Antioxidants and Functional Components in Aquatic Foods [1 ed.] 9780813813677, 0813813670

Citation preview

OH HO HO

OH HO

Written by the top researchers in the field, this volume covers:

HO

• lipid oxidation in aquatic foods • the pro- and antioxidant systems in aquatic foods • key pro- and antioxidants derived from aquatic foods • innovative ways to control lipid oxidation in aquatic foods and food systems with fish oils

OH HO

• the effects of aquatic foods on oxidative stress in the human body

OH HO

• the neurological effects of components derived from aquatic sources • the effects of fish consumption and components from aquatic foods on cardiovascular health.

HO

Antioxidants and Functional Components in Aquatic Foods addresses topics of crucial importance in maintaining the quality of aquatic foods and other muscle foods. It also offers an understanding of the health effects of marine food antioxidants on the human body. The Editor

OH HO

ANTIOXIDANTS AND FUNCTIONAL COMPONENTS IN AQUATIC FOODS

The consumption and popularity of fish and other seafoods has grown significantly in the past few years. One of the principle drivers of this has been their perceived health benefits, which arise mostly from the beneficial fatty acid profile and content of many fish. However, because of their high concentration of polyunsaturated omega-3 fatty acids and active prooxidants marine lipids are highly vulnerable to oxidation. Lipid oxidation is one of the primary causes of deterioration of fish muscle during storage and negatively affects color, odor and flavor, protein functionality, and the overall nutritional content of fish muscle. It is therefore critical to understand the processes behind oxidation and how the natural antioxidant defenses of the fish can be exploited to minimize oxidation.

OH

Dr Hordur G. Kristinsson is Chief Science Officer at Matis Ltd, Reykjavik, Iceland.

Dried Fruits: Phytochemicals and Health Effects Edited by Cesarettin Alasalvar and Fereidoon Shahidi ISBN 978-0-8138-1173-4 Coffee: Emerging Health Effects and Disease Prevention Edited by Yi-Fang Chu ISBN 978-0-470-95878-0

ISBN 978-0-8138-1367-7

Kristinsson

Also available from Wiley Blackwell

OH HO

OH HO

HO

OH OH HO

ANTIOXIDANTS AND FUNCTIONAL COMPONENTS IN AQUATIC FOODS Edited by Hordur G. Kristinsson

HO

HO

O HO

HO

HO O HO

OH HO

9 780813 813677

Kristinsson_Antioxidants_9780813813677_hb.indd 1

11/03/2014 08:54

Antioxidants and Functional Components in Aquatic Foods

Antioxidants and Functional Components in Aquatic Foods Edited by

Hordur G. Kristinsson Matis Ltd, Reykjavik, Iceland

This edition first published 2014 © 2014 by John Wiley & Sons, Ltd Registered office John Wiley & Sons, Ltd, The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK Editorial offices 9600 Garsington Road, Oxford, OX4 2DQ, UK The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK 111 River Street, Hoboken, NJ 07030-5774, 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/wiley-blackwell. The right of the author to be identified as the author of this work has been asserted in accordance with the UK 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. 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. Limit of Liability/Disclaimer of Warranty: While the publisher and author(s) have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose. It is sold on the understanding that the publisher is not engaged in rendering professional services and neither the publisher nor the author shall be liable for damages arising herefrom. 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 has been applied for. ISBN 978-0-8138-1367-7 (cloth) A catalogue record for this book is available from the British Library. Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic books. Cover image: Fresh Rainbow trout and fish oil capsules © Shutterstock/tab62 Underwater background © iStock/Nastco Cover design by Andy Meaden Set in 10.5/12.5pt Times by SPi Publisher Services, Pondicherry, India

1 2014

Contents

List of contributors

ix

Prefacexi 1 Oxidation in aquatic foods and analysis methods Magnea G. Karlsdottir, Holly T. Petty, and Hordur G. Kristinsson

1

1.1 Introduction 1 1.2 Analysis of lipid oxidation 2 1.3 Conclusions 16 References16

2 Protein oxidation in aquatic foods Caroline P. Baron

23

2.1 Introduction 23 2.2 Mechanisms involved in protein oxidation 24 2.3 Impact of protein oxidation on aquatic food 30 2.4 Case studies 33 2.5 Conclusions and perspectives 38 References38

3 Influence of processing on lipids and lipid oxidation in aquatic foods Sivakumar Raghavan and Hordur G. Kristinsson 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8

Effect of freezing on lipid oxidation Effect of salting and drying on lipid oxidation Effect of fermentation on lipid oxidation Effect of smoking on lipid oxidation Effect of high-pressure processing on lipid oxidation Effect of irradiation on lipid oxidation Effect of microwave processing on lipid oxidation Effect of modified atmosphere on lipid oxidation

43 43 49 53 55 58 61 63 65

vi

Contents 3.9 Effect of pH shift extraction method on lipid oxidatio 67 3.10 Effect of canning on lipid oxidation 70 References73

4 Strategies to minimize lipid oxidation of aquatic food products post harvest Huynh Nguyen Duy Bao and Toshiaki Ohshima

95

4.1 Introduction 95 4.2 Lipid oxidation and quality deterioration in post-harvest aquatic food products 96 4.3 Post-harvest control of oxidative deterioration in aquatic food products 106 4.4 Conclusions and prospects 117 References118

5 Antioxidative strategies to minimize oxidation in formulated food systems containing fish oils and omega-3 fatty acids Charlotte Jacobsen, Anna Frisenfeldt Horn, Ann-Dorit Moltke Sørensen, K. H. Sabeena Farvin, and Nina Skall Nielsen

127

5.1 Introduction 127 5.2 The lipid oxidation process 128 5.3 Factors affecting lipid oxidation in omega-3-enriched foods 129 5.4 Introduction to antioxidants 131 5.5 Antioxidant effects in different omega-3-enriched food products 132 5.6 Other strategies to protect omega-3 products against oxidation 145 5.7 Conclusions 145 References146

6 Methods for assessing the antioxidative activity of aquatic food compounds Holmfridur Sveinsdottir, Patricia Y. Hamaguchi, Hilma Eidsdottir Bakken, and Hordur G. Kristinsson

151

6.1 Background 151 6.2 Oxidation and antioxidants 153 6.3 Methods for determining antioxidant activity 157 References169

7 Influence of fish consumption and some of its individual constituents on oxidative stress in cells, animals, and humans Britt Gabrielsson, Niklas Andersson, and Ingrid Undeland

175

7.1 Introduction 7.2 What is oxidative stress? 7.3 Why is oxidative stress of importance and how does it link to diet? 7.4 How is oxidative stress measured? 7.5 Do components in fish affect oxidative stress?

175 176 177 178 182

Contents

vii

7.6 Effects of fish intake on biomarkers used to evaluate oxidative stress 195 7.7 Methodological considerations 200 7.8 Conclusion and need for future studies 202 References204

  8 Marine antioxidants: polyphenols and carotenoids from algae Kazuo Miyashita

219

8.1 Introduction 219 8.2 Chain-breaking antioxidants 220 8.3 Antioxidants and their beneficial health effects 221 8.4 Seaweeds as a rich source of antioxidants 222 8.5 Algal polyphenols 222 8.6 Marine carotenoids 224 8.7 Antioxidant activity of carotenoids 225 8.8 Astaxanthin and fucoxanthin 226 8.9 Conclusions 228 References229

  9 Fish protein hydrolysates: production, bioactivities, and applications Soottawat Benjakul, Suthasinee Yarnpakdee, Theeraphol Senphan, Sigrun M. Halldorsdottir, and Hordur G. Kristinsson

237

9.1 Introduction 237 9.2 Source of fish protein hydrolysates 238 9.3 Production of fish protein hydrolysate 241 9.4 Properties of hydrolysate 255 9.5 Applications of fish protein hydrolysates 263 References266

10 Antioxidant properties of marine macroalgae Tao Wang, Rosa Jonsdottir, Gudrun Olafsdottir, and Hordur G. Kristinsson

283

10.1 Introduction 283 10.2 Antioxidant properties of algal polyphenols 284 10.3 Antioxidant activity of algal sulfated polysaccharides 298 10.4 Antioxidant activities of fucoxanthin 302 10.5 Antioxidant activities of sterols from marine algae 304 10.6 Antioxidant activities of peptides derived from marine algae 306 10.7 Antioxidant activity of mycosporine-like amino acids 307 10.8 Concluding remarks 310 References311

Index319

List of Contributors

Niklas Andersson Division of Life Sciences/Food Science, Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden

Britt Gabrielsson Division of Life Sciences/Food Science, Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden

Hilma Eidsdottir Bakken Division of Biotechnology and Biomolecules, Matis Ltd, Saudarkrokur, Iceland

Sigrun M. Halldorsdottir Division of Biotechnology and Biomolecules, Matis Ltd, Reykjavik, Iceland

Huynh Nguyen Duy Bao Faculty of Food Technology, Nha Trang University, Vietnam

Patricia Y. Hamaguchi Division of Biotechnology and Biomolecules, Matis Ltd, Saudarkrokur, Iceland

Caroline P. Baron National Food Institute, Technical University of Denmark, Kgs. Lyngby, Denmark Soottawat Benjakul Department of Food Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla, Thailand K. H. Sabeena Farvin Section for Aquatic Lipids and Oxidation, National Institute of Aquatic Resources, Technical University of Denmark, Kgs. Lyngby, Denmark

Anna Frisenfeldt Horn Section for Aquatic Lipids and Oxidation, National Institute of Aquatic Resources, Technical University of Denmark, Kgs. Lyngby, Denmark Charlotte Jacobsen Section for Aquatic Lipids and Oxidation, National Institute of Aquatic Resources, Technical University of Denmark, Kgs. Lyngby, Denmark Rosa Jonsdottir Division of Biotechnology and Biomolecules, Matis Ltd, Reykjavik, Iceland

x

List of Contributors

Magnea G. Karlsdottir Division of Biotechnology and Biomolecules, Matis Ltd, Reykjavik, Iceland Hordur G. Kristinsson Division of Biotechnology and Biomolecules, Matis Ltd, Reykjavik, Iceland Department of Food Science and Human Nutrition, University of Florida, Gainesville, Florida, USA Kazuo Miyashita Faculty of Fisheries Sciences, Hokkaido University, Hakodate, Japan Nina Skall Nielsen Section for Aquatic Lipids and Oxidation, National Institute of Aquatic Resources, Technical University of Denmark, Kgs. Lyngby, Denmark Gudrun Olafsdottir School of Engineering and Natural Sciences, University of Iceland, Reykjavik, Iceland Toshiaki Ohshima Department of Food Science and Technology, Tokyo University of Marine Science and Technology, Japan Holly T. Petty Department of Food Science and Human Nutrition, University of Florida, Gainesville, Florida, USA Sivakumar Raghavan Department of Food Science and Human Nutrition, University of Florida, Gainesville, Florida, USA

Theeraphol Senphan Department of Food Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla, Thailand Ann-Dorit Moltke Sørensen Section for Aquatic Lipids and Oxidation, National Institute of Aquatic Resources, Technical University of Denmark, Kgs. Lyngby, Denmark Holmfridur Sveinsdottir Division of Biotechnology and Biomolecules, Matis Ltd, Saudarkrokur, Iceland Ingrid Undeland Division of Life Sciences/Food Science, Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden Tao Wang School of Food Science and Technology, Dalian Polytechnic University, Dalian, China Center for Excellence in Post-Harvest Technologies, North Carolina A&T State University, Kannapolis, NC, USA Suthasinee Yarnpakdee Department of Food Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla, Thailand

Preface

The consumption and popularity of fish and other aquatic animals in the world ­continues to grow. One of the reasons for this is the health benefits of seafood, which traditionally have been linked to the beneficial fatty acid profile and content of many fish (i.e. omega-3 fatty acids). Recent research, however, points to many more compounds that can have beneficial health effects. Aquatic animals, particularly fish, have long been known to be very susceptible to lipid oxidation, which leads to off-odors/flavors, reduced nutritional value, undesirable appearance and loss of quality. Omega-3 fatty acids are particularly prone to oxidation and if ­oxidized they lose their nutritional value. It is therefore important to understand the processes behind oxidation and how we can delay it. Only relatively recently have food scientists truly started to understand the role, function, and magnitude of different endogenous pro-oxidants. Also, we have in recent years started to understand better the natural antioxidative systems found in fish muscle and made the discovery that they are particularly potent and may even have the potential to be developed into ingredients for other food products. This goes beyond just fish, as there are many other aquatic animals that contain interesting and highly functional compounds that can find uses in the food, pharmaceutical and feed industries, including marine algae. This book, written by leading experts in the field, provides comprehensive coverage of the oxidative processes associated with aquatic foods, antioxidant mechanisms and procedures, the influence of processing on oxidation, and the health effects of consuming aquatic foods and their components. It also gives an overview of various natural antioxidants found in aquatic animals, or that can be extracted from them, in addition to techniques for analysing their activity.

1 Oxidation in aquatic foods and analysis methods Magnea G. Karlsdottir1, Holly T. Petty2, and Hordur G. Kristinsson1,2 Division of Biotechnology and Biomolecules, Matis Ltd, Reykjavik, Iceland

1 

Department of Food Science and Human Nutrition, University of Florida, Gainesville, Florida, USA

2 

1.1  Introduction Lipid oxidation in muscle food is one of the major deteriorative reactions causing a loss in quality during storage. Marine lipids are natural and good sources of ­polyunsaturated omega-3 fatty acids (PUFAs), which have been reported to have beneficial health effects. However, because of the high amount of PUFAs (Ackman 1980; Shewfelt 1981; Gandemer 1999), along with highly active pro-oxidants (Hultin 1994), marine lipids are highly vulnerable towards oxidation. Lipid oxidation is therefore one of the primary causes of deterioration of fish muscle during storage (Ackman 1980) and negatively affects color (Wasasundara and Shahidi 1994), odor and flavor (Bateman et al. 1953), protein functionality and conformation (Gutteridge 1988), and the overall nutritional content of fish muscle (Pearson et al. 1983; Gray 1987). Lipid oxidation can be divided into three types of initiation reactions, including non-enzymatic and enzymatic reactions. Non-enzymatic mechanisms include autoxidation (free radical mechanism) and photogenic oxidation (singlet oxygen mediated). Enzymatic mechanisms include actions by lipoxygenase and cyclooxygenase. Lipid oxidation most commonly occurs by a free radical mechanism involving the formation of a reactive peroxyl radical (Erickson 2002). The autoxidation mechanism occurs when the unsaturated fatty acids are exposed to oxygen and undergo an autocatalytic chain reaction (Figure 1.1). This mechanism is deemed to be the primary cause of lipid oxidation in post-mortem fish (Erickson 2002) and is historically referred to as lipid peroxidation (Mead 1976). The process has three main phases: initiation, propagation, and termination. Antioxidants and Functional Components in Aquatic Foods, First Edition. Edited by Hordur G. Kristinsson. © 2014 John Wiley & Sons, Ltd. Published 2014 by John Wiley & Sons, Ltd.

2

CH1 Oxidation in aquatic foods and analysis methods LH (fatty acid acyl chain) Initiation O2

H

L

(antioxidant) AH

A

LOO

LOOH

Propagation

LOOH (hydroperoxide)

LH Secondary products (aldehydes, ketones, alcohols, small acids, alkanes)

Figure 1.1  Autoxidation of polyunsaturated fatty acids. LH; AH.

Initiation begins with hydrogen abstraction from an unsaturated fatty acid, p­ articularly those having a pentadiene structure (Erickson 2002). The lipid radical (L∙) then reacts with molecular oxygen, resulting in peroxyl radical (LOO∙). Propagation is the phase in which the peroxyl radical (LOO∙) abstracts hydrogen from neighboring lipid molecules, thereby forming more lipid radicals and lipid hydroperoxide (LOOH) molecules. Lipid hydroperoxides cannot be detected through sensory a­ nalysis, which is the reason why peroxides do not correlate with “off-flavor”. The degradation of these lipid hydroperoxides, due to metal catalysts, results in ­unfavorable secondary intermediates of shorter chain length: ketones, aldehydes, alcohols, and small alkanes. The secondary products give rise to offaromas, ­flavors, and yellow color in fish (Khayat and Schwall 1983). Termination occurs when a radical reaction is quenched by a reaction with another radical or antioxidant. It is important to note that these reactions occur simultaneously and that the mechanism of lipid oxidations can become quite complex (Huss 1995).

1.2  Analysis of lipid oxidation Several methods have been developed to measure different compounds as they form or degrade during lipid oxidation (Figure 1.2). Since the system is dynamic, it is recommended that two or more methods are used to obtain a more complete understanding of lipid oxidation. In order to follow the lipid oxidation process, reactants, intermediates, and final products can be measured. Many of these compounds are unstable and they are often variously affected by the presence of pro-oxidants, ­antioxidants, and oxygen. Also, since various food systems have different compositions, the pattern of intermediates and products formed differs greatly. It is therefore recommended that more than one stage of the oxidation process is monitored since



PV

Oxygen electrode

Concentration

3

1.2 Analysis of lipid oxidation

Oxygen uptake

1°

Acid value, FFA Anisidine value, TBA, sensory, GC

Peroxide Acids

Polymers

Aldehydes 2°

3° Fluorescense, colorimetry, sensory

Time

Figure 1.2  An example of the progress of lipid oxidation and breakdown of lipid oxidation products as assessed by different methods. Oxygen uptake is with Oxipress or an oxygen electrode. The primary products are measured as peroxide values (PVs). Measurement of free fatty acids (FFA) gives the acid value. Secondary products (e.g., aldehydes) are measured by the thiobarbituric acid (TBA) test, and sensory evaluation and gas chromatography (GC). Tertiary products are polymers, which can be measured with fluorescence, colorimetric and sensory evaluation. Rosa Jonsdottir, Matis-Icelandic Food Research. Reproduced with permission of Rosa Jonsdottir.

using only one method might give rise to results that are difficult to interpret and highly misleading (Undeland 1997). The available methods for monitoring and evaluate lipid oxidation in foods can be divided into few groups based on what they measure: oxygen consumption, loss of initial substrates, formation of free radicals, and formation of primary, secondary, and tertiary oxidation products. Table 1.1 summarizes the main methods that have been employed in laboratories and the industry for the measurement of various lipid ­oxidation parameters.

1.2.1  Reactants and initiation of lipid oxidation 1.2.1.1  Oxygen consumption The major reactants in lipid oxidation are oxygen and unsaturated fatty acids, but exposure of lipids to atmospheric oxygen results in unstable intermediates (Erickson 2002). Inasmuch as lipid oxidation results in the uptake of oxygen from the surroundings, measuring the time required for the onset of the rapid disappearance of oxygen in a closed system provides a means of determining oxidative stability (Pike 2003). Oxygen consumption can also be measured by electrochemical detection of changes in oxygen concentration and can be useful in simple lipid systems (Eriksson and Svensson 1970; Halliwell and Gutteridge 1985). This technique has been used to analyze the activity of lipoxygenases isolated from fish (German et al. 1986), for example, but the analysis of the graphical data obtained creates a bottleneck for it.

• Sensitive to the detection of free radicals (intermediate)

• Sensitive and simple method • Most common measurement of primary oxidation in fish • Small sample amounts needed • Lipid hydroperoxides can also be analyzed in more specific ways using HPCL

Electron spin resonance

Peroxide value

Fatty acid composition

• Can be used as a chemical marker of lipid oxidation

Loss of antioxidants • e.g., α-tocopherol Iodine value

• Highly sophisticated way of measuring intermediates • Radicals not detectable at low concentration • Misleading results: a low value may represent either the beginning of or advanced oxidation • Poor correlation with sensory evaluation • Hazardous solvents used • Need to extract lipid from the samples

• Not a rapid method • Lipid extraction and esterification of fatty acids needed before GC analysis • Species dependant

• Not a rapid method • Lipid extraction needed

• The function and relevance of each pro-oxidant largely depends on its concentration and close surrounding • The presence of other compounds may enhance or inhibit the catalytic effect • These compounds may also be consumed by protein oxidation • Have to be carefully evaluated

• May be used as predictors of lipid oxidation

Pro-oxidants • e.g., metals and heme protein

• Measure of degree of unsaturation • Can be used as an indication of lipid oxidation since there is a decline in unsaturation during oxidation • May be used as an indicator of lipid oxidation • May correlate with PV and TBARS

• Useful in simple lipid systems

• Good correlation with rancidity shelf-life test

Oxygen consumption

Disadvantages

Advantages

Methods

Wheeler 1932; Lea 1946; Asakawa and Matsushita 1978; Gray 1978; Akasaka et al. 1987; Yamamoto and Ames 1987; Francisco and Munch 1989; Ohshima et al. 1996; Undeland 1997; Yasuda and Narita 1997; Pike 2003; Chotimarkorn et al. 2006

Shono and Toyomitzu 1971; Beltran and Moral 1990; Polvi et al. 1991; Xing et al. 1993; Verma et al. 1995; Castrillón et al. 1996; Milo and Grosch 1996; Aubourg et al. 1997; Undeland 1997; Frankel 1998; Sarma et al. 2000; Aranda et al. 2006 Halliwell and Gutteridge 1985; Halliwell and Chirico 1993; Undeland 1997; Carlsen et al. 2001; Andersen and Rinnan 2002

Hudson and Gordon 1999; Pike 2003

Eriksson and Svensson 1970; Halliwell and Gutteridge 1985; German et al. 1986; Undeland 1997; Pike 2003 Hultin 1988; Decker et al. 1989; Harris and Tall 1989; Decker and Hultin 1992; Schaich 1992; Hultin 1994; Undeland 1997; Lauritzsen et al. 1999; Lauridsen et al. 2000; Thanonkaew et al. 2006 Deng et al. 1978; Banda and Hultin 1983; Philippy 1984; Erickson 1993a,b; Brannan and Erickson 1996; Watanabe et al. 1996

References

Table 1.1  Overview of the main methods used to monitor lipid oxidation products and potential predictors of lipid oxidation

• Useful for monitoring the early stages of oxidation • Rapid and simple method

• Sensitive and simple method • Gives good information about lipid oxidation • Correlates well with the amount of total volatile substances

• Sensitive method • Lipid extraction is not required • Many modifications of the test have been developed • Correlates well with sensory evaluation • Sensitive technique for lipid oxidation • Correlates with TBARS, FFA and TVB • Comparable with sensory analysis and GC • Potential for on-line/at-line determination of lipid oxidation • Quantification at low concentration, high analysis speed and rapid sample preparation • May be used for direct determination of lipid hydroperoxides in fish muscle, without the need for extraction of lipid

Conjugated dienes and trienes

Anisidine value

TBA value

Fluorescent image analysis

Fluorescence spectroscopy

Advantages

Methods

• Non-specific to the vast amount of different oxidation products • A new approach

• Non-specific to the vast amount of different oxidation products • A new approach

• Fairly unspecific method • The magnitude of the changes in absorption is not easily related to the extent of oxidation in the advanced stage • At 268 nm, other oxidation products are also absorbing, in addition to the trienes • Does not measure the concentration of specific compounds (no unit; measure group of chemicals) • The absorption depends on the substrate combination and the value is only comparable within the same oil type • Need to extract lipids from the sample • Other compounds can react with TBA • A measure of a transient product of oxidation

Disadvantages

(continued )

Marcuse and Johansson 1973; Gray 1978; Ke and Woyewoda 1979; Robbles-Martinez et al. 1982; Pokorny et al. 1985; Shahidi et al. 1985, 1987; Tomas and Fumes 1987; Harris and Tall 1989; Schmedes and Holmer 1989; Undeland 1997; Pike 2003 Pokorny et al. 1974; Erickson 1993a; Aubourg et al. 1997, 1998; Undeland 1997; Frankel 1998; Undeland et al. 1998; Aubourg 1999; Wold et al. 2002; Veberg et al. 2006 Akasaka et al. 1987; Francisco and Munch 1989; Aubourg et al. 1997, 1998; Undeland 1997; Frankel 1998; Aubourg 1999; Chotimarkorn et al. 2006; Veberg et al. 2006

White 1995; Doleschall et al. 2002; Guillén and Cabo 2002; Pike 2003; van der Merwe et al. 2004

Gray 1978; Brown and Snyder 1982; Madhavi et al. 1996; Undeland 1997; Pike 2003

References

• Lipid extraction is not required • May correlate well with sensory determination of lipid oxidation

• Best method to monitor quality changes in fish caused by lipid oxidation

• Fast and ­non-destructive method • Simultaneous determination of multiple components per measurement • Can provide real-time information from the process stream

Volatile organic compounds

Sensory analysis

Near-intrared spectroscopy

• The quantity of other volatile compounds resulting from lipid oxidation can be obtained simultaneously and may enhance the characterization of lipid oxidation in various food commodities • Costly method • Poor reproducibility, depends on level of panelist training and skill • Requires large samples • A secondary method, depends on less-precise reference methods • Need to develop appropriate multivariate calibration approaches to build accurate model systems before it can be used for routine analysis

Disadvantages

Frankel 1998; Coppin and Pike 2001; Macfarlane et al. 2001; Broadbent and Pike 2003; Timm Heinrich et al. 2003; Rustad 2010 Li et al. 2000; Yildiz et al. 2001; Gerde et al. 2007; Roggo et al. 2007; Poon 2009

McGill et al. 1974; Undeland 1997; Pike 2003; Olafsdottir and Jonsdottir 2009

References

basic nitrogen

PV, peroxide value; TBARS, thiobarbituric acid reactive substances; GC, gas chromatography, TBA, thiobarbituric acid; FFA, free fatty acids; TVB, total volatile

Advantages

Methods

Table 1.1  (continued )



7

1.2 Analysis of lipid oxidation

Table 1.2  Important pro-oxidants present in fish tissues (Underland, 1997. Reproduced with permission of the International Institute of Refridgeration.) Low molecular weight metals Iron Copper

Reducing systems

Heme protein

Enzymes

Superoxide Ascorbate Mitochondial systems Microsomal systems

Myoglobin Hemoglobin Cytochromes

Lipoxygenases Cyclooxygenase Peroxidases

1.2.1.2  Pro-oxidants There are many compounds that are naturally present in fish muscle and can serve as pro-oxidants by interfering with the reactants at different stages of the oxidation process (Hultin 1988). They could therefore serve as predictors of lipid oxidation. These compounds are in nature both enzymatic and non-enzymatic (Table  1.2), such as transition metals, heme proteins, reducing agents, peroxidases, and ­lipoxygenases (Harris and Tall 1989; Hultin 1994). Transitional metals and heme proteins have been reported as one of the major pro-oxidants in muscle foods (Decker and Hultin 1992) and are thought to play a key role in the initiation of the autoxidation process. It has been reported that the detrimental effects of transitional metals and heme proteins are greater than effects due to lipoxygenase, mainly because of longer-lasting pro-oxidative activity (Medina et al. 1999). Both Fe and Cu are known to promote oxidative reactions  (Walling 1975), resulting in highly reactive hydroxyl radicals that cause oxidative damage to lipid membranes (Decker et al. 1989; Lauritzsen et al. 1999; Lauridsen et al. 2000). Thanonkaew et al. (2006) studied the effects of various metal ions (Fe, Cu, and Cd) at various concentrations on lipid oxidation, discolorations, and p­ hysicochemical properties of muscle protein in cuttlefish subjected to multiple freeze–thaw cycles. The rate of thiobarbituric acid reactive substances (TBARS) increases varied depending on concentration, type, and valency of the metal ion. Fe(II) induced lipid oxidation most effectively and its pro-oxidative effect was in a concentration-dependent manner, while Cu(I), Cu(II), and Cd(II) showed negligible effects on lipid oxidation. Detection of different pro-oxidants could be a valuable way to predict lipid ­oxidation, but the relevance and function of each pro-oxidant largely depends on its close surroundings and its concentration.

1.2.1.3  Antioxidants Antioxidants have the potential to act as chemical markers of lipid oxidation (Table 1.3). Following the dynamics of antioxidants degradation has been suggested as a convenient way to predict when the exponential phase of lipid oxidation would start, given that the various antioxidants deteriorate at different times

8

CH1 Oxidation in aquatic foods and analysis methods Table 1.3  Important inhibitors of lipid oxidation located in fish muscle. (adapted from Jonsdottir et al. 2008) Inhibitor/antioxidant Lipophilic antioxidants –– Phenol compounds –– α-tocopherol –– Carotenoids Antioxidants enzymes –– Superoxide dismutase –– Glutathione peroxidase –– Q-10 (ubiquinone)

Hydrophilic antioxidants –– Phenol compounds –– Glutathione –– Ascorbate (vitamin C) –– Peptide, polyamine –– Free amino acids (histidine) –– Urea

(Buettner 1993). Loss of antioxidants could therefore serve as an early indicator of lipid o­ xidation, and this method has gained more and more attention (Deng et al. 1978; Banda and Hultin 1983; Philippy 1984; Erickson 1993a,b; Watanabe et al. 1996). It is known that α-tocopherol may decrease significantly during cold and frozen storage of fatty fish such as sardine, trout, and mackerel (Pozo et al. 1988; Ackman and Timmins 1995), leaner fish like saithe (Dulavik et al. 1998), and c­ hannel catfish (Brannan and Erickson 1996). The study by Erickson (1993a) showed that following the decrease in α-tocopherol level was the best way to distinguish between farm-raised and hybrid striped bass during the induction period of the lipid oxidation process in comparison with TBARS, volatiles, dienes, fluorescence, and ascorbic acid. According to earlier studies, aqueous antioxidants appear to be more sensitive predictors of early oxidation changes in muscle since they seems to be consumed faster than their l­ ipid-­soluble counterparts. In channel catfish, antioxidants decreased in the following order at −6 °C: glutathione > ascorbic acid > α-tocopherol > γ-tocopherol (Brannan and Erickson 1996). However, before using ­antioxidants as predictors of lipid oxidation, the correlation between their degradation and the formation of lipid oxidation products should be carefully evaluated since these compounds can also be consumed by protein oxidation (Srinivasan and Hultin 1994; Undeland 1997).

1.2.1.4  Iodine value The iodine value is a measure of the degree of unsaturation, that is, the number of carbon–carbon double bonds in relation to the amount of fat or oil. The higher the amount of unsaturation, the more iodine is absorbed, therefore the higher the iodine value and the greater the degree of unsaturation. The iodine value can be used as an index of lipid oxidation since there is a reduction in unsaturation during oxidation (Hudson and Gordon 1999). This method is not rapid, however, and requires lipid extraction of muscle samples.



1.2 Analysis of lipid oxidation

9

1.2.1.5  Fatty acid composition The fatty acid composition, or fatty acid profile, of food products is determined by quantifying the kind and amount of fatty acids that are present, usually by extracting the lipids and analyzing them using gas chromatography (GC). Changes in fatty acid composition provide an indirect measure of the extent of lipid oxidation. Initiation of lipid oxidation in fish is generally associated with PUFAs in the phospholipids of muscle cell membranes, which are known to be more susceptible to oxidation than triacylglycerols in fat deposits. Studies are rather conflicting, however, concerning the use of fatty acid loss as an indicator of lipid oxidation. Considerable loss of PUFAs has been found after storage of jack mackerel light muscle and carp at 5 °C (Shono and Toyomitzu 1971). Castrillón et al. (1996) also found a link between a drop in the C22:6/C16:0 ratio and the rate of peroxide value (PV) and TBARS increase in frozen sardines. In contrast, no consistent pattern of change in omega-3 fatty acids was found in frozen sardines (Beltran and Moral 1990), in frozen and iced cod and mackerel (Xing et al. 1993) or in Atlantic salmon (Polvi et al. 1991).

1.2.2  Intermediate products of lipid oxidation Free radicals are important short-lived intermediates involved in the initial steps of lipid oxidation. The level of oxidation can be measured directly by detecting the formation of radicals, but methods based on this detection provide a good indication of initiation of lipid oxidation (Carlsen et al. 2001; Andersen and Skibsted 2002). Electron spin resonance (ESR) spectrometry is the only analytical technique that can specifically detect free radicals involved in autoxidation and related processes (Holley and Cheeseman 1993; Sharma and Buettner 1993; Milic et al. 1998). This method has been used to detect the formation of lipid radicals during oxidation in biological systems as well as the presence of superoxide and hydroxyl radicals (Halliwell and Gutteridge 1985; Halliwell and Chirico 1993). It can be rather difficult to quantify these radicals at low concentration, however, due to their short lifetimes.

1.2.3  Lipid oxidation products (primary, secondary, and tertiary) 1.2.3.1  Peroxide value The most common method of measuring primary oxidation products in muscle foods and oils is PV (Gray 1978; Pike 2003). The PV represents the total hydroperoxide and peroxide oxygen content of lipids or lipid-containing material, and is one of the most common quality indicators of fats and oils (Ruiz et al. 2001; Antolovich et al. 2002). A number of methods have been developed for determination of PV, but

10

CH1 Oxidation in aquatic foods and analysis methods

the iodometric (Wheeler 1932; Lea 1946; Asakawa and Matsushita 1978) and ferric thiocyanate (Santha and Decker Eric 1994) methods are the most frequently used. Lipid hydroperoxides can also be analyzed in a more specific way using HPCL (Yamamoto and Ames 1987; Ohshima et al. 1996; Yasuda and Narita 1997). The iodometric method is based on the ability of hydroperoxide to produce iodine (I2) from potassium iodide by a titration process using sodium thiosulfate (Na2S2O3). The chemical reactions involved in this method are given below, where ROOH is lipid hydroperoxide and ROOR is lipid peroxide: ROOH + 2H + 2I – → I 2 + ROH + H 2 O ROOR + 2H + + 2I – → I 2 + 2 ROH I 2 + 2S2 O32 – → S2 O32 – + 2I – Potential limitations of this method are well recognized and include poor sensitivity and selectivity, absorption of iodine at unsaturation sites of fatty acids leading to low results, liberation of iodine from potassium iodide by oxygen present in the solution to be titrated, and variation in the reactivity of different peroxides. This method also fails to adequately measure low PV because of difficulties in determination of the titration end point. For determination in foodstuffs, a disadvantage of this technique is the large amount of sample required, leading both to a significant amount of waste and difficulty in obtaining sufficient quantities from foods low in fat. Despite several disadvantages, the iodometric method still remains the standard procedure. The ferric thiocyanate method is a colorimetric method based on the ability of hydroperoxide to oxidase ferrous ion (Fe2+) to ferric ion (Fe3+) and has been widely accepted. This technique is simple, reproducible and more sensitive than the standard iodometric method and can detect very low peroxide concentrations. The small sample size requirement is also an advantage, making this method convenient for studying a large number of samples. The unstable and intermediate nature of peroxides and their sensitivity to temperature makes PV an approximate indicator of the state of oxidation, but particularly in the early stage of oxidation it serves as a good tool for the measurement of degree of oxidation. The PV method, however, generally shows little correlation with sensory evaluation as the lipid hydroperoxides are odor and flavorless, and as such it is not a useful practical method for detecting rancidity levels in aquatic foods as they are perceived by people.

1.2.3.2  Conjugated dienes and trienes Double bonds in polyunsaturated lipids are changed from non-conjugated to conjugated bonds on oxidation. Primary products containing conjugated double bonds can be measured with simple spectrometry at 234 (dienes) and 268 nm (trienes) (Figure 1.3). This is a rapid method and useful for monitoring the early stage of



11

1.2 Analysis of lipid oxidation Hydroperoxydiene H3C

Oxodiene CH3

H3C

O OH

CH3 O

Reduction

H3C

CH3

Hydroxydiene

OH H3C

Conjugated triene

CH3

H3C

CH3

Conjugated tetraene

Figure 1.3  Chemical reaction steps in the conjugable oxidation products assay. Shahidi & Wanasundra, 2002. Reproduced with permission of Taylor and Francis.

oxidation, but at the same time it is a rather unspecific method (Gray 1978). High background absorbance originating from the native lipid interferes with that arising from the conjugated diene structure at 234 nm. At 268 nm, other oxidation products are also absorbing in addition to the triens, such as ethylenic di-ketones and ­oxo-dienes (Brown and Snyder 1982; Pike 2003). This method can often not be performed directly on muscle samples because many other interfering substances are present (Madhavi et al. 1996), such as heme proteins, carotenoids, and chlorophylls, which absorb strongly in the UV region. Extraction of lipids into organic solvents before analysis is therefore a common approach to this problem.

1.2.3.3  p-anisidine value and totox value The p-ansidine value determines the amount of α- and β-unsaturated aldehydes (principally 2-alkenal and 2,4-alkadienals), which are secondary oxidation products, in fats and oils. The aldehydes react with p-ansidine to form chromogen, resulting in yellowish products that absorb at 350 nm. Since colored products from unsaturated aldehydes are more strongly absorbed at this wavelength, the method is more sensitive to unsaturated aldehydes compared to the saturated aldehydes. The method correlates well with the amount of total volatile substances (Doleschall et al. 2002) and has been shown to be a reliable indicator of lipid oxidation in oils and fatty foods (van der Merwe et al. 2004). However, studies have shown that the p-ansidine value is only comparable within the same oil type due to variation of the initial value among oil sources (White 1995; Guillén and Cabo 2002).

12

CH1 Oxidation in aquatic foods and analysis methods

The Totox value indicates the total oxidation of a sample using both the peroxide (primary oxidation product) and p-ansidine (secondary oxidation product) values: Totox value = p − ansidine value + (2 × PV) Since the PV measures hydorperoxide (which increases and then decreases) and the p-ansidine value measures aldehydes (decay products of hyroperoxides which continually increase), the Totox value usually rises continually during the course of lipid oxidation (Pike 2003). The main disadvantage of the Totox value is its lack of scientific basis because it combines variables with different dimensions (Shahidi and Wanasundra 2002).

1.2.3.4  Thiobarbituric acid test The thiobarbituric acid (2-thiobarbituric acid; TBA) test measures a secondary product of lipid oxidation, in particular malonaldehyde (Gray 1978), and is one of the most commonly used methods of detecting rancidity in some foods and oxidation products in biological systems (Frankel 1998). It is a colorimetric method based on the absorbance at 530–535 nm of the pink color formed between TBA and oxidation products of polyunsaturated lipids, in particular malonaldehyde (or malonaldehyde-type products) (Figure 1.4). This reaction is not specific to malonaldehyde, and results are often reported as TBARS. The tissue samples may be reacted directly with TBA or on a tissue distillate to eliminate interfering substances. One advantage of this method, therefore, is that the lipid does not have to be extracted from the ­tissue (Harris and Tall 1989; Undeland 1997). There are a few limitations when using the TBA test for evaluation of the oxidative state of foods and biological systems due to their chemical complexity. Components such as protein and sugar degradation products, amino acids, and nucleic acids interfere with the formation of the TBA color complex. Many modifications of the test have therefore been developed (Marcuse and Johansson 1973; Ke and Woyewoda 1979; Robbles-Martinez et al. 1982; Pokorny et al. 1985; Shahidi et al. 1985, 1987; Tomas and Fumes 1987; Schmedes and Holmer 1989).

OH

2 HS

N

OH +

N TBA

OH

O

O H

H MA

OH

HN S

NH N

OH

O

N H

TBA-MA complex

Figure 1.4  Reaction of 2-thiobarbituric acid (TBA) and malonaldehyde (MA).

S



1.2 Analysis of lipid oxidation

13

Despite its limitations, the TBA test, with minor modifications, is frequently used to measure lipid oxidation in a wide range of food products (Pike 2003). The test provides a good means of evaluating the relative oxidative state of food systems, especially on a comparative basis (Shahidi and Wanasundra 2002).

1.2.3.5  Fluorescence spectroscopy The formation of tertiary lipid oxidation products can be followed using fluorescence spectroscopy. Hydroperoxides (primary oxidation products) and aldehydes such as malonaldehyde (secondary oxidation products) can interact with proteins, phospholipids, and nucleic acids, forming Schiff bases (Figure 1.5). This reaction can lead to the formation of chromophores, which are brown-colored compounds (Frankel 1998). The fluorescent compounds formed from lipids are the result of the oxidation of phospholipids or are formed from oxidized fatty acids in the presence of phospholipids (Rustad 2010). Fluorescence spectroscopy has been demonstrated to yield good indices of lipid oxidation in biological materials such as fish (Aubourg et al. 1998). Formation of both aqueous and lipid-soluble fluorescence has been followed in fish (Pokorny et al. 1974; Erickson 1993a; Undeland et al. 1998; Aubourg and Medina 1999; Aubourg et al. 2007). Evaluations of organic fluorescence have been proved to be better than aqueous fluorescence in monitoring the initiation and propagation phases of lipid oxidation in minced bass (Erickson 1993a). Fluorescence with excitation/ emission settings of 327/415 and 393/463 nm have been demonstrated to be a more effective index of changes in fish quality than other commonly used methods (Aubourg and Medina 1999), and correlate well with TBARS after frozen storage and TVB-N after chilled storage (Aubourg and Medina 1997). Front-face fluorescence is a relatively new approach in which intact samples are measured. This method has been shown to be a sensitive technique with regard to lipid oxidation and comparable with sensory analysis and gas chromatography (Wold et al. 2002). It is therefore one of few methods with an on-line/at-line ­potential for determination of lipid oxidation. O HC CH CHOH

RNH2

RN CH CH CHOH Amine-malonaldehyde adduct (Non-fluorescent)

RNH2 O

O

HC CH2 CH Malonaldehyde

RN CH CH CH NHR Conjugated Schiff base (Fluorescent)

Figure 1.5  Reaction of lipid oxidation products and amines.

14

CH1 Oxidation in aquatic foods and analysis methods

The fluorescence techniques are very sensitive and are 10–100 times more s­ensitive for the detection of malonaldehyde compared to the TBA method. However, the fluorescence technique is not specific since it evaluates a complex mixture ­resulting from interactions of oxidized lipids, phospholipids, malonaldehyde, and ­unsaturated a­ldehydes with proteins, peptides, amino acids, nucleic acids, and DNA (Frankel 1998).

1.2.3.6  Volatile organic compounds Most secondary lipid oxidation products are volatile and can be responsible for the off-flavors and odors of oxidized oils and fats. Volatile compounds are therefore highly related to flavor, quality, and oxidative stability. Oxidative ­processes ­occurring during the storage of fish result in the accumulation of aldehydes, which contribute to the development of rancid cold-store flavors such as hexanal, ­cis-4-heptenal, 2,4-heptadienal, and 2,4,7-decadienal (McGill et al. 1974). Studies by Olafsdottir and Jonsdottir (2009) on the development of volatile compounds in chilled cod ­ ­ fillets showed that oxidatively formed, ­lipid-derived saturated aldehydes such as hexanal, heptanal, and decanal were detected in the fillets throughout the storage time. These oxidation products contributed to the overall characteristic fish-like odors of chilled cod fillets in combination with other carbonyls (3-hydroxy-2-­butanone, 3-methyl-butanal, 2-butanone, 3-pentanone, and 6-methyl-5-heptene-2-one). Aldehydes generally  have low odor thresholds therefore their impact was greater than that of alcohols and ketones, although their overall levels were lower (Olafsdottir and Jonsdottir 2009). Headspace gas chromatographic methods are excellent tools for determining the volatile oxidation products that are directly responsible for or serve as markers of the flavor development in oxidized lipids. Beside correlation with flavor scores from sensory analyses, these GC analyses also provide a sensitive method of detecting low levels of oxidation in various oils and food lipids. The most common GC method for detecting and quantifying volatile oxidation products is static headspace analysis. Samples are held in a closed container until the volatile compounds diffuse and vaporize into the gas phase, and reach or approach equilibrium. An aliquot of the gas phase headspace is then injected directly into the gas chromatograph. This method is rapid and complex food systems can be analyzed directly without manipulations or extraction. The main disadvantage of the static headspace method is the difficulty of reaching complete equilibrium with viscous and semi-solid samples. Solid-phase ­ ­microextraction (SPME) is an absorption technique that has gained extensive acceptance in the analysis of volatile compounds. This technique is rapid and has vanquished the difficulties experienced with traditional headspace methods. Volatile compounds are absorbed onto fused silica fiber and directly desorbed into the gas chromatograph.



1.2 Analysis of lipid oxidation

15

1.2.4  Other methods of monitoring lipid oxidation 1.2.4.1  Near-infrared spectroscopy Near-infrared (NIR) spectroscopy has become the alternative quality control method to traditional chemical and sensory methods in the food industry because of its advantages over other analytical techniques. It is used routinely for the compositional, functional, and sensory analysis of food ­ingredients, process intermediates, and final products. NIR spectroscopy has several advantages compared with traditional analytical methods. It is fast, non-destructive, and requires little or no sample preparation. It is also economic and environmentally friendly because no reagents are required, labor requirements are low, and no chemical wastes are produced. One of the strengths of NIR spectroscopy is that it can provide simultaneous determination of multiple components per measurement with a remote sampling capability and hence can provide real-time ­information from the process stream. NIR spectroscopy has been successfully used to enhance or replace classical methods in the determination of PV, conjugated dienes, and p-anisidine value in fats and oils (Li et al. 2000; Yildiz et al. 2001; Gerde et al. 2007; Poon 2009). The major limitation of NIR spectroscopy in food analysis is its dependence on less-precise reference methods (Roggo et al. 2007), and it cannot therefore not be considered a primary method. The main challenge of utilizing NIR technology for  the determination of lipid oxidation is to develop appropriate multivariate ­calibration approaches to build accurate model systems so that it can be used for routine analysis.

1.2.4.2  Sensory analysis The most powerful way of evaluating rancid odor and flavor is sensory analysis by trained panel. Sensory evaluation is defined as “the scientific discipline used to evoke, measure, analyze and interpret human reactions to characteristic of food perceived through the sense of sight, smell, taste, touch and hearing” (Huss 1995). Sensory analysis of lipid oxidation has been demonstrated by many researchers (e.g., Coppin and Pike 2001; Broadbent and Pike 2003; Timm Heinrich et al. 2003). Typical terms generally used to describe the oxidized flavor in foods include painty, cardboard, fishy, oily, and nutty. The flavor perceived as rancid flavor depends also on the composition of the product. Fish is known to be rich in long-chain omega-3 fatty acids. Lipid oxidation products from these fatty acids are known to have a great impact on odor and flavor, even at very low concentration. The detection of these low levels is not easy with general oxidation measurements methods. The primary oxidation product is hydroperoxides, which can degrade further into secondary oxidation products. These compounds are generally volatile products that are responsible for off-flavors and odor in, for

16

CH1 Oxidation in aquatic foods and analysis methods

e­ xample, fish oils. Frankel (1998) reported that sensory panels can detect off-flavors in oils with PVs lower than 1 meq/kg. This has been supported by the study of Macfarlane et al. (2001), which showed that freshly refined fish oil samples with PVs lower than 1 meq/kg had a strong fishy taste. The main limitations of sensory analysis are its cost and the requirement for a well-trained taste and odor panel. Even though sensory methods can give conclusive information, it can be difficult to compare data from different panels using different vocabularies to describe sensory attributes or data from the same panel analyzed at different times. Sensory methods also require a considerable number of samples, and the use of other chemical methods is recommended to support and complement the sensory data (Frankel 1998; Rustad 2010).

1.3  Conclusions There are several methods that exist for the analysis of lipid oxidation in aquatic food products, among which formation of oxidation products is the most commonly used. The diversity and abundance of methods used to assess lipid oxidation reflect the complexity of this issue and confirm the fact that multiple methods should be applied to get the maximum information available. Each method has both a­ dvantages and disadvantages, therefore is it of great importance to select the most appropriate method depending on the system under investigation and the state of the oxidation.

References Ackman, R. G. 1980. Fish lipids. Part 1, in Advances in Fish Science and Technology (ed. J. Connell). Farnham, Surrey: Fishing News (Books) Ltd, pp. 86–103. Ackman, R. G. and Timmins, A. 1995. Stability of α-tocopherol in frozen smoked fish. Food Lipids 2(1): 65–71. Akasaka, K., Suzuki, T., Ohrui, H. and Meguro, H. 1987. Study on aromatic phosphines for novel fluorometry of hydroperoxides (I) – synthesis and spectral properties of diphenyl aryl phosphines and their oxides. Analytical Letters 20(5): 731–745. Andersen, C. M. and Rinnan, Å. 2002. Distribution of water in fresh cod. Food Science and Technology. 35(8): 687–696. Andersen, M. L. and Skibsted, L. H. 2002. Detection of early events in lipid oxidation by electron spin resonance spectroscopy. European Journal of Lipid Science and Technology 104(1): 65–68. Antolovich, M., Prenzler, P. D., Patsalides, E., McDonald, S. and Robards, K. 2002. Methods for testing antioxidant activity. Analyst 127(1): 183–198. Aranda, M., Mendoza, N. and Villegas, R. 2006. Lipid damage during frozen storage of whole Jack mackerel (Trachurus symmetricus murphyi). Journal of Food Lipids 13(2): 155–166. Asakawa, T. and Matsushita, S. 1978. Colorimetric determination of peroxide value with potassium iodide–silica gel reagent. JAOCS 55(8): 1948–1950.

references 17

Aubourg, S. 1999. Recent advances in assessment of marine lipid oxidation by using fluorescence. JAOCS 76(4): 409–419. Aubourg, S. and Medina, I. 1997. Quality differences assessment in canned sardine (Sardina pilchardus) by detection of fluorescent compounds. Journal of Agricultural and Food Chemistry 45(9): 3617–3621. Aubourg, S. and Medina, I. 1999. Influence of storage time and temperature on lipid deterioration during cod (Gadus morhua) and haddock (Melanogramus aeglefinus) frozen storage. Journal of the Science of Food and Agriculture 79(13): 1943–1948. Aubourg, S., Sotelo, C. G. and Gallardo, J. M. 1997. Quality assessment of sardines during storage by measurement of fluorescent compounds. Journal of Food Science 62(2): 295–298, 304. Aubourg, S., Sotelo, C. G. and Pérez-Martin, R. 1998. Assessment of quality changes in frozen sardine (Sardina pilchardus) by fluorescence detection. JAOCS 75(5): 575–580. Aubourg, S., Lago, H., Sayar, N. and González, R. 2007. Lipid damage during frozen storage of Gadiform species captured in different seasons. European Journal of Lipid Science and Technology 109(6): 608–616. Banda, M. C. M. and Hultin, H. O. 1983. Role of cofactors in breakdown of TMAO in frozen red hake muscle. Journal of Food Processing and Preservation 7(4): 221–236. Bateman, L., Hughs, H. and Morris, A. L. 1953. Hydroperoxide decomposition in relation to the initation of radical chain reactions. Discussions of the Faraday Society. 14: 190–194. Beltran, A. and Moral, A. 1990. Gas chromatographic estimation of oxidative deterioration in sardine during frozen storage. Lebensmittel-Wissenschaft und Technologie. 23(6): 499–504. Brannan, R. G. and Erickson, M. C. 1996. Quantification of Antioxidants in Channel Catfish during Frozen Storage. Journal of Agricultural and Food Chemistry 44(6): 1361–1366. Broadbent, C. and Pike, O. 2003. Oil stability index correlated with sensory determination of oxidative stability in canola oil. JAOCS 80(1): 59–63. Brown, H. G. and Snyder, H. E. 1982. Conjugated dienes of crude soy oil: detection by UV spectrophotometry and separation by HPCL. JAOCS 59(7): 280–283. Buettner, G. R. 1993. The pecking order of free radicals and antioxidants: Lipid peroxidation, α-tocopherol, and acorbate. Archives of Biochemistry and Biophysics 300(2): 535–543. Carlsen, C., Andersen, M. and Skibsted, L. 2001. Oxidative stability of processed pork. Assay based on ESR-detection of radicals. European Food Research and Technology 213(3): 170–173. Castrillón, A. M., Alvarez-Pontes, E., García-Arias, M. T. and Navarro, P. 1996. Influence of frozen storage and defrosting on the chemical and nutritional quality of sardine (Clupea pilchardus). Journal of the Science of Food and Agriculture 70(1): 29–34. Chotimarkorn, C., Ohshima, T. and Ushio, H. 2006. Fluorescent image analysis of lipid hydroperoxides in fish muscle with 3-perylrne diphenylphosphine. Lipids 41(3): 295–300. Coppin, E. and Pike, O. 2001. Oil stability index correlated with sensory determination of oxidative stability in light-exposed soybean oil. JAOCS 78(1): 13–18.

18

CH1 Oxidation in aquatic foods and analysis methods

Decker, E. A. and Hultin, H. O. 1992. Lipid Oxidation in Muscle Foods via Redox Iron, in Lipid Oxidation in Food (ed Allen J. St. Angelo) Washington, DC: American Chemical Society, pp. 33–54. Decker, E. A., Huang, C., Osinchak, J. E. and Hultin, H. O. 1989. Iron and copper: Role in enzymic lipid oxidation of fish sarcoplasmic reticulum at is situ concentrations. Journal of Food Biochemistry 13(3): 179–186. Deng, J. C., Watson, M., Bates, R. P. and Schroeder, E. 1978. Ascorbic acid as an ­antioxidant in fish flesh and its degradation (ed Allen J. St. Angelo). Journal of Food Science 43(2): 457–460. Doleschall, F., Kemény, Z., Recseg, K. and Kővári, K. 2002. A new analytical method to monitor lipid peroxidation during bleaching. European Journal of Lipid Science and Technology 104(1): 14–18. Dulavik, B., Sörensen, K. R., Barstad, H., Horvli, O. and Olsen, R. L. 1998. Oxidative stability of frozen light and dark muscles of saithe (Pollachius virens L.). Journal of Food Lipids 5(3): 233–245. Erickson, M. C. 1993a. Ability of chemical measurements to differentive oxidative ­stabilities of frozen minced muscle tissue from farm-raised striped bass and hybrid striped bass. Journal of Food Chemistry 48(4): 381–385. Erickson, M. C. 1993b. Compositional parameters and their relationship to oxidative stability of channel catfish. Journal of Agriculture and Food Chemistry 41(8): 1213–1218. Erickson, M. 2002. Lipid oxidation of muscle foods, in Food lipids: Chemistry, ­nutrition and biotechnology (eds C. C. Akoh and D. B. Min). New York: Marcel Dekker, Inc., pp. 383–429. Eriksson, C. E. and Svensson, S. G. 1970. Lipoxygenase from peas, purification and properties of the enzyme. Biochimica et Biophysica Acta – Enzymology 198(3): 449–459. Francisco, A. D. and Munch, L. 1989. Practical applications of fluorescence image ­analysis, in Fluorescence Analysis in Foods (eds L. Munck and A. D. Francisco). New York: John Wiley & Sons, pp. 110–124. Frankel, N. 1998. Methods to determine extent of oxidation, in Lipid Oxidation (ed. N. Frankel). Dundee: Oily Press, pp. 79–98. Gandemer, G. 1999. Lipids and meat quality: Lipolysis, oxidation, maillard reaction and flavour. Science Des Aliment 19: 439–458. Gerde, J. A., Hardy, C. L., Hurburgh, C. R. and White, P. J. 2007. Rapid determination of degradation in frying oils with near-infrared spectroscopy. JAOCS 84(6): 519–522. German, B., Geza, G. and Kinsella, J. E. 1986. Lipoxygenase in trout gill tissue acting on arachidonic, eicosapentaenoic and docosahexaenoic acids. Biochimica et Biophysica Acta 875(1): 12–20. Gray, J. I. 1978. Measurement of lipid oxidation: A review. JAOCS 55(6): 539–546. Gray, J. I. 1987. Rancidity and warmed-over flavor. Advances in Meat Research 3: 221–269. Guillén, M. D. and Cabo, N. 2002. Fourier transform infrared spectra data versus ­peroxide and anisidine values to determine oxidative stability of edible oils. Journal of Food Chemistry 77(4): 503–510.

references 19

Gutteridge, J. M. C. 1988. Lipid peroxidation: Some problems and concepts, in Oxygen Radicals and Tissue Injury Symposium (ed. B. Halliwell). Bethesda, MD: Federation of American Societies of Experimental Biology, pp. 9–19. Halliwell, B. and Chirico, S. 1993. Lipid peroxidation: its mechanism, measurement, and significance. American Journal of Clinical Nutrition 57(5): 715S–724S. Halliwell, B. and Gutteridge, J. M. C. 1985. The chemistry of oxygen radicals and other oxygen derived species, in Free Radicals in Biology and Medicine (eds B. Halliwell and J. M. C. Gutteridge). New York: Oxford University Press, pp. 21–66. Harris, P. and Tall, J. 1989. Rancidity in fish, in Rancidity in Foods (eds J. C. Allen and R. J. Hamilton). London: Blackie A&P, pp. 256–272. Holley, A. E. and Cheeseman, K. H. 1993. Measuring free radical reactions in vivo. British Medical Bulletin 49(3): 494–505. Hudson, B. J. F. and Gordon, M. H. 1999. Evaluation of oxidative rancidity, in Rancidity in Foods (eds J. C. Allen and R. J. Hamilton). Gaithersburg: Aspen Publisher, Inc., pp. 54–67. Hultin, H. O. 1988. Potential lipid oxidation problems in fatty fish processing, in Fatty Fish Utilization: Upgrading From Feed to Food (ed. N. Davis). UNC Sea Grant College Program, Raleigh, NC, pp. 185–223. Hultin, H. O. 1994. Oxidation of lipids in seafoods, in Seafoods: Chemistry, Processing, Technology and Quality (eds F. Shahidi and J. R. Botta). London: Blackie A&P, pp. 49–74. Huss, H. H. 1995. Quality and changes in fresh fish. FAO Fisheries Technical Paper T348: 195. Jonsdottir, R., Bragadóttir, M. and Ólafsdóttir, G. (2008). Oxidation of fish muscle: Role of phospholipids, proteins, antioxidants and the effects of cooking on oxidation. Reykjavik. Matis Report 08–08. Ke, P. J. and Woyewoda, A. D. 1979. Micro determination of thiobarbituric acid values in marine lipids by direct spectrophotometric method with mono-phasic reaction ­systems. Analytica Chimica Acta 106(2): 279–284. Khayat, A. and Schwall, D. 1983. Lipid oxidation in seafood. Food Technology 7: 130–140. Lauritzsen, K., Martinsen, G. and Olsen, R. L. 1999. Copper-induced lipid oxidation during salting of cod (Gadus morhua L.). Journal of Food Lipids 6(4): 299–315. Lauridsen, C., Jensen, S. K., Skibsted, L. H. and Bertelsen, G. 2000. Influence of supranutritional vitamin E and copper on [alpha]-tocopherol deposition and susceptibility to lipid oxidation of porcine membranal fractions of M. Psoas major and M. Longissimus dorsi. Meat Science 54(4): 377–384. Lea, C. H. 1946. The determination of the peroxide value of edible fats and oils: The iodometric method. Journal of the Society of Chemical Industry 65: 286–291. Li, H., Van De Voort, F. R., Ismail, A. A. and Cox, R. 2000. Determination of peroxide value by fourier transform near-infrared spectroscopy. JAOCS 77(2): 137–142. Macfarlane, N., Salt, J. and Birkin, R. 2001. The FAST index – a fishy scale. Inform 12: 244–249. Madhavi, D. L., Deshpande, D. K. and Salunkhe, D. K. 1996. Food Antioxidants: Technology, toxicological and health perspectives. New York: Marcel Dekker 1–4.

20

CH1 Oxidation in aquatic foods and analysis methods

Marcuse, R. and Johansson, L. 1973. Studies on the TBA test for rancidity grading: II TBA reactivity of different aldehyde classes. JAOCS 50: 387–391. McGill, A. S., Hardy, R., Burt, J. R. and Gunstone, F. D. 1974. Hept-cis-4-enal and its contribution to the off-flavour in cold stored cod. Journal of the Science of Food and Agriculture 25(12): 1477–1489. Mead, J. F. 1976. Free radical mechanisms of lipid damage and consequence for cellular membranes, in Free radicals in biology. (ed W. A. Pryor). New York: Academic Press, p. 51–56. Medina, I., Saeed, S. and Howell, N. 1999. Enzymatic oxidative activity in sardine (Sardina pilchardus) and herring (Clupea harengus) during chilling and correlation with quality. European Food Research and Technology 210(1): 34–38. Milic, B. L., Djilas, S. M. and Canadanovic-Brunet, J. M. 1998. Antioxidative activity of phenolic compounds on the metal-ion breakdown of lipid peroxidation system. Food Chemistry 61(4): 443–447. Milo, C. and Grosch, W. 1996. Changes in the odorants of boiled salmon and cod as affected by the storage of the raw material. Journal of Agriculture and Food Chemistry 44(8): 2366–2371. Ohshima, T., Hopia, A., German, B. and Frankel, N. 1996. Determination of hydroperoxides and structure by high-performance liquid chromatography with post column detection with diphenyl-1-pyrenylphosphine. Lipids 31(10): 1091–1096. Olafsdottir, G. and Jonsdottir, R. 2009. Volatile aroma compounds in fish, in Handbook of Seafood and Seafood Products Analysis (eds L. M. L. Nollet and F. Toldrá). CRC Press, Boca Raton, pp. 97–117. Pearson, A., Gray, J. I., Wolzak, A. M. and Horenstein, N. A. 1983. Safety implications of oxidized lipids in muscle foods. Food Technology 37(7): 121–129. Philippy, B. Q. 1984. Characterization of the in situ TMAOase system iin red hake muslce. Ph.D thesis, University of Massachusetts. Pike, A. O. 2003. Fat characterization, in Food Analysis, 3rd edn (ed. S. S. Nielsen). New York: Kluwer Academic/Plenum Publishers, pp. 227–246. Pokorny, J., El-Zeany, B. A. and Janicek, G. 1974. Browning reactions of oxidized fish lipids with proteins. Proceedings of the IV International Congress on Food Science and Technology 1: 217–223. Pokorny, J., Valentová, H. and Davidek, J. 1985. Modified determination of 2-TBA value in fats and oils. Die Nahrung 29: 31–38. Polvi, S. M., Ackman, R. G., Lall, S. P. and Saunders, R. L. 1991. Stability of lipids and omega-3-fatty acids during frozen storage of Atlantic salmon. Journal of Food Processing and Preservation 15(3): 167–181. Poon, C. L. 2009. Studies of lipid oxidation in Salmon by near infrared spectroscopy. M.Sc. thesis, Norwegian University of Science and Technology. Pozo, R., Lavety, J. and Love, R. M. 1988. The role of dietary α-tocopherol (vitamin E) in stabilising the canthaxanthin and lipids of rainbow trout muscle. Aquaculture 73: 165–175. Robbles-Martinez, C., Cervantes, E. and Ke, P. J. 1982. Recommended method for ­testing the objective rancidity development in fish based on TBARS formation. Canadian Technical Reports of Fisheries and Aquatic Sciences No 1089.

references 21

Roggo, Y., Chalus, P., Maurer, L., Lema-Maritinez, C., Edmond, A. and Jent, N. 2007. A review of near infrared spectroscopy and chemometrics in pharmaceutical ­technologies. Journal of Pharmaceutical and Bimedical Analysis 44: 683–700. Ruiz, A., Canada, M. J. A. and Lendl, B. 2001.A rapid method for peroxide value determination in edible oils based on flow analysis with Fourier transfer infrared spectroscopic detection. Analyst 126: 242–246. Rustad, T. 2010. Lipid oxidation, in Handbook of Seafood and Seafood Products Analysis (eds L. M. L. Nollet and F. Toldrá). New York: CRC Press, pp. 87–95. Santha, N. C. and Decker E. A. 1994. Rapid, sensitive, iron-based spectrophotometric methods for determination of peroxide values of food lipids. Association of Official Analytical Chemists International 77(2): 421–424. Sarma, J., Reedy, G. V. S. and Srikar, L. N. 2000. Effect of frozen storage on lipid and functional properties of proteins of dressed Indian oil sardine (Sardinella longiceps). Food Research International 33(10): 815–820. Schaich, K. M. 1992. Metals and lipid oxidation. Contemporary issues. Lipids 27(3): 209–218. Schmedes, A. and Holmer, G. 1989. A new thiobarbituric acid (TBA) metod for ­determining free malonaldehyde and hydroperoxides selectively as a measure of lipid peroxidation. JAOCS 66(6): 813–817. Shahidi, F. and Wanasundra, U. N. 2002. Methods for measuring oxidative rancidity in fats and oils, in Food Lipids: Chemistry, nutrition, and biotechnology (eds C. C. Akoh and D. B. Min. New York: Marcel Dekker, Inc. 465–487. Shahidi, F., Rubin, L. J., Diosady, L. L. and Wood, D. F. 1985. Effect of sulfanimide on the TBA values of cured meats. Journal of Food Science 50(1): 274–275. Shahidi, F., Yun, J., Rubin, L. J. and Wood, D. F. 1987. Control of lipid oxidation in cooked ground pork with antioxidants and dinitrosyl ferrohemochrome. Journal of Food Science 52: 564–567. Sharma, M. K. and Buettner, G. R. 1993. Interaction of vitamin C and vitamin E during free radical stress in plasma: An ESR study. Free Radical Biology and Medicine 14(6): 649–653. Shewfelt, R. 1981. Fish muscle lipolysis – a review. Journal of Food Biochemistry 5(2): 79–100. Shono, T. and Toyomitzu, M. 1971. Changes in fatty acids constituting lipids in fish muscle during storage at low temperature (5°C): decreased rate of C22:6 acid as a criterion for the oxidative deterioration of lipids. Bulletin of the Japanese Society of Scientific Fisheries 37(9): 912–918. Srinivasan, S. and Hultin, H. O. 1994. Hydroxyl radical modification of fish muscle proteins. Journal of Food Biochemistry 18(6): 405–425. Thanonkaew, A., Benjakul, S., Visessanguan, W. and Decker, E. A. 2006. The effect of metal ions on lipid oxidation, colour and physicochemical properties of cuttlefish (Sepia pharaonis) subjected to multiple freeze–thaw cycles. Food Chemistry 95(4): 591–599. Timm Heinrich, M., Xu, X., Nielsen, N. S. and Jacobsen, C. 2003. Oxidative stability of structured lipids produced from sunflower oil and caprylic acid. European Journal of Lipid Science and Technology 105(8): 436–448.

22

CH1 Oxidation in aquatic foods and analysis methods

Tomas, M. C. and Fumes, J. 1987. Application of 2-thiobarbituric acid reaction to exudates of frozen and refrigerated meats. Journal of Food Science 52(3): 575–579. Undeland, I. 1997. Lipid oxidation in fish – causes, changes and measurements, in Methods to Determine the Freshness of Fish in Research and Industry (eds G. Olafsdottir, J. Luten, P. Dalgaard et al.). Paris: International Institute of Refrigeration, pp. 241–257. Undeland, I., Ekstrand, B. and Lingnert, H. 1998. Lipid oxidation in minced herring (Clupea harengus) during frozen storage. Effect of washing and precooking. Journal of Agricultural and Food Chemistry 46(6): 2319–2328. van der Merwe, G. H., du Plessis, L. M. and Taylor, J. R. N. 2004. Changes in chemical quality indices during long-term storage of palm-olein oil under heated storage and transport-type conditions. Journal of the Science of Food and Agriculture 84(1): 52–58. Veberg, A., Vogt, G. and Wold, J. P. 2006. Fluorescence in aldehyde model systems related to lipid oxidation. Food Science and Technology 39(5): 562–570. Verma, J. K., Srikar, L. N., Sudhakara, N. S. and Sarma, J. 1995. Effects of frozen ­storage on lipid freshness parameters and some functional properties of oil sardine (Sardinella longiceps) mince. Food Research International 28(1): 87–90. Walling, C. 1975. Fenton´s reagent revised. Accounts of Chemical Research 8: 125–131. Wasasundara, U. U. and Shahidi, F. 1994. Canola extracs as an alternative antioxidant for canola oil. JAOCS 71(8): 817–822. Watanabe, F., Goto, M., Abe, K. and Nakano, Y. 1996. Glutathione peroxidase activity during storage of fish muscle. Journal of Food Science 61(4): 734–735. Wheeler, D. H. 1932. Peroxide formation as a measure of autoxidative deterioration. Oil Soap 9: 89–97. White, P. J. 1995. Conjugated diene, anisidine value, and carbonyl value analysis, in Methods to Assess Quality and Stability of Oils and Fat-containing Foods (eds K. Warner and N. A. M. Eskin). Champaign, IL: AOCS Press, pp. 159–178. Wold, J. P., Mielnik, M., Pettersen, M. K., Aaby, K. and Baardseth, P. 2002. Rapid assessment of rancidity in complex meat products by front face fluorescence ­spectroscopy. Journal of Food Science 67(6): 2397–2404. Xing, Y., Yoo, Y., Kelleher, S. D., Nawar, W. and Hultin, H. O. 1993. Lack of changes in fatty acid composition of mackerel and cod during iced and frozen storage. Journal of Food Lipids 1(1): 1–14. Yamamoto, Y. and Ames, B. N. 1987. Detection of lipid hydroperoxides and hydrogen peroxide at picamole levels by an HPLC and isoluminol chemiluminescence assay. Free Radical Biology and Medicine 3(5): 359–361. Yasuda, M. and Narita, S. 1997. Simultaneous determination of phospholipid hydroperoxides and cholesteryl ester hydroperoxide in human plasma by high performance liquid chromatography with chemiluminescence detection. Journal of Chromatography B: Biomedical Sciences and Applications 693(1): 211–217. Yildiz, G., Wehling, R. L. and Cuppett, S. L. 2001. Method for determining oxidation of vegetable oils by near-infrared spectroscopy. JAOCS 78(5): 495–502.

2 Protein oxidation in aquatic foods Caroline P. Baron National Food Institute, Technical University of Denmark, Kgs. Lyngby, Denmark

2.1  Introduction Reactive oxygen species (ROS) such as hydrogen peroxide, superoxide, hydroxyl radicals, peroxynitrite, and peroxyl radicals are formed in living cells via metabolic processes such as those in electron transport systems, and can act as cell signaling molecules. They can also be produced as a response to external stimuli, for example irradiation, stress, and pollution (Davies and Dean 1997). In general, the living cell is able to deal with ROS using complex enzymatic and antioxidant defenses and repair systems, resulting in the control of ROS in vivo. Antioxidant systems, ­including reducing enzymes and low molecular weight antioxidant compounds, ensure minimal damage to the cell constituents and maintain optimal cellular ­function. Indeed, ROS are strong oxidants able to induce oxidation of the cell ­constituents, but under normal conditions oxidized cellular compounds such as lipids and proteins do not accumulate. Oxidation products are removed by ­enzymatic systems such as cathepsins in the lysosomes or by the proteosome. During aging both the metabolism and the enzymatic systems are impaired, resulting in ­accumulation of ROS and as a consequence accumulation of oxidized lipid (lipofusin) and protein (amyloid plaque), which result in the pathology of several diseases (Dean et al. 1997; Grimm et al. 2012). In post-mortem muscle the lack of ATP and co-factors as well as the anaerobic conditions lead to cessation of any controlled enzymatic reactions and results in the consumption of antioxidants, leading to an unbalanced system and oxidation of the muscle cell constituents. Lipid oxidation in food has been extensively studied due to the sensitivity of the long-chain fatty acids (omega-3) to oxidation, which results in the formation of significant off-flavor. For fish and fish products, oxidation of longchain omega-3 fatty acids and detailed mechanisms have been described in Chapter 1.

Antioxidants and Functional Components in Aquatic Foods, First Edition. Edited by Hordur G. Kristinsson. © 2014 John Wiley & Sons, Ltd. Published 2014 by John Wiley & Sons, Ltd.

24

CH2 Protein oxidation in aquatic foods

In contrast, protein oxidation in food in general and in aquatic food has received little attention in the last decade, perhaps because it results in subtle changes that are not immediately p­ erceived by the human senses. In addition, ­proteins are very large and complex molecules organized in different structures, and oxidation may lead to a large ­number of modifications either on the protein side chains or on the protein backbone. These modifications include amino acid modification, ­protein breakdown, and protein cross-linking. Proteins are the main constituent of cells and tissues. In muscle tissues, proteins represent an average 20% of the total weight, which can be larger than the lipid contribution. In fish muscle the lipid composition can vary from 1% to 30% depending on the type of fish and its physiological status. In addition, studies of reaction constants have reported that the most reactive hydroxyl radical often reacts faster with proteins than with lipids, or 9.109 for linoleic acid and 4.1011 for collagen (Davies and Dean 1997). In foods, the mechanisms describing protein ­oxidation are barely explored, probably due to the complexity of the reaction p­ roducts and the limitations of the poor methods available to study ­protein oxidation and protein modifications. However, recently several research groups have started to investigate protein oxidation and its consequences for food quality more thoroughly. It is now accepted that not only lipids but also proteins are oxidized and this can have consequences for food product quality, modifying protein functionality, and nutritional implications. The present chapter focuses on general considerations about protein oxidation and reviews not only the mechanisms involved in protein oxidation but also the consequences of protein oxidation on fish proteins. In addition, some of our findings will be presented via essentially two case studies. The first case study deals with protein and lipid oxidation in frozen rainbow trout while the second deals with oxidation in salted herring.

2.2  Mechanisms involved in protein oxidation 2.2.1  A free radical mechanism The mechanisms responsible for initiation of protein oxidation are not clear but it is generally accepted that free radical species initiating lipid oxidation are also able to initiate protein oxidation. ROS are able to abstract a hydrogen atom from the ­protein, leading to the formation of an initial carbon-centered radical. In model systems, hydrogen abstraction on the protein backbone has been shown to take place at the alpha carbon and to lead to protein fragmentation. At the side chain, hydrogen atom abstraction is believed to take place on (the) aliphatic side chain while addition is expected at the aromatic side chain. Oxidation of proteins at the side chain leads to protein carbonyls, alcohols, and peroxides. Several amino acid side chains are susceptible to hydroperoxide formation but as they are not easily identified few reports exist on their exact structure and formation (Davies et al. 1999). The amino acids that are generally most susceptible to oxidation are cysteine, tyrosine, p­ henylalanine, tryptophan, histidine, proline, arginine, lysine, and methionine (Stadtman and



2.2 Mechanisms involved in protein oxidation

25

Table 2.1  Amino acids susceptible to oxidation and some of their most common oxidation products Amino acid

Oxidation products

Cys Met Tyr Tryp

Disulfide, cystine, cysteic acid Methione sulfoxide, sulfone Dityrosine, 3,4-dihydrophenylalnine (DOPA) N-Kynurenine, N-formylkynurenine, 3-hydroxylkynurenine Hydroxyproline, γ-glutamic semialdehydes γ-Glutamic semialdehydes α-Aminoadipic semialdehyde

Pro Arg Lys

Levine 2003). The amino acids most susceptible to oxidation and their most c­ ommon oxidation products are listed in Table 2.1. The most reactive amino acids are cysteine and methionine, which have a sulfur atom in their side chain. Oxidation of cysteine residue will result in the formation of a series of radicals and intra- or inter-molecular dimerization occurs rapidly, leading to cysteine sulfonic acid and disulfide, whilst oxidation of methionine will lead to methionine sulfoxide and sulfone (Xu and Chance 2005). However, it is noteworthy that oxidation of cysteine and methionine is the only amino acid modification that may be reversed in vivo and in vitro either by enzymatic systems or reducing ­compounds. If these amino acids are reduced in muscle foods post mortem, and under which c­ onditions they are reduced, remain uninvestigated. However, it was suggested several decades ago that ­methionine is a modulator of oxidative attack protecting the protein from more severe damage in vivo (Levine et al. 1999). Specific amino acid oxidation products from oxidized protein have so far not been unambiguously detected in fish and fish products. This might be because of their very low abundance, their possible t­ransient character, and the complexity of the analytical methods that need to be available in order to precisely identify modification of protein-oxidized amino acids. In ­addition, it is also thought that interaction between protein and lipid oxidation products as well as protein and sugar oxidation products leads indirectly to protein oxidation, but only the former will be described in this chapter. More details about protein and reducing sugar interactions can be found in review papers on Maillard reactions (e.g., Baltes 1993; Martins et al. 2001). Carbonyl groups, disulfide bridge/free thiols, and protein dityrosine are the most common markers of protein oxidation in foods and are relatively easily measured protein oxidation reaction products. Measuring protein carbonyls groups determination using dinitrophenylhydrazyne (DNPH) is the most popular method used to follow oxidation of protein (Levine et al. 1994). Formation of carbonyl compounds on amino acid side chains can be described as the result of metal-initiated protein oxidation but it is also believed that other oxidative reactions can lead to the formation of protein carbonyl groups. Nevertheless, the content of protein carbonyl groups is often used as an estimation of protein oxidation, especially in biological tissues

26

CH2 Protein oxidation in aquatic foods Table 2.2  Protein carbonyls measured for nine rainbow trout immediately after killing and after storage for 48 hours on ice (from Baron et al. 2007) Fish 1 2 3 4 5 6 7 8 9

Protein carbonyl nmol/mg at killing

Protein carbonyl nmol/mg after 48 hours on ice

3.9 (±0.4) 3.5 (±0.4) 2.9 (±0.7) 2.3 (±0.7) 1.8 (±0.5) 2.2 (±0.3) nd nd nd

4.2 (±1.1) 3.4 (±0.7) 2.9 (±0.5) nd nd nd 2.3 (±0.5) 2.1 (±1.0) 2.3 (±0.2)

nd, not detected.

and in food, as it is an accessible method. However, protein carbonyl groups are also present in proteins under normal conditions and they can vary at the i­ ndividual level, as illustrated in Table 2.2, which reports protein carbonyls ­measured in individual trout pre and post rigor (Baron et al. 2007). At the time of slaughter protein carbonyl groups ranged from 1.8 to 3.9 nmol of carbonyl/mg of protein and post rigor no changes in the protein carbonyl levels were observed. The mean value for the six individuals did not reveal any significant ­difference for pre and post rigor, reaching 2.8 and 2.9 nmol of carbonyl/mg, respectively, which is in agreement with data reported for healthy biological tissue samples (Reznick and Packer 1994). It has been estimated by Reznick and Packer that protein have an average molecular weight of 50 kDa, and that for a sample containing 2.7–2.8 nmol of carbonyl/mg of protein, protein carbonyls could represented 14% of total protein fraction. However, the average molecular weight of muscle protein is expected to be much higher, with a contribution of myosin and other large proteins such as titin and nebulin of 50% and therefore the basal level of protein carbonyls can be easily estimated to be around 10%. In addition, protein carbonyls can also be formed or added to protein structures via indirect oxidative reactions such as i­ nteraction with aldehydes or reducing sugars, which may cause overestimation of direct protein oxidation. Despite its poor sensitivity and low reproducibility protein carbonyl group measurement is today the most widely used method for estimating protein oxidation in foods and has been used in fish muscle.

2.2.2  Initiation of protein oxidation in aquatic foods 2.2.2.1  Fenton: metals and peroxides Transition metals, especially iron, have been shown to play a central role in lipid oxidation and are also believed to be involved in the initiation steps of protein oxidation (Stadtman 1990). Some proteins are able to chelate metals and some proteins



2.2 Mechanisms involved in protein oxidation

27

have specific iron or copper-binding sites, such as heme protein, but more generally proteins can chelate metal when primary amine groups are present on their side chains, for example lysine or glutamine. The amount of free iron in fish muscle has been estimated to range from approximately 3 to 20 mg/kg depending on the fish species, the muscle and the processing condition, such as bleeding and washing (Venugopal and Shahidi 1996; Richards and Hultin 2002; Maqsood and Benjakul 2011). It is therefore expected that iron either free or bound contributes significantly to protein oxidation in fish and fish products. Metal-induced protein oxidation has been proposed to result in site-specific protein oxidation where the radical is generated next to the site of attack (Stadtman and Levine 2003). However, metal-induced oxidation is particularly relevant in the presence of peroxides, especially hydrogen peroxide. Indeed, decomposition of hydrogen peroxide via Fenton chemistry leads to the formation of the hydroxyl radical, which is a strong pro-oxidant and is able to oxidize all amino acids, although not all amino acids are reactive. Hydrogen peroxide is present in cells at concentrations ranging from 0.01 to 0.1 μM and accumulates in muscle tissue post mortem, reaching up to 0.6 mM under ischemia (Harel and Kanner 1985; Halliwell and Gutteridge 2007). The level of hydrogen peroxide in muscle tissue post mortem is most likely low. However, during meat aging hydrogen peroxide has been reported to be produced in significant amounts (Harel and Kanner 1985), although it has not been measured in fish. During fish processing utilization of hydrogen peroxide to whiten fish flesh and improve its general appearance is a common practice (Jafarpour et al. 2008), but addition of hydrogen peroxide to fish muscle is likely to induce oxidation of proteins. Indeed, this might significantly affect iron-initiated oxidation and promote Fenton chemistry, leading to severe protein oxidation, but this has not been investigated thoroughly. According to Stadtman and Levine (2003), the reaction between iron, peroxides, and proteins takes place at the iron binding site. The iron is released as iron 3+ and after ­deamination of the protein side chain a carbonyl group is formed (Figure 2.1). Recent investigations by Estevez et al. (2009) in meat demonstrated the importance of iron-mediated oxidation in the formation of oxidation products of Arg and Lys side chains, resulting in the formation of α-aminoadipic and β-glutamic acid semialdehydes. These oxidation products have been shown to be formed via ­ ­metal-catalyzed oxidation in meat, and have recently also been detected in fish ­muscle (Timm-Heinrich et al. 2013). In model systems metal-catalyzed oxidation of muscle proteins has been shown to result in loss of protein solubility and ­functionality (Srinivasan and Hultin 1997). In addition, some of the changes in fish texture and water-holding capacity during storage might also be related to ­iron-induced protein but more systematic investigations are needed.

2.2.2.2  Radical transfer: heme proteins Heme proteins such as hemoglobin (Hb) and myoglobin (Mb) are present in fish muscle tissue. Depending on the bleeding conditions, Hb is usually present in fish light muscle in small quantities whereas Mb is predominant in dark muscle. For example, a study performed on mackerel reported that Hb ranges between 6 and

28

CH2 Protein oxidation in aquatic foods CH2

CH2

Fe2+

H2N

H2N

H2O2

CH2 H2N

Fe2+

OH Fe3+

OH–

H2O C

H O

H2O

CH HN

NH3,Fe2+

Protease

CH Fe2+

H2O

H2N

Fe3+

OH~

Amino acids peptides

Figure 2.1  Mechanism for metal-catalyzed protein oxidation leading to the formation of ­carbonyl groups on protein. (Stadtman, 1990. Reproduced with permission of Elsevier.)

12 µmol/kg in light muscle whereas myoglobin reaches 120–150 µmol/kg in dark muscle (Richards and Hultin 2002). Heme proteins are able to initiate protein ­oxidation and the mechanism is believed to involve peroxides, especially hydrogen peroxides and activation of heme proteins to hypervalent heme species such as ­ferryl and perferryl heme proteins (Baron and Andersen 2002). It has recently been shown that the radical present on hypervalent myoglobin can be rapidly transferred to myosin, causing the generation of long-lived myosin radicals and formation of reversible and irreversible protein cross-linking between myosin molecules (Lund et al. 2008). The exact mechanisms are unknown but radial transfer from heme proteins to another molecule is a form of reaction that has received some attention, although the mechanisms are rather poorly understood (Irwin et al. 1999; Østdal et al. 2002). It has been shown, for example, that heme proteins can generate a longlived protein radical on bovine serum albumin (BSA) that is able to further transfer its radical to small antioxidative molecules such as urate, leading to speculation that radical transfer could be part of an antioxidant mechanism (Østdal et al. 2002). This indicates further that this mechanism might be relevant for fish products and could affect fish and fish product quality. It is clear that even if the mechanisms are not fully understood the radical transfer mechanism in fish muscle should be investigated further. A recent investigation performed in a washed cod model system revealed that Mb was inducing protein oxidation and affecting cathepsin, an enzyme that is important in fish muscle degradation post mortem (Egelandsdal et al. 2010). No mechanism was proposed but earlier investigation reported inactivation of cathepsin via hypervalent myoglobin species (Miura et al. 1995). The significance of free radical transfer as an initiating step in protein oxidation needs to be investigated in more detail, especially with the finding that Hb from fish has been shown to be much more susceptible in promoting free radical formation than its mammalian



2.2 Mechanisms involved in protein oxidation

29

counterpart and that reduction of hypervalent heme protein was crucial in order to prevent oxidation initiated by hemoglobin (Maestre et al. 2009).

2.2.3  Interaction between lipid and protein 2.2.3.1  Lipid vs protein Whether lipids or proteins oxidize first and to what extent oxidation of one leads to oxidation of the other are matters of controversy. In most cases protein oxidation followed by measuring protein carbonyls, and lipid oxidation followed by measuring peroxides seem to follow the same kinetics. However, considering that lipid hydroperoxides are primary oxidation products and protein carbonyls might be a later event or oxidation products in the oxidation of the protein it is difficult determine the exact time course of protein and lipid oxidation in relation to each other. In addition, ­available methods for measuring protein oxidation are not very sensitive and lack accuracy, and therefore the development of analytical methods will probably lead to more fundamental understanding of the interaction between lipids and proteins, and their kinetics of oxidation. Others have reported that in cod membrane systems using xanthine oxidase as free radical generating system oxidation of the muscle proteins seemed to occur before oxidation of the membrane lipids (Srinivasan and Hultin 1997). Furthermore, Srinivasan and Hultin reported d­ ifferences between the enzymatic and the non-enzymatic free radical generating system, and pointed out that the enzymatic seemed to be more efficient at initiating lipid oxidation whilst the nonenzymatic was more efficient at initiating protein oxidation (Soyer and Hultin 2000). Moreover, protein radicals can transfer radicals to lipids efficiently and this supports the general idea that the initiating species, the environment, and the type of mechanisms involved might have important implications with respect to the kinetics of protein and lipid oxidation (Eymard et al. 2009). It has also been suggested that protein radicals are intermediate reactive species and are involved in the antioxidative processes in muscle tissues (Østdal et al. 2002). It has been demonstrated in several foods that protein and polypeptides possess a­ ntioxidant potential by scavenging free radicals, and fish proteins and peptides are well described in the literature as good antioxidants (Najafian and Babji 2012). The antioxidant activity of proteins could be attributed to stabilization by the protein structure or burial of the radical in the protein structure, which would lead to a­ ntioxidant activity of proteins thereby preventing oxidation of the lipid fraction (Elias et al. 2008). In contrast, radicals on the surface of the protein could lead to further transfer of free radical processes onto the lipid fraction. The proximity of the target as well as the location of the protein radical on the protein surface might lead to radical transfer, as previously observed for BSA and lipids, and are important parameters to consider (Østdal et al. 2002). All these results seem to indicate that the initiating agent, the type of oxidizing radical, the environment, and the target are equality important for oxidation of proteins and lipids in relation to each other, as also suggested by others (Dean et al. 1997).

30

CH2 Protein oxidation in aquatic foods

2.2.3.2  Aldehydes Besides radical transfer, indirect oxidation of proteins via interaction between ­protein and lipid oxidation products can take place. Secondary lipid oxidation products, especially α,β-unsaturated aldehydes such as 4-hydroxynonenal (4-NHE), are very reactive. In fish, oxidation of omega-3 fatty acid usually leads to the formation of trans-4-hydroxy-2-hexenal (HHE), which has been reported to be formed in ­significant amounts (ranging from 1.5 to 40 nml/g) in several fish species during storage (Sakai et al. 1997). These aldehydes are not only toxic but also react readily with proteins or peptides. Amino groups on the side chains of proteins (e.g., Lys, Gln, Arg) may lead to the formation of Schiff bases via nucleophilic attack on the aldehyde. These reactions are reversible but further rearrangements lead to the ­formation of irreversible adducts that in general result in loss of protein surface charge. In addition, α,β-unsaturated aldehydes can undergo Michael addition at the protein amino group side chain (Berlett and Stadtman 1997). Strictly speaking, such mechanisms are not considered to be direct oxidation of the protein but result in the addition of carbonyl groups to the protein via covalent binding between the fatty acid moiety and the protein. In model systems it has been shown that increasing aldehyde unsaturation results in more severe damage of fish myosin, with consequences such as loss of protein solubility (Chopin et al. 2007). These interactions are important to consider not only in relation to protein functionality but also with respect to the potential toxicity of these protein/lipid complexes and the loss in nutritional value imparted by such reactions via, for example, loss of essential amino acids. These protein/lipid adducts might be formed in fish muscle under ­specific conditions but so far no studies have investigated their presence.

2.3  Impact of protein oxidation on aquatic food 2.3.1  Protein functionality Loss of protein functionality has been extensively reported in the literature and entails losses in protein emulsifying properties, solubility, gelling properties, and water-binding capacity (Xiong 2000). However, very few reports have linked loss of functionality explicitly to oxidation of proteins. Loss of protein functionality has been reported to be during frozen storage and during processing of fish proteins. For example, loss of functionality has been shown to have detrimental consequences for fish protein gelation, which is an important step in the transformation of lean fish into surimi-like products (Park 2000). In surimi processing, oxidation during the washing step was found to result in poorer gel-forming abilities, which was directly linked to protein oxidation (Tunhun et al. 2001). In addition, oxidized actomyosin from fish has been reported not to respond well to enzymatic cross-linking via transglutaminase, further confirming that oxidation can impair enzymatic reaction and gelation (Visessanguan et al. 2003). A decrease in protein functionality



2.3 Impact of protein oxidation on aquatic food

31

and protein solubility correlates with a decrease in free thiol groups and the importance of thiol group oxidation in protein functionality has been described in other types of food, such as dough (Maforimbo et al. 2007). In washed fish mince, a loss of 50% of thiol groups was reported after washing, but during storage of fish products washed d­ ifferently the decrease in thiol groups varied between approximately 0 and 40% of the initial thiol group content (Eymard et al. 2009). Similarly, in frozen fish, free thiol groups have been shown to decrease during storage whilst disulfide bridges have been shown to increase (Owusu-Ansah and Hultin 1986). Oxidation of thiol group residues might be part of a protective mechanism because they introduce reversible cross-linking and thereby might protect other amino acids against oxidation (Stadtman and Levine 2003). Few investigations have suggested that oxidation of other amino acid side chain residues apart from cysteine could affect protein functionality. Oxidation of tyrosine has been reported to be involved in the ability of myofibrillar proteins to retain water (Ooizumi and Xiong 2004; Bertram et al. 2007), which is also ­supported by a study by Morzel (Morzel et al. 2006) demonstrating not only loss of thiol groups but also formation of dityrosine after oxidation of myofibrillar proteins with free radical generating systems. The importance of myosin oxidation in reduced protein functionality is evident and supported by many studies, including the finding that both myosin light and heavy chains are heavily oxidized in fish muscle during frozen storage whereas actin and sarcoplasmic proteins seem to be more stable towards oxidative damage (Kjærsgaard et al. 2006). Contradictions exist in the literature with respect to the impact of protein oxidation on protein functionality. Improvement of functional properties such as gelation and the emulsifying abilities of fish myofibrillar proteins by oxidation with a free radical generating system has been reported (Srinivasan and Hultin 1997). Similarly, Parkington et al. (2000) reported increased gel strength for proteins exposed to ­oxidation. Bertram demonstrated that the pH as well as the ionic strength were ­critical factors in relation to the extent of protein oxidation in the myofibrillar fraction, which could explain the discrepancy between the different studies. In 1986 Hultin proposed that changes in protein functionality were due to minor modifications of the protein in combination with other conditions such as salt, pH, and temperature. He reported that protein oxidation alone was insufficient to induce any significant changes in the functional properties of proteins. In frozen fish, loss of functional properties is expected to be multifactorial and the combination of ­freezing and thawing may result in changes in protein surface properties due to oxidation. Hambly and Gross (2009) recently reported that hydrogen peroxide was a more potent protein oxidant during cold storage and showed evidence of cold solid-state oxidation. They reported that protein oxidation was strongly accelerated when the protein was in an ice lattice, with the amino acids methionine, tryptophan, and tyrosine being the most susceptible to cold solid-state oxidation. Cold solid-state oxidation of proteins has not been investigated in fish muscle but must be investigated thoroughly since a large proportion of fish today is sold frozen. It is therefore likely that protein oxidation contributes to a large extent to quality loss in fish and

32

CH2 Protein oxidation in aquatic foods

fish products during frozen storage and this deserves further attention. As far as protein gelation is concerned, Liu and Xiong (2000) suggested that gel formation from mildly oxidized proteins could be used to increase protein functionality, an aspect that also deserves further attention. Indeed, better control and understanding of the protein oxidation mechanism might lead to manipulation of protein functionality, which could have important consequences for fish and fish products. More systematic investigations need to be performed to further assess the impact of storage and processing on protein oxidation and its relation to fish protein quality and functionality.

2.3.2  Texture Protein oxidation leads to either protein cross-linking or protein breakdown, which beside protein functionality can to some extent affect fish muscle texture. In myofibrillar-based systems it has been shown that myosin, the most abundant muscle protein, is highly susceptible to oxidation and can easily oxidize, leading to polymerization (Xiong et al. 2010). In addition, oxidation has also been shown to induce protein fragmentation (Baron et al. 2006; Lui and Xiong 2000). The importance of protein oxidation and myosin cross-linking in the development of the characteristic texture of salted fish has recently been revealed (Andersen et al. 2007; Christensen et al. 2011). Similarly, a tough texture in meat was reported to be due to myosin cross-linking, which was found to occur in meat stored under a high oxygen atmosphere (Lund et al. 2007). The formation of cross-linking and aggregation of oxidized proteins has often been observed in fish muscle by the presence of unresolved protein complexes by one- (1D) and two-dimensional (2D) gel electrophoresis (Kjærsgard et al. 2006; Eymard et al. 2009). The difficulties reside in elucidating the structure of such complexes, as it is often a mixture of cross-linked proteins perhaps even associated with sugars and lipid oxidation products, generating a very complex network. Texture in fish is associated with the activity of the proteolytic enzymes calpains and cathespins (Cheret et al. 2007). Cathepsins have been shown to be oxidized and inactivated by oxidation products in biological systems (Headlam et al. 2006). In addition, Miura et al. (1995) reported inactivation of cathespin by hypervalent heme proteins. It has been reported for meat that the enzyme calpain can be inactivated via oxidative reactions, affecting muscle texture (Rowe et al. 2004). However, the extent to which oxidation of calpains and cathepsins in fish muscle can impact fish texture is not known. In addition to inactivation of enzymes and protein cross-­ linking, which can result in increased toughness, protein oxidation also results in protein breakdown. This has been reported in the literature and illustrated on SDSPAGE with the appearance of low molecular weight fragments on oxidation of fish muscle proteins (Liu and Xiong 2000; Baron et al. 2006). Fish muscle protein ­degradation post mortem, has been reported to be due to the activity of proteolytic enzymes. However, it might also be the result of oxidative damage to proteins. A large body of evidence suggests that oxidation of myofibrillar proteins enhances



2.4  Case studies

33

their degradation by proteolytic systems such as calpain, caspases, cathepsins, and the proteaosome (Goll et al. 2007; Smuder et al. 2010). However, the interaction between protein oxidation, protein degradation, and fish texture has not been investigated extensively and deserves further attention.

2.3.3  Nutritional value The impact of protein oxidation on food nutritional value has not been clearly reported. It seems evident that oxidation of Trp, Met, Lys, Val, Leu, Phe, and Thr may have important consequences for food nutritional properties since these are essential amino acids. In fish muscle the essential amino acids Trp, Met, and Phe are abundant but more investigation is needed to reveal to what degree their oxidation impacts nutritional ­properties, digestibility, and the bioavailability of the fish we eat. In addition, only a minor fraction of the total protein we eat in food may be oxidized and this might have no significant impact on human nutrition because amino acid availability is a non-­ limiting factor. Some oxidation products of Met and Cys have been shown to be ­unavailable nutritionally in poultry and mammals due to their inability to be reduced in vivo (Rutherfurd and Moughan 2012). However, in general the bioavailability and the biological significance of most amino acid oxidation products are still unexplored. It has recently been shown that oxidative damage to proteins from pork reduces their susceptibility to proteolysis by digestive enzymes and it might be that this is also the case for fish proteins (Santé-Lhoutellier et al. 2007). Previous investigations have shown that the susceptibility of myosin to proteolysis was either increased or decreased after oxidation (Liu and Xiong 2000). It is also possible that differences in the extent of protein damage are responsible for such discrepancies as it is known from biological studies that ­oxidized proteins are targets for degradation (Davies et al. 1987). However, highly ­oxidized proteins accumulate as they are not able to be degraded and eliminated by proteases or by the proteosome complex (Bader and Grune 2006). More systematic investigations on oxidized fish protein digestibility, oxidized amino acid bioavailability, and their biological significance need to be performed to clearly obtain information about the impact of protein oxidation on food nutritional value. This could be of ­relevance not only for human nutrition but also for animal feed, especially fish feed, in order to evaluate the impact of feed quality on muscle quality post mortem. In addition, this could provide relevant information for better utilization of our resources.

2.4  Case studies 2.4.1  Protein and lipid oxidation during frozen storage of rainbow trout In an attempt to investigate the development of protein and lipid oxidation in frozen fish a prolonged frozen storage investigation was performed with rainbow trout (Kjaersgard et al. 2006; Baron et al. 2009). Some of the fundamental questions we

34

CH2 Protein oxidation in aquatic foods

aimed to answer were (i) to what extent are protein and lipid oxidation linked in ­frozen fish, (ii) which proteins oxidize, and are all proteins susceptible to oxidation, (iii) could feed composition have an impact on the development of protein oxidation during storage, and finally (iv) how does protein oxidation affect fish eating quality? Rainbow trout were fed a diet containing either fish oil or vegetable oil and carotenoids pigments, that is, canthaxanthin or astaxathin (at a level of 200 ppm), and stored frozen for a prolonged period (22 months). Irrespective of the feeding regime, samples stored at −80 °C had lower levels of oxidized lipid and protein during ­storage compared to samples stored at −20 °C, which showed clear signs of protein and lipid oxidation after 8 months. The results indicated that both protein and lipid oxidation seemed to follow the same kinetics. Our results also showed that available methods to measure protein oxidation are rather unspecific. The development of analytical methods to study protein oxidation will also undoubtedly result in more precise understanding of the kinetics of protein and lipid oxidation in fish muscle. Immunoblotting of oxidized protein after DNPH derivatization indicated that ­proteins oxidized at the beginning of the storage period were also oxidized after 22 months. Quantification was not possible with the 2D immunoblot and no significant difference was observed between the different samples and the different storage temperatures. No newly oxidized proteins were detected even after the prolonged storage period (22 months), indicating that proteins that were oxidized at the ­beginning of the storage period were also oxidized at a more advanced storage time. This indicates that some proteins are more susceptible to oxidation than others. The oxidized proteins identified using LC-MS/MS in trout are presented in Table 2.3. Myosin was the primary target for oxidation and also appeared as an unresolved high molecular weight band on the immunoblot, indicating cross-linking (not shown). Cross-linking via disulfide bond formation and aggregation may be responsible for the high molecular weight protein bands observed. After 22 months, ­carbonyl groups for samples fed canthaxanthin were significantly lower compared to the trout fed astaxathin or no pigment (Table 2.4) even if no differences were observed using the 2D immunoblot. Table 2.3  Proteins identified as oxidized in fish muscle after storage at −20 °C for 22 months using immunoblotting and LC-MS/MS sequencing (modified from Kjærsgaard et al. 2006) Salt-soluble protein

Non-salt-soluble protein

Adenylate kinase Carbonic anhydrase, cytoplasmic Enolase-α-1 Enolase-α-2 Guanidinoactetate methyltransferase Myosin binding protein Nucleoside phosphate kinase Pyruvate kinase Tropomyosin

Actin Creatine kinase Myosin binding protein Myosin heavy chain Myosin light chain 1 Myosin light chain 2 Myosin light chain 3 Nucleoside phosphate kinase Tropomyosin



35

2.4  Case studies

Table 2.4  Protein carbonyls in rainbow trout fed fish oil and stored at −20 °C measured at the beginning of the storage period (T0) and after 22 months (T22) (from Baron et al. 2009) Frozen storage

T0

T 22

Astaxanthin Canthaxanthin No pigment

1.83 (±0.40) 1.66a(±0.30) 2.16a(±0.14)

5.26b(±0.64) 1.87a(±0.16) 4.24b(±1.38)

a

0,3

Fish oil

0,2 Astaxanthin

p[2]

0,1

Canthaxanthin

–0,0

Carbonyls α-Tocopherol

–0,1 γ-Tocopherol

–0,2 Peroxide and volatiles

Vegetable oil –0,3 –0,2

–0,1

–0,0

0,1

0,2

p[1] Oxidative stability

Figure 2.2  Loading plot from the principal component analysis performed on the data set obtained from the chemical analysis of frozen trout for the entire storage period. The arrow ­indicates increasing oxidative stability. (Modified from Baron et al. 2009.)

This seems to indicate that canthaxanthin was efficient at preventing protein o­ xidation. A protective effect of canthaxanthin on protein oxidation has previously been reported (Baron et al. 2003) but so far no mechanism has been proposed. As for the eating quality, fish muscle containing vegetable oil was the most stable toward oxidation as well as samples containing canthaxanthin and tocopherol when compared to the other samples but the impact of protein oxidation on fish eating quality was more difficult to assess. Protein carbonyls were not located close to lipid oxidation (peroxides and volatiles) on the loading plot from the principal component analysis, indicating that these reactions might not completely follow each other and might not be affected by a­ ntioxidants in a similar way (Figure 2.2). It has been shown in model systems that protein oxidation and lipid oxidation are not necessarily linked and that protecting lipids from oxidation does not necessarily

36

CH2 Protein oxidation in aquatic foods

prevent proteins from oxidizing (Baron et al. 2005). However, further investigations are needed to reveal to what extent these two reactions are linked in muscle food and how they affect fish eating quality.

2.4.2  Protein and lipid oxidation during ripening of salted herring To gain a better understanding of the biochemical reactions taking place during ripening of old-fashioned salted herring, lipid oxidation, protein oxidation, and ­texture changes were evaluated in herring muscle for up to 371 days of ripening (Andersen et al. 2007; Christensen et al. 2011). No significant development of lipid oxidation was detected during ripening as measured using both primary and secondary lipid oxidation products. However, using the protein carbonyl assay and immuneblotting, proteins were found to be highly susceptible to oxidation after 17 days of ripening and a maximum level of protein carbonyl groups was detected after 151 days. In addition, it was revealed that high molecular weight proteins in the fish tissue were heavily oxidized during ripening. SDS-PAGE electrophoresis (Figure 2.3A) revealed that extensive protein degradation, which was also reported by ­others (Nielsen and Børrensen 1997) and a combination of both protein oxidation and proteolysis might be responsible for fish muscle degradation during ripening. However, as already mentioned above the link between protein oxidation and proteolytic degradation of muscle protein in muscle food is unclear. The two main structural proteins, that is, myosin heavy chain and actin, disappeared during ripening, and extensive protein breakdown occurred as observed by the smearing pattern on the gel with increasing ripening time. Surprisingly, the fish texture as measured using texture profile analysis revealed that during ripening fish muscle was tougher than in fresh fish and at 371 days its texture was similar to the texture of fresh fish (Figure 2.3C). This contrasts with extensive protein degradation observed during ripening, since it is generally believed that proteolysis of muscle cytoskeletal proteins is usually associated with muscle softening rather than toughening. Immunoblotting of the myosin heavy chain revealed formation of myosin aggregates already after 2 days of ripening (Figure 2.3B). Such aggregates were present at days 85 and 151 but completely disappeared at day 371. After 371 days the presence of myosin fragments with low molecular weight was also detected. In contrast, immunoblotting against actin did not reveal any actin degradation and did not reveal the presence of any actin high molecular weight aggregates (not shown). A link between myosin cross-linking and fish muscle toughening during ripening was clearly established. A previous investigation revealed that beside proteolysis, oxidative reactions were taking place in fish muscle during ripening. It was ­suggested that Hb could play a major role in the ripening process due to its peroxidase-like activity, which was shown to be persistent during salting (Andersen et al. 2007). The ability of heme protein to induce cross-linking is also well documented but little investigation has been performed in food systems. The peroxidase activity of heme protein has been shown to be able to induce myosin cross-linking



37

2.4  Case studies

(b)

(a)

Immuno-blot myosin

SDS-PAGE

F

F 2d 85d 151d 371d

2d

85d 151d 371d

188 62 49 38 28 18 14

200 97 68 55

36 31 14

(c) Texture analysis (TPA)

45 40 35 Force (N)

30 25 20 15 10 5 0

0

2

85

151

371

Days of ripening

Figure 2.3  A, SDS-PAGE of herring muscle; B, immunoblot against myosin heavy chain; C, texture profile analysis with solid bars representing the first compression and empty bars representing the second compression. F/0 represents fresh herring and the numbers (2, 85, 151 and 371) represent days of ripening. (Christensen et al. 2011. Reproduced with permission of Wiley.)

38

CH2 Protein oxidation in aquatic foods

(Frederiksen et al. 2008). The peroxidase activity of Hb might be exacerbated ­during the ripening process via cleavage of the heme protein and/or release of the heme group, and these events might be partly responsible for protein oxidation and formation of myosin cross-linking during ripening. In summary, our findings ­suggest that Hb might play an important role in the ripening process and in the development of protein oxidation in marinated fish products. It has clearly been shown that during ripening cross-linking of myosin was taking place, as observed by the formation of high molecular weight cross-linking, which probably explains the increase in hardness of the myofibrillar component observed during ripening. The present study indicates that proteolysis might not be the only factor responsible for the ripening of salted herring and that oxidation can contribute to the texture of food products.

2.5  Conclusions and perspectives Protein oxidation in food is a new field of research that has only recently received some attention. This might be due to the fact that not only lipids but also proteins can oxidize. Research in protein oxidation is in its infancy and significant efforts are needed to fully evaluate its implications for fish quality. Devoted research efforts by several research groups around the world working not only with fish but with other types of foods, together with the development of new analytical methods, will undoubtedly generate a better understanding of the impacts of protein oxidation in foods. The effects of protein oxidation on food functional quality and nutritional quality, such as how food processing and storage affect protein oxidation, might be revealed. However, protein oxidation by itself might not have a significant impact on food, but in combination with specific processing and storage condition it might lead to detrimental effects on fish quality. In addition, protein oxidation may not only induce quality losses but may be desirable in some type of foods, such as salted herring. A better understanding and control of the oxidative reactions affecting food proteins might therefore lead to better control of these reactions in food and the development of proteins with functional properties suitable for specific applications.

References Andersen, E., Andersen, M. L. and Baron, C. P. 2007. Characterization of oxidative changes in salted herring (Clupea harengus) during ripening. Journal of Agriculture and Food Chemistry 55: 9545–9553. Bader, N. and Grune, T. 2006. Protein oxidation and proteolysis. Biological Chemistry 387: 1351–1355. Baltes, W. 1993. Maillard reactions in foods. Lebensmittelchemie 47: 9–14. Baron, C. P. 2011. Textural and biochemical changes during ripening of salted herrings. Journal of the Science of Food and Agriculture 91: 330–336.

references 39

Baron, C. P. and Andersen, H. J. 2002. Myoglobin-induced lipid oxidation. A review. Journal of Agriculture and Food Chemistry 50: 3887–3897. Baron, C. P., Berner, L. and Jacobsen, C. 2003. Effect of astaxanthin and canthaxanthin on lipid and protein oxidation of fish feed exposed to UVA. Free Radical Research 37: 61–61. Baron, C. P., Berner, L., Skibsted, L. H. and Refsgaard, H. H. F. 2005. Evaluation of activity of selected antioxidants on proteins in solution and in emulsions. Free Radical Research 39: 777–785. Baron, C. P., Pham, K. P. and Jacobsen, C. 2006. Oxidation of sarcoplasmic proteins from rainbow trout feed different diet, in Proceedings of Institute of Food Technologist. Orlando, FL: IFT. Baron, C. P., Kjaersgard, I. V. H., Jessen, F. and Jacobsen, C. 2007. Protein and lipid oxidation during frozen storage of rainbow trout (Oncorhynchus mykiss). Journal of Agriculture and Food Chemistry 55: 8118–8125. Baron, C. P., Hyldig, G. and Jacobsen, C. 2009. Does feed composition affect oxidation of rainbow trout (Oncorhynchus mykiss) during frozen storage? Journal of Agriculture and Food Chemistry 57: 4185–4194. Berlett, B. S. and Stadtman, E. R. 1997. Protein oxidation in aging, disease, and oxidative stress. Journal of Biological Chemistry 272: 20313–20316. Bertram, H. C., Kristensen, M., Østdal, H., Baron, C. P., Young, J. F. and Andersen, H. J. 2007. Does oxidation affect the water functionality of myofibrillar proteins? Journal of Agriculture and Food Chemistry 55: 2342–2348. Cheret, R., Delbarre-Ladrat, C., Lamballerie-Anton, M. de, and Verrez-Bagnis, V. 2007. Calpain and cathepsin activities in post mortem fish and meat muscles. Food Chemistry 101: 1491–1496. Chopin, C., Kone, M. and Serot, T. 2007. Study of the interaction of fish myosin with the products of lipid oxidation: The case of aldehydes. Food Chemistry 105: 126–132. Christensen, M., Andersen, E., Christensen, L., Andersen, M. L. and Baron, C. P. 2011. Textural and biochemical changes during ripening of old-fashioned salted herrings. Journal of the Science of Food and Agriculture 91: 330–336. Christensen, M., Andersen, M. and Serot, T. 2007. Study of the interaction of fish myosin with the products of lipid oxidation: The case of aldehydes. Food Chemistry 105: 126–132. Davies, M. J. and Dean, R. T. 1997. Radical-mediated protein oxidation. From Chemistry to Medicine, New York: Oxford University Press. Davies, M. J., Fu, S. L., Wang, H. J. and Dean, R. T. 1999. Stable markers of oxidant damage to proteins and their application in the study of human disease. Free Radical Biology and Medicine 27: 1151–1163. Dean, R. T., Fu, S., Stocker, R. and Davies, M. J. 1997. Biochemistry and pathology of radical-mediated protein oxidation. Biochemical Journal 324: 1–18. Egelandsdal, B., Ren, L. P., Gong, Y. S., Greaser, M. and Richards, M. P. 2010. Factors affecting solubilisation and oxidation of proteins during equine metmyoglobin-mediated lipid oxidation in extensively washed cod muscle. Food Chemistry 122: 1102–1110. Elias, R. J., Kellerby, S. S. and Decker, E. A. 2008. Antioxidant activity of proteins and peptides. Critical Reviews in Food Science and Nutrition 48: 430–441.

40

CH2 Protein oxidation in aquatic foods

Estévez, M., Ollilainen, V. and Heinonen, M. 2009. Analysis of protein oxidation ­markers α-aminoadipic and γ-glutamic semialdehydes in food proteins using liquid chromatography (LC) − electrospray ionization (ESI) − multistage tandem mass spectrometry (MS). Journal of Agriculture and Food Chemistry 57: 3901–3910. Eymard, S., Baron, C. P. and Jacobsen, C. 2009. Oxidation of lipid and protein in horse mackerel (Trachurus trachurus) mince and washed minces during processing and storage. Food Chemistry 114: 57–65. Frederiksen, A. M., Lund, M. N., Andersen, M. L. and Skibsted, L. H. 2008. Oxidation of porcine myosin by hypervalent myoglobin: The role of thiol groups. Journal of Agriculture and Food Chemistry 56: 3297–3304. Goll, D. E., Neti, G., Mares, S. W. and Thompson, V. F. 2007. The non-lysosomal Ca2+dependent protein degradation pathway: The calpains, proteasome, and myofibrillar protein turnover. Poultry Science 86: 445–446. Grimm, S., Höhn, A. and Grune, T. 2012. Oxidative protein damage and the proteasome. Amino Acids 42: 23–38. Halliwell, B. and Gutteridge J. M. C. 2007. Free Radicals in Biology and Medicine, 4th edn. New York: Oxford University Press. Hambly, D. M. and Gross, M. L. 2009. Cold chemical oxidation of proteins Analytical Chemistry 81: 7235–7242. Harel, S. and Kanner, J. 1985. Hydrogen peroxide generation in ground muscle tissue. Journal of Agriculture and Food Chemistry 33: 1186–1188. Headlam, H. A., Gracanin, M., Rodgers, K. J. and Davies, M. J. 2006. Inhibition of cathepsins and related proteases by amino acid, peptide, and protein hydroperoxides. Free Radical Biology and Medicine 40: 1539–1548. Irwin, J. A., Ostdal, H. and Davies, M. J. 1999. Myoglobin-induced oxidative damage: Evidence for radical transfer from oxidized myoglobin to other proteins and antioxidants. Archives of Biochemistry and Biophysics 362: 94–104. Jafarpour, A., Sherkat, F., Leonard, B. and Gorczyca, E. M. 2008. Colour improvement of common carp (Cyprinus carpio) fillets by hydrogen peroxide for surimi production. International Journal of Food Science and Technology 43: 1602–1609. Kjærsgaard, I. V. H., Norrelykke, M. R., Baron, C. P. and Jessen, F. 2006. Identification of carbonylated protein in frozen rainbow trout (Oncorhynchus mykiss) fillets and development of protein oxidation during frozen storage Journal of Agriculture and Food Chemistry 54: 9437–9446. Levine, R. L., Williams, J. A., Stadtman, E. R. and Shacter, E. 1994. Carbonyl assays for determination of oxidatively modified proteins. Methods in Enzymology 233: 346–357. Levine R. L., Berlett, B. S., Moskovitz, J., Mosoni, L. and Stadtman, E. R. 1999. Methionine residues may protect proteins from critical oxidative damage. Mechanims os Ageing and Development 107: 323–332. Liu, G. and Xiong, Y. L. 2000. Electrophoretic pattern, thermal denaturation, and in vitro digestibility of oxidized myosin. Journal of Agriculture and Food Chemistry 48: 624–630. Lund, M. N., Lametsch, R., Hviid, M. S., Jensen, O. N. and Skibsted, L. H. 2007. Highoxygen packaging atmosphere influences protein oxidation and tenderness of porcine longissimus dorsi during chill storage. Meat Science 77: 295–303.

references 41

Lund, M. N., Luxford, C., Skibsted, L. H. and Davies, M. J. 2008. Oxidation of myosin by heme proteins generates myosin radicals and protein cross-links. Biochemical Journal 410: 565–574. Maestre, R., Pazos, M., Iglesias, J. and Medina, I. 2009. Capacity of reductants and chelators to prevent lipid oxidation catalyzed by fish hemoglobin. Journal of Agriculture and Food Chemistry 57: 9190–9196. Maforimbo, E., Skurray, G. R. and Nguyen, M. 2007. Evaluation of l-ascorbic acid oxidation on SH concentration in soy-wheat composite dough during resting period. LWT – Food Science and Technology 40: 338–343. Maqsood, S. and Benjakul, S. 2011. Effect of bleeding on lipid oxidation and quality changes of Asian seabass (Lates calcarifer) muscle during iced storage. Food Chemistry 124: 459–467. Martins, S. I. F. S., Jongen, W. M. F. and Boekel, M. A. J. S. van 2001. A review of Maillard reaction in food and implications to kinetic modelling. Trends in Food Science and Technology 11: 364–373. Miura, T., Muraoka, S. and Ogiso, T. 1995. Relationship between protein damage and ferrylmyoglobin. Biochemistry and Molecular Biology International 36: 587–594. Morzel, M., Gatellier, P., Sayd, T., Renerre, M. and Laville, E. 2006. Chemical oxidation decreases proteolytic susceptibility of skeletal muscle myofibrillar proteins. Meat Science 73: 536–543. Najafian, L. and Babji, A. S. 2012. A review of fish-derived antioxidant and antimicrobial peptides: Their production, assessment, and applications. Peptides 33: 178–185. Nielsen, H. H. and Borrensen, T. 1997. The influence of intestinal proteinases on ripening of salted herring, in Seafood from Producer to Consumer: An Integrated Approach to Quality (eds Luten, J. B., Børresen, T. and Oehlenschlager, J.) Amsterdam: Elsevier Science Publishers, pp. 293–304. Ooizumi, T. and Xiong Y. L. 2004. Biochemical susceptibility of myosin in chicken myofibrils subjected to hydroxyl radical oxidizing systems. Journal of Agriculture and Food Chemistry 52: 4303–4307. Østdal, H., Davies, M. J. and Andersen, H. J. 2002. Reaction between protein radicals and other biomolecules. Free Radical Biology and Medicine 33: 201–209. 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. Park, J. W. 2000. Surimi and Surimi Seafood. New York: Marcel Dekker,Inc. Parkington, J. K., Xiong, Y. L., Blanshard, S. P., Xiong, S., Wang, B., Srinivasan, S. and Froning, G. 2000. Chemical and functional properties of oxidatively modified beef heart surimi stored at 2 °C Journal of Food Science 65: 428–433. Reznick, A. Z. and Packer, L. 1994. Oxidative damage to proteins: spectrophotometric method for carbonyl assay. Methods in Enzymology 233: 357–363. Richards, M. P. and Hultin, H. O. 2002. Contribution of blood and blood components to lipid oxidation in fish muscle. Journal of Agriculture and Food Chemistry 50: 555–564. Rowe, L. J., Maddock, K. R., Lonergan, S. M. and Huff-Lonergan, E. 2004. Oxidative environments decrease tenderization of beef steaks through inactivation of ­milli-­calpain. Journal of Animal Science 82: 3254–3266.

42

CH2 Protein oxidation in aquatic foods

Rutherfurd, S. M. and Moughan, P. J. 2012. Available versus digestible dietary amino acids. British Journal of Nutrition 108: S298–S305. Sakai, T., Matsushita, Y., Sugamoto, K. and Uchida, K. 1997. Lipid peroxidation-derived hepatotoxic aldehyde, 4-hydroxy-2-hexenal, in fish. Bioscience, Biotechnology and Biochemistry 61: 1399–1400. Smuder, A. J., Kavazis, A. N., Hudson, M. B., Nelson, W. B. and Powers, S. K. 2010. Oxidation enhances myofibrillar protein degradation via calpain and caspase-3. Free Radical Biology and Medicine 49: 1152–1160. Soyer, A. and Hultin, H. O. 2000. Kinetics of oxidation of the lipids and proteins of cod sarcoplasmic reticulum. Journal of Agriculture and Food Chemistry 48: 2127–2134. Srinivasan, S. and Hultin, H. O. 1997. Chemical, physical, and functional properties of cod proteins modified by a non enzymatic free radical generating system. Journal of Agriculture and Food Chemistry 45: 310–320. Stadtman, E. R. 1990. Metal ion-catalyzed oxidation of proteins: Biochemical mechanism and biological consequences. Free Radical Biology and Medicine 9: 315–325. Stadtman, E. R. and Levine, R. L. 2003. Free radical-mediated oxidation of free amino acids and amino acid residues in proteins. Amino Acids 25: 207–218. Subramanian, S. and Hultin, H. O. 1995. Hydroxyl radical modification of fish muscle proteins. Journal of Food Biochemistry 18: 405–425. Timm-Heinrich, M., Eymard, S., Baron, C. P., Nielsen, H. H. and Jacobsen, C. 2013. Oxidative changes during ice storage of rainbow trout (Oncorhynchus mykiss) fed different ratios of marine and vegetable feed ingredients. Food Chemistry 136: 1220–1230. Tunhun, D., Itoh, Y., Morioka, K. and Kubota, S. 2001. Oxidation during washing of fish meat induces a decrease in gel forming ability. Developments in Food Science 42: 357–374. Venugopal, V. and Shahidi, F. 1996. Structure and composition of fish muscle. Food Review Internationl 12: 175–197. Visessanguan, W., Benjakul, S. and Tanaka, M. 2003. Effect of microbial transglutaminase on rheological properties of oxidised and non-oxidised natural actomyosin from two ­species of bigeye snapper. Journal of the Science of Food and Agriculture 83: 105–112. Xiong, Y. L. 2000. Protein oxidation and implication for muscle food quality, in Antioxidants in Muscle Foods (eds Decker, E., Faustman, C. and Lopez-Bote, C. J.). Chichester: John Wiley & Sons, pp. 85–111. Xiong, Y. L, Blanchard, S. P., Ooizumi, T. and Ma, Y. 2010. Hydroxyl radical and ­ferryl-generating systems promote gel network formation of myofibrillar protein. Journal of Food Science 75: 215–221. Xu, G. and Chance, M. R. 2005. Radiolytic modification of sulfur-containing amino acid residues in model peptides: fundamental studies for protein footprinting. Analytical Chemistry 77: 2437–49.

3 Influence of processing on lipids and lipid oxidation in aquatic foods Sivakumar Raghavan1 and Hordur G. Kristinsson1,2 Department of Food Science and Human Nutrition, University of Florida, Gainesville, Florida, USA 2  Division of Biotechnology and Biomolecules, Matis Ltd, Reykjavik, Iceland 1 

Seafoods are commonly subjected to several processing conditions, including freezing, canning, smoking, salting, fermentation, pressurization, irradiation, ­ ­modified atmosphere packaging, and pH-shifting. These processing techniques could either promote or reduce lipid oxidation. This chapter will review research work done in these areas on oxidation in general and will also focus on the effect of the above processes on specific pro-oxidants, antioxidants, and lipids.

3.1  Effect of freezing on lipid oxidation 3.1.1  Introduction Freezing is one of the many preservation methods used for slowing down biochemical and chemical changes in a post-mortem seafood product. As freezing involves sub-zero temperatures, it is generally believed that preservation of food by freezing originated in the colder climates of the northern hemisphere. Although freezing of food products has been practiced for centuries, it was not until the advent of ice boxes or refrigerators in the late nineteenth century that freezing became a popular method of food preservation. Back in 1842, H. Benjamin of England was granted a patent for the method of freezing foods by immersion in ice and salt brine. In 1923, Clarence Birdseye of the USA experimented with freezing fish fillets using dry ice,

Antioxidants and Functional Components in Aquatic Foods, First Edition. Edited by Hordur G. Kristinsson. © 2014 John Wiley & Sons, Ltd. Published 2014 by John Wiley & Sons, Ltd.

44

CH3 Influence of processing on lipids and lipid oxidation in aquatic foods

and later in 1926 developed a quick-freeze machine. Since then freezing has rapidly evolved as a popular food preservation technique. The reader will find numerous books, book chapters, and research articles explaining the principles behind the processes of freezing, the various methods of freezing, and applications in food and seafood industries (Desrosier and Tressler 1977; FAO 1977; Jeremiah 1996; Erickson 1997; Fellows 2000). Although freezing can slow down enzymatic and microbial activity, it may also initiate several physical and physiochemical changes that might lead to quality deterioration. Chief among them is lipid oxidation. This chapter will focus on the effect of freezing on various pro-oxidants and antioxidants in seafoods as well as on lipid oxidation.

3.1.2  Effect of the freezing process on lipid oxidation The major constituent of fish or any seafood product is water, whose composition varies with the amount of fat and the season. Typically, fish contains around 60–83% water. This water can exist in three different forms: (i) bound water, which is ­typically bound or closely associated with muscle proteins through various ionic interactions and polar bonds (Offer and Knight 1988), (ii) water entrapped or immobilized within the muscle structure but not bound to proteins (Huff-Lonergan and Lonergan 2005), and (iii) free extracellular water, which is usually held by capillary forces within muscle fibers and accounts for the majority of water in muscle tissues. It is this free water that is affected during freezing operations. During freezing, extracellular water freezes first because of its low ionic strength and solute concentrations. There is also a gradual movement of water out of the muscle cells into the extracellular regions, leading to the concentration of non-aqueous muscle constituents in the non-frozen phase. This can lead to the concentration of pro-oxidants and antioxidants, as well as changes in pH, ionic strength, viscosity, and other colligative properties associated with an accumulation of solutes. Freezing can reduce the rate of chemical and enzymatic reactions responsible for lipid oxidation. For ­example, Saeed et al. (2002) reported that Atlantic mackerel stored at −30°C for 24 months had lower lipid oxidation compared to those stored at −20°C, while Baron et al. (2007) reported that lipid oxidation in frozen rainbow trout decreased with an increase in freezing temperature, that is, –20°C > −30°C > −80°C. Researchers have shown that deep freezing of Mediterranean fish at −80°C significantly lowers ­degradation of antioxidants such as vitamin C and ubiquinol compared to storage at −30 °C (Passi et al. 2005). However, increased concentration of solutes in the ­non-frozen phase of seafoods may sometimes offset the benefits obtained during freezing. Freezing can lead to protein denaturation (Ota and Tanaka 1978), loss of Ca2+-ATPase activity (Jiang and Lee 1985), and an increase in the content of lipid hydroperoxides and free fatty acids (Refsgaard et al. 1998). Lipid oxidation in ­seafoods can also be affected by freezing time. Freezing time is dependent on the temperature of freezer, product shape, type, and thickness, packaging, and the lipid content of the fish species (FAO 1977). A fatty fish, for example, will have a lower



3.1  Effect of freezing on lipid oxidation

45

percentage of water and hence could be frozen faster than a lean fish. However, a frozen fatty fish may oxidize faster due to its higher lipid content (Kurade and Baranowski 1987) and polyunsaturated fatty acid (PUFA) composition. With respect to size, seafood products of smaller dimensions will have larger surface areas and hence will freeze faster compared to bulk products of larger dimensions but having the same weight. However, an increase in the surface area will also increase the propensity of the product to lipid oxidation (Nair et al. 1987). Frozen seafood products are subjected to a variety of pre-treatments. These treatments may have a positive or negative influence on the storage stability of frozen aquatic products. Some common pre-treatments include addition of ice (Olafsdottir et al. 1997), chilled storage (Aubourg et al. 2002), steam cooking (Calder et al. 2005), addition of refrigerated seawater (Kraus 1992), chemicals (Woyewoda and Bligh 1986; Hwang and Regenstein 1995), and brine treatment (Aubourg and Ugliano 2002). The size of ice crystals and the number of freeze–thaw cycles could all have an impact on the quality of frozen fish and seafood. Srinivasan et al. (1997) showed that prawns subjected to repeated freeze–thaw cycles had higher level of lipid ­oxidation products compared to fresh prawns. Tseng et al. (2003) reported an increase in thiobarbituric acid reactive substances (TBARS), a secondary product of lipid oxidation, with an increase in freeze–thaw cycles. Slow freezing can lead to relatively large ice crystals which may cause structural damage within muscle cells and tissues (Bello et al. 1982). Also, large ice crystals can damage cellular ­membranes and release compartmentalized enzymes such as lipases and lipoxygenases, which can catalyze oxidation during thawing (Sahagian and Goff 1996; Benjakul and Bauer 2001). Nilsson and Ekstrand (1993) reported that rainbow trout subjected to repeated freezing and thawing showed increased enzyme activity in press juice (the soluble fraction of fish muscle), an indicator of enzyme leakage. The study also reported that slow freezing led to higher leakage of enzymes ­compared to fast freezing. Tomas and Anon (1990) studied the effect of freezing rate on lipid oxidation in salmon and reported no significant difference between fast and slow freezing methods. They also reported, however, that during the thawing cycle slow frozen muscle tissue had greater drip loss and loss of water-soluble antioxidants.

3.1.3  Nature of lipids in frozen seafoods The nature of lipids in seafoods can vary widely among different species of fish (Kurade and Baranowski 1987). Even within any particular fish species, the amount and type of lipid can vary with factors such as age, reproductive status, diet, size, geographic location, temperature, habitat, etc. (Nettleton 1985; Ackman 1989; Kelly and Kohler 1999). For example, in Cornish mackerel, the maximum and minimum levels of total lipids were recorded in December and June, respectively, while the highest and lowest lipid unsaturation levels were recorded in November and May, respectively (Hardy and Keay 1972; Aubourg et al. 2005). In king salmon,

46

CH3 Influence of processing on lipids and lipid oxidation in aquatic foods

spawning can result in a loss of muscle fat content from 15.5 to 2.2 % (Greene 1926). Within any particular fish, the nature of the lipid varies with its location inside the muscle. Adipose tissue contains primarily triacylglycerols, while membranes contain phospholipids and cholesterol (Body and Vlieg 1989). ­ Membrane lipids are more susceptible to oxidation than triacylglycerols due to ­polyunsaturation and their large surface area (Hultin 1995). The oxidative susceptibility of membrane phospholipids varies with the nature of their polar head groups and the type of unsaturated fatty acids (Vossen et al. 1993). The susceptibility of triacylglycerols to oxidation depends on the degree of unsaturation. Some parts of fish are more susceptible to lipid oxidation than others. For example, high lipid content and the presence of lipoxygenases (German and Kinsella 1985) makes the skin of a fish as well as the dark muscle beneath the skin susceptible to oxidation. When the oxidative stability of various horizontal layers of skinned herring during frozen storage was studied, Undeland et al. (1998a) reported that the layers under the skin oxidized first, followed by the inner part of the herring and then the middle part. When the fillets were stored frozen with the skin on, the layer under the skin still underwent oxidation first, but not to the same degree as in skinned herring. In frozen mackerel, the lipids from the skin were reported to oxidize eight times faster than the white and dark muscles. When the fatty acid content of herring was analyzed, the amount of C20:5, C18:1, and C20:1 decreased and the amount of C22:6 increased from under the skin layer toward the inner layer (Undeland et al. 1998a). Among different muscle types, dark muscles are more susceptible to oxidation than white muscles because of their high content of lipids (Body and Vlieg 1989), ­mitochondria, pro-oxidants, heme proteins (Richards and Hultin 2002), and oxidative enzymes. In fatty fish species such as herring, enzymic lipid oxidation in dark muscle was reported to be three to four times higher than that in light muscle (Hultin 1988). In lean fish species such as saithe, frozen storage led to more pronounced lipid oxidation and loss of polyunsaturated fatty acids in dark muscle compared to white muscle (Dulavik et al. 1998).

3.1.4  Pro-oxidants in frozen seafoods Seafoods contain a variety of pro-oxidants. During frozen storage, a balance between pro-oxidants and antioxidants plays an important role in determining the final quality of frozen fish products. The major pro-oxidants present in frozen seafoods are heme proteins (Richards and Hultin 2002), lipases and phospholipases (Geromel and Montgomery 1980), free ferrous iron released from the degradation of heme and from iron binding proteins such as ferritin and transferrin (Decker and Welch 1990; Gomez-Basauri and Regenstein 1992), copper (Decker and Hultin 1990a), and reducing agents such as ascorbate (Decker and Hultin 1990b) and cysteine (Searle and Willson 1983; Decker and Welch 1990). When frozen mackerel was analyzed, the press juice obtained from the white muscle was found to contain around 529 parts per billion (ppb) of iron and 118 ppb of copper, while the dark



3.1  Effect of freezing on lipid oxidation

47

muscle was found to contain around 2405 ppb of iron and 302 ppb of copper (Decker and Hultin 1990a). Of the total amount of iron and copper in the press juice, almost 10% was reported to be