after heat treatment to 50 °C a new small peak appeared at a lower temperature (around 28 °C), which resulted probably from denaturation of collagen to gelatine (Schubring 2007). Figure 8.4 displays the DSC curves of several invertebrates and shows the species-specific pattern.

8.4.3

DSC measurements on connective tissue and collagen

In Japan, a great amount of fish scales is produced in sardine-processing factories and has potential as an important collagen source. The DSC curve of soluble collagen obtained from sardine scales indicated one peak, with Tmax at about 44 °C. This denaturation temperature was about 10 °C lower than that of pig collagen gel and is therefore interesting for food application (Nomura et al. 1996). Isinglass, a substance used to clarify alcoholic beverages, is derived from the swim bladder of certain tropical fish and consists predominantly of collagen. It exists as a rod-like triple helical molecule and is thermally labile. A Tmax of 29.8 °C and ΔH of 58.1 J/g were measured by DSC (Hickman et al. 2000). Collagens of skin and bone from bigeye snapper were classified as type I collagen. An endothermic peak, with Tmax at 31.0 and 31.5 °C, was observed for collagens from the skin and bone rehydrated in water, respectively. For collagens rehydrated in acetic acid, Tmax shifted to lower temperatures, 28.7 and 30.8 °C for collagens from skin and bone, respectively (Kittiphattanabawon et al. 2005).

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–5.8 Scallop –6.0

–6.2

–6.4

Shrimp

Squid

–6.6

–6.8 ↑ Exo 25

30

35

40

45

50

55

60

65

Furnace temperature (°C) Figure 8.4 DSC curves of several invertebrates (scallop, Patinopecten spp.; shrimp, Parapenaeus longirostris; squid, Todarodes sagittatus).

Skin collagen from bigeye snapper has high thermal stability compared with those reported for some cold-water fish species (Jongjareonrak et al. 2005). The effect of frozen storage (−10 and −30 °C), formaldehyde and fish oil on collagen, isolated from cod muscle, was investigated (Badii and Howell 2003). DSC showed a highly cooperative transition at 28.2 °C for isolated collagen. Changes in the thermodynamic properties of collagen were observed on frozen storage at −10 °C compared with the control at −30 °C because of changes in structure. In the presence of formaldehyde, there were no changes in the DSC collagen transition; however, in the presence of fish oil, there was an increase in enthalpy and an extra peak was observed at 44.6 °C, indicating collagen–fish oil interaction. Interaction of gelatine obtained from the skin of North Sea horse mackerel (Trachurus trachurus) with egg albumin proteins was investigated (Badii and Howell 2006). Horse mackerel gelatine solutions of 3, 5, 7 and 10% w/w in distilled water denatured at Tmax 14.7 °C, 14.7, 15.1 and 15.0 °C, which were not significantly different; however, ΔH values were. The samples were heat–reversible, with minor changes in Tmax and ΔH on a second scan of each gelatine sample confirming that gelatine undergoes a helix to coil transition on heating and on cooling refolds, recovering most of the helical structure. DSC curves of egg albumin showed three transitions, which were not reversible. In the mixture of 3% gelatine and 10% egg albumin, gelatine showed one cooperative transition, with Tmax 15.0 °C and ΔH 1.5 J/g. Even in the presence of egg albumin, this transition was reversible.

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Gelatines were prepared from the skins of the tropical fish, sin croaker (Johnius dussumeiri) and shortfin scad (Decapterus macrosoma), and compared for their thermal stability with bovine gelatine. The values of Tmax of gelatine gels were 28.9, 24.6 and 18.5 °C respectively for bovine, shortfin scad and sin croaker gelatines. The melting point of bovine gelatine was significantly higher than that of the other gelatines. These melting points were far higher than those reported for cod skin (Cheow et al. 2007).

8.4.4

Heat-induced gelation process

Heat-induced gelation of surimi, an intermediate product produced by repeated washing of minced fish and mixing with cryoprotectants to extend its frozen shelf life, is an important step in the manufacture of a variety of surimi-based seafoods such as kamaboko, fish meat gel, and crab and other shellfish analogues. Heat-induced gelation of surimi is a complex physicochemical process involving structural and functional changes of myofibrillar proteins. DSC measurements were an obvious application for investigating these processes. Arrowtooth flounder myosin was found to undergo a multistage denaturation process, as characterised by an endothermic trough and peaks. Tonset was observed at 25 °C whereas Tmax was seen at 36 °C with one transition peak detected at 30 °C. This indicates that myosin of arrowtooth flounder is highly unstable to heat (Visessanguan et al. 2000). To investigate how proteolysis affects heat-induced gelation, papain was added to arrowtooth flounder myosin. This significantly decreased the enthalpy required to induce myosin denaturation without significant changes in Tonset and Tmax. Thermal denaturation kinetics indicated decreases in both activation energy and the rate of myosin denaturation (Visessanguan and An 2000). DSC curves of silver hake and mackerel surimi showed three endothermic peaks. When starch was added to surimi samples, an endothermic peak having a large area at a temperature of about 70 °C was observed, which overlapped the third actin transition. No significant shifts in the endothermic peaks of myofibrillar proteins were detected with increasing starch content. Transition temperatures for the starch–surimi system were higher than those for the starch–water system. There were deviations in the apparent heat capacity function calculated from DSC measurements in surimi samples containing starch. These are attributed to gelatinisation of starch and modification of water structure (Belibagli et al. 2003). Protein structural changes during preparation and storage of surimi investigated by DSC revealed a loss of myofibrillar proteins from surimi after three washing cycles and indicated greater protein stability in surimi compared with minced fish (Moosavi-Nasab et al. 2005). The use of polysaccharide gum such as hydroxypropylmethylcellulose in horse mackerel surimi as a possibility to reduce fat content resulted in variation of thermal stability (Chen et al. 2005). Medium-grade Alaska pollock surimi was used to investigate the effects of functional protein additives on calorimetric properties. The myosin peak temperature was shifted to higher values with addition of protein additives. These protein additives appeared to delay denaturation (unfolding) of fish proteins. Protein additives reduced the enthalpy of endothermic peaks. The reduction in enthalpy was possibly due to increased protein aggregation enhanced by protein additives (Park 1994). The thermal denaturation of tilapia (Oreochromis nilotica) surimi was shown to occur through three independent processes, and the temperatures at which these processes took place were 57.1 °C, 61.6 °C and 65.7 °C.

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Addition of 0.2% of carrageenan raised the gelatinisation temperatures of tapioca and modified waxy maize starches by 8 °C and 4 °C, respectively. However, it decreased the reaction enthalpy of waxy maize from 101.1 to 10.4 cal/g, and tapioca from 184.1 to 4.4 cal/g. The addition of starch and carrageenan to surimi was investigated by DSC. It was observed that for the surimi/tapioca and surimi/tapioca/carrageenan, the transition temperature increase was 20 °C compared with pure surimi (Barreto et al. 2000). Mixtures of κcarrageenan plus other hydrocolloids (locust bean, guar, xanthan, í-carrageenan, sodium carboxymethylcellulose, and sodium alginate) were examined for their effects on the thermal behaviour of heat-induced gels made from washed blue whiting mince. DSC revealed faint interactions for the mixtures of κ-carrageenan with locust bean and with xanthan, and weakly synergistic gelling effects between the last two hydrocolloids. The blend of κ-carrageenan with sodium alginate exhibited thermally strong synergistic interactions, but no particular effects were induced on corresponding functional properties (Perez-Mateos et al. 2001). Changes in thermal properties of ribbonfish (Trichiurus spp.) meat during different periods of ice storage were investigated (Dileep et al. 2005). The DSC profile of fresh ribbonfish meat revealed transitions at 33.2 °C, 48.9 °C and 61.0 °C, indicating the denaturation temperature of different protein fractions. The gelatinisation temperature of tapioca starch solution was found to be in the range 60–65 °C, and for cornstarch 67–70 °C. The viscoelastic properties of ribbonfish meat were altered significantly, both due to the addition of starch and ice-storage period (Dileep et al. 2005). DSC was used to study the effect of fish protein, salt, sugar and monosodium glutamate (MSG) on gelatinisation of tapioca and sage starches in fish cracker mixtures. One endothermic transition was observed for fishstarch mixtures (10–90% wet fish) if the moisture content was more than 61%. The effect of the salt on the starch gelatinisation was greater than sugar and MSG. Sugar and MSG addition to the mixture had little effect on gelatinisation of starch in the system. Two per cent salt increased the gelatinisation temperature by 4–5 °C. Tonset and Tmax increased with increases in fish content in fish-starch mixtures but the conclusion temperature (the temperature at which the DSC signal ceases to deviate from the baseline) remained relatively constant. Increases in fish content also narrowed the gelatinisation temperature ranges (Cheow and Yu 1997).

8.4.5

Antifreeze activities

Antifreeze glycoproteins originating from fish were analysed for their activities by DSC. These proteins prevent the growth of ice crystals in the supercooled organisms. In polar fish, antifreeze glycoprotein consists of eight glycoproteins, sequentially numbered based on the mobility in polyacrylamide gel electrophoresis. DSC revealed that the low molecular mass glycoprotein 8 was sensitive to cooling rate, whereas the high molecular mass glycoproteins 1–5 were not. DSC curves revealed an initial shoulder in the exotherm direction upon cooling, which correlates with observed c-axis ice growth. DSC further revealed that glycoprotein antifreezes have a linear increase in thermal hysteresis or antifreeze activity with a decrease in sample ice content (Hansen et al. 1991). Recently, it was shown that the smaller antifreeze glycoproteins 7 and 8 from Antarctic fish showed reduced levels of inhibition of ice growth, as indicated by the absence of an initial exotherm and the absence of a lag at the start of each run (Ramløv et al. 2005).

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The cryoprotective properties of proline in cod muscle were studied during freezing to − 60 °C and subsequent heating to 10 °C. No exothermic or endothermic transitions due to the crystallisation of proline were observed, suggesting that proline has the ability to stay in solution when the freezable water fraction of cod muscle was converted to ice. Therefore, proline may represent a promising ingredient as a cryoprotective in fish products (Rasmussen et al. 1997). Protein denaturation during frozen storage at −18 °C for 16 weeks was studied in jack mackerel (Trachurus murphyi) actomyosin. The cryoprotective effect of different additives was evaluated at a level of 8% (w/w): sucrose/sorbitol (1 : 1), maltodextrin 25 DE, milk whey and sodium lactate. DSC curves showed two endothermic transitions with Tmax at 46.8 °C and 68.9 °C assigned to myosin and actin, respectively. The best cryoprotective effect was achieved with sucrose/sorbitol and maltodextrin 25 DE. These additives showed Tmax and ΔH values for myosin and actin significantly higher than the control (Dondero et al. 1996). The antifreeze activities of saccharides that consisted of glucose measured by DSC were higher than those of the other food components. In salts, those that possessed high ionic charge had high antifreeze activities. In water-soluble amino acids, a few amino acids (threonine, arginine and proline) that formed no eutectic mixture above −20 °C had especially high antifreeze activities (Mizuno et al. 1997). A DSC heat denaturation study on the effects of various maltodextrins and sucrose on protein changes in minced blue whiting muscle during frozen storage at −10 and −20 °C revealed that all maltodextrins slowed the decreases in ΔH ascribed to myosin and actin, making evident a noticeable effectiveness against protein denaturation, especially at −20 °C. Sucrose was as effective as maltodextrins at −20 °C, but was the least effective treatment at −10 °C. Significant correlations between both ΔH and either protein solubility or formaldehyde production were found at each storage temperature (Herrera et al. 2001a, b). Unlike conventional surimi processing, a novel method of fish protein isolate chemically induces denaturation by altering pH during the process. The new process for fish protein isolate, where protein structures are intentionally unfolded/refolded by pH-shift, is almost ready for commercialisation. However, it is unknown whether cryoprotectant will be required or not. This process has been investigated to improve gelation properties and yield using acid- or alkali-aided treatment. The aims were to determine the effect of pH on fish protein isolate during frozen storage (pH 5.5 and pH 7.0) and the effect of cryoprotectant on the functional properties of the fish protein isolate. The DSC curve of Pacific whiting conventional surimi contained four endothermic transitions, with Tmax of 35.4 °C, 41.2 °C, 51.1 °C, and 68.8 °C, respectively. All alkali-treated protein isolates, with cryoprotectants with and without freeze/thaw treatments, showed three endothermic peaks, with Tmax about 33.5 to 34.7, 46.2 to 47.8 and 66.5 °C to 68 °C, respectively. Alkali-treated protein isolates kept frozen at pH 5.5 were relatively less stable than those stored at pH 7.0. Actin was highly sensitive to the pH-shift method, particularly samples without cryoprotectants. DSC curves of actin appeared at Tmax of 66–68 °C with very small endothermic transition (<0.04 J/g) in the samples with cryoprotectants, and disappeared in the samples without cryoprotectants. (Thawornchinsombut and Park 2006). To use fishery waste products as functional food material, shrimp head protein hydrolysate was produced from shrimp wastes by enzymatic hydrolysis. Their effects on the state of water and the denaturation of myofibrils during dehydration were evaluated by DSC. Results revealed that the amount of unfrozen water increased significantly after addition of

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Heat flow

Glass transition temperature

Temperature Figure 8.5 Schematic presentation of glass transition measured by DSC.

shrimp head protein hydrolysate, and implied that this hydrolysate can be used as an alternative food preservative to suppress the dehydration-induced denaturation of myofibrils (Ruttanapornvareesaku et al. 2006).

8.4.6

Glass transition in fish meat

Food is in the frozen state when a high proportion of freezable water exists as ice. From a physical aspect, as major food systems are cooled, ice formation takes place but a fraction of the water does not undergo phase transition and remains in the liquid phase to a very low temperature. In this non-freezing water, various chemical changes such as denaturation and discoloration may take place. The importance of relationships between glass transition and food processing, product properties, quality and stability have been increasingly recognised by food scientists. From such studies, it was established that in aqueous solution systems, glass transition could take place below ice crystallisation temperature and that glass transition temperature (Tg) depends on the solute concentration of the freeze concentrated phase (Cg). Glass transition (Figure 8.5) is hypothesised to be important for storage stability and product quality of frozen foods. Glass transition of fresh red meat of bigeye tuna (Thunnus obesus) and its filtrate occurred between −71 and −68 °C, independent of cooling rate from 1 K/min up to 50 K/min. Glass transition at low temperature appeared to occur in the liquid part. Neither dilution nor concentration of the filtrate affected Tg, but Cg was affected. Addition of salt to the filtrate caused a decrease of about 20 K in Tg. Results suggested the possibility of improvement in quality of fish by lowering the storage temperature by 15 K below the present storage temperature (−55 °C) (Inoue and Ishikawa 1997). Reports of Tg values for frozen muscle tissue are not common, and reported values are mostly much lower than would be expected. Tg values for muscle tissue and isolated proteins have been studied using DSC. The apparent Tg of mackerel, cod and beef were similar (about −11 to −13 °C) and substantially higher

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than most published values (−58 to −77 °C for tuna and beef), but in accord with expectations for substances of high molecular mass. Dialysed insoluble and soluble protein fractions from mackerel yielded apparent Tg values (about −7 °C) that were similar, with both being higher than those for whole muscle (Brake and Fennema 1999). Physicochemical characterisation of water in tuna muscle was performed using DSC at very low temperature. The temperature dependence of this reaction was analysed by an Arrhenius plot that resulted in two break points. The first break point occurring at freezing point might be due to the freeze effect. The second was at −10 °C. The reaction rate change steeply declined at the temperature range of −70 to −84 °C, and was thought to be related to glass transition that may occur in the fish sample (Agustini et al. 2001). Fresh tuna meat was dried in a freeze drier to vary the moisture content from 73.3% to 6.0% (wet basis). The cooling curve method was used to measure the freezing point and end point of freezing. The state diagram yielded maximally freeze-concentrated solutes at 61% solids with the characteristic temperature of glass formation being −54.2 °C (Rahman et al. 2003). DSC measurements have revealed different thermal transitions in cod and tuna samples. Transition temperatures detected at −11, −15 and −21 °C were highly dependent on the annealing temperature. In tuna muscle, an additional transition was observed at −72 °C. This transition appeared differently than the thermal events observed at higher temperatures, as it spanned a broad temperature interval of 25 °C. The transition was comparable to low-temperature glass transitions reported in protein-rich systems. No transition at this low temperature was detected in cod samples. The transitions observed at higher temperatures (−11 to −21 °C) may possibly stem from a glassy matrix containing muscle proteins. However, the presence of a glass transition at −11 °C was in disagreement with the low storage stability at −18 °C during practical time scales. It was proposed that freezing of cod could be associated with more than one glass transition, with a Tg at a temperature lower than −11 °C being too small to be detectable with instruments, yet governing important deterioration processes. To optimise frozen storage conditions, the relationship between deterioration processes important for preservation of quality and Tg still needs to be established (Jensen et al. 2003). The glass transition state of Katsuobushi (boiled and smoke-dried bonito) was studied by DSC. Tg was about 33 °C in 14.8% moisture. The moisture content of Katsuobushi on the market is approximately 12–15%, and Tg of katsuobushi containing such moisture was approximately 10–30 °C, which was within room temperature range. Tg of katsuobushi showed strong dependence on moisture content, and the Tg value varied from 11 to 165 °C with moisture levels from 18.04 to 0% (Hashimoto et al. 2003). Several dried fish muscles pre-boiled before freeze-drying, such as bonito, tuna, mackerel, cod and sea bream, showed clear glass transition phenomena in DSC curves. In contrast, bonito and cod muscles freezedried without any heat-treatment did not show the detectable glass transition, but after heat denaturation they did so. These experimental results demonstrated that the glass transition is a general characteristic for any dried and heat-treated fish muscles and that the heat denaturation process gives an important effect to its glass transition behaviour. Furthermore, it became clear that the glass transition of fish muscles is affected by the different composition of muscle of different fish species: the Tg of white meat fishes (cod and sea bream) tended to be higher than that of red meat fishes (bonito, tuna and mackerel). Such differences in Tg value dependent on fish species were caused by the difference in the properties of protein fractions, especially myofibrillar protein. Furthermore, it became clear that the Tg values of whole muscle were considerably lower than those of extracted protein fractions because

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of the plasticising effect of low molecular materials contained in the muscle (Hashimoto et al. 2004). The thermal behaviour of fresh tuna muscle, rehydrated freeze-dried tuna muscle and tuna sarcoplasmic protein fraction was studied by DSC. The relationship between Tg and water content was established. Only a low-temperature glass transition was detected for fresh tuna and freeze-dried tuna rehydrated to high water contents, whereas for sarcoplasmic protein fraction both a low-temperature and an apparent high-temperature glass transition were detected for samples of high water content. Construction of the supplemented state diagrams for whole tuna muscle and for tuna sarcoplasmic protein fraction confirmed the lowtemperature transition to be glass transition of the maximally freeze-dehydrated phase. Tg and the concentration of the maximally freeze dehydrated tuna muscle are −74 °C and 79% (w/w), respectively (Orlien et al. 2003). Tg of five protein hydrolysates from Nile tilapia (Oreochromus niloticus) were determined by DSC. Tg diminished with increasing moisture content, showing values between 1 and 55 °C for moisture values between 17 and 6 g water/100 g solids. However, the increase in degree of hydrolysis of the hydrolysates was not reflected in a decrease in Tg (Jardim et al. 1999).

8.4.7

DSC measurements on high-pressure-treated fish and fishery products

Thermal processing of foods has mainly been a two-dimensional process, time and temperature being the two process variables. With the commercialisation of high-pressure processes, pressure may now be used as a ‘third dimension’ to improve food quality and safety. Very recently, the application of DSC to investigate the changes occurring in muscle proteins of fish under the influence of high-pressure processing has been reviewed (Schubring 2005a). The following effects of high-pressure treatment on DSC curves could be outlined. When fresh fish fillet was treated in the pressure range 100–380 MPa for 20 min, the DSC curves showed a shift of the myosin transition towards lower temperature as pressure increased. Protein denaturation was found to be very weak in fish treated at 100 MPa, whereas it was important when pressure was equal or greater than 200 MPa. After pressure treatment, a new low melting transition was seen around 32 °C. This new structure appeared to form at pressures of 100 MPa or more and appeared to be little effected by the degree of pressure (to 800 MPa) to which the fish was subjected. At pressures of 300 MPa and higher, further new structures were formed which appeared to melt in the range 40–60 °C. It was shown that conventional freezing followed by frozen storage for two days had little effect on the characteristic thermal transitions. However, after pressure shift freezing (140 MPa, −14 °C) and frozen storage for 2 days, the DSC curve showed an important reduction of the myosin peak and the appearance of a new important peak at lower temperature connected with a significant decrease in total enthalpy. On the other hand, the actin peak was almost uninfected. High-pressure treatment up to 200 MPa was used to thaw frozen fish and compared with thawing at ambient pressure. Changes in muscle proteins were observed by DSC. Independent of the species, there were remarkable differences between differently treated samples. Whereas the conventionally thawed muscles showed patterns comparable to native proteins of fresh fish muscle, the patterns of high-pressure thawed muscles confirmed a far-reaching denaturation of the muscle proteins as a result of applying high pressure.

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Single peaks, representing the different protein fractions like myosin, sarcoplasmic proteins and collagen, and actin, disappeared almost completely in high-pressure thawed samples. This behaviour, caused by denaturation, could explain the differences in texture and waterbinding capacity seen in the differently treated samples (Schubring et al. 2003). Further observation on thawing included the comparison between pressure-assisted thawing up to 300 MPa and conventionally thawed samples of commercially important species such as herring, ocean perch and saithe. It was found that pressure-assisted thawing of frozen fish fillets in the range 0.1–300 MPa affected progressively the denaturation of muscle proteins with higher pressure. DSC curves mainly showed that myosin denatured with increasing pressure, whereas actin was the more resistant myofibrillar protein. This behaviour appeared to be species specific, as in herring the actin was shown to be the most sensitive muscle protein. Also, the appearance of new low-temperature melting peaks caused by pressure treatment during thawing has been observed for both saithe and herring, indicating that responses to high pressure of the different fish were species specific (Schubring 2005a). In the meantime, some papers were published to the same subject. When hydrostatic pressures (50–300 MPa) were applied at 0 °C for 50 min to determine the aggregation of tilapia (Orechromis niloticus) myosin fragments, it was found that Tmax of tilapia S-1 and rod were approximately 47 and 55 °C. The enthalpy of the native S-1 and rod were 0.18 and 0.09 J/g. With a 200 MPa treatment, only 6% of S-1 enthalpy remained, which decreased with increasing pressure. Notably, when S-1 was treated beyond 250 MPa, no remaining enthalpy was detected. These findings probably indicated that during a 200 MPa treatment, unfold and aggregation occurred in S-1. No enthalpy change was found in rod during pressurisation owing to the compact α-helix structure, although heat caused a helix–coil transition of entangled rod (Ko et al. 2004). High-pressure runs at 275 and 310 MPa for 2, 4 and 6 min, and heat treatments at 90 °C for 35 min, were applied to minced albacore muscle (Thunnus alalunga). Effects on thermal stability were measured. Pressure treatment increased thermal stability of most proteins, shifting their endotherms towards higher temperatures, but only myosin showed an enthalpy change higher than control. Heated proteins did not exhibit an endotherm peak, indicating total protein denaturation (Ramirez-Suarez and Morrissey 2006). Normally, calorimetric experiments are performed with a DSC at a constant (usually atmospheric) pressure using temperature as the working parameter. A high-pressure DSC was examined by Zhu et al. (2004) for the evaluation of high-pressure phase-transition behaviour of pure water (in the melting mode). Two techniques (T-scan and P-scan) were used. Results obtained were statistically different between the techniques, as well as between the experimental and some published models. The pressure-induced phase transition of ice I–water was well illustrated by P-scan. The latent heat of ice melting obtained by isothermal P-scan showed no significant difference from reference data. This good agreement validated the reliability and accuracy of the high-pressure DSC system during the isothermal P-scan test. High-pressure calorimetry requires large cell mass to resist pressure, which could reduce the accuracy/efficiency of calorimetric signal during isobaric T-scan test. T-scan tests produced good and consistent results but were slightly less accurate than P-scan counterparts or published models. The results obtained showed that the system could be successfully used for gathering high-pressure phase-transition information, which is essential for better understanding of pressure-dependent phase transition during high-pressure freezing/thawing of foods. Later, high-pressure DSC was used to evaluate and compare the pressure-dependent

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phase transition behaviour of water in different foods (tylose, potato, salmon, pork and pure water). Small samples (0.48–0.72 g, vacuum-packaged in polyethylene pouches) of these materials were tested through isothermal pressure scan (P-scan, 0.3 MPa/min) at various sub-zero temperatures. P-scan tests produced reliable information on phase transition and latent heat in test foods at elevated pressure. It was indicated that phase transition point (either temperature or pressure) in foodstuffs was significantly depressed compared with that in pure water. Latent heat measured during P-scan of food materials had a trend different from that of pure water owing to the temperature dependence of ice content in frozen foods. Ice-mass-based latent heat showed an insignificant difference from the latent heat between pure water and ice I under pressure. Moisture content was a major factor affecting phase transition and latent heat during pressure processing of foods. The P-scan technique is a powerful technique for successfully understanding pressure-dependent phase-transition phenomena in food products during high-pressure processing (Zhu et al. 2006a). Rapid depressurisation can create uniform, small and abundant ice nucleation during pressure-shift freezing (PSF) which can then protect the frozen food structure from cell damage. The amount of depressurisation-formed ice was evaluated using a high-pressure calorimeter for different food products (tylose, potato, salmon, pork and water). Experiments were conducted at an initial pressure of 62, 82, 112, 156, 180 and 196 MPa, at temperatures set at −5, −7, −10, −15, −18 and −20 °C, respectively (slightly above the phase diagram of water-ice I). Calorimetric curves recorded during PSF tests were used to compute the quantity of ice formed based on heat balance. A polynomial relationship was established for each product to compute the depressurisation-formed ice ratio as a function of the initial pressure applied. This model accurately predicted the maximum ice ratio for PSF at a given pressure (0.1–210 MPa) or the minimum ice ratio for PSF at a given temperature (−22 to 0 °C) (Zhu et al. 2006b).

8.4.8

Processing effects revealed by DSC

In the following section, prominent papers are discussed in more detail to inform the reader about goals of the experiments and conditions applied as well as the results obtained. Post mortem changes and influence of chilled storage DSC was shown to be a rapid method for monitoring the rigor progress and the corresponding formation of actomyosin complex of chub mackerel during ice storage. The heat-induced rigor shortening temperature (Ts) of the ‘in rigor’ muscle observed by DSC in terms of the exothermic peak correlated well with the rigor index and ATP content of the muscle. Ts dropped from 46 to 28 °C and its exothermic enthalpy fell from a maximum value to zero. Endothermic peaks of protein denaturation observed to be above 50 °C could be used to identify the states of actomyosin which changed from a relaxation (52 and 76 °C) state to a contraction (62 and 88 °C) state in response to rigor development. Both the exothermic and endothermic peaks of DSC could be applied to evaluate the progress of rigor. The presence of multiple small endothermic peaks below 40 °C was found to be a sign of severe proteolytic autolysis (Chen and Kong 1997). Figure 8.6 displays DSC curves taken on frozen/thawed ordinary muscle of several fish species in pre rigor (left) and in rigor conditions (right).

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–6.35 Whiting –6.55 Saithe Heat flow

–6.75 Haddock –6.95

Horse mackerel Redfish

–7.35

← Endo

–7.15

27

32

37

42

47

52

57

62

67

72

Temperature (°C) –12.37

Haddock

–12.62 Saithe

–12.87

Heat flow

–13.12 Whiting

–13.37 –13.62

Redfish –13.87

–14.37

Horse mackerel

← Endo

–14.12

–14.62 29

34

39

44

49

54

59

64

69

Temperature (°C) Figure 8.6 DSC curves taken on frozen/thawed ordinary muscle of several fish species in pre rigor (above) and in rigor (below) conditions.

When observing early post mortem structural changes in salmon as well as in wild and fed cod by DSC, the denaturation characteristics of myosin, actin and sarcoplasmic protein differed between salmon and cod, indicating species-specific differences, in particular in the stability of the myosin–actomyosin complex. Differences in DSC curves between wild and fed cod were largely explained by variation in pH (Ofstad et al. 1996).

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Recently, comparative iced storage experiments using flake ice and ice slurry called StreamIce® were performed on board ship using sardine, herring and horse mackerel caught in early autumn as raw material. DSC measurements were performed on samples frozen on board at the given sampling day and thawed after frozen storage by defrosting overnight in a refrigerator. DSC curves taken on specimens of sardine and herring at the various storage times exhibited almost no differences between both the flake ice and StreamIce® samples. DSC curves of sardine were characterised by two main peaks at 39.1 and 67.9 °C, with two smaller peaks at 50.8 and 56.5 °C in between. On the other hand, the DSC of herring exhibited only two main peaks at 38.1 and 69.0 °C. Both species belong to the Clupeidae family and therefore show comparable thermal stability. A remarkable influence of the storage time in ice on DSC pattern was also not detectable. This testifies to the fact that the muscle proteins were not denatured during ice storage. Some breakdown in protein may have occurred with formation of breakdown compounds such as peptides; however, the conformation of the existing proteins did not change as in the case of denaturation (Schubring and Meyer 2006a; 2007). In contrast, when horse mackerel was stored, DSC curves taken from samples after different storage time revealed some signs of protein denaturation, particularly in the StreamIce® batch at the end of storage by shifting the Tmax of myosin to lower temperature and decreasing the transition enthalpy. DSC curves of horse mackerel at the beginning of storage were characterised by two main peaks, with Tmax at 40.6 and 67.7 °C, which were in same range as those for herring and sardine living in the same environment (Schubring and Meyer 2006b). Freezing, double freezing and frozen storage Thawing, refreezing and frozen storage are processing steps that are widespread in fish processing. DSC analysis indicated that after 9 months storage the thermal profile of the −70 °C reference had not changed but treatments stored at −22 °C exhibited a slight broadening of the complex endothermic peak between 30 and 50 °C. There was no evidence that denaturation enthalpy differed significantly between once-frozen, ‘slow thawed’ refrozen and ‘fast thawed’ refrozen treatments. There was some evidence for a slightly higher −70 °C reference ΔH. Protein solubility decline was not caused by complete protein denaturation, as an endothermic event (although less cooperative) was clearly visible in samples with reduced protein solubility. The endothermic peak with Tmax of 75 °C was relatively unaffected by frozen storage or treatment. This suggests that primarily the thermal stability of myosin changed on frozen storage, whereas actin was unaffected (Hurling and McArthur 1996). The possibility of differentiating between single- and double-frozen fillets, both used as raw material for processing frozen breaded and battered fish portions like ‘fish fingers’, was investigated by applying two different types of DSC. Tmax and ΔH of both fillets types were not significantly different. Furthermore, no influence of the rigor state before freezing on thermal stability of the fish muscle proteins in frozen fillets could be detected. The differences in Tmax measured by using the various calorimeters could be explained by the differences in scanning rates applied (Schubring 1999b). In frozen stored hake fillet, the relationship between thermal stability changes and functionality loss was monitored over 40 weeks at −10 and −30 °C. DSC showed differences with storage time and temperature in both Tonset and ΔH, also mostly affecting the myosin transitions. Some protein denaturation occurred with little or no functionality loss. A large fraction of hake muscle proteins remained in the native-like condition, even at the higher

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frozen storage temperature (Careche et al 2002). DSC curves of cod and haddock fillets stored at −10 and −30 °C indicated changes in the protein conformation from their native to denatured state owing to freezing. A direct comparison indicated a noticeable change in the energy required for protein denaturation in fish muscle stored at −10 °C compared with −30 °C for both cod and haddock fillets. The DSC pattern of both species of fish indicated three main transitions. Tmax shifted and ΔH decreased for the myosin peak for cod at −10 °C compared with control at −30 °C owing to protein aggregation in fish fillets. Both cod and haddock fillets indicated similar levels of protein denaturation on storage especially at −10 °C (Badii and Howell 2002a). This suggests that formaldehyde plays a limited role in muscle toughening during frozen storage of gadoid fish. Formaldehyde production in cod was much higher (845 and 1065 nmol/g at 20 and 30 weeks respectively) than in haddock (93 and 101 nmol/g after 20 and 30 weeks respectively) (Badii and Howell 2002b). DSC was used to measure thermal denaturation (30–80 °C) in cod fillets stored at −20 °C and −30 °C for up to 12 months and subsequently from 0 to 21 days in modified atmosphere at +2 °C. The denaturation profiles were subjected to multivariate analysis to determine the effects of the various storage conditions. The main effect of the storage conditions was the difference in the denaturation profiles between short-term chill storage for all samples and long-term chill storage for samples stored at −30 °C. There was also a smaller effect due to the difference between short time frozen storage and long time frozen storage for samples stored at −20 °C. The variation in the DSC curves was primarily due to changes in size and position of the myosin, sarcoplasmatic protein and actin denaturation peaks, where variation in position could be related to changes in pH. Frozen storage at both temperatures and chill storage for samples previously stored at −30 °C caused a decrease in the myosin denaturation peak (Jensen and Jørgensen 2003). In paddlefish (Polyodon spathula) meat, thermal scan with a DSC showed two major endothermic transitions, which were ascribed to denaturation of myosin (55–57 °C) and actin (77–79 °C), respectively. There were no significant changes in Tmax and ΔH for myosin and actin during either refrigerated or frozen storage. These results indicated that myofibrillar proteins of paddlefish meat were relatively stable under the storage conditions examined (Lou et al. 2000). DSC was also used to investigate thermal stability of bighead carp (Aristichthys nobilis), freshwater fish muscle myosin. Three endothermic peaks were observed in the DSC curve of fresh muscle. After addition of salt, transition temperatures shifted to lower temperatures. Pre-heating samples at 70 °C caused virtual disappearance of all transition peaks. Storage at −18 °C for 5 weeks caused changes in myosin transition (Radicˇevic´ et al. 2002). During frozen storage of cod at different temperatures, DSC measurements were performed at regular intervals to observe whether DSC curves were influenced by frozen storage conditions (Schubring 2004b). It became obvious that within a given storage temperature all curves were comparable and mainly independent of the duration of storage. Small differences were observed between the different storage temperatures. This also became obvious from the Tmax for single peaks and ΔH appertaining to these peaks. Although Tmax for the first (myosin) and third peak (actin) were quite comparable and almost independent of the storage temperature, Tmax of the second peak, if measurable, appeared to be influenced by storage conditions. At −10 °C, the second peak had almost disappeared, whereas at −20 and −30 °C the peak was clearly visible and the same transition temperature was measured. For the twice-frozen fillets, however, the peak maximum had changed to lower temperatures by

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approximately 7 °C. It seemed that the second peak in the DSC curve of cod muscle is most sensitive to freeze denaturation. At a storage temperature of −10 °C, which caused the strongest changes in texture and water-holding capacity, the second peak was not detectable, presumably because of pronounced protein denaturation. After refreezing and storage at −20 °C, the transition maximum of the second peak is changed to lower temperatures and can be rated as sign of stronger denaturation compared with storage at −20 and at −30 °C for single frozen fillets. In a repetition of the storage experiment, cod caught in the Barents Sea was used (Schubring 2005b). During storage at −14 °C, Tmax of the myosin peak decreased, obviously indicating a loss of thermal stability at this high frozen storage temperature. However, ΔH, as an indication of the degree of denaturation of this protein, only slightly decreased during the 7 month storage period. However, almost no changes were found for actin in Tmax and ΔH. The minor peak between myosin and actin was sometimes difficult to detect and did not show significant changes. The same tendencies as discussed above were also seen at the single-frozen fillet stored at −20 °C. The decrease in Tmax of the myosin fraction, however, was less pronounced. As expected, during storage of cod fillet at −28 °C, proteins were least influenced. Results obtained on Baltic Sea cod (Schubring 2004b) evidenced higher Tmax for myosin (about 5–6 °C) as well as for actin (about 4–5 °C), indicating that thermal stability of muscle proteins is highly dependent on the environmental conditions. Averaged water temperature in the Baltic Sea is reportedly about 10 °C higher than that of the Barents Sea. The effects of freezing and frozen storage on difference in the specific heat capacity of native and heat-denatured fillet (Δd/nCP) of trout and herring have been studied (Beyrer and Rüsch 2007). Freezing of fillet induced a clear reduction of Δd/nCP of up to 15 mJ/g/K. Frozen storage at −20 °C reduced the Δd/nCP of herring fillet but not of trout fillet. Δd/nCP is suggested as a new parameter for the evaluation of protein stability at freezing and frozen storage of fish. Figure 8.7 shows DSC curves of commercially important fish species. Effects of salting During investigation of the changes in myofibrillar proteins during processing of heavily salted cod by DSC, the following denaturation temperatures for the fresh cod muscle were measured: Tmax for myosin was determined as 43.5 °C, for actin as 73.6 °C and for sarcoplasmic proteins as 59.3 °C, respectively. The salt-curing led to some shifts in transition temperatures and decrease in peak area. The transition peaks became lower, broader and less separable than the DSC curve of the fresh cod. After brine-salting, significant changes in the transition peaks were observed. The peaks shifted to lower Tmax and it was not possible to distinguish between the respective transitions of sarcoplasmic proteins and myosin. After the dry-salting step, the two main peaks observed were at approximately 50 and 70 °C, and a small transition was seen at 60 °C. The transition peak at 70 °C was from the actin molecule; the other two peaks were likely from the heavy meromyosin transitions. After storing of the dry-salted cod, a slight decrease in moisture content was observed. The peaks from myosin and actin were very low and broad, overlapping each other, and were displayed as one major transition peak. The actin peak could, however, be observed at approximately 72 °C, and transitions at approximately 60 °C were believed to display denaturation of myosin. Some restoration of the transition peaks could be seen after rehydration of the fish. However, all peaks were lower and broader, which led to the conclusion that the conformational stability

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–11.65

Herring

–11.90 Cod –12.15 –12.40 Sardine –12.65

Horse mackerel

–12.90 –13.15 –13.40

↑ Exo 30

35

40

45 50 55 Furnace temperature (°C)

60

65

70

75

Figure 8.7 DSC curves of commercially important fish species.

of the proteins had decreased compared with fresh cod and that some changes in protein structure occurred, particularly in the myosin molecule. The large myosin heavy chain peaks had all disappeared. A significant decrease in the peak size was observed, indicating breakdown or denaturation of the molecule. Actin was found to be less affected than myosin (Thorarinsdottir et al. 2002). Changes in thermal stability during processing and ripening of salted herring were investigated by DSC of both the fish muscle and pyloric caeca, which is the main source of enzymes responsible for ripening (Schubring 1997, 1999a). The salting process completely changed the DSC pattern of both the white and red muscle one month after salting. In both cases the cooperative pattern was reduced to only one main transition, with a shift of Tmax to lower temperature. In contrast, Tmax of skin from salted herring shifted to higher temperature. These changes observed after one month of processing remained stationary during further storage of heavily salted herring for up to one year. Not only was the thermal stability of the muscle proteins influenced by salting but also that of pyloric caeca. It was recognised that the salting itself leads to a remarkable increase of Tmax compared with raw herring. An influence of the salt : fish ratio could be observed. The higher the salt content, the higher the increase in Tmax. During ripening, Tmax remained high or showed only a slight decrease during the investigation period. The dependency on the salt content remained evident. The increase in Tmax was accompanied by a decrease in ΔH. The increase in thermal stability was connected with a decrease in the general proteolytic activity of pyloric caeca. Possibly,

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the enzymes were diffusing from the pyloric caeca into muscle and caused an increase in enzymatic activity there that was observable in North Sea herring accompanied by a decrease in activity of the pyloric caeca itself. Simultaneously, the thermal stability of pyloric caeca was lowered. Smoking of fish Retail samples of vacuum-packed sliced cold-smoked salmon were investigated by DSC for alteration in proteins caused by processing and refrigerated storage changes approximately 1 week before expiry date and on the ‘best before’ date (Schubring 2006a). For comparison, retail samples of gravlax were also investigated. DSC measurements were also performed on hot-smoked salmon and frozen raw material, Salmo salar. DSC curves taken from the different salmon products verified the influence of different processing steps and additives on them. Corresponding transition temperatures and enthalpies clarified the dramatic changes of muscle proteins caused by salting and smoking. DSC curves of farmed and wild salmon did not show any significant differences. The DSC curve of cold-smoked salmon made clear that both myosin and actin peaks had almost disappeared. Because cold-smoking temperature varies from 20 to 30 °C, the main reason for denaturation of myosin and actin can be seen in the influence of salt. Surprisingly, the DSC curve of ‘Stremellachs’, the hot-smoked product, exhibited a peak from 54 to 59 °C, with a Tmax of 57.3 °C. This led to the conclusion that during processing of this hot-smoked product, the minimum temperature of 60 °C postulated in the Guidelines of the German Food Book were not followed. This policy has to be seen as critical from the microbial point of view, and can only be explained by the effort of the processor to gain higher yield. The DSC curves of retail samples obtained at first and second investigation did not verify a significant difference between both measurements. The strong influence of salting in connection with cold-smoking became visible. The curves taken on gravlax samples seemed to suggest more pronounced changes of myofibrillar proteins compared with cold-smoked samples. A possible explanation can be seen in higher water activity found in gravlax because its processing does not include a drying step as it does in processing of cold-smoked salmon, which benefits enzymatic and microbial degradation of muscle proteins. To determine if different farming conditions like conventional and organic farming of rainbow trout cause differences in quality that are detectable by physical methods such as DSC, measurements were performed on both types of these farmed fish. First, DSC curves of smoked trout were used to verify that the core temperature of smoked fish had reached at least 60 °C during the hot-smoking process. In the DSC curves, only the actin peak was still visible. All other proteins were obviously denatured during hot smoking. When DSC curves were taken from smoked trout after different durations of chilled storage, it could be seen that the denaturation temperature of actin decreased almost linearly with progression of storage time. An influence of farming conditions on DSC pattern was not found (Schubring 2006c). Figure 8.8 shows DSC curves of tropical and farmed fish species. Myoglobins and CO treatment of tuna muscle Myoglobin, which is widely present in animal red muscle, has an important role in the uptake and release of oxygen. Its susceptibility to heat was investigated by DSC. Myoglobins from

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–5.43 Swordfish

–5.53 –5.63

Yellowfin tuna

Heat flow

–5.73 Salmon

–5.83

Rainbow trout

–5.93

–6.13 –6.23

← Endo

–6.03

30

35

40

45

50

55

60

65

70

75

Temperature (°C) Figure 8.8 DSC curves of tropical and farmed fish species.

yellowfin tuna, bonito and yellowtail exhibited two distinct endothermic peaks at values of Tmax of 65 and 74–78 °C, 60 and 65.6 °C and 43 and 48 °C, respectively, reflecting multiple states of structural unfolding. Fish myoglobins were found to be more susceptible to heat denaturation than those of mammals (Chanthai et al. 1996a). Apomyoglobin, known as an intermediate in the biosynthesis of myoglobin, from the same fish species, exhibited a single endothermic peak in the range 36–38 °C, reflecting a two-state process of thermal unfolding (Chanthai et al. 1996b). When the range of fish species investigated for the transition temperature of their myoglobins was enlarged to migratory, demersal and aquaculture fish as well as elasmobranchs, it became evident that spotted shark myoglobin showed the highest thermal stability and was followed by chub mackerel and tilapia myoglobin, whereas horse mackerel myoglobin showed the lowest thermal stability (Chen et al. 2004). Various tuna species (bigeye tuna, bluefin tuna, yellowfin tuna and skipjack) were investigated for thermostability of myoglobin by DSC. Measurements revealed that thermostability of bigeye tuna myoglobin (75.7 °C) was lowest. Of all the scombrid fish myoglobins examined, the highest value of Tmax (79.9 °C) occurred in skipjack. Tmax values of bluefin (78.6 °C) and yellowfin (78.2 °C) tuna myoglobins were very close to each other (Ueki and Ochiai 2004). Thermostability of bullet tuna (Auxis rochei) myoglobin is lowest among all the scombrid myoglobins so far examined. The highest Tmax value (72.8 °C) was obtained at pH 6.52 (Ueki et al. 2005). Carbon monoxide (CO), as a component of filtered smoke, is frequently used to stabilise the colour of red-fleshed fish such as tuna, although the addition of CO is not legally justified in Europe. Its influence on the thermal stability of treated tuna muscle has been investigated (Heyer and Schubring 2005). DSC patterns revealed that attachment of CO to blood and

Differential scanning calorimetry

Endothermic heat flow (W/g)

0.15 0.14

203

A, Sevruga; B, Ossetra; C, Beluga; D, Sevruga; E, Beluga; F, Sevruga; G, artificial caviar

0.12 0.10 0.08 A B 0.06 0.04 C D E 0.02 F G 0.00 28

40

50

60

70 80 90 100 Temperature (°C)

110

120

130

Figure 8.9 DSC curves of original and artificial caviar.

muscle pigments did not obviously affect their thermal stability. Only the freezing step caused modification to the myosin peak. The shoulder seen on the low-temperature side of the peak disappeared, causing lower differentiation and broadening of the myosin peak. However, transition temperatures as well as transition enthalpies did not change significantly (Schubring 2006b). Maturation and technological treatment of fish roe DSC was also used to investigate the influence of maturation and technological treatment on the roe of rainbow trout (Schubring 2004a). The DSC curves were markedly affected during the first phase of maturation, whereas on the whole almost no changes were visible in later stages. DSC curves measured on yolk proteins of rainbow trout were clearly discernible from those of milt and muscle proteins of the same species. Applying different technological treatments on roe, it became clear that freezing and frozen storage only have a minor influence on thermal behaviour of yolk proteins. On the other hand, lightly salting, heating at 90 °C and high-pressure processing (thawing) changed DSC curves markedly, indicating a stronger denaturation of yolk proteins by these treatments. However, compared with muscle proteins of rainbow trout, the influence of high pressure was less. DSC curves of yolk proteins are influenced by the particular species of fish. However, it did not seem possible to use them to discriminate species of the same family. However, discrimination of illegal (artificial) samples from original caviar did appear possible (Figure 8.9). Fish protein hydrolysate and lipid oxidation Finally, some other interesting applications of DSC in fish processing should be mentioned. With the goal of preparing low-cost functional food, a protein hydrolysate was prepared

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from fish scraps (fish protein hydrolysate, FPH) as well from by-products of squid processing (squid protein hydrolysate, SPH). The effect of FPHs on the state of water and denaturation of lizard-fish myofibrils was evaluated by DSC. Myofibrils with FPH from fish scraps had higher amounts of unfrozen water than the control, suggesting that FPH suppressed dehydration-induced denaturation (Khan et al. 2003). Comparable results were also obtained when SPH was used (Hossain et al. 2003a, b). Lipid oxidation is one of the main processes responsible for the loss of food quality. A major secondary product from lipid oxidation is malonaldehyde, which can react with proteins. In studying the influence of malonaldehyde on myofibrillar proteins of sea salmon (Pseudopercis semifasciata), results showed a different thermal behaviour, evidencing a decrease in thermal stability, changes in ΔH and the appearance of new molecular species with a loss in the cooperativity of the myosin denaturation (Tironi et al. 2002). Proteins are not the only subject of DSC application in fish. When lipid classes of salmon oil were modified by a two-step lipase procedure, two main peaks with low- melting temperature could be observed in crude oil, −38.4 and −11.5 °C, whereas the action of lipase resulted in the occurrence of a third peak, either in the permeate or retenate fractions (Linder et al. 2002).

8.5

Summary

Examples given in this chapter indicate the suitability of DSC in playing an important part in assessing quality and safety in fish processing. It is therefore expected that future application of DSC will intensify in research as well as in control.

8.6

References

Agustini, T.W., Suzuki, T., Hagiwara, T., Ishizaki, S., Tanaka, M. and Takai, R. (2001) Change of K-value and water state of yellowfin tuna Thunnus albacares meat stored in a wide temperature range (20 to −84 °C). Fisheries Science 67: 306–313. Akahane, T., Chihara, S., Yoshida, Y., Tsuchiya, T., Noguchi, S.F., Ookami, H. and Matsumoto, J.J. (1981) Application of differential scanning calorimetry to food technological study of fish meat gels. Bulletin of the Japanese Society of Scientific Fisheries 47: 105–111. Akahane, T., Chihara, S., Niki, T.P., Sano, T., Tsuchiya, T., Noguchi, S.F., Ookami, H. and Matsumoto, J.J. (1985) Differential scanning calorimetric studies on thermal behaviors of myofibrillar proteins. Bulletin of the Japanese Society of Scientific Fisheries 51: 1841–1846. Badii, F. and Howell, N.K. (2002a) Changes in the texture and structure of cod and haddock fillets during frozen storage. Food Hydrocolloids 16: 313–319. Badii, F. and Howell, N.K. (2002b) A comparison of biochemical changes in cod (Gadus morhua) and haddock (Melanogrammus aeglefinus) fillets during frozen storage. Journal of the Science of Food and Agriculture 82: 87–97. Badii, F. and Howell, N.K. (2003) Elucidation of the effect of formaldehyde and lipids on frozen stored cod collagen by FT-Raman spectroscopy and differential scanning calorimetry. Journal of Agricultural and Food Chemistry 51: 1440–1446. Badii, F. and Howell, N.K. (2006) Fish gelatin: structure, gelling properties and interaction with egg albumen proteins. Food Hydrocolloids 20: 630–640.

Differential scanning calorimetry

205

Barreto, P.L.M., Beirao, L.H., Soldi, M.S. and Soldi, V. (2000) Studies on differential scanning calorimetry and thermogravimetry of tilapia (Oreochromis nilotica) surimi, surimi/starch and surimi/ starch/carrageenan systems. Journal of Food Science Technology 37: 265–271. Beas, V.E., Wagner, J.R., Crupkin, M. and Anon, M.C. (1990) Thermal denaturation of hake (Merluccius hubbsi) myofibrillar proteins. a differential scanning calorimetric and electrophoretic study. Journal of Food Science 55: 683–686, 696. Beas, V.E., Wagner, J.R., Anon, M.C. and Crupkin, M. (1991) Thermal denaturation in fish muscle protein during gelling: effect of spawning condition. Journal of Food Science 56: 281–284. Belibagli, K.B., Speers, R.A. and Paulson, A.T. (2003) Thermophysical properties of silver hake and mackerel surimi at cooking temperatures. Journal of Food Engineering 60: 439–448. Benoist, L. (1990) Recent developments in bio-calorimetry with micro-DSC. Thermochimica Acta 163: 111–116. Beyrer, M. and Rüsch, M. gen. Klass. (2007) Influence of freezing and of frozen storage on the specific heat capacity of trout and herring fillet. European Food Research and Technology 224: 349–353. Biliarderis, C.G. (1983) Differential scanning calorimetry in food research – a review. Food Chemistry 10: 239–265. Boye, J.I., Ma, C.-Y. and Harwalkar, V.R. (1997) Thermal denaturation and coagulation of proteins. In: S. Damodaran and A. Paraf (Eds) Food Proteins and their Applications. Marcel Dekker, New York, Basel and Hong Kong, pp. 25–56. Brake, N.C. and Fennema, O.R. (1999) Glass transition values of muscle tissue. Journal of Food Science 64: 10–15. Brandts, J.F. and Lin, L.N. (1990) Study of strong to ultratight protein interactions using differential scanning calorimetry. Biochemistry 29: 6927–6940. Burjanadze T.V. (1992) Thermodynamic substantiation of water-bridged collagen structure. Biopolymers 32: 941–949. Careche, M., del Mazo, M.L. and Fernández-Martín, F. (2002) Extractability and thermal stability of frozen hake (Merluccius merluccius) fillets stored at −10 and −30 °C. Journal of the Science of Food and Agriculture 82: 1791–1799. Carey, P.P. and Surewicz, W.K. (1996) Spectroscopic and calorimetric methods for characterizing proteins and peptides. In: P.R. Carey (Ed) Protein Engineering and Design. Academic Press, San Diego, pp. 233–263. Chanthai, S., Ogawa, M., Tamiya, T. and Tsuchiya, T. (1996a) Studies on thermal denaturation of fish myoglobins using differential scanning calorimetry, circular dichroism, and tryptophan flourescence. Fisheries Science 62: 972–932. Chanthai, S., Ogawa, M., Tamiya, T. and Tsuchiya, T. (1996b) Studies on thermal denaturation of fish apomyoglobins using differential scanning calorimetry, circular dichroism, and flourescence. Fisheries Science 62: 933–937. Chen, H.-H. (1995) Thermal stability and gel-forming ability of shark muscle. Journal of Food Science 60: 1237–1240. Chen, H.-H. (1996) Changes in the thermal stability of silvertip shark (Carcharhinus albimarginatus) during frozen storage. Food Science 23: 56–65. Chen, H.-H., Lou, S.-N. and Chen, T.-Y. (1996a) Changes in freshness and gelation characteristics of small hammer-head shark during cold storage. Journal of the Chinese Agricultural Chemical Society 34: 174–187. Chen, H.-H., Lou, S.-N. and Chen, T.-Y. (1996b) Biochemical properties, thermal stability and gelation properties of thresher shark (Alopias pelagicus) during frozen storage. Journal of the Chinese Agricultural Chemical Society 34: 309–322. Chen, H.-H., Ferng, L.-H., Chen, S.-D., Sun, W.-C. and Lee, Y.-C. (2005) Combination model for the spatial partition of surimi protein and hydroxylpropylmethylcellulose. Food Hydrocolloids 19: 761–768.

206

Fishery Products: Quality, safety and authenticity

Chen, L.-C., Lin, S.-B. and Chen, H.-H. (2004) Thermal stability and denaturation rate of myoglobin from various species of fish. Fisheries Science 70: 293–298. Chen, T.-Y. and Kong, M.-S. (1997) Combined use of traditional and DSC methods in monitoring rigor mortis development of iced chub mackerel. Journal of the Chinese Agricultural Chemical Society 35: 333–341. Cheow, C.S. and Yu, S.Y. (1997) Effect of fish protein, salt, sugar, and monosodium glutamate on the gelatinization of starch in fish-starch mixtures. Journal of Food Processing and Preservation 21: 161–177. Cheow, C.S., Norizah, M.S., Kyaw, Z.Y. and Howell, N.K. (2007) Preparation and characterisation of gelatins from the skins of sin croaker (Johnius dussumieri) and shortfin scad (Decapterus macrosoma). Food Chemistry 101: 386–391. Chung, K.H. and Lee, C.M. (1991) Water binding and ingredient dispersion pattern effects on surimi gel texture. Journal of Food Science 56: 1263–1266. Collet, L.A., Brown, M.E. (1998) Biochemical and biological applications of thermal analysis. Journal of Thermal Analysis 51: 693–726. Cooper, A. (1999) Thermodynamics of protein folding and stability. In: G. Allen (Ed) Protein: A Comprehensive Treatise. JAI Press, Greenwich, Connecticut, pp. 217–270. Davies, J.R., Bardsley, R.G., Ledward, D.A. and Poulter, R.G. (1988) Myosin thermal stability in fish muscle. Journal of the Science of Food and Agriculture 45: 61–68. Davies, J.R., Ledward, D.A., Bardsley, R.G. and Poulter, R.G. (1994) Species dependence of fish myosin stability to heat and frozen storage. International Journal of Food Science & Technology 29: 287–301. Dileep, A.O., Shamasundar, B.A., Binsi, P.K., Badii, F. and Howell, N.K. (2005) Effect of ice storage on the physicochemical and dynamic viscoelastic properties of ribbonfish (Trichiurus spp.) meat. Journal of Food Science 70: E537–E545. Dondero, M., Araya, M. and Curotto, E. (1996) Prevention of protein denaturation of jack mackerel actomyosin (Trachurus murphyi) during frozen storage. Food Science and Technology International 2: 79–86. Freire, E. (1995) Differential scanning calorimetry. In: B.A. Shirley (Ed) Methods in Molecular Biology, Volume 40 (Protein Stability and Folding: Theory and Practice). Humana Press, Totowa, NJ, pp. 191–218. Fukushima, H., Satoh, Y., Nakaya, M., Ishizaki, S. and Watabe, S. (2003a) Thermal effects on fast skeletal myosins from Alaska pollock, white croaker, and rabbit in relation to gel formation. Journal of Food Science 68: 1573–1577. Fukushima, H., Yoon, S.H. and Watabe, S. (2003b) Differences in polymer formation through disulfide bonding of recombinant light meromyosin between white croaker and walleye pollack and their possible relation to species specific differences in thermal unfolding. Journal of Agricultural and Food Chemistry 51: 4089–4095. Hägerdal, B. and Martens, H. (1976) Influence of water content on the stability of myoglobin to heat treatment. Journal of Food Science 41: 933–937. Hansen, T.N., DeVries, A.L. and Baust, J.G. (1991) Calorimetric analysis of antifreeze glycoproteins of the polar fish, Dissostichus mawsoni. Biochimica et Biophysica Acta 1079: 169–173. Harwalkar, V.R. and Ma, C.-Y. (1990) Thermal Analysis of Foods. Elsevier Science Publishers, Essex, New York. Hashimoto, T., Hagiwara, T., Suzuki, T. and Takai, R. (2003) Study on the glass transition of Katsuobushi (boiled and dried bonito fish stick) by differential scanning calorimetry and dynamic mechanical analysis. Fisheries Science 69: 1290–1297. Hashimoto, T., Suzuki, T., Hagiwara, T. and Takai, R. (2004) Study on the glass transition for several processed fish muscles and its protein fractions using differential scanning calorimetry. Fisheries Science 70: 1144–1152.

Differential scanning calorimetry

207

Hastings, R. and Rodger, G. (1985) Differential scanning calorimetry of frozen cod: pH effects. I.I.F.-I.I.R.-Commissions C2, D3-Aberdeen, United Kingdom, pp. 337–341. Hastings, R.J., Rodger, G.W., Park, R., Matthews, A.D. and Anderson, E.M. (1985) Differential scanning calorimetry of fish muscle: the effect of processing and species variation. Journal of Food Science 50: 503–506, 510. Herrera, J.J., Pastoriza, L. and Nesvadba, P. (2001a) Effect of the addition of maltodextrins and sucrose on the ice-melting onset of minced fish muscle. Journal of the Science of Food and Agriculture 81: 305–310. Herrera, J.J., Pastoriza, L. and Sampedro, G. (2001b) A DSC study on the effects of various maltodextrins and sucrose on protein changes in frozen-stored minced blue whiting muscle. Journal of the Science of Food and Agriculture 81: 377–384. Heyer, H. and Schubring, R. (2005) Entwicklung einer einfachen und schnellen Analysenmethode für Kohlenmonoxid in Fisch – Teil 2: Methodenoptimierung. Informationen aus der Fischereiforschung 52: 106–114. Hickman, D., Sims, T.J., Miles, C.A., Bailey, A.J., de Mari, M. and Koopmans, M. (2000) Isinglass/ collagen: denaturation and functionality. Journal of Biotechnology 79: 245–257. Höhne, G.W.H., Hemminger, W.F. and Flammersheim, H.-J. (1996) Differential Scanning Calorimetry. Springer, Berlin, Heidelberg and New York. Hossain, A., Ishihara, T., Hara, K., Osatomi, K., Khan, A. and Nozaki, Y. (2003a) Effect of proteolytic squid protein hydrolysate on the state of water and dehydration-induced denaturation of lizard fish myofibrillar protein. Journal of Agricultural and Food Chemistry 51: 4769–4774. Hossain, M.A., Khan, M.A., Ishihara, T., Hara, K., Osatomi, K., Osako, K. and Nozaki, Y. (2003b) Effect of squid protein hydrolysate on freeze-induced denaturation of lizardfish (Saurida wanieso) myofibrillar protein. Food Science and Technology Research 9: 353–356. Howell, B.K., Matthews, A.D. and Donelly, A.P. (1991) Thermal stability of fish myofibrils: a differential scanning calorimetric study. International Journal of Food Science Technology 26: 283–295. Huang, M.C. and Ochiai, Y. (2005) Fish fast skeletal muscle tropomyosins show species-specific thermal stability. Comparative Biochemistry and Physiology B 141: 461–471. Hurling, R. and McArthur, H. (1996) Thawing, refreezing and frozen storage effects on muscle functionality and sensory attributes of frozen cod (Gadus morhua). Journal of Food Science 61: 1289–1296. Inoue, C. and Ishikawa, M. (1997) Glass transition of tuna flesh at low temperature and effects of salt and moisture. Journal of Food Science 62: 496–499. Iso, N., Mizuno, H., Ogawa, H., Mochizuki, Y. and Masuda, N. (1991) Differential scanning calorimetry on fish meat paste. Nippon Suisan Gakkaishi 57: 337–340. Jardim, D.C.P., Candido, L.M.B. and Netto, F.M. (1999) Sorption isotherms and glass transition temperatures of fish protein hydrolysates with different degrees of hydrolysis. International Journal of Food Properties 2: 227–242. Jensen, K.N. and Jørgensen, B.M. (2003) Effect of storage conditions on differential scanning calorimetry profiles from thawed cod muscle. Lebensmittel-Wissenschaft und -Technologie 36: 807–812. Jensen, K.N., Jørgensen, B.M. and Nielsen, J. (2003) Low-temperature transitions in cod and tuna determined by differential scanning calorimetry. Lebensmittel-Wissenschaft und -Technologie 36: 369–374. Jongjareonrak, A., Benjakul, S., Visessanguan, W. and Tanaka, M. (2005) Isolation and characterization of collagen from bigeye snapper (Priacanthus macracanthus) skin. Journal of the Science of Food and Agriculture 85: 1203–1210. Khan, M.A., Hossain, M.A., Hara, K., Osatomi, K., Ishihara, T. and Nozaki, Y. (2003) Effect of enzymatic fish protein hydrolysate from fish scrap on the state of water and denaturation of lizard

208

Fishery Products: Quality, safety and authenticity

fish (Saurida wanieso) myofibrils during dehydration. Food Science and Technology Research 9: 257–263. Kim, B.Y., Hamann, D.D., Lanier, T.C. and Wu, M.C. (1986) Effects of freeze–thaw abuse on the viscosity and gel-forming properties of surimi from two species. Journal of Food Science 51: 951–956, 1004. Kittiphattanabawon, P., Benjakul, S., Visessanguan, W., Nagai, T. and Tanaka, M. (2005) Characterisation of acid-soluble collagen from skin and bone of bigeye snapper (Priacanthus tayenus). Food Chemistry 89: 363–372. Ko, W.C., Hwang, J.S., Jao, C.L. and Hsu, K.C. (2004) Denaturation of tilapia myosin fragments by high hydrostatic pressure. Journal of Food Science 69: C604-C607. Levitsky, D.I., Nikolaeva, O.P., Orlov, V.N., Pavlov, D.A., Ponomarev, N.A. and Rostkova, E.V. (1998) Differential scanning calorimetric studies on myosin and actin. Biochemistry (Moscow) 63: 322–333. Linder, M., Matouba, E., Fanni, J. and Parmentier, M. (2002) Enrichment of salmon oil with n-3 PUFA by lipolysis, filtration and enzymatic re-esterification. European Journal of Lipid Science and Technology 104: 455–462. Lorinczy, D. (2004) The Nature of Biological Systems as Revealed by Thermal Methods. Kluwer Academic Publishers, Dordrecht, Boston and London, pp. 351. Lou, X., Wang, C., Xiong, Y.L., Wang, B., Liu, G. and Mims, S.D. (2000) Physicochemical stability of paddlefish (Polyodon spathula) meat under refrigerated and frozen storage. Journal of Aquatic Food Product Technology 9: 27–39. Lund, D.B. (1983) Applications of differential scanning calorimetry in foods. In: M. Peleg and E.B. Bagley (Eds) Physical Properties of Foods. AVI, Westport, pp. 125–143. Lundgren, C.H., Williams, T.R. and Nunnari, J.M. (1986) Determination of the state and content of water in normal avian, fish, porcine, bovine, and human lenses as studied by differential scanning calorimetry. Ophthalmic Research 18: 90–97. Ma, C.-Y. and Harwalkar, V.R. (1991) Thermal analysis of food proteins. Advances in Food and Nutrition Research 35: 317–366. Menashi, S., Finch, A., Gardner, P.J. and Ledward, D.A. (1976) Enthalpy changes associated with the denaturation of collagens of different imino acid content. Biochimica et Biophysica Acta 444: 623–625. Mizuno, A., Mitsuiki, M., Toba, S. and Motoki, M. (1997) Antifreeze activities of various food components. Journal of Agricultural and Food Chemistry 45: 14–18. Mochizuki, Y., Saito, T., Iso, N., Mizuno, H., Aochi, A. and Noda, M. (1987) Effects of adding fat on rheological properties of fish meat gel. Nippon Suisan Gakkaishi 53: 1471–1474. Mochizuki, Y., Mizuno, H., Ogawa, H. and Iso, N. (1995a) Trial determination of the ratio of myosin and actin in raw meat by differential scaning calorimetry. Fisheries Science 61: 723– 724. Mochizuki, Y., Mizuno, H., Ogawa, H., Ishimura, K., Tsuchiya, H. and Iso, N. (1995b) Changes of rhelogical properties of cuttlefish and squid meat by heat treatment. Fisheries Science 61: 680–683. Moosavi-Nasab, M., Alli, I., Ismail, A.A. and Ngadi, M.O. (2005) Protein structural changes during preparation and storage of surimi. Journal of Food Science 70: C448–C453. Nagai, T., Kurata, M., Nakamura, T., Ito, T., Fujiki, K., Nakao, M. and Yano, T. (1999) Properties of myofibrillar protein from Japanese stingfish (Sebastes inermis) dorsal muscle. Food Research International 32: 401–405. Niwa, E., Wang, T.-T., Kanoh, S. and Nakayama, T. (1988) Differential scanning calorimetry of the kamaboko added with various natural high polymers. Nippon Suisan Gakkaishi 54: 2139–2142. Nomura, Y., Sakai, H., Ishii, Y. and Shirai, K. (1996) Preparation and some properties of type I collagen from fish scales. Bioscience Biotechnology and Biochemistry 60: 2092–2094.

Differential scanning calorimetry

209

Ofstad, R., Egelandsdal, B., Kidman, S., Myklebust, R., Olson, R. and Hermansson, A.-M. (1996) Liquid loss as effected by postmortem ultrastructural changes in fish muscle: cod (Gadus morhua L) and salmon (Salmo salar). Journal of the Science of Food and Agriculture 71: 301–312. Ogawa, M., Ehara, T., Tamiya, T. and Tsuchiya, T. (1993) Thermal stability of fish myosin. Comparative Biochemistry and Physiology B 106: 517–521. Ogawa, M., Tamiya, T. and Tsuchiya, T. (1994) Structural changes of carp myosin during heating. Fisheries Science 60: 723–727. Orlien, V., Risbo, J., Andersen, M.L. and Skibsted, L.H. (2003) The question of high- or lowtemperature glass transition in frozen fish. Construction of the supplemented state diagram for tuna muscle by differential scanning calorimetry. Journal of Agricultural and Food Chemistry 51: 211–217. Paredi, M.E., Tomas, M.C., Crupkin, M. and Anon, M.C. (1994) Thermal denaturation of Aulacomya ater ater (Molina) myofibrillar proteins: a differential scanning calorimetric study. Journal of Agricultural and Food Chemistry 42: 873–877. Paredi, M.E., Tomas, M.C., de Vido de Mattio, N., Crupkin, M. and Anon, M.C. (1995) Postmortem changes in adduktor muscles from Aulacomya ater ater (Molina) stored at 2–4 °C. A differential scanning calorimetric study. Journal of Agricultural and Food Chemistry 43: 1758–1761. Paredi, M.E., Tomas, M.C., Crupkin, M. and Anon, M.C. (1996) Thermal denaturation of muscle proteins from male and female squid (Illex argentinus) at different sexual maturation stages. A differential scanning calorimetric study. Journal of Agricultural and Food Chemistry 44: 3812–3816. Paredi, M.E., Tomas, M.C., Anon, M.C. and Chrupkin, M. (1998) Thermal stability of myofibrillar proteins from smooth and stiated muscled of scallop (Chlamys tehuelchus): a differential scanning calorimetric study. Journal of Agricultural and Food Chemistry 46: 3971–3976. Paredi, M.E., Tomas, M.C. and Crupkin, M. (2002) Thermal denaturation of myofibrillar proteins of striated and smooth adductor muscles of scallop (Zygochlamys patagonica). A differential scanning calorimetric study. Journal of Agricultural and Food Chemistry 50: 830–834. Park, J.W. (1994) Functional protein additives in surimi gels. Journal of Food Science 59: 525– 527. Park, J.W. and Lanier, T.C. (1987) Combined effects of phosphates and sugar or polyol on protein stabilization of fish myofibrils. Journal of Food Science 52: 1509–1513. Park, J.W. and Lanier, T.C. (1989) Scanning calorimetric behavior of tilapia myosin and actin due to processing of muscle and protein purification. Journal of Food Science 54: 49–51. Park, J.W. and Lanier, T.C. (1990) Effects of salt and sucrose addition on thermal denaturation and aggregation of water-leached fish muscle. Journal of Food Biochemistry 14: 395–404. Perez-Mateos, M., Hurtado, J.L., Montero, P. and Fernandez-Martin, F. (2001) Interactions of kappacarrageenan plus other hydrocolloids in fish myosystem gels. Journal of Food Science 66: 838–843. Poulter, R.G., Ledward, D.A., Godber, S., Hall, G. and Rowlands, B. (1985) Heat stability of fish muscle proteins. Journal of Food Technology 20: 203–217. Privalov, P.L. (1989) Thermodynamic problems of protein structure. Annual Review of Biophysics and Biophysical Chemistry 18: 47–69. Privalov, P.L. and Plotnikov, V.V. (1989) 3 generations of scanning microcalorimeters for liquids. Thermochimica Acta 139: 257–277. Privalov, P.L. and Potekhin, S.A. (1986) Scanning microcalorimetry in studying temperature-induced changes in proteins. Methods in Enzymology 131: 4–51. Radicevic, T., Raicevic, S. and Niketic, V. (2002) Bighead carp myosin stability to heat and frozen storage. Acta Veterinaria 52: 151–161. Raemy, A. and Lambelet, P. (1991) Thermal behaviour of foods. Thermochimica Acta 193: 417–439.

210

Fishery Products: Quality, safety and authenticity

Raghavan, S. and Kristinsson, H.G. (2007) Conformational and rheological changes in catfish myosin as affected by different acids during acid-induced unfolding and refolding. Journal of Agricultural and Food Chemistry 55: 4144–4153. Rahman, M.S., Kasapis, S., Guiuzani, N. and Al-Amri, O.S. (2003) State diagram of tuna meat: freezing curve and glass transition. Journal of Food Engineering 57: 321–326. Ramirez-Suarez, J.C. and Morrissey, M.T. (2006) High hydrostatic pressure and heat treatment effects on physicochemical characteristics of albacore tuna (Thunnus alalunga) minced muscle. Journal of Aquatic Food Product Technology 15: 5–17. Ramløv, H., DeVries, A.L. and Wilson, P.W. (2005) Antifreeze glycoproteins from the Antarctic fish Dissostichus mawsoni studied by differential scanning calorimetry (DSC) in combination with nanolitre osmometry. CryoLetters 26: 73–84. Rasmussen, P.H., Jørgensen, B. and Nielsen, J. (1997) Cryoprotective properties of proline in cod muscle studied by differential scanning calorimetry. CryoLetters 18: 293–300. Rehbein, H. and Schubring, R. (1996) Blanchiert oder gegart? – Die Einstufung erhitzter Muscheln (Perna canaliculus) durch Eiwei ßuntersuchung. Informationen für die Fischwirtschaft 43: 29– 36. Richards, M.P., Nelson, N.M., Kristinsson, H.G., Mony, S.S. J., Petty, H.T. and Oliveira, A.C. M. (2007) Effects of fish heme protein structure and lipid substrate composition on hemoglobinmediated lipid oxidation. Journal of Agricultural and Food Chemistry 55: 3643–3654. Rivas-Vega, M.E., Rouzaud-Sanchez, O. and Ezquerra-Brauer, J.M. (2001) Effect of feed protein levels on digestive proteolytic activity, texture, and thermal denaturation of muscle protein in reared blue shrimp. Journal of Aquatic Food Product Technology 10: 25–38. Rodger, G., Weddle, R.B. and Craig, P. (1980) Effect of time, temperature, raw material type, processing and use of cryoprotective agents on mince quality. In: J.J. Connel (Ed) Advances in Fish Science and Technology. Fishing News Books Ltd, Farnham, Surrey, UK, pp. 199–217. Rodger, G., Weddle, R.B. and Craig, P., Hastings, R. (1984) Effect of alkaline protease activity on some properties of comminuted squid. Journal of Food Science 49: 117–119, 123. Ruttanapornvareesakul, Y., Ikeda, M., Hara, K., Osatomi, K., Osako, K., Kongpun, O. and Nozaki, Y. (2006) Concentration-dependent suppressive effect of shrimp head protein hydrolysate on dehydration-induced denaturation of lizardfish myofibrils. Bioresource Technology 97: 762–769. Schiraldi, A., Piazza, L., Fessas, D. and Riva, M. (1999) Thermal analysis in foods and food processes. In: R.B. Kemp (Ed) Handbook of Thermal Analysis and Calorimetry, Volume IV (From Macromolecules to Man). Elsevier Science B.V., Amsterdam, pp. 829–921. Schubring, R. (1997) DSC, TPA, and CIELAB – Tools for quality determination during enzymatic ripening of salted herring. In: J. Luten, T. Börresen and J. Oehlenschläger (Eds) Seafood from Producer to Consumer, Integrated Approach to Quality. Elsevier, Amsterdam, pp. 331–348. Schubring, R. (1999a) Differential scanning calorimetric investigations on pyloric caeca during ripening of salted herring products. Journal of Thermal Analysis and Calorimetry 57: 283– 291. Schubring, R. (1999b) DSC studies on deep frozen fishery products. Thermochimica Acta 337: 89–95. Schubring, R. (2004a) Differential scanning calorimetric (DSC) measurements on the roe of rainbow trout (Oncorhynchus mykiss): influence of maturation and technological. Thermochimica Acta 415: 89–98. Schubring, R. (2004b) Instrumental colour, texture, water holding and DSC measurements on frozen cod fillets (Gadus morhua) during long-term storage at different temperatures. Deutsche Lebensmittel-Rundschau 100: 247–254. Schubring, R. (2005a) Characterizing protein changes caused by application of high hydrostatic pressure on muscle food by means of DSC. Journal of Thermal Analysis and Calorimetry 82: 229–237.

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Schubring, R. (2005b) Changes in texture, water holding capacity, colour, and thermal stability of frozen cod (Gadus morhua) fillets: effect of frozen storage temperature. Deutsche LebensmittelRundschau 101: 484–493. Schubring, R. (2006a) Thermal stability, texture, liquid holding capacity and colour of smoked salmon on retail level. Thermochimica Acta 445: 168–178. Schubring, R. (2006b) Use of ‘filtered’ smoke and carbon monoxide with fish: a review. In: J.B. Luten, C. Jacobsen, K. Bekaert, A. Saebø and J. Oehlenschläger (Eds) Seafood Research from Fish to Dish. Quality, Safety and Processing of Wild and Farmed Seafood. Wageningen Academic Publishers, Wageningen, pp. 317–345. Schubring, R. (2006c) Veränderungen der Farbe und thermischen Stabilität der Muskelproteine von Räucherforellen während der Kühllagerung. Informationen aus der Fischereiforschung 53: 52–58. Schubring, R. (2007) DSC measurements on sharks. Thermochimica Acta 458: 124–131. Schubring, R. and Meyer, C. (2006a) Ice storage of fish, new aspects: comparison between flake ice and stream ice – part I: sardine (Sardina pilchardus). Deutsche Lebensmittel-Rundschau 102: 405–415. Schubring, R. and Meyer, C. (2006b) Iced storage of fish, new aspects: comparison between flake ice and stream ice – part II: horse mackerel (Trachurus trachurus). Deutsche Lebensmittel-Rundschau 102: 508–517. Schubring, R. and Meyer, C. (2007) Iced storage, new aspects: comparison between flake ice and stream ice – part III: herring (Clupea harengus). Deutsche Lebensmittel-Rundschau 103: 203–212. Schubring, R., Meyer, C., Schlüter, O., Boguslawski, S. and Knorr, D. (2003) Impact of high pressure assisted thawing on the quality of fillets from various fish species. Innovative Food Science and Emerging Technology 4: 257–267. Shnyrov, V.L., Sanchez-Ruiz, J.M., Boiko, B.N., Zhadan, G.G. and Permyakov, E.A. (1997) Applications of scanning microcalorimetry in biophysics and biochemistry. Thermochimica Acta 302: 165–180. Smith, D.M. (1994) Protein interactions in gels: protein–protein interactions. In: N.S. Hettiarachchi and G.R. Ziegler (Eds) Protein Functionality in Food Systems. Marcel Dekker, New York, pp. 209–224. Sriket, P., Benjakul, S., Visessanguan, W. and Kijroongrojana, K. (2007) Comparative studies on chemical composition and thermal properties of black tiger shrimp (Penaeus monodon) and white shrimp (Penaeus vannamei) meats. Food Chemistry 103: 1199–1207. Srinivasan, S., Xiong, Y.L. and Blanchard, S.P. (1997a) Effects of freezing and thawing methods and storage time on thermal properties of freshwater prawns (Macrobrachium rosenbergii). Journal of the Science of Food and Agriculture 75: 37–44. Srinivasan, S., Xiong, Y.L., Blanchard, S.P. and Tidwell, J.H. (1997b) Physicochemical changes in prawns (Machrobrachium rosenbergii) subjected to multiple freeze-thaw cycles. Journal of Food Science 62: 123–127. Stanley, D.W. and Hultin, H.O. (1984) Proteolytic activity in North American squid and its relation to quality. Canadian Institute of Food Science and Technology Journal 17: 163–167. Stanley, D.W. and Yada, R.Y. (1992) Physical consequences of thermal reactions in food protein systems. In: H.G. Schwartzberg and R.W. Hartel (Eds) Physical Chemistry of Food. Marcel Dekker, New York, pp. 669–733. Sturtevant, J.M. (1987) Biochemical applications of differential scanning calorimetry. Annual Review of Physical Chemistry 38: 463–488. Sych, J., Lacroix, C., Adambounou, L.T. and Castaigne, F. (1990a) Cryoprotective effects of lactitol, palatinit and polydextrose on cod surimi proteins during frozen storage. Journal of Food Science 55: 356–359.

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Sych, J., Lacroix, C., Adambounou, L.T. and Castaigne, F. (1990b) Cryoprotective effects of some materials on cod surimi proteins during frozen storage. Journal of Food Science 55: 1222–1227, 1263. Sych, J., Lacroix, C. and Carrier, M. (1991a) Determination of optimal level for lactitol for surimi. Journal of Food Science 56: 285–290, 298. Sych, J., Lacroix, C., Adambounou, L.T. and Castaigne, F. (1991b) The effect of low- or non-sweet additives on the stability of protein functional properties of frozen cod surimi. International Journal of Food Science & Technology 26: 185–197. Thanonkaew, A., Benjakul, S. and Visessanguan, W. (2006) Chemical composition and thermal property of cuttlefish (Sepia pharaonis) muscle. Journal of Food Composition and Analysis 19: 127–133. Thawornchinsombut, S. and Park, J.W. (2006) Frozen stability of fish protein isolate under various storage conditions. Journal of Food Science 71: C227–C232. Thorarinsdottir, K.A., Arason, S., Geirsdottir, M., Bogason, S.G. and Kristbergsson, K. (2002) Changes in myofibrillar proteins during processing of salted cod (Gadus morhua) as determined by electrophoresis and differential scanning calorimetry. Food Chemistry 77: 377–385. Tironi, V.A., Tomás, M.C. and Añón, M.C. (2002) Structural and functional changes in myofibrillar proteins of sea salmon (Pseudopercis semifasciata) by interaction with malonaldehyde (RI). Journal of Food Science 67: 929–935. Togashi, M., Kakinuma, M., Nakaya, M., Ooi, T. and Watabe, S. (2002) Differential scanning calorimetry and circular dichroism spectrometry of walleye pollack myosin and light meromyosin. Journal of Agricultural and Food Chemistry 50: 4803–4811. Uddin, M., Ahmad, M.U., Jahan P. and Sanguandeekul, R. (2001) Differential scanning calorimetry of fish and shellfish meat. Asian Journal of Chemistry 13: 965–968. Ueki, N. and Ochiai, Y. (2004) Primary structure and thermostability of bigeye tuna myoglobin in relation to those of other scombridae fish. Fisheries Science 70: 875–884. Ueki, N., Chow, C.-J. and Ochiai, Y. (2005) Characterization of bullet tuna myoglobin with reference to the thermostability–structure relationship. Journal of Agricultural and Food Chemistry 53: 4968–4975. Venugopal, V. (2006) Seafood processing. Adding value through quick freezing, retortable packaging, and cook-chilling. CRC Press, Boca Raton, FL, pp. 386–387, 428. Visessanguan, W. and An, H. (2000) Effects of proteolysis and mechanism of gel weakening in heat-induced gelation of fish myosin. Journal of Agricultural and Food Chemistry 48: 1024–1032. Visessanguan, W., Ogawa, M., Nakai, S. and An, H. (2000) Physicochemical changes and mechanism of heat-induced gelation of arrowtooth flounder myosin. Journal of Agricultural and Food Chemistry 48: 1016–1023. Watabe, S., Hirayama, Y., Nakaya, M., Kakinuma, M., Kikuchi, K., Guo, X.F., Kanoh, S., Chaen, S. and Ooi, T. (1998) Carp expresses fast skeletal myosin isoforms with altered motor functions and structural stabilities to compensate for changes in environmental temperature. Journal of Thermal Biology 22: 375–390. Wright, D.J. (1982) Application of scanning calorimetry to the study of protein behaviour in foods. In: B.J.F. Hudson (Ed) Developments in Food Proteins – 1. Applied Science Publishers, London, pp. 61–89. Wu, M.C., Lanier, T.C. and Hamann, D.D. (1985) Thermal transitions of admixed starch/fish protein systems during heating. Journal of Food Science 50: 20–25. Zhu, S., Bulut, S., Le Bail, A. and Ramaswamy, H.S. (2004) High-pressure differential scanning calorimetry (DSC): equipment and technique validation using water ice phase-transition data. Journal of Food Process Engineering 27: 359–376.

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Zhu, S., Le Bail, A. and Ramaswamy, H.S. (2006a) High-pressure differential scanning calorimetry: Comparison of pressure-dependent phase transition in food materials. Journal of Food Engineering 75: 215–222. Zhu, S., Ramaswamy, H.S. and Le Bail, A. (2006b) Calorimetry and pressure-shift freezing of different food products. Food Science and Technology International 12: 205–214, 2006b.

Chapter 9

Instrumental texture measurement Mercedes Careche and Marta Barroso

9.1

Introduction

Textural properties have been defined as ‘that group of physical characteristics that arise from the structural elements of the food, are sensed primarily by the feeling of touch, are related to the deformation, disintegration, and flow of the food under a force, and are measured objectively by functions of mass, time and distance’ (Bourne 2002). Texture has also been defined as the ‘sensory and functional manifestation of the structural and mechanical properties of foods detected through the senses of vision, hearing, touch, and kinesthetics’ (Szczesniak 1963, 2002). The sensory quality of fish depends largely on their texture characteristics, which in turn are related to their structural features. In this respect, fish muscle is composed of fibres lying parallel to the lengthwise axis of the body, crossed by sheets of connective tissue, forming fibre segments known as myotomes. The structure of the fibres is similar to that of striated, voluntary muscle (Howgate 1977). The main protein components in the muscle are the myofibrillar proteins, which form the myofibrils, the basic contractile element of the musculature, the sarcoplasmic proteins mostly composed of enzymes, and the connective tissue proteins, mainly consisting of collagen. Both connective tissue and muscle fibres contribute to the texture properties of raw fish (Dunajski 1980). An inverse correlation between collagen content and tenderness of raw fish muscle has been found (Hatae et al. 1986; Sato et al. 1986), but no such correlation exists in cooked fish and it is considered that in this case, muscle fibres account for this texture property (Howgate 1977; Dunajski 1980; Hatae et al. 1986). Textural differences in cooked muscle among species are due to structural factors such as fibre diameter, so that the smaller the diameter the higher the firmness of the muscle (Hatae et al. 1990; Hurling et al. 1996). For a given species, significant positive correlations have been found between muscle fibre density and texture characteristics such as chewiness and firmness (Johnston et al. 2000). Also, differences in the content of the sarcoplasmic proteins that coagulate, impeding the sliding of the fibres (Hatae et al. 1990), have been proposed to account for the firmness of the fish muscle. The collagen is melted upon cooking, and therefore does not contribute to the hardness of the fillets. However, the gelatine formed from the collagen renders succulence to the fish products (Sato et al. 1986). In addition, it results in the fillet being easily separated into its 214

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myotomes, conferring a characteristic ‘flaky’ texture. Also, fat and water have been regarded as important in imparting the specific mouth-feel characteristics to fish flesh (Dunajski 1980). Edible parts from seafood other than finfish muscle have structural and compositional characteristics rendering particular textural attributes to the products. There are many factors that may affect the texture of fish as food, ranging from species differences to the biological condition of the fish, or from methods of catch or slaughter to culinary treatments. In particular, after the death of the fish, several biochemical changes associated with the onset and resolution of rigor mortis occur. Before the onset of rigor the muscle is soft and elastic. In rigor, muscle becomes hard, owing to the contraction of the fibres forming the actomyosin complex, and with its resolution the muscle becomes soft and less elastic. An opposing process to the stiffness occurring in rigor, called tenderisation, begins within hours post mortem and continues during storage. This tenderisation affects key structural proteins in the myofibrils and extracellular matrix, as well as proteins involved in myofibril–myofibril and myofibril–sarcolemma linkages (Delbarre-Ladrat et al. 2006). The extent of these changes and their effect on muscle texture depend on factors including species, pre-slaughter conditions or catching methods, post mortem handling and treatments, and storage time and temperature. Freezing and frozen storage are among the most used technologies for long-term storage. During frozen storage, fish muscle may suffer from a series of unwanted changes in texture characteristics, which result in a hard and dry product. This is important in some lean species where, under prolonged storage time or high temperatures, myofibrillar proteins aggregate and become inextractable in salt solutions. In species of high susceptibility to toughening during frozen storage such as hake, the secondary structure of muscle proteins changes towards higher proportion of β-sheets at the expense of α-helix occur (Careche et al. 2002). Cross-links between the myosin thick filaments within the myofibrils can be observed. However, most notably, the sarcoplasmic reticulum degrades and myofibrils fuse to each other (Howgate 1977; Herrero et al. 2005). Many factors affect texture changes during frozen storage including biological condition of the fish, pre-processing, degree of comminution, or time and temperature of storage. Other technologies applied to fish may affect the textural properties in different ways, either provoking biochemical, structural or microbiological changes, or preventing some detrimental changes that otherwise would occur. Cooking conditions, for example time, temperature or ingredients, will render different textural changes that account for the quality of the product. The complexity of textural parameters in foods, the perception of texture, and consumer attitudes towards this property, as well as the most complete sensory method, the Sensory Texture Profile, have been reviewed by Szczesniak (1998, 2002). In addition to oral assessment, non-oral methods are used, and are commonly performed in the fish sector by pressing parts of the fish body with the finger. They are very useful because they are fast, non-destructive, and require little training. These non-oral tests have been used to define some of the parameters that comprise the quality index method (QIM) (Bremner et al. 1987). In many cases, sensory analysis of texture is difficult to perform for reasons including time and costs (Szczseniak 1987). Thus, there has been an effort to design instrumental methods that could be correlated with either the oral or non-oral sensory texture.

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Many instrumental methods have been developed for measuring the textural properties of foods (Bourne 2002; Dobraszcyk and Vincent 1999; Vliet 1999) and fish in particular (Barroso et al. 1997, 1998a; Hyldig and Nielsen 2001). They have been classified in three groups (Bourne 2002): (1) fundamental, when well-defined rheological properties are measured; (2) empirical, when instrumental parameters correlate with texture measured by sensory tests; (3) imitative, which are those tests that resemble the conditions to which the food material is subjected in practice. Most of the reported data on fish flesh texture for quality assessment are based on mechanical tests that are empirical or imitative. The fish industry has shown interest in the development of non-destructive tests to evaluate the textural properties in a rapid and non-expensive way. Attempts have been made to design methods either with large instrumental equipment or with portable instruments. The shape of the fish and fillets, the complex, non-uniform structure of fish muscle, and the slippage of the myotomes upon cooking, make many instrumental methods difficult to apply in fish. Within the same batch there can be considerable variation from fish to fish, and texture properties may vary along the location of the fish or fish fillets, thus adding difficulty to the measurement. Some authors consider making use of this variability as a parameter in itself, but in most cases this brings an additional difficulty to the measurement. Unlike fish muscle, surimi gels have in general homogeneous structures, the size and shape of the samples to be analysed are easier to control, and, therefore, more meaningful results can be obtained from fundamental tests, although empirical and imitative methods are also widely used. Despite the problems for the analysis of fish samples, the literature contains developments or adaptations of instrumental tests for the study of, or application on, specific situations. This chapter will describe the different instrumental texture measurements as applied in fish and some fish products, and their reported relation with sensory analysis, with examples on their use in different situations. Attempts to find a quality index based on texture analysis alone, or with other instrumental methods, will also be addressed. The methodology for the study of rheological and texture properties of surimi gels has been recently reviewed (Kim et al. 2005), and will not be addressed as such in this chapter.

9.2

Instrumental texture

The methods described here are the Kramer test, Warner-Braztler, puncture, tensile and compression tests, texture profile analysis, and viscoleastic methods such as stress relaxation, creep and oscillatory measurements.

9.2.1

Kramer test

A comprehensive description of the Kramer cell can be found in Bourne (2002). The standard cell contains an upper part with blades that penetrate a box with slots. Upon application of

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30

Maximum force (N)

25 20 15 10 5 0 0

5 10 Sample weight (g)

15

Figure 9.1 Maximum force from the Kramer test as a function of sample weight in Alaska pollack surimi.

force, the food materials undergo shearing, compression and extrusion. Factors affecting the performance of the cell including friction, sample size (Bourne 2002), or cell volume (Voisey and Kloek 1981) have been investigated. Many authors have used the original 10-blade cell (Borderías et al. 1983; Krueger and Fennema 1989), although modified cells have also been used with six (Racicot et al. 1984), four blades (Gill et al. 1979; Kramer and Peters 1981; LeBlanc et al. 1988; Manthey et al. 1988; Rehbein and Orlick 1990), or thinner blades with narrower slits (Kong et al. 2007). Most of the analyses have been performed on cooked samples, but also on raw fish. Parameters usually measured include maximum force at a given sample weight, slope, and energy of the force-deformation curve. Some food products do not display a linear relationship between maximum force and sample weight, and thus it has been advised to use a constant weight of sample in the test cell unless a linear relationship is demonstrated between sample weight and maximum force for that food (Bourne 2002). A highly linear relationship has been shown for surimi, in the range 5–14 g when samples are cut in parallelepipeds of 1 cm height, and with lengths and widths varying from 4 to 7 cm and 1 to 2 cm respectively (see Figure 9.1). However, this linear relationship was not found for fillets. Thus, a constant weight is preferred for these fish samples. Some authors have applied the force perpendicular to the lengthwise axis of the muscle fibres (Borderías et al. 1983; McKenna et al. 2003); others have applied either perpendicular or parallel, with the perpendicular setup giving better results (Taylor et al. 2002). Also, fillets can be flaked by hand once cooked, mixed thoroughly, and placed as a uniform layer in the cell before performing the test (Kramer and Peters 1981; Botta et al. 1987). Another alternative is evenly to spread a fixed amount of diced and cooked fish muscle in random fashion on the Kramer cell for measurement (Krivchenia and Fennema 1988; Krueger and Fennema 1989). The Kramer cell has been used to study several factors that affect the quality of fish, such as method of capture and the time of year (Botta et al. 1987), pH (Kramer and Peters 1981;

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Licciardello et al. 1982), pre-freezing treatment (Kramer and Peters 1981), formaldehyde formation (Gill et al. 1979), oxidised lipids (Rehbein and Orlick 1990; Careche and Tejada 1991), different cooking methods (Madeira and Penfield 1985), cooking temperatures and enzyme hydrolysis (Deng 1981), blanching time (Marshall et al. 1987), use of carbonates (Kolakowski et al. 1994), ionic radiation and high hydrostatic pressure (McKenna et al. 2003), effect of cryoprotectants (Krivchenia and Fennema 1988), or the effect of starvation and re-feeding (Bugeon et al. 2004). A decrease of Kramer shear force with resolution of rigor mortis in farmed Atlantic salmon (Salmo salar) fillets stored at refrigerated temperatures (Isaksson et al. 2001; Taylor et al. 2002) was found. Raw catfish fillets (Silurus glanis) stored at 0–2 °C up to 30 days displayed the highest changes in peak force value in the first 6 days whereas no significant changes could be found thereafter (Manthey et al. 1988). Many species display increased values for this test over frozen storage time or at high storage temperatures. In general, higher values were found along storage when the degree of muscle integrity was lower. Examples of use of the Kramer cell include studies in red hake (Urophycis chuss) (Racicot et al. 1984; Gill et al. 1979; Licciardello et al. 1982), hake (Merluccius spp.) (Careche and Tejada 1991; Barroso et al. 1998b; Herrero et al. 2005; Herrero and Careche 2006), cod (Gadus morhua) (LeBlanc et al. 1988; Kolakowski et al. 1994), Alaska pollack (Theragra chalcogramma) (Krueger and Fennema 1989; Boer and Fennema 1989) and haddock (Melanogrammus aeglefinus) (Gill et al. 1979). An agreement between Kramer test force and sensory evaluation of firmness was found for the effect of blanching time in ice-stored crawfish meat (Procambarus clarkii) (Marshall et al. 1987). However, sensory analysis of catfish (Silurus glanis) stored in ice revealed a loss in elasticity and firmness with storage time that could not be confirmed by Kramer test force (Manthey et al. 1988). Correlations were found between Kramer and sensory firmness in minced muscle of various species stored at −25 °C (Borderías et al. 1983), hardness in minced haddock (Melanogrammus aeglefinus) and red hake (Urophycis chuss) (Gill et al. 1979) or sensory toughness in red hake (Urophycis chuss) stored at different freezing temperatures (Licciardello et al. 1982). Thus the Kramer test can be considered a suitable method for the study of a wide variety of conditions affecting the overall texture quality of fish (Barroso et al. 1998a). It has even been used as the instrumental parameter to investigate the predictive and classification ability of some non-destructive measurements, such as UV/VIS spectroscopy (Isaksson et al. 2001). However, it may be unsuitable for some applications because of the relatively high amount of muscle required for the analysis, as well as the need for preparation of the probes, which can be considered as time consuming.

9.2.2

Warner-Bratzler test

The Warner-Bratzler shear cell consists of a blade with two cutting edges forming an angle of 60°, which penetrates another device with a slot. The blade cuts the sample like a guillotine and is thus subjected to a complex combination of tension, compression and shearing (Bourne 2002). Most authors have measured the maximum force exerted during the shearing action; but others, following the method proposed by Möller in beef samples (1980–81), have considered that in raw fish, the first peak of the force–deformation

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curve was due to the muscle fibres, and the second and much sharper and larger peak was due to the connective tissue (Montero and Borderías 1990a, b, 1992; Chamberlain et al. 1993). Chamberlain et al. (1993) used a shearing method similar to the Warner-Bratzler called a fish-shearing device (FSD), which consists of a blade that cuts the sample as it traverses a rectangular or circular device. Other devices similar to the Warner-Bratzler have been developed, for example for studying the influence of factors such as different cooking temperatures (Kanoh et al. 1988). Shear resistance has been used, for example, in the study of restructured adductor muscles of the Pacific calico scallop (catarina scallop; Argopecten ventricosus), and the giant lionspaw scallop (Nodipecten subnodosus) (Beltran-Lugo et al. 2005), studies on ice storage of Australian red claw crayfish (Cherax quadricarinatus) (Yen-Chang et al. 2002), characterisation of fish fingers (Schubring 2000), extruded snack foods based on pink salmon (Oncorhynchus gorbuscha) (Choudhury et al. 1998; Gautam et al. 1997), or dried atka mackerel (Pleurogrammus azonus) (Iseya et al. 1996). Examples of fish products whose shear resistance increases during frozen storage include sticks and blocks of fillets of red hake (Urophycis chuss) (Buck et al. 1986), whole muscle, or chopped fillets or minced hake (Merluccius merluccius) (Montero and Borderías 1990b, 1992; Barroso et al. 1998b). Correlations between data from sensory toughness and the fish shearing device, have been obtained for flathead (Platycephalus richardsoni), trevally (Pseudocaranx dentex) and morwong (Nematactylus douglassi) (Chamberlain et al. 1993) and between Warner-Bratzler maximum force and sensory hardness for smoked Atlantic salmon (Morkore and Einen 2003). Also, a strong correlation between sensory elasticity and firmness as measured by the Warner-Bratzler shear cell was found in frozen blue squat lobster (Cervimunida johni) tails (Pérez-Won et al. 2006). However, as discussed previously (Barroso et al. 1998a), in this test it is also important to take into account how the fibres are oriented with respect to the blade, and therefore the Warner-Bratzler method is difficult to perform, especially with small pieces of fish muscle. The shearing takes place in a very localised area of the muscle, causing distortion of the muscle fibres (Borderías et al. 1983). The device requires frequently dismounting for cleaning, as well as frequent calibration (Chung and Merritt 1991). Nevertheless, when comparing shear devices with other instrumental methods such as texture profile analysis (TPA), the shear test has been considered slightly more sensitive for some applications (Veland and Torrissen 1999; Sigurgisladottir et al. 1999).

9.2.3

Puncture test

During the puncture test a plunger is pushed into a food sample, so that there is an initial rapid increase in force as the probe moves into the food, which is being deformed under the load. After that, the punch begins to penetrate into the food, and this leads to a sudden change in slope called the yield point, which marks the instant where the punch begins to break the food. The direction of the force changes after the yield point, representing the force required to penetrate into the food (Bourne 2002). During puncture, the sample is subjected to a combination of compression and shearing in proportion to the area and perimeter of the

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cross-section of the plunger (Bourne 1975). The parameters measured are force at the point of rupture, slope and energy of the force–deformation curve (Barroso et al. 1998a). In addition, the shear or compression coefficients can be obtained by using several plungers with different perimeter/area ratios, and extracting their values from the linear graphs resulting from plotting the force/area (or force/perimeter) versus 1/perimeter (or perimeter). This was proven by Bourne (1975) for some foods, and could in principle be useful for some fish products. In this test, the sample size should be much larger than the punch. Thus, when a sample is thin, such as some fish fillets, there is a risk of compressing it against the support plate. If this is the case, then the test becomes a combination of puncture and compression, or even full compression (Bourne 2002). The test has been applied to raw or cooked samples. In fish fillets, samples have been measured perpendicularly (Orban et al. 1997) or parallel (Ando et al. 1991a) to the orientation of the muscle fibres, and regarded as an estimation of fish firmness or cohesiveness respectively (Izquierdo-Pulido et al. 1992). The measurement has been interpreted as an estimation of the fibres blocks toughness, when performed in conditions where care was taken to avoid direct contact of the puncture probe with the myocommata (Morzel et al. 2000). One variation of the puncture test is the ‘punch and die test’ (Segars et al. 1975), which is applied when the sample is thin and the support plate contains a hole whose diameter is about the same size as the punch diameter. Three characteristic parameters are measured such as: (a)

maximum shear stress which is related to the maximum force generated by the punch, (b) stiffness, related to the initial slope of the force-deformation curve, and (c) strain at failure, which depends on the deformation of the sample when the punch force is maximum (Segars et al. 1975; Sawyer et al. 1984). For a given punch diameter, these parameters are dependent on the sample thickness and volume, which are taken into account for obtaining normalised data (Segars et al. 1975). The test has been applied both in flakes extracted from the fillet and the fish fillet (Johnson et al. 1981). The puncture test is very widely applied in surimi gels, where it is easy to perform because of the homogeneity of the samples. In fish muscle, it has been used in the study of texture changes during storage at chilled temperatures. For example, an initial increase, corresponding to the onset of rigor mortis, and a subsequent decrease in puncture force values, have been found for raw cod fillets stored in ice (Careche et al. 2003); a decrease in penetration force values of hake muscle (Merluccius hubbsi) was reported after 18 days of storage at 3 °C (Kairiyama et al. 1990). Ando and co-workers (1991a, b, 1992) were able to show early softening (0–72 h) in rainbow trout muscle (Oncorhynchus mykiss) and in other seven different fishes (plaice, parrot bass, yellowtail, carp, red sea-bream, striped grunt, tiger puffer) (Ando et al. 1991b) by measuring the maximum force with a cylindrical plunger. This softening, measured in parallel to the orientation of the muscle fibres, was attributed to the disintegration of collagen fibres of the pericellular tissue. With this method, it could be

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observed that bleeding caused the delay of muscle softening in some pelagic fish species, whereas it did not have any influence in demersal fish (Ando et al. 1999). The puncture test has been used for the study of the double freezing on the quality of battered and breaded fish portions prepared from saithe (Pollachius virens) fillets and gutted and headed cod (Gadus morhua) (Schubring 2001, 2002a). Other applications include the study of stress previous to slaughter in sturgeon (Acipenser transmontanus) (IzquierdoPulido et al. 1992), cooking temperature and type of muscle (ordinary or dark) in yellowfin tuna (Thunnus albacares) (Kanoh et al. 1988) or the effects of high pressure in carp (Ciprinus carpio) (Yoshioka and Yamamoto 1998) which gave higher values of hardness as compared with the heated, non-pressurised muscles. Good correlations of the puncture test with sensory analysis have been observed when assessing the textural differences among species, such as maximum puncture force and sensory evaluation of hardness, chewiness and fibrousness of 26 finfish fillets when analysed cooked (Johnson et al. 1981). Maximum shear stress obtained by the punch and die test on cooked fillets presented very good correlations with sensory hardness or chewiness in 18 fish species (Sawyer et al. 1984). Sensory evaluation of firmness, hardness and cohesiveness in five different species stored at −25 °C correlated well with maximum force in cooked mince, where the sample was more uniform, but not in fish fillets (Borderías et al. 1983). Correlations have also been found for a given species in conditions known to render different texture values. Ando et al. (1991a), in raw common trout muscle (Oncorhynchuss mykiss), showed a good correlation between sensory evaluation of firmness and the instrumental parameter breaking strength measured by puncturing the fish muscle parallel to the orientation of the muscle fibres. The puncture test can be strongly influenced by the degree of separation of the myotomes when cooked, irrespective of the overall texture (Barroso et al. 1998a), unless it is performed by the punch and die test in flakes (Johnson et al. 1981; Sawyer et al. 1984).

9.2.4

Tension analysis

This test is exerted by holding strips or dumb-bell-shaped samples, with two parallel clamps, one of which moves away from the other at a constant rate (Bourne 2002). The parameters normally measured are maximum force or tensile strength and energy. The force deformation curves can be corrected to true stress–strain relationship and linearised so that the regression parameters of the resulting curve can be calculated. From them, the constant is considered a measure of the overall stiffness and the slope has been adopted as a hardening index (Kuo et al. 1990). The tensile stress–strain relationships in fish muscle show two types of deformation pattern (Segars et al. 1981), so that at relatively low strains, fillets behave as rubbery material, and at larger strains an increase in stiffness and irrecoverable deformation occurs. The tensile properties of strips of muscle on failure have been used for the study of the difference between rested and exhausted live Chinook salmon (Oncorhynchus tshawytscha) (Jerret et al. 1996). It has been applied to detect and quantify texture changes in mantle of squid (Illex illecebrosus and Loligo pealei), and relate the mechanical behaviour to the

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structural features of the mantle in both the longitudinal and transversal orientation (Kuo et al. 1990). Similarly, the study of tenderisation of broadtail shortfin squid (Ilex coindetii) mantle by marination at different pH and temperatures (Collignan and Montet 1998) has been addressed with this method. The texture changes in the early stages of storage at 4 °C of oval squid (Sepioteuthis lessoniana), Japanese common squid (Todarodes pacificus) and arrow squid (Heterololigo bleekeri) were analysed by several instrumental methods including tensile strength (Kagawa et al. 2002). The tension test of Japanese cockle (Fulvia mutica) revealed that both tensile strength and strain of cooked samples were significantly higher than those of raw samples and no changes in these parameters were observed upon storage at 4 °C for 10 days (Yoneda et al. 2002). Upon application of the tensile test, scattering of the rupture parameters if the sample is not the right shape can occur, as well as slipping or premature breaking in the clamps (Bourne 2002). However, this type of test can be of much interest in the study of different aspects of the quality of some seafood, such as squid.

9.2.5

Compression test

Compression analysis is performed upon application of a uniaxial compression force, between two parallel flat surfaces, one fixed and the other moveable. It is a non-destructive test when the force applied is insufficient to cause any irreversible damage to the sample (Bourne 2002). It can be performed either by compressing the sample to a fixed distance, percentage deformation or at a previously set force. During compression, there is a change in shape of the sample, and for uncompressible foods such as fish, there is no change in volume. For a true compression test, the probe should be much larger than the sample. The parameters normally measured from the force–deformation curves include slope, the degree of deformation produced by a set force, energy calculated by the area under the forcedeformation curve, and breaking strength when the compression is performed until breakdown of the sample (Barroso et al. 1998a). The deformation rate, the friction at the contact surfaces, and the physical dimensions of the samples can have a high influence on the force deformation curves (Calzada and Peleg 1978; Johnson et al. 1980; Chu and Peleg 1985). These can be important factors for fish and fish fillets that have special size and shape characteristics making often difficult to prepare standard samples. To avoid mechanical artefacts due to the specimen dimensions in uniaxial deformation, the force–time curves can be transformed to true stress–strain relationships to account for the expansion of the specimen cross-sectional area (Johnson et al. 1980). These authors proposed the use of a compressive deformability modulus for fish fillets, which is representative of a material’s overall resistance to deformation, derived from the true stress–strain relationships. This measurement is still dependent on the test conditions such as sample dimensions and deformation rate, but it can be a practical parameter when used within a certain experimental range (Jonhson et al. 1980). The compressive behaviour of several solid food samples with small height to diameter ratios uniaxially compressed to failure was studied by Chu and Peleg (1985) and revealed that the flatter the specimen the stiffer it appeared. This suggests that caution should be taken

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with interpretation of data when preparing samples with different height to diameter ratios. Another factor to be considered is the influence of the shape of the contact bodies. Several methods have been used to calculate the apparent modulus of elasticity, which accounts for the shape of the specimen and the contact surface (plate or indenter). These apply when the compression is at low deformations and it can be assumed that the contacting stresses vanish at the opposite ends of the body (semi-infinite body). They are based on the Hertz or Boussinesq solutions for contact stresses, the former for convex body pressing against another convex body or against a flat plate, the latter when the sample is under a rigid die, and were developed for agricultural products such as cereal grains (Mohsenin 1970a ; Arnold and Mohsenin 1971). In fish muscle, many methodological conditions have been assayed in order to choose the best one for the study of factors or technologies affecting the quality of fish. For example, the effect of compression speed, deformation level and thickness of the material was investigated for a fish product of minced and cooked silver carp with different salts added (Hypophthalmichthys molitrix) (Weinberg and Angel 1985). Deformations ranging from 30 to 80% were used in the study of the sea scallop (Placopecten magellanicus) adductor cooked muscle (Chung and Merrit 1991), with 50% deformation being chosen as better correlated with sensory analysis. Other authors have used 30% deformation for seabream (Sparus aurata) fillets (Orban et al. 1997). Slippage of the myotomes in cooked fish samples, when compressed in the texturometer, can occur (Hatae et al. 1990) and because of this, some authors do not consider this method suitable for analysis of cooked products (Borderías et al. 1983). Feinstein and Buck (1984) avoided this problem by coating the machine surfaces with an abrasive material. Increased force of compression has been reported in red hake (Urophycis chuss) stored at −18 °C as minced muscle (Knorr and Regenstein 1983) and increased force of the modulus of deformability at −7 °C as fillets (Owusu-Ansah and Hultin 1986). A high correlation between compression force and tensile strength measurements of carp (Hypophthalmichthys molitrix) was found (Weinberg and Angel 1984). A good agreement between compression measurements and texture scores with frozen storage time in fillets of mackerel (Scomber scombrus) and white hake (Urophycis tenuis) has been found (Santos and Regenstein 1990). Some methodological conditions do not comply with all the requirements for a true compression test, but they cannot be fully classified as puncture tests either. They use sample geometries that are larger than the probe compressing the food, often performing the tests directly in the fillets or the whole fish, with varying and sometimes small thickness along the sample being measured. In many cases, spherical probes, rather than cylinders, are used. The above conditions are usually applied to resemble the ‘finger test’, that is, in nondestructive conditions. It can be considered that the forces derive from compression and from shear (Pons and Fiszman 1996; Bourne 2002), but their contribution may vary from sample to sample, because their dimensions (for example thickness, shape of the contact surface) may not be constant. Despite this, the methods are reported to work well for the desired applications. Examples of tests performed directly on the whole fish or fish fillets include the analysis of the effect of ice storage, feeding strategy and slaughtering method of rainbow trout (Oncorhynchus mykiss), compressed with a 16 mm diameter probe

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the anterior part of the fish body by 10%, and the posterior part of the raw fillets by 30% Faergemand et al. (1995). Good correlations between maximum force recorded with these compression tests and a texture score, evaluated by one person pressing his forefinger and thumb were found. Also, texture properties along the fillet length of Atlantic salmon (Salmo salar) have been monitored by using devices such as sphere (30%) and cylinder probes (30%) (Casas et al. 2006). According to these authors, the slope of the force–deformation curve from the assay with a cylindrical probe was the most appropriate method to monitor the differences among the fillet locations. Nielsen et al. (2005) examined the correlations of sensory texture parameters of marinated fillets from Atlantic herring (Clupea harengus) with uniaxial compression measurements (60%) of the raw fish by a spherical plunger. In addition to the maximum compression force, and work (area under the curve), the x–y data from the compression curves, as well as their fit to polynomials, were used to relate to sensory data by multivariate analysis. Data from all three analyses were correlated with sensory firmness. Elasticity could be predicted from the maximum compression force. The curve contained information that was correlated with all the measured sensory texture parameters, whereas the fitted polynomials, in addition to firmness, could predict fatty mouth-feel. The authors concluded that the compression curves gave the highest information about the sensory texture. In addition to large, non-portable instruments, hand-held devices can also be applied. Sensory firmness of texture was well correlated with a Zwick hardness tester, which is a hand-held device that compresses the fish at a fixed load with an 18 mm indenter and measures the depth of compression on a 0 to 100 scale (Schubring 2002b). The compression test can be considered very suitable for measuring the overall resistance to deformation. However, as discussed by Hyldig and Nielsen (2001), one of the problems is the difficulty of comparing the results between authors, in part because different conditions of analysis are often not reported, and because, as discussed previously, many methodological factors can have an influence on the measurements.

9.2.6

Texture profile analysis

Texture profile analysis (TPA) (Friedman et al. 1963; Szczesniak et al. 1963) consists of compressing a sample twice, mimicking the action of the jaw. The classification of textural terms for solids and semi-solid foods by Szczesniak (1963) was the starting point for the development of a profiling method of texture, which was applicable to both instrumental (Szczesniak et al. 1963) and sensory (Brandt et al. 1963) measurements. Instrumental TPA was first applied in the General Foods Texturometer, devised especially for this purpose. Excellent correlations between instrumental and sensory ratings were obtained (Szczesniak et al. 1963). This method was adapted to the Instron Universal Testing Machine (Bourne 1968). The force–deformation curve is analysed to determine several texture parameters, originally defined as hardness, cohesiveness, elasticity, adhesiveness, brittleness, chewiness and gumminess (Szczesniak et al. 1963). These have been examined over the years and several modifications to their names or definitions have been proposed (Szczesniak 1998; 2002; Bourne 2002).

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Fish and fish products including surimi gels have been analysed using TPA. Breene (1975) and Pons and Fiszman (1996) reviewed the TPA analysis in terms of instrumentation, testing conditions, terminology and the time evolution of the main concepts. TPA has been applied to fish and fish products with different sizes of compressing units. This, and the sample size, affects the types of force measured. As discussed in the previous section, if the test is done in conditions where the sample size is much smaller than the probe, the forces derive mostly from uniaxial compression. In the opposite case, the forces derive mostly from puncture, that is, compression and shearing. For the analysis of whole fish, fillets and mince products, both flat-ended plungers (see, for example, Kolakowski et al. 1994; Chang and Regenstein 1997; Sigurgisladottir et al. 1999; Morzel et al. 2000; Morkore and Einen 2003; Gines et al. 2004; Pérez-Won et al. 2006) and spherical probes (see, for example, Sigurgisladottir et al. 1999; Veland and Torrissen 1999; Morkore and Einen 2003) have been used. The compression percentages also varied, depending on whether the test was performed in destructive or non-destructive conditions. The effect of different depths of compression by a spherical probe, both in absolute values irrespective the sample thickness (6–12.5 mm) and percentages of deformation (20–40%) were studied in raw salmon fillets, as affected by the temperature assay, fish size and feed/starvation status before slaughter (Veland and Torrisen 1999). TPA has been used to monitor seasonal variations in North Atlantic cod mince (Gadus morhua) (Ingólfsdóttir et al. 1998). Higher values of chewiness, gumminess, hardness and fracturability were found in arctic char (Salvelinus alpinus) reared at 15 °C compared with 10 °C (Gines et al. 2004) with 80% deformation. During frozen storage, increases in hardness were found for minced cod (Kim and Heldman 1985; Hsieh and Regenstein 1989; Kolakowski et al. 1994; Chang and Regenstein 1997), perch (Sebastes marinus) (Hsieh and Regenstein 1989), mackerel (Scomber scombrus) and white hake (Urophycis tenuis) (Chapman et al. 1993). Increased cohesiveness has been reported in cod (Gadus morhua), perch (Sebastes marinus) (Hsieh and Regenstein 1989), white hake (Urophycis tenuis) fillets and minced mackerel (Scomber scombrus) (Chapman et al. 1993). In contrast, decreased cohesiveness was found in minced muscle of cod (Kim and Heldman 1985; Kolakowski et al. 1994). Authors have compared the performance of TPA with other instrumental methods with factors known to render different texture values, and in some cases with sensory analysis. For example, TPA was compared with the Warner-Bratzler cell in raw salmon fillets (Salmo salar) and the latter was found to be more sensitive than the TPA (Veland and Torrisen 1999). Similarly, Sigurgisladottir et al. (1999) concluded that the shear force measurement was more sensitive than TPA with flat-ended or spherical probes when analysing changes as a function of the location of the salmon (Salmo salar) fillet. However, mechanical analysis using cylindrical probes of 12.5 or 23 mm diameter compressing 90% the sample height of raw and smoked salmon (Salmo salar) fillet strips at different pre-slaughter treatments rendered a higher correlation with sensory hardness than both the Warner-Bratzler blade and TPA exerted with a sphere (Morkore and Einen 2003). Morzel et al. (2000) used both TPA and puncture test in cured and fermented salmon (Salmo salar) analysed at different storage times after processing, and found some differences in the trend of hardness over time, which decreased in TPA and increased with the puncture test during the first days. The authors

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interpreted that each method measured a different property, i.e. muscle blocks toughness (puncture) and overall toughness (TPA). The effect of frozen storage of blue squat lobster (Cervimunida johni) tails was analysed by TPA, Warner-Bratzler and sensory methods. A strong correlation was found between sensory and TPA chewiness, cohesiveness and hardness parameters (Pérez-Won et al. 2006). TPA has also been performed in whole raw and frozen stored gilthead sea bream (Sparus aurata) (Carbonell et al. 2003) by 7 mm compression. A high percentage of the variance in the sensory parameters such as firmness, chewiness or juiciness was explained by some of the TPA parameters. Thus the authors suggest that it could be used as a fast non-destructive test. The extent of the correlation between instrumental and sensory analysis depends on the parameter, so that hardness in general correlates very well whereas springiness and cohesiveness render low correlations (Szczesniak 1998). In this sense, some authors question the need for a double compression for most applications in fish (Hyldig and Nielsen 2001) because hardness and fracturability are obtained from the first compression cycle.

9.2.7

Viscoelastic methods

Many food materials exhibit viscoelasticity, because they show simultaneously elastic properties from which they can be regarded as solids, and some flow properties, characteristic of liquids. In a viscoelastic solid there is an instantaneous deformation upon application of the force, and this deformation continues during the time the force is being applied. Viscoelasticity can be linear when the rheological properties are only dependent on time, whereas nonlinear viscoelasticity is shown when they depend also on parameters such as magnitude of the stress applied. Most foods behave as linear viscoelastic in a short range of strain (Mohsenin 1970b; Bourne 2002). Stress relaxation test In stress relaxation, the specimen is suddenly subjected to a given deformation and the force required holding the deformation constant is measured as a function of time. This rheological test can provide several parameters such as relaxation times, viscous and elastic moduli, as well as the ‘degree of solidity’ of the food. One problem associated with this test is how to analyse the relaxation curves to obtain the above parameters. When fitted to nonlinear regression, two or three exponential terms are sufficient to achieve a good fit of the experimental values (Mohsenin 1970b; Peleg 1979). The relaxation process can also be described by a linear model (Peleg 1980). Relaxation time distribution spectra rather than fixed relaxation times can also be derived from stress relaxation curves (Mao et al. 2000). Rheological properties have been studied using the stress relaxation in a variety of foods, and among them in surimi gels, fish myofibrillar protein films and meat. The applications in fish fillets include the study of raw, heated and ice stored carp (Cyprinus carpio) (Iso et al. 1984a, 1987), yellowtail Seriola dorsalis, (Iso et al. 1984b), or the effect of cooking temperature and immersion in water in different species of salted jellyfish (Kimura et al. 1991). The rheological properties have been used to characterise and distinguish between

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Figure 9.2 Stress relaxation test performed in whole fish.

the cuttlefish (Sepia esculenta) and four squid, Loligo bleekeri, Illex argentinus, Toradores pacificus and Nototodaus sloani sloani (Mochizuki et al. 1994), or the study of the relationships between composition and changes during heating (Mochizuki et al. 1995). Rheological properties of raw, steamed or boiled Abalone meat (Haliotis discus) have been analysed (Xin et al. 2001, 2002a, b). They have also been used to study the changes of meat-textured fish protein concentrate from Alaska pollack with storage time and temperature (Okazaki et al. 1984). The possibility of using the stress relaxation test for developing a non-destructive method to monitor post mortem textural changes in cod (Gadus morhua) (Herrero et al. 2004) and in frozen stored hake (Merluccius capensis and M. paradoxus) (Herrero and Careche 2005) has been addressed (Figure 9.2). Whole fish were compressed by 5% and deformation was kept constant for 60 seconds, rather than keeping it relaxing for several minutes as it is normally performed to study the rheological properties. The results were compared with the texture parameters of the QIM showing a similar trend. The same evolution with storage time has been found for fillet loins stored at −23 °C (unpublished data, Figure 9.3). Stress relaxation is widely used in the rheological characterisation of food products. It is an easy to perform method, which requires little sample preparation and could be used for quality assessment in those scenarios where the correlation with non-oral, low deformation, sensory assessment is pursued.

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0

5 10 15 Storage time (months)

20

Figure 9.3 Evolution with storage time of principal component 1 extracted from the stress relaxation parameters in hake (Merluccius capensis and M. paradoxus).

Creep test During the creep test, the sample is subjected to a given stress; the displacement required to hold the stress constant is measured as the function of time. Then the stress is removed and the recovery over time is recorded (Mohsenin 1970b). The creep test has mostly been used to study the rheological behaviour of food gels. Examples of the use of this method include the study of the rheological properties of channel rock fish, plaice, flying fish, common horse mackerel and skipjack (Hatae et al. 1988), and the effect of high pressure on carp (Cyprinus carpio) fillets (Yoshioka and Yamamoto 1998). A portable instrument was developed by Botta (1991, 1995) to determine a texture (firmness and resilience) index rapidly in raw Atlantic cod (Gadus morhua) fillets, which can be considered to be based on a very fast creep test. The resulting texture index was correlated with the sensory analysis performed by trained fish inspection officers using the ‘finger test’. The instrument measured the deformation distance by compressing the fillets at a set force, reducing the force and measuring the rebound distance. The ratio of rebound and deformation distances rendered the texture index. Different probe sizes, initial contact, deformation, and rebound forces, probe speeds, depression and rebound times, as well as different locations in the fillet were assayed. Conditions selected were those that were faster and with a closer agreement to the finger test. Nesvadba (2002) measured the creep compliance with a new hand held prototype for cod (Gadus morhua) that had been stored in ice. The softness of the fish was found to increase from day 1 to day 17 of storage; the author showed that the instantaneous deformations of frozen hake (Merluccius merluccius) stored at −20 °C for 0 and 18 months decreased, indicating an increase of hardness with frozen storage. It was concluded that it is possible to construct, based on the creep test, a hand-held portable instrument for determining the texture of fish in industrial settings. Thus, creep, as well as stress relaxation can be valuable methods for assessing the quality of fish with low deformations.

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Oscillatory test The small-amplitude oscillatory test is based on the application of a sinusoidal strain and the measurement of the resulting stress, or vice versa. When a linear viscoelastic material is subjected to this sine-wave stress, normally applied in shear, the strain will vary out of phase. The resulting frequency dependent modulus can be separated into an in phase or real component associated with the storage of energy associated to elastic behaviour and an out of phase associated with the loss of energy due to viscous behaviour. The phase angle (d) between stress and strain can be obtained. Perfectly elastic or viscous materials would render d = 0 or 90° respectively (Mohsenin 1970b; Gunasekaran and Ak 2000; Nesvadba 2002). This test is widely used for surimi gels (Kim et al. 2005) and it has been applied in the study of glass transition temperature in abalone (Rahman et al. 2003; Sablani et al. 2004). A hand-held probe for oscillatory measurement was constructed and applied in the study of cod unfrozen and frozen/thawed cod (Gadus morhua) stored in ice, as well as in hake that had been frozen for up to 18 months (Nesvadba 2002). The author proposed the use of this test for the study of fish freshness and frozen storage.

9.3 Texture measurement for quality classification or prediction 9.3.1

Classification of fish in quality categories

Some authors have attempted to predict changes in instrumental texture values occurring in fish fillets as a consequence of frozen storage temperature fluctuations by developing a model that would predict these textural changes in terms of the analysed chemical quality indexes (Leblanc et al. 1988). Also, the classification in quality categories of frozen stored hake (Merluccius spp.) with different time–temperature histories can be performed using a combination of data obtained by Kramer, Warner-Bratzler and puncture tests. These were three out of the four instrumental measurements required for this classification, among a list of physical–chemical methods applied (Barroso et al. 1998b). Results were analysed by principal component analysis, and it was shown that three factors accounted for 86.5% of the variance. The samples were separated in four clusters ranging from excellent to very poor quality, and they could be correctly classified by discriminant analysis in each of the quality groups in 95% of cases. The parameters required for classification were apparent viscosity, maximum force from the Kramer test, and maximum force and energy from the puncture test (Barroso et al. 1998b).

9.3.2

Prediction of storage time

Authors have attempted to find regression models based on instrumental measurements that could predict the storage time, as an indirect measurement of quality assessment. This can be done alone or in combination with other instrumental techniques.

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Predicted values

20 15 10 5 0

Calibration Validation 0

5

10

15

20

Storage time (months) Figure 9.4 Predicted values versus storage time in hake fillets (Merluccius capensis and M. paradoxus) stored at −23 °C using the Kramer test and apparent viscosity.

Kramer test for prediction of frozen storage time It is well accepted that among the considerable variations in the handling, freezing and storage conditions for commercial fish, most changes in parameters related to quality occur during storage, which for some species can span from 0 to 24 months, and temperature is one of the most important factors affecting them. The quality of frozen stored Cape hake (M. capensis and M. paradoxus) was indirectly estimated by a regression model based on instrumental analysis that predicts storage time at −20 °C (Herrero and Careche 2006). It was possible to achieve a simple regression model based on just two instrumental measurements such as the Kramer test and apparent viscosity measurement of homogenates of muscle extracted with 5% NaCl. The typical error of the regression was 2 months. The regression method can be constructed for different storage temperatures and products, and has been successfully adapted in the industry for fish fillets or pieces of fillets (Figure 9.4). Stress relaxation test as part of an ‘Artificial Quality Index’ Another approach to use instrumental texture analysis in assessing the quality of fish was done by creating an artificial quality index (AQI) (Di Natale 2003; Olafsdottir et al. 2004), based on similar principles of those of the QIM. The QIM is a non-oral sensory method designed in a way so that the sum of demerit points or defects given to each of the parameters correlates linearly with storage time, reaching a maximum at the reject threshold fixed by oral sensory analysis. The parameters are designed so that no special weight is given to one compared with the others. Thus, in a similar approach, data from several instrumental devices such as texturometer, colourmeter and electronic nose were calibrated for cod (Gadus morhua) stored in ice with their corresponding sensory attributes from the QIM. The instrumental texture method used was the stress relaxation test (Herrero et al. 2004) performed in the TA.XT2i SMS Stable Micro Systems Texture Analyser, but the measurements could in principle be done in smaller instruments so that a portable multisensor could be created (Nesvadba 2002).

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The AQI can be as accurate and precise as the QIM (Olafsdottir et al. 2004), because it described the freshness of cod, with the uncertainty of the predicted storage time being less than 0.5 days. Thus, the introduction of instruments, mimicking human senses, seems a promising approach to obtain a comparable judgement with trained panels. This concept was also applied to build a fish freshness indicator of sardine during refrigerated storage, with good results (Macagnano et al. 2005).

9.4

Conclusions

The different measurement methods described here have advantages and disadvantages, and most of the latter deal with the difficulties arising from the complex structure of fish muscle, and the changes occurring upon cooking that prevent the correct measurement of some of the properties. Some of the methods are preferred for estimating the overall texture of the sample, whereas others can be additionally applied to analyse some of the structural parts of the products. The importance of combining more than one method is stressed, as well as the more widespread use of fundamental tests, and standardised conditions that are reproducible in different laboratories. The search for rapid tests, if possible, with portable instruments is another area of great interest for the quality assessment of fish. The implementation of these texture methods in combination with instruments that measure other aspects of the fish quality, such as colour and odour, as for example in the artificial quality index, should be pursued.

Acknowledgements Thanks are due to the Food Quality Department of Pescanova S.A. for provision of data for Figures 9.3 and 9.4.

9.5

References

Ando, M., Toyohara, H., Shimizu, Y. and Sakaguchi, M. (1991a) Validity of a puncture test for evaluating change in muscle firmness of fish during ice storage. Nippon Suisan Gakkaishi 57: 2341. Ando, M., Toyohara, H., Shimizu, Y. and Sakaguchi, M. (1991b) Postmortem tenderization of fish muscle proceeds independently of resolution of rigor-mortis. Nippon Suisan Gakkaishi 57: 1165–1169. Ando, M., Toyohara, H. and Sakaguchi, M. (1992) Postmortem tenderization of rainbow-trout muscle caused by the disintegration of collagen-fibers in the pericellular connective-tissue. Nippon Suisan Gakkaishi 58: 567–570. Ando, M., Nishiyabu, A., Tsukamasa, Y. and Makinodan, Y. (1999) Post mortem softening of fish muscle during chilled storage as affected by bleeding. Journal of Food Science 64: 423– 428. Arnold, P.C. and Mohsenin, N.N. (1971) Proposed techniques for axial compression tests on intact agricultural products of convex shape. Transactions of the American Society of Agricultural Engineers 14: 78–84.

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Barroso, M., Careche, M. and Borderias, A.J. (1998a) Quality control of frozen fish using rheological techniques. Trends in Food Science & Technology 9: 223–229. Barroso, M., Careche, M., Barrios, L. and Borderias, A.J. (1998b) Frozen hake fillets quality as related to texture and viscosity by mechanical methods. Journal of Food Science 63: 793– 796. Barroso, M., Careche, M. and Borderias, A.J. (1997) Evaluation of fish freshness using mechanical methods. In: G. Olafsdottir, J.B. Luten, P. Dalgaard, M. Careche, V. Verrez-Bagnis, E. Martinsdottir and K. Heia, K. (Eds) (1997) Methods to Determine the Freshness of Fish in Research and Industry. International Institute of Refrigeration, Paris, pp. 355–362. Beltran-Lugo, A.I., Maeda-Martinez, A.N., Pacheco-Aguilar, R. and Nolasco-Soria, H.G., OcanoHiguera, V.M. (2005) Physical, textural, and microstructural properties of restructured adductor muscles of 2 scallop species using 2 cold-binding systems. Journal of Food Science 70: E78–E84. Boer, G. and Fennema, O. (1989) Effect of mixing and moisture modification on toughening and dimethylamine formation in Alaska pollock mince during frozen storage at –10 °C. Journal of Food Science 54: 1524–1529. Borderias, A.J., Lamua, M. and Tejada, M. (1983) Texture analysis of fish fillets and minced fish by both sensory and instrumental methods. Journal of Food Technology 18: 85–95. Botta, J.R. (1991) Instrument for nondestructive texture measurement of raw atlantic cod (Gadus morhua) fillets. Journal of Food Science 56: 962–964, 968. Botta, J.R. (1995) Physical methods of evaluating freshness quality. In: J.R.Botta (Ed.) Evaluation of Seafood Freshness Quality. VCH Publishers Inc., Weinheim, Germany, pp. 35–63. Botta, J.R., Bonnell, G. and Squires, B.E. (1987) Effect of method of catching and time of season on sensory quality of fresh raw atlantic cod (Gadus morhua). Journal of Food Science 52: 928–931. Bourne, M.C. (1968) Texture profile of ripening pears. Journal of Food Science 33: 223–226. Bourne, M.C. (1975) Method for obtaining compression and shear coefficients of foods using cylindrical punches. Journal of Texture Studies 5: 459–469. Bourne, M.C. (2002) Food texture and viscosity: concept and measurement. Food Texture and Viscosity: Concept and Measurement. Academic Press, London, pp. 423. Brandt, M.A., Skinner, E.Z. and Coleman, J.A. (1963) Texture profile method. Journal of Food Science 28: 404–409. Brune, W.M. (1975) Application of texture profile analysis to instrumental food texture evaluation. Journal of Texture Studies 6: 53–82. Bremner, H.A., Olley, J. and Vail, A.M.A. (1987) Estimating time–temperature effects by rapid systematic sensory methods. In: D.E. Kramer and J. Liston (Eds) Seafood Quality Determination. Elsevier, Amsterdam, pp. 413–434. Buck, E.M., Borhan, M., Hultin, H.O. and Damon, R.A. (1986) Changes in texture and flavor of red hake sticks as affected by frozen storage form. Journal of Food Science 51: 1063–1064. Bugeon, J., Lefevre, F. and Fauconneau, B. (2004) Correlated changes in skeletal muscle connective tissue and flesh texture during starvation and re-feeding in brown trout (Salmo trutta) reared in seawater. Journal of the Science of Food and Agriculture 84: 1433–1441. Calzada, J.F. and Peleg, M. (1978) Mechanical interpretation of compressive stress–strain relationships of solid foods. Journal of Food Science 43: 1087–1092. Carbonell, I., Duran, L., Izquierdo, L. and Costell, E. (2003) Texture of cultured gilthead sea bream (Sparus aurata): instrumental and sensory measurement. Journal of Texture Studies 34: 203– 217. Careche, M. and Tejada, M. (1991) Effect of added lipids on the texture of minced hake (Merluccius merluccius), megrim (Lepidorhombus whiffiagonis) and sardine (Sardina pilchardus) during frozen storage. Zeitschrift fur Lebensmittel-Untersuchung und -Forschung 193: 533–537.

Instrumental texture measurement

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Careche, M., Garcia, M.L., Herrero, A., Solas, M.T. and Carmona, P. (2002) Structural properties of aggregates from frozen stored hake muscle proteins. Journal of Food Science 67: 2827– 2832. Careche, M., Tryggvadottir, S.V., Herrero, A., Lägel, B., Petermann, U., Schubring, R. and Nesvadba, P. (2003) Instrumental methods for measuring texture of fish. In: J.B. Luten, J. Oehlenschager and G. Olafsdottir (Eds) Quality of Fish from Catch to Consumer: Labeling, Monitoring and Traceability, Wageningen Academic Publishers: Wageningen, pp. 189–200. Casas, C., Martinez, O., Guillen, M.D., Pin, C. and Salmeron, J. (2006) Textural properties of raw Atlantic salmon (Salmo salar) at three points along the fillet, determined by different methods. Food Control 17: 511–515. Chamberlain, A.I., Kow, F. and Balasubramaniam, E. (1993) Instrumental method for measuring texture of fish. Food Australia 45: 439–443. Chang, C.C. and Regenstein, J.M. (1997) Textural changes and functional properties of cod mince proteins as affected by kidney tissue and cryoprotectants. Journal of Food Science 62: 299–304. Chapman, K.W., Ifat, S., Keum, T.H. and Regenstein, J.M. (1993) Extra–cold storage of hake and mackerel fillets and mince. Journal of Food Science 58: 1208–1211. Choudhury, G.S., Gogoi, B.K. and Oswalt, A.J. (1998) Twin-screw extrusion pink salmon muscle and rice flour blends: effects of kneading elements. Journal of Aquatic Food Product Technology 7: 69–91. Chu, C.F. and Peleg, M. (1985) The compressive behavior of solid food specimens with small height to diameter ratios. Journal of Texture Studies 16: 451–464. Chung, S.L. and Merritt, J.H. (1991) Physical measures of sensory texture in thawed sea scallop meat. International Journal of Food Science and Technology 26: 207–210. Collignan, A. and Montet, D. (1998) Tenderizing squid mantle by marination at different pH and temperature levels. Lebensmittel-Wissenschaft und -Technologie 31: 673–679. Curran, D.M., Tepper, B.J. and Montville, T.J. (1990) Use of bicarbonates for microbial control and improved water-binding capacity in cod fillets. Journal of Food Science 55: 1564–1566. Delbarre-Ladrat, C., Cheret, R., Taylor, R. and Verrez-Bagnis, V. (2006) Trends in postmortem aging in fish: understanding of proteolysis and disorganization of the myofibrillar structure. Critical Reviews in Food Science and Nutrition 46: 409–421. Deng, J.C. (1981) Effect of temperatures on fish alkaline protease, protein-interaction and texture quality. Journal of Food Science 46: 62–65. Di Natale, C. (2003) Data fusion in MUSTEC: towards the definition of an Artificial Quality Index. In: J.B. Luten, J. Oehlenschager and G. Olafsdottir (Eds) Quality of Fish from Catch to Consumer: Labeling, Monitoring and Traceability. Wageningen Academic Publishers, Wageningen, pp. 273–282. Dobraszczyk, B.J. and Vincent, J.F.V. (1999) Measurement of mechanical properties of food materials in relation to texture: the materials approach. In: A.J. Rosenthal (Ed) Food texture: Measurement and Perception. Aspen Publishers Inc., Gaithersburg, Maryland, pp. 99–151. Dunajski, E. (1980) Texture of fish muscle. Journal of Texture Studies 10: 301–318. Faergemand, J., Ronsholdt, B., Alsted, N. and Borresen, T. (1995) Fillet texture of rainbow-trout as affected by feeding strategy, slaughtering procedure and storage post-mortem. Water Science and Technology 31(10): 225–231. Feinstein, G.R. and Buck, E.M. (1984) Relationship of texture to pH and collagen content of yellowtail flounder and cusk. Journal of Food Science 49: 298–299. Friedman, H.H., Whitney, J.E. and Szczesniak, A.S. (1963) The texturometer – a new instrument for objective texture measurement. Journal of Food Science 28: 390–396. Gautam, A., Choudhury, G.S. and Gogoi, B.K. (1997) Twin screw extrusion of pink salmon muscle: effect of mixing elements and feed composition. Journal of Muscle Foods 8: 265–285.

234

Fishery Products: Quality, safety and authenticity

Gill, T.A., Keith, R.A. and Lall, B.S. (1979) Textural deterioration of red hake and haddock muscle in frozen storage as related to chemical-parameters and changes in the myofibrillar proteins. Journal of Food Science 44: 661–667. Gines, R., Valdimarsdottir, T., Sveinsdottir, K. and Thorarensen, H. (2004) Effects of rearing temp. and strain on sensory characteristics, texture, colour and fat of Arctic charr (Salvelinus alpinus). Food Quality and Preference 15: 177–185. Gunasekaran, S. and Ak, M.M. (2000) Dynamic oscillatory shear testing of foods – selected applications. Trends in Food Science & Technology 11: 115–127. Hatae, K., Tobimatsu, A., Takeyama, M. and Matsumoto, J.J. (1986) Contribution of the connective tissues on the texture difference of various fish species. Bulletin of the Japanese Society of Scientific Fisheries 52: 2001–2007. Hatae, K., Nakayama, T., Matsui, Y., Shimada, A. and Matsumoto, J.J. (1988) Creep compliance behaviours of raw fish muscles in five species. Bulletin of the Japanese Society of Scientific Fisheries 54: 1595–1599. Hatae, K., Yoshimatsu, F. and Matsumoto, J.J. (1990) Role of muscle-fibers in contributing firmness of cooked fish. Journal of Food Science 55: 693–696. Herrero, A.M. and Careche, M. (2005) Stress relaxation test to evaluate textural quality of frozen stored Cape hake (M. capensis and M. paradoxus). Food Research International 38: 69–76. Herrero, A.M. and Careche, M. (2006) Prediction of frozen storage time of Cape hake (Merluccius capensis and Merluccius paradoxus) by instrumental methods. Journal of the Science of Food and Agriculture 86: 2128–2133. Herrero, A.M., Heia, K. and Careche, M. (2004) Stress relaxation test for monitoring post mortem textural changes of ice–stored cod (Gadus morhua). Journal of Food Science 69: FEP178– FEP182. Herrero, A.A., Carmona, P., Garcia, M.L., Solas, M.T. and Careche, M. (2005) Ultrastructural changes and structure and mobility of myowater in frozen-stored hake (Merluccius merluccius) muscle: Relationship with functionality and texture. Journal of Agricultural and Food Chemistry 53: 2558–2566. Howgate, P. (1977) Aspects of fish texture. In: G.G. Birch, J.G. Brennan and K.J. Parker (Eds) Sensory properties of food. Applied Science Publishers, London, UK, pp. 249–269. Hsieh, Y.L. and Regenstein, J.M. (1989) Texture changes of frozen stored cod and ocean perch minces. Journal of Food Science 54: 824–826. Hurling, R., Rodell, J.B. and Hunt, H.D. (1996) Fiber diameter and fish texture. Journal of Texture Studies 27: 679–685. Hyldig, G. and Nielsen, D. (2001) A review of sensory and instrumental methods used to evaluate the texture of fish muscle. Journal of Texture Studies 32: 219–242. Ingolfsdottir, S., Stefansson, G. and Kristbergsson, K. (1998) Seasonal variations in physicochemical and textural properties of North Atlantic cod (Gadus morhua) mince. Journal of Aquatic Food Product Technology 7: 39–61. Isaksson, T., Swensen, L.P., Taylor, R.G., Fjaera, S.O. and Skjervold, P.O. (2002) Non–destructive texture analysis of farmed Atlantic salmon using visual/near-infared reflectance spectroscopy. Journal of the Science of Food and Agriculture 82: 53–60. Iseya, Z., Sugiura, S. and Saeki, H. (1996) Procedure for mechanical assessment of textural change in dried fish meta. Fisheries Science 62: 772–775. Iso, N., Mizuno, H., Saito, T., Ohzeki, F. and Yang, L.C. (1984a) Studies on the rheological properties of heated carp meats. Bulletin of the Japanese Society of Scientific Fisheries 50: 349– 353. Iso, N., Mizuno, H., Saito, T., Ohzeki, F. and Wang, Z. (1984b) Studies on the rheological properties of the heated yellowtail meat. Bulletin of the Japanese Society of Scientific Fisheries 50: 2061–2064.

Instrumental texture measurement

235

Iso, N., Mizuno, H., Saito, T., Mochizuki, Y., Ishii, K., Okunuki, H. and Miyata, K. (1987) The relationships between the rheological properties and the freshness of fish meat. Bulletin of the Japanese Society of Scientific Fisheries 53: 1231–1235. Izquierdo-Pulido, M.L., Hatae, K. and Haard, N.F. (1992) Nucleotide catabolism and changes in texture indices during ice storage of cultured sturgeon, Acipenser transmontanus. Journal of Food Biochemistry 16: 173–192. Jerrett, A.R., Stevens, J. and Holland, A.J. (1996) Tensile properties of white muscle in rested and exhausted chinook salmon (Oncorhynchus tshawytscha). Journal of Food Science 61: 527–532. Johnson, E.A., Segars, R.A., Kapsalis, J.G., Normand, M.D. and Peleg, M. (1980) Evaluation of the compressive deformability modulus of fresh and cooked fish flesh. Journal of Food Science 45: 1318–1320. Johnson, E.A., Peleg, M., Sawyer, F.M., Segars, R.A. and Cardello, A. (1981) Mechanical methods of measuring textural characteristics of fish flesh. Refrigeration Science and Technology I.I.F./I.I.R. 1981–4, pp. 103. Johnston, I.A., Alderson, R., Sandham, C., Dingwall, A., Mitchell, D., Selkirk, C., Nickell, D., Baker, R., Robertson, B., Whyte, D. and Springate, J. (2000) Muscle fibre density in relation to the colour and texture of smoked Atlantic salmon (Salmo salar L.). Aquaculture 189: 335–349. Kagawa, M., Matsumoto, M., Yoneda, C., Mitsuhashi, T. and Hatae, K. (2002) Changes in meat texture of three varieties of squid in the early stage of cold storage. Fisheries Science 68: 783–792. Kairiyama, E., Lescano, G., Narvaiz, P. and Kaupert, N. (1990) Studies on the quality of radurized and non–radurized fresh hake (Merluccius merluccius hubbsi) during refrigerated storage. Lebensmittel-Wissenschaft und -Technologie 23: 45–48. Kanoh, S., Polo, J.M.A., Kariya, Y., Kaneko, T., Watabe, S. and Hashimoto, K. (1988) Heat-induced textural and histological changes of ordinary and dark muscles of yellowfin tuna. Journal of Food Science 53: 673–678. Kim, Y.J. and Heldman, D.R. (1985) Quantitative analysis of texture change in cod muscle during frozen storage. Journal of Food Process Engineering 7: 265–272. Kim, Y.J., Park, J.W. and Yoon, W.B. (2005) Rheology and texture properties of surimi gels. In: J.W. Park (Ed) Surimi and Surimi Seafood, 2nd edition. CRC Press, Boca Raton, pp. 491– 582. Kimura, H., Saito, T., Mizuno, H., Ogawa, H., Mochizuki, Y., Suyama, Y. and Iso, N. (1991) The rheological properties of salted jellyfish during cooking and dipping in water. Bulletin of the Japanese Society of Scientific Fisheries 57: 463–466. Knorr, D. and Regenstein, J.M. (1983) A simple method for evaluating textural changes of frozen fish minces. Journal of Food Science 48: 292–293. Kolakowski, E., Kolakowska, A., Lachowicz, K., Bortnowska, G. and Wianecki, M. (1994) The use of sodium carbonates to improve textural properties of cod minces. Journal of the Science of Food and Agriculture 66: 429–437. Kong, F.B., Tang, J.M., Rasco, B., Crapo, C. and Smiley, S. (2007) Quality changes of salmon (Oncorhynchus gorbuscha) muscle during thermal processing. Journal of Food Science 72: S103–S111. Kramer, D.E. and Peters, M.D. (1981) Effect of pH and prefreezing treatment on the texture of yellowtail rockfish (Sebastes flavidus) as measured by the Ottawa Texture Measuring System. Journal of Food Technology 16: 493–504. Krivchenia, M. and Fennema, O. (1988) Effect of cryoprotectants on frozen whitefish fillets. Journal of Food Science 53: 999–1003. Krueger, D.J. and Fennema, O.R. (1989) Effect of chemical additives on toughening of fillets of frozen Alaska pollack (Theragra chalcogramma). Journal of Food Science 54: 1101–1106.

236

Fishery Products: Quality, safety and authenticity

Kuo, J.D., Peleg, M. and Hultin, H.O. (1990) Tensile characteristics of squid mantle. Journal of Food Science 55: 369–371. LeBlanc, E.L., LeBlanc, R.J. and Blum, I.E. (1988) Prediction of quality in frozen cod (Gadus morhua) fillets. Journal of Food Science 53: 328–340. Licciardello, J.J., Ravesi, E.M., Lundstrom, R.C., Wilhelm, K.A. and Correia, F.F. (1982) Allsup, M.G. Time–temperature tolerance and physical–chemical quality tests for frozen red hake. Journal of Food Quality 5: 215–234. Macagnano, A., Careche, M., Herrero, A., Paolesse, R., Martinelli, E., Pennazza, G., Carmona, P., D’Amico, A. and Di Natale, C. (2005) A model to predict fish quality from instrumental features. Sensors and Actuators B – Chemical 111: 293–298. Madeira, K. and Penfield, M.P. (1985) Turbot fillet sections cooked by microwave and conventional heating methods: objective and sensory evaluation. Journal of Food Science 50: 172–177. Manthey, M., Karnop, G. and Rehbein, H. (1988) Quality changes of European catfish (Silurus glanis) from warm-water aquaculture during storage on ice. International Journal of Food Science & Technology 23: 1–9. Mao, R., Tang, J. and Swanson, B.G. (2000) Relaxation time spectrum of hydrogels by CONTIN analysis. Journal of Food Science 65: 374–381. Marshall, G.A., Moody, M.W., Hackney, C.R. and Godber, J.S. (1987) Effect of blanch time on the development of mushiness in ice-stored crawfish meat packed with adhering hepatopancreas. Journal of Food Science 52: 1504–1505. McKenna, D.R., Nanke, K.E. and Olson, D.G. (2003) The effects of irradiation, high hydrostatic pressure, and temperature during pressurization on the characteristics of cooked-reheated salmon and catfish fillets. Journal of Food Science 68: 368–377. Mochizuki, Y., Mizuno, H., Ogawa, H., Ishimura, K., Tsuchiya, H., Fukuzawa, M. and Iso, N. (1994) Rheological properties of cuttlefish and squid raw meat. Fisheries Science 60: 555– 558. Mochizuki, Y., Mizuno, H., Ogawa, H., Ishimura, K., Tsuchiya, H. and Iso, H. (1995) Changes of rheological properties of cuttlefish and squid meat by heat treatment. Fisheries Science 61: 680–683. Mohsenin, N.N. (1970a) Contact stresses between bodies in compression. In: N.N. Mohsenin (Ed) Physical Properties of Plant and Animal Materials, Volume 1 (Structure, Physical Characteristics and Mechanical Properties). Gordon & Breach Science Publishers Inc., New York, pp. 278– 308. Mohsenin, N.N. (1970b) Some basic concepts of rheology. In: N.N. Mohsenin (Ed) Physical Properties of Plant and Animal Materials, Volume 1 (Structure, Physical Characteristics and Mechanical Properties). Gordon & Breach Science Publishers Inc., New York, pp. 88–173. Moller, A.J. (1981) Analysis of Warner-Bratzler shear pattern with regard to myofibrillar and connective tissue components of tenderness. Meat Science 5: 247–260. Montero, P. and Borderias, J. (1990a) Influence of age on muscle connective tissue in trout (Salmo irideus). Journal of the Science of Food and Agriculture 51: 261–269. Montero, P. and Borderias, J. (1990b) Behavior of myofibrillar proteins and collagen in hake (Merluccius merluccius) muscle during frozen storage and its effect on texture. Zeitschrift fur Lebensmittel-Untersuchung und -Forschung 190: 112–117. Montero, P. and Borderias, J. (1992) Influence of myofibrillar proteins and collagen aggregation on the texture of frozen hake muscle. In: H.H. Huss, M. Jakobsen and J. Liston (Eds) Quality Assurance in the Fish Industry. Elsevier Science Publishers B.V., Amsterdam, The Netherlands, pp. 149–156. Morkore, T. and Einen, O. (2003) Relating sensory and instrumental texture analyses of Atlantic salmon. Journal of Food Science 68: 1492–1497.

Instrumental texture measurement

237

Morzel, M., Heapes, M.M., Reville, W.J. and Arendt, E.K. (2000) Textural and ultrastructural changes during processing and storage of lightly preserved salmon (Salmo salar) products. Journal of the Science of Food and Agriculture 80: 1691–1697. Nesvadba, P. (2002) Quality control by instrumental texture measurements. In: C. Alsavar and T. Taylor (Eds) Seafoods – Quality, Technology and Nutraceutical Applications. Springer, Berlin, pp. 43–57. Nielsen, D., Hyldig, G., Nielsen, J. and Nielsen, H.H. (2005) Liquid holding capacity and instrumental and sensory texture properties of herring (Clupea harengus) related to biological and chemical parameters. Journal of Texture Studies 36: 199–138. Okazaki, E., Tsukada, K., Kanna, K. and Suzuki, T. (1984) Changes of properties of meat-textured fish-protein concentrate during frozen storage. Bulletin of the Japanese Society of Scientific Fisheries 50: 307–312. Olafsdottir, G., Nesvadba, P., Di Natale, C., Careche, M., Oehlenschager, J., Tryggvadottir, S.V., Schubring, R., Kroeger, M., Heia, K., Esaiassen, M., Macagnano, A. and Jorgensen, B.A. (2004) Multisensor for fish quality determination. Trends in Food Science & Technology 15: 86–93. Orban, E., Sinesio, F. and Paoletti, F. (1997) The functional properties of the proteins, texture and the sensory characteristics of frozen sea bream fillets (Sparus aurata) from different farming systems. Lebensmittel-Wissenschaft und -Technologie 30: 214–217. Owusu-Ansah, Y.J. and Hultin, H.O. (1986) Chemical and physical changes in red hake fillets during frozen storage. Journal of Food Science 51: 1402–1406. Peleg, M. (1979) Characterization of stress relaxation curves of solid foods. Journal of Food Science 44: 277–281. Peleg, M. (1980) Linearization of relaxation and creep curves of solid biological-materials. Journal of Rheology 24: 451–463. Perez-Won, M., Barraza, M., Cortes, F., Madrid, D., Cortes, P., Roco, T., Osorio, F. and TabiloMunizaga, G. (2006) Textural characteristics of frozen blue squat lobster (Cervimunida johni) tails as measured by instrumental and sensory methods. Journal of Food Process Engineering 29: 519–531. Pons, M. and Fiszman, S.M. (1996) Instrumental texture profile analysis with particular reference to gelled systems. Journal of Texture Studies 27: 597–624. Racicot, L.D., Lundstrom, R.C., Wilhelm, K.A., Ravesi, E.M. and Licciardello, J.J. (1984) Effect of oxidizing and reducing agents on trimethylamine N-oxide demethylase activity in red hake muscle. Journal of Agricultural and Food Chemistry 32: 459–464. Rahman, M.S. and Sablani, S.S. (2003) Structural characteristics of freeze-dried abalone. Porosimetry and puncture test. Food and Bioproducts Processing 81: 309–315. Rahman, M.S., Kasapis, S., Guizani, N. and Al Amri, O.S. (2003) State diagram of tuna meat: freezing curve and glass transition. Journal of Food Engineering 57: 321–326. Rehbein, H. and Orlick, B. (1990) Comparison of the contribution of formaldehyde and lipid oxidation products to protein denaturation and texture deterioration during frozen storage of minced ice-fish fillet (Champsocephalus gunnari and Pseudochaenichthys georgianus). International Journal of Refrigeration 13: 336–341. Sablani, S.S., Kasapis, S., Rahman, M.S., Al Jabri, A. and Al Habsi, N. (2004) Sorption isotherms and the state diagram for evaluating stability criteria of abalone. Food Research International 37: 915–924. Santos, E.E.M. and Regenstein, J.M. (1990) Effects of vacuum packaging, glazing, and erythorbic acid on the shelf-life of frozen white hake and mackerel. Journal of Food Science 55: 64–70. Sato, K., Yoshinaka, R., Sato, M. and Shimizu, Y. (1986) Collagen content in the muscle of fishes in association with their swimming movement and meat texture. Bulletin of the Japanese Society of Scientific Fisheries 52: 1595–1600.

238

Fishery Products: Quality, safety and authenticity

Sawyer, F.M., Cardello, A.V., Prell, P.A., Johnson, E.A., Segars, R.A., Maller, O. and Kapsalis, J. (1984) Sensory and instrumental evaluation of snapper and rockfish species. Journal of Food Science 49: 727–733. Schubring, R. (2000) Instrumental and sensory evaluation of the texture of fish fingers. Deutsche Lebensmittel-Rundschau 96: 210–221. Schubring, R. (2001) Double freezing of saithe fillet. Influence on sensory and physical attributes. Nahrung 45: 280–285. Schubring, R. (2002a) Double freezing of cod fillets: influence on sensory, physical and chemical attributes of battered and breaded fillet portions. Nahrung 46: 227–232. Schubring, R. (2002b) Texture measurement on gutted cod during storage in ice using a hand-held instrument. Informationen für die Fischwirtschaft aus der Fischereiforschung 49: 25–27. Segars, R.A., Johnson, E.A., Kapsalis, J.G. and Peleg, M. (1981) Some tensile characteristics of raw fish flesh. Journal of Texture Studies 12: 375–387. Segars, R.A., Hamel, R.G., Kapsalis, J.G. and Kluter, R.A. (1975) A punch and die test cell for determining the textural qualities of meat. Journal of Texture Studies 6: 211–225. Sigurgisladottir, S., Hafsteinsson, H., Jonsson, A., Lie, O., Nortvedt, R., Thomassen, M. and Torrissen, O. (1999) Textural properties of raw salmon fillets as related to sampling method. Journal of Food Science 64: 99–104. Szczesniak, A.S. (1963) Classification of textural characteristics. Journal of Food Science 28: 385–389. Szczesniak, A.S. (1987) Correlating sensory with instrumental texture measurements – an overview of recent developments. Journal of Texture Studies 18: 1–15. Szczesniak, A.S. (1998) Sensory texture profiling – Historical and scientific perspectives. Food Technology 52: 54–57. Szczesniak, A.S. (2002) Texture is a sensory property. Food Quality and Preference 13: 215– 225. Szczesniak, A.S., Brandt, M.A. and Friedman, H.H. (1963) Development of standard rating scales for mechanical parameters of texture and correlation between the objective and the sensory methods of texture evaluation. Journal of Food Science 28: 397–403. Taylor, R.G., Fjaera, S.O. and Skjervold, P.O. (2002) Salmon fillet texture is determined by myofibermyofiber and myofiber-myocommata attachment. Journal of Food Science 67: 2067–2071. van Vliet, T. (1999) Rheological classification of foods and instrumental techniques for their study. In: A.J. Rosenthla (Ed) Food Texture: Measurement and Perception. Aspen Publishers Inc., Gaithersburg, Maryland, pp. 65–96. Veland, J.O. and Torrissen, O.J. (1999) The texture of Atlantic salmon (Salmo salar) muscle as measured instrumentally using TPA and Warner-Brazler shear test. Journal of the Science of Food and Agriculture 79: 1737–1746. Voisey, P.W. and Kloek, M. (1981) Effect of cell-size on the performance of the shear-compression texture test cell. Journal of Texture Studies 12: 133–139. Weinberg, Z.G. and Angel, S. (1984) Stress relaxation and tensile strength testing of a processed fish product. Journal of Texture Studies 15: 59–66. Weinberg, Z.G. and Angel, S. (1985) Behavior of a formed fish product under the stress–relaxation text. Journal of Food Science 50: 589–591, 638. Westad, F., Nilsen, B.N. and Rodbotten, R. (2001) Prediction and classification of food quality using VIS/NIR spectroscopy and parsimonious models. New Food 4: 52–57. Xin, G., Ogawa, H., Tashiro, Y. and Iso, N. (2001) Rheological properties and structural changes in raw and cooked abalone meat. Fisheries Science 67: 314–320. Xin, G., Tashiro, Y. and Ogawa, H. (2002a) The correlation between rheological properties and characteristic values of structure for steamed abalone meat. Food Science and Technology Research 8: 304–310.

Instrumental texture measurement

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Xin, G., Tashiro, Y. and Ogawa, H. (2002b) Rheological properties and structural changes in steamed and boiled abalone meat. Fisheries Science 68: 499–508. Yen-Chang, T., Xiong, Y.L., Webster, C.D., Thompson, K.R. and Muzinic, L.A. (2002) Quality changes in Australian red claw crayfish, Cherax quadricarinatus, stored at 0 °C. Journal of Applied Aquaculture 12: 53–66. Yoneda, C., Kasamatsu, C., Hatae, K. and Watabe, S. (2002) Changes in taste and textural properties of the foot of the Japanese cockle (Fulvia mutica) by cooking and during storage. Fisheries Science 68: 1138–1144. Yoshioka, K. and Yamamoto, T. (1998) Changes of ultrastructure and the physical properties of carp muscle by high pressurization. Fisheries Science 64: 89–94.

Chapter 10

Image processing Michael Kroeger

10.1

Introduction

Until now, sensory methods have been considered the most reliable system for quality grading and assessment of fishery products. However, increasing demands on quality monitoring requires an increasing support of sensory methods by instrumental sensors. The artificial quality index is a concept for the integration of instrumental sensors into a sensory system. A visual inspection system based on the analysis of hierarchical tissue or skin structures enables an embedding in this concept. Disorganisation of fine structures causes a disorganisation of larger structures. An image processing system designed for larger structures takes advantage of this chain for an efficient way to monitor quality attributes from surface patterns. In extensive trials, the variations of patterns with storage time at different temperatures were examined. Pattern analysis based on the measurement of local orientation provides data with a linear relationship to storage time and enables the assessment of freshness from images. In addition, the orientation data enable an approach to firmness of muscle tissue if they are used as input for discrete elastic equations. Quality demands have increased continuously in many areas of industrial production within the past few years. Online inspection of geometric accuracy, colour and texture of products, an identification of material defects and the robot control in a processing line are increasingly operated by image processing systems. The technology of optical measurement and the development of efficient algorithms in image analysis have enabled visual inspection systems to be a part of the industrial production process. In processing and the trade of fishery products, an increasing demand on quality monitoring during all steps in the fishery chain has been established. The necessity for machine inspection systems increases (Jörgensen et al. 2003). However, the definition of inspection details is a difficult task. Sensory methods, supported by physical, chemical and microbiological examinations are regarded as the most reliable methods for the determination of food quality (Clucas and Ward 1996). An approach to the link from sensory data and data from instruments is provided by the artificial quality index (AQI) (Di Natale 2003). The AQI gives instructions for the design of a visual inspection system. A necessary but insufficient property for replacing sensors by an inspection system is a linear relationship of artificial sensor data 240

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with (post mortem) storage time for samples in ice. The artificial sensor has to be a natural candidate, too. Measurements of samples with known storage history enable a test of the linearity of data. However, making the artificial sensor a natural candidate is difficult to realise (Mallot 1998; Girardin et al. 2002; Olzak and Thomas, 1986). An image processing system used as a visual sensor has to realise the linear relationship of output data with storage time and the overlap with human sensors. Because of the rapid development of vision systems for industry today, many optical components, cameras, framegrabbers and types of image processing software are available. For a seafood inspection system, the design of an illumination-camera ensemble and the use and development of efficient algorithms for the different analysis steps remains an essential task. In fish processing or trade with seafood products, there are additional quality criteria that are independent from the sensory features. Determination of the size and geometry of samples, position of parasites, colour variability or uniformity, surface defects, and orientation of myotomes for cutting machines are requirements of image processing. Different tasks require an adaptation of the image processing systems based on extensive knowledge of properties of biological material. An extensive review of different vision equipment for fish quality control has been compiled by Pau and Olafsson (1991). Stereo and three-dimensional image applications for food processing have been compiled by Russ (2004).

10.2

Quality characteristics from images

Sensory characteristics are classified by the human senses in appearance and colour, smell, taste and texture (Clucas and Ward 1996). An essential task of visual inspection is to measure appearance as a quality attribute. In the quality index method (QIM) (depending on species), appearance is a function of the attributes of skin and firmness (www.qim-eurofish.com). This only can be reproduced by an artificial vision system if the features measured from images have a sufficient overlap with impressions from human vision. Suitable image data have to show a linear correlation with storage time and have to enable the simulation of real sensory impressions such as firmness or colour. ‘Most of the mechanical and chemical properties of foods depend in various ways on the surfaces that are present, and it is, therefore important to measure them’ (Russ 2004). A structure–statistical analysis of image texture (Zheng et al. 2006) from the surface realises this overlap. On the skin of fish or the surface of muscle tissue, large-scale structures can be recognised without microscopic techniques. ‘Hierarchical structures are found in practically all complex system in nature, including cartilage, skin, wood, nacre, and other natural material’ (Aguilera and Stanley 1999). For fish muscle tissue and skin, all the structures have linearity as an essential feature. Consequently, linear hierarchical structures give a natural basis for the link of image features and sensory features. A local anisotropic fibre-matrix model (Figure 10.1) is a suitable specification for the link. The fibres, embedded in a matrix, represent the linear structures and are perceived on the surface of the sample as a typical pattern. Fibres and matrix differ in their material properties. Images from a small surface area – comparable to the area of a fingertip – are sufficient to derive meaningful data. To extract features, images are subdivided into cells by filtering and folding techniques. A cell is a small elementary pattern representing the local arrangement of fibres. It is the only information source apart from spectroscopic signatures with a link to appearance.

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Figure 10.1 Anisotropic fibre-matrix model of a local pattern cell on the surface of tissue. Orientation and diameter of the fibres within the cell are estimated by image processing.

The local micro-pattern shows a weak variation with the wavelength of incident light. So the spectroscopic signature of images is necessary either by use of a multi-spectral camera or by a grey-level camera combined with a suitable illumination technique. The use of bilateral telecentric lenses decreases the dependence of the sample distance relative to the sensor plane and avoids errors by shading from surface micro-topography. The same evaluation is performed for all the different spectroscopic channels. As a first step in pattern analysis, the grey-level image of a channel is transformed into a vector image by calculating the gradients within a neighbourhood for all the positions (pixels) of the image: g (r ) ⇒ ∇g(r ) = ( ∂g (r ) ∂x , ∂g (r ) ∂y ) g (r ) grey-level image → r image vector with components x, y The mean local gradient within the neighbourhood U points in the direction of the most changing of local grey-level. Perpendicular to the gradient, the orientation points parallel to constant grey-level. In the local neighbourhood, the local orientation is determined by the symmetric structure tensor (Jähne 1997): J (r ) = ∫ ∇g (r ′ ) ∇gT (r ′ ) dr ′ U

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By rotation of the local coordinate system, the structure tensor is transformed into a diagonal form resulting in the eigenvalue problem: JeW = λ W eW where J is the structure tensor, ew is the eigenvector, lw is the eigenvalue and w is the image dimension (here, w = 2). The two eigenvalues of the structure tensor of all the cells in the image classify the local microstructures and enable access to relevant quality information. The size of the surface area on the sample, the resolution of the area sensor of the camera (CCD or CMOS chip) and the size of the neighbourhood is a unity deciding the significance of the local orientation cell for the quality inspection. As a lower limit, a size on the surface of 10 mm × 10 mm, a sensor resolution of 512 × 512 pixels and a neighbourhood of 5 × 5 pixels was found to recognise real structures from muscle tissue and skin patterns of most fish species. Image processing not far from the lower limit guarantees a low data stream for processing without violation of the sampling theorem. The local eigenvalues l1 and l2 of the two-dimensional image enable a description of the local surface structure. For a rapid classification, the local coherence is suitable information from tissue or skin. The coherence is defined as (Bazen and Gerez 2000): coh = (λ1 − λ 2 ) (λ1 + λ 2 ) Coherence ranges from coh = 0 for an isotropic grey value to coh = 1 for an ideal oriented structure. For all the fish samples investigated, a decay of parallel arrangement of muscle structures and of the sharpness of skin pattern with storage time was found (Delbarre-Ladrat et al. 2006). On the same resolution scale, coherence histograms show a shift of peaks to lower coherence. Coherence denotes the ratio of the mean square magnitude of the orientation vector to the mean square magnitude of the gradient vector. Both quantities are assigned to local biological structure features of the sample.

10.3

Spectral signature of images

The micro-pattern of samples shows a weak dependence of the wavelength of incident light (Andersen and Wold 2003; Dufour et al. 2003). This fact has to be considered in the pattern recognition process. To receive reliable inspection data, the variation of micro-patterns with storage time has to be checked for more than 10 channels from ultraviolet to near infrared. An effective way to realise this is a step-by-step illumination of samples with different monochromatic light combined with the generation of images. A step-by-step illumination from 350 to 950 nm in steps of 50 nm can be performed by light-emitting diode (LED) clusters. LEDs have known irradiation characteristics, narrow bandwidth and low selfheating. They enable the design of an optimal illumination system that takes into account the different light-scattering properties of fish skin or muscle. Within a few seconds, a set of 12–16 spectroscopic signed images are generated from the same position of the sample surface. For reliable orientation data, a radiometric calibration of all single images is necessary. A two-point calibration by a standard material with constant reflection qualities over

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the wavelength range is essential for the analysis. The calibration step considers the spectral sensitivity of the camera and the different intensities and distances of the LED. For all the spectroscopic signed images, the same analysis is performed. The link from image data to storage time by a multivariate analysis uses only a few spectroscopic signed images (Kroeger 2004). Suitable wavelength combinations for different species are determined by test data from images with known storage history.

10.4

Elastic properties from images

Coherence is a certainty measure for the existence of a local linear structure. Image analysis based on coherence formalism involves insufficient structure information for a derivation of mechanical properties. The structure operator contains two components of the orientation vector of the local pattern and the mean square gradient of the local structure. Including all the components of the structure operator enables the modelling of an equivalent to the local stiffness of the surface of tissue. Mechanical properties such as firmness involve the total surface covered by the image. This requires a link of all the local stiffnesses in consideration of physical laws. The membrane equation (Landau and Lifschitz 1975) realises the link. ‘Membranes represent the main structural component for the complex architecture of biological systems’ (Lipowski 1991). Membrane modelling is a useful tool in determining quality of seafood, too. To simulate mechanical properties, the image is regarded as an elastic membrane (Jähne 1997; Aubert and Kornprobst 2006). Local stiffness data from images control the conformation of the membrane. The effect of a fingertip of an expert (Bazen and Gerez 2002) or the action of an indenter from a texture analyser (Bourne 2002; Nesvadba 2002) is comparable to the deformation of a membrane. For numerical calculations of mechanical properties, the membrane is approximated by a two-phase fibre-matrix material (Feughelman 1959; Garboczi et al. 1999). Oriented lines in the patterns represent fibres that are embedded in a matrix comparable to muscle fibres embedded in connectivity tissue. Fibres and matrix have different elastic properties and different ageing. Membranes are described by differential geometry, in particular in terms of the mean and the Gaussian surface curvature (David 2004). A suitable parametrisation for membrane deformation and fluctuations can be carried out by the unit vectors normal to the surface and by vertical displacements of discrete points on the surface (Sornette and Ostrowsky 1994). A deformation is simulated by the action of a constant virtual force on a flat membrane. The ratio of volume to the surface of the deformed membrane is a useful indicator for firmness and ageing. So a combination of image processing and membrane modelling enables a snapshot of the momentary firmness.

10.5

Analysis of image data

Coherence histograms for different wavelengths are the basis of a rapid analysis of image data for freshness identification. From coherence histograms, a set of about 10 equidistant data are fused to a feature vector. A classification of storage time is performed by multivariate regression or neural networks. In all investigations, partial least-square regression (Martens and Naes 1989) has been used as part of the image analysing system. The standard

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error for calibration (SEC), the standard error for prediction (SEP) and the coefficient of determination, R2, are used to assess the regression fit. R2 ranges from 0 for missing correlation to 1 for an ideal correlation. All the different steps in image processing are performed by the development tool Heurisko (AEON, Hanau). Multivariate calculations use Matlab (The MathWorks, Natick, Massachusetts) as a platform. The Finite Element system Biomem-image (Technet, Berlin) is adapted for image analysis to calculate conformation, firmness, and intrinsic and free energy of biomembranes.

10.6

Results and discussion

Fish quality is a complex concept (Olafsdottir et al., 2004). Consequently, a visual inspection system can only be designed for particular aspects of quality. Integration into a sensory system or into the context of the AQI is an essential part of its design. The analysis of surface patterns due to local linear symmetry has proven to be a suitable inspection method. Within two research projects, ‘Multi-sensor techniques for monitoring the quality of fish – MUSTEC’ (Luten et al. 2003) and ‘Seafood Quality Identification – SEQUID’ (Kent et al. 2005), both partly financed by the European Commission, a visual sensor based on orientation analysis of micropatterns was developed and tested. Most samples were investigated at the beginning of storage time on board research vessels after the catch and during the whole storage period. From all the samples, a set of spectroscopic signed images was generated for at least 11 different wavelengths in the range 400– 940 nm. The inspection window on the surface covered a rectangle with an 18 [mm] diagonal. Images were generated by an analogue grey-level CCD-camera with a resolution of 744 × 576 pixels and known spectral sensitivity. For the analysis, an area of interest (AOI) of 512 × 512 pixels was used to avoid inaccuracies caused by the boundaries of the sensor. The assigned coherence images involve 256 × 256 single data performing a typical density distribution, depending on species. A set of 10 equidistant data from the density distribution was used as an input for multivariate calculations. Measurements on skin and muscle tissue have a similar shift to lower coherencies with ageing. However, a slightly lower coefficient of determination was found for skin. This was caused by double-oriented structures of the skin, which are considered less accurate for the analysis method (Aach et al. 2004). Extensive investigations have confirmed the significance of orientation analysis for freshness determination. Image patterns from the surface require an approximately parallel orientation of muscle structures relative to it. The sensor plane needs to be approximately parallel to the sample surface, too. However, this prerequisite is insiginficant for the practice. Altogether, image analysis based on coherence of local patterns is robust against uneven illumination and is independent of horizontal rotation of the sample relative to the plane of the CCD or CMOS chip. More detailed orientation analysis gives additional, fundamental local-structure features as a basis for firmness calculations. However, the use of more features increases the time required for evaluation. The design of a visual inspection system has to take into account the advantages and disadvantages of a varying ratio with information abundance and evaluation time. Quality examinations for freshness and/or firmness only use a small part of the surface as representative of the whole sample. To identify surface defects, the whole sample has to be

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Table 10.1 Determination of storage time from image data for temperatures 0, −10, −20, −20 doublefrozen and −30 °C.

Sample Gadus morhua skin Gadus morhua fillet Gadus morhua fillet Gadus morhua fillet Gadus morhua fillet Gadus morhua fillet Sebastes marinus fillet Sebastes mentella fillet Dicentrarchus labrax fillet

T (°C)

Number of samples

Range of storage (days)

R²

SEC

SEP

0 0 −10 −20 −20 −30 0 0 0

75 75 39 40 40 34 140 140 54

0–15 0–15 38–234 30–301 38–260 30–314 0–14 0–14 2–16

0.915 0.922 0.885 0.869 0.882 0.801 0.886 0.915 0.935

1.53 1.46 26.2 33.2 28.7 43.0 1.48 1.27 0.946

1.65 1.46 26.1 33.3 27.4 45.2 1.59 1.55 1.39

SEC and SEP, standard error of calculation and prediction; R2, coefficient of determination (Johnson and Wichern 1992).

considered by scanning for information gaps in orientation patterns. This requires another optical system and supplementary analysis steps for contour recognition.

10.7

Freshness determination from images

The determination of storage time from image data is possible only for a constant temperature during the total storage interval. Temperature fluctuations during measurements blur the assignment of data to storage time. Image data involve this relationship between time and temperature. For constant temperature, an unambiguous relationship between image data derived from coherence distributions and storage time was checked in extensive tests. Table 10.1 summarises the accuracy of the assignment of image data to storage time for different species and different storage temperatures. The summary proves that determination of freshness by a visual inspection system is practicable if the prerequisites for image generation and image analysis are satisfied.

10.8

Firmness information from images

The relationship of coherence data with storage time has proved the usefulness of the analysis of patterns due to local linear symmetry. For a more comprehensive integration of image data into a sensory system, local fibre orientations and local gradients of patterns are a suitable basis for simulating elastic properties. Figure 10.2 demonstrates the result simulating the deformation of tissue by a virtual indenter. A function of gradients and orientation from images of cod for a single wavelength were mapped on an elastic membrane. The depth of indentation was simulated by a constant virtual force acting on the membrane. A comparison with texture measurements (Careche et al. 2003, p. 193) shows the significance of structure data for the simulation of mechanical properties of the sample.

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Depth (abitrary units)

130 120 110 100 90 80 70 60 50

0

2

6

4

8

10

12

14

Storage time (d) Figure 10.2 Depth of indentation for cod from a virtual texture analyser. Data from images by mapping local stiffness information to a membrane. Storage temperature T = 0 °C, spectroscopic signature l = 505 nm.

23.5

Volume/surface

23.0

22.9041

22.5

22.5559

22.0

21.9030 21.5676

21.5

20.9188

21.0 20.5

0

10

20

30

40

50

60

Storage time (h) Figure 10.3 Ratio of volume to surface for deformation simulation of cod muscle tissue. Identification of the rigor by the peak bend at 21 h. Storage temperature T = 20 °C, spectroscopic signature l = 505 nm.

A calculation of mechanical properties from images by membrane formalism requires a compression of coherence images for rapid calculation by personal computers. Images as they are used for prediction of storage time were compressed to 128 × 128 pixels. A simulation of firmness properties and internal local stresses from the compressed image leads to 49,152 nonlinear equations and exactly the same number of unknowns. This equation system is solved by efficient algorithms within about 150 seconds, depending on the surface of the tissue (Gründig et al. 2000; Ströbel and Singer 2005). Figure 10.3 represents the ratio of volume to surface of the deformed image membrane for cod. For faster ageing, the sample was stored at fixed temperature 20 °C. All the images were generated from the same position of the same sample in time steps of several hours.

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14 days

0 days

Figure 10.4 Deformation simulation of the assigned membrane for cod, controlled by local stiffness from image. Storage time 0 days and 14 days, storage temperature T = 0 °C, spectroscopic signature l = 505 nm.

Figure 10.5 Visualisation of local firmness for muscle tissue from redfish (Sebastes mentella). Storage time 0 days (left) and 14 days (right). The density of the membrane grid characterizes the internal stress. Increasing storage time causes increasing low-stress areas as an expression for aging.

The simulation of elastic properties enables the calculation of internal stresses in the muscle tissue. Figure 10.4 simulates the indentation for constant virtual forces acting on the plane of an image from cod muscle. Taking into account the topological properties from images in different analysis steps reduces the dependence on the spectroscopic signature. Figure 10.5 visualises the internal stresses for redfish (Sebastes mentella) at wavelength 505 nm for storage times of 0 (left) and 14 days (right). The reduction of grid density shows the loss of firmness within 14 storage days as well as the variation of stresses in the tissue. By introducing a numerical threshold, low-stress areas in tissue can be recognised as a candidate area for gaping.

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10.9

249

Conclusions

An image processing system has to consider the particular aspects of the complex quality concept. For the QIM, the artificial quality index is a link for the integration of artificial sensors into a human sensory system. The linear relationship of image data and storage time is implemented by a combination of structure analysis and statistical methods. Analysis of pattern structure registers the local disorganisation of fibres of muscle tissue and the blurring of skin patterns with storage time. In addition, structure data combined with membrane formalism enable access to local and global physical properties such as firmness. This authorises image processing as a natural candidate for the concept of the artificial quality index. Thus a visual inspection system is capable of investigating particular aspects of quality assessment for fishery products.

10.10

References

Aach, T., Stuke, I., Mota, C. and Barth, E. (2004) Estimation of multiple local orientations in image signals. In Proceedings of ICASSP-2004, Montreal, Volume III, pp. 553–556. Aguilera, J.M. and Stanley, D.W. (1999) Microstructural Principles of Food Processing and Engineering. AN Aspen Publication, Gaithersburg. Andersen, C.M. and Wold, J.P. (2003) Fluorescence of muscle and connective tissue from cod and salmon. Journal of Agricultural and Food Chemistry 51: 470–476. Aubert, G. and Kornprobst, P. (2006). Mathematical Problems in Image Processing, Partial Differential Equations and the Calculus of Variations. Springer, Berlin. Bazen, A.M. and Gerez, S.H. (2000) Directional field computation for fingerprints based on the principal component analysis of local gradients. In: ProRISC 2000, 11th Annual Workshop on Circuits, Systems and Signal Processing, Veldhoven, The Netherlands. Bazen, A.M. and Gerez, S.H. (2002) Thin-plate spline modelling of elastic deformations in fingerprints. In: Proc. 3rd IEEE Benelux Signal Processing Symposium, Leuven, Belgium, March 21–22. Bourne, M. (2002) Food Texture and Viscosity. Academic Press, London. Careche, M., Tryggvadottir, S.V., Herrero, A., Lägel, B., Petermann, U., Schubring, R. and Nesvadba, P. (2003) Instrumental methods for measuring texture of fish. In: J.B. Luten, J. Oehlenschläger, and G. Olafsdottir, (Eds) Quality of Fish from Catch to Consumer. Wageningen Academic Publishers, Wageningen, pp. 189–199. Clucas, I.J. and Ward, A.R. (1996) Post-Harvest Fishery Development: A Guide to Handling, Preservation, Processing and Quality. Natural Resources Institute, Chatham, UK. David, F. (2004) Geometry and field theory of random surfaces and membranes. In: D. Nelson, T. Piran, T. and S. Weinberg (Eds) Statistical Mechanics of Membranes and Surfaces. World Scientific, New Jersey, pp. 149–208. Delbarre-Ladrat, C., Cheret, R. Taylor, R. and Verrez-Bagnis, V. (2006) Trends in postmortem aging in fish: understanding of proteolysis and disorganization of the myofibrillar structure. Critical Reviews in Food Science and Nutrition 46: 409–421. Di Natale, C. (2003) Data fusion in MUSTEC: towards the definition of an artificial quality index. In: J.B. Luten, J. Oehlenschläger, and G. Olafsdottir, (Eds) Quality of Fish from Catch to Consumer. Wageningen Academic Publishers, Wageningen. Dufour, E., Frencia, J.P. and Kane, E. (2003) Development of a rapid method based on front-face fluorescence spectroscopy for the monitoring of fish freshness. Food Research International 36: 415–423.

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Feughelman, M. (1959) A two-phase structure for keratin fibers. Textile Research Journal 29: 223–228. Garboczi, E.J., Bentz, D.P. and Martys, N.S. (1999) Digital images and computer modelling. In: P. Wong, (Ed) Methods in the Physics of Porous Media. Academic Press, San Diego, pp. 1– 41. Girardin, C.C., Kiper, D.C. and Martin, K.A.C. (2002) The effect of moving textures on the responses of cells in the cat’s dorsal lateral geniculate nucleus. European Journal of Neuroscience 16: 2149–2156. Gründig, L., Moncrieff, E., Singer, P. and Ströbel, D. (2000) A history of the principal developments and applications of the force density method in Germany 1970–1999. In: IASS–IACM 2000, Fourth International Colloquium on Computation of Shell & Spatial Structures, June 5–7, Chania, Crete, Greece. http://www.technet-gmbh.com/download/Publikationen/Density2.pdf. Jähne, B. (1997) Digital Image Processing. Springer, Berlin. Jörgensen, B.M., Oehlenschläger, J., Olafsdottir, G., Tryggvadottir, S.V., Careche, M., Heia, K., Nesvadba, P., Nunes, M.L., Poli, B.M., Di Natale, C., Perez-Villareal, B., Ballo, H., Luten, J.B., Smelt, A., Denton, W., Bossier, P., Hattula, T. and Akesson G. (2003) A study of the attitudes of the European fish sector towards quality monitoring and labelling. In: J.B. Luten, J. Oehlenschläger, and G. Olafsdottir, (Eds) Quality of Fish from Catch to Consumer. Wageningen Academic Publishers, Wageningen, pp. 57–74. Johnson, R.A. and Wichern, D.W. (1992) Applied Multivariate Statistical Analysis. Prentice Hall, New Jersey. Kent, M., Knöchel, R., Barr, U.-K., Tejada, M., Nunes, L. and Oehlenschläger, J.(Eds.) (2005). SEQUID – A New Method for Measurement of the Quality of Seafood. Shaker Verlag, Aachen. Kroeger, M. (2004). Structural characterization of fish muscle tissue by image processing. In: H. Rehbein, H. Karl, M. Manthey-Karl, J. Oehlenschläger and R. Schubring (Eds) Proceedings of the WEFTA Conference, Hamburg. Federal Research Centre for Nutrition and Food, Department for Fish Quality, Hamburg, pp. 192–195. Landau, L.D. and Lifschitz, E.M. (1975). Lehrbuch der Theoretischen Physik, Volume VII. Akademie Verlag. Lipowsky, R. (1991). The conformation of membranes. Nature 349: 457. Luten, J.B., Oehlenschläger, J. and Olafsdottir, G. (Eds) 2003. Quality of Fish from Catch to Consumer. Wageningen Academic Publishers, Wageningen. Mallot, H.A. (1998) Sehen und Verarbeitung visueller Information. Vieweg, Braunschweig. Martens, H. and Naes, T. (1989) Multivariate Calibration. John Wiley, Chichester. Nesvadba, P. (2002) Quality control by instrumental texture measurements. In: C. Alasalvarand T. Taylor (Eds) Seafoods, Quality, Technology and Nutraceutical Applications. Springer, New York. Pau, L.F. and Olafsson, R. (1991) Fish Quality by Computer Vision. Marcel Dekker Inc., New York. Olafsdottir, G., Nesvadba, P., Di Natale, C., Careche, M. Oehlenschläger, J., Tryggvadottir, S., Schubring, R., Kroeger, M., Heia, K., Esaiassen, M., Macagnano, A. and Joergensen, B. (2004) Multisensor for fish quality determination. Food Science and Technology 15: 86–93. Olzak, L.A. and Thomas, J.P. (1986) Seeing spatial patterns. In: K.R. Boff, L. Kaufmann and J.P. Thomas (Eds) Handbook of Perception and Human Performance, Volume I (Sensory Processes and Perception). John Wiley, Chichester. Russ, C.R. (2004) Image Analysis of Food Microstructure. CRC Press, Boca Raton. Sornette, D. and Ostrowsky, N. (1994). Lamellar phases: effect of fluctuations (theory). In: by W.M. Gelbart, A. Ben-Shaul and D. Roux (Eds) Micelles, Membranes, Microemulsions, and Monolayers. Springer, Berlin, p. 251.

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Ströbel, D. and Singer, P. (2005) Computational modelling of lightweight structures; formfinding, load analysis and cutting pattern generation. Paper presented at Textile Roofs 2005, Tenth International Workshop on the Design and Practical Realisation of Architectural Membrane Structures, May 26–28, 2005, Berlin, Germany. http://www.technet-gmbh.com/download/Publikationen/ Computational_Modelling.pdf. Zheng, C., Sun, D-W. and Zheng, L. (2006) Recent applications of image texture for evaluation of food qualities – a review. Trends in Food Science and Technology 17: 113–128.

Chapter 11

Nuclear magnetic resonance Marit Aursand, Emil Veliyulin, Inger B. Standal, Eva Falch, Ida G. Aursand and Ulf Erikson

11.1

Introduction

When a sample containing nuclei with a non-zero spin is placed in a constant magnetic field    B 0, all the spins will start precessing around its direction with a Larmor frequency ω 0 = g B 0, where g is a nucleus-specific constant  called the gyromagnetic ratio. The nuclear spins can only align either along the field B 0 or in the opposite direction. Populations of the corresponding energy levels will be slightly different, and a small but detectable net magnetiza tion along B 0 will be created. The frequency of the nuclear precessing ω 0 is typically in the radio frequency (RF) range (megahertz). Therefore, using an RF coil operating at exactly  the same frequency ω 0, one can transfer energy of the external RF radiation to the nuclei causing a resonant absorption. This phenomenon is called nuclear magnetic resonance (NMR). Having absorbed the extra energy, the nuclear system will no longer be in the energetic equilibrium and will re-emit RF radiation and return to the lower-energy state. This process is called spin-lattice relaxation and is characterized by a time constant, T1. Spins taken out of the equilibrium at a certain time moment by an RF pulse will first precess coherently and there will be a non-zero component of the net magnetization in the plane transversal to B 0. This oscillating at the resonance frequency magnetic field will induce current in the same RF coil, which can be detected as an NMR signal. Small differences in the precessing frequency in the ensemble of nuclei will cause loss of the transversal coherence, a process called spin-spin relaxation with its characteristic time T2. Both T1 and T2 relaxation times can be related to several physical properties of the sample, which makes a basis for NMR relaxometry. An excellent introduction to the basics of NMR as well as T1 and T2 relaxation mechanisms can be found in Farrar and Becker (1971). In every NMR experiment, the local magnetic field exerted on each nucleus will always be slightly perturbed owing to the local variations in the molecular environment, which will affect its exact resonance frequency. This dependence of the resonance frequency on the position of a particular atom in a molecule is the basis of NMR spectroscopy, which is an extremely powerful technique for determining the structure of molecules. A good introduction to NMR spectroscopy was written by Farrar (1987), and a comprehensive account of most aspects of the NMR technique was given by Abragam (1961). In both fisheries and aquaculture, freshness and high product quality are important issues. Furthermore, because marine raw materials contain components with great dietary benefits 252

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for human health, they should be exploited. One of the most important challenges for the related industry is to be able to maintain quality through the whole value chain. It is necessary to develop basic knowledge about the product composition, degradation processes, effect of processing on product quality and shelf life. A considerable amount of research is being done in this field, where both traditional and more sophisticated techniques are in use. Modern NMR techniques are of interest partly because they open up possibilities to study foodstuffs non-destructively and non-invasively. NMR has eventually become an indispensable analytical technique in medicine, chemistry, physics, biology and food science. NMR-based methods have several inherent advantages. The NMR instrument communicates with the investigated object by electromagnetic waves in the radiofrequency range. This makes most NMR techniques non-invasive and nondestructive for the sample, rapid, not harmful for the operator and non-polluting for the environment. The applications of NMR methods in food research may be divided into three main groups according to the type of equipment used, as they can provide versatile information about the chemical composition and structure of biological systems at various levels. These are: magnetic resonance imaging (MRI), low-field (LF) NMR, and high-resolution NMR (HR NMR).

11.2

Magnetic resonance imaging

MRI is a technique that offers a unique opportunity to produce cross-sectional images of intact whole fish. Many different types of contrast in MR images can be achieved by applying specific MR pulse sequences. Depending on the experimental requirements, the NMR response of the nuclei localized in molecules with different mobility, relaxation times or chemical environments can be differentiated. Because MRI is non-destructive for the sample, it is also a powerful tool to follow various dynamic processes in time. Thus, MRI can be used in fish processing as a research tool for the optimization of various unit operations such as drying, freezing, thawing, rehydration and salting. In aquaculture, MRI can be useful to study various issues related to feed composition and flesh quality.

11.2.1

1

H NMR imaging of fish

1

H MRI can be used in several ways to obtain useful information about different seafoods. For instance, it is possible to produce ‘diffusion weighted’ MR images showing only molecules with low mobility (Mulkern et al. 1988) or high-resolution images of connective tissue (Bonny et al. 2001). MRI methods based on double-quantum filtering can suppress the signal from isotropic fluids and only detect molecules associated with ordered tissue structures (Tsoref et al. 1998). Spatial distribution and quantification of fat and water Distribution of fat in fish has been studied by Collewet et al. (2001), who showed that higher contrast between muscle and adipose tissues can be obtained by strong longitudinal

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(a)

(b)

(c)

Figure 11.1 Three types of MR image of a salmon fillet piece: (a) ‘proton density’ image, (b) ‘fat image’ and (c) ‘water image’. Two glass tubes filled with MnCl2-doped distilled water visible in (c) and 100% fish oil visible in (b) were used as references.

relaxation time (T1) weighing. Although this way of suppressing the water signal is sufficient for visualization purposes, more sophisticated MRI techniques must be used for full water or fat suppression, enabling quantification of the relevant component. Differences in fat distribution in the flesh of brown trout as affected by a low- or a high-energy diet have been demonstrated by using MRI (Toussaint et al. 2005). Using chemical shift selective excitation, Veliyulin et al. (2005a) were able to produce separate fat and water images of salmon fillet pieces (Figure 11.1) for accurate quantification of fat and water contents. Tingle et al. (1995) have written an interesting overview of different fat and water selective MRI techniques. Assessment of fish quality changes after freezing and thawing As an NMR signal is generally sensitive to water mobility and its interaction with other molecules, the technique is suitable to study changes in flesh texture as affected by freezing and thawing. Freezing and thawing of a lean fish species (cod) and a fatty species (mackerel) have been studied by Nott et al. (1999a). They showed that the effects of frozen storage in fish could be assessed by the relaxation time T1sat (measured during saturation transfer) and magnetization transfer rate. Although T1sat decreased as a result of the freezing-thawing cycle, compared with fresh fish, the magnetization transfer rate and T1 increased in both lean and fatty species. Increasing frozen storage time (2–12 weeks) resulted in the same effect. The same authors (Nott et al. 1999b) have also performed MRI studies on fresh and frozen– thawed trout where the effects of freezing method, repeated freeze–thawing and storage time on the MR detectable parameters were investigated. Howell et al. (1996) studied partly frozen fish and demonstrated that MRI can be used to show the presence of frozen and unfrozen zones. Using a cylindrical sample carrier in the MRI magnet, Foucat et al. (2001) simultaneously investigated fresh and frozen/thawed trout with increasing frozen storage times from 1 to 41 days. The combined MRI protocol contained information about magnetization transfer ratio, relaxation time (T2) and diffusion constants parallel (D||) and perpendicular (D⬜) to the

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muscle fiber orientation. Compared with fresh fish, the freezing–thawing cycle significantly affected the mean T2 and the D⬜ values only, whereas only D⬜ was affected after frozen storage for 41 days. The results were explained by partial protein denaturation due to the freezing–thawing cycle and appearance of the extracellular spaces between fibers. In addition, loss of the diffusion anisotropy (D|| ≈ D⬜) as a result of a great disorder of the fiber arrangement was observed after 41 days of frozen storage. In another MRI study of freezing of cod fillet pieces, Veliyulin et al. (2006a) determined T1 and T2 relaxation times and spin densities at several predefined constant temperatures between +10 °C and −10 °C. The fraction of unfrozen water in the fish sample was calculated as a function of temperature. It was shown that below the freezing point, the T1, T2 and unfrozen water fraction decreased rapidly (approximately four to five times) with the temperature in the range from 0 °C down to about −4 °C. Anatomical studies Blackband and Stoskopf (1990) first demonstrated the feasibility of using MRI for anatomical studies of aquatic organisms. A similar technique has been applied by Bock et al. (2002) to study non-anesthetized marine fish in vivo by MRI using a flow-through animal chamber. The authors could clearly distinguish between different organs and anatomical structures in fish. Detection of the backbone deformations in farmed salmon using MRI has been demonstrated by Veliyulin et al. (2006a). Backbone deformities were clearly seen in MR images revealing deformed or partly missing vertebrae. Another study (E. Veliyulin, unpublished results) suggested that MRI could be used for reliable detection of the skeletal deformities in salmon fry (weight 20 g), i.e. before the deformities were visible externally. MRI was also used to monitor anatomical changes occurring as a result of belly bursting in herring (Veliyulin et al. 2007a). From high-resolution images of the belly region, it was found that neither the stomach, nor zooplankton in the stomach, are sources of enzymes responsible for belly bursting in this species. Water dynamics in post mortem white rainbow trout muscle was studied by MRI and other methods to investigate ‘the soft flesh problem’, a phenomenon occurring apparently at random in farmed fish (Foucat et al. 2004). 23

Na imaging of fish: salt distribution and quantification challenges

Curing is one of the oldest methods of muscle-based food preservation. Addition of salt is also used for the preservation of smoked fish and a final salt concentration around 3–4% largely inhibits the activity of microorganisms. The preservation effect depends on the salt reaching all parts of the product and monitoring of salt distribution in a product is therefore of interest. At the same time, it is known that excessive use of salt has negative health effects. Therefore, cured foods should ideally have homogeneously distributed salt at the lowest possible concentration for effective inhibition. Measuring salt contents with traditional chemical methods (Association of Official Analytical Chemists 1990) may be a tedious and time-consuming procedure, and the development of rapid and non-destructive methods to measure salt contents is important. Direct imaging of 23Na nuclei using MRI is an efficient way to visualize salt distribution in cured foods. Figure 11.2 demonstrates a sodium MR

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(a)

(b)

Figure 11.2 Sodium MRI image of a salted salmon fillet piece (a) with the average salt content of 1.5% and a corresponding 1H image (b) of exactly the same slice. The 23Na image clearly shows the salt distribution in the sample and greatly reduced salt penetration in areas with high lipid content.

image of a salted salmon fillet piece (average salt content 1.5%) and a corresponding 1H image of exactly the same slice. The 23Na image clearly shows the salt distribution as well as a lower salt content in areas with high lipid content (myosepta: bright areas in the 1H image). Gallart-Jornet et al. (2006) compared brine salting of lean cod and fatty salmon fillets (skin on) at two different brine concentrations. The 23Na MR images revealed that salt diffusion took place only from the flesh side of the salmon fillets, whereas the cod fillets were salted from both sides. This difference was explained by the subcutaneous fat layer of salmon fillets, which acted as a barrier against salt diffusion whereas the skin did not seem to impair salt diffusion. Quantification of the sodium content in muscle foods by MRI is, however, not trivial because of the so-called partial NMR ‘invisibility’ of sodium nuclei. This phenomenon arises from the fact that the 23Na nucleus has an electric quadrupolar moment, which may result in quadrupolar splitting in the presence of local field gradients typical for muscle tissues. An explanation of the partial 23Na NMR ‘invisibility’ was given by Springer (1987), who presented both theoretical and experimental evidence of this phenomenon, resulting in 40% sodium visibility at low physiological salt concentrations. At considerably higher salt concentrations (5–25%), a salt visibility ranging from 21 to 96% has been reported in homogeneously salted cod and salmon fillet pieces when using 23Na MRI-based spin-echo detection (Veliyulin et al. 2007b), demonstrating that the MRI sodium visibility depends on the brine concentration and muscle composition. On the other hand, 100% sodium visibility has been reported in salted cod fillet pieces using 23Na NMR relaxometry at a sodium nucleus frequency of 25 MHz (Erikson et al. 2004). A better understanding of the partial sodium NMR ‘invisibility’ phenomenon might open up possibilities to account for the ‘lost’ part of the sodium signal, and thus make it possible to measure salt contents in foods accurately. An advanced MRI SPRITE technique (Balcom et al. 1996) based on a pure phase encoding and detection of the free-induction decay made it possible to obtain quantitative density

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257

profiles of materials with very short relaxation times (Deka et al. 2006). Using this approach, Romanzetti et al. (2006) obtained sodium three-dimensional images of a human brain, demonstrating its potential for quantitative 23Na MRI of various tissues.

11.3

Low-field NMR

Low-field (LF) NMR (often also called time-domain NMR) is a fast and powerful method that can be used for non-invasive measurements of mobility of water and fat in different tissues or compartments in muscle-based foods. The main advantages of the LF NMR method compared with the other NMR techniques are low investment costs, small size, no maintenance costs (permanent magnet), high stability, high degree of automation and easy operation. LF NMR can be implemented at-line under comparatively harsh industrial conditions. The structure of muscles can be studied indirectly using LF 1H NMR. Different tissue compartments, or muscle structures, as affected by fish processing, can be studied because protons in different environments exhibit different T1 and T2 relaxation properties. The relaxation data may be interpreted in terms of structural changes occurring after fish processing operations.

11.3.1

Low-field NMR instruments

LF NMR instruments are based on use of permanent magnets covering a range of proton resonance frequencies from about 2 to 60 MHz. Magnet bore openings of typical LF NMR equipment range from 5 to 52 mm in diameter. An inside-out NMR instrument – the minispec Bruker ProFiler® (Bruker Optik GmbH, Rheinstetten, Germany) – has been developed and commercialized to circumvent the restriction of the sample size. Bruker ProFiler® is an LF mobile NMR analyzer for near-surface volume measurements of samples unrestricted in size. Design of the mobile NMR analyzer and the principles of operation have been described by Eidmann et al. (1996).

11.3.2

Data processing and applications

There are several approaches to processing the NMR relaxation data to extract information that can be related to the muscle structure. The simplest model is to assume a fixed number of proton pools in the sample (typically two or three), each of them having a characteristic relaxation component and a corresponding exponential decay. Converting NMR relaxation data into a distribution of relaxation times is another widely used approach. One of the most common algorithms for doing this was originally implemented by Provencher (1982). The algorithm is called CONTIN and is based on the inverse Laplace transformation of the relaxation time curves. A more sophisticated two-dimensional version of the inverse Laplace transform (Song 2002; Callaghan 2003) has been applied by Veliyulin et al. (2005a) to a set of diffusion weighted NMR relaxation data from fresh and frozen–thawed salmon to produce two-dimensional relaxation time versus diffusion

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Figure 11.3 (a) LF NMR T2 relaxation curve measured by CPMG pulse sequence; (b) PCA score plot of T2 relaxation raw data obtained from salmon (S) and cod (C) fillets salted in 15 and 25% (w/w) brines (S15, C15, S25 and C25, respectively) for 14 days; samples were taken along the salting period (unpublished data).

coefficient maps. It has been shown that resolving conventional T2 distribution curves in the second dimension of diffusion can separate overlapping fat and water relaxation pools and give a better understanding of structural changes in fish tissues. Multivariate data analysis is yet another approach to processing the data. This technique has the advantages of being faster and more robust than the exponential fitting procedures, as well as being able to facilitate detection of outliers (relaxation and/or sample abnormalities).

11.3.3 Fish muscle quality and composition Fat and water contents as well as the water holding capacity of intact fish muscle have been determined by Jepsen et al. (1999) using multivariate data analysis applied to the transversal relaxation data. The effects of frozen storage on cod have been studied by exponential curve fitting (Lambelet et al. 1995) and the effect of both chill and frozen storage were studied by using multivariate three-way modeling of NMR relaxation profiles (Jensen et al. 2002). Other functional properties of fish flesh, i.e. texture changes in frozen cod (Steen and Lambelet 1997), distribution of water in cod (Andersen and Rinnan 2002), herring (Jensen et al. 2005), as well as in minced cod (Lillford et al. 1980) have also been investigated by the use of LF NMR. Moreover, the technique has been used to study the water-holding capacity (Andersen and Jørgensen 2004) and salting of cod (Erikson et al. 2004; Aursand I.G. et al. 2008) and salmon (Aursand M. et al. 2006; Aursand I.G. et al. 2008). Figure 11.3 shows an example of multivariate data analysis (principal component analysis) (b) applied to T2 relaxation raw data (a). It can be seen that the water mobility changes during the salting process (0, 4, 20, 25, 44, 68, 140, 188 and 332 h salting time), and that clusters occur related both to fish species and brine concentration. Furthermore, it has been demonstrated that LF NMR is well suited for rapid quantification of lipids in Atlantic salmon (Toussaint et al. 2001; Aursand, I.G. et al. 2006) and herring (Nielsen et al. 2005). Using the mobile NMR analyzer, truly non-destructive measurements of the fat content in live salmon have been demonstrated by Veliyulin et al. (2005b).

Nuclear magnetic resonance

11.3.4

259

Feed composition, at-line method

From a combined LF NMR technique applied to fish feed and its raw ingredients, it has been demonstrated that rapid simultaneous quantification of solid protein, fat and moisture is possible (Veliyulin et al. 2006b). The technique is based on a combination of the ‘solid-echo’ (Slichter 1990), ‘free induction decay’ (Farrar et al. 1971) and ‘Hahn echo’ (Hahn 1950) NMR pulse sequences.

11.4

High-resolution NMR

HR NMR has been shown to be particularly well-suited in the profiling of biological material (Nicholson et al. 1995; Fan 1996), and it has emerged as a popular technique for the analysis of foodstuff including fish and fish products (Table 11.1). HR NMR gives a ‘fingerprint’ of the composition of the sample analyzed and allows identification/quantification of metabolites with relevance for the quality and nutritional value. The most common magnetic nuclei for analysis of foods are 1H, 13C and 31P. Nuclei in different chemical environments have slightly different resonance frequencies. The resonances obtained from NMR are expressed as chemical shift values (δ) in units of parts per million (p.p.m.) relative to a reference compound (tetramethylsilane; dTMS = 0.00 p.p.m.) for 1 H. NMR spectra are presented as signals at different chemical shift values and are dependent on the molecular structure (Hunter et al. 2005), type of solvent and its concentration (Gunstone 2004) and pH (Fan 1996). NMR in chemistry has traditionally been limited to analysis of liquid samples. High-resolution magic angle spinning (MAS) NMR is a strong tool for studying heterogeneous systems (Bollard et al. 2000; Rooney et al. 2003) and it enables studies of a wide range of chemical components in intact materials.

11.4.1

Chemical composition and deterioration

During the past decade, HR NMR has become an important tool in quality assessment of fish and fish products in research. It has been found valuable particularly for studies of marine lipids because the pure extracts can be analyzed non-destructively and noninvasively. D-chloroform, which is the most commonly used solvent (Gunstone 2004), is easily evaporated and the sample may therefore be subjected to further analysis. The sample size of a routine lipid NMR analysis is 50–100 mg, but by increasing the number of scans (longer sampling time), it possible to decrease the sample amount. Table 11.1 gives an overview of HR NMR used in connection with compositional studies of fish and fish products. Lipids Lipid extracts have been previously analyzed by NMR to determine lipid class composition, i.e. triacylglycerols, phospholipids (including individual phospholipids such as phosphatidylcholine, phosphatidylethanolamine, and so on), cholesterol and free fatty acids.

Table 11.1 Applications of HR NMR in studying the composition of fish and fish products. Sample material

Intact/extract

References

13

C

Fish oil

Lipid extracts

Fatty acids and acylstereospecific positions of fatty acids in phospholipid molecules Methylesters Ethylesters Plasmalogen (alk-1-enylphosphatidylethanolamine) Individual phospholipids (phosphatidylcholine and phosphatidylethanolamine) Cholesterol

13

C

Cod roe and milt Tuna

Lipid extracts Lipid extracts

Tuna and reference standards Commercial fish oil Different fish samples

Lipid extracts Lipid extracts

Sacchi et al. 1994 Siddiqui et al. 2003 Sacchi et al. 1995

Glycerol-, methyl- and wax esters, acids, alcohols, nitriles, amides and acetates Cis/trans ratio of fatty acids Mobility of fatty acids at different freezing temperatures Quantification of n − 3 and DHA

13 1

C H, 13C

13c

Aursand and Grasdalen 1992; Sacchi et al. 1994; Aursand et al. 1995, 1997; Siddiqui et al. 2003; Broadhurst et al. 2004 Falch et al. 2006 Medina et al. 1994a

13

C

Cod roe and milt

Lipid extracts

Falch et al. 2006

13

C

Lipid extracts

13

C

Cod roe and milt Commercial n − 3 concentrates Different sample material

Lipid extracts

Falch et al. 2006 Siddiqui et al. 2003 Gunstone 1993

13

C C

Salmon and trout Salmon muscle

Lipid extracts Lipids

Aursand et al. 1997 Grasdalen et al. 1995

H H

Fish lipids Cod roe and milt

Lipid extracts Lipid extracts

H

Salmon muscle

Intact muscle

Cod roe and milt Tuna Canned tuna Cod roe and milt DHA (ethyldocosahexaenoate)

Lipid Lipid Lipid Lipid Lipid

Sacchi et al. 1993; Igaraschi et al. 2000, 2002 Falch et al. 2007 Aursand M. et al. 2006a; Gribbestad et al. 2005 Falch et al. 2007 Sacchi et al. 1993; Medina et al. 1994b, 1995 Falch et al. 2005, 2007 Falch et al. 2004, 2007

13

1 1

1

Cholesterol/cholesteryl esters Free fatty acids during lipolysis of fish

13

Specific oxidation products (aldehydes, ketones, oxygene bridges, hydroperoxides)

1

13

C C

H

extracts extracts extracts extracts extracts

Fishery Products: Quality, safety and authenticity

Chemical composition and deterioration Fatty acids and acylstereospecific positions of fatty acids in the triacylglycerol molecule

NMR method

260

Applications

Lipid oxidation (changes in proton ratios)

1

Changes in lipids during cooking

13

Authentication/origin testing of fish Characterization of fish oils and lipids Authentication of fish oil capsules

13 C SNIF-NMR

Multicomponent analysis of encapsulated marine oil supplements Changes in fish muscle during frozen storage

1

H and

1

Quality evaluation during ice storage Quality assessment and compositional analyses

Atlantic mackerel Different fish species Wild and farmed salmon Wild and farmed salmon, various fish oil Commercial marine oil supplements Commercial marine oil supplements

Lipid Lipid Lipid Lipid

H, MRI

Cod and haddock

1

H

Atlantic halibut

1

H, MRI

Atlantic salmon

Water- and saltsoluble extracts Perchrolic acid extract Whole fish, intact muscle, lipidand perchloric acid extracts Perchloric acid extracts

C

13

C 13

C

Cod

Cod and haddock

extracts extracts extracts extracts

Saito and Udagawa 1992a, b Saito 1987; Saito and Nakamura 1990, 1997 Saeed and Howell 1999 Aursand and Axelson 2001 Aursand, et al. 2000 Aursand, et al. 2006b Siddiquie et al. 2004

Howell and Shavila 1996

Sitter et al. 1999 Gribbestad et al. 2005

Martinez et al. 2005

13

Atlantic salmon

Perchloric acid extracts Perchloric acid extracts Minced muscle

31

Cod

Intact muscle

Jørgensen and Grasdalen 1986

31

Carp muscle

Intact muscle

Yokoyama et al. 1996

31

Oyster tissue

Oyster tissue

Yokoyama et al. 1996

1

H C P P P

Mussels

Standal et al. 2006 De Vooys and Geenevasen 2002 Aursand et al. 1995

261

Quantification of anserine and lactate Studies of phosphate metabolites in fish muscles Post mortem changes in carp muscle Post mortem changes in oyster

Lipid extracts Lipid extracts

Nuclear magnetic resonance

Changes in bioactive components following freezing, thawing, cooking and salting of cod. Assignments of low molecular mass metabolites in fish muscle Quantification of betaine in mussels

Fish meal Fish oil

H

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Also, specific esterified fatty acids and their individual acyl-positions of the fatty acids in lipid molecules have been interpreted (Falch et al. 2006). 1 H NMR has been developed to determine the concentration (milligrams per gram or molar per cent) of docosahexaenoate (DHA) and n − 3 fatty acids in fish oils/lipids (Igarashi et al. 2002; Sacchi et al. 2006). The DHA and n − 3 fatty-acid contents can also be quantified directly from intact material (for example fish tissues) by using the MAS technique (Aursand, M. et al. 2007). Another possible application of 31P NMR is to quantify phospholipids in intact tissues such as fish eggs (Grasdalen and Jørgensen 1987). Schiller and Arnold (2002) have reviewed the applications of 31P NMR to characterize the phospholipid composition of tissues and body fluids. Lipid oxidation and degradation Fish products are susceptible to degradation processes because of their high enzymatic, microbial and oxidative activities. These changes might affect the quality by reducing the nutritional value or organoleptic properties as well as the safety of the products. Marine lipids are degraded primarily by enzymatic activity and lipid oxidation. One of the important enzymatic changes in marine lipids is due to the action of lipases and phospholipases causing hydrolysis of acylglycerols into free fatty acids and related compounds. These changes are observable non-destructively by HR NMR (Falch et al. 2005; Falch et al. 2007). Other enzymatic changes such as esterification of cholesterol have also been reported during storage of fish raw materials (Falch et al. 2005, 2007). Lipid oxidation products are other important changes affecting the organoleptic properties of marine products. Among the NMR techniques, 1H has been most widely used to study lipid oxidation. Specific oxidation products such as different aldehydes, hydroperoxides and ketones produce unique resonances in the spectra. They have been previously reported in various studies of vegetable oils. Some of these reaction products have also been reported in studies of DHA, a characteristic fatty acid in fish and fish products (Falch et al. 2004; Zamora et al. 2006). The changes in these reaction products correlated well with the peroxide value and thiobarbutur acid reactive substances. Hydroperoxides from polyunsaturated fatty acids are easily decomposed into a complex set of secondary products, leading to a decrease of olefinic protons (Saito 1987). Differences in the ratio of different protons (olefinic-, aliphatic- and diallylmethylene protons) due to lipid oxidation have been demonstrated in 1H NMR spectra during storage of dried fish, fish meal and fish oils (Table 11.1). Multivariate data analysis of specific parts of the NMR spectra has great potential in studies of lipid oxidation. Contents of low molecular mass metabolites The contents of low molecular mass metabolites in muscle (such as phosphocreatine, nucleotides, taurine, betaine, anserine, TMAO, TMA, DMA) are also of relevance to the quality and nutritional value of fish products. The type and amount of low molecular mass metabolites in fish muscle is affected by physiological factors and ante mortem stress, post mortem storage and fish processing conditions.

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263

Several authors have used 1H NMR to study low molecular mass metabolites in fish muscle. Detailed peak assignments of the 1H NMR spectra from extracts of Atlantic salmon (Gribbestad et al. 2005) and cod and haddock (Standal et al. 2006) have been performed. The technique has been used to study changes in fish flesh during freezing (Howell et al. 1996; Sitter et al. 1999; Martinez et al. 2005), thawing, cooking and salting (Martinez et al. 2005). 1H NMR has also been applied to quantify betaine in mussels (de Vooys and Geenevasen 2002) and cod (Martinez et al. 2005). Intact fish tissues can also be analyzed. Aursand et al. (1995) analyzed minced muscle from Atlantic salmon, without any additional pre-treatment, by 13C NMR spectroscopy, whereas Gribbestad et al. (2005) demonstrated that HR MAS spectroscopy can identify single chemical components (such as hypoxanthine, anserine, and so on) in salmon muscle non-destructively. By using 31P NMR, phosphocreatine, inorganic phosphate, ATP, ADP, AMP/IMP and intracellular pH can be determined in perchloric extracts or intact tissues. The method is basically confined to more physiologically oriented in vivo studies where the effects of various stressors on muscle activity are assessed. However, 31P NMR has been suggested as a method for studying fish (Jørgensen and Grasdalen 1986; Chiba et al. 1991; Yokoyama et al. 1996a) or oyster (Yokoyama et al. 1996b) quality during the early post mortem phase.

11.4.2

Authentication of fish and fish products by NMR

At present, there is no reliable method either to distinguish wild from farmed fish, or to verify geographical origin. Reliable authentication methods are important to secure consumers’ rights for having the correct information, to discourage commercial fraud and to prevent illegal capture of protected stocks. As described elsewhere in this book (Chapter 18), analysis of lipids and stable isotope distributions provides information about both, species and breeding stock, as well as wild/ farmed- and geographic origins. NMR techniques give valuable information about the lipid profiles (13C NMR, 1H NMR) and stable isotope distributions (2H SNIF-NMR). (For a discussion of the 2H SNIF-NMR method see Chapter 10.) Lipid profiles: wild/farmed and geographical origin classifications Since September 2001, a European consortium of partners from France, Italy, the UK and Norway has been working on the development of a validated procedure for the authentication of the origin of salmon. The suitability of the NMR techniques 1H NMR, 2H NMR and 13C NMR (in addition to other analytical methods) has been tested. 1 H NMR data in combination with super vector machines as a new classification algorithm was able to distinguish correctly between wild and farmed salmon (Masoum et al. 2007). 13C NMR combined with the multivariate technique probabilistic neural network enabled the identification of farmed and wild Atlantic salmon and classification according to geographic origin. Products that had been inappropriately labeled could readily be identified (unpublished results). Preliminary studies on cod liver oils show that 13C NMR in

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combination with multivariate techniques is able to discriminate cod liver oil of wild and farmed origin. In addition, the NMR data enabled differentiation between cod of different geographical origins (Norway and Scotland). In these studies, 13C NMR appears to be a superior technique compared with gas chromatography for classification purposes. The reason for this is most likely that 13C NMR gives a lipid profile, which, in addition to the fatty-acid composition (Aursand and Grasdalen 1992), contains further information about, for example, lipid classes (Gunstone 1991; Falch et al. 2006) and positional distribution of fatty acids in triacylglycerols (Aursand et al. 1995).

Fish oil capsules to improve health For marine oils, there is a need for methods to verify composition and authenticity. Recently, 13C and 1H NMR spectroscopy have been used to detect and identify a wide range of compounds in encapsulated cod liver oil (Siddiqui et al. 2003). Natural sources of cod liver oils could be distinguished from those subjected to chemical modifications (Siddiqui et al. 2003). 13C NMR spectroscopy, in conjunction with multivariate analysis of commercial fish-oil-related health food products, has been used to discriminate the nature, composition, refinement and adulteration or authentication of the products (Aursand, M. et al. 2007). Quantitative class predictions with accuracies greater than 95% were achieved with probabilistic neural network analysis. Trout, salmon and cod oils were completely and correctly classified. Samples reported to be salmon oils and cod liver oils did not cluster with true salmon and cod liver oil samples, indicating mislabeling or adulteration.

Fatty-acid positional distribution for authentication The fatty-acid composition of fish is a net result of a wide range of factors (Tocher 2003), including dietary fatty-acid intake, season, age, size, stage of sexual maturity, genetic and environmental factors (Tocher and Sargent 1990; Hemre 2004). However, the positional distribution of the fatty acids in triacylglycerols seems to be species specific (Aursand et al. 1995) and not so much influenced by dietary factors. 13C NMR is a unique technique that opens up the possibility of studying the positional distribution of fatty acids in intact triacylglycerols non-invasively. Studies show that lipids from the muscle of salmon, herring and mackerel could be distinguished by the positional distribution of 22 : 6 n − 3, 20 : 5 n − 3 and saturated fatty acids in triacylglycerols (our unpublished results). The fatty-acid composition of phospholipids in fish muscle is less influenced by the diet than the composition of triacylglycerols (Dos Santos et al. 1993, Pickova et al. 1997). As reported elsewhere in this book (‘Authenticity assessment based on other principles’), fattyacid analysis of phospholipids has been used to study different fish species and stocks (Joensen et al. 2001, Grahl-Nielsen 2004). In addition to the fatty-acid profile, 13C NMR provides information about the positional distribution of fatty acids in phospholipids (that is, the distribution of 22 : 6 n − 3, sn1 versus sn2 position) in phosphatidylcholine and phosphatidylethanolamine (Falch et al. 2006). Chemometric treatment of 13C NMR data of phospholipids may therefore be a powerful tool for studying differences among fish species and populations.

Nuclear magnetic resonance

11.5

265

The future of NMR in seafood

Today, both fisheries and aquaculture are focusing on freshness and high product quality, in addition to the fact that marine raw materials should contain components with great dietary benefits for human health. One of the most important challenges for the industry is to be able to maintain the quality through the whole value chain. It is necessary to obtain basic knowledge about the product composition, degradation processes, and effects of processing and product quality preservation. There are many examples of ongoing research in this field, where both traditional and sophisticated invasive techniques are in use. However, modern NMR opens up possibilities for studying foodstuffs non-destructively and non-invasively. LF NMR is proving to be indispensable for rapid processing and quality control. This method has been implemented at-line in fish-feed production plants. Structural changes in the fish muscle, water holding capacity and its distribution within the muscle can be assessed by LF NMR. Use of advanced multivariate methods for extracting the information from the NMR relaxation signals will be important in the further development of new applications. LF NMR has great potential as a rapid and non-invasive method for routine measuring of important quality attributes of seafood. Unilateral LF NMR circumvents the restrictions of the sample size and offers a truly non-destructive analytical approach. HR NMR has been demonstrated to be a unique tool in marine lipid research, where many components can be studied simultaneously in a relatively short time without any extraction involved, producing a total picture of the structure. It is possible to quantify lipid classes, fatty-acid composition, and to study the positional distribution of the fatty acid in the triacylglycerol molecule as well as lipolysis and lipid oxidation in the same sample. Knowledge of the chromatographic concentration of the minor or polar components improves the information from NMR. Additionally, this technique will be of importance in developing databases that would include ‘fingerprint’ information about the composition of seafood products for verification of traceability. Highresolution MAS NMR can be used to study intact muscle. NMR on intact cells or muscle opens up possibilities to have information on degradation processes or biochemical processes related to biosynthesis from the same sample with only one simple preparation. This method will allow unique information and new knowledge to be obtained about the influence of processing and storage on seafood quality. MRI as a research tool in food science is unique. Owing to high investment costs, the size of the instrument and infrastructure needed, it cannot currently be considered as a standard analytical tool in aquaculture or the fish processing industry. However, as a research tool, taking advantage of the unique features of the method, we can obtain a basic insight into several issues related to anatomical studies, composition and structure of tissues, distribution of fat, water and salt as well as temperature profiles. Combined with theoretical transport models, MRI provides experimental data for quantifying transport phenomena such as salting, drying and freezing. For the fish industry, MRI studies are valuable for studying the effects of feeding regimes during on-growth phase and for optimizing unit operations in fish processing.

Acknowledgments Some of the presented results, as well as the preparation of the manuscript, were partly financially supported by the Research Council of Norway through the Matforsk-SINTEF

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Strategic Institute Programme ‘Production improvements of salted/cured meat and fish: Development of rapid and non-destructive salt analyses related to production, yield and quality (Project no. 153381/140)’, ‘Use of NMR spectroscopy in combination with pattern recognition techniques for elucidation of origin and adulteration of foodstuff’ (Project 146932/130) and ‘Conformation of the species, origin, processing and nutritious values of fishery products’ (Project 154137/130).

11.6

References

Abragam, A. (1961) Principles of Nuclear Magnetism. Oxford University Press, New York. Andersen C.M. and Rinnan Å. (2002) Distribution of water in fresh cod. Lebensmittel-Wissenschaft und -Technologie 35: 687–696. Andersen C.M. and Jørgensen B.M. (2004) On the relation between water pools and water holding capacity in cod muscle. Journal of Aquatic Food Product Technology 13: 13–23. Association of Official Analytical Chemists (1990) Official Methods of Analysis, Volume 2, 15th edition. Association of Official Analytical Chemists, Arlington, VA, pp. 810. Aursand, I.G., Veliyulin, E. and Erikson, U. (2006) Low field NMR studies of Atlantic Salmon (Salmo salar). In: G. Webb (Ed.) Modern Magnetic Resonance. Springer, The Netherlands, pp. 895–903. Aursand, I.G., Gallart-Jornet, L., Erikson, U., Axelson D.E. and Rustad, T. (2008) Water distribution in brine salted cod (Gadus morhua) and salmon (Salmo salar): a low-field 1H NMR study. Journal of Agricultural and Food Chemistry 56: 6252–6260. Aursand, M. and Axelson, D.E. (2001) Origin recognition of wild and farmed salmon (Norway and Scotland) using 13C NMR spectroscopy in combination with pattern recognition techniques. In: G.A. Webb, P.S. Belton, A.M. Gil, and I. Delgadillo (Eds) Magnetic Resonance in Food Science: A View to the Future. The Royal Society of Chemistry, Cambridge, pp. 227–231. Aursand, M. and Grasdalen, H. (1992) Interpretation of the 13C NMR spectra of omega-3 fatty acids and lipid extracted from the white muscle of Atlantic salmon (Salmo salar). Chemistry and Physics of Lipids 62: 239–251. Aursand, M., Jørgensen, L. and Grasdalen, H. (1995) Positional distribution of w3 fatty acids in marine lipid triacylglycerols by high-resolution 13C Nuclear magnetic resonance spectroscopy. Journal of the American Oil Chemists’ Society 72: 293–297. Aursand, M., Gribbestad, I.S., Skjetne, T., Grasdalen, H. and Jørgensen, L. (1997) Assessment of lipid quality in salmon and trout by quantitative NMR techniques. In: J.B. Luten, J. Oehlenschläger and T. Børresen (Eds) Seafood from Producer to Consumer, Integrated Approach to Quality. Elsevier, The Netherlands, pp. 367–377. Aursand, M., Mabon, F. and Martin, G.J. (2000) Characterization of farmed and wild salmon (Salmo salar) by a combined use of compositional and isotopic analyses. Journal of the American Oil Chemists’ Society 77: 659–666. Aursand, M., Gribbestad, I.S. and Martinez, I. (2006) Omega-3 fatty acid content of intact muscle of farmed Atlantic salmon (Salmo salar) examined by 1H MAS NMR spectroscopy. In: G. Webb (Ed.) Modern Magnetic Resonance. Springer, The Netherlands, pp. 941–945. Aursand, M., Standal, I.B. and Axelson, D.E. (2007) High-resolution 13C nuclear magnetic resonance spectroscopy pattern recognition of fish oil capsules. Journal of Agricultural and Food Chemistry 55: 38–47. Balcom, B.J., MacGregor, R.P., Beyea, D.P., Green, D.P., Armstrong, R.L. and Bremner, T.W. (1996) Single-point ramped imaging with T1 enhancement (SPRITE). Journal of Magnetic Resonance A 123: 131–134.

Nuclear magnetic resonance

267

Blackband, S.J. and Stoskopf, M.K. (1990) In vivo nuclear magnetic resonance imaging and spectroscopy of aquatic organisms. Magnetic Resonance Imaging 8: 191–198. Bollard, M.E.S., Garrod, E., Holmes, J.C., Lindon, E., Humpfer, M., Spraul, M. and Nicholson, J.K. (2000) High-resolution 1H and 1H–13C magic angel spinning spectroscopy of rat liver. Magnetic Resonance in Medicine 44: 201–207. Bock, C., Sartoris, F.-J. and Pörtner, H.-O. (2002) In vivo MR spectroscopy and MR imaging on non-anaesthetized marine fish: techniques and first results. Magnetic Resonance Imaging 20: 165–172. Bonny, J.-M., Laurent, W., Labas, R., Taylor, R., Berge, P. and Renou, J.-P. (2001) Magnetic resonance imaging of connective tissue: a non-destructive method for characterising muscle structure. Journal of the Science of Food and Agriculture 81: 337–341. Broadhurst, C.L., Schmidt, W.F., Crawford, M.A., Wang, Y. and Li, R. (2004) 13C Nuclear magnetic resonance spectra of natural undiluted lipids: docosahexaenoic-rich phospholipid and triacylglycerol from fish. Journal of Agricultural and Food Chemistry 52: 4250–4255. Callaghan, P.T., Godefory, S. and Ryland, B.N. (2003) Use of second dimension in PGSE NMR studies of porous media. Magnetic Resonance Imaging 21: 243–248. Carr, H.Y. and Puncell E.M. (1954) Effects of diffusion on free precession in nuclear magnetic resonance experiments. American Journal of Physiology 94: 630–638. Chiba, A., Hamaguchi, M., Kosaka, M., Tokuno, T., Asai, T. and Chichibu, S. (1991) Quality evaluation of fish meat by 31-phosphorus-nuclear magnetic resonance. Journal of Food Science 56: 660–664. Collewet, G., Toussaint, C., Davenel, A., Akoka, S., Médale, F., Fauconneau, B. and Haffray, P. (2001) Magnetic resonance in food science: a view to the future. In: G.A. Webb (Ed.) Special Publication 262. The Royal Society of Chemistry, Cambridge, pp. 252. Deka, K., MacMillan, M.B., Ouriadov, A.V., Mastikhin, I.V., Young, J.J., Glover, P.M., Ziegler, G.R. and Balcom, B.J. (2006) Quantitative density profiling with pure phase encoding and a dedicated 1D gradient. Journal of Magnetic Resonance 178: 25–32. de Vooys, C.G.N. and Geenevasen, J.A.J. (2002) Biosynthesis and role in osmoregulation of glycinebetaine in the Mediterranean mussel Mytilus galloprovincialis LMK. Comparative Biochemistry and Physiology B 132: 409–414. Dos Santos, J., Burkow, I.C. and Jobling, M. (1993) Patterns of growth and lipid deposition in cod (Gadus morhua L) fed natural prey and fish-based feeds. Aquaculture 110: 173–189. Eidmann G., Savelsberg R., Blümler P. and Blümich B. (1996) The NMR mouse, a mobile universal surface explorer. Journal of Magnetic Resonance A 122: 104–109. Erikson, U., Veliyulin, E., Singstad, T. and Aursand, M. (2004) Salting and desalting of fresh and frozen-thawed cod (Gadus morhua) fillets: a comparative study using 23Na NMR, 23Na MRI, low-field 1H NMR, and physicochemical analytical methods. Journal of Food Science 69: 107–114. Falch, E., Størseth, T.R. and Aursand, M. (2005) HR NMR to study quality changes in marine by-products. In: S.B. Engelsen, P.S. Belton and H.J. Jakobsen (Eds) Magnetic Resonance in Food Science. The Multivariate Challenge. The Royal Society of Chemistry, Cambridge, pp. 11–19. Falch, E., Størseth, T.R. and Aursand, M. (2006) Multi-component analysis of marine lipids in fish gonads with emphasis on phospholipids using high resolution NMR spectroscopy. Chemistry and Physics of Lipids 144: 4–16. Falch, E., Størseth, T. R. and Aursand, M. (2007) High resolution NMR for studying lipid deterioration in cod (Gadus morhua) gonads. Chemistry and Physics of Lipids 147: 46–57. Falch, E., Anthonsen, H., Axelson, D. and Aursand, M. (2004) Correlation between 1H NMR and traditional analytical methods for determining lipid oxidation in ethylesters of docosahexaenoic acid. Journal of the American Oil Chemists’ Society 81: 1105–1109.

268

Fishery Products: Quality, safety and authenticity

Fan, T.W.M. (1996) Metabolite profiling by one-and two-dimensional NMR analysis of complex mixtures. Progress in Nuclear Magnetic Resonance Spectroscopy 28: 161–219. Farrar, T.C. and Becker, E.D. (1971) Pulse and Fourier Transform NMR. Academic Press, London, pp. 18–20. Farrar, T.C. (1987) An Introduction to Pulse NMR Spectroscopy. Farragut Press, Chicago. Foucat, L., Taylor, R.G., Labas, R. and Renou, J.P. (2001) Characterization of frozen fish by NMR imaging and histology. American Laboratory August, 38–43. Foucat, L., Taylor, R.G., Labas, R. and Renou, J.P. (2004) Soft flesh problem in freshwater rainbow trout investigated by magnetic resonance imaging and histology. Journal of Food Science 69: C320–C327. Gallart-Jornet L., Barat J.M., Rustad T., Erikson U., Escriche I. and Fito P. (2007) A comparative study of brine salting of Atlantic cod (Gadus morhua) and Atlantic salmon (Salmo salar). Journal of Food Engineering 79: 261–270. Grahl-Nielsen, O. (2004) Fatty acid profiles as natural marks for stock identification. In: S.X. Cadrin, K.D. Friedland and J.R. Waldman Stock Identification Methods. Academic Press, New York, pp. 239–261. Grasdalen, H. and Jørgensen, L. (1987) In vivo NMR studies of fish eggs, monitoring of metabolite levels, intracellular pH, and the freezing of water in developing eggs of plaice, Pleuronectes platessa L. Sarsia 72: 359–361. Grasdalen, H., Aursand, M. and Jørgensen, L. NMR study of lipid fluidity of frozen red muscle of Atlantic salmon (Salmo salar): relation to autoxidation of lipids? In: P. Betlon, I. Delgadillo, A.M. Gil and G.A. Webb (Eds) Magnetic Resonance in Food Science. Bookcraft Ltd, UK, pp. 206–215. Gribbestad, I.S., Aursand, M. and Martinez, I. (2005) High-resolution 1H magnetic resonance spectroscopy of whole fish, fillets and extracts of farmed Atlantic salmon (Salmo salar) for quality assessment and compositional analyses. Aquaculture 250: 445–457. Gunstone, F.D. (1993) High-resolution 13C NMR spectra of long-chain acids, methyl esters, glycerol esters, wax esters, nitriles, amides, alcohols and acetates. Chemistry and Physics of Lipids 66: 189–193. Gunstone, F.D. (2004) The Chemistry of Oils and Fats Sources, Composition, Properties and Uses. Blackwell Publishing, CRC Press, Oxford. Hahn, E.L. (1950) Spin echoes. Physical Review 80: 580–594. Hemre, G.I., Karlsen, O., Eckhoff, K., Tveit, K., Mangor-Jensen, A. and Rosenlund, G. (2004) Effect of season, light regime and diet on muscle composition and selected quality parameters in farmed Atlantic cod, Gadus morhua L. Aquaculture Research 35: 683–697. Howell, N., Shavila, Y., Grootveld, M. and Williams, S. (1996) High resolution NMR and magnetic resonance imaging (MRI) studies on fresh and frozen cod (Gadus morhua) and Haddock (Melanogrammus aeglefinus). Journal of the Science of Food and Agriculture 72: 49–56. Hunter, C.A., Packer, M.J. and Zonta, C. (2005) From structure to chemical shift and vice-versa. Progress in Nuclear Magnetic Resonance Spectroscopy 47: 27–39. Igarashi, T., Aursand, M., Hirata, Y., Gribbestad, I.S., Wada, S. and Nonaka, M. (2000) Nondestructive quantitative determination of docosahexaenoic acid and n-3 fatty acids in fish oils by high-resolution H-1 nuclear magnetic resonance spectroscopy. Journal of the American Oil Chemists’ Society 77: 737–748. Igarashi, T., Aursand, M., Sacchi, R., Paolillo, L., Nonaka, M. and Wada, S. (2002) Determination of docosahexaenoic acid and n − 3 fatty acids in refined fish oils by 1H NMR Spectroscopy: IUPAC Interlaboratory Study. Journal of AOAC International 85: 1341–1354. Jensen, K.N., Guldager, H.S. and Jørgensen, B.M. (2002) Three-way modelling of NMR relaxation profiles from thawed cod muscle. Journal of Aquatic Food Product Technology 11: 201–214.

Nuclear magnetic resonance

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Jensen K.N., Jørgensen B.M., Nielsen H.H. and Nielsen J. (2005) Water distribution and mobility in herring muscle in relation to lipid content, season, fishing ground and biological parameters. Journal of the Science of Food and Agriculture 85: 1259–1267. Jepsen, S.M., Pedersen, H.T. and Engelsen, S.B. (1999) Application of chemometrics to low-field 1H NMR relaxation data of intact fish flesh. Journal of the Science of Food and Agriculture 79: 1793–1802. Joensen, H. and Grahl-Nielsen, O. (2001) The redfish species Sebastes viviparus, Sebastes marinus and Sebastes mentella have different composition of their tissue fatty acids. Comparative Biochemistry and Physiology B 129: 73–85. Jørgensen, L. and Grasdalen, H. (1986) 31P-NMR studies of phosphate metabolites in red and white swimming muscles of cod (Gadus morhua L.). Comparative Biochemistry and Physiology B 84: 447–450. Lambelet, P., Renevey, F., Kaabi, C. and Raemy, A. (1995) Low-field nuclear magnetic resonance study of stored or processed cod. Journal of Agricultural and Food Chemistry 43: 1462–1466. Lillford P.J., Jones D.V. and Rodger G.W. (1980) Water in fish tissue – a proton relaxation study of post rigor minced cod. In: J.J. Connell (Ed.) Advantages of Fish Science and Technology. Fishing News Books, Farnham, UK, pp. 495–497. Martinez, I., Bathen, T., Standal, I. B., Halvorsen, J., Aursand, M., Gribbestad, I.S. and Axelson, D.E. (2005) Bioactive compounds in cod (Gadus morhua) products and suitability of H-1 NMR metabolite profiling for classification of the products using multivariate data analyses. Journal of Agricultural and Food Chemistry 53: 6889–6895. Masoum, S., Malabat, C., Jalali-Heravi, M., Guillou, C., Rezzi, S. and Rutledge, D.N. (2007) Application of support vector machines to 1H NMR data of fish oils: methodology for the confirmation of wild and farmed salmon and their origins. Analytical and Bioanalytical Chemistry 387: 1499–1510. Medina, I. and Sacchi, R. (1994) Acyl stereospecific analysis of tuna phospholipids via high resolution 13 C NMR spectroscopy. Chemistry and Physics of Lipids 70: 53–61. Medina, I., Sacchi, R. and Aubourg, S. (1994) 13C Nuclear magnetic resonance monitoring of free fatty acid release after fish thermal processing. Journal of the American Oil Chemists’ Society 71(5): 479–482. Medina, I., Sacchi, R. and Aubourg, S.P. (1995) A C-13-Nmr study of lipid alterations during fish canning – effect of filling medium. Journal of the Science of Food and Agriculture 69: 445–450. Meiboom, S. and Gill, D. (1958) Modified spin-echo method for measuring nuclear times, Review of Scientific Instruments 29: 688–691. Mulkern, R.V. and Spencer, R.G.S. (1988) Diffusion imaging with paired CPMG sequences. Magnetic Resonance in Medicine 6: 623–631. Nicholson, J.K. and Foxall, P.J.D. (1995) 750 MHz 1H and 1H–13C NMR spectroscopy of human blood plasma. Analytical Chemistry 67: 793–811. Nielsen, D., Hyldig, G., Nielsen, J. and Nielsen, H.H. (2005) Lipid content in herring – influence of biological factors and comparison of different methods of analyses: solvent extraction, Fatmeter, NIR and NMR. Lebensmittel-Wissenschaft und -Technologie 38: 537–548. Nott, K.P., Evans, S.D. and Hall, L.D. (1999a) The effect of freeze-thawing on the magnetic. resonance imaging parameters of cod and mackerel. Lebensmittel-Wissenschaft und -Technologie 32: 261. Nott, K.P., Evans, S.D. and Hall, L.D. (1999b) Quantitative magnetic resonance imaging of fresh and frozen-thawed trout. Magnetic Resonance Imaging 17: 445–455. Pickova, J., Dutta, P.C., Larsson, P.O. and Kiessling, A. (1997) Early embryonic cleavage pattern, hatching success, and egg-lipid fatty acid composition: comparison between two cod (Gadus morhua) stocks. Canadian Journal of Fisheries and Aquatic Sciences 54: 2410–2416.

270

Fishery Products: Quality, safety and authenticity

Provencher S.W. (1982) A constrained regularization method for inverting data represented by linear algebraic of integral equations. Computer Physics Communications 27: 213–227. Romanzetti, S., Halse, M., Kaffanke, J., Zilles, K., Balcom, B.J. and Shah, N.J. (2006) A comparison of three SPRITE techniques for the quantitative 3D imaging of the 23Na spin density on a 4T wholebody machine. Journal of Magnetic Resonance 179: 56–64. Rooney, O.M., Troke, J., Nicholson, J.K. and Griffin, J.L. (2003) High-resolution diffusion and relaxation-edited magic angle spinning 1H NMR spectroscopy of intact liver tissue. Magnetic Resonance in Medicine 50: 925–930. Sacchi, R., Medina, I., Aubourg, S.P., Addeo, F. and Paolillo, L. (1993) Proton nuclear-magneticresonance rapid and structure-specific determination of omega-3 polyunsaturated fatty-acids in fish lipids. Journal of the American Oil Chemists’ Society 70: 225–228. Sacchi, R., Medina, I., Paolillo, L. and Addeo, F. (1994) High-resolution C-13-NMR olefinic spectra of DHA and EPA acids, methyl-esters and triacylglycerols. Chemistry and Physics of Lipids 69: 65–73. Sacchi, R., Medina, I. and Paolillo, L. (1995) One-dimensional and 2-imensional NMR-Studies of plasmalogens (alk-1-enyl-phosphatidylethanolamine). Chemistry and Physics of Lipids 76: 201–209. Sacchi, R., Savarese, M., Falcigno, L., Giudicianni, I. and Paolillo, L. (2006) Proton NMR of fish oils and lipids. In: G. Webb (Ed.) Handbook of Modern Magnetic Resonance. Kluwer/Plenum Publishers, London, pp. 919–923. Saeed, S. and Howell, N. (1999) High-performance liquid chromatography and spectroscopic studies on fish oil oxidation products extracted from frozen Atlantic mackerel. Journal of the American Oil Chemists’ Society 76: 391–397. Saito, H. (1987) Estimation of the oxidative deterioration of fish oils by measurement of nuclear magnetic resonance. Agricultural and Biological Chemistry 51: 3433–3435. Saito, H. (1997) Evaluation method for lipid oxidation by nuclear magnetic resonance. In: K.R. Cadwallader (Ed.) Flavor and Lipid Chemistry of Seafoods, American Chemical Society Symposium no. 674. American Chemical Society, Washington, DC, pp. 218–239. Saito, H. and Nakamura, K. (1990) Application of the NMR method to evaluate the oxidative deterioration of crude and stored fish oils. Agricultural and Biological Chemistry 54: 533– 534. Saito, H. and Udagawa, M. (1992a) Application of NMR to evaluate the oxidative deterioration of brown fish meal. Journal of the Science of Food and Agriculture 58: 135–137. Saito, H. and Udagawa, M. (1992b) Use of NMR to evaluate the oxidative deterioration of niboshi, boiled and dried fish. Bioscience, Biotechnology, and Biochemistry 56: 831–832. Schiller, J. and Arnold, K. (2002) Application of high resolution 31P NMR spectroscopy to the characterisation of the phospholipids composition of tissues and body fluids – a methodological review. Medical Science Monitor 8: 205–222. Siddiqui, N., Sim, J., Silwood, C.J.L., Toms, H., Iles, R.A. and Grootveld, M. (2003) Multicomponent analysis of encapsulated marine oil supplements using high-resolution H-1 and C-13 NMR techniques. Journal of Lipid Research 44: 2406–2427. Slichter, C.P. (1990) Principles of Magnetic Resonance. Springer-Verlag, New-York, pp. 371–379. Song, Y.Q., Venkataramanan, L., Hürlimann, M.D., Flaum, M., Frulla, P. and Straley, C. (2002) T1–T2 correlation spectra obtained using a fast two-dimensional Laplace inversion. Journal of Magnetic Resonance 154: 261–268. Springer, C.S. (1987) Measurement of metal cation compartmentalization in tissue by high-resolution metal cation NMR. Annual Review of Biophysics and Biophysical Chemistry 16: 375–399. Sitter, B., Krane, J., Gribbestad, I. S., Jørgensen, L. and Aursand, M. (1999) Quality evaluation of Atlantic halibut (Hippoglossus hippoglossus L.) during ice storage using 1H NMR spectroscopy.

Nuclear magnetic resonance

271

In P.S. Belton, B.P. Hills, and G.A. Webb (Eds) Advances in Magnetic Resonance in Food Science. The Royal Society of Chemistry, Cambridge, pp. 226–237. Standal, I.B., Gribbestad, I.S., Bathen, T.F., Aursand, M. and Martinez, I. (2006) Low molecular weight metabolites in white muscle from cod (Gadus morhua) and haddock (Melanogrammus aeglefinus) analyzed by high resolution 1H NMR spectroscopy. In: I.A. Farhat, P.S. Belton, G.A. Webb (Eds) Magnetic Resonance in Food Science, from Molecules to Man, pp. 55–62. Steen, C. and Lambelet, P. (1997) Texture changes in frozen cod mince measured by low-field nuclear magnetic resonance spectroscopy. Journal of the Science of Food and Agriculture 75: 268–272. Tingle, J.M., Pope, J.M., Baumgartner, P.A. and Sarafis, V. (1995) Magnetic Resonance Imaging of fat and muscle distribution in meat. International Journal of Food Science & Technology 30: 437–446. Tocher, D.R. (2003) Metabolism and functions of lipids and fatty acids in teleost fish. Reviews in Fisheries Science 11: 107–184. Tocher, D.R. and Sargent, J.R. (1990) Effect of temperature on the incorporation into phospholipid classes and metabolism via desaturation and elongation of n-3 and n-6 polyunsaturated fatty-acids in fish cells in culture. Lipids 25: 435–442. Toussaint, C., Fauconneau, B., Médale, F., Collewet, G., Akoka, S., Haffray, P. and Davenel, A. (2005) Description of the heterogeneity of lipid distribution in the flesh of brown trout (Salmo trutta) by MR imaging. Aquaculture 243: 255–267. Toussaint, C.A., Médale, F., Davenel, A., Fauconneau, B., Haffray, P. and Akoka, S. (2001) Determination of the lipid content in fish muscle by a self-calibrated NMR relaxometry method: comparison with classical chemical extraction methods. Journal of the Science of Food and Agriculture 82: 173–178. Tsoref, L., Shinar, H., Seo, Y., Eliav, U. and Navon, G. (1998) Proton double quantum filtered MRI – a new method for imaging ordered tissues. Magnetic Resonance in Medicine 40: 720– 726. Veliyulin, E., Aursand, I.G. and Erikson, U. (2005a) Study of fat and water in Atlantic salmon muscle (Salmo salar) by low field NMR and MRI. In: S.B. Engelsen, P.S. Belton and H.J. Jakobsen (Eds) Magnetic Resonance in Food Science. The Royal Society of Chemistry, Cambridge, pp. 148– 155. Veliyulin, E., van der Zwaag, C., Burk, W. and Erikson, U. (2005b) In-vivo determination of fat content in Atlantic salmon (Salmo salar) with a mobile NMR spectrometer. Journal of the Science of Food and Agriculture 85: 1299–1304. Veliyulin, E., Borge, A., Singstad, T., Gribbestad, I. and Erikson, U. (2006a) Post-mortem studies of fish using magnetic resonance imaging. In: G.A. Webb (Ed.) Modern Magnetic Resonance. Springer, The Netherlands, pp. 949–956. Veliyulin, E., Østerhus, K., Burk, W., Singstad, T. and Skjetne, T. (2006b) Comprehensive compositional analysis of fish feed by time domain NMR. In: G.A. Webb (Ed.) Modern Magnetic Resonance. Springer, The Netherlands, pp. 887–893. Veliyulin, E., Felberg, H.S., Digre, H. and Martinez, I. (2007a) Non-destructive nuclear magnetic resonance image study of belly bursting in herring (Clupea harengus). Food Chemistry 101: 1562–1568. Veliyulin, E. and Aursand, I.G. (2007b) 1H and 23Na MRI studies of Atlantic salmon (Salmo salar) and Atlantic cod (Gadus morhua) fillet pieces salted in different brine concentrations, Journal of Science of Food and Agriculture 87: 2676–2683. Yokoyama, Y., Azuma, Y., Sakaguchi, M., Kawai, F. and Kanamori, M. (1996b) Non-destructive phosphorus-31 nuclear magnetic resonance study of postmortem changes in oyster tissues. Fisheries Science 62: 416–420.

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Fishery Products: Quality, safety and authenticity

Yokoyama, Y., Azuma, Y., Sakaguchi, M., Kawai, F. and Kanamori, M. (1996a) P-31 NMR study of bioenergetic changes in carp muscle with cold-CO2 anesthesia and non-destructive evaluation of freshness. Fisheries Science 62: 267–271. Zamora, R. and Hidalgo, F.J. (2006) Determination of fatty acid composition and oxidation in fish oil by high resolution nuclear magnetic resonance spectroscopy. In: G. Webb (Ed.) Handbook of Modern Magnetic Resonance. Springer, The Netherlands, pp. 925–931.

Chapter 12

Time domain spectroscopy Michael Kent and Frank Daschner

12.1 12.1.1

Introduction Historical background

With the development and commercialisation of the Fischtester and the Torrymeter, further instrumental innovation for the measurement of fresh fish quality seemed to have halted, although a concerted action in evaluating fish freshness in the AIR programme of the 3rd Framework of the European Community (AIR3 CT94 2283) reported much experimental work in this area (Olafsdottir et al. 1998). That international project examined in detail the various methods available for estimating freshness, and electrical methods were among the physical methods considered. Among other things, it was concluded that, ‘more effort has to be put into making reliable instrumentation for measuring freshness. The need for developing methods for measuring freshness is real, but for all mentioned techniques more research is needed’. It was also concluded that all physical methods ‘need to be significantly improved to fulfil the expectations of the users’. Given that both the commercial instruments discussed above are generally limited in applicability to fish kept on ice and unaltered in any way by, for example, handling, irradiation or freezing (even partly), it was clear that a new approach was required. To advance the notion of a new approach, several European research laboratories* were awarded funding under the European Communities’ 5th Framework Programme, (specific RTD programme Quality of Life and Management of Living Resources, project QLK1-200101643), to develop ‘a new method for measurement of the quality of seafood’. The starting point was the work of an earlier project under the 4th Framework Programme (CT97 3020), which had been concerned principally with the measurement of added water in foods using a broadband microwave method (Kent et al. 2000a). That methodology relied on measuring the dielectric properties in the microwave region as a function of frequency and the subsequent multivariate analysis of the spectra so measured. A preliminary test of this approach had also been performed to distinguish different aspects of quality in seafood products, for example whether raw materials had been frozen and how well they had been stored (Kent et al. 2000b). Furthermore, mixtures of fish and other materials such as potato flour and milk protein were successfully allocated to categories containing 273

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fresh or previously frozen fish with overall success rates of around 90%. These results were unique. By using such high frequencies (100 MHz to 10 GHz), the problem of electrode polarisation, which both the Torrymeter and the Fischtester had been designed to eliminate, does not arise. Furthermore, by making measurements at several frequencies and using a multivariate technique of analysis, more variables were available for the elimination of other variations due to unspecified factors. The multivariate method most commonly used was that of principal component analysis (PCA) (Chatfield and Collins 1980; Martens and Næs 1989). The use of multivariate techniques in this application has the advantage that it avoids the creation of a physical model to explain the dielectric behaviour of the tissues: owing to the complexity of such a model, this is practically impossible. The new project, called ‘SEQUID’ (Seafood Quality Identification), dealt with determining the quality of seafood using ultrawideband pulses. A discussion of some of the results of this project (Kent et al. 2005) is presented in this chapter.

12.1.2

Theory

Because in water molecules and many organic molecules the positive and negative charges (atomic nuclei and orbiting electrons, respectively) have different centres of distribution, the molecules are polar; that is, they have an electric dipole moment. Such a property means that they interact with electric fields. For a rotating field of low frequencies, the water molecules are able to follow changes in the direction of the field. As the frequency increases, however, because molecules have inertia and because their motion is hindered by collisions with other molecules, in which energy is dissipated, this becomes more difficult. Above a characteristic frequency called the relaxation frequency (20 GHz at 25 °C for water), at which the dielectric losses have a maximum, the polar molecules are increasingly unable to follow the field and such losses decrease. This behaviour can be described by a complex permittivity (that is, a dielectric ‘constant’), mathematically having real and imaginary components, e* = ε′ − je″. The ability of material to store electrical field energy is expressed by the real part of the permittivity e′ and its dielectric loss by its imaginary part e″. The dependence of the complex permittivity on frequency is called the dielectric spectrum. Water being the largest component of fish tissue (or indeed any organism), its influence dominates the microwave dielectric spectrum. Of course, there are also many smaller contributions from other constituents. In addition, dissolved ions create an electrical conductivity, the effects of which are to create an additional dielectric loss term inversely related to frequency. Despite this inverse dependence, however, if the conductivity is large enough, there will be an effect even at microwave frequencies, and this is seen in the shape of the loss factor spectrum. Often, water molecules can be considered as more or less bound to the molecular surfaces of tissue components such as proteins. Other water molecules and larger organic molecules, through effects, which are manifestly viscous, hinder any rotational movement caused by an external electric field. The average relaxation frequency of tissue is then lower by (approximately 1 GHz) than that of normal liquid water. In relation to fish technology the abstract term quality is ultimately influenced by changes at the molecular level in the tissue of the fish. For instance, among many deteriorative

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changes, the denaturation of proteins leads to the release of liquid water, thus changing the dielectric spectrum. Other degradation processes lead to the formation of polar compounds, which may also have a high relevance to the dielectric spectrum. Breakdown of cell-wall structure leads to release of solutes into the intracellular region, changing the overall electrical conductivity. Hence the dielectric spectrum in the microwave frequency range contains information about the state of the tissue. The challenge is to extract this hidden information.

12.2 12.2.1

Measurement system Dielectric measurements using the open-ended coaxial line

The more broadband the measured dielectric spectrum is, the more information is available. However, the effort of any device required to measure this spectrum will increase proportionately. A well-established probe for broadband dielectric spectroscopy is the open-ended coaxial line (Fellner-Feldegg 1969). This simple and easy to use structure consists of nothing more than a transversely cut coaxial line. An electromagnetic wave of a given wavelength (frequency) fed into the line will be reflected at the cut end because the propagation conditions for the wave change abruptly at that point. Whatever material lies beyond that point, the properties of the reflected wave depend on its permittivity. Furthermore, not only the permittivity of the surface of the material has an influence, but because the field also penetrates it for some millimetres, this depth also must play a part. This is a very important advantage of this type of microwave measurement system because the surface is often already dried or damaged. It is also a disadvantage in that the larger bulk of the material is not measured. Some measurement device (vector network analyser) then must compare the incident and the reflected wave to calculate the reflection coefficient. Knowing the reflection coefficient, the complex permittivity then can be calculated if required. With this system, to measure the dielectric spectrum a broadband signal generator is needed and measurements must be made at discrete frequencies. An alternative is to make measurements in the so-called time domain.

12.2.2

Time domain reflectometry

Instead of applying the spectral frequencies sequentially to the probe (frequency domain), it is also possible to create a time-dependent signal that is composed of all these frequencies (time domain) (Fellner-Feldegg 1969). In theory, using this approach, the whole spectrum can be measured in one step. However, the most interesting advantage of the application is that the hardware effort of an instrument working in the time domain is lower than a vector network analyser working in the frequency domain. One possible time domain broadband signal is a voltage step having a sufficiently short rise time (here: tr ∼ 60 ps). The reflected pulses are shown in Figure 12.1. The shape of the reflected pulse depends on the complex permittivity of the material under test. In principle, the dielectric spectrum could be calculated from this time domain signal. A family of several mathematical transformations links the frequency and time domains, the

276

Fishery Products: Quality, safety and authenticity 1000

Pulse amplitude

800 600 400 200 0 –200

0

0.1

0.2

0.3

0.4 0.5 Time (ns)

0.6

0.7

0.8

Figure 12.1 Dashed line: reflected pulses at the probe interface in air. Solid lines: typical reflected pulse from the surface of some fish samples. Circles: selected data points for the subsequent data analysis.

most well known being the Fourier transform and its inverse. For time-varying data, such as are acquired by this method, the inverse Fourier transform in its most general form can be written as in equation (1). h (t ) =

1 π π g(ω )e − iω t dω 2 −∫π

(1)

where h(t) is a time-dependent function, the Fourier transform of which is a frequencydependent spectrum g(w). Examination of this equation shows that at any instant t, every component part of the spectrum contributes to the value of h. Because the subsequent PCA depends only on variations in g(w), then transformation of h(t) to the frequency domain for PCA is not required because the same sources of variation are present in h(t). In the method described here, truncation of the pulse is allowed at any time (see Figure 12.1); the same information on variation is still contained in the collected data.

12.2.3

Prototype instrument

To meet the requirements of the potential users of the instrument, the costs for the time domain reflectometer have to be kept low. To this end, and to ensure a good reproducibility, the instrument is predominantly built using readily available low-cost devices. Because the system needs to operate with pulses that have extremely fast rise times, a method has to be devised to sample the waveforms at different increments of time. The method is familiar to many people as that used in optical strobing to image rapidly rotating objects and ‘freeze’ their motion.

Time domain spectroscopy

Strobe pulse generator

277

0°

180°

6 dBPowersplitter

Clock 5 MHz

Delay

Data-processing and Display

Step generator

Microcontroller

Sampling gate

A

er(f) Open-ended coaxial line probe Material under test

D

Figure 12.2 Block-diagram of the TDR instrument.

The time domain reflectometer that was designed and constructed (Figure 12.2) has been described in detail elsewhere (Schimmer and Knöchel 2003; Kent et al. 2005). Initially, a crystal oscillator operating at 5 MHz provides the system clock for all the relevant radio frequency circuitry of the system. The clock signal is divided into two channels by a resistive 6 dB power splitter. In one channel the strobe signal is generated, providing sufficiently short pulses to trigger the sampling gate. The output from the second channel can be shifted in time with increasing delay of 10 ps increments, by redirecting the signal over delay lines, controlled by the microcontroller. Subsequently, the adjusted clock signal triggers the step generator, producing a step-signal with less than 100 ps rise-time. The measurement signal propagates through the sampling gate towards the open-ended coaxial line probe and is reflected at the interface with the material under test. The reflected signal superimposes with the incident step and is acquired in the sampling gate. The strobe pulses in the first channel trigger the sampling diodes of a six-diode travelling-wave sampling gate (Frye 1971). A holding capacitor is charged during the sampling period and stores the instantaneous voltage for analogue–digital conversion. The conversion is triggered and read by a microcontroller. This controller also buffers the measurements and passes them to a computer for high-level data processing and display; the final result is a sampled waveform such as that shown in Figure 12.1. The computer permanently communicates with the instrument through the microcontroller, in order to control all high-level hardware actions.

12.2.4

Data processing using multivariate analysis

As mentioned above, it is difficult, if not impossible, to describe completely in terms of some physical model, the dielectric behaviour of complex materials like fish tissue. The reduction of quality is explicable by degeneration of the tissue: as this happens, its permittivity changes also and the shape of the reflected pulse is coupled with these changes.

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To extract the information contained in the dielectric spectrum or in the time domain pulse, multivariate analysis is applied. Of the methods available for multivariate analysis, PCA seems as suitable as any (Martens and Næs 1989). It applies a linear transformation to any multidimensional set of correlated variables to produce a new set of uncorrelated, standardised and orthogonal variables called ‘principal components’ or scores. Each of these ‘explains’ a fraction of the variance of the data. This transformation can be written as in equation (2), Y j = a1 j X1 + a2 j X 2 + … a pj X p

(2)

where Yj is the jth principal component and the aij are loadings for the input vector X of length p. Often in PCA applications, the principal components and their loadings can be identified with the variation in particular variables, but in this application it is not possible. This is due to the complexity of the input data set; that is, a sampled time domain pulse, which itself is an incomplete convoluted function (Fourier transform) of the frequency domain spectrum. Within the parameters of the spectrum are the sources of variation. Instead the principal components may be considered as shape descriptors for the time domain pulse. As shown in Figure 12.1, a limited number of points are selected for data processing from the reflected pulse. PCA effectively reconstructs the information in the input data into a new form in which the sources of variation are ‘modelled’, rather than some attempt at a physical model of the system. The principal component scores can then be used either as descriptor variables for discriminant analysis (assigning samples to predefined groups) or as variables in regression equations designed to predict desired quality variables (principal component regression (PCR)). A different approach to multivariate analysis, which also can be used, is that of artificial neural networks (ANNs). The purpose of the ANN is to approximate the unknown (nonlinear) function, which describes the relationship between the inputs, in this case the sample time domain data, and the output, the desired quality variables. It is of some interest to note that if a linear function is used, then the results obtained by PCA are reproduced. In the literature, the use of multi-layer feed forward (MLFF) networks has been suggested for such ANNs (Patterson 1996). In the system described here, the nonlinear activation function chosen for the ANN was a tansig function (tansig (n) = 2/(1 + exp(−2n)) − 2)). Perhaps the only disadvantage of this type of approach is that very many samples are required for the training process. On the other hand, the acquisition of more data with use of the system allows for a gradual improvement over time in the training.

12.3

Time domain reflectometry measurements

The first experiments of the SEQUID project were made using expensive laboratory instruments to develop the method described here. These measurements led to the development of the required specifications of the prototype instrument. A photograph of this prototype is shown in Figure 12.3. Most of the results discussed in this chapter were obtained using this

Time domain spectroscopy

279

Figure 12.3 Photograph of the SEQUID prototype being used with fish.

instrument. A complete description of all results, including those made using laboratory instruments, has been published elsewhere (Kent et al. 2005). As examples of the results obtained for chilled fish, cod (Gadus morhua), sea bass (Dicentrarchus labrax) sardine (Sardina pilchardus) and Senegalese sole (Solea senegalensis) were selected. These fish were stored in ice until the end of their shelf life (up to 27 days for Senegalese sole but only 3–4 days for sardine). For frozen fish the results obtained with Alaska pollock (Theragra chalcogramma), cod and hake (Merluccius capensis, M. paradoxus) are reviewed. Several of the cod (20 out of 100 samples) were frozen twice. These measurements were processed both separately and together with the other single-frozen samples. The frozen samples were stored up to 406 days at temperatures ranging from −10 °C to −40 °C. The sample probe was used to make measurements directly on the skin, on the flesh side of fillets and on minced or blended samples. For some of the species an attempt was made to determine if there was an optimum position for the measurement, for example the ventral, dorsal or middle of the fish. Several replicate measurements were made on each fish/sample and such measurements were repeated at regular intervals on new samples. When a complete data series had been collected, for example when the fish had deteriorated to its rejection level after several days storage in ice, the data were subjected to the multivariate analyses discussed above. The aim of these analyses was to produce regression equations capable of predicting the values of the various quality related factors. These calibration equations were usually validated by the method of internal cross-validation (ICV) where one or more data points are removed in sequence and the process of calibration is repeated. The value of each removed point is then calculated (predicted) from the appropriate calibration equation obtained. In the case of ANNs, repeated calculations of the calibration data in this manner can introduce further variation. This was avoided by randomly selecting a validation data

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set from the complete data set. Thus, two-thirds of the data were used for calibration and one-third for validation. Predictive equations were found for variables such as time of storage (in ice or in the frozen state), QIM, various sensory variables and others related to change in quality. Only the results for the prediction of the storage period and the summarised quality score are discussed here. For PCR, the results are normally expressed in terms of the regression parameters; the coefficient of determination adjusted for degrees of freedom (R2adj), the standard error of calibration (SEC) and standard error of prediction (SEP) (equations 3 and 4 respectively). SEC =

∑ (ΔYi )2

(n − p − 1)

(3)

i

where ΔYi are the differences between predicted values calculated from the calibration equation and actual values (the residuals), n is the number of samples (observations) and p is the number of coefficients (principal components) in the regression equation. SEP is a comparable variable to the SEC but is calculated from the predicted values generated by the validation data set. It is a direct measure of the prediction error. SEP expresses the error to be expected in future predictions. SEP =

∑ (ΔYi )2

(n − 1)

(4)

i

Up to 10 principal components were normally calculated, but those used in the regression were selected by observing how R2adj, SEP and SEC varied as the number of principal components in the regression was increased. The optimum number chosen was that above which the SEP and SEC diverged. This usually meant that the SEC continued to decrease as the number of principal components increased, demonstrating that over-fitting was occurring, while the SEP reached a minimum. Also, R2adj was seen to stabilise at a constant value after this point was reached. Over-fitting is always a danger, but in this manner the problem was minimised. For the ANN, the degrees of freedom are unknown, so we must resort to the use of the unadjusted coefficient of determination (R2) and the root mean square error (RMSE). The latter is defined as the square root of the mean of the squared residuals, namely RMSE =

∑ (ΔYi )2

n

(5)

i

As can be seen, it is effectively identical to the SEC and SEP for large population sizes. Thus, in the following tables, owing to the relatively large number of samples, the coefficients of determination R2, and the root mean square errors (RMSE) for the calibration and validation groups, are documented for both PCR and ANN. The range of the calibration variables has also been included to give some idea of the magnitude of the uncertainty represented by RMSEc and RMSEv. The results for frozen fish are shown in Table 12.1. In general, it is observable that ANN has a better performance than PCA, but ANN is only applicable for large test series (more than about 30 samples are necessary for a useful calibration). The results for cod were

Time domain spectroscopy

281

Table 12.1 Regression parameters for frozen fish calibrations to predict time (days) of storage. The different measurement laboratories are indicated.

Range (days)

Number of samples

Number of pcs

110–353

136

8

110–353

77

8

110–406

144

8

110–406

139

9

38–260

100

8

PCR ANN

38–260

100

PCR ANN PCR ANN PCR ANN

38–260

R2

RMSEc

75.7 94.7 70.6 82.6 72.5 95.0 76.6 88.2 89.0 98.8

40.0 29.1 39.3 31.3 32.0 19.6 38.6 20.3 36.8 25.3

42.8 33.4 44.5 46.0 34.2 21.6 41.5 25.8 40.2 33.0

7

83.2 99.5

45.6 30.7

50.0 38.1

20

6

38–260

80

7

30–406

244

8

96.6 99.3 87.3 99.1 83.8 94.4

15.0 19.9 41.3 32.1 39.6 35.9

27.6 10.6 45.8 41.1 41.0 42.6

PCR ANN

38–406

209

8

70.8 98.0

54.0 31.0

56.2 32.6

PCR ANN

69–310

55

8

84.2 99.8

28.6 7.0

34.0 23.5

Sample Alaska Pollock (fillet)1 Alaska Pollock (mince)a Cod (mince)a Cod (fillet)a Cod (mince, twice and once frozen)b Cod (fillet, twice and once frozen)b Cod (mince, twice frozen)b Cod (mince, once frozen)b Cod (minced, twice and once frozen)a,b Cod (fillet, twice and once frozen)a,b Hake (mince)c

PCR ANN PCR ANN PCR ANN PCR ANN PCR ANN

RMSEv

a

Swedish Institute for Food and Biotechnology, Gothenburg, Sweden Federal Research Centre for Nutrition and Food, Department for Seafood Research, Hamburg, Germany c Instituto del Frio, Spanish Council for Scientific Research, Madrid, Spain b

obtained in two different laboratories, but combining the two independent sets of data still yielded similar predictive power, especially using the ANN. There is no absolute consistency in whether the measurements made on skin are better than those on minces. However, surprisingly, it is usually the case that the skin measurements are slightly better. It is possible that mincing introduces variable amounts of air into the samples. It had also been reported (Kent et al. 2000b) that such measurements could be used with other statistical procedures, such as discriminant analysis, to classify samples into groups. Thus, in the preliminary studies of the method (Kent et al. 2004), among the frozen cod studied, those that had been frozen twice could be readily distinguished from those frozen once only (see Table 12.2). For chilled fish samples, the selected data are presented in Table 12.3 in terms of days in ice. For some of these fish the R2 values are low, but this is possibly due to the small number of samples measured at each sampling time. The ANN approach appears better although the number of data points is marginal for success.

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Table 12.2 The results of discriminant analysis on once and twice frozen cod. Ten principal components were used as discriminators. The numbers show how many of each group were placed into the correct group. Data taken from Kent et al. (2004). Group

Number in group

1: once frozen

85

2: twice frozen

25

Training

True group Predicted group

1 2

Per cent correct Overall correct

1 82 3 96.5

2 0 25 100.0 97.3

Validation

True group Predicted group Per cent correct Overall correct

1 2

1 82 3 95.0

2 0 25 100.0 96.4

For both chilled and frozen fish it is possible to construct QIM scores based on various aspects of appearance and sensory assessments. Some results using this variable are shown in Table 12.4. Because of the smaller numbers of samples used, SEC and SEP are shown in this table. QIM has the advantage that it is a system based on the summation of scores of individual attributes. This has a tendency to smooth out anomalous assessments of any particular variable. Such results are important because they mean that the instrument can be calibrated to relate directly to the consumers’ appreciation of quality. It is also perhaps significant that the errors of prediction are often comparable in magnitude to those inherent in the subjective determination of quality indices by expert panels. (Because these are used to calibrate the instrument, then obviously it can never give a more accurate result than that provided by the calibration data.) It would be even more convenient if a single calibration could be found for all fish rather than for individual species. In Figure 12.4 the same QIM data for a combination of three species, each with comparable shelf lives, has been normalised by division with the maximum score attainable for each species. In the prediction of days in ice for the same data set (Figure 12.5) there is also generally good accuracy, marred only by two apparent outliers for sea bass and salmon at the end of the storage period. This is not a problem, because fish at that stage of loss of quality need no instrumental method to identify them! This was a preliminary attempt to produce a generalised calibration for all species and could be improved by a better knowledge of the potential shelf life of each species and by the collection of much more data. Much information is still being clarified for species that were not generally exploited before. The inclusion of fish with much shorter shelf life in this generalised analysis was not successful.

Time domain spectroscopy

283

Table 12.3 Regression parameters for chilled fish calibrations to predict time of storage in ice. The different measurement laboratories are indicated.

Sample and measurement position Coda

mince skin

Salmon (2–3 kg)b

skin

Sea bassc

skin

Sea breamc

skin

Senegalese solec

skin

Sardined

skin

PCR ANN PCR ANN PCR ANN PCR ANN PCR ANN PCR ANN PCR ANN

Range (days)

Number of samples

Number of principal components

0–18

75

8

0–18

75

8

0–18

40

3

2–23

50

8

1–17

40

8

1–27

60

8

0–4

12

8

R2

RMSEc

RMSEv

87.8 99.5 88.7 99.7 70.6

2.1 2.3 0.9 1.6 2.0 2.3 1.2 2.0 3.2 3.3 Not calculated 82.0 2.7 3.4 98.7 1.3 2.2 68.4 3.1 3.8 82.7 3.5 3.3 68.4 4.6 5.8 87.6 4.9 4.2 85.0 0.2 0.8 Not calculated

a

Swedish Institute for Food and Biotechnology, Gothenburg, Sweden Federal Research Centre for Nutrition and Food, Department for Seafood Research, Hamburg, Germany c Instituto del Frio, Spanish Council for Scientific Research, Madrid, Spain d Insituto de Investigação das Pescas e do Mar, Lisbon, Portugal b

Table 12.4 PCR parameters for chilled and frozen fish calibrations to predict QIM scores. The different measurement laboratories are indicated.

Sample Codb Salmon (2–3 kg)a Sea bassc Sea breamc Sardined

Range of QI

Number of samples

Number of principal components

R2adj

SEC

SEP

0–20 3–24

75 40

5 5

86.0 73.6

2.4 3.2

2.5 3.5

2–21 0–17 1–17

50 40 24

4 4 9

80.4 61.2 72.7

2.8 3.2 3.0

2.9 3.4 4.0

a

Swedish Institute for Food and Biotechnology, Gothenburg, Sweden Federal Research Centre for Nutrition and Food, Department for Seafood Research, Hamburg, Germany c Instituto del Frio, Spanish Council for Scientific Research, Madrid, Spain d Insituto de Investigação das Pescas e do Mar, Lisbon, Portugal b

12.4

Conclusions

It must be stressed that when attempts are made to correlate the time domain reflectometry measurements with any other quality-related variables, by generating multivariate predictive equations, this is not in the belief that the dielectric properties are directly related to them but simply as a device for showing their common dependence on time and/or temperature and to exploit that dependence. The effects of deteriorative changes in stored fish, on the shape of the time domain reflected pulses collected by this method, are not usually evident

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Predicted QIM/QIMmax

0.8

0.6 Sea bass 0.4

Salmon

0.2

Cod

0 0

0.2

0.4 0.6 Mean QIM/QIMmax

0.8

1

Figure 12.4 Predicted normalised QIM using ANN for a combined set of data of cod, sea bass and salmon, stored in ice for up to 20 days. R2 = 75.2, RMSEC = 0.13, RMSEP = 0.11.

Mean predicted days in ice

20

15

10

Cod

5

Sea bass Salmon 0 0

5

10 Days in ice

15

20

Figure 12.5 Predicted storage time using ANN for a combined set of data of cod, sea bass and salmon, stored in ice for up to 20 days. R2 = 70.8, RMSEC = 2.9, RMSEP = 2.6.

to the eye. Nevertheless, the information is there and the application of multivariate analysis techniques can extract it. Sound multivariate predictive equations for quality parameters can thus be generated from these waveforms. In general, the SEQUID prototype has managed to predict storage times of both chilled and frozen fish to a reasonably high accuracy. Prediction of the QIM variables is equally satisfying and shows the way forward in the prediction of remaining shelf life, perhaps with more generalised calibrations. The overall accuracy of

Time domain spectroscopy

285

such predictions is of the same order as for the subjective scores. Obviously, it can never be better. The fact that the instrument can be used on fresh or frozen fish is an advance on the previously available electronic devices. If that were not enough, then its usability with different types of fish sample, namely whole fish, fillets or mince, gives it a much wider applicability than its competitors. Other useful applications of the same instrument must also be considered as advantages: firstly, its potential for the discrimination of prior treatment, namely whether fish has been frozen once or twice; secondly, its ability for composition measurement, notably added water.

Note * Electrical Engineering Microwave Group, Christian Albrechts University of Kiel, Kiel, Germany; Swedish Institute for Food and Biotechnology, Gothenburg, Sweden; Department for Seafood Research, Federal Research Centre for Nutrition and Food, Hamburg, Germany; Instituto del Frio, Spanish Council for Scientific Research, Madrid, Spain; Insituto de Investigação das Pescas e do Mar, Lisbon, Portugal.

12.5

References

Chatfield, C. and Collins, A.J. (1980) Introduction to Multivariate Analysis. Chapman and Hall, London. Fellner-Feldegg H. (1969) The measurement of dielectric in the time domain. Journal of Physical Chemistry 73: 616–623. Frye, G.J. (1971) Sampling System. Patent US3629731. Portland, Oregon, USA. Kent, M., Knöchel, R., Daschner, F. and Berger, U.-K. (2000a). Composition of foods using microwave dielectric spectra. European Food Research Technology 210: 359–366. Kent, M., Mackenzie, K., Berger, U.-K., Knöchel, R. and Daschner, F. (2000b) Determination of prior treatment of fish and fish products using microwave dielectric spectra. European Food Research Technology 210: 427–433. Kent, M., Oehlenschlager, J., Mierke-Klemeyer, S., Knöchel, R., Daschner, F. and Schimmer, O. (2004) A new multivariate approach to the problem of fish quality estimation. Food Chemistry 87: 531–535. Kent, M., Knöchel, R., Barr, U.-K., Tejada, M., Nunes, L. and Oehlenschläger, J. (Eds) (2005) SEQUID: A New Method for Measurement of the Quality of Seafood. Shaker Verlag GmbH, Aachen. Martens, H. and Næs, T. (1989) Multivariate Calibration. John Wiley and Sons, Chichester, UK. Olafsdottir, G., Luten, J., Dalgaard, P., Careche, M., Verrez-Bagnis, V., Martinsdottir, E. and Heia, K. (1998) (Eds) Methods to Determine the Freshness of Fish in Research and Industry. IIR, Paris. Patterson, D.W. (1996) Artificial Neural Networks, Theory and Applications. Prentice Hall, New Jersey. Schimmer, O. and Knöchel, R. (2003) A hand-held TDR-system with a fast system-rise time and a high resolution bandwidth for moisture measurements in the microwave frequency range. In: K. Thakur (Ed.) Proceedings of the 5th International Conference on Electromagnetic Wave Interaction with Water and Moist Substances, Rotorua, New Zealand, 2003. Industrial Research Ltd, Auckland, New Zealand, pp. 171–179.

Chapter 13

Measuring electrical properties Michael Kent and Jörg Oehlenschläger

13.1

Introduction

The completely satisfactory and objective instrumental measurement of the quality of fish has been a goal of fish technologists for many years. Ideally, a ‘black box’ is desired that could unambiguously determine the quality or remaining shelf life of a product. It is now nearly 90 years since it was first noted that injury, death and decay profoundly affect the electrical properties (conductivity and capacitance) of biological materials (Osterhout 1922). If one considers the structure of typical cells, they have a lipid bilayer cell wall and contain electrically conducting fluids and further large surface-area-to-volume structures such as the endoplasmic reticulum. Added to this, they are surrounded by other structures and fluids, all of which disposes to a very large electrical capacitance. It is analogous to the way in which large capacitances are created in the construction of electrolytic capacitors. The gradual disruption of these cell structures with tissue decay causes large changes in the overall electrical conductivity and even larger changes in the capacitance. The optimum use of these effects in the measurement of storage time in ice, for example, requires that tissue damage does not occur by any other process such as irradiation, freezing or rough handling. During storage, several other post mortem changes in the fish muscle affect quality through mechanisms that could also alter the dielectric properties. Most obvious are changes affecting interactions between water and proteins, and degradation processes leading to the formation of polar compounds. Subsequent applications of the electrical properties to the measurement of fish quality have largely been in the audio- and radio-frequency regions (Hennings 1965; Jason and Lees 1971; Jason and Richards 1975; Niu and Lee 2000), and have only examined the spectra in these regions as a means of choosing optimum frequencies at which to make measurements. Those applications, which have reached the marketplace, will be discussed here.

13.2

Fischtester

The basis for electrical measurements on fish, using instruments like the Fischtester, is the fact that the resistance of fresh fish measured immediately after catch is about 2000 Ω 286

Measuring electrical properties

287

whereas spoiled fish has only 50 Ω of resistance left. The conductivity of fresh fish is approximately 500 mS and that of spoiled fish around 20,000 mS (Oehlenschläger and Nesvadba 1998; Botta 1995). Polarisation impedance is the problem that besets all measurements of electrical properties at low frequencies, where there are boundaries between materials of very different electrical properties: electrical charges accumulate at these boundaries. This is true not only of the interface between electrodes and sample but also any internal structures in the samples measured. At the electrodes its effect is to introduce very large reactive impedances, the magnitude of which completely swamps the variables to be measured. It is fortunate that this impedance is frequency dependent because measurement at two or more frequencies allows the elimination of its effect. This was the basis on which the Intellectron Fischtester was designed. Hennings (1965) divided the complex resistance of the cell tissue into different components: the resistance of the interstitial liquid, the resistance of the cell content, the resistance of the cell wall and the capacity of the cell wall. In 1963, a practical application based on the dielectric properties of fish was developed (Hennings 1960, 1961, 1962, 1963, 1964). The conductance of fish tissue changes more rapidly than the dielectric properties, and simple alternating current measurements of impedance are not satisfactory. The Intellectron Fischtester VI overcame this problem and that of polarisation by measuring purely capacity changes from a simultaneous determination of impedance at two different frequencies (Hennings 1963, 1964; Schwan 1963). The instrument worked on the basis of a Wheatstone bridge. The Intellectron Fischtester VI measures transversally through the entire fish; the electrodes are applied on the lateral line of the body close to the anal opening. The readings range from 0 to 100 and in a few species, rarely, the readings exceed 100. The Fischtester VI is equipped with an analogue indicator and the latest version, the VIa, with a digital one.

13.3 13.3.1

Torrymeter Background

At the same time as the Fischtester was being developed in Germany, a different approach to counter the effects of electrode polarisation was being taken in the UK. A long, unpublished study of the effects of spoilage on the electrical properties of fish tissues had opted for the use of a four-electrode system as used in biological studies to eliminate these effects (Jason and Lees 1971). In such a system, the applied current is through two electrodes at which, of course, polarisation develops. Positioned between these current electrodes a further pair of electrodes connected to a very high input impedance, draw no current and merely measure electric potential. Because there is negligible current there is also no polarisation at these two electrodes. Typical variations with days-in-ice of measurements of resistance and capacitance using a four-electrode system are shown in Figure 13.1 (Jason and Lees 1971). There it can be seen that the resistance, after falling rapidly in the first week or so, gradually changes more slowly as time progresses. The capacitance, however, hardly changes at all in the first stage, and in fact appears to increase slightly. Thereafter, as the rate of change of resistance decreases, that for

Fishery Products: Quality, safety and authenticity 2500

0.1

Resistance (Ω)

Q-factor

0.2

Resistance Capacitance Q-factor

2000

0.4 0.3

0.025

0.020

1500

0.015

1000

0.010

500

0.005

0

0

5

10 Days-in-ice

15

Capacitance (μF)

288

0 20

Figure 13.1 Typical variation with storage time of resistance, capacitance, and Q-factor of cod skin and muscle kept in ice (After Jason and Lees 1971).

capacitance increases considerably. From these observations it was concluded that neither variable on its own was entirely suited for measurement of time of storage. Some product of capacitance, C, and resistance, R, would be better in this respect and such a factor is the electrical Q-factor (not to be confused with the Q in QIM) defined as Q = 2πfCR, where f is the frequency of measurement. The Q-factor of the fish is also related to the reactive lag created between current flowing and voltage dropped across the ‘potential’ electrodes (current leads the voltage by phase angle f). At 2 kHz, the frequency of operation chosen for the Torrymeter (as it is now called), this phase angle is rarely in excess of some 0.4 radians. Defining Q as tan f gives an equivalent value of 0.42 for the Q-factor. It is thus a small approximation to redefine Q for the range expected as Q = f. The separation, size and shape of the electrodes are not critical because the phase lag is independent of such variables. The electrode design opted for is shown in Figure 13.2. The potential electrodes are made of stainless steel and the concentric current electrodes are of graphite. A further big difference in approach between the Fischtester and the Torrymeter is that with the four-electrode design, measurements are made only on one side of the fish whereas the Fischtester measures through the body of the fish. Both methods have their advocates, but a comparative study (Burt et al. 1976) of early prototypes showed only that the Fischtester was slightly more sensitive to days-in-ice, and for similar results the Torrymeter required some 50% more samples to be measured. The basic circuit of the Torrymeter is shown in Figure 13.3 (Jason and Richards 1975). The output from a 2 kHz sinusoidal oscillator is applied to the current electrodes P1 and P4 through a resistance R1. The voltage dropped across this resistance is proportional to, and in phase with, the current flowing through the circuit and is fed to an amplifier A2. Z1 and Z4 constitute both polarisation impedance and so-called fringing field impedance at each current electrode. Z2 and Z3 are polarisation impedances due to any current drawn by the electrodes P2 and P3. Z2 and Z3 may be ignored if the input impedance to the amplifier A1

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P1

Z1

P2

C1

Z2

R2

C

R

Z4

A1

C1

Z3

R2

NAND

P3

Lowpass filter

A2

P4 R1

Figure 13.2 Block diagram of Torrymeter. All impedances associated with the electrode assembly in contact with fish are enclosed within the broken line (After Jason and Richards 1975).

I

D

F A C

G

H

B

E

Figure 13.3 Four-electrode assembly: A, stainless steel potential electrodes; B, epoxy resin insulation; C, graphite current electrode; D, polycarbonate case; E, Tufnol block; F, neoprene seal; G, thermistor (when installed); H, thin metal disk; I, thermistor leads (After Jason and Richards 1975).

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is sufficiently large and negligible current is drawn. Here the input impedance was set at 10 MΩ by the resistors R2 and R3. The impedance of the sample (fish) is Z and is both resistive and capacitive as described previously. The potential measured across P2 and P3 is therefore out of phase, lagging behind the potential across R1, by a phase angle of f. It is fed by small blocking capacitors (to eliminate direct current potentials due to the electrolytic nature of the samples) to amplifier A1. The amplifiers A1 and A2 have extremely high gain, which means that their outputs are approximately square waves. If the two outputs are combined in a logic gate (NAND), then the output of this gate is a train of pulses, whose length is proportional to f. It is readily shown that the direct current component of such a pulse train is proportional to f and can be obtained by simple integration. If the temperature of the fish is not constant, then f shows temperature dependence. In some versions of the instrument, temperature compensation was provided by insertion of a thermistor between the electrodes (see Figure 13.2). A simple compensatory circuit was coupled to this to modify the output accordingly. Conveniently, thermistors with similar temperature characteristics to fish can be found and incorporated. Because the response as a function of days-in-ice was found to be logarithmic in nature, the output was transformed to provide a linear function F ranging from 0 to 19 according to the equation F = 10 log( Aφ ) The adjustable arbitrary factor A is found empirically to be 81. The instrument is easily calibrated using a resistance/capacitive network of similar values to those of fish and is used by placing the electrodes firmly on a position close to the lateral line, in the middle portion of the fish.

13.3.2 Use of Torrymeter From its inception and the first experimental data collected, it was clear that factors other than spoilage contributed in part to the meter readings (Jason and Lees 1971), variation in readings occurring for a given number of days on ice, due to provenance, season and species. The observed dependence on species is accounted for in use by the provision of different calibrations for each species. A study of both the Torrymeter and the Fischtester (Burt et al. 1976) revealed some interesting and important variations. Within the range of sizes of the fish studied, there was no significant effect of fish size for the Torrymeter, and only a variation equivalent to ±¼ day for the Fischtester. This compared well with sensory and chemical methods such as trimethylamine and hypoxanthine, which showed a difference in apparent freshness of ±½, ¾ and 2 days, respectively. When dealing with boxed fish there was a pronounced effect of the layer from which the fish were taken. Compared with fish at the bottom, the top layer appeared fresher by about 1½ days for the Torrymeter and 1 day for the Fischtester. This effect was only observed in one other aspect of the samples and that was in the appearance scores. Undoubtedly it is due to superficial damage arising from the weight of fish above the lower layers. There were also between box variations for which no explanation was given. A method for defining the

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Table 13.1 Sensitivity of methods expressed in units of days-in-ice. The estimate is that obtained directly for the calibration data using the known samples whereas the prediction is the sensitivity expected using unknown samples. Method Torrymeter Fischtester TMA General appearance Raw odour Cooked odour Cooked flavour

Estimate

Prediction

2.6 2.1 1.4 0.9 0.7 0.7 0.7

2.8 2.3 1.7 1.1 0.9 1.0 1.0

Sources: Hennings 1961; Botta 1995

Table 13.2 The number of samples required to achieve a sensitivity of ±1 day with 95% confidence. Method Torrymeter Fischtester TMA General appearance Raw odour Cooked odour Cooked flavour

Estimate

Prediction

27 17 7 3 2 2 2

31 20 10 4 3 4 4

Sources: Hennings 1961; Botta 1995

sensitivity of a technique (Baines and Shewan 1965) was used for these results. This sensitivity criterion is calculated as the ratio of the standard deviation to the regression coefficient (gradient) of the instrument readings versus days-in-ice. In other words, the standard deviation is expressed in units of days-in-ice enabling a direct comparison between methods. The consequence of such calculations gave a table of method sensitivities, shown in Table 13.1, where the trimethylamine test is included for comparison and results relating to various sensory assessments (Burt et al. 1975). The data in Table 13.1 refer to single measurements. It was also calculated how many measurements would need to be taken (how many fish to measure) to predict the age-in-ice to ±1 day with 95% confidence. For an estimation to lie within ±n days-in-ice, the number of fish is given by the value of (t × s/n)2, where s is the sensitivity and t is the Student’s t-value corresponding to the desired significance level and the number of samples used to calculate the standard deviation. These results are shown in Table 13.2. As can be seen, a large number is indeed required for the Torrymeter compared with using sensory scores. To a slightly lesser extent, this is also true for the Fischtester. However, it can be argued that the ease and speed of measurement of both instrumental methods greatly exceeds that for any chemical or sensory method. In addition, some may choose to ignore the poorer performance of the Torrymeter in exchange for its ease of use. This is why the

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device is provided with the facility to average 16 results on different fish. In this way the mean of the readings is related to the age of the sample and the uncertainty is reduced. To use the Torrymeter, or any other instrument, to grade individuals is a mistake sometimes made. For each ‘quality’ or age-in-ice of a group of specimens there will be a statistical distribution of readings due to biological variation and other factors as discussed above. To select the extremes of such a distribution as being high or low quality is to assign them to the wrong population and degrade the sensitivity of the method considerably.

13.3.3 Applications to freshness using the Torrymeter, successful or otherwise In the decades since their introduction, many experiments have been reported using electronic fish testers. Most such experiments have found the method useful and in many cases superior to alternative methods. Even for eels (Anguilla anguilla) (Rehbein and Hinz 1983), with which excessive slime could possibly cause problems for electrode contact, removing this slime reduced the sensitivity of prediction of storage time. There have also been some failures, notably purple emperor (Lethrinus lentjan), Spanish mackerel (Scomberomorus commerson), red snapper (Lutjanus sanguineous) (Curran et al. 1981) and Senegalese sole (Solea senegalensis) (Tejada et al. 2006). Such failures often, however, have a reason unconnected with the performance of the meter. With such a large amount of data to summarise, it is best to tabulate everything, as in Table 13.3.

Table 13.3 All the species of fish studied with the Torrymeter and other variables correlated with the meter readings. Species

Eel (Anguilla anguilla) Purple emperor perch (Lethrinus lentjan) Spanish mackerel (Scomberomorus commerson) Red snapper (Lutjanus sanguineous) Senegalese sole (Solea senegalensis) Sea bream (Pagelus caupei) Chub mackerel (Rastrelliger brachysoma) Grouper (Plectropoma maculatum) Fusilier (Caesio cuning) Vara-vara (Lutjanus bohar) Cordonnier blanc (Siganus abhortanti) Lascar (Lethrinus enigmaticus)

Days-in-ice

Sensory variables

Other quality variables

Reference

Y N

Rehbein and Hinz 1983 Curran et al. 1981

N

Curran et al. 1981

N

Curran et al. 1981

N

Tejada et al. 2006

Y Y

Amu and Disney 1973 Poulter et al. 1978

Y

Poulter et al. 1978

Y Y Y

Poulter et al. 1978 Hoffman 1981 Hoffman 1981

Y

Hoffman 1981

Table 13.3

Continued

Species

Days-in-ice

Sensory variables

Southern blue whiting (Micromesistius australis) Cod (Gadus morhua)

Y

Haddock (Melanogrammus aeglefinus) Whiting (Melanges merlangus) Redfish (Sebastes sp) Saithe (Pollachius virens) Plaice (Pleuronectes platessa) Herring (Clupea harengus)

Y

Mackerel (Scober scombrus)

Y

Yellowtail flounder (Limanda feruginea) Scup (Stenotomus chrysops) Butterfish (Peprilus triacanthus) Oil sardine (Sardinella longiceps) Mackerel (Rastrelliger Kanagurta) Threadfin bream (Nemipterus japonicus) Catfish (Tachysurus sp.) Atlantic croaker (Micropogon undulates) Grey trout (Cynoscion regalis)

Y

Y

Y Y Y

Y Y

Y Y

Y

Y

Y

Red hake Urophycis chuss

Y

Winter flounder (Pseudopleuronectes americanus Waldbaum) European catfish (Silurus glanis) Northern squawfish (Ptychocheilus oregonensis) Gilthead seabream (Sparus aurata) Vendace (Coregonus albula L) Lemon sole (Microstomus kitt) Cassava fish (Otolithus brachygnathus) Chisawasawa (Lethrinops praeorbitalis) Lady fish (Otolithus senegalensis) Sea bream (Dentex canariensis) Sea bream (Pagellus coupei) Spanish mackerel (Scomber colias) Tilapia (Tilapia lidole)

Y

Y

Other quality variables

Reference

Barassi et al. 1981 Y

Y

Y

Y Y Y Y Y

Jason and Lees 1971; Burt et al. 1975, 1976; Cheyne 1975; Anon.; Connell et al. 1976 Burt et al. 1975; Cheyne 1975; Anon. Cheyne 1975; Anon. Cheyne 1975; Anon. Cheyne 1975; Anon. Cheyne 1975; Anon. Anon.; Pivarnik et al. 1990 Anon.; Pivarnik et al. 1990 Pivarnik et al. 1990 Pivarnik et al. 1990 Pivarnik et al. 1990 Lakshmanan et al. 1984

Y

Lakshmanan et al. 1984

Y

Sakaguchi et al. 1989

N Y

N Y

Lakshmanan et al. 1984 Townley and Lanier 1981 Townley and Lanier 1981 Fey and Regenstein 1982 Field et al. 1986

Y

Manthey et al. 1988 Lin and Morrissey 1994

Y

Lougovois et al. 2003

Y Y Y

Muje et al. 2002 Anon. Anon.

Y

Anon.

Y

Anon.

Y Y Y

Anon. Anon. Anon.

Y

Anon.

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Fishery Products: Quality, safety and authenticity

13.3.4 Distinguishing fresh from frozen From early days, it was known that the disruption caused to the tissue structure by freezing decoupled electronic meter readings from other aspects of quality. This in itself, however, could be made a virtue in applications where it was desired to differentiate previously frozen from fresh fish. For example, K-value measurements on yellowtail fillets (Seriola quinqueradiata) showed low values both for freshly chilled and for frozen fillets (Kim et al. 1987). Torrymeter readings, however, were high for the chilled fish and very low for the frozen fish. This combination of variables readily differentiated fresh from frozen. In addition, the appearance of the fish in terms of skin colour could also be combined with the Torrymeter reading to provide this information. Repeated freeze–thaw cycles of carp (Cyprinus carpio) were also shown to increase the difference marginally between fresh and frozen (Sakaguchi et al. 1989). This ability to differentiate is common to both electronic freshness meters. A comparison of Fischtester and Torrymeter on hake (Caracciolo 1988) demonstrated this. More recently, a comparison between enzyme activity measurement and Torrymeter readings used plaice, whiting and mackerel, and demonstrated the efficacy of the Torrymeter in differentiating fresh from frozen whole fish (Duflos et al. 2002).

13.3.5

Other applications

Of all the applications of the Torrymeter, perhaps the most arcane is that found in competition fishing, notably in the USA. It would appear that the pressure to win such contests, which carry large amounts of prize money, has led some competitors in the past to reinterpret the rules in their own favour and present previously caught, or even frozen fish, as fish caught during the competition. The novel Double Whammy by Carl Hiaasen, exposing corruption at a Florida bass fishing contest, seems less a work of fiction in this light. Clearly the Torrymeter can identify such rogues by measurement either of freshness or by the clear indication that such a healthy looking fish must have been frozen. To this end, many competitions now include in the rules that either the Torrymeter reading must be above a specified level for the fish to be accepted as caught during the event, or even merely threaten the possibility of the fish being checked (Anon. 2006a, b, c). It is no doubt encouraging for the manufacturers to see such confidence placed in their instrument.

13.4

Use of the Fischtester

Shortly after the introduction of the Fischtester, it was tested by many scientists and fish technologists (Castell 1965; Wittfogel and Schlegel, 1965b) and the following citation shows what the scientific community thought about this new device for fish freshness quality measurement: ‘The findings demonstrate that the electronic resistance values for these three species, stored from death under fairly standard conditions, being neither badly pressed, bruised, damaged,

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bleached, contaminated, desiccated, heavily parasitized, temporarily frozen and re-thawed, nor in contact with electrolytic substances or solutions (salt, acids, alkalis) show a good correlation to the organoleptic classification results. The average values of the electronic measurement of a larger number of tested fish of the same species, even disregarding their source of supply and the fishing grounds they come from, are reliable scores for wet fish quality grading.’

The use of the Intellectron Fischtester VI was reviewed (Oehlenschläger and Nesvadba 1988; Oehlenschläger 2003; Oehlenschläger 2005) and was thoroughly investigated and found to be suitable for fresh fish grading (Münkner 1965; Wittvogel and Schlegel 1965a; Gibson and Shewan 1971). Electronic meters were used for the determination of days in ice and the prediction of days in ice left for lean fish species (Nunes et al. 1992; Meyer and Oehlenschläger 1996; Oehlenschläger 1998a, b, 2003, Oehlenschläger and Rehbein 2001; Ferri et al. 1995), for medium fatty and fatty fish species (Damoglou 1980; Nunes et al. 1992; Rehbein et al. 1994), for slimy and de-slimed eel (Rehbein and Hinz 1983) and for aquacultured fish species (Guidotti et al. 1996). The instruments have also been used to differentiate between fresh and frozen/thawed fish (Rehbein and Aust 1980) and to examine the influence of catching methods on fish quality. A prerequisite for using instruments like the Fischtester, however, is a sound knowledge of baseline readings for freshly caught fish from different species. Readings from samples of unknown history and origin can only be assessed with this knowledge base. In the years 1981–1985 and in 2002, these basic data were collected during numerous cruises with German research vessels. Table 13.4 gives selected information on Fischtester VI readings typical for marine species tested immediately after hauling. Similar calibration work was also corrected out with the Torry meter on UK research vessels. Because Fischtester measurements are based on the existence of intact cell membranes, they fail when the cells are disrupted or broken. In frozen/thawed fish, where no intact cell membranes have survived the freezing/thawing cycle, Fischtester readings are always 0 because of very high conductivity. This phenomenon was used to differentiate fresh fish from frozen/thawed fish that was offered as fresh. Frozen/thawed fish could be rated highly by QIM but a Fischtester measurement of 0 would show, reliably and instantly, that the perfect looking fish had undergone a freezing/thawing cycle. The skin and the mucus of fish contribute most to the impedance measured. This explains the fact that also in stale fish, where the Fischtester readings should be down to 0, readings above 0 can be found, which are produced by resistance of skin and any remaining slime. Therefore, electrical measurements give poor results in skin-on fillets (one skin lacking), skinless fillets (both skins lacking) and de-slimed fish (for example eel). In fillets, these instruments can only be used for a short time after catch/harvest (up to 7 days). Hennings himself described the limitations of the Fischtester (Hennings 1963), leading to erroneous results that are similar also for the Torrymeter as: z z z z z z

skin injuries at measuring point scaled fish skin fish which has been in contact with electrolytes (salts, acids) fish partly or completely frozen fish undergone strong mechanical influences warmer than 10 °C when measured

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Table 13.4 Basic Fischtester data of different fish species from North Atlantic, all data measured on freshly caught fish (less than 1 hour post mortem). Species

Year

Catching area

N

Arithmetic mean

Median

Min.

Max.

SD

marinus marinus marinus marinus marinus

1984 1984 1984 1984 1982

98 99 100 100 108

67.72 65.68 69.96 68.06 66.92

67 66 70 68 66

59 57 60 57 59

87 74 80 78 79

5.61 3.56 3.58 3.53 4.31

Sebastes mentella Sebastes mentella

1984 1982

100 101

76.16 71.50

77 72

56 58

94 80

7.98 4.92

Sebastes mentella Sebastes mentella Sebastes marinus

1981 1982 2002

81 99 155

43.91 74.35 66.27

44 74 66

32 66 57

54 86 78

5.56 3.67 4.10`

Gadus morhua Gadus morhua Gadus morhua Melanogrammus aeglefinus Melanogrammus aeglefinus Melanogrammus aeglefinus Pollachius pollachius Micromesistius poutassou Coryphaenoides rupestris Coryphaenoides rupestris Macrurus berglax Aphanopus carbo Clupea harengus

1984 1984 1982 1984

WBW WBW WBW WBW East Greenland WBW East Greenland WBW Barents Sea East Greenland WBW WBW Barents Sea Rockall Bank

100 74 108 100

78.86 74.61 76.88 85.71

79 75 77 86

70 62 64 68

90 87 85 100

3.21 5.27 3.72 4.68

1982

Rockall Bank

100

87.54

88

74

98

4.77

1982

Rockall Bank

57

80.98

82

59

93

7.79

1984 1983

Rockall Bank WBW

101 99

68.71 84.90

68 86

44 68

76 99

4.68 5.32

1981

WBW

100

52.42

53

34

63

5.39

1983

WBW

100

57.84

58

34

72

6.83

1983 1981 1983

WBW WBW St Kilda

99 56 100

42.48 88.34 72.08

42 88 72

30 74 54

64 98 80

5.54 5.19 4.51

Sebastes Sebastes Sebastes Sebastes Sebastes

WBW, West British waters

z z

dried out skin (shrinking when bent) surface of electrodes covered by slime or scales.

13.5

Summary

The two instruments described in detail are the only extant devices based on electrical properties known to the authors at the time when the chapter was written. There have been others in the past but they are no longer available and at least one was based on the Torrymeter and manufactured under licence. However, recently a new instrument, the Kiel Fish Meter (KFM), was developed in Germany and tested successfully on board a research vessel (Mierke-Klemeyer et al. 2008). Though indisputably useful for quality control, there are many interfering factors of which the user should be aware. Anything that causes damage at the cellular level may be consid-

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ered such a factor. This includes prior total or partial freezing, irradiation, rough handling, or storage in chilled seawater. In addition there are influences due to species and provenance of specimens (fishing ground), and seasonal physiological changes due to spawning, feeding cycle, maturity, and so on.

13.6

References

Amu, L. and Disney, J.G. (1973) Quality changes in West African marine fish during iced storage. Tropical Science 15: 125–138. Anon. Fish Freshness Meter Operators Handbook, Distell.com, Fauldhouse, UK. Anon. (2006a) 3rd Annual Texas Kingfish Classic, rules and regulations. Texas King Mackerel Association, Houston, Texas. Anon. (2006b) Alabama Deep Sea Fishing Rodeo. Mobile, Alabama, USA. Anon. (2006c) Pensacola Bud Light King Mackerel-Cobia Tournament and USADigital/Nextel In-Shore Classic, Florida. Baines, C.R. and Shewan, J.M. (1965) Lab Practise 14: 160. Barassi, C.A., Boeri, R.L., Crupkin, M., Davidovich, L.A., Giannini, D.H., Soulé, C.L., Trucco, R.E. and Lupín, H.M. (1981) The storage life of iced southern blue whiting (Micromesistius australis). Journal of Food Technology 16: 185–197. Botta, J.R. (1995) Evaluation of Seafood Freshness. VCH, New York, Weinheim and Cambridge, pp. 61–62. Burt, J.R., Gibson, D.M., Jason, A.C. and Sanders, H.R. (1975) Comparison of methods of freshness assessment of wet fish. I. Sensory assessments of boxed experimental fish. Journal of Food Technology 10: 645–656. Burt, J.R., Gibson, D.M., Jason, A.C. and Sanders, H.R. (1976) Comparison of methods of freshness assessment of wet fish. II. Instrumental and chemical assessments of boxed experimental fish. Journal of Food Technology 11: 73–89. Caracciolo, S. (1988) L’impiego di strumenti elettronici nel controllo del pesce. Il Pesce V: 73– 79. Castell, C.H. (1965) Preliminary studies of quality assessment with the Intelectron Fischtester V. In: R. Kreuzer (Ed.) The Technology of Fish Utilisation. Fishing News Books, London, pp. 158–161. Cheyne, A. (1975) How the GR Torrymeter aids quality control in the fishing industry. Fishing News International 14(12): 117–118. Connell, J.J., Howgate, P.F., Mackie, I.M., Sanders, H.R. and Smith, G.L. (1976) Comparison of methods of freshness assessment of wet fish. IV. Assessment of commercial fish at port markets. Journal of Food Technology 11: 297–308. Curran, C.A., Nicolaides, L. and Al-Alawi, Z.S. (1981) Quality changes during iced storage of three commercially important fish from Bahrain. Tropical Science 23: 253–268. Damoglou, A.P. (1980) A comparison of different methods of freshness assessment of herring. In: J.J. Connell (Ed.) Advances in Fish Science and Technology. Fishing News Books Ltd, Farnham, Surrey, UK, pp. 394–399. Duflos, G., Le Fur, B., Mulak, V., Becel, P. and Malle, P. (2002) Comparison of methods of differentiating between fresh and frozen-thawed fish or fillets. Journal of the Science of Food and Agriculture 82: 1341–1345. Ferri, M., Mattei, P., Civera. T. and Gili, S. (1995) Considerations on the employment of electrical conductivity in freshness assessment of nine Mediterranean fish species. Industrie Alimentari 34: 1277–1282.

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Fey, M.S. and Regenstein, J.M. (1982) Extending shelf-life of fresh wet red hake and salmon using CO2–O2 modified atmosphere and potassium sorbate ice at 1 °C. Journal of Food Science 47: 1048–1054. Field, C.E., Pivarnik, L.F. Barnett, S.M. and Rand, A.G. Jr (1986) Utilization of glucose oxidase for extending the shelf-life of fish. Journal of Food Science 51: 66–70. Gibson, D.M. and Shewan, J.M. (1971) Some recent results on the use of the Intelectron Fish Tester MK 5. In: R. Kreuzer (Ed.) Fish Inspection and Quality Control. Fishing News Books, London, pp. 208–210. Guidotti, P., Geri, G., Mecatti, M., Parisi, G., Lupi, P. and Poli, B.M. (1996) Changes of dielectric properties in rainbow trout (Oncorhynchus mykiss) and sea bass (Dicentrarchus labrax) at different storage temperatures. Refrigeration and Aquaculture. IIR Commission C2, Bordeaux, pp. 387–392. Hattula, T., Kiesvaara, M. and Moran, M. (1993) Freshness evaluation in European whitefish (Coregonus wartmanni) during chill storage. Journal of Food Science 58: 1212–1215. Hennings, C. (1960) Neues Verfahren zur objektiven Schnellbestimmung des Frischegrades von Fisch. Informationen für die Fischwirtschaft 7: 164–165. Hennings, C. (1961) Neues Verfahren zur objektiven Schnellbestimmung des Frischegrades von Nutzfischen. Informationen für die Fischwirtschaft 8: 65–67. Hennings, C. (1962) Neues elektronisches Verfahren und Gerät zur objektiven Schnellbestimmung des Frischegrades von Nutzfischen. Informationen für die Fischwirtschaft 9: 115– 117. Hennings, C. (1963) Ein elektronisches Schnellverfahren zur Ermittlung der Frische von Seefischen. Zeitschrift für Lebensmittel Untersuchung und Forschung 1: 462–477. Hennings, C. (1964) Der Fischtester. AFZ Jahresheft der Fischwirtschaft, 1.1.1994. Hennings, C. (1965) The ‘Intelectron fish tester V’. A new electronic method and device for the rapid measurement of the degree for the freshness of ‘wet’ fish. In: R. Kreuzer (Ed.) Fish Inspection and Quality Control. Fishing News Books, London, pp. 154–158. Hoffman, A. (1981) The use of the GR Torrymeter for the assessment of freshness of iced tropical fish from the Indian Ocean. Tropical Science 23: 283–291. Jason, A.C. and Lees, A. (1971) Estimation of Fish Freshness by Dielectric Measurement. Department of Trade and Industry, London. Jason, A.C. and Richards, J.C.S. (1975) The development of an electronic fish freshness meter. Journal of Physics E: Scientific Instruments 8: 826–830. Kim, J.-B., Murata, M. and Sakaguchi, M. (1987) A method for the differentiation of frozen-thawed from unfrozen fish fillets by a combination of Torrymeter readings and K-values. Nippon Suisan Gakkaishi 53: 159–164. Lakshmanan, P.T., Mathen, C., Varma, P.R.G. and Iyer, T.S.G. (1984) Assessment of quality of fish landed at the Cochin fisheries harbour. Fishery Technology 21: 98–105. Lin, D. and Morrissey M.T. (1994) Iced storage characteristics of northern squawfish (Ptychocheilus oregonensis). Journal of Aquatic Food Product Technology 3: 25–43. Lougovois, V.P., Kyranas, E.R. and. Kyrana, V.R. (2003) Comparison of selected methods of assessing freshness quality and remaining storage life of iced gilthead sea bream (Sparus aurata). Food Research International 36: 551–560. Manthey, M., Karnop, G. and Rehbein, H. (1988) Quality changes of European catfish (Silurus glanis) from warm-water aquaculture during storage on ice. International Journal of Food Science and Technology 23: 1–9. Meyer, C. and Oehlenschläger, J. (1996) Sensorische Bewertung, Mikrobiologie und chemische Kenngrößen von eisgelagertem Wittling (Merlangius merlangus). Informationen für die Fischwirtschaft 43: 89–94.

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Mierke-Klemeyer, S., Oehlenschläger, J., Knöchel, R., and Taute, W. (2008) Schnelle Qualitätsbeurteilung von Frischfisch: Das Kiel Fish Meter (KFM) in Praxistest (Rapid quality evaluation of fresh fish: The Kiel Fish Meter (KFM) tested in practical application. Archiv für Lebensmittelhygiene 59: 227–232. Muje, P., Pursiainen, J., Hyvönen, P., von Wright, A. and Raatikainen, O. (2002) Seasonal variation of storage properties of vendace (Coregonus albula L). Journal of Aquatic Food Product Technology 11: 21–29. Münkner, W. (1965) Untersuchungen über die Aussagefähigkeit des FISCHTESTER V zur objektiven Qualitätsbestimmung bei Seefisch. Fischerei-Forschung Wissenschaftliche Schriftenreihe 3: 59–64. Niu, J. and Lee, J.Y. (2000) A new approach for the determination of fish freshness by electrochemical impedance spectroscopy. Journal of Food Science 65: 780–785. Nunes, M.L., Batista, I. and Moraode Campos, R. (1992) Physical, chemical and sensory analysis of sardine (Sardina pilchardus) stored in ice. J Sci. Food Agric. 59: 37–43. Oehlenschläger, J. (1995a) Bewertung von Frische- und Verderbsindikatoren bei der Eislagerung von Schollen (Pleuronectes platessa). Informationen für die Fischwirtschaft 42: 94–102. Oehlenschläger, J. (1995b) Haltbarkeit von Nordsee-Wittling (Merlangius merlangus) bei Lagerung in schmelzendem Eis. Informationen für die Fischwirtschaft 42: 42–49. Oehlenschläger, J. (1998) Detailed experimental iced-storage characteristics of Barents Sea cod (Gadus morhua). Informationen für die Fischwirtschaft 45: 35–42. Oehlenschläger, J. (2003) Measurement of freshness of fish based on electrical properties. In: J.B. Luten, J. Oehlenschläger and G. Olafsdottir (Eds) Quality of Fish from Catch to Consumer: Labelling, Monitoring and Traceability. Wageningen Academic Publishers, Wageningen, pp. 237–249. Oehlenschläger, J. (2005) The Intellectron Fischtester VI – an almost forgotten but powerful tool for freshness and spoilage determination of fish at the inspection level. In: J. Rydes and L. Ababouch (eds) Fifth World Fish Inspection and Quality Control Congress, FAO Fisheries Proceedings No. 1, pp. 116–122. Oehlenschläger, J. and Nesvadba, P. (1998) Methods for freshness measurement based on electrical properties of fish tissue. In: G. Olafsdottir, J. Luten, P. Dalgaard, M. Careche, V. Verrez-Bagnis, E. Martinsdottir and K. Heia (Eds) Methods to Determine Freshness of Fish in Research and Industry. IIR, Paris, pp. 363–368. Oehlenschläger, J. and Rehbein, H. (2001) Shelf life of gutted and ungutted ocean perch (Sebastes marinus and S. mentella) during ice-storage. Annales Societatis Faeroensis Suppl. XXVIII: 51–61. Osterhout, W.J.V. (1922) Injury, Recovery and Death in Relation to Conductivity and Permeability. J.B. Lipincott, London. Pivarnik, L.F., Kazantzis, D., Karakoltsidis, P.A., Constantinides, S., Jhaveri, S.N. and Rand, A.G. Jr (1990) Freshness assessment of six New England fish species using the Torrymeter. Journal of Food Science 55: 79–82. Poulter, R.G., Nicolaides, L. and Hector, D.A. (1978) Quality changes in fish from the South China Sea. 1. Iced storage of chub mackerel, grouper and fusilier. Proceedings of the Indo-Pacific Fisheries Commission 18(3): 169–185. Rehbein, H. and Aust, M. (1980) Einsatzmöglichkeiten des Torrymeters und enzymatischer Analysenverfahren zur Untersuchung eisgelagerter Fische und Filets auf Auftauware. Archiv für Fischerei Wissenschaft 30: 181–188. Rehbein, H von and Hinz, A. (1983) Vergleich zwischen physikalischen und chemischen Methoden zur Bestimmung des Verderbs unbehandelter oder entschleimter Aale bei Eislagerung. Archiv für Lebensmittelhygiene 34: 53–80.

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Rehbein, H., Martinsdottir, E., Blomsterberg, F., Valdimarsson, G. and Oehlenschläger, J. (1994) Shelf life of iced-stored redfish, Sebastes marinus and S. mentella. International Journal of Food Science Technology 29: 303–313. Sakaguchi, M., Murata, M. and Kim, J.-B. (1989) The effects of repeated freeze–thaw cycles on Torrymeter readings of carp fillets. Nippon Suisan Gakkaishi 55: 1665–1669. Schwan, H.P. (1963) Determination of biological impedances. In: W.L. Nastuk (Ed.) Physical Techniques in Biological Research, volume 6, part B. Academic Press, London, pp. 323– 406. Tejada, M., De las Heras, C. and Kent, M. (2006) Changes in the quality indices during ice storage of farmed Senegalese sole (Solea senegalenisis). European Food Research and Technology 225: 225–232. Townley, R.R. and Lanier, T.C. (1981) Effect of early evisceration on keeping quality of Atlantic croaker (Micropogon undulates) and grey trout (Cynoscion regalis) as determined by subjective and objective methodology. Journal of Food Science 46: 863–867. Wittvogel, H. and Schlegel, H.L. (1965a) The suitability of the Intelectron fish tester V in the daily quality control and grading of wet fish landings. In: R. Kreuzer (Ed.) Fish Inspection and Quality Control. Fishing News Books, London, pp. 162–164. Wittfogel, H. and Schlegel, H.L. (1965b) Brauchbarkeit des Intellectron Fisch-Testers V bei der täglichen Qualitätskontrolle und Klassifizierung von Seefischanlandungen. Zeitschrift für Lebensmittel Untersuchung unt Forschung 127: 85–92.

Chapter 14

Two-dimensional gel electrophoresis Flemming Jessen

14.1

Introduction

The use of two-dimensional gel electrophoresis (2DE) for the assessment of quality and safety is only in its initial stages, although 2DE technology has been available for the past 30 years and has been used in biological and medical research. However, this technology offers a powerful analytical potential that can help solve many problems within seafood quality and safety. 2DE is ‘only’ an analysis of proteins, but it should be borne in mind that the complex expression of proteins in cells or tissues of an animal reflects the total homeostasis (regulation of internal environment to maintain stable) in the live animal, and how it is influenced by environments to the final metabolic outcome. 2DE can thus be essential when studying nearly every aspect of a live animal that has influence on quality or safety of the products made from it. The central role of proteins in the description of tissues applies throughout the entire production chain transforming the live animal to seafood. Accordingly, 2DE is able to depict this transformation and to be a tool for investigating how applied industrial processes influence the seafood. Figure 14.1 lists some parameters that influence live animals and seafood meat in relation to quality and it illustrates the central role of 2DE in research concerning quality, safety, health and nutrition. Initially 2DE was a technology for analytically separating proteins with no further analysis of them. In recent years, 2DE technology has gained increased interest within medicine owing to the advances made in microscale protein sequencing and mass spectrometry (Roepstorff 1997). Protein identification based on the picomolar amounts of protein that can be separated by and extracted from two-dimensional (2D) gels has made 2DE very usable for proteomics. This is also reflected by the fact that in most of the recently published studies, 2DE has been used as part of the proteomic approach for characterising cellular protein expression or for characterising changes in complex protein mixtures. Owing to its high power of separation, 2DE has been a cornerstone in proteomic work since the early 1990s when the proteome concept (the protein complement expressed by a genome) was established (Wilkins et al. 1996). However, current proteomics is based as much on non-gel-based separation technologies (e.g. liquid chromatography (LC)) as on gels. This chapter does not deal with proteomics as such, but focuses specifically on 2DE and 2DE approaches in seafood science. 301

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Figure 14.1 2DE, quality, health and nutrition. 2DE constitutes a scientific link between heredity and many environmental parameters on one side, and seafood quality, human health and human nutrition on the other. The influence of heredity and environmental factors on a fish is depicted in the protein expression profile as analysed by 2DE. Consumption of seafood has important effects on human health and nutrition, which can be studied by 2DE as a part of nutrigenomics.

14.2

Two-dimensional gel electrophoresis (2DE)

The 2D electrophoretic separation of proteins is the only technique that can separate thousands of proteins in one single analysis. Proteins are separated in the first dimension according to isoelectric point and in the second dimension according to molecular mass. Afterwards the proteins can be visualised in the gel by different staining procedures and the individual proteins are now recognised by their specific coordinates in the gel. When 2DE is run on cellular proteins, the result reflects a snapshot of the protein status in a cell or a tissue type. By analysing cells either under different experimental conditions or from different genotypes, it is possible to investigate the molecular basis of a cellular mechanism or a genetic trait by comparing the differentially expressed proteins.

14.2.1

Methodology of 2DE

In its initial stages, 2DE, as introduced by O’Farrel (1975), was not a robust method that could be reproduced. Moreover, at that time, the performance of 2DE could actually be

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regarded as a kind of art, only available as a useful method for the few skilled and committed laboratories. Many would say it still is, but 2DE has been considerably improved during the past 10–15 years. This is due mainly to the introduction and implementation of immobilised pH gradients (IPGs) in the first dimensional (1D) electrofocusing step (Görg et al. 1988), making the reproducibility of the gels much better. High-quality IPG strips covering broad or narrow pH ranges within pH 3–12 are now commercially available but separation of basic proteins is still problematic. The work by Angelika Görg’s group has contributed substantially to the development of procedures for 2DE that have made it possible to produce comparable gels in different laboratories when the same type of samples is analysed (Görg et al. 1999, 2000, 2004). A silver-stained 2D-gel after analysis of sarcoplasmic proteins from rainbow trout is shown in Figure 14.2, and the applied procedures are described in the figure legend.

14.2.2

Gel staining

One thing is to produce the gels. Another thing is to extract usable information from them. The first crucial step in this process lies in the visualisation of the proteins. Frequently used types of protein staining are based on Coomassie, silver or fluorescence dyes, but there are several other ways for detecting and quantifying proteins. For reviews, see Patton (2002) and Miller et al. (2006)). Coomassie and fluorescence-based methods are quantitative with a good dynamic range, whereas different methods using silver have a weak dynamic range. However, silver staining is still very usable because good quantification is not always essential because many of the sought-for differences between gels might be very evident: for example the presence or not of a protein spot; or a many-fold increase or decrease in the amount of a certain protein in the gels.

14.2.3

Image and data analysis

To compare 2D gels, it must be possible to recognise with absolute certainty the same protein spot in different gels. Computer-based image analysis is used for such an alignment by evaluating relative spot positions in the gels. Individual spots are either quantified before, during or after gel alignment. Several types of dedicated software based on different principles are on the market (Wheelock and Goto 2006) and their applicability has lately increased considerably. In the light of all the improvements that have already been made, what is currently being done and what will be done in the near future on the technological side of 2DE, the really huge challenge for the future use of 2DE is the extraction of comprehensive complex information from the gel images. Then 2DE has the potential to provide a substantial increase in our level of knowledge. Standard statistical methods such as Student’s t-test, ANOVA and non-parametric tests are the tools mainly used for comparing 2DE data, and multivariate data analysis seems to be one of the future ways of obtaining more information from the gels (Jessen et al. 2002; Faergestad et al. 2004; Jacobsen et al. 2007; Jia et al. 2006; Kjaersgard et al. 2006).

4.2

pI

7.0 Molecular mass (kDa) 200

8

97

7

2

66 55

6 1

4

36 31 5

3

21

Figure 14.2 Two-dimensional gel of water soluble proteins from rainbow trout muscle. Protein extraction. 200 mg muscle was homogenized (Polytron PT 1200, KINEMATICA, Switzerland) in 2 ml buffer A (50 mM Tris-HCl, pH 7.4 and 1 mM ethylenediaminetetraacetic acid (EDTA)) three times for 30 s each with a pause on ice for 30 s. The homogenate was centrifuged for 20 min. at 0°C at 11.200 g, and the supernatant was collected as water-soluble proteins (sarcoplasmic proteins). Protein concentration was determined according to Peterson (1977), a simplification of the (Lowry et al. (1951) method with the modification that precipitation by tri-chloroacetic acid (TCA) was performed in the presence of 0.7% (w/v) sodium dodecyl sulphate (SDS) and 90 mM Tris-HCl, pH 7.5 as introduced by Kaplan and Pedersen (1989). The sample extracts were stored at −80°C until electrophoresis. Two-dimensional polyacrylamide gel electrophoresis. First-dimensional isoelectric focusing (IEF) was performed in 18 cm linear immobilized pH gradients 4–7 (IPG; Immobiline Drystrips, Amersham Biosciences). Samples were diluted to 0.29 μg protein μl−1 in 8 M urea, 2 M thiourea, 1.5% (w/v) 3-3-cholamidopropyl-dimethylammonio-1-propane sulphonate (CHAPS), 1% Pharmalyt 4-6.8, 1% Pharmalyt 5-8, 50 mM dithiothreitol (DTT), 10 mM Tris-HCl, pH 8.3, and Orange G as Dye. Protein (100 μg) in 350 μl sample was applied by rehydration of the IPG strip overnight at room temperature. Isoelectric focusing was performed at 21°C (Multiphor II flatbed, Amersham Biosciences). The separation was performed at 100 V for 5 h, followed by 5 h where voltage was gradually raised to 3500 V, which was maintained for 18 h (74,000 Vh). Upon termination of IEF, the focused 1D IPG strip was frozen and stored at −80°C. Second-dimensional separation was performed as 12% (w/v) SDS – PAGE. The gel (19 cm × 23 cm) was run as one of ten using the Hoefer DALT System (Amersham Biosciences). The IPG strip was quickly brought to room temperature and reduced for 20 min in 10 ml equilibration buffer (6 M urea, 50 mM Tris-HCl, pH 8.8, 30% (v/v) glycerol, 2% (w/v) SDS) with 1% (w/v) DTT, followed by a 20 min alkylation with 4.5% (w/v) iodoacetamide in 10 ml equilibration buffer. The strip was then placed on top of the second-dimensional gel and sealed with 0.5% (w/v) agarose, 25 mM Tris-base, 192 mM glycine and 0.1% (w/v) SDS. Electrophoresis was performed at 12°C at a maximum current of 40 mA per gel. The gels were silver stained by a modification of Hochstrasser et al. (1988). Each gel was fixed in 250 ml 11.5% (w/v) TCA and 3.45% (w/v) sulphosalicylic acid for 30 min followed by fixing in 250 ml 40% (v/v) ethanol and 10% (v/v) acetic acid for 30 min. After a wash in 500 ml water for 10 min the gel was incubated in 250 ml sensitizer (0.5 M Na-acetate and 0.125 % (v/v) glutaraldehyde) for 30 min and washed two times 10 min in 500 ml water. Then the gel was incubated in 250 ml silver solution (24 mM AgNO3, 9 mM NaOH, and 0.14% (v/v) NH3) for 20 min and again washed 5 min in 500 ml water. Development was in 250 ml 750 μM citric acid and 0.037% (v/v) formaldehyde (4–5 min) with formaldehyde added just before use. The silver reaction was stopped in 250 ml 30% (v/v) ethanol, and 7% (v/v) acetic acid. The marked proteins are: 1, glycogen phosphorylase; 2, enolase; 3, nucleoside diphosphate kinase; 4, carbonic anhydrase (cytoplasmic); 5, adenylate kinase; 6, tropomyosin (fast myotomal muscle); 7, actin (fast myotomal muscle); 8, myosinbinding protein.

Two-dimensional gel electrophoresis

14.3

305

2DE applications in seafood science

The use of 2DE in seafood science has not been extensive. However, as shown in the following, the limited number of works where it has been applied have actually covered a broad spectrum of quality-related aspects.

14.3.1

Authenticity

No 2DE or proteomic-based method has yet been officially accepted as an analytical technique with respect to authentication. This will probably be the case in the near future because the potential of 2DE-based methodologies for several aspects has already been well documented. Methods for molecular identification of species used in seafood are vital because of labelling regulations, cases of fraud and the occurrence of poisonous species. Methods targeting either proteins or DNA are already well established and widely used. For a review, see Martinez et al. (2003). In fresh or frozen products the main protein-based techniques are isoelectric focusing (IEF) and SDS–polyacrylamide gel electrophoresis (SDS–PAGE) of water-soluble protein fractions (Rehbein 1990; Piñeiro et al. 1999a). In heat-processed products, water-soluble parvalbumins can be used in some cases (EsteveRomero et al. 1996; Etienne et al. 2000), but solubilisation (e.g. by urea or detergents) of heat denatured proteins before analysis is often used (Etienne et al. 1999; Piñeiro et al. 1999a; Mackie et al. 2000). IEF and SDS–PAGE provide a substantial power of discrimination between most species and it is therefore not surprising that 2DE, having a much higher analytical resolution, also has this ability (Huang et al. 1995; Piñeiro et al. 1999b; Berrini et al. 2006). When it comes to closely related species such as hake (Merluccius spp.), where no clear differences between IEF protein patterns from two of five investigated hake species could be found, further analysis by 2DE of the water soluble protein fraction made it possible to differentiate between all five species (Piñeiro et al. 1998). 2DE discrimination between closely related species has also been demonstrated in five different Lagocephalus spp. from the puffer fish family, including both poisonous and harmless species (Chen et al. 2004). Puffer fish look very much alike, with only few morphological differences. They are often sold as fillets, which are even more indistinguishable than whole fish. Mistaking one species for another might have lethal consequences, making species identification a very important safety aspect (Civera 2003). A protein expression profile is the complex outcome of the interaction between the genes, the specific differentiation level of the actual analysed cells and the environmental influence on these cells. Therefore a protein expression profile has the potential to differentiate not only between species, but also between tissues (Martinez et al. 1991) within the same species or even between cell culture lines derived from the same species (Wagg and Lee 2005). Determination of geographical origin is very relevant in relation to stock management and fisheries regulations. Owing to the nature of a protein expression profile, 2DE has a clear potential here. To the knowledge of the author, only one attempt has been made on seafood (canned crabmeat) and this was unsuccessful (Gangar et al. 1996). Moreover, analysis

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of lipid composition, minerals or isotopes seems to be even more suitable than 2DE for determining geographical origin (Martinez et al. 2003). Pollution is an aspect of geographical origin and 2DE has demonstrated that it can offer an outstanding possibility for measuring the nature of the pollution (the toxic compound) that a fish has been exposed to, because pollution leads to changes in protein expression when the organism attempts to adapt to the new conditions and/or to counteract the effects of the compound. A protein expression profile might even be able to determine the extent of pollutant and perhaps the duration of exposure. So far, specific differences in protein expression have been found in mussels when exposed to copper, arsenic, tributyltin, polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs) and a general unspecified pollution (Shepard et al. 2000; Shepard and Bradley 2000; Rodriguez-Ortega et al. 2003; Olsson et al. 2004; Mi et al. 2005). Moreover, exposure of Chinese mitten crab (Eriocheir sinensis) to cadmium (Silvestre et al. 2006), of rainbow trout to zinc (Hogstrand et al. 2002) and of zebrafish to oestrogens (Shrader et al. 2003) has also resulted in differential protein expression profiles. For a general review on toxicoproteomics, see Wetmore and Merrick (2004). For genetically modified organisms (GMOs), 2DE technology will definitely play a central role in verifying whether an organism is transgenic or not through the detection of gene products from new, introduced genes. However, another and perhaps even more crucial role for 2DE is within safety evaluation of new transgenic organisms. In addition to the actually introduced gene, there may be unintended effects resulting in an alteration of the existing metabolism of the organism, which can be revealed by 2DE (Kuiper et al. 2001, 2002). Until now, no analysis of this kind has been performed in relation to seafood, whereas studies on plants have been recently published (Ruebelt et al. 2006).

14.3.2 Characterisation of post mortem metabolism With the long-term objective of relating hereditary and environmental factors to fish quality, it is important to study biochemical processes involved in post mortem quality changes in order to understand these processes and to find markers that enable the prediction of fish quality. Fresh seafood is most commonly cold stored on/in ice. The effects of ice storage on fish have been studied extensively for several decades to prevent deterioration and to prolong storage life. We are still far from a good understanding of the processes involved, but 2DE has the potential to provide a much more detailed knowledge of post mortem metabolism. Protein changes in cod muscle have, for example, been followed during ice storage, and 11 protein spots were found to change significantly. In nine spots the amount of protein (spot volume) increased, and for eight of these it happened within the first 2 hours post mortem. The intensity of the remaining two spots was found to decrease after 8 days. Finding different rates of change indicates that in fish muscle several independent biochemical processes are involved in the observed post mortem protein changes (Kjaersgard and Jessen 2003). Verrez-Bagnis et al. (2001) used SDS–PAGE and 2DE to find specific peptides and proteins as indicators of fish freshness. They showed that proteins from sea-bass muscle changed very little during post mortem ice storage, especially among the high molecular mass proteins. However, the 2DE revealed some changing proteins, in particular a sarcoplasmic

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(low salt extract) 16 kilodalton (kDa) protein component that started to disappear immediately after death and had almost completely disappeared after 96 hours (Verrez-Bagnis et al. 2001). This protein was denoted as nucleoside diphosphate kinase (Ladrat et al. 2003). Post mortem texture changes might involve degradation of connective tissue by different proteinases, of which matrix metalloproteinases (MMPs) are potentially involved. It is therefore highly relevant to study MMPs in combination with TIMPs (specific tissue inhibitors of MMPs) in relation to texture. An MMP is present in cod (Lodemel et al. 2004b) and a TIMP of 21 kDa has been found using 2DE in a design called two-dimensional real-time reverse zymography (Lodemel et al. 2004a). In reverse zymography the separation gel is co-polymerised with gelatine labelled with a fluorescent dye, and a monocyte cultureconditioned media is included in the gel as well as a source of MMP activity. After electrophoresis the gel is incubated to allow degradation of gelatine, and presence of TIMPs in the sample will then appear as light fluorescent spots owing to a reduced degradation of the labelled gelatine. Gels can be monitored repeatedly in ultraviolet light and therefore followed over time, so named real-time (Hattori et al. 2002). To elucidate quality aspects, another specialised application is the use of 2DE in combination with immunoblotting for generating new knowledge of protein oxidation in seafood. After fractionation of muscle proteins from rainbow trout in low- and high-salt buffers, protein oxidation was evaluated by labelling protein carbonyls with 2,4-dinitrophenyl hydrazine (DNPH), after which the proteins were separated by 2DE. Gels were then western blotted and DNPH-labelled proteins were detected by antibodies (Kjaersgard and Jessen 2004). This study revealed that only certain proteins, constituting 1% of the proteins present in the 2D gel, seem prone to oxidation. These proteins were oxidised at different levels showing a protein specificity of the oxidative modifications. A tainting storage at room temperature for 48 hours resulted in only a limited increase in the number of oxidised proteins, but the extent of oxidation was increased for several of the proteins already oxidised, especially tropomyosin, actin and other high-salt soluble proteins. Future identification of the oxidised proteins revealing their specific cellular or biochemical function might indicate whether they are linked to lipid oxidation (Kjaersgard and Jessen 2004).

14.3.3

Technological quality

Many aspects of processing conditions can be studied by 2DE. It is potentially possible to find and assess effects of even small changes in manufacturing procedures to optimise product quality. Some examples using 2DE approaches in this context can be found in the literature. When producing surimi, the inclusion of Ca2+ and Mg2+ salts in the washing processes has an effect on the functional properties of surimi-based products (Solberg et al. 1990). Involvement of Ca2+-activated proteolysis in surimi products has been suggested on basis of 2DE studies (Martinez et al. 1992). Fermentation with lactic acid bacteria and its influence on proteolysis in salmon muscle has also been studied by 2DE, revealing that the main proteolytic events are primarily independent of the added starter culture, although some proteolysis, mainly on alkaline to slightly acidic proteins, could be ascribed to proteases from the bacteria (Morzel et al. 2000).

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It is well known that solubility of muscle proteins decreases during frozen storage (Sikorski et al. 1976; Shenouda 1980; Mackie 1993) and several studies of this phenomenon have used 1D electrophoresis (Jiang et al. 1987; LeBlanc and LeBlanc 1989; Careche et al. 1998; Huidobro and Tejada 1995; Jasra et al. 2001; Saeed and Howell 2002; Tejada et al. 2003a, b ). Only a few studies have used 2DE in investigation of protein changes in fish muscle during frozen storage (Jessen 1996; Kjaersgard et al. 2006). Two-dimensional gels of cod muscle, frozen and stored at a stable temperature of −20°C for one week, were compared with 2D gels of cod stored at fluctuating freezing temperatures for two weeks. Eighty-three potential protein markers were able to distinguish between the two groups (Jessen 1996) showing that frozen temperature abuse has a marked influence on many muscular proteins. A recent study of frozen storage conditions comparing storage temperatures of −20°C and −30°C showed no effect of the temperature on the 2DE profiles, whereas the profiles were significantly influenced by storage time making it possible to discriminate between cod stored for 12 months and cod stored from 3 and 6 months (Kjaersgard et al. 2006). Several protein spots correlating with storage time were found, and 15 spots were identified by liquid chromatography/mass spectrometry/mass spectrometry (LC–MS/MS). Contractile proteins from both thick and thin myofilaments (myosin light chains and α-actin) and enzymes (triose-phosphate isomerase, aldolase A and glyceraldehyde-3-phosphate-dehydrogenase) associated with the filaments were degraded during frozen storage (Kjaersgard et al. 2006). The results give an important basis for obtaining a deeper knowledge of the correlation between frozen storage time and textural damage of fish muscle.

14.3.4

Sensory quality

Studies on seafood combining 2DE and quality as evaluated by a detailed sensory assessment have never been published. However, the potential is enormous because it opens a way to knowledge of the nature and the underlying mechanisms of specific sensory attributes. In an unpublished investigation (Flemming Jessen, Erling Larsen and Bo Jørgensen (Danish Institute for Fisheries Research) and Niels Bøknæs (Royal Greenland A/S)), 2DE profiles of batches of frozen and heat treated shrimp (Pandalus borealis) muscle were correlated to the sensory profile of a final brined product after 6 weeks of storage at 8°C. It was found that groups of protein spots from the 2D gels could be correlated to specific sensory attributes by multivariate data analysis. The complex pattern of 27 spots was, for example, found to correlate highly with the sensory attribute ‘sweet taste’. It was possible from the ‘27 spot’ patterns of shrimps to predict how the attribute ‘sweet taste’ will develop in the final product (Figure 14.3). Identification of the involved proteins might reveal knowledge about underlying mechanisms for ‘sweet taste’ in brined shrimps.

14.3.5 Nutritional and health aspects 2DE has hitherto not played any role in nutritional aspects of seafood, but as an important part of the coming nutrigenomic boom, it will definitively have an impact (see section 14.4). For health, seafood is actually one of the leading causes of food hypersensitivity because fish and shellfish contain some major allergens and potential cross-reacting allergens, namely

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X=Y

Predicted ‘sweet taste’ (line scale)

9 8 7 6 5 4 3 3

4

5 6 7 8 Measured ‘sweet taste’ (line scale)

9

10

Figure 14.3 Prediction of ‘sweet taste’ in brined shrimps by 2DE. Twenty batches of shrimps were used for production of a brined product. The plot shows how the attribute ‘sweet taste’ of the individual batches can be predicted from 27 spots in all the 2D-gels from the remaining 19 batches. The quality was assessed after storage for 6 weeks by sensory profiling using specific attributes of odour, flavour and texture as descriptors of the quality. The sensory profiling was performed by a tested and specifically trained panel consisting of 10 assessors evaluating descriptors on a 15 cm unstructured line scale, with two anchor points: ‘little’ and ‘much’. Two samples for 2DE were taken from all batches before production of the brined product. Shrimp muscle (120 mg) was homogenized in 2 ml 1% (w/v) SDS, 60 mM Tris-HCl, pH 8.3 and 100 mM DTT. The samples were boiled for 2 min., incubated for 30 min. at room temperature, homogenised again and centrifuged for 15 min. at 20°C at 20,000g. The supernatant was collected as sample extract. For protein determination, 2DE and silver staining, see legend to Figure 14.2. The stained 2D-gels were digitised using a 420 OE (PDI, Bio-Rad, Hercules, CA, USA) flatbed scanner using PDQuest 7.1.0, Discovery Series (Bio-Rad). PDQuest was also used to locate, match and quantify protein spots. Multivariate data analysis of the spot quantities was performed in The Unscrambler Programme version 7.6 (CAMO ASA, Oslo, Norway). Partial least square regression (PLSR) and Jack Knifing (variable selection) found the 27 spots that correlated to the sensory attribute ‘sweet taste’. Unpublished results (Flemming Jessen, Erling Larsen and Bo Jørgensen (Danish Institute for Fisheries Research) and Niels Bøknæs (Royal Greenland A/S)).

parvalbumins, tropomyosins and arginine kinases (Wild and Lehrer 2005). A few studies have used 2DE to characterise and identify these allergens (Lin et al. 1993; Yu et al. 2003; Jeong et al. 2004).

14.3.6

The live fish and product quality

For many years, fish biology/physiology has been a central aspect for quality-related research. It will become even more central in the future as a benefit from the expected revolutionised understanding of fish physiology created by the post-genomic technologies (Parrington and Coward 2002). By means of 2DE, aspects such as embryogenesis of salmonids (Kanaya

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et al. 2000), maturation of medaka (Oryzias latipes) oocytes (Yoshikuni et al. 2003), muscle development in herring larvae (Johnston et al. 1997) and Arctic charr (Salvelinus alpinus) (Martinez and Christiansen 1994), the relationships between biochemical and morphological changes in larvae, juveniles and adults of African catfish (Heterobranchus longifilis) (Huriaux et al. 1999), myosin isoforms in more teleost species (Martinez et al. 1990, 1993) and effects of low oxygen (hypoxia) on skeletal muscle of zebrafish (Danio rerio) (Bosworth et al. 2005) have been studied. These are all research areas where a further understanding of the molecular mechanisms involved has a potential impact on quality aspects of fish. Culturing seafood animals influences their physiology compared with their wild counterparts, as illustrated by the differences in protein expression and composition between wild and farmed sea bass (Dicentrarchus labrax) (Monti et al. 2005), or by different protein expression profiles characterised by 2DE between wild and farmed gilthead sea bream (Sparus aurata) (Carpene et al. 1998), or between intertidal and cultured mussels (Mytilus galloprovincialis) (Lopez et al. 2001). Such differences are strongly influenced by the living conditions of the animals. In aquaculture, conditions may vary greatly between fish farms. Physical and chemical parameters are of course important, but feed and handling also affect or stress the animals in an either chronic or acute way, depending on extent and exposure time of the stress factor. For feed aspects, 2DE-based proteome analysis has shown that during starvation of rainbow trout, where protein degradation is increased partly to provide amino acids for energy, the lysosomal protease cathepsin D is increased in abundance in the liver (Martin et al. 2001). It has been shown that a diet composed of a mixture of plant proteins compared with a fishmeal-based diet resulted in changes in several metabolic pathway proteins in rainbow trout liver. This included proteins involved in primary energy generation, maintenance of reducing potential, bile acid synthesis and transport and cellular protein degradation. In addition, metabolic effects of plant protein substitution varied according to the source of plant protein (Martin et al. 2003;Vilhelmsson et al. 2004). It is self-evident that such diet-induced metabolic changes can potentially change the quality of fish, and it is therefore important to know how and by which mechanisms fish are affected by feed. Several studies have shown that acute stress of fish before slaughter has an impact on quality (Sigholt et al. 1997; Robb et al. 2000; Skjervold et al. 2001; Morzel and van de Vis 2003; Kiessling et al. 2004). To establish the mechanisms between a pre-slaughter stress factor and product quality, Morzel et al. (2006) have used 2DE to characterise the modification of protein expression in rainbow trout white muscle as induced by a pre-slaughter crowding. They found 29 protein spots differentially expressed in crowded and un-crowded fish. The identified spots were mainly proteins from energy-producing pathways or structural proteins (Morzel et al. 2006). Finding exactly these proteins is very much in line with what could be hypothesised, because one of the main quality-related effects found due to crowding is the earlier onset of rigor that is closely related to both energy metabolism and structural proteins.

14.4

2DE-based seafood science in the future

The future potential of 2DE within seafood science is huge, and its impact will be on a broad spectrum of quality and safety features. As mentioned earlier, 2DE is not a fast and easy

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method, but at the scientific level in terms of providing understanding of underlying mechanisms and of identifying biomarkers for development of specific and fast methods or even patented kits, it is an outstanding methodology. In combination with matrix-assisted laser desorption/ionization–time of flight–mass spectrometry (MALDI–TOF–MS) and nESIIT-MS, 2DE has already revealed peptides from hake (Piñeiro et al. 2001) and marine mussels (Lopez et al. 2002) with species-specific amino-acid sequences. In the mussels, a species-specific sequence is found in the prominent and highly abundant protein tropomyosin, opening the way for production of monoclonal antibodies suitable for a highly accurate and fast-performed species identification using crude tissue extracts. A wide range of seafood aspects will potentially benefit from biomarkers found by 2DE studies: evaluation of freshness, determination of remaining shelf life, optimisation and control of production processes (e.g. freezing, thawing, smoking, fermentation, marinating, drying and heating), optimisation of frozen storage conditions, determination of frozen storage history and development of products using beneficial inherent characteristics of fish, to mention just a few. Improved use of fish resources towards higher-quality production and reduced discards and waste during production will also potentially benefit from 2DE research. For example in mussel (Mytilus galloprovincialis), a calponin-like protein was found (Funabara et al. 2001). Calponin is one of the essential proteins in the regulation of ‘catch’. ‘Catch’ is the maintenance of tension in the adductor muscle of mussels for a long period with low energy consumption. An understanding of ‘catch’ and its regulation could therefore be very important if it proved possible to control the process. In aquaculture, a combination of biomarkers for different growth characteristics and for quality can be used in selective breeding. Moreover, biomarkers relating production parameters as diet composition, feeding regime and temperature to different qualities or production parameters can be used in the design of fish having properties relevant for certain products. Likewise, 2DE might potentially link human health aspects to aquaculture. Allergenic properties of fish proteins could perhaps be influenced by breeding and/or by parts of the fish-farming production procedure. Also, the impact of fish health on product quality or safety will most probably be a subject for 2DE research. Very recently, a plasma acute-phase protein response induced by injection of bacterial products in rainbow trout has been demonstrated by 2DE (Russell et al. 2006). This shows that it is possible to provide knowledge by 2DE that may be used in applications that can show whether a fish or other seafood organisms have suffered from an illness (or infection), and perhaps even determine how long has elapsed since the event and whether this is sufficiently long enough ago for the fish to be used as food.

Acknowledgements I acknowledge the Danish Ministry of Agriculture and Fisheries, which through several grants (Biot 99-6; 3310-04-00017; 93s-2466-A01-01451; FSK03-DFU-11; 3310-04-00104) has provided a financial basis for implementation and use of two-dimensional gel electrophoretic methodologies within the field of seafood quality. The Danish Council for Strategic Research has supported the development of new data analytical approaches in proteomics (grant 2101-04-0013). I am grateful to Hanne Jacobsen for technical assistance in providing

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2D-gels, and to Kristina K. Jensen, Bo M. Jørgensen, Inger V.H. Kjaersgaard, Helene Godiksen and Jette Nielsen for many discussions.

14.5

References

Berrini, A., Tepedino, V., Borromeo, V. and Secchi, C. (2006) Identification of freshwater fish commercially labelled ‘perch’ by isoelectric focusing and two-dimensional electrophoresis. Food Chemistry 96: 163–168. Bosworth, C.A., Chou, C.W., Cole, R.B. and Rees, B.B. (2005) Protein expression patterns in zebrafish skeletal muscle: initial characterization and the effects of hypoxic exposure. Proteomics 5: 1362–1371. Careche, M., Mazo, M.L.D., Torrejón, P. and Tejada, M. (1998) Importance of frozen storage temperature in the type of aggregation of myofibrillar proteins in cod (Gadus morhua) fillets. Journal of Agricultural and Food Chemistry 46: 1539–1546. Carpene, E., Martin, B. and Libera, L.D. (1998) Biochemical differences in lateral muscle of wild and farmed gilthead sea bream (Sparus aurata L.). Fish Physiology and Biochemistry 19: 229–238. Chen, T.Y., Shiau, C.Y., Wei, C.I. and Hwang, D.F. (2004) Preliminary study on puffer fish proteome – Species identification of puffer fish by two-dimensional electrophoresis. Journal of Agricultural and Food Chemistry 52: 2236–2241. Civera, T. (2003) Species identification and safety of fish products. Veterinary Research Communications 27: 481–489. EsteveRomero, J.S., Yman, I.M., Bossi, A. and Righetti, P.G. (1996) Fish species identification by isoelectric focusing of parvalbumins in immobilized pH gradients. Electrophoresis 17: 1380–1385. Etienne, M., Jerome, M., Fleurence, J., Rehbein, H., Kundiger, R., Mendes, R., Costa, H., Perez-Martin, R., Pineiro-Gonzalez, C., Craig, A., Mackie, I., Yman, I.M., Ferm, M., Martinez, I., Jessen, F., Smelt, A. and Luten, J. (2000) Identification of fish species after cooking by SDS–PAGE and urea IEF: a collaborative study. Journal of Agricultural and Food Chemistry 48: 2653–2658. Etienne, M., Jerome, M., Fleurence, J., Rehbein, H., Kundiger, R., Yman, I.M., Ferm, M., Craig, A., Mackie, I., Jessen, F., Smelt, A. and Luten, J. (1999) A standardized method of identification of raw and heat-processed fish by urea isoelectric focusing: a collaborative study. Electrophoresis 20: 1923–1933. Faergestad, E.M., Flaete, N.E.S., Magnus, E.M., Hollung, K., Martens, H. and Uhlen, A.K. (2004) Relationships between storage protein composition, protein content, growing season and flour quality of bread wheat. Journal of the Science of Food and Agriculture 84: 877–886. Funabara, D., Nakaya, M. and Watabe, S. (2001) Isolation and characterization of a novel 45 kDa calponin-like protein from anterior byssus retractor muscle of the mussel Mytilus galloprovincialis. Fisheries Science 67: 511–517. Gangar, V., Huang, T.S. and Wei, C.I. (1996) Comparison of crabmeat protein patterns by isoelectric focusing. Food Control 7: 295–307. Görg, A., Obermaier, C., Boguth, G., Harder, A., Scheibe, B., Wildgruber, R. and Weiss, W. (2000) The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 21: 1037–1053. Görg, A., Obermaier, C., Boguth, G. and Weiss, W. (1999) Recent developments in two-dimensional gel electrophoresis with immobilized pH gradients: wide pH gradients up to pH 12, longer separation distances and simplified procedures. Electrophoresis 20: 712–717. Görg, A., Postel, W. and Gunther, S. (1988) The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 9: 531–546.

Two-dimensional gel electrophoresis

313

Görg A, Weiss W, Dunn MJ (2004) Current two-dimensional electrophoresis technology for proteomics. Proteomics 4: 3665–3685. Hattori, S., Fujisaki, H., Kiriyama, T., Yokoyama, T. and Irie, S. (2002) Real-time zymography and reverse zymography: a method for detecting activities of matrix metalloproteinases and their inhibitors using FITC-labeled collagen and casein as substrates. Analytical Biochemistry 301: 27–34. Hochstrasser, D.F., Harrington, M.G., Hochstrasser, A.C., Miller, M.J. and Merril, C.R. (1988) Methods for increasing the resolution of two-dimensional protein electrophoresis. Analytical Biochemistry 173: 424–435. Hogstrand, C., Balesaria, S. and Glover, C.N. (2002) Application of genomics and proteomics for study of the integrated response to zinc exposure in a non-model fish species, the rainbow trout. Comparative Biochemistry and Physiology B – Biochemistry and Molecular Biology 133: 523–535. Huang, T.S., Marshall, M.R. and Wei, C.I. (1995) Identification of red snapper (Lutjanus-campechanus) using electrophoretic techniques. Journal of Food Science 60: 279–283. Huidobro, A. and Tejada, M. (1995) Alteration of the electrophoretic pattern of myofibrillar proteins in fish mince during frozen storage. Zeitschrift für Lebensmittel Untersuchung und Forschung 200: 247–251. Huriaux, F., Vandewalle, P., Baras, E., Legendre, M. and Focant, B. (1999) Myofibrillar proteins in white muscle of the developing African catfish Heterobranchus longifilis (Siluriforms, Clariidae). Fish Physiology and Biochemistry 21: 287–301. Jacobsen, S., Grove, H., Jensen, K.N., Sørensen, H.A., Jessen, F., Hollung, K., Uhlen, A.K., Jørgensen, B.M., Færgestad, E.M. and Søndergaard I. (2007) Multivariate analysis of 2-DE protein patterns – Practical approaches. Electrophoresis 28: 1289–1299. Jasra, S.K., Jasra, P.K. and Talesara, C.L. (2001) Myofibrillar protein degradation of carp (Labeo rohita (Hamilton)) muscle after post-mortem unfrozen and frozen storage. Journal of the Science of Food and Agriculture 81: 519–524. Jeong, K.Y., Lee, J., Lee, I.Y., Ree, H.I., Hong, C.S. and Yong, T.S. (2004) Analysis of amino acid sequence variations and immunoglobulin E-binding epitopes of German cockroach tropomyosin. Clinical and Diagnostic Laboratory Immunology 11: 874–878. Jessen, F. (1996) Freezing induced protein changes in fish evaluated by two-dimensional gel electrophoresis. In: Refrigeration and Aquaculture. Proceedings of the meeting of Commission C2, Bordeaux, March 20–22, 1996. Institut international du froid, Paris, pp. 363–370. Jessen, F., Lametsch, R., Bendixen, E., Kjaersgard, I.V.H. and Jorgensen, B.M. (2002) Extracting information from two-dimensional electrophoresis gels by partial least squares regression. Proteomics 2: 32–35. Jia, X.H., Hildrum, K.I., Westad, F., Kummen, E., Aass, L. and Hollung, K. (2006) Changes in enzymes associated with energy metabolism during the early post mortem period in Longissimus thoracis bovine muscle analyzed by proteomics. Journal of Proteome Research 5: 1763–1769. Jiang, S.T., Hwang, B.S. and Tsao, C.Y. (1987) Protein denaturation and changes in nucleotides of fish muscle during frozen storage. Journal of Agricultural and Food Chemistry 35: 22–27. Johnston, I.A., Cole, N.J., Vieira, V.L.A. and Davidson, I. (1997) Temperature and developmental plasticity of muscle phenotype in herring larvae. Journal of Experimental Biology 200: 849–868. Kanaya, S., Ujiie, Y., Hasegawa, K., Sato, T., Imada, H., Kinouchi, M., Kudo, Y., Ogata, T., Ohya, H., Kamada, H., Itamoto, K. and Katsura, K. (2000) Proteome analysis of Oncorhynchus species during embryogenesis. Electrophoresis 21: 1907–1913. Kaplan, R.S. and Pedersen, P.L. (1989) Sensitive protein assay in presence of high-levels of lipid. Methods in Enzymology 172: 393–399. Kiessling, A., Espe, M., Ruohonen, K. and Morkore, T. (2004) Texture, gaping and colour of fresh and frozen Atlantic salmon flesh as affected by pre-slaughter iso-eugenol or CO2 anaesthesia. Aquaculture 236: 645–657.

314

Fishery Products: Quality, safety and authenticity

Kjaersgard, I.V.H. and Jessen, F. (2003) Proteome analysis elucidating post-mortem changes in cod (Gadus morhua) muscle proteins. Journal of Agricultural and Food Chemistry 51: 3985–3991. Kjaersgard, I.V.H. and Jessen, F. (2004) Two-dimensional gel electrophoresis detection of protein oxidation in fresh and tainted rainbow trout muscle. Journal of Agricultural and Food Chemistry 52: 7101–7107. Kjaersgard, I.V.H., Norrelykke, M.R. and Jessen, F. (2006) Changes in cod muscle proteins during frozen storage revealed by proteome analysis and multivariate data analysis. Proteomics 6: 1606–1618. Kuiper, H.A., Kleter, G.A., Noteborn, H.P.J.M. and Kok, E.J. (2001) Assessment of the food safety issues related to genetically modified foods. Plant Journal 27: 503–528. Kuiper, H.A., Noteborn, H.P.J.M., Kok, E.J. and Kleter, G.A. (2002) Safety aspects of novel foods. Food Research International 35: 267–271. Ladrat, C., Verrez-Bagnis, V., Noel, J. and Fleurence, J. (2003) In vitro proteolysis of myofibrillar and sarcoplasmic proteins of white muscle of sea bass (Dicentrarchus labrax L.): effects of cathepsins B, D and L. Food Chemistry 81: 517–525. LeBlanc, E.L. and LeBlanc, R.J. (1989) Separation of cod (Gadus morhua) fillet proteins by electrophoresis and HPLC after various frozen storage treatments. Journal of Food Science 54: 827–834. Lin, R.Y., Shen, H.D., Han, S.H. (1993) Identification and characterization of a 30-kD major allergen from Parapenaeus fissurus. Journal of Allergy and Clinical Immunology 92: 837–845. Lodemel, J.B., Egge-Jacobsen, W. and Olsen, R.L. (2004a) Detection of TIMP-2-like protein in Atlantic cod (Gadus morhua) muscle using two-dimensional real-time reverse zymography. Comparative Biochemistry and Physiology B – Biochemistry and Molecular Biology 139: 253–259. Lodemel, J.B., Maehre, H.K., Winberg, J.O. and Olsen, R.L. (2004b) Tissue distribution, inhibition and activation of gelatinolytic activities in Atlantic cod (Gadus morhua). Comparative Biochemistry and Physiology B – Biochemistry and Molecular Biology 137: 363–371. Lopez, J.L., Marina, A., Alvarez, G. and Vazquez, J. (2002) Application of proteomics for fast identification of species-specific peptides from marine species. Proteomics 2: 1658–1665. Lopez, J.L., Mosquera, E., Fuentes, J., Marina, A., Vazquez, J. and Alvarez, G. (2001) Two-dimensional gel electrophoresis of Mytilus galloprovincialis: differences in protein expression between intertidal and cultured mussels. Marine Ecology-Progress Series 224: 149–156. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the folin phenol reagent. Journal of Biological Chemistry 193: 265–275. Mackie, I., Craig, A., Etienne, M., Jerome, M., Fleurence, J., Jessen, F., Smelt, A., Kruijt, A., Yman, I.M., Ferm, M., Martinez, I., Perez-Martin, R., Pineiro, C., Rehbein, H. and Kundiger, R. (2000) Species identification of smoked and gravad fish products by sodium dodecylsulphate polyacrylamide gel electrophoresis, urea isoelectric focusing and native isoelectric focusing: a collaborative study. Food Chemistry 71: 1–7. Mackie, I.M. (1993) The effects of freezing on flesh proteins. Food Review International 9: 575–610. Martin, S.A.M., Cash, P., Blaney, S. and Houlihan, D.F. (2001) Proteome analysis of rainbow trout (Oncorhynchus mykiss) liver proteins during short term starvation. Fish Physiology and Biochemistry 24: 259–270. Martin, S.A.M., Vilhelmsson, O., Medale, F., Watt, P., Kaushik, S. and Houlihan, D.F. (2003) Proteomic sensitivity to dietary manipulations in rainbow trout. Biochimica et Biophysica Acta – Proteins and Proteomics 1651: 17–29. Martinez, I. and Christiansen, J.S. (1994) Myofibrillar proteins in developing white muscle of the arctic charr, Salvelinus alpinus (L). Comparative Biochemistry and Physiology B 107: 11–20.

Two-dimensional gel electrophoresis

315

Martinez, I., Ofstad, R. and Olsen, R.L. (1990) Electrophoretic study of myosin isoforms in white muscles of some teleost fishes. Comparative Biochemistry and Physiology B 96: 221–227. Martinez, I., Christiansen, J.S., Ofstad, R. and Olsen, R.L. (1991) Comparison of myosin isoenzymes present in skeletal and cardiac muscles of the Arctic charr Salvelinus alpinus (L.). Sequential expression of different myosin heavy chains during development of the fast white skeletal muscle. European Journal of Biochemistry 195: 743–753. Martinez, I., Solberg, C., Lauritzen, K. and Ofstad, R. (1992) Two-dimensional electrophoretic analyses of cod (Gadus morhua, L.) whole muscle proteins water-soluble fraction and surimi. Effect of the addition of CaCl2 and MgCl2 during the washing procedure. Applied and Theoretical Electrophoresis 2: 201–206. Martinez, I., Bang, B., Hatlen, B. and Blix, P. (1993) Myofibrillar proteins in skeletal muscles of parr, smolt and adult atlantic salmon (Salmo salar L). comparison with another salmonid, the arctic charr Salvelinus alpinus (L). Comparative Biochemistry and Physiology B 106: 1021– 1028. Martinez, I., Aursand, M., Erikson, U., Singstad, T.E., Veliyulin, E. and van der Zwaag, C. (2003) Destructive and non-destructive analytical techniques for authentication and composition analyses of foodstuffs. Trends in Food Science and Technology 14: 489–498. Mi, J., Orbea, A., Syme, N., Ahmed, M., Cajaraville, M.P. and Cristobal, S. (2005) Peroxisomal proteomics, a new tool for risk assessment of peroxisome proliferating pollutants in the marine environment. Proteomics 5: 3954–3965. Miller, I., Crawford, J. and Gianazza, E. (2006) Protein stains for proteomic applications: Which, when, why? Proteomics 6: 5385–5408. Monti, G., De Napoli, L., Mainolfi, P., Barone, R., Guida, M., Marino, G. and Amoresano, A. (2005) Monitoring food quality by microfluidic electrophoresis, gas chromatography, and mass spectrometry techniques: Effects of aquaculture on the sea bass (Dicentrarchus labrax). Analytical Chemistry 77: 2587–2594. Morzel, M. and van de Vis, H. (2003) Effect of the slaughter method on the quality of raw and smoked eels (Anguilla anguilla L.). Aquaculture Research 34: 1–11. Morzel, M., Verrez-Bagnis, V., Arendt, E.K. and Fleurence, J. (2000) Use of two-dimensional electrophoresis to evaluate proteolysis in salmon (Salmo salar) muscle as affected by a lactic fermentation. Journal of Agricultural and Food Chemistry 48: 239–244. Morzel, M., Chambon, C., Lefevre, F., Paboeuf, G. and Laville, E. (2006) Modifications of trout (Oncorhynchus mykiss) muscle proteins by preslaughter activity. Journal of Agricultural and Food Chemistry 54: 2997–3001. O’Farrell, P.H. (1975) High resolution two-dimensional electrophoresis of proteins. Journal of Biological Chemistry 250: 4007–4021. Olsson, B., Bradley, B.P., Gilek, M., Reimer, O., Shepard, J.L. and Tedengren, M. (2004) Physiological and proteomic responses in Mytilus edulis exposed to PCBs and PAHs extracted from Baltic Sea sediments. Hydrobiologia 514: 15–27. Parrington, J. and Coward, K. (2002) Use of emerging genomic and proteomic technologies in fish physiology. Aquatic Living Resources 15: 193–196. Patton, W.F. (2002) Detection technologies in proteome analysis. Journal of Chromatography B – Analytical Technologies in the Biomedical and Life Sciences 771: 3–31. Peterson, G.L. (1977) A simplification of protein assay method of Lowry et al. which is more generally applicable. Analytical Biochemistry 83: 346–356. Piñeiro, C., Barros-Velázquez, J., Pérez-Martín, R.I., Martínez, I., Jacobsen, T., Rehbein, H., Kündiger, R., Mendes, R., Etienne, M., Jerome, M., Craig, A., Mackie, I.M. and Jessen, F. (1999a) Development of a sodium dodecyl sulfate-polyacrylamide gel electrophoresis reference method for the analysis and identification of fish species in raw and heat-processed samples: a collaborative study. Electrophoresis 20: 1425–1432.

316

Fishery Products: Quality, safety and authenticity

Piñeiro, C., Barros-Velázquez, J., Sotelo, C. and Gallardo, J.M. (1999b) The use of two dimensional electrophoresis in the characterization of the water-soluble protein fraction of commercial flat fish species. Food Research and Technology 208: 342–348. Piñeiro, C., Barros-Velázquez, J., Sotelo, C.G., Pérez-Martín, R.I. and Gallardo, J.D. (1998) Two-dimensional electrophoretic study of the water-soluble protein fraction in white muscle of gadoid fish species. Journal of Agricultural and Food Chemistry 46: 3991–3997. Piñeiro, C., Vázquez, J., Marina, A.I., Barros-Velázquez, J. and Gallardo, J.M. (2001) Characterization and partial sequencing of species-specific sarcoplasmic polypeptides from commercial hake species by mass spectrometry following two-dimensional electrophoresis. Electrophoresis 22: 1545–1552. Rehbein, H. (1990) Electrophoretic techniques for species identification of fishery products. Lebensmittel-Untersuchung und -Forschung 191: 1–10. Robb, D.H.F., Kestin, S.C. and Warriss, P.D. (2000) Muscle activity at slaughter: I. Changes in flesh colour and gaping in rainbow trout. Aquaculture 182: 261–269. Rodriguez-Ortega, M.J., Grosvik, B.E., Rodriguez-Ariza, A., Goksoyr, A. and Lopez-Barea, J. (2003) Changes in protein expression profiles in bivalve molluscs (Chamaelea gallina) exposed to four model environmental pollutants. Proteomics 3: 1535–1543. Roepstorff, P. (1997) Mass spectrometry in protein studies from genome to function. Current Opinion in Biotechnology 8: 6–13. Ruebelt, M.C., Lipp, M., Reynolds, T.L., Schmuke, J.J., Astwood, J.D., DellaPenna, D., Engel, K.H. and Jany, K.D. (2006) Application of two-dimensional gel electrophoresis to interrogate alterations in the proteome of genetically modified crops. 3. Assessing unintended effects. Journal of Agricultural and Food Chemistry 54: 2169–2177. Russell, S., Hayes, M.A., Simko, E. and Lumsden, J.S. (2006) Plasma proteomic analysis of the acute phase response of rainbow trout (Oncorhynchus mykiss) to intraperitoneal inflammation and LPS injection. Developmental and Comparative Immunology 30: 393–406. Saeed, S., Howell, N.K. (2002) Effect of lipid oxidation and frozen storage on muscle proteins of Atlantic mackerel (Scomber scombrus). Journal of the Science of Food and Agriculture 82: 579–586. Shenouda, S.Y.K. (1980) Theories of protein denaturation during frozen storage of fish flesh. Advances in Food Research 26: 275–311. Shepard, J.L. and Bradley, B.P. (2000) Protein expression signatures and lysosomal stability in Mytilus edulis exposed to graded copper concentrations. Marine Environmental Research 50: 457–463. Shepard, J.L., Olsson, B., Tedengren, M. and Bradley, B.P. (2000) Protein expression signatures identified in Mytilus edulis exposed to PCBs, copper and salinity stress. Marine Environmental Research 50: 337–340. Shrader, E.A., Henry, T.R., Greeley, M.S. and Bradley, B.P. (2003) Proteomics in zebrafish exposed to endocrine disrupting chemicals. Ecotoxicology 12: 485–488. Sigholt, T., Erikson, U., Rustad, T., Johnson, S., Nordtvedt, T.S. and Seland, A. (1997) Handling stress and storage temperature affect meat quality of farmed-raised Atlantic salmon (Salmo salar). Journal of Food Science 62: 898–905. Sikorski, Z., Olley, J. and Kostuch, S. (1976) Protein changes in frozen fish. Critical Reviews in Food Science and Nutrition 8: 97–129. Silvestre, F., Dierick, J.F., Dumont, V., Dieu, M., Raes, M. and Devos, P. (2006) Differential protein expression profiles in anterior gills of Eriocheir sinensis during acclimation to cadmium. Aquatic Toxicology 76: 46–58. Skjervold, P.O., Fjaera, S.O., Ostby, P.B. and Einen O (2001) Live-chilling and crowding stress before slaughter of Atlantic salmon (Salmo salar). Aquaculture 192: 265–280. Solberg, C., Ofstad, R. and Solberg, T. (1990) The influence of CaCl2 and MgCl2 in washing water on functional properties of cod (Gadus morhus) surimi. A multivariate data analysis. In: E.G. Bligh

Two-dimensional gel electrophoresis

317

(Ed) Proceedings of the International Conference Seafood Year 2000, Halifax. Canada Fishing New Books. Tejada, M., Huidobro, A. and Mohamed, G.F. (2003a) Comparison of gilthead sea bream (Sparus aurata) and hake (Merluccius merluccius) muscle proteins during iced and frozen storage. Journal of the Science of Food and Agriculture 83: 113–122. Tejada, M., Mohamed, G.F., Huidobro, A. and Garcia, M.L. (2003b) Effect of frozen storage of hake, sardine and mixed minces on natural actomyosin extracted in salt solutions. Journal of the Science of Food and Agriculture 83: 1380–1388. Verrez-Bagnis, V., Ladrat, C., Morzel, M., Noel, J. and Fleurence, J. (2001) Protein changes in post mortem sea bass (Dicentrarchus labrax) muscle monitored by one- and two-dimensional gel electrophoresis. Electrophoresis 22: 1539–1544. Vilhelmsson, O.T., Martin, S.A.M., Medale, F., Kaushik, S.J. and Houlihan, D.F. (2004) Dietary plantprotein substitution affects hepatic metabolism in rainbow trout (Oncorhynchus mykiss). British Journal of Nutrition 92: 71–80. Wagg, S.K. and Lee, L.E.J. (2005) A proteomics approach to identifying fish cell lines. Proteomics 5: 4236–4244. Wetmore, B.A. and Merrick, B.A. (2004) Toxicoproteomics: proteomics applied to toxicology and pathology. Toxicologic Pathology 32: 619–642. Wheelock, A.M. and Goto, S. (2006) Effects of post-electrophoretic analysis on variance in gel-based proteomics. Expert Review of Proteomics 3: 129–142. Wild, L.G. and Lehrer, S.B. (2005) Fish and shellfish allergy. Current Allergy and Asthma Reports 5: 74–79. Wilkins, M.R., Pasquali, C., Appel, R.D., Ou, K., Golaz, O., Sanchez, J.C., Yan, J.X., Gooley, A.A., Hughes, G., Humphery-Smith, L., Williams, K.L. and Hochstrasser, D.F. (1996) From proteins to proteoms: large-scale protein identification by two-dimensional electrophoresis and amino acid analysis. Bio/technology 14: 61–65. Yoshikuni, M., Sagegami, R. and Nagahama, Y. (2003) Proteome analysis: a new approach to identify key proteins involved in acquisition of maturational competence and oocyte maturation of medaka oocytes. Fish Physiology and Biochemistry 28: 379–380. Yu, C.J., Lin, Y.F., Chiang, B.L. and Chow, L.P. (2003) Proteomics and immunological analysis of a novel shrimp allergen, Pen m 2. Journal of Immunology 170: 445–453.

Chapter 15

Microbiological methods Ulrike Lyhs

15.1

Microorganisms in fish and fish products

The microbial status of fish after the catch is related to environmental conditions and the microbiological quality of the water, including temperature, salt content, natural bacterial flora in the water, and so on (Feldhusen 2002). During further processing of the fish and storage at particular conditions (temperature, atmosphere, salt, preservation methods) the composition of the microflora changes. The microflora on a fish product is composed of the indigenous flora and the microflora of the processing environment. Any handling of fish from the point of harvesting to the table of the consumer has the potential to contribute to the microflora on the final product. Table 15.1 shows the dominating microflora and the specific spoilage organisms in different fish products. Among the large variety of seafood, lightly preserved fish products have a special position. Being highly perishable and of limited shelf life, they must be stored and distributed under chilled temperatures. They are preserved by a low salt content (less than 6% NaCl (w/w) in the water phase). Preservatives like sorbate, benzoate or smoke may be added. The pH of these products is high (greater than 5.0), and they are often vacuum-packaged. These products are normally consumed as ready-to-eat (RTE) products without prior heating (Gram and Huss 1996). This group includes salted and fermented, cold-smoked, sugar-salted (termed ‘gravad’) and marinated fish products. Maatjes herring is a lightly salted and fermented RTE fish product that is very popular in the Netherlands and Germany. The term ‘maatjes’ refers to herring (Clupea harengus) caught just before its first spawning between May and July. It is characterised by a distinct level (16–20%) of subcutaneous fat. The caught herring is partly gibbed and lightly cured. The remaining intestines produce enzymes, which stimulate a fermentation process resulting in the typical maatjes product characteristics (Lyhs et al. 2006). ‘Gravad’ fish products (salmon (Salmo salar), whitefish (Coregonus lavaretus), rainbow trout (Oncorhyncus mykiss), halibut (Hippoglossus hippoglossus)) are very popular dishes traditionally manufactured in the Nordic countries. The fish is preserved by addition of sugar and salt, and sometimes dill is added. Before consumption it undergoes a short ripening process of 1–2 days at chilled temperatures (Lyhs et al. 2001a). Vacuum-packaged cold-smoked fish (salmon, whitefish, rainbow trout) is widely consumed in Europe. The cold-smoking process includes salting of 318

Table 15.1 Dominating microflora and specific spoilage organisms (SSO) in different fish products).

Fish product Fresh chilled fish (0–5 °C) Fresh fish (>10–15 °C) Fresh chilled fish (0–5 °C)

Packaging atmosphere Aerobic Aerobic

Dominating microflora

Typical SSO

Shewanella putrefaciens, Pseudomonas spp., Shewanella putrefaciens, Pseudomonas spp. Moraxella, Acinetobacter Shewanella putrefaciens, Vibrionaceae Aeromonas spp., Shewanella putrefaciens

Semi-preserved Lactic acid bacteria marinated fish Lightly salted and Modified Data not available fermented fish atmosphere Sources: Huss 1995; Gram and Huss 1996

Shewanella putrefaciens, Photobacterium phosphoreum, Lactic acid bacteria Photobacterium phosphoreum, Lactic acid bacteria Lactic acid bacteria (Lactobacillus, Lactococcus, Carnobacterium), Enterobacteriaceae (Serratia spp., Hafnia alvei, Enterobacter spp.), Photobacterium, Brochothrix thermospacta Lactic acid bacteria (Carnobacterium)

Lactic acid bacteria (Lactobacillus, Leuconostoc, Weissella, Carnobacterium) Lactic acid bacteria (Lactobacillus) Lactic acid bacteria (Lactobacillus, Enterococcus, Lactococcus)

Microbiological methods

Shewanella putrefaciens, Vibrionaceae, Photobacterium Photobacterium, Shewanella putrefaciens, Modified Pseudomonas spp. atmosphere Cold–smoked fish Vacuum Pseudomonas spp., Enterobacteriaceae, Acinetobacter, Staphylococcus spp., Shewanella putrefaciens, Vibrionaceae, Photobacterium Pseudomonas spp., Hot-smoked fish Vacuum Enterobacteriaceae, Staphylococcus spp., Lactic acid bacteria ‘Gravad’ fish Vacuum Data not available Vacuum

319

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the fish (brining or salting by injection) and smoking at temperatures not higher than 28°C. Among the countries producing cold-smoked salmon, France is the leading country followed by Denmark, Germany and the United Kingdom (Cardinal et al. 2004).

15.2

General aspects of microbiological methods

The aims of microbiological examination of fish and fish products are to evaluate the hygienic quality of fish and to detect possible pathogenic microorganisms. They consist of the measurement of total aerobic bacteria, spoilage bacteria and pathogenic bacteria (Huss 1995). The food microbiologist can choose from a wide variety of conventional microbiological and new molecular biological methods to analyse fish and fish products. Traditional microbiological methods require much manual labour, are time-consuming and costly. They require skills in the execution and interpretation of the results (Huss 1995). Furthermore, these methods are slow and the results can only be obtained after 1–5 days. However, incubation and agar-based detection methods are still the main methods used for determination of bacterial counts. They include qualitative tests (absence or presence), often used for detection of pathogenic bacteria, and quantitative methods, used for enumeration of viable cells of all kinds of microorganism (Holbrook 2000). Table 15.2 presents recommendations for agar types, incubation temperatures and times for different general bacterial counts in different products. As for all kinds of food product, there are two basic classical approaches to determine the microorganisms in a fish product: (1) direct plating of specimens of the fish product onto a selective agar; and (2) incubation of the extract of a fish product in an enrichment medium followed by plating onto a selective agar. Direct plating from the food includes the standard plate count (pour plating) and the surface count technique (spread plating), which are both used for different bacterial counts. The standard plate count method is the most widely used method for determination of numbers of viable cells or colony-forming units (cfu) (Jay 2000). Exposure of the inocula to the molten agar, which has a temperature of 45–49°C, is generally acceptable for determining of mesophilic and thermophilic bacteria (Holbrook 2000). Disadvantages are, for example, the long incubation time and limitations due to a specific culture medium and incubation conditions like atmosphere and temperature. Furthermore, both viable single cells and cell clumps are counted, which might lead to wrong numbers of bacteria (Jay 2000; Robinson 2002). In the determination of the heat-sensitive psychrotrophic bacteria, the surface count technique has been used. This has the advantage that the bacteria do not come into contact with the melted hot agar (Holbrook 2000). Strict aerobes are obviously favoured by this technique, whereas microaerophilic bacteria grow slower (Jay 2000). There are several disadvantages to the surface count technique (Jay 2000; Robinson 2002): It is time-consuming. Only 0.1 ml of dilution can be plated per conventional Petri dish. Undetected contaminant colonies occurring on the prepared plate can spread over the whole plate during the spreading procedure. A resulting film of growth (also when the agar surface is not dry enough before plating) can mask the development of colonies to be counted and make enumeration difficult. Incubation of the food product in an enrichment medium is often used for opportunistic pathogenic and pathogenic microorganisms, for example Listeria, Clostridia and Salmonella.

Table 15.2 Detection media, incubation temperature and incubation time used for determination of total number of bacteria including mesophilic, psychrotrophic and hydrogen-sulphide-producing bacteria in fish and fishery products.

Microorganisms Total viable count (including mesophilic bacteria)

Recommended incubation temperature (°C)

Plate count agar (PCA), pour plating

20 °C/30 °C

Iron agar (IA), pour platinga

Tryptic soy agar (TSA), pour plating Plate count agar (PCA), pour plating

Recommended incubation time (days)

Fish and fishery product

References

Remarks

1–3 days

Fresh fish stored in ice, vacuum-packaged coldand hot-smoked fish, modified-atmospherepackaged hot-smoked fish, fish stored in sous-vide

Addition of 0.5% NaCl (Leroi et al. 1998)

21 °C

3 days

21 °C/25 °C

3 days

Vacuum-packaged ‘gravad’ fish, salted and fermented fish packaged in modified atmosphere and in air Vacuum-packaged coldsmoked fish

Civera et al. 1995; Leroi et al. 1998; Lyhs et al. 1998a; Paarup et al. 2002; Taliadourou et al. 2003; Chytiri et al. 2004; González-Fandos et al. 2005; Cakli et al. 2006; Hozbor et al. 2006; Nordic Committee on Food Analysis 2006 Lyhs et al. 2002, 2007

5 °C/7 °C

10–14 days

Fresh fish stored in ice, fish stored in sous-vide, vacuum- and modifiedatmosphere-packaged hot-smoked fish

Addition of 1% NaCl (Magnússon et al. 2006)

Truelstrup Hansen et al. 1995, 1998 Cakli et al. 2006; GonzálezFandos et al. 2005; Huss et al. 2006

Continued

Microbiological methods

Total viable count (including psychrotrophic bacteria)

Medium (pour plating/spread plating)

321

322

Table 15.2

Continued

Hydrogensulphide (H2S)producing bacteria

a

Recommended incubation temperature (°C)

Recommended incubation time (days)

Long and Hammer’s agar (LH), spread plating

10 °C/15 °C

3–7 days

Tryptic soy agar (TSA), spread plating Iron agar (IA), pour plating

10 °C

5 days

20 °C/21 °C/ 22 °C/25 °C

2–4 days

Counting white and black colonies

Fish and fishery product

References

Dalgaard et al. 1997b, 2003; Fresh fish stored in air, vacuum-packaged coldKoutsoumanis et al. 1998; smoked fish, salted and Jørgensen et al. 2000; fermented fish packaged in Leroi et al. 2001; Emborg modified atmosphere and in et al. 2002, 2005; Pournis air, modified- atmosphereet al. 2005; Nordic packaged fish, cooked and Committee on Food brined modifiedAnalysis 2006; Lyhs et al. atmosphere-packaged 2007 shrimps Truelstrup Hansen et al. Vacuum-packaged ‘gravad’ and cold-smoked fish 1995, 1998; Lyhs et al. 2001a Fresh fish stored in air and in ice, vacuum-packaged ‘gravad’ and cold-smoked fish, salted and fermented fish packaged in modified atmosphere and in air, modified-atmospherepackaged fish

Gram et al. 1987; Rosnes et al. 1997; Koutsoumanis et al. 1998; Leroi et al. 1998; Emborg et al. 2002; Paarup et al. 2002; Sveinsdóttir et al. 2002; Taliadourou et al. 2003; Chytiri et al. 2004; Lyhs et al. 2001a, 2007; Pournis et al. 2005; Nordic Committee on Food Analysis 2006

Remarks Addition of 1% NaCl (Dalgaard 2000)

Fishery Products: Quality, safety and authenticity

Microorganisms

Medium (pour plating/spread plating)

Microbiological methods

323

When microorganisms have been through stress by freezing, drying, smoking, heating, and so on, they made have been killed or injured. Some might have survived but require optimal conditions to grow again. Therefore they should be grown in enrichment first and then plated on a medium selective for the target microorganism. To substitute for the enrichment step, techniques such as filtration, centrifugation or magnetic separation might be applied. Enrichment is a critical step in enhancing the growth of certain bacterial species while inhibiting the development of unwanted microorganisms. After the culturing step, identification of isolated and purified colonies using morphological, biochemical and immunological tests is still widely used. However, owing to the possibility of misidentifications, difficulties and unreliability of the results, DNA-based methods are increasingly used for these phenotypic tests.

15.2.1

Total viable count or aerobic plate count

The total number of microorganisms – called the total viable count (TVC) or aerobic plate count (APC) if performed by traditional methods – is the number of bacteria (cfu/g) in a food product that are capable of forming visible colonies on a culture medium at a given temperature (Huss 1995). TVC is not the measure of the ‘total’ bacterial population, only a measure of the fraction of the microflora that is able to produce colonies under the conditions of the growth medium and the incubation. It has long been known that the incubation temperature greatly influences the number of colonies developing from the same sample. Many marine microorganisms in seafood are psychrotolerant and heat labile. It has been shown that bacterial counts in fish products stored at chilled temperatures determined at 20–25°C are much higher than counts determined at 37°C (Liston 1957; Huss 1995). Therefore, to determine TVC including psychrotrophic microorganisms in seafood, pour plating with agar at about 45°C or incubating the plates at 30–37°C must be avoided. To determine TVC including mesophilic microorganisms in fish and fish products, common plate count agars (PCAs) on pour plates have been the substrates mostly used. Nowadays, iron agar (IA), a non-selective and indicative medium, is used to an increasing degree. It was developed to enumerate hydrogen sulphide (H2S)-producing microorganisms, which are important spoilage organisms of chilled and aerobically stored fresh fish (Gram et al. 1987). For aerobic bacterial counts including (mesophilic) H2S-producing and other non-heat-labile bacteria, pour plating on IA and 3–4 days’ incubation at 25°C is relevant. IA gives significantly higher counts than PCA (Gram 1990). H2S-producing microorganisms appear as black colonies owing to precipitation of iron sulphide (FeS), which is formed by decomposition of thiosulphate and/or l-cysteine (Gram et al. 1987; Huss et al. 1997; NCFA 2006). Both thiosulphate and l-cysteine are constituents of the IA and facilitate H2S production. NonH2S-producing microorganisms appear as white colonies. An overlay is usual poured on top of the IA to pretend fading of the black colonies appearing when FeS is oxidised. Lauzon (1997), studying shelf life and spoilage of American plaice (Hippoglossoides platessoides), concluded that the addition of the overlay was not necessary as the number of black colonies was generally greater for spread-plated than pour/overlaid plates. Spread-plating of IA usually gave higher total counts. However, the use of IA has disadvantages like any other agar plate method: long detection time and a relatively high detection level (50–100 cfu/g).

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Thus, it is not possibile to get an early warning of high numbers of H2S-producing microorganisms in the fish product (Skjerdal et al. 2004). However, for qualitative determination of these microorganisms this is not relevant. Skjerdal et al. (2004) describe a simple, rapid and quantitative method based on a patent of Lorentzen et al. (2003) for estimating the sulphideproducing bacteria in aerobically and cold-stored fish from Arctic and temperate areas. It detects sulphide-producing bacteria at levels above 105.5–106 cfu/g fish within 8 hours, and has a detection level of 16 cfu/g fish. The method is based on iron sulphide formation leading to fluorescence reduction, which is measured as changes in background medium fluorescence in a liquid medium incubated at 30°C during the growth of sulphide-producing bacteria. The sulphide production in fish of Arctic and temperate waters is dominated by Shewanella putrefaciens, which has an optimum growth temperature of 30–32°C and is known as the specific spoilage organism in marine temperate-water fish. It is possible to predict the remaining shelf life of fish of unknown time–temperature history with this method. Many marine microorganisms in seafood require sodium chloride (NaCl) for growth. Therefore, van Spreekens (1974) modified Long and Hammer’s agar (LH) (Long and Hammer 1941) by the addition of 0.5% NaCl to the medium. According to Dalgaard (2000), spread plating on pre-chilled plates of LH with 1% NaCl and subsequent incubation at 15°C for 5–7 days is a useful procedure for enumerating TVC of heat-labile and psychotolerant marine bacteria (dominating the spoilage microflora for fresh marine fish, particularly when vacuum – and modified-atmosphere-packed). Cardinal et al. (2004), characterising the quality of vacuum-packaged cold-smoked salmon products from supermarkets in different European countries, also recommended the use of spread plates of LH instead of pour PCA for determinating total psychrotrophic counts. Counts on LH have been generally one logarithm higher than counts on PCA (Joffraud and Leroi 2000). Koutsoumonis and Nychas (1999) noted that there was no significant difference between bacterial counts on LH and IA when all colonies (black and white) were counted on IA. Furthermore, on LH, luminous colonies can be counted after 4 days (NCFA 2006). Another agar used for determining TVC including mesophilic and psychrotrophic bacteria, for example in vacuum-packaged coldsmoked fish, is tryptic soy agar (TSA), sometimes containing 0.5% NaCl (Truelstrup et al. 1995 1998; Lyhs et al. 2001a).

15.2.2

Spoilage bacteria

Fresh, untreated seafood harbours a quite heterogeneous microflora. The flesh of healthy, live fish is generally thought to be sterile as the immune system prevents bacteria from growing in the flesh (Huss et al. 1997; Gram and Huss 2000). When the fish dies, the immune system stops functioning and bacteria can proliferate freely. The microflora of a fish product is composed of the natural, indigenous flora and the microflora typical of the processing environment. Depending on the type of preservation during storage, few bacteria of this microflora outgrow the others, depending on which is capable of rapid growth under the imposed conditions (temperature, atmosphere, microbial interactions, and so on). Spoilage is defined as the sensory changes resulting in a fish product being unacceptable for human consumption. It is caused by autolytic and chemical changes or off-odours and off-flavours due to bacterial metabolism. When a fish product is rejected based on sensory assessment, the microflora present, the so-called spoilage microflora, can still consist of

Microbiological methods

325

different species (Huss et al. 1997). Typically, one or two species of the group of bacteria called specific spoilage organisms (SSO) will cause the production of metabolites associated with the off-flavours and off-odours (Huss et al. 1997; Gram and Huss 2000). SSOs are typically present in low numbers and constitute only a very small fraction of the microflora on newly processed seafood (Gram and Dalgaard 2002). Table 15.3 shows recommendations for agar types, incubation temperatures and times for SSO counts in fish and fishery products. Pseudomonas spp. Pseudomonas spp. have been reported to be the SSO in ice-stored tropical freshwater fish (Gram et al. 1990). Together with S. putrefaciens, they are also spoilers in tropical marine fish (Gram 1992) and in fish obtained from temperate waters in the Mediterranean Sea (Koutsoumanis and Nychas 1999; Tryfinopoulou et al. 2000) both stored in ice aerobically. Gennari et al. (1999) used tryptone–peptone-extract (TPE) agar for determinating Pseudomonas spp. in fresh and spoiled sardines stored in ice. Pseudomonas agar base supplemented with three antibiotics, cetrimide, fusidin and cephaloridine (CFC), has been used by more researchers during studies of fresh fish stored aerobically in ice (Koutsoumanis and Nychas 1999; Talidourou et al. 2003; Chytiri et al. 2004) and packaged under modified atmosphere (Pournis et al. 2005). The antibiotics added to the CFC agar have been considered as good selective agents for most Pseudomonas spp. (Mead and Adams 1977). Many manufacturers state that cetrimide, a quaternary ammonium compound, is inhibitory to most bacterial species, except for Pseudomonas aeruginosa. However, the CFC agar has been found to be the most appropriate medium supporting the growth of Pseudomonas. Tryfinopoulou et al. (2001) studied the selectivity of the CFC agar during the enumeration of fish samples stored aerobically and under modified atmosphere at 0, 10 and 20°C. The selectivity of the agar was affected by storage temperature of the fish and type of packaging. In samples stored in air at low temperatures (0 and 10°C), most isolates were identified as Pseudomonas whereas isolates identified as Enterobacteriaceae were negligible. In fish stored at 20°C, the proportion of Enterobacteriaceae was higher than those of Pseudomonas and S. putrefaciens regardless of the storage atmosphere condition. The selectivity of the agar decreased with an increase in the interfering microorganisms (for example S. putrefaciens and Enterobacteriaceae), and had almost disappeared when the background flora was more robust than the organisms to be detected (Mossel et al. 1995). The differentiation of Enterobacteriaceae from Pseudomonas can be achieved by the oxidase test, but there is no effective and rapid test to differentiate Pseudomonas from Shewanella. Therefore, the enumeration of Pseudomonas directly from CFC agar is not adequate with fish samples. H2S-producing bacteria H2S-producing microorganisms are found to be important spoilage organisms of chilled and aerobically stored fresh fish (Gram et al. 1987; Koutsoumanis and Nychas 1999; Taliadourou et al. 2003; Chytiri et al. 2004; Hozbor et al. 2006) and in chilled, packaged, fresh and preserved fish (Truelstrup Hansen et al. 1998; Lyhs et al. 2001a; Emborg et al. 2002; Paarup et al. 2002; Magnusson et al. 2006). Different bacteria such as S. putrefaciens,

326

Recommended incubation temperature (°C)

Recommended incubation time (days) Fish and Fishery product

Microorganisms

Medium

Photobacterium phosphoreum

Iron agar (IA), spread plating

15 °C

5 days

Shewanella putrefaciens Pseudomonas spp.

Iron agar (IA), spread plating Cetrimide fusidin cephaloridine (CFC) agar

25 °C

3–4 days

20 °C

2 days

Fresh fish stored in ice, modified-atmosphere packaged fish

Pseudomonas isolation agar PseudomonasAeromonas selective agar

30 °C

2 days

25 °C

3–4 days

Vacuum-packaged coldsmoked fish Fresh fish stored in ice

Fresh fish stored in ice, vacuum- and modified-atmospherepackaged fish, vacuum-packaged cold-smoked fish Fresh fish stored in ice

References

Remarks

Emborg et al. 2002, 2005; Giménez. and Dalgaard 2004; Lopez-Caballero et al. 2002 Hozbor et al. 2006 Cato et al. 1986; Civera et al. 1995; Koutsoumanis et al. 1998; Taliadourou et al. 2003; Pournis et al. 2005 Civera et al. 1995 Hozbor et al. 2006

Addition of supplements (cetrimide, fusidin and cephaloridine)

Fishery Products: Quality, safety and authenticity

Table 15.3 Detection media, incubation temperature and incubation time used for determination of counts of specific spoilage bacteria in fish and fishery products.

Lactic acid bacteria

3–7 days

Nitrate Actidion Polymyxin agar (NAP), pH 6.2, spread plating

20 °C/25 °C

3 days

Rogosa SL agar, spread plating

20 °C/25 °C

5 days

Slanetz and Bartley agar (SB), spread plating

44 °C

2 days

KanamycinEsculine Azide Agar

37 °C

2 days

Vacuum-packaged fish, vacuum-packaged cold- and hot-smoked fish, modifiedatmosphere-packaged hot-smoked fish, fish stored in sous-vide, acetic-acid preserved fish, salted and fermented fish packaged in modified atmosphere and in air Vacuum- and modifiedatmosphere-packaged fish, vacuumpackaged coldsmoked fish, cooked and brined modifiedatmosphere-packaged shrimps Acetic-acid fish preserve, vacuumpackaged coldsmoked fish Vacuum-packaged coldsmoked fish, cooked and brined modifiedatmosphere-packaged shrimps Vacuum-packaged coldsmoked fish

Nordic Committee on Only anaerobic incubation Food Analysis 1991; Civera et al. 1995; González-Fandos et al. 2005; Lyhs et al. 2001a, 2001b, 2004, 2007; Pournis et al. 2005; Cakli et al. 2006

Jørgensen et al. 2000; Leroi et al. 2001; Emborg et al. 2002, 2005; Dalgaard et al. 2003

Only anaerobic incubation

Leroi et al. 2000; Lyhs et al. 2001b

Only anaerobic incubation

Dalgaard et al. 2003; Giménez and Dalgaard 2004

Civera et al. 1995

Microbiological methods

Enterococci

20 °C/25 °C/30 °C

De Man, Rogosa and Sharpe agar (MRS), pour plating

Continued

327

Medium

Recommended incubation temperature (°C)

Brochothrix thermospacta

Streptomycin thallium acetate actidion agar (STAA), spread plating Violet red bile glucose agar (VRBG), pour plating

20 °C

3 days

30 °C/37 °C

1–2 days

TSA, 1–2 h at room temperature, pour plating, overlaid by VRBG agar

25 °C/30 °C

2 days

Casein-peptone soymealpeptone (CASO) agar, pour plating overlaid by VRBG agar

30 °C

2 days

Enterobacteriaceae

Recommended Fish and Fishery product incubation time (days) Fresh fish stored in ice, fresh fish stored in air, Vacuum-packaged cold-smoked fish, modified-atmospherepackaged fish Fresh fish stored in ice, fresh fish stored in air, vacuum- and modified-atmospherepackaged hot-smoked fish, modifiedatmosphere-packaged fish, salted and fermented fish packaged in modified atmosphere and in air Vacuum-packaged coldsmoked fish, fish stored in sous-vide, vacuum- and modified-atmospherepackaged fish

Vacuum-packaged coldsmoked fish

References

Koutsoumanis et al. 1998; Leroi et al. 2001; Taliadourou et al. 2003; Chytiri et al. 2004; Pournis et al. 2005 Koutsoumanis et al. 1998; Lyhs et al. 2001a, 2007; Taliadourou et al. 2003; Chytiri et al. 2004; Pournis et al. 2005; Cakli et al. 2006

Civera et al. 1995; Jørgensen et al. 2000; Paarup et al. 2002; Giménez and Dalgaard 2004; Emborg et al. 2005; González-Fandos et al. 2005 Leroi et al. 2001

Remarks

Fishery Products: Quality, safety and authenticity

Microorganisms

328

Table 15.3 Continued

Microbiological methods

329

types of Enterobacteriaceae, Aeromonaceae, Vibrionaceae and some lactobacilli such as Lactobacillus sakei are able to produce hydrogen sulphide (Gram et al. 1987; Truelstrup Hansen 1995; Gram and Huss 1996; Joffraud et al. 2001; Leroi et al. 2001; NCFA 2006). In contrast, neither Pseudomonas nor Photobacterium phosphoreum produce significant amounts of H2S (Gram and Huss 1996). Good results in enumerating H2S-producing microorganisms were obtained in fresh and spoiled fish, for example, stored aerobically in ice (Koutsoumanis and Nychas 1999; Sveinsdóttir et al. 2002; Taliadourou et al. 2003; Chytiril et al. 2004) and packaged under vacuum (Leroi et al. 1998; Lyhs et al. 2001a) or modified atmosphere (Emborg et al. 2002; Pournis et al. 2005). However, Lyhs et al. (2006) reported a total absence of H2S-producing microorganisms when studying maatjes herring stored in air and under MAP at 4°C and 10°C. It is known that H2S-producing bacteria are favoured by a low oxygen level, but inhibited in environments with high or increasing carbon dioxide levels as in modifiedatmosphere or vacuum packaging (Jørgensen et al. 1988; Rosnes et al. 1997; Lyhs et al. 2001a). Studying the effect of smoke and salt on the microflora of vacuum-packaged cold-smoked salmon, Leroi et al. (2000) reported that salt produced a strong linear inhibition on H2S-producing bacteria. The salt used in the processing of maatjes herring might have influenced these bacteria by suppressing their growth. Shewanella spp. S. putrefaciens is considered to be the SSO in marine temperate-water fresh fish stored in ice (Gram and Huss 1996; Debevere and Boskou 1996; Koutsoumanis and Nychas 1999). Gram et al. (1987) identified it as the major spoilage organism in vacuum-packaged cod fillets stored in ice. On the other hand, it has been found to be of little importance in spoiled, modified-atmosphere packaged cod fillets (Dalgaard et al. 1993; Dalgaard 1995; Debevere and Boskou 1996). S. baltica has been identified as the main H2S-producing organism growing on iced marine fish caught in the Baltic Sea (Fonnesbech Vogel et al. 2005). There is no selective medium for culturing of S. putrefaciens (Rudi et al. 2004). Shewanella spp. have usually been identified by standard phenotypical key tests (Stenstrøm and Molin 1990). However, phenotypic characteristics do not allow always for species differentiation, for example between S. putrefaciens and S. algae (Fonnesbech Vogel et al. 1997), or between some psychrophilic Shewanella (Ziemke et al. 1997). Phenotypic limits of and within the psychrotropic Shewanella group are currently being redefined (Fonnesbach Vogel et al. 2005; Satomi et al. 2006). Recently, combining phenotypic and genetic analyses has shown success in determining novel species within the genus Shewanella (Satomi et al. 2006). Enterobacteriaceae Mesophilic Enterobacteriaceae (Gram and Huss 1996) have been found from fish caught in polluted waters. A mixture of lactic acid bacteria and Enterobacteriaceae may develop at the end of the chilled storage of vacuum-packaged cold-smoked salmon and rainbow trout, respectively (Truelstrup Hansen 1995; Lyhs et al. 1998a; Leroi et al. 2001). During study of different fish products packaged under vacuum and modified atmosphere (Pournis et al. 2005; Cakli et al. 2006; Lyhs et al. 2006) and unpacked stored in ice

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(Koutsoumanis and Nychas 1999; Taliadourou et al. 2003; Chytiri et al. 2004) Enterobacteriaceae have been successfully determined on violet red bile glucose (VRBG) agar using the pour plate technique. Typical Enterobacteriaceae colonies are counted after 1–2 days at incubation temperatures between 25 and 37°C (NCFA 2005). Other authors (Jørgensen et al. 2000; Paarup et al. 2002; Gimenez and Dalgaard 2004; Gonzalez-Fandos et al. 2005; Mejlholm et al. 2005) used pour plates of TSA for determination of Enterobacteriaceae, which after 1–2 hours at room temperature was overlaid by VRBG agar. Leroi et al. (2001), studying different microbiological parameters in vacuum-packaged coldsmoked salmon, used pour plates of casein-peptone-soymeal-peptone agar (CASO) overlaid by VRBG agar. For phenotypic identification of Enterobacteriaceae, different tests, for example absence of cytochrome oxidase, fermentative metabolism of glucose, DNAase production and the ability to ferment sorbitol using Hugh and Leifson’s basal medium, have been used (Lyhs et al. 1998a; Paludan-Müller et al. 1998). The API 20E system is another reliable method of high accuracy used for identification of Enterobacteriaceae isolated from vacuumpackaged, cold-smoked fish (Truelstrup Hansen 1995; Lyhs et al. 1998a; Paludan-Müller et al. 1998). Lactic acid bacteria Lactic acid bacteria (LAB) comprise the major component of the spoilage flora, which develops on vacuum-packaged, lightly preserved fish products (Civera et al. 1995; Truelstrup Hansen 1995; Leroi et al. 1998; Lyhs et al. 1998a; Paludan-Müller et al. 1998; Lyhs et al. 2001a; Leroi et al. 2001; Jørgensen et al. 2001; Joffraud et al. 2001) and hot-smoked fish products (Jöckel et al. 1986; Paleari et al. 1990; Zorn et al. 1993) after a few weeks’ storage at chilled temperatures. Important members of spoilage LAB are, among others, the genera Lactobacillus, Carnobacterium and Enterococcus. Lactobacillus spp. have been identified as the SSO in marinated raw or cooked herring preserved in vinegar and salt (Meyer 1956; Kreuzer 1957; Lerche 1960; Reuter 1965; Erichsen 1967; Sharpe and Pettipher 1983; Lyhs et al. 2001b 2004). Smoked and charred Baltic herring with high levels of enterococci have been related to food poisoning outbreaks in Finland (Korkeala and Pakkala 1988). Enterococci together with carnobacteria were dominant members of spoilage associations of cooked and brined modified-atmosphere-packaged shrimps when stored at high and low storage temperatures, respectively (Dalgaard and Jørgensen 2000; Dalgaard et al. 2003). For determination of LAB counts in vacuum- and modified-atmosphere packaged fish and fish products (Civera et al. 1995; Lyhs et al. 2001a 2004 2007; Pournis et al. 2005; Gonzalez-Fandos et al. 2005), the De Man, Rogosa and Sharpe (MRS) agar, originally introduced by De Man et al. (1960), has been successfully used. This agar is also recommended by the NCFA (1991). Other authors (Leroi et al. 2001; Jørgensen et al. 2000; Emborg et al. 2002; Dalgaard et al. 2003) recommended nitrate actidion polymyxin (NAP) agar, which was originally developed by Davidson and Cronin (1973). For enumeration of lactobacilli in vacuum-packed cold-smoked salmon (Leroi et al. 2000) and in an acetic-acid herring preserve (Lyhs et al. 2001b), Rogosa SL agar (Rogosa et al. 1951) has been used. This agar is selective for lactobacilli and inhibits Carnobacterium spp. Enterococci were detected in several fish products on Slanetz–Bartley agar (Dalgaard et al. 2003; Gimenez

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and Dalgaard 2004), forming colonies with a red–pink centre after 2 days’ incubation at 44°C. The identification of LAB has mainly been based on traditional biochemical and physiological tests, which often produce very controversial results. Moreover, there are certain species that cannot readily be distinguished by phenotypic characters (Schleifer et al. 1995; Gancel et al. 1997; Lyhs et al. 1998a). Several molecular methods have been used for strain typing or characterisation of LAB. Paludan-Müller et al. (1998), studying the role of Carnobacterium species in the spoilage of vacuum-packaged, cold-smoked salmon, identified carnobacteria by whole-cell-protein patterns. Ribotyping (Grimont and Grimont 1986), which is based on patterns generated from the ribosomal RNA genes only, has been applied with success for the identification of the main spoilage LAB in vacuum-packaged, cold-smoked and ‘gravad’ rainbow trout (Lyhs et al. 1999 2001a) and marinated herring (Lyhs et al. 2001b 2004; Lyhs and Björkroth 2008). Photobacterium phosphoreum Members of Photobacteria can be luminous, occurring as light-organ symbionts in several fish (Dalgaard et al. 1997a). Photobacterium phosphoreum, a bioluminescent species, is the SSO in marine fish from temperate waters stored in modified-atmosphere (Dalgaard 1995; Gram and Huss 1996; Dalgaard et al. 1997b; Dalgaard et al. 1998; Emborg et al. 2002). In vacuum-packaged cold-smoked salmon at the end of chilled storage, P. phosphoreum has been found with LAB (Truelstrup Hansen 1995). P. phosphoreum is heat labile and can be killed in agar of 45°C. Thus pour plating and spread plating methods with incubation temperatures of 23–25°C are inappropriate for its detection (van Spreekens 1974; Dalgaard et al. 1993 1997b). There is a lack of selective media and methods for enumeration and specific detection of P. phosphoreum in fish products (Dalgaard et al. 1997b; Rudi et al. 2004). P. phosphoreum, belonging to the Vibrionaceae, does not grow on thiosulphate citrate bile sucrose (TCBS) agar, a vibrioselective agar (Dalgaard et al. 1997b). However, various nutritionally rich media have allowed the detection of this bacterium. Dalgaard et al. (1997b) compared different kind of agars for detecting TVC and luminous colonies in spoiled, chilled cod stored in air and under modified atmosphere: LH and IA (both with the addition of 1% NaCl) and a luminescence medium (LM) (Baumann and Baumann 1984). All media were incubated at 15°C for 7 days. LM gave significantly lower counts than LH and IA. Most P. phosphoreum strains from chilled fish products were non-luminous (van Spreekens 1974; Dalgaard 1995; Dalgaard et al. 1997b). Therefore, the number of luminous colonies cannot be used for enumerating P. phosphoreum. Lopez-Cababello et al. (2002) isolated P. phosphoreum from spoiled hake stored in ice plated on IA with 1% NaCl stored for 5 days at 15°C. Agars without NaCl are inappropriate for detecting P. phosphoreum (Dalgaard et al. 1997b). The NCFA (2006) recommends the addition of 1% NaCl to the LH to improve the detection of P. phosphoreum. Furthermore, spread plating and incubation at 15°C for 5–7 days should be used (Huss 1995a). However, several national and international authorities recommend pour plating on IA and PCA (NCFA 1994). Direct microscopic counting of spread plates incubated at 15°C allowed quantitative enumeration of P. phosphoreum in fish products (van Spreekens 1974; Dalgaard et al. 1996). With the conductance method, quantitative and selective detection of even low levels of P. phosphoreum in various fresh

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fish products packaged in modified atmosphere has been possible (Dalgaard et al. 1996). To identify P. phosphoreum a scheme including tests, like growth in different NaCl concentrations and at different temperatures, gas production from glucose, ammonia (NH3) production from arginine, lysine and ornithine, reduction of trimethylamine oxide (TMAO) and nitrate (NO3−) and bioluminescence, have been used in different studies (Dalgaard 1995; Paludan-Müller et al. 1998). Brochothrix thermospacta Brochothrix thermospacta is the dominant spoilage bacterium of Mediterranean sea fish stored under modified atmosphere packaging (Drosinos and Nychas 1996; Koutsoumanis et al. 1998). It also may develop with LAB at the end of chilled storage in vacuum-packaged cold-smoked salmon (Leroi et al. 2001). With Carnobacterium maltaromaticum, it is responsible for spoilage in cooked and peeled modified-atmosphere packaged shrimps (Mejlholm et al. 2005). B. thermospacta is usual enumerated on streptomycin thallium acetate actidion (STAA) agar (Gardner 1966) using spread plates followed by identification using Gram staining, catalase and oxidase tests (Leroi et al. 2001; Mejlholm et al. 2005).

15.2.3

Pathogenic bacteria

Fish and fish products are known vehicles for transmission of foodborne diseases (Huss et al. 1997). Pathogenic bacteria associated with seafood can be categorised into three general groups: (1) bacteria (indigenous bacteria) that belong to the natural microflora of fish (Clostridium botulinum, pathogenic Vibrio spp., Aeromonas hydrophila); (2) enteric bacteria (non-indigenous bacteria) that are present due to faecal contamination (Salmonella spp., Shigella spp., pathogenic Escherichia coli, Staphylococcus aureus); and (3) bacterial contamination during processing, storage or preparation for consumption (Bacillus cereus, Listeria monocytogenes, Staphylococcus aureus, Clostridium perfringens) (Feldhusen 2000). However, indigenous pathogenic bacteria present in fresh cultured fish products are usually found at fairly low levels. When these products are adequately cooked before consumption and afterwards no recontamination takes place, food safety hazards are insignificant (Feldhusen 2000). Possible high levels of these bacteria are due to growth and might produce disease in humans (Huss 1997). For most RTE products like lightly preserved fish, the growth of pathogenic bacteria (especially L. monocytogenes and C. botulinum) is a serious safety concern. In cold-smoked and ‘gravad’ fish the amounts of smoke and salt, respectively, are not sufficient to prevent the growth of pathogenic bacteria (Lyhs et al. 1998b). Listeria spp. Owing to their processing method and extended shelf-lives at refrigeration temperatures, vacuum-packaged cold-smoked and ‘gravad’ fish products have to be considered as potential high-risk foods for L. monocytogenes (Civera et al. 1995; Loncarevic et al. 1996; Lyhs

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et al. 1998b; Jorgensen and Huss 1998; Johannson et al. 1999). Few sporadic cases of listeriosis have been reported associated with the consumption of contaminated fish and shellfish (Facinelli et al. 1989; Ericsson et al. 1997; Miettinen et al. 1999; Brett et al. 1998). Traditional isolation methods of Listeria spp. and L. monocytogenes are based on selective enrichment followed by plating on selective media (Lunden 2004). During a two-stage enrichment procedure, the food samples are homogenised and incubated in a selective liquid enrichment medium with reduced concentration of selective agents (primary enrichment). After 24 hours a culture obtained from the first enrichment broth is transferred into a second selective liquid enrichment medium with full concentration of selective agents (secondary enrichment). The Nordic Committee on Food Analyses (NCFA) (2004) recommends the use of Listeria enrichment broth LBI and LBII or Fraser enrichment broth, and the International Organization for Standardisation (ISO) (Anonymous 1997) half-Fraser and Fraser enrichment broths. Plating out of cultures and identification from the enrichment broths is performed on Listeria-selective agar (polymyxin-acriflavine-LiCl-ceftazidime-aesculin-mannitol (PALCAM) or Oxford agar). The presence of Listeria spp. and/or L. monocytogenes is confirmed by appropriate morphological, physiological and biochemical tests performed on several presumptive colonies (Anonymous 1997; Scotter et al. 2001; NCFA 2004; Autio 2003; Lunden 2004). Because a differentiation of L. monocytogenes colonies from other non-pathogenic Listeria species is not possible on PALCAM or Oxford agar, other plating media like chromogenic culture media (Agar Listeria Ottaviani and Agosti (ALOA), RAPID’ L. mono) (Karpiskova et al. 2000; Vlaemynck et al. 2000; Polivka 2001; Leclerq 2004; Becker et al. 2006) or Listeria monocytogenes blood agar (LMBA) (Johansson 1998) have been developed and used in recent years. Clear advantages (rapid and specific differentiation of L. monocytogenes from other Listeria spp. by specific reactions directly on the agar plate, lower costs, time reduction) let most authors recommend the substitution of the standard media by these other selective media or to use a combination of both. In addition, molecular typing methods should be used as an alternative to the classical confirmation test (Scotter et al. 2001). Clostridium botulinum C. botulinum type E is the most common type that is mainly associated with fish and fish products. It is of particular concern because of its ability to grow at temperatures between 3 and 5°C (Feldhusen 2000). Vacuum-packaged hot-smoked, air-dried and salted fish products have been identified as causes of botulinum (Slater et al. 1989; Weber et al. 1993; Korkeala et al. 1998; Hyytiä et al. 1998; Lindström et al. 2004 2006). Furthermore, vacuumpackaged unprocessed, ‘gravad’ and cold-smoked fish and smoked river lampreys from the Baltic Sea have been shown to be a serious health risk for C. botulinum type E (Hyytiä et al. 1998). Conventional detection and isolation of C. botulinum are based on culturing in a liquid medium. The standard method for detection of the botulinum toxin in the culture supernatant is the mouse bioassay (Kautter and Salomon 1977). This method is not only time consuming and expensive, but it also raises ethical concerns about due to the use of experimental animals (Lindström et al. 2001). A broad variety of routine liquid media are available but they are all non-selective, allowing other bacteria to grow (Lindström and Korkeala 2006). As plating media, blood agar and egg yolk agar (EYA) are mostly used (Hausschild and Hilsheimer

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1977). EYA enables the lipase reaction which is typical for C. botulinum. However, other clostridia produce lipase as well and may therefore confuse the identification (Cato et al. 1986). The development of selective media is complicated by the fact that the species C. botulinum consists of four physiologically and genetically distinct groups of organisms (Lindström and Korkeala 2006). To identify Clostridium spp., commercial tests based on biochemical reactions have been developed. However, different tests are able to identify clostridia to species level (Head and Ratnam 1988; Marler et al. 1991), but other tests fail owing to the diverse physiology of C. botulinum (Brett 1998; Lindström et al. 1999). They are time-consuming, laborious and often susceptible to technical bias affecting the success of identification (Lindström and Korkeala 2006). Aeromonas spp. Aeromonas spp. can be found in most aquatic environments (Huss et al. 1997), but also in fish and fish products like cold- and hot-smoked fish (Gobat and Jemmi 1993), fresh fish, fish-eggs and shrimps (Hänninen et al. 1997) and pre-packaged fresh fish (GonzàlezRodriguez et al. 2002a; Herrera et al. 2006). They are thought to be part of the spoilage flora of chilled freshwater fish stored in air (ICMSF 1998) and might originate from contamination after processing (Gobat and Jemmi 1993; Hänninen et al. 1997). Palumbo et al. (1985) developed the starch ampicillin (SA) agar to allow a rapid quantitative recovery of A. hydrophila from foods in the presence of many competing bacteria. Havelaar et al. (1987) combined the advantages of two selective agars in a new medium, ampicillin–dextrin (AD) agar. Gobat and Jemmi (1995) studied seven selective agars and two enrichment broths for their suitability for isolating mesophilic Aeromonas spp. from meat, fish and shellfish samples. For qualitative isolation they recommended enrichment in alkaline peptone water (APW, pH 8.7) followed by consecutive plating onto ampicillin sheep blood agar (ASBA 30; 30 mg/l ampicillin) and bile salts-irgasan-brillant green (BIBG) agar. Hänninen et al. (1997) cultured different fish samples either directly on AD agar or enriched the samples first in tryptic soy broth containing 30 μg/ml ampicillin followed by culturing onto AD agar. It should be noted that plates must be incubated at temperatures of 28–30°C because many Aeromonas spp. do not grow above 35°C (Hänninen et al. 1997). Suspected Aeromonas spp. must be confirmed and can be subdivided by morphological, physiological and biochemical tests, for example acid production from l-arabinose and salicin in O/F-medium, esculin hydrolysis, Voges–Proskauer test, lysine decarboxylase in Falkow’s medium, motility and growth in BHI + 1% NaCl at 37°C (Paludan-Müller et al. 1998). Also, commercial tests as different API systems (20E, 20NE, NE) have been used to identify Aeromonas spp. (Hänninen et al. 1997). However, many commercial test kits do not always recognise all Aeromonas spp. correctly (Altwegg et al. 1990). Those species that may grow at temperatures higher than 35°C can also show untypical biochemical characteristics (Hänninen and Siitonen 1995). Vibrio spp. Several Vibrio spp. are human pathogens which may also be transmitted to humans by the consumption of undercooked seafood or shellfish (Feldhusen 2000). In Western countries seafood-related illness caused by pathogenic Vibrio spp. is commonly associated with

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crustaceans or molluscan shellfish, whereas finfish are common vehicles for outbreaks in Japan or other Asian countries (ICMSF 1998). V. parahaemolyticus is an important cause of gastrointestinal disease, most often after the consumption of seafood (Dalgaard 1998). A qualitative enrichment procedure is normally performed for detection of foodborne Vibrio spp. when microbiological methods are used (Donovan and Netten 1995). For isolation of V. parahaemolyticus, APW, glucose teepol salt broth (GSTB), salt polymyxin broth (SPB) or salt colistin broth (SCB) have been recommended as enrichment media. For plating, Donovan and Netten (1995) recommended the use of thiosulphate citrate bile salt (TCSB) agar in parallel with polymyxin mannose tellurite (PMT) or sodium dodecylsulphate polymyxin sucrose (SPS) agar. Plates should be incubated for 24 hours at 37°C to prevent overgrowth of competitors. Su et al. (2005) examined the selectivity and specifity of a chromogenic medium, Bio-Chrome Vibrio medium (BCVM) which is based on the formation of purple colonies. It was possible to differentiate V. parahaemolyticus from other Vibrio spp. However, they concluded that further studies are needed. Alam et al. (2001) used a nutrient agar with 2% NaCl (V. parahaemolyticus is halophilic) as a primary culture medium and transferred thereafter colonies to TCBS agar. There is a possibility of failing in the detection of Vibrio spp. when TCBS or APW are used directly. Thus, non-selective media can be used because they have the advantage of recovering injured or weak cells which are very sensitive to selective agents in selective media (Alam et al. 2001). Hayashi et al. (2006) described the soft-agar-coated filter method for detecting pathogenic V. parahaemolyticus. It is possible to detect contamination levels of five viable cells per gram sample within 2 working days regardless of the background flora. In principle, only viable V. parahaemolyticus cells are able to penetrate through the soft-agar-coated paper owing to their mobility. DNA released from dead, virulent V. parahaemolyticus cells into an enrichment medium are eliminated by a DNAse pretreatment and can thus not interfere with the followed polymerase chain reaction (PCR). Two genes for two haemolysins (thermostable direct haemolysin, TDH, and TDH-related haemolysin, TRH) which are considered to be major virulence factors of V. parahaemolyticus are then detected by a multiplex PCR assay. This method is a great improvement in the detection of pathogenic V. parahaemolyticus in seafood and helps to decrease time and laboratory work. Salmonella spp. Generally, disease surveillance reports from public health authorities indicate that human salmonellosis associated with consumption of farmed freshwater or marine fish and crustaceans occurs very rarely compared, for example, with poultry meat (Feldhusen 2000). According to Huss (2003), when seafood is harvested in a clean environment and handled hygienically, cross-contamination is the most common cause of findings of Salmonella. In raw seafood products mainly from tropical climates, there is a high prevalence of Salmonella (Dalsgaard 1998; Reilly and Käferstein 1999), whereas low prevalence or absence can be common in temperate regions (González-Rodriguez et al. 2002). Several authors studied prevalence and growth of Salmonella in seafood, for example D’Aoust (2000), GonzálezRodriguez et al. (2002), Brands et al. (2005) and Herrera et al. (2006). Usually, for detecting Salmonella, a two-stage enrichment procedure including pre-enrichment and enrichment in Rappaport-Vassiliadis-soy-peptone broth is used (NCFA 1999). Plating out of cultures and identification from the enrichment broth is performed on different agars, for example,

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xylose-lysine-decarboxylase (XLD) agar (Nychas and Tassou 1996; González-Rodriguez et al. 2002) or bismuth-sulphite agar (Brands et al. 2005). After incubation at 37°C for 24 hours, the presence of characteristic colonies must be checked and confirmed by biochemical and serological tests.

15.3

Most probable number method

The prevalence of pathogenic bacteria in samples can be very low and it is possible that there is a lack of proper selective microbiological media for their detection. Therefore, enrichment in liquid media is often needed. In such cases, serial dilution tests measuring the concentration of a target microbe in a sample with an estimate – the most probable-number (MPN) method – can be used (Lindström and Korkeala 2006). In principle, several 10-fold dilutions of the sample are prepared. Three serial aliquots or dilutions are inoculated in test tubes containing the suitable liquid medium. They are then incubated and read for positive reactions. The number of positive tubes in each dilution is registered and the most probable number of bacteria per mass volume unit is read from the MPN table (Guyer and Jemmi 1991; Jemmi and Keusch 1992; Jay 2000; NCFA 2003; Lindström and Korkeala 2006). This method is simple, and specific microorganisms can be determined by using appropriate selective media. However, the disadvantages are that the morphology of colonies cannot be observed and a large amount of glassware is needed. Furthermore, this method lacks of precision (Jay 2000).

15.4

Molecular methods

Standard methods for recovering microorganisms from foods may include enrichment culture, streaking out onto selective or differentiating media or direct plating onto these, and identification of colonies by morphological, biochemical and immunological tests (Hill and Jinneman 2000). This all requires a lot of manual labour, is costly and usually needs between 2 and 5 days. Pathogenic microorganisms may be present at very low levels and they can be difficult to detect. Their detection may also be interfered with by components of the food or other bacteria present (Hill and Jinneman 2000). Furthermore, identification of microorganisms using phenotypical tests can often give incorrect results due to the influence of cultivation conditions (morphology, colour, and so on). The medium and the conditions (temperature and atmosphere) for culturing may also not accurately reflect the conditions given in the food (Cambon-Bonavita et al. 2001), or certain bacteria might be unable to grow on the medium used (Rudi et al. 2004). Molecular methods allow a rapid detection and identification of specific bacterial strains and/or virulence genes without the need for pure cultures. They are mainly based on oligonucleotide probes, PCR or antibody techniques. The advantages of molecular methods are: high sensitivity and specificity in detection and identification of bacteria; providing results within one working day; detection of the target microorganism at a very low detection level; a combination of the indication of the presence of the bacterium and the confirmation of its identity in one step; and identification of bacteria to species level. Many individual articles

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in research journals and books have described the different methods, their mechanisms and application in fish and fish products. Within the scope of this chapter, only a few publications can be referred to as examples for the use of molecular methods in detecting and identifying pathogenic and spoilage bacteria in fish and fish products, without claim for completeness and without valuation. Species-specific macromolecular patterns, such as lipopolysaccharides, fatty acids, proteins or DNA, can be used for bacterial identification. Strains belonging to the same species share characteristic bands and can be compared with type or reference strains in a database. Computer-assisted large-scale grouping analysis (species-specific cluster analysis) involves, for example, the creation of dendrograms to reveal groups of related organisms. Patterns of an unknown isolate can be identified against libraries containing corresponding patterns of known species. Two methods have been applied with some success for species-level identification of microbial strains in different fish products. Sodium dodecylsulfate– polyacrylamide gel electrophoresis (SDS–PAGE) of whole-cell proteins or cell envelope proteins has been applied to the identification of Carnobacterium spp. in spoiled vacuumand modified-atmosphere-packed cold-smoked salmon (Paludan-Müller et al. 1998). Determination of 16 + 23S rRNA gene restriction patterns (ribotyping) is based on patterns generated from the ribosomal RNA genes only. It has been used for identification of spoilage LAB in vacuum-packaged cold-smoked and ‘gravad’ rainbow trout and in semi-preserved marinated herring (Lyhs et al. 1998 2001a b, 2004) and for Aeromonas spp. in different fish (herring, rainbow trout, vendace and others) and fishery (shrimps and fish-eggs) samples (Hänninen et al. 1997). The use of probes and PCR has increased dramatically in recent years. Gene probes and PCR primers for detecting and identifying almost every foodborne pathogenic bacterial species have been developed. A probe is a fragment of a single-stranded nucleic acid that will specifically bind (hybridise) to complementary regions of a target nucleic acid. There are three different approaches to the design of nucleic acid probes: randomly cloned probes; probes complementary to specific genes and gene fragments (16S or 23S rRNA targeted oligonucleotide probes); and rRNA target probes (Schleifer et al. 1995). In addition to DNA hybridisation techniques, specific areas of the genes encoding rRNA can be used as templates for primer design for detecting nucleic acid hybridisation (Van Belkum 1994). PCR is a method for the in vitro amplification of a given region of DNA. A pair of specific oligonucleotides bracketing the region serves as primers for polymerase to initiate the DNA replication (Hill and Jinneman 2000). For fish products, PCR assays have been used to detect L. monocytogenes, for example in cold-smoked salmon (Simon et al. 1996; Rodriguez-Lazaro et al. 2005) and in ‘gravad’ rainbow trout (Ericsson and Stalhandske 1997). Furthermore, using PCR-based techniques, pathogenic Aeromonas spp. have been detected in raw and coldsmoked trout and salmon (González-Rodríguez et al. 2002b) and C. botulinum in rainbow trout (Hielm et al. 1996), in modified-atmosphere packaged fish (Kimura et al. 2001) and in whitefish (Lindström and Korkeala 2006). Cambon-Bonavita et al. (2001) performed PCR amplification coupled with amplification ribosomal DNA restriction analyses (ARDRA) in vacuum-packed cold-smoked salmon, and Rudi et al. (2004) examined partial 16S rRNA gene sequences of bacteria from modified-atmosphere-packaged salmon and coalfish. Giacomazzi et al. (2004) found the nested-PCR/temporal temperature gradient gel electrophoresis (TTGE) technique on the rpoB gene on pure cultures of reference strains to be a promising way for describing the microbial diversity in cold-smoked salmon.

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The use of a culture-independent molecular biology approach as an alternative method to cultivation has been found more and more promising for evaluating microbial communities in foods (Rudi 2003). However, as a powerful tool in future research, the combination of both traditional and molecular methods for the direct description of microbial communities with traditional methods for enrichment and isolation of important strains has been recommended (Cambon-Bonavita et al. 2001; Rudi et al. 2002). The question whether bacterial cells are dead or alive is important in monitoring foods and water safety (Nocker and Camper 2006). With traditional culture methods it is not possible to differentiate between dead and living bacteria or their DNA, or to detect intermediate states like cell injury (Berney et al. 2007). The most commonly used strategy is to focus on the presence of rapidly degrading RNA (Nocker and Camper 2006). Another possibility is the use of dyes and microscopy for differentiation between dead and living cells. Nowadays the LIVE/DEAD® Bac Light™ kit has been increasingly used by researchers in different fields (Berney et al. 2007). In a study about the changes of bacterial composition in spoiled oysters during storage, Romero et al. (2002) used this kit for determinating viable bacteria. Other alternatives have been described by Nogva et al. (2003) and Rudi et al. (2005) using ethidium monoazide bromide (EMA)-PCR. This diagnostic DNA-based method combines live–dead staining dye and real-time PCR. However, Vaitilingo et al. (1998) showed that reverse transcriptase PCR (RT–PCR) offers the greatest potential for the detection/discrimination of live and dead cells. Many seafood-related infections are caused by norovirus and hepatitis A virus. Electron microscopy was the most used laboratory diagnostic method for direct detection of viruses (Butt et al. 2004). Nowadays, enteric viruses are detected in shellfish concentrates by cell-culture infectivity assays, but increasingly by molecular techniques like PCR (Shieh et al. 1999). Gene probes and PCR can detect fewer than 10 virus particles (Hill and Jinneman 2000). RT–PCR has become available commercially and show greater sensitivity and specificity than electron microscopy (Butt et al. 2004).

15.5

References

Alam, M.J., Tomochika, K., Miyoshi, S. and Shinoda, S. (2001) Analysis of seawaters for the recovery of culturable Vibrio parahaemolyticus and some other vibrios. Microbiology and Immunology 45: 393–397. Altwegg, M., Steigerwalt, A.G., Altwegg-Bissig, R., Luthy-Hottenstein, J. and Brenner, D.J. (1990) Biochemical identification of Aeromonas genospecies isolated from humans. Journal of Clinical Microbiology 28: 258–264. Anonymous (1997) EN ISO 11290–1 Microbiology of food and animal feedingstuffs – Horizontal method for the detection and enumeration of Listeria monocytogenes – Part 1: Detection. International Organisation for Standardisation, Geneva. Autio, T. (2003) Tracing the sources of Listeria monocytogenes contamination and listeriosis using molecular tools. Thesis, Department of Food and Environmental Hygiene, Faculty of Veterinary Medicine, University of Helsinki, Finland. Baumann, P. and Baumann, L. (1984) Genus II. Photobacterium Beijernik 1889: 402AL. In: N.R. Krieg and J.G. Holt (Eds) Bergey’s Manual of Systematic Bacteriology, Volume 1. Williams and Wilkins, Baltimore, MD, pp. 539–545.

Microbiological methods

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Becker, B., Schuler, S., Lohneis, M., Sabrowski, A., Curtis, G.D. and Holzapfel, W.H. (2006) Comparison of two chromogenic media for the detection of Listeria monocytogenes with the plating media recommended by EN/DIN 11290–1. International Journal of Food Microbiology 109: 127–131. Berney, M., Hammes, F., Bosshard, F., Weilenmann, H.-U. and Egli, T. (2007) Assessing and interpreting bacterial viability using LIVE/DEAD® Bac Light™ kit in combination with cytometry. Applied and Environmental Microbiology 73: 3283–3290. Brands, D.A., Inman, A.E., Gerba, C.P., Mare, C.J., Billington, S.J., Saif, L.A., Levine, J.F. and Joens, L.A. (2005) Prevalence of Salmonella spp. in oysters in the United States. Applied and Environmental Microbiology 71: 893–897. Brett, M.M. (1998) Evaluation of the use of the bioMerieux Rapid ID32 A for the identification of Clostridium botulinum. Letters in Applied Microbiology 26: 81–84. Brett, M.S., Short, P. and McLauchlin, J. (1998) A small outbreak of listeriosis associated with smoked mussels. International Journal of Food Microbiology 43: 223–229. Butt, A.A., Aldridge, K.E. and Sanders, C.V. (2004) Infections related to ingestion of seafood. Part I: viral and bacterial infections. The Lancet Infectious Diseases 4: 201–212. Cakli, S., Kilinc, B., Dincer, T. and Tolasa, S. (2006) Comparison of shelf-lives of modifiedatmosphere- and vacuum-packaged hot-smoked rainbow trout (Onchoryncus mykiss) European Food Research and Technology 224: 19–26. Cambon-Bonavita, A.-M., Lesongeur, F., Menoux, S., Lebourg, A. and Barbier, G. (2001) Micorbial diversity in smoked salmon examined by a culture-independent molecular approach – a preliminary study. International Journal of Food Microbiology 70: 179–187. Cardinal, M., Gunnlaugsdottir, H., Bjornevik, M., Ouisse, A., Vallet, J.L. and Leroi, F. (2004) Sensory characteristics of cold-smoked Atlantic salmon (Salmo salar) from European market and relationship with chemical, physical and microbiological measurements. Food Research International 37: 181–193. Cato, E.P., George, W.L. and Finegold, S.M. (1986) Genus Clostridium. In: P.H.A. Sneath, N.S. Mair, M.E. Sharpe and J.G. Holt, (Eds) Bergey’s Manual of Systematic Bacteriology, Volume 2. Williams and Wilkins, Baltimore, MD, pp. 1141–1200. Chytiri, S., Chouliara, I., Savvaidis, I.N. and Kontominas, M.G. (2004) Microbiological, chemical and sensory assessment of iced whole and filleted aquacultured rainbow trout. Food Microbiology 21: 157–165. Civera, T., Parisi, E., Amerio, G.P. and Giaccone, V. (1995) Shelf-life of vacuum-packed smoked salmon: microbiological and chemical changes during storage. Archiv fur Lebensmittelhygiene 46: 1–24. D’Aoust, J.-Y. (2000) Salmonella. In: B.M. Lund, T.C. Baird-Parker and G.W. Gould (Eds) The Microbiological Safety and Quality of Foods. Aspen Publishers, Gaithersburg, MD, pp. 1233–1299. Dalgaard, P. (1995) Qualitative and quantitative characterisation of spoilage bacteria from packed fish. International Journal of Food Microbiology 26: 319–333. Dalgaard, P. (2000) Freshness, quality and safety in seafoods. Flair-Flow Europe Technical Manual F-FE 380A/00. Dalgaard, P. and Jørgensen, L.V. (2000) Cooked and brined shrimps packed in a modified atmosphere have a shelf-life of >7 months at 0°C, but spoil at 4–6 days at 25°C. International Journal of Food Science and Technology 35: 431–442. Dalgaard, P., Gram, L. and Huss, H.H. (1993) Spoilage and shelf-life of cod fillets packed in vacuum or modified atmospheres. International Journal of Food Microbiology 19: 283–294. Dalgaard, P., Mejlholm, O. and Huss, H.H. (1996) Conductance method for quantitative determination of Photobacterium phosphoreum in fish products. Journal of Applied Bacteriology 81: 57–64.

340

Fishery Products: Quality, safety and authenticity

Dalgaard, P., Manfio, G.P. and Goodfellow, M., (1997a) Classification of Photobacteria associated with spoilage of fish products by numerial taxonomy and pyrolysis mass spectrometry. Zentralblatt fur Bakteriologie 285: 157–168 Dalgaard, P., Mejlholm, O., Christiansen, T.J. and Huss, H.H. (1997b) Importance of Photobacterium phosphoreum in relation to spoilage of modified atmosphere-packed fish products. Letters in Applied Microbiology 24: 373–378. Dalgaard, P., Garcia, M.L. and Mejlholm, O. (1998) Specific inhibition of Photobacterium phosphoreum extends the shelf-life of modified-atmosphere-packed cod fillets. Journal of Food Protection 61: 1191–1194. Dalgaard, P., Vancanneyt, M., Euras, N., Swings, J., Fruekilde, P. and Leisner, J.J. (2003) Identification of lactic acid bacteria from spoilage associations of cooked and brined shrimps stored under modified atmosphere between 0°C and 25°C. Journal of Applied Microbiology 94: 80–89. Dalsgaard, A. (1998) The occurrence of human pathogenic Vibrio spp. and Salmonella in aquaculture. International Journal of Food Science and Technology 33: 127–138. Davidson, C.M. and Cronin, F. (1973) Medium for the selective enumeration of lactic acid bacteria from foods. Journal of Applied Microbiology 26: 439–440. Debreve, J. and Boskou, G. (1996) Effect of modified atmosphere packaging on the TVB/TMAproducing microflora of cod fillets. International Journal of Food Microbiology 31: 221–229. De Man, J.D., Rogosa, M. and Sharpe, M.E. (1960) A medium for the cultivation of Lactobacilli. Journal of Applied Bacteriology 23: 130–135. Donovan T.J. and van Netten P. (1995) Culture media for the isolation and enumeration of pathogenic Vibrio species in foods and environmental samples. International Journal of Food Microbiology 26: 77–91. Drosinos, E., and Nychas, G.-J.E. (1996) Brochothrix thermosphacta, a dominant organism in Mediterranean fresh fish (Sparus aurata) stored under modified atmosphere. Italian Journal of Food Science 4: 323–329. Emborg, J., Laurensen, B.G., Rathjen, T. and Dalgaard, P. (2002) Microbial spoilage and formation of biogenic amines in fresh and thawed modified atmosphere-packed salmon (Salmo salar) at 2°C. Journal of Applied Microbiology 92: 790–799. Emborg, J., Laurensen, B.G. and Dalgaard, P. (2005) Significant histamine formation in tuna (Thunnus albacares) at 2°C – effect of vacuum- and modified atmosphere packaging on psychrotolerant bacteria. International Journal of Food Microbiology 101: 263–279. Erichsen, I. (1967) The microflora of semi-preserved fish products III. Principal groups of bacteria occurring in tibits. Antonie van Leeuwenhoek 33: 107–112. Ericsson, H. and Stålhandske, P. (1997) PCR detection of Listeria monocytogenes in ‘gravad’ rainbow trout. International Journal of Food Microbiology 35: 281–285. Ericsson, H., Eklöw, A., Danielsson-Tham, M.-L., Loncarevic, S., Mentzing, L.-O., Persson, I., Unnerstad, H. and Tham, W. (1997) An outbreak of listeriosis suspected to have been caused by rainbow trout. Journal of Clinical Microbiology 35: 2904–2907. Facinelli, B., Varaldo, P.E., Toni, M., Casolari, C. and Fabio, U. (1989) Ignorance about Listeria. British Medical Journal 299: 738. Farber, J.M., Daley, E.M., Mackie, M.T. and Limerick, B. (2000) A small outbreak of listeriosis potentially linked to the consumption of imitation crab meat. Letters in Applied Microbiology 131: 100–104. Feldhusen, F. (2000) The role of seafood in bacterial foodborne diseases. Microbes and Infection 2: 1651–1660. Fonnesbech Vogel, B., Jorgensen, K., Christensen, H., Olsen, J.E. and Gram, L. (1997) Differentiation of Shewanella putrefaciens and Shewanella algae on the basis of whole-cell protein profiles, ribotyping, phenotypic characterization, and 16S rRNA gene sequence analysis. Applied and Environmental Microbiology 63: 2189–2199.

Microbiological methods

341

Fonnesbech Vogel, B., Venkateswaran, K., Satomi, M. and Gram, L. (2005) Identification of Shewanella baltica as the most important H2S-producing species during iced storage of Danish marine fish. Applied and Environmental Microbiology 71: 6689–6697. Gancel, F., Dzierszinski, F. and Tailliez, R. (1997) Identification and characterization of Lactobacillus spp. isolated from fillets of vacuum-packed smoked and salted herring (Clupea hargenus) Journal of Applied Microbiology 82: 722–728. Gardner, G.A. (1966) A selective medium for the enumeration of Microbacterium thermosphactum in meat and meat products. Journal of Applied Bacteriology 29: 455–460. Gennari, M., Tomaselli, S. and Cotrona, V. (1999) The microflora of fresh and spoiled sardines (Sardina pilchardus) caught in Adriatic (Mediterranean) sea and stored in ice. Food Microbiology 16: 15–28. Giacomazzi, S., Leroi, F., L’Henaff, C. and Joffarud, J.-J. (2004) rpoB-PCR amplified gene and temporal temperature gradient gel electrophoresis: a rapid tool to analyse bacterial strains representative of cold-smoked salmon microflora. Letters in Applied Microbiology 38: 130–134. Giménez, B. and Dalgaard, P. (2004) Modelling and predicting the simultaneous growth of Listeria monocytogenes and spoilage microorganisms in cold-smoked salmon. Journal of Applied Microbiology 96: 96–109. Gobat, P.F. and Jemmi, T. (1993) Distribution of mesophilic Aeromonas species in raw and ready-to-eat fish and meat products in Switzerland. International Journal of Food Microbiology 20: 117–120. Gobat, P.F. and Jemmi, T. (1995.) Comparison of seven selective media for the isolation of mesophilic Aeromonas species in fish and meat. International Journal of Food Microbiology 24: 375–384. González-Fandos, E., Villarino-Rodríguez, A., García-Linares, M.C., García-Arias, M.T. and GarcíaFernández, M.C. (2005) Microbiological safety and sensory characteristics of salmon slices processed by the sous vide method. Food Control 16: 77–85. González-Rodríguez, M.N., Sanz, J.J., Santos, J.A., Otero, A., García-López, M.L. (2002a) Numbers and types of microorganisms in vacuum-packed cold-smoked freshwater fish at the retail level. International Journal of Food Microbiology 77: 161–168. González-Rodríguez, M.N., Santos, J.A., Otero, A. and García-López, M.L (2002b) PCR detection of potentially pathogenic aeromonads in raw and cold-smoked freshwater fish. Journal of Applied Microbiology 93: 675–680. Gram, L. (1990) Spoilage of three Senegalese fish species stored in ice and at ambient temperature. Paper presented at SEAFOOD 2000 in Halifax, Canada, 12–16 May 1990. Gram, L. (1992) Evaluation of bacteriological seafood. In: J. Lista (Ed.) Quality Assurance in the Fish Industry. Elsevier, Amsterdam, pp. 269–282. Gram, L. and Dalgaard, P. (2002) Fish spoilage bacteria – problems and solutions. Current Opinion in Biotechnology 13: 262–266. Gram, L. and Huss, H. (1996) Microbiological spoilage of fish and fish products. International Journal of Food Microbiology 33: 121–137. Gram, L. and Huss, H.H. (2000) Fresh and processed fish and shellfish. In: B.M. Lund, T.C. BairdParker and G.W. Gould (Eds) The Microbiological Safety and Quality of Foods, Volume 1. Aspen Publishers, Gaithersburg, MD, p. 488. Gram, L., Trolle, G. and Huss, H.H. (1987) Detection of specific spoilage bacteria from fish stored at low (0°C) and high (20°C) temperatures. International Journal of Food Microbiology 4: 65–72. Gram, L., Wedell-Neergaard, C. and Huss, H.H. (1990) The bacteriology of fresh and spoiling Lake Victorian Nile perch (Lates niloticus) International Journal of Food Microbiology 10: 303–316. Grimont, F. and Grimont, P.A.D. (1986) Ribosomal ribonucleic acid gene restriction as potential taxonomic tools. Annales de l’Institute Pasteur/Microbiologie B 137: 165–175. Guyer, S. and Jemmi, T. (1991) Behaviour of Listeria monocytogenes during fabrication and storage of experimentally contaminated smoked salmon. Applied and Environmental Microbiology 57: 1523–1527.

342

Fishery Products: Quality, safety and authenticity

Hänninen, M.L. and Siitonen, A. (1995) Distribution of Aeromonas phenospecies and genospecies among strains isolated from water, foods or from human clinical samples. Epidemiology and Infection 115: 39–50. Hänninen, M.L., Oivanen, P. and Hirvelä-Koski, V. (1997) Aeromonas species in fish, fish-eggs, shrimp and freshwater. International Journal of Food Microbiology 34: 17–26. Hausschild, A.H.W. and Hilsheimer, R. (1977) Enumeration of Clostridium botulinum spores in meats by a pour-plate procedure. Canadian Journal of Microbiology 23: 829–832. Havelaar, A.H., During, M. and Versteegh, J.F. (1987) Ampicillin-dextrin agar medium for the enumeration of Aeromonas species in water by membrane filtration. Journal of Applied Bacteriology 62: 279–287. Hayashi, S., Okura, M. and Osawa, R. (2006) Soft-agar-coated filter method for early detection of viable and thermostable direct hemolysin (TDH)- or TDH-related hemolysin-producing Vibrio parahaemolyticus in seafood. Applied and Environmental Microbiology 72: 4576–4582. Head, C.B. and Ratnam, S. (1988) Comparison of API ZYM system with API AN-Ident, API 20A, Minitek Anaerobe II, and RapID ANA systems for identification of Clostridium difficile. Journal of Clinical Microbiology 26: 144–146. Herrera, F.C., Santos, J.A., Otero, A. and García-López, M.-L.(2006) Occurrence of foodborne pathogenic bacteria in retail prepackaged portions of marine fish in Spain. Journal of Applied Microbiology 100: 527–536. Hielm, S., Hyytiä, E., Ridell, J. and Korkeala, H. (1996) Detection of C. botulinum in fish and environmental samples using polymerase chain reaction. International Journal of Food Microbiology 31: 357–365. Hill, W.E. and Jinneman, K.C. (2000) Principles and applications of genetic techniques for detection, identification, and sub-typing of food-associated pathogenic microorganisms. In: Lund, B., BairdParker, T.C. and Gould, G.W. (Eds.) Microbiological safety and quality of food. Vol 1–2, Aspen Publishers, Gaithersburg, Maryland. pp. 1813–1851. Holbrook, R. (2000) Detection of microorganisms in foods-principles of culture methods. In: B.M. Lund, T.C. Baird-Parker and G.W. Gould (Eds) The Microbiological Safety and Quality of Foods. Aspen Publishers, Gaithersburg, MD, pp. 1761–1789. Hozbor, M.C., Saiz, A.I., Yeannes, M.I. and Fritz, R. (2006) Microbiological changes and its correlation with quality indices during aerobic iced storage of sea salmon (Pseudopercis semifasciata) LWT – Food Science and Technology 39: 99–104. Huss, H.H. (1995) Quality and quality changes in fresh fish. In: H.H. Huss (Ed.), FAO Fisheries Technical Paper No. 348. FAO, Rome, Italy. Huss, H.H. (1995) Assurance of seafood quality. In: H.H. Huss (Ed.), FAO Fisheries Technical Paper No. 334. FAO, Rome, Italy. Huss, H.H. (1997) Control of indigenous pathogenic bacteria in seafood. Food Control 8: 91–98. Huss, H.H., Dalgaard, P. and Gram, L. (1997) Microbiology of fish and fish products. In: J. Luten, T. Børresen and J. Oehlenschläger (Eds) Seafood from Producer to Consumer, Integrated Approach to Quality. Elsevier Science B.V., pp. 413–430. Huss, H.H. and Gram, L. (2003) Pathogenic bacteria. In: H.H. Huss, L. Ababouch and L. Gram (Eds), Assessment and Management of Seafood Safety and Quality (FAO Fisheries Technical Paper No. 444). FAO, Rome, Italy. Hyytiä, E., Hielm, S. and Korkeala, H. (1998) Prevalence of Clostridium botulinum type E in Finnish fish and fishery products. Epidemiology and Infection 120: 245–250. International Commission on Microbiological Specifications for Food (ICMSF) (1998) Fish and fish products. VIII. Cured, smoked and dried seafood. Microorganisms in food 6. Microbial ecology of food commodities, 1st edition. Blackie Academic and Professional, London, p. 138. Jay, J.M. (2000) Culture, microscopic and sampling methods. In: Modern Food Microbiology, 6th edition. Springer-Verlag, p. 186.

Microbiological methods

343

Jemmi, T. and Keusch, A. (1992) Behaviour of Listeria monocytogenes during processing and storage of experimentally contaminated hot-smoked trout. International Journal of Food Microbiology 15: 339–346. Jöckel, J., Kirschfeld, R. and Dickertmann, D. (1986) Vakuumverpackter Räucherfisch – Mikrobiologischer Status von Handelsproben und technologische Modellversuche zur Histaminbildung bei Makrelenfilets. Archiv für Lebensmittelhygiene 37: 109–128. Joffraud, J.J., Leroi, F., Roy, C. and Berdagué, J.L. (2001) Characterisation of volatile compounds produced by bacteria isolated from the spoilage flora of cold-smoked salmon. International Journal of Food Microbiology 66: 175–184. Johansson, T. (1998) Enhanced detection and enumeration of Listeria monocytogenes from foodstuffs and food-processing environment. International Journal of Food Microbiology 40: 77–85. Johansson, T., Rantala, L., Palmu, L. and Honkanen-Buzalski, T. (1999) Occurrence and typing of Listeria monocytogenes strains in retail vacuum-packed fish products and in a production plant. International Journal of Food Microbiology 47: 111–119. Joffraud, J.J. and Leroi, F. (2000) Final report of European contract FAIR-PL-95–1207 – spoilage and safety of cold-smoked fish. Jørgensen, B.R., Gibson, D.M. and Huss, H.H. (1988) Microbiological quality and shelf-life prediction of chilled fish. International Journal of Food Microbiology 6: 295–307. Jørgensen, L.V. and Huss, H.H. (1998) Prevalence and growth of Listeria monocytogenes in naturally contaminated seafood. International Journal of Food Microbiology 42: 127–131. Jørgensen, L.V., Dalgaard, P. and Huss, H.H. (2000) Multiple compound quality index for cold-smoked salmon (Salmo salar) developed by multivariate regression of biogenic amines and pH. Journal of Agricultural and Food Chemistry 48: 2448–2453. Karpiskova, R., Pejchalovam, M., Mokrosova, J., Vytrasova, J., Smuharova, P. and Ruprich, J. (2000) Application of a chromogenic medium and the PCR method for rapid confirmation of L. monocytogenes in foodstuffs. Journal of Microbiological Methods 41: 267–271. Kautter, D.A. and Salomon, H.M. (1977) Collaborative study of a method for the detection of Clostridium botulinum and its toxins in foods. Journal of AOAC INTERNATIONAL 60: 541– 545. Kimura, B., Kawasaki, S., Nakano, H. and Fujii, T. (2001) Rapid, quantitative PCR monitoring of growth of Clostridium botulinum type E in modified-atmosphere-packaged fish. Applied and Environmental Microbiology 67: 206–216. Korkeala, H.J. and Pakkala, P.K. (1988) Microbiological changes in smoked and charred Baltic herring during storage. Journal of Food Protection 51: 197–200. Korkeala, H., Stengel, G., Hyytiä, E., Vogelsang, B., Bohl, A., Wihlman, H., Pakkala, P. and Hielm, S. (1998) Type E botulism associated with vacuum-packaged hot-smoked whitefish. International Journal of Food Microbiology 43: 1–5. Koutsoumonis, K. and Nychas, G.-J.E. (1999) Chemical and sensory changes associated with microbial flora of Mediterranean boque (Boops boops) stored aerobically at 0, 3, 7, and 10°C. Applied and Environmental Microbiology 65: 698–706. Koutsoumanis, K., Taoukis, P., Drosinos, E.H. and Nychas, G.-J.E. (1998) Lactic acid bacteria and Brochothrix thermosphacta – the dominant spoilage microflora of Mediterranean seafish stored under modified atmosphere packaging conditions. In: G. Olafsdottir, J. Luten, P. Dalgaard, M. Careche, E. Verrez-Begnis, E. Martinsdottir and K. Heia, K. (Eds) Proceedings of the Final Meeting of the Concerted Action – Evaluation of Fish Freshness Methods to Determine the Freshness of Fish in Research and Industry. International Institute of Refrigeration, pp. 158–165. Kreuzer, R. (1957) Untersuchungen über den biologisch bedingten Verderb von Fischwaren und seine Verhinderung. I. Kaltmarinaden-Organismem und Milieu. Archiv für Fischerei Wissenschaft 8: 104–139.

344

Fishery Products: Quality, safety and authenticity

Lauzon, H.L. (1997) Shelf-life and bacteriological spoilage of American plaice (Hippoglossoides platessoides). M.Sc. thesis, Department of Food Science, Faculty of Science, University of Iceland, Iceland. Leclerq, A. (2004) Atypical colonial morphology and low recoveries of Listeria monocytogenes strains on Oxford, Palcam, Rapid ‘L. mono and Aloa solid media. Journal of Microbiological Methods 57: 251–258. Lerche, M. (1960) Bombage-Ursache in Fischpräserven. Berliner und Münchener tierärztliche Wochenschrift 75: 12–14. Leroi, F., Joffraud, J., Chevalier, F. and Cardinal, M. (1998) Study of the microbial ecology of coldsmoked salmon during storage at 8°C. International Journal of Food Microbiology 39: 111–121. Leroi, F., Joffraud, J.J. and Chevalier, F. (2000) Effect of salt and smoke on the microbiological quality of cold-smoked salmon during storage at 5°C as estimated by the factorial design method. Journal of Food Protection 63: 502–508. Leroi, F., Joffraud, J.J., Chevalier, F. and Cardinal, M. (2001) Research of quality indices for coldsmoked salmon using a stepwise multiple regression of microbiological counts and physicochemical parameters. Journal of Applied Microbiology 90: 578–587. Lindström, M.K., Jankola, H., Hielm, S., Hyytiä, E. and Korkeala, H. (1999) Identification of Clostridium botulinum with API 20A, Rapid ID 32 A and RapID ANA II. FEMS Immunology and Medical Microbiology 24: 267–274. Lindström, M., Keto, R., Markkula, A., Nevas, M., Hielm, S. and Korkeala, H. (2001) Multiplex PCR assay for detection and identification of Clostridium botulinum types A, B, E and F in food and fecal material. Applied and Environmental Microbiology 67: 5694–5699. Lindström, M., Hielm, S., Nevas, M., Tuisku, S. and Korkeala, H. (2004) Proteolytic Clostridium botulinum type B in the gastric content of patient with type E botulism due to whitefish eggs. Foodborne Pathogens and Disease 1: 53–57. Lindström, M. and Korkeala, H. (2006) Laboratory Diagnostic of botulism. Clinical Microbiology Reviews 19: 298–314. Lindström, M., Vuorela, M., Hinderink, K., Korkeala, H., Dahlsten, E., Raahenmaa, M. and Kuusi, M. (2006) Botulism associated with vacuum-packed smoked whitefish in. Eurosurveillance 11, E060720.3. Available from: http://www.eurosurveillance.org/ew/2006/060720.asp#3. Liston, J. (1957) The occurrence and distribution of bacterial types on flatfish. Journal of General Microbiology 16: 205–216. Loncarevic, S., Tham, W. and Danielsson-Tham, M.-L. (1996) Prevalence of Listeria monocytogenes and other Listeria spp. in smoked and ‘gravad’ fish. Acta Veterinaria Scandinavica 37: 13–18. Long, H.F. and Hammer, B.W. (1941) Classification of the organisms important in dairy products. III. Pseudomonas putrefaciens. Research Bulletin of Iowa Agricultural Experiment Station 285. Lopez-Caballero, M.E., álvarez Torres, M.D., Sánchez-Fernández, J.A. and Moral, A. (2002) Photobacterium phosphoreum isolated as a luminescent colony from spoiled fish, cultured in model system under controlled atmospheres. European Food Research and Technology 251: 390–395. Lorentzen, G., Skjerdal, O.T. and Tryland, I., (2002) New and rapid method for detection and enumeration of sulphide-producing bacteria in fish products. Food Micro 2002: 18th International ICFMH Symposium, Lillehammer, Norway: 18–23 August 2002. Lorentzen, G., Skjerdal, O.T. and Berg, J.D. (2003) Rapid method for detection and enumeration of sulphide-producing bacteria in food products. US-patent 6: 632,632 B1: 14 October 2003. Lundén, J. (2004) Persistent Listeria monocytogenes contamination in food processing plants. Thesis, Department of Food and Environmental Hygiene, Faculty of Veterinary Medicine, University of Helsinki, Finland. Lyhs, U. and Björkroth, J. (2008) Lactobacillus Sake/curvatus is the prevailing lactic acid bacterium group in spoiled maatjes herring. Food Microbiology 25: 529–533.

Microbiological methods

345

Lyhs, U., Björkroth, J., Hyytiä, E. and Korkeala, H. (1998a) The spoilage flora of vacuum-packaged, sodium nitrite or potassium nitrate treated, cold-smoked rainbow trout stored at 4°C or 8°C. International Journal of Food Microbiology 45: 135–142. Lyhs, U., Hatakka, M., Mäki-Petäys, N. and Korkeala, H. (1998b) Microbiological quality of vacuum-packaged Finnish fishery products at retail level. Archiv für Lebensmittelhygiene 49: 46–150. Lyhs, U., Björkroth, J. and Korkeala, H. (1999) Characterisation of lactic acid bacteria from spoiled, vacuum-packaged, cold-smoked rainbow trout using ribotyping. International Journal of Food Microbiology 52: 77–84. Lyhs, U., Lahtinen, J., Fredriksson-Ahomaa, M., Hyytiä-Trees, E., Elfing, K. and Korkeala, H. (2001a) Microbiological quality and shelf-life of vacuum-packaged ‘gravad’ rainbow trout stored at 3 and 8°C. International Journal of Food Microbiology 70: 221–230. Lyhs, U., Korkeala, H., Vandamme, P., and Björkroth, J. (2001b) Lactobacillus alimentarius: a specific spoilage organism in marinated herring. International Journal of Food Microbiology 64: 355–360. Lyhs, U., Korkeala, H. and Björkroth, J. (2002) Identification of lactic acid bacteria from spoiled, vacuum-packaged ‘gravad’ rainbow trout using ribotyping. International Journal of Food Microbiology 72: 147–133. Lyhs, U., Koort, J.M., Lundström, H.S. and Björkroth, K.J. (2004) Leuconostoc gelidum and Leuconostoc gasicomitatum strains dominated the lactic acid bacterium population associated with strong slime formation in an acetic-acid herring preserve. International Journal of Food Microbiology 90: 207–218. Lyhs, U., Lahtinen, J. and Schelvis-Smit, R. (2007) Microbiological quality of maatjes herring stored in air and under modified atmosphere at 4°C and 10°C. Food Microbiology 24: 508–516. Magnússon, H., Sveinsdóttir, K., Lauzon, H.L., Thorkelsdóttir, À. and Martinsdóttir, E. (2006) Keeping quality of desalted cod fillets in consumer packs. Journal of Food Science 71: M69–M76. Marler, L.M., Siders, J.A., Wolters, L.C., Pettigrew, Y., Skitt, B.L. and Allen, S. (1991) Evaluation of the new RapID ANA system II for the identification of clinical anaerobic isolates. Journal of Clinical Microbiology 29: 874–878. Mead, G.C. and Adams, B.W. (1977) A selective medium for the rapid isolation of pseudomonads with poultry meat spoilage. British Poultry Science 18: 661–670. Mejlholm, O., Bøknæs and dalgaard, P. (2005) Shelf life and safety aspects of chilled cooked and peeled shrimps (Pandalus borelias) in modified atmosphere packaging. Journal of Applied Microbiology 99: 66–76. Meyer, V. (1956) Probleme des Verderbens von Fischkonserven in Dosen. II. Aminosäuredecarboxylase durch Organismen der Betabacterium-Buchneri-Gruppe als Ursache bombierter Marinaden. Veroeffentlichungen des Instituts fuer Meeresforschung in Bremerhaven 4: 1–16. Miettinen, M.K., Siitonen, A., Heiskanen, P., Haajanen, H., Björkroth, K.J. and Korkeala, H.J. (1999) Molecular epidemiology of an outbreak of febrile gastroenteritis caused by Listeria monocytogenes in cold-smoked rainbow trout. Journal of Clinical Microbiology 37: 2358–2360. Mossel, D.A.A., Corry, J.E.L., Struijk, C.B. and Baird, R.M. (1995) Essentials of the Microbiology of Foods: A Textbook for Advanced Studies. Wiley, London. Nocker, A. and Camper, A.K. (2006) Selective removal of DNA from dead cells of mixed bacterial communities by use of ethidium monoazide. Applied and Environmental Microbiology 72: 1997–2004. Nogva, H.K., Dromtorp, S.M., Nissen, H. and Rudi, K. (2003) Ethidium monoazide for DNA-based differentiation of viable and dead bacteria by 5′-nuclease. PCR BioTechniques 810: 812–813. Nordic Committee on Food Analysis (1991) Lactic acid bacteria. Determination in meat and meat products. NCFA method no. 140, Espoo, Finland.

346

Fishery Products: Quality, safety and authenticity

Nordic Committee on Food Analysis (1999) Salmonella. Detection in foods. NCFA method no. 71 (5th edition) Espoo, Finland. Nordic Committee on Food Analysis (2003) Bacterial examinations in fresh and frozen seafood. NCFA method no. 9 (3rd edition), Espoo, Finland. Nordic Committee on Food Analysis (2004) Listeria monocytogenes. Determination in Foods. NCFA method no. 136 (3rd edition), Espoo, Finland. Nordic Committee on Food Analysis (2005) Enterobacteriaceae. Determination in foods and feeds. NCFA method no. 144 (3rd edition), Espoo, Finland. Nordic Committee on Food Analysis (2006) Aerobic count and specific spoilage organisms in fish and fish products. NCFA method no. 184, Espoo, Finland. Nychas, G.-J.E. and Tassou, C.C. (1996) Growth/survival of Salmonella enteriditis on fresh poultry and fish stored under vacuum or modified atmosphere. Letters in Applied Microbiology 23: 115–119. Paarup, T., Sanchez, J.A., Peláez, C. and Moral, A. (2002) Sensory, chemical and bacteriological changes in vacuum-packed pressurised squid mantle (Todaropsis eblanae) stored at 4°C. International Journal of Food Microbiology 74: 1–12. Paleari, M.A., Soncini, G. and Beretta, G. (1990) Smoked tuna, sliced and vacuum-packed, a relatively new product. Zeitschrift für Lebensmittel-Untersuchung und -Forschung 190: 118–120. Paludan-Müller, C., Dalgaard, P., Huss, H.H. and Gram, L. (1998) Evaluation of the role of Carnobacterium piscicola in spoilage of vacuum- and modified-atmosphere-packed cold-smoked salmon at 5°C. International Journal of Food Microbiology 39: 155–166. Palumbo, S.A., Maxino, F., Williams, A.C., Buchanan, R.L. and Thayer, D.W. (1985) StarchAmpicillin Agar for the quantitative detection of Aeromonas hydrophila. Applied and Environmental Microbiology 50: 1027–1030 Polivka, C. (2001) Identification of Listeria with a new chromogenic medium Rapid ‘L. mono. Archiv fur Lebensmittelhygiene 52: 22–23. Pournis, N., Papavergou, A., Badeka, A., Kontominas, M.G. and Savvaidis, I.N. (2005) Shelf-life extension of refrigerated Mediterranean mullet (Mullus surmuletus) using modified atmosphere packaging. Journal of Food Protection 68: 2201–2207. Reilly, A. and Käferstein, F. (1999) Food safety and products from aquaculture. Journal of Applied Microbiology (Symp. Supplement) 85: 249S–257S. Reuter, G. (1965) Das Vorkommen von Laktobazillen in Lebensmitteln und ihr Verhalten im menschlichen Intestinaltrakt. Zentralblatt fur Bakteriologie, Parasitenkunde, Infektionskrankheiten und Hygiene Abteilung 1 197: 468–487. Robinson, R.K. (2002) Quality control in the dairy industry. In: R.K. Robinson (Ed.) Dairy Microbiology Handbook. John Wiley and Sons, New York, pp. 686–687. Rodriguez-Lazaro, D., Jofre, A., Aymerich, T., Garriga, M. and Pla, M. (2005) Rapid quantitative detection of Listeria monocytogenes in salmon products: evaluation of pre-real-time PCR strategies. Journal of Food Protection 68: 1467–1471. Rogosa, M., Mitchel, J.A. and Wiseman, R.F. (1951) A selective medium for the isolation and enumeration of oral and fecal lactobacilii. Journal of Bacteriology 62: 132–133. Romero, J., González, N. and Espejo, R.T. (2002) Marine Pseudoalteromonas sp. composes most of the bacterial population developed in oysters (Tiostrea chilensis) spoiled during storage. Journal of Food Science 67: 2300–2303. Rosnes, J.T., Sivertsvikand, M. and Bergslien, H. (1997) Distribution of modifiedatmosphere packaged salmon (Salmo Salar) products. In: J. Luten, T. Børresen, and J. Oehlenschläger (Eds) Seafood from Producer to Consumer, Integrated Approach to Quality. Elsevier Science B.V., pp. 305–317. Rudi, K. (2003) Application of 16S rDNA arrays for analyses of microbial communities. In: Recent Research Developments in Bacteriology, Volume 1, pp. 35–44.

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Rudi, K., Flateland, S.L., Hanssen, J.F., Bengtsson, G. and Nissen, H. (2002) Development and evaluation of a 16S ribosomal DNA array-based approach for describing complex microbial communities in ready-to-eat vegetable salads packed in a modified atmosphere. Applied and Environmental Microbiology 68: 1146–1156. Rudi, K., Maugesten, T., Hannevik, S.E. and Nissen, H. (2004) Explorative multivariate analyses of 16S rRNA gene data from microbial communities in modified-atmosphere-packed salmon and coalfish. Applied and Environmental Microbiology 70: 5010–5018. Rudi, K., Moen, B., Drømtorp, S.M. and Holck, A.L. (2005) Use of ethidium monoazide and PCR in combination for quantification of viable and dead cells in complex samples. Applied and Environmental Microbiology 71: 1018–1024. Satomi, M., Vogel, B.F., Gram, L. and Venkateswaran, K. (2006) Shewanella hafniensis sp. nov. and Shewanella morhuae sp. nov., isolated from marine fish of the Baltic Sea. International Journal of Systematic and Evolutionary Microbiology 56: 243–249. Scotter, S.L., Langton, B., Lombard, B., Schulten, S., Nagelkerke, N., In’t Veld, P.H., Rollier, P. and Lahellec, C. (2001) Validation of ISO method 11290 Part 1 – Detection of Listeria monocytogenes in foods. International Journal of Food Microbiology 64: 295–306. Schleifer, K.H., Ehrmann, M., Beimfohr, C., Brockmann, E., Ludwig, W. and Amann, R. (1995) Application of molecular methods for the classification and identification of lactic acid bacteria. International Dairy Journal 5: 1081–1094. Schmidt, C.F., Lechovich, R.V. and Folinaccio, J.F. (1961) Growth and toxin production by type E Clostridium botulinum type E below 40°F. Journal of Food Science 26: 626–630. Sharpe, M.E. and Pettipher, G.L. (1983) Food spoilage by lactic acid bacteria. In: A.H. Rose (Ed.) Economic Microbiology, Volume 8. Academic Press, pp. 199–223. Shieh, Y.-S.C., Calci, K.R. and Baric, R.S. (1999) A method to detect low levels of enteric viruses in contaminated oysters. Applied and Environmental Microbiology 65: 4709–4714. Simon, M.C., Gray, D.I. and Cook, N. (1996) DNA extraction and PCR method for the detection of Listeria monocytogenes in cold-smoked salmon. Applied and Environmental Microbiology 62: 822–824. Skjerdal, O.T., Lorentzen, G., Tryland, I. and Berg, J.D. (2004) New method for rapid and sensitive quantification of sulphide-producing bacteria in fish from arctic and temperate waters. International Journal of Food Microbiology 93: 325–333. Slater, P.E., Addiss, D.G., Cohen, A., Lebventhal, A., Chasis, G., Zehavi, H., Bashari, A. and Costin, C. (1989) Foodborne botulism: an international outbreak. International Journal of Epidemiology 18: 693–696. Stenstrøm, M. and Molin, G. (1990) Classification of the spoilage flora of fish, with special reference to Shewanella putrefaciens. Journal of Applied Bacteriology 68: 601–618. Su, Y.C., Duan, J. And Wu, W.H. (2005) Selectivity and specificity of a chromogenic medium for detecting Vibrio parahaemolyticus. Journal of Food Protection 68: 1454–1456. Sveinsdóttir, K., Martinsdóttir, E., Hyldig, G., Jørgensen, B. and Kristbergsson, K. (2002) Application of quality index method (QIM) scheme in shelf-life study of farmed Atlantic salmon (Salmo salar) Journal of Food Science 64: 1570–1579. Taliadourou, D., Papadopoulos, V., Domvridou, E., Savvaidis, I.N. and Kontominas, M.G. (2003) Microbiological, chemical and sensory changes of whole and filleted Mediterranean aquacultured sea bass (Dicentrarchus labrax) stored in ice. Journal of the Science of Food and Agriculture 83: 1373–1379. Truelstrup Hansen L. (1995) Quality of chilled, vacuum-packed cold-smoked salmon. Thesis, Danish Institute of Fisheries Research, Department of Seafood Research, Technical University, Denmark. Truelstrup Hansen, L., Gill, T. and Huss, H.H. (1995) Effects of salt and storage temperature on chemical, microbiological and sensory changes in cold-smoked salmon. Food Research International 28: 123–130.

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Truelstrup Hansen, L., Drewes Røntved, S. and Huss, H.H. (1998) Microbiological quality and shelf-life of cold-smoked salmon from three different processing plants. Food Microbiology 15: 137–150. Tryfinopoulou, P., Drosinos, E.H. and Nychas, G.-J.E. (2001) Performance of Pseudomonas CFC-selective medium in the fish storage ecosystems. Journal of Microbiological Methods 47: 243–247. Tryfinopoulou, P., Tsakalidou, E. and Nychas, G.-J.E. (2002) Characterization of Pseudomonas spp. associated with spoilage of gilt-head sea bream stored under various conditions. Applied and Environmental Microbiology 68: 65–72. van Belkum, A. (1994) DNA fingerprinting of medically important microorganisms by use of PCR. Clinical Microbiology Reviews 7: 174–184. van Spreekens, K.J.A. (1974) The suitability of a modification of Long and Hammer’s medium for the enumeration of more fastidious bacteria from fresh fishery products. Archiv für Lebensmittelhygiene 10: 213–219. Vaitilingo, M., Gendre, F. and Brignon, P. (1998) Direct detection of viable bacteria, molds, and yeasts by reverse transcriptase PCR in contaminated milk samples after heat treatment. Applied and Environmental Microbiology 64: 1157–1160. Vlaemynck, G., Lafarge, V. and Scooter, S. (2000) Improvement of the detection of Listeria monocytogenes by the application of ALOA, a diagnostic chromogenic isolation medium. Journal of Applied Microbiology 88: 430–441. Weber, J.T., Hibbs, R.G. Jr, Darwish, A., Mishu, B., Corwin, A.L., Rakha, M., Hatheway, C.L., el Sharkawy, S., el-Rahim, S.A. and al-Hamd, M.F. (1993) A massive outbreak of type E botulism associated with traditional salted fish in Cairo. Journal of Infectious Diseases 167: 451–454. Ziemke, F., Brettar, I. ans Hofle, M.G. (1997) Stability and diversity of the genetic structure of a Shewanella putrefaciens population in the water column of the central Baltic. Aquatic Microbial Ecology 13: 63–74. Zorn, W., Greuel, E. and Krämer, J. (1993) Beurteilung des Hygienestatus geräucherter, vakuumverpackter Forellenfillets. Archiv für Lebensmittelhygiene 44: 81–104.

Chapter 16

Protein-based methods Hartmut Rehbein

16.1

Introduction

Electrophoretic, immunological and enzymatic methods have a long tradition in seafood analysis. A comprehensive overview of the application of protein-based techniques for identifying the species in fishery products has been given by Rehbein (1990) and Mackie (1996). However, in the past two decades, the polymerase chain reaction (PCR) has extensively replaced isoelectric focusing (IEF) and sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) as tools for species identification. Nevertheless, IEF of sarcoplasmic proteins is still in use for authentication of raw products (Berrini et al. 2006), mainly fresh or frozen fish fillets, because it is a very fast and cost-effective method with a high power of differentiation. Recently, protein- and DNA-based techniques have been reviewed for their suitability for identifying gadoid fish species (Hubalkova et al. 2007). Dipstick immunoassays offer a simple and rapid possibility for species identification, which may be used by fish processors, traders and food control authorities ‘in the field’, for example at the auction. Several immunological test systems have been developed for identifying fish species (see below), but to the knowledge of the author no kit or stick is commercially available. Beside species identification, the following topics will be dealt with: detection of allergenic proteins, determination of the heating temperature of smoked fish and differentiation between fresh and frozen/thawed fish fillets. Authenticity assessment by two-dimensional electrophoresis (2DE) or differential scanning calorimetry (DSC) are described in Chapter 14 and Chapter 8 of this book, respectively. In the following, the term ‘fish’ also denotes ‘mollusc’ and ‘crustacean’, if not indicated otherwise.

16.2

Fish muscle proteins

In most cases ‘seafood’ means muscle food, exceptions being caviar, canned cod liver and mussels. The two main classes of fish muscle proteins (Sikorski et al. 1994), myofibrillar 349

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Table 16.1 Protein-based techniques for species identification in fishery products. Analytical method Native IEF (isoelectric focusing) Urea IEF Cellulose acetate electrophoresis SDS-PAGE (sodium dodecylsulphate polyacrylamide gel electrophoresis) 2-DE (2-dimensional electrophoresis) Native IEF combined with enzyme specific staining Capillary zone electrophoresis High-performance liquid chromatography (HPLC) Immunoassays

Fishery product Raw, cold-smoked, hot-smoked, pre-fried, cooked, canned, formed, high-pressure treated products Raw, cold-smoked, hot-smoked, pre-fried, cooked, formed, high-pressure treated, surimi-based products Raw products (Yearsley et al. 1999, 2003) Raw, cold-smoked, hot-smoked, pre-fried, cooked, formed, high-pressure treated, surimi-based products Raw, cold-smoked, hot-smoked, pre-fried, cooked, formed, high-pressure treated, surimi-based products Raw products (Pineiro et al. 2000; Jiang and Xiong, 2006) Raw products (Gallardo et al. 1995) Raw products (Knuutinen et al. 1998) Raw, smoked, canned surimi-based products

and sarcoplasmic proteins, have been used to develop methods to authenticate declaration of species or processing technology. Differences in electrical charge, size (molecular mass) and amino-acid sequence of proteins result in different electrophoresis patterns, highperformance liquid chromatography (HPLC) chromatograms or matrix-assisted laser desorption/ionization–time of flight (MALDI–TOF) mass spectra for different fish species. An overview of the application of analytical methods is given in Table 16.1. Two classes of proteins, parvalbumins and myosin light chains (MLCs), are of great significance for fish species identification. Parvalbumins are water-soluble, acidic, calciumbinding proteins of low molecular mass (10–12 kilodaltons (kDa)) (Gerday 1982). Light muscle of fish may contain high concentration of parvalbumins (up to 5 mg/g wet weight), consisting of several isoforms with low isoelectric point (pI) values (between pH 4 and 5) (Rehbein et al. 2000). Parvalbumins from various fish species have been identified as major food allergens, possessing a high allergenicity (Bugajska-Schretter et al. 2000). Owing to their great heat stability, parvalbumins can be used not only for identification of raw fish by IEF (Figure 16.1), but also in the case of cooked or even canned fish products (Rehbein et al. 1990). The MLCs, which belong to the myofibrillar proteins, are useful for species identification for those fish products where the sarcoplasmic proteins have been removed during processing by washing steps, such as for surimi and surimi-based products, like kamaboko or imitation crab-meat. During fish processing and storage of products, fish muscle proteins are degraded to a varying extent. However, the statement that the proteins in canned fish or other fishery products have become irreversibly denatured, which is often found in papers about PCRbased species identification (Infante et al. 2004), is not quite correct. Products like canned salmon or herring are good examples for the stability of muscle proteins. To develop a method for identification of canned fish, canned salmon flesh had been treated with cyanogen bromide and the peptides obtained were separated by native IEF resulting in species-specific

Protein-based methods 8.45

7.35

6.55

5.85

5.20 4.55

3.50

351

pI value

pI calibration kit heated Turbot raw heated Norway pout raw heated Pollack raw heated Red mullet raw heated Anglerfish raw heated Brill raw heated Sprat raw <Parvalbumins>

Figure 16.1 Differentiation of fish species by IEF of sarcoplasmic proteins. Proteins were extracted from raw fillet (white muscle) and run on Servalyt Precote® 3-10; raw: original extract, heated: extracted heated to 70°C and clarified by centrifugation. Turbot, Psetta maxima; Norway pout, Trisopterus esmarki; pollack, Pollachius pollachius; red mullet, Mullus barbatus; anglerfish, Lophius piscatorius; brill, Scophthalmus rhombus; sprat, Sprattus sprattus.

peptide patterns (Mackie 1996). Differentiation between canned herring, sprat and sardine was achieved by native IEF of a protein fraction enriched in the heat-stable parvalbumins (Rehbein et al. 1990). On the other hand, DNA in canned fish is heavily degraded into fragments of 100–200 base pairs in length (Quinteiro et al. 1998). The main reason for preferring DNA over protein for fish species identification is the ease of selective amplification and characterisation (by sequencing, restriction-fragment length polymorphism (RFLP), single-strand conformation polymorphism (SSCP) and similar techniques of mutation detection) of DNA sequences using PCR-based techniques.

16.3

Electrophoretic methods for fish species identification

For more than two decades isolelectric focusing of sarcoplasmic proteins has been the most popular technique for fish species identification of raw products, like fresh, frozen or formed fillets. In IEF, proteins are separated according to the electrical charge of their surface,

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producing narrow bands within a preformed pH gradient. IEF of sarcoplasmic proteins has a high discriminating power and reproducibility. Occasionally, capillary electrophoresis methods have been described to analyse fish sarcoplasmic proteins (LeBlanc et al. 1994; Gallardo et al. 1995; Valenzuela et al. 1999).

16.3.1 Native isoelectric focusing Species identification by native isoelectric focusing (nIEF) consists of the following steps: z

z

z z z z

Extraction of water-soluble fish muscle proteins by mixing light muscle with three- to fourfold amounts of water, followed by a short-time centrifugation to remove nondissolved material. Loading of extract by means of sample application pieces or applicator strips on the gel, preferentially on the cathodic site for most fish species. Ready-to-use IEF gels containing ampholytes for different pH gradients are offered by several vendors. Run of the IEF until 3000–5000 volt hours have been reached. Ampholytes are removed and protein bands are fixed by shaking the gel in an acidic solution. Protein bands are visualised by staining with Coomassie dye. Comparison of protein profiles of unknown samples with patterns obtained from reference fish run on the same gel or from databases.

By using this protocol, 20–40 samples can be analysed within one working-day. Unknown samples may be identified either by comparison with the pattern of reference fish run on the same gel or by calculating the pI values of protein bands. Pattern and pI value for several commercially important fish species are available on the internet from databases like www. cfsan.fda.gov/~frf/rfe0.html (fish relevant for the North American market) or www.fischdb. de (fish relevant for the European market). Also, a library of IEF patterns of 43 commercial fish species belonging to the orders Pleuronectiformes and Gadiformes has been published (Tepedino et al. 2001). Protein patterns for scombroid, salmonid and gadoid fishes are shown in Figure 16.2. Each family exhibits a characteristic type of pattern: the red-fleshed scombroids have prominent bands mostly in the cathodic part of the gel, whereas salmonids give protein bands of high and medium pI value (pI 6–9), and gadoids possess strong bands in the anodic part of the IEF gel. Protein patterns are affected by the type of muscle used (Rehbein and Kündiger, 1984), the chemical composition of ampholytes, and the position of the sample within the pH gradient (see Figure 16.2). It is recommended placing the sample in a region devoid of strong protein bands. Numerous examples of application of nIEF for identification of fish species have been compiled previously (Rehbein 1990). The reliability of the technique was demonstrated by collaborative studies using one protocol (Lundstrom 1980) or several modifications of the technique. When each of eight European laboratories had applied its own method of IEF to identify 10 unknown samples of raw muscle by means of reference material, the

Protein-based methods Cathode ↓ sample application

353

Anode

P a r v

Oncorhynchus nerka O. gorbuscha Salmo salar O. mykiss Scomber scombrus Theragra chalcogramma Melanogrammus aeglefinus Pollachius virens Gadus morhua pI marker pI marker Onocrhynchus nerka O. gorbuscha Salmo salar O. mykiss Scomber scombrus Theragra chalcogramma Melanogrammus aeglefinus Pollachius virens Gadus morhua

sample application ↑

Figure 16.2 Characteristics of protein patterns of different fish species. Extract of fillet (light muscle) from salmonid species (Oncorhynchus spp., S. salar), mackerel (S. scombrus) and gadoid species (T. chalcogramma, M. aeglefinus, P. virens, G. morhua) was applied to the IEF gel either at the cathodic or anodic side. The position of parvalbumin bands is indicated, but with this type of gel (FocusGel 3-10, ETC) no sharp parvalbumin bands were obtained as with Servalyt Precotes®.

assignment between sample and reference was correct in 93% of cases (Rehbein et al. 1995). Recently nIEF has been found suitable to identify Aegean fish species (Ataman et al. 2006), freshwater fish commercially labelled ‘perch’, Perca fluviatilis, Lates niloticus, Stizostedion lucioperca, Morone chrysops x saxatilis, (Berrini et al. 2006), six species of puffer fish (Chen et al. 2003), and to distinguish steaks of blue marlin, Mediterranean spearfish and swordfish (Renon et al. 2005).

16.3.2

Urea IEF

Raw or denatured fish muscle proteins can be solubilised by mixing the fish flesh with 8 M buffered urea, and analysed by IEF with gels containing the same concentration of urea. The extract contains a mixture of sarcoplasmic and myofibrillar proteins. The method has been applied for identification of the fish species in surimi-based products, as well as in cooked, smoked or high-pressure treated fish. A standard operation procedure for urea IEF (uIEF) has been developed comprising the following steps (Etienne 1999; Rehbein et al. 1999):

354 z

z z z z z z z

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Extraction of fish muscle protein by mixing light muscle with fourfold amount of 8 M urea/0.1 M 1,4-dithiothreitol (DTT)/20 mM sodium phosphate pH 6.5. The mixture is kept at room temperature for about half an hour to complete solubilisation of protein, then undissolved material is removed by high-speed centrifugation. CleanGels (obtainable from GE Healthcare or other vendors) are rehydrated with a solution containing 8 M urea and ampholytes. After prefocusing, extract is loaded on the gel by means of sample application pieces or applicator strips, which are placed at the cathodic site of the gel. Run of IEF until 5000 volt hours have been reached. Ampholytes and urea are removed and protein bands are fixed by shaking the gel in an acidic solution. Protein bands are visualised by staining with Coomassie dye. The gel is impregnated with glycerol and dried. Comparison of protein profiles of unknown samples with patterns obtained from reference fish run on the same gel or from databases. If pI values of proteins are calculated, the pI shift of proteins caused by urea has to be taken into account (Rehbein et al. 2000).

Instead of CleanGels, immobiline dry plates (Etienne et al. 1999) or home-made gels (An et al. 1989a, b) have been applied for uIEF with good results. From the protocol described above it is clear that uIEF is a technically demanding method, which needs experience to avoid pitfalls like crystallisation of urea during the run. Several applications of uIEF for species identification and analysis of protein changes during fish processing and storage are listed in Table 16.2. In several of these studies, the results obtained by uIEF had been compared with the results from nIEF and SDS–PAGE.

16.3.3

SDS–PAGE

Whereas differences in electrical charge are used for separation of proteins by IEF, all proteins carry a negative charge in SDS–PAGE. The negative charge is produced by loading proteins with the anionic detergent sodium dodecylsulphate; on average, 1.4 g of SDS is taken up by 1 g of protein. Secondary and tertiary structures are disrupted and protein-detergent-ellipsoids move through the polyacrylamide gel according to the molecular mass of the protein. The field of application of SDS–PAGE is nearly the same as it is for uIEF. A standard operation procedure for SDS–PAGE with ExcelGels has been developed previously, comprising the following steps (Pineiro et al. 1999): z

z z

Solubilisation of fish muscle by boiling in an SDS-containing solution (2% w/v SDS/ 0.1 M DTT, 60 mM Tris-HCl pH 7.5), and centrifugation to remove non-dissolved material. Flat-bed electrophoresis with ExcelGels (GE Healthcare). Silver staining of proteins.

This method has been applied to the identification of smoked salmon and eel (Mackie et al. 2000), cooked fish (Pineiro et al. 1999) and high-pressure treated products (Etienne

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355

Table 16.2 Urea IEF as a tool for identification of fish and shellfish species and analysis of protein changes during processing. Objective of study Differentiation of shrimps Identification of fish species in surimi-based products Identification of sturgeon caviar Identification of blue crab (Callinectes sapidus); effect of season, location and processing on protein patterns Differentiation of raw and cooked bivalves (Meretrix lyrata, Ruditapes decussatus, Cerastoderma edule) Differentiation of cooked gadoids, flatfish and other species: collaborative study Differentiation of cooked salmonids and other species Identification of smoked salmonids, gravad salmonids and smoked eels Parvalbumins as marker proteins for uIEF Identification of formed and high pressuretreated fish Protein changes after irradiation and storage of horse mackerel (Trachurus trachurus)

Method

Reference

uIEF with home-made gel uIEF with home-made gel

An et al. 1989a An et al. 1989b

uIEF with home-made gel uIEF with home-made gel

Chen et al. 1996 Gangar et al. 1996

uIEF with CleanGel; extracts from adductor muscle uIEF with CleanGel and immobiline dry plate uIEF with CleanGel

Etienne et al. 2000b Rehbein et al. 1999

uIEF with CleanGel

Etienne et al. 2000a Mackie et al. 2000

uIEF with CleanGel uIEF with CleanGel

Rehbein et al. 2000 Etienne et al. 2001

uIEF with CleanGel

Silva et al. 2006

et al. 2001). The comparison of the efficiency of SDS–PAGE and uIEF for differentiation of closely related species demonstrated a good performance for both methods in flatfishes and hakes, but in salmonids and tunas several species could not be distinguished by uIEF (Etienne et al. 2000a). For differentiation of hot-smoked eel, uIEF was preferred to SDS–PAGE (Mackie et al. 2000). Many other gel types can be used for SDS–PAGE, either in horizontal or vertical electrophoresis chambers. The composition of the gel must be adapted to the size of proteins to be separated. As most of the proteins relevant for differentiation of fish species, like the MLCs and troponins, possess a molecular mass below 40 kDa, a total polyacrylamide concentration of 12–15% is recommended (Pineiro et al. 1999). SDS–PAGE has not only been applied to analysis of muscle food, but also to characterisation of fish roe and caviar. Figure 16.3 shows the protein patterns for gadoid roe and sturgeon caviar, which allow differentiation between these types of products. However, the technique was not suitable for differentiating caviar (Beluga, osietra, sevruga) of different sturgeon species (Chen et al. 1996). The main field of application of SDS–PAGE for the study of fish as food is analysis of protein changes during fish processing. Recently published examples are given by the analysis of protein changes after irradiation and ice storage of horse mackerel (Silva et al. 2006) and the study of denaturation of myofibrillar proteins during preparation of kamaboko gels (Shikha et al. 2006).

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1

2

3

4

5

6 7

8

9

Figure 16.3 SDS–PAGE of roe and caviar. Lanes 1–5: sturgeon caviar; (1) Osietra, Acipenser gueldenstaedtii; (2 and 3) Beluga, Huso huso; (4 and 5) Osietra, A. baeri; lanes 6 and 7: cod roe, Gadus morhua; extract of lane 6 had twice the protein concentration as extract of lane 7; lanes 8 and 9: marker proteins of known of molecular mass (kilodaltons) are indicated.

16.4

High-performance liquid chromatography

In the last decades of the 20th century, several attempts were made to establish HPLC as an alternative to electrophoresis techniques for fish species identification (Ashoor and Knox 1985; Osman et al. 1987). Possible advantages of HPLC versus electrophoresis may be the short time required for sample preparation, and easier automation (for example by sample application systems). In most cases, reversed-phase HPLC has been used, but Rehbein (1990b) successfully tested anion-exchange chromatography as an alternative. For reversed-phase HPLC of sarcoplasmic proteins, preparation of samples is straightforward: light muscle of fish is extracted with water, extracts are acidified to pH 2 with trifluoracetic acid, then filtered through a 0.2 or 0.45 μm membrane filter and injected. In all cases, a gradient with an increasing concentration of acetonitril is applied, and the time of the HPLC run is varied between 30 and 60 minutes. This is much shorter than the time needed for IEF with slab-gels, but by HPLC one sample after the other has to be run whereas 20 samples can be applied simultaneously on an IEF gel. Identification of four gadoid fish species has been reported by Pineiro et al. (1997). Protein profiles did not change during storage of refrigerated fish for 10 days. In another study, 16 of the most common Finnish freshwater fish species were differentiated by species-specific HPLC chromatograms obtained using photodiode-array detection at 200–350 nm (Knuutinen and Harjula 1998).

Protein-based methods

16.5

357

Immunological methods and detection of allergenic proteins

Methods for identification of the fish or shellfish species are not only relevant for prevention of fraud, but are also important for detection of the presence of seafood as one of the most common food allergens. Several types of immunoassay have been developed for detection of seafood allergens, for process control and authentication of fish and shellfish. In most cases an enzyme-linked immunosorbent assay (ELISA) has been used owing to the sensitivity, short time of analysis, and specificity of the method. In the dip-stick format, ELISA can be applied in the ‘field’, for example by customers or food controllers. Recently an ELISA has been developed for identifying three clam species using polyclonal antibodies against soluble proteins extracted from whole clam. The polyclonal antibodies were made species-specific by blocking them with soluble proteins from heterologous clam species. Two formats of indirect ELISA, microtitre plates and immunostick tubes, were found to be suitable to differentiate between Ruditapes decussatus (grooved carpet shell), Venerupis rhomboides (yellow carpet shell) and V. pullastra (pullet carpet shell) (Fernandez et al. 2002). Work in the same laboratory on authentication of fish by immunoassay comprised production of polyclonal and monoclonal antibodies to be used in an indirect ELISA (Asensio et al. 2003a, b, c). Differentiation of grouper (Epinephelus guaza), wreck fish (Polyprion americanus) and Nile perch (Lates niloticus) was achieved using polyclonal antibodies as described above for clams (Asensio et al. 2003a). A monoclonal antibody specific to grouper and wreck fish (Asensio et al. 2003b) was developed and tested against raw, cooked and sterilised protein extracts of 15 fish species using indirect ELISA. The monoclonal antibody recognised only grouper and wreck fish samples and did not show any cross-reactivity with other fish species. Heated extracts performed just as well as raw extracts. The development of a specific monoclonal antibody for grouper allowed identification of this species by indirect ELISA with microtitre plates or immunostick tubes. Antisera against myosin alkali light chain 1 (MLC A1) have been used by Ochia et al. (2003) for identification of fish species in dried fish products (fushi and noboshi) by immunoblotting of MLC separated by SDS–PAGE. Owing to the heat stability of MLC, fish processed to fushi and noboshi could be identified by immunostaining with anti-mackerel-A1 antiserum. Earlier applications of immunological methods for species identification have been reviewed previously (Rehbein 2003). Fish and crustacean shellfish are common food allergens which have to appear on the ingredient list if present in the food. Control of correct labelling may be performed by immunoassays using antibodies against parvalbumin (for fish) or tropomyosin (for shellfish and crustaceans) (Swoboda et al. 2002; Fuller et al. 2006).

16.6

Determination of heating temperature

Fishery products are heated for several reasons: (1) to achieve the desired sensory properties by cooking, frying or smoking; (2) to produce a storable product; or (3) to destroy pathogenic

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organisms like nematode larvae or bacteria. Information about the temperature the product has experienced during processing can be obtained by several methods of protein analysis. The following methods have been used, either alone or in combination: z z

z z z

Measurement of soluble protein. Determination of the temperature necessary to coagulate extracted protein. The coagulation test is performed by extracting the muscle sample with water or buffer, heating the extract and measuring the temperature at which protein coagulation takes place (Doesburg and Papendorf 1969). IEF of water-soluble protein. SDS–PAGE. Measurement of enzyme activity.

In search of suitable methods for determination of the heating temperature of products made from herring (Clupea harengus), mackerel (Scomber scombrus), saithe (Pollachius virens), haddock (Melanogrammus aeglefinus) and tuna (Thunnus alalunga), the coagulation test, IEF and measurement of water-soluble protein were compared (Rehbein 1992). It was found that most (about 80%) of the sarcoplasmic protein had become insoluble at the temperature of 60°C. IEF demonstrated that the heat stability of sarcoplasmic proteins varied considerably. Proteins of gadoid species were denatured sequentially from high to low pI value. In later studies, a similar result was obtained for rainbow trout (Oncorhynchus mykiss) (Figure 16.4). Three different methods, coagulation test, SDS–PAGE and measurement of activities of lactate dehydrogenase and glycerolaldehyde dehydrogenase, were applied to assess the endpoint temperature (EPT) of heated fish and shellfish meats (Uddin et al. 2002). Proteins had been extracted with NaCl solution. The coagulation test was able to determine EPT for meat

Cathode

Anode 55°C

60°C

65°C

70°C

Figure 16.4 Changes in the protein pattern of sarcoplasmic proteins caused by heating of extracts. Extract from light muscle of rainbow trout was heated to the temperatures indicated, clarified by centrifugation and run on Servalyt Precote 3-10®.

Protein-based methods

359

of skipjack tuna (Katsuwonus pelamis), red sea bream (Pagrus major), Kuruma prawn (Penaeus japonicus) and scallop (Patinopecten yessoensis) in the range 60–67°C. SDS–PAGE revealed the presence of a few thermostable proteins in extract of tuna meat (32–34 kDa, approximately14 kDa) and scallop adductor muscle (35 kDa, approximately14 kDa). As expected, enzyme activities decreased during heating of muscle tissue, but the extent of variability of enzyme activities for different specimens was not determined. The thermal inactivation of acid and alkaline phosphatases in fish and shellfish muscle was measured to determine the suitability of heating temperature (Kuda et al. 2004; Johnsen et al. 2007). The results suggest that measurement of residual phosphatase activity may be used to check the temperature seafood experienced during processing.

16.7

Differentiation of fresh and frozen/thawed fish fillets

Fresh fish and fillets have different sensory properties and spoilage characteristics compared with frozen/thawed products (Rehbein et al. 1978). The cells of fish muscle and their organelles are destroyed by freezing and thawing, and particle-bound enzymes are released into thaw drip and press-juice (Rehbein 1992b). Enhanced enzyme activity in these liquids has been measured, allowing differentiation between fresh and frozen/thawed fish fillets (Duflos et al. 2002).

16.8

References

An, H., Marshall, M.R., Otwell, W.S. and Wei, C.I. (1989a) Species identification of raw and boiled shrimp by a urea gel isoelectric focusing technique. Journal of Food Science 54: 233–236, 257. An, H., Wei, C.I., Zhao, J., Marshall, M.R. and Lee, C.M. (1989b) Electrophoretic identification of fish species used in surimi products. Journal of Food Science 54: 253–257. Asensio, L., Gonzalez, I., Rodriguez, M.A., Mayoral, B., Lopez-Calleja, I., Hernandez, P.E., Garcia, T. and Martin, R. (2003a) Identification of grouper (Epinephelus guaza), wreck fish (Polyprion americanus), and Nile perch (Lates niloticus) by polyclonal antibody-based enzyme-linked immunosorbent assay. Journal of Agricultural and Food Chemistry 51: 1169–1172. Asensio, L., Gonzalez, I., Rodriguez, M.A., Hernandez, P.E., Martin, R. (2003b) Development of a monoclonal antibody for grouper (Epinephelus guaza), wreck fish (Polyprion americanus) authentication using an indirect ELISA. Journal of Food Science 68: 1900–1903. Asensio, L., Gonzalez, I., Rodriguez, M.A., Mayoral, B., Lopez-Calleja, I., Hernandez, P.E., Garcia, T. and Martin R. (2003c) Development of a specific monoclonal antibody for grouper (Epinephelus guaza) identification by an indirect enzyme-linked immunosorbent assay. Journal of Food Protection 66: 886–889. Ashoor, S. and Knox, M. (1985) Identification of fish species by high-performance liquid chromatography. Journal of Chromatography 324: 199–202. Ataman, C, Celik, U, Rehbein, H (2006) Identification of some Aegean fish species by native isoelectric focusing. European Food Research Technology 222: 99–104. Berrini, A., Tepedino, V., Borromeo, V. and Secchi, C. (2006) Identification of freshwater fish commercially labelled ‘perch’ by isoelectric focusing and two-dimensional electrophoresis. Food Chemistry 96: 163–168. Bugajska-Schretter, A., Grote, M., Vangelista, L., Valent, P., Sperr W.R., Rumpold, H., Pastore, A., Reichelt R., Valenta R. and Spitzauer, S. (2000) Purification, biochemical, and immunological

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characterisation of a major food allergen: different immunoglobulin E recognition of the apo- and calcium-bound forms of carp parvalbumin. Gut 46: 661–669. Chen, I.-C., Chapman, F.A., Wei, C.I., O’Keefe, S.F. (1996) Preliminary studies on SDS–PAGE and isolelectric focusing identification of sturgeon sources of caviar. Journal of Food Science 61: 533– 535, 539. Chen, T.-Y., Shiau, C.-Y., Noguchi, T., Wie, C.-I. and Hwang, D.-F. (2003) Identification of puffer fish species by native isoelectric focusing technique. Food Chemistry 83: 475–479. Doesburg, J.J. and Papendorf, D. (1969) Determination of degree of heating of fish muscle. Journal of Food Technology 4: 17–26. Duflos, G., Le Fur, B., Mulak, V., Becel, F. and Malle, P. (2002) Comparison of methods of differentiating between fresh and frozen–thawed fish or fillets. Journal of the Science of Food Agriculture 82: 1341–1345. Etienne, M., Jerome, M., Fleurence, J., Rehbein, H., Kündiger, R., Malmheden Yman, I., Ferm, M., Craig, A., Mackie, I., Jessen, F., Smelt, A. and Luten, J. (1999) A standardized method of identification of raw and heat-processed fish by urea-isoelectric focusing: a collaborative study. Electrophoresis 20: 1923–1933. Etienne, M., Jerome, M., Fleurence, J., Rehbein, H., Kündiger, R., Mendes, R., Costa, H., Perez-Martin, R., Pineiro-Gonzales, C., Craig, A., Mackie, I., Malmheden Yman, I., Ferm, M., Martinez, I., Jessen, F., Smelt, A. and Luten, J (2000a) Identification of fish species after cooking by SDS-PAGE and urea IEF: a collaborative study. Journal of Agricultural and Food Chemistry 48: 2653– 2658. Etienne, M., Jerome, M. and Fleurence, J (2000b) Species identification of raw and cooked bivalves using electrophoresis. Science des Aliments 20: 367–377. Etienne, M., Jerome, M., Fleurence, J., Rehbein, H., Kündiger, R., Mendes, R., Costa, H. and Martinez, I. (2001) Species identification of formed fishery products and high pressure treated fish by electrophoresis: a collaborative study. Food Chemistry 72: 105–112. Fernandez, A., Garcia, T., Asensio, L., Rodriguez., M.A., Gonzalez, I., Lobo, E., Hernandez, P.E. and Martin, M. (2002) Identification of the clam species Ruditapes decussatus (grooved carpet shell), Venerupis rhomboides (yellow carpet shell) and Venerupis pullastra (pullet carpet shell) by ELISA. Food and Agricultural Immunology 14: 65–71. Fuller, H.R., Goodwin, P.R. and Morris G. (2006) An enzyme-linked immunosorbent assay (ELISA) for the major crustacean allergen, tropomyosin, in food. Food and Agricultural Immunology 17: 43–52. Gallardo, J.M., Sotelo, C.G., Pineiro, C. and Perez-Martin, R.I. (1995) Use of capillary zone electrophoresis for fish species identification. Differentiation of flatfish species. Journal of Agricultural and Food Chemistry 43: 1238–1244. Gerday, C. (1982) Soluble calcium-binding proteins from fish and invertebrate muscle. Molecular Physiology 2: 63–87. Hubalkova, Z., Kralik, P., Tremlova, B. and Rencova, E. (2007) Methods of gadoid fish species identification in food and their economic impact in the Czech Republic. Veterinarni Medicina 52: 273–292. Infante, C., Catanese, G., Ponce, M. and Manchado, M. (2004) Novel method for the authentication of frigate tunas (Auxis thazard and Auxis rochei) in commercial canned products. Journal of Agricultural and Food Chemistry 52: 7435–7443. Jiang, J.-G. and Xiong, Q.-W. (2006) An isozyme analysis of four species in the family Cyprinidae: genetic, taxonomic and germplasm significance. Journal of the Science of Food and Agriculture 86: 465–472. Johnsen, S., Skipnes, D., Skara, T. and Hendrickx, M.E. (2007) Thermal inactivation kinetics of avid phosphatase (ACP) in cod (Gadus morhua). European Food Research Technology 224: 315–320.

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Knuutinen, J. and Harjula, P. (1998) Identification of fish species by reversed-phase high-performance liquid chromatography with photodiode-array detection. Journal of Chromatography B 705: 11–21. Kuda, T., Tsuda, N. and Yano, T. (2004) Thermal inactivation characteristics of acid and alkaline phosphatase in fish and shellfish. Food Chemistry 88: 543–548. LeBlanc, E., Singh, S. and LeBlanc, R.J. (1994) Capillary zone electrophoresis of fish muscle sarcoplasmic proteins. Journal of Food Science 59: 1267–1270. Lundstrom, R.V. (1980) Fish species identification by thin layer polyacrylamide gel isoelectric focusing: collaborative study. Journal – Association of Official Analytical Chemists 63: 69–73. Mackie, I.M. (1996) Authenticity of fish. In: P.R. Ashurst and M.J. Dennis (Eds) Food Authentication. Blackie Academic and Professional, London, pp. 140–170. Mackie, I., Craig, A., Etienne, M., Jerome, M., Fleurence, J., Jessen, F., Smelt, A., Kruijt, A., Malmheden Yman, I., Ferm, M., Martinez, I., Perez-Martin, R., Pineiro, C., Rehbein, H. and Kündiger, R. (2000) Species identification of smoked and gravad fish products by sodium dodecylsulphate polyacrylamide gel electrophoresis, urea isoelectric focusing and native isoelectric focusing: a collaborative study. Food Chemistry 71: 1–7. Ochiai, Y. and Watabe, S. (2003) Identification of fish species in dried fish products by immunostaining using anti-myosin light chain antiserum. Food Research International 36: 1029–1035. Osman, M., Ashoor, F. and Marsh, P. (1987) Liquid chromatographic identification of common fish species. Journal – Association of Official Analytical Chemists 70: 618–625. Pineiro, C., Sotelo, C.G., Medina, I., Gallardo, J.M., Perez-Martin, R.I. (1997) Reversed-phase HPLC as a method for the identification of gadoid fish species. Zeitschrift für Lebensmittel-Untersuchung und -Forschung 204: 411–416. Pineiro, C., Barros-Velazquez, J., Perez-Martin R.I., Martinez, I., Jacobsen, T., Rehbein, H., Kündiger, R., Mendes, R., Etienne, M., Jerome, M., Craig, A., Mackie, I.M. and Jessen, F. (1999) Development of a sodium dodecyl sulphate-polyacrylamide gel electrophoresis reference method for the analysis and identification of fish species in raw and heat-processed samples: a collaborative study. Electrophoresis 20: 1425–1432. Pineiro, C., Barros-Velasquez, J., Perez–Martin, R.I. and Gallardo, J.M. (2000) Specific enzyme detection following isoelectric focusing as a complimentary tool for the differentiation of related gadoid fish species. Food Chemistry 70: 241–245. Quinteiro, J., Sotelo, C.G., Rehbein, H., Pryde, S.E., Medina, I., Perez-Martin, R.I., Rey-Mendez, M. and Mackie, I.M. (1998) Use of mtDNA direct polymerase chain reaction (PCR) sequencing and PCR-restriction fragment length polymorphism methodologies in species identification of canned tuna. Journal of Agricultural and Food Chemistry 46: 1662–1669. Rehbein, H. (1990a) Electrophoretic techniques for species identification of fishery products. Zeitschrift für Lebensmittel-Untersuchung und -Forschung 191: 1–10. Rehbein, H. (1990b) Fish species identification by HPLC of sarcoplasmic proteins. Fresenius Journal of Analytical Chemistry 337: 106. Rehbein, H. (1992a) Determination of the heating temperature of fishery products. Zeitschrift für Lebensmittel-Untersuchung und -Forschung 195: 417–422. Rehbein, H. (1992b) Physical and biochemical methods for the differentiation between fresh and frozen-thawed fish or fillets. Ital. Journal of Food Science 2: 75–86. Rehbein, H (2003) Identification of fish species by protein- and DNA-analysis. In: R.I. Perez-Martin and C.G. Sotelo (Eds) Authenticity of Species in Meat and Seafood Products. Instituto de Investigaciones Marinas, CSIC, Vigo, Spain, pp. 83–101. Rehbein, H., Kress, G. and Schreiber, W (1978) An enzymic method for differentiating thawed and fresh fish fillets. Journal of the Science of Food Agriculture 29: 1076–1082. Rehbein, H. and Kündiger, R. (1984) Comparison of the isoelectric focusing patterns of the sarcoplasmic proteins from red and white muscle of various fish species. Archiv für Fischereiwissenschaft 35: 7–16.

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Rehbein, H., Kress, G. and Kündiger, R. (1990) Determination of species of fish in long-life canned products by isoelectric focusing. [In German.] Fleischwirtschaft 70: 706–709. Rehbein, H., Etienne, M., Jerome, M., Hattula, T., Knudsen, L.B., Jessen, F., Luten J.B., Bouquet, W., Mackie, I.M., Ritchie, A.H., Martin R., Mendes, R. (1995) Influence of variation in methodology on the reliability of the isoelectric focusing method of fish species identification. Food Chemistry 52: 193–197. Rehbein, H., Kündiger, R., Malmheden Yman, I., Ferm, M., Etienne, M., Jerome, M., Craig, A., Mackie, I., Jessen, F., Martinez, I., Mendes, R., Smelt, A., Luten, J., Pineiro, C. and Perez-Martin, R. (1999) Species identification of cooked fish by urea isoelectric focusing and sodium dodecylsulfate polyacrylamide gel electrophoresis: a collaborative study. Food Chemistry 67: 333–339. Rehbein, H., Kündiger, R., Pineiro, C. and Perez-Martin, R.I. (2000) Fish muscle parvalbumins as marker proteins for native and urea isoelectric focusing. Electrophoresis 21: 1458–1463. Renon, P., Bernardi, C., Malandra, R. and Biondi, P.A. (2005) Isoelectric focusing of sarcoplasmic proteins to distinguish swordfish, blue marlin and Mediterranean spearfish. Food Control 16: 473–477. Shikha, F.H., Hossain, M.I., Morioka, K. and Kubota, S. (2006) Effect of pH-shifting on the gel forming characteristics of salt-ground meat from walleye pollack. Fisheries Science 72: 870–876. Sikorski, Z.E., Sun Pan, B. and Shahidi, F. (1994) Seafood Proteins. Chapman and Hall, London. Silva, H.A., Mendes, R., Nunes, M.L. and Empis, J. (2006) Protein changes after irradiation and ice storage of horse mackerel (Trachurus trachurus). European Food Research Technology 224: 83–90. Swoboda, I., Bugajska-Schretter, A., Verdino, P., Keller, W., Sperr, W.R., Valent, P., Valenta, R. and Spitzauer, S. (2002) Recombinant carp parvalbumin, the major cross-reactive fish allergen: a tool for diagnosis and therapy of fish allergy. Journal of Immunology 168: 4576–4584. Tepedino, V., Berrini, A., Borromeo, V., Gaggioli, D., Cantoni, C., Manzoni, P. and Secchi, C. (2001) Identification of commercial fish species belonging to the orders Pleuronectiformes and Gadiformes: library of isoelectric focusing patterns. Journal of AOAC International 84: 1600–1607. Uddin, M., Ishizaki, S., Ishida, M. and Tanaka, M. (2002) Assessing the end–point temperatue of heated fish and shellfish meats. Fisheries Science 68, 768–775. Valenzuela, M.A., Gamarrra, N., Gomez, L., Kettlun, A.M., Sardon, M., Perez, L.M., Vinagre, J. and Guzman, N.A. (1999) A comparative study of fish species identification by gel isoelectric focusing, two-dimensional gel electrophoresis, and capillary zone electrophoresis. Journal of Capillary Electrophoresis and Microchip Technology 6: 85–91. Yearsley, G.K., Last, P.R. and Ward, R.D. (Eds) (1999) Australian Seafood Handbook: Domestic Species. CSIRO Marine Research, Hobart, Australia. Yearsley, G.K., Last, P.R. and Ward, R.D. (Eds) (2003). Australian Seafood Handbook: Imported Species. CSIRO Marine Research, Hobart, Australia.

Chapter 17

DNA-based methods Hartmut Rehbein

17.1

Introduction

In the course of globalisation of food production and distribution, trade with fishery products has steadily increased over recent decades. In many countries the situation on the market is characterised by shortage of traditional fish species, introduction of new species and products, and substitution of highly prized species by cheaper ones. As an example, on the German market cod (Gadus morhua) has been replaced with Alaska Pollock (Theragra chalcogramma) as raw material for deep-frozen products (fillets, fillet portions, fish sticks), and in Spain the North Atlantic hake (Merluccius merluccius) has been substituted by other hake species. It seems to become increasingly difficult to fulfil the consumer’s demand for high-quality fishery products, food safety and authenticity at a reasonable price. In September 2006 at the 28th Session of the Codex Committee on Fish and Fishery Products in Beijing, China, procedures for the inclusion of additional species in standards for fish and fishery products were discussed (Codex Committee on Fish and Fishery Products 2007). Cases of substitution and fraud have been uncovered in several countries. Food Standards Australia has published results of a pilot survey performed in 2003 on the identity of fish species as sold through outlets in Australia and New Zealand (http://www.foodstandards. gov.au/mediareleasespublications/publications/). Samples (n = 138) were analysed by DNA fingerprinting (PCR–RFLP: polymerase chain reaction–restriction fragment length polymorphism) to check products for correct labelling either as Australian barramundi (Lates calcarifer) or red emperor (Lutjanus sebae). Overall the survey found that 87% of all samples sold as barramundi had been labelled correctly. However, in the case of products sold as red emperor, about 41% of all samples were identified as being mislabelled. Recently, similar experiences were reported from the USA for groupers, red snapper and walleye. For example, DNA testing revealed that in 11 restaurants located in the Tampa Bay area, Florida, grouper was substituted by cheaper fish (hake, tilapia, catfish and other species) in more than 50% of cases (St. Petersburg Times, Tampa Bay 2006). The FDA list of examples of substituted seafood has 19 entries (http://www.cfsan.fda.gov/~frf/econ.html). In Europe, annual reports of food control laboratories regularly describe cases of mislabelled fish. 363

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Black caviar, a costly sturgeon product with a long tradition, represents another example of product shortage caused by over-exploitation of fish resources and destruction of natural habitats. Owing to reduction of sturgeon stocks in the Black Sea and Caspian Sea, caviar trade is restricted by Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) regulations and national rules (htpp://www.fws.gov). PCR-based methods have been developed for identification and differentiation of sturgeon caviar and successfully applied to detect fraud in the caviar trade (Birstein et al. 1997). The main objectives for authenticity assessment by DNA analysis are: z z z z z z z

To protect the consumer against fraud. To protect the consumer against health risks (for example allergic reaction against fish and shellfish). To safeguard fair trade. To prevent mislabelling of substitutive products. To ensure correct labelling of ethnic food. To protect endangered species. To support customs examinations.

The topic has been reviewed frequently over recent years (Bossier 1999; Civera 2003; Sotelo and Perez-Martin 2003; Martinez et al. 2005; Teletchea et al. 2005), and new publications about fish or shellfish identification are steadily appearing in scientific journals. The many aquatic animal species used for human consumption, several thousand species worldwide, which are often closely genetically related, is a considerable analytical challenge for food inspection laboratories. Currently, PCR-based methods deliver the best tools for species identification. The different techniques, which have been originally developed and applied for mutant detection in medical research in most cases (Taylor and Day 2005), are discussed in the following part of this chapter. Results obtained for several commercially important fish families (tunas, gadoids, hakes, flatfishes, salmonids, sturgeons, snapper, groupers and sharks) have been compiled in Tables 17.6–17.14. In the case of crustaceans and molluscs, the situation is less satisfactory, as for many species PCR-based techniques are lacking. A selection of publications dealing with shellfish authentication has been compiled in Tables 17.15 and 17.16. In the following part of this chapter the term ‘fish’ is used for fish and shellfish (crustaceans and molluscs), if not indicated otherwise.

17.2

DNA in fishery products

The edible part of most fishery products consists of fillet, which contains white and red muscle tissue. Other tissues used for human consumption are liver, roe, milt and skin. Mussel meat is an exception, as it is composed of different kinds of muscle and several other tissues (gills, digestive tract, and so on). The DNA content of whole fish was found to range from 140 to 900 μg/g wet weight, the mean value for 31 North Atlantic fish species being about 255 μg/g wet weight (Horstkotte and Rehbein 2006). The DNA content of fish fillet, fish eggs and other tissues is given in Table 17.1. The DNA content of a single grain of sturgeon or trout caviar was determined

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Table 17.1 DNA content of fishery products as determined with the fluorescence enhancement assay using the dye Hoechst 33258 (Rehbein unpublished results; Rehbein and Horstkotte 2003).

Fish species Carp, Cyprinus carpio

Whiting, Merlangius merlangus Rainbow trout, Oncorhynchus mykiss

Mackerel, Scomber species

Cod, Gadus morhua

Atlantic salmon, Salmo salar Eel, Anguilla anguilla North Atlantic hake, Merluccius merluccius Sevruga, Acipenser stellatus Osietra, Acipenser gueldenstaedtii Beluga, Huso huso Herring, Clupea harengus Skipjack, Katsuwonus pelamis

Type of tissue/product White muscle, raw Red muscle, raw Liver, raw Kidney, raw Spleen, raw White muscle, raw Red muscle, raw White Muscle, raw White muscle, cooked White muscle, sterilised Caviar (egg) White muscle, raw Red muscle, raw Kidney, raw Spleen, raw Fillet, hot smoked White muscle, raw Red muscle, raw Kidney, raw Spleen, raw White muscle, raw White muscle, hot smoked Roe, raw Caviar (egg) Caviar (egg) Caviar (egg) Bismarck herring Rollmops Light muscle, canned

DNA content (μg/g wet weight) 672 1177 4221 18,479 18,490 63 304 839 739 535 85 545 1705 8478 9062 177 478 870 4757 9040 261 180 1590 450 244 236 261 309 303

to be about 6 μg (Rehbein and Horstkotte 2003). Generally the DNA content increases from white to red muscle to innards (liver, spleen, kidneys). During processing (for example cooking, smoking, marinating) of fish, DNA may be degraded to some extent, but the reduction in DNA content and size does not hamper most of the PCR-based methods. However, in canned fish the DNA is fragmented into short pieces, making it necessary to use sequences of fewer than 200 base pairs (bp) for PCR (Quinteiro et al. 1998). Like other animals, fish and shellfish possess two different types of DNA in their tissues, belonging to nuclear and mitochondrial genomes. The mean haploid nuclear genome size for teleost fish is about 1.15 × 109 bp (www.genomesize.com) (for example 1.7 × 109 bp for zebra fish, Danio rerio), whereas the mitochondrial genome comprises about 1.65 × 104 bp (Meyer 1993). In muscle and other tissues of fish, each cell has a high number of mitochondrial genomes, but because of the greater size of the nuclear genome only 2 × 10−2% of total DNA belongs to mitochondrial DNA (Battersby and Moyes 1998). However, in fish

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Table 17.2 Comparison of copy numbers of nuclear and mitochondrial genomes in different cells.

Cell type

Mitochondria: nucleus

Mitochondrial DNA : total DNA

DNA content per cell (ng)

∼103 ∼107 – – 7 × 1010 3 × 1011

1% 99% – – >99.9% >99.9%

– – 150 18,250a 140 6500

Liver cell (rat) Egg (frog) Egg (Alaska Pollack) Egg (Alaska pollack) Egg (herring) Egg (rainbow trout) a

Sum of DNA

+ RNA (Aranishi 2006)

Table 17.3 Number of copies of nuclear single-copy genes in 1 gram of fish muscle.

Fish species Atlantic salmon, Salmo salar Rainbow trout, Oncorhynchus mykiss Carp, Cyprinus carpio Cod, Gadus morhua a

DNA content of white muscle of fish (μg/g wet weight)

C value (amount of DNA (pg) contained in a haploid nucleus)a

Calculated gene copy number per gram fish muscle

261 839

3.10 2.80

0.084 × 109 0.30 × 109

672 478

1.82 0.93

0.37 × 109 0.51 × 109

Data from Gregory 2008

eggs having only one haploid nuclear genome per egg, the share of nuclear DNA is very much less than 0.1%, as eggs contain very high amounts of mitochondrial DNA (Table 17.2). The number of single-copy nuclear genes in raw fish muscle can be calculated by dividing the DNA content of muscle through the DNA content of haploid genomes. The values given in Table 17.3 show that one gram of white muscle of fish contains 108–109 copies on average.

17.3

Genes used for species identification

Genes of fish and shellfish are classified as belonging to the nuclear or mitochondrial genome. The following types of nuclear genes have been used for species identification: z z z z

Introns and exons of protein coding genes. Ribosomal RNA genes (5S, 5.8S, 18S, 28S rRNA) (S: Svedberg units). ITS (internal transcribed spacer) of ribosomal genes. Microsatellites.

Protein coding genes are composed of DNA segments being either translated into proteins (exons) or removed during RNA processing (introns). Amplification of exon sequences has

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Table 17.4 Species identification of food fish and shellfish by PCR of nuclear genes. Species

a b

Gene

Hakes Tunas Cyprinids

ITS1-rRNA ITS1-rRNA ITS1-rRNA

Mackerels Sturgeons Sole and Greenland halibut

NTS-5S rRNAa Not specified 5S rRNA

Atlantic salmon and brown trout

5S rRNA

Trachurus spp.

5S rRNA

Mytilus spp. Shrimps 230 fish species Salmonids Salmonids

ITS, PLIIab Not specified Rhodopsine Parvalbumin Growth hormone

Method RFLP, sequencing Sequencing Species-specific primers RFLP RAPD Agarose gel electrophoresis Agarose gel electrophoresis Agarose gel electrophoresis RFLP RAPD Sequencing SSCP SSCP

Reference Perez et al. 2005 Chow et al. 2006 Wyatt et al. 2006 Aranishi 2005 Urbanyi et al. 2004 Cespedes et al. 1999 Pendas et al. 1995 Karaiskou et al. 2003 Heath et al. 1995 Phongdara et al. 1999 (http://fishtrace.org) Rehbein 2005 Rehbein 2005

NTS: non-transcribed spacer PLIIa: protamine-like sperm-specific protein

been rarely applied to species differentiation (Asensio et al. 2001) as exon sequences are considered to be relatively conservative. On the other hand, introns have been shown to yield substantial variability, resulting in polymorphism of sequence and length. This variability has been used to develop exon-primed intron-crossing (EPIC) PCR systems for population and species differentiation of fish and shellfish (Bierne et al. 2000; Chow and Nakadate 2004; Hubert et al. 2006). Ribosomes of eukaryotes have four types of RNA, named according to their size 5S, 5.8S, 18S and 28S rRNA. The genes for these RNAs are arranged in transcription units containing also internal transcribed spacer sequences (ITS). All cells contain multiple copies of the rRNA genes (Cooper and Hausman 2004), Microsatellites are short stretches of DNA arranged in tandem consisting of repeated units of one to six base pairs, mainly di-, tri- or tetra-nucleotides. They are mainly applied for characterisation of populations, parentage assignment, brood stock selection, constructing dense linkage maps and marker-assisted selection (Chistiakov et al. 2006). Moderately variable microsatellite markers may also be candidates for species identification, as recently demonstrated for differentiation between Anguilla species (Maes et al. 2006). By multiplexPCR of four microsatellite loci, identification of individuals belonging to A. anguilla, A. rostrata, A. japonica or A. marmorata was achieved using a Bayesian individual assignment technique. Examples of application of nuclear genes for species identification by various PCR-based techniques are given in Table 17.4. However, in most studies, mitochondrial genes have been preferred for identification of fish and shellfish. For several reasons mitochondrial genes are excellent tools for species identification of fishery products:

368 z

z z

z

Fishery Products: Quality, safety and authenticity

The number of mitochondrial genomes exceeds the number of nuclear genomes by 10to 100-fold in muscle and other tissues. Thus PCR systems based on mitochondrial DNA have a very low detection limit. Mitochondrial DNA generally evolves much faster than single-copy nuclear genes, making it easier to differentiate between closely related species (Meyer 1993). GenBank and other databases contain many more sequences of mitochondrial genes than of nuclear genes of fishes and other animals. For example, the combination ‘cytochrome b and Teleostei’ gave 16,955 entries for core nucleotides, whereas ‘actin and teleostei’ resulted in 671 entries for core nucleotides (August 2006). In recent years, the whole mitochondrial genome of more than 240 teleost fish species has been sequenced and deposited in GenBank. However, usage of mitochondrial DNA has also some disadvantages:

z

z

Differentiation between hybrids and maternal species is not possible. Hybridisation is observed quite often for wild fish (Wyatt et al. 2006) and applied in aquaculture to improve traits (Urbanyi et al. 2004). Quantification of tissue amounts by real-time PCR is difficult if mitochondrial genes are used, as the copy number of mitochondrial genes per gram of muscle may vary.

17.4

Methods

At present, seafood authentication by DNA analysis is performed mainly by PCR-based methods, but DNA micro-arrays are under development (see section 17.4.4). The protocol consists of the following steps: z z z z

Isolation of DNA. Amplification of a selected sequence of mitochondrial or nuclear DNA by specific or universal primers. Characterisation of the amplicon. Interpretation of results.

17.4.1

DNA isolation

Extraction and isolation of DNA from raw or processed fish may be performed by standard methods used in molecular biology (Hoelzel 1992). As an alternative one of the numerous commercially available kits can be used. A short, simple and inexpensive DNA extraction procedure comprises solubilisation of tissue (muscle, roe) by urea and detergent followed by removal of PCR inhibitors by binding them to the Chelex resin (Aranishi et al et al. 2006; Chakraborty et al. 2006). A comparison of DNA extraction methods for food analysis has been published recently (Di Pinto et al. 2007). Many protocols do not include digestion of contaminating RNA by RNase treatment, resulting in a mixture of DNA and RNA as final product. In most cases, presence of residual RNA in DNA preparations will not pose problems for PCR analysis, but in quantitative PCR

DNA-based methods

369

Table 17.5 Fish species identification by PCR using species-specific primers. Fish species analysed Sturgeons Tunas and bonitos Flatfishes

Frigate tunas Eels (A. anguilla, A. rostrata) Eels (A. anguilla, A. japonica) Grouper, wreck fish, Nile perch Mackerel, Scomber colias

Comments (Rehbein 2003) Specificity depended on PCR conditions Specificity depended on PCR conditions Primers for sole were specific, but primers for Pacific halibut reacted with other flatfishes

Species-specific TaqMan probes

Reference Birstein et al. 1997 Lockley and Bardsley 2000 Cespedes et al. 1999

Infante et al. 2004 Trautner 2006 Itoi et al. 2005 Asensio et al. 2001a Infante and Manchado 2006

RNA may act as a competitor to DNA in primer binding, thus lowering the efficiency of the PCR (Pikaart and Villeponteau 1993). Suitable methods for quantification of isolated DNA are measurement of the optical density at 260 nm (OD260 nm) (if RNA has been removed) or fluorescence enhancement assays (Downs and Wilfinger 1983; Gorokhova and Kyle 2002).

17.4.2

Polymerase chain reaction with species-specific primers

The fastest and most convenient PCR-based method for fish species identification is usage of species-specific primers or probes. However, owing to the large number of closely related species used for human consumption, for example more than 15 species of the family Scombridae, 12 species from the genus Merluccius or about 25 sturgeon species, it may be difficult to find primers of sufficient specificity. Examples for application of specific primers are given in Table 17.5. When testing the PCR system originally developed for differentiation of blue fin tuna (Thunnus thynnus) and bonito (Sarda sarda), it was found that the primers claimed to be specific for blue fin tuna reacted with another member of the genus Thunnus as well (Figure 17.1). Additional examples of limited specificity of fish PCR systems have been given previously (Rehbein 2003). These results demonstrate that many fish species have to be tested before the statement of specificity is justified.

17.4.3

Polymerase chain reaction with universal primers

The great number of PCR methods developed for identification of fish or shellfish species in seafood relies on so-called ‘universal’ primers. Universal primers may react with any fish species (Dalmasso et al. 2004), or only with members of certain families or genera if preselection is desired (Perez et al. 2005). Universal primers are selected from conservative regions of the nuclear (Perez et al. 2005) or mitochondrial genome (Wolf et al. 2000).

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T. thynnus T. orientalis T. albacares T. alalunga T. maccoyii T. obesus T. tonggol S. orientalis K. pelamis E. affinis A. thazard A. rochei 100 bp ladder T. thynnus T. orientalis T. albacares T. alalunga T. maccoyii T. obesus T. tonggol S. orientalis K. pelamis E. affinis A. thazard A. rochei

Figure 17.1 Specificity of PCR primers. Twelve tuna and bonito species of the genera Thunnus, Sarda, Katsuwonus, Euthunnus and Auxis were analysed by the PCR system described by Lockley and Bardsley (2000). In the upper part amplicons obtained by using the annealing temperature of 60°C are shown; in the lower part results of PCR with annealing at 69°C are presented. Amplicons were run on 10% polyacrylamide gel and visualised by silver staining. Sample application position and cathode are on the left side. The amplicon of the mitochondrial cytochrome b gene has a size of 207 bp.

The distance between primer binding sites should be a few hundred nucleotides for most types of fishery product, but less than 150 nucleotides in canned tuna (Quinteiro et al. 1998 ). If presence or absence of ‘fish’ as such should be proved in a given food, formation of PCR product can be detected by simple agarose gel electrophoresis or by more sophisticated real-time PCR analysis. The second method is used by the SureFood® Allergen Fish Real-Time PCR Kit (www.congen.de). The identification of a single genus, species or even population needs further characterisation of the amplicon by DNA sequencing or secondary methods of mutation detection like RFLP and single-strand conformation polymorphism (SSCP) analysis, which are more straightforward, faster and less costly than sequencing. Sequencing of amplicons The combination of PCR and DNA sequencing was introduced by Bartlett and Davidson (1992) to identify animal species including fish under the name of FINS (forensically informative nucleotide sequencing). In FINS analysis, an unknown sample sequence is compared by a genetic distance measurement method with a set of reference sequences. The sequence

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371

of the unknown sample will be clustered to those sequences from reference species to which the sample is phylogenetically most related (Quinteiro et al. 1998). A more convenient way of identifying an unknown sample is by aligning the obtained sequence to nucleotide sequences deposited in a database like GenBank using the program BLAST (basic local alignment search tool) (Parson et al. 2000; Brodmann et al. 2001). Several points have to be taken into consideration before starting BLAST or FINS analysis: (1) Does the sample consist of a single species or does it contain a mixture of species? In the second case, the PCR product has to be cloned before sequencing. Information about the number of species present in a product can be obtained by RFLP. (2) Selection of a sequence being as short as possible, but containing sufficient variation between species. (3) Selection of a gene having a large number of entries in databases (for example cytochrome b). The identity of sequences necessary to assign an unknown sample to a reference depends on the genus and the gene. Closely related fish species of the genus Sebastes or Acipenser have nearly identical cytochrome b sequences, whereas the differences in other genera and genes may be greater.

Restriction-fragment length polymorphism analysis RFLP analysis is currently the most popular method of species identification of fishery products. The technique comprises the following steps: PCR, cutting of the amplicon with one or more restriction endonucleases, separation of fragments by agarose or polyacrylamide gel electrophoresis, capillary electrophoresis (Dooley et al. 2005) or high-performance liquid chromatography (HPLC) (Horstkotte and Rehbein 2003). The fragment patterns can be compared with published results or with the patterns of references treated along with samples. A precondition for rational choice of suitable restriction endonucleases is knowledge of the sequence to be cut. The method has been successfully validated for several commercially important fish species (Dooley et al. 2005; Hold et al. 2001). The same 464 bp sequence of the mitochondrial cytochrome b gene was amplified in both studies, followed by restriction digestion and separation of fragments. Hold et al. (2001) used native polyacrylamide gel electrophoresis (PAGE) for resolving DNA restriction fragments of 36 species of the genera Merluccius, Oncorhynchus, Anguilla and others; DNA bands were visualised by silver staining. The advanced method applied by Dooley et al. (2005) was based on lab-on-a-chip capillary electrophoresis (CE) for fragment separation. Single species samples of 10 white fish species (gadoids and others) were correctly identified by five food control laboratories. By using a fast DNA extraction method, for example boiling of tissue with Chelex resin, a high-speed thermocycler and ready-to-run agarose gel (for example E-Gel, Invitrogen; Figure 17.2) or a CE system, results can be obtained within one working day. A multitude of restriction endonucleases is available on the market, which may be applied in most cases without the necessity of purifying the amplicon to be digested.

Single-strand conformation polymorphism analysis SSCP analysis is an inexpensive, convenient and sensitive method for detecting genetic variation by electrophoretic separation of single-stranded nucleic acids (Sunnucks et al.

372

Fishery Products: Quality, safety and authenticity 4% Agarose (HR) 1

2

3

4

5

6

7

8

9

10 11 12

uncut amplicon 464bp

primer dimer

Figure 17.2 Identification of gadoid roe product by RFLP using an E-Gel (Invitrogen). A 464 bp sequence of the mitochondrial cytochrome b gene was amplified according to Hold et al. (2001), and digested with the restriction endonuclease Dde I. Lane 1: DNA marker; lanes 2–5: different brands of Tarama, a fish roe product; lane 6: Atlantic cod (Gadus morhua); lane 7: Pacific cod (Gadus macrocephalus); lane 8: North Atlantic hake (Merluccius merluccius); lanes 9 and 11: Greenland cod (Gadus ogac); lane 10: hoki (Macruronus novaezelandiae); lane 12: untreated amplicon. The products contained varying amounts of cod roe, together with roe from other fish species.

2000). The principle of the technique is shown in Figure 17.3. It comprises the following steps: z z z z

PCR to amplify DNA segments of 100–500 bp. Generation of single-stranded DNA (ssDNA) by heat, and alkali and/or organic solvents (ssDNA can be produced also by asymmetric PCR (Rehbein et al. 1998)). Separation of ssDNA strands by electrophoresis in slab-gels or capillaries (Binz et al. 2001) under non-denaturing conditions. The ssDNA strands in slab-gels can be visualised by staining with silver or fluorescence dyes; in the case of CE, PCR is performed with primers labelled with fluorescence dyes. Former usage of radioactive labelling for detection was less convenient.

The mobility of ssDNA in native gel electrophoresis is affected by the sequence-depending conformation of the nucleic acid strand, the type of gel matrix, the temperature and the composition of electrophoresis buffer (Hayashi et al. 1998). For better comparison of ssDNA patterns, it is recommended to run unknown samples and references on the same gel. The reliability of the method performed under controlled electrophoretic conditions has been demonstrated by collaborative study (Rehbein et al. 1999). Examples of seafood authentication by SSCP analysis are given in Tables 17.6–17.16. As prediction of the ssDNA pattern from the sequence is hardly possible, the development of a protocol needs several

Sequence 1: ATGCCAGTCA TACGGTCAGT

_____ _____

dsDNA

Sequence 2: ATGCAAGTCA TACG TTCAGT

_____ _____

Mix with formamide, heat & chill in iced water

Ø

Single-strand DNA

Native polyacrylamide gel electrophoresis and silver staining of DNA Lane 1: DNA marker, lane 2: PCR product; Lane 3–13: ssDNA patterns for different fish

ssDNA

dsDNA

Figure 17.3 Principle of SSCP analysis.

Table 17.6 Differentiation of tunas and bonitos. Species, product 8 species of the genus Thunnus and Katsuwonus pelamis, Sarda orientalis T. albacares, T. obesus, T. alalunga, K. pelamis Auxis thazard, A. rochei, canned fish Thunnus alalunga, T. albacares 17 species of tuna and bonito, canned fish T. albacares, T. thynnus T. obesus, T. alalunga, K. pelamis, canned fish 8 species of tuna and bonito, canned fish

Gene

Method

Reference

ITS1 (first internal transcribed spacer)

Sequencing of amplicons (∼600 bp)

Chow et al. 2006

Cytochrome b

SSCP

Cytochrome b, 12S rRNA, ATPase 6 16S rRNA

Sequencing, specific primers Specific TaqMan systems RFLP, SSCP

Colombo et al. 2005 Infante et al. 2004

Cytochrome b Cytochrome b

Nested PCR, sequencing, RFLP

Cytochrome b

SSCP

Lopez and Pardo 2005 Mackie et al. 1999 Pardo and BegoniaVillareal 2004 Rehbein et al. 1999

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Table 17.7 Differentiation of gadoid fish species. Species, product

a

Gene

Method

Reference

8 species of the family Gadidae, 3 Lotidae, 3 Phycidae, Raniceps raninus, M. merluccius, Coryphaenoides rupestris, raw muscle 16 gadoids, raw or salted muscle

rRNA, cytochrome b

Sequencing

Bakke and Johansen 2005

Cytochrome b

Sequencing, RFLP

8 gadoids, raw muscle

Cytochrome b

3 gadoids

ATPase 6/8

25 species of the family Gadidae, 4 species of the family Merlucidae

Cytochrome b, cytochrome oxidase I

RFLP, SSCP, DGGEa TaqMan MGB probe Sequencing

Calo-Mata et al. 2003 Comi et al. 2005 Taylor et al. 2002 Teletchea et al. 2006

Denaturating gradient gel electrophoresis

Table 17.8 Differentiation of hakes (Merluciidae). Species, product

Gene

Method

Reference

9 species of hake 9 species of hake

Cytochrome b (a) 5S rRNA, (b) cytochrome b ITS1 rRNA Mitochondrial control region

RFLP (a) electrophoresis (b) RFLP Sequencing, RFLP Sequencing, RFLP

Hold et al. 2001 Perez and Garcia-Vazquez 2004 Perez et al. 2005 Quinteiro et al. 2001

Method

Reference

12 species of hake 11 species of hake

Table 17.9 Differentiation of flatfish. Species, product Solea solea, Pleuronectes platessa, Platichthys flesus, Reinhardtius hippoglossoides Solea solea, Pleuronectes platessa, Platichthys flesus 5 flatfish species 9 flatfish species 24 flatfish species 10 sole species 6 sole species

Gene Cytochrome b

SSCP

Cespedes et al. 1999a

Cytochrome b

RFLP

Cespedes et al. 1998

12S rRNA Cytochrome b

RFLP RFLP

Cytochrome b Cytochrome b, 12S rRNA, 16S rRNA U1 and U2 snRNA

Sequencing, RFLP Sequencing

Comesana et al. 2003 Sanjuan and Comesana 2002 Sotelo et al. 2001 Infante et al. 2004a

Electrophoresis

Manchado et al. 2006

DNA-based methods

375

Table 17.10 Differentiation of salmonids. Species, product 10 salmonid species 10 salmonid species 10 salmonid species, roe and cold-smoked fillet Oncorhynchus mykiss, Salmo salar, Brama raii, smoked fish Oncorhynchus mykiss, Salmo salar

Gene

Method

Reference

Cytochrome b Cytochrome b Cytochrome b, parvalbumin, growth hormone 5S rRNA

RFLP RFLP, validation study SSCP

Russell et al. 2000 Hold et al. 2001a Rehbein 2005

Electrophoresis

Carrera et al. 2000

Not identified

AFLP-derived primers specific for O. mykiss

Zhang and Cai 2006

Table 17.11 Differentiation of sturgeons. Species, product

Gene

Huso huso, Acipenser stellatus, A. gueldenstaedtii, caviar 22 sturgeon species 11 sturgeon species 22 sturgeon species 6 sturgeon species, caviar 12 sturgeon species

Method

Reference

Cytochrome b

Species-specific primers

Birstein et al. 1998

Cytochrome b, 12S rRNA, 16S rRNA Cytochrome b Cytochrome b Cytochrome b ND4 gene

Sequencing

Birstein and De Salle 1998 Wolf et al. 1999 Ludwig et al. 2002 Rehbein et al. 1999a Zhang et al. 2000

RFLP Sequencing, RFLP SSCP Sequencing

Table 17.12 Differentiation of snappers (Lutjanidae). Species, product

Gene

13 western Atlantic snapper species Red snapper, L. campechanus L. sanguineus, L. erythopterus, L. argentimaculatus, L. malabarius, Lethrinus leutjanus, Pinjalo pinjalo

Method

Cytochrome b, 12S rRNA Cytochrome b 12S rRNA

Reference

RFLP

Chow et al. 1993

Sequencing RFLP

Marko et al. 2004 Zhang et al. 2006

Table 17.13 Differentiation of groupers. Species, product

Gene

Method

Reference

Epinephelus guaza Epinephelus guaza

Not specified 5S rRNA

Asensio et al. 2002 Asensio et al. 2001a

6 grouper species

16S rRNA

RAPD Species-specific primers, electrophoresis Multiplex PCR, real-time PCR

Trotta et al. 2005

Table 17.14 Differentiation of sharks. Species, product Great white shark, Carcharodon carcharias 35 shark species from western North Atlantic Squalus acanthias, Scyliorhinus caniculus, Prionace glauca, Mustelus mustelus, Mustelus asterias Carcharhinus obscurus, C. plumbeus 16 Lamniform species

7 Carchariniform species 11 Carchariniform species

Gene

Method

ITS2, cytochrome b 12S RNA, 16S rRNA, valine tRNA 16S rRNA

ITS2 Cytochrome b, NADH2

Cytochrome b Cytochrome b

Reference

Species-specific primers Sequencing

Chapman et al. 2003 Greig et al. 2005

RFLP

Vicari et al. 2001

Species-specific primers Sequencing, universal shark primers, speciesspecific primers RFLP Sequencing, RFLP

Pank et al. 2001 Hoelzel, AE (2001)

Chan et al. 2003 Heist and Gold 1999

Table 17.15 Differentiation of molluscs. Species, product Bivalve species, raw Perna species, raw 15 mussel species of the genera Mytilus, Perna and others Mytilus spp., Pecten maximus and others Crassostrea and Ostrea oysters 8 Cephalopod species 10 Cephalopod species 5 species of the families Loliginidae and Ommastrephidae Pecten jacobaeus, P. maximus 4 scallop species, canned products

Gene

Method

Reference

18S rRNA COX I, NADH 4 18S rRNA, ITS 1, polyphenolic adhesive protein 18S rRNA, 16S rRNA 5S rRNA

SSCP. sequencing Specific primers Sequencing, RFLP

Livi et al. 2006 Blair et al. 2006 Santaclara et al. 2006

Specific primers

Bendezu et al. 2005

Specific primers

Cross et al. 2005

Cytochrome b 16S rRNA 16S rRNA

Sequencing, RFLP Sequencing RFLP

Chapela et al. 2003 Chapela et al. 2002 Colombo et al. 2002

16 S rRNA

Specific primers

Colombo et al. 2004

IST 1, ITS 2

RFLP

Lopez-Pinon et al. 2002

Table 17.16 Differentiation of shrimps. Species, product Shrimp, crab, lobster and crawfish species 5 Penaeid shrimps 7 shrimp species

Gene

Method

Reference

16S rRNA

RFLP

Brzezinski et al. 2005

16S rRNA COX I

SSCP, RFLP SSCP, RFLP

Khamnamtong et al. 2005 Rehbein 2001

DNA-based methods

Sample 1 Sample 2 Sample 3: Sample 4 Sample 5 Sample 6 Sample 7 Sample 8: Sample 9 Sample 10 Cathode ssDNA

377

Cod

Ling

dsDNA

Figure 17.4 Differentiation between five specimens of cod (Gadhus morhua) and ling (Molva molva) by RFLP–SSCP. A 464 bp sequence of the mitochondrial cytochrome b gene was amplified according to Hold et al. (2001), and digested with the restriction endonuclease Taq I yielding fragments of 243 (double-band) bp for cod and 215 and 251 bp for ling. The fragments obtained were further analysed by SSCP using CleanGel HP 15% as described by Rehbein (2005).PCR–SSCP strengthened the differences between cod and ling, and revealed some intra-species variability.

experiments to select a suitable amplicon and optimised electrophoretic conditions, which may be considered a disadvantage of the method. RFLP–SSCP analysis The combination of RFLP and SSCP was found to make it easier to detect sequence variations in large amplicons (Barros et al. 1997) and to enable the differentiation between species exhibiting the same restriction fragment pattern (Rehbein 2002). For fish species identification, the specificity of cutting sites of restriction endonucleases can be used in the first step to differentiate several species by distinct fragment pattern, whereas the enhanced sensitivity of SSCP in case of short sequences can be used to detect sequence differences between fragments (Figure 17.4). The number of restriction enzymes necessary for identification of a species may be reduced considerably by SSCP analysis of fragments. Random amplified polymorphic DNA analysis Random amplified polymorphic DNA (RAPD) analysis can be performed without prior knowledge of specific DNA sequences of the species under study. RAPD is a PCR-based method using one or two primers, in most cases 10 bp in length, binding at non-stringent annealing temperatures (35–40°C) at various sites of the genome resulting in DNA fingerprints. Elucidation of population structure or kinship analysis has been successfully performed in fish and shellfish research by RAPD (Ali et al. 2004). RAPD has been used in several studies for identification of fish species products. Partis and Wells (1996) investigated the potential of RAPD by analysing 116 specimens from eight species of fish important to the Australian market (barramundi, Nile perch, John Dory and other species). According to their results, the method was determined to be reasonably specific. In another study, products made from cod (Gadus morhua, raw and cooked fillet,

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different types of roe), Atlantic salmon (Salmo salar, fresh and cold-smoked fillet) or Arctic charr (Salvelinus alpinus, fresh and hot-smoked fillet) gave species-specific fingerprints (Martinez et al. 2001). It is highly recommended to include suitable references (species, products) in the RAPD analysis, because the state of integrity of DNA, conditions of PCR and type of electrophoresis may influence the resulting DNA pattern.

17.4.4 Quantitative polymerase chain reaction A considerable number of fishery products contain muscle or other tissue of more than one species. Examples are fish cakes, pies, pastries, soups, baby food products and fish meal. Recently, PCR-based methods have been developed to determine the share of a certain fish species relative to the total amount of fish in the product. The copy numbers of nuclear genes (calmodulin, myostatin, parvalbumin) (Rehbein and Horstkotte 2003) have been determined for raw fish muscle (Rehbein and Horstkotte 2003) and baby food by real-time PCR. In the case of raw muscle, the three genes gave similar copy numbers (0.5–1.1 × 108 per gram wet weight) for several fish species. Hird et al. (2005) developed a method to quantify haddock (Melanogrammus aeglefinus) in commercial products using the nuclear transferrin gene for real-time PCR. The CT values (cycle number when fluorescence intensity crosses the set threshold) for haddock-specific primers and probes were related to CT values for a universal fish system. The relationship between haddock muscle weight and gene copy number (CT value) was found to remain relatively constant, throughout the year and across several fishing grounds. Applied to analysis of model samples consisting of mixtures of DNA of haddock with DNA of various other fish species, the PCR system turned out to be specific for haddock, being suited to quantifying the share of haddock within 7% of the true percentage. However, when processed fish was analysed, the CT value increased, owing to degradation of DNA by heat and pressure, and the relationship between the CT value and amount of tissue had changed. In another study, the mitochondrial 16S rRNA gene was used to construct fish speciesspecific primers and probes, as well as universal fish primers, for relative quantification. To quantify the relative amounts of albacore tuna (Thunnus alalunga) and yellow fin tuna (Thunnus albacares) in mixtures of raw muscle, two specific TaqMan systems were devised. Another system specific to scombroid species was taken as a consensus system. Good results were obtained for raw samples, whereas measurements of canned samples were highly inaccurate (Lopez and Pardo 2005). To summarise the experiences described above, it seems possible to determine by real-time PCR the share of a given fish species relative to the total amount of fish in raw products. The uncertainty of measurement by real-time PCR was similar to uncertainty of determination of nitrogen content (Hird et al. 2005).

17.4.5

Microarrays

DNA microarrays, also known as DNA chips, consist of a solid support with bound oligonucleotides (probes). Samples, for example PCR products labelled with fluorophores, are hybridised to the targets, and finally detected by scanning. The first high-density DNA

DNA-based methods

379

chip for the multi-detection of animal species in food and feed is obtainable from bioMerieux (www.biomerieux.com). The chip can be used to identify samples of several salmonid, scombroid and eel species, as well as a few other fish species. New DNA chips to identify marine organisms are under development (www.fish-and-chips.uni-bremen.de; www.tuat. ac.jp), but not yet available for routine food analysis. However, DNA microarray technology is already used in toxicology and ecotoxicology (Lettieri 2006), and has recently been applied to genetic stock identification of chum salmon (Oncorhynchus keta) (Sato et al. 2004).

17.5

Conclusions and outlook

Since the invention of PCR, numerous methods have been developed to identify all kinds of seafood, and the information is scattered in hundreds of publications. However, only a few methods have been validated by ring trials and have become official methods suitable for food control laboratories (Hold et al. 2001). What is urgently needed for making the work of seafood control laboratories more efficient is an international database covering fish and shellfish species traded worldwide, with open access to everybody engaged in seafood authentication. In recent years several differently structured databases have been created: z z z z z z

Fishtrace (http://www.fishtrace.org). Fishgen (http://fishgen.jrc.it/). FischDB (www.fischdb.de). Fish Barcode of Life Initiative (www.fishbol.org). AFLP database ([email protected]). Validation (www.seafoodplus.org; http://www.azti.es/dna_database/).

Fish Trace, initiated by a project funded by the European Union and now located at the Joint Research Centre in Ispra, Italy, has the following objectives: 1. To draw up a genetic catalogue of representative European marine fish species as indisputable evidence for the origin of the fish and fish products. 2. To pool reference biological materials for cross-referencing in fish traceability. 3. To establish a public accessible database compiling the new standardised data generated in the network. 4. To validate the information compiled in the database for its applicability for end-users in the analysis, characterisation and diagnosis of marine fish species. 5. To use the collection of standardised information to lend support to European policies for fishery stocks, food traceability and environmental protection. At the moment, the data (sequences for the mitochondrial cytochrome b gene and nuclear rhodopsine gene) of 230 fish species have been collected. Unfortunately, the database is restricted to European marine fish species. Fishgen is a small database, with data for 11 species available. The data have been collected from literature, and are not very useful for product analysis.

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Fishery Products: Quality, safety and authenticity

The aim of FischDB is to support food control laboratories by listing sequences of the cytochrome b gene, fragment sizes to be used in RFLP analysis, SSCP patterns and patterns and pI values obtained by isoelectric focusing of sarcoplasmic proteins. The explanations are in German, thus restricting the benefit of the database. The Fish Barcode of Life Initiative (FISH-BOL) is a global effort to establish a standardised reference sequence library for all fish species. An approximately 655 bp sequence of the mitochondrial cytochrome oxidase subunit I gene has been selected for amplification, and up November 2006 the database contained sequences of 2522 species (Ward et al. 2005). In the future, FISH-BOL can make PCR-based species identification much easier, but at the moment the practical application is limited by the nature of the gene (COX I). Most of the work previously performed in fish species identification is based on PCR of cytochrome b or the 12S or 16S rRNA gene respectively. Amplified restriction fragment length polymorphism (AFLP) is a PCR-based method widely used in population studies. Without prior knowledge of DNA sequences of the species under study, it is possible to obtain hundreds of genetic markers (Vos et al. 1995). AFLP gives more reproducible results than RAPD, but the limiting point is the necessity to use non- or slightly degraded DNA. Nevertheless, recently a database with AFLP patterns of 32 species of fish, molluscs and crustaceans (frozen and fresh products) has been generated (Maldini et al. 2006). Validation is a sub-project within the large European project SEAFOODplus with following objectives: z z

To validate the traceability systems developed and implemented in different fish production chains across Europe. To validate the traceability data coming from the chains testing different tools, such as PCR-based DNA analysis.

The database contains sequences of more than 50 fish species for several mitochondrial genes.

17.6

References

Ali, B.A., Huang, T.-H., Qin, D.-N. and Wang, X.-M. (2004) A review of random amplified polymorphic DNA (RAPD) markers in fish research. Reviews in Fish Biology and Fisheries 14: 443–453. Aranishi, F. (2005) PCR-RFLP analysis of nuclear nontranscribed spacer for mackerel species identification. Journal of Agricultural and Food Chemistry 53: 508–511. Aranishi, F. (2006) Single fish egg DNA extraction for PCR amplification. Conservation Genetics 7: 153–156. Aranishi, F., Okimoto, T. and Ohkubo, M. (2006) A short-cut DNA extraction from cod caviar. Journal of the Science of Food and Agriculture 86: 425–428. Asensio, L., Gonzalez, I., Fernandez, A., Rodriguez, M.A., Lobo, E., Hernandez, P.E., Garcia, T. and Martin, R (2001) Genetic differentiation between wreck fish (Polyprion americanus) and Nile perch (Lates niloticus) by PCR-RFLP analysis of an alpha actin gene fragment. Archiv für Lebensmittelhygiene 52: 131–134. Asensio, L., Gonzalez, I., Fernandez, A., Rodriguez, M.A., Lobo, E., Hernandez, P.E., Garcia, T. and Martin, R. (2002) Application of random amplified polymorphic DNA (RAPD) analysis for

DNA-based methods

381

identification of grouper (Epinephelus guaza), wreck fish (Polyprion americanus), and Nile perch (Lates niloticus) fillets. Journal of Food Protection 65: 432–435. Asensio, L., Gonzalez, I., Fernandez, A., Cespedes, A., Rodriguez., A, Hernandez, P.E., Garcia, T. and Martin, R. (2001a) Identification of Nile perch (Lates niloticus), grouper (Epinephelus guaza) and wreck fish (Polyprion americanus) fillets by PCR amplification of the 5S rDNA gene. Journal – AOAC International 84: 777–781. Bakke, I. and Johansen S.D. (2005) Molecular phylogenetics of Gadidae and related Gadiformes based on mitochondrial DNA sequences. Marine Biotechnology 7: 61–69. Bartlett, S.E. and Davidson, W.S. (1992) FINS (forensically informative nucleotide sequencing) a procedure for identifying the animal origin of biological specimens. Biotechniques 12: 408–411. Barros, F., Laren, M.V., Salas, A. and Carracedo, A. (1997) Rapid and enhanced detection of mitochondrial DNA variation using single-strand conformation analysis of superposed restriction enzyme fragments from polymerase chain reaction. Electrophoresis 18: 52–54. Battersby, B.J. and Moyes, C.D. (1998) Influence of acclimation temperature on mitochondrial DNA, RNA, and enzymes in skeletal muscle. American Journal of Physiology – Regulatory Integrative and Comparative Physiology 275: R905–R912. Bendezu, I.F., Slater, J.W. and Carney, B.F. (2005) Identification of Mytilus spp. and Pecten maximus in Irish waters by standard PCR of the 18S rDNA gene and multiplex PCR of the 16S rDNA gene. Marine Biotechnology 7: 687–696. Bierne, N., Lehnert, S.A., Bedier, E., Bonhomme, F. and Moore, S.S. (2000) Screening for intron-length polymorphisms in penaeid shrimps using exon-primed intron-crossing (EPIC)-PCR. Molecular Ecology 9: 233–235. Binz, T., Reusch, T.B.H., Wedekind, C. and Milinski, M. (2001) SSCP analysis of Mhc class IIB genes in the threespine stickleback. Journal of Fish Biology 58: 887–890. Birstein, V.J. and DeSalle, R. (1998) Molecular phylogeny of Acipenserinae. Molecular Phylogenetics and Evolution 9: 141–155. Birstein, V.J., Doukakis, P., Sorkin, B. and DeSalle, R. (1998) Population aggregation analysis of three caviar-producing species of sturgeons and implications for the species identification of black caviar. Conservation Biology 12: 766–775. Blair, D., Waycott, M., Byrne, L., Dunshea, G., Smith-Keune, C. and Neil, K.M. (2006) Molecular discrimination of Perna (Mollusca: Bivalvia) species using the polymerase chain reaction and species-specific primers. Marine Biotechnology 8: 380–385. Bossier, P. (1999) Authentication of seafood products by DNA patterns. Journal of Food Science 64: 189–193. Brezezinski, J.L. (2005) Detection of crustacean DNA and species identification using a PCR-restriction fragment length polymorphism method. Journal of Food Protection 68: 1866–1873. Brodmann, P.D., Nicholas, G., Schaltenbrand, P. and Ilg, E.C. (2001) Identifying unknown game species: experience with nucleotide sequencing of the mitochondrial cytochrome b gene and a subsequent basic local alignment search tool search. European Food Research and Technology 212: 491–496. Calo-Mato, P., Sotelo, C.G., Perez-Martin, R.I., Rehbein, H., Hold, G.L., Russell, V.J., Pryde, S., Quinteiro, J., Rey-Mendez, M., Rosa, C. and Santos A.T. (2003) Identification of gadoid fish species using DNA-based techniques. European Food Research and Technology 217: 259–264. Carrera, E., Garcia, T., Cespedes, A., Gonzalez I., Fernandez, A., Asensio L.M., Hernandez, P.E. and Martin, R. (2000) Differentiation of smoked Salmo salar, Oncorhynchus mykiss and Brama raii using the nuclear marker 5S rDNA. International Journal of Food Science & Technology 35: 401–406. Cespedes, A., Garcia, T., Carrera, E., Gonzalez, I., Sanz, B., Hernaz P.E. and Martin, R. (1998) Identification of flatfish species using polymerase chain reaction (PCR) amplification and restriction analysis of the cytochrome be gene. Journal of Food Science 63: 206–209.

382

Fishery Products: Quality, safety and authenticity

Cespedes, A., Garcia, T., Carrera, E., Gonzalez, I., Fernandez, A., Hernandez, P.E., Martin, R. (1999) Identification of sole (Solea solea) and Greeland halibut (Reinhardtius hippoglossoides) by PCR amplification of the 5S rDNA gene. Journal of Agricultural and Food Chemistry 47: 1046–1050. Cespedes, A., Garcia, T., Carrera, E., Gonzalez, I., Fernandez, A., Hernandez, P.E. and Martin, R. (1999a) Application of polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) to identification of flatfish species. Journal – AOAC International 82: 903–907. Chakraborty, A., Sakai, M. and Iwatsuki, Y. (2006) Museum fish specimens and molecular taxonomy: a comparative study on DNA extraction protocols and preservation techniques. Journal of Applied Ichthyology 22: 160–166. Chan, R.W.K., Dixon, P.I., Peperell, J.G. and Reid, D.D. (2003) Application of DNA-based techniques for the identification of whaler sharks (Carcharhinus spp.) caught in protective beach meshing and by recreational fisheries off the coast of New South Wales. Fisheries Bulletin 101: 910–914. Chapela, M.J., Sotelo, C.G., Calo-Mata, P., Perez-Martin R.I., Rehbein, H., Hold, G.L., Quinteiro, J., Rey-Mendez, M., Rosa, C. and Santos, A.T. (2002) Identification of cephalopod species (Ommastrephidae Loliginidae) in seafood products by forensically informative nucleotide sequencing (FINS). Journal of Food Science 67: 1672–1676. Chapela, M.J., Sotelo, C.G. and Perez-Martin, R.I. (2003) Molecular identification of cephalopod species by FINS and PCR-RFLP of a cytochrome b gene fragment. European Food Research and Technology 217: 524–529. Chapman, D.D., Abercrombie, D.L., Douady, C.J., Pikitch, E.K., Stanhope, M.J. and Shivji, M.S. (2003) A streamlined, bi-organelle multiplex PCR approach to species identification: application to global conservation and trade monitoring of great white shark, Carcharodon carchias. Conservation Genetics 4: 415–425. Chistiakov, D.A., Hellemans, B., Volckaert, F.A.M. (2006) Microsatellites and their genome distribution, evolution, functions and applications: a review with special reference to fish genetics. Aquaculture 255: 1–29. Chow, S. and Nakadate, M. (2004) PCR primers for fish G6PD gene intron and characterization of intron-length variation in the albacore Thunnus alalunga. Molecular Ecology Notes 4: 391–393. Chow, S., Clarke M.E. and Walsh, P.J. (1993) PCR-RFLP analysis on thirteen western Atlantic snappers (subfamily Lutjaninae) a simple method for species and stock identification. Fishery Bulletin 91: 619–627. Chow, S., Nakagawa, T., Suzuki, N., Takeyama, H. and Matsunaga, T. (2006) Phylogenetic relationships among Thunnus species inferred from rDNA ITS1 sequence. Journal of Fish Biology (Supplement A) 68: 24–35. Civera, T. (2003) Species identification and safety of fish products. Veterinary Research Communications 27 (Supplement 1): 481–489. Codex Alimentarius Commission (2007) Report of the twenty-eighth session of the Codex Committee on Fish and Fishery Products. Discussion Paper on the Procedure for the Inclusion of Additional Species in Codex Standards on Fish and Fishery Products. www.codexalimentarius.net. Colombo, F., Cerioli, M., Colombo, M.M., Marchisio, E., Malandra, R. and Renon, P. (2002) A simple polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for the differentiation of cephalopod mollusc families Loliginidae from Ommastrephidae, to avoid substitutions in fishery field. Food Control 13: 185–190. Colombo, F., Trezzi, I., Bernardi, C., Cantoni, C. and Renon, P. (2004) A case of identification of pectinid scallop (Pecten jacobaeus, Pecten maximus) in a frozen and seasoned food product with PCR technique. Food Control 15: 527–529. Colombo, F., Mangiagalli, G. and Renon P. (2005) Identification of tuna species by computer-assisted and cluster analysis of PCR-SSCP electrophoretic patterns. Food Control 16: 51–53. Comesana, A.S., Abella, P. and Sanjuan, A. (2003) Identification of flatfish by PCR-RFLP. Journal of the Science of Food and Agriculture 83: 752–759.

DNA-based methods

383

Comi, G., Iacumin, L., Rantsiou, K., Cantoni, C. and Cocolin, L. (2005) Molecular methods for the differentiation of species used in production of cod-fish can detect commercial frauds. Food Control 16: 37–42. Cooper, G.M. and Hausman, R.E. (2004) The Cell-A molecular Approach, 3rd edition. ASM Press, Washington, DC, pp. 339. Cross, I., Rebordinos, L. and Diaz, E. (2005) Species identification of Crassostrea and Ostrea by polymerase chain reaction amplification of the 5S rRNA gene. Journal – AOAC International 89: 144–148. Dalmasso, A., Fontanella, E., Piatti, P., Civera, T., Rosati, S. and Bottero, M.T. (2004) A multiplex PCR assay for the identification of animal species in feedstuffs. Molecular and Cellular Probes 18: 81–87. Di Pinto, A., Forte, V.T., Guastadisegni M.C., Martino, C., Schena, F.P. and Tantillo, G. (2007) A comparison of DNA extraction methods for food analysis. Food Control 18: 76–80. Dooley, J.J., Sage, H.D., Clarke, M.-A.L., Brown, H.M. and Garrett, S.D. (2005) Fish-species identification using PCR-RFLP analysis and lab-on-a-chip capillary electrophoresis: application to detect white fish species in food products and an interlaboratory study. Journal of Agricultural and Food Chemistry 53: 3348–3357. Downs, T.R. and Wilfinger, W.W. (1983) Fluorometric quantification of DNA in cells and tissues. Analytical Biochemistry 131: 538–547. Gorokhova, E. and Kyle, M. (2002) Analysis of nucleic acids in Daphnia: development of methods and ontogenetic variations in RNA-DNA content. Journal of Plankton Research 24: 511–522. Gregory, T.R. (2008) Animal Genome Size Database. http://www.genomesize.com. Greig, T.W., Moore, M.K., Woodley, C.M. and Quattro, J.M. (2005) Mitochondrial gene sequences useful for species identification of western North Atlantic sharks. Fishery Bulletin 103: 516–523. Hayashi, K., Kukita, Y., Inazuka, M. and Tahira, T. (1998) Single-strand conformation polymorphism analysis. In: R.G.H. Cotton, E. Edkins and S. Forrest (Eds) Mutation Detection – A Practical Approach. IRL Press, Oxford, pp. 7–24. Heath, D.D., Rawson, P.D. and Hilbish, T.J. (1995) PCR-based nuclear markers identify alien blue mussel (Mytilus spp.) genotypes on the west coast of Canada. Canadian Journal of Fisheries and Aquatic Sciences 52: 2621–2627. Heist, E.J. and Gold J.R. (1999) Genetic identification of sharks in the U.S. Atlantic large coastal shark fishery. Fishery Bulletin 97: 53–61. Hird, H.J., Hold, G.L., Chisholm, J., Reece, P., Russell, V.J., Brown, J., Goodier, R. and MacArthur, R. (2005) Development of a method for the quantification of haddock (Melanogrammus aeglefinuis) in commercial products using real-time PCR. European Food Research & Technology 220: 633–637. Hoelzel, A.R. (Ed) (1992) Molecular Genetic Analysis of Populations – A Practical Approach. IRL Press, Oxford. Hoelzel, A.R. (2001) Shark fishing in fin soup. Conservation Genetics 2: 69–72. Hold, G.L., Russel, V.J., Pryde, S.E., Rehbein, H., Quinteiro, J., Vidal, R., Rey-Mendez, M., Sotelo, C.G., Perez-Martin, R.I., Santos, A.T. and Rosa, C. (2001) Development of a DNA-based method aimed at identifying the fish species present in food products. Journal of Agricultural and Food Chemistry 49: 1175–1179. Hold, G.L., Russel, V.J., Pryde, S.E., Rehbein, H., Quinteiro, J., Rey-Mendez, M., Sotelo, C.G., PerezMartin, R.I., Santos, A.T., Rosa, C. (2001a) Validation of a PCR-RFLP based method for the identification of salmon species in food products. European Food Research and Technology 212: 385–389. Horstkotte, B. and Rehbein, H. (2003) Fish species identification by means of restriction fragment length polymorphism and high-performance liquid chromatography. Journal of Food Science 68: 2658–2666.

384

Fishery Products: Quality, safety and authenticity

Horstkotte, B. and Rehbein, H. (2006) Determination of DNA content of whole fish. Fisheries Science 72: 429–436. Hubert, N., Duponchelle, F., Nunez, J., Rivera, R. and Renno, J.-F. (2006) Evidence of reproductive isolation among closely related sympatric species of Serrasalmus (Ostariophsii, Characidae) from the upper Madeira river, Amazon, Bolivia. Journal of Fish Biology (Supplement A), 69: 31–51. Infante C., Catanese, G. and Manchado, M. (2004a) Phylogenetic relationships among ten sole species (Soleidae, Pleuronectiformes) from the Gulf of Cadiz (Spain) based on mitochondrial DNA sequences. Marine Biotechnology 6: 612–624. Infante, C., Catanese, G., Ponce, M. and Manchado, M. (2004) Novel method for the authentication of frigate tunas (Auxis thazard and Auxis rochei) in commercial canned products. Journal of Agricultural and Food Chemistry 52: 7435–7443. Infante C. and Manchado, M. (2006) Muliplex-polymerase chain reaction assay for the authentication of the mackerel Scomber colias in commercial canned products. Journal – AOAC International 89: 708–711. Itoi, S., Nakaya, M., Kaneko, G., Kondo, H., Sezaki, L. and Watabe, S. (2005) Rapid identification of eels Anguilla japonica and Anguilla anguilla by polymerase chain reaction with single-nucleotide polymorphism-based specific probes. Fisheries Science 71: 1356–1364. Khamnamtong, B., Klinbunga, S. and Menasveta, P. (2005) Species identification of five penaeid shrimps using PCR-RFLP and SSCP analyses of 16 S ribosomal DNA. Journal of Biochemistry and Molecular Biology 38: 491–499. Karaiskou, N., Triantafyllidis, A. and Triantafyllidis, C. (2003) Discrimination of three Trachurus species using both mitochondrial- and nuclear-based DNA approaches. Journal of Agricultural and Food Chemistry 51: 4935–4940. Lettieri, T. (2006) Recent applications of DNA microarray technology to toxicology and ecotoxicology. Environmental Health Perspectives 114: 4–9. Livi, S., Cordisco, C., Damiani, C., Romanelli, M. and Crosetti, D. (2006) Identification of bivalve species at an early developmental stage through PCR-SSCP and sequence analysis of partiaö 18S rDNA. Marine Biology 149: 1149–1161. Lockley, A.K. and Barsdley, R.G. (2000) Novel method for the discrimination of tuna (Thunnus thynnus) and bonito (Sarda sarda). Journal of Agricultural and Food Chemistry 48: 4463–4468. Lopez, I. and Pardo, M.A. (2005) Application of relative quantification TaqMan real-time polymerase chain reaction technology for the identification and quantification of Thunnus alalunga and Thunnus albacares. Journal of Agricultural and Food Chemistry 53: 4554–4560. Lopez-Pinon, M.J., Insua, A. and Mendez, J. (2002) Identification of four scallop species using PCR and restriction analysis of the ribosomal DNA internal transcribed spacer region. Marine Biotechnology 4: 495–502. Ludwig, A., Debus, L. and Jenneckens, I. (2002) A molecular approach to control the international trade in black caviar. International Review of Hydrobiology 87: 881–674. Mackie, I.M., Pryde, S.E., Gonzales-Sotelo, C., Medina, I., Perez-Martin, R., Quinteiro, J., Rey-Mendez, M. and Rehbein, H. (1999) Challenges in the identification of species of canned fish. Food Science and Technology 10: 9–14. Maes, G.E., Pujolar, J.M., Raeymaekers, J.A.M., Dannewitz, J. and Volckaert, F.A.M. (2006) Microsatellite conservation and Bayesian individual assignment in four Anguilla species. Marine Ecology Progress Series 319: 251–261. Maldini, M., Marzano, F.N., Gonzalez Fortes, G., Papa, R. and Gandolfi, G. (2006) Fish and seafood traceability based on AFLP markers: elaboration of a species database. Aquaculture 261: 487–494. Manchado, M., Rebordinos, L. and Infante, C. (2006) U1 and U2 small nuclear RNA genetic linkage: a novel molecular tool for identification of six sole species (Soleidae, Pleuronectiformes). Journal of Agricultural and Food Chemistry 54: 3765–3767.

DNA-based methods

385

Marko, P.B., Lee, S.C., Rice, A.M., Gramling, J.M., Fitzhenry, T.M., McAlister, J.S., Harper, G.R. and Moran, AL (2004) Mislabelling of a depleted reed fish. Nature 430: 309–310. Martinez, I., Jakobsen Friis, T. and Seppola, M. (2001) Requirements for the application of protein sodium dodecyl sulfate-polyacrylamide gel electrophoresis and randomly amplified polymorphic DNA analyses to product speciation. Electrophoresis 22: 1526–1533. Martinez, I., James, D. and Loreal, H. (2005) Application of modern analytical techniques to ensure seafood safety and authenticity. FAO Fisheries Technical Paper 455. FAO, Rome. Meyer, A. (1993) Evolution of mitochondrial DNA in fishes. In: P.W. Hochachka and T.P. Mommsen (Eds) Biochemistry and Molecular Biology of Fishes, volume 2. Elsevier, pp. 1–38. Pank, M., Stanhope, M., Natanson, L., Kohler, N. and Shivji, M. (2001) Rapid and simultaneous identification of body parts from the morphologically similar sharks Carcharhiuns obscurus and Carcharhiuns plumbeus (Carcharhinodae) using multiplex PCR. Marine Biotechnology 3: 231–240. Pardo, M.A. and Perez-Villareal, B. (2004) Identification of commercial canned tuna species by restriction site analysis of mitochondrial DANN products obtained by nested primer PCR. Food Chemistry 86: 143–150. Parson, W., Pegoraro, K., Niederstätter, H., Föger, M. and Steinlechner, M. (2000) Species identification by means of the cytochrome b gene. International Journal of Legal Medicine 114: 23– 28. Partis, L. and Wells, R.J. (1996) Identification of fish species using random amplified polymorphic DNA (RAPD). Molecular Cellular Probes 10: 435–441. Pendas, A.M., Moran, P., Martinez, J.L. and Garcia-Vazquez, E. (1995) Applications of 5S rDNA in Atlantic salmon, brown trout, and in Atlantic salmon × brown trout hybrid. Molecular Ecology 4: 275–276. Perez, J. and Garcia-Vazquez E. (2004) Genetic identification of nine hake species for detection of commercial fraud. Journal of Food Protection 67: 2792–2796. Perez, M., Vieites, J.M. and Presa, P. (2005) ITS1-rDNA-based methodology to identify worldwide hake species of the genus Merluccius. Journal of Agricultural and Food Chemistry 53: 5239–5247. Phongdara, A., Chotigeat, W., Chandumpai, A., Tanthana, C. and Duangtong, P. (1999) Identification of Penaeus merguiensis and Penaeus indicus by RAPD-PCR derived DNA markers. Science Asia 25: 143–151. Pikaart, M.J. and Villeponteau, B. (1993) Suppression of PCR amplification by high levels of RNA. Biotechniques 14: 24–25. Quinteiro, J., Sotelo, C.G., Rehbein, H., Pryde S.E., Medina, I., Perez-Martin, R.I., Rey-Mendez, M. and Mackie, I.M. (1998) Use of mtDNA direct polymerase chain reaction (PCR) sequencing and PCR-restriction fragment length polymorphism methodologies in species identification of canned tuna. Journal of Agricultural and Food Chemistry 46: 1662–1669. Quinteiro, J., Vidal, R., Izquierdo, M., Sotelo, C.G., Chapela, M.J., Perez-Martin R.I., Rehbein, H., Hold, G.L., Russell, V.J., Pryde, S.E., Rosa, C., Santos, A.T. and Rey-Mendez, M. (2001) Identification of hake species (Merluccius genus) using sequencing and PCR-RFLP analysis of mitochondrial DNA control region sequences. Journal of Agricultural and Food Chemistry 49: 5108–5114. Rehbein, H. (2002) Identification of fish species processed to fish meal. Journal of Aquatic Food Product Technology 11: 45–46. Rehbein, H. (2003) Identification of fish species by protein- and DNA-analysis. In: Perez-R.I. Martin and C.G. Sotelo (Eds) Authenticity of Species in Meat and Seafood Products. Instituto de Investigaciones Marinas, Vigo, Spain, pp. 81–101. Rehbein, H. (2005) Identification of the fish species of raw or cold-smoked salmon and salmon caviar by single-strand conformation polymorphism (SSCP) analysis. European Food Research and Technology 220: 625–632.

386

Fishery Products: Quality, safety and authenticity

Rehbein, H. and Horstkotte B. (2003) Determination of the composition of multi-species fishery products by PCR-based techniques. In: Proceedings of the TAFT 2003 Conference, The Icelandic Fisheries Laboratories, Reykjavik 2003, pp. 190–192. Rehbein, H., Mackie, I.M., Pryde, S., Gonzales-Sotelo, C., Perez-Martin, R., Quinteiro, J. and Rey-Mendez, M. (1998) Comparison of different methods to produce single-strand DNA for identification of canned tuna by single-strand conformation polymorphism analysis. Electrophoresis 19: 1381–1384. Rehbein, H., Gonzales-Sotelo, C., Perez-Martin, R., Quiteiro, J., Rey-Mendez, M., Pryde, S., Mackie, I.M. and Santos, A.T. (1999a) Differentiation of sturgeon caviar by single strand conformation polymorphism (PCR-SSCP) analysis. Archiv für Lebensmittelhygiene 50: 13–17. Rehbein, H., Mackie, I.M., Pryde, S., Gonzales-Sotelo, C., Medina, I., Perez-Martin, R., Quinteiro, J. and Rey-Mendez, M. (1999b) Fish species identification in canned tuna by PCR-SSCP: validation by a collaborative study and investigation of intra-species variability of the DNA patterns. Food Chemistry 64: 263–268. Russell, V.J., Hold, G.L., Pryde, S.E.; Rehbein, H., Quinteiro, J., Rey-Mendez, M., Sotelo, C.G., Perez-Martin R.I., Santos, A.T. and Rosa, C. (2000) Use of restriction fragment length polymorphism to distinguish between salmon species. Journal of Agricultural and Food Chemistry 48: 2184–2188. Sanjuan, A. and Comesana A.S. (2002) Molecular identification of nine commercial flatfish species by polymerase chain reaction-restriction fragment length polymorphism analysis of a segment of the cytochrome b region. Journal of Food Protection 6: 1016–1023. Santaclara, F.J., Espineira, M., Cabado A.G., Aldasoro, A., Gonzalez-Lavin, N. and Vieites, J.M. (2006) Development of a method for the genetic identification of mussel species belonging to Mytilus, Perna, Aulacomya, and other genera. Journal of Agricultural and Food Chemistry 54: 8461–8470. Sato, S., Moriya, S., Azumaya, T., Suzuki, O., Urawa, S., Abe, S. and Urano, A. (2004) Genetic stock identification of chum salmon in the central Behring Sea and adjacent North Pacific Ocean by DNA microarray during the early falls of 2002 and 2003. (NPAFC Doc. 793). National Salmon Resources Centre, Sapporo, Japan, pp. 21. Sotelo, C.G. and Perez-Martin R.I. (2003) Species identification in processed seafood. In: M. Lees (Ed.) Food Authenticity and Traceability. Woodhead Publishing Ltd., Cambridge, UK, pp. 323–346. Sotelo, C.G., Calo-Mata, P., Chapela, M.J., Perez-Martin R.I., Rehbein, H., Hold, G.L., Russell, V.J., Pryde, S., Quinteiro, J., Izquierdeo, M., Rey-Mendez, M., Rosa, C. and Santos, A.T. (2001) Identification of flatfish (Pleuronectiformes) species using DNA-based techniques. Journal of Agricultural and Food Chemistry 49: 4562–4569. Sunnucks, P., Wilson, A.C.C., Beheregaray, L.B., Zenger, K., French, J. and Taylor, A.C. (2000) SSCP is not so difficult: the application and utility of single-stranded conformation polymorphism in evolutionary biology and molecular ecology. Molecular Ecology 9: 1699–1710. Taylor, G.R. and Day, I.N.M. (Eds) (2005) Guide to Mutation Detection. Wiley, Hoboken, New Jersey. Taylor, M.I., Fox, C. and Rico, C. (2002) Species-specific TaqMan probes for simultaneous identification of (Gadus morhua L.), haddock (Melanogrammus aeglefinus L.) and whiting (Merlangius merlangus L.). Molecular Ecology Notes 2: 599–601. Teletchea, F., Laudet, V. and Hänni, C. (2006) Phylogeny of the Gadidae (sensu Svetovidov, 1948) based on their morphology and two mitochondrial genes. Molecular Phylogenetics and Evolution 38: 189–199. Teletchea, F., Maudet, C. and Hänni, C. (2005) Food and forensic molecular identification; update and challenges. Trends in Biotechnology 23: 359–366.

DNA-based methods

387

Trautner, J. (2006) Rapid identification of European (Anguilla anguilla) and North American eel (Anguilla rostrata) by polymerase chain reaction. Informationen aus der Fischereiforschung 53: 49–51. Trotta, M., Schönhuth, S., Pepe, T., Cortesi, M.L., Puyet, A. and Bautista, J.M. (2005) Multiplex PCR method for use in real-time PCR for identification of fish fillets from grouper (Epinephelus and Mycteroperca species) and common substitute species. Journal of Agricultural and Food Chemistry 53: 2039–2045. Urbanyi, B., Horvath, A. and Kocas, B. (2004) Successful hybridization of Acipenser species using cryopreserved sperm. Aquaculture International 12: 47–56. Vicari, N., Arvangeli, G., Tisato, E., Gambarin, P. and Spagna, G. (2001) Identification of some squaliform species for human consumption by PCR-RFLP technique. Industria Conserve 76: 33–43. Vos, P., Hogers, R., Bleeker, M., Reijans, M., van de Lee, T., Hornes, M., Frijters, A., Pot, J., Peleman, J., Kuiper, M. et al. (1995) AFLP: a new technique for DNA fingerprinting. Nucleic Acids Research 23: 4407–4414. Ward, R.D., Zemlak, T.S., Innes, B.H., Last, P.R. and Hebert, P.D.N. (2005) DNA barcoding Australia’s fish species. Philosophical Transactions of the Royal Society B 360: 1847–1857. Wolf, C., Burgener, M., Hübner, P. and Lüthy, J. (2000) PCR-RFLP analysis of mitochondrial DNA: differentiation of fish species. Lebensmittel-Wissenschaft und -Technologie 33: 144–150. Wolf, C., Hübner, P. and Lüthy, J. (1999) Differentiation of sturgeon species by PCR-RFLP. Food Research International 32: 699–705. Wyatt, P.M.W., Pitts, C.S. and Butlin R.K. (2006) A molecular approach to detect hybridization between bream Abramis brama, roach Rutilus rutilus and rudd Scardinius erythrophthalmus. Journal of Fish Biology 69 (Suppl. A), 52–71. Zhang, J. and Cai, Z. (2006) Differentiation of the rainbow trout (Oncorhynchus mykiss) from Atlantic salmon (Salmo salar) by AFLP derived SCAR. European Food Research and Technology 223: 413–417. Zhang, J., Huang, H., Cai, Z. and Huang, L. (2006) Species identification in salted products of red snappers by semi-nested PCR-RFLP based on the mitochondrial 12S rRNA gene sequence. Food Control 17: 557–563. Zhang, S., Zhang, Y., Zheng, X., Chen, Y., Deng, H., Wang, D., Wei, Q., Zhang, Y., Nie, L. and Wu, Q. (2000) Molecular phylogenetic systematics of twelve species of Acipenseriformes based on mtDNA ND4L-ND4 gene sequence analysis. Science in China C 43: 129–137.

Chapter 18

Other principles: analysis of lipids, stable isotopes and trace elements Iciar Martinez

18.1

Introduction

Dennis (1998) refers to food authentication as ‘the process by which a food is verified as complying with its label description’. Information on seafood compulsory in the European Union (EU) includes specification of the commercial designation and scientific name, method of production (‘caught’, ‘caught in freshwater’, ‘farmed’ or ‘cultivated’) and the area in which it was caught. For cultivated species, a reference should be made to the country in which the product undergoes the final developmental stage (EU Commission regulation No 2065/2001 of 22 October 2001 laying down detailed rules for the application of Council Regulation EC No 104/2000 as regards informing consumers about fishery and aquaculture products). Thus, issues that are relevant to authentication are the species, production method and geographic origin, in addition to new production methods for highly priced or exclusive products such as ‘organically produced’. Japan has legislation similar to Europe. The Law on Standardization and Proper Labeling of Agricultural and Forestry products (also known as Japanese Agricultural Standards – JAS – Law) of 1999 demands that the name and place of origin be indicated in fresh foods and some processed foods. This law was modified in October 2006 to make compulsory the inclusion on the label of information about the place of origin. In addition, and, for several processed foods such as boiled, dried or seasoned fish and surface roasted fillets, the main ingredients have to be listed. The USA applies the Federal Food, Drug, and Cosmetic Act and The Fair Packaging and Labeling Act Title 15 – Commerce and Trade, Chapter 39 – Fair Packaging and Labeling Program §145. This makes mandatory a statement of the identity of the commodity that shall be in terms of the name specified in, or required by, any applicable Federal law or regulation, or by using the common or usual name of the food. For the correct labeling of seafood, the US Food and Drug Administration (FDA) has elaborated, in cooperation with the National Marine Fisheries Service (NMFS), ‘The Seafood List’, which is a compilation of existing acceptable market names for imported and domestically available seafood. The list is an extensive, although not necessarily complete, listing of seafood commonly sold in the United States, which can be found at http://www.cfsan.fda.gov/~frf/seaintro.html. There does not seem to be a need to label products with the geographic origin or production method in the USA. This chapter will address the issues of authentication of the species, geographic origin and production 388

Other principles: analysis of lipids, stable isotopes and trace elements (a)

O

389

*

H2C

O

x O *

O

z

C

H O

H2C O

* y

(b) R

R

O C

CH2

O O C

C

H

H2 C

P–

H2C O

CH3

O

O

O O

N+ C H2

CH3 CH3

Figure 18.1 (a) Triglyceride general structure. From http://en.wikipedia.org/wiki/Image: TriglycerideGeneralStructure.png. The copyright holder grants any entity the right to use this work for any purpose, without any conditions, unless such conditions are required by law. (b) Structural formula of the phospholipid phosphatidyl choline. From http://en.wikipedia.org/wiki/Image: Phosphatidyl-Choline. png. Both (a) and (b) have been released into the public domain by the copyright holder. This applies worldwide.

methods by targeting lipid composition, stable isotope distributions and trace element signatures.

18.2

Species and breeding stock identification by lipid analysis

Triacylglycerols are neutral lipids in which the glycerol is esterified with three fatty acids (Figure 18.1A). They are the main constituent of vegetable oil and animal fats, and constitute the main energy reserves in fish (Cowey and Sargent 1977; loc. cit.; Dalsgaard et al. 2003). Although the fatty-acid composition of triglycerides closely resembles that of the fish diet (Dalsgaard et al. 2003) and has been used to identify the production method (that is, farmed versus wild, discussed below), the positional distribution of the fatty acids in the carbons of the glycerol (α for the first and third carbons or β for the second carbon, in the middle position) has been used to identify species and adulterations in oils and oil mixtures. High-resolution 13C nuclear magnetic resonance (NMR) spectroscopy, described in detail in Chapter 11, provides information about the different types of carbon atom (their chemical shift value) and their relative amounts in the sample (as the relative intensities of the peaks). The technique has been applied to oils to elucidate their composition (triacylglycerol, oils, waxes, and so on), the fatty acids contained in the sample (which fatty acids and their relative amounts) and the position of the fatty acids in the triglyceride and phospholipid molecules (Aursand and Grasdalen 1992; Hidalgo and Zamora 2003, Siddiqui et al. 2003).

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Fishery Products: Quality, safety and authenticity

170

160

150

140

130

120

110

100

90

80

70

60

50

40

30

20

ppm

Figure 18.2 High-resolution 13C NMR of mackerel, Scomber scombrus (top spectrum), and herring Clupea harengus (lower spectrum), oils. The peaks in the region 15–40 ppm are due to carbons in aliphatic chains (methylene region); the region 60–80 ppm to the carbons in the glycerol; 120–140 ppm to the olefinic region; and the peaks at 170–180 ppm are due to the carbonyl carbons in the oil. The figure was kindly provided by Inger Beate Standal.

Aursand and Grasdalen (1992) and Aursand et al. (2007) published the interpretation of the 13 C-NMR spectra of lipids extracted from Atlantic salmon, cod, trout, and several fish oil capsules; Siddiqui et al. (2003) published the spectra of encapsulated marine oil supplements. Figure 18.2 shows an example of a 13C NMR spectra of mackerel and herring oils. By using 13C NMR to examine the fatty-acid positional distribution in triacylglycerols, it has been possible to identify the composition of oils and the presence of contaminants (Aursand et al. 2007; Pfeffer et al. 1977; Ng 1985, Hidalgo and Zamora 2003). In plant oils, Zamora et al. (2001) were able to detect a 5% hazelnut oil contamination in virgin olive oil; Siddiqui et al. (2003) were able to distinguish natural fish oils from oils subjected to industrial refining as well as the addition of synthetic ethyl esters of docosahexaenoic acid (DHA, C22:6n–3) and eicosapentaenoic acid (EPA; C205n–3) to encapsulated oil supplements. Aursand et al. (2007) analyzed fish oils from different species (Atlantic salmon, cod and trout), different geographic origins of cod (Iceland, Barents Sea, Norway) and different degrees of processing, as well as several fish-oil health products, by 13C NMR and several multivariate data analyses techniques. They were able to classify correctly over 95% of the samples. In this work, the carbonyl region of the spectrum was shown to give information on whether natural oil had been chemically modified or oil from another species had been added (Aursand et al. 2007). Aursand et al. (1995a) identified the species of farmed Atlantic salmon, cod liver and seal oils by using differences in the positional distribution of n–3 fatty acids on the glycerol backbone of triacylglycerols by 13C NMR: 73–74% of the C22:6n–3 was found to be preferentially esterified at the β position of the triacylglycerols in depot fat of farmed Atlantic salmon and cod liver oil respectively. However, only about 38–40% of the C20:5n–3 was

Other principles: analysis of lipids, stable isotopes and trace elements

391

located in the β position in lipids of cod liver oil and farmed Atlantic salmon respectively, giving an almost random distribution of this fatty acid in triacylglycerols compared with C22:6n–3. C22:5n–3 was preferentially esterified (69%) at the β position of the triacylglycerols in muscle lipids of farmed Atlantic salmon. However, the amount of this fatty acid in cod liver oil was too low to allow analysis of its positional distribution. Those findings confirmed previous works by Brockerhoff et al. (1968) and Litchfield (1969) which demonstrated the general tendency of C20:5n–3, C22:6n–3 and C22:5n–3 to be preferentially esterified at the β position of fish and invertebrate triacylglycerols. Ando et al. (1992) showed that the positional distribution of C22:6n3 and C22:5n3 is related to the amount of C22:1 and C20:1 fatty acids in the triacylglycerols: in fish lipids with high contents of C20:1 and C22:1, nearly 70–80% of the C22:6n3 was in the β position of the glycerol moiety. The distribution in marine mammals was quite different: in harp seal oil, nearly 100, 97 and 95% of C22:5n3, C22:6n3 and C20:5n3, respectively, were esterified to the α (1 and 3) positions of the glycerol moiety, in accordance with previously obtained positional data for triacylglycerols of harp seal blubber (Brockerhoff et al. 1968; Litchfield 1969). The positional distribution of the fatty acids in triglycerides can be used as a speciesspecific marker in zooplankton as well. In the rotifer Brachionus plicatilis and in Artemia franciscana nauplii, whose feed had been enriched with fish oils, once the fatty acids were released from the dietary oils they were incorporated into both animals in a consistent manner, independent of their origin: although C22:6n–3 was preferentially esterified in the β position in the fish oils, it ended up in the α position in both Artemia and B. plicatilis (Ando et al. 2004a, b). That was not, however, the case in bovine adipose tissues (Smith et al. 1998). Long-term feeding of cattle with fatty acids of variable degrees of unsaturation induced significant alterations in the fatty-acid composition, consisting of changes both in the distribution and in the composition of the triacylglycerol species, which, in turn, accounted for marked differences in melting points among treatment groups. Phospholipids are made up of four components: fatty acids, a negatively charged phosphate group, a nitrogen-containing alcohol and a glycerol backbone (Figure 18.1b). With glycolipids and cholesterol, phospholipids are a major component of all biological membranes. Some work has shown the value of analyzing the fatty-acid type and their position in phospholipids for species identification. Medina et al. (1997) used multivariate data analysis on the phospholipid fraction for species identification in tuna products, comprising specimens of three different species, caught during different seasons, which had been either commercially processed or overprocessed. The three species were correctly classified by using a forward discriminant function analysis on 14 variables, including the phospholipid content, the proportions of each phospholipid class, and the content of the total phospholipid fatty acids (Figure 18.3). C16:0 and C18:1n–9 were the major saturated and mono-unsaturated fatty acids, respectively, in the three species, with a preponderance of C16:0 over C18:0, and differences in minor fatty acids, such as C17:0 and C20:1n–9. The major differences between species were on the amounts of polyunsaturated fatty acids, with important variations in the magnitudes of C20:4n–6, C20:5n–3, and C22:6n–3. The most relevant variables for the classification were the C20:4n–6 and C18:1n–7 contents for the first and second function respectively: all the samples were correctly classified according to the species.

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Fishery Products: Quality, safety and authenticity 10

Second discriminant function

6

2

–2

–6

–10 –10

–6

–2

2

6

10

14

18

First discriminant function Figure 18.3 Two discriminant function scores for canned tuna species: 䊏, albacore; 䊉, bonito; 䉱, big eye tuna. Reprinted with permission from Medina, I., Aubourg, S.P. and Martin, R.P. (1997). Species differentiation by multivariate analysis of phospholipids from canned Atlantic tuna. Reproduced with permission from the Journal of Agricultural and Food Chemistry 45: 2495–2499. Copyright (1997) American Chemical Society.

Fatty-acid profiling of several tissues has proved useful for differentiating among several species of Sebastes for which protein (Rehbein 1983; Nedreaas and Nævdal 1991a, b; Nedreaas et al. 1994;) and DNA analyses (Danielsdottir 1998; Sundt and Johansen 1998; Roques et al. 1999a, b) did not render clear results. Joensen and Grahl-Nielsen (2000, 2001) performed principal component analysis (PCA) and soft independent modeling of class analogies (SIMCA) on the fatty-acid content obtained by gas chromatography of oils extracted from the heart, gills, skull and otoliths of Sebastes viviparous, S. marinus and S. mentella (Figure 18.4). They found that the fatty-acid profiles appeared to be species-specific despite considerable individual variability and large differences in the tissues. Interestingly, the method could be used to identify species even when the specimens came from different locations, namely Norwegian or Faroese waters (Joensen and Grahl-Nielsen 2000), even though the fatty acids that contributed to the distinction among the species varied according to the geographic origin of the fish. This suggested that the composition of fatty acids may be population-dependent in some species. Therefore, as for genetic analysis, this may be useful for identifying the geographic origin when the populations discriminated by the analysis also segregated in space. Indeed, that proved to be the case for the Sebastes species (Joensen and Grahl-Nielsen 2004) and herring (Grahl-Nielsen and Ulvund 1990) in the Atlantic, striped bass stocks in American rivers (Grahl-Nielsen and Mjaavatten 1992) and between two stocks of cod reared under identical conditions on the Faroe Islands (Joensen et al. 2000). Fatty-acid profiling is now a method included in the ICES Stock Identification Methodology (Grahl-Nielsen 1997).

Other principles: analysis of lipids, stable isotopes and trace elements (b)

PC2 (14%)

PC2 (21%)

(a)

393

PCI (60%)

PCI (35%)

PC2 (28%)

(c)

PCI (54%) Figure 18.4 (a) PC-plot of the three redfish species, S. viviparus, S. marinus, and S. mentella, based on the fatty-acid composition in the heart tissues. Each symbol represents one individual fish. The percentage of the total variance along each of the principal components is given. (b) PC-plot of two redfish species, S. marinus and S. mentella, based on the fatty-acid composition in the heart tissues. Each symbol represents one individual fish. The percentage of the total variance along each of the principal components is given. (c) PLS-plot of two redfish species, S. marinus, and S. mentella, based on the fatty-acid composition in the heart tissues. Each symbol represents one individual fish. The percentage of the total variance along each of the principal components is given. Reprinted with permission from Joensen, H. and Grahl-Nielsen, O. (2000) Discrimination of Sebastes viviparus, S. marinus, and S. mentella from Faroe Islands by chemometry of the fatty acid profile in heart and gill tissues and in the skull oil. Comparative Biochemistry and Physiology 126B: 69–79. Copyright (2000), with permission from Elsevier.

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Fishery Products: Quality, safety and authenticity

18.3

Verification of the production method

18.3.1

Verification of the production method by lipid analysis

The main energy requirements of marine fish are supplied by lipids and proteins (Shul’man 1960; loc. cit.; Dalsgaard et al. 2003). As already mentioned, fish store lipids in the neutral triglycerides (Figure 18.1) and many studies have shown that the fatty-acid composition reflects that of the diet in both wild and farmed fish (Hardy et al. 1987; Jobling et al. 2002; Bell et al. 2003a, b; Dalsgaard et al. 2003; Jobling 2004). A review of the verification of the production method of Atlantic salmon has recently been published by Martinez (2006). Owing to the high prices of fish oils and the danger of overexploitation of pelagic species, alternative oils, mostly vegetable but also from zooplankton, are being investigated in the formulations of fish feeds. Dietary inclusion of vegetable oils (50–100% substitutions: rapeseed or olive oil) does not seem to have any major impact on growth rate, feed efficacy or mortality in post-smolt salmon fed experimental diets for 42 weeks (Torstensen et al. 2004a, b). Moreover, these data accord with previous experiments with dietary rapeseed oil replacement (Bell et al. 2001, 2003a, b). However, the fatty-acid composition of tissue lipids is closely influenced by dietary fatty-acid composition in salmonids (Dosanjh et al. 1988, Lie et al. 1988, Torstensen et al. 2000). This is particularly the case for the triacylglycerols, which are the main lipid class found in salmon flesh. Ng et al. (2004) showed that there were no significant effects of the diet on growth, feed utilization efficiency, total lipid content in muscle or pigment concentration. The diets had been supplemented with 50 : 40 : 10 and 50 : 25 : 25 blends of fish oil : rapeseed oil : crude palm oil. It was notable in this work that, despite having high concentrations of saturated fatty acids, mainly C16:0, Atlantic salmon fed crude palm oil diets did not deposit a significant amount of this fatty acid in the muscle tissue, results that were corroborated by additional studies in salmon (Bell et al. 2002) and in tropical fish (Ng et al. 2001, 2003). However, specific fatty acids are selectively retained or utilized. This has been shown by studies demonstrating a selective deposition and retention of C22:6n–3, so that the concentrations in flesh were always higher than in the diet (Bell et al. 2001, 2003a). The Atlantic salmon analyzed by Nichols et al. (2002) had higher levels of C18:1n–9 and C22:6n–3 and less C20:5n–3 than the diets. It seems that C18:1n–9, C22:1n–11, C18:2n–6 and C18:3n–3 were selectively utilized in the flesh when present at high concentrations in the diet, so for these fatty acids, dietary concentrations were higher than in the fillet (Bell et al. 2001). Bell et al. (2003a) suggested that the latter fatty acids oxidize swiftly when present at high concentrations. The fatty acids C18:1n–9 and 18:2n–6 may act as markers for vegetable oils. In particular, the latter seems to be the most persistent (Bell et al. 2003a): after a dietary switch to fish oil and after the levels of C20:5n–3 and C22:6n–3 recuperate the original high levels, the ratio n3:n6 is not restored. Indeed, inclusion of the n3:n6 ratio as a variable on the data published by Aursand et al. (2000) for wild and farmed Atlantic salmon produced a statistical model in which the two production methods were clearly separated (Figure 18.5). Feeding pikeperch, Stizostedion lucioperca, diets formulated with linseed oil induced a decrease in saturated fatty acids and an increase in the C18:1n9 and the C18:2n–6 amounts in the fillet. However, the total polyunsaturated fatty-acid proportion remained constant (Molnar et al. 2006). Similarly, the fatty-acid composition analyses of juvenile Russian

Other principles: analysis of lipids, stable isotopes and trace elements

PC2

395

Scores

10 W-1 F-1 F-1 F-1 F-1 F-1

5

W-1 W-1

F-2 F-2F-1 F-1 F-2 F-2 F-2 F-2 F-1

0

W-1

F-1 F-1

–5

W-1 W-1

F-1 W-1

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–10

PC1

–10 –5 Result1, X-expl: 73%, 17%

0.6

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X-loadings

15

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C22:1 n3/n6 0.3 C20:5n3 C20:1n9 C22:5n-3 C18:1n6 C25:1n9 0 C16:1

C18:0

C14:0 C16:0 C18:1n7

–0.3 –0.8 –0.6 Result1, X-expl: 73%, 17%

PC1 –0.4

–0.2

0

0.2

0.4

0.6

0.8

Figure 18.5 Principal component analysis on the fatty-acid composition of oil extracted from the white muscle of Norwegian wild Atlantic salmon (W-01) and of specimens farmed in Norway (F-01) and Scotland (F-02) estimated by gas chromatography. In addition to the fatty acids, the ratio n3/n6 [(C20:5n3 + C22:5n–3 + C22:6n3)/C18:2n6] was included as a variable. The model was fully cross-validated and each variable was given a weight of 1. The scores plot is shown on the top and the loadings plot on the bottom. The first and second principal components explained 73% and 17% of the total variability, respectively. The original data are from Aursand et al. (2000).

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Fishery Products: Quality, safety and authenticity

sturgeon (Acipenser gueldenstaedtii) fed feeds including fish oil, soybean oil and sunflower oil showed that the total n–3 and n–6 in the whole body and the liver fatty acids varied significantly depending on the oil included in the feed. As expected, in the groups fed vegetable oil, the total amount of n–6 fatty acids was higher and the n–3/n–6 fatty-acid ratio was lower than in the sturgeon fed fish oil, both in muscle and liver fatty-acid composition (Sener et al. 2006). In several works where farmed shrimp species, including Penaeus monodon (Deering et al. 1997; Kumuraguru Vasagam et al. 2005) and Litopenaeus vannamei (Gonzalez-Felix et al. 2002), had been fed diets containing vegetable oils such as sunflower oil, peanut oil, palm oil, coconut oil, soybean oil, linseed oil, canole oil, animal (lard) or fish oils (sardine oil, menhaden oil or cod oil), the fatty-acid composition of the test diets was reflected to a certain extent in the fatty-acid composition of whole shrimp (Deering et al. 1997; GonzalezFelix et al. 2002; Kumuraguru Vasagam et al. 2005). However, as in the case of Atlantic salmon, certain fatty acids appeared to be actively synthesized and/or retained, because they were present in small amounts in some diets, but in relatively higher amounts in tissue of whole shrimp. These fatty acids were C20:4n–6; C20:5n–3 and C22:6n–3 (Kumuraguru Vasagam et al. 2005). Sparing or preferential retention of specific polyunsaturated fatty acids at the expense of saturated and monounsaturated fatty acids had also been demonstrated in previous works with shrimps (Deering et al. 1997; González-Félix et al. 2002). Thus, the fatty-acid profile, owing to the alteration it suffers in farmed fish to mirror that of the diet, can be considered a marker with clear potential for discriminating farmed from wild fish. However, care must be taken in particular to classify fish captured in areas were farming is practised, because it has been shown that feeding around farms affects the fattyacid composition and taste of fish. Skog et al. (2003) investigated claims by local fishermen that saithe captured in the vicinity of fish farms tasted worse than saithe from other areas. Their results showed that saithe collected near farms had a higher condition factor, that is, they were larger, than those from control sites of the same age, that they had been eating pellets and that the fatty-acid composition of their fillet was more similar to the composition of pellets than the composition of fish fillets from reference sites. In a sensory analysis, the test panel found that saithe from the fjord without fish farms tasted better than saithe collected near the cages, thus confirming the claims of the local fishermen (Skog et al. 2003). Fernandez-Jover et al. (2007) performed a similar study after previous observations (Dempster et al. 2002) of horse mackerel (Trachurus mediterraneus) feeding around sea-cages fish farms of sea bass and sea bream in the Mediterranean Sea. This study revealed that while the control fish not associated with fish farms had juvenile fish and small cephalopods in the stomachs, most horse mackerel associated with farms had pellets in their guts. They were also larger than the controls and had a markedly higher fat content and condition factor, although the total protein content did not differ between the two groups. The fatty-acid composition of the dorsal white muscle of horse mackerel associated with farms was similar to that of the feed pellets. The main differentiating fatty acid between the control horse mackerel and those around farms was C22:6n–3 present in higher amounts in the control fish, followed by C18:2n6 and C18:1n9 present in high levels in the commercial feed pellet and in the horse mackerel associated with farms. This work did not register a significant difference between the two groups in the amount of C20:5n–3, which may not be surprising because its level was very low in all groups (about 1.3%). Although in general n–3 and n–6 fatty acids were higher in control and in farm-associated fish respectively,

Other principles: analysis of lipids, stable isotopes and trace elements

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C20:4n–6 was an exception because it was higher in the control fish. The study also revealed large individual variations in the farm-associated horse mackerel, which the authors attributed to differences in the residence times of individuals around the cages, and to different feeding habits during short migrations, whereas the fat levels in control fish were relatively stable. Therefore, one may expect intermediate fatty-acid profiles in wild fish feeding around farms and in escaped fish. Finally, as indicated by Refsgaard et al. (1998) and confirmed by Fernandez-Jover et al. (2007), one must always consider the very wide variation in the concentrations of lipid components that can be found in apparently homogeneous populations of farmed salmon – a statement that can easily be extended to other species – which brings in the necessity of analyzing many fish when an estimate of a population average is wanted. This large variability, and the fact that escaped farmed fish and wild fish eating around farms may display intermediate lipid profiles (Skog et al. 2003; Fernandez-Jover et al. 2007), may contribute to the difficulty of performing correct classifications as wild or farmed based solely on the fatty-acid composition. This claims the need to develop extensive databases with the profiles of known samples.

18.3.2

Use of the carotenoids in flesh to differentiate wild, farmed and organically produced salmon

Bacteria, algae, yeasts, molds and some plants can synthesize carotenoids, a group of natural fat-soluble pigments that have antioxidant properties. Most animals, on the other hand, except for copepods, which have been shown to be able to synthesize astaxanthin (Andersson et al. 2003), cannot synthesize carotenoids de novo, and must obtain these pigments from their diet (Prache et al. 2005). Astaxanthin is essential for the proper growth and survival of salmonids and it must be added to their feed to make up for the lack of a natural dietary source of the pigment in formulated feeds (Torrissen and Christiansen 1995). Natural astaxanthine is the main carotenoid found in the flesh of wild Atlantic (Salmo salar) and Pacific (Oncorhynchus spp.) salmon species. Astaxanthin is produced principally by plants, yeast and microalgae. It occurs in several different forms: stereoisomers, geometric isomers, and free or esterified forms. In its natural state, astaxanthin is usually associated with other molecules, it may be dissolved in the lipid fraction or it may be bound chemically to molecules such as fatty acids to form esters (Bernhard 1990). Free, unbound astaxanthin is less stable and its occurrence is rare. The most thermodynamically stable all-E (all-trans) isomer is also the most common geometric configuration in both synthetic and natural astaxanthin. However, although astaxanthin from natural sources tends to occur predominantly as either the 3S,3′S or 3R,3′R stereoisomers, the meso (3R,3′S) isomer is the most abundant in synthetic astaxanthin, produced as the free (unesterified) xanthophyll and as a 1 : 2 : 1 mixture of the three stereoisomers: 3S,3′S, 3R,3′S and 3R,3′R (Bernhard 1990). The major form currently being used in fish feeds is synthetic astaxanthin (McCoy 1999). The higher abundance of the isomer 3R,3′S fed to farmed fish is reflected in its composition: the proportion of the (3R, 3′S)-isomer is 10 times higher in farmed than in wild salmon, and it is higher in the eggs and alevins of farmed salmon fed synthetic astaxanthin, which also allows this method to be used to determine the spawning success of escaped farmed female

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Fishery Products: Quality, safety and authenticity

Atlantic salmon (Lura and Sægrov 1991). Sægrov et al. (1997) used this criterion to identify whether Atlantic salmon populations were wild native or colonizing from escaped farmed fish in a Norwegian river. Astaxanthin deposition in Atlantic salmon tissues has also been shown to be influenced by the type of dietary oil used in the feed (Bjerkeng et al. 1999). Ostermeyer and Schmidt (2004) analyzed the astaxanthin and cantaxanthin contents in wild, farmed and organically farmed Atlantic salmon (currently being produced in Ireland, Scotland and Norway). These authors found the detection of canthaxantin to be a good indicator for conventionally farmed salmon. They could also distinguish salmon fed with synthetic astaxanthin from organically produced salmon fed the yeast Xanthophyllomyces dendrorhous, and these two from wild fish. However, when the astaxanthin used in the feed was produced from shrimp shells, it was not possible to differentiate conventionally from organically farmed salmon. They also noted that if, in the future, astaxanthin was to be produced from the alga Haematococcus pluvialis, it would no longer be possible to use the analytical method they describe in their work to differentiate farmed from wild salmon.

18.4

Identification of the geographic origin

European legislation establishes that the FAO area (Table 18.1) in which wild fish is caught should be part of the information available to consumers (EC regulation No 2065/2001 of 22 October 2001). The FAO areas are wide regions, and very often marketing is based on a narrower and even local area where the fish comes from, because consumers usually appreciate it more and are willing to pay higher prices for their local products. Similarly, Japan’s Law concerning Standardization and Proper Labelling of Agricultural and Forestry Products (JAS Law, Law No. 175 of 1950) requires for perishable, fresh fish the indication on the label (among other information) of the ‘product name’, ‘place of origin’ and, for marine products, whether it is ‘frozen-thawed’ or ‘cultured’. For processed foods, the label must Table 18.1 Catch area and identification of the area. (Commission Regulation (EC) No 2065/2001 of 22 October 2001 Laying down detailed rules for the application of Council Regulation (EC) No. 104/2000 as regards informing consumers about fishery and aquaculture products. Published in the Official Journal of the European Communities, 23 October 2001, pp. L278/6–L278/8)).

a b

Catch area

Identification of areaa

North-West Atlantic North-East Atlanticb Baltic Sea Central-Western Atlantic Central-Eastern Atlantic South-West Atlantic South-East Atlantic Mediterranean Sea Black Sea Indian Ocean Pacific Ocean Antarctic

FAO FAO FAO FAO FAO FAO FAO FAO FAO FAO FAO FAO

FAO Yearbook. Fishery Statistics. Catches. Volume 86 (1), 2000 Excluding the Baltic Sea

area area area area area area area area area area area area

21 27 27.IIId 31 34 41 47 37.1, 37.2 and 37.3 37.4 51 and 57 61, 67, 71, 77, 81 and 87 48, 58 and 88

Other principles: analysis of lipids, stable isotopes and trace elements

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indicate the ‘product name’, ‘ingredients’ and, in many products, the ‘place of origin’ of the main ingredients. Additional reasons to establish methods to confirm the geographic origin lie in the perceived quality, or toxicity of the fish: some areas are considered to be ‘clean’ and others ‘polluted’, especially for the content of some metals (mercury, cadmium, arsenic, lead), elements (radioactive elements) and/or environmental pollutants (pesticides, PCBs, dioxine-like product, and so on) (Jacobs et al. 2002; Madeniian et al. 2002; Foran et al. 2004; Hites et al. 2004). Thus fish may be marketed according to how pristine and unpolluted the farming region is perceived by the customers, which in turn makes these customers more demanding about information of the geographic origin of the fish. Finally, customers tend to favor their own fish and ‘traditional’ fish, and are willing to pay higher prices for them. It is evident that as long as there are no methods to demonstrate the geographic origin of fish, false labels will be found in markets, offering fish from less attractive areas labeled as their more expensive counterpart.

18.4.1.

Geographic origin determined by the distribution of stable isotopes and trace elements

The determination of the geographic origin of fish was reviewed by Martinez et al. (2003). Techniques aimed at identifying the geographic origin of samples make use of the different distribution of isotopes in different geographic regions. The isotopes may be from the most common elements making up the organic material such as H, C, O, N, S, or isotopes of trace elements that nonetheless are either essential for normal functioning of organisms, such as zinc, selenium, magnesium, manganese, or contaminants picked up from the environment such as mercury, cadmium, lead, and so on. The ratios of stable isotopes are usually given as proportions (0/00) or as excess (delta (δ) values, which are also given as 0/00). These delta values (for example δ15N, δ13C) are the difference between the value of the sample and that of widely used natural standards which are considered to have a δ value of zero. The transformation of absolute (percentage) values into relative (to a certain standard) delta values is used because the absolute differences between samples and standard are quite small at natural abundance levels and might appear only in the third or fourth decimal place if the percentage values were reported. Examples of standards are: air for N, which has a percentage of 15N of 0.3663033% relative to the total (14N + 15N) and Pee Dee Belemnite for C, which has a content of 1.1112328% of 13C relative to the total (12C + 13C + 14C). An example of calculation of δ15N, would be (from http://www.uga.edu/~sisbl/stable.html#trace): δ15N 0/00 versus [std] = ((Rsample − Rstd)/Rstd) (1000 δ 0/00); where R = (At%15N/At%14N) The application of this approach is based on the fact that organisms accumulate in their tissues the elements present in their environment (water, air and soil) as well as those contained in their foods. Because there are differences in the isotope distribution of trace elements in different geographic locations (depending on the composition of soils, the weather conditions, fauna and flora, proximity to cities, industries or farms, and so on) these differences will be reflected in the composition of the fauna and flora of that particular location, and the fingerprint of the organisms can be used as a marker of their geographic provenance. For example, δ13C and δ15N increase from canopied to open forests or agriculture; δ13C and

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δD vary according to the photosynthetic pathway, and so on (Hobson 2005). The application of using isotope and trace-element analyses to determine the geographic origin of food, mainly meat, diary, beverages, and wines and cereals, has recently been reviewed by Kelly et al. (2005). It must be noted, however, that to make sensible comparisons, one should know and be aware of differences in the deposition and metabolic rates of the different elements in the different tissues of the species under consideration (Hobson 2005) The two main techniques used to determine the isotope ratios of natural products are isotope ratio mass spectrometry (IRMS) and site-specific natural isotope fractionation analyzed by nuclear magnetic resonance (SNIF–NMR). IRMS has the advantage over NMR that all except 12 elements can be analyzed by the technique; and SNIF–NMR has the advantage over IRMS that it allows the precise and accurate quantification of the natural abundance of 2H isotopomers (Martin and Martin 1991), whereas IRMS only gives a mean value of the deuterium content of a given chemical species. The SNIF–NMR® technology was developed to detect the adulteration of wines in the early 1980s by Gerard and Mar Yvonne Martin (Martin and Martin 1988, 1991; Martin et al. 1988). Today this method has been adopted as an official European method for authentication of wine (Official Journal of the European Community (1990) 33 L-272, 3 October 1990) and an AOAC approved technique for control of sugar addition in fruit juices (Martin et al. 1996). SNIF–NMR produces a unique and distinctive isotopic fingerprint for a variety of substances. The fingerprint is created on the basis of the isotopic composition of biomolecules because different isotopes of certain atoms (2H/1H, 13C/12C, 15N/14N, 18O/16O) occur at certain relative frequencies; these frequencies and the proportion of each isotope or the relative position of each isotope in a given molecule will vary depending on the geographic origin and the processing and production techniques applied to the sample. For example, it has been shown that it is possible to follow the primary photosynthetic metabolism of plant products by analyzing the 13C/12C ratio (O’Leary 1981), whereas the ratios of the stable isotopes of oxygen (16O/18O) and hydrogen (2H/1H) give a better indication of environmental conditions (Ziegler et al. 1976). One condition necessary to be able to apply SNIF–NMR satisfactorily for authentication of foodstuffs is the construction of large and representative databases containing the fingerprints of all the relevant products, preferably both authentic ones and those that may be used fraudulently. This is because identification is performed by multivariate data analysis classification techniques, which require the processing of the fingerprint obtained from the unknown sample with as many fingerprints as possible (usually in their thousands) of samples representative for all the possible species, tissues, origins, products, and production and processing conditions. As mentioned, SNIF–NMR is the official method to authenticate European wines, which can be done because the European Community has implemented the construction of a large databank containing approximately 12,000 isotopic profiles of Quality Wines Produced in Specific Regions (Martin 2005 and references therein). Every year, all wine-producing countries in the EU harvest several samples proportional to the area of their vineyards, which are analyzed by SNIF–NMR. The databank contains the SNIF–NMR parameters of 2H/1H of ethanol and the 13C and 18O determined by IRMS of ethanol and water, respectively. The method is also widely used to authenticate fruit juices, aromas and perfumes (Martin, 2005). Stable isotope variation was used by Boner and Förstel (2004) to differentiate meat from Europe and America by using isotopes of O and H, cattle from two German regions by using

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isotopes of N and S, and organically produced meat from conventional cattle by using 13 C/12C. However, these authors concluded that controlling the authenticity of cattle required additional data such as slaughtering day, region or local geographic origin and fodder base, so that the stable isotope analyses could be used to confirm or negate the declared information. Based on C and N isotope ratios, Schmidt et al. (2005) were able to differentiate American from European beef as well as organically produced from conventional beef, both Irish, by the isotopic composition of C, N and S, conventional beef having higher δ15N values than organic beef. In any case, it must be noted that meat from animals originating from different areas that have a similar climatology or geology may produce indistinguishable isotope signatures. Studies on fish have mostly been performed on oil extracts. The non-statistical distribution of 2H analysed by NMR was used to differentiate Atlantic salmon from different sources (Aparicio et al. 1998; Aursand and Axelson 2001; Aursand et al. 1995b, 2000, 2007). The ‘fingerprint’ that resulted from the chemical shift position and peak height of 13C NMR spectra of lipids was used to identify the species and origin of purified marine oils (Aursand et al. 1995b,c, 2007). Using three analytical techniques, namely gas chromatography, IRMS and high-resolution 2H SNIF–NMR spectroscopy on fish oils and lipids extracted from Atlantic salmon, Aursand et al. (2000) were able to classify correctly samples of wild and farmed salmon from Norway and Scotland according to both the geographic origin and the production method. Although the authors were not able to find significant differences between the (2H/1H)tot isotope ratios of feeds from Norway and Scotland, the feeds were correctly assigned to their corresponding fish group. It was therefore concluded that the observed differentiation between farmed salmon from Norway and Scotland may not be directly related to the composition of the feeds, but rather to the distribution of 2H in the fatty acids (Aursand et al. 2000). A combination of fatty-acid and δ15N composition was successfully used by Moltenkin et al. (2007) to differentiate organical from farmed salmon, and their geographic origin (Norway or Ireland). Currently, there are no standardized official methods to verify the geographic origin of fish. Establishment of such methods has been one of the aims of the European project COFAWS (EC, DG RTD, contract number G6RTD-CT-2001-00512). The five partners in the project tested the suitability of a series of analytical techniques including 1H-NMR, 2 H-NMR, 13C-NMR, 13C-IRMS, 18O-IRMS, GC and GC–IRMS, but only a few results have been published. Rezzi et al. (2003) were able to discriminate the geographic origin (Scotland, Iceland, Norway) of farmed Atlantic salmon by analyzing oil extracts from the white skeletal muscle by 1H-NMR. Specimens from six farms were separated into five clusters: three clusters, one for each farm from Scotland, one cluster comprising two Norwegian farms and one cluster for the only farm from Iceland. In addition to the C and H contained in the fatty acids, the water contained in the muscle tissue of fish reflects the aqueous environment in which the fish has been harvested. The two isotopic parameters that can be determined from this water, 2H and 18O NMR, also have potential as markers for the geographic origin. In a recent review, Franke et al. (2005) examined the suitability of stable isotope ratios and trace element signatures to identify the geographic origin of meat. Trace element signatures are usually analyzed by mass spectrometry, particularly IRMS or inductively coupled plasma mass spectrometry (ICP–MS). Franke et al. (2005) concluded that some elements are of particular interest to discriminate among products from different small-scale

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geographic regions, for example different regions in Germany, whereas others had the potential to differentiate meat from different continents (for instance Se is consistently higher in meat produced in America than in meat produced in Europe). However, the fact that most feeds are enriched with essential elements and minerals, that the animals may switch areas during fattening, and that customary changes in feeds are made during breeding (all of them concepts that with small variations apply to fish farming) did not allow use of a single element as a marker; rather, one should use those trace elements characteristic for the local water, soil and air as the most promising markers. As trace elements, stable isotopes are incorporated in local feeds and in the body of animals. Therefore the ratios of the rare to the abundant isotope may be specific for the given areas. The ratios 2H/1H and 16O/18O in the body tissues are primarily influenced by the (drinking) water, whereas the ratios of 12C/13C; 14 N/15N, 32S/34S and 86Sr/87Sr are usually markers for soil and feed (Franke et al. 2005 and references therein). Interestingly, animal products are usually enriched in 13C and 15N, depending on their diet, and the enrichments proceeds stepwise along the trophic chain from one level to the next. This helps to link the meat to its diet and if the diet is unique to a certain area, then to its geographic origin (Franke et al. 2005 and references therein). Hobson (2005) has recently published a review illustrating the use of stable isotopes, primarily δ13C; δ15N; δ34S; δ2H and δ87S, to trace nutritional origin and migration in terrestrial and aquatic animals, and in bats. Trace-element signatures in otoliths by inductively coupled plasma mass spectrometry (ICP-MS) have been used to distinguish between Atlantic and Mediterranean tuna (Secor et al. 2002) and different spawning aggregations of cod (Campana et al. 1994). Otolith microconstituent analysis has also been applied recently to study stock structure (Edmonds et al. 1989; Campana et al. 1994, 1995; Proctor et al. 1995) and migration rates (Secor 1992; Secor and Piccoli 1996) in a variety of fish species. The premise of this approach is that trace elements are incorporated into otoliths in direct proportion to their availability in surrounding water or food. Few laboratory experiments have been conducted to verify the assumption that otoliths can record environmental histories, but such studies have supported this assumption for uptake of strontium (Fowler et al. 1995; Limburg, 1995; Secor et al. 1995; Farrell and Campana 1996). Physiological factors, temperature and genetics may also affect the uptake of specific elements into otoliths (Kalish 1989; Thresher et al. 1994). In a study by Secor and Zdanowicz (1998) to differentiate bluefin tuna (Thunnus thynnus) from the Atlantic and the Mediterranean, larvae or young-of-the-year exposed to either the Gulf of Mexico or Mediterranean waters were expected to incorporate different mixtures of elements into their otoliths. Multivariate data analysis of the results obtained by ICP–MS of microconstituents measured in whole otoliths of juvenile northern bluefin tuna from the Mediterranean Sea and western Pacific Ocean showed a clear separation between the two groups, and that sodium, magnesium, manganese and zinc were most useful in differentiating Mediterranean and Pacific samples (Secor and Zdanowicz 1998). Most of the work on this subject has been based on the analyses of otholites and/or scales (Campana 1999; Campana and Thorrold 2001), but Born et al. (2003) and Yamashita et al. (2006) have shown that the approach is also valid when applied to soft tissues. Thus, Born et al. (2003) determined regional variations in long-term elemental diagnostics of stock differences among 159 minke whales harvested in west Greenland, the northeast Atlantic Ocean and the North Sea in 1998. The diagnostics tested included mercury, selenium and cadmium, 15N and 13C and stable lead-isotope ratios in muscle liver and kidney, and the trace and major element

Other principles: analysis of lipids, stable isotopes and trace elements

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composition of baleen. Yamashita et al. (2006) applied trace-element analyses to identify the origin of eels. They analyzed the levels of six elements in eel muscle: selenium, mercury, zinc, copper, manganese and arsenic. They found that the first four of them were the main determinants of the first two factors in the multivariate data analysis that allowed identification of the origin of eels from three Japanese prefectures (Miyazaki, Kagawa and Shizuoka), Taiwan and China. The authors concluded that the use of techniques with high sensitivity, such as ICP–MS, would allow inclusion in the analysis of certain rare trace elements such as uranium, lead, cadmium and vanadium, which may be particularly useful for discriminating the geographic origin of fish. Interestingly, some of these very same elements are also highly relevant from the point of view of food safety, for example cadmium, mercury, lead or arsenic. If, in addition, the accumulation of some elements presents a species-specific pattern, as shown for mercury in the muscle of tuna and alfonsino (Yamashita et al. 2005), that would give the potential to identify the species, as well as determining the geographic origin and the potential detection of toxic levels of certain elements in seafood.

18.5

Future prospects

Analyses of the astaxanthin content and isomers and their pattern of deposition in different tissues has been useful in discriminating wild from farmed salmonids. However, the application is currently limited to species whose feed may contain these pigments, such as some crustaceans, salmonids and the breeding of some other species. If the addition of these compounds, which seem to have a positive effect on human health (Baker and Günther 2004), was to be allowed to all fish feeds, then these analytes could have a wider application in the identification of the production method. Incidentally, carotenoids have been mentioned as potential markers to verify traceability information on meat and milk of small ruminants (Prache et al. 2005). On the other hand, the use of alternative sources of carotenoids, indistinguishable from the natural sources, as mentioned by Ostermeyer and Schmidt (2004), may make the current analytical methods obsolete for differentiating these three production methods. To enforce legislation about species, production methods and geographic origin of seafood will need the collaboration of all relevant actors involved in the harvesting and production of seafood, to contribute the authentic samples necessary for the construction, updating and maintenance of databases of raw materials and of sampling methods and analyses. This has already been done for the authentication of wines in Europe, where all producers contribute samples of their annual production to the database. It is also the approach proposed by Hobson (2005) and Smith et al. (2003) to track migratory organisms and to link populations through their annual cycle – namely, to collect feathers from birds, exoskeletons from insects, and hairs from mammals in an archive – in order to carry out meaningful studies of longdistance dispersal in wild animals. The challenge in all cases is to identify the markers or combination of markers with discriminatory value and to have a database for each analysis comprehensive enough to allow the classification of as many samples as possible. International cooperation in the construction of the databases and the sharing of the results will be essential to cover the wide spectrum of samples and geographic origins that the current seafood trade comprises.

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Acknowledgments This work was performed with the financial support of the Research Council of Norway and of the European Union to the Integrated Research Project SEAFOODplus, contract No. FOOD-CT-2004-506359, and to the Specific Targeted Research Project SigmaChain, contract No. FP6-518451. Dr Marit Aursand and Inger Beate Standal (SINTEF Fisheries and Aquaculture Ltd, Trondheim, Norway) are gratefully acknowledged for allowing the use of their unpublished material and manuscript in press, and Dr Isabel Medina (Instituto de Investigaciones Marinas, CSIC, Vigo, Spain) for providing Figure 18.4. The author is also very grateful to Dr Michiaki Yamashita, Dr Yumiko Yamashita (National Research Institute of Fisheries Science, Yokohama, Japan) and MrYasuharu Takashima (IAA Center for Food Quality, Labeling and Consumer Services, CFQLCS, Tokyo, Japan) for information about legislation in Japan and results in the field of seafood authentication.

18.6

References

Andersson, M., Van Nieuwerburgh, L. and Snoeijs. P. (2003) Pigment transfer from phytoplankton to zooplankton with emphasis on astaxanthin production in the Baltic Sea food web. Marine Ecology Progress Series 254: 213–224. Ando, T., Nishimura, K., Aoyanagi, N. and Takagi, T. (1992) Stereospecific analysis of fish oil triacylsn-glycerols. Marine Ecology Progress Series 69: 417–424. Ando, Y., Kobayashi, S., Sugimoto, T. and Takamaru, N. (2004a) Positional distribution of n–3 highly unsaturated fatty acids in triacylglycerols (TAG) of rotifers (Brachionus plicatilis) enriched with fish and seal oils TAG. Aquaculture 229: 275–288. Ando, Y., Samoto, H. and Murayama, Y. (2004b) Positional distribution of DHA and EPA in triacylsn-glycerols (TAG) of Artemia franciscana nauplii enriched with fish oils ethyl esters and TAG. Aquaculture 233: 321–335. Aparicio, R., MaIntyre, P., Aursand, M., Eveleigh, L., Marighetto, N., Rossell, B., Sacchi, R., Wilson, R. and Woolfe, M. (1998) Oils and fats. In: M. Lees (Ed.) Food Authenticity, Issues and Methodologies. FAIM Concerted Action no. AIR3-CT94-2452. Eurofins Scientific, Nantes, France. pp. 211–269. Aursand, M. and Axelson, D. (2001) Origin recognition of wild and farmed salmon (Norway and Scotland) using 13C NMR spectroscopy in combination with pattern recognition techniques. In: G.A. Webb, P.S. Belton, A.M. Gil, and I. Delgadillo (Eds) Magnetic Resonance in Food Science: A View to the Future. The Royal Society of Chemistry, pp. 227–231. Aursand, M. and Grasdalen, H. (1992) Interpretation of the 13C-NMR spectra of omega-3 fatty acids and lipid extracted from the white muscle of Atlantic salmon (Salmo salar). Chemistry and Physics of Lipids, 62: 239–251. Aursand, M., Jørgensen L. and Grasdalen, H. (1995a) Positional distribution of ω-3 fatty acids in marine lipid triacylglycerols by high resolution 13C nuclear magnetic resonance spectroscopy. Marine Ecology Progress Series 72: 293–297. Aursand, M., Jørgensen, L. and Grasdalen, H. (1995b) The use of high resolution 1H and 13C nuclear magnetic resonance (NMR) spectra to obtain information on the composition of fats and oils. LIPIDFORUM 49: 14–17. Aursand, M., Jørgensen, L. and Grasdalen, H. (1995c) Information on the composition of fats and oils from their high resolution 1H and 13C nuclear magnetic resonance (NMR) spectra. In: LIPIDFORUM, Proceedings of the 18th Nordic Lipid Symposium, Reykjavik, Iceland.

Other principles: analysis of lipids, stable isotopes and trace elements

405

Aursand, M., Mabon, F. and Martin, G.J. (2000) Characterization of farmed and wild salmon (Salmo salar) by a combined use of compositional and isotopic analyses. Journal of the American Oil Chemists Society 77: 659–666. Aursand, M., Standal, I.B. and Axelson, D. (2007) High-resolution 13C nuclear magnetic resonance spectroscopy pattern recognition of fish oil capsules. Journal of Agricultural and Food Chemistry 55: 38–47. Baker, R. and Gunther, C. (2004) The role of carotenoids in consumer choice and the likely benefits from their inclusion into products for human consumption. Trends in Food Science and Technology 15: 484–488. Bell., J.G., MsEnvoy, J., Tocher, D.R., McGhee, F., Campbell, P.J. and Sargent, J.R. (2001) Replacement of fish oil with rapeseed oil in diets of Atlantic salmon (Salmo salar). Journal of Agricultural and Food Chemistry 46: 119–127. Bell, J.G., Henderson, R.J., Tocher, D.R., McGhee, F., Dick, J.R., Porter, A., Smullen, R. and Sargent, J.R. (2002) Substituting fish oil with crude palm oil in the diet of Atlantic salmon (Salmo salar) affects tissue fatty acid compositions and hepatic fatty acid metabolism. Journal of Nutrition 132: 222–230. Bell, J.G., McGhee, F., Campbell, P.J. and Sargent, J.R. (2003a) Rapeseed oil as an alternative to marine fish oil in diets of post-smolt Atlantic salmon (Salmo salar): changes in flesh fatty acid composition and effectiveness of subsequent fish oil ‘wash out’. Aquaculture 218: 515–528. Bell, J.G., Tocher, D.R., Henderson, R.J., Dick, J.R. and Crampton. V.O. (2003b) Altered fatty acid compositions in Atlantic salmon (Salmo salar) fed diets containing linseed and rapeseed oils can be partially restored by a subsequent fish oil finishing diet. Journal of Nutrition 133: 2793–2801. Bernhard K. (1990) Synthetic astaxanthin. The route of a carotenoid from research to commercialization. In: N.I. Krinski, M.M. Mathews-Roth and R.F. Taylor (Eds) Carotenoids: Chemistry and Biology. Plenum Press, New York, pp. 337–364. Bjerkeng, B., Hatlen, B. and Wathne, E. (1999) Deposition of astaxanthin in fillets of Atlantic salmon (Salmo salar) fed diets with herring, capelin, sandeel, or Peruvian high PUFA oils. Aquaculture 180: 307–319. Brockerhoff, H., Hoyle, R.J., Hwang, P.C. and Litchfield, C. (1968). Positional distribution of fatty acids in depot triglycerides of aquatic animals. Lipids 3: 24–29. Boner, M. and Förstel, H. (2004) Stable isotope variation as a tool to trace the authenticity of beef. Analytical and Bioanalytical Chemistry 378: 301–310. Born, E.W., Outridge, P., Riget, F.F., Hobson, K.A., Dietz, R., Oien, N. and Haug, T. (2003) Population substructure of North Atlantic minke whales (Balaenoptera acutorostrata) inferred from regional variation of elemental and stable isotopic signatures in tissues. Journal of Marine Systems 43: 1–17. Campana, S.E. (1999) Chemistry and composition of fish otoliths: pathways, mechanisms and applications. Marine Ecology Progress Series 188: 263–297. Campana, S.E., Fowler, A.J. and Jones, C.M. (1994) Otolith elemental fingerprinting for stock identification of Atlantic cod (Gadus morhua) using laser ablation ICPMS. Canadian Journal of Fisheries and Aquatic Sciences 51: 1942–1950. Campana, S.E., Gagne, J.A. and McLaren, J.W. (1995) Elemental fingerprinting of fish otoliths using ID ICPMS. Marine Ecology Progress Series 122: 115–120. Campana, S.E. and Thorrold, S.R. (2001) Otoliths, increments, and elements: keys to a comprehensive understanding of fish populations? Canadian Journal of Fisheries and Aquatic Sciences 58: 30–38. Cowey, C.B. and Sargent, J.R. (1977) Minireview: lipid nutrition in fish. Comparative Biochemistry and Physiology 57B: 269–273. Dalsgaard, J., St John, M., Kattner, G., Müller-Navarra, D. and Hagen, W. (2003) Fatty acid trophic markers in the pelagic marine environment. Advances in Marine Biology 46: 225–340.

406

Fishery Products: Quality, safety and authenticity

Danielsdottir, A.K. (1998) Genetic studies on redfish species and stocks. Past–Ongoing–Future. ICES 1998; SGRS: WD3. Deering, M.J., Fielder, D.R. and Hewitt, D.R. (1997) Growth and fatty acid composition of juvenile leader prawns, Penaeus monodon, fed different lipids. Aquaculture 151: 131–141. Dempster, T., Sanchez-Jerez, P., Bayle-Sempere, J.T., Gimenez-Casalduero, F. and Valle, C. (2002) Attraction of wild fish to sea-cage fish farms in the south-western Mediterranean Sea: spatial and short-term temporal variability. Marine Ecology Progress Series 242: 237–252. Dennis, M.J. (1998) Recent developments in food authentication. The Analyst 123: 151R– 156R. Dosanjh, B.S., Higgs, D.A., Plotnikoff, M.D., Markert, J.R., and Buckley, J.T. (1988) Preliminary evaluation of canola oil, pork lard and marine lipid singly and in combination as supplemental dietary lipid sources for juvenile fall chinook salmon (Oncorhynchus tshawytscha). Aquaculture 68: 325–343. Edmonds, J.S., Moran, M.J., Caputi, N. and Morita, M. (1989) Trace element analysis of fish sagittae as an aid to stock identification: pink snapper (Chrysophrys auratus) in Western Australian waters. Canadian Journal of Fisheries and Aquatic Sciences 46: 50–54. Farrell, J. and Campana, S.E. (1996) Regulation of calcium and strontium deposition on the otoliths of juvenile tilapia, Oreochromis niloticus. Comparative Biochemistry and Physiology 115A: 103–109. Fernandez-Jover, D., Lopez-Jimenez, J.A., Sanchez-Jerez, P., Bayle-Sempere, J., Gimenez Casalduero, F., Martinez-Lopez, F.J. and Dempster, T. (2007) Changes in body condition and fatty acid composition of wild Mediterranean horse mackerel (Trachurus mediterraneus, Steindachner, 1868) associated to sea cage fish farms. Marine Environmental Research 63: 1–18. Foran, J.A., Hites, R.A., Carpenter, D.O., Hamilton, M.C., Mathews-Amos, A. and Schwager, S.J. (2004) A survey of metals in tissues of farmed Atlantic and wild Pacific salmon. Environmental and Toxicological Chemistry 23: 2108–2110. Fowler, A.J., Campana, S.E., Jones, C.M. and Thorrold, S.R. (1995) Experimental assessment of the effects of temperature and salinity on elemental composition of otoliths using laser ablation ICPMS. Canadian Journal of Fisheries and Aquatic Sciences 52: 1431–1441. Franke, B.M., Gremaud, G., Hadorn, R. and Kreuzer, M. (2005) Geographic origin of meat – elements of an analytical approach to its authentication. European Food Research and Technology 221: 493–503. González-Félix, M.L., Lawrence, A.L., Gatlin, D.M. and Perez-Velazquez, M. (2002) Growth, survival and fatty acid composition of juvenile Litopenaeus vannamei fed different oils in the presence and absence of phospholipids. Aquaculture 205: 325–343. Grahl-Nielsen, O. (1997) Fatty acid profiles as natural marks for stock identification. ICES CM 1997/ M:2, Annex 3. Grahl-Nielsen, O. and Mjaavatten, O. (1992) Discrimination of striped bass stocks: a new method based on chemometry of the fatty acid profile in heart tissue. Transactions of the American Fisheries Society 121: 307–314. Grahl-Nielsen, O. and Ulvund, K.A. (1990) Distinguishing populations of herring by chemometry of fatty acids. American Fisheries Society Symposium 7: 566–571. Hardy, R.W., Scott, T.M. and Harrell, L.W. (1987) Replacement of herring oil with menhaden oil, soybean oil, or tallow in the diets of Atlantic salmon raised in marine net-pens. Aquaculture 65: 267–277. Hidalgo, F.J. and Zamora, R. (2003) Edible oil analysis by high-resolution nuclear magnetic resonance spectroscopy: recent advances and future perspective. Trends in Food Science and Technology 14: 499–506. Hites, R.A., Foran, J.A., Carpenter, D.O., Hamilton, M.C., Knuth, B.A. and Schwager, S.J. (2004) Global assessment of organic contaminants in farmed salmon. Science 303: 226–229.

Other principles: analysis of lipids, stable isotopes and trace elements

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Hobson, K.A. (2005) Using stable isotopes to trace long-distance dispersal in birds and other taxa. Diversity and Distributions 11: 157–164. Jacobs, M.N., Covaci, A. and Schepens, P. (2002) Investigation of selected persistent organic pollutants in farmed Atlantic salmon (Salmo salar), salmon aquaculture feed, and fish oil components of the feed. Environmental Science and Technology 36: 2797–2805. Jobling, M. (2004) Are modifications in tissue fatty acid profiles following a change in diet the result of dilution? Test of a simple dilution model. Aquaculture 232: 551–562. Jobling, M., Larsen, A.V., Andreassen, B., Sigholt, T. and Olsen, R.L. (2002) Influence of a dietary shift on temporal changes in fat deposition and fatty acid composition of Atlantic salmon post-smolt during the early phase of seawater rearing. Aquaculture Research 33: 875–889. Joensen, H. and Grahl-Nielsen, O. (2000) Discrimination of Sebastes viviparus, S. marinus, and S. mentella from Faroe Islands by chemometry of the fatty acid profile in heart and gill tissues and in the skull oil. Comparative Biochemistry and Physiology 126B: 69–79. Joensen, H. and Grahl-Nielsen, O. (2001) The redfish species Sebastes viviparus, Sebastes marinus and Sebastes mentella have different composition of their tissue fatty acids. Comparative Biochemistry and Physiology 129B: 73–85. Joensen, H. and Grahl-Nielsen, O. (2004) Stock structure of Sebastes mentella in the North Atlantic revealed by chemometry of the fatty acid profile in heart tissue. ICES Journal of Marine Science 61: 113–126. Joensen, H., Steingrund, P., Fjallstein, I. and Grahl-Nielsen, O. (2000) Discrimination between two reared stocks of cod (Gadus morhua) from the Faroe Islands by chemometry of the fatty acid composition in the heart tissue. Marine Biology 136: 573–580. Kalish, J.M. (1989) Otolith microchemistry: validation of the effects of physiology, age, and environment on otolith composition. Journal of Experimental Marine Biology and Ecology 132: 151–178. Kelly, S., Heaton, K. and Hoogewerff, J. (2005) Tracing the geographical origin of food: the application of multi-element and multi-isotope analysis. Trends in Food Science and Technology 16: 555–567. Kumaraguru Vasagam, K.P., Ramesh, S. and Balasubramanian, T. (2005) Dietary value of different vegetable oil in black tiger shrimp Penaeus monodon in the presence and absence of soy lecithin supplementation: effect on growth, nutrient digestibility and body composition. Aquaculture 250: 317–327. Lie, Ø., Waagbø, R. and Sandnes, K. (1988) Growth and chemical composition of adult Atlantic salmon (Salmo salar) fed dry silage based diets. Aquaculture 69: 343–353. Limburg, K.E. (1995) Otolith strontium traces environmental history of subyearling American shad Alosa sapidissima. Marine Ecology Progress Series 119: 25–35. Litchfield, C. (1968) Predicting the positional distribution of cocosahexaenoic and docosapentaenoic acid sin aquatic animal triglycerides. Lipids 3: 417–419. Lura, H. and Sægrov, H. (1991) A method of separating offspring from farmed and wild Atlantic salmon (Salmo salar) based on different ratios of optical isomers of astaxanthine. Canadian Journal of Fisheries and Aquatic Sciences 48: 429–433. Madeniian, C.P., O’Connor, D.V., Stewart, D.J., Miller, M.A. and Masnado, R.G. (2002) Field estimate of net trophic transfer efficiency of PCBs to Lake Michigan chinook salmon from their prey. Environmental Science and Technology 36: 5029–5033. Martin, G. (2005) Advances in the authentication of food by SNIF-NMR. In: S.B. Engelson, P.S. Belton and H.J. Jakobsen (Eds) Magnetic Resonance in Food Science. The Multivariate Challenge. The Royal Society of Chemistry, Cambridge, UK, pp. 31–38. Martin, G.J. and Martin, M.L. (1988) The site-specific natural isotope fraction-NMR method applied to the study of wines. In: H.F. Linkens and J.F. Johnsen (Eds) Modern Methods of Plant Analysis, Wine analysis, Volume 6. Springer Verlag, Berlin and Heidelberg, Germany, pp. 258–275.

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Martin, G.J. and Martin, M.L. (1991) Deuterium labelling at the natural abundance level as studied by high field quantitative 2H-NMR. Tetrahedron Letters 22: 3525–3528. Martin, G.J., Guillou, C., Martin, M.L., Cabanis, M.T., Tep, Y. and Aerny, J. (1988) Natural factors of isotopic fractionation and the characterization of wines. Journal of Agricultural and Food Chemistry 36: 316–322. Martin, G.G., Wood, R. and Martin, G.J. (1996) Detection of added beet sugar in concentrated and single strength fruit juice by deuterium NMR (SNIF-NMR method). Collaborative study. Journal of AOAC International 79: 917–928. Martinez, I. (2006) Revision of analytical methodologies to verify the production method of fish. In: J.B. Luten, C. Jacobsen, K. Bekaert, A. Sæbø, and J. Oehlenschlager (Eds) Seafood from Fish to Dish, Quality, Safety and Processing of Wild and Farmed Fish. Wageningen Academic Publishers, Wageningen, The Netherlands, pp. 541–550. Martinez, I., Aursand, M., Erikson, U., Singstad, T.E., Veliyulin, E. and van der Zwaag, C. (2003) Destructive and non-destructive analytical techniques for authentication and composition analyses of foodstuffs. Trends in Food Science and Technology 14: 489–498. McCoy, M. (1999) Astaxanthin market a hard one to crack. Chemical and Engineering News 77: 15–17. Medina, I., Aubourg, S.P. and Martin, R.P. (1997) Species differentiation by multivariate analysis of phospholipids from canned Atlantic tuna. Journal of Agricultural and Food Chemistry 45: 2495–2499. Molnar, T., Szabo, A., Szabo, G., Szabo, C. and Hancz, C. (2006) Effect of different dietary fat content and fat type on the growth and body composition of intensively reared pikeperch Sander lucioperca (L.). Aquaculture Nutrition 12: 173–182. Moltenkin, J., Meisel, H., Lehman, I. and Rehbein, H. (2007) Identification of organically farmed Atlantic salmon by analysis of stable isotopes and fatty acids. European Food Research and Technology 224: 535–543. Nedreaas, K. and Nævdal, G. (1991a) Genetic studies of redfish (Sebastes spp.) along the continental slopes from Norway to East Greenland. ICES Journal of Marine Science 48: 173–186. Nedreaas, K. and Nævdal, G. (1991b) Identification of 0- and 1-group redfish (genus Sebastes) using electrophoresis. ICES Journal of Marine Science 48: 91–99. Nedreaas, K., Johansen, T. and Nævdal, G. (1994) Genetic studies of redfish (Sebastes spp.) from Iceland and Greenland waters. ICES Journal of Marine Science 51: 461–467. Ng, S. (1985) Analysis of positional distribution of fatty acids in palm oil by 13C NMR spectroscopy. Lipids 20: 778–182. Ng, W.K., Lim, P.K. and Sidek, H. (2001) The influence of a dietary lipid source on growth, muscle fatty acid composition and erythrocyte osmotic fragility of hybrid tilapia. Fish Physiology and Biochemistry 25: 301–310. Ng, W.K., Lim, P.K. and Boey, P.L. (2003) Dietary lipid and palm oil source affects growth, fatty acid composition and α-tocopherol concentration in African catfish, Clarias gariepinus. Aquaculture 215: 229–243. Ng, W.K., Sigholt, T. and Bell, J.G. (2004) The influence of environmental temperature on the apparent nutrient and fatty acid digestibility in Atlantic salmon (Salmo salar L.) fed finishing diets containing different blends of fish oil, rapeseed oil and palm oil. Aquaculture Research 35: 1228–1237. Nichols, P.D., Mooney, B.D. and Elliot, N.G. (2002) Nutritional Value of Australian Seafood II. CSIRO Marine Research and FRDC, Hobart, Tasmania, Australia. 198 pp. O’Leary, M. (1981) Carbon isotope fraction in plants. Phytochemistry 20: 553–567. Ostermeyer, U. and Schmidt, T. (2004) Differentiation of wild salmon, conventionally and organically farmed salmon. Deutsche Lebensmittel-Rundschau 100: 437–444. Pfeffer, P.E., Sampugna, J., Schwartz, D.P. and Schoolery, J.N. (1977) Analytical 13C NMR: detection, quantitation and positional analysis of butyrate in butter oil. Lipids 12: 869–871.

Other principles: analysis of lipids, stable isotopes and trace elements

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Prache, S., Cornu, A., Berdague, J.L. and Priolo, A. (2005) Traceability of animal feeding diet in the meat and milk of small ruminants. Small Ruminants Research 59: 157–168. Proctor, C.H., Thresher, R.E., Gunn, J.S., Mills, D.J., Harrowfield, I.R. and Sie, S.H. (1995) Stock structure of the southern bluefin tuna Thunnus maccoyii: an investigation based on probe microanalysis of otolith composition. Marine Biology 122: 511–526. Refsgaard, H.H.F., Brockhoff, P.B. and Jensen, B. (1998) Biological variation of lipid constituents and distribution of tocopherols and astaxanthin in farmed Atlantic salmon (Salmo salar). Journal of Agricultural and Food Chemistry 46: 808–812. Rehbein, H. (1983) Differentiation of redfishes from the Northeast Atlantic (Sebastes marinus L., S. mentella Travin, S. viviparus Krøer and Helicolenus dactylopterus D. Delaroche 1809) by isoelectric focusing of sarcoplasmic proteins. ICES CM G: 40, p. 11. Rezzi, S., Guillou, C.G., Reiniero, F., Holland, M. and Ghelli, S. (2003) Natural abundance 2H-NMR spectroscopy. Application to food analysis. In: P.A. de Groot (Ed.) Handbook of Stable Isotope Analytical Techniques, Chapter 5. Elsevier Science, pp. 103–121. Roques, S., Pallotta, D., Sévigny, J.M. and Bernatchez, L. (1999a) Isolation and characterization of polymorphic microsatellite markers in the North Atlantic redfish (Teleostei: Scorpaenidae, genus Sebastes). Molecular Ecology 8: 685–702. Roques, S., Duchesne, P. and Bernatchez, L. (1999b) Potential of microsatellites for individual assignment: the North Atlantic redfish (genus Sebastes) species complex as a case study. Molecular Ecology 8: 1703–1717. Sægrov, H., Hindar, K. Kålås, S. and Lura, H. (1997) Escaped farmed Atlantic salmon replace the original salmon stock in the River Vosso, western Norway. ICES Journal of Marine Science 54: 1166–1172. Schmidt, O., Quilter, J.M., Bahar, B., Moloney, A.P., Scrimgeour, C.M., Begley, I.S. and Monahan, F.J. (2005) Inferring the origin and dietary history of beef from C, N and S stable isotope ratio analysis. Food Chemistry 91: 545–549. Secor, D.H. (1992) Application of otolith microchemistry analysis to investigate anadromy in Chesapeake Bay striped bass. Fisheries Bulletin 90: 798–806. Secor, D.H. and Piccoli, P.M. (1996) Age- and sex-dependent migrations of the Hudson River striped bass population determined from otolith microanalysis. Estuaries 19: 778–793. Secor, D.H. and Zdanowicz, V.S. (1998) Otolith microconstituent analysis of juvenile bluefin tuna (Thunnus thynnus) from the Mediterranean Sea and Pacific Ocean. Fisheries Research 36: 251–256. Secor, D.H., Henderson-Arzapalo, A. and Piccoli, P.M. (1995) Can otolith microchemistry chart patterns of migration and habitat utilization in anadromous fishes? Journal of Experimental Marine Biology and Ecology 192: 15–33. Secor, D.H., Campana, S.E., Zdanowicz, V.S., Lam, J.W.H., Yang, L. and Rooker, J.R. (2002) Interlaboratory comparison of Atlantic and Mediterranean bluefin tuna otolith microconstituents. ICES Journal of Marine Science 59: 1294–1304. Sener, E., Yildiz, M. and Savas, E. (2006) Effects of dietary lipids on growth and fatty acid composition in Russian sturgeon (Acipenser gueldenstaedtii) juveniles. Turkish Journal of Veterinary and Animal Sciences 29: 1101–1107. Skog, T.E., Hylland, K., Torstensen, B.E. and Berntssen, M.H.G. (2003) Salmon farming affects the fatty acid composition and taste of wild saithe Pollachius virens L. Aquaculture Research 34: 999–1007. Shul’man, G.E. (1960) Dynamics of the fat content of the body of fish. Russian Review of Biology 49: 209–222. Siddiqui, N., Sim, J., Silwood, C.J.L., Toms, H., Iles, R.A. and Grootveld, M. (2003) Multicomponent analysis of encapsulated marine oil supplements using high-resolution 1H and 13C techniques. Journal of Lipid Research 44: 2406–2427.

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Smith, T.B., Marra, P., Webster, M.S., Lovette, I., Gibbs, L., Holmes, R.T., Hobson, K.A. and Rohwer, S. (2003) A call for feather sampling. Auk 120: 218–221. Sundt, R.C. and Johansen, T. (1998) Low levels of interspecific DNA sequence variation of the mitochondrial 16S rRNA in North Atlantic redfish Sebastes (Pisces, Scorpaenidae). Sarsia 83: 449–452. Torrissen, O.J. and Christiansen, R. (1995) Requirements for carotenoids in fish diets. Journal of Applied Ichthyology 11: 225–230. Torstensen, B.E., Froyland, L. and Lie, Ø. (2004a) Replacing dietary fish oil with increasing levels of rapeseed oil and olive oil – effects on Atlantic salmon (Salmo salar L.) tissue and lipoprotein lipid composition and lipogenic enzyme activities. Aquaculture Nutrition 10: 175–192. Torstensen, B.E., Froyland, L., Ørnsrud, R. and Lie, Ø (2004b) Tailoring of a cardioprotective muscle fatty acid composition of Atlantic salmon (Salmo salar) fed vegetable oils. Food Chemistry 87: 567–580. Torstensen, B.E., Lie, Ø. and Froyland, L. (2000) Lipid metabolism and tissue composition in Atlantic salmon (Salmo salar L.) – effects of capelin oil, palm oil and oleic acid-enriched sunflower oil as dietary lipid sources. Lipids 35: 653–664. Thresher, R.E., Proctor, C.H., Gunn, J.S. and Horrowfield, I.R. (1994) An evaluation of electron-probe microanalysis of otoliths for stock delineation and identification of nursery areas in a southern temperate groundfish, Nemadactylus macropterus (Cheilodactylidae). Fisheries Bulletin 92: 817–840. Yamashita, Y., Omura, Y and Okazaki, E. (2005) Total mercury and methylmercury levels in commercially important fishes in Japan. Fisheries Science 71: 1029–1035. Yamashita, Y., Omura, Y and Okazaki, E. (2006) Distinct regional profiles of trace element content in muscle of Japanese eel Anguilla japonica from Japan, Taiwan, and China. Fisheries Science 72: 1109–1113. Zamora, R., Alba, V. and Hidalgo, F.J. (2001) Use of high-resolution 13C nuclear magnetic resonance spectroscopy for the screening of virgin olive oils. Marine Ecology Progress Series 78: 89–94. Ziegler, H., Osmond, C.B., Stichler, W. and Trimborn, P. (1976) Hydrogen isotope discrimination in higher plants: correlations with photosynthetic pathway and environment. Planta 128: 85–92.

Chapter 19

Sensory evaluation of seafood: general principles and guidelines Emilia Martinsdóttir, Rian Schelvis, Grethe Hyldig and Kolbrun Sveinsdóttir

19.1

General principles for sensory analysis

Sensory evaluation is the scientific discipline that evokes, measures, analyses and interprets human reactions to characteristics of food perceived through the senses of sight, smell, taste, touch and hearing. Scientific sensory evaluation methods must be performed under carefully controlled conditions in order to reduce the effects of test environment, personal bias, and so on. It is extremely important to define the problem to be solved or what is to be measured. Sensory evaluation is quantitative: numerical data are collected to establish relationship between product characteristics and human perception. It is critical to use proper analysis of the sensory data and interpret the results. The sampling system, methods and procedures for sensory evaluation must be very well defined to serve its purpose. In sensory evaluation, a sensory panel is established and panellists or inspectors trained to perform sensory analysis with clear and descriptive guidelines. The role of the panel leader is to select and train the panellists, manage product samples and references/standards, supervise preparation and presentations of the samples, maintain the technical skills and motivation of the panellists and compile the sensory data. Analysis and interpretation of the sensory data require understanding of the methods used and are performed by the panel leader or in cooperation by the panel leader/sensory staff and project leaders. The panellists must be monitored for their ability to perform the analysis by the panel leader. Textbooks on sensory evaluation of foods often describe the facilities required for sensory evaluation. The recommendations in these publications are intended for establishments or situations where sensory evaluation is a major activity, for example R&D laboratories of food companies and research institutes. Sensory evaluation can be practised at different levels in the fish processing industry. However, sensory evaluation for quality control must be performed no less accurately and conscientiously than in R&D laboratories, but the requirements need not be as elaborate. 411

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Specifically for fish and other seafood, these guidelines describe the tools for sensory evaluation: the senses, selection and training of panellists, facilities for sensory evaluation and sample preparation. In this chapter, application of sensory evaluation for different purposes is described, such as in research, quality control, product development, studies of shelf life and consumer studies. Referral to appropriate sensory methods is given, but each method is covered in chapter 20. We discuss the various application areas where sensory evaluation is used and different methods are chosen to serve the purpose of the measurement. Recommendations on which methods are practical in each situation are given.

19.1.1

The senses

Sensory evaluation is a systematic assessment of the appearance, odour, flavour and texture of food. During perception, most or all of the attributes overlap. However, with training, independent evaluation of each attribute is obtained (Meilgaard et al. 2006). In sensory evaluation of seafood, vision is very important. We see defects such as bloodstains, bones and parasites. The appearance of the fish, including the gills and eyes, gives information about the freshness of the fish. Odour of both raw and cooked fish is also important in sensory evaluation of freshness. People are very sensitive to various compounds produced in fish during storage, and especially spoilage, such as several sulphur and nitrogen compounds. Odours are described as reminding us of something else. Thus, the odour of fish can even be described as an odour of milk or cucumber, as can be seen in the sensory evaluation scheme for whole salmon (Table 20.3). There are four classical basic tastes: sour, salt, bitter and sweet. Various others have been proposed to belong to these basic tastes, such as metallic, astringent and umami. Flavour is the olfactory perception caused by volatile substances released from a product in the mouth as well as the basic tastes caused by soluble substances in the mouth. The chemical feeling factors stimulating the trigeminal nerve ends, for example those in mint and chilli peppers, are also a part of the flavour. In sensory evaluation of fish, the tactile sense is mainly used to evaluate the texture of fish flesh, for example by pressing a fingertip on the fish flesh to observe if the fish is still stiff or soft. The texture of seafood can also be sensed through chewing. References for further reading can be found in Meilgaard et al. (2006).

19.1.2 Selection and training of sensory panels Selection of participants in a sensory panel is based on their basic sensory acuity and ability to describe perceptions analytically. The panellist must know the procedures, improve their ability to identify sensory attributes and improve their sensitivity and memory to provide precise, consistent and standardised reproducible measurements. Personal characteristics are very important when selecting people for the sensory group, such as conscientiousness and accuracy. They should also be interested in sensory evaluation and food. Panellists must also be healthy and normally sensitive (taste and odour senses).

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They should know the nature and limits of the sense organs, and learn how to recognise and evaluate the sensory attributes of fish and seafood products. Basic selection tests and training guidelines can be found in Meilgaard et al. (2006), ISO 8586-1 (1993) and, more specifically for seafood, in Martinsdottir et al. (2001). The persons participating in a panel may be recruited outside the company or institute (external panels), as is more used in research institutes, or they may be recruited among the staff in the company or institute (internal panels), as is more common in industrial companies. Outside panellists maybe more focused, have a more neutral attitude to the samples and probably have more time to concentrate on the evaluation. The advantages of internal panels are that they are easy to reach at short notice and have product knowledge; however, they may be biased by having too much knowledge of the project or products. The training of the sensory panel should begin by describing the procedures of the sensory evaluation, what is expected of the panellists, and so on. The nature and limits of the sense organs are described, such as the importance of breathing deeply and resting between samples during odour evaluation. The schemes intended for use in the sensory evaluation must be carefully explained. Training results should be evaluated. The average and standard deviation of each sample should be calculated and a comparison made between the panellists, namely by performing statistical analysis (analysis of variance, for example). The ability of the panellists can be examined during repeated evaluation of the same samples. Repetition of the training will show the qualifications of the panellists. Regular training of the sensory panel should be done and performance of the panellists monitored. Psychological factors can influence the performance in sensory evaluation. Samples should be coded and randomly presented among the panellists, because given information with samples may lead to preconceived ideas as people may find what they expect to find. As an example, presenting a sample of fish fillet to a panellist with the information that the fish has been kept for a prolonged period in ice before filleting may influence that panellist not to use his or her senses adequately before judging the sample with negative attributes. An error can also occur if panellists associate different sensory attributes. An example is that a fish fillet from a species with a naturally darker muscle than fish fillet from a white fish species may be judged as not having fresh sensory characteristics because of the darker colour. It is also a source of error if the panellists let their hedonic personal judgement interfere with the evaluation. The panellists should be monitored to ensure they are adequately interested or whether they have become bored with or tired of the sensory evaluation. Some kind of encouragement (reward of some kind) and information about performance may be needed to keep the panellists interested. They are curious about their performance in sensory evaluation, and about what has been achieved by their work as a sensory panel. Sensory panels in research In research, both internal and external panels are used. For external panels, the recruitment can be done by announcement in a local newspaper. A first screening can be done by a telephone interview, where people for the test are picked out based on the requirements. All panel members must pass a test of their capability to use their senses and to express the response. According to ISO 5496 (2006), ISO 8586-1 (1993), ISO 8586-2 (1994), ISO 3972

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(1991), ISO/CD 13300 – 1 (2002) and ISO/CD 13300 – 2 (2002), the tests should contain training in detection and recognition of tastes and odours, sensitivity, ranking and/or triangle tests of basic tastes, odour, texture and appearance tests as well as scale- and product training. Based on the results from the test, the opportune candidates for the sensory panel are chosen. All assessors must be re-evaluated once a year during a similar procedure, but with some variation and with focus on the specific product, species, that the sensory panel are working with. The number of assessors depends on the sensory methods and the training of panellists. Depending on the sensitivity, the number of assessors for difference tests may vary from five to 60 or more (ISO 4120 (2004); ISO 5495 (2005)). For descriptive tests, a minimum of five to eight selected assessors or experts are required, specially trained in the method, though the usual number is 8–12 (ISO 6564 (1985); Meilgaard et al. 2006). Sensory panels in industry The method for sensory evaluation in the fish industry is to perform sensory analysis on the daily production. Therefore a sensory panel or trained inspectors should be established, usually within the compary. To avoid errors in the sensory evaluation in daily quality control, it is necessary to follow well-defined grading systems or guidelines and standards. The assessors must be selected, trained and have clear and descriptive guidelines to produce reliable results from sensory evaluation. The Codex guidelines for sensory evaluation of fish and shellfish in laboratories (Codex Standards 1999) can be regarded as helpful for selecting and training panellists in the industry. Generally, the selection and training methods for sensory panels in research (see previous section) apply for sensory panels in industry as well. Depending on the methods used in the industry, the selection criteria and norms for selection may not be as strict as for panels in research. Panellists for industry sensory panels are usually recruited within the company. This is logical but has some drawbacks as the staff may be too involved in the process or product, which might bias their evaluation. However, a company panel is more neutral to the sensory evaluation of the incoming raw material than the production manager. A panel member should not be pressed to make decisions about the product deposition. One of the advantages of an internal panel in a processing company is that the assessors are more likely to be motivated to perform the testing at inconvenient hours of the day (i.e. nightshift). The selection criteria for industry panels (Codex Standards 1999) are the ability to perceive and recognise odours in general and those relevant for the product, and ability to perceive colours and recognise tastes (if tasting is involved in one of the methods used). Another criterion is also to be able to rely on sensory perceptions and to report them appropriately, and to disconnect personal preferences and work-related preference. Training an industry sensory panel is similar to training a research panel. Most likely, the methods used during the training are grading and scaling instead of descriptive tests, and they are more species- or product specific. In any situation, the panel will eventually become an expert panel because of the limited variation in products and methods. This has the advantage that the results will become more precise and accurate; on the other hand, it has the disadvantage that experts tend to skip important steps within the evaluation process and become biased. In daily quality control there is a risk of repeating the same scores and the possibility of missing a developing trend or even defects in samples.

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19.1.3

415

Facilities for sensory evaluation of seafood

The physical settings should be designed to reduce disturbing factors. The facilities should be centrally located, temperature controlled, have adequate light and be free from odour and noise. Sensory facilities in research Textbooks on sensory evaluation of foods often describe the facilities required for sensory evaluation. There are international and national standards and guidelines for the design and construction of sensory assessment rooms (ISO 8589 (1988); NMKL Procedure No. 6 (1998); Meilgaard et al. 2006). The recommendations in these publications are intended for establishments or situations where sensory evaluation is a major activity, for example R&D laboratories of food companies and research institutes. Sensory facilities in industry Sensory evaluation can be practised at different levels in fish processing. For quality control, it must be performed no less accurately and conscientiously than in R&D laboratories, but the requirements need not be as elaborate. Sensory evaluation of whole fish is generally done by trained assessors (experienced experts or fish inspectors) in the reception or processing halls of fish factories or at auction sites. However, sensory evaluation of cooked fillets in quality control must be performed in rooms with special facilities. General requirements can be stated about the sensory facilities according to ISO 8589 (1988). A special room for sample preparation is preferred adjacent to, but separate from, the testing area. At least some separation should be provided so that the panellists cannot see the samples being prepared because this might enhance expectation error. The testing area should be located near the preparation room. In the testing area, noise level must be kept to a minimum and no unauthorised persons allowed to enter. Lighting is very important. Light must be adequate and appropriate: daylight (either real daylight or artificial daylight with a colour temperature of more than 5000 K). The ambient lighting in the testing area must be uniform, shadow free and controllable. The room should be free of any foreign odour. This can be achieved by installing an air conditioner with activated carbon filters. If necessary, a slight positive pressure may be created in the testing area to reduce the inflow of air from other areas. The testing area must be readily accessible to all panellists. A separation between panellists is necessary to avoid any distraction during the evaluation. No eating, drinking or smoking should be allowed in the testing area. The testing area must be easy to clean and disinfect. Regular cleaning and disinfecting must take place. The cleaning agents used must not leave odours in the testing area.

19.1.4

Seafood sample preparation

Preparation of seafood samples depends on the type or nature of the sample and the sensory method applied, for example whether the fish/seafood is evaluated whole, as raw fillets or cooked.

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Sampling for sensory analysis must be as representative as possible. Consideration must be given not only to how and where the samples are collected or cut, but also that the preparation must have a minimal influence on sensory characteristics of the samples. The sampling system, methods and procedures for sensory evaluation must be very well defined for sensory evaluation to serve its purpose in quality management. The sampling plan for pre-packaged food as described in the Codex sampling plans for pre-packaged foods (Codex Standards 1969) might be used as a basis for sampling plans. Lots and batches have to be defined before deciding the number of samples taken from each batch. A sample must be collected randomly from a defined homogeneous lot, and the number of fish/fillets or units within a sample will depend on the size of each unit. All samples should be individually blind-coded, with three-digit codes.

Whole fish and seafood, raw fillets For medium-sized or large fish (for example cod, red fish or salmon), at least three to five should be included within a sample, but 10 for small fish species, such as herring (Martinsdóttir et al. 2001). Each fish sample is blind coded and placed in random order on a clean table. The fish should be kept cool (2–7 °C) and constant during the evaluation.

Heat treated/cooked seafood samples For fat fish species, it is important to consider if the samples should be with or without skin. The dark muscle is immediately under the skin, so if the skin is removed from the fillets some of the dark muscle will be removed. This can influence intensity of rancid flavour because of the high lipid content in the dark muscle. Each sample serving should be 40–50 g for each assessor. For very small fish, the whole fillet might be required to obtain the right amount of sample. However, for large fish, to ensure that all assessors get their sample from the same part of the fish, samples are collected from the loin part of the fillets. Shrimp and prawns can be very different in size. Some may be big enough to be assessed as one sample (one ‘mouthful’), but others may be so small that several need to be included as one sample. The samples must be cooked or heated sufficiently, but not too much, and served at appropriate consumption temperatures. Cooking time and temperature needed should be tested using samples in appropriate containers and defined before the actual sensory evaluation. Fillets may be cooked in convection ovens at 100 °C in porcelain or heat-tolerant glass containers covered with lids, or aluminium boxes covered with aluminium foil, to a core temperature of 70 °C. The type of aluminium foil used must be controlled to ensure that it does not give a metal taste to the sample. If convection steam ovens are used, containers do not need to be covered during the heat treatment. Cooking in a water bath will require diffusiontight plastic bags under vacuum and heating at lower temperatures (70–85 °C) to a core temperature of 70 °C. Prawns and shrimps are cooked in fresh water for 3 minutes and served at normal consumption temperature. Samples can be cut into portions before or after heat treatment. If the texture and appearance is of no importance, it is possible to make minced samples to eliminate the differences between individuals.

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Sensory evaluation of samples is usually done in duplicate, and sometimes more replications are included. Different types of seafood product Different types of seafood product demand different sample preparation, depending on how the products are normally consumed. Some products might require heating before serving (see the section on heat treated/cooked samples), but other products are served cold or at room temperature. It is important to take into account the normal serving temperature and to ensure that the temperature remains constant during assessment. The usual serving size should be around 40–50 g, but the sample cut depends on the product. As an example, smoked fish samples may be prepared in different ways; cold smoked salmon may be collected as thin slices (0.5–1.0 cm) cut at the right angle to the fillet surface, or as fillets, served on a plate with lid or aluminium foil, at room temperature. Hot smoked rainbow trout may be collected as a fraction from the anterior, mid-dorsal part of the fillet and served at room temperature.

19.2

Application of sensory evaluation to fish and other seafood

Sensory evaluation is applied in research, in quality control, in product development and consumer studies. Seafood freshness is of special interest as seafood is a very perishable food with a limited shelf life.

19.2.1

Seafood research

In seafood research, sensory analysis is often used to monitor effects of different methods of handling, changes in processes or storage conditions on sensory quality and storage life of fish and seafood products. For wild-caught fish, this might be different fishing gear, new icing methods, and so on. For aquaculture, this might be differences in rearing conditions, feeding conditions, dietary modulation, (pre)slaughter handling, post-slaughter processing, and so on. Depending on the research questions, different methods can be used. As an example, for studying the effect of fishing gear and icing on quality and shelf life, the quality index method (QIM) (see Chapter 20.4.) is recommended. For other changes, descriptive methods can be used, such as quantitative descriptive analysis (QDA) (see Chapter 20.5.). Both methods will result in quantitative data and will form a basis for explanatory research, based on small differences. A difference test will be more simple but less useful for explanatory research. For some purposes an intensity scale or ranking test is more simple than QDA, but is sufficient to measure the effect of dietary modulation gradients.

19.2.2

Freshness of seafood

Bremner and Sakaguchi (2000) put forward an approach to the overall idea of freshness, as the total set of characteristics of a recently harvested product that bear on its ability to meet

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stated or implied requirements. Quality evaluated by the senses not only describes freshness sensory properties of the fish but also includes factors such as bleeding and storage that may normally be considered as good manufacturing practices. Those factors are described in section 19.2.4. Freshness makes a major contribution to the quality of fish and fish products (Oehlenschläger 1997). Sensory evaluation is the most important method and is widely used for freshness assessment in the fish sector. Many schemes have been developed for sensory evaluation of raw fish. Grading methods have been most common for buying and selling fish (Anonymous 1996). The former Torry Research Station (Shewan et al. 1953) developed the first modern and detailed method for freshness evaluation. In the article, score sheets for sensory evaluation of white raw fish are described and the sensory factors classified. The general raw appearance and odour, texture of the fish and the flesh, including belly flaps, are described as well as the odour, flavour and texture of cooked fish. The Torry scheme has been commonly used by producers and buyers of fish, especially in the UK. It is critical for a sensory system used in quality management that it reflects the different quality levels in a simple, reliable and documented way. Therefore improved seafood freshness systems that are both rapid and more objective have been developed for various fish species. The QIM is such system and has several unique characteristics. It is based upon a scheme originally developed by the Tasmanian Food Research Unit (Bremner 1985). QIM has to be adapted to each fish species. More information about QIM is described in Chapter 20.3.

19.2.3

Shelf-life studies

Sensory methods are a very important part of shelf-life studies. In these, the questions to be answered mainly deal with determining how long a product can be stored before the sensory quality changes make the product unacceptable and how the products change with storage time at a given temperature (Lyon et al. 1992). For the fish industry, shelf life is based on how long the company is prepared to accept the product in the marketplace before sale (Barbosa and Bremner 2002). For consumers, the end of shelf life is when the product no longer has acceptable sensory characteristics. The end of shelf life for whole fish is defined as the number of days that whole, fresh (gutted) fish can be stored in ice until it becomes unfit for human consumption. Shelf life of fish and fish products is thus the whole period of time in which it is regarded as being fit for human consumption. Spoilage due to microbial activity is the main limitation of the shelf life. Another cause of spoilage may be rancidity, especially in fat fish species. The QIM for whole fish can be used in shelf-life studies to predict storage time in ice and remaining shelf life. Estimated storage time in ice is defined as the number of days the fish has been stored in ice. From these results, a prediction can be calculated for the remaining shelf life (equal to the shelf–life estimated storage time). In the QIM reference manual (Martinsdóttir et al. 2001), an estimated shelf life for 12 fish species is given, assuming optimal storage conditions, namely storage in ice without fluctuations in temperature. The shelf life and the estimated storage time in ice are based upon the outcome of well-controlled storage experiments with whole, fresh fish (gutted) stored in ice under good manufacturing conditions on board the vessel, which implies proper gutting, washing and use of the appro-

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priate fish/ice ratio. The end of storage time is often defined when a trained sensory panel detects spoilage flavour in cooked samples of the fish. A linear relationship between the quality index and storage time in ice has been found, and the best fit of the regression lines calculated for each species are also shown in the manual. The regression lines are used to predict storage time in ice after evaluation of the quality index and estimate the remaining shelf life.

19.2.4

Quality control in fish industry

The application of sensory evaluation in the fish industry is mostly in quality control that considers several aspects like product variability, processing conditions and marketing objectives. The objective of using sensory evaluation in companies should be to develop an efficient way to understand the key attributes that affect consumer liking. Further, it should be to assess which raw-material and processing variables affect the final sensory properties and to use a system to measure and control the sensory attribute (Munoz et al. 1992). The methods best suited in quality control are those measuring variability like ratings and overall conformance to a product concept. Methods like overall difference tests and hedonic tests should be avoided. Sensory evaluation systems can very well be placed within the hazard assessment critical control point (HACCP) or within any quality control system as incoming control, process control and end-product control. They are most important when sensory analysis is used to define strict and exact procedures, standards (norms) and tolerances. Using the results of sensory evaluation as a part of a quality control program, analysis of the data might consist of comparing the results with a lower and upper limit that have previously been established by the management and the buyer. When QIM results are used, these limits refer to the total of demerit points or the quality index. When control charts are used to monitor seafood freshness of specific seafood over time, they permit immediate detection of trends and outof-control conditions, thus allowing appropriate handling and processing procedures to be corrected and the variability to be reduced (Martinsdóttir 2002). In quality control, standards for fish sampling are rarely available for specific fish products. This makes it difficult to set norms and tolerances. However, for use in training assessors for fish, freshness or other specifications of incoming goods (for example whole fish/fillets) with known history of handling, time and temperature from catch can be used. In specific process control and/or product control, detection of defects such as bruising, bones, scales, parasites, blood-spots, parts of skin and intestines, ruptured fillets, freezer burn, and so on is a part of the inspection. Defects can be detected by visual methods. All defects have to be defined and described in instructions and guidelines for the inspectors, like the number of areas damaged by guts and visible cod-worms. Defects like bruises and blood-spots can be measured and counted, and parasites (for example anisakis) can be counted and compared with standards. Colour cards are available for colour estimation of some fish products, such as for salmon grading and selection. Descriptions, eventually with photographs such as how a fillet should be placed in a consumer package, could be used. Taints like naturally occurring muddy, earthy off-odours can be detected in seafood. Sensory evaluation is an effective way of measuring such taints. Suitable methods for these

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types of quality control are described in Chapter 20.2 (difference tests) and 20.3 (grading methods).

19.2.5

Regulation

The EU regulation 2406/96 (Anonymous 1996) refers to only one sensory method for seafood: the EU-quality grading scheme for fresh fish. This method is to be used at the first point of sale and implies freshness and other quality items (parasites, pressure marks, injuries, blemishes and bad discolouration). There are different schemes for whitefish, bluefish, selachii, cephalopods and crustaceans. This method is to be used by experts or by the competent authority (inspection body). For HACCP, no specific sensory methods are described. For every critical control point, the best control method must be defined; sometimes sensory methods to estimate the freshness of, for example, incoming goods are very useful.

19.2.6

Product development

Sensory analysis can help in finding the answers to many of the questions in product development. In developing new products, modifying existing products or the production process, some of the questions are: does the product satisfy a consumer need? Will it be acceptable to consumers, wholesalers and retailers? Is it possible to change an ingredient? Is it unique? Will the production technology influence the sensory properties? What products does it replace or compete against? These questions are important for a food business to contribute to the product’s success. Sensory analysis may help to shorten development time and contribute to a better understanding of the product’s behaviour (Lyon et al. 1992). The differences test can be used for a fast sensory screening, the descriptive sensory analysis to make a sensory characterisation of the product, and the consumer test to evaluate the preferences and acceptability. Sensory analysis by a trained panel is often used to explain and predict consumer preferences, as described in this chapter (Sveinsdóttir et al. 2009).

19.2.7

Consumer studies

Eating-quality preference decisions are ultimately made during consumption. The structural and flavour-active components of the seafood are perceived by the sensory systems.This information is integrated with the consumer’s recollection of previous seafood-eating experiences, with the expectations created for the consumer by the retailer, and by the means of presenting the seafood as a product. Eating quality will vary from one species of seafood to another, and again by the choice of storage, handling, packaging, transportation, and so on

Sensory evaluation of seafood: general principles and guidelines Correlation Loadings (X and Y)

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• • O-Sour • • F-Frozen storage • F-Sour • O-Frozen storage

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Cod products stored for 10 days at 0–2°C –1.0

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•• •• • • •• •• • •• • • • • F-Fresh/sweet • • • • • • O-Fresh/sweet • • • ••••••• • •• • • •• Freshness (Torry Score) •• • • • T-Juicy • • • T-Tender • •• • • •

Cod products stored for 2 days at 0–2°C

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Figure 19.1 Multivariate analysis (PCR) of consumer liking (䊉) in relation to sensory characteristics of cod products; F, flavour; O, odour; T, texture.

made at each point in the chain from seafood catch, or slaughter, to consumption. Different consumers will have different experiences with seafood, related to availability and frequency of consumption, that will determine individual preferences. Consumer preferences or acceptability can be measured with hedonic methods (see Chapter 20.6). Consumer acceptance of fish is very much related to its freshness, even though consumer criteria for freshness have not yet been established. The demand for freshness or knowledge of the freshness stage has been from retailers. Retailers play a significant role in influencing consumer perception of quality (Bisogny et al. 1987). Several authors have studied the consumer awareness of quality factors related to fish. A high correlation was found between descriptions of trained panels and the opinions of consumers, even though the trained panel used a wider range of intensity scales (Sawyer et al. 1988; Bech et al. 1997). Further, these authors showed that it is possible to establish a link between consumers’ demand for taste quality and attributes from sensory profiling. Perception of taste determines a major part of overall attitudes to buying fish (Bredahl and Grunert 1997). Therefore consumer studies with tasting of products are very important. Sveinsdóttir et al. (2003) studied acceptability to Icelandic consumers and the sensory quality of fresh, thawed and modified-atmosphere-packed cod fillets of different storage time. Fish products with odours and flavours characteristic for very fresh fish were overall more accepted by consumers than products approaching the end of shelf life (Figure 19.1). However, consumers were also influenced by other sensory attributes such as texture. The

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reasons for the consumers’ acceptability are most reliably identified by sensory evaluation outperformed in parallel on the tested products, with methods such as QDA (chapter 20.5). Although different sensory attributes influence consumers’ acceptability of products, consumers often find it difficult to explain why they prefer one product to another, and the results may be difficult to interpret. However, descriptive sensory analysis performed by trained sensory panels provides accurate and detailed description of the products under study. The consumer acceptance or preference may then be related to the sensory characteristics of products by preference mapping (Greenhoff and MacFie 1994; McEwan 1996). Preference mapping has been used to study acceptability of various food products such as meat (Helgesen et al. 1997), beverages (Guinard et al. 2001; Geel et al. 2005), fruits (DaillantSpinnler et al. 1996; Thybo et al. 2003 ) and cheese (Murray and Delahunty 2000; Westad et al. 2004). A recent study on consumer acceptability in four European countries of different cod products (Sveinsdóttir et al. 2009) showed that consumers have very different preferences. Preference mapping showed that one group of consumers had particularly high preferences for sensory attributes characteristic for farmed cod, such as rubbery and meaty texture, meat odour and flavour, whereas another group preferred freshness characteristics of wild cod, such as sweet and metallic flavours. A third group preferred storage characteristics, for example hints of frozen storage and tablecloth odour, dark and discoloured appearance. The fourth group had generally low preferences, but had a tendency to prefer juicy, soft and tender cod. Further, a fifth group was identified, with generally high preferences, regardless of cod product.

19.3

References

Anonymous (1996) Council Regulation (EC) No 2406/96 of 26 November 1996 laying down common marketing standards for certain fishery products. Barbosa, A. and Bremner, A. (2002) The meaning of shelf-life. In: H.A. Bremner (Ed.) Safety and Quality Issues in Fish Processing. Woodhead Publishing Limited, UK, pp. 173–190. Bech, A.C., Kristensen, K., Juhla, H.J. and Poulsen, C.S. (1997) Development of farmed smoked eel in accordance with consumer demands. In: J. Luten, T. Børresen and J. Oehlenschläger (Eds) Seafood from Producer to Consumer, Integrated Approach to Quality. Proceedings of the International Seafood Conference on the occasion of the 25th anniversary of the WEFTA, Noordwijkerhout, The Netherlands, 13–16 November, 1995. Amsterdam, Elsevier Science B.V., pp. 21–30. Bisogny, C.A., Ryan, J. and Regenstein, J.M. (1987) What is fish quality? Can we incorporate consumer perceptions. In: D.E. Kramer and J. Liston (Eds) Seafood Quality Determination, Proceedings of the International Symposium on Seafood Quality Determination, Coordinated by the University of Alaska Sea Grant College Program, 10–14 November 1986, Anchorage, Alaska, U.S.A. New York, Elsevier Science B.V., pp. 547–573. Bjerkeng, B., Refstie, S., Fjalestad K.T., Storebakken, T., Røbotten, M. and Roem, A.J. 1997. Quality parameters of the flesh of Atlantic salmon (Salmo salar) as affected by dietary fat content and full-fat soybean meal as a partial substitute for fish meal in the diet. Aquaculture 157: 297–309. Bredahl, L. and Grunert, K.G. (1997) Determinants of the consumption of fish and shellfish in Denmark: an application of the theory of planned behaviour. In: J. Luten, T. Børresen and J. Oehlenschläger (Eds) Seafood from Producer to Consumer, Integrated Approach to Quality. Proceedings of the International Seafood Conference on the occasion of the 25th anniversary of the WEFTA,

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Noordwijkerhout, The Netherlands, 13–16 November, 1995. Amsterdam, Elsevier Science B.V., pp. 3–19. Bremner, H.A. (1985) A convenient easy to use system for estimating the quality of chilled seafood. Fish Processing Bulletin 7: 59–70. Bremner, A. and Sakaguchi, M. (2000) A critical look at whether ‘freshness’ can be determined. Journal of Aquatic Food Product Technology 9(3): 5–25. Codex Standards (1969) Codex standards for methods of analysis and sampling, ‘Sampling Plans for Prepackaged Foods (AQL 6.5)’, XPT 13-1969, Rome, FAO/WHO Codex Alimentarius. Codex Standards (1999) Codex standards for fish and fishery product, ‘Guidelines for the sensory evaluation of fish and shellfish in laboratories’, CAC-GL 31-1999, Rome, FAO/WHO Codex Alimentarius. Daillant-Spinnler, B., MacFie, H.J.H., Beyts, P.K. and Hedderley, D. (1996) Relationships between perceived sensory properties and major preference directions of 12 varieties of apples from the Southern Hemisphere. Food Quality and Preferences 7: 113–126. Geel, L., Kinnear, M. and de Kock, H.L. (2005) Relating consumer preferences to sensory attributes of instant coffee. Food Quality and Preferences 16: 237–244. Greenhoff, K. and MacFie, H.J.H. (1994) Preference mapping in practice. In: H.J.H. MacFie and D.M.H. Thomson (Eds) Measurements of Food Preferences. Academic and Professional, London, pp. 137–166. Guinard, J.X., Uotani, B. and Schlich, P. (2001) Internal and external mapping of preferences for commercial lager beers: comparison of hedonic ratings by consumers blind versus with knowledge of brand and price. Food Quality and Preferences 12: 243–255. Helgesen, H., Solheim R. and Næs, T. (1997) Consumer preference mapping of dry fermented lamb sausages. Food Quality and Preference 8: 97–109. ISO 3972 (1991) Sensory analysis – Methodology – Method of investigating sensitivity of taste. ISO 4120 (2004) Sensory analysis – Methodology – Triangle test. ISO 5495 (2005) Sensory analysis – Methodology – Paired comparison test. ISO 5496 (2006) Sensory analysis – Methodology – Initiation and training of assessors in the detection and recognition of odours. ISO 6658 (2005) Sensory analysis – Methodology – General guidance. ISO 8586-1 (1993) Sensory analysis – General guidance for selection, training and monitoring of assessors – Part 1: Selected assessors. ISO 8586-2 (1994) Sensory analysis – General guidance for selection, training and monitoring of assessors – Part 2: Experts. ISO 8589 (1988) Sensory analysis – General guidance for the design of test rooms. ISO 11035 (1994) Sensory analysis – Identification and selection of descriptors for establishing a sensory profile by a multidimensional approach. International Standard, 1st edition. ISO/CD 13300 – 1 (2002) Sensory analysis – General guidance for the staff of a sensory evaluation laboratory – Part 1: Staff responsibilities. ISO/CD 13300 – 2 (2002) Sensory analysis – General guidance for the staff of a sensory evaluation laboratory – Part 2: Recruitment and training of panel leaders. Lyon, D., Francombe, M.A., Hasdell, T.A. and Lawson, K. (1992) Guidelines for Sensory Analysis in Food Product Development and Quality Control. Chapman & Hall, London, UK, pp. 131. Martinsdóttir, E. (2002) Quality management of stored fish. In A. Bremner (Ed.) Safety and Quality Issues in Fish Processing. Woodhead Publishing Ltd, UK, pp. 360–378. Martinsdóttir, E., Sveinsdóttir, K., Luten, J., Schelvis-Smit, R. and Hyldig, G. (2001) Reference Manual for the Fish Sector: Sensory Evaluation of Fish Freshness. QIM-Eurofish, The Netherlands. McEwan, J.A. (1996) Preference mapping for product optimization. In: T Næs and E Risvik (Eds) Multivariate Analysis of Data in Sensory Science. Elsevier Science B.V., London, pp. 71–102.

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Meilgaard, G., Civille, V. and Carr, B.T. (2006) Sensory Evaluation Techniques, 4th edition. CRC Press, New York. Munoz, A.M., Civille, G.V. and Carr, B.T. (1992) Sensory Evaluation in Quality Control. Van Nostrand Reinhold, New York. Murray, J.M. and Delahunty, C.M. (2000) Mapping consumer preference for the sensory and packaging attributes of Cheddar cheese. Food Quality and Preferences 11: 419–435. NMKL Procedure No. 6 (1998) Generelle retningslinier for kvalitetssikring af sensorsike laboratorier. Norsk metodikkomitee for levnedsmidler. Oehlenschläger, J. (1997) Sensory evaluation in inspection. In: G. Ólafsdóttir, J. Luten, P. Dalgaard, M. Careche, V. Verrez-Bagnis, E. Martinsdóttir and K. Heia K (Eds) Methods to Determine the Freshness of Fish in Research and Industry. Proceedings of the Final Meeting of the Concerted Action ‘Evaluation of Fish Freshness, AIR3CT942283, Nantes Conference, November 12–14. International Institute of Refrigeration, Paris, pp. 339–344. Sawyer, F.M., Cardello, A.V. and Prell, P.A. (1988) Consumer evaluation of sensory properties of fish. Journal of Food Science 53: 12–24. Shewan, J.M., Macintosh, R.G., Tucker, C.G. and Ehrenberg, A.S.C. (1953) The development of a numerical scoring system for the sensory assessment of the spoilage of wet white fish stored in ice. Journal of the Science of Food and Agriculture 4: 283–298. Sveinsdóttir, K., Thorkelsdóttir, Á. and Martinsdóttir, E. (2003) Consumer survey: cod fillets packed in air and modified atmosphere (MAP). In Proceedings of the TAFT 2003 Conference, 10–14 June 2003, Reykjavik, Iceland. The Icelandic Fisheries Laboratories. Sveinsdóttir, K., Martinsdóttir, E., Green Petersen, D., Hyldig, G., Schelvis, R. and Delahunty, C. (2009) Sensory characteristics of different cod products and consumer preferences. Food Quality and Preference 20: 120–132. Thybo, A.K., Kühn, B.F., Martens, H. (2003) Explaining Danish children’s preferences for apples using instrumental, sensory and demographic/behavioural data. Food Quality and Preferences 15: 53–63. Westad, F., Hersleth, M. and Lea, P. (2004) Strategies for consumer segmentation with applications on preference data. Food Quality and Preferences 15: 681–687.

Chapter 20

Sensory evaluation of seafood: methods Emilia Martinsdóttir, Rian Schelvis, Grethe Hyldig and Kolbrun Sveinsdóttir

20.1

Introduction

Analytical objective sensory tests can be divided into two groups: discriminative tests and descriptive tests. Discriminative testing is used to determine if a difference exists between samples (triangle test, ranking test). Descriptive tests are used to determine the nature and intensity of the differences (profiling and quality tests). The subjective test is an affective test based on a measure of preference or acceptance of consumers (hedonic tests). Sensory responses can be variously measured and can be assigned to sensory impression in different ways: nominal data, ordinal data, interval data and ratio data. Nominal data are obtained if the samples are placed in groups that differ in name but do not have a quantitative relationship. The panellist places the samples in groups belonging to ordered series (namely slight, moderate and strong). Interval data are obtained if the samples are placed into numbered groups separated by an interval. In sensory evaluation of seafood, grading, ranking and scaling methods are the most frequently used methods. However, difference tests can be relevant to use in selected cases. Grading is a useful method of evaluation and is often used in commerce. It depends on one or two product experts. Graders usually learn the scale from other graders. Grading schemes usually have four or five steps. Grading suffers from the drawback that it is difficult or impossible to correlate other, measurable properties statistically. The EU-scheme (Chapter 20.3.1) is an example of a grading scheme. In ranking, three or more samples are arranged in order of intensity or degree of some specific attribute. For example, the colour of four different smoked salmon samples can be ranked in order of intensity. These methods are useful for research but less so for industry. A category scaling is a method where the panellists are asked to rate the intensity of a particular stimulus by assigning a value on a limited numerical scale.

20.2

Difference tests

Difference tests can be used to determine whether a difference exists in a single sensory attribute or in several. They determine whether there exists a perceptible difference in a given attribute, and the specification of the direction of difference. However, they do not 425

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give any indication of the extent of the difference. If the difference between the samples is very large and thus obvious, difference tests are not useful. The most frequently used difference tests are ranking (ISO 8587, 1988), the triangle test (ISO 4120, 2004) and the paired comparison test (ISO 5495, 2005).

20.2.1 Attribute difference tests: ranking In ranking, assessors receive three or more (two is a paired comparison test) samples. Their task is to arrange them in order according to the degree to which they exhibit some specified characteristics, for example four samples of herring ranked for the degree of rancidity. The number of assessors is chosen based on the sensitivity desired for the test. The rank numbers received by each sample are summed, and the resulting rank sums indicate the overall rank order of the samples. Rank orders cannot meaningfully be used as a measure of intensity, but they are amenable to significance tests such as the χ2-test and Friedman’s test. Note that the lack of a difference between samples in one attribute does not imply that no overall difference exists. Usually ranking can be done more quickly and with less training than evaluation by other methods. Thus ranking is often used for preliminary screening. The method gives no individual differences among samples and it is not suited for sessions where many criteria have to be judged simultaneously.

20.2.2 Overall difference test Triangle test The most frequently used difference test in sensory analysis of fish products is the triangle test. The number of assessors is chosen based on the sensitivity desired for the test. The assessors receive a set of three coded samples, are informed that two of the samples are identical in their sensory properties and that one is different. The assessors report which sample they believe to be different, even if the selection is based only on a guess. The triangle test is applicable even when the nature of the difference is unknown (that is, it determines neither the size nor the direction of difference between samples, nor is there any indication of the attribute(s) responsible for the difference). The method is applicable only if the products are fairly homogeneous. Analysis of results from the triangle test is done by comparing the number of correct identifications to the number expected by use of a statistical table. For example, if the number of responses is 12, there must be 9 correct responses to achieve a significant answer (1% level). Paired comparison This method can be used if the objective is to determine in which way a particular sensory attribute differs between two samples. In the paired comparison test, the assessors receive a set of two samples. They designate the sample that they consider the most intense for the sensory attribute under consideration, even if this choice is based only on a guess. The absence of difference for the attribute under study does not necessarily mean that no

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difference exists between the samples. As an example, dipping a fish fillet into a weak salty solution before freezing may influence the salty taste of the fillet. The assessors could be asked which sample is saltier than the other. The number of times that each sample is selected is counted, and the significance is determined by reference to a statistical table, taking into consideration the results obtained for the expected sample (one-sided test) or the highest number of responses obtained for either of the samples (two-sided test). For example, if the number of responses is 12, there must be 11 correct responses to achieve a significant answer (1% level).

20.2.3

Difference from control

Difference from control (degree of difference) was originally described by Aust et al. (1985). This is used when the objective is the combination of determining if a difference exists between samples and a reference or control sample, and estimating the size of such a difference. Quality assurance/quality control and storage studies are cases in which the relative size of the difference may affect the decision making. This difference test is appropriate if a simple difference cannot be used because of the normal heterogeneity of products like seafood products. Difficulties can occur in using the method for seafood because it requires a consistent reference sample or standard. Reference samples are usually very difficult to keep stable. The method described as a sensory quality control test may be categorised as a descriptive qualitative and quantitative method. This is described in the standard International IDF Standard 99B (1995) which applies to dairy products. This method has also been developed for drinking water (NMKL Method, 2005) and is very thoroughly described in the standard. Panellists are trained to recognise the different attributes occurring in the product during production or storage. In some cases, attributes can be rated in addition to the overall difference. When the panellists detect a difference between the reference samples, the deviation is indicated, eventually using a scale to indicate how much the sample deviates from the reference. Different forms of rating scales exist (Munoz 1992). It is useful to have a blind control within each session, which is a blind-labelled sample of the reference. This additional sample could be from a product of a different batch, which should be very similar. This would provide information about the test product’s variability. Terms from a nomenclature list describing deviations from appearance, odour, flavour and texture should be developed for each product. When the panellist detects a difference, he or she should choose a word from the nomenclature list as described. The method could be recommended for use with some seafood products like fish oil.

20.3

Grading schemes

Grading is the process of applying a categorical value to a lot or group of products. Although this may be based on sensory information, it is no longer generally considered an example of sensory evaluation methodology. Sensory grading most often involves a process of integration of perceptions by the grader. The grader is asked to give one overall rating of the combined effect of the presence of the positive attributes, the blend or balance of those

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attributes, the absence of negative characteristics and the comparison of the products being graded with some written or physical standard. Grading has the advantage that it offers the possibility of selecting products for different qualities. However, statistical correlation with measurable physical or chemical properties is difficult or impossible. Consequently, many of the time-honoured grading scales are being replaced by the methods described below. Examples of grading schemes for fish are the EU quality grading scheme (EU-scheme) and the Torry scheme.

20.3.1

EU-scheme

Specific for seafood, the EU regulation: ‘Council Regulation (EC) No 2406/96 of 26 November 1996 (Anonymous 1996), laying down common marketing standards for certain fishery products’ has only one sensory method in place: the EU-scheme for fresh fish. This method is to be used at the first point of sale and implies freshness and other quality items (parasites, pressure marks, injuries, blemishes and bad discolouration). There are different schemes for whitefish, bluefish, selachii, cephalopods and crustaceans. It is to be used by experts or by the competent authority (inspection body). A drawback of this method is that no training, practical procedures and sampling plan are available. Therefore it is hardly possible for outsiders to use this method in a reliable and reproducible way. Furthermore, the method only uses general parameters to describe freshness quality, it does not take different sensory characteristics of different species into account, nor does it provide useful information about the past or remaining storage time as it is too general, applying to many different species that spoil at different rates. In addition, the assessor is forced to grade the fish based on several quality parameters, which increases the risk of overemphasis on one single criterion. The method is not suitable for predicting the shelf life, nor for statistical analyses of reliability and reproducibility.

20.3.2

Torry scheme

For sensory evaluation of fish fillets, it is common to cook the fillets and evaluate their odour and flavour. The Torry scale is the first detailed scheme developed for evaluating the freshness of cod (Shewan et al. 1953). Many schemes used for sensory evaluation of cooked fillets by a trained panel are based on this original work with some modifications. The Torry scale (Table 20.1) is the most frequently used industry scale for evaluating the freshness of cooked fish. It is used both by producers and buyers. It is a descriptive 10-point scale that has been developed for lean, medium fat and fat fish species. Scores are given from 10 (very fresh in taste and odour) to 3 (spoiled). It is considered unnecessary to have descriptions below 3, as the fish is then not fit for human consumption. The average score of 5.5 has been used as the limit for ‘fit for consumption’ (Martinsdóttir et al. 2001). Members of the sensory panel detect evident spoilage characteristics, such as sour taste and hints of ‘off’ flavours. In Martinsdóttir et al. (2001), results from storage experiments on cod and haddock from two different seasons showed a high correlation between quality index and Torry scores (Figure 20.1). It could be of importance for the industry to compensate the sensory method

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Table 20.1 Torry scoresheet for freshness evaluation of cooked lean fish such as cod, haddock and pollock. Odour Initially weak odour of sweet, boiled milk, starchy, followed by strengthening of these odours Shellfish, seaweed, boiled meat Loss of odour, neutral odour Wood shavings, wood sap, vanillin Condensed milk, boiled potato Milk jug odours, reminiscent of boiled clothes Lactic acid, sour milk, TMA Lower fatty acids (for example acetic or butyric acids) decomposed grass, soapy, turnipy, tallowy

Flavour

Score

Watery, metallic, starchy. Initially no sweetness but meaty flavours with slight sweetness may develop Sweet, meaty, characteristic Sweet and characteristic flavours but reduced in intensity Neutral Insipid Slight sourness, trace of “off”-flavours Slight bitterness, sour, “off”-flavours, TMA Strong bitterness, rubber, slight sulphide

10

Quality index, QI

20

9 8 7 6 5 4 3

10 9

15

8 10

7 6

5

QI, May QI, December Torry, May Torry, December

5 0 0

5 10 Storage time (days)

15

4

Figure 20.1 Relationship between quality index (QI) of whole cod and Torry scores of cooked cod fillets versus storage time.

of cooked fillets with the use of a sensory evaluation of whole raw fish with the same result. This would be beneficial as evaluation of the whole raw fish is more rapid and performed much earlier in the production chain. It is also of great importance to have a method that can show a linear relationship with storage time in ice, as the results can be used for product management when remaining shelf life can be predicted.

20.3.3

Taint

Contamination and taint can be naturally occurring (such as the muddy, earthy off-flavour) or man-made (for example, petroleum or other processing effluents). Off flavours in seafood can occur as a result of many factors. The farm-raised catfish industry considers environmental off-flavours associated with blooms of blue–green algae and other microbes to be its

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Table 20.2 Intensity rating scheme for off-flavours. Rating 0 1 2 3 4

Definition No off-flavour can be detected in the sample Off-flavour at threshold level can be detected by tasting the cooked sample Distinct off-flavour can be detected by tasting the cooked sample Off-flavour can be detected in the odour of the cooked sample Off-flavour can be detected in the odour of raw fish

Sources: Bett (1997), modified from Johnsen and Bett (1996).

most significant problem. Algal blooms can produce geosmin and 2-methylisoborneol (MIB), which impart a muddy, musty flavour in seafood. Microbial production of the muddy metabolites geosmin and MIB can be a problem in freshwater fish such as trout, bream and pike. Based on the values presented in the literature (Howgate 2004), the estimated odour detection threshold in water at normal ambient temperature of natural geosmin is 0.015 μg 1−1 and 0.035 μg 1−1 for MIB. Because geosmin and MIB are much more soluble in lipid than in water, the concentration of the chemical in the lipid phase will be much greater than in water phase. This will influence the threshold in fish with different lipid content. Sensory evaluation is the accepted method for monitoring off-flavours in seafood where detection of unknown compounds or compounds at low concentrations is necessary (for example 1 part per billion (ppb) for geosmin and MIB) (Bett and Dionigi 1997). In research, the most appropriate method would be a descriptive method to determine the intensity of the off-flavour. However, in industry a more practical method is needed to discern off-flavoured fish from acceptable fish. Bett (1997) has developed a quality rating system for microwaved cooked fish (Table 20.2). This paper includes well-defined panel selection, training and sample preparation procedures. Extensive training is necessary for assessors to evaluate specific taints (Codex Standards 1999).

20.4 20.4.1

Quality index method Introduction

The quality index method (QIM) is a freshness grading system for seafood. It is based on a scheme originally developed by the Tasmanian Food Research Unit (TFRU) (Bremner 1985). QIM is widely accepted as a reference method in research. For further development it is important that the fish sector implements the method (Martinsdóttir et al. 2003). In quality management it is important to be able to apply a sensory system that reflects the different quality levels in a simple, reliable and documented way. QIM has those advantages, in addition to being rapid, cheap to use, non-destructive and objective compared with other sensory methods. Further, it is easy to work with as it includes instructions. It is a convenient method to teach inexperienced people to evaluate fish, and to train and monitor performance of panellists. As the quality index (the total sum of scores, referred to as the QI) is designed to increase linearly with storage time, the information may be used in production management (Figure 20.2).

Sensory evaluation of seafood: methods 25

Cod Salmon

20 Quality index, QI

431

15 10 Remaining shelf life

Remaining shelf life

5 0 0

5

10

Predicted storage time in ice (days)

15

20

Estimated maximum storage time

Figure 20.2 Use of QIM in estimating past and remaining storage time.

Several papers and book chapters describe the advantages of QIM and its use in research and industry, such as Hyldig and Nielsen (1997), Luten and Martinsdóttir (1997), Martinsdóttir et al. (2001), Schelvis-Smit and Luten (2003) and Martinsdóttir et al. (2003). Further, Hyldig et al. (2007) provide a detailed overview of QIM, such as the principle of QIM, development, training of QIM inspectors, application of the method and interpretation of results. The QIM-Eurofish Foundation (www.qim-eurofish.com) is an alliance between three fish research institutes in the Netherlands, Iceland and Denmark, which have been the most involved in research concerning QIM, development and allocation of the method. The aim of QIM-Eurofish is to promote and implement the use of QIM as a versatile quality tool within fisheries distribution or production chains in Europe. Manuals covering QIM schemes for 13 commercially important species have been published in 11 European languages (Danish, Dutch, English, French, German, Greek, Icelandic, Italian, Norwegian, Portuguese and Spanish; Martinsdóttir et al. 2001, 2004). The manuals include relevant high-quality photographs of attributes, and QIM calibration curves which offer the opportunity to estimate the remaining shelf life on the basis of a QIM assessment. Furthermore, guidelines for sensory evaluation, sample preparation, selection and training of panellists are given.

20.4.2

The principle of the quality index method

QIM is based on characteristic changes that occur in seafood with storage time in ice. A score from 0 to 3 points is given for changes of parameters in outer appearance (for example of eyes, skin and gills) and changes that occur in odour and texture (Tables 20.3–20.5). The colour of blood and fillets (or the cut surface at the flaps) is evaluated in gutted fish. For some fish species that are not gutted, such as redfish, dissolution of viscera is evaluated. The descriptions corresponding to each score for each parameter evaluated are listed in the QIM

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Table 20.3 QIM scheme for farmed salmon (Salmo salar). Quality parameter Skin

Colour/appearance

Mucus

Odour

Texture

Eyes

Pupils

Form

Gills

Colour

Mucus

Odour

Abdomen

Blood in abdomen Odour

Quality Index (QI)

Description

Score

Pearl-shiny all over the skin The skin is less pearl-shiny The fish is yellowish, mainly near the abdomen Clear, not clotted Milky, clotted Yellow and clotted Fresh seaweed, neutral Cucumber, metal, hay Sour, dish cloth Rotten In rigor Finger mark disappears rapidly Finger leaves mark over 3 seconds Clear and black, metal shiny Dark grey Matt, grey Convex Flat Sunken Red/dark brown Pale red, pink/light brown Grey-brown, brown, grey, green Transparent Milky, clotted Brown, clotted Fresh, seaweed Metal, cucumber Sour, mouldy Rotten Blood red/not present Blood more brown, yellowish Neutral Cucumber, melon Sour, fermenting Rotten/rotten cabbage

0 1 2 0 1 2 0 1 2 3 0 1 2 0 1 2 0 1 2 0 1 2 0 1 2 0 1 2 3 0 1 0 1 2 3 0–24

Source: Sveinsdóttir et al. 2002

scheme. Each parameter within the QIM scheme is evaluated independently. The description for score zero (0) should be characteristic for the parameter at the very beginning of the shelf life/storage time (for example the day of catch/slaughter), but higher scores are given for later stages of storage according to the changes occurring for that parameter. Using as an example the parameter eye-form for a fish species such as farmed salmon, three scores describe the changes of the form of the eyes during storage time in ice. At the very beginning of storage time the eye is convex (score = 0), then it becomes flat (score = 1), and at last concave (sunken) (score = 2). As the descriptions in the QIM scheme are species-dependent, the descriptions may be different for other species, as for brill, where at the beginning

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Table 20.4 QIM scheme for herring (Clupea harengus). Quality parameter General appearance

Skin

Bloodspot on gill cover

Stiffness

Belly

Smell

Eyes

Clarity Shape

Gills

Colour Smell

Quality index (QI)

Description

Score

Bright, shining Bright Dull None Small (10–30%) Big (30–50%) Very big (50–100%) Stiff, in rigor mortis Elastic Firm Soft Firm Soft Belly burst Fresh, seaweed/metallic Neutral Musty/sour Stale meat/rancid Clear Cloudy Normal Plain Sunken Characteristic, red Faded, discoloured Fresh, seaweed/metallic Neutral Sweaty/slightly rancid Sour stink/stale, rancid

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 0–20

Source: Jónsdóttir 1992

of storage, the eye form is flat (score = 0), but then it becomes slightly sunken (score = 1), and sunken (score = 2) with the storage time. Yet other species may require a different number of scores for the same parameter: for example, the QIM scheme for plaice has a fourth score (score = 3) for the form of eyes. Typically, the number of parameters in a QIM scheme is around 10, each with two to four score categories. Therefore, it is important that the description behind each score for a parameter is concise, simple and not too long. Otherwise, the assessor might get confused and the evaluation might require too much time. When all parameters in the scheme have been given a score, the scores for all the characteristics are summarised to give an overall sensory score, the so-called quality index (QI). The scientific development of QIM schemes for various species aims at having the QI to increase linearly with storage time in ice (Figure 20.2). In theory, the ideal curve or line starts at zero (zero scores at storage day zero); that is, the intersection is at (0,0) and its maximum (the maximum sum of scores) is reached at the

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Table 20.5 QIM scheme for plaice (Pleuronectes platessa). Quality parameter Appearance

Skin (both dark and white side)

Mucus

Eyes

Form

Brightness

Gills

Odour

Colour

Mucus

Flesh, fillets

Colour

Quality index (QI)

Description Fresh, bright, metallic, no discolouration Bright, but without shine Mat, rather dull, slight green/blue or purple discolouration Dull, green/blue, purple discolouration Clear, not clotted Slightly clotted and milky Clotted and slightly yellow Yellow and clotted Convex Convex but slightly sunken Flat or swollen (like a balloon) Flat, sunken in the middle Clear, black shining pupil Rather mat, black pupil Mat, opaque pupil Milky, grey pupil Fresh oil, seeweedy, metallic, peppery Neutral, oily, grassy, slightly musty Musty, bread, beer, malt, slightly rancid Rancid, sour, rotten, sulphurous Bright, light red Slightly discoloured, especially at the end of gill filaments Discoloured Yellowish, brown, grey No mucus Clear Yellowish, slightly clotted Yellow, brown, clotted Fresh, translucent, bluish Waxy, milky Dull, slightly discoloured, yellowish Opaque, discoloured, yellow, brown

Score 0 1 2 3 0 1 2 3 0 1 2 3 0 1 2 3 0 1 2 3 0 1 2 3 0 1 2 3 0 1 2 3 0–24

Source: Martinsdóttir et al. 2001

day of sensory rejection. However, in practice the intersection is usually slightly above zero, as can been seen in the literature (see, for example, Martinsdóttir et al. 2001; Sveinsdottir et al. 2002, 2003; Vaz-Pires and Seixas 2006; Bonilla et al. 2007; ). Also, in many cases the maximum sum of QI scores does not correspond to the maximum shelf life (see, for example, Martinsdóttir et al. 2001; Sveinsdóttir et al. 2002; 2003; Bonilla et al. 2007). Despite coming from the same defined lot, for example the same storage time and same handling, individual fish may spoil at slightly different rates, owing to several factors such as different individual biological condition, treatment onboard the vessel and size. In addition, the fish continues to spoil, despite being past its shelf life.

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The definition of shelf life may also vary. Barbosa and Bremner (2002) discussed the various definitions of the term ‘shelf life’, and emphasised that the definition of the beginning and end of shelf life must be clear and well defined. It is important to consider how the product is presented to consumers. In some areas, such as Northern Europe, fillets are the most common product, whereas in Mediterranean countries consumers usually buy whole fish, which would be likely to result in different shelf life for the same fish. Usually, the end of shelf life is defined when the fish is not fit for human consumption. Spoilage due to microbial activity is the main limitation of the shelf life. Another cause of spoilage may be rancidity, especially in fat fish species. The flesh of newly caught fish is free of bacteria. However, considerable amounts of bacteria may be in the viscera, the gills and on the skin. When the fish is stored whole in ice, the deterioration caused by bacteria is minimal for the first days of storage. The flavour and odour compounds that characterise newly caught fish decrease and disappear in the first few days during storage in ice, and the fish flesh becomes almost flavourless and odourless. The number of bacteria increases rapidly in the flesh, using various compounds to grow, resulting in increasingly bad-smelling sulphurous and nitrogenous volatiles. The end of shelf life is most often determined by sensory evaluation (see the paragraphs on quantitative descriptive analysis (QDA; section 20.5) and the Torry scale (section 20.3.2). Trained sensory panellists evaluate cooked samples of the fish. Usually the end of shelf life is determined when the panel scores for those bad-smelling volatile off odours that have reached a certain limit. At these limits consumers are also likely to reject the fish. Estimated storage time in ice is defined as the number of days the fish has been stored in ice. If the end of shelf life has been defined, an estimate can be calculated for the remaining shelf life (=shelf life – predicted storage time). It is emphasised that the term ‘remaining shelf life’ should be used with some caution becasue of the uncertainty in the estimation. Various factors can affect the remaining shelf life. It depends on the handling of the fish. Rapid cooling after the catch and an uninterrupted cold storage, different fishing gear, bleeding and gutting methods are important. The season and catching ground can also have an effect. Before freshness is evaluated with QIM, a sample has to be taken from a homogenous lot. A homogenous lot could be a catching day or a batch. It should be kept in mind that individual fish spoil at different rates. Martinsdóttir et al. (2001) provide guidelines on how to collect samples for QIM evaluation, and recommend that three to ten fish, collected randomly from different places in fish boxes from a homogenous lot, are included in one sample. Controlled storage experiments suggest that increasing the amount of fish per sample increases the precision of the evaluation. Sveinsdottir et al. (2002) concluded that by assessing three fish in a sample, the storage time could be estimated with an accuracy of 2 days (at the 95% significance level), but examining more fish per sample might increase the precision.

20.4.3

Development of quality index method schemes

Different types of seafood have different characteristics and spoilage patterns, and QIM schemes must be to be adapted to each species incorporating their respective characteristics. QIM schemes have mainly been developed for whole raw fish, but several schemes have

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also been developed for other types of seafood, and products, such as raw, frozen/thawed fillets and cooked fillets. QIM has been developed for, or adapted to, several seafood species and products. Currently the literature offers publications on cod (Gadus morhua) (Larsen et al. 1992), herring (Clupea harengus) (Jónsdóttir 1992), Atlantic mackerel (Scomber scombrus), horse mackerel (Trachurus trachurus), European sardine (Sardina pilchardus) (Andrade et al. 1997), thawed whole cod, thawed cod fillets, thawed cooked cod fillets (Gadus morhua) (Warm et al. 1998), redfish (Sebastes mentella/marinus), deep water shrimp, fjord shrimp and peeled shrimp (Pandalus borealis), plaice (Pleuronectes platessa), brill (Rhombus laevis), dab (Limanda limanda), haddock (Melanogrammus aeglefinus), pollock (Pollachius virens), sole (Solea vulgaris), turbot (Scophthalmus maximus) (Martinsdottir et al. 2001), gilthead seabream (Sparus aurata) (Huidobro et al. 2000), farmed Atlantic salmon (Salmo salar) (Sveinsdottir et al. 2003), frozen hake (M. capensis and M. paradoxus) (Herrero et al. 2003), Mediterranean Hake (Merluccius merluccius) (Baixas-Nogueras et al. 2003), octopus (Octopus vulgaris) (Barbosa and Vaz-Pires 2004), cod fillets (Gadus morhua) (Bonilla et al. 2007), flounder (Paralichthys patagonicus) (Massa et al. 2005), air and modified-atmosphere-packed maatjes herring (Clupea harengus) (Lyhs and Schelvis-Smit 2005), Mediterranean anchovies (Engraulis encrasicholus) (Pons-Sánchez-Cascado et al. 2006), cuttlefish (Sepia officinalis), broadtail shortfin squid (Illex coindetii) (Vaz-Pires and Seixas 2006), farmed Atlantic halibut (Hippoglossus hippoglossus) (Guillerm-Regost et al. 2006) and tub gunard (Chelidonichthys lucernus) (Bekaert 2006). Additionally, schemes similar to QIM, based on the originally developed TFRU scheme, have been developed for other species, such as cultured sea bream (Sparus aurata) (Alasalvar et al. 2001) and cultured and wild sea bass (Dicentrarchus labrax) (Alasalvar et al. 2002). Several authors have suggested modifications to improve QIM schemes already developed. Sveinsdottir et al. (2002) introduced a slightly modified QIM scheme for farmed Atlantic salmon for more precise evaluation. Inácio et al. (2003) published a modified QIM scheme for horse mackerel (Trachurus trachurus). Nielsen and Hyldig (2004) introduced a modified QIM scheme for herring, and suggested further modification to improve its usability. They also demonstrated the different spoilage patterns of herring stored in ice or in a tank before landing, resulting in different QI scores. There are some concerns that the total QI may be influenced and reduced by washing the fish. This has been studied by Huidobro et al. (2001), who showed that the QI was significantly reduced at later storage stages by washing gilthead seabream (Sparus aurata). On the contrary, Inácio et al. (2003) found no such effects by washing horse mackerel (Trachurus trachurus). Meticulousness and thorough planning is needed to develop new QIM schemes. Useful information and guidelines on this are given in Hyldig et al. (2007), Martinsdóttir (2002) and Hyldig and Nielsen (1997). Whenever a QIM scheme is to be developed or adapted for new species, controlled ice storage studies must be conducted to ensure that the appropriate criteria and their corresponding defined characteristics are included in the QIM scheme. There are mainly three steps in the development of a new QIM scheme (Sveinsdottir et al. 2003). First, a pre-observation is conducted, where one or two experts in sensory evaluation of fish, preferably with some specific knowledge of the fish species, observe fish of different storage times, ranging from the beginning of storage time to past the expected end of shelf

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life. All changes occurring in appearance, odour and texture during storage are listed in a draft scheme. The next step is development of the QIM scheme and training of a QIM sensory panel, which should be done in several sessions. In each session, three or four different groups of fish that have been stored different periods within the expected shelf life are observed using the draft scheme. During the first session, the draft scheme is explained to the panellists while they evaluate fish identified with their storage time. The panellists evaluate each fish independently and are encouraged to comment on the scheme. Suggestions for changes are discussed and should be taken into consideration. In the following sessions, the panellists are trained by evaluating the samples without knowledge of the storage time before the sessions. During the development of the scheme, some parameters might be removed if the evaluation of those parameters is considered destructive to the sample. In addition, the descriptions should be checked to ensure that they are sufficiently clear and descriptive, and that the number of scores for each evaluated parameter span the changes occurring with storage time. In the final training session, the QIM scheme should be finalised. The results are analysed, such as observing the linear relationship between the storage time and the maximum QI scores. The performance of the QIM panellists is observed, by analysis scoring of individual panellists against the average scores and standard deviations to check the robustness of the QIM scheme. In parallel to the training of the QIM panellists, a second sensory panel could be trained in sensory evaluation of the cooked samples. In the third step, a full-scale shelf-life study is conducted. Throughout the storage trial, the fish must not be handled, so each time a new sample is taken. The QIM panel evaluates unknown coded samples in random order using the QIM scheme. The fish should be evaluated at least every third day during the shelf life of the fish. The day interval of course depends on the length of the expected shelf life, and if it is only a few days, samples should be evaluated each day or every other day. Preferably five fish should be evaluated on each of the storage days. During storage experiments, chemical and microbiological indices might also be measured to follow the spoilage pattern and may be used for comparison. In parallel, a trained sensory panel should conduct a sensory evaluation of cooked samples to estimate the reasonable maximum shelf life. The shelf life study should be repeated to observe if the same slope is obtained between the QI and the storage time. Analysis of the results from the shelf-life studies is an important part of the development. The linearity of the QI with storage time should be checked. The scores for each attribute in the QIM scheme should be studied against the storage time as well. The weight of scores might be changed to obtain a QI with a higher correlation to storage time.

20.4.4

Selection and training of panellists for evaluation of fish freshness by QIM

For the QIM for evaluation of whole fish kept in ice, a manual has been made that includes a section on panel selection and training. During training courses (standardised by QIMEurofish, www.qim-eurofish.com), candidates are appointed by the management of the company, selected on being conscientious and accurate, interested in sensory evaluation and readily available for testing on regular basis. The participants are chosen from the whole company, not necessarily persons from just the quality or incoming-goods departments.

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On the first day of the course these candidates are tested for colour blindness by Ishihara’s colour deficiency test (Ishihara 1998) and the smell identification test from Sensonics (http:// www.smelltest.com). The norms are that no colour deficiency is allowed. A minimum smell identification score of 29 out of 40 represents a ‘normal smeller’. For the selection of an industry panel dealing with specific off-flavours (that is, earthy off-flavour in farmed fish), participants should be able to identify these specific flavours as well. For QIM, the panel must be trained for three to six sessions (depending on previous experience with seafood sensory evaluation). For training, three or four samples of fish of different freshness with known storage time in ice and treatment are used. The storage time of the fish is introduced to the panellists before they start evaluation. They are asked if they can agree on the scores that should be given for each sample. A discussion is allowed between the panel leader and panellists. Next, the panellists separately evaluate blind coded samples of different freshness, and preferably the same samples three times. The samples are numbercoded. All panellists should become very familiar with fish of all freshness stages: that is, not only raw material that is on the borderline of production.

20.5

Descriptive sensory analysis

Sensory attributes, such as appearance, odour, flavour and texture, are highly species- and product-specific and can be measured in detail using descriptive sensory analysis. Descriptive sensory analysis is a very useful tool both for research and industry. It can be very simple and used to assess the single attributes of appearance, odour, flavour and texture, or many sensory attributes can be evaluated in each sample. The flavour profile is described in ISO 6564 (1985). QDA provides a detailed description of all or selected sensory characteristics in a qualitative and quantitative way (Meilgaard et al. 2006). Trained assessors should always be used in descriptive sensory analysis. The attribute to be assessed must be clearly defined and understood. Special care must be taken with several factors: some flavors are very special, like iodine (from bromophenols) and muddy (from geosmin or MIB), and must be known to the assessors; there might be considerable differences between the individual fish, and it can be a challenge to have homogeneous samples and even more complicated to get replicates for a panel of 12. In all cases the assessors require intensive training and a detailed briefing before each session. Continuous training during long-term projects, preferably using reference material, can reduce the risk of drifting. If the training is interrupted, the assessors might forget descriptor meanings and/or rating levels with time. The words used for describing different attributes must be cognitively clear. To make reference samples, it is good to have a definition of the sensory attribute. Reference samples for the different attributes can be found not only in seafood samples, but also in other foods like cucumber and boiled potato. Another example is the sensory descriptor ‘warm milk’. It is then important to know that milk should only be heated and not boiled, because when it boils a sulphurous odour develops (Hyldig 2007; Hyldig and Nielsen 2007). To see if these demands are fulfilled, the sensory data can be analysed for the signal-to-noise relation for each assessor and attribute. The results can then be evaluated with multivariate data analysis (Thybo and Martens 2000). Examples on how sensory attributes of fish can be described are given in Table 20.6.

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Table 20.6 Sensory descriptors of cooked salmon and cod products. Sensory attribute Appearance Discoloured Homogenous/ heterogeneous Light/dark colour White precipitation Odour Boiled milk Boiled potatoes Butter Frozen storage Meaty Putrid Rancid Sea/seaweed Sour Sourish Sulphur Sweet Table cloth TMA Vanilla Flavour Butter Cooked potatoes Fresh fish oil Frozen storage Meaty Metallic Mushroom Pungent Putrid Rancid Salt Sour taste Sourish Sweet TMA Texture Clammy Dry/juicy Firm/soft Flakiness Juicy Meaty Mushy Oily Rubbery Tough/tender

Description and Scaling

Brown or yellow spots, dark areas Left end: homogenous, even colour. Right end: discoloured, heterogeneous, stains Left end: light, white colour. Right end: dark, yellowish, brownish, grey White precipitation in the broth or on the fish Boiled milk, fruity/mushy odour Odour of boiled potatoes Butter odour, popcorn Reminds of odour found in refrigerator and/or freezing compartment Meaty odour, reminds of boiled meat Putrid odour Rancid fish, paint, varnish Fresh seaweed, fresh sea smell Sour dishcloth/sour sock, spoilage sour Acidic, fresh citric acid Sulphur, matchstick Sweet odour Reminds of a table cloth (damp cloth to clean kitchen table, left for 36 h) TMA odour, reminds of dried salted fish, amine Vanilla odour, sawdust, timber Butter flavour, popcorn Cooked peeled potatoes Fresh oil, fresh green hazelnut Reminds of food which has soaked in refrigerator/freezing odour Meaty flavour, reminds of boiled meat, meat sour, farmed fish Metallic flavour Mushroom flavour Pungent flavour, bitter Putrid flavour Rancid fish, paint, varnish Salt taste Sour taste, spoilage sour Acidic, fresh citric acid Sweet flavour, warm milk TMA flavour, reminds of dried salted fish, amine Clammy texture, tannin Evaluated after chewing several times: dry – pulls juice from the mouth Evaluate how firm or soft the fish is during the first bite. Force required to compress the sample between the molars The fish portion slides into flakes when pressed with the fork The samples ability to hold water after two or three chews Meaty texture, meaty mouth feel Mushy texture Amount of fat coating in the mouth Rubbery texture, chewing gum Evaluated after chewing several times

Sources: Green-Petersen et al. 2006; Sveinsdóttir et al. 2009

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The results from the descriptive sensory analysis can be presented as a spider plot for means of the different sensory attributes. For more detail, the sensory properties can be related to assessors, samples and trials by either principal component analysis (PCA) or partial least square regression (PLSR) using, for example, Unscrambler®, (Camo ASA, Norway) (Martens and Næs 1989). Both discriminant-PLSR (DPLSR) and ANOVA-PLSR (APLSR) models can be calculated (Martens and Martens 1999, 2001). Data from the sensory descriptive analysis can be corrected for the ‘level effect’ (that is, assessors using different parts of the line scale) by the method described by Thybo and Martens (2000).

20.6

Consumer tests (hedonic)

Acceptability of seafood, the degree of liking and disliking, is usually estimated using a scalar method, the most common being the nine-point structured hedonic scale. Validity of data generated using this method can be influenced by factors such as unequal size category intervals in the scale, and the tendency of consumers to avoid extreme values on the scale and to score close to the midpoint. Taking this into account, the method, or variations of it, is still recommendable for estimating hedonic quality of seafood. The most common hedonic scales are seven- or nine-point scales. They are simple to use and easy to implement. If the consumers are children, the scales are often more simple, fivepoint scales, and the hedonic scaling can also be achieved using face scales, such as simple ‘smiley’ faces, or more realistic pictures of adults. Hedonic scales have been widely studied and have been shown to be useful in the hedonic assessment of food. The hedonic scale assumes that consumer preferences exist on a continuum and that preference can be categorised by responses based on liking and disliking. The samples are served to the consumer in random order and the consumers are asked to indicate their hedonic response to the sample on the scale. The words chosen for each scale option are based on equal interval spacing. The categories on a nine-point scale are: dislike extremely, dislike very much, dislike moderately, dislike slightly, neither like nor dislike, like slightly, like moderately, like very much, like extremely. The result can be used for ranking the samples or for preference mapping (see Chapter 19.2.7). Another commonly used and very simple method is the paired comparison test, where the consumer is asked only to indicate which one of the two samples she or he prefers. This method does not give any information about the degree or intensity of the preference.

20.7

References

Alasalvar, C., Taylor, K.D.A., Öksüz, A., Garthwaite, T., Alexis, M.N. and Grigorakis, K. (2001) Freshness assessment of cultured sea bream (Sparus aurata) by chemical, physical and sensory methods. Food Chemistry 72: 33–40. Alasalvar, C., Taylor, K.D.A., Öksüz, A., Shahidi, F. and Alexis, M. (2002) Comparison of freshness quality of cultured and wild sea bass (Dicentrarchus labrax). Journal of Food Science 67: 3220–3226. Andrade, A., Nunes, M.L. and Batista, I. (1997) Freshness quality grading of small pelagic species by sensory analysis. In: G. Ólafsdóttir, J. Luten, P. Dalgaard, M. Careche, V. Verrez-Bagnis,

Sensory evaluation of seafood: methods

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E. Martinsdóttir and K. Heia K (Eds) Methods to Determine the Freshness of Fish in Research and Industry. Proceedings of the Final Meeting of the Concerted Action Evaluation of Fish Freshness, AIR3CT942283, Nantes Conference, November 12–14. International Institute of Refrigeration, Paris, pp. 333–338. Anonymous (1996) Council Regulation (EC) No 2406/96 of 26 November 1996 laying down common marketing standards for certain fishery products. Aust, L.B., Gacula, M.C., Beard, S.A. and Washam, I.I. (1985) Degree of difference test method in sensory evaluation of heterogenous product types. Journal of Food Science 50: 511–513. Baixas-Nogueras, S., Bover-Cid, S., Veciana-Nogués, T., Nunes, M.L. and Vidal-Carou, M.C. (2003) Development of a quality index method to evaluate freshness in Mediterranean hake (Merluccius merluccius). Journal of Food Science 68: 1067–1071. Barbosa, A. and Bremner, A. (2002) The meaning of shelf-life. In: H.A. Bremner (Ed.) Safety and Quality Issues in Fish Processing. Woodhead Publishing Limited, UK, pp. 173–190. Barbosa, A. and Vaz-Pires, P. (2004) Quality index method (QIM): development of a sensorial scheme for common octopus (Octopus vulgaris). Food Control 15: 161–168. Bekaert, K. (2006) Development of quality index method scheme to evaluate freshenss of tub gunard (Chelidonichthys lucernus). In: J.B. Luten, C. Jacobsem, K. Bekaert, A. Sæbö and J. Oehlenschläger (Eds) Seafood from Fish to Dish. Wageningen Academic Publishers, Wageningen, The Netherlands, pp. 289–296. Bett, K.L. (1997) Flavour-quality control in freshwater aquaculture. The Progressive Fish-Culturist 59: 149–154. Bett, K.L. and Dionigi, C.P. (1997) Detecting seafood off-flavours: limitations of sensory evaluation. Food Technology 51(8): 70–79. Bonilla, A.C., Sveinsdottir K. and Martinsdottir, E. (2007) Development of quality index method (QIM) scheme for fresh cod (Gadus morhua) fillets and application in shelf life study. Food Control 18: 352–358. Bremner, H.A. (1985) A convenient easy to use system for estimating the quality of chilled seafood. Fish Processing Bulletin 7: 59–70. Codex Standards (1999) Codex standards for fish and fishery product, ‘Guidelines for the sensory evaluation of fish and shellfish in laboratories’ CAC-GL 31–1999 Rome, FAO/WHO Codex Alimentarius. Green-Petersen, D.M.B., Nielsen, J. and Hyldig, G. (2006) Sensory profiling of the most common salmon products on the Danish market. Journal of Sensory Studies 21: 415–427. Guillerm-Regost, C., Haugen, T., Nortvedt, R., Carlehög, M., Lunestad, B.T., Kiessling, A. and Rora, A.M.B. (2006) Quality characterization of farmed Atlantic halibut during ice storage. Journal of Food Science 71: 83–90. Herrero, A.M., Huidobro, A. and Careche, M. (2003) Development of a quality index method for frozen hake (Merluccius capensis and M. paradoxus). Journal of Food Science 68: 1086–1092. Huidobro, A., Pastor, A., Lopez-Caballero, M.E. and Tejada, M. (2001) Washing effect of the quality index method (QIM) developed for raw gilthead seabream (Sparus aurata). European Food Research and Technology 212: 408–412. Huidobro, A., Pastor, A. and Tejada, M. (2000) Quality index method developed for raw gilthead seabream (Sparus aurata). Journal of Food Science 65: 1202–1205. Howgate, P. (2004) Tainting of farmed fish by geosmin and 2-methyl-iso-borneol: a review of sensory aspects and of uptake/depuration. Aquaculture 234: 155–181. Hyldig, G. (2007) Sensory profiling of fish, fish products, and shellfish. In: L.M.L. Nollet (Ed.) Handbook of Meat, Poultry and Seafood Quality. Blackwell Publishing, Iowa, USA, pp. 511–528. Hyldig, G. and Nielsen, J. (1997) A rapid sensory method for quality management. In: G. Ólafsdóttir, J. Luten, P. Dalgaard, M. Careche, V. Verrez-Bagnis, E. Martinsdóttir and K. Heia K (Eds) Methods to Determine the Freshness of Fish in Research and Industry. Proceedings of the Final Meeting of

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the Concerted Action Evaluation of Fish Freshness, AIR3CT942283, Nantes Conference, November 12–14. International Institute of Refrigeration, Paris, pp. 297–305. Hyldig, G. and Nielsen, D. (2007) Texture of fish, fish products, and shellfish. In: L.M.L. Nollet (Ed.) Handbook of Meat, Poultry and Seafood Quality. Blackwell Publishing, Iowa, USA, pp. 549–561. Hyldig, G., Bremner, A., Martinsdóttir, E. and Schelvis, R. (2007) Quality index methods handbook of meat, poultry and seafood quality. In: L.M.L. Nollet (Ed.) Handbook of Meat, Poultry and Seafood Quality. Blackwell Publishing, Iowa, USA, pp. 529–561. International IDF Standard 99B (1995) Sensory evaluation of dairy products. Reference method. Inácio, P., Bernardo, F. and Vaz-Pires, P. (2003) Effect of washing with tap and treated seawater on the quality of whole scad (Trachurus trachurus). European Food Research and Technology 217: 406–411. Ishihara, S. (1998) Ishihara’s Tests for Colour Deficiency, concise edition. Tokyo, Kanehara & Co., Ltd. ISO 10399: 2004 Sensory analysis – Methodology – Duo–trio test. ISO 4120: 2004 Sensory analysis – Methodology – Triangle test. ISO 5495: 2005 Sensory analysis – Methodology – Paired comparison test. ISO 6564: 1985 Sensory analysis – Methodology – Flavour profile methods. ISO 8587: 1988 Sensory analysis – Methodology – Ranking. Johnsen, P.B. and Bett, K.L. (1996) Sensory evaluation of the off-flavours geosmin and 2methylisoborneol (MIB) in farm raised catfish. Journal of Applied Aquaculture 6(2): 21–37. Jónsdóttir, S. (1992) Quality index method and TQM-system. In R. Olafsson and A.H. Ingthorsson (Eds) Quality Issues in the Fish Industry. Proceedings from the COMETT Conference Course: Quality Issues in the Fish Industry, European Cooperation, Reykjavik, Iceland, September 1992, pp. 81–94. Larsen, E., Heldbo, J., Jespersen, C.M. and Nielsen, J. (1992) Development of a method for quality assessment of fish for human consumption based on sensory evaluation. In: H.H. Huss, M. Jakobsen and J. Liston (Eds) Quality Assurance in the Fish Industry. Elsevier Science Publishing, Amsterdam, pp. 351–358. Luten, J.B. and Martinsdóttir, E. (1997) QIM: a European tool for fish freshness evaluation in the fishery chain. In: G. Ólafsdóttir, J. Luten, P. Dalgaard, M. Careche, V. Verrez-Bagnis, E. Martinsdóttir and K. Heia K (Eds) Methods to Determine the Freshness of Fish in Research and Industry. Proceedings of the Final Meeting of the Concerted Action Evaluation of Fish Freshness, AIR3CT942283, Nantes Conference, November 12–14. International Institute of Refrigeration, Paris, pp. 287–296. Lyhs, U. and Schelvis-Smit, R. (2005) Development of a quality index method (QIM) for maatjes herring stored in air and under modified atmosphere. Journal of Aquatic Food Product Technology 14: 63–76. Martens, H. and Næs, T. (1989) Multivariate Calibration. John Wiley and Sons Ltd, Chichester, UK. Martens, H. and Martens, M. (1999) Modified jack-knife estimation of parameter uncertainty in bilinear modelling by partial least squares regression (PLSR). Food Quality and Preference 11: 5–16. Martens, M. and Martens, H. (2001) Multivariate Analysis of Quality. An Introduction. John Wiley and Sons Ltd, Chichester, UK. Martinsdóttir, E. (2002) Quality management of stored fish. In: H.A. Bremner (ed.) Safety and Quality Issues in Fish Processing. Woodhead Publishing Limited, UK, pp. 360–378. Martinsdóttir, E., Luten, J.B., Schelvis-Smit, A.A.M. and Hyldig, G. (2003) Developments of QIM – past and future. In: J.B. Luten, J. Oehlenschläger and G. Ólafsdóttir (Eds) Quality of Fish from Catch to Consumer, Labelling, Monitoring and Traceability. Wageningen Academic Publishers, Wageningen, The Netherlands, pp. 265–272.

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Martinsdóttir, E., Sveinsdóttir, K., Luten, J., Schelvis-Smit, R. and Hyldig, G. (2001) Reference Manual for the Fish Sector: Sensory Evaluation of Fish Freshness. QIM-Eurofish, The Netherlands. Martinsdóttir, E., Sveinsdóttir, K., Luten, J., Schelvis-Smit, R. and Hyldig, G. (2004) Reference Manual for the Fish Sector: Sensory Evaluation of Fish Freshness. QIM-Eurofish, The Netherlands. Massa, A.E., Palacios, D.L., Paredi, M.E. and Crupkin, M. (2005) Postmortem changes in quality indices of ice-stored flounder (Paralichthys patagonicus). Journal of Food Biochemistry 29: 570–590. Meilgaard, M., Civille, G.V. and Carr, T. (2006) Descriptive analysis techniques. In: Sensory Evaluation Techniques, 4th edition. CRC Press, New York. Munoz, A.M., Civille, G.V. and Carr, B.T. (1992) Sensory Evaluation in Quality Control. Van Nostrand Reinhold, New York. Nielsen, D. and Hyldig, G. (2004) Influence of handling procedures and biological factors on the QIM evaluation of whole herring (Clupea harengus L.). Food Research International 37: 975–983. NMKL method, No. 183, (2005) Sensory quality control of drinking water. http: //www.nmkl.org/. Pons-Sánchez-Cascado, S., Vidal-Carou, M.C., Nunes, M.L. and Veciana-Nogués, M.T. (2006) Sensory analysis to assess the freshness of Mediterranean anchovies (Engraulis encrasicholus) stored in ice. Food Control 17: 564–569. Sanders, H.R. and Smith, G.L. (1976) The construction of grading schemes based on freshness assessment of fish. Journal of Food Technology 11: 365. Schelvis-Smit, A.A.M. and Luten, J.B. (2003) Catch index: development of a tool for measurement the quality of the catch handling at sea. In: J.B. Luten, J. Oehlenschläger and G. Ólafsdóttir (Eds) Quality of Fish from Catch to Consumer, Labelling, Monitoring and Traceability. Wageningen Academic Publishers, Wageningen, The Netherlands, pp. 137–144. Shewan, J.M., Macintosh, R.G., Tucker, C.G. and Ehrenberg, A.S.C. (1953) The development of a numerical scoring system for the sensory assessment of the spoilage of wet white fish stored in ice. Journal of the Science of Food and Agriculture 4: 283–298. Sveinsdóttir, K., Martinsdottir, E., Hyldig, G., Jorgensen, B. and Kristbergsson, K. (2002) Application of quality index method (QIM) scheme in shelf-life study of farmed Atlantic salmon (Salmo salar). Journal of Food Science 67: 1570–1579. Sveinsdóttir, K., Hyldig, G., Martinsdottir, E., Jorgensen, B. and Kristbergsson, K. (2003) Quality index method (QIM) scheme developed for farmed Atlantic salmon (Salmo salar). Food Quality and Preference 14: 237–245. Sveinsdóttir, K., Martinsdóttir, E., Green Petersen, D., Hyldig, G., Schelvis, R. and Delahunty, C. (2009) Sensory characteristics of different cod products and consumer preferences. Food Quality and Preference 20: 120–132. Thybo, A.K. and Martens, M. (2000) Analysis of sensory assessors in texture profiling of potatoes by multivariate modelling. Food Quality and Preference 11: 283–288. Vaz-Pires, P. and Seixas, P. (2006) Development of new quality index method (QIM) schemes for cuttlefish (Sepia officinalis) and broadtail shortfin squid (Illex coindetii) Food Control 17: 942–949. Warm, K., Boknæs, N. and Nielsen, J. (1998) Development of quality index methods for evaluation of frozen cod (Gadus morhua) and cod fillets. Journal of Aquatic Food Product Technology 7: 45–59.

Chapter 21

Data handling by multivariate data analysis Bo M. Jørgensen

21.1

Introduction

One of the main features of nature is the covariance structure linking various properties or phenomena. Not many entities exist or function independently of at least some others and exploration of physical or biochemical phenomena is incomplete without taking this into account. Recognition of, for example, which family, species or stock a certain individual belongs to is in general based on much more than one attribute, and concepts like quality or safety cannot themselves be quantified by a single figure although certain aspects may. Thus in most research, and in most routine supervision, several quantities are determined, providing the scientist or technician with a set of measurements on each entity. The primary purpose of collecting these data may be to describe a concept like quality, but an extra issue could be to gain new information on covariance structures. As valuable as classical univariate statistics may be, both of these outcomes will indisputably take advantage of a subsequent data analysis designed for data vectors with many or at least several measured quantities, namely for multivariate data.

21.2

What is multivariate data analysis?

The phrase ‘multivariate data analysis’ covers a wide range of mathematical methods for simultaneous treatment of several measurements on a sample. Some are developed and optimised to treat specific problems, and some are generic in nature. Several variants of what is named multi-linear modelling belong to the second type and have the further advantage of being understandable to laymen. Thereby the food scientist him- or herself is able to analyse their results without having to lean on experts in statistics and mathematics (see, for example, Martens and Martens (2001) for an argument of the advantage of that). Multivariate data analysis aims to take advantage of the correlation structure by substituting patterns of measurements for the single values. A sample is then characterised by an amount of each member of a usually small set of such patterns, and the difference between samples expressed as the difference in amounts. In that way, the patterns serve as fundamental characteristics and the amounts summarise even a high degree of variability in a few 444

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figures. These may be considered as ‘generalised’ attributes by which, for example, quality or authenticity assessment can be based. When using the type of multivariate data analysis named multi-linear modelling, the patterns are also sometimes named latent structures because they are initially hidden behind the physical measurements but may be disclosed as a result of the data analysis. The latent structures provide information on the correlation of the measured quantities, which is often at least as interesting as the values of the quantities themselves. Multi-linear modelling, mostly bi-linear, but also frequently tri-linear and sometimes of even higher order, has turned out to be a very efficient generic approach. At first sight, this may be surprising as the world definitely is nonlinear in nature. However, locally, linear models are often sufficient (like Taylor series expansions), and if so, they are to be preferred at least because of their relative simplicity. This again leads to a high degree of interpretability, in contrast to nonlinear methods like artificial neural networks which are often applied to calibrations based on, for example, near-infrared (NIR) spectroscopy. These provide better fit but at the expense of lack in transparency (they act as ‘black boxes’). Also, in the multivariate frame, nonlinear variables may, if necessary, conveniently be introduced in linear modelling by just adding functions of the variables, for example squares (xi2) and cross-terms (xixj) as new columns in the data matrix. In many investigations, the main difference between measurements is related to the difference in the property of interest, for example, species or quality. It is therefore probably not surprising that one of the most useful bi-linear methods is principal component analysis (PCA). This method resolves the data matrix (see later) into latent structures named principal components of which the first sums up as much of the total sample variance as possible. The residual data may contain further structure, and the next principal component then catches as much as possible of that under the constraint that it must be independent of the former component(s). This data matrix decomposition continues until the residuals apparently contain nothing but noise. Because the main variation between samples is contained in the first few principal components, and that variation is likely to be the most interesting, these few components often suffice to describe the experimental results in an easily interpretable way. Occasionally, the main variation between samples is not the most interesting (relevant) in the actual context. In that case, another very useful and widely applicable method, PLS regression, may advantageously be substituted for PCA. The acronym PLS comes from partial least squares, focussing on the mathematics, but has also been explained as projection on latent structures, expressing relevance rather than main sample variance. Here, two blocks of data are in play. The second block is used as a means of rotating the bi-linear solution (model) away from the principal axes, to axes where the variation of interest is more evident. Apart from adding to the interpretability of results, the method has proven very successful in multivariate calibration where a certain quantity, for example fat or protein content, is determined from a fast instrumental measurement, for example an NIR spectrum (see, for example, the monograph by Næs et al. 2002). These two bi-linear methods, PCA and PLS, have proven useful in many fields of investigation and are generally applicable to almost all types of measurement, although specialised methods may give marginally better results. A good piece of advice to the fisheries scientist is therefore to get familiar with PCA and PLS and routinely apply the most relevant of those to the data at hand. Most often, they will turn out to be sufficient. In cases where the single

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Sampled curves (data vectors) e.g. spectra and chromatograms

Single measurements

Sample 1 Sample 1 Sample 2 Sample 2

Sample N Sample N Figure 21.1 Schematic structure of the data matrix in bi-linear modelling. Each column contains the same type of variable, either a design variable indicating to what group in an experimental design a sample belongs, a single measurement (for example pH, salt percentage or an assessment of a sensory attribute) or a point of a sampled curve (for example a spectrum). Each row contains all variables belonging to a single sample. In the figure, duplicates are arranged as separate samples.

measurements produce higher-dimensional output, multi-linear extensions should be considered, though, because of their further advantages (see, for example, Smilde et al. 2004).

21.3

Arrangement of data for bi-linear modelling

The first step in bi-linear modelling is to construct a data matrix consisting of one row for each sample and one column for each measured quantity. A measured quantity may be an isolated value like pH, salt content or assessment of a sensory attribute. It may also be one of many sampling points of a curve (for example, a spectrum or a chromatogram). Figure 21.1 illustrates a data set containing both types of measured quantity supplemented by columns describing the experimental design. In that way, each row (excluding the design variables) is mathematically a vector containing all measurements on a single sample, and each column is a vector containing a single sort of measurement made on all the samples involved. The columns are traditionally named variables. Replicates are normally considered as, and therefore assigned to, separate samples (rows) in the data matrix rather than extending the variable space. This has several advantages: (a) it is easier to calculate averages over replicates if considered appropriate or advantageous; (b) too many variables with trivial correlations are avoided; (c) some error structure in the sample mode (direction) is immediately apparent. However, to make sense, this organisation demands that all measurements in a row are made on the same replicate. Otherwise, one should average over replicates before constructing the data matrix. Although it is straightforward to arrange measurements of a certain sample in one row in the data table, the other requirement, to put similar measurements in the same column, is not always that easy. This statement applies not to single measurements of, for example, pH

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or salt concentration, but to sampled curves that may behave differently from measurement to measurement. Perhaps the most obvious example is if one wants to use a chromatogram directly without having to identify peaks and calculate peak areas. Owing to small but significant changes in the stationary phase during each run, the same compound present in several samples is eluted at different times, presenting its signal to different sampling points, i.e. in different variables. In that way, two identical samples, analysed for example first and last in an experimental series, will appear as different. This difference obviously blurs the data analysis and may prevent real differences of interest from being acknowledged. The existence of what may be named x-axis misalignment thus has to be taken care of, and recently developed methods (Malmquist and Danielsson 1994; Nielsen et al. 1998; Tomasi et al. 2004) seem to do this quite well. The problem is normally not that severe for spectroscopic measurements because of wavelength calibration. That is if the signal is sampled at the same nominal wavelengths from measurement to measurement, of course. This obvious requirement may pose a practical problem, for example when instruments have to be replaced with newer versions or software is upgraded. A misalignment may, however, be apparent and be caused by phenomena of interest. For example, unresolved overlapping peaks may change in relative magnitude between two samples, giving rise to a shift in the maximum of the composite peak. An attempt to align the signals would in this case remove relevant information. A situation like this should be recognised by replicate measurements showing equal behaviour, though. Only one data matrix is needed for PCA, and design variables are normally not included in calculations but may be useful for interpretation of results. To perform a PLS regression one must define two data matrices or two blocks in the large matrix and define which one is X and which is Y. For a calibration, the normal procedure is to define the sampled spectrum or equivalent as X and the variable to be determined from the model as Y (often a one-column block). For classification, the design variables in binary form (0 or 1) may function as either X or Y depending on the purpose of the calculations. If the model is to be used for classifying samples from multivariate measurements, these are put in the X block. If the effect of controlled experimental conditions on measured values is to be studied, the binary design variables make up the X block. The difference may seem a bit subtle and the results often reflect that in being rather similar.

21.4

The outcome of bi-linear modelling

The bi-linear modelling algorithms decompose the data matrix (X block) as outlined in Figure 21.2 into a product of two matrices, the scores matrix, T, and the transpose of the loadings matrix, P. The columns of the loadings matrix contain the patterns or generalised attributes as mentioned before, and the columns of the scores matrix contain the amounts of each pattern. For PCA, both matrices have orthogonal columns; that is, the patterns as well as their amounts are independent. For PLS regression, this applies to only one of the matrices depending of the algorithm used. The valid number of columns depends on the number of independent features. The maximal value is the number of rows or columns in the data matrix X, whichever is the smallest. However, often a few columns suffice to capture the systematic variation between samples making the residual matrix (Figure 21.2) containing mostly noise.

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Fishery Products: Quality, safety and authenticity p1′ X

= t1

p2′ + t2

+...+

Residual

X = TP¢ + Residual Figure 21.2 Bi-linear modelling. The data matrix X is decomposed into a sum of outer products of vectors ti whose elements are named scores and transposed vectors pi whose elements are named loadings. The decomposition may also be expressed as a matrix product where the scores vectors are columns in matrix T and the loadings vectors columns in matrix P. The model is linear in T as well as in P and thus bi-linear.

Samples are distinguishable by having different set of scores values (row elements in T) and variables by having different set of loadings values (row elements in P). Points representing samples that are similar for the measured values are thus relatively close to each other in scores plots of, for example, the second scores vector versus the first. Likewise, points representing measured values that co-vary through the sample set are close in the loadings plot of, for example, the second loadings vector versus the first. The two plots are linked in that the scores plot may reveal sample groupings and the loadings plot may show which of the measurements have mainly influenced the grouping. When investigating aspects of nature, it is a rule with only few exceptions that one cannot measure the whole population but usually a set of samples. These should be representative in the sense that the variability in measured quantities reflects the variability in the population. Likewise, the sample mean should be a good, or at least fair, estimate of the population mean. Bi-linear modelling focuses on the sample variability and rarely on the absolute values of the means. The clearest picture is therefore obtained by changing the zero points of the measured variables by subtracting their sample mean. As a rule of thumb, this means centring of the data matrix always ought to be done before bi-linear modelling. Another consideration is about scaling of the variables. Because the focus is on sample differences in the full pattern of measured variables, high absolute values tend to dominate. This is especially apparent when measurements are expressed in different units. And change in unit of a single variable, for example a relative fat content of 0.15 expressed in per cent as 15, will substantially change the loadings and thereby the emphasis of which variables are the most important for distinguishing between the various samples. It is therefore common practice to scale the measured values with the aim of balancing their contribution to the total variance. Unfortunately, the choice of scaling is not straightforward. The most popular choice is to divide each value in a data matrix column by the standard deviation of the values. This possibility, named ‘auto scaling’ in several multivariate data analysis software packages, makes sense when the main contribution to the standard deviation of the variables comes from genuine differences between samples. If, however, a variable is almost constant and its standard deviation thus is low and mainly caused by noise, it will be given a relatively high weight and may blur the result. A variable of that type is of course not very informative and should be excluded from the data matrix before model calculation. If only one type of measurement, for example sensory profiling data or sampled values of absorption spectra, is included in the data matrix, scaling is often omitted.

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PC2

PC1

Figure 21.3 Example of a scores plot. The second scores vector from a PCA is graphed against the first. The data set contains eight samples from four treatments (codes) analysed in duplicate. Replicates are represented with equal symbols. Open and closed symbols of the same shape represent two individuals within the same code. Different shapes indicate different codes.

An example of a graphical representation of the outcome of a PCA is shown in Figures 21.3 and 21.4. It is a simulated case showing sensory profiles of four different types (codes) of fish sample. There were two individuals in each code, and each sample was analysed in duplicate. The sensory profile consisted of eight attributes: white and brown colour, sweet, marine, amine and rancid smell (or taste), and fibrous and flaky texture. The data matrix thus contained 4 × 2 × 2 = 16 rows and 8 columns. Data were mean centred, which explains why negative values of scores and loadings occur despite each attribute being assessed on a scale from zero to fifteen. The calculated model showed two principal components together explaining 93% of the total variance; the rest was considered as being due to measurement noise. The resultant scores matrix T thus contained 16 rows and 2 columns, and the loadings matrix P contained 8 rows and 2 columns. Figure 21.3 shows principal component 1 and 2 scores for the 16 determinations. The four codes are clearly separated into four groups; that is, the distance between points representing samples from the same code is much smaller than the distance between points representing samples from different codes. It is also seen that the duplicates are closer together than are the two individuals in the same code. From the distance between replicates one may estimate the error of determination, and the distance between replicate means within the same group informs about the size of the variation between individuals. To obtain a meaningful grouping, these two types of variance must of course be substantially smaller than the inter-code variance. This is a general requirement that applies to bi-linear modelling too. Outlying measurements may disclose themselves by, for example, replicates being far apart. In that case it is important to identify the outlier, correct (if caused by a data transfer

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Sweet Marine

Fibrous Amine PC1

Flaky

Rancid Brown

Figure 21.4 Example of a Loadings plot. The second loadings vector from the same PCA as in Figure 21.3 is graphed against the first. The points are labelled according to the measured variables (a sensory profiling taken as an example).

error) or remove the ‘bad’ row from the data matrix and recalculate the model. This is because a large part of the variance is caused by the outlier which at least partly has defined the first principal component(s) and thereby made the model invalid. Having found a clear sample grouping, one may be interested in what variables caused this. Figure 21.4 shows principal component 1 and 2 loadings for the same model as in Figure 21.3. Points are shown as labels (variable names) according to the attributes assessed. Two almost independent phenomena are revealed having a main influence on one axis each. The first is related to microbiological activity (marine for fresh, amine for spoiled), and the second to lipid oxidation (white for fresh, rancid and brown for oxidised). Sweet and flaky are other characteristics of the unspoiled samples, whereas fibrous follows amine. Like in the scores plot where points that are close together have almost equal variable patterns, closely spaced variables in the loadings plot are correlated through the samples in the data set. If these samples are representative for the population from which they were drawn, ‘global’ conclusions on variable correlation may be drawn. That type of information is usually valuable, and in some situations it may even point out a possibility of avoiding difficult or expensive analyses in future experiments in favour of easier or cheaper ones providing an equally useful model. The scores plot and the loadings plot of the same model may be superimposed (with proper scaling of the axes). In the example in Figures 21.3 and 21.4, one may deduce that the group of samples marked with circles were of the highest quality, having higher values for sweet, marine and white than the average. The group of samples marked with squares were spoiled (high amine and fibrous), but not oxidised. The triangles pointing upwards represent samples that were rancid but not microbiologically spoiled, having high values of rancid and brown,

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but not of amine. And the last group (triangles pointing downwards) contains samples that were of the lowest quality with respect to spoilage as well as to rancidity.

21.5

Validation and prediction

The bi-linear model X = TP, (Figure 21.2) may be rearranged to T = XP if the algorithm used scales P to containing orthonormal columns, which is often the case. That is, scores T2 from a new set of measurements may be placed in the scores plot by right-multiplying the new (mean-centred and possibly scaled) data matrix X2 with the loadings matrix of the original model. This provides two possibilities: the model validity may be evaluated by using new samples with known properties; or the properties of unknown samples may be estimated from their scores. The basic assumption when calculating the model in the first place, that the set of samples involved should be representative for the population from which they were drawn, may efficiently be tested by drawing a new, independent set of samples, a socalled test set. The samples must have known properties either measured by another, reliable method, or though their design. In the example in Figures 21.3 and 21.4, one might make another experiment with at least some of the same four treatments and apply exactly the same measurements on the new samples as were done on the calibration samples, that is the original samples on which the model is based. The scores of the new samples should place themselves in the right groups; otherwise, the model is of limited general applicability and the conclusions only valid for the original set of samples. A lack of representativeness may be due to a phenomenon that accidentally was not included in the original sample set. In our example, the total fat content might influence the importance of attributes connected to lipid oxidation. And the texture attributes would be affected by the pre-treatment conditions, for example if frozen-stored fish were frozen pre- or post-rigor. In such cases, one may recalculate the model including the new samples. Including more measurements, for example the fat content, in the data should also be considered. A new model should then be validated by a third sample set, a new test set. There are cases where drawing an independent test set is not practicable for some reason, for example a long storage experiment, fish caught under a rare event like an expedition, or the use of very expensive and tedious measurements. An internal validation, cross-validation (Stone 1974; see also Martens and Næs 1989), is then the best one can do and has to do. The samples are split into groups; one group is put aside as a test set and the (sub)-model calculated on the rest. The test set is then used as if it were an independent one and its scores matrix calculated. After the previously removed samples have been put back, another set is withdrawn as a test set, a new sub-model made and a new set of scores calculated. The procedure is continued until all samples have been used as a test sample once. Some consideration is necessary when segmenting the data matrix for the purpose of cross-validation. The one sample per segment choice, used for what is named full crossvalidation, is frequently met in the literature. It is not recommended though, unless only a few samples are available, because it costs many calculations without paying off compared with other options. Neither should one make sets consisting of one replicate each of all samples except when the measurement error is to be estimated, or in special cases where physically meaningful latent structures are to be validated (see, for example, Jensen et al. (2002) for an example). For experiments like the one illustrated in Figure 21.3, an obvious

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choice would be to divide the data matrix into segments containing the duplicate measurements on one fish, namely eight segments in total. In that way, all four codes are represented in all sub-models so that they span the inter-code variation. Also, the requirement that differences between individuals must be less than differences between codes in order for the result to be meaningful is probed. Cross-validation may also be used for estimating the valid number of principal components or latent structures (Wold 1978). Over-fitting, namely including too many components (columns in T and P), often shows up when ‘explained total validation variance’ is calculated as a function of the number of components included in the model. After a certain number, the explained variance stops to increase and may even decrease, because the over-fitted submodels fit the test samples less well than the model with the right number of components. Unless the full sample set is far from being representative, cross-validation seems to be efficient in estimating model size (number of components or latent structures), and the present author recommends it for that purpose and, if possible, collecting an independent set of samples as a test set for model validation. During the cross-validation procedure, each sub-model produces a set of scores and loadings. If these are stored, one may examine how much a given sample scores values or a given variable loadings value change from one sub-model to another. This provides an estimate of the uncertainty of the scores and loadings, namely how well their placement in the scores or loadings plot is defined. It is a very useful diagnostic, especially in cases where the grouping is not as pronounced as it is in Figure 21.3. The principle applies not only to the scores and loadings but also to all parameters of a model. In multivariate calibration using a PLS regression model, for example, the result may be expressed as y = Xb where the coefficient vector b has as many elements as there are variables (columns) in the data matrix X. The sub-model values of b can be used to estimate standard deviations of the coefficients, and to find by the well-known t-test which ones are significantly different from zero (Martens and Martens 2000). Only variables whose corresponding b-coefficients are significantly different from zero contribute to the determination of y. The other variables are not important to the model and may be left out, often with a decrease in prediction error as an extra benefit. The case presented in Figures 21.3 and 21.4 was chosen to illustrate some general points of bi-linear modelling. It is common to see plots of values of principal component 2 values versus principal component 1, either scores or loadings or both together in a so-called biplot. However, that is not the only way to illustrate results. For example, if the measured variables were sampled curves, for example spectra, there would be many points in the loadings plot, and it is often more illustrative to plot the loadings vectors against sample point number or its ‘natural’ quantity, for example wavelength, rather than to plot loadings vector 2 against vector 1. In calibration, it may be more informative to look at the coefficients, the b-vector as mentioned above, rather than the loadings. A third variant is to look at the so-called correlation loadings, which are defined as the correlation between the scores vectors and the measured variables (columns in the data matrix). This plot illustrates how much each measured quantity contributes to the scores and thereby to the parameters used to distinguish between samples. The quality of a calibration model for determining a quantity y from measurements on a set of samples X may also be judged by inspecting the plot of predicted values of the test set of samples against the known values determined by a standard method (or known

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Table 21.1 Some references to the use of multivariate data analysis in quality, safety or authenticity assessment of fish or seafood. Model use Mainly discrimination based on measurement of distinct quantities Mainly discrimination based on sampled signals Mainly calibration

References 2, 4, 10, 15, 19, 20, 22, 26, 30, 39, 40, 44, 47, 48, 51, 54, 58, 63 5, 6, 12, 14, 16, 25, 27, 28, 33, 37, 46, 55, 57, 59, 60 1, 7, 8, 9, 11, 13, 17, 18, 21, 24, 29, 28, 42, 45, 52, 61

from designed samples if possible). Points in that plot should spread around the line y = x, because, in effect, two methods determining the same quantity are compared. The sum of squared distances from the points to this line is an estimate of the sum of variances of the two methods if an independent test set were used. The plot may also reveal when the linear model is insufficient. The points are then often not randomly distributed around the line.

21.6

Real examples and further reading

It is not the purpose of this chapter to present a review of cases where multivariate data analysis has been used in fisheries research. However, for the reader who would like a more complete overview of the wide applicability of this analytical technique, some references are listed in Table 21.1. A recent review by Arvanitoyannis and van HouwelingenKoukaliaroglou (2003) covers a wide range of applications within food research, including a couple of fish-related examples. There are several excellent textbooks on the subject, some rather heavily founded on the mathematical aspects and some more easily readable by the layman. The first category includes Malinowski (1991), Vandeginste et al. (1998) and Smilde et al. (2004), whereas Martens and Martens (2001) and Næs et al. (2002) belong to the second. One of the classics, the monograph by Martens and Næs (1989), lies in between the two categories. Despite its mathematical language, Smilde et al. (2004) is highly recommended to readers interested in tri-linear or higher models.

21.7

References

Andersen, C.M. and Jørgensen, B.M. (2004) On the relation between water pools and water holding capacity in cod muscle. Journal of Aquatic Food Product Technology 13: 13–23. Antignac, J.-P., Marchand, P., Gade, C., Matayron, G., El Qannari, M., Le Bizec, B. and Andre, F. (2005) Studying variations in the PCDD/PCDF profile across various food products using multivariate statistical analysis. Analytical and Bioanalytical Chemistry 384: 271–279. Arvanitoyannis, I.S. and van Houwelingen-Koukaliaroglou, M. (2003) Implementation of chemometrics for quality control and authentication of meat and meat products. Critical Reviews in Food Science and Nutrition 43: 173–218.

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Arvanitoyannis, I.S., Tsitsika, E.V. and Panagiotaki, P. (2005) Implementation of quality control methods (physic-chemical, microbiological and sensory) in conjunction with multivariate analysis towards fish authenticity. International Journal of Food Science & Technology 40: 237–263. Aubourg, S.P., Sotelo, C.G. and Perez-Martin, R. (1998) Assessment of quality changes in frozen sardine (Sardina pilchardus) by fluorescence detection. Journal of the American Oil Chemists’ Society 75: 575–580. Barroso, M., Careche, M., Barrios, L. and Borderias, A.J. (1998) Frozen hake fillets quality as related to texture and viscosity by mechanical methods. Journal of Food Science 63: 793–796. Bassompierre, M., Munck, L., Bro, R. and Engelsen, S.B. (2004) Rapid dioxin assessment in fish products by fatty acid pattern recognition. Analyst 129: 553–558. Bechmann, I.E. and Jørgensen, B.M. (1998) Rapid assessment of quality parameters for frozen cod using near-infrared spectroscopy. Journal of Food Science & Technology 31: 648–652. Bechmann, I.E., Jensen, H.S., Bøknæs, N., Warm, K. and Nielsen, J. (1998) Prediction of chemical, physical and sensory data from process parameters for frozen cod using multivariate analysis. Journal of the Science of Food and Agriculture 78: 329–336. Boe, B. (1983) Quantitative separation of species in fish mixtures by multivariate analysis of electrofocused protein bands. Food Chemistry 11: 127–137. Bøknæs, N., Jensen, K.N., Andersen, C.M. and Martens, H. (2002) Freshness assessment of thawed and chilled cod fillets packed in modified atmosphere using near-infrared spectroscopy. Lebensmittel-Wissenschaft und -Technologie 35: 628–634. Cozzolino, D., Chree, A, Scaife, J.R. and Murray, I. (2005) Usefulness of near-infrared reflectance (NIR) spectroscopy and chemometrics to discriminate fishmeal batches made with different fish species. Journal of Agricultural and Food Chemistry 53: 4459–4463. Cozzolino, D., Murray, I., Chree, A. and Scaife, J.R. (2005) Multivariate determination of free fatty acids and moisture in fish oils by partial least-squares regression and near-infrared spectroscopy. Lebensmittel-Wissenschaft und -Technologie 38: 821–828. Cubadda, F., Raggi, A. and Coni, E. (2006) Element fingerprinting of marine organisms by dynamic reaction cell inductively coupled plasma mass spectrometry. Analytical and Bioanalytical Chemistry 384: 887–896. Duflos, G., Coin, V.M., Cornu, M., Antinelli, J. and Malle, P. (2006) Determination of volatile compounds to characterize fish spoilage using headspace/mass spectrometry and solid-phase microextraction/gas chromatography/mass spectrometry. Journal of the Science of Food and Agriculture 86: 600–611. Girard, B. and Nakai, S. (1993) Species differentiation by multivariate analysis of headspace volatile patterns from canned Pacific salmon. Journal of Aquatic Food Product Technology 2: 51–68. Hatae, K., Yoshimatsu, F. and Matsumoto, J.J. (1988) An integrated quantitative correlation of textural profiles of fish. Journal of Food Science 53: 679–683. Huang, Y., Cavinato, A.G., Mayes, D.M., Kangas, L.J., Bledsoe, G.E. and Rasco, B.A. (2003) Nondestructive determination of moisture and sodium chloride in cured Atlantic salmon (Salmo salar) (teijin) using short-wavelength near-infrared spectroscopy (SW/NIR). Journal of Food Science 68: 482–486. Ingemansson, T., Kaufmann, P. and Ekstrand, B. (1995) Multivariate evaluation of lipid hydrolysis and oxidation data from light and dark muscle of frozen stored rainbow trout (Oncorhynchus mykiss). Journal of Agricultural and Food Chemistry 43: 2046–2052. Ingemansson, T., Olsson, N.U. and Kaufmann, P. (1993) Lipid composition of light and dark muscle of rainbow trout (Oncorhynchus mykiss) after thermal acclimation: a multivariate approach. Aquaculture 113: 153–165. Isaksson, T., Togersen, G., Iversen, A. and Hildrum, K.I. (1995) Non-destructive determination of fat, moisture and protein in salmon fillets by use of near-infrared diffuse spectroscopy. Journal of the Science of Food and Agriculture 69: 95–100.

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Jensen, H.S. and Jørgensen, B.M. (1997) A sensometric approach to cod-quality measurement. Food Quality and Preference 8: 403–407. Jensen, K.N., Guldager, H.S. and Jørgensen, B.M. (2002) Three-way modelling of NMR relaxation profiles from thawed cod muscle. Journal of Aquatic Food Product Technology 11: 201–214. Jepsen, S.M., Pedersen, H.T. and Engelsen, S.B. (1999) Application of chemometrics to low-field 1H NMR relaxation data of intact fish flesh. Journal of the Science of Food and Agriculture 79: 1793–1802. Karoui, R., Thomas, E. and Dufour, E. (2006) Utilisation of a rapid technique based on front-face fluorescence spectroscopy for differentiating between fresh and frozen-thawed fish fillets. Food Research International 39: 349–355. Kent, M., MacKenzie, K., Berger, U.K., Knoechel, R. and Daschner, F. (2000) Determination of prior treatment of fish and fish products using microwave dielectric spectra. European Food Research and Technology 210: 427–433. Kent, M., Oehlenschlager, J., Mierke-Klemeyer, S., Manthey-Karl, M., Knoechel, R., Daschner, F. and Schimmer, O. (2004) A new multivariate approach to the problem of fish quality estimation. Food Chemistry 87: 531–535. Kent, M., Knoechel, R., Daschner, F., Schimmer, O., Tejada, M., Huidobro, A., Nunes, L., Batista, I. and Martins, A. (2005) Determination of the quality of frozen hake using its microwave dielectric properties. International Journal of Food Science & Technology 40: 55–65. LeBlanc, E.L., LeBlanc, R.J. and Blum, I.E. (1988) Prediction of quality in frozen cod (Gadus morhua) fillets. Journal of Food Science 53: 328–340. Li-Chan, E., Nakai, S. and Wood, D.F. (1987) Muscle protein structure-function relationships and discrimination of functionality by multivariate analysis. Journal of Food Science 52: 31–41. Malinowski, E. (1991) Factor Analysis in Chemistry. John Wiley & Sons Inc., New York, USA. Malmquist, G. and Danielsson, R. (1994) Alignment of chromatographic profiles for principal component analysis: a prerequisite for finger printing methods. Journal of Chromatography A 687: 71–88. Marquardt, B.J. and Wold, J.P. (2004) Raman analysis of fish: a potential method for rapid quality screening. Lebensmittel-Wissenschaft und -Technologie 37: 1–8. Martens, H. and Martens, M. (2000) Modified jack-knife estimation of parameter uncertainty in bilinear modelling by partial least squares regression (PLSR). Food Quality and Preference 11: 5–16. Martens, H. and Martens, M. (2001) Multivariate Analysis of Quality. John Wiley & Sons Ltd., Chichester, UK. Martens, H. and Næs, T. (1989) Multivariate Calibration. John Wiley & Sons Ltd., Chichester, UK. Martinez, I., Bathen, T., Standal, I.B., Halvorsen, J., Aursand, M., Gribbestad, I.S. and Axelson, D.E. (2005) Bioactive compounds in cod (Gadus morhua) products and suitability of 1H NMR metabolite profiling for classification of the products using multivariate data analyses. Journal of Agricultural and Food Chemistry 53: 6889–6895. Lin, M., Mousavi, M., Al-Holy, M., Cavinato, A.G. and Rasco, B.A. (2006) Rapid near infrared spectroscopic method for the detection of spoilage in rainbow trout (Oncorhynchus mykiss) fillet. Journal of Food Science 71: S18-S23. Morita, K., Kubota, K. and Aishima, T. (2003) Comparison of aroma characteristics of 16 fish species by sensory evaluation and gas chromatographic analysis. Journal of the Science of Food and Agriculture 83: 289–297. Jensen, K.N., Jørgensen, B.M., Nielsen, H.H. and Nielsen, J. (2005) Water distribution and mobility in herring muscle in relation to lipid content, season, fishing ground and biological parameters. Journal of the Science of Food and Agriculture 85: 1259–1267. Nielsen, N.-P.V., Carstensen, J.M. and Smedsgaard, J. (1998) Aligning of single and multiple wavelength chromatographic profile for chemometric data analysis using correlation optimized warping. Journal of Chromatography A 805: 17–35.

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Nilsen, H., Esaiassen, M., Heia, K. and Sigernes, F. (2002) Visible/near-infrared spectroscopy: a new tool for the evaluation of fish freshness? Journal of Food Science 67: 1821–1826. Næs, T., Isaksson, T., Fearn, T. and Davies, T. (2002) Multivariate Calibration and Classification. NIR Publications, Chichester, UK. Ofstad, R., Egelandsdal, B., Kidman, S., Myklebust, R., Olsen, R.L. and Hermansson, A.M. (1996) Liquid loss as effected by post mortem ultrastructural changes in fish muscle: cod (Gadus morhua L.) and salmon (Salmo salar). Journal of the Science of Food and Agriculture 71: 301–312. Olafsdottir, G., Lauzon, H.L., Martinsdottir, E. and Kristbergsson, K. (2006) Influence of storage temperature on microbial spoilage characteristics of haddock fillets (Melanogrammus aeglefinus) evaluated by multivariate quality prediction. International Journal of Food Microbiology 111: 112–125. Pink, J., Naczk, M. and Pink, D. (1998) Evaluation of the quality of frozen minced red hake: use of Fourier transform infrared spectroscopy. Journal of Agricultural and Food Chemistry 46: 3667–3672. Rodrigues, M.J., Ho, P., Lopez-Caballero, M.E., Bandarra, N.M. and Nunes, M.L. (2005) Chemical, microbiological, and sensory quality of cod products salted in different brines. Journal of Food Science 70: M1-M6. Sferlazzo, G., Franco, M.A., del Caro, A., Madau, M.E., Cristini, A. and Menghini, V. (1995) Discrimination of tuna (Neothynnus albacora) fishing-sites using chemical parameters elaborated by multivariate statistical techniques. Italian Journal of Food Science 7: 395–402. Smilde, A., Bro, R. and Geladi, P. (2004) Multi-way Analysis. John Wiley & Sons Ltd., Chichester, UK. Stone, M. (1974) Cross-validatory choice and assessment of statistical prediction. Journal of the Royal Statistical Society B 39: 111–133. Sveinsdottir, K., Hyldig G., Martinsdottir, E., Jørgensen, B. and Kristbergsson, K. (2003) Quality index method (QIM) scheme developed for farmed Atlantic salmon (Salmo salar). Food Quality and Preference 14: 237–245. Svensson, V.T., Nielsen, H.H. and Bro, R. (2004) Determination of the protein content in brine from salted herring using near-infrared spectroscopy. Lebensmittel-Wissenschaft und -Technologie 37: 803–809. Tomasi, G., van den Berg, F. and Andersson, C. (2004) Correlation optimized warping and dynamic time warping as preprocessing methods for chromatographic data. Journal of Chemometrics 18: 231–241. Tritt, K.L., O’Bara, C.J. and Wells, M.J.M. (2005) Chemometric discrimination among wild and cultured age-0 largemouth bass, black crappies, and white crappies based on fatty acid composition. Journal of Agricultural and Food Chemistry 53: 5304–5312. Uddin, M., Okazaki, E., Turza, S., Yumiko, Y., Tanaka, M. and Fukuda, Y. (2005) Non-destructive visible/NIR spectroscopy for differentiation of fresh and frozen-thawed fish. Journal of Food Science 70: C506-C510. Vandeginste, B.G.M., Massart, D.L., Buydens, L.M.C., de Jong, S., Lewi, P.J. and Smeyers-Verbeke, J. (1998) Handbook of Chemometrics and Qualimetrics: Part B (Data Handling in Science and Technology 20B). Elsevier, Amsterdam, The Netherlands. Vazquez, M.J., Lorenzo, R.A. and Cela, R. (2003) The use of an ‘electronic nose’ device to monitor the ripening process of anchovies. International Journal of Food Science & Technology 38: 273–284. Warm, K., Nelsen, J., Hyldig, G. and Martens, M. (2000) Sensory quality criteria for five fish species. Journal of Food Quality 23: 583–601. Warm, K., Martens, H. and Nielsen, J. (2001) Sensory quality criteria for five fish species predicted from near-infrared (NIR) reflectance measurement. Journal of Food Quality 24: 389–403.

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Westad, F., Nilsen, B.N. and Rødbotten, R. (2001) Prediction and classification of food quality using VIS/NIR spectroscopy and parsimonious models. New Food 4: 52–57. Wold, S. (1978) Cross-validatory estimation of the number of components in factor analysis and principal component models. Technometrics 20: 397–406. Wold, J.P., Jakobsen, T. and Krane, L. (1996) Atlantic salmon average fat content estimated by nearinfrared transmittance spectroscopy. Journal of Food Science 61: 74–77. Yamashita, Y., Omura, Y. and Okazaki, E. (2006) Distinct regional profiles of trace element content in muscle of Japanese eel Anguilla japonica from Japan, Taiwan, and China. Fisheries Science 72: 1109–1113.

Chapter 22

Traceability as a tool Erling P. Larsen and Begoña Pérez Villarreal

22.1

Introduction

In recent years, it has been very amusing to look up the word ‘traceability’ in all the dictionaries that we have come across. In the Concise Oxford Dictionary of Current English, sixth edition 1976, twelfth impression 1981, the word ‘traceability’ does not appear, but the word ‘trace’ appears with 11 different distinct meanings. If the word traceability is ‘Googled’ on the Internet, it gives around 7 million hits. A few years ago it only gave a couple of hundred. There are several definitions of traceability, but the site www.Wikipedia.org is a good place to start. It states that ‘Traceability refers to the completeness of the information about every step in a process chain.’ In the USA, the National Institute of Standards and Technology (NIST) has the following definition: ‘Traceability requires the establishment of an unbroken chain of comparisons to stated references.’ The most useful statement from NIST is: ‘In logistics, traceability refers to the capability for tracing goods along the distribution chain on a batch number or series number basis. Traceability is an important aspect for example in the automotive industry, where it makes recalls possible, or in the food industry where it contributes to food safety.’ There are several other definitions that are useful when working with the food sector. ISO 9000 (ISO 2000) defines traceability as ‘the ability to trace the history, application or location of that which is under consideration . . . when considering a product, traceability can relate to the origin of materials and parts, and the processing history.’ In recent years the European Union (EU) has been working on drawing up legislation to cover all aspects of the food sector, commonly called the ‘the General Food Law’ (EU 2002). Here the EU defines traceability as ‘the ability to trace and follow a food, feed, food-producing animal or substance intended to be, or expected to be incorporated into a food or feed, through all stages of production and distribution.’ The Codex Alimentarius Commission, which was created in 1963 by FAO and WHO to develop food standards, guidelines and related texts, put forward at its meeting in July 2004, in Geneva, the following definition of traceability to be added to its standard: ‘Traceability/Product tracing: the ability to follow the movement of a food through specified stage(s) of production, processing and distribution.’ As can be seen from these different definitions, the area of traceability is still not mature and a finally agreed definition has not yet been found. There are still differences between 458

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A supply chain with four links P

B-ID

P

B-ID

P

B-ID

P

B-ID

Internal traceability in each link One link in the chain External traceability between the links or across the links P

Product with product data: details of raw material, etc.

B-ID

Batch identification

Figure 22.1 Internal and external traceability with the most important activities in the supply chain.

the USA and the EU in the way the concept is perceived (Larsen 2003). This is caused mainly by different attitudes in placing traceability in its proper context. This will be discussed later in the chapter. Traceability is normally divided into two categories: internal traceability and external traceability (Derrick and Dillon 2004). Internal traceability is related to the product and the information relating to it internally in a factory, company and even inside a conglomerate of companies. External traceability relates to the product information that a company either receives or provides to the next links in the chain from primary producer to the end-user. There is an continuing discussion about whether internal traceability is of importance in the total supply chain. As long as the batches that enter and leave the company can be clearly identified, then traceability is maintained. What has happened to the product within the company is of no interest as long as the final product can be identified with the proper information attached to it. There is no limit to how large or small a batch can be. A company can choose to have a batch size of one product unit, one day or one year’s worth of production, or even an unlimited batch size of the total production (Frederiksen et al. 2004). It reduces to an economic calculation by the company, where the cost of having a finemeshed traceability system can be compared with the possibility of having to recall all the products belonging to the same batch. See Figure 22.1 for an illustration of internal and external traceability. The introduction of traceability in the fish sector is spreading slowly. Internal traceability is expanding most, whereas external traceability comes in fits and starts depending on the pressure from the authorities for a system to be put in place. Food scares are a constant threat and it is here that traceability has its justification in the future. The fish sector has traditionally been considered as something special compared with other food sectors, not to mention other industrial processing. The fresh fish trade handles highly perishable foods and special operations have to be managed to maintain an acceptable eating quality. The only comparable sector is the dairy sector, where fresh milk is handled. The shelf life of milk – normally pasteurised – and fresh fish in the fish retailers shops is

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generally less than 10 days old, although many fish species have a longer shelf life, for example up to 20 days for farmed salmon and 18 days for redfish (Pérez Villarreal and Pozo 1990; Sveinsdottir et al. 2003). The dairy sector is characterised by the same ownership of many of the links in the chain from the primary producer to final sale to the consumer. There are individual differences between the European countries: from the Scandinavian model with full ownership from producer, through dairy processing to transportation to the wholesaler, to the British model where some of the dairy processing plants still deliver to the customer’s address. In these cases, the supply chain is fairly simple and there is full traceability due to a high degree of internal traceability. The customer is used to seeing all the important data on the milk bottle or the carton. This has only recently been the case in the fresh fish sector. Fresh fish was sold unpacked, wrapped in paper or other suitable material, with no information, not even the name of the fish species. This has changed in recent years (Hansen and Fischer 2003). Multiple retail stores have entered the market for selling fresh fish, starting with the introduction of modified atmosphere packaging (MAP) technology (technology). This technology has increased the total shelf life, while giving space on the packaging for introducing information such as: the name of the fish species, the date of packaging, the weight and often a recipe to help with preparing the fish. This has lead to the ‘shop in shop’ concept, which is a staffed counter selling fresh fish inside the multiple. This is presumably going to be one of the strongest driving forces towards having full traceability in the fish sector, because the multiple is generally used to having traceability for all other goods in the shop and will, of course, consider it necessary to have it for the fish as well.

22.2

Traceability from older times to the present

Historically, traceability was an important part of trade and social organisation. Taxation on actual money income is a relatively new invention, dating back three centuries. Before that, taxes on different goods, or more precisely customs fees, were an integral part of daily life. Of course there were individual taxes such as land tax, tax on the number of fireplaces and even on coins where a piece was cut off. If goods were being transported, and that includes foods such as fish and fish products, and they were crossing a border, a city limit, or maybe just a bridge or a specific road, a fee had to be paid. This fee could go to the local owner, the local community or to the ruler of the area or country. One of the more famous examples is from Northern Europe, where wars have been fought over the income from the trade with codfish and herring. In Figure 22.2 the herring are so numerous that, according to legend, an axe could stand upright if it was stuck in a shoal of them. The King of Denmark taxed the number of people allowed to fish, the salt coming primarily from Germany and even the barrels where the herring was salted. This system demanded a very well-functioning traceability system. Different kinds of salt meant different taxes, which required a specific quality control. The barrels with the salted fish each had an individual number – a batch number in a bigger lot – which could be traced to the fisherman, the community he belonged to, the salt supplier and the transporter. This system created an organisation of quality controllers, tax collectors, customs officials, civil servants in the central administration, and so on. There was, of course, an ongoing fight between the producers to try to pay as little tax as possible and to sell the goods at as high a price as possible and the authorities/tax collectors who wanted full control and high tax levels.

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Figure 22.2 An old engraving from the 13th century showing the abundance of herring in the Ore Sound between Denmark and Sweden.

These systems operated for centuries, but were lost when mass production was introduced in the 18th century. The next big step was the extensive mass production of foods, which began during the two World Wars in the 20th century, especially after the Second World War. It was, of course, important to have information about the place of production, but the lot numbers covered huge quantities of batches produced. The development towards the present situation possibly started with the introduction of the hazard assessment critical control point (HACCP) concept in the 1960s. HACCP was a new way of handling food production, originally developed to ensure absolutely safe foods for astronauts orbiting the Earth and later going to the Moon (the Pillsbury Company 1973). During recent decades, it has always been in the interests of the authorities to have safe and healthy food products for the population. However, a conflict has been building up between the wish to have safe products on the one hand and cheap products that can be mass produced on the other. In the 1980s and 1990s, some major scares occurred in the food sector. During these years, bovine spongiform encephalopathy (BSE)/Creutzfeldt–Jakob disease (CJD) spread from the UK to the rest of the world; there was foot-and-mouth disease in the UK; and in Belgium the dioxin issue was affecting chicken feed. The fish sector has until now avoided big food scares. So there has been a good opportunity to develop traceability systems adapted to the fish sector. This sector is confronted by some rough conditions: salty water, high humidity, high temperature variation and the multiple ownerships of the products – just to mention just a few of the obstacles. The one big challenge for future traceability systems is the matter of who owns the information that is sent back and forth. The major traceability research projects in the past

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decade have dealt with an ‘open’ approach, whereby the necessary information is shared along the entire chain. Developments in the commercial area have focused more on the ‘closed’ approach, the development of a central information management system which gives the ‘caretaker’ of this central system an advantage. One of the open approach research projects was the Danish ‘Info-Fish’, which operated by an Internet solution based on barcodes as information carriers (Frederiksen et al. 2001). At that time the use of barcodes required that practical solutions be found for use in the harsh saltwater and climate with changes in temperature. This meant not only finding labels that could withstand these conditions, but also on finding equipment that could operate for longer periods. The ‘Info-Fish’ project developed and performed a full-scale test of a traceability system in a fresh fish chain, starting on board a fishing vessel in the North Sea, going through the operation of collecting (unloading, sorting and repacking the catch), auction, wholesale and ending at the retail counter. The result of this project is that today, in the 21st century, more than 50 Danish fishing vessels are using systems that allow them to sort, weigh and pack their catch onboard the vessel and then sell it at auction with all the relevant information included. The information includes: fish species, weight, size, catch date, vessel identity, and even more information if necessary. The buyers of the fresh, gutted fish at auctions are paying more per kilogram than for traditional fresh fish, where only the name of the vessel is given in the fish boxes (personal comment). Fish auctions around Europe are currently one of the most visible places where traceability is actively used. At a normal, traditional auction, a key item of information is the number or name of the fishing boat that has caught and landed the boxes of fish that are for sale. The auctioneer calls out the name of the fishing vessel when the sale begins. This, combined with the name of the fish species, the weight-class/grading of the fish, the total weight of the fish for sale and the quality grading according to EU regulations (EU 1996), gives the buyer the necessary information to give a bid at the auction. In the past 10 years, electronic auction systems have been introduced in fish salerooms. Starting with auctions specialising in the sale of flowers and vegetables, electronic systems are now spreading throughout Europe. One of the first electronic auctions of fish was the Pefa system (http://www.pefa. com/), which now operates nine different auctions (Krott 2003). This system is based on an electronic clock that counts down, and the buyers stop the clock when they make their bid. In the clock itself, which can be seen in a selling room or on a computer screen anywhere in the world, several types of information are given: the fishing vessel, fish species, weight, sorting according to weight and, last but not least, the quality grading. All the information is kept electronically and can be used, for example, if a lot number is recalled. The concept of electronic auctions is spreading across European countries. In Spain there are several companies supplying slightly different systems. The Autec (Automatismes Electrònics i Control, SL) system is chosen as an example (www.autex.com). In 2006, there were already 52 systems running, placed in fishing port auctions along the Mediterranean cost of Spain and on the Azores. The auction system is a combination of an electronic auction and a traditional auction with an auctioneer. To illustrate how it works, and the integration of traceability elements, the auction in La Garrucha (Almería province in Spain) is illustrated. The buyers have their individual remote key to the auction system and press a button to make their bid. When the auctioneer calls the bids, the buyers use the remote control. The same procedure is followed when the electronic auction clock system is used. When the

Traceability as a tool

463

Figure 22.3 Auction of swordfish in La Garrucha (Spain), as a combination of a traditional auction and an electronic auction, with a screen over the gate to the quayside and remote keys for the buyers to give their bid. Photograph by Erling Larsen.

clock starts, the price goes down until a buyer has made a bid. At the same time, all the traceable data is shown on a screen. Figures 22.3–22.5 show the electronic auction in operation. TraceFish is the short title for the ‘Traceability of Fish Products’ concerted action project, which ran from 2000 to 2002, coordinated by the Norwegian Institute of Fisheries and Aquaculture (www.tracefish.org). The TraceFish premise is that with increasing demands for information from buyers and consumers of food products, it is no longer practical to convey all the relevant data physically with the product. A more sensible approach is to mark each package with a unique identifier, and then transmit or extract all the relevant information electronically. The main outcome of TraceFish comprised three consensus-based standards for recording and exchanging traceability information in the seafood chains: one for the electronic transmission of the data; and two other standards for the information data to be traced along the different links of the fish chains, one for farmed fish and one for wild-caught fish. The two last standards can be seen on the homepage of GS1 (GS1 Standard (2006) www.gs1.org/ docs/traceability/GS1_fish_traceability.pdf.).

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Fishery Products: Quality, safety and authenticity

Figure 22.4 The electronic auction, showing boxes on a conveyer belt and the screen showing the most important data such as quality grade, fish species, name of fishing boat and starting price. Photograph by Erling Larsen.

Figure 22.5 After the auction is finished, a label with all the traceable data is printed out and placed in the fish box. This label is used by the buyer in the next link in the chain. Photograph by Erling Larsen.

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These standards now form the basis of numerous traceability implementations in the industry, both privately funded projects and pilot R&D projects with public funding. Plans are being developed to duplicate the TraceFish process in other food chains that are to develop standards for the recording and electronic interchange of traceability information in other sectors (TraceFood: www.tracefood.org). Today, one of the major marketing subjects in the commercial trade with seafood products is the use of traceability. This stretches from shrimps (Traceshrimp: www.thaitraceshrimp. com), to full traceability in the production of fish products guaranteed by one the major suppliers of equipment to the sector (Marel hf: www.marel.com).

22.3

Traceability research in the seafood sector and other EU-funded food traceability projects

The implementation of traceability in the seafood sector is in progress worldwide. Research is taking place in scientific communities that deal with seafood research, but it has opened up to several new research areas such as information technology and consumer science. In recent years the interest has been to involve sustainability and resource management in the concept of traceability (Thompson et al. 2005). The ethical aspects of traceability have increased owing to the growing interest in, for example, the ‘Fair Price’ concept or in animal welfare (Gouveia 2007) In the EU-financed integrated research project SEAFOODplus (http://www.seafoodplus. org/), the traceability part is concentrated in three major areas: development of methods, testing and implementation of specific elements, and development of validated traceability systems in the seafood sector. The strategic impact of having validated traceability systems is to supply the European consumer with seafood of the required quality and functionality. At the same time, fraud can be reduced. The validated traceability systems developed will also be working on imported seafood products from countries outside the EU, improving the delivery of seafood to the European market and making the choice of retail products much larger for European consumers. In addition to reinforcing consumer confidence in safe and healthy seafood, the introduction of validated traceability systems will also improve the competitiveness and trustworthiness of the seafood processing industry and the whole seafood sector as such. Major elements in the delivered new traceability systems will be the development of an agreed vocabulary and the description of guidelines for good traceability practice, testing and developing technology for automated data capture, developing methods for traceability surveys, and estimating costs and benefits from implementating traceability practice and systems. Moreover, an improved overview of the diverse methods for authentication will be given, and selected methods will be tested and validated for general use in the seafood production chain. Since 2006, the EU has been funding a Specific Support Action project ‘Promoting European Traceability Excellence & Research’ (PETER: http://www.eu-peter.org/) (Table 22.1). This project will provide an international forum for focusing and disseminating the results of the European Commission’s 1100 million investment into research into food traceability during the first decade of the 21st century. There is an urgent need for rapid consolidation and dissemination of European expertise to developing countries and SMEs so that they can

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Fishery Products: Quality, safety and authenticity

Table 22.1 Projects of which the coordinators are members of the PETER project, and other projects where traceability is part of the scope and results are exchanged with PETER.

Projects of which the coordinators are members of PETER

Projects with traceability as a main subject or part of their scope

TRACE (IP) Co-Extra (IP) SEAFOODPlus (IP) GTIS CAP (SSA) GeoTraceAgri (FPV-RTD) DNA-Track (FPV-RTD) Oliv-TRACK (FPV-RTD) ALCUEFOOD (SSA) FoodTrace (FPV-CA)

TRACEBACK P-2-P Chill-On Σ-Chain SAFEED-PAP Ethical Traceability ERMES EUROLATSEA TraceFood

gain access to the global markets that now exist. After consolidation of the European research in the field, the main outcome of this project will be to promote international activities through workshops and conferences. A web-based communication vehicle will serve as a dialogue forum, and specific platforms will be created for discussion between the industry, the consumer and standardisation stakeholders.

22.4

Validation of traceability data

Now that some time has passed since the enforcement of the EU’s General Food Law regulation No. 178/2002 on 1 January 2005, we can observed that the food sector is now accustomed to hearing and speaking about traceability, and even putting into practice some internal traceability issues. Nevertheless, there is still much misunderstanding about what traceability is, and even the full meaning of traceability for the sector is still not entirely understood (EC 178/2002). In particular, for chain traceability – which is the scope of the EU’s General Food Law – the level of doubt increases with information issues and the way in which this information is handled, managed and interchanged. These problems must be solved specifically in the different food chains. One aspect that is generally not taken fully into account for the practical implementation of traceability in a food chain is the validation of the information and the way it is transmitted through the chain. Information can be separated into two main areas: z z

collection and handling of data; verification of the data.

In internal traceability, it can be assumed that the verification of data issued during the production process is not a difficult task and can be put into practice by applying regular quality management tools. This has been proven by several tests in the fish industry in Norway, Spain and Denmark.

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467

However, there are some drawbacks or missing points on the practical implementation of traceability issues in a food chain. These are related to the guarantee of traceability or the verification of data, which can be called a validated traceability. A validated traceability chain is a system that can ensure clients’ and consumers’ confidence in the information given.

22.4.1

Traceable data

Multiple traceable data are important for the fish sector, and for each type of data a diverse methodology has been described for measuring it. In most cases, there are no standards that allow easy or simple comparison of data between interested parties, and not every methodology is suitable for each link in the fish chain. One of the main parameters important for traceability is the authentication of seafood products. Authenticity can be defined as the quality or condition of being authentic, trustworthy or genuine. If this definition is applied to the seafood sector, a distinction can be made between: z z z

identification of fish species; identification of geographic origin; discrimination between some production methods (for example farmed from wild fish) and/or processing conditions.

The identification of fish species is an important concern in order to label seafood products following EU regulations (EC 104/2000). Morphological characterisation is used in fishing vessels, harbours and retailers. However, when the external characteristics are removed, DNA-based methodologies, instead of those that are protein based, are now preferred for their discriminatory power over very closely related fish species and highly processed seafood products (see Chapters 16 and 17). Genetic techniques are somehow still limited in routine control laboratories owing to the cost of the equipment and the need to have qualified personnel, although this will change in the near future thanks to the reliability of the methods and the development of more simplified and rapid kits and probes (Martínez et al. 2005). The best method to validate these techniques is by the direct sequencing of DNA fragments amplified by polymerase chain reaction with forensically informative nucleotide sequencing (PCR–FINS) and then comparing them with sequences compiled in a reliable database (Pardo 2005) (AZTI: www.azti.es/dna_database/). Identification of the geographic origin of fish can be important when dealing with sustainability issues in protecting exploited commercial species, giving appropriate information to consumers (EC No. 2065/2001) and avoiding possible fraud in the labelling. Here, there is an even greater need for research because there is a lack of suitable methodologies to deal with regulations and labelling. Among them, isotope signatures analysed by isotope ratio mass spectrometry have been used for certain foodstuffs such as wines (official EU method) and water (being developed in an R&D EU project, TRACE), but not fish. Molecular techniques using microsatellites for differentiating between fish stocks (Pardo and Estonba 2006) seem to be a very promising methodology.

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Fishery Products: Quality, safety and authenticity

Nevertheless, some of the most important traceable data are related to fish safety and quality parameters, including: z z z z

volatile amines as criteria for chemical quality assessment; histamine and biogenic amine analysis; microbiological and sensory quality assessment methods; and biotoxins, viruses and other biological hazards.

For some of them, official methods exist, although most are time consuming and quite slow, showing results that could be used to make decisions along the fish chain. In general, some specific sensors and probes have recently appeared, but there is a lack of validation, and reference methods are needed to see the possibility of having them as rapid quality control tools for the fish industry. As the development of new, faster and simpler methods continues, identity, safety and quality parameters will be more frequently controlled, contributing to a safer and more reliable fish distribution chain. The establishment of a validated system for traceability management, including establishing standards for analysing relevant traceability parameters and inspection procedures in each link of the fish chain, is of primary importance in these circumstances.

22.5

Traceability in a global perspective

Food products are an integral part of the global trading system. Food is an international commodity: in the past 25 years the cost of transporting it from one end of the world to the other has fallen because of the increased use of containerisation. It costs as much to send a 20-foot freezer container from Europe to China as it does to send the same container from Northern Europe to Italy. At the borders, the World Trade Organization has been responsible for reducing previous obstacles in the form of import–export customs declaration papers and health certificates, the main function of which was to prevent imports that could compete with nationally produced products. The world is not yet free of these artificial barriers, but the major trading organisations such as the EU and the North American Free Trade Agreement (NAFTA: http://www.nafta-sec-alena.org/) are creating free-trade areas. This development puts more pressure on the need for a reliable traceability system for two reasons: first, for reasons of food safety so an effective recall procedure can be operational; secondly, because of the growing threat from terrorist activities, this demands a 100% certainty of the origin of the products in question. One of the best definitions of globalisation is given by the OECD: ‘a dynamic and multidimensional process of economic integration whereby national resources become more and more internationally mobile while economies become increasingly interdependent’ (OECD 2005). The global transportation of foods can be a risky business for the transporter. Traditionally the transporter guarantees that nothing untoward will happen to the load or cargo. Today, live fish and shellfish are transported in containers, both by plane and by cargo vessel. This demands much more management than transporting at a specific temperature in the container. The humidity and/or water flow must be managed very accurately, and the facilities in the port of destination must be able to support this kind of transport.

Traceability as a tool

5,000,000

Consumers

5000

Retailers Outlets Buying

6

salers

Processing Fishermen

600

desks

Whole-

industry and

469

aquaculture

500 2000 4000

Figure 22.6 The supply chain funnel in Denmark, illustrating the concentration of power over the supply chain with seafood. The number people are estimated in each segment.

Nowadays, the trend is for fish caught in, for example, Europe to be transported to Southeast Asia or China for processing and then brought back to the European market. This demands not only a strict quality control, but also a well-functioning traceability system, where the individual batches can be easily recognised. The reason for this long transportation is not only a matter of saving on labour costs. The indirect reason is that the rationalisation and mechanisation of the fish-processing industry cannot keep up with the capability of developing countries to produce products with a higher yield and individual adaptation. On the other hand, several transportation links in the entire chain from the fisherman to the consumer are more the rule than the exception. So the total transportation time now takes 6 months in a reefer container instead of a total of 6 days in a refrigerated lorry. However, the traceability requirements are the same. If the same international standard is used for example a 128 barcode labelling and the TraceFish standard including a lot number, the level of traceability can just be as high or even higher than for fish sold the very day after it is caught. It is a myth that fish caught by an inshore fisherman is of a higher quality and a better traceability standard than fish caught in bigger quantities and produced in large fish-processing factories. There is an continuing discussion about which of the links is the most powerful. This is setting the agenda for the whole sector. Figure 22.6 clearly illustrates that the retail link is very important – in 2006 only six people at the buying desk of the major multi-retailers were taking decisions on what to buy. In future, perhaps only five big multiple retail chains will dominate Europe. Their influence will be significant. At present, everybody is on the threshold of introducing traceability based on radio frequency identification (RFID) tag technology. The biggest American multiple chain, Wal-Mart, has just postponed the introduction of RFID tags for the second time. It is striking that a low-price multiple chain such as Wal-Mart wants to introduce this technology, but a short look at the history of the company shows that its success has been based on non-food goods coming from the Third World. It has thereby developed a very strong management system to deal with imports. That has created a very good logistical system that the company now wants to apply to the food sector of its shops. In Europe, one of the biggest

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Fishery Products: Quality, safety and authenticity

multiple chains, Carrefour in France, has adopted the strategy that every supplier must put its information on traceability into the format of the Trace-One system, and Carrefour must have access to all the information. It is difficult to say how traceability will find its place among all the other documentation systems. However, a good estimate is that it will be integrated within safety systems. Traceability is on the one hand a very old business, and on the other a very young system that needs to mature and find its own place in the overall management system.

22.6

References

Derrick, S. and Dillon, M. (2004) A Guide to Traceability within the Fish Industry EUROFISH and Swiss Import Promotion Programme (SIPPO), Copenhagen. EC (European Commission) 1996 Regulation No. 2406/96 of the European Parliament and of the Council of 26 November 1996. Laying down common marketing standards for certain fishery products, Official Journal, No. L334, pp. 1–14. EC (Council Regulation) 2000 Regulation No 104/2000 of 17 December 1999. On the common organisation of the markets in fishery and aquaculture products. Official Journal L 017, 21/01/2000, pp. 22–52. EC (European Commission) 2002 Regulation No. 178/2002 of the European Parliament and of the Council of 28 January 2002. Laying down the general principles and requirements of food law, establishing the European procedures in matters of food safety. Official Journal L 031, 01/02/2002, pp. 1–24. Frederiksen, M., Østerberg, C., Silberg, S., Larsen, E. and Bremner, H.A. (2002) Info-fisk. Development and validation of an Internet based traceability system in a Danish domestic fresh fish chain. Journal of Aquatic Food Product Technology 11: 13–34. Frederiksen, M., Larsen, E. and Børresen, T. (2004) European traceability legislation in the context of seafood. In: The International Review of Food Science and Technology. International Union of Food Science and Technology (IUFoST), Sovereign Publications Limited, London, UK, pp. 38–41. Gouveia, R. (2007) The Consumer co-operatives’ perspective. In Trace 3rd Annual Meeting, Crete, Greece. www.trace.eu.org/je/greece/meeting/trace_m3_gouveia.php. Hansen, H.E. and Fischer, K. (2003) Demand for documentation of freshness of loose fresh fish. In: J.B. Luten, J. Oehlenschhläger, and G. Ólafsdóttir (Eds) Quality of Fish from Catch to Consumer. Wageningen Academic Publishers, Wageningen, The Netherlands, pp. 361–365. ISO (International Organization for Standardization) (2000) Quality Management Systems – Fundamentals and Vocabulary. European Standard (EN ISO 9000:2000, Pint 3.5.4) Committee for Standardisation, Brussels, Belgium. Krott, W. (2003) PEFA: Selling fish on the Internet across Europe – bridge between suppliers and remote demand for fresh fish. In: J.B. Luten, J. Oehlenschhläger, and G. Ólafsdóttir (Eds) Quality of Fish from Catch to Consumer. Wageningen Academic Publishers, Wageningen, The Netherlands, pp. 165–173. Larsen, E. (2003) Traceability in fish processing. In: M. Lees (Ed.) Food Authenticity and Traceability. Woodhead Publishing Limited, Cambridge, UK, pp. 507–517. Larsen, E. and Olesen, E. (2002) Life cycle analysis on farmed fish. Network of LCA in the Nordic Fish Sector, Nordic Council of Ministers, final workshop, Roskilde, Denmark. Martínez, I., James, D. and Loréal, H. (2005) Application of modern analytical techniques to ensure seafood safety and authenticity. FAO Technical Paper No. 455. OECD (2005) Measuring Globalisation: OECD Economic Globalisation Indicators. OECD, Paris, France, pp. 198.

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Pardo, M.A. (2005) Report on the methods to authenticate seafood products. SEAFOODplus-VALID internal report. Pardo, M.A. and Estonba, E. (2006) Authentication of the European anchovy from the Biscay Bay: an approach based on the use of molecular techniques. In: Proceedings of the 2006 TAFT-WEFTA Meeting. Pérez Villarreal, B. and Pozo, R. (1990) Chemical composition and ice spoilage of albacore (Thunnus alalunga). Journal of Food Science 55: 678–682. The Pillsbury Company (1973) ‘Food Safety Through the Hazard Analysis and Critical Control Point System’, Contract No. FDA 72–59, Research and Development Dept., The Pillsbury Company, Minneapolis, USA. Sveinsdottir, K., Hyldig, G., Martinsdottir, E., Jørgensen, B.M. and Kristbergsson, K. (2003) Quality index method (QIM) scheme developed for farmed Atlantic salmon (Salmo salar). Food Quality and Preference 14: 237–245. Thompson, M., Sylvia, G. and Morrissey, M.T. (2005) Seafood traceability in the Unites States: current trends, system design, and potential applications. Comprehensive Reviews in Food Science and Food Safety 1: 1–5.

Index

Acinetobacter spp, 319 adenosine triphosphate, 16, 72, 263 Aeromonas spp, 332, 334 alcohols, 112 actin, 184 actomyosin, 69 alkanes, 114 allergens, 357 amines, 112 amine decarboxylase activity, 44 amination, 42 amino acids, 32 decarboxylation, 17, 42 ammonia, 22 anisopropic model, 242 antifreeze glycoproteins, 189 antioxidant, 13 appearance, 412 aquaculture, 1, 2, 132 production, 2 artificial neural network, 278 artificial quality index (AQI), 131, 230, 240 astaxanthin, 134, 397 Atlantic cod, 16, 137 Atlantic salmon, 132, 135–136 QIM scheme, 432 authentication, 98, 263 DNA based methods, 363–387 protein based methods, 349–362 autolysis, 69 Bacillus cereus, 332 bacteria, 17 heat labile, 323 hydrogen sulphide producing, 321, 323, 325 lactic acid, 330 mesophilic, 321, 323 472

pathogenic, 332–336 psychrotrophic, 321 basic local alignment search tools (BLAST), 371 bilinear modelling, 446–451 biogenic amines, 42–67, 468 chemical quality index, 50 metabolism, 43 regulatory issues, 54 bony fish, 4 Brochothrix thermaspacta, 319, 332 bruises, 100 cadaverine, 42 calibration substances, 178 calorimeter, 174–178 cantaxanthin, 134, 153, 398 capillary electrophoresis, 59, 352 zone, 59 carbonyls, 112 Carnobacterium, 330, 332 carotenoids, 132, 397 cartilaginous tissue, 4 caviar, 203, 355 chemical composition, 92 chemical sensors, 105 chemiluminescence, 80 chilled storage, 111, 195, 279 Clostridium botulinum, 332–333 Clostridium perfrigens, 45, 332 chewiness, 214 chondrichthyes, 4 chroma, 128 CIE system, 127 CIELAB space, 127 clupeidae, 54 coagulation test, 358

Index Codex guidelines for sensory evaluation, 415 coherence, 243 collagen, 186 colorimeter, 130 colorimetric techniques, 108 colour measurement, 127 composition, 258 feed, 259 compression test, 222 connective tissue, 4, 10, 186, 214 consumer studies, 421 consumer tests, 440 Coryphaenidae, 54 CO-treatment, 201–203 creatine, 10 phosphate, 16 Creep test, 228 crustacean species, 1 cryoprotectants, 149–150 dark musculature, 4 data analysis, 91–92 data fusion, 116 data processing, 257 degree of difference, 427 denaturation, 180 deoxyribonucleic acid (DNA), 363 content, 364–366 databases, 379 isolation, 368 mitochondrial, 365 nuclear, 365 Descriptive sensory analysis (QDA), 438 deterioration, 259 developing countries, 1 dielectric measurements, 275 difference from control test, 427 difference test, 425 differential scanning calorimetry (DSC), 173 curves, 177, 187, 196, 200, 202 dimethyl trisulphide, 114 dimethylamine, 22, 25–26, 114 docosahexenoic acid, 10 double-frozen fish block, 150, 282 drip loss, 71 eicosapentaenoic acid, 10 electrical properties, 286–300 conductivity, 287 resistance, 286

473

electronic auction, 462 electronic nose, 105 electronic tongue, 105 Engraulidae, 54 Enterobacteriaceae, 319, 329 Enterococcus spp, 319, 330 enthalpy, 173 enzyme-linked immunosorbent assay (ELISA), 56, 357 enzymes, 16 Escherichia coli, 44, 332 EU quality grading scheme, 428 exon, 366 eye fluids, 33 FAO, 1 fat content, 3 fat determination, 93 fat soluble vitamins, 11, 13 fatty acids, 264 authenticity, 264, 389 positional distribution, 389 profiles, 392 fatty fish species, 3 firmness, 214, 246–248 Fischtester, 286, 294–296 fish, 4 autolysis, 42 captured, 1–2 chilled, 114 decomposition, 51 farmed, 310, 396 fillet, 4 freshwater, 3 geographic origin, 398–403 marine, 3 meal, 3 microflora, 50 mince, 139 muscle, 4, 349 odour, 109–111 oil, 95, 264, 401 organically farmed, 401 proteins, 349 protein hydrolysate, 203 roe, 13, 203 species, 1 Fish Barcode of Life Initiative (FISH-BOL), 380 flavour, 412

474

Index

food-deficient countries, 1 forensically informative nucleotide sequencing (FINS), 370 formaldehyde, 17, 24 freezing/thawing, 254, 294, 359 freshness, 79, 96, 114, 116, 246, 418 frozen storage, 144, 180, 197, 281, 308 gas chromatography. 56 gelation, 188 gels, 143 geosmin, 430 glass transition, 191 glycogen, 68 glycolysis, 16 glycoproteins, 189 grading schemes, 427–430 Gram-negative, 17 groundfish, 4 HACCP, 51, 420, 461 Hafnia alvei, 319 hardness, 214 headspace, 107 heating temperature, 357–359 heat stability, 179 high-resolution NMR, 259–260 histamine determination, 55, 57–58 histamine, 42 fish poisoning, 48 methods of analysis, 57 rapid methods, 58 High Performance Liquid Chromatography (HPLC), 56, 356 high-pressure processing, 156–159, 193 histidine, 17 H NMR imaging of fish, 253 hue, 128 Hunter space, 127 hydrocolloids, 141 hydrolysis, 32 hypoxanthin, 16, 72 indole, 31 image analysis, 244–247 image processing, 240 immunoassay, 357 inosine monophosphate, 26, 73 intron, 366 internal transcribed spacer (ITS), 366

invertebrates, 183 iodine, 15 irradiation, 149 isinglass, 186 isoelectric focusing (IEF), 352 native, 352 urea, 353 isotope ratio mass spectrometry (IRMS), 400 Klebsiella pneumoniae, 44 Kramer-test, 216 k-value, 68, 75 lactic acid, 68 bacteria, 319, 330 Lactobacillus spp, 319, 330 Lactococcus spp, 319 lean fish species, 3 light musculature, 4 lightness, 128 lipids, 10, 259–262 authentication, 388 profiles, 263 oxidation, 203, 262 profiles, 263 lipolysis, 17 Listeria monocytogenes, 322 low-field NMR, 257, 259 magnetic field, 252 magnetic resonance imaging (MRI), 253, 255 SPRITE technique, 256 marine fish, 4 medium fatty species, 3 metabolism, 16 metabolites, 262 phosphocreatine, 263 phosphate, 263 methylamines, 23 2-methylisoborneol, 430 microarrays, 378 microfurnaces, 178 microwave dielectric spectrum, 274 microbiological methods, 318–348, 468 aerobic plate count, 323 detection media, 321 direct plating, 320 incubation conditions, 320 molecular techniques, 336–338 most probable number, 336

Index

475

surface count technique, 320 total viable count, 323 microorganisms, 42 microsatellites, 366 minerals, 15 mitochondrial genes, 367 modified atmosphere packaging, 78, 145–148 moisture determination, 92 molluscan species, 1 Moraxella spp, 319 Morganella morganii, 44 Morganella psychrotolerans,, 45 multivariate data analysis, 91, 278, 413, 444–457 myofibrillar proteins, 180, 214 myoglobin, 201 myosin, 184 light chains, 350

31 P NMR, 262 polyamines, 42 Polymerase Chain Reaction (PCR), 336 quantitative, 378 species-specific primers, 369 universal primers, 369 Pomatomidae, 54 post mortem changes, 15–17, 69–76 Principal Component Analysis (PCA), 392–395, 445 cross validation, 451 loading plot, 450 score plot, 449 proteolysis, 16, 32, 70 proteome, 301 Pseudomonas spp, 44, 319, 325 puncture test, 219 putrescine, 42

23 Na imaging, 255 Natural Colour System (NCS), 132 nematode detection, 99 near infrared (NIR) reflectance spectroscopy. 92, 95 NIR spectroscopy, 87, 95 non-protein-nitrogen, 10 nuclear magnetic resonance (NMR), 252–253 nucleotide, 68–69 degradation, 72 nutritional composition, 4–9, 308

quality, 34 assessment, 49 index method (QIM), 116, 145, 242, 419, 430–438

odour, 412 off-flavours, 430 olfaction, 105–106 oscillatory test, 229 osteichthyes, 4 paired comparison test, 426 paramyosin, 184 partial least square model, 117–119 parasites, 99 parvalbumins, 350 pattern analysis, 242 pelagic fish, 4 β-phenylethylamine, 42 Photobacterium phosphoreum, 45, 319, 331 pH value, 33 phospholipids, 391 pI value, 352 pigmentation, 133

rainbow trout, 132 random amplified polymorphic DNA analysis (RAPD), 377 ranking, 426 receptor, 105 reducing substances, 30 refractive index, 33, 100 refrigerated storage, 144 relaxation curve, 258 restriction-fragment length polymorphism analysis (RFLP), 371 -SSCP, 377 ribonucleic acid (RNA), 366 ribosomal, 366 ribotyping, 331 rigor mortis, 16, 70, 215 safety, 47, 98 Salmonella spp, 335 salting, 199 salt distribution, 255 sarcoplasmic proteins, 184 scombridae, 54 sea bream, 137 seafood, 425 selenium, 15

476

Index

sensors, 105, 240 metal-oxide chemoresistor, 114 MOSFET, 114 amperometric, 115 conducting polymer, 115 quartz microbalance, 115 sensory analysis, 411–424 sensory attributes, 19 sensory characteristics, 413 sensory descriptors, 439 sensory evaluation, 34, 79, 411 consumer studies, 418, 421, 440 facilities, 415 methods, 425–443 product development, 418 research, 418 quality control, 308, 418 sample preparation, 416–417 sensory panel, 412 senses, 127, 412 Serratia spp, 319 shark, 185–186 shelf life, 149, 419, 431, 435 Shewanella putrefaciens, 53, 319, 324, 329 Shigella spp, 332 shortening of fillets, 16, 71 shrimps, 367, 396 single-strand conformation polymorphism and analysis (SSCP), 371–373 site-specific natural isotope fractionation analysis by NMR (SNIF-NMR), 400 smoking, 201 sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS PAGE), 354–356 species differentiation, 373–377 flatfishes, 374 gadoids, 374 groupers, 375 hakes, 374 molluscs, 376 salmonids, 375 sharks, 376 shrimps, 367 snappers, 375 sturgeons, 375 tunas and bonitos, 373 Specific Spoilage Organisms (SSO), 325–328 spermidine, 42 spermine, 42 spin, 252

spoilage, 19, 24–25, 69 bacteria, 324 cod fillets, 111 microbial, 110 odours, 112 squid, 184 stable isotopes, 388, 399–402 stale, 112 Staphylococcus spp, 319, 332 stiffness, 220 storage time, 229–231 stress, 310 stress relaxation test, 226 structure tensor, 243 sulphur compounds, 112, 114 supply chain, 459 surimi, 139–144, 159, 180, 189 taint, 429 tarama, 372 taste, 412 tension analysis, 221 texture, 214, 412 measurement, 214 profile analysis (TPA), 224 thaw rigor, 71 thermal processing, 98, 152 thin-layer chromatography, 56 thiobarbituric acid, 49 Time Domain Spectroscopy (TDI), 273–285 time domain reflectrometry, 275–277 TMA-index, 27 Torrymeter, 287–294 Torry scheme, 428 total volatile basic nitrogen (TVB-N), 17, 19, 20–21, 114 ammonia, 22 distillation, 21 traceability, 458–471 external, 459 internal, 459 validation, 466 verification, 466 trace elements, 388 signatures, 402 traditional methods, 19–39 training, 413 transamination, 42 transflection measurement, 90, 96 transmission measurement, 90, 94

Index transmittance measurement, 90 triangle test, 426 trimethylamine, 22, 24–25, 114 index, 27 oxide, 17, 114 tristimulus, 128 Two-Dimensional Gel Electrophoresis (2DE), 301–317 authenticity, 305 gel staining, 303 image and data analysis, 303 methodology, 302 post mortem metabolism, 306 umami, 72 urea, 10, 353 uric acid, 72 urocanic acid, 50

Vibrio spp, 332, 334 Vibrionaceae, 319 VIS spectroscopy, 87 viscoelastic methods, 226 vitamins, 10–13 volatile acids, 29–30 volatile amines, 468 volatile compounds, 105, 109–110 wavelength, 90, 244 Warner-Bratzler test, 218 water holding capacity, 258 water soluble vitamins, 11, 14 whiteness, 139 world fishery production, 1–2 xanthine, 72

477