Food Science and Security

FOOD SCIENCE AND SECURITY

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FOOD SCIENCE AND SECURITY

LOUIS AMSEL AND

LENA HIRSCH EDITORS

Nova Science Publishers, Inc. New York

Copyright © 2009 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers’ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works. Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. LIBRARY OF CONGRESS CATALOGING-IN-PUBLICATION DATA Amsel, Louis. Food science and security / Louis Amsel and Lena Hirsch. p. cm. Includes index. ISBN 978-1-61728-423-6 (E-Book) 1. Agricultural innovations--Developing countries. 2. Biotechnology--Developing countries. 3. Food industry and trade--Technology transfer--Developing countries. 4. Food supply--Developing countries. 5. Food industry and trade--Quality control. I. Hirsch, Lena. II. Title. S494.5.I5A49 2009 338.1'9--dc22 2009004754

Published by Nova Science Publishers, Inc.

New York

CONTENTS Preface

vii

Research and Review Studies

1

Chapter 1

The Contribution of Plant Biotechnology to Food Security in the 21st Century Koreen Ramessar, Shaista Naqvi, Svetlana Dashevskaya, Ariadna Peremarti, Dawei Yuan, Sonia Gomez-Galera, Sol Maiam Rivera Velez, Gemma Farre, Maite Sabalza, Bruna Miralpeix, Richard Twyman, Changfu Zhu, Ludovic Bassie, Teresa Capell and Paul Christou

Chapter 2

Bio (Single Cell) Protein: Issues of Production, Toxins and Commercialisation Status Pogaku Ravindra, Ravinder Rudravaram, Anuj Kumar Chandel, Linga Venkateswar Rao and Yim Zhi Hui

73

Chapter 3

Food Security and Materialism Michael W. Allen

99

Chapter 4

An Examination of Gender Differences in the Relationship between Reporting a Food Hardship and Physical Health Colleen Heflin

113

Chapter 5

Investigating Viral Hepatitis A Dynamics in Campania, Italy Marco Ajelli and Stefano Merler

125

Chapter 6

Seafood as a Functional Food in Decreasing the Risk for the Metabolic Syndrome Helen Hermana Miranda Hermsdorff, Itziar Abete, María Ángeles Zulet, Inga Thorsdottir and José Alfredo Martínez

147

Chapter 7

Underutilised Fish Species: Their Potential as Fresh Fillets and Processed Products T. Ronan Gormley and John D. Fagan

161

3

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Contents

Chapter 8

Improvement of Poultry Meat Nutritional Value and Quality through Different Natural Sources and Mineral Dietary Supplements Ricard Bou, Eric A. Decker, Francesc Guardiola and Rafael Codony

187

Chapter 9

Livestock and Food Security with Special Reference to the Developing Countries R. Trevor Wilson

231

Chapter 10

Organic Meat: Market Development and Consumer Willingness to Pay Fabio Napolitano, Ada Braghieri and Antonio Girolami

253

Chapter 11

Meat Consumption and Anemia in Chinese Adults in Jiangsu Province Zumin Shi , Xiaoqun Pan and Gerd Holmboe-Ottesen

267

Short Communications

Index

281

Gas Chromatography-Olfactometry: An Efficient Tool for the Monitoring of Seafood Quality Vincent Varlet and Xavier Fernandez

283

Determination of Inorganic Arsenic in Seafood by HG-ICP-OES after Conventional Wet Digestion Khalid Boutakhrit, Fabien Bolle and Leo Goeyens

295

305

PREFACE Food security is taken for granted in the industrialized world, where stable political and social structures ensure that everyone has access to safe and nutritious food sufficient to maintain a healthy and active lifestyle. The picture in the developing world is very different. This important topic is the focus of our new book. One of the persistent myths concerning food insecurity is that it is caused by a shortfall in food production. Quite to the contrary, there is plenty of food to go around. The reasons for food insecurity are complex, but one of the main factors is poverty. Biotechnology provides a range of tools that can be used to improve agriculture in the developing world. This book considers some of the biotechnologybased approaches to improving agriculture and food security in the developing world. Despite the wealth present in American society, high rates of food insecurity, food insufficiency, and hunger are a significant problem in the United States. Some adults have obsessive concerns about their food supply. A study in this book suggests that this may, in part, be a symptom of materialistic tendencies developed in childhood in response to an insufficient food supply. A further study presents the effect of reports of food hardships on the presence of chronic health conditions. This book continues with specific research in seafood and meat regarding the abundance of these products, food security concerns as well as health related issues. Any long-term strategy to address food insecurity in the developing world must tackle the underlying problem of poverty by increasing the level of rural employment-based income through increased agricultural productivity (Christou and Twyman, 2004). Given projected increases in the world’s population, increases in the cost of oil, falling reserves of fresh water and increased urbanization, the only solution to the above problem is to increase the yields of major food crops, particularly cereal grains, using currently available land and less water, and to diversify the uses to which crops are put to facilitate the creation of wealth. A variety of approaches can be envisaged, including the efficient use of organic and inorganic fertilizers, irrigation strategies, soil and water conservation, pest and disease management and the production of improved plant varieties with higher yields or novel products. Biotechnology provides a range of tools that can be used to improve agriculture in the developing world and we consider some of the biotechnology-based approaches to improving food security in Chapter 1. The alarming rate of population growth and a regular depletion in food production and food resources are the important reasons which make the dire need of hour to look out new viable options for food and feed sources. Based on the scientific developments particularly in industrial microbiology, one feasible solution could be the consumption of microorganisms as

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human food and animal feed supplements. Human has used microbial based products like alcoholic beverages, curd, cheese, yogurt, soya sauce and idly even before the starting of civilization. Due to research developments caused in scientific arena in last two decades, (Bio) Single cell protein (SCP) has roused new attentions towards its supplement as human food, feed or supplements as staple diet. There are several benefits using SCP as food or feed, viz their rapid growth rate and high protein content. The microorganisms involved as SCP have the ability to utilize cheap and plentiful available feedstock for their growth and energy have made them an attractive option. However, in spite of lab based successful stories, only a limited number of commercial SCP production plants have been seen worldwide. Chapter 2 analyzes the possibility of SCP production, various raw materials for its production, and available microorganisms with cultivation methods, the toxicity assessment and their removal. Also, new developments and risk assessment using SCP along with worldwide industrial SCP production have been discussed. To better understand and address food insecurity, it may help to consider the motivations that drive some people to feel unusually worried/anxious about their food supply, resulting in hoarding and overeating. That is, why might someone, when food supplies are not genuinely threatened, be obsessed about the state of his or her food supply, and make food security a top life goal? Materialism may play a role. In Chapter 3, it is posited that individuals may cope with childhood deprivation of material goods and a reliable, regular, and sufficient supply of food by developing materialistic tendencies. This may include, in addition to efforts to attain social security (e.g., improve socio-economic status), leanings toward the excessive accrual or consumption of material goods including food items. In short, some adults’ obsessive concerns about their food supply may, in part, be a symptom of materialistic tendencies developed in childhood in response to an insufficient food supply (and/or other goods), rather than current threats to food supply per se. A recent series of surveys and experiments reported by Allen and Wilson (2005) support this position; these are elaborated in the present chapter, and new findings that have not been previously reported are also presented. Despite the wealth present in American society, high rates of food insecurity, food insufficiency, and hunger are a significant problem in the United States. The persistently high reports of food security are troubling given that experiencing food hardships is not a social problem with isolated consequences. A lack of an adequate food supply can potentially affect mental well-being and overall quality of life, as well as physical health outcomes. However, an important challenge facing research on nutrition and health is to distinguish the consequences of food hardship from those of its common risk factors, such as poverty and low socioeconomic status. Chapter 4 used data from the National Survey of America’s Families to model the effect of reports of food hardships on the presence of chronic health conditions and to examine if gender differences exist in this relationship. Using instrumental variable linear probability models to adjust for endogeneity between food hardship and health, I find that reporting food hardship increases the risk of having a chronic health condition for both men and women. However, men who report a food hardship face a much higher risk of chronic health conditions than do women who report hardships. Viral Hepatitis A is an acute infection caused by Hepatitis A Virus (HAV). Although most infected persons recover completely, this infection imposes a large economic burden, mainly due to its relevant morbidity and its high symptomaticity in adult individuals (while a significant proportion of children and adolescents remain asymptomatic). Thus, Viral

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Hepatitis A represents a priority for public health agencies. This infection is transmitted via the fecal–oral route. Person–to–person transmission is typical in low hygiene areas, while ingestion of contaminated water or food (mainly shellfish) is the main risk factors in lowintermediate endemicity regions (as the case of Italy). The aim of Chapter 5 is the investigation of Viral Hepatitis A epidemic in the Italian region of Campania (Naples region). Building on a previous study, here we consider an age– structured network model, based on the explicit sociodemographic composition of the population. Since Hepatitis A is an endemic disease, and therefore the time scale of the model is of the order of several years, we employ a dynamic network of contacts in order to take into account the vital dynamics of the population. The model allows accounting for age–specific interventions (e.g. vaccination) and for all the main risk factors for HAV infections: person– to–person transmission, travels to high endemicity foreign areas and ingestion of infected food/water. The last kind of transmission plays a key role in the explanation of HAV epidemic: in fact, more than 60% of the Italian infections occur in this way (and the number increases if only the most affected areas are considered). For this reason, the process of the virus excretion in the environment and the consequent seafood contamination is explicitly modeled. Ourmodel is able to reproduce the Campania notification data and it allows the prediction of the effects of intervention options. In presence of a vaccination programme of unlimited duration, Hepatitis A epidemic can be controlled, even by performing a very mild vaccination programme. Improvements in hygienic conditions, leading to a reduction of seafood contamination, are also able to mitigate the epidemic even in the absence of vaccination programmes. As discussed in Chapter 6, the increased incidence of the metabolic syndrome, characterized by insulin resistance, visceral adiposity, dyslipidemia, hypertension, and a systemic proinflammatory state, greatly increases the risk of cardiovascular disease. These clinical conditions are multifactorial in origin and treatment, having a strong dietary determinant. Currently, the most promising approach to mitigate obesity and metabolic syndrome is lifestyle intervention, weight reduction, decreased total and saturated fat consumption, and increased physical activity. In this sense, traditional foods, considered as functional foods, could be an important choice helping in the treatment of obesity and metabolic syndrome features. Functional foods consumed in an adequate amount on a healthy diet could improve the benefits associated to weight loss and reduce the metabolic syndrome features. Specifically, seafood has been considered as a functional food since their properties are not only nutritional, but also have been related with the improvement of several cardiovascular risk factors. Benefits include lower risk of primary cardiac arrest, reduced cardiovascular mortality, particularly sudden cardiac death, reduced triglyceride levels, increased high density lipoprotein levels, improved endothelial functions, reduced platelet aggregability, and lower blood pressure. Thus, healthy diets that avoid excess energy intake, reduce saturated fat and include seafood consumption may be a key component in the nutritional treatment of obesity and metabolic syndrome features. The term underutilised fish species (UUFS) is used in Chapter 7 to describe species that are outside mainstream use. However, all species are utilised somewhere in the world. As fish stocks dwindle and quotas tighten the use of UUFS for consumption as fillets/portions, and also in a range of fish products, may assume a new importance. For this reason, the evaluation and test processing of UUFS has been an ongoing topic of R and D at Ashtown Food

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Research Centre (AFRC) and six separate studies were conducted as fish became available. Tests on silver smelt (Study 1) indicated that this bland white-fleshed fish is useful for a range of products including enrobed nuggets, fingers and fishcakes; it also produced strong gels. Sensory tests (Study 2) on 15 UUFS indicated that all were liked when tasted as breaded nuggets, and all compared favourably with cod. Study 3 was on 21 UUFS and taste panellists preferred six of these to cod as fillets, ten as nuggets and eight as fish-cakes. The best species were orange roughy, black scabbard, morid cod and siki shark. Study 4 was conducted on steamed fillets of 15 species and none had sensory scores better than cod. However, samples of morid cod, bluemouth rockfish, ling, forkbeard, black scabbard, silver roughy, orange roughy and roundnose grenadier compared favourably with cod. Study 5 involved adding value to selected UUFS using sous vide, marinating, and freeze-chilling coupled with modified atmosphere packaging (MAP). Orange roughy, cardinal fish and redfish gave well textured sous vide/frozen products, but the flesh of sous vide/frozen roundnose grenadier, blue ling and Greenland halibut was considered too soft. Tests with salt-based marinades and albacore tuna indicated sensory acceptability scores in the order Cajun>arrabbiata>tandoori> lemon+lime>southern fried>smoke. Trials on freeze-chilling combined with MAP indicated that both technologies were suitable for extending the shelf life of redfish, cardinal fish, and roundnose grenadier portions. In study 6, breaded products were prepared from blue whiting and received good acceptability scores. The overall outcome from the six trials indicated that a number of the species have potential for sale as chilled fillets or as processed products. They may provide an alternative to over-exploited fish species using sustainable fishing practices and could be processed to produce high quality nutritious meal solutions for human consumption. Poultry meat is widely consumed and its total fat content is medium to relatively low although its relatively poor in n-3 polyunsaturated fatty acids (PUFA) when in animals are fed with standard diets (19). However, poultry meat has also been reported to be successfully enriched in several compounds through dietary practices (19-21). Chapter 8 summarizes the different dietary strategies that have been used to improve the nutritional value as well as the oxidative stability and sensory properties of poultry meats. The keeping of livestock is the only source of livelihood for at least 20 million pastoral families across the world. Livestock are also an important – indeed often the main -- source of income for a minimum of 200 million smallholder families in Asia, Africa and Latin America. Domestic animals provide power for the cultivation of more than 300 million hectares or some 25 per cent of the world’s total cropped area. This land would otherwise have to be cultivated by hand (with the concomitant drudgery, especially for women) or by tractor power with the inevitable consequences of depletion of fossil fuels and loss of valuable foreign exchange for developing countries. Ownership of livestock provides one of the very few opportunities for the landless to engage in agricultural activities. Livestock are often the only asset for many people who do not own the land they farm and that they can take with them when their leases expire. The single cow, the goat, a few scavenging poultry or a donkey are the mainstay of survival for many of the really poor. It is becoming more generally accepted that the smaller the farming enterprise the greater the importance of the contribution of livestock to its viability and to its sustainable production. Solving the challenge of global food security is not possible without a focus on animals. The contribution of domestic animals to sustainable development could be greatly enhanced not only by technical innovation but by provision of an appropriate enabling environment and recognition

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in policy formulation of the dramatic changes that have recently transformed the developing livestock sector. With these developments and these provisos livestock will continue to feed future generations with the type and quality of food they need without overuse of other natural resources. Chapter 9 provides a largely qualitative overview of the role of domestic livestock in assuring food security together with a series of detailed quantitative case studies. Chapter 10 provides an overview on the evolution of global market demand for organic meat and factors affecting it. Future prospects for development of organic meat consumptions and possible problems concerning trading are analysed. The price that people are willing to pay is the major determinant of the market share of organic meat. Although intent to purchase depends upon the interactions of quality attributes such as appearance and colour, it has been hypothesized a consistent effect of organic labelling as compared to some sensory characteristics on the price offered by consumers for organic meat. Previous experiments have shown that expectations induced by the information can affect the quality perception. Therefore, consumers’ behaviour and attitudes toward organic meat are tested through the study of the effect of information about organic farming practices on product liking and consumer willingness to buy organic beef and pay the extra production costs. Our results indicate that this information, if given to the consumers, can be a major determinant of beef liking, thus providing a potential tool for meat differentiation to traditional farms where husbandry is based on extensive rearing systems and high animal welfare standards. Accordingly, consumers are prepared to spend more for organic beef, thus indicating that reliable information about the organic farming system may markedly increase consumer willingness to pay. Background: China is undergoing rapid nutrition transition towards higher intake of animal foods. The prevalence of anemia has decreased during the past decade but still remains high. The objective of Chapter 11 is to describe the sociodemographic differences in meat consumption and the association between different types of meat consumption and anemia in Chinese adults. Method: Data from a national household nutrition survey carried out in year 2002 including 2849 adults aged ≥ 20 years from Jiangsu province were used. Dietary information was assessed by three days weighed food records. Fasting blood specimens were collected for assessment of hemoglobin and serum ferritin. Results: The overall prevalence of anemia was 25.5%. Across quartiles of serum ferritin (1-4), the prevalences of anemia were: 23.9, 15.5, 17.9, and 15.8% in men; 38.6, 29.5, 27.1, and 30.3% in women. The mean intake of total meat was 101.9 g/day in men and 74.9g/day in women, Pork and poultry were the main sources of meat. Meat consumption varied according to socioeconomic status. Higher educational level, higher income and urban residency were associated with higher intake of meat. A positive association between beef and lamb consumption and hemoglobin level was observed. Pork, poultry, and total meat consumption was negatively associated with hemoglobin level and positively associated with prevalence of anemia. In multivariate analysis, the prevalence ratios (OR) for anemia across pork intake quartiles were: 1, 1.20(0.99-1.46), 1.39(1.14-1.70), and 1.37(1.09-1.73) (p for trend: <0.001). Total meat consumption was positively related to serum ferritin level adjusting for age and gender. Conclusion: Socioeconomic conditions were positively associated with meat consumption. Pork, poultry and total meat consumption was significantly and positively associated with the risk of anemia among Chinese. Further research is needed to interpret this association.

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In the first Short Communication, the four main groups of gas chromatographyolfactometric (GC/O) methods were presented, i.e. dilution analysis methods, detection frequency methods, time-intensity methods and posterior intensity methods. A compilation of the applications of gas chromatography-olfactometry in seafood odour analysis was carried out. The interpretations of the results obtained with these different methods applied to seafood matrices allowed to consider olfactometry as an efficient tool for the monitoring of seafood quality from organoleptic, sanitary and nutritional point of views. The quantity and the nature of odorant volatile compounds determined by this analytical method can provide reliable indicators on the freshness and the stability of such reactive food. A method for the determination of total and inorganic arsenic in seafood by inductively coupled plasma atomic emission spectrometry with continuous hydride generation is described in the second Short Communication.. Several analytical parameters have been investigated and optimised (recovery, precision, accuracy and limit of detection). A practical detection limit of 3.6 µg/kg fresh weight for As is reached. The precision of the method, expressed as relative standard deviation (RSD), ranges from 2.7 to 3.7 % and the recovery percentage ranges from 98.4 to 101.8 %. Various procedures for the mineralisation or extraction of inorganic arsenic were tested; acid digestion with nitric acid at 180°C was retained as a best compromise. The interference of some arsenical compounds, present in seafood, was studied as arsenobetaine (present at more than 90% of total As), monomethylarsonic acid and dimethylarsinic acid. Real samples of seafood from local market and reference materials of seafood were analysed and the obtained values agree well with that given in the literature by other authors. Despite the high levels of total arsenic found, the inorganic arsenic, highly toxic form, was present in percentages lower than 10% of total As, thus, there is no direct hazard for human health due to the consumption of these fish products.

RESEARCH AND REVIEW STUDIES

In: Food Science and Security Editors: L. Amsel and L. Hirsch, pp. 3-71

ISBN: 978-1-60692-977-3 © 2009 Nova Science Publishers, Inc.

Chapter 1

THE CONTRIBUTION OF PLANT BIOTECHNOLOGY TO FOOD SECURITY IN THE 21ST CENTURY Koreen Ramessar1, Shaista Naqvi1, Svetlana Dashevskaya1, Ariadna Peremarti1, Dawei Yuan1, Sonia Gomez-Galera1, Sol Maiam Rivera Velez1, Gemma Farre1, Maite Sabalza1, Bruna Miralpeix1, Richard Twyman2, Changfu Zhu1, Ludovic Bassie1, Teresa Capell1 and Paul Christou1,3 1 2

Universitat de Lleida, Av. Alcalde Rovira Roure, 191, E-25198 LLEIDA, Spain Department of Biology, University of York, Heslington, York YO10 5DD, UK 3 Institucio Catalana de Recerca i Estudis Avancats

I. Overview of Food Security and Poverty Food security is taken for granted in the industrialized world, where stable political and social structures ensure that everyone has access to safe and nutritious food sufficient to maintain a healthy and active lifestyle. The picture in the developing world could not be more different: almost one billion people are chronically undernourished, regularly consuming less than 2000 calories per day (FAO, 2006). There are also further two billion people who, despite having access to an adequate source of calories, nevertheless lack essential nutrients. This means that up to half the world’s population at any one time may suffer from malnutrition (Graham et al., 2001). One of the persistent myths concerning food insecurity is that it is caused by a shortfall in food production. Quite to the contrary, there is plenty of food to go around. The reasons for food insecurity are complex, but one of the main factors is poverty, reflecting the fact that more than one billion people live on less than $US1 per day and another two billion are only marginally better off (World Bank, 2000). The world’s poorest people tend to be rural farmers in developing countries, depending entirely on small-scale agriculture for their own subsistence and to make their livings. In these countries, 70% or more of the population lives in rural areas and depends on subsistence agriculture, compared to the US, for example, where less than 1% of the population are farmers. Because of limited purchasing power, the

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poorest farmers generally cannot irrigate their crops nor buy fertilizers, herbicides and pesticides. This leads to soil exhaustion and falling yields, and the crops become susceptible to pests, diseases and natural disasters such as drought. Many poor farmers are eventually forced to abandon their land and move to cities, adding to the growing problem of urban poverty and hunger (DFID, 2002). It is now thought that half the world’s population lives in cities, so any disruption to agriculture could precipitate an urban food crisis in a matter of days. Any long-term strategy to address food insecurity in the developing world must tackle the underlying problem of poverty by increasing the level of rural employment-based income through increased agricultural productivity (Christou and Twyman, 2004). Given projected increases in the world’s population, increases in the cost of oil, falling reserves of fresh water and increased urbanization, the only solution to the above problem is to increase the yields of major food crops, particularly cereal grains, using currently available land and less water, and to diversify the uses to which crops are put to facilitate the creation of wealth. A variety of approaches can be envisaged, including the efficient use of organic and inorganic fertilizers, irrigation strategies, soil and water conservation, pest and disease management and the production of improved plant varieties with higher yields or novel products. Biotechnology provides a range of tools that can be used to improve agriculture in the developing world and we consider some of the biotechnology-based approaches to improving food security in this article.

II. How Can Biotechnology Help? 1. Conventional and Biotechnological Approaches to Micronutrient Fortification The fortification of food with vitamins and minerals provides an excellent basis to compare conventional and biotechnology-based approaches to improve nutrition. Fortification has been highly successful in the developed world, and has significantly reduced the incidence of deficiency diseases. Many processed foods, including bread, packaged cereals, milk and soft drinks, are fortified with vitamins and minerals so the average diet contains micronutrients well in excess of requirements. In developing countries, the less robust and less reliable food distribution infrastructure, poor governance and the lack of funding renders such programs inefficient and unsustainable, especially when trying to reach remote areas (Darton-Hill and Nalubola, 2002; Timmer, 2003). Only iodine fortification and, to a lesser extent, vitamin A supplementation have met some degree of success, but other programs have failed, especially at the local level when dealing with small and remote villages (Underwood, 2003). An alternative approach to adding nutrients directly to food products is to enhance the incorporation of nutrients in the field, a process known as biofortification. There are both conventional and biotechnological approaches to biofortification, each of which has met with some success (Zhu et al., 2007). Conceptually the simplest is biofortification through mineral fertilization, which involves the application of inorganic salts of the target minerals directly to the land. This can help to improve the mineral content of plants when the deficiency arises because the soil is exhausted; the plant itself is not modified in any way. The major drawback

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5

of this approach is the expense (it has been successful in developed countries, with particularly good results for selenium fortification in Finland and New Zealand, but the cost of mineral fertilizers is often too much for poor rural farmers in Africa and Asia) and that it can only be used for mineral – not vitamin – enhancement (Lyons et al., 2004). The minerals also have to be mobile in the soil (i.e. they must exist in a form that can readily be absorbed by plant roots), so the best candidates are selenium, iodine and zinc. Other important minerals such as iron have poor mobility and fertilization strategies are unsuccessful. To address the full spectrum of micronutrients obtained from plants, another widely considered approach is conventional breeding. Conventional in this context means without the introduction of foreign genes by transgenesis (see below), but it does allow for the use of accelerated mutation techniques and forced hybridization methods to modify endogenous genes and introgress genes from distant relatives; it also allows the use of molecular markers to facilitate the selection of productive alleles. Thus, in some cases, the boundary between a ‘conventional’ crop and a transgenic crop carrying a ‘foreign’ gene is blurred. Limitations of conventional breeding include the dependence on a compatible gene pool (i.e. the collection of species with which the crop can be forced to interbreed) and on the degree of available natural variation. If sufficient natural variation in mineral or vitamin levels can be identified within a breeding population, then selection strategies can be used to produce lines that have elevated nutrient levels. Even so, programs to exploit such variation have met with only marginal success. For example, a recent attempt to use variation at the lycE locus in maize to increase β-carotene levels succeeded in raising levels approximately five-fold (Harjes et al., 2008), compared to transgenic strategies in rice and maize that achieved greater than 100-fold enhancement (see below). Further disadvantages include the long time required to generate nutritionally improved lines, especially if a trait has to be introgressed into an elite local breeding line, and the fact that such approaches would increase exponentially in complexity if fortification for multiple vitamins and minerals were required (White and Broadley, 2005). No ‘conventional’ biofortified crops have yet reached the market. Biofortification at source can be accelerated by transferring genes directly into elite breeding lines by transgenesis. Most of this chapter is dedicated to transgenic approaches because these have produced the quickest and most impressive results and have the greatest potential impact. Transgenic varieties of numerous crops have been developed that are enhanced for primary nutrients (carbohydrates, proteins, fats) as well as micronutrients (vitamins and minerals). Compared to conventional breeding and mineral fertilizer use, transgenesis has the advantages of speed, direct modification of breeding lines, simplicity, the potential for multiple simultaneous biofortification and unrestricted access to genetic diversity (i.e. genes from very distant species including bacteria and animals, and even completely synthetic or artificially modified genes that do not exist in nature) (Zhu et al., 2007).

2. Molecular/Marker-Assisted Breeding Before leaving the subject of conventional breeding all together, it is worth pointing out once again that even conventional breeding strategies are often dependent on molecular biology and recombinant DNA technology. Plant improvement requires patience; it can take 12-15 years from the first cross to produce a registered variety. Breeding techniques rely on the production and use of genetic variation followed by selection of the most suitable

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phenotypes, and this is a slow process at the best of times. Marker-assisted selection (MAS) involves the use of molecular markers, i.e. polymorphic DNA sequences that can be detected easily and rapidly, to assist the selection of desirable lines without having to wait for the morphological or nutritional phenotype to manifest (Collard and MacKill, 2008; Xu and Crouch, 2008; Dwivedi et al., 2008). It is a shortcut that allows selection to be accelerated while improving the precision and efficiency of the selection process. Some molecular markers can even be used to distinguish homozygous and heterozygous individuals in a breeding population. The usefulness of molecular markers is particularly evident when dealing with quantitative traits, since these tend to reflect the activities of multiple genes contributing to the same phenotype. Breeding without markers can only identify the most productive combinations of alleles by chance, whereas MAS allows particular alleles, identified by the linked markers, to be stacked in the same line without any need for phenotypic analysis. Many of the characteristics of plants that affect their agronomic performance and nutritional properties are quantitative traits, e.g. height, fiber content, disease resistance, yield and nutrient levels. Due to the complex genetics, such traits have traditionally been difficult to dissect and modify, but MAS has led to the construction of saturated linkage maps for many crops and has made it possible to map the quantitative trait loci (QTLs) that control them. Understanding how QTLs affect crop performance under different environmental conditions and in different genetic backgrounds can facilitate the development of enhanced crop varieties. Consequently, the mapping of QTLs for agronomic traits is an important component of ‘conventional’ nutritional improvement programs (Dwivedi et al., 2008).

3. Accelerated Mutagenesis Natural variation in nutrient levels is the cornerstone of selective breeding, and natural variation arises through the occurrence of spontaneous mutations in genes controlling nutrient accumulation or synthesis. Therefore, one way to accelerate the development of ‘conventional’ nutritionally-enhanced crops is to accelerate the mutation rate by subjecting plants to deliberate mutagenesis. This can be achieved by irradiating seeds with X-rays or exposing them to chemical mutagens such as ethyl methanesulfonate (EMS), each of which causes random damage to DNA and usually generates point mutations. Any phenotypic effects of such mutations are observed in subsequent generations, depending on whether the effect is dominant or recessive, and must be mapped to identify the affected gene. An advanced method for identifying such point mutations is TILLING (Targeting Induced Local Lesions IN Genomes), a high-throughput method based on conformational electrophoresis for the detection of point mutations in large populations of plants (Comai and Henikoff, 2006). TILLING can identify genetic variation in elite germplasm without the need to acquire variation from exotic cultivars, thus avoiding the introduction of agriculturally undesirable traits. Once a TILLING library is set up, it can be used for the analysis of many different gene targets. TILLING is a powerful reverse genetics approach that has the unique advantage of allowing the generation of an allelic series for any target gene, including essential genes (Slade and Knauff, 2005). If a variety developed by TILLING has commercial potential, it is not subject to the same regulatory approval requirements as transgenic crops.

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Even with methods such as TILLING making the process of mapping novel mutations easier, it is still preferable to be able to identify mutated genes with a unique DNA signature. This is the benefit of mutagenesis using unique DNA tags such as transposons or T-DNA, the former endogenous to plants, the latter introduced artificially, but both achieving the same aim which is insertional mutagenesis. In this approach, randomly integrating DNA sequences disrupt genes and cause a loss of function. The identification of desirable mutant phenotypes is followed by DNA analysis using the insertional mutagen sequence as a probe, or as the basis for PCR primer design, allowing flanking gene sequences to be identified. Another useful approach is RNA interference (RNAi), in which short double stranded RNA molecules are used to induce a defense pathway that destroys homologous mRNAs and leads to potent post-transcriptional gene silencing (Lawrence and Pikaard, 2003; Mansor et al., 2006). RNAi has the advantage of being able to knock down the expression of multiple related genes with one construct if they share a homologous core sequence, whereas conventional mutagenesis/TILLING and insertional mutagenesis only affect single genes. The ability to silence several genes simultaneously using RNAi can speed up the creation of novel nutritional traits in crop plants.

4. Genetic Engineering Genetic Engineering (GE) involves the transfer of genes directly into the plant genome, a process known as transformation, which is an indispensable tool for the experimental investigation of gene function and for the improvement of plants either by enhancing existing traits or introducing new ones (Birch, 1997). It is now possible to stably introduce and express DNA in at least 200 different plant species. Many aspects of plant physiology and biochemistry that cannot be addressed easily by any other experimental means can be investigated by the analysis of gene function and regulation in transgenic plants. This offers an unprecedented opportunity to study the molecular basis of important processes that have been intractable to conventional analysis, including nutrition. The same techniques that are used to analyze the genetic basis of nutrition can also be used to enhance it by offering a rapid way to introduce such traits into elite varieties. The chosen approach depends predominantly on whether the plant synthesizes the nutritional compound de novo or obtains it from the environment. Organic molecules such as amino acids, fatty acids and vitamins are synthesized by the plant, and increasing nutritional value requires some form of metabolic engineering with the aim of increasing the amount of the desirable compound, decreasing the amount of a competitive compound (e.g. one that uses the same precursors as the desirable compound) or even extending an existing metabolic pathway to generate a product that is not usually made in that species (Capell and Christou, 2004). In contrast, mineral nutrients are obtained by the plant from the environment and mineral enhancement therefore involves strategies to increase uptake, transport and/or to increase accumulation in harvestable tissues (Welch and Graham, 2004). As discussed earlier, the advantages of genetic engineering over conventional breeding include the speed with which elite varieties can be improved, and the ability to transfer genes from species that cannot breed naturally with the host crop. However, recent developments have advanced the state of the art significantly, showing that transgenic strategies far outperform conventional alternatives. For example, the simultaneous transfer of multiple

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genes has made it possible not only to enhance and extend metabolic pathways, but also to develop a combinatorial transformation system that allows libraries of transgenic metabolic variants to be generated, and novel metabolic traits to be selected for by screening for appropriate phenotypes (Zhu et al., 2008). When applied to the carotenoid pathway in maize, this system generated numerous phenotypes comprising different carotenoid profiles, not only providing a series of nutritionally enhanced variants but also allowing the basis of carotenoid metabolism in maize to be investigated (Zhu et al., 2008). One of the most ambitious recent reports describes the simultaneous modification of three metabolic pathways in maize, generating a novel variety with elevated levels of three key vitamins (A, C and B9) (Naqvi et al., 2009). Such achievements would be next to impossible with conventional breeding, since tens or even hundreds of QTLs would need to be monitored simultaneously.

III. The Targets of GE Technology 1. Improving Yields and Nutrition in Food/Feed Crops Food security can be addressed directly through the genetic engineering of food and feed crops to make them more agronomically robust (resistant to pests and diseases, and tolerant of harsh environments), to increase the yield of harvestable products (e.g. grain, fruit) and to enhance the nutritional properties of the edible parts (e.g. higher content of vitamins and minerals). The ‘GE revolution’ which promises to achieve these improvements in quantity and quality is not the first revolution in agricultural productivity – this was the Green Revolution of the latter half of the 20th century, which significantly increased food production, albeit at the expense of high inputs of fertilizers, and irrigation. During this period, high yielding varieties of rice and wheat were widely adopted by Asian farmers, but the success of these varieties was mainly limited to irrigated and rain-fed areas, leaving drier regions no better off. Over time, excessive fertilizer use caused soil degradation, the over-use of pesticides affected human health and resulted in the emergence of resistant pests, and heavy irrigation depleted groundwater and increased soil salinity (Ho, 2008). The GE revolution promises to match the aims of the Green Revolution without causing such serious environmental impacts. High-quality agricultural land is becoming scarce, particularly in developing countries where farmers struggle with productivity because of poor soil quality and the unfavorable climate. The ability to produce enough food for a hungry population is severely limited when key agricultural inputs such as water, fertilizers and pesticides are unaffordable. As water shortages become more acute and soil conditions deteriorate, conventional breeding alone is insufficient to address food insecurity. The potential benefits of GE crops include the lower production costs, higher yields and the ability to use low-quality land such as the marginal lands in developing countries. The better yields will allow farmers to take the step away from subsistence agriculture and begin to use their food/feed as cash crops. The higher quality will command better prices at local markets, and this will eventually provide the farmers with the opportunity to get involved in further income-generating activities. Since the commercialization of the FlavrSavr™ tomato, in 1994, 12 million farmers in 23 countries have grown GE food/feed crops, increasing their income by more than $US 34 billion (James, 2008). To date, more than 1.7 billion acres have been used to grow GE crops

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(James, 2008). The US, Argentina, Canada, Brazil, Paraguay, China and South Africa are the major GE-growing countries. It has been predicted that the full adoption of GE crops globally could increase the world’s income by $US 210 billion per year within the next decade (Abdalla et al., 2003).

2. Non-subsistence Commodity Crops In agriculture, a cash crop is grown for money instead of subsistence, and where a rural farmer’s productivity has advanced enough to allow the sowing of cash crops, a whole new vista of opportunities becomes available in the form of commodities such as cocoa, coffee, tea, fiber crops (cotton, flax), rubber trees, cut flowers and tobacco. Developing countries depend on the export of a few agricultural commodities. Therefore, fluctuation in world prices can have a significant impact on household income (Bourguignon, 2004). Global price fluctuations affect small farmers most severely, since they often concentrate on the production of a single cash crop. When a country’s economy depends on a single crop, price fluctuation or crop failure can have disastrous and systemic effects. For example, Ghana depended on the export of cocoa for many years, with severe economic repercussions when the price of cocoa fell. Since then, the government has promoted diversification of the export market and has also expanded the amount of land dedicated to cash crop production and strengthened the country’s infrastructure, making it more competitive (Ghana Investment Promotion Centre; http://www.gipc.org.gh/home). Although Ghana’s cash crop production and export have been successful, critics believe that food production for domestic consumption should take precedence because the most fertile lands are allocated to cash crops whereas food production, especially subsistence cultivation, is marginalized. Similar situations have arisen in other countries. An example of how biotechnology could impact on cash crops and their commodities is the modification of flower color. Although breeders have been creating new flower color varieties for centuries using conventional processes, biotechnology allows novel colors to be produced that are outside nature’s repertoire. The blue rose is a spectacular case, created through a joint venture involving a major Japanese breeder (Suntory Ltd) and the Australian biotechnology company Florigene Ltd (http://www.csiro.au/resources/bluerose.html). The creation of a new rose variety is important in the global cut flower market, and developing countries now have the opportunity to compete in this trade. Traditional non-subsistence commodity crops such as tobacco, sugar, cotton and rubber take advantage of abundant natural products in those plants. GE technology has allowed the development of a new generation of cash crops, where standard crops are modified to produce value-added products. These fall into three somewhat overlapping categories – small molecules (secondary products, so-called because they are the products of secondary metabolic pathways, i.e. those not essential for survival), recombinant proteins and polymers. The categories are overlapping because some valuable small molecules can polymerize to form useful products such as rubbers and plastics, and some proteins can also form polymers (e.g. plants making collagen, or spider silk). Plants engineered to produce value added products are sometimes food crops where the additional product is nutritionally beneficial (e.g. rice and maize with elevated carotene levels, potato with higher protein levels) and sometimes commodity crops where the product is destined to be isolated and purified (e.g.

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plants producing pharmaceutical proteins, small-molecule drugs or industrial enzymes). In many cases the products are immensely valuable and difficult and/or expensive to produce using other methods, providing a novel and potentially more generous source of additional income than traditional commodities.

3. Biofuels Biofuels are solid or liquid fuels derived directly from plants, as opposed to fossil fuels that originate from finite deposits of ancient organic material. The two most important biofuels are biodiesel and bioethanol, the former comprising methyl esters of fatty acids (usually derived from oilcrops such as oil palm, rapeseed or soybean), the latter produced by fermentation of carbohydrates, predominantly from sugar cane and maize and, to a lesser degree, from wheat, sugar beet and cassava (Sánchez and Cardona, 2008). Sugar from Brazil and maize from the US dominate bioethanol production, accounting for approximately 80% of global supply. China, the European Union (EU) and India are other significant bioethanol producers. In energy terms, ethanol accounts for almost 90% of the biofuel currently used in the world. The major producers of biodiesel are Germany and France, although biodiesel from oil palm grown in Southeast Asia is gaining an increasing share (FAO, 2008). First generation biofuel feedstock competes directly with food crops for land and resources, and therefore some believe it has a direct and negative impact on global food security. Second generation biofuels are derived from cultivated lignocellulosic crops or straw waste, and these could coexist with food crops (or even constitute the waste material from food crops) without harm. Unfortunately, second-generation biofuel technology is not yet commercially viable (Gressel, 2008). Third generation biofuels include those derived from algae, and these have probably the greatest likelihood of replacing fossil fuels without compromising food and feed production (Chisti, 2008). Ideally, biofuel crops would have a positive impact on food security by providing additional sources of income for rural farmers, protecting the environment from damage caused by petrodiesel products, and increasing energy security and sustainability. However, the potential negative impact of biofuels, particularly the direct effects on food/feed agriculture and land use, must be evaluated carefully. Many of the most popular species used for biofuel production exacerbate two of the major causes of biodiversity loss on the planet: clearing and conversion of yet more natural areas for monoculture, and invasion by non-native species (Chapin et al., 2000). The Global Invasive Species Programme (GISP) reports that some of the most commonly recommended species for biofuel production, particularly for biodiesel, are also major invasive alien species in many parts of the world (GISP, 2008). A major issue with the use of first generation biofuels is the direct competition between energy needs for industrialized countries and severe increases in food prices in the developing world (Zilberman and Rajagopal, 2007). In order to mitigate the potential negative impacts of biofuels, it will be important to reduce competition with food supplies by focusing on the use of organic wastes and residues such as cereal straw and third generation biofuels. Biotechnology will play a key role in this context, for example by developing efficient microorganisms and enzymes to convert the hemi-celluloses in straw and stover to sugars, replacing the use of edible parts of crop plants. It will also be necessary to carry out a science-

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based risk-benefit analysis to avoid the introduction of invasive species for biofuel projects and to select native and/or non-native species that can proliferate on marginal lands that pose the lowest risks to biodiversity.

IV. GE Strategies to Improve Food Security 1. Protecting Crops from Pests and Diseases Weeds Weeds are plants that compete with crops for resources, in some cases by parasitism. The cost of weeds, measured in terms of reduced yields, the application of herbicides, and the mechanical and manual labor required to control them, is probably the largest single input into agriculture. For this reason, weed control has been identified as the primary target for first generation GE technology and most of the transgenic plants grown in the world today have been modified for herbicide tolerance, allowing the use of safe, broad-spectrum herbicides such as glyphosateTM and bastaTM. Herbicide tolerant crops provide increased management flexibility that comes from a combination of the ease of use associated with broad-spectrum, post-emergent herbicides and the larger time window for spraying. Post-emergent herbicide application may ‘knock-back’ conventional crops but this is unlikely in GE crops. The deployment of herbicide tolerant crops also allows the adoption of no/reduced tillage practices, which can save time and equipment use during the harvest, and there is little risk of damage caused by soilincorporated residual herbicides in follow-on crops. Because GE technology in the West is driven predominantly by the potential for commercial gain, research has focused on the weed problems facing farmers in the industrialized nations. There has been little interest in developing crops with resistance to the weed species that plague subsistence crops in the developing world, even though this would have an immediate impact on food security. For example, Striga is a genus of parasitic flowering plants that infests cereal and legume crops throughout Africa. It is particularly difficult to control in maize crops and causes yield losses estimated at $US 7 billion every year (Berner et al., 1995). Genes allowing the selective control of this weed by herbicide application have been identified (Joel et al., 1995) but thus far no transgenic varieties have been produced (Gressel, 2002). Progress towards the selective control of Striga in Africa has been made solely through mutation and conventional breeding for imazapyr resistance in maize (Kanampiu et al., 2001; 2002).

Insect Pests Many of our crop plants are also food for insect pests, and devastating losses occur throughout the world due to pest infestations either in the field or in stored products such as cereal and legume grains. In the developing world, about half of all crop production is thought to be lost to insects, 15% of these losses occurring due to post-harvest consumption and spoilage (Christou et al., 2006). Insects not only cause direct yield losses by damaging

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and consuming plants, they also act as vectors for many viral diseases and the damage they inflict encourages microbial infections. In the industrialized world, pest control is heavily dependent on chemical inputs, which are expensive and damaging to the environment. The chemicals are non-selective, killing harmless and beneficial insects as well as pests, and accumulating in water and soil. In the developing world, many farmers are too poor to afford pesticides and are left at the mercy of nature. In any case, chemical control measures are ineffective against some of the worst pests, such as the rice brown planthopper (Nilaparvata lugens) which feeds by sucking sap from the phloem. Another major problem is that insects may evolve resistance to chemical insecticides (Schuler et al., 1998). Because of these difficulties, insect pests represent another primary target of GE technology (Estruch et al., 1997; Schuler et al., 1998). The genetic engineering of plants to express insect-resistance genes offers the potential to overcome all of the shortcomings listed above, since genes that show exquisite specificity towards particular pest species have been isolated from bacteria and other sources, thus minimizing the threat towards non-target organisms. Furthermore, the expression of such proteins within plants allows effective control of insects that feed or shelter within the plant, and the degree of protection is not influenced by the weather, as is the case for topical chemical pesticide applications. Finally, the likelihood of insects becoming resistant to the transgenic plants can be reduced by a number of strategies, such as pyramiding resistance genes affecting different receptors in the target insect, conditional expression and the provision of ‘safe-havens’ or refuges, to reduce selection pressure. Several different transgenic approaches are discussed below. Although many microbial species are potential sources of insecticidal genes, the most popular source by far is Bacillus thuringiensis (Peferoen et al., 1997; de Maagd et al., 1999). This is a spore-forming bacterium that produces insecticidal toxins during the sporulation process. Transgenic plants (Bt plants) that express B. thuringiensis insecticidal δ-endotoxins (also known as Cry proteins) were first commercialized in 1996. Nearly 40% of all the commercially grown transgenic plants in the world contain synthetic insecticidal toxin genes from B. thuringiensis (James, 2008). More than 150 Cry proteins have been identified in various strains of B. thuringiensis and B. cereus, each functioning in essentially the same manner but displaying specificity to a narrow taxonomic group of insect pests. The functional similarity among the Cry proteins reflects their highly conserved tertiary structure and the presence of five blocks of conserved sequences. Otherwise, the protein sequences are highly diverse, and this accounts for the different ranges of target insects. All Cry proteins are expressed as inert protoxins that are activated by proteinases in the highly alkaline environment of the insect gut. This provides an important safety barrier since the environment in which the toxins are activated is unique to insects, and thus it is safe for other animals, and humans, to consume plants expressing Bt toxins. Once activated, the toxins interact with receptors on the midgut epithelium cells creating pores in the plasma membrane by disrupting osmotic balance. This results in paralysis and the ultimate death of the insect. Over time and according to exposure levels, insects can evolve resistance to Bt toxins. Three resistance mechanisms have been identified, all of which result from single gene mutations. The predominant and most characterized resistance mechanism is mutation of the toxin receptors on midgut epithelial cells, resulting in a lower binding affinity or a reduction in the number of available binding sites. The second resistance mechanism involves a

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mutation in one of the components of the proteolytic cascade that processes the Cry toxin (Brookes, 2008). Such mutations may reduce protoxin solubilization, inhibit toxin activation or introduce new proteolytic pathways that degrade the toxin. A third mechanism has been proposed in which damaged midgut cells are rapidly replaced, preventing septicemia. A number of recent developments in the field of Bt technology have helped to introduce strategies that reduce the risk of resistance evolving in insect pest populations, and have also helped to broaden the range of insect pests that can be targeted by Bt genes. Natural Bt genes do not affect sap-sucking pests, which are among the most prevalent field pests especially in rice and until now the only solution has been to stack Bt genes with other insecticidal proteins, such as lectins and enzyme inhibitors (anti-feedants). A number of such strategies are outlined in TABLE 1, and are discussed in more detail below. One way to reduce the likelihood of resistance is to introduce multiple Bt genes into one plant line. This can achieve two different aims – it can target multiple pests each of which is sensitive to a different Bt gene (this is known as resistance stacking, and suffers from the same challenges as discussed above, i.e. each target pest can evolve resistance to its cognate toxin) or it can target different receptors in the same pest (this is known as resistance pyramiding, and makes resistance much less likely to evolve because simultaneous mutations would be required in different genes in the target pest). When used in combination with ‘refuges’, i.e. set-aside plots of non-Bt plants that allow the survival of non-resistant insects, and therefore reduce selection pressure, pyramiding resistance can be a highly effective strategy for sustainable pest management (Ferry et al., 2006). A distinct approach is to recombine Bt toxins in order to make fusion proteins with combined, augmented or novel properties. Even ‘wild type’ Bt toxins need to be mutated to work in plants, since the codon preference and wild type regulatory sequences in bacteria combine to generate unstable and virtually unreadable mRNAs in the context of a plant cell. Table 1. Novel approaches to increase the potency and range of Bt toxins in transgenic plants, and delay the evolution of resistance in target pests Novel Bt Toxins Binary toxins require two components for activity Single-chain Vip protein Modified reengineering of coding sequences Addition of heterologous binding domain

Examples

Target

Transgenic crop

Cry34/35

Corn rootworm

Maize (Zea mays)

Vip1/2

Corn rootworm

Under development

Vip3

Lepidopteran larvae

Under development

Cry3Bb1

Corn rootworm

Maize

Cry1Ac

Expanding target range

Maize, rice (Oryza sativa)

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Koreen Ramessar, Shaista Naqvi, Svetlana Dashevskaya et al. Table 2. The Use Of Fusion Bt Toxins with Combined or Novel Properties

Protein engineering Method Domain exchange in three-domain Cry toxins

Principle Combining domains from different proteins could generate active toxin with novel specificities

Mutagenesis of three-domain Cry toxins

Modification of Bt toxins by site-directed mutagenesis

Fusion proteins

A gene construct containing a single translationally fused coding sequence encoding one Cry protein and an additional binding partner

Examples

Target pests

Cry1Ab-Cry1C

Armyworm

Cry1Ca III

Spodoptera

Cry1Ba I-III Cry1IaII

Lepidopteran

Mutation Cry1Ab

Larvae of gypsy moth Coleopteran Rootworm

Mutation Cry3A MON863 Ribosomeinactivating protein ricin Gal-binding lectin domain fused C- Cry1Ac domain III

Larvae of stemborers and leaf armyworm

However, these new strategies take the concept of Bt mutagenesis a step further, aiming not to stabilize a single toxin and retain its activity in a heterologous setting, but to create completely novel toxins that do not exist in nature. Some of these principles, such as domain exchange, domain combination and the development of fusion toxins, are outlined in Table 2. In addition to the development of novel Bt strategies, additional non-Bt insecticidal proteins have been expressed in transgenic plants. One example is the common bean (Phaseolus vulgaris) α-amylase inhibitor gene, which was expressed in seeds of transgenic garden pea (Pisum sativum) and other grain legumes, using a strong seed-specific promoter (Shade et al., 1994). The transgenic seeds contained up to 3% of the foreign protein and were resistant to stored product pests, such as larvae of bruchid beetles, and field pests, such as larvae of the pea weevil Bruchus pisorum (Shade et al., 1994). For sap-sucking pests, researchers have used lectin genes such as gna from snowdrop (Galanthus nivalis). Transgenic rice plants expressing gna under constitutive or phloem-specific promoters were partially resistant to rice brown planthopper (Nilaparvata lugens) and other hemipteran pests. Similar results have been achieved by expressing a mannose-specific lectin from garlic (Allium sativum) leaves (ASA-L) in transgenic rice (Saha et al., 2006). Recent developments in GE pest-resistance have focused on the discovery and development of novel insecticidal proteins. For example, nematodes from the genus Heterorhabditis contain symbiotic bacteria that are widely used for small-scale biological control of insect pests. Toxins from the nematode gut are released into insect hosts, resulting in lethal septicemia. These bacterial toxins are useful candidates for the next generation of pest-resistant plants (Ffrench-Constant, 2007). Similarly, bacterial cholesterol oxidase activity is thought to promote membrane destabilization, and has an insecticidal activity comparable to Bt toxins in transgenic tobacco (Corbin et al., 2001). Avidin also has a significant impact

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on insect survival, and has been investigated as an insecticidal protein in transgenic plants (Kramer et al., 2000; Murray et al., 2002). As well as specific toxic proteins, GE strategies have also been used to modify plant secondary metabolism to enhance the synthesis of defensive compounds. For example, the introduction of genes encoding two cytochrome P450 oxidases and a UDP glycosyltransferase into sorghum enhances the synthesis of the cyanogenic glycoside dhurrin from tyrosine (Kristensen et al., 2005). The resulting transgenic plants synthesize hydrogen cyanide following tissue damage and therefore show enhanced resistance to the flea beetle Phyllotreta nemorum. A relatively new approach in the development of pest-resistant transgenic plants is the use of RNAi to deter coleopteran and lepidopteran pests (Tang and Galili, 2004; Gordon and Waterhouse, 2007; Mao et al., 2007; Baum et al., 2007). The observation that feeding insects with double stranded RNAs (dsRNA) can achieve potent gene silencing (Turner et al., 2006) has led to two recent articles in which transgenic plants producing dsRNAs are shown to exhibit partial resistance to insect pests. Transgenic maize producing dsRNA directed against V-type ATPase of corn rootworm showed suppression of mRNA in the insect and reduction in feeding damage compared to controls (Baum et al., 2007). Similarly, transgenic tobacco and Arabidopsis (Arabidopsis thaliana) expressing dsRNA directed against the gene for a detoxification enzyme (CYP6AE14) for gossypol in cotton bollworm caused the insect to become more sensitive to gossypol in the diet (Mao et al., 2007).

Microbial Diseases Many plants have evolved resistance mechanisms that protect them either generally from pathogens or against particular pathogen species. The transfer of genes from resistant to susceptible species is one GE strategy that can be used to accelerate conventional breeding for disease resistance (Salmeron and Vernooij, 1998; Stuvier and Custers, 2001). The plant cell has a number of general defense responses that occur in the presence of any pathogen. One of the first responses is a burst of reactive oxygen species, resulting in the activation of signal transduction pathways that lead to programmed cell death (Lamb and Dixon, 1997). This is known as the hypersensitive response and prevents the spread of infection by killing cells at the infection site. Metabolic changes in adjacent tissues then occur to generate physical and chemical barriers to further infection. The cell wall is strengthened, antimicrobial molecules called phytoallexins are synthesized, defense peptides such as defensins and thionins are expressed and there may also be synthesis of pathogenesis-related proteins (PR-proteins) such as hydrolytic enzymes that break down bacterial and fungal cell walls (Bowles, 1990; Kuc, 1995). Plant cells surrounding the infection site also produce longrange signals that induce the synthesis of PR-proteins throughout the plant. This is known as systemic-acquired resistance and protects the plant from subsequent attacks by the same pathogen and others (Sticher et al., 1997). As well as these general responses, plants may synthesize specific resistance proteins (Rproteins) that interact with particular components of a given pathogen. These components are known as avirulence (avr) factors, and they are neutralized by contact with the R-proteins (Bent, 1996).

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Koreen Ramessar, Shaista Naqvi, Svetlana Dashevskaya et al. Table 3. Strategies for tackling microbial diseases in transgenic plants

Approach Constitutive expression

Examples Pyramiding R genes

PR genes

Antimicrobial peptides

Local expression

Pre-formed barriers Master switch genes

Elicitor or Avr genes

Toxic genes

RNAi

Silencing of pathogen essential genes

Gene knockouts

Knockouts or mutations of negative regulators of defense

Advantages Could build more durable resistance; R genes successfully used by breeders Many reports of increased resistance; do not activate the whole defense response Could increase durability by ‘stacking’; can target the pathogen by linking to antibodies Could lead to durable resistance Activate banks of genes; might confer resistance without activating all defense responses Trigger to activate successful defense; could be enough to change susceptibility to resistance Could stop pathogen growth and lead to resistance Could potentially target all pathogens Targets specific pathogens Unlikely to have any fitness penalty Does not activate defenses Mutations in genes such as Mlo could provide durable resistance

Disadvantages Requires knowledge of specificity; might come with a fitness penalty; overexpression might activate defense genes Might only be effective against a few pathogens Overexpression might reduce yield and/or fitness Requires a range of active peptides; might come with a fitness penalty Altering the cell wall might reduce size and yield Requires a pathogen-inducible promoter

Requires a pathogen-inducible promoter

Requires a pathogen-inducible promoter; public perception of ‘toxic’ gene product

Need to identify negative regulators of defense; not localized; might come with a fitness penalty

In addition to exploiting natural plant defenses, foreign genes can be expressed in plants to prevent or delay pathogen infections. Two major strategies are utilized: pathogen-derived resistance, in which a pathogen gene is expressed in the protected plant, and pathogentargeted resistance, in which a foreign gene is used to interfere with pathogen infection or reproduction. Some strategies for tackling microbial diseases in plants are outlined in Table 3.

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2. Protecting Crops from Abiotic Stress and Maximizing Land Use After pests and diseases, harsh environmental conditions (abiotic stresses) are the next most important constraint on crop productivity. The most significant abiotic factors affecting food production are drought, salinity, poor soil quality, adverse temperatures (heat or cold) and flooding. The development of crops with an inbuilt capacity to withstand these effects could help to stabilize crop production and hence significantly contribute to food security (Holmberg and Bulow, 1998; Zhang et al. 2000). Furthermore, since only 35% of the world’s potential arable land is currently used, the modification of plants to prosper in marginal environments could help to expand agricultural production to ensure continuing food security in the coming decades. The consequences of abiotic stress are reduced growth, abnormal development and ultimately death. However, many plants show a degree of tolerance to certain types of stress, and in each case that has been studied it has been shown that the stress induces a cascade of gene expression culminating in a physiological response that counters the stress and allows near normal growth. The identification of the genes involved in such responses, and their introduction into susceptible plants, can transfer stress resistance traits to crops making them more productive in harsh environments. Stress response genes can be placed in two general categories: those encoding specific enzymatic or structural proteins involved in the direct response to stress (e.g. enzymes for the biosynthesis of osmoprotectants, detoxifying enzymes, and proteins involved in the maintenance of homeostasis), and those involved in the regulation of stress pathways (receptors, intracellular signaling molecules and transcription factors) (Shinozaki and Yamagushi-Shinosaki, 2000). The latter group may be more useful as targets for GE technology because they, in turn, regulate a large number of protective genes (Bhatnagar-Mathur et al., 2007).

Direct Response – Detoxification Plant cells produce reactive oxygen species (ROS) such as oxygen free radicals and peroxide ions continuously as part of normal metabolism, but stress can lead to elevated ROS levels and this causes damage to the structural components of cells as well as to DNA, resulting in the typical damage seen in stressed plants (stunting, chlorosis, wilting and ultimately death). Plants produce a number of enzymes whose specific function is to detoxify ROS, and these are induced by stress usually as a direct response to elevated ROS levels. Such enzymes include superoxide dismutase (SOD), ascorbate peroxidase (APX), peroxidase (POD), catalase (CAT), and enzymes that synthesize low molecular weight antioxidants such as ascorbic acid, glutathione and phenolic compounds. SOD is a very important enzyme that participates in the first step of ROS scavenging. SODs are classified according to their metal cofactor, which can be copper, zinc, manganese or iron. Plants generally contain Cu/ZnSOD in the cytosol, FeSOD and/or Cu/ZnSOD in the chloroplasts and MnSOD in mitochondria. The overexpression of SOD in stress-sensitive plants should improve their tolerance of a number of environmental stresses. For example, Nicotiana plumbaginifolia MnSOD and Arabidopsis FeSOD were overexpressed in the mitochondria and chloroplast of alfalfa and this resulted in ~20% more photosynthetic activity in transgenic plants compared to wild type plants under stress (Rubio et al., 2002). In another study, Cu/Zn SOD and APX were expressed in potato chloroplasts under the control

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of the oxidative-stress inducible promoter SWPA2, and the transgenic plants were four times more resistant to stress than control plants (Tang et al., 2006). Cu/Zn SOD and APX were also expressed in tall fescue plants, resulting in less chlorophyll degradation, low peroxide accumulation and low solute leakage compared to controls under oxidative stress (Lee et al., 2007). Similarly, tomato Cu/Zn SOD protected sugar beet from oxidative stress (Tertivanidis et al., 2004). Additional studies are compared in TABLE 4. It had been shown that SOD participates not only in combating oxidative stress but also drought and salt stress. Some plants known as halophytes thrive in saline environments and tend to have much higher levels of SOD than sensitive plants. For example, the mangrove Avicennia marina has a SOD activity 40 times higher than pea. Transgenic rice plants expressing cytosolic SOD1 from this species were much more tolerant towards oxidative, salt and drought stress than untransformed plants (Prashanth et al., 2007). The overexpression of SOD in the chloroplast provides more effective oxidative stress tolerance than expression in the cytosol, perhaps because chloroplasts and mitochondria are rich sources of ROS. Cytosolic SOD is more effective against drought and salt stress (Prashanth et al., 2007). Table 4. Overexpression of detoxifying genes in different food and feed plants Genes

Subcellular location Mitochondria, chloroplast

Promoter

Stress

Host plant

References

CaMV35S (constitutive)

Flooding

Alfalfa (Medicago sativa)

Rubio et al., 2002

Chloroplast

SWPA2 (inducible)

Oxidative, high temperature

Potato (Solanum tuberosum)

Tang et al., 2006

Chloroplast

SWPA2 (inducible)

Oxidative

Lee et al, 2007

Pea MnSOD

Chloroplast

Tomato (Lypersicon esculentum) Cu/ZnSOD Avicennia marina Cu/ZnSOD

Cytosol, chloroplast

SWPA2 (inducible) CaMV35S (constitutive)

Drought, oxidative Oxidative

Tall fescue (Festuca arundinacea) Rice (Oryza sativa) Sugar beet (Beta vulgaris)

Ubiquitin (constitutive)

Oxidative, salt, drought

Nicotiana plumbaginifolia MnSOD and Arabidopsis (Arabidopsis thaliana) FeSOD Cassava (Manihot esculenta) Cu/Zn SOD and pea (Pisum sativum) APX Cassava SOD and pea APX

Cytosol

Rice

Wang, 2004 Tertivanidi s et al., 2004 Prashanth et al., 2007

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Direct Response – Antiporters As well as inducing oxidative stress, salinity can directly affect plant survival by raising the intracellular concentration of sodium ions to the point where this interferes with normal physiological processes. Plants use three strategies to maintain Na+ at physiologically normal levels: sodium exclusion, sodium compartmentalization and sodium export, the last two being achieved by the activity of membrane channels known as Na+/H+ antiporters. There are two classes: plasma membrane antiporters, which export Na+ from the cytosol to the apoplast or external medium, and vacuolar antiporters, which transfer Na+ ions across the tonoplast into the vacuole, wherein the Na+ accumulates. Several reports describe the overexpression of antiporters in transgenic plants as a means to enhance tolerance to salinity. For example, transgenic canola plants expressing a vacuolar Na+/H+ antiporter from A. thanliana (AtNHX1) were able to grow normally and produce normal seeds in 200 mM NaCl, which would be sufficient to kill wild type plants after a few days (Zhang et al., 2001). The leaves and roots of transgenic plants contained higher levels of NaCl than normal, but the seeds were unaffected. Tall fescue plants transformed with the same gene germinated more successfully and grew more robustly than wild type plants under salt stress (Zhao et al., 2007).

Direct Response – Enzymes for the Synthesis of Osmoprotectants Osmoprotectants are small molecules such as amino acids, sugars and polyamines that increase the osmotic potential of the cytosol and prevent water moving out of cells by osmosis (Wang et al., 2003). Many halophytes synthesize large amounts of such molecules to protect them against dehydration, but many crops lack this ability (Bhatnagar et al., 2008). The transfer of genes for osmoprotectant synthesis can confer stress tolerance on susceptible crops, and some examples of this strategy are listed in TABLE 5. One of the most widely used osmoprotectants is glycinebetaine (GB) a quaternary amine that has been used to increase the salt tolerance of numerous crops (Sakamoto et al., 1998; 2002; Mohanty et al., 2002). The most efficient strategy for synthesizing GB in transgenic plants was described by Park et al. (2004; 2007). They generated tomato plants constitutively expressing a bacterial choline oxidase gene (codA), which converts choline to GB in a single step, in contrast to the typical eukaryotic GB pathway where two steps are required. The recombinant enzyme was targeted to the chloroplast, since this provides better tolerance of salinity than the same enzyme accumulating in the cytosol (Park et al., 2007). Polyamines (PAs) can also be used as osmoprotectants, and since these nitrogen-rich molecules also have nutritional value the manipulation of PA biosynthesis has multiple benefits in GE technology. Rice plants expressing arginine decarboxylase were more resistant to drought stress than wild type plants (Roy and Wu, 2001; Capell et al., 2004), and pear trees expressing S-adenosylmethionine decarboxylase were resistant to high salinity, high osmolarity and heavy metals (Wen et al., 2008). The ability of these plants to withstand high concentrations of metal ions including Cu2+ indicates they could also be developed for the recovery of metal ions (phytomining) or their removal from contaminated sites (phytoremediation). Sugars and sugar-alcohols such as mannitol and trehalose are used as osmoprotectants by a number of halophytes, and the transfer of the corresponding metabolic pathways to cereal crops has been successful in conferring stress tolerance in a number of cases (Penna, 2003). For example, transgenic wheat plants accumulating mannitol showed enhanced salt tolerance,

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Koreen Ramessar, Shaista Naqvi, Svetlana Dashevskaya et al.

although excessive mannitol synthesis was harmful (the plants were stunted and infertile) perhaps because the mannitol disrupted the normal osmolytic balance of the cells (Abebe et al., 2003). Trehalose synthesis in plants has been achieved by importing two enzyme activities from Escherichia coli – trehalose-6- phosphate synthase/phosphatase (TPS and TPP). These act consecutively in the E. coli pathway and generate trehalose from the precursors UDP-glucose and glucose-6-phosphate. In plants, the simplest way to implement this is to express a fusion gene (tpsp) encoding a protein with both enzyme activities (Garg et al., 2002; Jang et al., 2003). The overexpression of this gene in rice under the control of a stress-inducible or light-regulated promoter conferred a high degree of salt, drought and freezing tolerance concomitant with a modest increase in trehalose levels (Garg et al., 2002). Transgenic plants were also more tolerant of solarization, with a 5-10% higher photosynthesis rate than controls. Trehalose is less successful as an osmoprotectant in dicot plants (Stiller et al., 2008; Schluepmann et al., 2004; Goddijn et al., 1997). Finally, proline also protects plants against water deficit, as shown by Vendruscolo et al. (2007) who created transgenic wheat plants overexpressing pyrroline-5-carboxylate synthetase under the control of a stress-inducible promoter. Although proline levels correlated with stress tolerance, the underlying mechanism appears to involve protection against oxidative stress rather than osmotic adjustment. Table 5. Overexpression of enzymes that synthesize osmoprotectants to enhance stress tolerance Enzyme

Expression

Osmolyte accumulation Glycinebetaine

Stress tolerance Chilling, salinity, oxidative

Secondary effects Not reported

Salinity, chilling Salinity, heavy metals Drought

Poor growth

Choline oxidase

Constitutive CaMV 35S

Choline monooxygenase Spermidine synthase

Constitutive Ubi-1 Constitutive CaMV 35S

Glycinebetaine

Arginine decarboxylase Mannitol-1-Pdehydrogenase

Constitutive Ubi-1 Constitutive Ubi-1

Polyamines Mannitol

Trehalose-6-Psynthase/phosphatase

Stressinducible StDS2 Constitutive Ubi-1 Stressinducible ABA, tissuespecific rdcS Stressinducible AIPC-ABA

Trehalose

Pyrroline-5carboxylate synthetase

Polyamines

Not reported

Host plant

References

Tomato (Lycopersic on esculentum) Rice (Oryza sativa) Pear (Pyrus communis)

Park et al., 2004; 2007

Shirasawa et al., 2006 Wen et al., 2008

Not reported

Rice

Drought, salinity

Not reported

Trehalose

Drought

None

Trehalose

Drought, salinity, chilling Drought, salinity, chilling

None

Wheat (Triticum aestivum) Potato (Solanum tuberosum) Rice

None

Rice

Garg et al., 2002

Not reported

Wheat

Vendruscolo et al., 2007

Proline

Drought

Capell et al., 2004 Abede et al., 2003 Stiller et al., 2008 Jang et al., 2003

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Regulatory Genes – Signal Transduction All stress responses involve signal transduction and recent work to elucidate the underlying signaling pathways has identified many of the regulatory factors involved (TABLE 6). For example, many Ca2+-sensing protein kinases operate in stress responses, such as calcineurin B-like protein-interacting protein kinase (CIPK) which confers enhanced tolerance to cold, drought and salt stress when overexpressed in rice (Xiang et al., 2007). Rice plants expressing CIPK accumulated significantly more proline and soluble sugars than control plants, indicating that the signaling pathway induces antioxidant and osmoprotectant activities. Another example is stress-activated protein kinase 4 (SAPK4) which increases salt tolerance in rice. This signaling pathway reduces Na+ levels and maintains photosynthetic activity under stress by activating a raft of response genes such as the vacuolar H+-ATPase, the Na+/H+ antiporter NHX1, the Cl- channel CLC1 and a catalase (Diedhiou et al., 2008). Since many stress pathways ultimately trigger oxidative stress, it is no surprise that many of the signaling components identified in recent experiments converge on genes whose function is to eliminate ROS. For example, the overexpression of Arabidopsis nucleoside diphosphate kinase 2 (AtNDPK2) in rice plants was shown to induce multiple antioxidant genes including those encoding peroxidase, thioredoxin reductase, peroxiredoxin reductase, glutathione reductase and glutathione transferase (Seong et al., 2007b). Further investigation has shown that NDPK2 is also involved in the phytochrome A response, UV-B signaling, heat stress and chilling stress. Indeed, two genes responsible for chilling tolerance (pBC442 and pBC601) are upregulated in the AtNDPK2 transgenic rice plants described above (Seong et al., 2007b). Promoter choice is important in the context of stress resistance to avoid the negative effects of a ‘permanent’ stress response. In transgenic potato plants expressing AtNDPK2, the oxidative-stress inducible SWPA2 promoter was much better than the constitutive CaMV35S promoter in protecting the plants against high temperatures and salinity stress (Tang et al., 2008). As stated earlier, signaling genes are perhaps more efficient in conferring stress tolerance than structural genes and enzymes because one signal can induce multiple stress response pathways. But how does AtNDPK2, for example, activate pathways that respond to different forms of stress? Recent research suggests this is achieved through activation of the mitogenactivated protein (MAP) kinase cascade, which can be induced by very small amounts of ROS. For example, MAP kinase kinase kinase (MAPKKK) is induced in Arabidopsis in the presence of H2O2 (Kovtun et al., 2000). Nicotiana protein kinase (NPK1) is a tobacco protein kinase homologous to MAPKKK and transgenic maize plants expressing NPK1 showed enhanced tolerance to drought stress (Shou et al., 2004b). NPK1 overexpression induced a number of downstream response genes including those encoding glutathione-S-transferase, heat shock proteins, pathogen response genes (showing cross-talk between biotic and abiotic stress responses), alcohol dehydrogenase, a blue copper protein, DREB1 and many others. MAPK is activated by MAPKKK through a phosphorylation cascade and numerous reports now invoke this protein in biotic and abiotic stress responses. When the rice MAPK5 gene was overexpressed in transgenic rice plants, the plants showed enhanced tolerance of chilling, drought and salinity, whereas suppressing the same gene by RNAi made the resulting plants hypersensitive to the same stress conditions (Xiong and Yang, 2003). Whereas RNAi generates plants with signaling processes suppressed, traditional mutagenesis can generate both loss-of-function and gain-of-function phenotypes.

22

Koreen Ramessar, Shaista Naqvi, Svetlana Dashevskaya et al. Table 6. Overexpression of plant protein kinases to enhance abiotic stress tolerance Gene

AtNDPK2

Genes induced pBC442, pBC601, OsAPX1, OsAPX2, OsSodB

Chilling, oxidative Chilling, drought, salt Chilling, drought, salt

OsCIPK OsMAPK5

SAPK4

Stress

Vacuolar H+ATPase, Na+/H+ antiporter Salt NHX1, Clchannel OsCLC1 and a catalase.

Source

Host

References

Arabidopsis (Arabidopsis thaliana)

Rice (Oryza Seong et al., sativa) 2007b

Rice

Rice

Rice

Rice

Rice

Rice

Xiang et al., 2007 Xiong andYang, 2003

Diedhiou et al.,

2008

RACB

Flooding

Barley (Hordeum vulgare)_

Barley

Schultheiss et al., 2005

NDPK2 (SWPA2 and CaMV35S) promoters) MAPKKK (NPK1) CaMV 35S promoter MAPKKK (NPK1) CaMV 35S promoter

Oxidative, high temperature, salt

Arabidopsis

Potato (Solanum tuberosum)

Tang et al., 2008

HSP

Drought

Tobacco (Nicotiana tobaccum)

Maize (Zea mays)

Shou et al., 2004a

GST, HSP, PR1

Chilling

Tobacco

Maize

Shou et al., 2004a

Transgenic barley plants expressing a constitutively active RACB mutant racb-G15V under the control of the maize ubiquitin-1 promoter lost more water than wild type plants due to increased transpiration (Schultheiss et al., 2005). Experiments in Arabidopsis showed that this phenotype reflected the mutant’s inability to respond to abscisic acid.

Regulatory Genes – Transcription Factors The mention of abscisic acid (ABA) in relation to stress tolerance brings us neatly to the discussion of transcription factors and their role in stress responses. At the end of every signal transduction pathway is either a direct response at the protein level which alters cell behavior,

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or a transcription factor that modulates gene expression. Stress response networks in plants appear to be divided into those dependent on ABA and those that do not involve this hormone (Shinozaki and Yamagushi-Shinosaki, 2005). Many of the transcription factors that are known to control stress tolerance are ABA-independent, particularly transcription factors of the DREB/CBF (dehydration responsive element binding protein/C-repeat binding factor) and NAC (NAM/ATAF1,2/CUC2) families (TABLE 7). An ankyrin repeat domain zinc finger transcription factor has also been implicated in stress tolerance (Seong et al., 2007a). Numerous reports have shown that the overexpression of DREB genes can enhance tolerance to drought, salinity and/or chilling in cereals (Oh et al., 2005; 2007; Ito et al., 2006; Pellegrineschi et al., 2004), tomato (Hsieh et al., 2002), peanut (Bhatnagar-Mathur et al., 2007) and forage grasses (James et al., 2008). Stress-inducible promoters are preferred for the regulation of DREB genes because the constitutive activity of DREB transcription factors can have unwanted effects such as stunting and chlorosis, although there were no such effects under constitutive expression in rice (Oh et al., 2005; 2007). Microarray experiments have demonstrated the upregulation of target genes containing the DRE, including 35 genes induced by the rice DREB1A protein (Ito et al., 2006). Rice plants overexpressing NAC family transcription factors are comparable to those expressing DREB1A (Hu et al., 2006; 2008; Nakashima et al. 2007). In addition, Hu et al. (2006) reported that, under field conditions, rice plants expressing the stress-responsive protein NAC1 were tolerant to severe drought and salt stress during the sensitive reproductive stage. Table 7. Overexpression of plant transcription factors to enhance abiotic stress tolerance Gene

Promoter

Stress tolerance

Secondary effects

Stressinduced genes Not reported

DREB1A

HVA1 (stressinducible)

Salinity, drought

Not reported

HvCBF4

Ubi-1 (constitutive )

None

15 identified

DREB1A

rd29A (stressinducible)

Salinity, drought, chilling Drought

None

Not reported

DREB1A

rd29A

Drought

Not reported

Not reported

OsDREB1A

35 identified

None

DREB1A

CaMV35S

Salinity, drought, chilling Drought, salinity Chilling, oxidative

Reduced growth

DREB1A

CaMV35S and Ubi-1 (constitutive) Ubi-1

12 identified 1 identified

Reduced growth

Host plant Forage grass (Paspalum notatum) Rice (Oryza sativa)

References James et al., 2008 Oh et al., 2007

Peanut (Arachis hypogaea) Wheat (Triticum aestivum) Rice

BhatnagarMathur et al., 2007 Pellegrines chi et al., 2007 Ito et al., 2006

Rice

Oh et al., 2005 Hsieh et al., 2002

Tomato (Lycopersicon esculentum)

24

Koreen Ramessar, Shaista Naqvi, Svetlana Dashevskaya et al. Table 7. Continued Gene

Promoter

Stress tolerance

Secondary effects Reduced growth and yield None

OsNAC6

Ubi-1

Drought, salinity

OsNAC6

Salinity

SNAC1

OsNAC6 and LIP9 (stressinducible) CaMV35S

SNAC2

Ubi-1

CaKR1

CaMV35S

ABF3

Ubi-1

Drought, salinity Drought, salinity, chilling Salinity, oxidative Drought

None None

Thick leaves None

Stressinduced genes 14 identified

Host plant

References

Rice

Nakashima et al., 2007

14 identified

Rice

Nakashima et al., 2007

~ 40 identified 36 identified

Rice

Hu et al., 2006 Hu et al., 2008

Not reported 7 identified

Tomato

Rice

Rice

Seong et al., 2007a Oh et al., 2005

3. Improving the Intrinsic Yields of Food/Feed Crops When discussing the yields of food and feed crops, it is necessary to differentiate between two concepts – the yield gap and the yield ceiling. The yield gap is the difference between the actual yield of a harvestable product (such as grain) and the maximum possible yield under ideal conditions. The yield gap is caused by pests, diseases and unfavorable environmental conditions. All the strategies discussed in the earlier part of this chapter aim to increase yields by reducing the yield gap, but in each case the yield ceiling remains the same. The yield ceiling is the maximum yield a particular plant can produce under ideal conditions with no pests, diseases and in a perfect environment free of any stress. A second way to increase yields in transgenic plants is to lift the yield ceiling, i.e. increase the plant’s intrinsic capacity to produce the harvestable product. Much of the success of the Green Revolution was due to the development of semi-dwarf varieties of rice and wheat with biomass accumulation diverted away from vegetative tissues and towards grains, thus lifting the yield ceiling (Chrispeels and Sadava, 2003). As yields are maximized using conventional techniques, GE approaches will be required to facilitate the next significant enhancement in crop yields. The principal targets for this strategy are the efficiency of photosynthesis, the efficiency of energy storage in carbohydrates and lipids, plant architecture and development (driving more plant biomass towards harvestable tissues) and the efficiency of nutrient acquisition from the environment (particularly mineral uptake from the soil).

The Efficiency of Photosynthesis (Carbon Assimilation from CO2) The four key photosynthetic enzymes are Rubisco, fructose-1,6-bisphosphatase (FBPase), sedoheptulose-1,7-bisphosphatase (SBPase) and phosphoribulokinase (PRK), so the upregulation of any or all of these enzymes is an obvious starting point to enhance photosynthesis. However, this strategy is frustrated by the multilevel controls that feedback to

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25

regulate photosynthetic enzyme activity (Paul et al., 2001). For example, the rice Rubisco small subunit gene (rbcS) was overexpressed in rice plants, resulting in a 1.3-fold increase in Rubisco protein content but light-saturated photosynthesis was not enhanced even under low CO2 conditions, in which Rubisco becomes limiting for photosynthesis (Suzuki et al., 2007). An alternative approach is to target genes that control the whole photosynthetic pathway rather than manipulating a single key enzyme. For example, sugar levels feed back to control the expression of genes, pathways and processes that determine photosynthetic capacities and source-sink balance. Trehalose, a non-reducing disaccharide, performs a signaling role and controls glycolysis in yeast, so a similar mechanism was proposed to operate in plants. Plants expressing the E. coli trehalose phosphate phosphatase gene (otsA) were therefore generated and the rate of photosynthesis per unit leaf area was shown to increase by up to a third under saturating irradiance (Paul et al., 2001). Trehalose may interact with hexokinase, modifying the plant’s perception of glucose content and indicating a carbon deficit where none exists, stimulating the plant to increase its photosynthetic capacity. A very successful strategy to enhance photosynthesis is the introduction of C4-like photosynthesis into C3 plants. Transgenic rice plants overexpressing the maize phosphoenolpyruvate carboxylase (PEPC) and pyruvate orthophosphate dikinase (PPDK) genes, which play key role in organic acid metabolism in the guard cells to regulate stomatal opening, demonstrated 10-30% and 30-35% yield increases, respectively, which was quite unexpected since only one C4 enzyme was expressed in each case (Ku et al., 1999; 2001). In the PEPC transgenic plants, there was also an unanticipated secondary effect in which Rubisco showed reduced inhibition by oxygen (Ku et al., 1999). Peterhansel and colleagues introduced the E. coli glycolate catabolic pathway into Arabidopsis chloroplasts to reduce the loss of fixed carbon and nitrogen that occurs in C3 plants when phosphoglycolate, a photosynthetic byproduct, is recycled by photorespiration. The generated transgenic plants by step-wise nuclear transformation with five chloroplasttargeted bacterial genes encoding glycolate dehydrogenase, glyoxylate carboligase and tartronic semialdehyde reductase, such that the plastidial glycolate was converted directly to glycerate. The transgenic plants grew faster, produced more biomass, and contained more soluble sugars than wild type plants, reflecting reduced photorespiration and enhanced photosynthesis (Kebeish et al., 2007).

Primary Carbon Metabolism (Sugar to Storage Carbohydrate) Starch is the most widespread carbon reserve stored in plants and is of considerable industrial significance for food and non-food use. Several successful approaches have been used to increase the conversion of sugars to starch in potato including overexpression of an upregulated bacterial AGPase (ADPglucose pyrophosphorylase), overexpression of Arabidopsis amyloplastidial ATP-ADP translocator, antisense inhibition of NAD-dependent malate dehydrogenase enzyme and antisense inhibition of the plastid stromal isoform of adenylate kinase (Nunes-Nesi, 2005). In the last case, adenylate kinase inhibition increased both starch yield and tuber yield in potato (Regierer, 2002) whereas in the other three cases only starch yield was increased. These studies highlight that in spite of our structural knowledge of the sucrose to starch conversion we lack a fundamental understanding of the complex interactions that occur in the regulation of the pathway.

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Koreen Ramessar, Shaista Naqvi, Svetlana Dashevskaya et al.

Another potential way to increase starch levels is to manipulate sugar transport in order to modulate the source-sink balance. Sucrose is transported from photosynthetic tissues to storage tissues via the phloem, a process depending on the SoSUT transporter. When spinach SoSUT1 was constitutively overexpressed in potato, less sucrose was detected in transgenic leaves even though there was little change in the rate of photosynthesis, but the level of tuber starch was unaffected (Leggewie et al., 2003). The modification of pyrimidine biosynthesis in potato by tuber-specific antisense suppression of the UMP synthase (UMPS) gene shifted the pyrimidine synthesis pathway in favor of uridine salvaging, resulting in increased uridine nucleotide levels, accompanied by increased rates of starch and cell wall synthesis in growing tubers (Geigenberger et al., 2005). In tree fruits, sorbitol rather than sucrose is the primary end-product of photosynthesis and aldose 6-phosphate reductase (A6PR) is the key enzyme in sorbitol synthesis. Antisense suppression of this enzyme in apple reduced A6PR activity to 15-30% of the norm, resulting in lower levels of sorbitol but higher levels of sucrose and starch in mature leaves (Cheng et al., 2005). Further examples of where carbohydrate yield has been increased in heterotrophic organs include the overexpression of a modified form of the large subunit of maize AGPase in wheat (Smidansky et al., 2002) and the seed-specific overexpression of a potato sucrose transporter in pea (Rosche et al., 2002). The metabolism of photosynthetic carbon also plays a major role in determining crop yields, and this is another target for GE technology. Two successful studies have been carried out involving cyanobacterial enzymes. The expression of fructose-1,6-biphosphotase /sedoheptulose-1,7-bisphosphotase in tobacco helped to increase the rates of photosynthesis and growth under normal conditions (Miyagawa et al., 2001) whereas the expression of ictB, a gene involved in HCO3- accumulation, in tobacco and Arabidopsis increased photosynthesis and growth under CO2-limiting conditions (Lieman-Hurwitz et al., 2003). The RNAi-based suppression of both the mitochondrial and cytosolic isoforms of aconitase in wild tomato resulted in a reduction of flux through the Krebs cycle and, unexpectedly, an increase in the rate of photosynthetic sucrose synthesis (Carrari et al., 2003). The mechanism responsible for this effect is still under investigation.

Plant Development, Architecture and Reproductive Cycle All crops are cultivated for specific harvestable tissues, e.g. seed in cereal and legume crops, fruit in tree species, specialized root tissue in the case of potatoes and carrots, leaves in salad crops. All these tissues vary in size and weight, yet surprisingly little is known of the mechanisms controlling these properties. Recently, a gene called GW2 has been characterized in rice that controls weight gain in the rice grain. Loss of GW2 activity results in the development of larger grains with more cells and wider spikelet hulls, representing a yield increase of up to 20% (Song et al., 2007). GW2 is a negative regulator of cell division and its loss allows cell division and growth to continue longer than normal, in turn extending the developmental window available for grain filling. The wheat Rht gene was identified in the 1960s during the Green Revolution, and allowed plant breeders to develop short-stem varieties that increased yield by diverting resources towards grain filling rather than in stem elongation. Not only did this increase the harvest index (the ratio of harvestable to waste material) but the shorter, thicker stems were also more resistant to lodging (breakage at or near ground level, leading to collapse of the

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27

canopy). GE technology has allowed the same characteristics to be transferred to other crops. For example, the dominant dwarfing allele of the GAI (gibberellic acid insensitive) gene in Arabidopsis was discovered during research into the gibberellin signaling pathway and was found to be orthologous to Rht in wheat (Peng et al., 1999). The introduction of Arabidopsis GAI into rice generated dwarf rice varieties, with the expression level determining the severity of the dwarfing phenotype (Fu et al., 2001). Whereas the study described above created dwarf plants by preventing the plant responding to gibberellin, Sakamoto et al. (2003) achieved the same objectives by removing excess gibberellin from developing rice plants by overexpressing the enzyme GA 2-oxidase, which converts gibberellin into an inactive derivative. When the GA2ox1 gene was constitutively expressed the transgenic plants showed profound dwarfism but also failed to set seeds because gibberellin also participates in seed development. In order to avoid this problem GA2ox1 was expressed under the control of the tissue specific GA3ox2 (D18) promoter, generating semi-dwarf plants with normal flowering and grain. The studies discussed above have focused on the control of development to produce harvestable tissues of the best quality. However, it is also necessary to consider what happens when this developmental process is complete. Many thousands of tons of fruits and vegetables are lost each year because they ripen too quickly and are difficult to sell, eventually becoming inedible. The control of ripening therefore has an important impact on food security since it increases the storability of fruit and vegetable products, thus extending the period of time during which farmers can generate income. Consumers would also benefit from the extended shelf-life of fruits and vegetables, which would also reduce the cost of production by doing away with artificial treatments and controlled environment storage facilities. Perhaps the most widely known example of ripening control is the Flavr Savr tomato, in which the enzyme polygalacturonase was suppressed by antisense RNA to delay softening. Unlike wild type tomatoes which are picked when green and artificially ripened using ethylene, Flavr Savr tomatoes could be allowed to ripen on the vine, without compromising their shelf-life. The product was ultimately unsuccessful, competing against abundant supplies of conventional tomatoes in the aggressive anti-GE environment of the industrialized world, but the control of ripening in developing countries could provide an important advantage to both subsistence farmers wanting to store their own produce, and for the full exploitation of fruit and vegetable cash crops. Finally, nutrient acquisition can be an important limiting factor in plant development and nitrogen availability is a major limiting factor in plant productivity. Crop plants, especially those grown for grain yield, are usually supplied with inorganic nitrogen fertilizers. In an attempt to develop nitrogen-efficient plants, rice was genetically engineered by introducing barley AlaAT (encoding alanine aminotransferase). This modification increased the biomass and grain yield significantly in comparison with control plants when plants were well supplied with nitrogen. Compared with controls, transgenic rice plants also demonstrated significant changes in key metabolites and total nitrogen content, indicating increased nitrogen uptake efficiency. The development of crop plants that take up and assimilate nitrogen more efficiently would not only improve the use of nitrogen fertilizers, resulting in lower production costs, but would also have significant environmental benefits (Shrawat et al., 2008).

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Koreen Ramessar, Shaista Naqvi, Svetlana Dashevskaya et al.

4. Nutritional Enhancement Food security depends not only on the quantity of food but also its nutritional quality. Unfortunately, the poorest people in the world are faced with a limited choice, and generally rely on a single staple food. Since most plants are deficient in certain vitamins, minerals and essential amino acids, a diet restricted to one major staple will tend to be nutritionally insufficient (Graham et al., 2001). As discussed at the beginning of this chapter, both conventional and GE strategies have been used to try to overcome micronutrient deficiency. In this section, we look in detail at some of the most exciting developments in GE approaches to nutritional enhancement.

Vitamin Enhancement Transgenic strategies have been used to increase the levels of several vitamins in the edible parts of plants, including vitamins A, B9 (folic acid), C and E. Vitamin A deficiency is prevalent in the developing world, and is probably responsible for the death of 2 million children every year. In surviving children, vitamin A deficiency is a leading, but avoidable, cause of blindness (WHO, 2001). Humans can synthesize vitamin A if provided with the precursor molecule β-carotene (also known as provitamin A), a pigment found in many plants but not cereal grains. Therefore, many researchers have attempted to elevate β-carotene levels in staple cereals using GE technology (TABLE 8). The first significant advance was “Golden Rice 1”, where the entire β-carotene biosynthetic pathway was reconstructed in the endosperm by expressing daffodil (Narcissus pseudonarcissus) phytoene synthase and lycopene β-cyclase, and a bacterial (Erwinia uredovora) phytoene desaturase; the resulting grains contained up to 1.6 μg/g of carotenoids by dry weight (Ye et al., 2000). In later experiments, the daffodil phytoene synthase gene was substituted by its maize ortholog resulting in “Golden Rice 2”, in which the total carotenoid content of the endosperm rose to 37 μg/g dry weight (Paine et al., 2005). More recently, Diretto et al. (2007) reported a potato variety expressing phytoene synthase, phytoene desaturase and lycopene β-cyclase all from E. uredovora, in which the total carotenoid content was 114 μg/g dry weight. Vitamin E is a group of eight compounds (four tocopherols and four tocotrienols) of which α-tocopherol has by far the most potent activity in humans. Dietary vitamin E is obtained mainly from seeds, which contain higher levels of γ-tocopherol than α-tocopherol, so increasing total Vitamin E levels or boosting the proportion of α-tocopherol in seeds is a useful strategy to increase total vitamin E activity. Deficiency leads to general wasting, kidney degeneration and infertility. TABLE 8 shows the results of different studies aiming to elevate total tocopherol levels by increasing the flux through the biosynthetic pathway, or increasing the proportion of α-tocopherol. The greatest success has been achieved by expressing the Arabidopsis vte3 and vte4 genes in soybean, leading to a shift in vitamer ratios from 10% to >95% α-tocopherol, equating to a fivefold increase in vitamin E activity (Eenennaam et al., 2003). Although there has been limited progress with other vitamins, a small number of studies have achieved success enhancing the levels of folate (vitamin B9) and ascorbate (vitamin C). Plants are the sole source of folate in human diets, and a deficiency is particularly dangerous in pregnancy, leading to severe neural tube defects such as spina bifida. Folates are tripartite

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molecules formed of pteridine, p-aminobenzoate (PABA), and one or more glutamate moieties. Folates are present in high levels in green leafy vegetables, legumes and some fruits, whereas in cereals and in root and tuber crops folate level is low (Hossain et al., 2004). Metabolic engineering has been used to increase folate levels but here a complicating factor is that the molecule is formed by the convergence of three metabolic pathways restricted to different cell compartments. Following early experiments in which very limited increases in folate were achieved by individually engineering the cytosolic and plastidial branches of the pathway (Diaz de la Garza et al., 2004) crossing to stack the traits in a single plant line resulted in both branches being enhanced simultaneously increasing the folate content 15-fold to 8.4 µg/g dry weight, providing the entire guideline daily amount of folate in a single portion (Diaz de la Garza et al., 2007). Vitamin C or ascorbate is a potent antioxidant with particularly important roles in the cardiovascular and immune systems. Humans are unable to synthesize ascorbate because the final enzyme in the biosynthetic pathway is missing, meaning the vitamin must be obtained from food. Transgenic lettuce expressing L-gulono γ-lactone oxidase (GLOase), the enzyme missing in humans, accumulated seven times normal levels of ascorbate, showing that the final enzyme step is limiting in other organisms too (Jain and Nessler, 2000). It is also possible to enhance ascorbate levels by controlling the way in which it is recycled and metabolized. For example, the antisense suppression of a mitochondrial malate dehydrogenase gene in tomato increased ascorbate levels up to 6-fold (Nunes-Nesi et al., 2005). A novel approach in vitamin fortification is the simultaneous enhancement of multiple vitamins without complex breeding programs. The roots of this approach lie in a technique called combinatorial genetic transformation, which can be used to modify any metabolic pathway regardless of its complexity. Essentially, the method involves transforming plants with a large number of genes encoding the enzymes in a given pathway or network, and then screening a library of random transformants for plants with appropriate or informative metabolic profiles. Since transgene integration is random, the expectation is a population of plants in which all possible combinations of transgenes are represented. The pilot study for this technique involved the introduction of five genes encoding enzymes in the carotenoid biosynthesis pathway (Zmpsy1, PacrtI, Glbch, Gllycb and ParacrtW), each under the control of an endosperm-specific promoter, into an elite white maize variety lacking carotenes in the endosperm. The result was a metabolically diverse library comprising plants with a range of carotenoid profiles, elegantly revealed by easily identifiable endosperm colors ranging from yellow to scarlet. The plants contained high levels of β-carotene and other nutritionallydesirable carotenoids such as lycopene, and additional commercially relevant carotenoids like astaxanthin (Zhu et al., 2008). The successful multiple gene transfer experiments encouraged the same group to attempt a more ambitious program involving the simultaneous modification of multiple metabolic pathways to increase the levels of three key vitamins (β-carotene, ascorbate and folate). This was achieved by transferring four genes into the same white maize variety described above, resulting in a 407-fold elevation of β-carotene levels (57 μg/g dry weight), a 6.1-fold increase in ascorbate (106.94 µg/g dry weight) and a two-fold increase in folate (200 μg/g dry weight) (Naqvi et al., 2009).

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Koreen Ramessar, Shaista Naqvi, Svetlana Dashevskaya et al.

Mineral Enhancement Mineral nutrient deficiencies are responsible for a number of preventable diseases even in industrial nations, including anemia, diarrhea and retarded development (WHO/FAO, 1998). However, people in developing countries are disproportionately affected because of the cereal-rich diets and the fact that milled cereal grains lack essential minerals including iron and zinc. Unlike vitamins, minerals are not synthesized but accumulated from the environment, so GE strategies to increase mineral accumulation in plants are distinct from those involving vitamins. Indeed, there are two main approaches, namely to improve mineral uptake, transport and storage, and to increase the bioavailability of the minerals once consumed (many plants, including cereals, synthesize antinutritional compounds like phytate that prevent adsorption by the human gut, and reducing the prevalence of such compounds will increase the nutritional value of plants without the need to change the mineral levels). These approaches have been reviewed in detail (Bauer and Bereczky, 2003; Ghandilyan et al., 2006) and some of the major studies involving the overexpression of genes for iron transport, chelation genes and deposition are summarized in TABLE 9. One approach worthy of special mention is the expression of ferritin, an iron storage protein. The overexpression of soybean ferritin in rice, using an endosperm specific promoter, resulted in rice grains with 3-4 times the normal levels of iron (Goto et al., 1999; Vasconcelos et al., 2003). In contrast, constitutive expression of the same protein in rice increased iron levels in vegetative tissues but not seeds (Drakakaki et al., 2000) whereas constitutive expression of the pea ferritin gene did increase the iron content of milled grains, by five-fold (Hong-Xia et al., 2008). The combined expression of ferritin and phytase (a fungal enzyme that breaks down phytate) doubled the iron levels of rice grains and also increased its bioavailability in simulated digestion/absorption trials (Lucca et al., 2002). Similar results were obtained in maize (Drakakaki et al., 2005). Table 8. Transgenic plants enhanced for vitamin content Species

Genes

BETA-CAROTENE psy1 and lycb (Nicotania Rice (Oryza pseudonarcissus) sativa) crtI (Erwinia uredovora) psy1 (Zea mays) Rice and crtI (E. uredovora) Canola crtB (E. (Brassica uredovora) napus) crtE, crtB, crtI Canola and crtY (E. uredovora) Tomato crtI (E. (Lycopersicon uredovora) esculentum)

Promoters

Content (and increase relative to wild type)

References

Rice Gt1 (seed specific; psy1 and lycb) and CaMV35S (constitutive; crtI)

1.6 μg/g DW

Ye et al., 2000

Rice Gt1

31 μg/g DW

Paine et al., 2005

Napin (seed specific)

1180 μg/g DW (50-fold)

Shewmaker et al., 1999

Napin

1028 μg/g DW (50-fold)

Ravanello et al., 2003

CaMV35S

520 μg/g DW (1.9-fold)

Romer et al., 2000

The Contribution of Plant Biotechnology to Food Security in the 21st Century Table 8. Continued Species

Genes

Tomato

Endogenous lycb over-expression

Tomato

lycb (A. thaliana)

Tomato

Tomato CRY2

Tomato Tomato Potato (Solanum tuberosum) Potato Potato

Content (and increase relative to wild type)

Promoters CaMV35S

References

2050 μg/g DW (31.7-fold) D’Ambrosio et al., 2004

det-1 gene silencing crtB (E. uredovora)

546 μg/g DW (7-fold) 101 μg/g DW CaMV35S (1.3-fold) P119, 2A11 and 130 c μg/g DW TFM7 (fruit specific) (8-fold) Tomato 825 μg/g DW polygalacturonase (2.5-fold)

crtB (E. uredovora)

Patatin (tuber specific)

11 μg/g DW (19-fold)

Ducreux et al., 2004

lyc-e antisense Or gene (Brassica oleracea var botrytis)

Patatin

0.043 μg/g DW (14-fold)

Diretto et al., 2006

GBSS (tuber specific)

25 μg/g DW Lu et al., 2007 (total carotenoid) (6-fold)

Pds (fruit specific)

Rosati et al., 2000 Giliberto et al., 2005 Davuluri et al., 2005 Fraser et al., 2002

Potato

bch antisense

Patatin

0.085 μg/g DW (38-fold)

Diretto et al., 2007

Potato

crtB, crtI, crtY (E. uredovora)

Patatin

47 μg/g DW (3600-fold)

Diretto et al., 2007

Maize (Zea mays)

Wheat (Triticum aestivum) LMW and psy1 (Z. mays), crtI barley (Horedeum (E. uredovora) vulgare) D-hordein (endosperm specific)

57 μg/g DW (407-fold)

Naqvi et al., 2008

Soybean (Glycine max)

tyrA (E. uredovora), hppd (Arabidopsis thaliana) and ggh (A. thaliana)

4.8 μg/mg seeds total crhomanols

Karunanandaa et al., 2005

Soybean

vte3 and vte4 (A. thaliana)

Napin

(8-fold of α-tocopherol)

Maize

Hggt (barley)

Embryo-specific

> 800nmol/g DW (6-fold)

Maize

dhar (T. aestivum)

Maize Ubi-1 (constitutive)

~ 80 nmol/g FW in kernel Chen et al., 2003 (2 to 4-fold)

Lettuce (Lactuca sativa)

GLOase (rat)

CaMV 35S

430 to 580 nmol/g (4 to 7-fold)

Jain and Nessler, 2000

Tomato

mMDH

CaMV 35S

(5 to 6-fold)

Nunes-Nesi et al., 2005

Maize

Dhar (rice)

Barley D-hordein

VITAMIN E

Lac (E. coli)

Eenennaam et al., 2003

Cahoon et al., 2003

ASCORBATE

607.2 nmol/g FW/ 106.94 µg/g FW (6.1folds)

Naqvi et al., 2009

31

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Koreen Ramessar, Shaista Naqvi, Svetlana Dashevskaya et al. Table 8. Continued Species

FOLATE Tomato Tomato

Maize

Genes Mammalian GTPCH1 Mammalian GTPCH1 and ADCS (A. thaliana) folE ( E. coli)

Content (and increase relative to wild type)

Promoters Tomato E8

0.8 to 2.3 nmol/g FW (2fold)

Tomato E8

References Diaz de la Garza et al., 2004 Diaz de la Garza et al., 2007

8.4 µg/g DW (15-fold) Barley D-hordein

200 μg/g DW(2-fold)

Naqvi et al., 2009

Table 9. Transgenic plants enhanced for mineral content Function of gene product

Genes

Iron transporter

irt1

Iron 3+ reductase enzyme

fro2

Phytosiderophore enzyme

bHLH protein regulator of genes involved in phytosiderophore biosynthetic pathway

Seed storage protein

Enzyme to degrade phytate

Source Arabidopsis (Arabidopsis thaliana) Arabidopsis

Host

References

Arabidopsis

Connolly et al., 2002 Connolly et al., 2003

Arabidopsis

Arabidopsis Soybean (Glycine max) Rice (Oryza sativa) Tobacco (Nicotiana tabaccum) Tobacco

naat-A and naat-B

Barley

Rice

Takahashi et al., 2001;2003

idef1

Rice

Tobacco Rice

Kobayashi et al., 2007

iro2

Rice

Rice

Ogo et al., 2007

nas1

ferritin

phytase

Arabidopsis Barley (Hordeum vulgare)

Vasconcelos et al., 2006 Higuchi et al., 2001 Takahashi et al., 2003 Douchkov et al., 2005

Soybean

Rice

Pea (Pisum sativum) Aspergillus fumigatus Aspergillus niger

Rice

Goto et al., 1999 Drakakaki et al., 2000 Vasconcelos et al., 2003 Qu et al., 2005 Hong-Xia et al., 2008

Rice

Lucca et al., 2002

Maize (Zea mays)

Drakakaki et al., 2005

Essential Amino Acids Eight of the 20 standard amino acids are described as essential because they cannot be synthesized de novo and must be obtained from the diet (another four are essential in children because they cannot be synthesized in sufficient amounts). Because cereal grains are naturally deficient in lysine and threonine (and in some cases tryptophan), while legume seeds tend to be deficient in methionine, tryptophan and/or cysteine, many of the world’s poorest people lack access to the full complement of amino acids and suffer from corresponding deficiency diseases.

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There are many approaches that can be used to improve the amino acid profiles of staple crops (TABLE 10), perhaps the simplest being the introduction of genes that encode heterologous or even artificial storage proteins with multiple codons for essential amino acids. One example of this approach is the expression of the seed albumin gene AMA1 from Amaranthus hypochondriascus in potato, completing the amino acid complement and increasing the total protein levels (Chakraborty et al., 2000). However, one drawback of this approach is that it relies on the availability of free amino acids, which in some cases may itself be limited. The free amino acid pool can be modulated by increasing metabolic flux towards the under-represented amino acids, which can then be incorporated into proteins or left as free amino acids in solution. For example, the expression in rice of a feedback insensitive subunit of rice anthranilate synthase resulted in increase of free tryptophan in seeds, to about twice the wild-type level (Wakasa et al., 2006).

Essential and Very-Long-Chain Fatty Acids The consumption of very-long-chain polyunsaturated fatty acids (VLC-PUFAs) has a variety of health benefits but such molecules are usually sourced from fish oils, which are often beyond the reach of the world’s poorest people (Ruxton et al., 2007; Truksa et al., 2007). Synthesis of the three ω-3 VLC-PUFAs arachidonic acid (ARA), eicosapentenoic acid (EPA) and docosahexenoic acid (DHA) in agronomically important oilseed plants such as soybean would provide a cost-effective and sustainable alternative (Damude and Kinney, 2008). Table 10. Transgenic plants enhanced for amino acid content

Species Wheat (Triticum aestivum) Potato (Solanum tuberosum)

Genes

Promoters

Protein content (and increase relative to wild type)

References

legA (pea; Pisum sativum)

Wheat LMW glutenin

500 mg/g DW

Stoger et al., 2001

AmA1 (Amaranthus hypochondriacus)

Potato GBSS

16.12 mg/g DW

Chakraborty et al., 2000

CaMV35S

16.58 mg/g DW

Rice (Oryza ssa sativa)

Wheat 1Dx5 and Bx17 HMWglutenin promoters

Rice

oasa1d

Maize Ubi-1

Maize (Zea mays)

Sb401(Solanum berthaultii)

P19Z

12.8 μmol/g DW Hagan et al., 2003 of methionine 19.8 μmol/g DW of lysine 21 μmol/g DW of threonine 8.1 to 48.5 Wakasa et al., 2006 μmol/g (1.9-11.6-fold) 17.2 mg/g total Yu et al., 2004 protein content

34

Koreen Ramessar, Shaista Naqvi, Svetlana Dashevskaya et al. Table 11. Transgenic plants enhanced for long chain fatty acid content Species

Arabidopsis (Arabidopsis thaliana) Linseed (Linum usitatissimum) Tobacco (Nicotioana tabaccum)

Arabidopsis

Soybean (Glycine max)

Mustard (Brassica juncea)

Genes (Source) Δ9-elongase (Isochrysis galbana) Δ8-desaturase (Euglena gracilis) Δ5-desaturase (Mortierella Alpine) Δ6-desaturase (Phaeodactylum tricornutum) Δ6-elongase (Physcomitrella patens) Δ5-desaturase (Phaeodactylum tricornutum) Δ5/Δ6-desaturase (Danio rerio) Δ6-elongase (Caenorhabditis elegans) Δ4-desaturase (Pavlova salina) Δ5-elongase (Pavlova salina) Δ6-desaturase (Saprolegnia diclina) Δ6-elongase (Mortierella alpina) Δ5-desaturase (Mortierella alpina) Fad3(Arabidopsis thaliana) Δ17-desaturase (Saprolegnia diclina) Δ4-desaturase (S. aggregatum) Elongase (Pavlova salina) Δ6-desaturase (P. irregulare) Δ6-elongase (Physcomitrella patens) Δ5-desaturase (Thraustochytrium sp.) Δ12-desaturase (C. officinalis) Elongase (Thraustochytrium sp.) ω3-desaturase (P. infestans)

Marchantia polymorpha Tobacco

Soybean

Δ6-desaturase (Marchantia polymorpha) Δ6-elongase (M. polymorpha) Δ5-desaturase (M. polymorpha)

Promoters Constitutive CaMV 35S

% Fatty Acids ARA: 6.6 EPA: 3

References

Qi et al., 2004

ARA: 1.5 EPA: 1 Seed-specific USP promoters

Abbadi et al., 2004 ARA: 2

Seed-specific Napin

ARA: 1 EPA: 2.4 DHA: 0.5

Different strong seed-specific promoters

EPA: 19.5 DHA: 3

Kinney et al., 2004

ARA: 25 EPA: 15 DHA: 0.5

Wu et al., 2005

Seed-specific Napin

Constitutive CaMV35S Constitutive modified CaMV35S Seed-specific α’subunit of βconglycinin promoter

Robert et al., 2005

ARA: 10.55 EPA: 10.2 ARA: 13.4 EPA: 3.2

Kajikawa et al., 2008

ARA: 3 EPA: 0.3

Some examples of transgenic plants fortified with essential fatty acids are shown in TABLE 11. In soybean, Kinney et al. (2004) achieved a striking change to the normal profile of oils, i.e. 19% EPA and 3% DHA, which is the best result published thus far for DHA. This involved the introduction of several genes involved in the conventional ω3 and ω6 pathways (Δ6-desaturase, Δ6-elongase and Δ5-desaturase) together with additional genes in order to reduce the accumulation of intermediate products and maximize the most desirable VLCPUFAs. In mustard, Wu et al. (2005) achieved high levels of ARA (25%) and EPA (15%), but DHA accounted for only 0.5% of the total oil content. Further investigation of pathway

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regulation and feedback will be necessary to identify the bottlenecks and generate even higher levels of DHA (Zhu et al., 2007).

V. GE Strategies to Reduce Poverty 1. Increasing the Potential of Biofuel Crops Although biofuel production creates competition for resources with food and other agricultural products, it is nevertheless worthwhile considering the potential impact of GE technology on biofuel production because biotechnology may provide key strategies to enhance the productivity of energy crops thus reducing the competition with food and feed. Notably, applying the principles discussed earlier to reduce the impact of pests and diseases, increase the use of marginal land and lift the yield ceiling, will maximize the amount of biofuel obtained from a given area of land. However, we do not consider these aspects further because they are essentially identical to the concepts described above for food/feed crops and indeed many of the plants are one and the same. In this section we consider GE strategies to increase the conversion of plant biomass to fuel, i.e. bioethanol or biodiesel. To produce cellulosic ethanol, lignocellulosic biomass is harvested from the feedstock crop, pretreated with heat and chemicals to break it down into intermediates and remove the lignin, then the solid residue is treated with enzymes to produce sugars that are fermented to ethanol. Currently, such enzymes are produced at great expense by microbial fermentation so one of the major GE strategies to improve bioethanol production is the expression of such enzymes in the plant itself. Accumulation in the cytosol is avoided because constitutive activity would be detrimental to the plant, so the currently preferred strategy is compartmentalization, which allows the enzyme to become active when the plant is harvested, generally by extracting it along with total soluble proteins and adding this back to the solid residue for treatment. Notable reports include expression of the E1 enzyme from Acidothermus cellulolyticus at almost 5% total soluble protein (TSP) in rice and 2% TSP in maize (Oraby et al., 2007; Ransom et al., 2007) although this needs to increase to approximately 10% before supplementation with microbial enzymes becomes unnecessary. The activity of hydrolytic enzymes expressed in plants can be increased by expressing just the catalytic domain, which was achieved in both the studies cited above. The yield of ethanol can also be increased by increasing the amount of substrate, i.e. promoting the synthesis of cellulose and hemicelluloses, but not enough is yet known about the corresponding metabolic pathways in today’s energy crops for this strategy to be implemented. An alternative is to increase overall biomass, which can be achieved as discussed above by manipulating plant development e.g. through gibberellin synthesis or carbon metabolism. For example, expression of ADP-glucose pyrophosphorylase (AGP) in rice endosperm resulted in a totally unexpected 20% increase in plant biomass, although the underlying mechanism is not yet understood (Smidansky et al., 2003). Perhaps a more realistic option at the current time is the modification of lignin synthesis to reduce the need for pre-treatment. Lignin is the main component of plant biomass that interferes with processing, and a reduction in lignin content or a structural modification that improved extraction would considerably accelerate bioethanol production. Lignin is derived from three precursors — p-coumaryl, coniferyl and sinapyl alcohols — which are synthesized

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in separate but interconnected pathways, all of which have already been subject to GE approaches such as antisense suppression and RNAi. The downregulation of 4-coumarate 3hydroxylase (C3H) in alfalfa (Medicago sativa) altered lignin structure and improved the digestibility of alfalfa by cattle (Reddy et al., 2005). The downregulation of cinnamyl alcohol dehydrogenase (CAD) in alfalfa resulted in modification of lignin residue composition and made the lignin more digestible, but did not alter overall levels (Baucher et al., 1999), while in Populus spp., CAD downregulation made lignin more soluble and reduced the need for pretreatment (Pilate et al., 2002). The suppression of O-methyl transferase (OMT) in tobacco increased biomass production without decreasing the overall lignin content (Blaschke et al., 2004). Further approaches for consideration in the future include modifying the structure of cellulose to make it more digestible, and diverting flux away from lignin synthesis (reviewed by Sticklen, 2008). In contrast to bioethanol crops where the focus is on extractability, GE strategies to improve biodiesel crops focus on overall oil yields and oil quality. The fuel characteristics of biodiesel can be modified by altering the seed’s fatty acid composition, e.g. low saturated fatty acid levels improve cold-temperature flow characteristics, whereas low unsaturated and polyunsaturated fatty acid levels result in higher oxidative stability and reduced nitrogen emissions. A very good compromise can be reached by considering a fuel high in monounsaturated fatty acids, such as oleate or palmitoleate (16:1Δ9), and low in saturated and polyunsaturated fatty acids. Soybean lines with high levels of oleic acid and low levels of saturated and polyunsaturated fatty acids have been developed using a transgenic strategy involving the downregulation of two genes involved in fatty acid synthesis. The first is FAD2-1, encoding a D12 fatty acid desaturase, whose suppression prevents the conversion of oleic acid to polyunsaturated fatty acids. The second is FatB, which encodes a palmitoyl ACP thioesterase, whose downregulation prevents the release of saturated fatty acids from ACP (Durrett et al., 2008).

2. Value Added Products Plants can be modified to express a range of value added products including specific small molecules (generally products of secondary metabolism) and recombinant proteins for industrial, technical or pharmaceutical use. The range of secondary metabolites produced by plants is immense, and GE strategies to enhance the production of specific molecules that may be useful as drugs, toxins, dyes or chemical precursors provides an enormous scope for wealth generation.

Alkaloids The alkaloids are perhaps the largest group of secondary products synthesized by plants and the pathways, enzymes and regulatory genes involved in their synthesis have been extensively reviewed (Hashimoto and Yamada, 2003). Several distinct types of alkaloids can be distinguished, including the indole, tropane and isoquinoline classes. Monoterpenoid indole alkaloids (MIAs) are probably the most studied secondary products in transgenic plants. Many of these compounds have important clinical uses, e.g. vinblastine and vincristine from the Madagascar periwinkle (Catharanthus roseus) are valuable anti-cancer drugs. MIAs

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are synthesized in plants through two convergent pathways. The indole moiety is derived through decarboxylation of the aromatic amino acid tryptophan by the enzyme tryptophan decarboxylase (TDC) to yield tryptamine. Then the tryptamine and an irregular monoterpene, secologanin, derived from the plastidial methylerythriol-4-phosphate (MEP) or mevalonateindependent pathway are joined together by strictosidine synthase (STR1) to give strictosidine, which is the parent compound of most monoterpene indole alkaloids. Most attempts to modify MIAs have focused on this early, common part of the pathway so increase flux towards the MIAs, and have ignored the plethora of additional enzymes that decorate the basic skeleton and produce thousands of diverse MIAs with different properties. Unfortunately, the tight regulation of the pathway has ensured that such efforts only serve to increase the accumulation of intermediates [e.g. Leech et al. (1998) expressed TDC and STR1 in tobacco and achieved much enhanced enzyme levels but no increase in alkaloid content despite an increase in the immediate product strictosidine]. Tropane alkaloids are also important secondary metabolites with useful properties – in particular scopolamine and hyoscyamine are anticholinergic agents, acting on the parasympathetic nervous system. A major biotechnological goal in tropane alkaloid biosynthesis is the increase in the accumulation of scopolamine over closely related alkaloids, but in whole plants this has only been achieved thus far when plants are fed with the immediate metabolic precursors. Transgenic tobacco plants expressing two early enzymes, tropinone reductase (TR) and hyoscyamine 6-hydroxylase (H6H), produced greater amounts of nicotine as well as many nicotine-related compounds (e.g. anatabine, nornicotine, bipyridine, anabasine and myosmine) suggesting pleiotrophic changes induced by the new enzyme activities (Rocha et al., 2002). The isoquinoline type alkaloids include a number of clinically important compounds such as the morphine alkaloids morphine (narcotic and analgesic properties) and codeine (cough suppressant). Despite the relatively large number of genes cloned in morphine and other isoquinoline alkaloid biosynthesis, very few reports have been published describing heterologous expression of such transgenes in cell cultures or intact plants. Park et al. (2002) have demonstrated antisense suppression of the berberine bridge enzyme (BBE) in poppy cells, reducing the amount of benzophenanthridine alkaloids but increasing the levels of several amino acids. RNA interference was also used to down-regulate bbe in E. californica cells (Fujii et al., 2007) leading to the accumulation of (S)-reticuline and a substantial reduction in the level of sanguinarine. A methylated derivative of reticuline, laudanine was also produced in the silenced lines, which is absent from wild type plants. Transgenic poppies with increased morphine content were generated following constitutive expression of cor, the penultimate step in morphine synthesis. The morphine alkaloid content was up to 30% more abundant in transgenic lines compared to high-yielding commercial wild type genotypes (Larkin et al., 2007).

Terpenoids Terpenoids, or isoprenoids, comprise the largest class of plant secondary metabolites. Structurally they are classified as monoterpenoids (C10), sesquiterpenoids (C15), diterpenoids (C20), and triterpenoids (C30). A number of volatile terpenoids are involved in interactions between plants and insect herbivores and are also implicated in general defense or stress responses (Dudareva et al., 2006). Two different biosynthetic pathways are responsible for the formation of the isoprenoids: the cytosolic mevalonic acid (MVA)

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Koreen Ramessar, Shaista Naqvi, Svetlana Dashevskaya et al.

pathway, and the plastidial MEP pathway. Both pathways form the two different C5 intermediates, isopentenyl diphosphate (IPP) and its allylic isomer dimethyl allyl diphosphate (DMAPP), which are precursors of farnesyl diphosphate (FPP), geranyl diphosphate (GPP), and geranylgeranyl diphosphate (GGPP). Thus far, there have been few examples of monoterpenoid metabolic engineering. In one study, petunia was transformed with the (S)-limonene synthase gene from Clarkia breweri resulting in the production of linalool, which repels aphids (Lucker et al., 2001). In another, the same gene was introduced into tomato, under the control of a fruit-specific promoter, resulting in the accumulation of linalool in tomato fruits (Lewinsohn et al., 2001). Lucker et al. (2004) also reported the transformation of tobacco plants with three different monoterpene synthases from lemon. When the three transgenes were stacked in one transgenic line, the tobacco plants produced more terpenoids and displayed a different terpenoid profile to wildtype plants, the first report of transgenic plants expressing multiple foreign enzymes competing for the same substrate. Artemisinin, a very potent antimarial drug, is a sesquiterpene produced by Artemisia annua in tiny amounts (0.001% to 0.5% dry weight) (Walaart et al., 2001). GE strategies to improve the production of artemisinin have focused on the overexpression of key enzymes such as farnesyl diphosphate synthase (FPS), and amorpha-4, 11-diene synthase (AMS) (Gómez-Galera et al., 2007).

Flavonoids The flavonoids are phenolic compounds derived ultimately from phenylalanine, and thus share a common metabolic source with lignin (see above). The first committed step in flavonoid biosynthesis is the conversion of the precursor 4-coumaroyl-CoA into chalcone by the enzyme chalcone synthase. Chalcone is then derivatized in a series of enzymatic steps to produce a variety of molecules that act as pigments, defense chemicals (phytoalexins) and regulatory molecules (Forkmann and Martens, 2001). Flavonoids have been a favorite target for metabolic engineering since the early 1990s, because the result of different modifications can be determined through alterations in flower color. Increasing the levels of flavonoids in food plants can provide health benefits, as these molecules often have antioxidant activity. This has been demonstrated in the case of tomatoes by overexpressing the petunia gene for chalcone isomerase, leading to an 80-fold increase in the flavonoid content of the tomato peel and a corresponding 20-fold increase in the flavonoid level in tomato paste (Muir et al., 2001). In addition, chalcone synthase and flavonol synthase transgenes were found to act synergistically to upregulate flavonol biosynthesis significantly in the flesh of tomato fruits (Verhoeyen et al., 2002).

Recombinant Proteins (Pharmaceuticals) The first recombinant plant-derived pharmaceutical protein was human serum albumin, initially produced in 1990 in transgenic tobacco and potato plants (Sijmons et al., 1990). Nearly 20 years later, the first technical proteins produced in transgenic plants are on the market, and proof of concept has been established for the production of many therapeutic proteins, including antibodies, blood products, cytokines, growth factors, hormones, recombinant enzymes and human and veterinary vaccines (Twyman et al., 2005).

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Furthermore, several pharmaceutical products for the treatment of human diseases are in late stage clinical trials and are approaching commercialization, and the first plant-derived vaccine for veterinary use has been approved (Spok et al., 2008). The major pharmaceutical products produced in plants are antibodies, vaccines and replacement enzymes/blood products. Some have been produced in staple crops such as rice and maize (TABLE 12). These are advantageous because plants represent an inexpensive and easily scalable production system that does not support the replication of human pathogens. In the context of food security, pharmaceutical plants could be beneficial in multiple ways because as well as providing a source of wealth generation for farmers in developing countries, the availability of locallyproduced inexpensive drugs and vaccines would increase wealth by helping to prevent ill health. This is supported by concepts such as Pharma-Planta (http://www.pharma-planta.org), which aim to transfer the technology for the production of drugs such as HIV microbicides to developing countries for humanitarian purposes (Ma et al., 2005a,b; Ramessar et al., 2008c).

Industrial And Technical Products Metabolites and proteins can also be used as bulk industrial products, and these fall under a general class of products known as plant made industrials (PMIs). Recombinant proteins in this category include industrial enzymes (e.g. α-amylases for starch liquefaction, glucanases and xylanases for the degradation of plant material, and phytase as a food/feed additive), technical reagents for laboratory work including avidin, β-glucuronidase, aprotinin, laccase and trypsin (Hood, 2002), bulk nutritional/nutraceutical products to add to dried foods (e.g. the milk proteins β-casein and lactoferrin; Chong and Langridge, 2000), and protein polymers such as collagen, elastin and spider silk (Scheller et al., 2001). The production of biopolymers in transgenic plants remains at an early stage of development as both yields and extraction methods need to be improved, but there have been some encouraging developments. Ruggiero and colleagues have shown that human collagen can be produced in transgenic tobacco plants and that the protein is spontaneously processed and assembled into its typical, triple-helical conformation (Ruggiero et al., 2000). The expression construct contained the human gene encoding the α1(I) pro-collagen chain. However, the recombinant protein was recovered as mature collagen, folded and assembled into a stable triple helix as normally found in animals, and with appropriatelypositioned disulphide bonds. The thermal stability of the protein was reduced compared to bovine collagen due to the lack of hydroxyproline residues but this has been addressed in a subsequent study in which the collagen transgene was co-introduced with genes encoding a chimeric animal proline-4hydroxylase (Merle et al., 2002). Collagen is widely used in the cosmetics industry and in the manufacture of surgical devices, and plants could become an economical new source for this sought after protein. A synthetic spider silk has also been expressed in transgenic plants (Scheller et al., 2001). Genes based on the endogenous silk protein genes of the spider Nephila clavipes were synthesized in the laboratory and introduced into tobacco and potato. Proteins up to 100 kDa in size and with 90% identity to the genuine silk protein were produced in tobacco and potato leaves and in potato tubers. The proteins were produced at levels up to 2% TSP and, because they were heat-stable, could be easily isolated from the transgenic tissue.

Table 12. Pharmaceutical and industrial proteins produced in transgenic cereals Plant used Maize

Product Avidin Trypsin Aprotinin GUS Peroxidase Laccase Cellulase Gastric lipase Antibody Lactoferrin Lt-B vaccine TGE vaccine Fusion gene Glycoprotein S Lactoferrin Lysozyme Lactoferrin

Preventative indication/Usage Diagnostic use Wound care / insulin manufacture Non clinical use Non clinical use Industrial enzyme - paper bleaching Industrial enzyme - paper bleaching Industrial enzyme - ethanol production Cystic fibrosis Cancer Gastrointestinal disorders Traveler’s diarrhea Piglet gastroenteritis Newcastle disease virus Porcine transmissible gastroenteritis Cell culture media For research purposes Antibacterial, antiviral and antifungal

Research/Clinical Stage

Company / Reference

Available in Sigma catalogue Available in Sigma catalogue

Prodigene Prodigene

No longer in Sigma catalogue No longer in Sigma catalogue Available to license

Prodigene Prodigene Applied Biotechnology Institute

Available to license

Applied Biotechnology Institute

Available to license

Applied Biotechnology Institute

Phase II Phase II ( failed) Phase 1 Phase I complete - not ongoing Phase I complete - not ongoing Chicken feeding experiments

Meristem Therapeutics NeoRx / Monsanto Meristem Therapeutics Prodigene Prodigene Guerrero-Andrade et al., 2006 Lamphear et al., 2002

Available from company Available from company USDA regulatory approval

Ventria Biosciences Ventria Biosciences Ventria Biosciences

Table 12. Continued Plant used Rice

Product Lysozyme Human serum albumin VP2 protein Cholera toxin B subunit SS1 protein, vaccine α-interferon Allergen-specific T cell epitope (Japanese cedar pollen) Growth factors and cytokines Human serum albumin, lactoferrin, lysozyme

Preventative indication/Usage

Research/Clinical Stage

Company / Reference

Antibacterial, antiviral and antifungal Replacement blood product

USDA regulatory approval

Ventria Biosciences

USDA regulatory approval

Ventria Biosciences

Bursal disease virus (IBDV) Cholera

Chicken challenge experiments Mice challenge experiments

Wu et al., 2007 Nochi et al., 2007

Hepatitis B

Mice challenge experiments

Qian et al., 2008

Wound healing Pollinosis (elicits allergic disorders)

Mice oral feeding experiments

Zhu et al., 1994 Takagi et al., 2005

Various

Available from company

ORF genetics

Cell culture media, Antibacterial, antiviral and antifungal properties

Field trials (third round)

Maltagen

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Plants can also produce the precursors of chemical polymers, either for polymerization after extraction or polymerization in situ followed by extraction of the polymer as a complete unit. Potential monomers include methacrylate, fructose, sorbitol and hydroxyalkanoates (reviewed by van Beilen and Poirier, 2008). By introducing genes that allow the production of metabolic precursors, plants can be made to synthesize polyhydroxyalkanoates, which represent an economical, sustainable and biodegradable alternative to petroleum-based plastics (although a similar microbial process is currently even more economical). Natural rubber is also a possible target for GE technology, providing alternatives to Hevea brasiliensis, the rubber tree. For example, dandelions can provide an alternative source of latex from which rubber can be obtained, but the extraction process is expensive due to its rapid coagulation. A recent study in which polyphenol oxidase activity was suppressed in transgenic dandelion significantly prolongs the coagulation time, allowing the extraction process to be completed (Wahler et al., 2008).

VI. Barriers to the Uptake of GE Technology The World Food Program estimates that 840 million people suffer from malnutrition and hunger (http://www.wfp.org). Improving food security at the national and household level is critical if hunger and malnutrition are to be reduced in developing countries. As stated above, the problems of food security go far beyond technological solutions – food insecurity is also a product of poverty, poor infrastructure, poor food storage, post harvest losses, and inequitable intra-household food distribution, but technological solutions can certainly be part of the strategies policymakers and farmers use. GE technology provides many benefits and can potentially play a major role in improving nutrition and enhancing food security in developing countries, which will influence crop production and price competitiveness globally. But the use of GE technology in global agriculture and the food supply chain is currently controversial. On one extreme, we have witnessed a remarkably rapid adoption of some innovative products, such as herbicide-resistant soybeans and cotton, and insectresistant maize and cotton. On the other hand, although GE crops currently cover 114.3 million hectares (James, 2008), large-scale adoption has essentially been limited to three countries: the United States, Argentina, and Brazil. Adoption in other countries has been prevented by a number of encroaching factors that directly affect the diffusion of biotechnology products at various market stages. This section will shed some light onto the heated debate on how such factors erect barriers to agricultural biotechnology, particularly in the developing world where the benefits of this technology are needed the most. We will discuss four major factors: (i) international trade barriers that generate a severe handicap preventing resource-poor countries and small-scale farmers extracting benefits from GE technology, especially with regard to trade in agricultural products; (ii) intellectual property rights and patents that cover GE technology, which creates a barrier to technology transfer and sustainability; (iii) biosafety regulation frameworks (or the lack of them in some countries) and how the absence of a harmonized international policy and overstrict regulations prevent biotechnology form achieving poverty reduction especially in developing countries; and (iv) how the media manipulates the public, causing scientific controversies that are rarely about science and how the public perceives and reacts to the information at hand. All these factors raise questions about how national and international markets for GM and

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non-GE derived crops will develop, and how negative, unscientific opinions surrounding biotechnology slow down the application of GEtechnology, and obstruct its potential to contribute to global poverty reduction and food security.

1. Trade Barriers GE crops were rapidly accepted when they appeared in the early 1990s. Industrialized countries, including those in the EU, made a positive effort to establish a safety assessment to commercialize them and 14 products were approved by the EU prior to 1998. The US, Canada and Argentina established national frameworks based on the scientific principle of substantial equivalence and GE crops have been grown and commercialized since 1996 with a great success. However, due to public opposition and consumer concerns, the EU started rejecting the new technology and established policies based on the precautionary approach (supported by the Cartagena Protocol), and has since been considered to be the main trade barrier for the development of GE crops worldwide. The barrier to international GE trade can be divided into tariff and non-tariff barriers. Tariff is a tax imposed on the import or export of a product, which is used mainly to restrict imports; non-tariff barriers include quotas, subsidies, technical barriers to trade (TBT), government health and safety standards, and administrative delays. These barriers are used not only to control the import of GE food but also to protect the wellbeing of a country. TBT are major barriers to the international trade of genetically engineered organisms (GEOs) and their products. Government health and safety standards are considered as important TBTs. National standards are imposed regulations provided either by government or non-government authorities based on producers, consumers or other market elements (Bhalla and Kennedy, 1998). Some countries may modify their standards in order to enjoy better trade with other countries using similar standards. However an absence of global harmonization in standards leads to market distortions and also blocks the entrance of foreign firms into the market, thus increasing international trade frictions. The trade between the EU and the US is strongly affected by differences in GE food standards. For instance, there is no longer any export of GE maize between the EU and US because the EU refuse to accept shipments that contain even a small amount of GE maize (USDA/FAS, 2004). Labeling of GE food products is another barrier to international trade. The US, Canada and Argentina have taken a step forward in order to file a formal dispute with the WTO against the EU moratorium on new approvals. Labeling of GE food cannot be applied without expenses, which will increase the price of GE food (Reiss, 2002). In addition this will unnecessarily alarm consumers and delay advances in biotechnology. Labeling combined with a lack of public understanding of GEOs can also deter consumers from buying GE foods and eventually, they would disappear off the market. The Food and Drug Administration (FDA) in January 2001 stated that the only GE foods that required labels were those that differed from their natural counterparts. Studies of consumer attitudes showed that the phrase “genetically modified” incorrectly led buyers to believe the food was different to its natural counterpart. Therefore the FDA prefers that foods should instead be labeled as “genetically engineered” or “made through biotechnology” (Rousu and Huffman, 2001). The EU on the other hand has strict policies for GM (GE) labeling. In January 2000, the Commission of the Council stated GM (GE) foods must be labeled “genetically modified” if at least 1% of GM

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(GE) material was detectable. In order to avoid a trade war with the US, the EU lifted its ban on GE food approvals in July 2003, but left its strict labeling policies in place, which continues to cause trade controversies with GE-producing countries. The Cartagena Protocol on Biosafety (CPB), considered a non-tariff barrier to GEO trade, was opened for signature in Nairobi in May 2000 and has affected the international trade of GEOs and their products ever since. The CPB is a legal document controlling the import, export, transit, handling and use of GEOs. The protocol implies that countries have the right to use the precautionary approach and refuse GE product imports for safety reasons, without giving scientific proof (Madeley, 2000). This protocol has divided the trade market into two blocks: the EU and most of the developing countries are in favor, while the US, Canada, Australia and Argentina do not support this biosafety protocol.

GE Trade: The Case of EU versus US During the mid-1990s, the consequence of weak political and governmental management of food crises in Europe (an example being the outbreak of bovine spongiform encephalopathy (BSE) also known as the “mad cow disease”) led to a societal food safety scare. Claims against the environmental and anti-globalization impacts of GE products by non-governmental organizations (NGOs) (such as Greenpeace) and consumers, raised concerns among the public thus provoking social non-acceptance of GE products. GE food was seen to be a risk rather than a benefit to society, and public concern for food security and consumer distrust of government regulatory agencies increased simultaneously. GEOs became politically unpopular due to these social pressures, and all open processes for the acceptance of new GE products became paralyzed, with calls for stricter regulations. In the US, GEOs are gaining popularity, but the EU’s strict precautionary approach towards trade of GEOs has increased the risk of conflict. Banning GEO imports was a major controversy in US-EU trade (initiated in 1998) when the EU imposed a moratorium on the approval of GE products (Capell, 2008). A de facto moratorium was promoted by some Member States that ignored the European Commission’s efforts to establish a common legislation for GEOs, without a scientific basis and inconsistent with the World Trade Organization (WTO) Agreement on the Application of Sanitary and Phytosanitary Measures (SPS). Consequently this caused the banning of GE crops and their derived products from 1998 to 2004 (no GEO approval within this period). The EU claims that its justification for the strictness of the regulations was based on the consumers’ “right to know” (this appeared to be a political decision, not based on actual risk) in order to restore consumers’ confidence. In 2003, the EU was accused of violating international free trade agreements with a formal complaint presented by US, Canada and Argentina to the WTO (the only global international organization dealing with the rules of trade between nations, www.wto.org). Under the pressure of this ongoing US-EU-WTO conflict, the EU finally approved new regulations (applied in 2004) to allow approvals for commercialization (but not cultivation) of GEO products, albeit under strict legislation of labeling and traceability. Some products have been approved since then, but the new regulations did not convince the US, which regarded them costly, unworkable, unenforceable, unnecessary and discriminatory. Even though the WTO (in 2006) ruled that the EU had broken international trade rules by blocking the import of GEOs (hailing US trade officials as victorious), the damage to public perception was done

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and the international debate on GEO acceptability was left open, thus the EU continues with its strict legislation, approving just a few GE products each year. Nowadays there are calls for a global and harmonized regulatory system which could start with a mutual recognition of the products approved in different countries. This would break down the existing oppressive trade barriers (especially in the EU) and allow the development of GE crops that could ensure food security since the benefits of GE crops are well known (reviewed by Ramessar et al., 2008a,b). But the conflict is not yet over, as despite the approval of EU legislations by all member states, some continue to reject GE crops and their products. Local policies (called “safeguard” clauses) in Austria, France, Germany and Luxemburg maintain the ban on GEOs and in some cases, there were requests for declaring GE-free areas. The European Commission rejected these state proposals and declared them illegal, but they are still enforced and thus discord among member states goes on.

Trade Barriers Affecting Industrialized and Developing Countries International trade conflicts affecting both industrialized and developing countries have arisen from the EU policies discussed above (Tencalla, 2006). Industrialized countries (e.g. the US, Canada and Argentina) that grow GE crops and trade their products have experienced economic losses due to the elimination of EU markets. Developing countries pay attention to such international conflicts before setting out their policies for GEO regulation, and fear the loss of foreign markets if they produce and export GE products (Paalberg, 2002). This explains why some countries (e.g. Zimbabwe and Uganda) reject GEOs despite the benefits that GE crops offer their national welfare and economy. Moreover, the Zambian government rejected food aid in the form of GE maize from the US despite an ongoing famine, and decided to adopt the ‘precautionary approach’. This sparked a huge political crisis both within Zambia and abroad, with opponents of the decision highlighting that people were facing starvation. Another example is China, whose population reached 1.3 billion in 2007. Despite a welldeveloped agricultural system, China still needs to import food for its population. Indeed, China imports food, feed and oil mainly from the US and Canada which export over 28 million tons of oilseed and 100 million tons of cereals each year (Hodgson, 2000). These countries do not label GE food and they export GE food mixed with conventional food, thus affecting China’s export of livestock products to Japan and the EU, since these countries demand the labeling of GE food and its products. Hence the EU cancelled tobacco imports from China due to fears that they might contain GE tobacco. Additionally, Indian activists protested when their government tried to import GE soybean and maize from the US, and India now attempts to block GE food imports from the US in order not to compromise its market with the EU. Thus, India seeks premium prices for GE-free commodities such as soybean, sunflower and rapeseed meal when these are exported to Japan, Thailand, Gulf countries and the Middle East. The EU invites economic decline by insisting on strict regulation and labeling policies. Not only is Europe progressively losing its share in the agriculture markets, it is also suffering a ‘brain drain’ as biotechnology research organizations, small businesses and larger companies (such as Syngenta and BASF) relocate to countries that accept GEOs (Tencalla, 2006). Furthermore, since European markets are isolated from global price fluctuation by way of the Common Agricultural Policy (CAP), which protects farmers’ income using flexible

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import tariffs and variable export subsidies to support the institutional price arrangements (van Mejil and van Tongeren, 2004), consumers have less variety to choose from and pay higher prices, and are deprived of the real benefits of GE crops.

Figure 1. Patents awarded on plant biotechnology by US Patent and Trademark Office between 1985 and 2000 (Delmer et al., 2003).

2. Intellectual Property Patents covering modified living organisms were first approved by the US Supreme Court in 1980 (Sullivan, 2004). Since then, the number of crop biotechnology patents has increased (FIGURE 1) and many organizations and agreements have been set up in the USA and Europe to legislate on the creation and protection of intellectual property (IP) (Sechley and Schroeder, 2002). Private companies were thus able to develop commercial interest in agricultural biotechnology, ultimately affecting the public sector (Streitz and Bennett, 2003). Traditionally all the data, research materials and discoveries were open and publicly available, but most institutes and universities now use the patent system to commercialize their inventions. Although free exchange is promoted by the National Science Foundation (NSF) and the National Institutes of Health (NIH), these are becoming exceptions rather than the rule. Many biotechnology patents are awarded each year but most new technologies are built on other innovations that are already patented, including vectors, selectable markers, transcription regulatory elements and sub-cellular targeting systems (Krattiger and Mahoney, 2007). Some of these pre-existing patents have extremely broad coverage and are all but unavoidable when working in the field (e.g. transformation methods) thus introducing intellectual property restrictions that can affect the development of transgenic crops (Dunwell, 2005). One of the successful examples of dealing with the problem of fragmented IP ownership is Golden Rice. As discussed earlier, this is a range of transgenic rice varieties that are able to

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synthesize β-carotene, providing a potential solution to vitamin A deficiency diseases. The creation of this variety required the use of many technologies that were patented by different companies, such as Bayer AG, Monsanto Company, Novartis AG, Orynova BV and Zeneca Mogen BV (Krattiger and Potrykus, 2007). However, since Golden Rice was produced in Europe, it could not be transferred to developing countries without proper licenses. Due to the humanitarian purpose of Golden Rice and the huge amount of publicity thus generated, all the companies agreed to provide access to their technologies free of charge for defined humanitarian research purposes only, allowing the use of Golden Rice in developing countries by resource-poor farmers earning less than $US 10,000 per year from farming (Krattiger and Potrykus, 2007). Although Golden Rice demonstrated that the IP problem can be resolved, biotechnology companies are generally uneasy about such humanitarian ventures because most developing countries lack enforceable IP policies that would ensure that companies’ IP rights are protected (Wendt and Izquierdo, 2000). Thus to establish and maintain international technology transfer agreements, industrialized and developing countries need to cooperate in the development of a manageable system for IP protection. This can be achieved in a number of ways, e.g. local ownership and the involvement of scientists from developing countries (e.g. Ugandan scientists involved in the development of nutritionally enhanced bananas to address food insecurity in Uganda; Enserink, 2008) or by the implementation of appropriate IP policies and effective enforcement procedures in developing countries (Kowalski, 2002). Several new organizations have been set up to promote biotechnology in developing countries, particularly in the area of subsistence agriculture, focusing on IP access and management, and some offering a complete set of biotechnology techniques ensuring freedom to operate (FTO): •

•

•

CAMBIA (Center for the Application of Molecular Biology to International Agriculture). This is a non-profit, private research organization based in Canberra, Australia, that provides access to and information on patents, tutorials and other white papers on key areas of biotechnology, for national and research institutions in developing countries. PIPRA (Public Intellectual Property Resource for Agriculture). This organization, based in California, was created to pool the resources of many public institutes, allowing them to collaborate and develop new crops with the technologies they invented. PIPRA also encourages the provision of general access to the underlying technologies and management of IP in the public sector. The anticipated benefits of a collective IP management regime include the effective assessment of FTO issues, overcoming the fragmentation of public sector IP rights to re-establish the necessary FTO in agricultural biotechnology for the public good, and the efficient identification of collective commercial licensing opportunities to enhance private sector interactions. AATF (African Agricultural Technology Foundation). This is similar to PIPRA but is based in Kenya and is directed more towards developing countries. The goal is to access advanced scientific and technological resources and adapt them for use in projects for the development of agricultural products in sub-Saharan Africa. The

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•

AATF does not fund projects itself but provides the matchmaking, stewardship and guidance at all levels to ensure the development of successful projects. SIPPI (Science and Intellectual Property in the Public Interest). This is a project developed by the AAAS (American Association for the Advancement of Science) that develops IP management to promote humanitarian access through different licensing strategies. These strategies are promoted from the initiation of a project or technology transfer process, or to meet an explicit goal in the patent policies of universities.

3. Regulation GE plants must satisfy pre-approval regulatory requirements before being considered for commercial approval, which includes thorough safety assessments for humans, animals and the environment. The biosafety regulations in different countries are based on five main international instruments (TABLE 13) which influence the development of national regulations in terms of field trial permit requirements, risk assessment criteria, labeling, traceability, transparency, public awareness, post-monitoring and import regulations. Although the existence of these instruments ensures a certain level of international harmonization (König et al., 2004; Jaffe, 2004), there remain major differences in interpretation, and therefore different countries regulate the approval of GE crops in different ways. The most widely cited example is the difference in approach between the EU and US, with the EU adopting a precautionary approach enshrining the consumers’ ‘right to know’ using stringent approval, labeling and traceability standards on any food produced from or derived from GE ingredients (Gruère, 2006), whereas the US regulations are based on differences in the end product, and include a voluntary safety consultation and voluntary labeling guidelines for GE food. Most other developed countries, including Japan, Canada and Australia, have introduced regulations that share features of both the EU and US systems (Carter and Gruère, 2006). There have been many calls for the global harmonization of regulations, which would make the requirements compatible and consistent. This does not necessarily imply that all countries should have identical policies, priorities or strategies. The aim is uniformity in requirements for data collection, testing procedures and the exchange of information. Eventually, the outcome of national regulations depends on public perceptions and public acceptance, as well as on cultural and institutional processes. Regulatory harmonization would help to remove artificial trade barriers, expedite the adoption of GE crops, foster technology transfer and protect developing countries from exploitation, instilling confidence and bringing the benefits of GE products to the consumer. Developing countries have unfortunately been drawn into the mire of international regulatory disunity. Although they want to ensure that GE products are tested to the same safety levels as in the developed world before they are put in the hands of farmers, this is hampered by the lack of resources (human, financial and sometimes institutional) to develop a science-based regulatory infrastructure similar to the industrialized economies. Therefore policy makers in developing countries often feel they cannot accept innovative GEcrops and must postpone decisions or adopt a very cautious approach to the products of GE technology (IPC, 2004). Many developing countries have implemented bans on GE products that not only

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affect market access but also make it more difficult for them to gain financial support from industrialized nations, particularly in order to conduct research and build human capital for biotechnology activities. Table 13. Key international instruments governing national biosafety regulations International biosafety agreements (i) World Trade Organization (WTO) agreements (www.wto.org) Agreement on the Applications of Sanitary and Phytosanitary Measures (SPS Agreement) 1994

Agreement on Technical Barrier to Trade (TBT Agreement) 1994

(ii) International Treaty on Plant Genetic Resources for Food and Agriculture by the UN FAO (http://www.fao.org/biodiversity/IPGR_en.asp) (iii) Codex Alimentarius 2001 (www.codexalimentarius.net)

(iv) Cartagena Protocol on Biosafety under the Convention on Biological Diversity (CBD) 2000 (http://www.cbd.int/biosafety/default.shtml) (v) Organization for Economic Cooperation and Development (OECD) 1961 (www.oecd.org)

Role in biotechnology Aims to control barriers to international trade Provides for the enactment of laws, decrees, regulations, requirements and procedures relating to sanitary and phytosanitary concerns that may affect trade Provides for standards of ensuring the elimination of unfavorable treatment of trading member countries’ products (biotechnological, industrial and agricultural products) Multilateral agreement relating to any genetic material of plant origin of value for food and agriculture Set of international codes of practice, guidelines and recommendations pertaining to food safety. The WTO currently relies upon the Codex in making its adjudication Multilateral agreement covering the transboundary movement of living modified organisms (LMOs) that might have an adverse effect on biological diversity Harmonization of international regulations, standards and policies

The complex regulations applying in international markets generate overlapping and sometimes contradictory requirements that are a burden to the developing country farmer. Different countries regulate according to different principles, e.g. some according to the use of GE technology, some on the amount of GE material present; some require labels on the final product alone, others also require labels on intermediate products; labeling is mandatory in some countries but voluntary in others, and some countries require proactive labeling (stating GE material is present) while others stipulate negative labeling (e.g. ‘does not contain GE material’). Trade becomes difficult when regulatory regimes vary so widely, particularly for developing countries that may lack the resources to comply with complex regimes. The cost of segregating GE and conventional crops (for commercial or regulatory reasons) can be

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daunting for developing countries that do not have a modern handling and transportation infrastructure (IPC, 2004). International trade becomes limited as it is more difficult for producers in developing countries to maintain their supply contracts with distributors in the developed world. The end result is that developing countries may decide it is simply easier to avoid the issue altogether by avoiding GE products, even if they are acknowledged to be safe. Consumer attitudes are influenced by the media, which generally promotes ‘avoidance’ and has a negative impact on the ‘acceptance’ of GE products. GE-free foods are portrayed as being ‘superior’ to GE foods without scientific evidence. Modern biotechnology may need to overcome problems that are not based on science, even though the application of the science could help to alleviate poverty and hunger.

4. Media Manipulation and Public Perception The public are predominantly exposed to science through the mass media, which therefore play a key role in communicating science and shaping public attitudes. Communicating effectively requires sensitivity to the audience, knowledge of the topic and skill in providing answers that are scientifically accurate, not subject to misinterpretation and that address public concerns. The media can be a good source of information for the public on current scientific discoveries and cutting edge technologies. However when we hear of GEfield trials in Europe being destroyed by protesters, and GE crops in India being burned, we cannot help but wonder what provoked these aggressive and inhumane actions. Media coverage varies widely in terms of accuracy, detail and advocacy. For example, newspapers may carry biotechnology stories and may devote anything from a short, nonbylined column piece to long, exhaustive series that include graphics and explanatory sidebars. Such pieces may emphasize the positive or the negative aspects of the story, whether justified or not, and may be well researched or not. Reporting biotechnology poses unique challenges because it is perceived by some as a controversial, evolving science, and it is difficult to produce quality news stories in an environment where science gets attention from activists and politicians. It is also clear that controversy itself attracts attention, which makes biotechnology a tempting opportunity for journalists good and bad. GE crops have allowed journalists to pander to very basic fears, because the consumption of food is seen as a low-risk activity so anything changing that status quo is bound to be controversial. Theories of risk were constructed with reference to environmental and technological hazards, such as nuclear power. However following a decade of “food scares” (TABLE 14), attention has moved toward the study of food risk and has focused almost exclusively on explaining the divergent opinions between experts and the lay public without actually addressing the underlying cause (Knox, 2000). Journalists turn an occurrence into a newsworthy event, and a newsworthy event into a story. In reporting a story, journalists and editors employ frames according to their own understanding, while conforming to practical limitations such as deadlines and space. Through frames, media highlight certain points of view and marginalize others, explaining how events are to be understood. The public can be manipulated by the media in two major ways: media structure (e.g. reach, accessibility, level of authoritarian control) and media content. In developed countries, media access is taken for granted, but in many developing countries the news travels slowly and can be subject to political interference. Local journalists, editors and managers also need

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to resist being manipulated by governments, and should ideally acquaint themselves with the topic independently before relaying stories to the public. This is not always possible for stateowned media outlets or those not granted freedom to operate. On the other hand, the way the media handles and communicates scientific information to the public can affect government decisions and policy (e.g. the 2002 GE food aid row that caused the Zambian government to ban GE aid from the US even though millions of its people faced starvation, and the unnecessarily strict regulations or bans on GE crops in the EU). Table 14. List of ‘food scares’ that caused the mass media to focus on food safety issues (adapted from Brian, 2004) Year 1996 1997 1998 1999 1999 2000 2001 2001 2002 2002 2006

Incident BSE/CJD link discovered Contagious swine fever in the Netherlands Arpad Pusztai and the GE potato hoax Contaminated Coca-Cola in Belgium GE maize and Monarch butterflies BSE hits continental Europe Antibiotics and growth hormones in German pigs Foot and mouth disease all over Europe E. coli in ConAgra Beef Listeria in Pilgrim’s Pride Chicken E. coli in organic spinach and tomato

Media content is important as it helps shape an individual's view by forming the lens through which all issues are viewed. As news is first and foremost a business, it is inevitable that the more controversial aspects of biotechnology are increasingly finding their way into the headlines – after all, topicality rather than significance tends to sell newspapers. With column inches and broadcast time at a premium, science is often reduced to sound bites, issues are oversimplified, and viewpoints are polarized into universal acceptance or rejection of a particular technology, with little room for reasoned discourse. Scientific data is often molded into ‘publication format’ which allows several different types of manipulation to take place (Parenti, 1998): •

•

Suppression by omission occurs where a complex story is reduced into a concise report, and important facts are left out. Many journalists would argue that selectivity is necessary when condensing complex stories, but if editing is carried out by a nonexpert it can lead to the accidental omission of key data. Furthermore, the selection process is ultimately subject to individual bias and can either unintentionally or intentionally lead to ‘spinning’ of the story to cast it in either an unrealistically positive or negative light. Preemptive assumption occurs when a publisher accepts information given to them without further examination, and publishes it as fact. An example is the immense media coverage initially given to Greenpeace’s call for a ban on chlorine and organochlorides, without seeking concrete data on the consequences of such a ban on drinking water safety.

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•

Framing refers to how facts are presented, either by emphasis or positioning. Examples include the amount of exposure, the placement (front page or buried within, lead story or last), the tone of presentation (sympathetic or antagonistic), the nature of headlines and photographs, and the representation of key facts. Labeling predefines a subject by giving it a positive or negative label without the benefit of any explanatory details. The media often use misleading terms like ‘Frakenfood’ or ‘killer corn’ for GE crops, thus telling their readers what to think before they get the chance to think about it themselves. The use of malevolent metaphors as headlines (e.g. ‘Frankenstein food: a boon for mankind or a timebomb?’), unfairly transfers a negative emotional burden to the reader who now approaches the article with a preconceived idea that such food is potentially dangerous (Roberts, 1999). This contrasts sharply with the description of novel recombinant cancer drugs as ‘magic bullets’ and ‘wonder-drugs’, and it is clear that medical stories are often labeled as ‘hope’ whereas GE is often labeled as ‘warning’. It is interesting to speculate how different the last 10 years in the EU may have unfolded had we seen more headlines such as ‘Superfoods to tackle global hunger’ or even ‘Frankenstein food beats starvation’ (Lavelle, 2007). Where GE and medicine combine, as in transgenic plants producing pharmaceutical proteins, the media has been cautiously upbeat: ‘Transgenic maize to cure HIV/AIDS’ (GMO Africa, 2008).

The public perception of GE crops and their products depends on the ways in which the public process the information presented to them. Most public communication about science is channeled through daily newspapers, special-interest magazines and television. However the problem lies in instances where articles written by researchers themselves are occasionally published in newspapers or in popular science magazines. About 90% of newspaper science articles are written by science journalists, most of whom do not have scientific qualifications. If there is a mistake or misinterpretation of the original data, this is transferred directly to the public who are driven by concerns about the impact that the technology will have on societal and social structures and relationships. In doing so, the media unnecessarily amplifies ‘perceived risk’ and these reports can heighten public fear of certain areas (e.g. biotechnology) according to the Eurobarometer (http://europa.eu.int/comm/public_opinion). There is further evidence that the broad and often negative media coverage of biotechnology has raised public awareness, influenced public perceptions, and altered the public agenda on GE foods in Europe and elsewhere (Bauer et al., 1998). Some of the public carefully weigh potential benefits more heavily than risks, while others form their biotechnology attitudes solely on media sound bites. Different cultural groups may address the risks and benefits of media-reported science in different ways and this can create conflict when such groups try to reconcile the complex health, environmental and social risk/benefit balance in the international exchange of GE products (Finucane and Holup, 2005). The public often employ social trust to cope with any lack of knowledge about a new technology (Earle and Cvetkovich, 1995; Siegrist and Cvetkovich, 2000). This refers to people’s willingness to rely on experts and institutions in the management of risks and technologies (Earle and Cvetkovich, 1995). Such trust in regulators, science and industry is particularly important if the public perceive that they have no control over a particular event or activity, but must confer responsibility for ensuring consumer protection or public welfare onto others, as arguably is the case with GE foods

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(Frewer et al., 2004). Trust in the government may be lost due to previous poor management of food scandals, for example the dioxin contamination in Belgium, the hormonal treatment of pigs in Bavaria, or the illegal use of certain herbicides in organic farming in Germany. All these led to the loss of trust in governments, and some see the public as a victim of misleading information from non-governmental organizations (NGOs) or newspapers that are seeking to increase their circulation numbers, both of which obstruct impartial scientific reasoning. In Europe, the ‘tabloids’ are seen as a primary news source by a large segment of the public, despite their tendency towards sensationalism. This is more widely recognized in the US, and most written news coverage of biotechnology tends to come from higher-quality publications which present a more balanced view (AgBioView, 2003). Furthermore, transatlantic attitudes are thought to differ because Americans place more trust in government, regulators and industry than Europeans, who have been discouraged by regular food crises (AgBioView, 2003).

Figure 2. The acceptance of GM (GE) food (adapted from Costa-Font et al., 2008).

Perceptions are generally assumed to affect behavior. Therefore the issue of consumer acceptance depends directly on how much trust is placed on the available risk-benefit information (FIGURE 2). Public concerns and associated risk-benefit behavior have direct long-term consequences for human health, food safety and security, economic expansion and international regulation, consequences the media often fail to understand.

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Breaking the Barriers The media have improved their biotechnology reporting over the last few years, but much remains to be done. Journalists and editors need to seek more knowledge on subjects such as biotechnology, and should be able to provide more detailed and balanced reports based more on science not politics in order to leave freedom of acceptance in the hands of the public. Meanwhile, scientists need to popularize their achievements through means of communication other than the media, such as public lectures, conferences and science open days, or through direct interactions with newspaper editors. Conveying accurate information can reduce fears of new technologies, and scientists should not ignore such opportunities.

VII. Conclusions and Outlook GE crops have the potential to address some of the major elements of food insecurity both directly (by increasing the availability of food) and indirectly (by reducing poverty in developing countries). Crop failure could be averted through the deployment of plants resistant to pests and diseases and tolerant of environmental stress, and the latter could also allow the development of marginal lands for agriculture. The modification of plant architecture could increase yields and sturdiness by diverting biomass from stems and stalks to the edible organs. Overall yields could be increased by manipulating photosynthesis, carbon and nitrogen metabolism, modifying plant development or improving nutrient uptake. It is thought that such measures could improve grain yields by up to 50% over the next 50 years (Christou and Twyman, 2004). GE strategies can also be used to improve nutritional properties of plants by altering primary metabolism to increase the levels of essential amino acids, long-chain fatty acids and vitamins, and by favoring the accumulation of minerals in a bioavailable form. Golden Rice, a GE strain of rice engineered to produce provitamin A, provides a model for the development of nutritionally enhanced crops for the subsistence farmers of developing countries. As well as addressing the yields of staple foods, GE strategies can also be used to develop other commodity crops, including fiber producers, biofuel crops and specialized GE varieties producing valuable secondary products, recombinant proteins and polymers. Before any of these advances can really achieve fruition, the world needs to focus on the political hurdles that currently frustrate attempts to bring much needed technological benefits to the people who most need them. Politics and trade have always been closely associated with agricultural production and distribution, and disagreements between the US and EU over GE crops and their products have resulted in regulatory disharmony and disarray, with unworkable rules and a stagnant international market. These policies have been influenced by public opinion, in turn influenced by a sensationalist and manipulative media, ultimately creating a vicious cycle in which general distrust and fear of GE technology means that more newspapers will be sold by running negative stories, which will put pressure on the government and regulators to provide a strict regulatory environment which will in turn restrict the basic research and applications of the technology. GE technology offers countless opportunities to provide rapid help to the world’s poorest people, allowing them to develop a sustainable subsistence agriculture that has the potential to expand to include cash crops and specialist products for wealth generation. It is our fond hope that the current stranglehold on

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GE development, deployment, trade and distribution will eventually be overcome, allowing the benefits of GE technology to be enjoyed by those most in need.

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In: Food Science and Security Editors: L. Amsel and L. Hirsch, pp. 73-97

ISBN: 978-1-60692-977-3 © 2009 Nova Science Publishers, Inc.

Chapter 2

BIO (SINGLE CELL) PROTEIN: ISSUES OF PRODUCTION, TOXINS AND COMMERCIALISATION STATUS Pogaku Ravindra11, Ravinder Rudravaram2, Anuj Kumar Chandel3, Linga Venkateswar Rao2, and Yim Zhi Hui1 1

School of Engineering and Information Technology, Universiti Malaysia Sabah, Kota Kinabalu, 88999, Malaysia 2 Department of Microbiology, Osmania University, Hyderabad-500 007 (India) 3 Department of Biotechnology, Jawaharlal Nehru Technological University, Hyderabad-500 007 (India)

Abstract The alarming rate of population growth and a regular depletion in food production and food resources are the important reasons which make the dire need of hour to look out new viable options for food and feed sources. Based on the scientific developments particularly in industrial microbiology, one feasible solution could be the consumption of microorganisms as human food and animal feed supplements. Human has used microbial based products like alcoholic beverages, curd, cheese, yogurt, soya sauce and idly even before the starting of civilization. Due to research developments caused in scientific arena in last two decades, (Bio) Single cell protein (SCP) has roused new attentions towards its supplement as human food, feed or supplements as staple diet. There are several benefits using SCP as food or feed, viz their rapid growth rate and high protein content. The microorganisms involved as SCP have the ability to utilize cheap and plentiful available feedstock for their growth and energy have made them an attractive option. However, in spite of lab based successful stories, only a limited number of commercial SCP production plants have been seen worldwide. This review analyzes the possibility of SCP production, various raw materials for its production, and available microorganisms with cultivation methods, the toxicity assessment and their removal.

1

E-mail address: dr_ [email protected]. Telephone: 006 088 320000 ext 3048. Mobile: 006 013, 87666634. Fax: 006 088 320348. Corresponding Author: Dr. Pogaku Ravindra School of Engineering and Information Technology, Universiti Malaysia Sabah, Kota Kinabalu, 88999, Malaysia.

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Pogaku Ravindra, Ravinder Rudravaram, Anuj Kumar Chandel et al. Also, new developments and risk assessment using SCP along with worldwide industrial SCP production have been discussed.

Keywords: Single cell protein (SCP), Solid state fermentation (SSF), Toxins, Nucleic acid.

1. Introduction Single Cell Proteins are microbiologically produced protein and has been termed at Massachusetts Institute of Technology (MIT) in 1966 by Carrol Wilson. It can be derived from a variety of micro-organisms, both unicellular and multicellular- namely, bacteria, yeast, fungi, or microscopic algae. These potentially important food substances are not pure proteins but are, rather, dehydrated cells consisting of mixtures of proteins, lipids, carbohydrates, nucleic acids and a variety of other non-protein nitrogenous compounds, vitamins, and inorganic compounds (Anupma and Ravindra, 2000). Earth has always seen fast and drastic changes towards progressive side. It has fulfilled all our needs and necessities, but the changes are so swift that the resources provided by it are falling short. First and foremost reason for it is geometric progression of population explosion (Kapitza, 1996). Population growth has ill effects on the economy of the country, and also in social and political aspects. Further it has direct impact on the availability of food (LevyCosta, Sichieri, dos Santos, Pontes and Monteiro, 2005). In the starting of the 20th century, population of the world was less than 2 billion people and the population had grown to 6 billion when this century ended. The increment of the human population in the last half-century is unprecedented. In the present scenario, world population is projected to continue to rise exponentially for next 50 years (Smil, 2005). India is the second most populous country in the world after China. Over the past four decades, the size of India's population has more than doubled and is projected to reach 1.22 billion by 2015 and 1.40 billion by 2030, despite the considerable slowdown in fertility and population growth rates. Three different scenarios of population explosion could be regardless of which projection one uses it is clear that at least 1.3 billion people will be added to the world population by next 25 years (Figure 1). Population spurt is seen both in developing and developed countries except for the difference in rate of explosion (Chamie, 2004). The developing countries owe about 98% of total population increase while developed countries add the rest of 2% difference. The population growth diverts our thinking towards the availability of basic needs for this enormous number. Apart from increased population, rapid urbanization and fast reduction of area under cultivation have further aggravated the problem of availability of food. Proteins are useful for body building, repair and maintenance of tissues, osmotic pressure, and chemical coordination (in the form of hormones). In extreme condition protein may also be used for the production of energy in human body (ZoBell, Olson, Wiedmeier, and Stonecipher, 2004). The nutritive value of proteins depends on its amino acid composition. Amino acids are the bricks with which tissue protein is built and replaced.

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Figure 1. Projected World Population (Chamie, 2004).

Table 1. Daily requirement of essential amino acids (Lehninger, 1990) Amino acid Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Valine

Quantity (g) 0+ Unknown * 1.30 2.02 1.50 2.02 2.02 0.91 0.46 1.50

+Required by infants and growing children * Essential but precise requirement not yet established

There are some 20 amino acids commonly found in dietary proteins, out of which 10 amino acids cannot be synthesized by human beings and they have to be supplied through diet. These are called essential amino acids (Koehnle, Russell and Gietzen, 2003). Table 1 shows the daily requirement of essential amino acids. Adults require 9 essential amino acids while children require 10, the extra amino acid being arginine. Although the liver normally makes arginine as a step in the synthesis of urea, children cannot make arginine fast enough to support both urea synthesis and the synthesis of body proteins. Dietary protein requirement varies from 30 to 92 g/day. To fulfill this minimum protein requirement, constant research work on providing alternative sources of proteins in form of microorganisms is in progress world over. In this direction the research activities have given some encouraging results over a decade (Anupma et al., 2000; Ravinder, Venkateshwar Rao and Ravindra, 2003; Ravinder,

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Chandel, Venkateshwer and Ravindra, 2006; Berge, Baeverfjord, Skrede and Storebakken, 2005). This review deals with single cell protein (SCP) production technologies, available microorganisms, toxins and their removal, applications and industries involved in production of SCP world wide.

2. Microorganisms: An Assest in SCP Production! Microorganisms have excelled at producing primary and secondary metabolites from a variety of raw carbohydrates since billions of years under varying cultivation processes for the production of value-added products. In current vogue, the results of studying giant ‘microbial libraries’ for microbial conversion of alternative and cheap carbohydrates can serve as a raw material for SCP production (Singh and Kumar, 2007). Over the centuries microorganisms have been used by human as biomass in production of various products such as alcoholic drinks, cheese, yogurt and soybean paste (Tuse, 1984). Today, there are many works in food and feed production using microorganisms in the world. Usefulness of SCP is determined by the parameters related to nutritional values and its contents. These parameters are nutrients, vitamins, nitrogen, carbohydrates, fats, cell wall, nucleic acids, and profile of protein and amino acids. Microbial protein is a non-traditional protein; it is not a palatable, desirable food and must be incorporated directly or indirectly into other foods (Anupma et al., 2000). Consumption of microorganisms as a source of protein seems an attractive and sustainable option due to several advantages. The period of doubling time of microorganisms is shorter than the other living beings for SCP production (Table 2). In recent years, SCP is being produced from many species of microorganisms. These are fungi, bacteria and algae, which are widely used practically. Use of bacteria and fungi for SCP production is more economical when grown on easily available wastes (Pandey, Soccol and Mithchell, 2000a; Pandey, Soccol, Nigam, Soccol, Vandenberghe and Mohan, 2000b; Rudravaram, Chandel, Linga, and Ravindra, 2006). The reason for preferring microorganisms is their fast growth with high protein ratio. The acceptance of a species as food or feed depends on growth rate, substrate utilization and toxicity (Ravinder PhD Thesis, 2003; Ravinder et al., 2003). Filamentous fungi are preferred in SCP production due to easy growth on lignocellulosic wastes and ease of separation from its media (Westhuizen and Pretorius, 1996). Table 2. The doubling period of mass for some living beings (Bailey and Ollis, 1986) ORGANISMS Bacteria and yeast Filamentous fungi and algae Grass and some plants Chicken Pig Cattle Human being

TIME FOR DOUBLING MASS 20-120 minutes 2-6 hours 1-2 weeks 2-4 weeks 4-6 weeks 1-2 months 3-6 months

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2.1. Yeast Yeast is commonly used organism in the production of biomass, probably because it is already accepted both in the human food and animal feed industries. Yeast-based processes are the farthest advanced towards commercial production, followed by bacterial processes (Kourkoutas, Sipsas, Papavasiliou and Koutinas, 2007). Yeasts have many convenient characteristics, such as the ability to use a wide variety of substrates like hexoses, pentoses, and hydrocarbons (Moeini, Nahvi and Tavassoli, 2004), susceptibility to induced and genetic variation, ability to flocculate (Labuza, 1975) and high nutritional value (Yanez, Ballester, Fernandez, Gattos and Mönckeberg, 1972). However, attention has often been drawn to the fact that yeast appears to be deficient in essential sulphur amino acids (Worgan, 1973).

2.2. Algae Unicellular algae are the smallest and simplest photosynthetic apparatus for effective utilization of plentiful solar energy (Venkatratnam, 1978). The advantages and disadvantages of growing algae as protein sources were discussed by several investigators (Chae, Hwang and Shin, 2006). The use of certain algae in foods in certain parts of the world has been well documented. The blue green algae Spirulina is collected and eaten by natives around Lake Chand in Africa traditionally. In countries like Tartary, Mangolia and China it was found that algae was eaten by people and also marketed. The autotrophic, mixotrophic and heterotrophic methods of production of microalgae (Fabregas and Herrero, 1985), Chlorella in Asia has been reviewed by Kawaguchi (1980). Algae processes are still short of full-scale development because they are limited by the requirements for light over a major portion of the year and for a continuous supply of CO2 or other carbon source (Venkatratnam, 1978). In Taiwan, however, plants for the production of Chlorella feeds using methane generated from manure are now in operation (Srinivasan and Fleenor, 1972).

2.3. Bacteria A number of different species of bacteria are used in SCP production. Bacteria have a slight advantage over the other microorganisms as a food source, because of their higher growth rates and relatively higher protein content and sulphur-containing amino acids (Kurbanuglu and Algur, 2002). Bacterial SCP is high in protein and certain essential amino acids. The crude protein content is around 80% of the total dry weight. The nucleic acid content, especially RNA, is very high on a dry weight basis and is reported to be 15-16%. Bacterial SCP is rich in methionine, around 2.2-3.0%, which is comparatively higher than that of algae (1.4-2.6%) and fungi (1.8-2.5%) (Singh, 1998). The essential amino acid composition of different Lactobacilli group appears comparable to that of the FAO reference protein and SCP from other sources (Erdman, Bergen and Reddy, 1977). The use of bacteria was objected because of their size, which makes harvesting difficult without the use of flocculants or thickeners (Dabbah, 1970). However, a very limited success has been achieved so far with bacterial biomass consumption as human food or animal feed. This is partly because of high nucleic acid content and partly due to the fear of bacterial toxins (Fossum and Almlid, 1977). Production

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of SCP on SSF by utilizing lignocellulosic waste is not practiced due to less moisture content in lignocellulosic material (Han and Srinivasan, 1968)

2.4. Fungi The filamentous fungi are extensively used in SCP production. Many strains such as Aspergillus, Fusarium, Chaetomium, Rhizopus, Tricoderma, are being used on commercial scale for this purpose (Solomans, 1985). Fungi have the ability to provide form and texture (Humphrey, 1975), and hence can be harvested with ease; also, the cost of production may be reduced. Like algae, fungi generally have low nucleic acid content, and accordingly, the dangers of kidney stones and gout are not great even without processing the biomass to lower nucleic acid content (Lipinsky, 1974). Another advantage is that fungi can prosper on a variety of carbohydrates and they are best suited for agroresidues, although growth rates vary considerably with different substrates (Anderson, 1975).

3. Sources for SCP Production and Cultivation Methods Bacteria and fungi utilize a variety of substrates (Tannenbaum and Wang, 1975) in SCP production. Table 3 shows the macromolecular composition and general properties of microorganisms. The main substrate required for algae are carbondioxide and sunlight (Kawaguchi, 1980). Fungal species are cultured on different substrates, mostly cheap wastes, which supply the carbon and nitrogen for growth (Smith, Fermor and Zadraz¡il, 1988). Among various substrates, lignocellulosic material such as wood from angiosperms and gymnosperms, grasses, leaves, wastes from paper manufacture, sugarcane bagasse, wheat straw, wheat bran, rice bran, groundnut shell and other agricultural wastes are cheap source for SCP production (Gupte and Madamwar, 1997; Chaudhary and Sharma, 2005). Lignocellulose is the most abundant organic material on earth, representing 50% of the total plant biomass and presenting an estimated annual production of 5.0 x 1010 tons (Kuhad and Singh, 1993). Plant production from agriculture contributes only a small part of this overall production, but still represents a considerable amount, 1.23 x 108 tons per year (Kuhad et al., 1993). Approximately half of the agricultural biomass produced is not used as food or feed (Smith et al., 1988). The major part of these agricultural residues, if not burned, is fragmented and added to soil as organic fertilizer (Zadrazil, Brunnert and Grabbe, 1983). Agro-industrial wastes from cereals, wood and fruit processing industry wastes have also been explored for SCP production worldwide. For example the availability of rice bran, which is almost 40 million metric tons rice bran produced every year, has been discarded as unfit for human consumption. After the oil extraction it is called deoiled rice bran (DOB) which contains 9% protein and 36% cellulose and has been thoroughly exploited for protein enrichment using Aspergillus oryzae MTCC1846 (Ravinder et al., 2003, 2006). Waste recycling has been advanced as a method for preventing environmental decay and increasing food supplies. The potential benefits from successful recycling of agricultural wastes are enormous. It may be the only method for large-scale protein production that does not require a concomitant increase in energy consumption. In addition, it may be the most effective method for producing animal and human food from lignocellulose materials apart

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from their use in bioethanol production (Chandel, Rudravaram, Narasu ML, Rao and Ravindra, 2007a; Chandel et al., 2007b). The lignocellulosic waste materials contain cellulose, hemicellulose and lignin. Cellulose is most abundant of these polysaccharides. These sources are attractive due to their easy availability and low cost (Lin and Tanaka, 2006). These materials however have to be pretreated with some physical or chemical methods since lignin-cellulose complex is tough to unravel and does not allow easy accessibility of enzymes in enzymatic hydrolysis of lignocellulose (Chandel et al., 2007a; Chandel, Kapoor, Singh and Kuhad, 2007c). Table 3. Macromolecular Composition and General Properties of Microorganisms (Champagnat, Vernet, Laine and Filosa, 1963) PARAMETERS

BACTERIA

YEAST

FUNGI

ALGAE

Doubling time (hours) Crude protein (% dry cell weight) Nucleic acids (%) Carbohydrates and fats (%) Ash content (%) Temperature range (°C) pH range

1-3 40-80 8-20 10-30 4-10 22-55 5-7

2-6 40-60 5-15 10-40 4-10 25-40 3-5

5-12 30-45 6-13 10-45 4-10 25-50 6-8

6-24 40-50 45-51a 34.6-45 5-8 25-32 6.9-9.6

a. Percentage

4. Toxins of Microflora and Their Removal Toxins are secondary metabolites produced by fungi (Bennett and Keller, 1997) and bacteria (Blancou, Calvet and Riviere, 1978). Algae in general do not produce harmful toxin (Leonard and Compere, 1967). The toxicity of SCP product must be assessed before marketing, since SCP is considered as animal feed and human foods (Anupama et al., 2000).

4.1. Toxins Produced by Bacteria and Their Removal Bacteria produce exotoxins such as enterotoxin, erythrogenic toxin, alpha-toxin, and neurotoxin (Ekenvall, Dolling, Gothe, Ebbinghaus, von Stedingk and Wasserman, 1983). Endotoxins are an integral part of the cell walls of gram-negative bacteria and are liberated upon lysis. Endotoxins are lipopolysaccharides in nature. Endotoxins cause fever in the host and are fatal for laboratory animals at slightly higher doses than exotoxins (Powar and Daginawala, 1995). SCP from Pseudomonas species and Methylomonas methanica, when used for animal feed purposes, caused febrile reaction and high titres of IgG and IgM due to endotoxins (Ekenvall et al., 1983). Exotoxins can be easily removed, as these are present in soluble form in the medium (Powar et al, 1995). They are sensitive to temperatures above 60 °C. Moreover, 50% alcohol, formaldehyde and dilute acids can denature exotoxins or convert them into non-toxic toxoids (Blancou et al., 1978). Toxoids are beneficial for artificial immunization (Powar et al, 1995). On the other side, removal of endotoxins is difficult as they are part of cellular components of few gram-negative bacteria. Formation of endotoxins can only be prevented by genetic engineering, where the activity of genes controlling the formation of the unwanted toxins can be modified

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or suppressed (Anupama et al., 2000). SCP produced by methanotropic and heterotrophic bacteria generally contains a large amount of nucleic acid making the product less suitable for human consumption.

4.2. Toxins Produced by Fungi (Mycotoxins) and Their Removal Minute of mycotoxins are able to induce allergies, diseases, and rashes on skin, neurotoxicity and other disorders. There are many reports on mycotoxins but aflatoxins are the most important and best understood (Eaton and Groopman, 1994). Prominent fungal toxins include aflatoxins of type B1, B2, G1 and G2 Aspergillus flavus, citrinin from Penicillium citrinum, trichothecenes and zearalanone from Fusarium species and ergotamine from Claviceps species (Bennett et al., 1997). Aflatoxins are produced by A. flavus, A. parasiticus and A. oryzae (Schlegel, 1996). Aflatoxins cause human liver cancer (Eaton et al., 1994). Apart from aflatoxins, ochratoxins are also important mycotoxins. Ochratoxin are structurally related groups of pentaketides. Ochratoxin A is the most abundant and toxic of the five metabolites in the Ochratoxin group. These metabolites have been found to occur in Aspergillus and Penicillium species and cause damage to the liver as well as kidneys (Varga, Kevel, Rinyu, Teren and Kozakiewicz, 1996). Trichothecenes are considered the next most important group among mycotoxins. Trichothecenes have a 12, 13 epoxytrichothec-9-ene nucleus which able to induce dermal toxicity and several hematopoietic effects (Joffe, 1986).

Figure 2. Pathways of nucleic acid metabolism (Calloway, 1974).

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Ammoniation is the most successful method to remove mycotoxins as this method can reduce aflatoxin levels by 99% (Park and Liang, 1993). Co-culture of A. flavus with other microbes results in reduction of toxins production (Cotty, Bayman and Egel, 1994). The biosynthetic pathways for both aflatoxins (Bennet et al., 1997) and trichothecenes were studied (Desjardins, Hohn and McCormick, 1993). Molecular biology techniques have been exploited for detoxification. Molecular dissection and elimination of genes responsible for mycotoxin synthesis is developing into an exciting area of contemporary mycotoxin research (Keller, Cleveland and Bhatnagar, 1992). The techniques of cloning, probing, sequencing, expression libraries, transcript mapping, gene disruption, and chromosome walking have been employed to isolate the aflatoxin pathway clusters from A. flavus and A. parasiticus (Bennett et al, 1997). Studies that identify the molecular determinants regulating mycotoxin production are promising rational control strategies. For example, aflR is the regulatory gene controlling the production of both aflatoxins and sterigmatocystin in Aspergillus. This forms the target gene to be inhibited for the future control of mycotoxin production in Aspergillus (Yu, Butchko and Fernandes, 1996). When Aspergillus sp is impaired in asexual reproduction, the production of aflatoxins is inhibited (Kale, Bhatnagar and Bennett, 1994). A report describes how a plasmid vector (pDEL2) was engineered to introduce a deletion within the aflatoxin biosynthesis gene cluster of A. parasiticus. Subsequent aflatoxin precursor feeding studies confirmed that the enzyme activities associated with the deleted genes were absent (Cary, Barnaby, Ehrlic, and Bhatnagar, 1999). There are also reports that products of plant defense pathways (i.e. lipoxygenase or the jasmonate pathway) can inhibit aflatoxin production (Zeringue, 1996).

5. Nucleic Acids and Its Removal Bacterial SCP products may have nucleic acids as high as 16% of dry weight. Human consumption greater than 2 g nucleic acid equivalent per day may lead to kidney stone formation and gout (Calloway, 1974). Figure 2 shows the metabolic pathway of nucleic acids. Uric acid, an important metabolite, is slightly soluble at physiologic pH and if the blood uric acid content is elevated, crystals may form in the joints, causing gout or gouty arthritis and urinary problems (Dabbah, 1970). In rapidly proliferating microbial cells RNA forms the bulk of the nucleic acids (Singh, 1998). The RNA content of yeast cells is known to be dependent on the culture conditions and carbon/nitrogen (C/N) ratios. The nucleic acid level can be reduced by several means. These include activation of endogenous RNAase by brief heat treatment up to 60-70 °C for 20 min, alkaline hydrolysis of nucleic acids, modifications of cultural conditions with respect to nitrogen, carbon, phosphorous and zinc content or chemical extraction and removal of nucleic acid (Worgan, 1973). Some researchers have extensively discussed safety evaluation of SCP products for human food and animal feed, and specific methods to reduce the nucleic acids are also reported (Taylor, Lucas, Gable and Graber, 1974). Purine content in baker's yeast can be reduced by chemical treatment and autolytic methods (Trevelyan, 1976a, b). Reduction of nucleic acids can be achieved by the endogenous polynucleotide phosphorylase and RNase in Brevibacterium (Yang, Thayer and Yang, 1979). Two derivatives of pancreatic RNase and an endonuclease of Staphylococcus aureus, immobilized on corncobs, have been used to reduce the percentage of nucleic acids in SCP concentrates of yeasts from 5-15% to 0.5% with a protein loss of only

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6% after treatment (Martinez, Sanchez-Montero, Sinisterra and Ballesteros, 1991). Immobilized nucleases like benzonases on corncobs were also used to reduce the nucleic acid content in protein concentrates. The percentage of DNA was reported to be reduced to 3-6% and RNA to 50% with loss of protein being only 1% (Moreno et al., 1991). An immobilized pancreatic RNase was also investigated for the degradation of yeast ribonucleic acid. The rapid reaction rates obtainable at relatively low temperatures offer a potential alternative method of purifying yeast SCP with minimal loss of derived protein (Dale and White, 1979). Methods for reduction of nucleic acid content in SCP obtained from gas oil are also reported (Abou-Zeid, Khan and Abulnaja, 1995).

6. New Developments for Improvement in SCP Production Timely interventions such as strain improvement through mutagenesis, gene cloning and expression, and optimization of potential fermentation parameters enhance the production of selective metabolites (Singh et al., 2007). Mutation has been a successful method for improving the yield of biomass. Pseudomonas methylotrophus was isolated by Imperial Chemical Industries (1CI) and then significantly improved through genetic engineering and physiological development. When glutamate dehydrogenase system isolated from Escherichia coli was transferred to GOGAT mutant of P. methylotrophus by genetic engineering, improved nitrogen assimilation was observed by the mutant. It was used commercially for yield improvement in terms of raw protein, pure protein, and cell dry weight and μmax was significant (Gow, Littlehailes, Smith, and Walter, 1975). Yields of the engineered organisms were about 5% greater than the parent organism (Balasubramanian, Bryce, Dharmalingam, Green and Jayaraman, 1998). The use of genetic engineering is not restricted to designing and production of microbes with high protein yield capability alone. It also finds an application in the improvement of downstream processing in post-fermentation stage. It is used in the production of 'designer' or 'recombinant' proteins of high nutritive value and their subsequent purification. Novel techniques like specifically designed tags or by modification of sequences with target-gene product are used for efficient recovery of native and modified proteins (Murby, Uhlen and Stahl, 1996). Improvement in protein recovery can also be achieved by suppression or elimination of protease enzyme activity in the microbial cells. Mutants with modified protease gene sequence can be isolated and used for the purpose. For example, LDHP1 strain of S. cerevisiae, a protease deficient mutant, was produced due to osmotic shock and showed an improved beta-galactosidase yield (Becerra, Cerdan and Gonzalez-Siso, 1997). Recombinant DNA technology has also been used to isolate a mutant gene which can enhance the overall protein yield or enables the organism to produce high amount of specific amino acids like glutamate, tryptophan, phenylalanine and others along with a high protein yielding capacity. Such genes can be combined together by recombination technique and incorporated in the organism, which has a wide substrate range. A mutant variety of S. cerevisiae produced high concentration of intracellular glutamate (Hill, 1994). Mutants of S. cerevisiae, which were resistant to toxic ethionine, showed accumulation of methionine as high as 163 times that of parental strain. Moeini et al., (2004) used mixed culture of the isolated yeast strains with S. cerevisiae was used in order to increase the biomass yield and BOD removal. The highest biomass yield (22.38 g/l) and

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reduction of initial BOD from 30000 to 3450 mg/l were obtained with the mixed culture of K. lactis (M2) and S. cerevisiae. The property of high accumulation in mutant was gained due to enhanced specific activity and reduced feedback inhibition by threonine of the enzyme aspartokinase and homoserine (Martinez-Force and Benitez, 1993). About 180,000 tons of LLysine, is needed annually to improve the nutritive value of food and SCP. Mutant Corynebacterium glutamicum, which is auxotrophic for alanine, sensitive to betafluoropyruvate and resistant to alpha-chlorocaprolactum, could produce 70 g/1 L-Lysine. On improvement of strain by recombinant DNA technique the yield was enhanced to 98 g/l of LLysine (Sahm, 1995). The yield of expensive amino acids like L-Threonine can also be improved by use of recombinant Escherichia coli (Tsukada and Sugimori, 1971) and that of phenylalanine by genetically manipulated strain of Rhodotorula rubra (De Boer and Dijkhuizen, 1990). High cost restricts the use of L-Tryptophan to humans. Enhanced yield can be achieved by classical mutation and genetic engineering. Unification of four mutations by recombination and their incorporation in S.cerevisiae helped to produce 12 g/l Ltryptophan (Moller, 1994). Strain improvement is done by complementing the auxotrophic mutants by wild type genes (Jungehulsing, Arntz, Smit and Tudzynski, 1994). The substrate-range of such strains can be extensively widened when compared to the auxotrophic variety. This would allow the utilization of a variety of unused substrates for the growth of microbes for SCP production.

6.1. Application of New Substrates in Solid State Fermentation (SSF) for SCP Production and Commercialization of SCP SSF has been traditionally followed for production of fermented foods since ancient times. Some of these foods have worldwide popularity and are used for preparation of delicious curries (Nigam and Vogel, 1990). SSF can be commercially applied for the production of organic acids, mushrooms, cheese and upgradation of lignocellulose, composting, dough fermentation, mycotoxins, starter inoculum, ethanol, cocoa, vanilla, coffee, insecticides and biodegradation of wastes (Pandey et al., 2000a). In recent times, SSF has applications in the field of SCP production in an extensive way due to its advantage of utilization of wastes to produce value-added product (Ravinder et al., 2003; 2006). Crop residues represent potential source of dietary energy to ruminants if the protein content can be increased (Robinson and Poonam, 2002). Crop residues are renewable and could be a potential raw material for SCP production. They represent a potential solution to feeding animals. Table 4 shows various substrates and micro organisms used for protein enrichment (SCP) by solid state fermentation. Cassava starch an important food for millions of Africa, Asia and South America contains low protein, vitamins and sulphur containing amino acid (Pandey et al., 2000b). Soccol, Leon, Rouses and Raimbault (1993) investigated biotranformation of cassava for nutritional improvement. Apple pomace has been used protein enrichment under SSF by A. niger as food and feed for pigs (Nigam and Singh, 1996). The use of protein enrichment of apple pomase by use of co-culture of moulds and yeast has been explored by Bhalla and Joshi (1994). Carob pods contain high amounts of tannins, which have adverse effect on animal growth.

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Pogaku Ravindra, Ravinder Rudravaram, Anuj Kumar Chandel et al. Table 4. Substrates and microorganisms used for protein enrichment by SSF

Substrate Deoiled rice bran Ram horn

Rice bran Apple pomace

Carob pods Canola meal Wheat straw Coffee pulp Rape-seed meal Sago starch Sugar beet pulp

Microorganism A. oryzae Bacillus cereus NRRL B3711, Bacillus subtilis NRRL NRS-744, Escherichia coli Aspergillus niger C.utilis sp. Kloeckera apicula, A .niger, Tricoderma sp. Agrocybe aegirata, Amellaria mellae Aspegillus carbonarius Chaetomium cellulolyticum Trichoderma reesei Aspergillus niger Aspergillus clavatus Fusarium oxysporum Rhizopus oligosporus

Sugar beet pulp

Trichoderma reesei Talaromyces Trichoderma viride

Manure

Chaetomium cellulolyticum

Soy beans Sawdust Lignocellulosic Substrates Cassava starch Banana wastes

Rhizopus oligosporus Chaetomium cellulolyticum Chaetomium cellulolyticum

Citrus peel extracts

Geotrichum candidum

Fodder beets

Saccharomyces cerevisiae

Maize

Aspergillus niger Saccharomyces sp. Schwanniomyces castelli

Bagasse

Citrus Pulp

Rhizopus oligosporus Aspergillus niger

Aspergillus niger, Trichoderma virideae

References Ravinder et al, 2003; 2006 Kurbanoglu et al., 2002

Anupama and Ravindra, 2001 Rahmat, Hodge, Manderson and Yu, 1995 Bhalla et al., 1994 Nicolini, Volpe, Pezzotti and Carilli, 1993 Alasheh and Duvnjak, 1995 Viesturs, Apsite, Laukevics, Ose and Bekers, 1981 Nicolini et al., 1993 Viesturs et al., 1981 Penbaloza, Molina, Brenes and Bressani, 1985 Cochrane, 1958 Mitchell, Gumbira-Said, Greenfield and Doelle, 1991 Considine, Mehra, Hackett, O'Rorke, Comerford and Coughlan, 1986 Reu, 1995 Durand and Chereau, 1988 Chahal, Vlach and Moo-Young, 1980 Mitchell and Lonsane, 1992 Baldensperger, Le Mer, Hannibal and Quinto, 1985 Ziino, Curto, Salvo, Signorino, Chifal and Giuffrida, 1999 Rodrguez, Echevarria, Rodriguez, Sierra, Daniel and Martinez, 1985 Han and Steinberg, 1986 Suacedo-Casteneda, Gutierrez-Rojas, Bacquet, Raimbault and VinegraGonzalez, 1992 De Gregorio, Mandalari, Arena, Nucita, Tripodo and Locurto, 2002

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Protein enrichment of 20% was obtained in 4 days of SSF of carob pods SSF with 83% of reduction in tannins (Smail, Salhi and Knapp, 1995). Recently some unconventional sources have been exploited for SCP production. Berge and colleagues (2005) used natural gas for production of bacterial protein meal (BPM) and evaluated growth. Ghaly and Kamal (2004) used cheese whey as source for SCP production from Kluyveromyces fragilis under the optimum conditions (air flow- 3 vvm, agitation- 400 rpm, temperature -31.6 0C. About 99% of lactose utilization was observed after 28 h of growth. Cheese whey was also used as carbon source for SCP production under fed batch culture using Kefir microflora in anaerobic conditions (Paraskevopaulou, Athanasiadis, Kanellaki, Bekatorou, Blekas and Kiosseoglou 2003). Deproteinized cheese whey concentrates were investigated for their suitability as substrates for the production of single-cell protein with Kluyveromyces marxianus CBS 6556 at 100 L fermenter scale (Schultz, Chang, Hauck, Reuss and Syldatk, 2006). Honda, Fukushi and Yamamoto (2006) optimized the waste water feeding for SCP production in anaerobic waste water treatment process utilizing purple non sulfur bacteria under mixed culture conditions. Maximum protein content was observed when waste water was fed as organism showed maximum growth. Tray bioreactors are very simple in design, with no forced aeration or mixing for solid substrate and can be excellent tool to be used in SCP production. These types of reactors are restricted in amount of substrate that can be fermented, as only thin layers can be used, in order to avoid over heating and to maintain aerobic condition (Nigam et al., 1996). Wooden trays were initially used for soy sauce production in Koji fermentations by A. niger (Pandey et al., 2000ab). The use of tray fermentors has remained largely unchanged, with only engineering advance being the use of modern materials such as plastic, alluminium and stainless steel. The advantage of modern trays over wooden trays is to make sterilization and cleaning easier and therefore reduce contamination or spoilage (Tengerdy and Szakacs, 2002). However the use of tray fermenters in large-scale production is limited as they require a large operational area and tend to labour intensive with mechanical handling also being difficult (Williams, 2002). There have been increasing demand for protein and recent high prices of fish meal and soya meal have shown that there is a definite market for SCP. Various companies such as British Petroleum, Ranks, Hovis, and McDougal-Dupont etc. have shown that pilot plant scale production of SCP is feasible (Table 5). Table 5. Some of the industrial establishments involved in the SCP Production Organization Ammoco, USA Bellyyeast, France British petroleum, France Hoechst, Germany ICI, U.K IFP, France Liquichemica, Italy Philips, USA Pekilo, Finland

Microorganisms applied C. utilis Kluveromyces Candida tropicalis Methylomonas clara Methylotrophus Candida tropicalis Candida maltosa Pichia sp, Torula sp. C. utilis

Used substrates for SCP production Ethanol Whey Hydrocarbons Methanol Methanol n-alkanes n-alkanes Sugar feed stock Starches

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8. Applications of SCP 8.1. As Feed The feed industry, through its technology in evaluating, processing and using new ingradients, is offering many opportunities to use substrates that were not used for feed production in the past, in order to minimize feed costs (Caldas, 1999). The acquisition of great volumes of these ingredients by feed industries has been an important factor in prompting vegetable processing industries to invest in the processing of by-products that were previously wasted. A feed is usually produced from agricultural products or by-products such as grains, cereals and residues. However, it is necessary to add micro-ingredients to improve levels of essential amino acids, vitamins and minerals (Edelstein, 1982). New additives such as metabolic modifiers, anti-microbial agents, probiotics and special minerals are also incorporated in order to supply essential nutrients, to enhance growth and to avoid diseases (Wenk, 2000). Even if the raw material is low cost, the addition of these micro-ingredients increases the final prices of feed. Therefore in searching for cheap raw materials to be used in feed production, it is crucial to obtain higher nutritional values than traditional substrates, in order to minimize the need for these high cost additives, and thereby maintain the economic viability of animal production (Villas-Boasa, Granato, Espositob and Mitchellc, 2002). On the other hand, many of the raw materials currently used in feed production represent products that would be advantageous if used in human nutrition (Brum, Bellaver, Zanotto, and Lima, 1999b). Therefore, it is desirable to use lower quality materials as the basic ingredient of animal feeds. However, most of the agro-industrial by-products and food industry wastes are poor in nutrients such as proteins and vitamins and are rich in fibres with low digestibility. Such materials are not suitable for non-ruminant animals and, in some cases; the digestibility is so low that they are not even suitable for ruminants (Brum Lima, Zanotto and Klein, 1999a; Brum et al., 1999b). In order to solve the problem, microorganisms (mainly fungi) are utilized to convert agroindustrial wastes in order to obtain products with higher nutritive value, especially in regard to protein and vitamin contents, and with increased digestibility (Kuhad, Singh, Tripathi, Saxena and Eriksson, 1997). Fungi, bacteria and seaweed, cultivated on a large scale, can be used as feed. These microorganisms are very attractive feed stuffs, because they can be cultivated on agroindustrial wastes, with production of large amounts of cells rich in proteins that commonly contain all the essential amino acids, in addition to favorably high vitamin and mineral levels (Kuhad et al, 1997; Brum et al, 1999 a,b). Further, the growth of microbes in lignocellulosic wastes is able to furnish all the hydrolytic enzymes often added in the preparation of feeds, and also makes the minerals more available for absorption by the animal. According to Pelczar, Chan and Krieg (1996), there are some advantages in using microorganisms in order to obtain nutritionally enriched raw materials for feed compared to the traditional way of formulating feeds. Microorganisms grow fast and produce protein in large amounts, because the protein content of microbial cells is very high, typically being around 600 g Kg -1. The substrate contain wastes or industrial by-products such as hydrocarbons from oil refineries, liquid effluents from the cellulose and paper industries, beet and sugarcane molasses and hydrolyzed wood wastes (Pelczar et al, 1996; Kuhad et al, 1997).

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Microbial by-products from traditional fermentation industries have already been used in various feed preparations for some time. These products are of major importance to the livestock feed industry. Fermentation of by-products from the brewing and distilling industries has long been utilized for livestock, particularly ruminants, mainly as protein and energy supplements. Numerous by-products of other fermentation industries are also used as sources of nutrients in compounded feed (Shaver and Batajoo, 1995). Nevertheless, some microbial products contain toxic metabolites that could represent serious risks for animal and human health. Therefore, the risk must be evaluated in all bioconversion processes in order to avoid possible toxicity to animals and humans (Brum et al., 1999a, b). SSF of lignocellulosic by-products has an advantage over submerged fermentation in that it requires only one-tenths of the fermenter size as compared with the latter. The dry matter content in the substrate of solid-state fermentation lies between 250 and 300 g Kg-1 and that in submerged fermentation between 20 and 30 g kg-1 (Moo-Young, Chahal, Swan and Robinson, 1977). The capacity of lignin degradation by fungi is influenced by the penetration of hyphae into the substrate and the extent of close physical contacts between the substrate and the degrading fungus. Fungal mycelium attaches physically to lignin (Tono, Tani and Ono, 1968) and agitation disrupts this contact (Kirk, Schultz, Conors, Lorenz, and Zeikus, 1978). Therefore, SSF or non-agitated culture in thin layers is always preferred over agitated submerged fermentation as far as lignin degradation is concerned.

8.2. SCP for Human Consumption Prepared Aerobacter aerogenes becomes unpleasantly slimy and palatable for food. In human feeding studies consumption of certain SCP sources lead to wide range of gastrointestinal complaints like bulky stools and flatulence, nausea, vomiting and diarrhea from killed and purified bacteria. Other pathological reactions are peeling of skin of the palm of the hand, soles of feet and itching, pain and edema of the toe; these symptoms are due to high nucleic acid. This is the major limitation to use SCP as food, unless safe process for removal of nucleic acid is established.

9. Risk Assessment of SCP in Food and Feed The purpose of toxicological testing is to define the potential hazard that the microbebased feed presents, not only to the animal but also to humans. As direct testing is impracticable, the evaluation is generally done with laboratory animals. It is recommended that a toxicological evaluation should involve both rodent and target species (Hoogerheid, Yamada, Littlehailes and Ohno, 1979), although the understanding of the basic pathology and biochemistry of far livestock is less well documented than that of the rodent. The extent of the testing procedure required depends upon the chemical analysis of the product. Materials that have a chemical composition close to that of natural occurring raw materials are unlikely to produce abnormal effects. In contrast, those products with abnormal composition resulting from the biosynthesis of compounds not present in natural feeds may need more careful evaluation. Thus, an extensive chemical analysis of the product is required. The potential for biological hazard is low for the microbially converted feeds so far evaluated (Banerjee, Azad, Vikineswary, Selvaraj and

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Mukherjee, 2000). Nevertheless, this hazard has to be continually evaluated by various biological studies when a new microbially fermented product is proposed. Tests involving rats are valuable during the development stages of the process. These tests may give some preliminary indication of possible cellular mutagenicity, potential carcinogenicity and teratogenicity (Sinskey and Batt, 1987).

Figure 3. Coordinated action for improvement in SCP production and its long term benefits.

The main problem associated with these types of investigation is the level of inclusion of the test material in the diet, and the subsequent effects that this has on the nutrient balance of the

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test diet (Hoogerheide et al, 1979). It is possible to induce a "toxicological change" due to simple alterations of dietary minerals or energy. These changes could be wrongly ascribed to microbially fermented feed and hence extreme care must be exercised in the formulation of the test diets. The toxicological evidence gained from the laboratory rodent species will form the principle basis for evaluation of hazard. Nevertheless, it is still necessary to test the converted substrate in the diets of farm animals. Long term life-span studies equivalent to those carried out with the rodent are not necessary. The normal economic life span of a farm animal forms a sensible basis for the evaluation of risk in the actual target species (Hoogerheide et al, 1979). These experiments with farm livestock should follow the same experimental concepts as those employed for the laboratory animal, namely biochemistry, hematology and terminal pathology (Hoogerheide et al, 1979). Mortality beyond that of normally experienced in farm livestock must be carefully evaluated. The experimental designs used in these studies should simulate those used for the laboratory animal, but due regard should be paid to the normal practices followed in animal production. The reproductive potential and the viability of the offspring are important both scientifically and commercially. Multigenerational experiments undertaken with both poultry and pigs need to have a sound statistical basis. Specific teratological investigations should also be made in at least one species apart the rodent (Hoogerheide et al., 1979).

Conclusion The review presents the technological assessments for SCP production along with the available microorganisms, various lignocellulosic substrates, toxins and their removal, risk assessment, and worldwide industrial scenario of SCP production. Among the class of microorganism used for SCP production, use of bacteria and fungi is more economical when grown on easily available substrates. The use of cheap and abundantly available sources viz. lignocellulosic residues, whey and petroleum products are attractive option over to costly sources (soymeal and fishmeal etc). The successful exploitation of these substrates is beneficial first, in terms of overall economics of SCP and secondly it would make less dependent on land for food production. The selection of microorganisms as food or feed depends upon their growth rate with high protein ratio, substrate utilization and toxicity profile. Recent biotechnology based tools and techniques has been exploited rationally less for SCP production in comparison to other microbial originated products. The beneficial aspects of SCP over conventional protein sources are- its independency on land, climate, and can be controlled genetically, cause less pollution while there are some factors like high nucleic acid content, cell wall indigestibility, high color and aroma which impair the use of SCP. Figure 3 presents coordinated actions which are crtically required for the improvement in SCP production and its long term benefits. However, despite lab based successful research, SCP could not achieve the desired level of success at industrial scale. To make program successful and economically viable, the above said reasons need to be curb upon for the successful implementation of SCP for food/feed consumption or as a supplement to make a staple diet.

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In: Food Science and Security Editors: L. Amsel and L. Hirsch, pp. 99-111

ISBN: 978-1-60692-977-3 © 2009 Nova Science Publishers, Inc.

Chapter 3

FOOD SECURITY AND MATERIALISM Michael W. Allen1 University of Sydney, Discipline of Marketing Economics and Business Building (H69) Room 506 Sydney NSW 2006, Australia

Abstract To better understand and address food insecurity, it may help to consider the motivations that drive some people to feel unusually worried/anxious about their food supply, resulting in hoarding and overeating. That is, why might someone, when food supplies are not genuinely threatened, be obsessed about the state of his or her food supply, and make food security a top life goal? Materialism may play a role. Here, it is posited that individuals may cope with childhood deprivation of material goods and a reliable, regular, and sufficient supply of food by developing materialistic tendencies. This may include, in addition to efforts to attain social security (e.g., improve socio-economic status), leanings toward the excessive accrual or consumption of material goods including food items. In short, some adults’ obsessive concerns about their food supply may, in part, be a symptom of materialistic tendencies developed in childhood in response to an insufficient food supply (and/or other goods), rather than current threats to food supply per se. A recent series of surveys and experiments reported by Allen and Wilson (2005) support this position; these are elaborated in the present chapter, and new findings that have not been previously reported are also presented.

Introduction Food insecurity is defined as a state of “limited or uncertain availability of nutritionally adequate and safe foods or limited or uncertain ability to acquire acceptable foods in socially acceptable ways” (Olson and Holben, 2002; pg 1841). Many practical methods for addressing food insecurity have been suggested, from combating poverty to improving distribution and increasing assistance by governments and local communities (e.g., American Dietetic Association, 2002). However, it may be beneficial to take a fresh and deeper look at food 1

E-mail address: [email protected]. Phone: +61 2 9036 6421, Fax: +61 2 9351 6732.

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insecurity. In particular, this chapter focuses on one pattern, a type of person with three characteristics:

1. The individual experiences food insecurity in childhood; 2. In adulthood, this person makes food security a top life goal, and later achieves this goal by having plenty of food;

3. Yet the person still has an anxious, emotional, and unhealthy psychological and physiological relationship with food, leading to hoarding, overeating, and using food for emotional comfort.

It is argued here that this pattern is common because it is associated with materialism. According to Dictonary.com, materialism is the “preoccupation with or emphasis on material objects, comforts, and considerations, with a disinterest in or rejection of spiritual, intellectual, or cultural values.” This chapter elaborates why I believe this tripartite pattern is associated with materialism, but it is worth noting that others have also observed a link between childhood food insecurity and adult issues with food. For instance, Fieldhouse (1995) noted that “emotional growth in infancy is nurtured by undisrupted routines, thus regular meals are necessary for a happy child. Early lack of security may give rise to fussiness and eating difficulties later in life.” (p. 191). Even more to the point, Cohen (1961) observed that in societies in which children are not fed regularly, the adults seem to hoard food and focus on wealth accumulation. In contrast, regular feedings in childhood lead to a greater impulse to share. In short, these studies, and more recent work by Allen and Wilson (2005), suggest that some people who experience food insecurity in childhood will turn to materialism in adulthood as a way to ensure that food and possessions are plentiful, but will continue to use food for psychological reasons, which in turn may lead to unhealthy outcomes. In philosophy, materialism is essentially the concept that only the physical world is real or explainable, while in psychology and consumer research, materialism refers to the general psychological investment in possessions and/or services (e.g., Belk, 1985; Claxton and Murray, 1994; Holt, 1995/1998; Inglehart, 1977, 1997; Richins and Dawson, 1992). In this latter view, materialism may be a set of personality traits such as envy and selfishness (Belk, 1984/1985); a personal value based on acquiring items as a measure of success or happiness (Richins and Dawson, 1992); a way of directly defining oneself socially by material goods and services (Claxton and Murray, 1994); or a consumption style in which people incorporate the symbolism of products and services into their self-concept, thereby using material goods to differentiate themselves from others (Holt, 1995/1998). One conception of materialism in particular may be most pertinent for assessing how materialism may relate to food insecurity, namely, that materialism may be a method for achieving a sense of survival security (Inglehart, 1977, 1997). This socio-political view posits that survival insecurity, which stems from a fear of military conflict and economic uncertainty, is a feature of modern culture that may influence or shape a whole host of domains, including politics (e.g., a need for social order and strong leaders), religion (e.g., emphasis on higher power and absolute rules), sexuality (e.g., maximize reproduction), and economics (e.g., a need for financial security). Individuals with a strong need for survival security would be deeply concerned about personal safety and ensuring that their basic needs

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are never threatened. Experiencing survival insecurity (say, via material deprivation) during one’s formative years could lead to this strong drive for survival security and thus a need to accrue or consume material goods (say, products that meet the basic needs) (Inglehart, 1977, 1997). In this way, childhood deprivation could result in adulthood materialism. Although materialism may arguably arise from a host of factors including disruptions to the family unit (e.g., Rindfleisch, Burroughs, and Denton, 1997), a lack of quality education (e.g., Duch and Taylor, 1993; Warwick, 1998), insufficient time to fully develop one’s identity during adolescence (e.g., Pfeiffer and Cote, 1991), whether or not one’s spouse shows materialistic tendencies (e.g., Claxton, Murray, and Janda, 1995), or the personal values and personality of one’s parents (e.g., Kasser, Ryan, Zax, and Sameroff, 1995; Marks, 1997), several studies have shown that childhood poverty is indeed associated with materialism later in life (e.g., Abramson and Inglehart, 1995; Cohen and Cohen, 1996; Kasser, Ryan, Zax, and Sameroff, 1995). Under this scenario, one may cope with survival insecurity by developing materialistic tendencies. This may include leanings toward the excessive accrual or consumption of material goods to achieve a sense of survival security. For instance, one may hoard products that meet the basic necessities (e.g., food, clothes), which would provide tangible, visual evidence of future survival security. Fieldhouse (1995) makes a similar observation about the Gurage people of Ethiopia, in that “during early milk feeding and weaning, Gurage children are exposed to a pattern of alternating glut and want; this, combined with the emotional detachment shown by parents toward their children, may explain the extreme anxiety exhibited by adults toward their food supply” (pp. 186). There is evidence in other fields that insecurity can lead to the pursuit of wealth and possessions (e.g., Fromm, 1976; Maslow, 1954). Of the products that meet basic needs, food is central. Thus, survival insecurity should trigger strong concerns about food supply in particular, and a desire to ensure that stores are stable and abundant. In summary, materialism may be one response to survival insecurity developed during childhood. Such materialism could manifest in part as hoarding behavior in adulthood, in particular of products that meet the basic needs. Of such products, food is paramount, and thus following this line of logic, adult materialists would show an elevated concern over the state of their food supply, make food security a primary life goal, and hoard food. Said another way, they would exhibit behaviors associated with food insecurity, even when food supplies are not genuinely threatened. For instance, materialists and adults who suffered food insecurity in childhood may use food for emotional reasons. Fieldhouse (1995) notes that “over-consumption and obesity among people living in a land of plenty have sometimes been ascribed to residual anxieties about hunger stemming from earlier experiences” (pp. 186). Similarly, Gifft, Washbon and Harrison (1972) observe that “adults too frequently use food as an emotional outlet – a crutch to help them handle and to live with anxiety, tension, frustration, unhappiness, irritability, disappointment, loneliness or boredom. No human can escape such emotions as these and thus he must find ways to cope with them” (pp. 100). This potential underlying motivation of food insecurity should be considered in future investigations or when attempting to address this issue in practice. A recent series of surveys and experiments reported by Allen and Wilson (2005) support these positions. This information, along with new findings not yet reported, are presented below.

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Allen and Wilson (2005) findings plus new analyses and data Study 1 Allen and Wilson (2005) consisted of three studies. The first one surveyed 95 adult Australians randomly chosen from the phone book. The survey measured each respondent’s endorsement of materialism (using Inglehart’s (1977) Materialism Scale), childhood food insecurity, present-day food insecurity, and food security as goal. Present-day Food Insecurity was measured using four items from the Bickel, Nord, Price, Hamilton, and Cook (2000) Food Insecurity Scale, rated on a three-point scale from “Never True” to “Often True” (e.g., “In the last 12 months, the food that I bought just didn’t last and I didn’t have money to get more”). The items from the same scale were used to measure Childhood Food Insecurity (e.g., “When I was a child, the food that my parents bought just didn’t last and I didn’t have money to get more”). Food Security as Goal was measured by having participants rate the following statement on the same three-point scale: “Ensuring that you have enough food to eat.” Although Allen and Wilson (2005) used regression to analyze the results, they are presented here more simply and in greater detail. Participants were first divided at the median (5.2) of the Inglehart Materialism Scale. As shown in Figure 1, high materialists have a stronger goal of food security than low materialists (t= -2.5, df=92, p<.01, two-tailed). On average, high materialists rate food security as goal 6.5 (out of 7), whereas low materialists only rate food security as goal 5.9. 6.8

6.6

6.4

Food security as goal

6.2

6.0

5.8

5.6 Low

High

Materialism

Figure 1. Study 1. Food Security as Goal by Materialism (Mean +- 1 SE).

High and low materialists did not significantly differ in their incidence of present-day food insecurity (high materialism mean = 1.2, low materialism mean = 1.1, t= -.7, df=92, p<.ns), but high materialists report a greater incidence of food insecurity during their childhood than low materialists (see Figure 2)( t= -2.9, df=92, p<.01, two-tailed). Table 1 lists these discrepancies at the item level. For instance, 20% of low materialists reported that in the last 12 months the food they bought did not last, compared to 28% of high materialists. However, the leading difference between high and low materialists is their childhood food

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insecurity. 10% of low materialists reported that when they were children the food their parents bought did not last, compared to a substantial 35% of high materialists. Likewise, 26% of high materialists reported that when they were children their family could not afford to eat balanced meals (compared to 6% of low materialists). Table 1. Prevalence of food insecurity among high and low materialists. Percent responding “sometimes” or “often” true Materialists Low High Present-day Food Insecurity In the last 12 months, the food that I bought just didn't last and I didn't have money to get more In the last 12 months, I couldn't afford to eat balanced meals In the last 12 months, I cut the size of my meals or skipped meals because there wasn't enough money for food In the last 12 months, I ate less than I felt I should because there wasn't enough money to buy food Childhood Food Insecurity When I was a child, the food that my parents bought just didn't last and we didn't have money to get more When I was a child, my family couldn't afford to eat balanced meals When I was a child, I cut the size of my meals or skipped meals because there wasn't enough money for food When I was a child, I ate less than I felt I should because there wasn't enough money to buy food

20.4%

28.9%

10.2% 16.3%

22.2% 17.8%

8.2%

15.6%

10.2%

35.6%

6.1% 2.0%

26.7% 6.7%

6.1%

15.6%

1.3

Childhood food insecurity

1.2

1.1

1.0 Low

High

Materialism

Figure 2. Study 1. Childhood Food Insecurity by Materialism (Mean +- 1 SE).

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Although not reported in Allen and Wilson (2005), their survey also included additional measures. For instance, hunger was measured using Stunkard and Messick’s (1985) Hunger Subscale (e.g., “I am usually so hungry that I eat more than three times a day”, “I often feel so hungry that I have to eat something”; four items, Cronbach’s Alpha = .77). High and low materialists did not significantly differ in present-day hunger (high materialism mean = 3.6, low materialism mean = 4.0, t= 1.4, df=91, p<.ns). This is consistent with the previous finding that high- and low-level materialists do not differ on present-day food insecurity.

Study 2 Study 1 showed that materialists had greater food insecurity in childhood, and more strongly considered food security a major life goal in adulthood. These results support the view that childhood deprivation may lead to materialism as a strategy to cope with survival insecurity. Interestingly, the results also suggested that the strategy may work, as materialists reportedly did not experience present-day hunger or suffer from an inability to acquire food. That is, materialists exhibited food insecurity (worried about food stores), even though they did not lack access to (or consumption of) food. Study 2 built on these results, to assess whether actions taken by materialists to achieve food security (e.g., hoarding food) affected their weight. Study 2 surveyed 130 adults (randomly selected from the phone book). As in Study 1, the survey included the Inglehart (1977) Materialism Scale, but also measured food hoarding and the height and weight of participants to calculate their body mass index (BMI). To gauge food hoarding/storing, participants indicated the total number of servings they had in their house and the number of adults and children in their household. A food hoarding index was then calculated for each participant by dividing the total number of stored servings by the number of household members. The results showed that overweight subjects (BMI > 30) more strongly endorsed materialism than those who were underweight or normal weight, and that high-level materialists stored on average 14 more servings of food per household member than low-level materialists (39 vs. 25 servings). Another finding, although not previously reported, is nevertheless important. Because the study developed a novel hoarding index based on the number of servings of food stored at home, I tested its validity. I quantified the calories, protein, fat, carbohydrates, sodium, vitamin A, vitamin C, saturated fat, and cholesterol each participant consumed in the seven days preceding the study (by taking the reported numbers of servings of meat, vegetables, fruit, dairy, seafood/fish, and cereals, and multiplying them by each food’s nutritional value). As shown in Table 2, the more overweight (the higher the BMI), the greater the consumed calories, protein, fat, saturated fat, and cholesterol. Similarly, the more servings of food stored at home, the greater the consumed calories, protein, fat, saturated fat, and cholesterol. These trends supported the validity of the hoard index, but perhaps more importantly, also showed that hoarding food at home is associated with negative health outcomes.

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Table 2. Partial correlations of BMI and Hoarding index with BMI and nutritional values of foods consumed in previous seven days (controlling for age and sex). DFS are 99

BMI Calories Protein Fat Carbs Sodium Vit A Vit C Sat Fat Colestl

BMI

Total Servings of Food Stored At Home (/hs)

1.00 .16 .16 .26 .07 .10 .08 .04 .26 .25

.28 .20 .22 .33 .08 .09 .08 .12 .37 .37

Study 3 The results of Studies 1 and 2, taken together, showed that materialists did not suffer from present-day hunger or a lack of access to food, but tended to store excessive amounts of food (i.e., hoard) and were more likely to be overweight. They also make food security a top life goal. These findings imply that, while these materialists had no apparent immediate threat to their food supply, they nonetheless showed behaviors consistent with persons who are excessively concerned about survival security. Study 3 examined whether materialism was indeed the vehicle for coping with survival security (of which food security is just one component). It was based on the following assumptions: If materialism were used in adulthood to cope with survival insecurity developed in childhood, increasing participants’ feelings of insecurity should increase their endorsement of materialism and Food Security as Goal. Allen and Wilson (2005) also predicted that increasing insecurity would increase the use of food for emotional comfort. To reduce survival security, participants were made to think about their own death, using the Mortality Salience manipulation from Terror Management Theory (Greenberg, Pyszczynski, and Solomon, 1986; also see Arndt, Solomon, Kasser and Sheldon, 2004; Kasser and Sheldon, 2000). Half of the participants were exposed to the Mortality Salience manipulation (in which they wrote about their own death). Then the Mortality Salience manipulation group and the control group completed four scales: General Survival Security as State (three items: “I feel secure about the money I have”, “I feel safe and secure”, “My income is sufficient to satisfy my needs”); the Inglehart (1977, 1997) Materialism Scale; Food Security as Goal; and Use of Food for Emotional Comfort (five items: e.g., “Eating gives me emotional satisfaction”, “I tend to get in a good mood after eating”). A series of ANOVA analyses, reported at length in Allen and Wilson (2005) supported the predictions. For instance, participants who felt insecure after the Mortality Salience

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manipulation more strongly endorsed materialism, Food Security as Goal, and Use of Food for Emotional Comfort than controls. Finally, it is worth reviewing some findings of Study 3 that were not published in Allen and Wilson (2005). As mentioned above, it was expected that the Mortality Salience manipulation would lead to feelings of insecurity by making people think about their own death. Indeed, Allen and Wilson (2005) showed that the manipulation group felt greater insecurity than the control group. Nevertheless, not all people have the same reaction to thinking about death. Table 3 lists some key responses of participants to the Mortality Salience manipulation questions. These quotes reveal that while some participants did feel insecure and fearful, others were seemingly in denial or had positive emotions about their death. Table 3. Sample quotes from participants in the Mortality Salience Manipulation Q1. In the space below, write about what will happen to you as you physically die. Apart from the obvious, I don’t feel anything happens afterwards Bodily functions will slow down to a stop, effectively shutting down brain function, resulting in loss of consciousness and death. Don't know or care Go into a deep sleep and never wake up I believe that I will be in fear, then I will feel peaceful I will cry I will die from a great fall at 70 years of age probably sky-diving I will have a peaceful sleep. My worries will be forgotten. I do not have to go to work and face my former boss in the hallway. I know my physical body will disintegrate but my mind will not know about it. My body will slow down, there will be some pain, I'll probably be emotional, then just stop. Organs shut down and can't breath

O2. In the space below, write about the emotions that the thought of your own death arouses in you. Fear, sadness, uncertainty Frightens me. I will hopefully will not feel any pain I would feel panic stricken and scared No emotions it happens to everyone

Conclusion Taken together, the results of Allen and Wilson (2005) and the new analyses and findings reported herein are illuminating. Study 1 revealed that materialists set a greater life goal of food security and felt more food insecurity during childhood, but were not more likely to lack access to food or be hungry. Study 2 found that materialists stored/hoarded more food at

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home, and that overweight people endorsed materialism more than under/normal weight people. Study 3 found that participants subjected to a Mortality Salience manipulation (in which they described their own death) felt less survival security, and more strongly endorsed materialism, food security as a goal, and using food for emotional comfort. These results imply that materialists attempt to overcome food insecurity developed in childhood by making food security a top life goal, but that they exhibit present-day concerns about food security, which may not wholly stem from genuine present-day threats to their food supply. That is, if thinking about death makes materialists place more importance on food security (Study 3) and materialists hoard more food and tend to be overweight (Study 2), food insecurity clearly stems from concerns beyond sufficient nutrient intake and likely involves deeper psychological forces. In conclusion, materialism appears to be one method for coping with survival insecurity developed in childhood. A lack of opportunity or deprivation of material goods and a stable and sufficient supply of food may lead one, in early adulthood, to develop materialistic tendencies. This may include, in addition to efforts to attain social security (e.g., improve socio-economic status), leanings toward the excessive accrual or consumption of material goods including food items. Interestingly, such efforts appear to pay off to an extent, as materialists reportedly did not experience present-day hunger or suffer from an inability to acquire food. Nonetheless, materialism should not be advocated as a method for solving food insecurity. For one, the studies also found that materialists exhibited behaviors associated with food insecurity (worried excessively about food stores), even though they did not lack access to or consume less food. That is, they may have appeared food secure, but were in fact still food insecure. While many studies have evaluated how products and services please materialists (e.g., Belk, 1985; Claxton and Murray, 1994; Holt, 1998; Inglehart, 1977, 1997; Richins, 1994; Richins and Dawson, 1992), the role of food has largely been ignored, despite its biological and socio-cultural importance. Food is not only necessary for survival, but is also a status symbol (e.g., Allen and Baines, 2002; Allen, Gupta and Monnier, in press; Allen and Ng, 2003; Levy, 1959) and a cultural vehicle through which societies shape social roles and structures (e.g., Fieldhouse, 1995; Rozin, 1996). Therefore, materialists seemingly use food as a tool to satisfy their socio-cultural as well as biological needs. In addition, there was a link between materialism and overeating. Overeating may be a way to avoid or disassociate oneself from unpleasant emotions (e.g., Heatherton and Baudmeister, 1991; Ruderman, 1985). For example, women with a history of eating issues such as binge eating or bulimia tend to eat more after their self-esteem is threatened (McManus, Waller and Chadwick, 1996) or after being subjected to threatening subliminal messages such as “Mum hates me” and “Mum hurts me” (Waller and Mijatovich, 1998). In Study 3, making participants feel less secure (via the Mortality Salience manipulation) led to greater endorsement of using food for emotional comfort (i.e., higher ratings for items such as “When I feel lonely I console myself by eating”). However, this response was only observed in high-level materialists. Furthermore, materialism is also associated with psychosocial issues such as dissatisfaction with life (e.g., Saunders and Allen, 2000; Wright and Larsen, 1993). Therefore, rather than a method for addressing food insecurity, materialism as a potential underlying motivation of food insecurity should be considered in future investigations or when attempting to address this issue in practice. Food insecurity is associated with socio-economic struggles such as racism, poverty, and economic forces (e.g., Nord, Andrews, and Carlson, 2003). For example, while food supply in

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developed countries has become more secure and stable in time (e.g., Mennell, 1987; Mennell, Murcott and van Otterloo, 1992), food distribution is still unequal, and not all members of society have the same access to goods. Boserup (1983) reported that “rapid growth of the population and rapid growth of per capita income go together with malnutrition and sometimes starvation among the poor” (pp. 208–209). Moreover, countries with more income inequality tend to have worse nutrition indicators, controlling for GNP (Foster, 1992), and these problems tend to get worse during economic downturns, crop losses, war, and other challenges. Although making food security a primary life goal seemed to result in some benefits (e.g., no present-day hunger, a large supply of accessible food), there were also disadvantages (e.g., tendency to be overweight, continued feelings of psychological insecurity). Therefore, future studies should further evaluate the efficacy of making food security a life goal. Goalsetting is known to augment performance (e.g., Locke and Latham, 1991) by motivating one to put forth more effort, have more focus, and persist in the face of challenge (e.g., Bartholomew, Parcel, Kok and Gottlieb, 2001). In addition, the link between materialism and overeating deserves further investigation. Although the link between materialism and food insecurity may have a psychological component, food insecurity itself is a real problem. The food insecure tend to lack a steady and stable supply of food and be of low socio-economic status (e.g., Nord, Andrews, and Carlson, 2003). It is estimated that about 10% of U.S. households, for example, suffer from food insecurity (e.g., Olson and Holben, 2002). Food insecurity does not necessarily involve hunger (e.g., Olson, 1999). Individuals may switch their diets to less expensive (and typically less diverse and lesser-quality) foods (e.g., Nord, Andrews, and Carlson, 2003); this may avoid starvation or chronic hunger, but can result in insufficient nutrient intake (e.g., Dixon, Winkleby, and Radimer, 2001; Kendall, Olson, and Frongillo, 1996; Rose and Oliveira, 1997), lower physical and mental health, and higher risk of disease (e.g., Pheley, Holben, Graham, and Simpson, 2002; Siefert, Heflin, Corcoran, and Williams, 2001). Therefore, it is imperative to develop a comprehensive strategy, based on sound science and smart policy, to address food insecurity on a global scale.

References Abramson, P.R., and Inglehart, R. (1995). Value change in global perspective. Ann Arbor: University of Michigan Press. Allen, M.W. and Baines, S. (2002). Manipulating the symbolic meaning of meat to encourage greater acceptance of fruits and vegetables and less proclivity for red and white meat. Appetite: Research on Eating and Drinking, 38(2), 118-130. Allen, M.W., Gupta, R., and Monnier, A. (in press). The interactive effect of cultural symbols and human values on taste evaluation. Journal of Consumer Research. Allen, M.W. and Ng, S.H. (2003). Human values, utilitarian benefits and identification: The case of meat. European Journal of Social Psychology, 33(1), 37-56. Allen, M.W. and Wilson, M. (2005). Materialism and food security. Appetite: Research on Eating and Drinking, 45(3), 314-323.

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American Dietetic Association (2002). position of the American Dietetic Association: Domestic food and nutrition security. Journal of the American Dietetic Association, 102, 1840-1847 . Arndt, J., Solomon, S., Kasser, T., and Sheldon, K.M. (2004). The urge to splurge: A terror management account of materialism and consumer behavior. Journal of Consumer Psychology, 14, 198-212. Bartholomew, K., Parcel, G., Kok, G., and Gottlieb, N. (2001). Intervention Mapping: Developing theory and evidence-based health education programs. Mountain View, CA: Mayfield . Belk, R. (1984). Three scales to measure constructs related to materialism: Reliability, validity, and relationships to measures of happiness. Advances in Consumer Research, 11, 291-297. Belk, R. (1985). Materialism: Trait aspects of living in the material world. Journal of Consumer Research, 12(3), 265-281. Bickel, G., Nord, M., Price, C., Hamilton, W., and Cook J.(2000). Guide to measuring household security revised. Alexandria VA: United States Department of Agriculture, Food and Nutrition Service. Boserup, E. (1983). The impact of scarcity and plenty on development. Journal of Interdisciplinary History, 14, 383-407. Claxton A., and Murray, J.B. (1994) Object Subject Interchangeability: A symbolic interactionist model of materialism. Advances in Consumer Research, 21, 422-426. Claxton A., Murray, J.B., and Janda, S. (1995). Spouses’ materialism: Effects of parenthood status, personality type and sex. Journal of Consumer Policy, 18, 267-291. Cohen, P. and Cohen, J. (1996). Life values and adolescent mental health. Mahwah, NJ: Erlbaum. Cohen, Y.A. (1961). Food and its vicissitudes: A cross cultural study of sharing and nonsharing. In (ed. Y.A. Cohen) Social Structure and Personality, (pp 312-350). Holt, Rinehart and Winstone, New York. Dictionary.com Unabridged (v 1.1). Retrieved May 31, 2008, from Dictionary.com website: http://dictionary.reference.com/browse/materialism. Dixon, L.B., Winkleby, M., and Radimer, K. (2001). Dietary intakes and serum nutrients differ between adults from food-insufficient and food-sufficient families: Third National Health and Nutrition Examination Survey, 1988-1994. Journal of Nutrition, 131, 12321246. Duch, R., and Taylor, M. (1993). Postmaterialism and the economic condition. American Journal of Political Science, 37, 747-779. Fieldhouse, P. (1995). Food and nutrition. London: Chapman and Hall. Foster, P. (1992). The world food problem. Boulder: Lynn Reiner. Fromm, E. (1976). To have or to be? New York: Harper and Row. Gifft, H., Washbon, M. and Harrison, G. (1972). Nutrition Behavior and Change. Englewood Cliffs: Prentice-Hall. Greenberg, J., Pyszczynski, T., and Solomon, S. (1986). The causes and consequences of the need for self-esteem: A terror management theory. In R.F. Baumeister (Ed.), Public Self and Private Self (pp 189-212). New York: Springer-Verlag. Heatherton, T.F., and Baudmeister, R.F. (1991). Binge eating as escape from self-awareness. Psychogical Bulletin, 110, 86-108.

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Holt, D.B. (1995). How consumers consume: A typology of consumption practices. Journal of Consumer Research, 22, 1-16. Holt, D.B. (1998). Does cultural capital structure American consumption? Journal of Consumer Research, 25, 1-15. Inglehart, R. (1977). Silent revolution: Changing values and political styles among Western publics. Princeton, N.J.: Princeton University Press. Inglehart, R. (1997). Modernization and post modernization: Cultural, economic, and political change in 43 societies. Lupton: Princeton University Press. Kasser, T., and Sheldon, K.M. (2000). Materialism, mortality salience, and consumption behavior. Psychological Science, 11(4), 348-351. Kasser, T., Ryan, R. M., Zax, M., and Sameroff, A. J. (1995). The relations of maternal and social environments to late adolescents’ materialistic and prosocial values. Developmental Psychology, 31, 907-914. Kendall, A., Olson, C.M., and Frongillo, E.A. Jr. (1996). Relationship of hunger and food insecurity to food availability and consumption. Journal of the American Dietetic Association, 96, 1019-1024. Levy, S. (1959). Symbols for sale. Harvard Business Review, 37, 117–119. Locke, E.A., and Latham, G.P. (1991). A theory of goal setting and take performance. Englewood Cliffs: NJ: Prentice Hall. Marks, G.N. (1997). The formation of materialist and postmaterialist values. Social Science Research, 26, 52-68. Maslow, A. (1954). Motivation and personality. New York: Harper. McManus, F., Waller, G., and Chadwick, P. (1996). Biases in the processing of different forms of threat in bulimic and comparison women. Journal of Nervous and Mental Disease, 184, 547-554. Mennell, S. (1987). On the civilising of appetite. Theory, Culture and Society, 43, 373-403. Mennell, S., Murcott, A., and van Otterloo, A.H. (1992). The sociology of food: Eating, diet and culture. London: Sage Publications. Nord, M., Andrews, M., and Carlson, S. (2003). Household food security in the United States. Food and Rural Economics Division, Economic Research Service, U. S. Department of Agriculture. Alexandria, VA. Olson, C.M. (1999). Nutrition and health outcomes associated with food insecurity and hunger. Journal of Nutrition, 129, 521S-524S. Olson, C.M., and Holben, D.H.(2002). Position of the American Dietetic Association: domestic food and nutrition security. Journal of the American Dietetic Association, 102(12), 1840-1847. Pfeiffer, M., and and Cote, J.E. (1991). Inglehart’s silent revolution thesis: An examination of life-cycle effects in the acquisition of postmaterialist values. Social Behavior and Personality, 19, 223-235. Pheley, A.M., Holben, D.H., Graham, A.S., and Simpson, C. (2002). Food security and perceptions of health status: A preliminary study in rural Appalachia. Journal of Rural Health, 18, 447-454. Richins, M. and Dawson, S. (1992). A Consumer Values Orientation for Materialism and its Measurement: Scale Development and Validation. Journal of Consumer Research, 19, 303-316.

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Richins. M. (1994). Special possessions and the expression of material values. Journal of Consumer Research, 21, 522-533. Rindfleisch, A., Burroughs, J.E., and Denton, F. (1997). Family structure, materialism, and compulsive consumption. Journal of Consumer Research, 23, 312-325. Rose, D., and Oliveira, V. (1997). Nutrient intakes of individuals from food-insufficient households in the United States. American Journal of Public Health, 87, 1956-1961. Rozin, P. (1996). Towards a psychology of food and eating: Motivation to module to model to marker, morality, meaning and metaphor. Current Directions in Psychological Science, 5, 18-24. Ruderman, A. J. (1985). Dysphoric mood and overeating: A test of restraint theory’s disinhibition hypothesis. Journal of Abnormal Psychology, 94, 78-85. Saunders, S., and Allen, M.W. (2000). Materialism and life satisfaction in Australia: Is more better? South Pacific Journal of Psychology, (1), 48-51. Siefert, K., Heflin, C.M., Corcoran, M.E., and Williams, D.R. (2001). Food insufficiency and the physical and mental health of low-income women. Women and Health, 32(1-2), 159177. Stunkard, A.J, Messick, S. (1985). The three-factor eating questionnaire to measure dietary restraint, disinhibition, and hunger. Journal of Psychosomatic Research, 29,71-83. Waller G, Mijatovich S. (1998). Preconscious processing of threat cues: impact on eating among women with unhealthy eating attitudes. International Journal of Eating Disorders, 24(1), 83-89. Warwick, P.V. (1998). Disputed cause, disputed effect: The postmaterialist thesis reexamined. Public Opinion Quarterly, 62, 583-603. Wright, N.D., and Larsen, V. (1993). Materialism and life satisfaction: A Meta - analysis. Journal of Consumer Satisfaction, Dissatisfaction and Complaining Behaviour, 6, 158 165.

In: Food Science and Security Editors: L. Amsel and L. Hirsch, pp. 113-124

ISBN: 978-1-60692-977-3 © 2009 Nova Science Publishers, Inc.

Chapter 4

AN EXAMINATION OF GENDER DIFFERENCES IN THE RELATIONSHIP BETWEEN REPORTING A FOOD HARDSHIP AND PHYSICAL HEALTH Colleen Heflin University of Missouri

Abstract Despite the wealth present in American society, high rates of food insecurity, food insufficiency, and hunger are a significant problem in the United States. The persistently high reports of food security are troubling given that experiencing food hardships is not a social problem with isolated consequences. A lack of an adequate food supply can potentially affect mental well-being and overall quality of life, as well as physical health outcomes. However, an important challenge facing research on nutrition and health is to distinguish the consequences of food hardship from those of its common risk factors, such as poverty and low socioeconomic status. This paper used data from the National Survey of America’s Families to model the effect of reports of food hardships on the presence of chronic health conditions and to examine if gender differences exist in this relationship. Using instrumental variable linear probability models to adjust for endogeneity between food hardship and health, I find that reporting food hardship increases the risk of having a chronic health condition for both men and women. However, men who report a food hardship face a much higher risk of chronic health conditions than do women who report hardships.

Introduction Despite the wealth present in American society, high rates of food insecurity, food insufficiency, and hunger are a significant problem in the United States [Alaimo et al. 1998; Nord et al. 2006]. It is currently estimated that more than 35.1 million people live in food insecure households, meaning that at some time during the previous year, they were unable to acquire or were uncertain of having enough food to meet basic needs due to inadequate household resources [Nord et al. 2006].

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Rates of food insufficiency, a measure indicating that households “sometimes” or “often” did not have enough food to eat, vary considerably by race, gender, and socioeconomic status. Analyzing data from the Third National Health and Nutrition Examination Survey (NHANES III), Alaimo et al. [1998] found rates of food insufficiency to be 11.8 percent among lowincome whites, 13.5 percent among low income non-Hispanic Blacks, and 24.8 percent among low-income Mexican Americans. More recently, the USDA found that while the overall household prevalence of food insecurity with hunger, a measure indicating an uneasy or painful sensation caused by a lack of food that results from not being able to afford enough food, in the United States was 3.9 percent, it was 8.6 percent among Black households, 5.3 percent among Hispanic households, 8.7 percent among families headed by a single woman, and 13.5 percent among households below the poverty line [Nord et al. 2006]. The persistently high rates of food insecurity are troubling given that experiencing food hardship is not a social problem with isolated consequences. As Olson [1999] observes, food insecurity and hunger can potentially affect mental well-being and overall quality of life, as well as health outcomes. However, an important challenge facing research on nutrition and health is to distinguish the consequences of food insecurity from those of its common risk factors, such as poverty and low socioeconomic status. Additional issues arise when trying to determine causality—is the food hardship causing the health problem or is the health problem causing the food hardship? Building on previous work by Siefert et al. [2000, 2001, 2004] and Heflin et al. [2005], I use the National Survey of America’s Families (NSAF) to model the effect of food hardship on one measures of physical health--the presence of chronic health conditions--in order to determine if there is a relationship between food hardship and physical health in a national sample of low-income households. Furthermore, I test for the presence of gender differences in the effects of food hardship on the health of low-income men and women. This research provides several important contributions to the literature on social determinants of health. First, by examining the relationship between one form of material well-being, food hardship, and physical health among a low-income population, this project confirms earlier research showing an association between food insufficiency and health in a sample of women on welfare. Furthermore, gender is examined explicitly to determine if experiencing food hardship has a differential effect for men and women. Finally, advanced statistical tools, instrumental variable models, are used in an attempt to advance the causal argument about the direction of the relationship between food hardship and health.

Food Hardships and Health There are several potential pathways whereby household experiencing food hardships could have a detrimental effect on the health of the household head. I draw on two main theoretical traditions. The first is sometimes termed the neomaterial view. Here, food harships could have a negative impact on health through a direct effect of nutritional shortfalls or reductions in positive health behaviors [Bhattacharya et al. 2004; Lynch et al. 2000]. Prior research suggests that food insufficiency can have important health consequences [Nelson et al. 1998; Olson 1999; Casey et al. 2004; Siefert et al. 2001; Seifert et al. 2004]. Other recent studies that have found that an inadequate household food supply is associated with lower energy and nutrient intake among women. Rose and Oliviera [1997] analyzed the diets of

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3,774 adult women who participated in the 1989-1991 Continuing Survey of Food Intake by Individuals and found that for adult women, household food insufficiency was significantly associated with low energy and nutrient intakes. Similarly, Dixon, Winkelby and Radimer [2001] found that adults in food insufficient households were more likely to have diets that could compromise their health than adults from food sufficient families, and in addition had lower serum concentrations of critical nutrients. Support for the second potential causal pathway between food hardships and poor chronic health can be found in studies linking socioeconomic disadvantage, chronic stress and health [Lantz et al. 2005]. Proponents of this view suggest that awareness of disadvantage in regard to relative social positioning creates feelings of shame and distrust that have negative biological consequences through the psycho-neuro-endocrine chain [Lynch et al. 2000; Kelly et al. 1997; McEwan and Seeman 1999]. In addition, stress caused by the cumulative burden of coping with limited resources may increase the allostatic load, the wear and tear accumulated as a result of physiological responses to environmental stress. When long-term psychological stress causes the activation of the hypothalamic- pituitary-adrenal axis and sympathetic nervous system, overexposure to stress mediators can have adverse effects on various organ systems, leading to disease [McEwen 1998; McEwen and Seeman 1999]. In practice, it is not possible to differentiate which of the two mechanisms are operating the neomaterial versus the psychosocial. However, by specifying two potential causal pathways from food hardship to health, we can identify several possible intervention points. For example, the neomaterial pathway can be remedied by providing financial resources to households experiencing food hardships. However, the psychosocial pathway may require larger structural changes in the degree of societal income inequality. While general patterns of gender disparities in health outcomes are well documented, less research has been done exploring the possibly gendered pathway of food hardships. While household levels of low food security are higher in national samples among women with children in the household than among men with children in the household, men living alone have higher rates than do women living alone [Nord et al. 2006]. Furthermore, most of the research on the consequences of food hardships has tended to focus on welfare samples of women or on children [Jyoti et al. 2005; Casey et. al 2005; Laraia et al. 2006]. The lack of data on the health consequences of food hardships for adult men is a potentially important omission in light of the evidence that the causal pathways of poor health may vary by gender. Denton and Walters [1999] report finding gender differences in the determinants of health. Of particular interest here are their findings that both income and overweight are important predictors of health for women but not men. Additionally, they find that compared to men who are an acceptable weight, men who are underweight are in poorer health. Thus, if weight is thought of as a neomaterial mechanism and income as a psychosocial mechanism, then there is some evidence that both causal pathways discussed above may operate differently for men and women in terms of health outcomes. Therefore, I hypothesize that food hardship will have a differential impact on the health of low-income men and women in terms of measured health outcomes in a nationally representative survey.

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Data, Measures and Methods This study used data come from the 1999 and 2002 rounds of the National Survey of America’s Families (NSAF). The NSAF is part of The Urban Institute's Assessing the New Federalism project. Its purpose is to track the effects of federal policy changes decentralizing many social programs. The survey provides quantitative measures of child, adult and family well-being in America, with an emphasis on persons in low-income families. The NSAF focuses on the economic, health, and social characteristics of adults under the age of 65 and their families and is designed to be representative of the United States as a whole, as well as thirteen individual states. The NSAF has information on both food hardship and adult health on 35,631 individuals in 1999, and 43,806 in 2002, for a pooled sample size of 79,437 cases. Table 1. Characteristics of Study Participants Food Hardship

9.3%

Percent (SD) (0.291)

Presence of Chronic Health Condition

16.2%

(0.368)

Female

69.3%

(0.461)

Education Less than high school

14.1%

(0.348)

High school graduate

60.8%

(0.488)

More than high school

25.1%

(0.434)

50.8%

(0.500)

Ever Married

22.1%

(0.415)

Never Married

29.1%

(0.454)

Black

12.8%

(0.334)

Hispanic

12.1%

(0.326)

75.2

(0.432)

Mean Age

38.605

(12.130)

Social Environmental Risk Factors Poverty Status

15.5%

(0.362)

Unemployed

20.4%

(0.403)

South Resident

26.7%

(0.443)

Martial Status Married

Race

White

N

79,437

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Table 1 presents descriptive statistics (means and standard deviations) for selected outcomes and demographics across each of the waves and for the pooled sample which combines data across the two rounds. The NSAF includes items which are a subset of the 18item Food Security Module designed by USDA and administered as a supplement to the Current Population Survey. I combine two measures which indicate whether (i) the food they bought did run out, and (ii) one or more adults ate less or skipped meals because there was not enough money for food. As shown in Table 1, about 9.3 percent of individual surveyed lived in households indicating that food stores were insufficient to cover need. For physical health, the NSAF includes a measure of the presence of a “physical, mental or other health condition that limits the kind or amount of work that you can do.” About 16.2 percent indicated that the presence of a chronic health condition. Female is dummy variable indicating if the respondent is female. Education level is captured at three levels: less than high school, high school graduates, more than high school. Marital status is grouped into three categories: married, ever married indicating that the head is currently divorced, widowed or separated, and never married. Race of the head is measured with a series of dummy variables as white if the respondent self-identified themselves as white; black indicates that the respondent self-identified themselves as having a primary racial identify of black; and other indicates that the respondent identified themselves as American Indian, Asian, Latino or some other combination. Age represents the age in years of the respondent. Poverty status indicates if the total family income reported is below the federal needs standard for the family size. Unemployed indicates that the respondent reported working zero weeks the prior year. South indicates that the respondent resides in the named region as defined by the US Census Bureau. In order to see if gender moderates the relationship between food hardship and health, I estimate the following model of health: HEALTHi = β0 + β1FDHRDi + β2 FDHRD *GENDER + β3 DEMOi +β4 RISKi + εi

(1)

where HEALTHi represents the presence of chronic health conditions for individual i, FDHRDi represents food hardship, FDHRD*GENDER represents the interaction between food hardship and gender, DEMOi is a vector of exogenous demographic explanatory variables, and RISKi is a vector of exogenous social and environmental risk factors. The interaction term allows for an examination of the relationship between food hardship and health to see if it varies systematically by gender within low-income populations [Baron and Kenny 1986]. Prior research has shown a strong relationship between food insufficiency and health among samples of women on welfare, but it is unknown if the affect is constant for males and females. Demographic variables include controls for gender, age, race, ethnicity, education, marital status and southern residence. Social and environmental risk factors include poverty status and whether employed in the prior year. One potential problem with this model, is that food hardship may be correlated with the error term through simultaneity (i.e. food hardship and chronic health may be jointly determined). This would be the case if there are shared unobservables such that not only does food hardship lead to poor physical health but poor physical health leads to food hardship. Since not addressing this problem leads to biased regression coefficients [Wooldridge 2003], this possibility leads to the need to adopt an instrumental variables procedure. As is true in all applications of instrumental variables, an identifying instrument is required. In this case I need

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variables that independently determine food hardship, but that affect current physical health only via its impact on current food hardship. Previous research indicates that state variation in the implementation of the Food Stamp Program policies affects program participation [Kabbani and Wilde 2003]. Therefore, one possible set of instruments for food hardship are state-level measures of characteristics of the Food Stamp Program such as the timing of adoption of electronic benefit transfers, the full vehicle exclusion, the level of the shelter deduction, the benefit level, and the error rate. The idea is that state level policy differences that affect participation in the Food Stamp Program may be correlated with food hardship but not health at the individual-level [Gunderson and Olivera 2001; Hofferth and Curtin 2005]. In order to allow comparison of the results between statistical methods that do and do not address the issue of possible endogeneity between reports of food hardships and chronic health, I first present results from linear probability models.1 Then, I present results using instrumental variable models (also known as two-stage least squares regression models). The appendix includes results from the first-stage regression models.

Results Figure 1 demonstrates the simple bivariate relationship between food hardship and the presence of chronic health conditions by gender. Results indicate that women have higher rates of food hardship than men but that men are more likely to have chronic health conditions. This 18.0% 16.7% 15.9%

16.0%

14.0%

12.0% 10.5% 10.0% Male Female 8.0% 6.7% 6.0%

4.0%

2.0%

0.0% Food Hardship

Chronic Health Condition

Source: Author’s calculations from the National Survey of America’s Families. Differences shown are statistically significant at the p<.01 level.

Figure 1. Presence of Food hardship and Chronic health conditions by sex. 1

Results are qualitatively consistent across models using logit and probit to the linear probability models results presented here in Table 2. I chose to present linear probability results because they are most easily compared to those from the instrumental variable model.

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is interesting since women made up 59.1 percent of the Food Stamp participants in fiscal year 2005 [Barret 2005]. In order to draw a connection between levels of food hardship and health, and the presence of gender difference in that relationship, I present results from multivariate regression analysis using STATA SE version 9. Table 2. Gender Differences in the Effects of Food Hardship on Presence of Chronic Health Conditions

Food Hardship

Linear Probability Model 0.174 (0.009)

**

Instrumental Variable Model 3.166 (0.988)

**

Female

-0.035 (0.003)

**

0.174 (0.069)

**

Food Hardship*Female

-0.029 (0.010)

**

-2.975 (0.973)

**

-0.050 (0.004)

**

-0.280 (0.009)

**

-0.110 (0.004)

**

-0.054 (0.019)

**

-0.077 (0.003)

**

-0.071 (0.006)

**

0.001 (0.004)

-0.044 (0.016)

**

0.001 (0.004)

-0.022 (0.009)

**

Education High school graduate More than high school Martial Status Married Ever Married Race Black Hispanic

-0.059 (0.004)

**

-0.0834 (0.010)

**

Age

0.006 (0.000)

**

0.006 (0.000)

**

0.059 (0.004)

**

-0.016 (0.026)

Unemployed

0.228 (0.003)

**

0.189 (0.014)

**

South

0.004 (0.003)

0.009 (0.005)

*

Constant

-0.019 (0.006)

-0.216 (0.066)

***

N

79,437

Social Environmental Risk Factors Poverty Status

**

79,437

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In the first column of Table 2, I present results from the OLS linear probability models of the effects of food hardship on the presence of chronic health conditions. This model does not correct for possible endogeneity between food hardship and having a chronic health problem. Model 1 in column 1 shows that food hardship is positively related to having a chronic health condition. The coefficient on food hardship suggests that individuals who experience food hardships are 17.4 percentage points more likely to also report a chronic health condition. All things equal, being female reduces the likelihood of reporting the presence of a chronic health condition, although the substantive size of the effect (3.5 percentage points) is not large. The sign on the interaction term between food hardship*female is also negative and this indicates that the risk associated with food hardship for women is lower than that for men. All else equal, men who experience food hardship face a probability of chronic health conditions of 17.4 percentage points, while women who experience food hardship face a probability of chronic health conditions of 11.0 percentage points (0.174 – 0.035 - 0.029*100)—this represents a 36.8 percent reduction in risk. Results for other control variables are consistent with past research. The relationship between health and education is positive, monotonic and statistically significant: Having education at the high school level or above is protective for physical health compared to those with less than a high school education. Being married appears to be protective while being ever married is negatively associated with physical health when compared to never married respondents. Having a racial identity classified as Black is positively associated with poor health. Having a racial identity of “other” is negatively associated with having a chronic condition compared to whites in this low-income sample. Age is positively associated with fair/poor health. Poverty status and not working the prior year are both positively associated with fair/poor health. Finally, southern residence is positively associated with fair/poor health. To test the sensitivity of my findings from the linear probability model to issues of endogeneity, the second model of Table 2 presents findings from the instrumental variable model. State-level measures of the characteristics of the Food Stamp Program are used to predict food hardship in order to control for possible endogeneity between food hardship and chronic health. Results from the first-stage regression can be found in the Appendix. The magnitudes of the coefficient of the two variables of interest changed significantly as does the sign on the female coefficient. This suggests that endogeneity is biasing the linear probability model results although the final conclusion is qualitatively similar across both models. In the instrumental variable model, having a food hardship is positively associated with the probability of reporting the presence of a chronic health condition but the magnitude of the affect is much larger after controlling for endogeneity. Men who report food hardships have, on average, a 3.166 greater risk of having a chronic health condition over those who do not report food hardships. For females, however, the effect of experiencing a food hardship is actually much lower—36.5 percentage points (3.166-2.975+.174=0.365)—representing an 88 percent reduction in the risk due to food hardship for males. When compared to females who do not report a food hardship, however, the risk can be seen to double from 17.4 percentage point risk of chronic health conditions without a food hardship to 36.5 percentage points with a food hardship. Clearly, there are health consequences associated with food hardship for both sexes but these findings suggest that the magnitude is much higher for males than females. The results for the remaining variables in the instrumental variable model are similar to those estimated with the linear probability model with the exception that since the model is estimated with greater precision, several additional variables in the model—ever married,

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Black and South—are now found to be statistically related to the risk of reporting a chronic health condition. Poverty status, which was related to the risk of reporting a chronic health condition, is no longer statistically significant after the state-level characteristics of food program policies are used to predict food hardships in the instrumental variable model.

Conclusion This research contributes to the literature on social determinants of health by exploring the relationship between one form of material hardship, food hardships, and adult health using nationally representative data. Controlling for demographic and social and environmental predictors of health, I investigate if the relationship between food hardships and health varies by gender. In both linear probability models and instrumental variable models correcting for endogeneity, I find consistent evidence that the effects of food hardships on the risk of chronic health conditions are higher for males than for females. This research moves the literature on social determinants of health forward along several dimensions. First, by examining determinants of health among a national sample of the lowincome population, this project confirms earlier research by Siefert et al. [2001, 2002, 2004] and Heflin et al. [2005] showing an association between food insufficiency and health in a welfare sample. Results shown here indicate that reporting a food hardship is a significant social determinant of the presence of chronic conditions both before and after correcting for endogeneity. Given the rise in rates of low food security from 1999 to 2005, from 10.1 to 11.9 percent [Nord 2006], researchers interested in social determinants of health as well as public health officials and policy-makers should be interested in furthering our understanding of the consequences of food hardships for different segments of the American population. Second, this project expands the research focus on the consequences of material hardships beyond women and children by examining gender differences in the consequences of food hardships. I find important gender differences in the affect of food hardships on health are present. Both before and after controlling for endogeneity, I find that females reporting a food hardship face a lower risk of reporting the presence of a chronic health condition. Stated differently, reporting a food hardship has more serious health consequences, at least in terms of chronic health conditions, among men than women.

Appendix A: First-Stage Estimate of Food Hardship Electronic Benefit Transfer

0.0016 (0.0013)

Shelter Deduction

-0.0000 (0.0000)

Average Food Stamp Benefit

-0.0001 (0.0001)

Error Rate

0.0001 (0.0002)

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Colleen Heflin Appendix A. Continued Full Vehicle Exclusion

0.0043 (0.0013)

Female

-0.0699 (0.0011)

Food * Female

0.9847 (0.0020)

High school graduate

-0.0073 (0.0015)

More than high school

-0.0177 (0.0018)

Married

-0.0022 (0.0014)

Ever Married

0.0147 (0.0016)

Black

0.0072 (0.0016)

Other

0.0084 (0.0016)

Age

0.0001 (0.0000)

Poverty Status

0.0252 (0.0015)

Unemployed

0.0130 (0.0013)

South

-0.0003 (0.0015)

References Alaimo, K., Beiefel, R.R., Frongillo, E. A. and Olson, C. M. (1998). Food insufficiency in the United States: Results from the Third National Health and Nutrition Examination Survey (NHANES III). American Journal of Public Health, Vol. 88, 419-426. Baron, R. M. and Kenny, D. A. (1986). “The Moderator-Mediator Variable Distinction in Social Psychological Research: Conceptual, Strategic, and Statistical Considerations.” J. Pers Soc Psychol. Vol 51(6), 1173-1182.

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Bhattacharya, J., Currie, J. and Haider, S. (2004). “Poverty, Food Insecurity, and Nutritional Outcomes In Children and Adults.” Journal of Health Economics. 23(4), 839-862. Casey, P., Goolsby, S., Berkowitz, C., Frank, D., Cook, J., Cutts, D., Black, M., Zaldivar, N., Levenson, S., Heeren, T., Meyers, A., and the Children’s Sentinel Nutritional Assessment Program Study Group. (2004). “Maternal Depression, Changing Public Assistance, Food Security, and Child Health Status.” Pediatrics, 113(2), 298-304. Denton, M. and Walters, V. (1999). “Gender differences in structural and behavioral determinants of health: An analysis of the social production of health.” Social Science and Medicine, 48, 1221-1235. Dixon, L.B., Winkleby, M.A and Radimer, K.L. (2001). “Dietary intakes and serum nutrients differ between adults from food-insufficient and food-sufficient families: Third National Health and Nutrition Examination Survey, 1988-1994.” Journal of Nutrition, Vol. 131, 1232-1246. Gundersen, C. and Oliveira, V. (2001). “The Food Stamp Program and Food Insufficiency.” American Journal of Agricultural Economics, Vol. 83(1), 875-887. Heflin, C., Siefert, K., Corcoran, M.E. and Williams, D.R. (2005). “Food Insufficiency and the Mental Health of Current and Recent Welfare Recipients: Findings from a Longitudinal Survey.” Social Science and Medicine. Vol. 61, 1971-1982. Hofferth, S. and Curtin, S. (2005). “Poverty, food programs and childhood obesity.” Journal of Policy Analysis and Management. Vol. 24(4), 703-26. Jyoti, D.F., Frongillo, E.A. and Jones, S.J. (2005). “Food insecurity affects school children's academic performance, weight gain, and social skills.” Journal of Nutrition Vol. 135, 2831-2839. Kabani, N., and Wilde, P. (2003). “Short Recertification Periods in the U.S. Food Stamp Program.” Journal of Human Resources, Vol. 38 Supplement, 1112–1138. Kelly, S., Hertman, C., and Daniels, M. (1997). “Searching for Biological Pathways between Stress and Health.” Annual Review of Public Health. Vol. 18, 437-62. Lantz, P.M., House, J.M., Mero, R.P. and Williams, D.R. (2005). “Stress, Life Events, and Socioeconomic Disparities of Health: Results from the Americans’ Changing Lives Study.” Journal of Health and Social Behavior. Vol. 46(3), 274-288. Laraia, B.A., Siega-Riz, A.M., Gundersen C. and Dole, N. (2006). “Psychological Factors and Socioeconomic Indicators are Associated with Household Food Insecurity Among Pregnant Women.” Journal of Nutrition. Vol. 136, 177-182. Lynch, J., Smith, G.D, Kaplan,G.A. and House, J.S. (2000). “Income Inequality and Mortality: Importance to Health of Individual Income, Psychosocial Environment, or Material Conditions.” BMJ Vol. 320, 1200-4. McEwen, B. S. (1998). “Protective and Damaging Effects of Stress Mediators.” New England Journal of Medicine. Vol. 338,171-179. McEwen, B. S. and Seeman, T. (1999). “Protective and Damaging Effects of Mediators of Stress: Elaborating and Testing the Concepts of Allostatis and Allostatic Load.” Annuls of the New York Academy of Science. Vol. 896, 30-47. National Center for Health Statistics. (1998). Health, United States, 1998 With Socioeconomic Status and Health Chartbook. Hyattsville, Maryland. Nelson, K., Brown, M.E. and Lurie, N. (1998). “Hunger in an Adult Population.” JAMA, Vol. 279(15), 1211-1214.

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Nord, M., Andrews, M. and Carlson, S. 2006. Household Food Security in the United States, 2004. Economic Research Report No. (ERR011). Washington, DC: Economic Research Service, U.S. Department of Agriculture. Olson, C.M. (1999). “Nutrition and health outcomes associated with food insecurity and hunger.” Journal of Nutrition, Vol. 129, 521S-524S. Rose, D. and Oliveira, D. (1997). “Nutrient intakes of individuals from food-insufficient households in the United States.” American Journal of Public Health, Vol. 87 (12), 19561961. Siefert, K., Bowman, P., Heflin, C.M., Danziger, S. and Williams, D.R. (2000). “Social and environmental predictors of major depression in current and recent welfare recipients,” American Journal of Orthopsychiatry, 2000, Vol. 70 (4), pp. 510-522. Siefert, K., Heflin, C.M., Corcoran, M. and Williams, D.R. (2001). “Food insufficiency and the physical and mental health of low-income women,” Women and Health, Vol. 32, 159177. Siefert, K, Heflin, C.M, Corcoran, M.E., and Williams, D.R. (2004). “Food Insufficiency and Women’s Health: Findings from a Longitudinal Study of Welfare Recipients” Journal of Health and Social Behavior, Vol. 45(2), 171-186. Wooldridge, J. (2002). Econometric Analysis of Cross Section and Panel Data. Cambridge: MIT Press.

In: Food Science and Security Editors: L. Amsel and L. Hirsch, pp. 125-145

ISBN 978-1-60692-977-3 c 2009 Nova Science Publishers, Inc.

Chapter 5

I NVESTIGATING V IRAL H EPATITIS A DYNAMICS IN C AMPANIA , I TALY Marco Ajelli1,2∗and Stefano Merler1 Fondazione Bruno Kessler, Trento, Italy. 2 Information Engineering and Computer Science Department, University of Trento, Italy. 1

Abstract Viral Hepatitis A is an acute infection caused by Hepatitis A Virus (HAV). Although most infected persons recover completely, this infection imposes a large economic burden, mainly due to its relevant morbidity and its high symptomaticity in adult individuals (while a significant proportion of children and adolescents remain asymptomatic). Thus, Viral Hepatitis A represents a priority for public health agencies. This infection is transmitted via the fecal–oral route. Person–to–person transmission is typical in low hygiene areas, while ingestion of contaminated water or food (mainly shellfish) is the main risk factors in low-intermediate endemicity regions (as the case of Italy). The aim of this chapter is the investigation of Viral Hepatitis A epidemic in the Italian region of Campania (Naples region). Building on a previous study, here we consider an age–structured network model, based on the explicit sociodemographic composition of the population. Since Hepatitis A is an endemic disease, and therefore the time scale of the model is of the order of several years, we employ a dynamic network of contacts in order to take into account the vital dynamics of the population. The model allows accounting for age–specific interventions (e.g. vaccination) and for all the main risk factors for HAV infections: person–to–person transmission, travels to high endemicity foreign areas and ingestion of infected food/water. The last kind of transmission plays a key role in the explanation of HAV epidemic: in fact, more than 60% of the Italian infections occur in this way (and the number increases if only the most affected areas are considered). For this reason, the process of the virus excretion in the environment and the consequent seafood contamination is explicitly modeled. Our model is able to reproduce the Campania notification data and it allows the prediction of the effects of intervention options. In presence of a vaccination programme of unlimited duration, Hepatitis A epidemic can be controlled, even by performing a E-mail address: [email protected]. (Corresponding author.) Address for correspondence: Fondazione Bruno Kessler, Via Sommarive 18, I–38100 Trento, Italy. ∗

126

Marco Ajelli and Stefano Merler very mild vaccination programme. Improvements in hygienic conditions, leading to a reduction of seafood contamination, are also able to mitigate the epidemic even in the absence of vaccination programmes.

Keywords: Individual–based model, Dynamical network of contacts, Sociodemographic model, Age–structured model, Endemic disease, Infected seafood

1.

Introduction

Hepatitis A Virus (HAV) is the cause of Viral Hepatitis A infection. The infection is an acute form of hepatitis. The patients recover completely and become immune against future HAV infections and they do not have to face any chronical stage (50). Nevertheless Viral Hepatitis A represents an important public health issue, causing remarkable economic burden worldwide (9; 16) and also in Italy (32). HAV is transmitted via the fecal–oral route associated to person–to–person transmission (direct contact) and to ingestion of contaminated food or water (indirect contact). Indirect contacts are the most frequent source of contagion (19), as the case of Italy (31). Specifically, shellfish and mussels are the main source of infection in Italy (34), especially for the most affected regions: Puglia and Campania (11; 12; 45). Another significant source of infection is represented by travels to high–endemicity areas (34) where both direct and indirect transmission can occur. Nowadays, even in the absence of vaccination, Hepatitis A epidemic is in a decay phase, mainly thanks to improved hygienic conditions as derived from economic development and higher standards of living (25; 26). Although it is true also for Italy, as documented by both notification (34; 36) and serological data (15), the South of Italy shows a completely different pattern. HAV infections are still common in Puglia, Campania and Basilicata, and a very large outbreak was observed in 1996–1997, despite the improvement of socio– economic conditions. This epidemic led Puglia to adopt, as a unique regional case in Italy, a vaccination programme consisting in the immunization of newborn and 12-year old individuals (30), which contribute to the decline of the incidence from ≈ 53 notified cases per year per 100,000 during the period 1992–1996 to ≈ 8 notified cases per year per 100,000 during the period 1998–2003, after the start of the vaccination campaign (36). Mathematical models have become valuable tools for describing the dynamics of infectious diseases and for assessing the effectiveness of intervention options (e.g., vaccination) (13; 14; 17; 23). Many models were applied also to Hepatitis A epidemic (8; 22; 46; 47; 49). The only model specific for describing the Italian epidemic and accounting for all the three most common sources of infection has been presented in (2). It is a basic model that gives a simple explanation of the temporal trends of Hepatitis A in Italy and allows the evaluation of the effects of the vaccination campaign implemented in Puglia. In this chapter we extend it by including the age structure. Following the suggestion of Riley (43), in this chapter we propose a network model, based on real sociodemographic data and accounting for millions of individuals, for investigating the dynamics of Viral Hepatitis A and evaluating the effectiveness of intervention strategies. In network models individuals are connected to other individuals by co–locating them

Investigating Viral Hepatitis A Dynamics in Campania, Italy

127

into groups (e.g., households, schools and workplaces, etc.). Additionally, the members of a group may not be connected equally well. For instance, in large schools or workplaces, subgroups of individuals (e.g. representing classmates or close colleagues) can be more strongly connected. Finally, the force of infection for a susceptible individual is non zero when there is an infectious in his/her network of contacts. Finally, in order to take into account the vital dynamics of the population, the adopted network of contact is dynamic. We focus this model on the Italian region of Campania to predict the effects of strategies similar to those already implemented in the region of Puglia. Moreover, the effects on the reduction of seafood contamination due to the improvement of hygienic conditions are also evaluated.

2.

Materials

Sociodemographic data Census data, sociodemographic data (35; 37; 38; 39) and survey data (e.g., on households composition) (40) on the Italian population are provided by the Italian Institute of Statistics. Data on schools and workplaces are provided by the Italian Ministry of University and Research (41; 42). Specific data on Campania region were employed whenever such information is available, otherwise average Italian data were used. Epidemic data Age stratified case notifications of HAV in Italy during 1992–2003 are provided by the Italian Institute of Statistics (36). Data on HAV risk factors (34) and serological data (5) are drawn from published materials of the surveillance system for Hepatitis. HAV case notifications data collected at the regional level cover the period 1992–2003 with a “hole” in 1997 (data missing). In the 1992–1996 period the national average number of notified Hepatitis A cases per 100,000 inhabitants per year was 7.9. Essentially all the regions exhibited incidences between 2 and 5 per 100,000, with two noteworthy exceptions, Puglia and Campania, with incidences of 52.6 and 14.3 per 100,000 respectively. Hepatitis A incidence in Puglia declined to 8 per 100,000 during 1998–2003 (see Figure 1). This decay is due to the starting of a vaccination campaign involving both newborns and adolescents (12 years–old). Nowadays, Puglia is the only Italian region performing Hepatitis A vaccination.

3.

Methods

One of the aims of this chapter is to propose a new methodological framework for describing the dynamics of endemic infectious diseases. Specifically, we propose a network model, based on real sociodemographic data and accounting for millions of individuals, for describing the dynamics of Viral Hepatitis A. Network models allow the explicit representation of both individuals and places where transmission can occur. Moreover, they allow the introduction of the age structure in a natural way. In mathematical models, the transmission rates are related to (i) the type of a contact and (ii) the frequency of a contact, which are both crucial to determine the probability of being infected. In network models, the type of contact and its frequency follow directly from the sociodemographic structure of the population. Therefore, it is sufficient to make

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0 2004

2003

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Puglia (scale on the left axis) Campania (scale on the right axis)

5

Monthly number of notified cases per 100,000 individuals

Figure 1. Monthly number of notified cases per 100,000 inhabitants in the two most af fected Italian regions: Puglia and Campania.

assumptions on the probability of transmitting the disease in the different environments where transmission can occur, thus avoiding the need of arbitrarily defining transmission rates among age classes, for which it is much more difficult to obtain reliable data. In the next section we explain in detail the network model employed in this chapter.

3.1.

Sociodemographic Model

The contact networks among individuals is adapted from the one proposed in (3). Basically, each individual is explicitly represented in the model and the network of contacts is generated by co–locating individuals in households, schools and workplaces on the basis of sociodemographic data. Population data Population data of the Italian region of Campania (5,701,931 inhabitants) are obtained from Italian census data (39). Individuals are hierarchically grouped within the region by municipalities (505) and provinces (5), according to the political borders of the study area. Number of individuals by municipality ranges from 400 to 1,004,500 (Naples municipality) with an average of 10,348 (95% CI 1,654 to 52,870). Households Census data on age structure and household type and size (39) are jointly used with survey data (40) to randomly assign age and co–locate individuals in households. Data on the age structure are specific for the region of Campania, while survey data refer to an analysis conducted at national level. Nine different types of household are considered in the model: single without children, single with children, single with children and an additional member, couple without children, couple without children and an additional member, couple with children, couple with children and an additional member, adults living together, more household groups. Note that the wording “without children” indicates both families without children or with children which do not live with the parents anymore and the wording “additional member” refers to an elderly person. The distribution of household type and size is summarized in Table 1. The following additional constraints are also considered:

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Table 1. Percentage of different household types and household size by type (in percentage). household type

percentage

household size 1 2 3 4 single without children 25.8 100 0 0 0 single with children 7.9 0 68.9 25 6.1 single with children⋆ 0.6 0 0 68.9 25 couple without children 19.7 0 100 0 0 couple without children⋆ 1.3 0 0 100 0 couple with children 40.0 0 0 45.1 43.8 couple with children⋆ 1.9 0 0 0 45.1 adults living together 1.6 0 78.5 21.5 0 more household groups 1.2 0 0 0 49.3 ⋆ with additional household member.

5 0 0 6.1 0 0 9.5 43.8 0 44.3

≥6 0 0 0 0 0 1.6 11.1 0 6.4

C1 any household must contain at least 1 adult (age ≥ 18); C2 the age of any child is between 43 and 18 years less than that of the youngest parent; C3 spouses age differs by no more than 15 years. The algorithm employed for generating individuals, assigning age and co–locating individuals in households is described in Figure 2. A comparison between real and simulated age structure is reported in Figure 3a. A comparison between real and simulated data on household size is reported in Figure 3b. To implement the dynamic network of contacts individuals are divided into two classes: those having the opportunity to create their own family and those that have already created one. The second class comprises household heads, his/her spouse (if any) and the aggregates (if any); all the other categories of individuals are members of the first class. Employment Demographic, school (41; 42) and industry (38) census data are used for randomly assigning an employment category to each individual on the basis of age (see Table 2). Simulated population is structured as follows: 2,154,137 workers (117,640 of which employed as teachers, and thus employed in schools), 1,111,204 students and 2,437,185 unemployed or retired. Students are deterministically assigned to a specific school type (6 types, from nursery school to university) on the basis of age. For workers there is no distinction for the type of work, except for those employed in schools (e.g., teachers) that are assigned to one of the six types of schools. Commuting Commuting destinations for both workers and students were randomly assigned by employing a gravity model (51). Basically, individuals tend to move away from small municipalities and travel to the most populate ones and the probability of traveling to a given destination decreases with the distance. The parameters of the gravity model are the

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⊲ Foreach municipality m ⊲ Let nm be the number of individuals living in m ⊲ na ← 0 ⊲ While na < nm ⊲ determine household type t ∼ M (pT ), where M (pT ) is a multinomial distribution with probabilities pT (see Table 1). ⊲ determine the household size s ∼ Mt (pS (t)), where M (pS (t)) is a multinomial distribution with probabilities pS (t) (see Table 1). ⊲ determine age class of household head c ∼ M (pC (t, s)), where M (pC (t, s)) is a multinomial distribution with probabilities pC (t, s) obtained from survey data. ⊲ determine age of household head a ∼ M (pA (c)), where M (pA (c)) is the multinomial distribution of age structure of the region of Campania in the interval c. ⊲ determine age of other members (see additional constraints C1, C2 and C3). ⊲ na ← na + s EndWhile EndForeach

Figure 2. Pseudocode of the algorithm employed for generating individuals, assigning age and co–locating individuals in households. Table 2. Schools/workplaces age group and employing rate (approximate value obtained by averaging on the specific employment rate by age). School category and workplace Day care Kindergarten Primary school Middle school High school University Workplace

age group (in years) 0–2 3–5 6–10 11–13 14–18 ≥ 19 ≥ 15

employing rate (in percentage) 14 90 97 96 82 31 43

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1.8 0.9 0

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a

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20 10 0

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30

b

1

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>5

Size

Figure 3. a. Age–structure of the region of Campania (black line) and age–structure simulated by the sociodemographic model (grey line). b. Italian household size (black bars) and household size simulated by the model (grey bars). Note that this data on household size are not directly employed in the algorithm briefly described in Figure 2.

same employed in (13) for Italy. Moreover, the gravity model is integrated with the data on the proportion of commuters specific for the Italian region of Campania (39). 3.1.1.

Dynamic Network of Contacts

Network models are used mainly for evaluating the effectiveness of intervention options for containing/mitigating a new influenza pandemic (13; 17; 18; 23; 27; 29) or a bioterroristic attack (e.g., by the employment of smallpox virus) (24; 28; 44). Since the time duration of these epidemics is “short” compared to the temporal scale of human social network changes, the network models proposed in literature are static, i.e., the connections among individuals remain constant over time. On the contrary, in this chapter we are interested in the analysis of an endemic disease, in which the temporal scale considered is of the order of many years. Therefore, we need to introduce the vital dynamics in the model and the network of contacts among individuals has to be kept updated. In particular, individuals come to life, grow, can generate a new household, can procreate and die; moreover they may go to school (following the scholastic career), have an employment and retire.

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Updating step The updating step of the population and of the network of contacts is one year. Vital dynamics Here we assume a closed population, i.e. without immigration and emigration, with a constant number of individuals, i.e. the number of deaths corresponds to the number of newborns. Newborns and deaths Newborns are determined by replacing randomly select individuals who are considered as dead, on the basis of the age of the individuals and the specific mortality rates of the region of Campania (35). Each newborn is located in an existing household, according to the data on households size and the age of the adults in the households. Finally, on the basis of the employment rate, it is determined whether the newborn go to a day care center or not. The age of the other individuals is simply incremented by one year. New household groups Each individual that has not already generated his/her own family and aged at least 18 years old has a positive probability of creating a new household. New households are generated according to the wedding rates per age provided in (37). Moreover, such kind of individuals has a positive probability of becoming single. New employments Every time the population is updated, on the basis of the employing rate per age, an employment (possibly, “unemployed”) is associated to each individual. This allows individuals to follow the scholastic career. More in detail, for each individual a new type of employment (see Table 2) is randomly chosen: if it is the same he/she had before the update he/she keeps his/her place, otherwise a new school/workplace is randomly chosen among the existing ones. In the latter case the commuting destination is chosen by employing the commuting model described in the previous section. Sociodemographic projections The model is therefore able to give us some indications on future characteristics of the sociodemographic structure of the population. In particular, estimates on the age structure of the population (see Figure 4a) and on the size of the households are obtained (see Figure 4b). They will be briefly discussed in the closing section.

3.2.

Epidemic Transmission Model

The epidemic transmission model is an adapted version of the one proposed in (2) which takes into account all the three main sources of HAV infection in Italy: the person–to– person transmission, the ingestion of infected food and the travels to high endemicity areas. Individuals are assumed to move among the following epidemiological states: susceptibles, i.e. individuals with no immunity to Viral Hepatitis A; exposed, i.e. individuals infected by HAV who are not able to transmit the disease yet; infectious, i.e. individuals able to transmit the disease; removed, i.e. individuals that have become immune after

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1.8 0.9 0

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a

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Size

Figure 4. a Age–structure of Campania (black line) and age–structures simulated by the sociodemographic model, scenarios at 5, 10 and 15 years (from dark grey to light grey). b Italian household size (black bars) and household size simulated by the model, scenarios at 5, 10 and 15 years (from dark grey to light grey).

having been infected by HAV, and vaccinated, i.e. individuals that have become immune after the administration of HAV vaccine. The flow chart of individuals is the following: a susceptible can become exposed after a contact with an infectious individual or after the ingestion of infected food1 , or can become a vaccinated after an administration of a dose of vaccine2 ; exposed become infectious after the latent period; infectious become removed after the infectious period. Finally, as a consequence of the excretions of HAV by infectious individuals, a quantity of virus also infects the water and the seafood (shellfish and mussels). Moreover, since network models allow considering the explicit representation of the places where the transmission can occur, the person–to–person contacts are divided into contacts within households, schools and workplaces. Therefore, the transmission process can be written down in terms of equations by associating to any susceptible individual, i, a probability of becoming exposed given by: pi = 1 − e−λi τ , where τ represents the time step of the simulation (1 week) and λi is the risk of infection 1 2

Both kinds of infection can occur in Campania or during a travel outside the region. After the administration of an HAV vaccine the probability of developing immunity is about 98% (7).

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of the susceptible i. The latter is the sum of the risk factors given by the three sources of contagion: S T λi = λD i + λi + λi , S where λD i represents the direct contact component, λi represents the indirect component T and λi the component associated to the travels.

Direct contacts Direct contacts account for the person–to–person transmission which can occur within households and schools/workplaces. In particular, the risk factor for any susceptible individual i is given by the equation: λD i

=

Hi X Ij (τ )βh j=1

Hi − 1

+

Pi X Ij (τ )βpϑ j=1

Pi − 1

,

where • Ij (τ ) is equal to 1 if individual j is infectious at time τ , 0 otherwise; • Hi is the number of individuals living in the same household of i3 ; • βh is the transmission rate within households; • Pi is the number of individuals working or studying in the same place of i4 ; • βpϑ is the transmission rate within the place of type ϑ, e.g., kindergarten, primary school, workplace (the complete list of the considered places can be found in Table 2). Indirect contacts Indirect contacts account for the infection occurred by ingesting contaminated seafood. The risk of infection due to this component is given by: λSi

= βs σs (ai )U (τ ) ,

where • βs is the transmission rate associated to the ingestion of infected seafood; • σs (ai ) represents the consumption of seafood. In order to take into account that the consumption of shellfish and mussels varies by age, σs is a function of the age of the individual i. • U (τ ) is the quantity of HAV virus in seafood at time τ , as a consequence of the excretions of infected individuals. 3 4

If Hi = 1 the first term in Equation (1) is defined to be 0. If Pi = 0 or Pi = 1 the second term in Equation (1) is defined to be 0.

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The variable U (τ ) depends on the number of individuals that were infective prior to time τ , basically because they excrete HAV virus in the environment during their entire period of infectivity. For simplicity, we decide to model the dynamics of U (τ ) by the ordinary differential equation: h i d U (τ ) = δ I(τ ) − U (τ ) , (1) dτ where δ is the decay rate of HAV virus in the environment and I(τ ) is the number of infectious individuals given by the network model at time τ . Thus, our model could be seen as a hybrid model, since it couples a network model with a classical ordinary differential equations one. All the details on the modeling of the indirect contacts component of the transmission can be found in (2). Travels to high–endemicity areas Travels to high–endemicity areas account for all the infections occurring outside the region. This component of the risk factor is simply modeled as λTi

= βt σt (ai ) ,

where • βt is the transmission rate associated to the travels; • σt (ai ) represents the frequency of the travels. In order to take into account that the probability of having a travel varies by age, σt is a function of the age of the individual i.

3.3.

Intervention Measures

Since Hepatitis A is a vaccine-preventable disease, we will evaluate the effectiveness of some vaccination programmes. In particular, the efficacy of the vaccination programme currently implemented in Puglia will be evaluated on the region of Campania. Moreover, since Hepatitis A is undergoing a global decline (even in the absence of vaccination) due to the worldwide improvements in standards of living and hygiene, we will draw different scenarios on possible hygienic improvements. Vaccination Vaccination target population for Hepatitis A is traditionally represented by children and young individuals. In particular we consider newborns and adolescents of 12 years old as the target population. Vaccination is modeled by reducing the proportion of susceptible individuals in the target population. This proportion depends on vaccination coverage (i.e., the proportion of target population that is adequately immunized) and vaccine effectiveness (i.e., the probability of developing immunity after the administration of a vaccine dose). The latter is kept fixed at 98% according to (7). Since vaccination coverage depends on the vaccination programme set by the public health and on the collaboration of the population, many scenarios are evaluated. More in detail, in the simulations individuals are treated during the whole course of the year in such a way to reach the vaccination coverage for each target class.

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Modeling hygienic improvements We assume that hygienic improvements are mainly related to the indirect contacts component. In fact, the main source of contagion is represented by the consumption of infected shellfish and mussels consumed raw (34). Often the contagion of the seafood is due to infected water used in fish market stands (48). Therefore, we model the improvement of hygienic conditions as a reduction of the infected seafood. More in detail, after solving Equation 1 we decrease the value of U (τ ) by a reduction factor. Some scenarios are drawn by changing this parameter.

3.4.

Model Parametrization

In mathematical modeling of infectious diseases the main parameter is the basic reproductive number R0 . It is related to the average number of secondary cases generated by an infectious individual assuming a completely susceptible population (4). In classical models with vital dynamics R0 can be computed as the inverse of the number of susceptibles at the endemic equilibrium. Therefore, we compute the basic reproductive number by adopting such definition. First of all, in order to parametrize the model, we want that the number of susceptibles at the endemic equilibrium is such that R0 = 2.6, i.e. consistent with the estimated value for the Italian region of Campania (2). The free parameters of the model are the transmission rates, i.e. βh , βpϑ , βs σs (a) and βt σt (a), while all the other epidemiological parameters (e.g., the latent period and the infectious period) are kept fixed from the literature (see Table 3). Let us notice that the probability of becoming symptomatic plays an important role in the parametrization process because only the symptomatic infectious have a positive probability of becoming notified cases. Finally, the simulations are initialized by assuming a proportion of immune individuals per age given by the serological analysis conducted on the Italian population in (5). Therefore, we choose the values of the transmission rates such that R0 ≈ 2.6 and the percentages of cases generated by each source of infection (direct, indirect and travels) complies with the risk factors by age reported in (34). Finally, we estimate the reporting rate in such a way that the simulated epidemic complies with the Hepatitis A notification data for Campania.

4.

Results and Discussion

Baseline scenario The risks of infection by age for the three sources of contagion are: 33.5% (direct contacts), 47.5% (indirect contacts), 19% (travels to high–endemicity areas), for individuals aged 0–14; 14.7% (direct contacts), 74.3% (indirect contacts), 11% (travels), for individuals aged 15–24; 21% (direct contacts), 53.4% (indirect contacts), 25.6% (travels), for individuals older than 24 years. These estimates agree well with the survey data reported in (34). A comparison is shown in Figure 5a. Fixing R0 = 2.6, the percentage of the removed at the endemic equilibrium is 61.6%. This estimate complies with the value obtained in (5) where a value of 60.1% is reported for Italy as a whole and 67.7% for South and Islands. The percentages of removed per age also comply with the serological analysis data (5). A comparison can be found in Figure 5b.

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Table 3. Epidemiological parameters and sources. Parameter Reproductive number Latent period Infectious period Decay of survival of HAV in the environment Probability of becoming symptomatic for individuals aged 0–6 for individuals aged 7–16 for individuals older than 16 Vaccine efficacy Vaccination coverage in Puglia for newborns for 12 years-old individuals

Unit adimensional weeks weeks weeks−1

Value 2.6 2 3 0.0833

Reference (2) (21; 50) (21; 50) (1; 2; 10; 33)

percentage percentage percentage percentage

4 16 80 98

(19; 50) (19; 50) (19; 50) (7)

percentage percentage

20 80

(30) (30)

The estimated value for the notification rate is about 2%. This estimation is quite consistent with the estimation of less than 10%, given by the Centers for Disease Control and Prevention for the Viral Hepatitis A epidemic in USA (20). Moreover, this value is partially justified by the low risk of Hepatitis A infection perceived by the population of Campania. As reported in (45), only 39% of the interviewed people thinks that it is necessary to go to the hospital if an icteric condition occurs. A deeper discussion on the reporting rate can be found in (6), where it is suggested that Hepatitis A symptomatic cases are seriously under–reported. By employing the estimated value for the notification rate, the average number of notified cases per year per 100,000 is 14.5, which is in excellent agreement with the value reported in (36). A comparison between the monthly notification data provided in (36) and an average of the simulations of the model is shown in Figure 5c. As discussed in (2), the yearly pattern observed in the data is probably due to the consumption of seafood in the region of South Italy, which shows a clear seasonal pattern. This kind of seasonality has not been considered in the model presented in this chapter: this is the reason why we do not fit the notification data. Effectiveness of vaccination programmes In presence of a vaccination programme of unlimited duration involving both newborns and 12–years–old adolescents, Hepatitis A epidemic can be controlled quite well. Performing a very mild vaccination programme (involving 50% of the 12–years–old and only 10% of the newborns) the proportion of removed becomes about one half of that observed in the baseline scenario (i.e., without any kind of intervention). The number of notified cases per 100,000 inhabitants also reduces to 8.5. The cost of this vaccination campaign is 557 vaccine doses per 100,000 individuals per year. Better results are obtained for higher vaccination coverages. In particular, for a very efficient vaccination strategy (90% of the 12–years–old and 80% of the newborns), the proportion of removed drastically declines to 8.2% at the cost of about 1,000 vaccine

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travel

direct

travel

direct

seafood

seafood travel

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seafood

80 60 40 0

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30−39

40−49

50−59

>59

Age class

5 4 3 2 1 0

Notified cases per 100,000 per month

c

Jan 1992

Jan 1993

Jan 1994

Jan 1995

Jan 1996

Jan 1997

Time (in months)

Figure 5. Baseline scenario. a Risk factors per age class. Comparison between the data reported in (34) (black bars) and the model (grey bars). Children account for individuals aged 0–14, Young for 15–24 and Adults for individuals older than 24 years old. b HAV seroprevalence per age class. Comparison between the data reported in (5) (black bars) and the model (grey bars). c Number of monthly notified cases per 100,000 in Campania between January 01, 1992 and December 31, 1996. Comparison between the data (black squares) and the average of the model simulations (dark grey line). Horizontal light grey line represents the average number of notified cases per 100,000 in Campania during the considered period.

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∗ † ‡

Maximum number of notified cases per year per 100,000 ∗

Number of vaccine doses per year per 100,000

Vaccinated (% of population)

Removed (% of population)

Average number of notified cases per year per 100,000

Reduction of seafood contamination (%)

Duration of the vaccination campaign (in years)

Vaccination coverage of 12 aged individuals

Vaccination coverage of newborns

Table 4. Effectiveness of intervention measures.

– – – – 14.5 61.6 – – – † 10 50 – 8.5 31.3 39.9 557 – † 20 80 – 5.6 19.1 56.5 788 – † 80 90 – 2.6 8.2 77.0 1,049 – 20 80 5‡ – 13.8 54.9 3.6 78 27.4 20 80 10‡ – 12.9 51.2 7.2 157 20.1 ‡ 20 80 20 – 11.4 44.5 13.7 296 19.6 – – – 20 13.9 45.6 – – – – – – 50 12.8 38.5 – – – – – – 80 11.2 36.9 – – – † 20 80 20 5.1 15.2 56.3 788 – † 20 80 50 4.8 14.1 56.3 788 – † 20 80 80 4.8 13.8 56.3 788 – due to the build up of susceptibles after the end of vaccination programme. for the entire duration of the simulation (i.e., 50 years). note that the duration of this simulation is 50 years.

doses per 100,000 individuals per year. The summary of these results is given in Table 4. Whatever vaccination programme is performed, the epidemic can not be eradicated because some sporadic cases can often be caused by travels to high endemicity areas. By fixing the vaccination coverage at 20% for newborns and 80% for 12 years–old individuals for mimicking the intervention implemented in Puglia (30), some scenarios on the duration of the vaccination programme are drawn. The effects of a very short vaccination campaign (5 years) can be counterproductive. After 50 years from the starting of the programme, the average number of notified cases is about the same observed in the baseline scenario, and the proportion of removed decreases only to 54.9%. The proportion of vaccinated individuals is only 3.6%, but the cost is limited: less than 800 doses per 100,000 individuals per year during the vaccination programme, i.e. about 78 doses per 100,000 individuals per year during the 50 years simulated. Finally, an epidemic wave due to the build up of susceptibles after the end of the vaccination programme is expected. During the epidemic wave, the average number of notified cases grows to 27.4 per year per 100,000, i.e. about 2 times larger than the average number in absence of vaccination. Quite the same results are obtained for the other scenarios considered. At the 50th year, the proportion of removed decreases to 51% and 44% and the proportion of vaccinated increases to 7% and

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14% for a duration of 10 and 20 years, respectively. The expected epidemic wave for a 10– years–vaccination campaign is still in its increasing phase after 50 years from the starting of the programme: this is the reason for a lower value of notified cases (20 per 100,000) than for a 5–years–programme. The same holds for a 20–years–vaccination campaign, in which the epidemic wave is expected after 50 years from the starting of the programme. Effects of improvements in hygienic conditions Improvements in hygienic conditions have a wide effect on the Hepatitis dynamics. Although the decrease in the number of notified cases is limited (from 14.5% to 13.2% in the case of 80% of hygienic conditions improvement) the proportion of removed drastically declines: from 61.6% of the baseline scenario to 45.6% for 20% of hygienic conditions improvement, 38.5% for 50% of hygienic conditions improvement and 36.9% for 80% of hygienic conditions improvement (see Table 4). These results suggest that, with a natural improvement in hygienic conditions, the epidemic will decline even in the absence of vaccination (as observed in (25; 26)). The estimated values of R0 in these scenarios are 1.8, 1.6, 1.6, respectively for 20%, 50% and 80% of hygienic conditions improvement. Combining a vaccination programme of unlimited duration with natural hygienic improvement leads to a very low proportion of removed (about 5%), just a bit less than considering an equivalent vaccination campaign without hygienic improvements (see Table 4).

5.

Conclusion

In this chapter a mathematical model has been proposed for investigating the dynamics of Viral Hepatitis A in the Italian region of Campania. This chapter also proposes a new methodological framework for describing endemic infectious diseases. The network model considered allows modeling a highly heterogeneous population, thus improving the characterization of contacts and representing age–structured population in a natural way. These improvements are due to the explicit representation of the individuals in the model. The model is able to reproduce the seroprevalence of HAV in the population, the proportion of the risk factors and the average number of notified cases. Moreover it allows analyzing the effectiveness of vaccination programmes and the effects of improvements in hygienic conditions. The results obtained show how a vaccination programme of unlimited duration is able to reduce Hepatitis A at a limited cost in terms of vaccine doses required and low vaccination coverage. If vaccination campaigns were interrupted an epidemic wave of at least 27 notified cases per 100,000 would be expected. Moreover, expected and desirable improvements in hygienic conditions would lead to a decrease in R0 from ≈ 2.6 to ≈ 1.8. Finally, the eradication of the disease is not possible, due to the importation of infected cases from high–endemicity areas. However, there are some critical issues. First of all, the model is based on the assumption of a constant population. Neither immigration nor emigration are considered and the number of newborns is assumed to be equal to the number of deaths. These are reasonable modeling choices, but certainly they can not be considered as the optimum. Nowadays in Italy the fertility rate seems in a decline phase (for the Campania region this phenomenon is less remarkable than for the rest of the country (35)) and the immigration is constantly

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increasing. Thus, the demographic projections resulting from our model can only be considered as a plausible scenarios. Therefore, the longer the temporal interval of the simulations, the less accurate the predictions are. Of course, these issues can be integrated in a future work. We focused on a single Italian region. It would be interesting to study the effects of the intervention measures implemented in a region on the rest of the country. In particular, by looking at the available notification data (36) it seems that the vaccination programme of region Puglia has an effect on the whole of Italy (2). In order to develop an Italian model specific to the Hepatitis A, epidemiological and sociodemographic data should be collected with a greater level of detail. In fact, it is easy to believe that the risk factors greatly vary from region to region (e.g., the consumption of raw seafood is not much common in the North of Italy). Therefore, model parametrization, which is a hard problem to tackle, may become a critical point. Finally, the low risk of Hepatitis A infection perceived by the population (especially in Campania (45)), makes the notification data very unreliable. In fact, it is common wisdom among public health operators that a high level of perceived risk significantly increases the reporting rate, and it is clear that during the epidemic phases the risk perceived by the population is higher than during the intra–epidemic periods. As discussed in (2), this may have occurred during the great epidemic in Puglia in 1996–1997.

Acknowledgments The authors thank Laura Fumanelli and Bruno Caprile for reading earlier versions of the manuscript and for their useful suggestions. The authors also thank Mimmo Iannelli and Piero Manfredi for helpful discussions on Hepatitis A modeling.

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[6] G. L. Armstrong and B. P. Bell, Hepatitis A virus infection in the Unites States: modelbased estimates and implications for childhood immunization, Pediatrics 109 (2002), 839–845. [7] F. Averhoff, C. N. Shapiro, B. P. Bell, I. Hyams, L. Burd, A. Deladisma, E. P. Simard, D. Nalin, B. Kuter, C. Ward, M. Lundberg, N. Smith, and H. S. Margolis, Control of Hepatitis A Through Routine Vaccination of Children, Journal of the American Medical Association 286 (2001), no. 23, 2968–2973. [8] C. T. Bauch, A. S. Srinivasa Rao, B. Z. Pham, M. Krahn, V. Gilca, B. Duval, M. H. Chen, and A. C. Tricco, A dynamic model for assessing universal Hepatitis A vaccination in Canada, Vaccine 25 (2007), no. 10, 1719–1726. [9] J. J. Berge, D. P. Drennan, R. J. Jacobs, A. Jakins, A. S. Meyerhoff, W. Stubblefield, and M. Weinberg, The cost of hepatitis A infections in American adolescents and adults in 1997, Hepatology 31 (2000), no. 2, 469–473. [10] E. Biziagos, J. Passagot, J. M. Crance, and Deloince R., Long-term survival of Hepatitis A virus and poliovirus Type 1 in mineral water, Applied and Environmental Microbiology 54 (1988), no. 11, 2705–2710. [11] M. Chironna, C. Germinario, D. De Medici, A. Fiore, S. Di Pasquale, M. Quarto, and S. Barbuti, Detection of hepatitis A virus in mussels from different sources marketed in Puglia region (South Italy), International Journal of Food Microbiology 75 (2002), 11–18. [12] M. Chironna, A. Grottola, C. Lanave, E. Villa, S. Barbuti, and M. Quarto, Genetic analysis of HAV strains recovered from patients with acute hepatitis from Southern Italy, Journal of Medical Virology 70 (2003), no. 3, 343–349. [13] M. L. Ciofi degli Atti, S. Merler, C. Rizzo, M. Ajelli, M. Massari, P. Manfredi, C. Furlanello, G. Scalia Tomba, and M. Iannelli, Mitigation measures for pandemic influenza in Italy: an individual based model considering different scenarios, PLoS ONE 3 (2008), no. 3, e1790. [14] V. Colizza, A. Barrat, M. Barthelemy, A.-J. Valleron, and A. Vespignani, Modeling the Worldwide Spread of Pandemic Influenza: Baseline Case and Containment Interventions, PLoS Medicine 4 (2006), no. 1, e13. [15] R. D'Amelio, A. Mele, A. Mariano, L. Roman, R. Biselli, F. Lista, A. Zanetti, and T. Stroffolini, Hepatitis A, Italy, Infectious Diseases 11 (2005), no. 7. [16] A. Das, An economic analysis of different strategies of immunization against hepatitis a virus in developed countries, Hepatology 29 (2003), no. 2, 548–552. [17] N. M. Ferguson, D. A. Cummings, S. Cauchemez, C. Fraser, S. Riley, A. Meeyai, S. Iamsirithaworn, and D. S. Burke, Strategies for containing an emerging influenza pandemic in Southeast Asia, Nature 437 (2005), 209–214.

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[48] H. Shuval, Estimating the global burden of thalassogenic diseases: human infectious diseases caused by wastewater pollution of the marine environment, Journal of Water and Health 1 (2003), 53–64. [49] A. S. Srinivasa Rao, M. H. Chen, B. Z. Pham, A. C. Tricco, V. Gilca, B. Duval, M. D. Krahn, and C. T. Bauch, Cohort effects in dynamic models and their impact on vaccination programmes: an example from hepatitis A, BMC Infectious Diseases 6 (2006), 174. [50] J. T. Stapleton and S. M. Lemon, Infectious Diseases, ch. Hepatitis A and hepatitis E, pp. 790–797, Philadelphia, US, Lippincott Co, 1994. [51] C. Viboud, O.N. Bjrnstad, D.L. Smith, L. Simonsen, M.A. Miller, and B.T. Grenfell, Synchrony, Waves, and Spatial Hierarchies in the Spread of Influenza, Science 312 (2006), no. 5772, 447–451.

In: Food Science and Security Editors: L. Amsel and L. Hirsch, pp. 147-160

ISBN: 978-1-60692-977-3 © 2009 Nova Science Publishers, Inc.

Chapter 6

SEAFOOD AS A FUNCTIONAL FOOD IN DECREASING THE RISK FOR THE METABOLIC SYNDROME Helen Hermana Miranda Hermsdorff1, Itziar Abete1, María Ángeles Zulet1, Inga Thorsdottir2 and José Alfredo Martínez1 1

Department of Nutrition and Food Science, Physiology and Toxicology, University of Navarra, Navarra, Spain. 2Unit for Nutrition Research, Department of Food Science and Human Nutrition, Landspitali University Hospital, University of Iceland, Reykjavik, Iceland

Abstract The increased incidence of the metabolic syndrome, characterized by insulin resistance, visceral adiposity, dyslipidemia, hypertension, and a systemic proinflammatory state, greatly increases the risk of cardiovascular disease. These clinical conditions are multifactorial in origin and treatment, having a strong dietary determinant. Currently, the most promising approach to mitigate obesity and metabolic syndrome is lifestyle intervention, weight reduction, decreased total and saturated fat consumption, and increased physical activity. In this sense, traditional foods, considered as functional foods, could be an important choice helping in the treatment of obesity and metabolic syndrome features. Functional foods consumed in an adequate amount on a healthy diet could improve the benefits associated to weight loss and reduce the metabolic syndrome features. Specifically, seafood has been considered as a functional food since their properties are not only nutritional, but also have been related with the improvement of several cardiovascular risk factors. Benefits include lower risk of primary cardiac arrest, reduced cardiovascular mortality, particularly sudden cardiac death, reduced triglyceride levels, increased high density lipoprotein levels, improved endothelial functions, reduced platelet aggregability, and lower blood pressure. Thus, healthy diets that avoid excess energy intake, reduce saturated fat and include seafood consumption may be a key component in the nutritional treatment of obesity and metabolic syndrome features.

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Introduction Overweight and obesity, resulting from an increase in visceral adipose tissue, are associated with type 2 diabetes mellitus and cardiovascular diseases (CVD) and with the development of metabolic complications such as dislipemias, hypertension, insulin resistance (IR), that characterize the metabolic syndrome (MS). In addition, a high adiposity increases the activity of adipokines, cytokines and other inflammatory markers, inducing a low-grade chronic inflammation and oxidative stress in obese individuals [1-5]. In this context, the consumption of foods with functional properties may help not only in body weight and fat mass reductions, but also improve MS features, inflammation and oxidative stress related to obesity. Nowadays, functional foods are products with nutritional or non-nutritional compounds able to specifically affect some body functions, improving the health status and reducing the risk for chronic diseases in addition to supply essential dietary nutrients [6, 7]. Interesting, the intake of traditional foods could be a relevant functional option in some circumstances. Indeed, fish and derivative compounds consumption have been associated with decreased risk of suffering MS features and CVD through several mechanisms [8-10]. Thus, the chapter was focussed on a brief review about obesity and related-diseases as well as about the concept and related-claims for functional foods. The examination of the role of fish and related-fish bioactive compounds with functional properties on the basis of a healthy diet to promote body weight regulation and beneficial metabolic and inflammatory changes was also discussed.

Obesity and Related-Diseases Earlier, infectious diseases were the greater cause of mortality in the world. Nowadays, chronic diseases are the main cause of death, according to World Health Organization [11]. This chronic disease prevalence not only affects industrialized countries, but also countries in economic transition. The estimation is that mortality by ischemic heart disease will increase 120% for women and 137% for men in these countries, between 1990 and 2020 [12]. Regarding to obesity, at least one billion adult people have overweight and 312 million have obesity, with a prevalence of 40-60% for overweight and obese in different countries [13]. Excessive weight gain and accompanying pathological disturbances such as IR, hypertension, hyperlipidemia are the major public health concerns. In this context, lifestyle has been implicated as an important risk factor for obesity, cancer, diabetes and CVD development, in addition to a genetic predisposition in some individuals [14]. Thus, inadequate dietary habits strongly influence obesity and associated disorders during the life course. Moreover, sedentarism and the consecutive weight gain are also related with the onset of MS features.

Seafood as a Functional Food in Decreasing the Risk for the Metabolic Syndrome 149 Indeed, the International Diabetes Foundation (IDF) defined in 2005 criteria for the diagnosis of the MS: central obesity must be present in addition to two or more of the following four factors: waist circumference ≥85 cm for women and ≥90 cm for men, systolic blood pressure ≥ 130 mmHg or diastolic blood pressure ≥ 85 mmHg; triglycerides ≥150 mg/dl, fasting blood glucose ≥ 100 mg/dl, high density lipoprotein cholesterol (HDL-c) < 40 mg/dl in males and <50mg/dl in females [15]. Other institutions such as the World Health Organisation [16], the European Group for the Study of Insulin Resistance (EGIR) [17] or the National Cholesterol Education Programme Adult Treatment Panel III [18] also have set up their own diagnosis criteria for MS. Inflammation and oxidative stress also are involved in the pathogenesis of major public health problems, overweight and obesity as well as type 2 diabetes mellitus, dyslipidemia, hypertension and CVD [1, 3, 4, 19, 20]. Furthermore, recent findings indicate that elevated levels of cytokines, endothelial function and acute-phase markers, such as tumour necrosis factor-alpha (TNF-α), interleukins (IL), soluble adhesion cellular molecules, selectins, Creactive protein (CRP), among others, are associated with features accompanying MS and increased risk of CVD [1, 4, 21, 22]. In obese individuals, an altered adipokines expression can lead to modification on glucose and lipid metabolism as well as on inflammatory and endothelial functions, by several mechanisms:

1. Lipolysis stimulation in adipose tissue and higher availability of non-esterified fatty 2. 3. 4.

5. 6.

acids (NEFA), resulting in hepatic insulin resistance, atherogenic lipid profile, reactive oxygen species production related to oxidative stress [1, 4, 19]. Impaired insulin signalling in adipose and muscle tissues, with likely IR in these tissues [23-26]. Increased hepatic production of acute-phase proteins such as CRP, fibrinogen, haptoglobin, amiloid A apolipoprotein, which are involved in inflammatory processes [3, 27, 28]. Increased production of adhesion cellular molecules such as soluble vascular cell (sVCAM-1) and intercellular adhesion molecule type-1 (sICAM-1), P and E selectins and, activation of nuclear transcriptional factor-κβ and increased platelet aggregation. All are mechanisms associated with atherogenic processes [20, 29-31]. Reduced production of nitric oxide, impairment in endothelial vasodilatation and alterations in renin angiotensin system, which are risk factors for hypertension [1, 4, 32]. Activated coagulation system and reduced fibrinolytic activity, increasing the risk for thrombosis [19, 27].

Thus, excessive adiposity induces adipokines, cytokines and other inflammatory markers activity resulting in a low-grade chronic inflammation, which is associated with MS development and higher risk for type 2 diabetes mellitus and CVD (Figure 1).

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OBESITY (Adipose Tissue Hypertrophy and Hyperplasia)

Changed insulin and catecholamine sensibility ↑ / ↓ of adipocytokines expression and secretion

↑ NEFA

↓ Insulin signalling (muscle/ liver/ adipocites) ↑ TG ↑ LDL-c ↓ HDL-c

Insulin Resistance

Atherogenic Lipid Profile

↑ Muscle and liver fat

Insulin Resistance

Changed renin angiotensin system ↓ Nitric oxid production

↑ Platelet activity ↑ reactive oxigen species and adhesion molecules secretion

Hypertension

ATHEROSCLEROSIS

Figure 1. Metabolic syndrome and obesity-related mechanisms. HDL-c: high density lipoprotein; LDLc: low density lipoprotein; NEFA: non-esterified fatty acids; TG: triglycerides.

Functional Food: Concepts and Claims The term functional food was coined in Japan, during the 80´s decade, and the Food for Specific Health Use (FOSHU) was established in 1991. Nowadays, functional foods are products containgin nutritional or non nutritional compounds able to specifically affect some body functions, improve the health and reduce risk for diseases in addition to supply essential dietary nutrients. In 1998, the European Union project Functional Food Science in Europe (FUFOSE) presented a Consensus document [7], suggesting that any claim for enhanced/improved function and reduced risk of disease should be scientifically justified. Enhanced function statements were defined as claims that concern specific beneficial effects of nutrients and other substances on physiological and psychological functions or biological activities beyond their established nutritional role in growth, development and other cellular physiological functions of the body. As regards to disease risk reduction claims, it was noted that the true disease endpoint often cannot be measured directly for ethical or practical reasons. Therefore, the identification and validation of suitable markers were considered as key issues. Markers were classified as related to 1) exposure, 2) a target function or biological response, and 3) an appropriate intermediate endpoint of an improved state of health and well-being, or reduction of the risk of disease, or both. The Consensus document of the project Process for the

Seafood as a Functional Food in Decreasing the Risk for the Metabolic Syndrome 151 Assessment of Scientific Support for Claims on Foods (PASSCLAIM) [6], published in 2005 is focussed on beneficial effects of foods and food components on health. The PASSCLAIM of products, together with the forthcoming regulation on foods, will facilitate that consumers could benefit from an improved and harmonised approach concerning the scientific assessment and communication of claims on foods. In this context, the PASSCLAIM Consensus document [6] has categorized seven different potential issues for claims of foods and food components: Diet related cardiovascular disease; bone health and osteoporosis; physical performance and fitness; body weight regulation, insulin sensitivity and diabetes risk; diet-related cancer; mental state and performance; and gut health and immunity. The potential food related-MS issues are diverse (Table 1). Table 1. Existing and potential claims for food effects on metabolic syndrome features Existing Claims Can help to lower/ to reduce cholesterol as part of a low-fat diet Lowers/ Regulates/ Controls cholesterol Helps maintain a healthy heart Potential claims HDL-c/ TG concentrations May increase HDL-c concentrations/ improve your lipoprotein profile May lower triacylglycerol concentrations (after a meal)/ the risk of cardiovascular diseases/ coronary heart disease Blood Pressure May lower systolic/ diastolic blood pressure May reduce left ventricular hypertrophy May lower the risk of stroke/ heart failure/ cardiovascular diseases/ coronary heart disease Overweight Reduces the risk of body weight gains/ abdominal fat/ appetite Contributes to body weight reduction Helps to reduce energy/food intake Increases satiety/ metabolic rate/energy expenditure/ lipid oxidation MS features Improves insulin sensitivity/ fat composition in the body Increases insulin-mediated glucose disposal Improves vascular function in individuals with IIS and/or hyperglycaemia Reduces saturated fat in the body/ inflammation associated with IIS and/or hyperglycaemia Lowers plasma NEFA IIS: impaired insulin sensitivity; HDL-c: high density lipoprotein cholesterol; MS: metabolic syndrome; NEFA: non-esterified fatty acids; TG: triglycerides. Source: Aggett PJ et al.[6]. PASSCLAIM: consensus on criteria. European Journal of Nutrition, v.44, p5-30, 2005.

Several ingredients such as fibre, ω3-fatty acid (FA), antioxidants and minerals may produce beneficial effects on body weight regulation, satiety, lipid and carbohydrate turnover and other positive healthy metabolic changes. Currently, there are many modified products with functional properties without clear information, which makes really difficult to choose

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the appropriate product. Traditional foods intake could be a relevant functional option in some circumstances by including selected fish and compounds, fruits, vegetables, legumes, dairy products, olive oil, nuts, which consumed in adequate amounts on a healthy dietary pattern may contribute in decreasing the risk of suffering MS features through effects on several mechanisms: weight reduction, lowering blood lipid levels, improving dietary compliance, reducing low-density lipoprotein cholesterol (LDL-c) oxidation, increasing HDL-c, decreasing blood pressure, improving insulin sensitivity and inhibiting inflammation and platelet aggregation.

Seafood as a Functional Food A reduction in the daily caloric intake is one of the most important components of dietary recommendations in those subjects suffering MS, taking into account that a modest weight loss (5 to 10%) has beneficial effects on plasma lipids, blood pressure and blood glucose. Hence, a number of approaches are being investigated affecting the inclusion of specific functional foods in the nutritional intervention treatment of this cluster of metabolic impairments such as unsaturated FA, fibre, antioxidants and others bioactive foodcompounds. Although the diet promotes a body weight and body fat loss and MS features improvement, nowadays, interventional studies try an to positively impact the inflammatory and oxidative state related to obesity [9, 33-36]. In recent years, a number of studies have clearly demonstrated the nutritional benefits of fish consumption or specific fish components such as proteins, vitamins, minerals, and especially ω3-FA, which may protect against several adverse health problems, including CVD mortality and stroke [37, 38]. During the last decade increasing evidences have suggested that the type of fat is more important than the amount of fat. Thus, saturated fatty acid (SFA) and trans FA intakes increase, while unsaturated fatty acids may decrease the risk of CVD and the cluster of MS symptoms. Consumption of long-chain ω3-FA from a seafood source, which is rich in α-linolenic acid (ALA), eicosapentanoic acid (EPA) and docosahexaenoic acid (DHA), may improve MS clinical manifestations, and thus, consequently, may reduce risk of CVD [39] (Table 2). In this context, a recent metaanalysis, including 14 randomized interventional studies, 25 cohort prospective studies and 7 case-control studies, has concluded that ω3-FA intake by fish and fish oil consumption is inversely associated with CVD mortality [38]. Moreover, it has also suggested that a regular intake of fatty fish produces changes in cardiac membranes, attenuates heart failure and cardiac hypertrophy, which reduces the risk of myocardial failure [40]. Other studies have evidenced that ω3-FA decrease adipose tissue mass and the development of obesity in rodents by targeting a set of key regulatory gene encoding involved in expression of lipogenic and lipolytic enzymes, transcriptional factors and leptin. Changes in these enzymes mRNA levels are closely related to the decrease of fat cell size by adipogenesis regulation and lipid homeostasis [41]. In addition, ω3-FA content of abdominal subcutaneous adipose tissue was negatively associated to cell size in overweight/obese humans. Part of this effect also may be related to changes in plasma membrane FA composition and EPA biosynthesis, suppression FA synthesis, increased β-oxidation and

Seafood as a Functional Food in Decreasing the Risk for the Metabolic Syndrome 153 decreased triglycerides synthesis [42]. Furthermore, ω3-FA also appears to modulate satiety, increasing the 2 hours postprandial fullness feeling [43]. Table 2. Metabolic syndrome features and the healthy benefits of fish and compounds consumption Metabolic syndrome criteria Abdominal obesity Hypercholesterolemia Hypertension Hypertriglyceridemia Insulin resistance Low HDL-cholesterol

Healthy mechanism of fish consumption Improve weight loss Modulate lipogenic enzymes and transcriptional factors, increasing lipid oxidation and decreasing fat accumulation Decrease total and LDL-cholesterol levels Increase systemic arterial compliance Improve endothelial function Stimulation of lipid oxidation and decrease triglyceride level Enhance insulin sensitivity Maintain or increase HDL-cholesterol levels

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In an interventional trial, overweight subjects receiving an energy-restricted diet (minus 30% of energy requirement) were followed 8 weeks. The diet contained 50%, 20% and 30% of energy from carbohydrate, protein and lipids, respectively and varied the fish and oil fish content: (1) control (sunflower oil capsules, no seafood); (2) lean fish (3x150 g portions of cod/week); (3) fatty fish (3x150 g portions of salmon/week); (4) fish oil (DHA/EPA capsules, no seafood), during eight weeks. The outcome was that the inclusion of either lean or fatty fish, or fish oil as part of an energy-restricted diet resulted in a significant higher weight loss, than did a similar diet without seafood or supplement of marine origin. Thus, this intervention study showed that a moderate calorie-restricted cod-based diet was found as a useful strategy to lose weight [9, 10]. These results also may indicate that other bioactive components of fish may be beneficial to human health beyond the ω3-FA. A plausible compound is taurine, an amino acid abundant in fish protein, because its consumption has been related to weight loss in mice and humans [51, 52]. Regard to dislipidemia-improvement effect of fish consumption, fish oil and ω3-FA intake effect on hypertriglyceridemia has been established [44, 47, 53, 54]. A meta-analysis of 36 crossover and 29 parallel-design studies demonstrated that ω3-FA lowered serum triglycerides levels in a dose-dependent manner, with the triglycerides lowering being generally proportional to baseline levels. In trials concerning subjects with hypertriglyceridemia taking the EPA and/or DHA in dosages of 3.4–4 g/day, triglycerides levels decreased by an average of 29% (range 16–45%) [47]. Several potential mechanisms have been proposed about to triglyceride-lowering mechanisms of the ω3-FA from oil fish, preferentially EPA and DHA. These FA can both reduce hepatic very-low-density lipoprotein cholesterol (VLDL-c) synthesis and secretion and enhance triglyceride clearance from chylomicrons and VLDL-c particles. A reduction of VLDL-c rich in triglyceride secretion is due to decreased expression of sterol regulatory element-binding proteins (SREBP)-1c or increased rates of mitochondrial and/or peroxisomal

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β-oxidation, leading to reduced substrate for triglyceride synthesis. A reduction in SREBP-1c expression may be mediated by inhibition of liver X receptor (LXR) and retinoid X receptor (RXR), while the increased rates of peroxisomal β-oxidation may be a consequence of peroxisome proliferator-activated receptor- alpha (PPARα), all transcriptional factors. On the other hand, decreased activity of triglycerides-synthesizing enzymes, decreased NEFA delivery from adipose tissue, and decreased availability of apoB also are potential mechanisms explaining reduced hepatic VLDL-c release. In the systemic periphery, increased lipoprotein lipase activity, by increasing PPARα activity and expression, may lead to increased triglyceride clearance from chylomicrons and VLDL-c [41, 48]. The effects on serum lipids also depend on patient and on the consumed SFA amount. In hyperlipidemic patients, ω3-FA consumption decreases LDL-c if the SFA content is also reduced. Otherwise, there is an increase in this lipoprotein concentration when SFA and trans FA intake is not decreased. Also, the consumption of ω3-FA from fish oil or fatty fish can maintain or increase the HDL-c levels [8, 44]. These effects are likely mediated through regulating the activity of transcriptional factors [8, 55]. Furthermore, increased intakes of fatty fish or supplements of ω3-FA marine may prevent alterations in glucose homeostasis and the further development of type 2 diabetes mellitus, likely by improving defects in insulin signalling, decreasing FAs accumulation in the muscle and in the adipose tissue and reducing insulin secretion in the pancreas [55]. In fact, nutritional intervention with calorie-restricted diet accompaying a regular fatty fish (450 g per week) or fish oil (6 capsules per day) consumption (mean of 3.0 g and 1.4 g of ω3-FA, respectively) has resulted in an improvement in insulin sensitivity, independently of weight loss in overweight and obese subjects [49, 50]. Regarding blood pressure values, a meta-analysis of 36 randomized trials found that fishoil intake (median dose 3.7 g/day EPA plus DHA) has resulted in a significant reduction of systolic blood pressure by 2.1mm Hg and diastolic blood pressure by 1.6 mm Hg [46]. Among of mechanisms proposed for explaining this effect, the first is an incorporation of EPA and DHA into membrane phospholipids that could increase systemic arterial compliance and second, EPA and DHA could improve endothelial function [56, 57]. Moreover, ω3-FAs may contribute to decreasing the burden of the MS, modulating inflammation, platelet activation and endothelial functions [38, 56] (Table 3). EPA and DHA reduce pro-inflammatory prostaglandin and leukotriene production from arachidonic acid metabolism, indicating its role on inflammatory state [58]. Thus, in an healthy people, a lower EPA intake was negatively associated with IL-6 levels, while total intake of ω3-FA was inversely associated with IL-6, TNF-α, IL-1 [59], CRP, sVCAM-1, sICAM-1 and E-selectin levels [56]. Other study with hypercholesterolemic subjects found a higher anti-inflammatory effect of a diet rich in ALA with linoleic acid (LA): ALA ratio of 1.5:1 by a significant reduction in CRP, sVCAM-1 and E-selectin- concentrations, compared to a diet rich in LA (LA:ALA ratio of 3.5:1) [45]. Thus, the fatty fish consumption appears to act through several mechanisms in reducing the risk of CVD (Figure 2). In this context, dietary recommendations have been made for ω3FA intake, including ALA, EPA and DHA to achieve nutrient adequacy and to prevent and treat CVD. The current evidence supports a dietary recommendation of approximately 500 mg/day of EPA and DHA for cardiovascular risk reduction. For treatment of existing CVD, 1g/day has been recommended [60].

Seafood as a Functional Food in Decreasing the Risk for the Metabolic Syndrome 155 Table 3. Summary of effects of fish and compounds consumption on inflammation and endothelial function markers obtained by clinical trials Change on inflammation and endothelial markers Hypertriglyceridemic ↓ sICAM-1 and E-selectin subjects (27) concentrations (P<0.05) Smokers with ↓ TM / vWF and hyperlipidemia (41) ↑ E-selectin concentrations (P<0.05) Subjects with CHD 5.1 g ω3-fatty acid ↓ TM / vWF and (54) (4 weeks) ↑ sVCAM-1/ E-selectin concentrations (P<0.05) Healthy 7 g safflower oil (placebo) ↓ CRP and IL-6 postmenopausal or 7 g safflower oil and 7 g concentrations with fish oil vs placebo (P<0.05) women (30) fish oil or 14 g fish oil (5 weeks) ALA-enriched (LA/ ALA: ↓ CRP concentrations in the Moderately ALA group vs LA (P<0.05) hypercholesterolemic 1.5/1) or LA-enriched (LA/ALA: 28/1) margarine subjects (103) (2 years) No effects on CRP Healthy subjects (80) 1.5 g EPA+DHA, with or concentrations (P>0.05) without 800 IU alphatocopherol (12 weeks) ↓ CRP, sVCAM-1, and EHypercholesterolemic ALA diet (LA/ ALA: selectin concentrations in subjects (23) 1.5/1), LA diet (LA/ALA ALA group vs LA; 3.5/1) or control (LA/ ↓ sICAM-1 concentrations ALA: 9/1) in ALA and LA groups vs (6 weeks) control (P<0.05) Studied subjects (n)

Type of dietary intervention (time) 4 g ω3-fatty acid (7-12 months) 4.8 g ω3-fatty acid (6 weeks)

References [61] [62]

[63]

[53]

[64]

[57]

[45]

ALA: α-linolenic acid; CHD: coronary heart disease; CRP: C-reactive protein; DHA: docosahexaenoic acid; EPA: eicosapentanoic acid; IL6: interleukin-6; LA: linoleic acid; sICAM-1, soluble intercellular adhesion molecule-1; sVCAM-1, soluble vascular cell adhesion molecule-1; TM, soluble thrombomodulin; vWF, von Willebrand factor.

A dietary strategy for achieving the 500 mg/day recommendation is to consume 2 fish meals per week (preferably fatty fish species). Foods enriched with EPA and DHA or fish oil supplements are also a suitable approach to achieve recommended intakes and may be necessary to assume intakes of 1 g/day or more. However, several intervention studies have shown that fish oil supplementation or modified foods including ω3-FA may not be as beneficial as fatty fish intake. It could be due to the fact that not only PUFA content has beneficial effects on health, but also the type of proteins or other fish components may be implicated in the effects of ω3-FA also per se [61].

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ω3-Fatty acid (ALA + EPA+ DHA) intake from seafood source

Modulation of expression of lipogenic and lipolytic enzymes and, transcription factors ↑ Satiety In liver:

In adipose tissue:

↓ VLDL-c secretion ↑ TG uptake from CM/ VLDL-c ↑ TG oxidation

Incorporation of ω3-FA into membrane

↓ Expression inflammatory and dysfunction

phospholipids

endothelial markers

↓ NEFA delivery

1.

Improving in the lipid profile

2.

Improving insulin signalling

3.

Reducing blood pressure values

4.

Improving endothelial function

5.

Decreasing fat accumulation and inflammatory state

Reduced risk of cardiovascular diseases

Figure 2. Effect of dietary ω3-fatty acid on the improvement of metabolic syndrome and cardiovascular disease risk. ALA: α-linolenic acid; CM: chylomicron; DHA: docosahexaenoic acid; EPA: eicosapentanoic acid; FA: fatty acid; NEFA: non-esterified fatty acids; TG: triglycerides; VLDL-c: very low density lipoprotein;

Conclusion In summary, it has been repeatedly suggested that including functional foods on the habitual diet may be more effective on metabolic improvements than modified foods, presenting functional properties. So, fish must be an important part of a balanced diet. Combination of regular fish consumption (twice a week) with important contribution of fatty fish species, in combination with regular consumption of other foods (enriched with EPA + DHA or naturally containing ω3-FA), can be advised to achieve the recommendation for ω3FA intake.

Acknowledgements Thanks are given to EU-Commission for financial support a grant from the 6th framework for the project SEAFOODplus: A better life with seafood (FOOD-CT-2004506359), and to Línea Especial about Nutrition, Obesity and Health (University of Navarra

Seafood as a Functional Food in Decreasing the Risk for the Metabolic Syndrome 157 LE/97) as well as IBERCAJA. The Capes Foundation, Ministry of Education of Brazil (nº 375605-0) and Government of Navarra also provided research grants.

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Helen Hermana Miranda Hermsdorff, Itziar Abete, María Ángeles Zulet et al. Ramel, A., Martinez, A., Kiely, M., Morais, G., Bandarra, N. M., Thorsdottir, I. (2008). Beneficial effects of long-chain n-3 fatty acids included in an energy-restricted diet on insulin resistance in overweight and obese European young adults. Diabetologia, 51, 1261-1268. Fujihira, E., Takahashi, H., Nakazawa, M. (1970). Effect of long-term feeding of taurine in hereditary hyperglycemic obese mice. Chem Pharm Bull (Tokyo), 18, 16361642. Zhang, M., Bi, L. F., Fang, J. H., Su, X. L., Da, G. L., Kuwamori, T., Kagamimori, S. (2004). Beneficial effects of taurine on serum lipids in overweight or obese nondiabetic subjects. Amino Acids, 26, 267-271. Ciubotaru, I., Lee, Y. S., Wander, R. C. (2003). Dietary fish oil decreases C-reactive protein, interleukin-6, and triacylglycerol to HDL-cholesterol ratio in postmenopausal women on HRT. J Nutr Biochem, 14, 513-521. Panagiotakos, D. B., Zeimbekis, A., Boutziouka, V., Economou, M., Kourlaba, G., Toutouzas, P., Polychronopoulos, E. (2007). Long-term fish intake is associated with better lipid profile, arterial blood pressure, and blood glucose levels in elderly people from Mediterranean islands (MEDIS epidemiological study). Med Sci Monit, 13, CR307-312. Carpentier, Y. A., Portois, L., Malaisse, W. J. (2006). n-3 fatty acids and the metabolic syndrome. American Journal of Clinical Nutrition, 83, 1499S-1504S. López-Garcia, E., Schulze, M. B., Manson, J. E., Meigs, J. B., Albert, C. M., Rifai, N., Willett, W. C., Hu, F. B. (2004). Consumption of (n-3) fatty acids is related to plasma biomarkers of inflammation and endothelial activation in women. J Nutr, 134, 1806-1811. Vega-López, S., Kaul, N., Devaraj, S., Cai, R. Y., German, B., Jialal, I. (2004). Supplementation with omega3 polyunsaturated fatty acids and all-rac alpha-tocopherol alone and in combination failed to exert an anti-inflammatory effect in human volunteers. Metabolism, 53, 236-240. Rallidis, L. S., Paschos, G., Liakos, G. K., Velissaridou, A. H., Anastasiadis, G., Zampelas, A. (2003). Dietary alpha-linolenic acid decreases C-reactive protein, serum amyloid A and interleukin-6 in dyslipidaemic patients. Atherosclerosis, 167, 237-242. Ferrucci, L., Cherubini, A., Bandinelli, S., Bartali, B., Corsi, A., Lauretani, F., Martin, A., Andres-Lacueva, C., Senin, U., Guralnik, J. M. (2006). Relationship of plasma polyunsaturated fatty acids to circulating inflammatory markers. J Clin Endocrinol Metab, 91, 439-446. Harris, W. S. (2007). International recommendations for consumption of long-chain omega-3 fatty acids. J Cardiovasc Med (Hagerstown), 8 Suppl 1, S50-52. Abe, Y., El-Masri, B., Kimball, K. T., Pownall, H., Reilly, C. F., Osmundsen, K., Smith, C. W., Ballantyne, C. M. (1998). Soluble cell adhesion molecules in hypertriglyceridemia and potential significance on monocyte adhesion. Arterioscler Thromb Vasc Biol, 18, 723-731. Seljeflot, I., Arnesen, H., Brude, I. R., Nenseter, M. S., Drevon, C. A., Hjermann, I. (1998). Effects of omega-3 fatty acids and/or antioxidants on endothelial cell markers. Eur J Clin Invest, 28, 629-635. Johansen, O., Seljeflot, I., Hostmark, A. T., Arnesen, H. (1999). The effect of supplementation with omega-3 fatty acids on soluble markers of endothelial function in patients with coronary heart disease. Arterioscler Thromb Vasc Biol, 19, 1681-1686. Bemelmans, W. J., Lefrandt, J. D., Feskens, E. J., van Haelst, P. L., Broer, J., Meyboom-de Jong, B., May, J. F., Tervaert, J. W., Smit, A. J. (2004). Increased alphalinolenic acid intake lowers C-reactive protein, but has no effect on markers of atherosclerosis. Eur J Clin Nutr, 58, 1083-1089.

In: Food Science and Security Editors: L. Amsel and L. Hirsch, pp. 161-186

ISBN: 978-1-60692-977-3 © 2009 Nova Science Publishers, Inc.

Chapter 7

UNDERUTILISED FISH SPECIES: THEIR POTENTIAL AS FRESH FILLETS AND PROCESSED PRODUCTS T. Ronan Gormley1 and John D. Fagan Ashtown Food Research Centre (Teagasc), Ashtown, Dublin 15, Ireland

Abstract The term underutilised fish species (UUFS) is used here to describe species that are outside mainstream use. However, all species are utilised somewhere in the world. As fish stocks dwindle and quotas tighten the use of UUFS for consumption as fillets/portions, and also in a range of fish products, may assume a new importance. For this reason, the evaluation and test processing of UUFS has been an ongoing topic of R and D at Ashtown Food Research Centre (AFRC) and six separate studies were conducted as fish became available. Tests on silver smelt (Study 1) indicated that this bland white-fleshed fish is useful for a range of products including enrobed nuggets, fingers and fishcakes; it also produced strong gels. Sensory tests (Study 2) on 15 UUFS indicated that all were liked when tasted as breaded nuggets, and all compared favourably with cod. Study 3 was on 21 UUFS and taste panellists preferred six of these to cod as fillets, ten as nuggets and eight as fish-cakes. The best species were orange roughy, black scabbard, morid cod and siki shark. Study 4 was conducted on steamed fillets of 15 species and none had sensory scores better than cod. However, samples of morid cod, bluemouth rockfish, ling, forkbeard, black scabbard, silver roughy, orange roughy and roundnose grenadier compared favourably with cod. Study 5 involved adding value to selected UUFS using sous vide, marinating, and freeze-chilling coupled with modified atmosphere packaging (MAP). Orange roughy, cardinal fish and redfish gave well textured sous vide/frozen products, but the flesh of sous vide/frozen roundnose grenadier, blue ling and Greenland halibut was considered too soft. Tests with salt-based marinades and albacore tuna indicated sensory acceptability scores in the order Cajun>arrabbiata>tandoori> lemon+lime>southern fried>smoke. Trials on freeze-chilling combined with MAP indicated that both technologies were suitable for extending the shelf life of redfish, cardinal fish, and roundnose grenadier portions. In study 6, breaded products were prepared from blue whiting and received good acceptability scores. The overall outcome from the six trials indicated that a number of the species have potential for sale as chilled fillets or as processed products. They may provide an alternative to over-exploited fish species using sustainable fishing practices 1

E-mail address: [email protected].

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T. Ronan Gormley and John D. Fagan and could be processed to produce high quality nutritious meal solutions for human consumption.

Introduction The term underutilised fish species (UUFS) is used here to describe fish species that are outside mainstream use. However, virtually all species are utilised to some extent somewhere in the world. The interest in UUFS has arisen due to dwindling fish stocks and tightening quotas for conventional species (Brennan and Gormley, 1999) and also the increasing demand (Gormley, 2006) for seafood as a result of greater awareness by consumers of fish as a healthy food (Gormley, 2008). This demand is resulting in increased fish farming worldwide but also an increasing focus on catching UUFS, and on their quality and suitability for use as fresh fillets, or in a range of seafood products. Some UUFS are slow breeding and their stocks may be easily over fished (Brennan and Gormley, 1999). Many are also deepwater and require special catching technology. For these reasons, the evaluation and test processing of UUFS has been an R and D topic at Ashtown Food Research Centre (AFRC) for a number of years (Gormley, 2008). The studies are listed and discussed in chronological order in this chapter and relate to: (1) silver smelt; (2) sensory tests on 15 UUFS; (3) tests on 21 UUFS; (4) tests on 15 UUFS; (5) adding value to UUFS by sous vide processing, marinating or by modified atmosphere packaging; (6) tests on blue whiting. Many of the data from studies 1, 2, 3, and 6 have been published (Gormley and Fagan, 2005) and are referenced here with a short discussion on the main outcomes. Most of the data from studies 4 and 5 have not been published and are dealt with in more detail. A major difficulty in testing UUFS is the nonavailability of replicate samples. Most UUFS are caught as a by-catch and so the philosophy of the testing laboratory must be ‘test when available’. This often results, therefore, in testing a range of spot samples on a once-off basis, and then repeating the activity weeks or months later when more fish have been sourced. This is the case for the data reported here. The conclusions section of the chapter brings together the outcomes from the six studies and lists the best species for use as fresh fillets/portions, and also those most suited to processing. The Latin names of the different species tested are given in Annexe 1.

Results and Outcomes Evaluation of Silver Smelt (Study 1) Silver smelt is potentially an important commercial species and the study reported here was part of a project part-funded by the EU FAR programme. The fish were assessed for their suitability for use fresh, or as processed products. Sourcing samples: Two lots of silver smelt, caught off the west coast of Ireland, were tested as whole fish, fillets and mince on the day of receipt. Samples were also frozen and were tested after a period in cold storage. A range of gels was prepared to assess water holding capacity and these were subjected to rheological testing. Commercial block-frozen fillets originating from the same catches were also received at AFRC for testing. Samples of ling, pollock and cod were used for some of the comparisons and were sourced from the

Underutilised Fish Species: Their Potential as Fresh Fillets and Processed Products 163 Dublin fish market. Sub-samples of the fillets were also blast frozen (-35°C/2h). Commercial samples of frozen cod fishcakes and fillets were purchased in a local supermarket for use in comparative tests with silver smelt nuggets, fishcakes and breaded fillets/portions. The sample sets were subjected to a series of physicochemical and sensory tests (Stoknes et al., 2006). Flesh composition, colour, odour and flavour: Skin-on portions had a higher fat content than-skin off (2.1 vs 0.6%) and Hunter colour values (Lab) showed that silver smelt whiteness was comparable to that of cod. Sensory tests (test 1) on steamed samples indicated that silver smelt odour ratings were good and remained relatively constant over days 3, 4, 5 and 6 postcatching with scores of 8.7, 8.1, 9.0 and 8.0 (10 = best; 0 = worst), respectively. Corresponding scores for flavour were 7.5, 7.3, 8.0 and 8.0. Paired comparison tests of steamed silver smelt versus steamed cod (test 2) showed a preference ratio of 9/3 (12 tasters) in favour of silver smelt. Most panellists agreed that silver smelt had a pleasant bland flavour and a firmer texture than the sample of cod (Stoknes et al., 2006). TVBN, TVC and centrifugal drip (CD): Total volatile basic nitrogen (TVBN) values were generally well below the upper limit of 35mg/100g flesh (EC Directive 95/149/EC). Total viable count (TVC) values were generally below the upper limit of log105 cfu/g (proposed by the Irish Department of Health and Children Guidelines 1992) on day 5 and suggest that silver smelt has a good shelf life at 2-4°C. Freezing caused an increase in CD for the four species and values increased by 71 (silver smelt), 118 (cod), 132 (pollock) and 205% (ling). Thawing frozen fillets and immediately refreezing did not influence CD values but storing frozen fillets for six months gave a large increase in CD. This agrees with the findings of Gormley et al. (1993) who showed that water binding properties of silver smelt decreased steadily during frozen storage for 150, 235 and 374 days. Properties of silver smelt gels: Full strength (no added-water) silver smelt gels had intermediate compression values, being softer than ling or cod but firmer than pollock gels. However, silver smelt gels were the softest of the added-water gels (Gormley et al., 1991; 1994). These gels were made from frozen mince, and silver smelt mince is particularly prone to freezing damage. Acceptability of silver smelt products: Fishcakes from silver smelt were preferred to those made from pollock, ling or cod and were much preferred to a commercial sample. Panel member comments for silver smelt and cod samples were favourable. Comments for commercial fishcakes included ‘lacks fish’, ‘bad mouth-feel’ and ‘poor flavour’. Silver smelt nuggets were preferred on all counts to commercial cod nuggets with the exception of crumb colour. Some tasters found the texture of the silver smelt nuggets to be rubbery or too firm. However, they still preferred the silver smelt nugget texture (Stoknes et al., 2006). Outcomes from study 1: Responses from sensory tests on steamed fillets, fishcakes and breaded nuggets from silver smelt were positive. Full strength silver smelt gels had high compression and penetrometer values indicating good water binding properties. Freezing increased centrifugal drip (CD) values in silver smelt fillets. However, frozen mince from silver smelt had a much higher CD value indicating that mince should not be stored frozen but as whole fillets that are then minced post-thawing (Gormley et al., 1993). These data suggest that silver smelt has potential for seafood products but that toughening during frozen storage could be a potential problem as could total bone removal during filleting.

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Assessment of 15 UUFS (Study 2) This study was part-funded by the Irish Marine Institute and was conducted to explore the potential of UUFS for the production of processed products. Sourcing samples: Nine UUFS were studied (sensory properties) in part 1, and the efficacy of cryoprotectants in improving water-holding capacity and texture of fish minces and gels, respectively, in part 2 (Maier et al., 1997). The species (Table 1) for part 1 were obtained from a deep-water survey conducted off the northwest coast of Ireland. Samples for part 2 (Table 2) were obtained as a by-catch by trawlers from Loughinver in Scotland. Some species were common to both parts 1 and 2, and there were 15 UUFS in all. Both sets of samples were received at AFRC as frozen fillets. Sensory evaluation: Because of the small number of fillets available for each species, it was decided to prepare the samples for tasting as deep fried breaded nuggets (circa 20g each) from fish mince using cod nuggets as control (Maier et al., 1997). Nine paired comparison taste panels were conducted and in all cases the UUFS nuggets were preferred to cod nuggets (Table 1). Four species had scores >4 on the 6-point scale and all samples had values well above the mid-point (3) of the acceptability scale. The scores for the cod nuggets were reasonably consistent except for panel 1 which had a lower value (3.11) (Table 1). Table 1. Taste panel1 acceptability scores2 (mean values) for breaded nuggets of nine underutilised fish species (UUFS) in comparison with cod nuggets Panel No. 1 2 3 4 5 6 7 8 9 1

Species Greenland halibut Silver smelt Roughhead grenadier Baird’s smoothhead Siki shark Blue ling Roundnose grenadier Orange roughy Kitefin shark Mean

UUFS nuggets

Cod nuggets

4.81 (1) 3 4.42 (2) 4.34 (3) 4.09 (4) 3.99 (5.5) 3.99 (5.5) 3.82 (7) 3.81 (8) 3.79 (9) 4.12

3.11 3.70 3.51 3.63 3.74 3.80 3.65 3.45 3.56 3.57

11-16 tasters/per panel 6-cm line with end-points of 0 (unacceptable) and 6 (very acceptable) 3 Rank order

% of tasters preferring UUFS samples 94 83 67 73 67 60 60 73 58 ---

2

Efficacy of cryoprotectants: Good water-holding capacity (WHC) and high gel compression values are key factors in the preparation of seafood products, especially for those made from minced/comminuted fish. Part 2 of this trial used sodium caseinate and whey protein concentrate (each included in the mince at 8% w/w) as potential cryoprotectants in fish mince and gels. Previous work showed that these were effective cryoprotectants in three conventional fish species (Anese and Gormley, 1996). The frozen fish was thawed, minced, the cryprotectants were added with salt; the mince was cooked in a sausage casing (Gormley et al., 1991). The mince samples were subjected to three freeze-thaw cycles to mimic long-

Underutilised Fish Species: Their Potential as Fresh Fillets and Processed Products 165 term frozen storage and the WHC of the thawed mince was assessed. WHC values were highest for minces with sodium caseinate, followed by whey protein concentrate, and no cryoprotectant. This order (i.e. 1, 2, 3) prevailed for five of the species, cardinal fish excepted, and indicated that sodium caseinate was the best of the two cryoprotectants in frozen fish minces in terms of WHC. Blue ling, cardinal fish and tusk had the firmest gels in the absence of cryoprotectants (Table 2), and forkbeard, lesser spotted dogfish and leafscale gulper shark the softest. Previous work with salmon, spent salmon, haddock and cod (Anese and Gormley, 1996) indicated that fish mince gels with whey protein concentrate were generally firmer than those with caseinates. This pattern was maintained for seven of the species (Table 2) but was reversed for orange roughy, lesser spotted dogfish, siki shark, leafscale gulper shark and bluemouth rockfish. The rank order (Table 2) indicated that the two cryoprotectants had widely different effects on gel firmness of some of the species. With the exception of tusk and roundnose grenadier, one or both cryoprotectants raised gel firmness values in comparison with gels made from mince without cryoprotectants. Gels from roundnose grenadier, orange roughy and black scabbard were the whitest (Table 2; values averaged over cryoprotectants), and those from blue ling, forkbeard and lesser spotted dogfish the least white. Table 2. Compression1 (Newtons) and colour (Hunter L/b; white/yellow ratio) values for full-strength gels made from thawed mince of UUFS and from cod (± cryoprotectants) Cryoprotectant2 Species

1

Siki shark Cardinal fish Orange roughy Bluemouth rockfish Lesser spotted dogfish Leafscale gulper shark Tusk Blue ling Black scabbard Cod Forkbeard Roundnose grenadier Mean

Sodium caseinate 147 (1)3 121 (2) 118 (3.5) 118 (3.5) 113 (5) 105 (6) 101 (7) 80 (8) 74 (9) 62 (10) 52 (11) 45 (12) 95

Whey protein concentrate 105 (5)3 154 (1) 84 (10) 98 (6) 91 (8) 70 (12) 107 (4) 148 (2) 120 (3) 85 (9) 96 (7) 72 (11) 103

None 79 (5.5)3 121 (2) 74 (7) 72 (9) 69 (11) 59 (12) 108 (3) 123 (1) 87 (4) 79 (5.5) 70 (10) 73 (8) 85

Gel colour: Hunter (L/b) 7.6 (9)3 7.7 (8) 9.6 (2) 8.2 (7) 4.7 (12) 8.7 (5) 9.0 (4) 6.3 (10) 9.1 (3) 8.3 (6) 4.9 (11) 10.8 (1) 7.9

Gel cylinders were compressed by 40% of their height Included at 8% (w/w) 3 Rank order 2

Both cryoprotectants reduced gel whiteness, i.e. mean Hunter L/b values of 6.7 (both cryoprotectants) versus 7.9 (no cryoprotectant). High gel whiteness (L/b) values are desirable in minced/comminuted products made with white fish.

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Outcomes from study 2: Nine of the UUFS tested were well liked by taste panels when evaluated as breaded nuggets, and all compared favourably with cod. Cardinal fish, siki shark, and lesser spotted dogfish had the most favourable WHC and gel compression values (full strength gels) and would be expected to give the highest product yields. Roundnose grenadier, forkbeard and leafscale gulper shark had the least favourable values. Gel whiteness values were best for roundnose grenadier, orange roughy and black scabbard, and were least good for lesser spotted dogfish, forkbeard and blue ling.

Assessment of 21 UUFS (Study 3) This study was conducted as part of the Marine Research Measure of the Operational Programme for Fisheries which was administered by the Irish Marine Institute and partfinanced by the EU European Regional Development Fund. It was the second study at AFRC which embraced a large number of UUFS and the goal was to investigate their performance and acceptability as fresh fillets/portions and as processed products. Sourcing samples: The 21 UUFS (Table 3) were caught by personnel from the Irish Fisheries Research Centre in two deep-water survey trips to the Rockall Trough in 1997 (Brennan and Gormley, 1999). The samples were gutted at sea and were received at AFRC as frozen fillets. Fresh cod fillets were obtained as control and were blast frozen (-35ºC) and stored at -20ºC until required. All samples were thawed and were given to a 12-member taste panel for assessment on a 6-cm line with end-points of 0 (unacceptable) and 6 (very acceptable). The samples were presented as steamed fillets, breaded deep fried nuggets, and as fish cakes (flaked cooked fish and mashed potato) as described by Brennan and Gormley, 1999. Sensory acceptability, colour and texture: Six species received higher scores than cod as fillets, nine as nuggets and eight as fish cakes (Table 3). Orange roughy, morid cod, black scabbard, silver smelt, siki shark and snake mackerel were the highest performing species for sensory acceptability. No fillet sample received a sensory score >3.9. This was to be expected as steamed whitefish has a bland flavour and the samples were presented without salt or sauce. Some nugget samples received higher acceptability scores than the corresponding fillets due, presumably, to a reduction in blandness as a result of deep frying. This was not the case for fish cakes but these were a different product with the fish content diluted by mashed potato. Leafscale gulper shark, siki shark and great lantern shark had the whitest cooked flesh colour values and wolf-fish and bluemouth rockfish the lowest (Table 3). Shear values above the range 800±200N are on the tough side (especially small-eyed rabbit fish, tusk, blue ling and snake mackerel samples) and below the range are soft (birdbeak dogfish and rabbit fish) (Table 3). It should be noted that the raw fish samples were frozen, stored at -20ºC for one month, and then thawed and tested. The shear values, therefore, indicate fillet softness/firmness and also how this was influenced by the frozen storage/thawing regimes. Outcomes from study 3: Six species scored more highly than cod for sensory acceptability as steamed fillets and most also received good scores as nuggets or cakes. The colour of these was intermediate relative to the other species, none being very white or dark. This set of six samples/species also had close to ideal shear values except for snake mackerel which was on the tough side.

Underutilised Fish Species: Their Potential as Fresh Fillets and Processed Products 167 Table 3. Sensory acceptability scores1 for UUFS fillets and products, and also colour (Hunter L/b) ratios and shear values for steamed (and cooled) fish fillets/portions

1

Species Orange roughy Morid cod Black scabbard Silver smelt Siki shark Snake mackerel Cod Roundnose grenadier Baird’s smoothhead Wolf fish Bluemouth rockfish Redfish Greater lantern shark Tusk Forkbeard Blue whiting Leafscale gulper shark Blue ling Longnose velvet dog. Birdbeak dogfish Small-eyed rabbit fish Rabbit fish No. of species tasted No. preferred to cod

Fillet2 3.9 3.9 3.9 3.9 3.8 3.8 3.6 3.5 3.4 3.4 3.3 3.3 3.2 3.0 3.0 3.0 2.8 2.8 2.7 2.6 2.3 1.6 21 6

Acceptability Nugget3 4.7 4.2 3.8 -4.1 -3.7 4.0 4.1 4.1 3.4 -3.1 4.0 3.0 -4.1 3.7 3.2 1.8 1.4 1.0 17 9

Cake4 2.7 3.7 3.6 3.4 2.8 2.7 3.0 -2.3 3.4 3.5 -3.0 3.3 3.5 3.2 2.7 2.9 2.8 ---17 8

Hunter L/b5 7.35 9.01 8.24 7.85 15.60 6.95 10.08 7.67 10.00 6.40 6.42 6.53 11.78 8.91 9.71 6.70 19.02 10.50 10.29 12.46 6.85 10.40 21 --

Shear (N)6 1010 816 1090 675 1330 1550 800 1333 976 741 1140 1250 625 1610 825 1220 474 1550 1420 539 2060 426 21 --

6-cm scale with end-points of 0 (unacceptable) and 6 (very acceptable); 12 tasters Steamed 3 Formed, breaded and deep-fried 4 Formulated and pan-fried 5 Hunter L/b (white/yellow ratio); steamed fillets 6 Shear value (N/50g flesh; Kramer shear press and standard test-cell; fillets steamed and cooled) 2

Sensory Evaluation of 15 UUFS (Study 4) This was the third trial (study 4) conducted at AFRC on a number (15) of UUFS, and followed two years after study 3. It was part funded by the Irish Marine Institute. The objective was similar to that in study 3, i.e. to investigate the acceptability of UUFS as fresh fillets/portions and as processed products. Sourcing samples: Four sets of samples were evaluated over a 9-month period. Sets 1 to 3 were supplied from an Irish Marine Institute deepwater survey programme in 2000, and the fourth set was supplied by the Irish Sea Fisheries Board (BIM) in 2001. Twelve of the

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samples (Table 5) were received at AFRC as whole round fish and were filleted (commercially), blast frozen (-35ºC/2.5h), stored (–22oC) until required, and then tested. The other species were already filleted when received. Only morid cod, blackmouth dogfish and birdbeak dogfish were obtained in quantities of 10kg or greater. These species were subjected to sensory and physicochemical tests, but for the other species, there were only sufficient amounts to conduct sensory tests. Ease of filleting: This was determined by an experienced filleter employed by a Dublin seafood company and his comments are presented in Table 4. These are important as a species is of limited use if it is difficult to fillet. Blue ling, wolf-fish, tusk and forkbeard were easy to fillet while most of the other species were not. Three species were deemed suitable for smoked coley. Sensory testing: No sample was rated statistically significantly better than cod but five species did receive higher sensory scores than cod (Table 5). However, a number of species were rated significantly less acceptable than cod. The order of species relative to cod was different to that found in study 3 and emphasises the difficulties of conducting trials with a series of spot samples. Sensory scores for the cod (control) samples varied from panel to panel (Table 5). This may be due to differences between the cod sub-samples used, but more likely because the response of tasters is influenced by the other samples being compared with cod in a particular panel. Table 4. Ease of filleting comments for 12 UUFS (filleted by hand) Species

Comments

Siki shark

Not usually filleted; mostly cooked on the bone

Blue ling

Easy to skin and fillet; used commercially for smoked coley

Morid cod

Skin usually left on; flesh similar to hake

Roughhead grenadier

Difficult to fillet; tough scales; very good fillets

Wolf-fish

Easily skinned and filleted; potential for smoked coley

Black scabbard

Very difficult to skin; should be sold skin-on; delicate flesh

Tusk

Easy to skin and fillet; excellent for smoked coley

Birdbead dogfish

Should be cooked on the bone; skin easily removed

Rabbitfish

Fillets very small; should be left on the bone; smoke skin-on

Blackmouth dogfish

Very difficult to fillet; long narrow fillets

Forkbeard

Easy to fillet; delicate flesh; skin best left on

Lesser spotted dogfish

Very difficult to fillet; fillets long and narrow

This was also the case for samples that were presented twice, either in the same panel or in different panels (Table 5). Sensory data for deep fried breaded nuggets (Table 6) indicated that morid cod nuggets received higher sensory scores than cod nuggets (not significant). Nuggets of one of the blackmouth dogfish samples were significantly downgraded relative to cod. The scores for morid cod and cod nuggets were above the mid-point of the 6-cm line indicating a reasonable level of acceptability for these samples.

Underutilised Fish Species: Their Potential as Fresh Fillets and Processed Products 169 Table 5. Sensory acceptability scores1 for steamed fillets/portions from 15 UUFS

1

Panel 1 Morid cod (1)2 Cod Morid cod (2)2 Blackmouth dog (1)2 Blackmouth dog (2)2 F-test LSD4

Score1 3.84 3.42 3.26 1.82 È3 1.79 È P<0.001 0.88

Panel 2 Bluemouth rockfish Cod Morid cod Siki shark Blue ling F-test LSD

Score1 3.27 2.98 2.62 0.57 È 0.47 È P<0.001 1.01

Panel 3 Cod Black scabbard Wolf-fish Tusk Roughhead grenadier F-test LSD

Score 4.16 2.64 È 2.61 È 1.14 È 1.12 È P<0.001 0.94

Panel 4 Tusk Blue ling Cod Birdbeak Forkbeard Rabbitfish F-test LSD

Score 2.96 2.82 2.73 2.25 2.04 0.69 È P<0.001 0.91

Panel 5 Cod Roundnose grenadier Orange roughy Longnose velvet dog. F-test LSD

Score 3.58 3.52 3.37 2.13 È P<0.01 0.90

Panel 6 Silver roughy Cod Black scabbard Siki shark F-test LSD

Score 3.89 3.79 3.36 2.42 È P<0.001 0.70

6-cm scale with end-points of unacceptable (0) and very acceptable (6) Sourced from different catching dates 3 (È) significantly less acceptable than cod; no arrow denotes no significant difference from cod 4 Least significant difference 2

Outcomes from study 4: Seven of the UUFS were equally acceptable to cod as steamed fillets based on taste panel results, i.e. morid cod, bluemouth rockfish, birdbeak dogfish, forkbeard, roundnose grenadier, orange roughy and silver roughy. No species scored significantly higher than cod. The remaining species received lower ratings than cod. Ease of filleting tests indicated that blue ling, wolf-fish, tusk and forkbeard were easy to skin and fillet. Morid cod and black scabbard filleted well but the skin should be left on. Siki shark, birdbeak dogfish and rabbitfish were deemed best cooked on the bone while roughhead grenadier and blackmouth dogfish were difficult to fillet. The lesser spotted dogfish was very difficult to fillet and gave long narrow fillets of poor quality. Breaded nuggets from morid cod compared favourably with cod nuggets.

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1

Morid cod (sample 2) Morid cod (sample 1) Cod Blackmouth dogfish (sample 2) Blackmouth dogfish (sample 1)

Score 3.71 3.44 3.39 2.38 1.98 È

F-test LSD

P<0.01 0.96

See footnotes in Table 5

Adding Value to UUFS by Sous Vide Processing (Study 5; Part 1) The objective was to study the effect of sous vide (SV) cooking on selected quality parameters of seven UUFS with and without 12 savoury sauces. The SV samples were subjected to freezing or chilling post-cooking and the context of the study was the potential of the fish/sauce samples as ready-meal components. This approach was taken in view of the importance of the ready-meal sector in Ireland (Anon., 2003). Sous vide technology has considerable potential as a method for processing value-added seafood products. Raw or parcooked food is vacuum-sealed in laminated plastic pouches or containers, heat treated by controlled cooking, rapidly chilled, and then reheated for service after a period of chilled storage (Fagan and Gormley, 2005a, b). The minimum recommended thermal process for SV products is 90oC for 10 minutes, or its time-temperature equivalent (SVAC, 1991). Sous vide/freezing technology has proved successful for cod and salmon portions as potential ready-meal components (Gormley et al., 2003). However, virtually no research has been conducted on the SV cooking of underutilised fish species. Sourcing samples: Orange roughy, albacore tuna, cardinal fish, deepwater redfish, roundnose grenadier, blue ling and Greenland halibut were sourced during fishing trials by the Irish Sea Fisheries Board (BIM). Fillets were delivered on ice to AFRC within 24h of landing and were vacuum packed, blast-frozen (–35oC/2.5 h), and stored (-20°C/5 weeks) until SV processing. Eleven oil-based and one water-based sauces were sourced from commercial companies. The sauces were: tomato+pesto, szechwan, Cajun, arrabbiata, béarnaise, hollandaise, mushroom (from Knorr, UBF Food Solutions, Ireland); toskana, tomato+basil, Italian, rosemary+garlic (from Raps, UK); tikka (water-based)(from All in All Ingredients, Ireland). The sauces were ready-to-use and were added to the fish at a 1:1 ratio. Fillets were thawed (2-4oC/12h), portioned (circa 200 g), vacuum packed (+/- sauce), and cooked (P90>10 min; Barriquand Steriflow retort). The cooked samples were blast frozen (35°C), stored (-20°C/1 week) and tested as described by Fagan and Gormley, 2005a. Results and outcomes from study 5, part 1: Sensory tests showed that SV cooked albacore tuna, cardinal fish and blue ling were the most acceptable species and tikka, tomato+pesto, arrabbiata and hollandaise the preferred sauces. Greenland halibut and roundnose grenadier were considered too soft after SV cooking. Freezing post SV cooking did not influence product quality and gave additional benefits over chilling of an extended shelf life and more

Underutilised Fish Species: Their Potential as Fresh Fillets and Processed Products 171 flexibility in relation to product safety. The pH of the sauces was in the range 3.96 (Cajun) to 5.42 (béarnaise) and mean pH values fell from 4.66 before SV cooking to 4.38 after cooking. Sauce colour also became lighter during SV cooking as indicated by Hunter Lab values. The outcomes from this study show that a number of UUFS are suitable for SV cooking in a range of savoury sauces and have a high level of acceptability. The inclusion of fish as a healthy option in ready-meals is increasing in parallel with the dynamic expansion of the ready-meal sector which offers quality and convenience to a wide spectrum of consumers including single households, working couples, the ageing population, and others. The results from these trials have been disseminated to seafood companies (Fagan and Gormley, 2005a).

Adding Value to Albacore Tuna by Marinading (Study 5; Part 2) Albacore tuna is an underutilised species in Ireland and in a number of other countries. The cost of processing equipment is often prohibitive and minimal processing methods such as marinating technology present an attractive alternative. This technology involves the introduction of flavours into raw fish fillets, which can then be vacuum/modified atmosphere packaged and retailed as chilled, frozen or freeze-chilled products. The conduit by which flavour components pass from marinade to fish is most likely osmotic dehydration. Therefore, this process can achieve simultaneous dewatering and direct formulation of a product through impregnation plus leaching (Raoult-Wack, 1994). Marinades can be purchased fully made (liquid) and as water/oil-soluble powders, and can be formulated to particular specifications, e.g. salt-based or sugar-based as required. The aim of this study was to investigate the suitability of albacore tuna portions for marinating with a range of commercially available salt-based marinades. The potential commercial outcome was a range of added value marinated fish products with high consumer acceptability. It is likely that the outcomes can be applied to other UUFS and to conventional species. Sourcing samples: Albacore tuna was sourced from ongoing fishing trials by the Irish Sea Fisheries Board (BIM) in the Irish Box location. The fish were filleted within 24h of landing, delivered to AFRC on ice, vacuum-packed, blast frozen (-35ºC/2.5h), stored (–30°C), thawed overnight (4oC) when required, portioned (120g), and then marinated. Applying the marinades: Six water-soluble commercial marinades (termed Cajun, smoke, tandoori, lemon+lime, arrabbiata, southern fried) were purchased from food ingredient suppliers in Dublin. Solutions of 10, 20 and 30% (w/v) of the six salt-based marinades were prepared by dissolving dry powder in water at 4oC. Tuna portions (3 x 120g) were placed in 500ml of each marinade concentration (marinade in excess) for 3h/4oC. The samples were removed, covered with cling-film, stored (4oC/18h) pending sensory and physicochemical testing. Sensory testing sequence: A preliminary sensory panel of four trained tasters carried out the first tests. Marinated fish samples were plated, covered (cling film) and microwaved (5 min/700W). Each panellist received three (circa 30g) pieces of fish, representing an individual marinade at 10, 20 and 30% concentration and was asked to mark flavour intensity on a 6-cm line with end-points of 0 (very weak flavour) and 6 (too strong a flavour) with a score of 3.0 indicating the ideal level of flavour. Thus, the optimum concentration was determined for each marinade and an expanded sensory analysis consisting of 6 marinades x 1

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concentration x 25 tasters (Table 7) was carried out. In this case, panellists were asked to mark a 6-cm line with end-points of 0 (unacceptable) and 6 (very acceptable). Physicochemical tests: Samples were tested for salt content, pH, moisture, centrifugal drip, ash, texture (Kramer shear values) and colour as outlined by Fagan et al. (2004). Mass transfer was calculated as the percentage difference in fish weight before and after immersion in each marinade solution. Determination of ideal marinade concentration: This was determined by 4 trained tasters as described above. A taste panel score of 3, i.e. the mid-point on the 6-cm flavour intensity scale, indicated the ideal level of marinade in the fish flesh for each type of marinade. In most cases a 20 to 30% marinade concentration gave the optimum sensory score (i.e. closest to the mid-point) (Table 7). However, panellists commented that the smoke marinade had an overwhelming salty taste (see levels below) indicating that a 10% concentration was preferable in this case. Table 7. Taste panel flavour intensity scores1 for marinated albacore tuna fish portions

1

Marinade Cajun Smoke Tandoori Lemon+lime Arrabbiata Southern fried Mean F-test: marinade F-test: conc. F-test: interaction

10 1.30 3.83 2.37 2.73 3.20 0.80 2.37 P<0.001 P<0.001 NS

Concentration (%) 20 2.27 4.30 3.40 3.57 3.33 2.27 3.19 LSD 0.59 LSD 0.42 LSD 1.02

30 2.57 4.97 4.27 3.40 4.80 2.50 3.75

Mean 2.04 4.37 3.34 3.23 3.78 1.86

6-cm line with end-points of 0 (very weak flavour) and 6 (too strong a flavour); 4 trained tasters; a score of circa 3 indicates ideal level of marinade flavour in the fish flesh

Table 8. Salt content (g/100g) of marinated albacore tuna fish portions Marinade Cajun Smoke Tandoori Lemon+lime Arrabbiata Southern fried Mean F-test: marinade F-test: conc. F-test: interaction

Marinade concentration (%) 10 20 0.20 0.24 1.25 2.10 0.67 1.22 0.43 0.83 0.77 1.52 0.30 0.39 0.60 1.05 P<0.001 LSD 0.15 P<0.001 LSD 0.11 P<0.001 LSD 0.26

30 0.28 2.68 1.67 1.25 2.24 0.58 1.49

Mean 0.24 2.01 1.18 0.84 1.51 0.42

Underutilised Fish Species: Their Potential as Fresh Fillets and Processed Products 173 Table 9. pH values of marinated albacore tuna fish portions Marinade Cajun Smoke Tandoori Lemon+lime Arrabbiata Southern fried Mean F-test: marinade F-test: conc. F-test: interaction

Marinade concentration (%) 10 20 5.82 5.77 5.93 5.92 5.84 5.81 5.80 5.67 5.66 5.61 5.76 5.76 5.80 5.76 P<0.001 LSD 0.08 P<0.001 LSD 0.06 NS LSD 0.15

30 5.70 5.85 5.80 5.57 5.52 5.74 5.70

Mean 5.77 5.90 5.82 5.68 5.60 5.75

Salt and pH values for marinated tuna portions: Salt is commonly used in marinades to enhance flavour, slow microbial growth, and to increase the osmotic effect. However, salt content of food must be also be minimised for reasons of consumer health. The Irish Heart Foundation (2004) suggested an intake ceiling of 6g/day. The salt (NaCl; g/100g) and pH values for a 10% (w/w) solution of the different marinades were, respectively: Cajun (0.59; 4.04), smoke (4.76; 4.71), tandoori (2.67; 5.78), lemon+lime (1.91; 3.62), arrabbiata (3.32; 4.53), southern fried (0.23%; 6.08). Marinade concentrations of 20 and 30% had double and treble the above salt concentrations, respectively, and gave a slight decrease in solution pH. As expected, the salt content of the marinated tuna flesh increased with the concentration of marinade used, and the marinades with the highest salt contents resulted in the highest content in the flesh (Table 8). Salt contents in the tuna flesh were positively correlated with ash content (0.92) and negatively correlated with centrifugal drip values (-0.81; Table 12). Thorarinsdottir et al. (2004) suggested that brine (salt) concentration can influence inter- and intra-molecular bonds of the muscle protein thereby affecting muscle structure and protein denturation. This in turn influences the water holding capacity of the muscle. There was a positive relationship between salt content x mass transfer values and a negative relationship between salt content x moisture content/centrifugal drip (Table 13). As marinade and salt concentration increased, tuna fish flesh pH decreased (Table 9). Thorarinsdottir et al. (2004) found that as brine concentration increased, pH and moisture content decreased in cod flesh with moisture losses occurring due to shrinkage of the myofibrils via denaturation in a high salt environment. Marinade type had a significant affect on pH values with smoke having the highest pH (most basic) followed by tandoori, Cajun, lemon+lime, southern fried and arrabbiata (most acidic). However, the correlation coefficient between salt content and pH was small. Mass transfer, moisture content and centrifugal drip (CD) values for tuna portions: Mass transfer is an important feature in marinated seafood products. Mass transfer consists of a simultaneous exchange of water and solids between the fish and surrounding solution (Raoult-Wack, 1994). This can influence the commercial feasibility of developing valueadded food products as marinades have the potential to increase or decrease final product weight (by dehydration) depending on the marinade used. In the current study, both marinade type and concentration influenced mass transfer values (Table 10) with values ranging from

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12.5 (southern fried) to 2.48 g/100g (Cajun). Marinade concentration increased mass transfer (weight gain) especially in going from the 20 to 30% level. The exception was for the smoke marinade where a decrease occurred (Table 10). The rank correlation coefficient between salt content of the marinade solutions and the corresponding mass transfer values was 0.50 in the absence of the southern fried marinade value. The latter was the exception in that it was ranked 6th for salt content and highest (1st) for mass transfer (Table 10). There was a positive relationship between mass transfer values x salt content and a negative relationship between mass transfer x moisture content/centrifugal drip (Table 13). Weight loss, due to dehydration, in tuna fish is undesirable as the texture of cooked tuna is meaty and any loss of moisture results in an even firmer product. Table 10. Mass transfer (weight gain; g/100g) values for marinated albacore tuna fish portions Marinade Cajun Smoke Tandoori Lemon+lime Arrabbiata Southern fried Mean F-test: marinade F-test: conc. F-test: interaction

Marinade concentration (%) 10 20 2.23 2.40 12.7 8.50 7.72 9.90 3.17 5.77 10.2 9.93 7.90 11.0 7.26 7.91 P<0.001 LSD 2.12 P<0.001 LSD 1.50 P<0.001 LSD 3.67

30 2.70 7.10 10.0 7.60 16.2 18.5 10.4

Mean 2.48 9.44 9.06 5.51 12.1 12.5

Table 11. Moisture content (g/100g) of marinated albacore tuna fish portions Marinade Cajun Smoke Tandoori Lemon+lime Arrabbiata Southern fried Mean F-test: marinade F-test: conc. F-test: interaction

Marinade concentration (%) 10 20 69.4 68.4 68.4 66.2 72.5 71.1 72.2 71.1 73.2 72.0 73.9 73.6 71.6 70.5 P<0.001 LSD 2.43 P<0.05 LSD 1.72 NS LSD 4.20

30 66.1 65.4 69.8 69.8 69.7 72.5 68.9

Mean 68.0 66.8 71.2 71.0 71.6 73.3

All tuna samples used in the current trials were blast-frozen after filleting and were tempered (2-4oC) before marinating. Freezing-thawing favours solids uptake and yields lower weight loss/solute uptake ratios. Moisture values of the tuna portions ranged from 66.8 (smoke) to 73.3 g/100g (southern fried) with no difference between tandoori, lemon+lime and arrabbiata marinades (Table11).

Underutilised Fish Species: Their Potential as Fresh Fillets and Processed Products 175 Moisture contents fell with increasing marinade concentrations (Table 11) and yet the tuna gained weight as a result of marinating (Table 10). This indicated that there was an influx of solids (including salt) from the marinating solutions into the fish tissue. There was a positive relationship between moisture content x centrifugal drip and a negative relationship between moisture content x mass transfer/salt content values (Table 13). Centrifugal drip (CD) is a convenient method to measure the amount of loosely bound water in food and the application of centrifugal force to marinated fish samples showed that the marinades had a variable affect on CD values (Table 12). Cajun marinade had the highest CD (indicating the highest amount of loosely bound water) and smoke and arrabbiata the lowest.There was a positive relationship between CD x moisture content and a negative relationship between CD x mass transfer/salt content values (Table 13). Centrifugal drip values generally decreased as marinade concentration increased. Table 12. Centrifugal drip values (g/100g) for marinated albacore tuna fish portions Marinade Cajun Smoke Tandoori Lemon+lime Arrabbiata Southern fried Mean F-test: marinade F-test: conc. F-test: interaction

Marinade concentration (%) 20 17.5 0.8 5.1 9.2 1.0 9.6 7.2 LSD 2.21 LSD 1.56 LSD 3.83

10 16.9 2.4 8.5 11.2 5.3 10.5 9.1 P<0.001 P<0.001 NS

30 16.2 0.9 1.0 5.6 0.9 6.7 5.2

Mean 16.8 1.39 4.87 8.67 2.4 8.9

Table 13. Interrelationship (Rank Orders From Tables 8, 10, 11, 12)1 between salt content, mass transfer, moisture content and centrifugal drip values ff marinaded albacore tuna fish portions

1

Parameter Salt Mass transfer Moisture Centrifugal drip

10 3 3 1 1

Marinade concentration (%) 20 2 2 2 2

30 1 1 3 3

Highest numerical value ranked 1.

Sensory acceptability of marinated tuna portions: All the marinated tuna samples were highly acceptable with the exception of the smoke marinade, which was downgraded. This was due to the high salt content of this sample. Tuna portions marinated in Cajun and arrabbiata marinades received the highest sensory scores followed closely by portions in tandoori, lemon+lime and southern fried (Table 14). Portions from smoke marinade were the only ones scoring below the mid-point of the acceptability scale. The Hunter L/b (white/yellow colour ratio) values for the marinaded tuna products were 3.46 (Cajun), 5.79

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(smoke), 2.22 (tandoori), 3.24 (lemon+lime), 5.40 (arrabbiata) and 5.30 (southern fried) indicating that tuna marinated in tandoori, lemon+lime and Cajun flavours had the darkest appearance while those in smoke and arrabbiata were brightest. Table 14. Sensory acceptability scores1 (25 tasters) for marinated2 albacore tuna fish portions3 Marinade

1

Cajun Smoke Tandoori Lemon+lime Arrabbiata Southern fried F-test: sensory score

Marinade concentration (%)2 30 10 20 30 10 30 P<0.001

6-cm line with end-points of 0 (unacceptable) and 6 (very acceptable) Ideal concentration selected from data in Table 7 3 Microwaved for 5 min/700W 4 Rank order

Sensory score1 4.3 (1)4 2.6 (6) 3.8 (3.5) 3.8 (3.5) 4.2 (2) 3.7 (5) LSD 0.78

2

Outcomes from study; part 2: Marinating technology was used to produce albacore tuna portions with good sensory and physicochemical attributes. Marinating can be applied to both UUFS and conventional fish species and is a relatively cheap and straightforward processing technique. The tuna portions had a wide range of flavours depending on choice of marinade and the concentration used. Samples were microwaved in the current trials to obtain reproducible time-temperature conditions. Other cooking methods that could be used include grilling or pan-frying. These methods may change acceptability scores, positively or negatively, by altering the mouth-feel of the product.

Adding Value to UUFS by Freeze-Chilling and MAP (Study 5; Part 3) The objective was to add value to fillets from UUFS by a combination of freeze-chilling (FC) and modified atmosphere packaging (MAP). Freeze-chilling of food involves freezing and frozen storage followed by controlled thawing and then retailing at chill storage temperatures, i.e. the consumer sees the product as ‘chilled’ (Fagan et al., 2003a,b; Fagan et al., 2004). It also confers a number of logistic advantages. A combination of MAP and freezechilling was shown to be beneficial for increasing the shelf life of whiting, mackerel and salmon portions (Fagan et al., 2003a) and the combination was applied in the current study to deep water redfish, cardinal fish and roundnose grenadier. This was with a view to retailing these species as ‘fresh fillets’ from chilled retail cabinets as added value products. Sourcing samples: Deep water redfish, cardinal fish and roundnose grenadier were caught by personnel from the Irish Sea Fisheries Board (BIM) during deepwater fishing trials in Irish waters off the eastern slopes on the Rockall Trough. The fish were iced on-board (4-5 days), filleted on-shore, and delivered on ice to AFRC. The fillets were vacuum packed (3-5kg lots),

Underutilised Fish Species: Their Potential as Fresh Fillets and Processed Products 177 blast-frozen (–35ºC/2.5h), stored (-20ºC) until required, and then thawed (4ºC) overnight followed by MAP. Modified atmosphere packaging (MAP) and freeze-chilling (FC): Thawed fillets were cut into consumer sized portions (circa 200g), placed in plastic trays (110x150x55mm; Dynopak limited, Ireland) which were sealed [MECAPAC 500 MAP machine (Mecaplastic, Bagnolet, France)] using a Dyno AF320 antifog film (Dynopak Limited) and a gaseous atmosphere of 30% oxygen, 40% carbon dioxide, 30% nitrogen, or alternatively, air. The MA packs were then FC by blast freezing (-30ºC/2.5h), storing (-20ºC/1d), and holding at 2-4ºC for 7 days with testing on days 0, 3, 5, and 7. In this instance the frozen component of the FC process was only one day. However, it could equally well have been days, weeks or even a month. Table 15. Effect of gaseous atmosphere and storage time (days at 4ºC) on odour scores1 for freeze-chilled samples of three UUFS Days Atmosphere Redfish Freeze-chill + air Freeze-chill + MAP2 Mean

1

0

3

5

7

Mean

5.23 4.70 4.97

4.03 4.17 4.10

1.37 4.13 2.75

1.13 4.10 2.62

2.94 4.27

Cardinal fish Freeze-chill + air Freeze-chill + MAP2 Mean

4.80 5.07 4.97

3.97 4.40 4.18

2.77 4.27 3.55

0.47 3.93 2.20

3.00 4.43

Roundnose grenadier Freeze-chill + air Freeze-chill + MAP2 Mean

4.87 5.23 5.05

4.23 4.40 4.32

2.27 4.23 3.25

2.63 3.60 3.12

3.50 4.37

F-test: atmosphere3

Redfish ***LSD 0.37

Cardinal ***LSD 0.38

F-test: days3

***LSD 0.51

***LSD 0.35

F-test: interaction3

***LSD 0.72

***LSD 0.50

Roundnose ***LSD 0.29 ***LSD 0.35 ***LSD 0.63

6-cm scale with end-points of 6 (fresh seaweed-like odour) and 0 (spoiled/rancid odour) 30% O2; 40% CO2; 30% N2 3 P<0.05(*), P<0.01(**), P<0.001(***); LSD (least significant difference)

2

Test procedures: The odour status of the MA and air-packed fish samples was determined on each test date (Table 15) by a sniff test using an eight-member trained taste panel. The packs (one sample portion per pack) were opened, sniffed immediately, and the score entered on 6-cm line with end-points of 6 (fresh seaweed-like odour) to 0 (spoiled/rancid odour). This was carried out for each packaging method at each storage time. Assessment of sensory

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acceptability was conducted on microwaved samples (4min/850W) by an 8-member panel using a 6-cm line with end-points of 0 (unacceptable) and 6 (very acceptable). Physicochemical tests were conducted as outlined by Fagan et al. (2003a) and embraced moisture, centrifugal drip (CD), gravity drip (GD), flesh pH, colour (Hunter Lab), total volatile basic nitrogen (TVBN), and total viable count (TVC). Odour scores: Fish odour is usually a strong indicator of spoilage and was used to good effect in these trials. The panel found no difficulty in assessing product odour and the option of opening and smelling packs immediately (i.e. the first whiff) gave consistent results. The efficacy of MAP in maintaining fresh odour was striking, especially from day 3 onwards (Table 15). MAP samples of the three species had a much fresher odour (high numerical scores = best) than the air-packed samples (Table 15), and the fresh odour remained over the 7 day storage period. In contrast, the odour of air-packed samples deteriorated rapidly (i.e. numerical values decreased over time). This led to a significant interaction indicating the different performance (in terms of odour) of the MAP versus air packs. The correlation coefficients between odour values of redfish/cardinal fish and the other test variables were relatively high (Table 20). As expected, the coefficients were negative with TVBN, TVC and flesh pH, i.e. odour scores declined as values for these variables increased. The coefficients for roundnose grenadier (see below) were of a smaller order of magnitude to those found for the other two species. Table 16. Effect of gaseous atmosphere and storage time (days at 4ºC) on centrifugal drip (CD) values (g/100g) for freeze-chilled samples of three UUFS Days

1

Atmosphere Redfish Freeze-chill + air Freeze-chill + MAP1 Mean

0

3

5

7

Mean

14.6 15.7 15.2

11.5 14.9 13.2

7.4 15.1 11.3

3.3 16.2 9.68

9.2 15.5

Cardinal fish Freeze-chill + air Freeze-chill + MAP1 Mean

4.50 12.4 8.43

5.37 8.10 6.73

4.73 9.67 7.20

2.60 10.0 6.32

4.30 10.0

Roundnose grenad. Freeze-chill + air Freeze-chill + MAP1 Mean

23.1 21.0 22.0

21.5 19.3 20.4

20.3 19.0 19.6

17.8 21.0 19.4

20.7 20.1

F-test: atmosphere2 F-test: days2 F-test: interaction2

Redfish ***LSD 0.74 ***LSD 2.06 ***LSD 2.91

Cardinal ***LSD 0.38 ***LSD 0.35 ***LSD 0.50

Roundnose NS; LSD 1.83 NS; LSD 2.43 NS; LSD 3.43

30% O2; 40% CO2; 30% N2 P<0.05(*), P<0.01(**), P<0.001(***); LSD (least significant difference); NS (not significant)

2

Underutilised Fish Species: Their Potential as Fresh Fillets and Processed Products 179 Centrifugal drip, gravity drip and moisture content: Centrifugal drip (CD) values were highest for the MAP treatment for redfish and cardinal fish (Table 16). This was anticipated as the CO2 component of MAP often increases drip in fish fillets (Fagan et al., 2004). The decrease in CD values for the air-packed samples over the storage period was not anticipated and may be due to denaturation of some of the protein resulting in greater water binding. However, this effect was not observed to the same extent with MAP resulting in a statistically significant interaction (Table 16). Centrifugal drip was negatively correlated (Table 20) with TVBN, TVC and pH, and positively correlated with fish odour score and sensory acceptability. However, the coefficients were small and in the range 0.30 to 0.64. Test days or the use of MAP had no influence on CD values of roundnose grenadier, but values (circa 20%) were high relative to the other two species. Gravity drip (GD) values were much lower than their CD counterparts. The MAP samples had a GD of 6.6 (redfish), 6.7(cardinal fish) and the air packs 3.6% (both species), while there was little difference between GD values on days 3, 5 and 7 for samples in either atmosphere. The GD values for roundnose grenadier were not influenced by the treatments (mean value 3.9%). Moisture content of the fish was similar for MAP versus air-stored samples, and between the test days, with overall mean values of 79.4 (redfish), 77.0 (cardinal fish) and 82.6% (roundnose grenadier). Flesh pH and colour values: Samples in MAP had lower flesh pH values than those in air presumably due to the presence of CO2 in the gas mixture. This effect was found for the three species and indicates a longer shelf life for the MAP samples which was the case (Tables 1719). Flesh pH values rose with storage time for both atmospheres but the increase was largest for the air-packed samples. This resulted in a significant (P<0.001) interaction. Flesh pH values were highly correlated with CD, TVBN, TVC, and odour scores but the correlation with acceptability score was smaller (Table 20). Fish whiteness was measured with a Hunter meter and the redfish samples in MAP were whiter than those in air with a difference of four Hunter L units being readily discernable by the eye. The L value of redfish portions in MAP (61.8) was similar to that of silver smelt (61) and only slightly lower than that of raw cod (65). Both these latter species are noted for their flesh whiteness. The samples became slightly whiter between days 0 and 3 (perhaps due to slight surface desiccation) but changed little thereafter. For cardinal fish, the samples in MAP were less yellow/brown than those in air. The MAP 'b' values remained relatively constant over storage days 0 to 5 whereas for air the values increased steadily with time. There were no statistically significant differences for Hunter colour values for samples of roundnose grenadier from the different treatments. The mean whiteness value (L = 65) compared favourably with that of silver smelt (61) and raw cod (65). Total volatile basic nitrogen: TVBN is a measure of protein degradation in fish and values in excess of 35mg/100g represent fish that is at, or near the end of its shelf life. The results (Table 17) indicate that both MAP and duration of storage influenced TVBN values of the three species. The mean TVBN value for MAP (redfish) was only half that of air, and the difference on day 7 was 3-fold, for cardinal fish 2-fold, and for roundnose grenadier 2.5 fold. These data suggest that MAP is particularly beneficial for shelf life extension of the three species and this is supported by the total viable count values (Table 18).

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T. Ronan Gormley and John D. Fagan

Table 17. Effect of gaseous atmosphere and storage time (days at 4ºC) on total volatile basic nitrogen (TVBN) values (mg/100g) for freeze-chilled samples of three UUFS Days Atmosphere Redfish Freeze-chill + air Freeze-chill + MAP1 Mean

0

3

5

7

Mean

10.1 10.3 10.2

12.9 11.4 12.2

27.8 11.5 19.7

40.6 13.3 27.0

22.9 11.6

Cardinal fish Freeze-chill + air Freeze-chill + MAP1 Mean

8.33 10.9 9.63

11.8 10.9 11.4

15.9 11.8 13.8

24.1 11.8 17.9

15.0 11.4

Roundnose grenad. Freeze-chill + air Freeze-chill + MAP1 Mean

16.4 15.2 15.8

18.7 16.4 17.5

21.7 14.2 17.9

41.7 16.7 29.2

24.6 15.6

Redfish ***LSD 1.30 ***LSD 1.29 ***LSD 1.82

Cardinal ***LSD 1.36 ***LSD 1.63 ***LSD 2.31

Roundnose ***LSD 2.02 ***LSD 1.65 ***LSD 2.33

2

F-test: atmosphere F-test: days2 F-test: interaction2

1

30% O2; 40% CO2; 30% N2 P<0.05(*), P<0.01(**), P<0.001(***); LSD (least significant difference)

2

The air-packed sample of redfish was near the end of its shelf life on day 5 (TVBN=28mg/100g) while the MAP sample still had a low TVBN (13mg/100g) on day 7 suggesting a much longer shelf life (Table 17). The air-packed samples of cardinal fish had a shelf life of 1-2 days beyond day 5 and the MAP samples an even longer shelf life. The airpacked sample of roundnose grenadier was at the end of its shelf life on day 7 (TVBN=42mg/100g) while the MAP sample still had a relatively low TVBN (17mg/100g) on day 7 suggesting a much longer shelf life (Table 17). TVBN values were strongly correlated with most of the other test variables (Table 20). Total viable count: The use of MAP resulted in much lower TVC values compared to air on days 3, 5 and 7 for the three species. The values for the air system were high by day 7 (>log108 cfu/g redfish and cardinal fish; >log107 cfu/g roundnose grenadier) (Table 18). TVCs of the samples in air generally increased throughout the storage period while values in the MAP packs remained relatively constant with time. The interaction (P< 0.001) is an indication of the different performance (in terms of TVC) of the air versus MAP packs. TVC values were strongly correlated with most of the other test variables (Table 20).

Underutilised Fish Species: Their Potential as Fresh Fillets and Processed Products 181 Table 18. Effect of gaseous atmosphere and storage time (days at 4ºC) on total viable count (log10cfu/g) values for freeze-chilled samples of three UUFS Days Atmosphere

0

3

5

7

Mean

5.40

7.23

7.00

8.67

7.08

5.77

4.90

5.57

5.90

5.53

5.58

6.07

6.28

7.28

4.17

5.13

5.63

8.40

5.83

5.43

3.70

5.13

6.23

5.13

4.80

4.42

5.38

7.32

3.80

6.43

6.17

7.63

6.01

4.73

4.47

4.80

4.67

4.67

4.27

5.45

5.48

6.15

Redfish

Cardinal

Roundnose

***LSD 0.07

***LSD 0.22

*LSD 0.85

***LSD 0.33

***LSD 0.43

***LSD 0.53

***LSD 0.46

***LSD 0.60

***LSD 0.76

Redfish Freeze-chill + air Freeze-chill + MAP

1

Mean Cardinal fish Freeze-chill + air Freeze-chill + MAP

1

Mean Roundnose grenad. Freeze-chill + air Freeze-chill + MAP Mean

2

F-test: atmosphere 2

F-test: days

2

F-test: interaction

1

1

30% O2; 40% CO2; 30% N2 2 P<0.05(*), P<0.01(**), P<0.001(***); LSD (least significant difference)

Sensory acceptability scores: The acceptability scores were good (i.e. generally above the mid-point of the scale) for the three species. Cardinal fish received the highest scores, followed by roundnose grenadier and redfish. Scores for MAP samples (Table 19) of redfish and cardinal fish were higher than for samples in air which is in agreement with the TVBN and TVC values. Scores for roundnose grenadier samples were most influenced by storing in MAP versus air (Table 19). The day 7 MAP and air samples were not tasted in view of high TVC values. Acceptability scores were strongly correlated with most of the other test variables (Table 20). Correlation matrix for selected test variables: Flesh pH was strongly correlated with most of the other test variables for redfish and cardinal fish (Table 20). The negative correlation with CD was anticipated since water binding improves (and hence less drip) as the pH moves from the isoelectric point (circa pH 5.2 for fish). The high correlations between flesh pH, TVBN, TVC and odour scores were expected since high TVBN means more free amines and a more alkaline pH.

182

T. Ronan Gormley and John D. Fagan Table 19. Effect of gaseous atmosphere and storage time (days at 4ºC) on sensory acceptability scores1 for freeze-chilled samples of three UUFS Days

Atmosphere Redfish Freeze-chill + air Freeze-chill + MAP2 Mean

0

3

5

7

Mean

4.57 4.70 4.63

3.93 4.40 4.15

NT4 2.97 2.97

NT NT NT

3.73 4.01

Cardinal fish Freeze-chill + air Freeze-chill + MAP2 Mean

4.77 5.00 4.90

4.53 4.60 4.57

3.67 4.63 4.15

NT NT NT

4.32 4.76

Roundnose grenad. Freeze-chill + air Freeze-chill + MAP2 Mean

4.77 4.50 4.63

4.60 4.63 4.60

3.30 3.87 3.58

NT NT

4.21 4.33

Redfish NS; LSD 0.28 **LSD 0.31 NS; LSD 0.44

Cardinal *LSD 0.33 **LSD 0.35 *LSD 0.50

Roundnose NS; LSD 0.23 ***LSD 0.37 NS; LSD 0.52

3

1

F-test: atmosphere F-test: days3 F-test: interaction3

6-cm scale with end-points of 0 (unacceptable) and 6 (very acceptable) 30% O2; 40% CO2; 30% N2 3 P<0.05(*), P<0.01(**), P<0.001(***); LSD (least significant difference); NS (not significant) 4 Not tested due to TVC>log105 cfu/g 2

Sensory acceptability scores for redfish and cardinal fish were negatively correlated with TVBN, TVC, and positively with odour score, but the magnitude of the coefficients was generally less than those found between some of the other test variables. The coefficients for roundnose grenadier were not as large as those for redfish or cardinal fish, partly due to the lower level of variability in the roundnose grenadier data, i.e. correlation coefficients >0.50 were 0.80 (sensory acceptability score x odour score), -0.70 (odour score x pH), -0.64 (odour score x TVBN), -0.63 (odour score x TVC), 0.60 (TVC x pH), 0.59 (TVBN x pH) and -0.57 (sensory acceptability x pH). Outcomes from study; part 3: MAP is a suitable technology for extending the shelf life of freeze-chilled portions of redfish, cardinal fish and roundnose grenadier in comparison with air-stored samples. The samples received good odour and acceptability scores, and this suggests that portions of these species are suitable for retailing as freeze-chilled samples. It is likely that the dual technology of MAP with freeze-chilling will also be suitable for extending the shelf life (and hence ‘retailability’) of other fish species.

Underutilised Fish Species: Their Potential as Fresh Fillets and Processed Products 183 Table 20. Correlation matrix for selected test variables (redfish = upper triangle; cardinal fish = lower triangle) CD1 TVBN TVC Odour Accept.2 pH3

CD1 -0.53 -0.30 0.64 0.56 -0.53

Centrifugal drip. 2 Sensory acceptability. 3 Flesh pH.

TVBN -0.92 0.83 -0.94 -0.71 0.94

TVC -0.84 0.85 -0.81 -0.83 0.88

Odour 0.86 0.92 -0.77 0.83 -0.93

Accept.2 0.07 -0.47 -0.18 0.57

pH3 -0.93 0.94 0.80 -0.94 -0.45

-0.83

1

Tests on Blue Whiting (Study 6) Products and outcomes: The findings suggested that blue whiting fillets originating as block-frozen or IQF (individually quick frozen) samples have potential for sale in retail packs at 2-4 °C, especially when packed in MA, and when pan fried. These have a high quality shelf life of about 5 days at 2-4 °C. Frozen breaded blue whiting goujons and nuggets were also well accepted (Oehlenschläger et al., 2008). Parasites were not present in the blue whiting samples examined from the commercial catch used in these trials. However, the incidence of the parasitic nematode Anisakis simplex varies widely between catches of blue whiting and can be as high as 90-100%, and as such could be a major obstacle to the use of this species.

Conclusions • •

•

•

Silver smelt is a useful species for a range of products including enrobed nuggets, fingers and fishcakes; it also produced good gels (study 1). In tests on 15 UUFS, nine were well liked by taste panels when evaluated as breaded nuggets, and all compared favourably with cod. Cardinal fish, siki shark, and lesser spotted dogfish had the most favourable WHC and gel compression values and roundnose grenadier, forkbeard and leafscale gulper shark had the least favourable values. Gel whiteness values were best for roundnose grenadier, orange roughy and black scabbard, and were least good for lesser spotted dogfish, forkbeard and blue ling (study 2). In tests on 21 underutilised species, taste panellists preferred six of these to cod as fillets, ten as nuggets and eight as fish-cakes. The best species were orange roughy, black scabbard, morid cod and siki shark, and the least good were rabbitfish, smalleyed rabbitfish, birdbeak dogfish and flying squid (study 3). Steamed fillets of 15 species were compared with steamed cod using taste panels. No sample was significantly better than cod in terms of sensory scores. However, samples of morid cod, bluemouth rockfish, ling, forkbeard, black scabbard, silver roughy, orange roughy and roundnose grenadier compared favourably with cod.

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•

•

•

Fillets of tusk, siki shark, blue hake, roughhead grenadier, rabbit fish and longnose velvet dogfish were downgraded (study 4). Sous vide, marinating, freeze-chilling and modified atmosphere packaging (MAP) were found to be highly suitable technologies for adding value to a range of fish species both in terms of processed products (sous vide and marinating), and extended shelf-life fresh fillets (freeze-chilling+MAP) (study 5). Added value breaded products were prepared from blue whiting and received good acceptability scores from taste panels. However, the presence of parasites can be a problem with this species (study 6). The overall outcome from the six trials indicated that a number of the species are suitable for potential exploitation as chilled fillets or processed products. Many of the UUFS studied are sensitive to fishing pressures and must be caught using sustainable fishing practices. The use of increasingly sophisticated fishing gear, the fact that many are deep water species, and are slow-growing, and in the case of shark-species, produce few offspring, must be taken into account if they are to provide sustainable alternatives to conventional commercial fish species.

Annexe 1. Glossary of Fish Species Tested Albacore tuna (Thunnus alalunga) Baird’s smoothhead (Alepocephalus bairdii) Birdbeak dogfish (Deania calceus) Black scabbard (Aphanopus carbo) Blue whiting (Micromesistius poutassou) Blue ling (Molva dipterygia) Bluemouth rockfish (Helicolenus dactylopterus) Cardinal fish (Epigonus telescopus) Cod (Gadus morhua) Flying squid (Todarodes sagittatus) Forkbeard (Phycis blennoides) Greater lantern shark (Etmopterius spinax) Greenland halibut (Reinhardtius hippoglossoides), Haddock (Melanogrammus aeglefinus) Kitefin shark (Dalatias licha) Leafscale gulper shark (Centrophorus squamosus) Lesser spotted dogfish (Scyliorhinus canicula) Ling (Rachycentron canadum) Longnose velvet dogfish (Centroscymnus crepidater) Mackerel (Scomber scombrus) Morid cod (Mora moro) Orange roughy (Hoplostethus atlanticus) Pollock (Pollachius virens) Rabbit fish (Chimaera monstrosa) Redfish (Sebastes mentella) Roughhead grenadier (Macrourus berglax)

Underutilised Fish Species: Their Potential as Fresh Fillets and Processed Products 185 Roundnose grenadier (Coryphaenoides rupestris) Salmon (Salmo salar) Siki shark (Centroscymnus coelolepis) Silver roughy (Hoplostethus mediterraneus) Silver smelt (Argentinus silus) Small eyed rabbit fish (Hydrolagus affinis) Snake mackerel (Ruvettus pretiosis) Tusk (Brosma brosma) Whiting (Merlangius merlangus) Wolf fish (Anarchichas lupus)

Acknowledgements Thanks to Martine Brennan, Michelle Mitchell, and many visiting foreign work experience students for assistance in conducting this R and D. Thanks are also extended to the European Union (FAR Programme; LEONARDO Programme; Regional Development Fund; SEAFOODplus project;), the Irish Sea Fisheries Board (BIM), the Irish Marine Institute, Enterprise Ireland, and various seafood companies for their support and part-funding of elements of this research.

References Anese, M. and Gormley, T.R. 1996. Effects of dairy ingredients on some chemical, physicochemical and functional properties of minced fish during freezing and frozen storage. Food Science and Technology, 29, 151-157. Anon. 2003. Prepared Consumer Foods Sector Strategy 2003-2006, Enterprise Ireland, Dublin, Ireland. Brennan, M.H. and Gormley, T.R. 1999. The quality of underutilised deep-water fish species. End of Project Report, ISBN 1-84170-149-1, Teagasc, The National Food Centre, Dublin, Ireland, 22 pages. Fagan, J.D., Gormley, T.R. and Uí Mhuircheartaigh, M.M. 2004. Effect of modified atmosphere packaging with freeze-chilling on some quality parameters of raw whiting, mackerel and salmon portions. Innovative Food Science and Emerging Technologies, 5, 205-214. Fagan, J.D. and Gormley, T.R. 2005a. Effect of sous vide cooking, with freezing, on selected quality parameters of seven fish species in a range of sauces. European Research and Technology, 220, 299-304. Fagan, J.D. and Gormley, T.R. 2005b. Underutilised fish species--the challenges of consumer acceptance and adding value. In: Culinary Arts and Sciences:-Global and National Perspectives. ISBN 1-85899-179-X, (Eds Edwards, J. S. A., Kowrygo, B. and Rejman, K.), Wydawnictwo SGGW, Warsaw, Poland, 312-321. Fagan, J.D., Gormley, T.R. and Uí Mhuircheartaigh, M.M. 2003a. Effect of freeze-chilling in comparison with fresh, chilling and freezing, on some quality parameters of raw whiting, mackerel and salmon portions. Lebensmittel-Wissenschaft und-Technologie, 36, 647-655.

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Fagan, J.D., Gormley, T.R. and Uí Mhuircheartaigh, M.M. 2003b. Freeze-chilling and gas flushing of raw fish fillets. End of Project Report, ISBN-1-84170-335-4, Teagasc, The National Food Centre, Dublin, Ireland, 28 pages. Gormley, T.R. and Fagan, J.D. 2005. Sensory acceptability of new fish species. Food Science and Technology, 19, 35-38. Gormley, T.R. 2006. New fish species: the three-way challenge of supply, adding value and consumer acceptance. Food Science Central (www.foodsciencecentral.com), (www.foodsciencecentral.com/fsc/ixid14338), May, 3 pages. Gormley, T.R. 2008. Overview of seafood research at Ashtown Food Research Centre (19902007). End of Project Report, ISBN 1-84170-509-5, Teagasc, Ashtown Food Research Centre, Dublin, Ireland, 30 pages. Gormley, T.R., Connolly, P.L. and Ward, P. 1994. Evaluation of deep water fish species. Farm and Food, 4, 8-11. Gormley, T.R., Duelk, C. and Tansey, F.S. 2003. Proceedings of the Transatlantic Fisheries Technology Conference (TAFT), Icelandic Fisheries Laboratories, Reykjavik, Iceland, 6667. Gormley, T.R., Ward, P. and Somers, J. 1991. Silver smelt: A valued non-quota fish. Farm and Food, 1, 8-10. Gormley, T.R., Ward, P. and Somers, J. 1993. A note on the effect of long-term frozen storage on some quality parameters of silver smelt (Agrentinus silus). Irish Journal of Agricultural and Food Research, 32, 201-204. Maier, K., Gormley, T.R., Connolly, P.L. and Auty, M. 1997. Assessment of underutilised fish species. Farm and Food, 7, 30-34. Oehlenschläger, J., Karl, H., Mitchell, M., Fagan, J. and Gormley, T.R. 2008. Evaluation of quality parameters of canned and frozen blue whiting (Micromesistius poutassou). Journal of Fisheries Sciences.com, D01:10.3153/jfscom.2008040, 11 pages. Raoult-Wack A.L. 1994. Recent advances in the osmotic dehydration of foods. Trends in Food Science and Technology, 5, 255-260. Stoknes, I. S., Oehlenschläger, J. and Gormley, T.R. 2006. Quality evaluation of silver smelt (Argentina silus) and its suitability for seafood products: Compilation of results from three European research centres. In: Seafood Research from Fish to Dish: Quality, safety and processing of wild and farmed fish. ISBN-10:90-8686-005-2, (Eds Luten, J. B., Jacobsen, C., Bekaert, K., Saebo, A. and Oehlenschläger, J.), Wageningen Academic Publishers, The Netherlands, 439-456. SVAC (Sous Vide Advisory Committee). 1991. Codes of Practice for Sous Vide. Thorarinsdottir, K.A, Arason. S., Bogason, S.G. and Kristbergsson, K. 2004. The effects of various salt concentrations during brine curing of cod (Gadus morhua). International Journal of Food Science and Technology, 39, 79-89.

In: Food Science and Security Editors: L. Amsel and L. Hirsch, pp. 187-230

ISBN: 978-1-60692-977-3 © 2009 Nova Science Publishers, Inc.

Chapter 8

IMPROVEMENT OF POULTRY MEAT NUTRITIONAL VALUE AND QUALITY THROUGH DIFFERENT NATURAL SOURCES AND MINERAL DIETARY SUPPLEMENTS Ricard Bou1,a, Eric A. Deckerb, Francesc Guardiola a and Rafael Codonya a

Nutrition and Food Science Department-XaRTA-INSA, Faculty of Pharmacy, University of Barcelona, Av. Joan XXIII s/n, 08028 Barcelona, Spain. b Department of Food Science, University of Massachusetts, Amherst, MA 01003, USA.

1. Introduction Nowadays, consumers demand food products with increased nutritional value and free of artificial preservatives or other food additives. In this frame the development of meat products with lower fat content and/or higher levels of unsaturated fatty acids, especially the n-3 fatty acids, would be desirable as a strategy to help control cardiovascular and inflammatory pathologies (1-4) since dietary fat intake in Western countries often exceeds the recommended intake and the level of saturated fatty acids intake is high in relation to the unsaturated fatty acids (5, 6). Meat is an excellent source of proteins but also is a good source of vitamins and minerals such as iron, selenium and zinc (7). These latter elements are important for health since they are essential for the activity of certain enzymes that are necessary for the normal function of the human body. Antioxidant enzymes such as superoxide dismutase (SOD) and glutatione peroxidase (GSH-Px) need mineral cofactors to be functional and protect cells against reactive oxygen species (8). The activity of these antioxidant enzymes is also important in meat quality since they impact of the shelf-life of foods such as meat products (9). 1

E-mail address: [email protected]. Telephone number: 34-93-4024511, Telefax number: 34-93-4021896, Corresponding author: Ricard Bou, Nutrition and Food Science Department, Faculty of Pharmacy, University of Barcelona, Av. Joan XXIII s/n, 08028 Barcelona, Spain.

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Lipid oxidation products have harmful biological effects and thus can adversely impact health (10-14). As a consequence, it is important to not only improve the nutritional value of foods but also to minimize lipid oxidation to provide healthier and higher quality food products. Control of lipid oxidation can be a challenge since not all of the antioxidants are accepted by consumers since natural products are often perceived as safer as well as more nutritious than food containing additives or antioxidants coming from animals feed ingredients of a non-natural origin. While natural products are desired by many consumers, these products can be difficult to define since some man-made food additives and feed ingredients can be completely identical to those present in the nature or slightly modified to increase their effectiveness. Various natural compounds can act as antibiotics, growth promoters, antioxidants and also as food colorants. Therefore, different strategies have been studied to improve the nutritional value and the quality (e.g. a higher oxidative stability, better sensory characteristics) of meat products through natural dietary supplements. Dietary strategies can be more challenging than the direct addition of nutrients into meat products but can present higher benefits than the simple addition because first the living animals may efficiently distribute the compounds throughout the tissues and second, the dietary supplementation is safer because the resulting enriched meat products ensure tolerable amounts in humans. Animal metabolism plays an important role on the effectiveness of any dietary supplementation and further meat enrichment. For instance, modification of the fatty acid profile of ruminants animals through diet has been proven to be more difficult than in nonruminant animals such as poultry or pigs due to the biohydrogenation of unsaturated fatty acids in the rumen (15). In addition, the presence of feed compounds such as flavonoids and phytates, which could have positive human health benefits, are not always desirable since these compounds can negatively affect the absorption of other desired compounds such as minerals thus negatively impacting the animal performance and/or animal health (16-18). The sensory characteristics of foods, especially appearance, texture and flavor influence consumers’ food purchasing decisions. Therefore, a major part of the goal is to increase the nutritional composition of meat products without negatively compromising meat yields and/or qualities. Poultry meat is widely consumed and its total fat content is medium to relatively low although its relatively poor in n-3 polyunsaturated fatty acids (PUFA) when in animals are fed with standard diets (19). However, poultry meat has also been reported to be successfully enriched in several compounds through dietary practices (19-21). This review summarizes the different dietary strategies that have been used to improve the nutritional value as well as the oxidative stability and sensory properties of poultry meats.

2. Dietary Fat Source Addition The fatty acid composition of skeletal muscle and adipose tissue of poultry is influenced by the dietary fat source (22, 23). In storage tissues, such as abdominal and subcutaneous fat, the fatty acid profile is easier to modify through diet than in skeletal muscle (23, 24). The difference to change fatty acid profile in muscle and storage lipids has been attributed to their different function in each tissue. Storage tissues reflect fairly well the fatty acid diet composition because they act as a fat reservoir (25). On the contrary, the fatty acids in lean

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skeletal muscle are predominately found in cell membranes and thus have a relatively constant fatty acid composition since they have to maintain the physical properties and biological activity. Therefore, the modification of membrane lipids through changes in dietary fatty acid composition is limited (23, 26). However, consumers generally eat meat which refers to the skeletal muscle with its associated fat. Thus, alterations in the fatty acid composition of the fat associated with meat can be more important in improving the nutritional composition of poultry. Poultry meat fatty acid composition reflects that given in the diet. For example, meats rich in saturated fatty acids can be obtained when animals are fed diets containing coconut oil (27, 28) or palm oil (29). Oleic acid concentrations can be increased by feeding animals with olive oil (27, 28, 30-33) and in linoleic acid can be increased when rapeseed (23, 34) sunflower (29, 32-34) or soybean oil (23, 34) is included in the diet. Likewise, the content in linolenic acid in poultry meat can be increased through the addition of linseed oil into the feeds (27-29, 32-37). These modifications in the fatty profile of meat can be achieved without altering the content in total lipids and the ratio of the main lipid classes (25, 38). However, diets supplemented with PUFA-rich oils compared with diets supplemented at the same level with animal fats rich in saturated fatty acids, lead to a lower body fat deposition in broilers (39).

2.1. Addition of n-3 PUFA As consumers demand healthier food, meat rich in n-3 PUFA are of interest because of their benefits for cardiovascular related effects (i.e. lowering triacylglycerol and cholesterol levels, reducing blood pressure), inflammatory diseases, behavioral disorders and mental issues (1-4, 21, 40, 41). Poultry is able to synthesize very long chain n-3 PUFA such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from linolenic acid. However, when animals were fed with vegetables sources rich in linolenic acid (i.e. linseed, flaxseed or their oils) only relatively small increases in EPA and DHA were observed (21, 22, 37, 39, 42, 43) (Figure 1). Therefore, in order to produce poultry meat enriched in EPA and DHA, these very long chain n-3 PUFA must be included in the animals diet (21, 34, 35, 37, 44). Fish oil has been mainly used for meat enrichment in EPA and DHA (19, 21, 23, 24, 29, 37, 42, 4447). However, we have to take into account that the very long chain n-3 fatty composition and concentration of fish oil is dependent on fish species. Therefore, the impact of fish oil supplementation on the fatty acid composition of poultry can vary accordingly to the fish oil origin. Other dietary sources like fish meal and algae have also been used to enrich poultry meat in very long chain n-3 fatty acids and their respectively fatty acid composition subsequently influence meat fatty acid composition (19, 21, 48-51). Equations that relate poultry meat fatty acid content for one single n-3 fatty acid with the fatty acids in the diet have been recently reported (22). Overall, animal feeding regime that leads to meat rich in n-3 PUFA is interesting from a nutritional point of view; however, this can cause oily carcasses due to the lower lipid melting points as a result of increasing the degree of unsaturation of the fatty acids resulting in consumer rejection (52). In addition, meat enriched with unsaturated fatty acids is more prone to oxidize leading to the formation of off-flavor and off-odors as well as the loss of nutritional value.

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% of the recommendation

50

40

30

20

10

0 6% BT

6% LO

2.5% FO

added fat source linolenic acid

EPA + DHA

Figure 1. Percentages of the Adequate Intakes for linolenic acid (5) and human recommended daily dietary intakes for linolenic acid and for eicosapentaenoic acid (EPA) plus docosahexaenoic acid (DHA) (283) provided by 100 g of raw chicken meat from animals fed 6% beef tallow (BT), 6% linseed oil (LO) or 2.5% fish oil (FO) (53, 284).

In relation to the unpleasant aromas, the presence of breakdown products from fatty acid oxidation and nitrogenated compounds such as trimetylamine which is formed during fish spoilage, are responsible of the typical fishy taints in marine products used in animal feeds (19, 21). These fishy and other off-odors aromas and flavors present in feeds can appear in poultry meat from animals that have been fed fish oils and fish meals (19, 21, 43, 53). The oxidative status of the fish oil and fish meal is a determinant factor in achieving enriched meat products without compromised sensory quality (19). The formation of unpleasant aromas and flavors are more pronounced in dark meat than in white meat (34, 44). This is explained by the higher iron content which favors fatty acid oxidation and the higher fat content in dark meat that concentrate fat soluble off-flavors. Selecting the appropriate fish oil dose is a determinant factor to successfully enrich poultry products with n-3 fatty acids while being accepted by the consumers (e.g. avoiding the presence of unpleasant odors, flavors or texture). To minimize unpleasant aromas and flavors, it has been recommended to add fish oil or fish meals to give an equivalent amount of fish oil of no more than 20 g fat / kg feed (21). Other strategies have also been described to enrich poultry meat with n-3 PUFA without negatively impacting the sensory quality of the product (21, 37, 42, 44, 48). These strategies are based on combining antioxidants with fish oil, mixing fish oil with vegetable oils and/or seeds, and substituting fish oil for other lipid sources shortly before slaughter (20, 37, 48, 54). However, it should be taken into account that the substitution of fish oil by other fat sources results in a decrease in the content of EPA and DHA in the final product (20, 21). Using the appropriate feeding strategy (dose of the n-3

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fatty acid, time of supplementation and level of antioxidant) it is possible to achieve chicken meat with skin rich in very long chain n-3 fatty acids (Figure 1) which is accepted by consumers and has a relatively long shelf-life (20, 53, 55).

2.2. Addition of Conjugated Linolenic Acid Weight loss is an important issue for many consumers so the observation that conjugated linolenic acid (CLA) dietary addition can reduce body fat without substantially reducing body weight in many animals has gained a lots of attention (56, 57). Apart from CLA’s capacity of reducing body fat, several animal and in vitro studies have also shown that CLA may have anticarcinogenic activity, protect against atherosclerosis, and have other positive physiological effects (56-59). CLA is a collective term for 28 different isomers with the cis-9,trans-11-CLA isomer being the predominate isomer naturally present in foods such as milk, milk products and meat from ruminant animals as a consequence of the incomplete biohydrogenation of linoleic acid by rumen bacteria (60, 61). Poultry lacks of rumen but can have the cis-9,trans-11-CLA and other CLA isomers which can be due to the formation of CLA and other trans fatty acids by heating, refining and hydrogenation of oils used in animal feeds (62-64). Synthetically commercial CLA, which is produced by heating linolenic acid under alkaline conditions or by partial hydrogenation, contains equal levels of the cis-9,trans-11-CLA and trans-10,cis-12CLA and a remarkable levels in other CLA isomers compared to natural sources that contain predominately the cis-9,trans-11-CLA isomer (56, 65-67). Health benefits of CLA have been mainly attributed to cis-9,trans-11-CLA and/or trans10,cis-12-CLA (57). The trans-10,cis-12-CLA isomer seems to be the responsible in reducing body fat and maintaining a lean muscle with mice being the most sensitive to CLA response among mammalians (57, 68). CLA isomers besides the cis-9,trans-11-CLA and trans-10,cis12-CLA may also have biological effects but they have been far less studied (66). In addition, it should be noted that the biological activity of CLA mixtures and individual CLA isomers can show considerable variation between species and strains of the biological systems where they are being tested (67). A few studies using CLA mixtures or individual isomers have been done in humans, which in turn are not consistent with animal studies (66, 67, 69). Because chickens do not have a digestive system that includes rumen, the presence of CLA isomers in poultry products is relatively low (70, 71). However, it is possible to enrich poultry meats and eggs through dietary supplementation with CLA isomers (72-78). Chicken meat with skin preferentially incorporates some trans fatty acids and CLA isomers, such as ditrans-CLA which can came from oxidized oils, in comparison to other CLA isomers or cis fatty acids (71). Before developing a meat product rich in CLA a reasonable level of enrichment must be determined. Enrichment should be less than < 500 mg / day which is the estimated daily consumption (60, 61). The health benefits of the trans-10,cis-12-CLA isomer and other CLA isomers in human health is not completely well known and remains controversial (66, 68, 7981). Therefore, the supplementation of humans with synthetically prepared CLA should be done carefully and further studied (66, 69).

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3. Supplementation with Antioxidant Vitamins Ascorbic acid and vitamin E are related vitamins in protecting cells from reactive oxygen species in vivo (8, 82). Sufficient dietary levels of these two vitamins are necessary to protect against radicals and avoid oxidative stress thus ensuring animal health. Poultry meat products that have a high content in PUFA or a low content in antioxidants result in meats more prone to oxidative deterioration. Addition of liposoluble antioxidants such as tocopherols into processed meats is not very effective at delaying lipid oxidation due to difficulty in distributing the antioxidant where it is needed especially when the meat is not ground. However, the α-tocopherol addition into the feeds has been clearly demonstrated to be very effective in decreasing lipid oxidation (9, 83). This high efficiency is because α-tocopherol is efficiently incorporated into cell membranes where the oxidation is initiated (30, 83).

3.1. Tocopherol Supplementation The term vitamin E is commonly used for that group of tocol and tocotrienol derivatives having vitamin E activity. The biological and antioxidant activity of the four tocopherols (α, β, γ and δ) and four tocotrienols (α, β, γ and δ) are different. Animals are not able to synthesize vitamin E so their presence in the tissues totally depends on the diet (83). The amount and ratio of the different tocopherol analogs in meat is dependent on the type of tocol compound present in the feed. Immediately after the intake of tocopherols, chicken plasma reflects the tocopherol composition of the diet (84). However, biological discrimination of the different analogs occurs because α-tocopherol (Figure 2) is the main analog found in skeletal muscle (84-87). Tocopherol analogs have three chiral carbons in its phytyl tail. The 2R,4’R,8’R isomer (Figure 2) is the natural form present in foods although eight stereoisomeric forms (RRR, RRS, RSR, RSS, SSS, SSR, SRS and SRR) exist in synthetic all-rac-α-tocopherol. The distribution of the different stereoisomers in rats and chicken has been studied after dietary supplementation with all-rac-α-tocopheryl acetate (88, 89). In chickens, Cortinas et al. (88) found that there is a positive biodiscrimation for the RRR-α-tocopherol and the other three 2R stereoisomers. These authors reported that chicken thigh and liver from animals fed all-rac-αtocopheryl acetate preferentially accumulated (69 - 100%) those isomers having the 2R stereoisomer form, including the 2R,4’R,8’R isomer, and the stereoisomer ratios were not affected by the α-tocopheryl acetate dose (88). The nutrient requirement of poultry for vitamin E supplementation is 10 mg of all-rac-αtocopheryl acetate/kg feed (90). Tocopherol is a very efficient free radical scavenger and their supplementation in the diet is normally done in the form of all-rac-α-tocopheryl acetate since α-tocopherol, once acetylated, is more stable against oxidation thus ensuring a higher delivery of this vitamin through the diet (83). Once in the intestinal lumen, the esterified acetate group is hydrolyzed and, subsequently, the different tocopherol analogs are absorbed by the animal (83). Increasing the amount of vitamin E in the diet favors the oxidative balance (ratio of prooxidant and antioxidant factors) in the meat and also increases the nutritional value of meat products (9, 28, 53, 83, 91-93).

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CH3 HO CH3 H H3C

CH3

H

CH3

O

CH3 CH3

CH3

Figure 2. The chiral 2R,4’R,8’R configuration of α-tocopherol.

% of the Recommended Dietary Allowances

100 90 80 70 60 50 40 30 20 10 0 α-tocopheryl acetate

organic selenium type of supplement control

supplemented

Figure 3. Percentages of the Recommended Dietary Allowances for tocopherol and selenium (94) respectively provided by 100 g of raw thigh chicken meat from animals fed 0 or 225 mg α-tocopheryl acetate / kg feed (96) and by 100 g of raw thigh quail meat from animals fed 0.2 mg selenite / kg feed (basal) or 0.5 mg organic selenium / kg feed (247).

A shown in Figure 3, the dietary supplementation of 225 mg of all-rac-α-tocopheryl acetate/kg feed throughout the whole feeding period of broilers led to meat that delivered 24% of the Recommended Dietary Allowances for vitamin E (53, 94). Dietary supplementation with α-tocopheryl acetate has been proved to protect fatty acids (30, 52, 83, 95-98) and cholesterol (92, 99) from oxidation in both raw and cooked poultry meat. In addition, supplementation with several tocopherol sources lowers the onset of formation of primary and secondary oxidation compounds (36, 83, 100, 101, 101-103) which positively decreases the perception of off-flavor and –odors that are responsible for rancidity in poultry meat (19, 21, 31, 55, 100, 104). In Table 1, the effects of dietary tocopherols on lipid oxidation in poultry meat products have been summarized.

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Source1 all-rac-α-TA

Dose (mg/kg) 100

Period 7 weeks

all-rac-α-TA, RRR-α-γ-δ-TA

100, 500

10-42 days

all-rac-α-TA

200

21-45 days

all-rac-α-TA

200

16 weeks

all-rac-α-TA

75, 150, 225

7-50 days

all-rac-α-TA

200

2-6 weeks

all-rac-α-TA

200

41-47 weeks

Effect2 Breast and thigh raw meat from animals fed the supplement recorded lower TBARS values during refrigerated and frozen storage (28). The α-tocopherol content in breast meat increased with dietary intake of αtocopherol irrespectively from the natural or synthetic source. The use of natural tocopherols had no influence on thigh αtocopherol content whereas animals fed 500 mg/kg of synthetic tocopherol recorded increased α-tocopherol in thigh meat (84). The dietary mixture of natural source of tocopherols was less effective in protecting dark and white either raw and cooked than the synthetic α-tocopherol when compared on a weight basis (93). Thigh and breast raw meat from chicken fed the supplement recorded better TBARS values and sensory scores. The concentration of volatile aldehydes is higher in those samples from animals not receiving the supplement (100). Turkey meat from animals fed the supplement had decreased radical production, lipid and protein oxidation (285). Raw and cooked dark chicken meat from animals fed longer periods of supplementation or higher supplement doses showed increased tocopherol content, decreased lipid hydroperoxides and lower TBARS values (96). Raw and cooked chicken meat from animals fed the supplement showed lower TBARS values and cooked meat lower cholesterol oxidation products (169). Raw and Cooked thigh chicken meat from animals fed the supplement recorded lower TBARS values which are related with higher meat α-tocopherol content and lower glutathione peroxidase activity in raw thigh meat (286).

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Table 1. Continued

1 2

Source1 all-rac-α-TA

Dose (mg/kg) 200

Period 1, 2, 3, 4, and 5 weeks

all-rac-α-TA

200

6 weeks

all-rac-α-TA

50-250

0-18 weeks

all-rac-α-TA

200

1-42 days

all-rac-α-TA

200, 800

1-42 days

all-rac-α-TA

200

12-16 weeks

all-rac-α-TA

400, 1000

1-42 days

Effect2 Breast and thigh raw chicken meat from animal fed longer periods of supplementation recorded higher meat αtocopherol content. Feeding the animals for at least 4 weeks prior slaughter is necessary to optimize oxidative lipid stability (287). Raw and cooked dark chicken meat from animals fed the supplement recorded increased tocopherol content and lower TBARS values (203). Turkey breast meat from animals fed higher levels of supplementation recorded higher acceptable meat flavors with fewer oxidized off-flavor notes and less pale color was recorded with higher levels. TBARS values and volatile aldehydes were inversely related with the dietary tocopherol (288). Breast and thigh raw chicken meat from animals fed the supplement recorded higher α-tocopherol content and the oxidative stability of the membranal fractions tended to increase with increasing concentrations of α-tocopherol in the membranes (30). Cooked breast and thigh chicken patties from animals fed higher supplements recorded higher α-tocopherol content in meat and recorded decreased TBARS values and cholesterol oxidation products (289). Breast and thigh raw and cooked turkey meat from animals fed the supplement recorded lower TBARS values and higher content in meat α-tocopherol content (155). Breast and thigh raw duck meat from animals fed higher supplements recorded increased α-tocopherol content and enhanced oxidative stability but did not improve meat color stability (290).

TA means tocopheryl acetate TBARS means thiobarbituric acid reactive substances

Several extensive reviews about the protective effects of tocopherol on inhibition of lipid oxidation in poultry and other animals have been reported elsewhere (83, 105, 106). In addition to the ability to dietary α-tocopheryl acetate to inhibit lipid oxidation it has also been reported to prevent myoglobin oxidation thus protecting meat color and increase the water

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holding capacity of meats since α-tocopherol is involved in the stabilization of the lipid membranes of different animals (83, 87, 103). The protective effect of α-tocopherol against oxidation and their concomitant effects on the sensory characteristics of the meat is dependent on factors such as the level of the tocopherol source added into the diet, the degree of unsaturation and oxidative status of the oils added into the feed, the physiological status of the animal and finally the net oxidative balance of the meat (83, 96, 103, 107). Therefore, the adequate amounts to be supplemented should be determined in each condition and feeding period although a dose of ≤ 150 mg/kg feed throughout the feeding period seems to be enough to ensure oxidative stability in raw and cooked chicken meat (93, 96).

3.2. Ascorbic Acid Supplementation Ascorbic acid is a water soluble vitamin that protects cellular components from free radical damage. Ascorbate acts as an antioxidant in vivo by two mechanisms (82). The first is through its direct interaction with free radicals to form dehydroascorbate radical which is much less reactive than are many other radicals. The second mechanism is based on ascorbic acid ability to reduce tocopheroxyl radicals back to their native form thus regenerating oxidized tocopherol in the tissues. However, ascorbic acid can also promote oxidation because it can release of iron bound to proteins such as ferritin and is able to reduce prooxidant metals resulting in rapid decomposition of hydrogen peroxide and lipid peroxides into free radicals (108, 109). The antioxidant activity of ascorbic acid predominates in vivo because the reactivity of metals is tightly controlled. However, in meat the prooxidant/antioxidant behavior of ascorbate is dependent on concentration (9, 82). The concentration of ascorbic acid in meats is relatively low and may only be important in the early stages of meat storage since is depleted quite rapidly. For instance, in mackerel skeletal muscle most ascorbate is lost within 48 hr post-mortem (9, 108). When it is added to meats at relatively low concentrations (0.02 – 0.03%) ascorbic acid has been reported to be either ineffective or prooxidative through its ability to reduce ferric ions to the ferrous form while at high concentrations (0.5%) it inhibits lipid oxidation (9, 109, 110). Although it is essential for humans, poultry, under normal conditions, can synthesize enough ascorbic acid for normal growth and development (111). As ascorbic acid is efficiently regulated under optimal breeding conditions this explains that overall the addition of this compound into the diet has no effect in enhancing storage shelf-life (111-113). However, controversial results have also been reported about the prooxidative or antioxidative effects in chicken meat from animals fed ascorbic acid supplements (101, 114). Ascorbic acid may be an essential nutrient when poultry are reared under stressful conditions (111). This explains why when animals are reared under conditions of heat and moisture stress, increased GSH-Px activity and lowered oxidation in erythrocytes were found in chickens fed ascorbic acid supplements (115, 116). Likewise, ascorbic acid supplementation increased GSH-Px and CuZnSOD activities in chicken liver and erythrocytes and led to lowered levels of malondialdehyde in these tissues (115). Moreover, dietary supplementation with ascorbic acid under those stressful conditions has been shown to enhance immunological responses and improve animal production (111).

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4. Addition of Bioactive Natural Compounds: Isoprenoids and Polyphenols Feed additives of natural origin are perceived as harmless and environmentally friendly satisfying consumer demands for more healthy foods. The addition of dehydrated leaves and/or other parts of plants such as oregano, rosemary and thyme have been added into poultry diets to study animal performance, meat oxidative stability and overall quality of poultry meat (98, 114, 117-119). These plants and other natural sources have received much more attention recently because of the in vitro antioxidant activity (120-123). As shown in Table 2, the addition of these plants in the diet (from 5 to 30 g/kg feed) is effective in improving the oxidative stability of poultry meat though it is less efficient than the addition of 170 mg/kg feed of all-rac-α-tocopheryl acetate. However, various compounds present in plants and plant extracts also showed antibacterial, anticoccidial, antifungal, antiviral and antitumor activity (123-132). Therefore, the antioxidant, antibiotic, growth-promoting and even immune enhancing activity of these sources of natural compounds represents an alternative for animal production facilities to synthetic antioxidants and antibiotics (133-140). These beneficial activities found in these natural sources can be attributed to several phenol- and isoprene-based compounds, which have been summarized respectively in Tables 3 and 4. Most of the phytochemicals with antibiotic activity are found in the Lamiaceae family. This explains the increased animal performance when plants containing those bioactive compounds have been added into the poultry diets (114, 118). However, several authors reported no positive effects on animal performance (97). These controversial results can be explained because well-nourished healthy poultry reared under clean and ideal conditions, such as those used in animal experiments, often do not respond to growth promoting supplements. This is confirmed by the reduction of the adverse effects by the dietary addition of oregano (133) and Olympus tea (136) on animal performance in chickens after infection with Eimeria tenella.

4.1. Addition of Essential Oils and Extracts: Phenyl Terpenoids In order to concentrate the desired bioactive compounds, plant and other natural sources can be subjected to different extraction processes. Essential oils from plants and herbs are obtained after a steam-distillation process which concentrates relatively volatile and hydrophilic compounds. The chemical compounds present in the essential oils fall into two primary groups: terpenes and oxygenated compounds such as phenols, terpene alcohols, esters and ethers (141). Among them, various terpenoid phenol derivatives such as thymol, carvacrol and eugenol are known as a potent antimicrobials and antioxidants (142). Clove essential oil is mainly rich in eugenol whereas oregano and thyme essential oils are rich in thymol, carvacrol and eugenol (124, 126, 143-145). The Trolox equivalent antioxidant activity of the essential oils is in the order of clove > oregano > thyme and is related with the amount and ratio of those compounds in the extract (142).

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Table 2. Effect of dietary supplementation with different natural sources on poultry meat oxidative stability and sensory quality Natural source Herbs Rosemary

5 and 10

12 – 16 weeks

Oregano

5 and 10

1-42 days

Oregano

5 and 30

1-42 days

0.50 and 1.00

1 - 42 days

Oregano

0.10 and 0.20

12 – 16 weeks

Oregano

0.10 and 0.20

12 – 16 weeks

Essential Oils Herbal mix

Dose (g/kg)

Period

Effect1 Turkey raw and cooked meat from animals fed rosemary delayed lipid oxidation. Dose dependent effect (119, 134). Chicken meat from animals fed the oregano showed better oxidative stability than the control though the lower dose is more effective. There was a synergic antioxidative effect with those animals fed 170 mg/kg α-TA supplement and oregano (118). Chicken meat from animals fed the higher dose showed no effect on meat oxidative status but led to meat with more yellow color. However, protein oxidation in drip loss fluid was increased in those animals fed the oregano supplement (86). Refrigerated chicken raw and cooked breast and thigh meat from animals fed 5 g/kg dose showed better oxidative stability than the control but lower than those animals that received 170 mg/kg α-TA supplement. There was a synergic antioxidative effect with those animals fed 170 mg/kg α-TA and oregano (114).

Raw and cooked thigh and breast meat from chickens fed the EO mix delayed lipid oxidation in comparison to the control. Dose dependent effect but always lower than those animals fed 200 mg/kg of α-TA supplement (152). Raw and cooked breast and thigh meat from turkeys fed oregano EO stored under different conditions showed better oxidative stability. Dose dependent effect. The higher EO dose is equivalent to 200 mg/kg of α-TA supplement. There was a synergic antioxidative effect with those animals fed 200 mg/kg α-TA and oregano EO (154, 155, 157). Refrigerated cooked breast and thigh patties from turkey fed oregano EO showed better oxidative stability and similar to those animals fed 200 mg/kg of α-TA supplement (158).

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Table 2. Continued Natural source Oregano

Dose (g/kg)

Period

0.05 and 0.10

1-38 days

Oregano

0.10 and 0.30

1 – 42 days

Rosemary

0.10 and 0.30

1 – 42 days

Extracts Rosemary

0.5

21-42 days

Rosemary

0.5

2-6 weeks

Sage

0.5

2-6 weeks

Effect1 Raw and cooked breast and thigh meat from chickens fed oregano EO stored under different conditions were less susceptible to oxidize. Dose dependent effect. However, those animals fed 200 mg/kg of α-TA supplement showed even lesser susceptibility (151, 153, 156). Chicken breast raw meat from animals fed oregano EO showed better oxidative stability than the control. Dose dependent effect in breast but equivalent in thigh. The higher EO dose is equivalent to 200 mg/kg of α-TA supplement. The combination with rosemary EO was synergistic. Dietary EO spoils meat taste and odor except the combination of oregano and rosemary both at 75 mg/kg (150). Chicken breast raw meat from animals fed rosemary EO showed better oxidative stability than the control. Both doses caused similar oxidative stability and higher than those animals fed 200 mg/kg of α-TA supplement. The combination with oregano EO was synergistic. Dietary EO spoils meat taste and odor except the combination of oregano and rosemary both at 75 mg/kg (150). Chicken breast and thigh raw meat from animals fed rosemary extracts showed a similar oxidative stability to those meats from animals fed 40 mg/kg of α-TA supplement and both higher than the control. Meat from animals fed rosemary very recognized in a triangle test and preferred (291). Either frozen or refrigerated breast or thigh raw meat from animals fed rosemary extract were less prone to lipid oxidation than the control but less effective than those that received 200 mg/kg of α-TA supplement (169). Either frozen or refrigerated breast or thigh raw meat from animals fed sage extract were less prone to lipid oxidation than the control but less effective than those that received 200 mg/kg of α-TA supplement (169).

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Natural source Sage

Period

Effect1

0.56

22-42 days

Coneflower

0.56

22-42 days

Thyme

0.56

22-42 days

Marian thistle

0.04 and 0.08

1-60 days

Tomato powder

25 and 50

10-42 days

Phaffia yeast

20

35-56 days

Dried tomato pulp

50 and 100

21-24 days

Green tea

5, 10 and 20

1 - 42 days

Grape pomace

5, 15 and 30

1-21 days

Chicken breast raw meat from animals fed sage extract showed the same oxidative stability than the control and also no changes in the sensory properties (172). Chicken breast raw meat from animals fed coneflower extract showed the same oxidative stability than the control and also no changes in the sensory properties (172). Chicken breast raw meat from animals fed thyme extract showed the same oxidative stability than the control and also no changes in the sensory properties (172). Chicken breast and thigh raw meat from animals fed the plant extract were less susceptible to oxidize. Both doses offered similar protection (170). Japanese quail breast and thigh raw meat from animals fed the supplement showed better oxidative stability when they are reared under normal and heat stress conditions (208). Chickens fed yeast rich in astaxanthin led to breast and thigh meat with increased red color (197). Japanese quail breast raw meat with skin was darker as the dose of the dietary supplement increases whereas in thigh meat was only darker at the higher dose. No differences in color when meat was without skin (196). Japanese quail raw and cooked breast meat from animals fed the 5% dose showed better oxidative stability than the control whereas the 10% dose was prooxidative or showed no effect on iron-induced lipid oxidation (209). Chicken mixed breast and thigh raw meat from animals fed the by-product showed a better oxidative stability than the control although there were no differences between doses (173). Chicken breast and thigh raw meat from animals fed the by-product showed lesser susceptibility to oxidation although higher than those animals fed 200 mg/kg of α-TA supplement. Dose dependent effect (174).

Dose (g/kg)

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Table 2. Continued Natural source Grape pomace

1

Dose (g/kg) 15, 30 and 60

Effect1

Period 21-42 days

Chicken breast raw meat from animals fed the by-product showed lesser susceptibility to oxidation than the control after 7 days of storage. There were not differences between treatments and neither with those animals fed 200 mg/kg of α-TA supplement (292).

EO means essential oil whereas TA means tocopheryl acetate

Figure 4. Effect of refrigerated storage on lipid oxidation of turkey thigh meat as a function of dietary supplementation with 100 and 200 mg oregano essential oil kg-1 (OR100 and OR200, respectively), 200 mg α-tocopheryl acetate kg-1 (VE200), and 100 mg oregano essential oil plus 100 mg α-tocopheryl acetate kg-1 feed (ORVE100) (154).

The presence of these active phytochemicals in the essential oils of oregano and other plants and herbs essential oils explain why their use as dietary supplements (0.1 - 0.3 g/kg) results in inhibition of lipid oxidation in poultry meat (146-158) (Table 2). However, similarly to what happens with the dietary addition of plants and herbs, the antioxidative effects recorded in meat from animals fed these essential oils are usually lower than those meats from animals fed about 200 mg of α-tocopheryl acetate/kg feed (Figure 4). These results seem to be in agreement with the in vitro lower antioxidant activity of these compounds in comparison with α-tocopherol (159, 160). It should also be noted that utilization of dietary essential oil to improve oxidative stability should be done carefully because high doses of these essential oils may led to meat with altered taste and odor (150).

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Several authors reported that the oregano and rosemary oil supplementation can increase the retention/protection of α-tocopherol in meat thus explaining the synergistic effect found when α-tocopheryl acetate and essential oils are added together (150, 155, 157). This synergistic antioxidative effect has also been reported in meat after adding plants and herbs into poultry feeds (114, 118). In oregano essential oil, around 80% of the total phenols are constituted by carvacrol and thymol (124), both of which have strong antioxidant activity (142). These latter compounds and other such as rosmarinic acid and other monoterpenes also present in the essential oil may be all involved in protecting meat against oxidation. The efficient distribution of these compounds or their active metabolites into the tissues could explain why the dietary supplementation with essential oils is more efficient than the addition of the essential oil into the muscle post-mortem (158). However, further research is required to elucidate the protective and synergistic effects of these minor compounds in vivo and postmortem since some compounds such as the phenyl terpenoids are usually rapidly excreted and not retained in the muscle (161-164). The phenol-based antioxidant compounds present in the essential oils can also have antibacterial and antifungal activity suggesting they could be used as an antibiotic replacement. However, when these essential oils are added into the diet, poultry performance of poultry is mainly unaffected (150-152, 157) although some positive effects have been reported (135). Similar to what happens when plants and herbs are added into the diet; the positive effects are likely masked when animals are reared under healthy environmental conditions. On the other hand, herbs but not essential oils contain a variety of glycosidically bound volatile and non-volatile constituents that exhibit biological activity after enzymatic or acid hydrolysis (144). These compounds could explain why the positive effects on animal performance are more commonly reported when plants are added into the diet rather than essential oils (118, 150). An alternative to essential oils to concentrate bioactive compounds for addition into poultry diets are extracts from plants and other natural sources. These extracts originate from a process in which solvents such as water, methanol, ethanol or their mixtures are used to produce plant extracts rich in bioactive compounds. After extraction the total mass of liquid is concentrated by evaporation or physical separation of the unneeded solvent with the desired thick oily matter rich in phytochemicals (165-168). Some of the compounds found in the extracts are the same as in essential oils although more lipophilic compounds can be isolated depending on the nature of the solvent used (168). Therefore, plant extracts can have different content of phenol-based molecules compared to essential oils and can also be relatively rich in pigments. Because plant extracts contain almost the same active compounds as plant essential oils their effectiveness as dietary supplements is similar. As shown in Table 2, dietary supplementation of natural extracts at doses around 0.5 g/kg feed resulted in inhibition of lipid oxidation (169, 170). Dietary plant extracts of rosemary, grape, citrus or marigold efficiently inhibited lipid oxidation in plasma of rats fed n-3 PUFA rich diet thus suggesting that the phytochemicals in extracts can be absorbed and further prevent from oxidation (171). However, some authors reported no significant effects of the dietary extracts on the oxidative stability of poultry meat (172-174) (Table 2). Therefore, the effectiveness of each source and type of product has to be tested because not all types of extracts showed the same effects on meat oxidative stability (173, 175).

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The composition of the extract and the feed nutrient bioavailability may produce controversial results in animal performance. For instance, the addition of chestnut wood extracts into poultry diets improved animal performance (176). This may be explained because these extracts are rich in tannins which have been reported as useful antimicrobial phytochemicals (177). However, the presence of tannins in the natural source extracts may also affect the digestibility and absorption of other compounds in the diet such as iron (16, 17, 178, 179) thus likely explaining why the addition of some extracts such as dietary grape pomace extracts (180) and Marian thistle fruits extracts (170) resulted in decreased animal performance and slaughtering yields.

O

Figure 5. Basic structure of flavonoids.

Other reports suggest that the addition of an extract into poultry diets has no deleterious effects on animal performance as reported for a green tea source (173) or the addition of an extract made from sage, thyme, and rosemary (181).

4.2. Addition of Polyphenols The majority of studies dealing with the effect of the dietary supplementation of a single phenolic compound on poultry meat are made with flavonoids (Table 5). Flavonoids are a class of polyphenol with a similar chemical structure (Figure 5) which exert antioxidant capacity in vitro (182-185). The activity of polyphenols depends on their respective intakes, bioavailability and body tissue retention, which can vary greatly but, in general, is known to be low (16, 18, 121, 186-188). Accordingly to these latter authors, isoflavones are the most well absorbed polyphenols whereas the least are proanthocyanidins and anthocyanins. For instance once absorbed, quercetin and some polyphenol metabolites may have longer halflives and accumulate in tissues after repeated ingestion (16, 186, 187). This could explain why the in vitro antioxidant activity of polyphenols is not necessarily correlated with in vivo antioxidant capacity (16, 171). While little is known about the physiological role of polyphenols, research suggests that the low concentrations into the tissues seem to be sufficient to exert several biological effects as well as potent antioxidant action (16, 171). In poultry, the dietary supplementation with polyphenols confirms that these latter compounds exert antioxidant effects on post-mortem skeletal muscle (146, 147, 189). Therefore, this antioxidant activity implies that the flavonoids or derived active compounds have some effects in vivo. Plant polyphenols are multifunctional antioxidants as they can act as reducing agents, hydrogen donating antioxidants, singlet oxygen quenchers and chelating

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agents (16, 190). The likely mechanisms that can be involved are first, reduction of free radical formation, second, protecting α-tocopherol from oxidation, third, regenerating the oxidized α-tocopherol and finally chelating metal ions. Further studies in determining the absorption, content and distribution of these phenols and metabolites in different tissues are necessary to better understand those antioxidant effects.

4.3. Addition of Carotenoids Depending on the nature of isolation some pigments such as carotenoids can be present in plant extracts. Carotenoids are lipid soluble pigments that include carotenes which contain carbon and hydrogen only and xantyhopylls which also contain oxygen. Table 4 shows carotenoid classifications. Table 4. Classification of carotenoids and their typical occurrence Carotenoid Carotenes Lycopene β-carotene α-carotene

Xanthophylls β-Cryptoxanthin Lutein Zeaxanthin Astaxanthin Canthaxanthin

Sources Tomatoes, watermelon, papaya, guava (182, 297, 298) Carrots, sweet potato, tomatoes, paprika, apricot, cantaloupe, mangoes, green vegetables (182, 297, 298) Tomatoes, carrots, pumpkin, corn, yellow pepper, green vegetables (182, 297, 298)

Mango, papaya, peaches, paprika, oranges (182, 297, 298) Banana, satsuma peel, egg yolk, green vegetables (182, 297) Paprika, satsuma peel (182) Salmon, crab (182) Carrots, kale, red peppers (182)

Table 5. Effect of dietary supplementation with flavonoids and carotenoids on poultry meat oxidative stability and sensory quality Pure compound

Dose (mg/kg)

Period

Flavonoid glycitein

10 to 80

21-42 days

tea catechins mix

50 to 300

1-42 days

Effect1 Breast raw meat from chickens fed glycitein showed decreased susceptibility to oxidation and increased superoxide dismutase and catalase activity. The addition of 20 or 40 mg/kg increased the pH of the meat whereas 40 or 80 mg/kg increased the lightness of meat color (189). Breast and thigh raw meat from chickens fed above 100 mg/kg showed improved oxidative stability. Thigh meat from animals fed 300 mg/kg showed better oxidative stability than those animals fed 200 mg/kg of α-TA. The supplementation at 50 mg/kg was found to be prooxidative in breast meat (146).

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Table 5. Continued Pure compound

Dose (mg/kg)

Period

Effect1

tea catechins mix

50 to 300

1-42 days

Frozen breast and thigh raw chicken meat from animals fed tea catechins showed better oxidative stability. After 9 months of frozen storage the supplementation of 300 mg/kg is equivalent to those meats from animals fed α-TA supplement at 200 mg/kg (147).

Carotenoid β-carotene

25

4-6 weeks

β-carotene

100

1-37 days

β-carotene

5.1 and 10.2

11-42 days

β-carotene

15

8-50 days

β-carotene

15

8-12 weeks

β-carotene

15 and 50

1-6 weeks

β-carotene

1.5

not specified

zeaxanthin

100

1-37 days

canthaxanthin

100

1-37 days

Raw and cooked thigh meat from chickens fed the supplement recorded higher TBARS values than the control and lower α-tocopherol content but no differences were found in meat smell and flavor (112). Chicken breast from animals fed this supplement showed neither a different content in this compound nor a reduced susceptibility to oxidation although the supplementation with 100 mg/kg of α-TA did it (201). Frozen and refrigerated breast and thigh raw chicken meat from animals fed this supplement showed no differences on susceptibility to oxidation. This compound was not detected in meat (205). Raw and cooked thigh meat from animals fed the supplement showed no differences on cholesterol oxidation products whereas the supplement with 200 mg/kg of α-TA better cholesterol oxidative stability (202). Turkey breast and thigh raw meat from animals fed this supplement did not present a synergistic effect on oxidative stability and sensory properties when this supplement is in combination with the addition of 50 mg/kg of αTA (299). Raw and cooked thigh chicken meat from animals fed 15 mg/kg of this supplement showed antioxidant activity in fresh and cooked meat whereas at 50 mg/kg showed prooxidant activity after 7 days of storage at 4 C (203). Chicken breast raw meat from animals this supplement did not improve meat iron-induced oxidative stability whereas 100 mg/kg α-TA did. Both supplements and their combination modified meat texture (204). Chicken breast from animals fed this supplement showed increased total carotenoid content and showed no effect on the susceptibility to oxidation although the supplementation with 100 mg/kg of α-TA did it (201). Chicken breast from animals fed this supplement showed increased total carotenoid content and showed no effect on the susceptibility to oxidation although the supplementation with 100 mg/kg of α-TA did it (201).

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Pure compound

Dose (mg/kg)

Period

canthaxanthin

2

11-42 days

astaxanthin

22.5

3-8 weeks

lutein

20

22-42 days

lutein and cantaxanthin mix

40

1-42 days

Effect1 Frozen and refrigerated breast and thigh raw chicken meat from animals fed this supplement showed no differences on susceptibility to oxidation. This compound was not detected in meat (205). Chicken breast raw meat from animals fed this carotenoid showed different color. The stability of the skin to oxidize was higher in meat coming from this dietary supplement (210). Chicken breast raw meat from animals fed marigold xantophylls (about 90% lutein) showed the same oxidative stability than the control and also no changes in the sensory properties. Synthetic yellow xantophylls led to meat with lower flavor and taste than the control (172). Chicken breast skin and abdominal fat from animals fed the mixtures deposited lutein and zeaxanthin thus influencing yellowness breast skin color (193).

1 TA means tocopheryl acetate whereas TBARS means thiobarbituric acid reactive substances

As several plants, herbs, yeasts as well as their extracts contain pigments. These natural sources have been used for altering the color of foods. Consumers, through a repeated dietary experience, evoke an anticipated set of sensations associated with flavors and odors in relation to the color of food. Therefore, animal breeders use these natural sources of pigments to modify the color of meat with skin to achieve more pigmented poultry meats as indication for the consumers of a maize-based diet and bird’s general health (191, 192). When oregano is added into the diet at high doses, the presence of lutein and β-carotene causes an increased yellowish color (86). Similarly, marigold extracts, rich in lutein, are often been used to modify poultry meat and egg yolk pigmentation (193-195). Tomato derived products are rich in lycopene and they have been reported to improve the skin pigmentation of meat (196). Other natural products such as alfalfa concentrate and Phaffia yeast, rich in the red carotenoid astaxanthin, or algae, rich in the yellowish carotenoid zeaxanthin, led to meat plus skin with improved color appreciated by consumers (197, 198). Carotenoids can exert an antioxidant effect because of their radical scavenging capacity but especially because of their singlet oxygen quenching activity (199, 200). Therefore, carotenes could be used in animal diets not only to improve meat sensory traits but also meat oxidative stability. In relation to this, various carotenoids have been added into poultry diets to study meat oxidative stability and sensory quality (Table 5). The addition of β-carotene in poultry diets has been reported to have a protective effect on the onset of lipid oxidation in liver (201) but in meat showed no effect (201-205) or even had a prooxidative effect (112). Measuring the susceptibility to oxidation through an iron-induced TBARS method, Ruiz et al. (203) reported that dietary supplementation with β-carotene acts as an antioxidant in fresh and recently cooked meat whereas it acted as a prooxidant when studied in meat that has been stored meat at 4 C during 7 days. This prooxidative effect is likely due to the decreased αtocopherol deposition as a result of high dietary carotene supplementation.

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Dietary supplementation with other carotenoids such as lycopene showed antioxidant effects in Japanese quail serum (206) by decreasing depletion of antioxidants and ameliorating the antioxidant status caused by heat stress (207). In addition, tomato-based products added into the animal diet have been reported to improve poultry meat oxidative stability when animals are reared under heat stress conditions (208) although, at high dietary doses, tomato-based products have been reported to have some prooxidant activity (209). Xanthophylls such as astaxanthin showed antioxidant activity in poultry skin (210). Conversely, lutein, extracted from marigold, showed some prooxidant activity in chicken breast (172). However, zeaxanthin, which is very similar to lutein, and canthaxanthin did not inhibit fatty acid oxidation in chicken meat (201, 205). In addition, the synthetic xanthophyll β-apo-8-carotenoic acid ethyl ester had no antioxidant effect (172). In general, carotenoids are believed to be distributed in the membranes where they can scavenge singlet oxygen thus exerting their protective effects (211). However, this protective mechanism is believed to be of minor importance in meat products because, in comparison to free radical attack, oxidation caused by singlet oxygen is usually a less important phenomenon. Therefore, this would explain why the antioxidant effect is sometimes not detected.

5. Mineral Supplementation Poultry nutritional deficiencies are avoided through dietary mineral supplements. However, few studies have been addressed to study the effect of dietary supplementation of different elements on nutritional value, oxidative stability and sensory quality of poultry meat. In general, when elements supplied in the diet are imbalanced or below the recommended levels, this lowered animal welfare (90, 212). At levels below toxicity, the addition to the diet of certain elements at high doses may cause nutrient interactions such as high iron loads leading to a lower Zn absorption or high Zn loads decreasing copper absorption (213, 214). This review is going to focus on the elements that can impact the antioxidant and prooxidant balance in meat. Iron is known to promote lipid oxidation, however; muscle iron levels seem to be relative insensitive to dietary modification (215-217). Despite that, several authors studied the effect of removing additional iron from a non-deficient diet on lipid oxidation. The removal of this supplemental iron resulted in an increased oxidative stability of raw and cooked poultry meat (92, 216, 218) and also has been reported to decrease copper content of the muscle (218). However, other authors recorded an increased susceptibility to oxidation in cooked meat when dietary iron was decreased (217). This latter effect could be explained because, apart from catalyzing lipid oxidation, iron and copper are also part of the antioxidant enzyme system. The enzyme catalase contains a heme iron group whereas the SOD present in the cytoplasm contains copper and zinc. As for the iron supplemented at a higher dose than usual, the additional load seems to have no effect on the oxidative stability of turkey meat (92). Even though copper is known to promote lipid oxidation, it is used as a dietary supplement because at high doses it has been reported to have antimicrobial and growth promoting effects (219-221). Skeletal muscle copper levels are relatively insensitive to dietary intake (215) but its dietary removal from non-deficient diets has been reported to cause a decrease in the meat copper content (218). Upon copper supplementation, the copper concentration of chicken thigh slightly increases (221). In addition, the supplement has been

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reported to reduce the ratio in saturated fatty acids in abdominal fat (220) and decrease the cholesterol content in eggs, plasma and chicken muscle (221-227). The relationship between copper and lipid metabolism can also be seen in the ability of copper deficiencies to cause hypercholesterolemia in rats (228, 229). The enzyme GSH-Px contains selenium and takes part in the antioxidant defense system. The addition of this element to the diet has to be done carefully because of its toxicity (230). Dietary supplementation of this element at doses higher than the nutritional requirements has been studied. Early, de Vore et al. (231) reported that the selenium supplementation in poultry diets lead to an increased activity of the GSH-Px which could be due to an increased content in selenoproteins. Moreover, several authors reported that animals fed a selenium deficient diet had a lower GSH-Px activity and higher susceptibility to oxidation in different tissues (115, 232-234). Selenium can be absorbed in both organic and inorganic forms (235, 236). The organic form selenomethionine follows the same pathways as methionine and is incorporated into body tissues in a non-specific and unregulated manner. Inorganic and organic selenium forms can be transformed into selenocysteine, the biologically active form of Se that forms part of the GSH-Px and other selenoproteins (237, 238). Therefore, the intake in selenite and other organic and inorganic forms of selenium other than selenomethionine are incorporated into the body pool for general use and appear to be under homeostatic regulation (236, 239). Therefore, inorganic and organic selenium forms such as selenomethionine can restore the activity of the GSH-Px (235, 240). However, selenomethionine can be retained in tissues and tissue proteins into a much greater extent than other organic and inorganic Se forms (239, 241). Selenium enriched products by dietary strategies is of interest especially in areas with selenium deficient soils because this in turn causes a low human and animal dietary intake of this element (242, 243). Careful supplementation can improve animal health and can be a safe way to increase selenium intake for humans in depleted areas. Use of inorganic selenium to supplement animals has led to controversial results in meat selenium enrichment. Some authors found that sodium selenite (1.2 mg Se /kg feed) did not increase selenium content in chicken mixed dark and white meat with their skin (20) whereas others (1 mg Se /kg feed as sodium selenite) found a slight increase in selenium content in breast muscle (244). Conversely to meat, other tissues like liver, kidney, spleen are easily enriched after supplementation with inorganic selenium (244-246). Selenium supplementation with organic forms is normally done with Se-enriched yeast or algae which are rich in selenomethionine. Dietary supplementation with selenomethionine (at doses of 0.2 mg Se/kg feed) led to meat with increased selenium content (Figure 3) though this seems to have had no effect on oxidative stability (20, 243, 244, 247). Conversely to these latter results, goose receiving selenium supplementation recorded an increased susceptibility to oxidation (248). Overall, supplementation with organic Se is preferred to other selenium forms because it is not only associated with an increased meat selenium concentrations but also results in higher water holding capacity of muscle, increased animal performance and better reproductive characteristics of poultry (241, 249, 250). In addition, a high selenium intake has been related with increased concentrations in EPA and DHA after dietary addition of vegetable fat sources (242). Zinc is another element that forms part of the antioxidant enzyme system as a cofactor for SOD (251). Dietary zinc and other cations induce the synthesis of metallothionein (252, 253).

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This protein is related with the Zn uptake from the diet and at the same time allows for homeostatic Zn control (252). This protein is also related with other functions such as metal storage, protection against heavy metals toxicity and scavenging free radicals (252-254). The zinc content in meat is mainly genetically determined and seems to be only slightly affected by feed composition (255). The addition of zinc into the poultry diet at doses of 20 g/kg feed reduces feed intake and body weight (256, 257). Furthermore, the addition of zinc from 0.2 to 20 g/kg chicken feeds during short periods of time (less than 3 weeks) caused the increase of this element in liver, kidney, pancreas and spleen (256-260). Zinc content in the skeletal muscle can be increased in young chicks (≤ 3 weeks of age) by dietary supplementation but when the animal gets older a poor linear regression between dietary zinc supplementation and muscle zinc content was observed (259) and at the end of the broiler growing period of broilers (42 days old) the supplementation has no effect on zinc content in meat with skin (20, 53). In addition, the supplementation with zinc can lead to some other positive effects because it can improve animal performance and alleviate lipid oxidation in animals reared under heat stress (261, 262). Finally, Zn supplementation has been reported to increase the selenium content in meat while not affecting oxidative stability (20).

6. Miscellaneous Additions Meat contains low amounts of the fat-soluble vitamins A, D and K in comparison to liver which regulates the metabolism of these compounds (215). Thus, the concentration of vitamin A, D and K in meat is difficult to modify through dietary treatments. Accordingly to this, the supplementation with retinol acetate led to increased retinol content in chicken breast and thigh though it is only significant in breast at the highest dose (16800 IU) (205). Moreover, the supplementation with the provitamin β-carotene was also not very efficient in increasing meat retinol content and, in addition, both retinol and β-carotene supplements showed no differences on susceptibility to oxidation in frozen and refrigerated breast and thigh raw chicken meat (205). As vitamin D synthesis is light induced, little attention has been addressed to its dietary manipulation though this vitamin as well as calcium, phosphorus and vitamin K are important to ensure a correct bone mineralization in fast growing broiler chickens (263-265). In general, water soluble vitamin concentrations are low in muscle meat and any excess is rapidly excreted (215). However, in 21-day-old chickens, folate has been increased in breast and thigh meat from animals fed the supplement throughout life but this increase was not significant after 42 days of age (266). Despite that, the supplementation with folate can attenuate the decline in performance and antioxidant status caused by heat stress in Japanese quail (267). Coenzyme Q, also known as ubiquinone, is a lipid-soluble phenolic compound that can be found in skeletal muscle and is capable of acting as an antioxidant (108, 268, 269). Dietary ubiquinone has been reported to increase ubiquinone concentrations in the plasma and liver of rats but not in kidney, heart, brain and skeletal muscle (270) thus suggesting that their supplementation would not be effective at increasing the oxidative stability of meat if included in animal feeds. Another phenolic compound that has been reported to protect against oxidative stress is the water-soluble pyrroloquinoline quinine, which is widely distributed in microorganisms, plants and animals, and found to be important in animal

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nutrition (271, 272). However, the effectiveness of its dietary supplementation on the oxidative stability has not been demonstrated. L-carnitine (β-hydroxy-γ-trimethylaminobutyrate) is a water-soluble quaternary amine that occurs naturally in microorganisms, plants and animals (273) and reported to have antioxidant activity in vitro (274). This could explain why quails supplemented with carnitine showed decreased lipid oxidation in the muscle (275). Supplementation with carnitine also seems to redistribute lipids by increasing intramuscular fat, decreasing subcutaneous fat deposits and by lowering plasma cholesterol and triglyceride concentrations (276). Reduced thiols such as cysteine, glutathione and lipoic acid, and several nitrogen based compounds found in skeletal muscle such as uric acid, polyamines, amino acids, peptides and proteins can inactivate free radicals (108). The antioxidant activity of these compounds is due to their capability of donating hydrogen to free radicals and to the reaction of amine or sulphur groups present with lipid hydroperoxides leading to the formation of imines, sulphides, thiosulphinates and sulphoxides, respectively (277). However, a thiols reducing potential can also promote metal-catalyzed oxidation by causing iron reduction (108). In general, little is known about the antioxidant role of these compounds in meat or the ability to change their concentrations by dietary manipulation though their supplementation seems to be ineffective since they are efficiently regulated, rapidly metabolized or hydrolyzed in the gastrointestinal tract by proteases (108). Despite that, it has recently been reported that free leucine and free glutamate content in chicken muscles were significantly increased by dietary leucine (278). Free glutamate is a major flavor component of meat explaining why chicken soup made from animals fed supplemental leucine showed better sensory scores for overall preference, umami taste and chicken-like taste compared to the control (278). Skeletal muscle is rich in histidine-containing dipeptides including carnosine (β-alanineL-histidine) and anserine (β-alanine-1-methyl-histidine) (87). These dipeptides have been postulated to protect against oxidation through metal chelation and/or free radical scavenging (87, 272, 279). Their efficiency as antioxidants has been demonstrated after post-mortem addition of carnosine to chicken meat (280, 281). In chicken muscle the content in anserine is higher than carnosine (272). The content of this later compound in the skeletal muscle can be affected by either deficiency or high dietary loads of histidine (272) but high dietary carnosine levels are required to significantly increase carnosine concentrations in muscle (87). Recently has been reported that the dietary supplementation with β-alanine resulted in increased content in anserine and carnosine in brain but is ineffective in increasing the anserine and carnosine concentrations broiler muscle (282).

7. Conclusions Meat fatty acid composition reflects, into some extent, the fatty acid composition of the diet. Therefore, the addition of very long chain n-3 PUFA into poultry feeds lead to meat with an increased nutritional value. However, this enrichment in polyunsaturated fatty acids may cause detrimental effects in meat quality such as an increased susceptibility to oxidization. The most efficient way to protect meat against oxidation is through the dietary supplementation with α-tocopheryl acetate. This supplementation not only protects meat against lipid oxidation but also leads to meat richer in tocopherol. Ascorbic acid has also been

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described to act as an antioxidant in vivo though its addition into poultry diets is, overall, considered ineffective. The addition of plants and their essential oils and extracts can be a useful approach to reduce lipid oxidation likely thanks to the presence of phenyl terpenoids and polyphenols in these natural sources. These compounds seem to be present in the tissues at very low concentrations though they are able to exert a potent antioxidative activity in vivo. In addition, this antioxidative effect can be synergistic with the addition of tocopherol. Further research is needed to fully exploit these antioxidants in animal diets and understand the antioxidative mechanisms involved in vivo and in post-mortem tissue. Moreover, natural sources may contain carotenoids thus modifying the color of poultry meat with skin and, in consequence, improve the sensory characteristics of the product. Overall, carotenoids and other several pure compounds such as ubiquinone, L-carnitine, peptides, amino acids and various thiol compounds which have antioxidant activity in vitro fail to improve the oxidative status of meats when they are added as feed ingredients. Their absorption, metabolism and mechanism of protection against oxidation explain this lack of efficiency. In addition, pure compounds are, in general, too expensive to be used as feed additives in comparison to plants, essential oils and other natural extracts. To achieve poultry meat rich in minerals is quite limited to selenium. This meat enrichment can be obtained through dietary supplementation with selenomethionine since this compound seems to follow the same metabolic pathway as methionine. In addition, the supplementation with this organic source of selenium results in meat with higher water holding capacity.

Acknowledgements The authors thank Cristina Andres-Lacueva for the revision of the manuscript.

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In: Food Science and Security Editors: L. Amsel and L. Hirsch, pp. 231-251

ISBN: 978-1-60692-977-3 © 2009 Nova Science Publishers, Inc.

Chapter 9

LIVESTOCK AND FOOD SECURITY WITH SPECIAL REFERENCE TO THE DEVELOPING COUNTRIES R. Trevor Wilson1 Independent International Consultant, Bartridge Partners, Bartridge House, Umberleigh, Devon EX37 9AS, UK.

Abstract The keeping of livestock is the only source of livelihood for at least 20 million pastoral families across the world. Livestock are also an important – indeed often the main -- source of income for a minimum of 200 million smallholder families in Asia, Africa and Latin America. Domestic animals provide power for the cultivation of more than 300 million hectares or some 25 per cent of the world’s total cropped area. This land would otherwise have to be cultivated by hand (with the concomitant drudgery, especially for women) or by tractor power with the inevitable consequences of depletion of fossil fuels and loss of valuable foreign exchange for developing countries. Ownership of livestock provides one of the very few opportunities for the landless to engage in agricultural activities. Livestock are often the only asset for many people who do not own the land they farm and that they can take with them when their leases expire. The single cow, the goat, a few scavenging poultry or a donkey are the mainstay of survival for many of the really poor. It is becoming more generally accepted that the smaller the farming enterprise the greater the importance of the contribution of livestock to its viability and to its sustainable production. Solving the challenge of global food security is not possible without a focus on animals. The contribution of domestic animals to sustainable development could be greatly enhanced not only by technical innovation but by provision of an appropriate enabling environment and recognition in policy formulation of the dramatic changes that have recently transformed the developing livestock sector. With these developments and these provisos livestock will continue to feed future generations with the type and quality of food they need without overuse of other natural resources. This paper provides a largely qualitative overview of the role of domestic livestock in assuring food security together with a series of detailed quantitative case studies.

1

E-mail address: [email protected].

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Introduction The 1972 Stockholm Conference introduced environmental concerns to the formal political development sphere [UNEP, 1972]. It was, however, the Brundtland report -commonly known as Our Common Future -- that placed environmental issues firmly on the political agenda. This report started discussions on the environment and development as a single issue [WCED, 1987]. In the years of ensuing debate livestock were widely seen as the main culprit in environmental degradation. The role of domestic animals in food security was little understood and was largely ignored. This applies in particular to the role of livestock in the “critical triangle” of development -- food security, poverty alleviation and environmental protection [Vosti, 1995]. Food production in many developing countries is insufficient for the needs of their people. This situation will be further exacerbated by rapidly expanding human populations. Food production cannot be allowed to stagnate but nor can it be increased at the expense of the environment or the natural resource base [Nel, 1998]. There is a clear need for increases in agricultural output in order to balance the adverse effects of intensive production and the exploitation of the resource base. Livestock contribute to value-added production through partial recycling of nutrients and as a source of energy, although not all of the energy supplied is free to the environment and to all the poor people of the world [Wilson, 2003]. This does not mean, however, that the unique complimentarity of livestock within many farming systems should not be fully exploited. It should also be understood from the foregoing that livestock’s contribution to human welfare is not only in the direct provision of food. Livestock also generate income, enable more crops to be produced, alleviate the drudgery of much of peasant agriculture in the developing world and contribute to gender equality.

General View There is an age-old relationship between people and animals. A major reason for domestication, beginning some 12 000 years ago, was to help overcome the problems of a fluctuating and weather-influenced food supply if subsistence depended on crops alone. Recognition of the role of animals in food production and food security is evident in that many ancient peoples incorporated them into their pantheons of gods. On both short and long term bases milk and eggs are virtually the only agricultural products that can be harvested every day and year in and year out. Lack of food security is a major dimension of poverty. Food poverty is now a common expression in the lexicon of development. Food poverty is an inability to afford the amount of food necessary to provide minimum energy and protein requirements. Domestic animals and their products contribute to both the quantity and quality of food required to ensure food security. The rearing of livestock is the sole source of livelihood for at least 20 million pastoral families. It is also an important – and often the main -- source of income for a minimum of 200 million smallholder farmer families in Asia, Africa and Latin America [de Haan et al, 1996]. Domestic animals provide power for the cultivation of more than 300 million hectares or some 25 per cent of the world’s total cropped area. This land would otherwise have to be cultivated by hand (with the concomitant drudgery, especially for women) or by tractor power with the inevitable consequences of depletion of fossil fuels and

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a loss of valuable foreign exchange for developing countries. Livestock return some of the plant nutrients they have previously removed from large areas of crop land. It has been estimated that manure provides nutrients valued at US$ 800 million per year to tropical irrigated areas [Jansen and de Wit, 1996]. Livestock ownership provides one of few opportunities for the landless to engage in agriculture. Animals are often the only asset of people who do not own the land they farm and that they can take with them when their leases expire. Many who have livestock do not, however, own them. On the other hand many who have nothing else have livestock. The single cow, the she goat, a few poultry or a donkey are the mainstay of survival for many really poor. Livestock are an asset for investment and insurance for hundreds of millions of rural poor living remote from banks and where banking systems are unreliable in safeguarding the meagre savings that might be accumulated. It is now accepted that the smaller the farming enterprise the greater the importance of the contribution of livestock to its viability and to its sustainable production. Domestic animals make major contributions to food security and income generation for people in the developing world. They contribute up to 20 per cent of agricultural Gross Domestic Product (GDP) in Southeast Asia [Devendra and Thomas, 2002]. In seven countries in northeast Africa the contribution of livestock to agricultural GDP in the 1990s was between 13 per cent and 30 per cent where agriculture’s contribution to total GDP was 42 per cent to 59 per cent [Wilson et al, 1999]. In Somalia the export of livestock and hides and skins was valued at 49 per cent of total exports and in Ethiopia hides and skins exports were second only to coffee in value [Wilson et al, 1999]. The contribution of livestock to the often difficult-to-obtain hard currency for these African countries can add to food security at the national level. Integrated animal-crop enterprises are important to many farmers. Livestock convert plant materials inedible to or of low nutritive value for humans to high quality protein products. In crop-livestock interactions ruminants make good use of low quality crop residues which their rumen flora and fauna can degrade in the process of converting them to food for humans. Were this not the case these waste products would mostly be burned and thus add to environmental pollution. Large ruminants and equines provide draught power, are used for transport and return some nutrients to the soil through their manure. Synergistic interaction between livestock and crops improves the possibilities of sustainable production and maintains or improves soil fertility. In general farmers understand the importance of having animals as the entry point to improve farm productivity. In many cases, however, limited resources (including land area and lack of capital) prevent the development of fully integrated and functional mixed farming systems. The contribution of livestock to food security is not, therefore, only direct. Domestic animals add to food security in other ways [Sancoucy et al, 1995]. These include a source of income, a generator of employment, a supplier of inputs and services to crop agriculture and (when properly managed) a contributor to environmental conservation. Increased livestock production keeps product prices down and therefore allows access to them by the poor. Paradoxically, producers should gain from lower prices as livestock products are both price and income elastic. Lower prices therefore increase demand, raise total production and lead to higher farm incomes. Increased production and low prices may help low income consumers to overcome both energy and protein deficiency simultaneously [Lipton, 1988]. In addition to the foregoing increased domestic production reduces imports and saves foreign exchange.

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This can then be diverted to productive investment and indirectly contribute to food security. Some countries generate revenue by taxing imports but taxing income from domestic production could serve the same purpose [Sancoucy et al, 1995].

Case Studies Livestock in the Holy Bible There is evidence throughout the Old Testament of the Holy Bible, written some 4000 years ago, of the importance attached to livestock in food security. The sheep is the first animal mentioned by name: “And Abel was a keeper of sheep and he also brought of the firstlings of his flock and of the fat thereof. And the Lord had respect unto Abel and his offering” [Genesis Ch 4 V 2 and 4]2. The sheep was amongst the most treasured possessions of the people of the Holy Land and its environs. Sheep meat was widely eaten but its skin and wool were also used for clothing and for making tents. Cattle were of immense importance in all agricultural work in ploughing and in treading out the corn (both functions have, of course been carried through to the present day, Figure 1). Butter, milk and cheese were prized foods as was meat. Livestock were specially fed in pens as well as allowed to forage for themselves, as is shown by “ten fat oxen and twenty oxen out of the pastures, and an hundred sheep, ....., and fatted fowl” [1 Kings Ch 4 V 23] and “Better is a dinner of herbs where love is, than a stalled ox and hatred therewith” [Proverbs Ch 15 V 17]. Goats ranked with cattle and sheep as the chief wealth of the Israelites in their pastoral days and their flesh but not the fat (of which there would in any case be little) was eaten. Goat kids were quickly prepared food items: “Go now to the flock, and fetch from thence two good kids of the goats; and I will make them savoury meat for thy father, such as he loveth” [Genesis Ch 27 V 9]. Goat milk was also a regular household food item: “And thou shalt have goats’ milk enough for thy food, for the food of thy household, and for the maintenance for thy maidens [Proverbs Ch 27 V 27]. All the ruminant species were used in sacrifice to propitiate the Lord. It seems, however, that at times many more were slaughtered than would appear prudent for food security. At the dedication of Solomon’s Temple, for example, 22 000 oxen and 120 000 sheep were sacrificed [1 Kings Ch 8 V 63]. These numbers nonetheless give an indication of the immense numbers of animals that were kept. Individual flocks ran into thousands of animals amongst the elders. Some students of the Bible are of the opinion, that even at this early stage some 4000-5000 years ago, livestock were already causing enormous damage to the local vegetation and were in large part responsible for deforestation. Because of their dependence on their livestock for food and sustenance the Israelites were compelled to wander over vast distances in search of animal feed. When Joseph (of the coat of many colours) was in Egypt he interceded with the Pharaoh on behalf of his father and his brothers: “And the men are shepherds, for their trade hath been to feed cattle; and they have brought their flocks and herds, and all that they have” [Genesis Ch 46 V 32]. Joseph also pleaded that they be allowed to settle:

2

The quotations here are from the Authorized King James Version of 1611 of the Holy Bible

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Figure 1. Oxen threshing barley near Asmara, Eritrea.

“My father and my brethren, [have come] and their flocks, and their herds, and all that they have .... For to sojourn in the land [Goshen] are we come; for thy servants have no pasture for their flocks; for the famine is sore in the land of Canaan [Genesis Ch 47 V 4]. But it was not only in Canaan that there was famine. In the first of the seven lean years the Egyptians asked Joseph for bread and he replied: “Give your cattle; and I will give you for your cattle, if money fail. And they brought their cattle unto Joseph: and Joseph gave them bread in exchange for horses, and for the flocks, and for the cattle of the herds, and for the asses; and he fed them with bread for all their cattle for that year [Genesis Ch 47 V 17-18]. The lives of cattle were more highly valued by the Israelites than those of their human enemies. At the Battle of Jericho the people of Joshua killed all living things. When they fought at Ai, however, the Lord instructed them to “do to Ai and her king as thou didst to Jericho and her king: only the spoil thereof, and the cattle thereof, shall ye take for a prey for yourselves” [Joshuah Ch 8 V 2]. Similarly in their wars against other peoples: “And we utterly destroyed them, as we did unto Sihon king of Heshbon, utterly destroying the men, women, and children, of every city. But all the cattle, and the spoil of the cities, we took for a prey to ourselves” [Deuteronomy Ch 3 V 6-7].

Rinderpest in Africa The “great African cattle plague” (rinderpest) probably arrived in subSaharan Africa between 1887 and 1889 by cattle brought into Eritrea from India by the Italian Army [Mack, 1970]. Caused by a virus that does not affect man – although it is similar to those of human measles and canine distemper and may have been their precursor [Schwabe, 1984] – the disease had caused havoc in Europe since ancient times. Between the 9th and 19th centuries rinderpest devastated European cattle populations every 40-50 years. Human deaths from

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starvation as a result of these cyclical epidemics cannot, of course, be calculated with any accuracy but were certainly very many millions. Rinderpest was largely responsible for the creation, by public subscription, of the first veterinary school at Lyon in France in 1762 [Schwabe, 1984]. Consequent on its introduction to Eritrea the disease spread quickly southwards in the totally naïve cattle population and was soon causing devastating losses in Ethiopia. The effects on the local economy were sudden and devastating and resulted in the greatest famine (at least until that time) in Ethiopia’s long recorded history. According to one source [quoted in Schwabe, 1984]: “the population was driven not only to the eating of traditionally forbidden food but also to the abandonment or sale of children ...[and]... selfenslavement, suicide, murder and cannibalism. The Emperor Menelik tied himself to a plough as a way of example and wept disconsolately ‘Oh! how my country has fallen in ruins! My people are finished!’”.

Before the end of 1890 cattle were dying on the shores of Lake Tanganyika far to the south. The Masai – the archetypal pastoralists of southern Kenya and northern Tanganyika – were on the verge of starvation and “some made faint attempts to engage in farming” [Schwabe, 1984]. It should be noted that in both the rinderpest pandemic and in later droughts and famines many Masai would rather die than kill their animals [Majok and Schwabe, 1996] and thus jeopardize future food security. The colonial administrator Lord Lugard lamented the loss of cattle in Kenya as “never before in the memory of man, or by the force of tradition, have cattle died in such numbers”. From his point of view, however, this was not all bad as he considered the powerful and warlike pastoral tribes had been humbled and colonial progress had been “facilitated by this awful visitation” [Lugard, 1922]. The effects of rinderpest on food security were rather different (or at least differently perceived) in southern Africa. Transport here, in Rhodesia (Zimbabwe) and the Cape Colony (later part of South Africa), was almost entirely dependent on ox-drawn waggons [Wilson, 2008]. Rinderpest crossed the Zambezi river – where it had been held up for some time – in February 1896 [Sinclair, 1922]. During its southward progression it “mowed down the whole bovine race in its passage” and “hundreds of carcasses lay here and there, on the roadside, or piled up in the fields” [Coillard, 1898] (Figure 2). By early March trek oxen were dying in vast numbers: healthy one minute and mortally sick the next, they dropped dead in their traces. As these waggon trains carried food mainly for the new white settlers many of these were unable to continue with their recently established farming businesses: in Melsetter district ”food was very short and Government supplies of rice, beef, tea and other necessities were ......rationed out each week in the township” [Sinclair, 1971]. As a result of the loss of cattle, meat requirements -- at least for the white minority -- had to be imported at very high prices from places as far away as Madagascar. At one time foodstuffs for the beleaguered whites of Bulawayo was brought from Mafeking through 500 miles (800 kilometres) of pest-ridden country by the few donkey and mule waggons that could be procured. The waggon owners in general were clearly adversely affected: “we could no longer sell cattle, and so could no longer buy them from natives, which meant, too, that the natives could no longer buy trading goods from us” [Hyatt, 1969]. There was little official concern for the vast majority of “natives” who lost their sources of meat and milk.

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Figure 2. Cattle dead from rinderpest in 1896-1898 in Rhodesia (courtesy of National Archives of Zimbabwe).

Indeed, Earl Grey, the joint administrator of Rhodesia, considered that the “worst difficulty is not the Matabele, [they were perhaps not surprisingly in revolt] but the Rinderpest, which is killing all the cattle on which we depend for our transport and meat. I may soon .... become a vegetarian, and not by choice” [letter, Grey to Viscount Howick [his son], 15 June 1896, quoted by Mutowo, 2001]. The African rinderpest panzootic not only affected food security in the short to medium term. It also had far-reaching social and economic consequences in the break down of family ties and in moves of pastoralists and peasants into paid forms of employment and perhaps even the beginnings of African urbanization.

Effects of Conflict on Livestock and Food Security Conflicts occur within a country in the form of a civil war and between or among countries. In any of these situations assets are destroyed or diminished as a result of enemy action or even from “friendly fire”. In the Rwanda genocide of 1994 it has been estimated that 50 per cent of the cattle stock was destroyed as was 12 per cent of the houses [Verpoorten, 2003]. Households that were not poor became so due to reductions in food security not only directly from loss of stock but also from market disruption and increased difficulties in getting to markets. A common response to shocks of several kinds (for example, drought or other extreme climatic events) is to sell cattle or other livestock [Fafchamps et al, 1998]. This was not generally the case in Rwanda. This was because people perceived livestock as an insecure asset likely to be targeted by violence. The lack of safety on the roads also prevented households from getting their animals to markets. The few animals that were sold received prices lower than their normal value

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[Verpoorten, 2005]. Household food security was thus compromised both directly and indirectly by reduced income. Following the first Gulf War livestock disease spread rapidly throughout Iraq [Arabic News, 1999]. The General Manager of the Veterinary Department in the Ministry of Agriculture announced that Iraq required urgent help to combat foot-and-mouth disease that had caused the deaths of more than one million sheep and 150 000 calves. In response to this the UN Food and Agriculture Organization (FAO) expressed grave concern "that the outbreak of animal diseases in Iraq may spread to other countries in the Near East seriously undermining food security in the region". FAO went on to warn that "The multitude of diseases afflicting livestock in Iraq threatens the health of people living in infested areas and could seriously undermine food security in the countries of the Near East". The UN agency said Iraq needed to import the vaccines required to fight the disease as well as vaccines against Peste des Petits Ruminants. The vaccines, which could not be used for other purposes, were vital to safeguarding animal health which was an essential component of food security in the region. The animal disease situation in Iraq was aggravated by the collapse of the veterinary infrastructure and disease investigation, surveillance and diagnostic services. The Government was unable to monitor and control diseases in large part because the inspection and supervision groups affiliated to the United Nation in charge of disarming Iraq had destroyed the facilities producing the vaccine for this disease. The Iraq Ministry of Health reported that more than 10 000 children and elderly people died of malnutrition in January 1999 against 766 lives claimed during the same month in 1989 before the enforcement of the UN sanctions against Baghdad in 1990 after the Gulf War. Figures for February 1999 were 9162 children and elderly dead against 835 recorded during February 1989.

Draught Animals The main corpus of the literature on draught power and the use of animals for transport is strongly supportive. It accentuates the perceived positive attributes of animal draught power on food security, household incomes and gender equity but almost entirely disregards possible negative ones [Wilson, 2003]. Many results show crop output to be generally greater by households that own or have ready access to draught animals and this can undoubtedly contribute to food security and higher incomes. Holdings that lack draught power may, however, be disadvantaged not only because they must resort to "purchasing" it but also because they are the main losers in the conflict between animals and crops for many types of resources including space (more people = more land for crops = more oxen to cultivate the land = need for a larger grazing area which is compressed by crop encroachment). Intensification, integration of crops and livestock, nutrient cycling and sustainable production are among the perceived benefits of animal power [Astatke and Mohamed-Saleem, 1996]. These benefits may not, however, be as substantial as they are often presented. Integration may be as much fallacy as fact as it mainly applies to oxen whose demands on the purported holistic system may be greater than the goods they supposedly deliver in return. Nutrient “cycling” [de Leeuw et al, 1994] is largely nutrient “transfer” and sustainable crop agriculture may be achieved only at the expense of unsustainable animal agriculture due to loss of grazing areas and accelerated depletion of resources in the remaining areas (Figure 3).

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Figure 3. Dung dried for fuel and crop residues for animal feed for home use and for sale in local markets in Ethiopia (in both cases plant nutrients are removed from the land).

Draught animals emit considerable quantities of methane mainly because they are large animals and because they are fed on poor quality feed and the cost of these emissions per unit of output is high. The use of animal energy in agriculture is widespread in developing countries. There are continuing cries for its expansion but the case for its contribution to overall food security is not proven. Detailed analyses of long-term economic impact (and not just short-term financial gain) that account not only for discernible benefits but also all implied costs, especially those related to the environment and renewable resources, are needed before an explicit statement can be made about the advantages or disadvantages of animal power [Wilson, 2003].

Urban Livestock Farming Small scale rearing of livestock in many cities and towns in the developing world is often forbidden but is then usually ignored by the local authorities [Waters-Bayer, 1996]. Such a system is, however, much more widespread than is generally acknowledged. Under conditions of rapid urbanization, a deteriorating economic situation and reduced food security, many urban families resort to raising of large (cattle, buffalo), medium (goats, sheep, pigs) and small or microlivestock (poultry, rabbits, guinea pigs, snails) as a general risk reduction strategy. Detailed studies have shown that: − −

more than 20 000 Bamako (Mali) households keep livestock and many thousands more supply inputs and marketing services [Centres, 1991]; over one-third of Harare (Zimbabwe) households keep domestic fowl, rabbits, pigeons, ducks and turkeys [ENDA-ZW, 1994];

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51 per cent of dwellers in six Kenya cities keep livestock [Lee-Smith and Memon, 1994]; urban farming is the second largest employer in Dar es Salaam (Tanzania) and 74 per cent of such farmers keep livestock [Mougeot, 1994].

It is not only the urban poor who keep livestock [Waters-Bayer, 1996]. The better-off also keep domestic animals as a source of highly valued food, to gain supplementary income and to reduce expenditure on bought in food. Livestock keeping is nonetheless more important for the poor as it provides food, income and security. The main functions of livestock for the poor are to provide high quality animal protein, generate income, accumulate capital, serve as a buffer against high rates of inflation, make productive use of “free” resources (kitchen waste, street garbage and water) and provide transport [Waters-Bayer, 1996]. Animals are also kept and nurtured for slaughter on festive or religious occasions (Figure 4). Food security is enhanced not only for the keepers of the animals but also for other people in the informal sector. These include casual labourers, suppliers of collected feed, middlemen and market traders.

Figure 4. A ram being fattened for slaughter at a Muslim religious festival at Niono in central Mali.

There are clear advantages for the urban environment in the keeping of livestock in towns. Amongst these are more efficient land use as land unsuitable for buildings or as yet undeveloped is put to productive use. Municipal maintenance costs can be reduced through the use of garbage and the grazing of areas under and around utility supplies which otherwise

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would have to be maintained at public expense. Transport and energy costs are reduced. Highly nutritious food is made available to other urban dwellers at a cost lower than that which would be incurred if it were to be imported from more distant rural areas.

Livestock and Crops in Tanzania In a study in Tarime District in northwest Tanzania 75.8 per cent of respondents indicated dependence on both livestock and crops for their livelihoods [Ngowi et al, 2008]. Most respondents ranked livestock as more important than crops for both household food security and for income (Table 1) although the difference for food security was marginal. In both highland and lowland zones cattle were regarded as the major contributor to income through selling of live animals and livestock products (mainly milk, butter and hides). Cash crops in the highland (coffee and banana) and food crops (maize, sorghum, rice and finger millet) in the lowland zone were considered as secondary enterprises for generating income. There is, however, a high degree of integration of livestock with crops. The former contribute to food security indirectly through provision of draught power and manure which in turn results in increased production of food crops. Livestock are sold during the period of food shortage to buy cereal grains and in this way they contribute directly to food security. This is similar to the situation in Kenya where livestock have a crucial role in coping with risk and providing livelihood options in the face of increasing climate variability [Krishna et al, 2004]. Table 1. Contribution of livestock and crops to household food security and income in Tanzania Parameter

Zone (n = 60 in each) Highland

Lowland

Household size (number)

6.9

5.7

Land holding (ha)

1.9

2.2

Livestock

51.7

50.0

Crops

48.3

50.0

Livestock

81.7

68.3

Crops

18.3

31.7

Contribution (%) to household food security

Contribution (%) to household income

Source: adapted from Ngowi et al, 2008.

Draught power (72.5 per cent of respondents) was second only to milk for home consumption (89.2 per cent) as a reason for keeping cattle with other uses being meat for home consumption (66.7 per cent), dowry payment (50.8 per cent), income (40 per cent) and savings and security against future uncertainties (23.3 per cent) [Ngowi et al, 2008]. These objectives are consistent with the general situation whereby indigenous cattle produce milk

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and meat for subsistence, supply draught power and manure for cropping, provide fibre and transport and are sold when cash needs for household necessities or purchase of farm inputs arise [Rege and Gibson, 2003].

Small Ruminants in Botswana Livestock are a major source of food, income and employment for most of the population of Botswana whether people live in towns or in rural areas. Livestock and particularly cattle are a major source of foreign exchange with which Government can buy essential foods not produced in the country [MoA, 1989]. Botswana is one of a handful of developing countries that has access to the lucrative European Union market for its beef. Cattle are owned by up to 60 per cent of households. Because of the contribution of cattle to foreign exchange Government has concentrated its policies and its research and development efforts on this species to the relative neglect of the goats and sheep that are owned by almost every household (Panin. 1996). Livestock and crops contribute 55 per cent to total household income of which 49 per cent is from animals and only 6 per cent is from the arable activities (Table 2). Cattle (33 per cent) were the major source of income from livestock with small ruminants providing the remaining 16 per cent. The low contribution of crops to income means that most output is for home consumption whereas much of the output from livestock is for sale. Cash derived from livestock can then be used to buy food to supplement low crop output and to stabilize fluctuating interannual food supplies due to the riskiness of crop production in the arid to semiarid environment. Table 2. Summary of farm household income for two districts in Botswana, 1991/1992 A.

Source of income Net crop revenue

Amount (Pula) 389.0

Contribution per cent 6.2

B.

Net livestock revenue

3102.4

49.1

Cattle

2102.7

33.3

Goats + sheep

921.0

14.6

Other

78.7

1.2

3491.4

55.4 44.6

C.

Net farm income (A+B)

D.

Off-farm income

2814.2

E.

Total household income (C+D)

6305.6

Source: Panin, 1996.

West African Dwarf Goats in Nigeria Goat rearing is related to the value of their production. Goats produce milk and provide skins but their main output is meat [Payne and Wilson, 1999]. Some eighty-five of one hundred farm households in Nigeria rear goats. General estimates of the contribution of goats to meat consumption in that country are of the order of 20 per cent but the real figure, which would take account of home and local slaughtering is probably much higher [Ikwuegbu et al,

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1994]. A 30-month study in the central subhumid belt of Nigeria showed that “offtake” (that is animals eaten on the farm, used as gifts or for dowry and sales for cash) was between 25 per cent and 50 per cent of all animals in the flock on an annual basis [Ikwuegbu et al. 1994]. Whereas slaughter and gifts took place all the year round sales were mostly made just before the start of the annual cropping season. This allows farmers to reduce labour requirements at a time when there is much work in the fields. The cash raised from the sale of a goat was sufficient to purchase 200 kg of mineral (inorganic) fertilizer, enough to render more fertile and greatly increase yields on 1 hectare of crop land.

Small Scale Producers in Cambodia About 90 per cent of livestock in Cambodia are owned by the small scale sector [Devendra and Thomas, 2002]. Average holdings per household in the rural areas are 1.6 large ruminants (cattle plus buffalo), 0.96 pigs and 13 poultry. These are mostly managed in scavenging or semiscavenging systems. Animals are kept as part of a risk management strategy and therefore the reasons for owning them are not necessarily oriented to increased production and higher incomes. Large ruminants are used to provide draught power whereas pigs and poultry contribute directly to food security. Most farmers raise local breeds although small and medium scale commercial farms close to the cities and towns keep exotic or “improved” breeds in an effort to meet the increasing demand for meat, milk and eggs from the fast growing and increasingly affluent urban populations. Many farmers are opportunistic and responsive to short term possibilities. Thus, pig numbers build up rapidly after rice is harvested due to the availability of by-products such as broken grains and bran. In addition many flocks of 200-500 ducks are released to eat paddy spilt or remaining in the field. Farmers who do not own large ruminants exchange rice straw with those who have them for manure to fertilize their crops. Farmers claim that plant growth responses are greater with manure-based compost than with fresh manure or inorganic fertilizers [Thorne and Tanner, 2002]. Appropriate management of animals within the farming system is important in order to maximize use of manure for crops as well as to optimize use of crop residues as animal feed and maintain an efficient “food-feed” system.

Pigs in Kiribati The Republic of Kiribati comprises a number of coral atoll archipelagos spread over 2000 miles along the equator in the central Pacific Ocean. The total population is small (65 883 in 1985) but is concentrated mainly in the western atolls, particularly on South Tarawa where the population density is 1354 per km² compared with 86 per km² for the country as a whole [AIDAB, 1989]. Livestock husbandry in Kiribati fulfils an important social role as well as contributing to human nutrition. Animals, particularly pigs, represent a valuable capital reserve which is important for fulfilling social obligations at times of births, marriages and deaths. The range of human foods in atoll countries is limited because of poor soils and little variety of ecosystems. Consumption of meat products increases this range. It also directly helps to combat malnutrition by providing high-quality protein to supplement the established diet of fish.

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Small and Microlivestock in Bolivia The Bolivian Department of Santa Cruz lies mainly at an altitude of 350-450 m above sea level to the east of the Andes and in the centre of the country. Rainfall is in the range 14001800 mm per year falling mainly between October and May [Paterson and Rojas, 2004]. Just over half the population lives in rural areas. In 1992 it was estimated that 74 per cent of farm families had incomes below US$ 1100 per annum and 25 per cent had less than US$ 300 [Roca, 1998]. Small and microlivestock contribute mainly directly to food security but some are sold in times of need. The poorer members of the rural community are unable to find the investment needed for cattle. Because of their long gestation period and low calving rates associated with poor nutrition and disease cattle make little contribution to household consumption and income. Almost all poultry, pigs and sheep scavenge for their food. Guinea pigs are kept indoors, often in the family kitchen, and are provided feed by their owners on household waste and cut fodder. Average numbers of animals kept were about 15 fowl (present on 90 per cent of holdings), six ducks, one or two sows, up to ten ewes and 15 guinea pigs [Paterson and Rojas, 2004]. No single farm, however, kept all these species. As inputs, other than a little labour provided by women and children, are negligible all production whether consumed at home or sold can be considered as profit. The return from small animal species overall is up to 30 per cent of annual family income [Paterson and Rojas, 2004]. Table 3. Annual output of Bolivian livestock before and after interventions

Item

Domestic fowl

Livestock species Hair Duck Pig Sheep

Guinea pig

Traditional management Live young per breeding event

7.1

6.7

8.1

1.2

2.3

Mortality before maturity (per cent)

30.5

54.1

38.2

30.8

10.5

Gross return/breeding female (US$/yr)

13.29

12.05

260.34

13.82

8.91

Live young per breeding event

7.1

6.7

8.1

1.2

2.3

Mortality before maturity (per cent)

18.2

27.4

28.3

19.5

11.0

Gross return/breeding female (US$/yr)

17.21

23.82

302.05

22.51

9.30

Increase in productivity (per cent)

29.5

97.7

16.0

62.9

4.4

Improved management

Source: adapted from Paterson and Rojas, 2004.

Most domestic fowl, especially young males and older females, are eaten by the household. On average two to four birds are consumed per month but more are eaten on family occasions and religious holidays. In contrast to the few sales of fowl almost all ducks are sold through intermediaries to the urban restaurant trade and thus provide supplementary income. Young pigs up to 40 kg live weight are in great demand for festivals and family celebrations. Of a typical litter of six pigs one or two would be consumed by the family and

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the remainder sold. A single sow that farrowed twice in a year could provide an income of US$ 400 or the equivalent of more than three months casual labour. Sheep are mostly sold but as for other species are eaten on special private and public occasions. Small animals are almost exclusively managed by women and children and many of the benefits associated with livestock accrue to them. This is true in terms of better nutrition and increased income. In both cases the social status of these normally marginalized groups is enhanced not only in the family but also in the wider community. Simple interventions in feeding, health care and management (Table 3) would further improve the contribution of small and microlivestock to food security and family welfare.

Small Scale Dairying in India and Kenya Faced with rapidly increasing human populations, especially in the developing world, and a finite or even diminishing land base, agriculture and livestock production need to intensify. In many parts of the tropics medium and high potential areas, usually but not always in highland zones, have opted for semi-intensive or intensive dairy production. In these systems it has been usual, but again not always universally, to introduce exotic breeds of proven high potential in their home areas. India and Kenya provide examples of how dairy production has improved food security at both the household and broader levels. In the early 21st century livestock contribute about 27 per cent of India’s agricultural Gross Domestic Product. Several programmes have been established in India since 1971 to improve the livelihoods of the poor. One of the best known of these is “Operation Flood” sponsored by the National Dairy Development Board. The programme provided much needed financial, technical and organizational support for dairy production mainly through a network of cooperatives. Families from all livelihood groups benefited from markedly higher incomes. Importantly, however, the proportion of dairy income was highest for landless families. This proportion deceased with increased size of holding [Shukla and Brahmanker, 1999]. Before Operation Flood became effective the availability of milk per person per day in 1968/1969 was 112 ml but this had increased to 240 ml in 2005/2006. India's milk production increased from 21.2 million tonnes in 1968/1969 to 97.1 million tonnes in 2005/06 and was expected to reach 100 million tonnes in 2006/2007. The 3.9 per cent annual growth of milk production between 1995/1996 and 2005/2006 surpassed the 2 per cent growth in population and provided a net increase in milk availability of about 2 per cent per year. Daily milk supply by cooperatives to each one thousand urban consumers increased from 37.3 litres in 1991 to 63.6 litres in 2007. Dairy cooperatives generate employment opportunities for around 12.96 million farm families. An estimated 80 per cent of Kenya’s people depend on agriculture for their livelihoods and employment. Agriculture is estimated to contribute more than 25 per cent of Kenya’s Gross Domestic Product (GDP) of which about half derives from livestock. Livestock kept for meat and subsistence milk production is the main economic activity in dryland Kenya. In rural areas with medium and high potential for agriculture, however, dairy production and marketing dominate livestock’s contribution to the household economy [Muriuki et al, 2001] Dairy production is Kenya’s largest livestock subsector. Kenya has the largest dairy output in eastern and southern Africa. These smallholder areas with crop-dairy systems – the maize crop and weeds and residues from other crops provided nearly half the fodder fed to dairy

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cattle [Lukuyu, 2001] -- support up to three quarters of Kenya’s rural population as well as many of its ruminant livestock [Muriuki, 2001]. The 85-90 litres per person of milk it delivers to Kenya’s people through a well established market oriented smallholder system is one of the highest in subSaharan Africa [Muriuki and Thorpe, 2001]. Production of milk for the market is a major source of income and food for an estimated 625 000 producer households and for those involved in milk marketing. Dairying also has a role in sustaining smallholder cropdairy systems through its contributions to nutrient cycling. The large aggregate volumes of marketed milk – often through informal channels – are made up of small quantities of 4-6 litres per household sold by hundreds of thousands of smallholder households (Figure 5). Producer households retain an average of about 2 litres of milk per day for home consumption which is 30-40 per cent of their production. Table 4. Jobs created for 100 litres of milk traded by small scale marketing and processing in Kenya Enterprise Mobile milk trader

Jobs created Direct Indirect 1.7 0.3

Total jobs 2.0

Milk bar

1.1

0.3

1.4

Small processor

0.2

0.1

0.3

Source: Cheng’ole Mulindo et al, 2001.

The dairy herd, which usually comprises no more than two or three animals, enables resource poor households to accumulate and then liquidize financial capital. The need for cash and not poor animal performance (10 per cent of sales) is the most common reason that smallholders sell dairy cows (60 per cent of voluntary exits) and heifers (85 per cent of voluntary exits). Cash generated from sales is most frequently used for financing school fees, hospital bills and household investments. Milk marketing has important implications with respect to the competitiveness of producer and consumer prices to the advantage of low income earners of both groups. Small scale production also provides considerable additional employment for small-scale market agents (Table 4). The economic efficiencies of small scale dairying and marketing are major contributions to poverty alleviation. At the same time they ensure that the high nutritive value of milk and derived products is accessible to the lowest income groups of society who could not afford higher cost pasteurized packaged milk through the formal marketing system [Ouma et al, 2000]. Both producer and non-producer households drink more milk than their counterparts in neighbouring countries. Marketing of forage is an important ancillary source of income as shown by the estimated value in 1996 of US$ 5 million of Napier and maize fodder in one district alone. This underlines the importance of dairy cattle as a means of accumulating capital assets and the critical role of dairying in alleviating poverty. Nonetheless, even in these commercially oriented systems, the primary objective of smallholder dairying is to produce milk for home consumption [Bebe et al, 2001]. In sum, smallholder dairying makes a major contribution to Kenya’s food security and poverty alleviation. In the context of continuing pressure on land from population expansion and the resulting intensification of land use it is expected to continue to do so.

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Figure 5. Marketing channels for smallholder milk in Kenya.

Conclusion There is general consensus among politicians, planners and professional livestock people that animal production in the developing countries is not expanding sufficiently fast to meet the needs of the world’s burgeoning population. Lack of knowledge among politicians and policy makers on the contribution of livestock and perhaps particularly that of the smaller species and their special place in gender access to food and income is a principal reason for the inadequate attention given to domestic animals. Solving the challenge of global food security will only be possible if there is sufficient focus on animals. Growth in output of animal products in many countries has been mainly the result of increased numbers rather than increased productivity. In spite of enormous investment by the international, bilateral and national communities the reality is that there has been little to no change in the efficiency nor the sustainability of animal production in the developing world. Livestock can contribute to the global development goal of meeting the needs of the present without compromising the ability of future generations to meet their own needs. If, however, livestock are to continue to provide food security, governments will need assistance in preparing appropriate strategies and policy frameworks. New technology will need to take

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full account of the multifaceted role of livestock in agriculture and food security. Technology must also be acceptable to producers in terms of appropriateness, affordability and benefits. Some available technology already assists the optimum use of local feed resources including crop residues, agroindustrial by-products and multipurpose crops such as sugar cane and cassava. Optimal use of different types of legume and their incorporation into existing farming practices will be critical in achieving sustainable agriculture. Two important animal products, manure and draught power, are not efficiently used at present and specific programmes are required to maximize their use. In view of increasing concern with regard to conservation of the environment and natural resources, high priority must be given to further development of both plant and animal agriculture. There remains, however, much negative emotional conjecture, lack of objectivity and over-simplification of the perceived damaging effects of livestock on the environment. There is a clear need to modify some livestock production systems. Strategies to achieve efficient livestock production will need to address conservation of the resource base, minimization of waste and maximization of nutrient recycling. It should nonetheless be accepted – and there is ample evidence for it – that the contribution of livestock to sustainable development could be greatly increased if an appropriate enabling environment were created and if full account were taken of the dramatic changes transforming the global livestock sector in future policies. Only then can it be expected that future generations will have access to the type and quality of food they desire without depleting natural resources.

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In: Food Science and Security Editors: L. Amsel and L. Hirsch, pp. 253-266

ISBN: 978-1-60692-977-3 © 2009 Nova Science Publishers, Inc.

Chapter 10

ORGANIC MEAT: MARKET DEVELOPMENT AND CONSUMER WILLINGNESS TO PAY Fabio Napolitanoa, Ada Braghierib and Antonio Girolami Dipartimento di Scienze delle Produzioni Animali - Università degli Studi della Basilicata – Viale dell’Ateneo Lucano, 10 – 85100 Potenza, Italy

Abstract This chapter provides an overview on the evolution of global market demand for organic meat and factors affecting it. Future prospects for development of organic meat consumptions and possible problems concerning trading are analysed. The price that people are willing to pay is the major determinant of the market share of organic meat. Although intent to purchase depends upon the interactions of quality attributes such as appearance and colour, it has been hypothesized a consistent effect of organic labelling as compared to some sensory characteristics on the price offered by consumers for organic meat. Previous experiments have shown that expectations induced by the information can affect the quality perception. Therefore, consumers’ behaviour and attitudes toward organic meat are tested through the study of the effect of information about organic farming practices on product liking and consumer willingness to buy organic beef and pay the extra production costs. Our results indicate that this information, if given to the consumers, can be a major determinant of beef liking, thus providing a potential tool for meat differentiation to traditional farms where husbandry is based on extensive rearing systems and high animal welfare standards. Accordingly, consumers are prepared to spend more for organic beef, thus indicating that reliable information about the organic farming system may markedly increase consumer willingness to pay.

Introduction The global market for organic food was valued at USD (United States dollars) 25 billion in 2003, which can be considered a niche (Willer and Yussefi, 2004). Although production of organic products is increasing across the globe, sales are concentrated in the industrialised a b

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parts of the world. North America and Western Europe account for 95 % of the world retail sales of organic food products with USD 13 and 10.4 billion, respectively. Although consumer interest is growing in other regions, the demand is confined to the industrialised world largely because of the price premium of organic products. Many developing countries have large sections of their populations below the poverty line, and this makes it difficult for a market for organic products to develop. A typical consumer of organic products has the following attributes: lives in urban areas, usually in a big city, in a medium to high-income household with relatively high purchasing power, belongs to middle-high social classes and is sensitive to factors like quality, provenance and production methods (Willer and Yussefi, 2004). The industrialised nations have a sizeable middle-class, and this is the reason why most organic food sales are concentrated in these countries. As more countries develop economically and as their populations become more affluent, demand for organic products is likely to rise. This will cause sales of organic products to become less concentrated in the world. Rapid economic growth in countries like China, Brazil, and South Africa is causing the upper social classes to expand, and this is creating a market for organic food. In other regions, there is an increase in organic farm land because farmers are attracted to the export benefits of organic production. Although most production in Asian and African countries will be for export markets, it is also creating regional markets to develop in which organic farmers market their organic crops to consumers in their region. Sales of organic products are slowing in certain countries, especially in Western Europe; however, the market is becoming increasingly global.

The Market of Organic Meat During 2005, while beef trade was growing at an average of 3.0 %, global pork export was led by the European Union, followed by USA, China, Brazil, Mexico and Canada, whereas Japan was the largest consumer market for pork followed by Russia, Mexico, Korea and USA (Organic Monitor Ltd., 2006). The Western European market for organic meat was traditionally the largest in the world, however it has now been equalled by North America. The European market has faced rapid expansion since the mid 1990s and is now reporting slowing growth rates as certain sectors approach maturity. Supply-demand imbalances have become a characteristic of the European organic food industry. A number of countries are showing oversupplies in sectors like organic meat whereas other sectors like organic cereals continue to suffer from product shortages. A reason for this is that much of the early converts to organic agriculture were dairy and beef cattle farmers. The average European expenditure rate on organic products is USD 27.2 per annum. There is much variation in the expenditure rate between European countries, ranging from USD 7.3 (Spain) to USD 105 (Switzerland) per capita. In 2003, EU, including 15 countries, certified non-dairy cattle (mainly suckler cows) amounted to about 1 million heads corresponding to 1.7 % of total non-dairy cattle herd (European Commission, 2005). Austria ranked first with 25 % of EU, followed by Italy with 15 %, the United Kingdom with 13 % and Germany with 12 %. Certified pigs amounted to 450,000 heads or about 0.4 % of total pig herd. Only a few member states, such as Germany with 8 % and France with 4 %, had a significant share of certified animals. The number of

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certified pigs is still relative low. The relative slow development may be linked with higher production costs in organic pig meat production. Certified broiler number amounted to more than 9 million, France ranked first with 5.1 million. Sheep and goats amounted to 2.4 million heads or 2.4 % of total sheep and goats herd. Of the about 2 million certified sheep, 35 % were located in the United Kingdom and 21 % in Italy. According to Hamm et al. (2002) beef had the highest market share (1.7 %) followed by sheep and goat meat (0.7 %), pork (0.3 %) and poultry (0.3 %). Only little and partial information is available on prices for organic meat and meat products as prices diverge depending on the country and on the product. For instance, farmer price for organic pork in 2002 was about 2.46 €/kg on EU-15 average, only Italy reported a price 20 % below this average, but some countries had significantly higher prices like Greece (5.00 €/kg), Ireland (3.49 €/kg), Luxembourg (3.27 €/kg) and the United Kingdom (3.22 €/kg). Price premiums for organic pork ranged from 45 % in Germany and Austria to 132 % in the Netherlands, while EU average was about 62 %. Consumer price for organic pork cutlet was about 13.17 €/kg but differed considerably between the 15 member states: the Danish price was about the double of the price in Spain, Portugal and Finland. Consumer price premium for organic pork cutlet (81 %) was higher than average farm gate price premium and ranged extremely from 0 % in Portugal to 165 % in Greece. The high divergence in consumer prices and price premiums reflect often the different sales chains used, i.e. consumer prices are usually much higher in organic food shops than in supermarket chains. There was a considerable variability among the 15 Member States in farm gate prices for beef as well as in consumer prices for minced beef. As for beef, farm gate prices for organic and conventional were very high in Greece resulting in a high consumer price for minced beef. Organic price premium at farm gate level varied from 17 % in Denmark to 190 % in Spain and was on average about 49 % (European Commission, 2005). The North American market for organic products is reporting the highest growth worldwide. The meat sector is the fastest growing organic food industry with sales expanding by 51 % in 2005 (Organic Monitor Ltd., 2006). The USA organic beef market has been showing the highest growth with sales doubling each year since BSE was reported in 2003. High market growth rates have led to organic meat supply shortages with producers unable to meet growing demand from retailers. The organic beef and pork markets are the most affected because of low production levels. American farmers have shown little interest in producing these organic meats due to high production costs and the lack of distribution infrastructure. Although production has increased since 2004, supply is expected to lag demand for a number of years. As a consequence prices are rising for a range of organic meat products and an increasing number of North American processors are looking overseas for supplies. In particular, organic meat products imports are mainly from Latin America, Australasia and Canada. Although organic beef has the fastest growing market, organic poultry is the most widely available in North America (nearly two-thirds of this sector). USA retail sales of organic poultry were USD 161 million in 2005, well under 1 % of conventional poultry sales. However, retail sales of organic poultry have almost quadrupled since 2003, and estimates of annual growth rates range from 23 to 38 % through the end of the decade, with annual sales reaching almost USD 600 million by 2010 (Nutrition Business Journal, 2006). Approximately half (51 %) of organic poultry sales were in natural food stores in 2003, 45 % in mass market grocery stores (including conventional grocery, mass merchandiser, and club stores), and 4 % through direct sales and other distribution channels (Nutrition Business Journal, 2006). As in

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the conventional food sector, sales of broilers account for the majority of organic poultry sales. The relatively short production cycle and low price premium are responsible for organic chicken to be the most popular organic meat with consumers. Prices for organic broilers were steady at an average of USD 2.17 per pound from July 2004 through early 2006, with a range of USD 1.89 to USD 2.45. Average prices for conventional broilers, on the other hand, ranged from USD 0.59 to USD 0.82 per pound during the same period (Oberholtzer et al., 2006). Organic beef in comparison is more rarely found in retailers because of small-scale production, and inadequate distribution infrastructure, although the success of competing products like “natural” beef is decreasing. USDA (United States Department of Agriculture) roles for “natural” beef are less strict than those for organic products: antibiotics, hormones and animal by-products are banned but conventional feedstuffs are allowed. The number of certified animals in 2005 were: over 36,000 beef cows, 10,000 hogs and pigs, 4,400 sheep and lambs and over 10,000,000 broilers. In comparison with 2002 the increase ranged from 9 % (sheep and lambs) to 264 % (hogs and pigs). The Argentine government has established national standards for organic products. These are at least as stringent as those of IFOAM and the European Union. The organic industry in Argentina is mainly export oriented (85 % by value is exported with an estimated value of USD 32 million). Organic meat production, and beef in particular, is growing as a consequence of climatic suitability and soil fertility and can be cited as an example of a developing country supplying external markets. However, a domestic market is being developed in Buenos Aires. In 2000, out of a total production of 40,000 tons of organic produce, meat and livestock products represented 8 %. Of this total, 35,000 tons were exported, of which meat and animal products accounted for 3 %. This would imply that a substantial proportion of organically produced meat was consumed in the domestic market. It is not known what proportion was sold as organic meat and what proportion went into conventional sales. The European Union is the principal destination of Argentina's organic beef exports. Meat exporting began in the mid 90s with beef, and more recently Patagonian lamb became the predominant export for international markets. In 2002 there were 754,000 sheep and 122,000 head of cattle certified in Argentina (Willer and Yussefi, 2004). Elsewhere, both Brazil and Uruguay are seeking to develop exports of organically produced meat (Willer and Yussefi, 2004). In the case of Brazil, organic beef production systems are being developed in the centre of the country. The authorities in Brazil see the organic production system as a way of boosting local cattle producers' income, while reducing the environmental damage that traditional methods of cattle raising have caused. While 90 % of Brazil's overall organic production is exported, Brazil's internal market for organic foods is growing at around 25 % a year. Although the Australasian continent comprises almost 40 % of global organic farmland with 12.1 million hectares, the market represents a fraction of the global total (Willer and Yussefi, 2004). Sales of organic products were estimated at about USD 200 million in 2002 with Australia comprising the most. Beef cattle farmers use much of the organic farmland in Australia as pastureland. The Australian and New Zealand organic food industry is exportoriented with significant quantities of primary products, including beef and lamb, going to other northern hemisphere countries and relatively low amounts sold in the domestic market. However, sales of organic products within Australia are growing at about 15 to 20 % per annum.

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Exporters are finding increasingly difficult to meet supply gaps because of the differences in organic standards between regions. For instance National Organic Program standards developed in USA for organic meat products differ from those of the EU, and, although trade liberalisation may be removing tariff barriers, standards are becoming the major impediment to free trade in the global organic meat industry.

Consumer Willingness to Buy Organic Meat Food quality is not an objective feature of the products as it is affected by consumer perception, and it is often referred to as perceived quality (Cardello, 1995). Many aspects can be used by consumers to perform their food choices. Intrinsic (e.g. cut, colour, fat rim) and extrinsic cues (price, origin, stamp of quality, production and nutritional information) are used to form expectations about product quality attributes, the latter can be classified in two categories: those experienced before or during consumption (experience quality attributes: e.g. price and sensory properties) and those not experienced directly, such as healthiness, naturalness, ethical aspects, etc., which should be communicated to be perceived as they are credence characteristics that can not be confirmed either before or after purchase (Grunert et al., 2004). Tangible aspects can markedly affect most purchasing decisions, independently from the knowledge and awareness of the consumer, whereas intangible attributes are important for high-involvement consumers possessing enough beliefs and attitudes (McEachern and Schroder, 2002). For these consumers, such intangible attributes play a central role in defining product quality. According to a recent on line survey (ACNielsen, 2005), organic alternatives are purchased mainly for health reasons. Over two thirds of survey respondents think that organic foods are healthier for them and their children. This healthy perception is consistently strong across Asia Pacific. European people seem to be more conscious of the wider benefits of organic foods, such as protecting the environment and animals. Nearly a fifth think that organic foods preserve the environment, and 12 % cites kindness to animals as the key reason for purchasing organic alternatives. However, in other regions, less than a fifth of consumers are aware of the environmental benefits of organic foods, and even less mentions animal protection. As to the barriers to purchasing organic alternatives, high prices are perceived as the biggest problem for one third of respondents in Asia Pacific and over 40 % of European and North American people. Across Latin America and in South Africa, availability is considered a problem. Interestingly, lack of availability prevails over lack of credibility as a reason for not purchasing organic foods globally. An element favouring the spread of organic meat into the general retail sector is that purchasers of organic food tend to be in the higher income segment (F.A.O., 2002). Consequently, supermarkets seek to attract such customers by providing a wide range of food, including organic meats. The increased involvement of supermarkets, with their centralised systems of purchasing and distribution, may result in pressure to reduce the current price differential between organic and conventional products. As an extension of this trend, some supermarkets have introduced "own brand" organic meat products, with such products being priced below those of competing brands. The general sense among consumers who would not buy organic products is that they are usually more costly. Manufacturers and retailers should take into account the

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geographical differences in terms of acceptance and perception of organic food. For example, in Thailand and China credibility is more an issue, while in Asia Pacific perceptions of price of organic foods is the major constraint in eight out of 13 markets studied. Meeting organic certification requirements usually implies higher production costs. For example, it is reported that the cost of producing organic beef in the United Kingdom is 20 % higher than under conventional methods. In some cases, the high cost of converting to organic meat and dairy production has led to subsidies being paid to the farmer. However, this is not the case in many countries (e.g. USA and developing countries). Thus the extra production costs have to be paid by consumers.

Study of the Effect of Information on Organic Beef Acceptability and WTP For consumers from western countries, price is not the only determinant behind animalfood purchases as they are acquiring an increasing interest in farming practices and the related organic standards. Consumers do not seek the cheapest food but the best value for money, i.e. the maximum benefit for what they are prepared to spend (McInerney, 2004). Information is a key consumer need. Consumers should be provided with the information they require, which should be presented in a simple form in order to enable them to make informed choices. In fact, it has been observed that consumers are willing to access to more information about organic food (Brennan et al., 2003). Numerous studies have been conducted on the effect of information on food liking (Aaron et al., 1994; Daillant and Issanchou, 1993; Kähkönen and Tuorila, 1995; Solheim, 1992; Westcombe and Wardle, 1997). In particular, information about the manufacturing process (organic vs. OGM) can affect product acceptability (Caporale and Monteleone, 2004). Recent studies have been conducted on the effect of information about animal welfare on lamb (Napolitano et al., 2007a) and beef liking (Napolitano et al., 2007b). All these experiments have shown that expectations induced by the information can affect the quality perception. Thus, if expectations are either positively (the liking score of the product tasted without external information is higher than expected) or negatively disconfirmed (the product is worse than expected), the assimilation model is generally applicable, which means that hedonic ratings move towards the expectations when an external information is given compared to tasting without external information (Anderson, 1973; Cardello and Sawyer, 1992). However, there is generally a significant difference between liking score with information and expected liking score, i.e. consumers do not completely assimilate (Lange et al., 1999; Siret and Issanchou, 2000). The price that people are willing to pay is the major determinant of the market share of organic meat. Although intent to purchase depends upon the interactions of quality attributes such as appearance and colour (Brewer and McKeith, 1999), it has been found a more consistent effect of organic labelling as compared to some sensory characteristics on the price offered by consumers for organic pork (Dransfield et al., 2005). Using questionnaires on organic foods in Spain, consumers appeared to be prepared to pay about 12 % more for organic red meats and chicken (Gil et al., 2000). In France and The

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Netherlands, questionnaire responses suggested that almost half of consumers would pay 20 % more for pork from pigs raised outdoors (Carpentier and Latouche, 2005). Dranfield et al. (2005) stated that those studies over-estimated the premiums consumers would be willing to pay, while their results suggest that people would offer 5 % extra, with about one-fifth of consumers willing to pay 20 % extra, for organic pork. However, hedonic and purchase intent measurements may be not representative of the real behaviour of consumers. They may declare high preferences and purchase intent for products with highperceived quality, albeit not buying them under economic constraints (Lange et al., 1999). Recently, Lange et al. (2002) and Napolitano et al. (2008) observed that, as for food liking, the assimilation model is applicable also to consumer willingness to pay. As a consequence, it can be hypothesized that information about the organic manufacturing process can increase both meat acceptability and willingness to pay thus providing a means to cover the extra production costs sustained by organic farmers. Little is known on the effect of the information about organic production on the real consumer willingness to pay for meat. Recent studies demonstrated that auctions are able to place consumers in real situations where they can show their true preferences. In particular, the Vickrey second price auction is widely used to assess consumer willingness to pay real goods (e.g. Melton et al., 1996), including foods (Lange et al., 2002), and the value consumers give to food safety (e.g. Hayes et al., 1995) and animal welfare (Napolitano et al., 2008). According to this specific type of auction consumers are individually asked to submit a sealed bid corresponding to the highest price they would agree to pay for a particular product. The highest bidder (i.e. the winner), by paying the second highest price, has the opportunity to buy a product at a price equal to or, more often, lower than the value he assigns to the product (Vickrey, 1961). The present study was aimed to verify whether consumers confirm their higher liking for organic meat by willing to pay extra costs in a situation where a potential purchase performed by consumers, such as the Vickrey auction, is included.

Methodology Beef was obtained from twelve Podolian young bulls aged 16 months and reared either in conventional (CB) or organic local farms (OB). Carcasses were aged 15 days in order to increase meat tenderness and make the product acceptable from a sensory point of view (Braghieri et al., 2005). Both products were obtained from the muscle Longissimus dorsi. Meat samples (10 x 10 x 1 cm) were grilled at 300°C to an internal temperature of 75°C assessed using a thermocouple probe inserted into the meat. Mean cooking time was 6 min. Samples were offered to the subjects immediately after cooking in booths where salt was available ad libitum. Subjects were recruited in Potenza (main town in the region of Basilicata, southern Italy). The consumer panel consisted of fifty subjects recruited on the basis of age and level of education. In addition, subjects were selected using predetermined screening criteria based on consumption frequency of beef and organic products.

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Table 1. Socio-demographic features of the subjects participating to the consumer test

Age

Sex

Education level

Number

Percentage

20-39 years

14

28

40-59 years

17

34

> 59

19

38

Female

22

44

Male

28

56

Primary school

3

6

Secondary school

6

12

High school

20

40

Graduated

17

34

Post-graduate

4

8

Table 2. Summary of the experimental design for the assessment of consumer liking (L) and willingness to pay (WTP) Test

Day

Stimulus presentation

Type of evaluation

Type of rating

1

1

Beef

Tasting without information

Perceived L

2

1

Information

Expectation

Expected L

3

2

Beef + information

Tasting with information

Actual L

4

2

Product display

Auction

WTP

65 subjects were interviewed and were asked their frequency of consumption of beef at home (1 = never; 2 = once a year or less; 3 = 3-5 times a year; 4 = less than once a month; 5 = 1-2 times a month; 6 = more than twice a month; 7 = at least once a week ). The selected consumer panel included subjects who reported to consume beef at least “12 times a month” and organic products occasionally (at least “once a year or less” but “less than once a week”). Subjects had a mean age of 49 years and were almost equally distributed for sex. The main features of the subjects participating to the consumer panel are depicted in Table 1. The experiment was planned in four tests (Table 2). In the first test the consumers were offered both CB and OB in a balanced order of presentation. They were asked to taste the meat and rate their liking receiving no information on the products (Perceived liking). In the second test the subjects received two sheets with the information concerning the farming systems (conventional or organic). They were asked to read carefully the information and give their liking expectation for that product (Expected liking). First and second tests were performed in the same day. The day after the third test was performed: the consumers were given OB only along with the information sheet. They were instructed to read the information before tasting the sample and express their liking score (Actual liking).

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Consumers rated their liking on a 9-point hedonic scale labelled at the left end with “extremely unpleasant”, at the right end with “extremely pleasant” and at the central point with “neither pleasant nor unpleasant” (Kähkönen, Tuorila and Rita, 1996). In tests 2 (expectations produced by information) and 3 (acceptability generated by information and tasting of the product) the following information concerning the farming systems were given to consumers:

1) Conventional beef: conventional beef cattle farming ensure standards of animal

welfare as set by the current legislation; the administration of pharmaceuticals is allowed within the suspension limits, as well as the use of GMO and chemicals for the production of animal feeds, in accordance with the current legislation; a high stocking density per hectare is allowed. 2) Organic beef: organic beef cattle farming practices ensure standards of animal welfare higher than those set by the current legislation by promoting grazing systems and the expression of species-specific natural behaviour; the use of pharmaceuticals is markedly reduced; the use of GMO and chemicals for the production of animal feeds is banned; stocking density per hectare is low in order to reduce the impact of faming on the environment. The second price Vickrey auction (Vickrey, 1961) was used to assess consumer willingness to pay organic beef. Participants attended a short presentation explaining the procedure to be followed for the auction. It was made clear that the submission of bids implied a commitment to buy the product. Participants agreeing to the procedure signed a consent and received 10 Euro in cash. Subsequently, a formal training on the use of the Vickrey second price auction was conducted. It was explained that the maximum price accepted to pay for 100 g of organic beef had to be written on paper, separately by each participant. The bid had to be comprised between 0.00 and 5.00 €). On each form provided to consumers there was a suggested price (ranging from 0.29 to 2.71 €; mean = 1.54 €, corresponding to the actual commercial value of organic beef), however it was explained that they could offer lower or higher prices. The participant submitting the highest price (winner) had to buy the product, not at the submitted price, but at the second highest price (i.e. the second highest submitted bid). In case of more consumers offering the same highest bid only one participant, randomly chosen by another consumer, would be selected as winner. This procedure allowed one of the participants to buy organic beef at a price lower than or equal to the price they would normally accept to pay. It was also explained that the study aimed to know the value that the product had for the consumers, not its commercial value, and that the best option for them was the submission of their real reservation price. In order to ascertain that all participants correctly interpreted the procedure, some practice was conducted using snacks. Subsequently, an auction was conducted where consumers submitted bids after the display of a 100 g slice of organic beef. The Student’s paired t-tests were used to evaluate differences between mean scores either obtained for the two products (OB and CB), or obtained for the same product under different conditions (tasting only, information only, tasting with information). The same test was used to compare the suggested price for organic beef and the actual bids offered by consumers.

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Results and Discussion Ratings given by consumers to OB and CB are summarised in Table 3. Consumers rated both products at scores above the central point (5 = neither pleasant nor unpleasant) for perceived liking. These results indicate that the meat from both organically and conventionally reared beef cattle was characterised by a good eating quality. However, mean scores of perceived liking were higher for OB as compared to CB (P<0.001). No information is available on the effect of organic farming on beef acceptability, although previous studies reported minor effects on flavour (Marino et al., 2006) and tenderness (Braghieri et al., 2005). In this study organic farming practices induced a much higher acceptability as compared with conventional techniques (P<0.001) with scores above 7 (pleasant). Table 3. Rating (± S.E.) given by the consumer panel during the three hedonic tests Type of rating

Organic beef

Conventional beef

Perceived liking

7.22 ± 0.16a

5.94 ± 0.20b

Expected liking

7.88 ± 0.12a

5.18 ± 0.24b

Actual liking

7.72 ± 0.15

NR

P-E

-0.66*** Negative disconfirmation1

0.76*** Positive disconfirmation2

0.50* Assimilation3 -0.16 Complete4

-

A-P A-E

-

P = perceived liking mean scores (blind condition); E = expected liking mean scores (only information); A = actual liking mean scores (with information); NR = not recorded. a,b = P<0.001; * = P<0.05; *** = P<0.001. 1 The product is worse than expected 2 The product is better than expected 3 Actual liking moves towards the expectations 4 Assimilation occurs, and actual liking is not different from expectations

Expected liking scores were higher for OB than for CB animals (P<0.001). These results indicate that consumers are aware of the possible positive effects of organic farming on product quality and safety. Our study also confirms previous reports stating that consumers use the application of organic standards as an indicator of product attributes such as food safety, food quality and food healthiness (Brennan et al., 2003; Michaelidou and Hassan, 2008). Fresh meat is usually commercialised as undifferentiated product. For instance, Grunert (1997) suggests that the butcher is considered as an expert by consumers and, therefore, reliable to predict meat quality and characteristics. As long as fresh meat is mainly sold as a commodity, there is also only a limited incentive for meat producers to differentiate their product (Grunert et al., 2004). Any form for improved or otherwise differentiated meat quality requires new ways to signal the quality to the consumer. Thus, meat product differentiation needs a constant and reliable signalling of quality through appropriate

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information given to consumers in order to motivate them and increase their willingness to buy and pay for meat (Bredahl, 2004). This differentiation can be based on both product or process characteristics. For animal-based products process characteristics may be represented by the farming practices and the related organic standards. In fact, consumers showed a WTP for OB higher than the suggested price (P<0.001), the latter corresponding to the commercial value for organic beef (Table 4). Results concerning the effect of information on expected and actual liking of beef are shown in Table 3. For both products (OB and CB) the expected acceptability was significantly different from the perceived liking expressed in blind conditions (P<0.001), thus indicating that a disconfirmation occurred. In particular, the consumers found OB worse than expected (negative disconfirmation), whereas CB was considered better than expected (positive disconfirmation). These results indicate that information about farming practice can have a marked impact on consumer expectancy with organic standards associated with high expected product quality and conventional farming associated with lower expected product quality. Accordingly, previous studies revealed a marked effect of positive information about farming practices and animal welfare on actual liking of meat (Napolitano et al., 2007a,b) and willingness to pay for yogurt (Napolitano et al., 2008). A significant difference between perceived and actual liking was observed for OB as the former was lower than the latter (P<0.001). Therefore, the information given about organic farming was able to affect the actual liking of beef. In this case, the effect of information can be explained on the basis of the assimilation model, which can be observed when the actual liking of the product moves in the direction of the expectations. In particular, the information concerning the use of organic farming techniques characterised by high levels of animal welfare, product safety and low impact on the environment had a positive impact on actual liking. Consumers completely assimilated their liking in the direction of expectations, as indicated by the fact that expectancy was not significantly different from actual liking (P>0.05), which in turn showed scores close to 8 (very pleasant). The complete assimilation observed for this product is likely to be due to the important role played by the information in the determination of actual liking of organic beef. In addition, the complete assimilation was possibly facilitated by the good eating quality of the organic product, as indicated by the high value of perceived liking observed in blind conditions. Table 4. Mean ratings (±S.E.) of WTP WTP Suggested price

1.54 ± 0.12a

Consumer bid

2.65 ± 0.13b

a,b = P<0.001.

Conclusion The main limit to purchasing organic meat remains price because of high production costs, which are affected by organic rules (higher space allowance, origin of feedstuffs, etc.) and small-scale production systems. Two strategies to overcome this constraint are: the

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spread of organic meat in supermarkets, which is likely to induce a reduction of current price (this approach may be suitable for meat which is organic but otherwise undifferentiated from conventional products); the induction of increased willingness to pay by constant and reliable quality signalling systems capable to provide an ethical value to the product, which may become even higher if associated to traditional farming systems and typical meat productions. The results concerning the study of the effect of information on beef liking show that consumers are influenced by information about organic production and move their actual acceptability in the direction of expected liking, possibly because consumers are aware of the ethical value of organic farming and its effects on product safety. In particular, the hedonic discrepancy was totally assimilated indicating that actual liking of organic beef was markedly affected by information. Conversely, the information concerning conventional production had a detrimental effect on expectancy. Therefore, the information about organic farming, if given to the consumers, can be a major determinant of beef liking, thus providing a potential tool for meat differentiation to traditional farms where husbandry is based on extensive rearing systems and high animal welfare standards. Accordingly, consumers are prepared to spend more for organic beef as compared to the suggested price, thus indicating that reliable information about the organic farming system may markedly increase consumer willingness to pay.

Acknowledgement We are grateful to the Regione Marche for supporting the program E.QU.I.ZOO.BIO. "Efficienza, Qualità e Innovazione nella Zootecnia Biologica" (SP 11), and thereby the effort of the authors.

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Melton, B., Huffman, W., Shogren, J. and Fox, J. (1996). Consumer preferences for fresh food items with multiple quality attributes: evidence from an experimental auction of pork chops. American Journal of Agricultural Economics, 78, 916–923. Michaelidou, N. and Hassan, L. M. (2008). The role of health consciusness, food safety concern and ethical identità on attitudes and intentions towards organic food. International Journal of Consumer Studies, 32, 163-170. Napolitano, F., Braghieri, A., Caroprese, M., Marino, R., Girolami, A. and Sevi, A. (2007a). Effect of information about animal welfare, expressed in terms of rearing conditions, on lamb acceptability. Meat Science, 77, 431-436. Napolitano, F., Caporale, G., Carlucci, A. and Monteleone, E. (2007b). Effect of information about animal welfare and product nutritional properties on acceptability of meat from Podolian cattle. Food Quality and Preference, 18, 305-312. Napolitano, F., Pacelli C., Girolami A. and Braghieri, A. (2008). Effect of information about animal welfare on consumer willingness to pay for yogurt. Journal of Dairy Science, 91, 910–917. Nutrition Business Journal (2006). Organic Foods Report, Penton Media Inc., San Diego, USA. Oberholtzer, L., Greene, C. and Lopez, E. (2006). Organic poultry and eggs capture high price premiums and share of specialty markets. USDA Outlook Report No LDPM-15001. http://wwwersusdagov/Publications/ Organic Monitor (2006). The North American Market for Organic Meat Products. Organic Monitor Ltd. Siret, F. and Issanchou, S. (2000). Traditional process: influence on sensory properties and on consumers’ expectation and liking. Application to ‘pâté de campagne’. Food Quality and Preference, 11, 217-228. Solheim, R. (1992). Consumer liking for sausages affected by sensory quality and information on fat content. Appetite, 19, 285-292. Vickrey, W. (1961). Counterspeculation, auctions, and competitive sealed tenders. Journal of Finance, 16, 8–37. Westcombe, A. and Wardle, J. (1997). Influence of relative fat content information on responses to three foods. Appetite, 28, 49-62.

In: Food Science and Security Editors: L. Amsel and L. Hirsch, pp. 267-280

ISBN: 978-1-60692-977-3 © 2009 Nova Science Publishers, Inc.

Chapter 11

MEAT CONSUMPTION AND ANEMIA IN CHINESE ADULTS IN JIANGSU PROVINCE Zumin Shi1,2,*, Xiaoqun Pan1 and Gerd Holmboe-Ottesen2 1

2

Jiangsu Provincial Center for Disease Control and Prevention, China Institute of General Practice and Community Medicine, University of Oslo, Norway

Abstract Background: China is undergoing rapid nutrition transition towards higher intake of animal foods. The prevalence of anemia has decreased during the past decade but still remains high. Objective: To describe the sociodemographic differences in meat consumption and the association between different types of meat consumption and anemia in Chinese adults. Method: Data from a national household nutrition survey carried out in year 2002 including 2849 adults aged ≥ 20 years from Jiangsu province were used. Dietary information was assessed by three days weighed food records. Fasting blood specimens were collected for assessment of hemoglobin and serum ferritin. Results: The overall prevalence of anemia was 25.5%. Across quartiles of serum ferritin (1-4), the prevalences of anemia were: 23.9, 15.5, 17.9, and 15.8% in men; 38.6, 29.5, 27.1, and 30.3% in women. The mean intake of total meat was 101.9 g/day in men and 74.9g/day in women, Pork and poultry were the main sources of meat. Meat consumption varied according to socioeconomic status. Higher educational level, higher income and urban residency were associated with higher intake of meat. A positive association between beef and lamb consumption and hemoglobin level was observed. Pork, poultry, and total meat consumption was negatively associated with hemoglobin level and positively associated with prevalence of anemia. In multivariate analysis, the prevalence ratios (OR) for anemia across pork intake quartiles were: 1, 1.20(0.99-1.46), 1.39(1.14-1.70), and 1.37(1.09-1.73) (p for trend: <0.001). Total meat consumption was positively related to serum ferritin level adjusting for age and gender. Conclusion: Socioeconomic conditions were positively associated with meat consumption. Pork, poultry and total meat consumption was significantly and positively associated with the risk of anemia among Chinese. Further research is needed to interpret this association.

*

E-mail address: [email protected]. Telephone number:+47-22850519, Fax number: +47-22850590. Address of corresponding author: Zumin Shi, MD, PhD, Institute of General Practice and Community Medicine, University of Oslo, PO Box 1130 Blindern, 0318 Oslo, Norway.

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Introduction Anemia is a worldwide health problem (1). In developing countries, 42.3% women and 30.0% men aged 15-59 years are anemic, with half having iron deficiency anemia (IDA) [1]. Even in developed countries, 10.3% of women are anemic [1]. The prevalence of anemia in China was 15.2%, according to the national health and nutrition survey conducted in 2002 [2]. Low iron content in the diet and low bioavailability from plant-based diets are main causes of IDA. Other main causes of IDA include pregnancy, menstruation, growth and development, malaria, intestinal parasites such as hookworm, and gastrointestinal blood loss caused by infections such as Helicobacter pylori [3]. Iron fortified wheat flour and soy sauce are used in population approaches to prevent IDA [1, 3, 4]. Meat consumption has a long history in human evolution [5]. Meat provides a good source of proteins, vitamins, and minerals. Heme-iron from red meat has high bioavailability and helps to prevent anemia. Meat intake is a known enhancing factor of nonheme iron absorption [6]. Nonetheless, meat consumption is often related to high intake of fat and energy, and consequently to overweight/obesity [5, 7], diabetes [8], and cancer [9]. A recent review concludes that social class predicts diet quality and that possible mechanisms may include food prices and diet costs, food access, food environmental factors, education and culture [10]. Meat consumption, especially lean meat, is usually positively related to socioeconomic status (SES) [10-13]. Gender differences in food consumption, including meat and fruit and vegetables, are substantial due to social norms and cultural beliefs [14] and this may partly explain the disparity in morbidity between genders. China is undergoing rapid nutrition transition. Due to quick economic growth, the affordability of food increases in China. A steady increase in meat consumption has been reported since 1980’s. The mean consumption of meat among adults aged 18-45 in 1989, 1991, 1993, 1997, 2000 was 75, 85, 94, 105, 117 g/day, respectively [15]. The prevalence of anemia has been decreasing steadily during the past decades but is still high [2]. Understanding the role of meat in association to disease like anemia is important for preventive action. The objective of the study is to 1) describe sociodemographic determinants of meat consumption 2) describe the association between different types of meat consumption and anemia in Chinese adults.

Subjects and Methods Sample In 2002, China launched a national survey in nutrition and health under the approval of the Chinese Ministry of Health. The data presented in this article are based on the sample from Jiangsu province, which is one of the economically prosperous areas of China with a population of 73.6 million. Notably, together with Shanghai, this area has the highest prevalence of anemia in China according to the previous three national nutrition surveys [16]. The prevalence of anemia was 43.2% and 21.9% in men, 54.8% and 43.4% in women in 1982 and 1992, respectively [16]. A multistage cluster sampling method was used to select the participants. The participants from rural areas were recruited from 6 counties (Jiangyin, Taicang, Suining, Jurong, Sihong and Haimen) across Jiangsu province. From each of the six

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counties, three towns were randomly selected. The participants from urban areas were selected from two prefecture capital cities (Nanjing and Xuzhou). From each city prefectures, three streets were randomly selected. The six counties and two city prefectures represented a geographically and economically diverse population with gross domestic product (GDP) ranging from approximately 403 US Dollars /capita/year to 4,396 US Dollars /capita/year (mean 1,993 US Dollars) [17]. In each town/street, two villages/neighborhoods were further randomly selected. In each village/neighborhood, 30 households were randomly selected. All members in the households were invited to take part in the study. Written consent was obtained from all participants. In the present study, we excluded those who were under 20 years. The sample included 1,308 men and 1,541 women. Of these, 711 participants were from urban areas. The response rate was 89.0%.

Measurement and Interview Participants were interviewed in their homes by trained health workers using a pre-coded questionnaire. Interviews took approximately two hours to complete and included questions on diet, socio-demographic information, medical history, drug treatment, health habits such as cigarette smoking and physical activity, and other lifestyle factors. Hemoglobin (HB) and Serum Ferritin (SF) All participants were requested to provide a fasting venous blood sample, which was preserved and analyzed in the local Centers for Disease Control and Prevention (CDCs) with quality control set by the National Centre for Disease Control and Prevention. Hemoglobin (Hb) was measured by the cyanmethemoglobin method [18]. Serum ferritin was analysed by the National CDC in Beijing using a commercially available radioimmunoassay kit (Beijing North Institute of Biological Technology). Anemia was defined as Hb level below 13 g/dl for men and 12 g/dl for women [19]. Iron deficiency anemia (IDA) was defined as the presence of both anemia and a serum ferritin level <15μg/l. Dietary measurements Food and nutrient intakes were measured by three day weighed food records. Participants were instructed to undertake this for 3 consecutive days including a weekend day. Food consumption data were analyzed using the Chinese Food Composition Table [20]. Other covariates measured Height was measured without shoes, and weight was measured with light clothing. BMI was calculated as weight in kilograms divided by height squared in meters. Education was recoded into three categories from the six originally in the questionnaire: ‘Low’: illiteracy, primary school; ‘Medium’: junior middle school; ‘High’: high middle school or higher. Occupation was recoded into manual or non-manual categories based on the 12 occupations recorded in the questionnaire.

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Statistical Analyses The participants were divided into quartiles according to their intake of pork, poultry, organ meat, beef and lamb, and total meat. The chi-square test was used to compare differences between categorical variables and ANOVA was used for the continuous variables. Poisson regression was used to examine intakes of meat in relation to anemia with adjustment for potential confounders including residence, age, and education, and intakes of total energy, protein and vitamin C. The association between meat intake and anemia was also adjusted for iron intake. P values were two-sided and statistical significance was considered when p<0.05. All the analyses were performed by using STATA 10 (Stata Corporation, College Station, TX).

Results Table 1 shows characteristics of the study population by gender. Mean Hb levels were 14.3 (SD 1.7) g/dl for men and 12.7 (SD 1.5) g/dl for women. Women had a higher prevalence of anemia than men. The prevalence of anemia was 18.3% (95% Confidence Interval [CI]: 16.3-20.6) in men and 31.5% (95% CI 29.2-33.9) in women. Approximately 9% of the sample had serum ferritin (SF) <15μg/l, while 12% had SF >200μg/l. The prevalence of iron deficiency anemia was relatively low (6.3% in women and 0.7% in men). Among the anemic patients, only 29.6% had SF <30 μg/l. The prevalences of anemia across quartiles of serum ferritin from low to high were 23.9, 15.5, 17.9, and 15.8% in men; 38.6, 29.5, 27.1, and 30.3% in women. The mean intake of total meat was 101.9(SD 90.1) g/day in men and 74.9(69.6) g/day in women (Table 2). Pork and poultry were the main sources of meat consumption in the sample. The intake of all four types of meat decreased with the increase of age. SES and education were positively associated with all types of meat consumption. The mean intakes of total meat were 49.1, 90.0, and 118.7 g/day among participants with low, medium and high SES. Similar trends were found between education and all types of meat consumption. Urban residents had a higher intake of all kinds of meat, except organ meat, than rural residents. Participants from the south of the province had higher intake of all kinds meat than those in the north. Across total meat intake quartiles, there were significant differences in intake of energy, fat, protein, iron, and vegetables in both men and women (Table 3). Percentage of energy intake from fat and protein increased with the increase of meat intake. Table 4 shows the association between meat intakes and hemoglobin levels and anemia. Across the total meat intake quartiles, the prevalences of anemia were 20.6, 25.1, 29.8, and 26.2% respectively (p<0.001). The mean Hb levels were significantly different among different meat intake groups.

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Table 1. Characteristics of a sample of adults >20yrs from Jiangsu province, China, who were surveyed to determine the relation between meat consumption and anemia, by sex

Age, yrs (mean, SD) Hemoglobin, g/dl (mean, SD) Anemia (%) a Serum ferritin, μg/l (mean, SD) First quartile (mean, SD, minimum) Second quartile Third quartile Fourth quartile BMI, kg/m2 (mean, SD) Central obesity (%) b Smoking (%) Never 1-19 cigarettes/day ≥20 cigarettes /day Alcohol consumption (%) 0 times/week 1-2 times/week 3-4 times/week Daily Urban dwellers (%) Manual jobs (%) Education (%) Low Medium High SES Low Medium High a b

Men (n=1308)

Women (n=1541)

47.2(14.6) 14.3(1.7) 18.3

46.8(14.4) 12.7(1.5) 31.5

131.9(90.1) 42.2(16.6), 3.7 89.1(12.8),66.7 140.3(18.3),111.1 257.4(77.7),175.6 23.4(3.2) 19.5

71.1(71.5) 12.7(5.8),0.21 33.8(7.2),22.3 68.6(13.8),47.3 170.2(74.8),95.6 23.6(3.7) 38.2

42.9 27.8 29.3

97.3 2.1 0.5

50.7 14.3 8.7 26.3 25.2 54.1

94.2 2.3 1.2 2.2 24.7 50.4

36.0 43.5 20.5

57.5 30.1 12.4

32.3 31.9 35.8

32.4 32.7 34.8

Hemoglobin level below 13 g/dl for men and 12 g/dl for women Defined as waist circumference: men ≥90 cm, women ≥80 cm

Table 2. Intakes of meat (mean, SD, g/day) by sociodemographic factors in adults in Jiangsu, China Pork Age group 20-29 30-39 40-49 50-59 60Gender Men Women SES low medium high Education Low Medium High Residence urban rural Region South North

54.5(61.5) 49.9(55.1) 50.9(54.5) 54.4(57.8) 43.6(52.3) 57.8(61.7) 43.8(49.4)

p

<0.001

<0.001

Lamb/beef 8.1(20.0) 7.2(20.4) 10.0(27.9) 5.6(19.4) 4.8(18.3) 9.3(25.4) 5.4(18.3)

p

<0.001

<0.001

Organ meat(liver, etc) 7.3(18.2) 5.9(20.6) 6.4(21.7) 4.3(15.5) 3.7(15.7) 6.6(21.0) 4.3(16.6)

p

0.018

0.001

Poultry 33.3(52.1) 24.4(41.5) 26.4(43.8) 26.1(45.6) 15.9(33.0) 28.2(47.5) 21.3(38.3)

p

<0.001

<0.001

Total meat 103.3(89.2) 87.3(81.9) 93.6(84.8) 90.4(80.6) 68(65.2) 101.9(90.1) 74.9(69.6)

p

<0.001

<0.001

29.3(44.5) 53.8(56.8) 64.9(56.4)

<0.001

3.3(15.7) 7.0(21.9) 10.9(26.0)

<0.001

4.1(19.2) 3.8(13.4) 8.2(22.3)

<0.001

12.4(32.6) 25.4(42.1) 34.7(49)

<0.001

49.1(66.4) 90.0(75.0) 118.7(82.7)

<0.001

42.5(51.9) 54.7(56.4) 63.2(61.9)

<0.001

3.2(14.3) 9.4(25.5) 13.8(28.6)

<0.001

4.2(17.9) 6.4(19.6) 6.5(19.4)

0.007

18.0(37.3) 28.5(45.1) 34.4(50)

<0.001

68.0(70.3) 99.1(84.1) 117.9(87.4)

<0.001

55(56.1) 48.6(55.6)

<0.001

14.7(32.6) 4.7(16.2)

<0.001

5.5(17.2) 5.4(19.3)

0.853

30.8(48.1) 22.3(40.8)

<0.001

106(85.7) 81(78.1)

<0.001

67.9(59.6) 30.5(43.4)

0.008

9.3(25.3) 4.9(17.2)

<0.001

6.5(19.9) 4.1(17.5)

<0.001

34.4(48.5) 13.4(32.3)

<0.001

118.1(82.5) 52.9(63)

<0.001

Table 3. Food and nutrient intakes across total meat intake quartiles among adults in Jiangsu China 2002 Men a

Women

Q1 (n=284)

Q2(n=284)

Q3(n=328)

Q4(n=404)

1.3

48.5

100.8

4.4

13.1

0

b

P

Q1(n=424)

Q2(n=432)

Q3(n=393)

Q4(n=283)

P

211

2.9

47.3

100.4

189.3

18.7

68.2

6.3

13.7

18.3

49.8

24.9

67.5

133.4

0

23.3

68.3

133.4

20.0

66.8

133.3

507.1

23.3

66.8

133.3

366.6

2571.3(40.0)

2540.8(40)

2512.7(37.2)

2742(33.6)

<0.001

2133.3(29.1)

2055.3(28.8)

2130.6(30.2)

2274.3(35.6)

<0.001

66.9(1.6)

83.4(1.6)

88.8(1.4)

110(1.3)

<0.001

58.2(1.0)

69.5(1.0)

79.8(1.1)

93.4(1.3)

<0.001

26.1(1.1)

30.1(1.1)

32.1(0.1)

35.1(0.1)

<0.001

27.1(0.1)

32.1(0.1)

32.1(0.1)

35.1(1.1)

<0.001

72.4(0.9)

74.5(0.9)

77.2(0.9)

92.4(0.8)

<0.001

60.9(0.6)

61.5(0.6)

67.8(0.7)

78.3(0.8)

<0.001

12.1(0.1)

13.1(0.1)

13.1(0.1)

14.1(0.1)

<0.001

12.1(0.1)

13.1(0.1)

14.1(0.1)

14.1(0.1)

<0.001

30.2(0.6)

27.1(0.6)

25.9(0.5)

29.5(0.5)

<0.001

24.9(0.3)

22.3(0.3)

22.1(0.4)

24.9(0.4)

<0.001

Intake of meat (g/day) Mean SD

c

Min Max Daily food and nutrient intake d (Mean, SE e) Energy (kcal/day) Fat (g/day)

d

Fat, %E Protein (g/day)

d

Protein, %E Iron (mg/day) Vitamin C (mg/day) d

d

66.3(2.2)

62.7(2.2)

61.8(2)

66.9(1.9)

0.614

63.1(1.8)

57.3(1.7)

57.5(1.8)

65.1(2.2)

0.066

31(4.7)

34.5(4.7)

33.8(4.4)

41.4(3.9)

0.554

37.8(4.6)

51.3(4.6)

42.1(4.8)

56.6(5.7)

0.050

305.5(8.6) 266.9(8.6) 283.5(8) 300(7.2) a Q1Stands for the lowest quartile b Chi-square test for categorical variables and ANOVA for continuous variables c SD-Standard deviation d Energy adjusted e SE-Standard error

0.026

282(7.3)

245.3(7.2)

266.2(7.6)

280.5(8.9)

0.020

Fruits (g/day) d Vegetables (g/day) d

Table 4. Association between meat intakes and hemoglobin and ferritin levels among adults in Jiangsu China a

Pork intake quartiles Q1 Q2 Q3 Q4 Beef/lamb intake (g/day) 0 1-50 >=50 Organ meat intake (g/day) 0 1-50 >=50 Poultry intake (g/day) 0 1-50 >=50 Total meat intake quartiles Q1 Q2 Q3 Q4

n

Hb Mean

SD

932 503 676 702

135.7 132.6 134.2 133.8

18.4 18.3 17.9 17.5

2381 265 167

133.7 136.3 140.0

17.8 19.6 17.9

2443 254 116

134.3 132.6 138.0

1816 344 653 708 709 715 681

p

0.011

Anemia (%) 22.4 26.9 26.2 27.8

p

0.056

SF Mean

SD

92.7 91.9 101.8 109.7

85.4 81.6 84.9 89.9 85.5 87.9 89.5

<0.001

26.0 24.5 19.6

0.179

97.2 108.5 109.8

18.1 17.7 18.6

0.029

25.1 30.3 22.2

0.138

98.4 101.6 106.3

134.7 132.3 134.3

18.2 17.8 17.8

0.069

24.2 30.6 26.2

0.041

135.9 133.6 132.4 135.4

17.9 18.5 17.9 17.9

<0.001

20.6 25.1 29.8 26.2

a Chi-square test for categorical variables and ANOVA for continuous variables b High SF: SF>=175.6 ng/ml in men and SF >=95.6 ng/ml in women

0.001

p

<0.001

High SF (%) b 22.9 23.5 26.4 26.9

p

0.174

0.032

24.4 27.9 26.5

0.409

86.2 84.4 86.5

0.550

24.5 27.8 25.2

0.520

96 95.1 109.4

85.4 79.4 90.3

0.002

23.7 23.8 28.6

0.045

89.7 92.5 97.8 116.8

83 86.8 79.4 92.3

<0.001

21.1 23.7 25.0 29.7

0.002

Table 5. Multivariate model of prevalence ratio of anemia and high ferritin for meat intake in Chinese adults (n=2849) Anemia High ferritin a b Age and gender adjusted Multivariate model Age and gender adjusted Pork intake quartiles Q1 Q2 Q3 Q4 p for trend Beef intake (g/day) 0 1-50 >=50 p for trend Organ meat intake (g/day) 0 1-50 >=50 p for trend Poultry intake (g/day) 0 1-50 >=50 p for trend Total meat intake quartiles Q1 Q2 Q3 Q4 p for trend

Multivariate modelb

1 1.21(1.00-1.45) 1.24(1.05-1.48) 1.35(1.15-1.60) <0.001

1 1.09(0.90-1.31) 1.10(0.92-1.32) 1.16(0.96-1.42) 0.139

1 1.09(0.90-1.32) 1.25(1.05-1.48) 1.23(1.04-1.46) 0.005

1 0.90(0.74-1.09) 0.97(0.81-1.15) 0.91(0.76-1.10) 0.470

1 1.02(0.82-1.26) 0.86(0.63-1.18) 0.462

1 0.99(0.80-1.23) 0.92(0.67-1.27) 0.64

1 1.21(0.99-1.48) 1.20(0.92-1.55) 0.043

1 1.04(0.85-1.26) 0.94(0.72-1.22) 0.788

1 1.25(1.03-1.51) 1.00(0.71-1.41) 0.207

1 1.19(0.98-1.44) 1.09(0.77-1.53) 0.173

1 1.22(0.99-1.50) 1.10(0.80-1.51) 0.128

1 1.10(0.89-1.36) 1.08(0.79-1.49) 0.382

1 1.30(1.09-1.54) 1.18(1.01-1.37) 0.010

1 1.16(0.97-1.39) 1.06(0.91-1.24) 0.326

1 1.07(0.87-1.31) 1.30(1.12-1.50) 0.001

1 0.94(0.77-1.16) 1.12(0.96-1.30) 0.191

1 1.26(1.05-1.53) 1.54(1.28-1.84) 1.49(1.23-1.80) <0.001

1 1.20(0.99-1.46) 1.39(1.14-1.70) 1.37(1.09-1.73) <0.001

1 1.20(0.99-1.45) 1.26(1.04-1.52) 1.57(1.31-1.88) <0.001

1 1.00(0.82-1.21) 0.94(0.77-1.15) 1.09(0.88-1.36) 0.513

a high ferritin defined as fourth quartile. SF>=175.6 ng/ml in men and SF >=95.6 ng/ml in women b Multivariate Poisson model adjusted for age, gender, SES, education, urban/rural, south/north, smoking, alcohol drinking, BMI, intake of energy, fat, vegetable, and iron

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Intake of pork was negatively while intake of beef/lamb was positively associated with Hb levels. A positive association between pork, lamb/beef, and total intake but not organ meat intake and serum ferritin was found. The prevalences of high ferritin were: 21.1, 23.7, 25.0, and 29.7% from quartile 1 to quartile 4 of total meat intake. In an age and gender adjusted model, intake of pork and poultry were still significantly and positively associated with the risk of anemia as well as high ferritin (Table 5). In multivariate analysis, after adjusting for sociodemographic variables, intake of alcoholic drinks, smoking, BMI, intake of energy, fat, vegetable, and iron, the prevalence ratio of anemia across total meat intake quartiles were 1, 1.20, 1.39, 1.37(1.09-1.73) (p for trend <0.001). Intake of beef/lamb and organ meat was not associated with anemia. The association between pork, beef/lamb, poultry, and total meat consumption and high SF disappeared when adjusted for sociodemographic and dietary factors. Also in multivariate linear regression, meat intake was not associated with SF (data not shown).

Discussion In this cross sectional study we found that pork and poultry were the two main sources of meat in the population. All types of meat intake were positively related to socioeconomic position and higher in urban than in rural areas. A positive association between pork, poultry, and total meat consumption and anemia were found. There was no such association with regard to organ meat, beef and lamb. Pork, beef/lamb, poultry, and total meat intake was also positively associated with serum ferritin level/high ferritin after adjusting for age and gender. However, this association disappeared when adjusted for sociodemographic, lifestyle, and other dietary factors. Over two decades, the prevalence of anemia in China decreased from more than 30% to 15.2%. Concomitantly, meat consumption at national level increased from 34 g/day to 79 g/day in 2002 [21]. The total meat intake in the sample was comparable to the national mean intake in 2002[15]. The positive association between meat intake and socioeconomic status is consistent with findings from China and other countries [10, 15]. The urban/rural difference in meat consumption could be attributed to the differences in both availability and purchase ability between urban and rural areas. However, this difference in meat intake is not big (25grams/day), possibly because this region of China is economically well-off and even the poor in the rural areas can afford to consume meat. The positive association between education and meat consumption is also seen in less developed counties, while an opposite association is often the case in more developed countries [14]. This positive association suggests that the region is in an early stage of nutrition transition [22]. Women consumed 27 gram/day less meat than men. Similar gender difference has been found in developed countries [14]. A high prevalence of anemia together with a high level of serum ferritin was observed in the study, while the prevalence of IDA was low. The prevalence of anemia in the group with highest quartile of serum ferritin was 15.8% and 30.3% in men and women, respectively. It is common to use SF as a marker of stored body iron [23], but SF is also increased by inflammation and infection [24, 25]. Since Thalassaemia is not a problem in this area with a prevalence of 0.09% [26], it is reasonable to assume that a considerable proportion of the anemia in the population is caused by inflammation or infection. Markers of inflammation

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such as c-reactive protein, were not measured in the study, but it has been reported that the prevalence of the hepatitis B virus marker is 42.6% and chronic hepatitis B surface antigen carriage is 10.2% in various parts of China [27]. Furthermore, helicobacter pylori (H. pylori) infection is very common in the study area. A recent study in the province shows that the prevalence of H. pylori seropositive was 62% [28]. Although there is no study from the area showing the association between H. pylori infection and anemia, such association could exist based on current knowledge. The association between sociodemographic factors and H. pylori infection in the reported study is similar to the distribution of anemia by sociodemographic factors [28, 29]. We have earlier suggested that inflammation and infection may be one of the important causes of anemia in the area (30, 31). Tofu and magnesium intake has been found to be associated with lower risk of anemia in the population [30, 31]. The mechanism of this association may be due to the anti-inflammatory effects of tofu and magnesium. No association between intake of organ meat, beef and lamb and anemia was found. This could be due to the low absolute intake of these types of meat. However, the positive association between pork, poultry and anemia and high serum ferritin is in-consistent with current knowledge. Meat intake can prevent anemia in two ways: heme iron in the meat has high bioavailability; and the meat factor can enhance the bioavailability of non-heme iron in the diet. It is unlikely that our findings imply that meat consumption as such increases the risk of anemia. If so we would have seen an increase in the prevalence of anemia in the region. Since this is a cross-sectional study, we cannot assume a causal relationship. With the improvement of the economic situation, people have become more conscious of their health. The possibility that the anemic participants may have increased their meat intake as a preventive measure can be ruled out, since the majority had mild anemia and thus would be unlikely to take preventive action through dietary change or use of iron supplement to control anemia. The finding is not consistent with our previous findings on the association between food pattern and anemia. A food pattern rich in meat and alcohol was not associated with risk of anemia [29]. This suggests that there could be an interaction between meat consumption and other components in the diets, such as microorganisms or chemical residuals etc. However, the current results are consistent with a study in Canada, which showed that the prevalence of anemia found was accompanied with a high intake of meat [32]. A multifactor etiology in regard to anemia was suggested including H. pylori infection [32]. In the Chinese population, a high level of serum ferritin was found to be associated with risk of diabetes in different studies [33-35]. A recent study from Spain suggested that ferritin was not a good marker of iron store, since elevated ferritin levels in type 2 diabetes was mainly a result of inflammatory mechanisms rather than iron overload [36]. In our study, a positive association between meat consumption (regardless of types of meat) and serum ferritin (as well as high ferritin) was found in univariate analysis. However, this association disappeared in the multivariate analysis. However, we cannot exclude that there is an association, since the variables included in this analysis are highly correlated with meat consumption. The findings are difficult to interpret. Nonetheless, the positive association between total meat consumption and high serum ferritin is of concern. Although we adjusted for some covariates, residual confounding cannot be excluded. The cross-sectional study design and the lack of measurement of some potential confounding factors including inflammation markers

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as well as information on helicobacter infection imply that no conclusions on the etiological association between meat consumption and Hb concentration can be drawn. No information on pig and poultry breeding including use of hormones and antibiotics is available. Such information is important in order to exclude the possibility of anemia caused by chemical residuals in meat. In conclusion, socioeconomic conditions were positively associated with meat consumption among Chinese in Jiangsu province. Furthermore, pork, poultry, and total meat consumption was significantly and positively associated with the risk of anemia, concomitantly with high serum ferritin. Further study is needed to investigate the association between inflammation and infection and iron status in the area. Iron supplementation or dietary intervention cannot be justified without a clear understanding of the etiology of anemia in the area.

References [1]

WHO. Iron Deficiency Anaemia. Assessment, Prevention and Control: A Guide for Programme Managers. WHO/NHD/01.3; 2001. [2] Li L, Rao K, Kong L, Yao C, Xiang H, Zhai F, et al. A description on the Chinese national nutrition and health survey in 2002. Zhonghua Liu Xing Bing Xue Za Zhi. 2005 Jul;26(7):474-84. [3] Zimmermann MB, Hurrell RF. Nutritional iron deficiency. Lancet. 2007 Aug 11;370(9586):511-20. [4] Mannar V, Gallego EB. Iron fortification: country level experiences and lessons learned. J Nutr. 2002 Apr;132(4 Suppl):856S-8S. [5] Larsen CS. Animal source foods and human health during evolution. J. Nutr. 2003 Nov;133(11 Suppl 2):3893S-7S. [6] Hallberg L, Rossander L. Improvement of iron nutrition in developing countries: comparison of adding meat, soy protein, ascorbic acid, citric acid, and ferrous sulphate on iron absorption from a simple Latin American-type of meal. Am. J. Clin. Nutr. 1984 Apr;39(4):577-83. [7] WHO/FAO. Diet, nutrition and the prevention of chronic diseases : report of a Joint WHO/FAO Expert Consultation. Geneva: World Health Organization; 2003. [8] Bantle JP, Wylie-Rosett J, Albright AL, Apovian CM, Clark NG, Franz MJ, et al. Nutrition recommendations and interventions for diabetes: a position statement of the American Diabetes Association. Diabetes Care. 2008 Jan;31 Suppl 1:S61-78. [9] Sandhu MS, White IR, McPherson K. Systematic review of the prospective cohort studies on meat consumption and colorectal cancer risk: a meta-analytical approach. Cancer Epidemiol Biomarkers Prev. 2001 May;10(5):439-46. [10] Darmon N, Drewnowski A. Does social class predict diet quality? Am. J. Clin. Nutr. 2008 May;87(5):1107-17. [11] Shi Z, Lien N, Kumar BN, Holmboe-Ottesen G. Socio-demographic differences in food habits and preferences of school adolescents in Jiangsu Province, China. European journal of clinical nutrition. 2005 Dec;59(12):1439-48.

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[12] Perrin AE, Simon C, Hedelin G, Arveiler D, Schaffer P, Schlienger JL. Ten-year trends of dietary intake in a middle-aged French population: relationship with educational level. European journal of clinical nutrition. 2002 May;56(5):393-401. [13] Hulshof KF, Brussaard JH, Kruizinga AG, Telman J, Lowik MR. Socio-economic status, dietary intake and 10 y trends: the Dutch National Food Consumption Survey. European journal of clinical nutrition. 2003 Jan;57(1):128-37. [14] Prattala R, Paalanen L, Grinberga D, Helasoja V, Kasmel A, Petkeviciene J. Gender differences in the consumption of meat, fruit and vegetables are similar in Finland and the Baltic countries. Eur. J. Public Health. 2007 Oct;17(5):520-5. [15] Zhai F, Wang H, Du S, He Y, Wang Z, Ge K, et al. Lifespan nutrition and changing socio-economic conditions in China. Asia Pacific journal of clinical nutrition. 2007;16 Suppl:374-82. [16] Chang S, Ge K, Zhai F, Jia F, Xu X. The analysis of nutritional factors on anemia in Chinese adults. Acta Nutrimenta Sinica. 1998;20(2):132-7. [17] Jiangsu Bureau of statistics. Statistical yearbook of Jiangsu. Beijing: China Statistics Press; 2002. [18] Dallman PR. Diagnosis of anemia and iron deficiency: analytic and biological variations of laboratory tests. Am. J. Clin. Nutr. 1984 Jun;39(6):937-41. [19] WHO. Nutritional anaemias. Report of a WHO scientific group. World Health Organ Tech. Rep. Ser. 1968;405:5-37. [20] Yang Y. Chinese Food Composition Table 2004. Beijing: Peking University Medical Press; 2005. [21] Zhai FY, He YN, Ma GS, Li YP, Wang ZH, Hu YS, et al. Study on the current status and trend of food consumption among Chinese population. Zhonghua Liu Xing Bing Xue Za Zhi. 2005 Jul;26(7):485-8. [22] Popkin BM, Gordon-Larsen P. The nutrition transition: worldwide obesity dynamics and their determinants. Int. J. Obes. Relat Metab. Disord. 2004 Nov;28 Suppl 3:S2-9. [23] Cook JD, Lipschitz DA, Miles LE, Finch CA. Serum ferritin as a measure of iron stores in normal subjects. Am. J. Clin. Nutr. 1974 Jul;27(7):681-7. [24] Pan Y, Jackson RT. Ethnic difference in the relationship between acute inflammation and serum ferritin in US adult males. Epidemiol. Infect. 2007 Mar 22:1-11. [25] Baynes RD. Assessment of Iron Status. Clinical Biochemistry. 1996;29(3):209-15. [26] Zeng YT, Huang SZ. Disorders of haemoglobin in China. J. Med. Genet. 1987 Oct; 24(10): 578-83. [27] Yao GB. Importance of perinatal versus horizontal transmission of hepatitis B virus infection in China. Gut. 1996;38 Suppl 2:S39-42. [28] Shi R, Xu S, Zhang H, Ding Y, Sun G, Huang X, et al. Prevalence and risk factors for Helicobacter pylori infection in Chinese populations. Helicobacter. 2008 Apr; 13(2): 157-65. [29] Shi Z, Hu X, Yuan B, Pan X, Dai Y, Holmboe-Ottesen G. Association between dietary patterns and anaemia in adults from Jiangsu Province in China. Br. J. Nutr. 2006; 96: 906-12. [30] Shi Z, Hu X, He K, Yuan B, Garg M. Joint association of magnesium and iron intake with anemia among Chinese adults. Nutrition. 2008 Jun 27;24(10):977-84.

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[31] Shi Z, Hu X, Yuan B, Pan X, Dai Y, Holmboe-Ottesen G, et al. Strong Negative Association between Intake of Tofu and Anemia among Chinese Adults in Jiangsu, China. J. Am. Diet Assoc. 2008 Jul;108(7):1146-53. [32] Jamieson JA, Kuhnlein HV. The paradox of anemia with high meat intake: a review of the multifactorial etiology of anemia in the Inuit of North America. Nutr. Rev. 2008 May;66(5):256-71. [33] Luan DC, Li H, Li SJ, Zhao Z, Li X, Liu ZM. Body iron stores and dietary iron intake in relation to diabetes in adults in North China. Diabetes Care. 2008 Feb;31(2):285-6. [34] Shi Z, Hu X, Yuan B, Pan X, Meyer HE, Holmboe-Ottesen G. Association between serum ferritin, hemoglobin, iron intake, and diabetes in adults in Jiangsu, China. Diabetes Care. 2006;29(8):1878-83. [35] Sun L, Franco OH, Hu FB, Cai L, Yu Z, Li H, et al. Ferritin concentrations, metabolic syndrome, and type 2 diabetes in middle-aged and elderly Chinese. J. Clin. Endocrinol. Metab. 2008 Sep 16. [36] Lecube A, Hernandez C, Pelegri D, Simo R. Factors accounting for high ferritin levels in obesity. International journal of obesity. 2008 Sep 9.

SHORT COMMUNICATIONS

In: Food Science and Security Editors: L. Amsel and L. Hirsch, pp. 283-294

ISBN: 978-1-60692-977-3 © 2009 Nova Science Publishers, Inc.

GAS CHROMATOGRAPHY-OLFACTOMETRY: AN EFFICIENT TOOL FOR THE MONITORING OF SEAFOOD QUALITY Vincent Varlet1 and Xavier Fernandez Université de Nice-Sophia Antipolis Laboratoire de Chimie des Molécules Bioactives et des Arômes UMR 6001 CNRS-UNSA, Faculté des Sciences, France

Abstract The four main groups of gas chromatography-olfactometric (GC/O) methods were presented, i.e. dilution analysis methods, detection frequency methods, time-intensity methods and posterior intensity methods. A compilation of the applications of gas chromatographyolfactometry in seafood odour analysis was carried out. The interpretations of the results obtained with these different methods applied to seafood matrices allowed to consider olfactometry as an efficient tool for the monitoring of seafood quality from organoleptic, sanitary and nutritional point of views. The quantity and the nature of odorant volatile compounds determined by this analytical method can provide reliable indicators on the freshness and the stability of such reactive food.

Introduction Gas chromatography is the technique of choice for volatile compounds analysis. They are commonly detected by chemical detectors which provide relevant information about their quantity and nature such as flame ionisation detector (FID) and mass spectrometric detector (MS). However, these detectors are not very useful for odour evaluation and not as efficient as the human nose. Indeed, even if the technology of electronic noses has been improved (Jonsdottir et al., 2004 ; Olafsdottir et al., 2005), these electronic detectors give only 1

E-mail address: [email protected], Fax : 0033 492 07 61 25. Corresponding author: Université de NiceSophia Antipolis Laboratoire de Chimie des Molécules Bioactives et des Arômes UMR 6001 CNRS-UNSA, Faculté des Sciences, FRANCE, 28, avenue Valrose, 06108 NICE Cedex 2, FRANCE.

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information on the intensity and not about the odour evalutation. Olfactometry was developed to investigate odorant compounds with human nose detection among volatile molecules identified by gas chromatography. The principle of Gas Chromatography-Olfactometry (GC/O) currently used today is not very different from its conception in 1964 (Fuller et al., 1964). It consists in the assessment by a judge of the perceived odour by sniffing of gas chromatographic effluent. However, important improvements of the gas chromatographic techniques have been carried out and particularly the possibility to acquire simultaneously several signals from several detectors after the chromatographic separation of the GC effluent. From an aromatic extract, it is now possible to obtain simultaneously the quantity (by FID detection), the identity (by MS detection) and odorant characteristics (by olfactometry), mainly aromatic note and odorant intensity (Varlet et al., 2006) Similarly, optimisations of olfactometric conditions have led to better reliability of olfactometric results. Indeed, all the actual GC/O analytical devices are equipped with a humidified air system to reduce nasal dehydratation during sniffing which result in a gain of reproducibility and a better comfort of the assessor (Dravnieks et al., 1971 ; Delahunty et al., 2006). Moreover, to increase the relevance and the intensity of odorant volatile compounds, the assessment by several judges is required. Until now, the same GC/O run was repeated and subjected to the evaluation of the judges. However, recent systems have allowed to avoid these repetitions and to obtain the evaluations of eight judges in a single GC/O by the split 8:1 of the GC effluent (Berdagué et al., 2004 and 2005).

Olfactometric Methods Several techniques have been developed over the last decades to collect and process GC/O data. According to the number, the composition and the training of the panellists, various olfactometric methods can be used. They can be sorted in four families: the dilution analysis methods, the detection frequency methods, the time-intensity methods and the posterior intensity methods. Qualitative olfactometric (QO) or GC/Sniffing, methods have been also used and consist in a qualitative description of the odours perceived but they are not very precise and reproductible.

Dilution Analysis Methods In dilution analysis, an aromatic extract is diluted, and each dilution is sniffed by the judges until there are no longer any odours detected (Van Ruth et al., 2001). Two dilution analysis methods were developed: Aromatic Extract Dilution Analysis (AEDA) in 1987 (Ullrich et al., 1987) and Combined Hedonic Aroma Response Measurement (CHARM) in 1984 (Acree et al., 1984). In AEDA, the odorant potency of odour-active compounds is illustrated by a dilution factor (FD factor) which represents the last dilution at which an odorant volatile compound is perceived. In CHARM, the durations of each odorant stimulus perceived for all the tested dilutions are taken into account and are graphed to yield a chromatogram illustrating the relative odour potency perceived during the GC/O run. These techniques were especially used to determine the most potent odorant volatile compounds in the aroma of an odorant matrix. The main criticism which can be formulated

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against these techniques is their contradiction with the psychophysical theories of odorant perception. Indeed, several authors have demonstrated the absence of linearity between the concentration of an odorant volatile compound and its odorant intensity (Le Guen et al., 2001a). As these methods are very time-consuming, a weaker number of judges is used. However, it increases the subjectivity of each judge in the final assessment. Finally, phenomena of “dilution hole” can appear in the assessment: a judge might not perceive an odour at a certain dilution and perceive it at a more important dilution. In order to obtain more reliable results less dependant of the judges, other methods as the frequency-of-detection method was developed.

Frequency-of-Detection Methods The frequency-of-detection techniques (FDT) use the number of judges who have perceived an odour at the same retention time of the GC/O run as an indicator of the odorant intensity of eluted compounds (Linssen et al., 1993). However, a minimal number of eight assessors is required and detection of an odour by a number of judges fewer than three is commonly considered as noise (Varlet et al., 2006). An aromagram is obtained giving the number of judges who have detected an odour according to the retention time. Another frequency-of-detection technique consists in the conversion of the heights of the frequency-of-detection peaks in Nasal Impact Frequency (NIF). The highest frequency-ofdetection peak corresponds to 100 % of the NIF. It is also possible to convert the areas of frequency-of-detection peaks in Surface of Nasal Impact Frequency (SNIF). The most important area of frequency-of-detection peak represents 100 % of the SNIF. These methods allow to obtain results independent from the number of judges but a minimal number of eight assessors is required (Pollien, P. et al., 1997 ; Debonneville, C. et al., 2002). These methods appear as the most in adequation with the psychophysical theories of human olfaction and the results are more reliable because the contribution of each judge corresponds to 1/n of the final result (n representing the number total of judges).

Posterior Intensity Detection Methods Posterior Intensity Detection (PID) methods consist in the assessment by the judges of the perceived odorant intensity after the detection of the odour. The scores are evaluated on a scale on which the assessors must be previously trained to avoid too large variability because if untrained, the use of the scale can differ considerably among assessors (Van Ruth, 2001). As result, these methods are not often employed individually but they can be used in complement of other olfactometric techniques (Varlet et al., 2007a and 2007b). However, they constitute a reliable source of information about the aromatic note and the odorant intensity of odorant volatile compounds.

Time-Intensity Methods Time intensity methods (TIM) constitute a direct measurement of the odorant intensity. The aromatic extract is evaluated only at a single dilution. The assessors record the beginning,

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the maximum and the end of the odorant perception during the GC/O run. An automatised version of this principle was developed under the name of OSME (which means olfaction in Greek) (McDaniel et al., 1989). This technique allows to obtain in real time a single aromagram of odorant intensity and to compile these chromatograms in an osmegram (Van Ruth, S.M., 2001). The main disadvantage of this technique is the subjectivity introduced by the judge assessment and the chromatographic optimisation. Indeed, this method requires trained panellists which must participate in the evaluation of short GC/O runs in order to remain alert.

Applications of Olfactometry in Seafood and Freshwater Food Odour Analysis Seafood products constitute very reactive food matrices. Therefore, the assessment of the odour is the main indicator for the consumer to assess the freshness of the product. Gas chromatography-olfactometry appears as very adequate technique to characterise the odorant volatile compounds of seafood matrices. Indeed, the treatment of olfactometric results allows to investigate seafood odour and quality further than the identification of odour-active compounds. Table 1. Olfactometric studies performed on shellfishes Shellfish

GC/O method FDT

Mussels

AEDA, TI and FDT FDT FDT FDT, TI

Oysters

FDT, TI AEDA FDT

Sea fig

Clams

CHARM, TI QO AEDA

Objectives of the studies References Characterisation of two types of mussels by their odorant volatile compounds Le Guen et al., 2001b compositions Comparison of olfactometric methods Identification of odorant volatile compounds according to their origin Characterisation of desalinisation process Chracterisation of the relationships between the aromatic profiles and the odorant volatile compounds composition Characterisation of lipid addition in the diet Characterisation of the odorant potential of oyster cooker effluent Identification of odorant volatile compounds Identification of odorant volatile compounds Identification of odorant volatile compounds Identification of odorant volatile compounds, Study of storage

Le Guen et al., 2000a Le Guen et al., 2000b Cros et al., 2004 Pennarun, et al., 2002 Pennarun et al., 2003 Kim et al., 2000 Piveteau et al., 2000 Senger-Emonnot et al., 2006 Tanchotikul et al., 1991 Sekiwa et al., 1997

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Table 2. Olfactometric studies performed on crustaceans Crustacean

Lobster

Crab

GC/O method AEDA AEDA AEDA AEDA QO

Crayfish

QO TI

Shrimps

AEDA AEDA

Objectives of the studies Identification of odorant volatile compounds Identification of odorant volatile compounds Identification of odorant volatile compounds Comparison of odorant volatile compounds of different parts of crab Identification of odorant volatile compounds Identification of odorant volatile compounds Characterisation of enzymatic hydrolysis Identification of odorant volatile compounds Characterisation of roasting process

References Lee et al., 2001 Cadwallader et al., 1995 Chung et al., 1994 Chung et al., 1995 Tanchotikul et al., 1989 Vejaphan et al., 1988 Baek et al., 1996 Tachihara et al., 2004 Ishizaki et al., 2005

Gas chromatography-olfactometry was firstly developed to characterise odorant volatile compounds in odorant matrices. The aroma compounds of shellfishes such as oysters (Piveteau et al., 2000), sea fig (Senger-Emonnot et al., 2006) or clams (Tanchotikul et al., 1991) were determined by various GC/O methods (Table 1). The odour-active compounds of crustaceans such as lobster (Cadwallader et al., 1995 ; Lee et al., 2001), crab (Chung et al., 1994) or freshwater crayfish (Vejaphan et al., 1988 ; Tanchotikul et al., 1989) were also analysed by olfactometry (Table 2). Finally, GC/O was also carried out to investigate the odorant volatile compounds of fishes as turbot (Prost et al., 1998), trout (Selli et al., 2007) or cod (Jonsdottir et al., 2004) and related products such as tuna sauce (Cha et al., 1998) or fish sauce (Fukami et al., 2002) (Table 3). However, according to the state of the matrix, the monitoring of odour-active compounds has allowed to characterise an industrial process or a storage. The odorant differences between raw and processed or stored matrices were carried out on salmon (Varlet et al., 2006) and mackerel (Alasalvar et al., 1997). Moreover, the efficiency of enzymatic hydrolysis of crayfish (Baek et al., 1996), desalinisation process of mussel juices (Cros et al., 2004), salting-drying process of herring (Chung et al., 2007), ripening of anchovy (Triqui et al., 1995a, 1995b, 1997 and 1999), salting-fermentation of anchovy (Cha et al., 1997), smoking processes of salmon (Varlet et al., 2007a and 2007b) have been also studied by GC/O techniques. The results obtained have provided important information to better control industrial processes.

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Herring

Sardine

Smoked salmon

Anchovy

Cod

Trout

GC/O method

Objectives of the studies Identification of odorant volatile AEDA, FD and compounds, Salting-Drying process PID characterisation TI Characterisation of storage conditions Chracterisation of the relationships between the aromatic profiles and the AEDA odorant volatile compounds composition during the storage Identification of odorant volatile QO compounds during cold storage Chracterisation of the relationships Electronic nose, between the aromatic profiles and the QO, PID odorant volatile compounds composition Identification of odorant volatile FDT and PID compounds of raw and smoked salmon Comparison of odorant volatile FDT and PID compounds between different smoked salmons TI Characterisation of ripening process AEDA Characterisation of ripening process AEDA Characterisation of ripening process Characterisation of odorant volatile TI compounds of salted-fermented anchovy AEDA Characterisation of ripening process Identification of odorant volatile QO, electronic compounds generated by nose microoraganisms during cold storage QO, electronic Identification of odorant volatile nose compounds AEDA Characterisation of storage conditions Identification of odorant volatile AEDA compounds during storage Detection of off-flavours during the AEDA storage Characterisation of lipid addition in the FDT diet Identification of off-flavour volatile FDT, TI compounds Detection of off-flavours during the AEDA storage AEDA Characterisation of storage conditions

References Chung et al., 2007 Prost et al., 2004 Triqui et al., 2003 Jørgensen et al., 2001 Jonsdottir et al., 2008 Varlet et al., 2006 Varlet et al., 2007a Triqui et al., 1995a Triqui et a1., 1995b Triqui et al., 1997 Cha et al., 1997 Triqui et al., 1999 Olafsdottir et al., 2005 Jonsdottir et al., 2004 Milo et al.., 1996 Milo et al., 1997 Milo etal., 1995 Sérot et al., 2002 Selli et al., 2006 Milo et al., 1995 Milo et al., 1993

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Table 3. Continued Fish

GC/O method QO

Salmon

QO AEDA

Sauce de poisson Turbot

AEDA FDT, AEDA FDT

Tuna sauce

AEDA

Hake

AEDA

Mackerel

TI

Catfish

FDT, TI

Carp

AEDA

Objectives of the studies Identification of odorant volatile compounds and the roles of their precursors Identification of odorant volatile compounds during cold storage Characterisation of storage conditions Identification of odorant volatile compounds Identification of odorant volatile compounds Characterisation of lipid addition in the diet Identification of odorant volatile compounds Characterisation of storage conditions Comparison of odorant volatile compounds between raw and stored mackerel Characterisation of the rearing conditions influences on odorant volatile compounds Characterisation of methionine addition in the diet

References Josephson et al., 1991 Refsgaard et al., 1998 Milo et al., 1996 Fukami et al., 2002 Prost et al., 1998 Sérot et al., 2001 Cha et al., 1998 Triqui, 2006 Alasalvar et al., 1997

Hallier et al., 2005 Schlüter et al., 1999

GC/O is also very helpful to determine the origin of the seafood analysed. The authentification of food material by the quantification of odorant volatile compounds thanks to gas chromatography coupled to olfactometry and chemical detectors has permitted to inform about the geographic origins of mussels (Le Guen et al., 2000b) or fishes (Chung et al., 2007), the age of fishes (Chung et al., 2007) and seafood purchasers (Jonsdottir et al., 2008). The nature and quantities of odorant volatile compounds in different parts of crab were also analysed (Chung et al., 1995). Thanks to a GC/O analysis, it was possible to determine in the final product the initial anatomic part of seafood raw material. Important improvements of knowledge about seafood storage conditions were done thanks to olfactometric results obtained on shellfishes (Sekiwa et al., 1997) or fishes as sardine (Prost et al., 2004), cod (Milo et al., 1996 and 1997), trout (Milo et al., 1993), salmon (Milo et al., 1996) and hake (Triqui et al., 2006). Indeed, the evaluation of freshness at a certain time or during storages and the assessment of different thermal storage conditions were performed by GC/O monitoring of odorant volatile compounds on smoked salmon (Jørgensen et al., 2001), salmon (Refsgaard et al., 1998), cod (Milo et al., 1995), and trout (Milo et al., 1995 ; Selli et al., 2006), Besides, thanks to the quantification of odour-active compounds, it was possible to study the influence of supplementation in seafood diet. It has been shown that variations of feed could be responsible for variations in overall final odour and odorant volatile compound compositions on turbot (Serot et al., 2001), trout (Serot et al., 2002), freshwater carp (Schlüter

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et al., 1999) and oysters (Pennarun et al., 2003). GC/O has allowed to identify the aromatic precursors of these kinds of seafood and to give reliable indicators to obtain required final overall odour. Olfactometric results were also useful to differentiate rearing conditions of freshwater European catfish (Hallier et al., 2005). Olfactometric results are also of major importance for the seafood quality control. Indeed, the concentrations of odorant compounds deriving from fatty acids through lipid oxidation can enlighten about the state of oxidation of the food (Josephson et al., 1991). Indeed, high amounts of lipid oxidation products illustrate a rancid overall odour, an oxidised aspect and nutritional loss by the oxidation of fatty acids. Similarly, once the odorant volatile compounds deriving from microbial activity identified, it becomes possible to investigate the spoilage of seafood (Olafsdottir et al., 2005). The latest studies applying olfactometry on seafood have related the odorant volatile compounds concentrations to the sensory analysis of the overall odour, in order to identify the most potent odorants and the roles that they play in the overall odour. The relationships of smoked salmon odour and its composition in odour-active compounds were investigated by Partial Least Squares (Varlet et al., 2007b ; Jonsdottir et al., 2008). Attempts of explanation of relationships had already been carried out on mussels (Le Guen et al., 2001), oysters (Pennarun et al., 2002) or anchovy (Triqui et al., 2003) but only with descriptive statistical tools.

Conclusion Gas chromatography-olfactometry is a good method to increase seafood quality. According to the extraction method (depending on the physical matrix state and its biochemical composition) and the objectives of the study, several olfactometric techniques are available. Olfactometric results have been employed for several decades to improve the industrial processes of seafood technology such as conditions of storage. Biochemical parameters such as pH, and the determination of odorant volatile compounds by GC/O coupled simultaneously with physico-chemical detectors constitute reliable indicators which appear strong enough to be used to optimise analytical methods.

References Acree, T.E., Barnard, J., Cunningham, D. (1984). A procedure for the sensory analysis of gas chromatographic effluents. Food Chem., 14, 273-286. Alasalvar, C., Quantick, P.C., Grigor, J.M. Aroma compounds of fresh and stored mackerel (Scomber scombrus). In Flavor and lipid chemistry of seafoods ; Shahidi, F., Cadwallader, K.R., Eds ; ACS Symp.ser. 674 ; American Chemical Society : Washington, DC, 1997 ; pp 39-54. Baek, H.H., Cadwallader, K.R. (1996). Volatile compounds in flavour concentrates produced from crayfish-processing byproducts with and without protease treatment. J. Agric. Food Chem., 44, 3262-3267. Berdagué, J.L., Tournayre, P. (2004).Dispositif d’analyse par chromatographie en phase gazeuse olfaction et procédé. Brevet FR0350257.

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Josephson, D.B., Lindsay, R.C., Stuiber, D.A. (1991). Influence of maturity on the volatile aroma compounds from fresh Pacific and Great Lakes salmon. J. Food Sci., 56(6), 15761579. Kim, D.S., Baek, H.H., Ahn, C.B., Byun, D.S., Jung, K.J., Lee, H.G., Cadwallader, K.R., Kim, H.R. (2000). Development and characterization of a flavouring agent from oyster cooker effluent. J. Agric. Food Chem., 48, 4839-4843. Lee, G.H., Suriyaphan, O., Cadwallader, K.R. (2001). Aroma components of cooked tail meat of American lobster (Homarus americanus). J. Agric. Food Chem., 49, 4324-4332. Le Guen, S., Prost, C., Demaimay, M. (2000a). Characterization of odorant compounds of mussels (Mytilus edulis) according to their origin using gas chromatography-olfactometry and gas chromatography-mass spectrometry. J. Chromatogr. A, 896, 361-371. Le Guen, S., Prost, C., Demaimay, M. (2000b). Critical Comparison of Three Olfactometric Methods for the Identification of the Most Potent Odorants in Cooked Mussels (Mytilus edulis). J. Agric. Food Chem., 48(4), 1307-1314. Le Guen, S., Prost, C., Demaimay, M. (2001a). Evaluation of the representativeness of the odor of cooked mussel extracts and the relationship between sensory descriptors and potent odorants. J. Agric. Food Chem., 49, 1321-1327. Le Guen, S. (2001b). Caractérisation de l’arôme des moules (Mytilus edulis) par analyse sensorielle, chromatographique et olfactométrique. Thèse de doctorat, Université de Nantes, Faculté des sciences et des techniques. Linssen, J.P.H., Janssens, J.L.G.M., Roozen, J.P., Posthumus, M.A. (1993). Combined gas chromatography and sniffing port analysis of volatile compounds of mineral water packed in laminated packages. Food Chem., 8, 1-7. McDaniel, M.R., Miranda-Lopez, R., Watson, B.T., Michaels, N.J., Libbey, L.M. (1989). Pinot noir: a sensory/gas chromatographic approach. In Flavors and off-flavors; Charalambous,G., Ed.; Elsevier Science B.V.: Amsterdam. Milo, C., Gosch, W. (1993). Changes in the odorants of boiled trout (Salmo fario) as affected by the storage of the raw material. J. Agric. Food Chem., 41, 2076-2081. Milo, C., Grosch, W. (1995). Detection of odor defects in boiled cod and trout by gas chromatography-olfactometry of headspace samples. J. Agric. Food Chem., 43, 459-462. Milo, C., Grosch, W. (1996). Changes in the odorants of boiled salmon and cod as affected by the storage of the raw material. J. Agric. Food Chem., 44, 2366-2371. Milo, C., Grosch, W. Potent odorants in boiled cod as affected by the storage of raw material. In Flavor and lipid chemistry of seafoods ; Shahidi, F., Cadwallader, K.R., Eds ; ACS Symp.ser. 674 ; American Chemical Society : Washington, DC, 1997 ; pp 110-119. Olafsdottir, G., Jonsdottir, R., Lauzon, H.L., Luten, J., Kristbergsson, K. (2005). Characterization of volatile compounds in chilled cod (Gadus morhua) fillets by gas chromatography and detection of quality indicators by an electronic nose. J. Agric. Food Chem., 53, 10140-10147. Pennarun, A.L., Prost, C., Demaimay, M. (2002). Identification and origin of the characterimpact compounds of raw oyster Crassostrea gigas. J. Sci. Food Agric., 82, 1652-1660. Pennarun, A.L., Prost, C., Haure, J., Demaimay, M. (2003). Comparison of Two Microalgal Diets. 2. Influence on Odorant Composition and Organoleptic Qualities of Raw Oysters (Crassostrea gigas). J. Agric. Food Chem., 51(7), 2011 – 2018.

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In: Food Science and Security Editors: L. Amsel and L. Hirsch, pp. 295-303

ISBN: 978-1-60692-977-3 © 2009 Nova Science Publishers, Inc.

DETERMINATION OF INORGANIC ARSENIC IN SEAFOOD BY HG-ICP-OES AFTER CONVENTIONAL WET DIGESTION Khalid Boutakhrit1, Fabien Bolle and Leo Goeyens Scientific Institute of Public Health, Section of Food, rue Juliette wytsman 14, 1050 Brussels, Belgium

Abstract A method for the determination of total and inorganic arsenic in seafood by inductively coupled plasma atomic emission spectrometry with continuous hydride generation is described. Several analytical parameters have been investigated and optimised (recovery, precision, accuracy and limit of detection). A practical detection limit of 3.6 µg/kg fresh weight for As is reached. The precision of the method, expressed as relative standard deviation (RSD), ranges from 2.7 to 3.7 % and the recovery percentage ranges from 98.4 to 101.8 %. Various procedures for the mineralisation or extraction of inorganic arsenic were tested; acid digestion with nitric acid at 180°C was retained as a best compromise. The interference of some arsenical compounds, present in seafood, was studied as arsenobetaine (present at more than 90% of total As), monomethylarsonic acid and dimethylarsinic acid. Real samples of seafood from local market and reference materials of seafood were analysed and the obtained values agree well with that given in the literature by other authors. Despite the high levels of total arsenic found, the inorganic arsenic, highly toxic form, was present in percentages lower than 10% of total As, thus, there is no direct hazard for human health due to the consumption of these fish products.

Introduction Chronic arsenic (As) exposure places people at risk to multiple adverse health effects. As is a recognized human carcinogen [1,2] and, moreover, several recent studies have shown As

1

E-mail address: [email protected].

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to be a potent endocrine disruptor, altering hormone-mediated cell signalling at very low concentration [3,4]. The majority of harmful exposure comes from drinking water, that contains primarily inorganic As. Other major sources are food, soil and air, with most people in fact being exposed to dietary organic As-forms. The inorganic As species arsenite or As(III) and arsenate or As (V) are generally held to be more acutely toxic. Moreover, As has a rich organic chemistry and this is reflected in the large diversity of identified organocompounds [5]. Methylated As species such as monomethylarsonic acid (MMA) and dimethylarsenic acid (DMA) are relatively non-toxic and are believed to contribute little to the overall risk associated with As exposure. Some researchers argue, however, that the organic metabolites may be the ultimate carcinogens. DMA has been shown to induce bladder cancer and to promote tumor development in other organs in rodents eg. [6]. Human dietary exposure to As is very often related to the consumption of seafood. It is known that marine organisms exhibit highly variable As profiles [7,8]. Arsenosugars are present in algae, whereas arsenobetaine (AB) is absent [9]. Otherwise, AB is the predominant species in fish and crustaceans [10] and bivalves contain both AB and arsenosugars [11]. Mostly, total As concentrations in marine food items are high and, therefore, they were considered a principal public health concern. Speciation analyses evidenced that merely a small fraction was in the inorganic form and several laboratory approaches to distinguish the inorganic As fraction or to specify the different As species were published in the scientific literature. As a safeguard for public health maximum permissible concentrations of metals in food were set to limit the dietary exposure. Since it is nowadays well accepted that also the chemical forms must be considered when assessing health effects, the separation, identification and quantification of different fractions become more important [5,12]. The present study focuses on the distinction between the inorganic and total As concentrations. It aims at the optimisation and validation of a simple and economic determination of the inorganic As fraction in marine matrices and illustrates its feasibility by a local market survey.

Materials and Methods Basically, the determination of both total and inorganic As relies on the reduction of As (V) into As (III), the subsequent conversion of As into AsH3 and atomization in the argon plasma [13]. The instrument used is a Perkin-Elmer ICP Optima 4300 DV inductively coupled Ar plasma optical emission spectrometer (Norwalk, CT, USA), equipped with an autosampler (AS 93 plus, PE) and with a hydride generation (HG) system (FIAS 400 PE) with two peristaltic pumps and regulated gas supply. The gas flow rate was set to 40 – 250 mL/min. Prior to HG, sample for total arsenic determination were mineralised in an open digestion system (2040 Digestor, Foss Tecator) for 40 test tubes (quartz, 100 mL), with air cooled condensers and built-in electronic temperature and time control [13]. Samples for inorganic arsenic were digested in a drying oven (Binder FD 115, Tuttlingen, Germany), equipped with thermostatisation and an integrated timer. Ultrasonic extractions were performed using a Branson 5510 E-DTA ultrasonic cleaner (Danbury, CT, USA) and extracts were isolated using an Eppendorf 5810 R centrifuge (Hambourg, Germany).

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All reagents and standards were prepared as before [13]. The certified reference materials (CRM) that were for this study include a dogfish muscle (DORM-2), dogfish liver (DOLT-3), lobster hepatopancreas (TORT-2), and non-defatted lobster hepatopancreas (LUTS-1), purchased from the National Research Council of Canada (NRCC, Ottawa, Ontario). BCR 278R (mussel tissue) and BCR 422 (Cod muscle) were purchased from the Institute for Reference Materials and Measurement (IRMM, EC, Geel, Belgium). Additionally, canned fish from a proficiency testing exercise (Crab fresh meat, FAPAS Series 7, Round 55, CSL, York, UK) was also used. Several natural seafood samples were purchased at local market. All CRMs and samples were homogenized by mechanical mixing in a high speed Blender and then frozen at –20°C.

Results and Discussion Optimisation of the Inorganic as Extraction A variety of methods from the literature [14-18] was tested for the extraction and solubilization of total As. Extraction with different methods: water or methanol : acetic acid mixture proved often insufficient. Only the methanol : water mixture (1:9,v:v) evidences sufficient extraction efficiency, with 74% of total arsenic being extracted from TORT-2 (table 1). More aggressive methods to solubilize the arsenic of TORT-2 were investigated using nitric and hydrochloric acid with variable concentration and by sonication for 1h at 40°C. The extraction efficiency ranged from 76-83% of the total arsenic content in TORT-2, with maximal extraction efficiency being obtained with HCl (0.5M). The remaining arsenic may be bound to thiol groups of proteins or may correspond to species such as arsenolipids that are not soluble [19]. The solubilization with nitric acid produces much foam when analysing inorganic As with HG-ICP-AES and this generates a technical problems, for concentration ranging from 1-5 M, an efficiency of 80% was observed. Therefore, HCl was extended to other CRMs and optimising parameters were performed such as the acid concentration, the sonicating time and temperature of the solubilisation. The extraction efficiencies in the six investigated fish species (CRMs) ranged from 74-98% of the total arsenic (table 2). As a result, hydrochloric acid extraction (0.5M) is better than methanol or nitric acid solubilisation, however, the extraction of inorganic arsenic is not quantitative especially for fatty matrix as FAPAS S7R55 (crab meat). These low efficiencies are in accordance with the findings reported by other authors [11, 20]. Table 1. Total As extracted with different solvent at several proportion Extractent Methanol/water HCl HNO3

Concentration or proportion

Efficiency (%)

1:9 (v:v)

74

(0.05 – 5.0) M

76 - 83

(1 – 5) M

80

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Khalid Boutakhrit, Fabien Bolle and Leo Goeyens Table 2. Total and Inorganic As found by the two procedures

Certified reference materials

Acid Extraction (HCl 0.5 M) a

b

Acid digestion (HNO3) a

b

Total As (%)

Inorg. As %

Total As (%)

Inorg. As %

TORT-2

76

4.1

101

5,82

DORM-2

88

0.32

98

0,74

DOLT-3

65

0.66

101

0,89

LUTS-1

98

2.65

123

3,02

BCR-422

86

0.22

93

0,22

FAPAS S7R55

74

0.25

98

0,74

a : Total As was determined by ICP-OES b : Inorganic As expressed as a percentage of the total arsenic

Optimisation of the Inorganic as Digestion Method In order to facilitate the dissolution of fatty and proteinaceous material, the development work was directed towards the use of acid digestion to improve the extraction efficiency of As. For this purpose, digestion with nitric acid was performed for six CRMs of different matricies. The effect of increasing the digestion temperature was investigated for the canadiens CRMs in the range of 80-180°C. The figure 1 illustrates a plot of the inorganic As content with the temperature, according to this diagram, the response behaviour depend on the CRMs matricies. For the same species, dogfish, we observed a difference in behaviour with the temperature between muscle matrix (DORM-2) and liver matrix (DOLT-3). The same goes for the defatted lobster hepatopancreas matrix (TORT-2) and the non defatted matrix (LUTS-1). These results can explain the difficulties to extract completely the inorganic arsenic by using conventional methods (sonication and centrifugation) except for the TORT2, it seems to release inorganic arsenic easily both for extraction and digestion methods. For lower temperatures as 60°C, the digested sample produces a lot of foam, in the hydride generation due to non-destroyed proteins, which disturb the plasma and causes an analytical errors. For higher temperatures, over 200°C, there is a risk to convert the organoarsenic compounds to inorganic arsenic. It was reported that, the behaviour of cationic arsenic as AC and AB, in a microwave system with nitric acid and hydrogen peroxide, shows a conversion to trimethylarsine oxide (TMAO) at 207°C and the later remain unaltered [21]. The same goes for MMA, DMA and As (V), in close pressurized digestion system. This is more aggressive than the conventional oven digestion used in our work. These compounds remain intact up to 160°C. Then at 190°C, MMA was partially decomposed to arsenate whereas DMA and As (V) were unaltered [22]. In our work, the optimised digestion procedure was applied to AB, MMA and DMA and no conversion to arsenate has been noticed but unfortunately, if MMA is significantly present in the sample such as the seaweed, the proposed method can overestimate the inorganic As value. For the arsenosugars, we have no data about their behavior in acid digestion but it is not the predominant compound in seafood.

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Inorganic As / ( µg/kg)

1400 1200 1000 800 TORT-2

600 400 200 0 0

20

40

60

80

100 120 140 160

180 200

Temperature / (°C)

Inorganic As /(µg/kg)

140 120 100 DORM-2

80

DOLT-3

60

LUTS-1

40 20 0 0

20

40

60

80

100 120 140 160 180 200

Temperature/ (°C)

Figure 1. Effect of temperature on the acid digestion of marine CRMs using concentrated nitric acid 2.5 mL.

Determination of Inorganic Arsenic in Reference Samples The two proposed methods (extraction and digestion) were applied to the following reference materials: TORT-2, DORM-2, DOLT-3, LUTS-1, BCR 422 and the FAPAS S7R55. The results obtained appear in table 2. The content of inorganic arsenic in these CRMs is not certified and there are few previous references in the literature for its quantification. The table 3 shows the results from the analysis of marine CRMs and also includes published results for inorganic arsenic in these samples (if data available). A large variability exists between the reported values. The results of inorganic arsenic for the acid extraction method are generally among the lowest reported for all CRMs with the exception for BCR 422, which is equal to that found by acid digestion and higher than that published.

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The values obtained in the present study for the acid digestion method are within the range given in the literature except for the TORT-2 where is higher both with the two proposed methods. Table 3. Inorganic arsenic content reported in the literature and in this study for some investigated CRMs CRMs

DORM-2

TORT-2

BCR 422 FAPAS S7R55 DOLT-3 LUTS-1

Reported result in mg/kg dry weight 0.088 0.10 ± 0.02 a 0.102 ± 0.029 0.145 ± 0.011 0.15 ± 0.01 0.19 ± 0.03 0.132 ± 0.007c 0.41 ± 0.03a 0.46 ± 0.07 0.506 ± 0.031 0.581 ± 0.055 0.680 ± 0.405 1.268 ± 0.080c < 0.002 < 0.026 0.043 ± 0.013c 0.0851 ± 0.0176b 0.083 ± 0.005c Not available 0.091 ± 0.006c Not available 0.105 ± 0.006c

References 27 10 28 23 24 29 present work 30 24 28 23 31 present work 29 24 present work FAPAS present work Not available Present work Not available Present work

a : Sum of As (III) and As (V) b : Assigned value; wet weight c : Standard deviation

The discrepancies of the values reported in the literature are due probably to the different modes of sample preparation and then to the used techniques of analysis. Indeed, after examining a several publications, it seems that HG-AAS technique gives a higher values for the inorganic arsenic than a coupled techniques as HPLC-ICP-MS used for the speciation of As (V) and As (III), where the later is often not detected.

Determination of Inorganic Arsenic in Real Samples Inorganic As were analysed in frozen seafood samples of various matricies such as fish, crustaceans and bivalves by using the acid wet digestion method. The results are given in table 4. Bivalves (Mussel, Oyster) show high levels of inorganic As comparing to the other species but we can not generalize this observation because only one sample of each species

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was analysed. The found values varied between 0.004 and 0.151 mg/kg fresh matter. Expressed as a percentage of total As, the inorganic As varied between 0.1-7.6%, which agrees with results reported by other authors [23-26]. Table 4. Determination of inorganic and total As in real samples

Mussel

1,99

Inorganic As found (mg/kg) 0,151

Oyster

1,85

0,073

3,9

Scallop

1,29

0,007

0,5

Prawn

0,75

0,023

3,1

Scampi

0,75

0,011

1,5

Cuttle fish

6,56

0,020

0,3

Squid

0,27

0,004

1,5

Ray

11,67

0,014

0,1

Cod

2,54

0,010

0,4

Shark

8,98

0,006

0,1

Trout

1,34

0,004

0,3

Plaice

6,55

0,004

0,1

Sole

2,44

0,004

0,2

Tuna

2,09



Seafood products

Total As found (mg/kg)

a

% of Inorg. As 7,6

a

a : Detection limit

Conclusion Despite the high levels of total arsenic found, the inorganic arsenic, highly toxic form, was presenting percentage lower than 10% of total As. Thus, there is no direct hazard for human health due to the consumption of these fish products. Comparing the two proposed methods, acid extraction and acid digestion, the latter shows a more better efficiencies and the results found for inorganic As in CRMs falls with that’s of literature. The accuracy of this method will be evaluated by participating in proficiency testing schemes in the future. If the reliability and accuracy were confirmed, it will be a good alternative for the other laborious method of extraction involving sonication, centrifugation, distillation and also the amount of sample handling. While in the proposed method, the preparation of the sample is easy, rapid and suitable for all matricies of seafood and can be used as a routine method. Since the level of inorganic arsenic is lower in seafood, the sample homogeneity must be taken in account and can also explain the discrepancies in the results found by authors, these are nearby the detection limit. The use of the proposed method shows a good mineralization of fatty seafood without convertion of the arsenical species to inorganic arsenic (arsenate).

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Acknowledgements Arsenobetaine, Monomethylarsonic acid and Dimethylarsinic acid solutions were offered by the department of Analytical and Environmental Chemistry of the Vrij Universiteit Brussel (VUB).

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Determination of Inorganic Arsenic in Seafood by HG-ICP-OES… [30] J. Kirby, W. Maher, J. Anal. At. Spectrom. 2002, 17, 838. [31] A. Brisbin, C. B. Hymer, J. A. Caruso, Talanta. 2002, 58, 133.

303

INDEX A abiotic, 17, 21, 22, 23, 55, 59, 61, 62, 64, 65, 67, 68, 70, 71 absorption, 30, 86, 188, 203, 204, 207, 211, 212, 225, 268, 278 academic, 123 academic performance, 123 access, vii, 3, 5, 32, 47, 48, 49, 50, 104, 105, 106, 107, 108, 233, 238, 242, 247, 248, 258, 268 accessibility, 50, 79 accidental, 51 accounting, ix, 10, 125, 126, 127, 280 accuracy, xii, 50, 236, 295, 301 acetate, 192, 193, 195, 197, 201, 202, 206, 209, 210, 214, 216, 217, 218, 220, 221, 224, 229, 230 acetic acid, 297 acid, xii, 17, 22, 25, 27, 28, 33, 34, 36, 37, 61, 71, 74, 75, 77, 78, 80, 81, 82, 83, 87, 89, 90, 91, 92, 93, 94, 95, 97, 151, 152, 153, 154, 155, 156, 158, 159, 160, 188, 189, 190, 191, 192, 195, 196, 202, 206, 207, 210, 212, 213, 214, 215, 216, 217, 218, 222, 223, 224, 225, 228, 229, 278, 294, 295, 296, 297, 298, 299, 300, 301, 302 acidic, 173 ACS, 217, 290, 292, 294 activation, 13, 15, 21, 81, 115, 149, 154, 160 acute, viii, 125, 126, 142, 144, 149, 279 acute infection, viii, 125 Adams, 65, 159 additives, 86, 96, 187, 188, 197, 211 adenylate kinase, 25 adhesion, 149, 150, 155, 160 adipocyte, 159 adipocytokines, 150, 158 adiponectin, 158 adipose, 148, 149, 152, 154, 156, 158, 159, 188 adipose tissue, 148, 149, 152, 154, 156, 158, 188 adiposity, 148, 149, 158 adjudication, 49 adjustment, 20, 270 administration, 133, 135, 222, 261

administrative, 43 adolescence, 101 adolescents, viii, 110, 125, 127, 135, 137, 142, 278 adsorption, 30 adult, viii, 100, 101, 102, 115, 116, 121, 125, 129, 148, 279 adulthood, 100, 101, 104, 105, 107 adults, vii, viii, xi, 99, 100, 101, 104, 109, 115, 116, 117, 123, 128, 129, 132, 142, 157, 158, 159, 160, 267, 268, 271, 272, 273, 274, 275, 279, 280 advocacy, 50 aerobic, 85 aflatoxins, 80, 81, 91, 92 Africa, x, 5, 9, 11, 47, 52, 55, 59, 77, 83, 231, 232, 233, 235, 236, 245, 246, 249, 250, 251, 254, 257 age, ix, xi, 59, 105, 106, 110, 116, 117, 125, 126, 127, 128, 129, 130, 132, 134, 135, 136, 138, 163, 209, 255, 259, 260, 267, 270, 275, 276, 289 ageing, 171, 264 ageing population, 171 agent, 292 agents, 37, 69, 86, 96, 203, 204, 223, 226, 246 aggregates, 129 aggregation, 149 agricultural, vii, x, 4, 8, 9, 17, 35, 42, 45, 46, 47, 49, 54, 62, 69, 78, 86, 91, 92, 94, 95, 96, 231, 232, 233, 234, 245 agricultural commodities, 9 agricultural residue, 78, 91, 95 agriculture, vii, 3, 4, 8, 9, 10, 11, 42, 45, 47, 49, 54, 57, 58, 61, 78, 232, 233, 238, 239, 245, 248, 249, 254 agroindustrial, 86, 248 aid, 51 air, 85, 144, 177, 178, 179, 180, 181, 182, 284, 296 alanine, 27, 68, 83 alanine aminotransferase, 27 albumin, 33, 41, 56 alcohol, 21, 36, 55, 79, 275, 277 alcohols, 35, 197 aldehydes, 194, 195, 212 alfalfa, 17, 36, 55, 66, 206 algae, 10, 74, 76, 77, 78, 189, 206, 208, 214, 296 algorithm, 129, 130, 131

306

Index

alien species, 10 alkali, 215 alkaline, 12, 69, 81, 181, 191 alkaline hydrolysis, 81 alkaloids, 36, 37 allele, 27 alleles, 5, 6 alpha, 154, 160, 212, 225 alpha-linolenic acid, 159, 160, 213 alpha-tocopherol, 160, 213, 214, 216, 217, 221, 224, 228, 229 alternative, x, 4, 25, 33, 35, 42, 61, 63, 75, 76, 82, 93, 94, 161, 171, 197, 202, 213, 301 alternatives, 7, 42, 184, 257 alters, 22, 216 amelioration, 211 American Association for the Advancement of Science, 48 American Diabetes Association, 278 American Indian, 117 amine, 19, 210 amines, 181 amino, 7, 19, 28, 32, 33, 37, 54, 74, 75, 76, 77, 82, 83, 86, 93, 94, 95, 153, 210, 211 amino acid, 7, 19, 28, 32, 33, 37, 54, 74, 75, 76, 77, 82, 83, 86, 93, 94, 95, 153, 210, 211 amino acids, 7, 19, 28, 32, 33, 37, 54, 75, 76, 77, 82, 83, 86, 93, 210, 211 AMR, 58 AMS, 38 Amsterdam, 292 amyloid, 160 anaemia, 278, 279 anaerobic, 85, 92 analgesic, 37 analog, 192 Andes, 244 anemia, xi, 30, 63, 267, 268, 269, 270, 271, 274, 275, 276, 277, 278, 279, 280 animal agriculture, 238 animal diseases, 238 animal health, 188, 192, 208, 238 animal husbandry, 251 animal studies, 191 animal welfare, xi, 207, 253, 258, 259, 263, 264, 266 animals, x, 5, 12, 39, 48, 79, 83, 86, 87, 89, 90, 188, 189, 190, 191, 192, 193, 194, 195, 196, 198, 199, 200, 201, 202, 204, 205, 206, 207, 208, 209, 210, 216, 222, 231, 232, 233, 234, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 251, 254, 256, 257, 262, 265 Animals, 192, 233, 238, 240, 243 ANOVA, 105, 270, 273, 274 antagonistic, 52 antibacterial, 197, 202, 219 antibiotic, 197, 202 antibiotics, 188, 197, 256, 278 anti-cancer, 36 anticholinergic, 37

antigen, 65, 277 antioxidant, 21, 29, 38, 56, 59, 66, 70, 159, 187, 191, 192, 196, 197, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 214, 218, 219, 220, 221, 222, 223, 224, 225, 228, 230 Antioxidative, 220 antioxidative activity, 211, 222 antisense, 25, 26, 27, 29, 31, 36, 37 antisense RNA, 27 antitumor, 197 antiviral, 40, 41, 197 ants, 9, 17 anxiety, 101 Appalachia, 110 appendix, 118 appetite, 110, 151 application, 4, 11, 43, 50, 55, 58, 60, 69, 82, 92, 96, 175, 262 Arabidopsis thaliana, 15, 34, 61, 65 arachidonic acid, 33, 154 Argentina, 9, 42, 43, 44, 45, 186, 256 arginine, 19, 75 argon, 296 argument, 114 arid, 242 Army, 235 arsenic, xii, 295, 296, 297, 298, 299, 300, 301 arsenite, 296 arsenobetaine, xii, 295, 296 artemisinin, 38, 70 ascorbic, 17, 61, 196, 214, 217, 218, 229, 278 ascorbic acid, 17, 61, 196, 214, 217, 218, 229, 278 asexual, 81 ash, 172, 173, 228 Asia, x, 5, 10, 77, 83, 93, 142, 231, 232, 233, 249, 251, 257, 258, 279 Asian, 8, 90, 96, 117, 222, 254 aspartate, 93 Aspergillus niger, 32, 84, 90, 95 assessment, viii, xi, 43, 47, 73, 89, 151, 166, 215, 222, 251, 260, 267, 284, 285, 286, 289 assets, 237, 246 assimilation, 56, 82, 258, 259, 263 assumptions, 105, 128 asymptomatic, viii, 125 atherogenesis, 158 atherosclerosis, 157, 160, 191 Atlantic, 90 atmosphere, x, 161, 162, 171, 176, 177, 178, 179, 180, 181, 182, 184, 185 atomic emission spectrometry, xii, 295 ATPase, 15, 22 attacks, 15 attitudes, xi, 43, 50, 52, 53, 57, 111, 253, 257, 264, 266 Australasia, 255 Australia, 44, 47, 48, 62, 99, 111, 256 Austria, 45, 254, 255 autoimmune, 214

Index autoimmune disease, 214 autoimmune diseases, 214 autotrophic, 77 availability, 27, 33, 39, 54, 69, 74, 78, 79, 93, 99, 110, 149, 154, 162, 222, 243, 245, 257, 276 averaging, 130 avoidance, 50 awareness, 48, 52, 115, 144, 162, 257

B Bacillus, 12, 57, 84 Bacillus subtilis, 84 Bacillus thuringiensis, 12, 57 bacteria, 5, 12, 13, 14, 58, 74, 76, 77, 79, 80, 85, 86, 87, 89, 90, 92, 93, 191, 291 bacterial, 14, 15, 19, 25, 28, 57, 66, 77, 85, 92 bacterium, 12, 95 Baghdad, 238 bananas, 47 Bangladesh, 248 banking, 233 banks, 16, 233 barley, 22, 27, 31, 60, 64, 67, 69, 235 barrier, 12, 42, 43, 44 barriers, 15, 16, 42, 43, 45, 48, 49, 257 basic needs, 74, 100, 101, 113 basic research, 54 beef, xi, 190, 218, 236, 242, 253, 254, 255, 256, 258, 259, 260, 261, 262, 263, 264, 265, 267, 270, 272, 276, 277 beer, 264 beetles, 14, 67 behavior, 22, 53, 101, 109, 110, 196, 298 behavioral disorders, 189 Beijing, 269, 279 Belgium, 51, 53, 251, 295, 297 beliefs, 257, 264, 268 beneficial effect, 150, 151, 152, 155 benefits, viii, ix, 8, 19, 27, 33, 38, 42, 45, 46, 47, 48, 52, 54, 55, 67, 73, 78, 88, 89, 91, 108, 147, 152, 153, 159, 170, 188, 189, 191, 211, 238, 239, 245, 248, 254, 257 Best Practice, 62 beta-carotene, 218, 220, 224, 230 beverages, viii, 73, 221 bias, 51 Bible, 234 binding, 12, 13, 14, 23, 60, 163, 179, 181 bioactive compounds, 148, 197, 202, 225 bioavailability, 30, 203, 223, 268, 277 biochemistry, 7, 87, 89, 212, 226 bioconversion, 87 biodegradable, 42 biodegradation, 83 biodiesel, 10, 35, 36 biodiversity, 10, 11, 49, 56, 60 bioethanol, 10, 35, 36, 56, 79

307

biofuel, 10, 11, 35, 54, 59, 68, 91 biofuels, 10, 58, 71 biological activity, 189, 191, 202, 212, 219 biological consequences, 115 biological systems, 191 biomarkers, 157, 160, 212 biomass, 24, 25, 27, 35, 36, 54, 55, 61, 64, 66, 68, 76, 77, 78, 82, 93, 96 biopolymers, 39 Bioreactor, 95 bioreactors, 85 biosafety, 42, 44, 48, 49 biosynthesis, 17, 19, 26, 29, 37, 38, 56, 61, 65, 67, 81, 87, 91, 93, 152 biosynthetic pathways, 37, 81 biotechnological, 4, 37, 49, 67 biotechnology, vii, 9, 35, 42, 43, 45, 46, 47, 49, 50, 51, 52, 53, 54, 61, 62, 67, 89, 90, 91, 93 biotic, 21, 64, 67 birds, 244 births, 243 Blacks, 114 bladder, 296 bladder cancer, 296 bleaching, 40 blends, 215 blindness, 28 blocks, 12, 43, 44 blood, ix, xi, 38, 39, 41, 81, 147, 149, 151, 152, 154, 156, 157, 159, 160, 189, 216, 227, 267, 268, 269 blood glucose, 149, 152, 160 blood pressure, ix, 147, 149, 152, 154, 156, 160, 189 BMI, 104, 105, 269, 271, 275, 276 body composition, 215, 216 body fat, 152, 189, 191 body fluid, 226 body mass index, 104 body weight, 148, 151, 152, 209 Bolivia, 244, 250 bomb, 52 bonds, 39, 173 boredom, 101 Boston, 56 Botswana, 242, 249, 250 bottlenecks, 35 bovine, 39, 44, 236 brain, 45, 106, 209, 210, 229 brain drain, 45 Brazil, 9, 10, 42, 90, 93, 157, 254, 256 breakdown, 190 breeder, 9, 220 breeding, 5, 6, 7, 8, 11, 15, 29, 56, 58, 71, 162, 196, 244, 278 British Petroleum, 85 broilers, 189, 193, 209, 213, 214, 215, 216, 221, 223, 224, 226, 229, 256 brothers, 234 Brundtland report, 232 Brussels, 295

308

Index

bryophyte, 61 Buenos Aires, 256 buffer, 240, 249, 251 buildings, 240 bulimia, 107 bypass, 61 by-products, 86, 87, 243, 248, 256

C Ca2+, 21 cabinets, 176 CAD, 36 calcium, 209, 228 caloric intake, 152 calving, 244 Cambodia, 243 campaigns, 140 Canada, 9, 43, 44, 45, 48, 60, 142, 249, 254, 255, 277, 297 cancer, 52, 148, 151, 215, 219, 268, 296 Candida, 85, 91, 94, 96 candidates, 5, 14 CAP, 45 capacity, 17, 24, 25, 62, 82, 87, 162, 173, 191, 196, 203, 206, 208, 211, 223 capacity building, 62 carbohydrate, 26, 151, 153, 211 carbohydrates, 5, 10, 24, 74, 76, 78, 104 carbon, 24, 25, 26, 35, 54, 56, 63, 77, 78, 81, 85, 91, 177, 204 carbon dioxide, 91, 177 carcinogen, 295 carcinogenicity, 88 carcinogens, 296 cardiac arrest, ix, 147 cardiovascular disease, ix, 147, 148, 151, 156, 157, 158, 159, 211, 212, 214 cardiovascular risk, ix, 147, 154 carnosine, 210, 228, 229 carob, 85 carotene, 9, 66, 206 carotenoids, 28, 29, 58, 67, 204, 206, 207, 211, 220, 223, 224, 225 case study, 62, 250 cash crops, 8, 9, 27, 54, 56 cast, 51 CAT, 17, 218, 226 catabolic, 25 catalase, 17, 21, 22, 204, 207 catalyst, 215 catechins, 204, 205, 220 catecholamine, 150 category a, 130 catfish, 290, 291 cattle, 36, 234, 235, 236, 237, 239, 241, 242, 243, 244, 246, 249, 250, 254, 256, 261, 262, 266 causal relationship, 277

causality, 114 CBS, 85, 93 cDNA, 57, 58, 71 cell, viii, 13, 15, 16, 22, 26, 29, 37, 65, 73, 74, 76, 79, 82, 84, 89, 90, 91, 92, 93, 94, 96, 97, 149, 152, 158, 159, 160, 189, 192, 296 cell adhesion, 155, 160 cell culture, 37, 65 cell death, 15 cell division, 26 cell membranes, 189, 192 cellulose, 35, 36, 78, 79, 86, 92, 96 cellulosic, 35, 64, 68 cellulosic ethanol, 35, 64 Census, 117, 127, 128 Census Bureau, 117 Center for Disease Control and Prevention, 267 Centers for Disease Control, 137, 143, 269 central obesity, 149 cereals, 4, 23, 28, 29, 30, 40, 45, 78, 86, 104, 230, 254 certification, 258 channels, 19, 246, 247, 255 chemical composition, 87, 219, 227 chemicals, 12, 35, 38, 221, 261 chicken, 157, 190, 191, 192, 193, 194, 195, 196, 198, 205, 206, 207, 208, 209, 210, 212, 213, 214, 216, 217, 218, 220, 221, 222, 224, 226, 228, 229, 256, 258 chickens, 70, 191, 192, 196, 197, 198, 199, 204, 205, 209, 213, 214, 216, 217, 218, 219, 220, 221, 222, 224, 225, 226, 227, 229, 230 chicks, 209, 217, 220, 222, 223, 225, 227, 228 childhood, vii, viii, 99, 100, 101, 102, 104, 105, 106, 107, 123, 142 children, viii, 28, 32, 75, 100, 101, 103, 104, 115, 121, 123, 125, 128, 129, 135, 235, 236, 238, 244, 245, 257 China, xi, 9, 10, 45, 74, 77, 254, 258, 267, 268, 271, 272, 273, 274, 276, 277, 278, 279, 280 chiral, 192, 193 Chi-square, 273, 274 chlorine, 51 chlorophyll, 18 chloroplast, 17, 18, 19, 25, 57 chloroplasts, 17, 18, 25, 65, 69 cholecalciferol, 228 Cholera, 41 cholesterol, 14, 57, 104, 149, 151, 152, 153, 189, 193, 194, 195, 205, 208, 210, 211, 212, 214, 216, 224, 225, 226 chromatograms, 286 chromatography, xii, 283, 284, 289, 291, 292, 293, 294 chromium, 226 chromosome, 81 chronic disease, 148, 278 chronic diseases, 148, 278 chronic stress, 115

Index cigarette smoking, 269 cigarettes, 271 circulation, 53 cis, 144, 191 citizens, 265 citrus, 95, 202 civil war, 237 clams, 287 classes, 19, 36, 128, 129, 189, 254 classical, 83, 135, 136, 158 classification, 158 cleaning, 85 clinical trial, 39, 155 clinical trials, 39, 155 cloning, 70, 81, 82, 92 cluster sampling, 268 clusters, 81 CO2, 24, 25, 55, 56, 77, 177, 178, 179, 180, 181, 182 coagulation, 42, 70, 149 Coca-Cola, 51 Cochrane, 84, 91 cocoa, 9, 83 coconut, 189 coconut oil, 189 codes, 49 coding, 13, 14 codon, 13 codons, 33 coenzyme, 228 cofactors, 187 coffee, 9, 83, 94, 233, 241 cohort, 152, 278 collaboration, 135 collagen, 9, 39, 63, 66 College Station, 270 colorectal cancer, 278 colors, 9, 29 combined effect, 218 commercialization, 8, 39, 44 commodity, 9, 54, 262, 265 communication, 52, 54, 151 communities, 99, 247 community, 244, 245 competition, 10, 35 competitiveness, 42, 246 compilation, xii, 283 complement, 32, 33, 285 complex interactions, 25 complexity, 5, 29 compliance, 152, 153, 154 complications, 148, 158 components, 13, 15, 17, 21, 79, 151, 152, 153, 155, 170, 171, 196, 223, 229, 277, 291, 292, 293, 294 composition, ix, 36, 55, 74, 77, 78, 87, 97, 125, 127, 151, 152, 159, 163, 188, 189, 192, 203, 209, 210, 212, 213, 214, 216, 217, 219, 220, 222, 224, 226, 227, 229, 284, 286, 288, 290 compost, 243 composting, 83

309

compounds, x, xii, 15, 17, 28, 30, 36, 37, 38, 74, 87, 148, 150, 152, 153, 155, 188, 190, 193, 197, 201, 202, 203, 209, 210, 211, 220, 225, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 298 concentrates, 81, 82, 85, 94, 95, 197, 290 concentration, 19, 82, 90, 154, 171, 172, 173, 174, 175, 176, 189, 194, 196, 207, 209, 227, 278, 285, 296, 297 conception, 100, 284 concrete, 51 confidence, 44, 48 configuration, 193 conflict, 44, 45, 52, 100, 238, 251 confounders, 270 conjecture, 248 consensus, 151, 157, 247 consent, 261, 269 conservation, vii, 4, 233, 248 constraints, 55, 128, 130, 259 construction, 6 consumer protection, 52 consumers, xi, 43, 44, 46, 48, 110, 151, 162, 171, 187, 188, 189, 190, 191, 206, 233, 245, 253, 254, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266 consumption, vii, viii, ix, x, xi, xii, 9, 11, 33, 50, 73, 77, 78, 80, 81, 87, 89, 99, 100, 101, 104, 107, 110, 111, 134, 136, 137, 141, 144, 147, 148, 152, 153, 154, 155, 156, 159, 160, 161, 162, 191, 211, 241, 242, 244, 246, 257, 259, 260, 267, 268, 269, 270, 271, 276, 277, 278, 279, 295, 296, 301 consumption frequency, 259 contaminants, 294 contaminated food, 126 contamination, ix, 53, 85, 125, 126, 127, 139 contracts, 50 control, 6, 11, 12, 14, 17, 18, 20, 21, 22, 23, 25, 27, 29, 38, 43, 49, 50, 52, 55, 56, 57, 58, 61, 81, 94, 105, 106, 120, 141, 144, 153, 155, 164, 166, 168, 187, 193, 198, 199, 200, 201, 205, 206, 209, 210, 238, 277, 287, 296 control group, 105, 106 convergence, 29 conversion, 10, 25, 35, 36, 38, 57, 64, 76, 96, 216, 285, 296, 298 cooking, 170, 171, 176, 185, 259, 291 coordination, 74 copper, 17, 21, 65, 207, 208, 225, 226 coral, 243 corn, 15, 52, 64, 66, 92, 204, 234 coronary heart disease, 151, 155, 158, 159, 160 correlation, 173, 174, 178, 179, 181, 182 correlation coefficient, 173, 174, 182 correlations, 105, 181 Corynebacterium, 83, 95 cosmetics, 39 cost-effective, 33

310

Index

costs, xi, 8, 27, 86, 239, 240, 241, 253, 255, 258, 259, 263, 268 cotton, 9, 15, 42, 63 cough, 37 cough suppressant, 37 country of origin, 265 couples, 135, 171 coverage, 46, 50, 51, 52, 135, 137, 139, 140 covering, 46, 49 cows, 246, 254, 256 CPB, 44 crab, 204, 287, 289, 291, 297 Crassostrea gigas, 292, 293 c-reactive protein, 277 C-reactive protein, 155, 160 credibility, 257, 258 criticism, 284 CRM, 297 crop production, 9, 11, 17, 42, 242 crop residues, 233, 239, 243, 248 crops, vii, 4, 5, 6, 8, 9, 10, 11, 17, 19, 24, 26, 27, 29, 33, 35, 36, 39, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 54, 56, 59, 60, 61, 63, 64, 65, 70, 232, 233, 238, 241, 242, 243, 245, 248, 254 cross-cultural, 265 cross-sectional, 277 cross-sectional study, 277 cross-talk, 21, 67 CRP, 149, 154, 155 crustaceans, 287, 296, 300 crystals, 81 cues, 111, 257 cultivation, viii, x, 9, 44, 73, 74, 76, 94, 231, 232 cultural factors, 58 cultural values, 100 culture, 40, 41, 81, 82, 83, 85, 87, 92, 93, 94, 97, 100, 110, 268 culture conditions, 81 culture media, 40, 41 curing, 186 Current Population Survey, 117 customers, 257 CVD, 148, 149, 152, 154 cyanide, 15 cyanmethemoglobin, 269 cycles, 164 cycling, 238, 246, 249, 251 cysteine, 32, 210 cytochrome, 15, 66 cytokines, 38, 41, 148, 149, 158 cytoplasm, 207 cytosol, 17, 18, 19, 35, 65 cytosolic, 18, 26, 29, 37, 65

D dairy, 104, 152, 185, 245, 246, 248, 250, 251, 254, 258, 265

dairy products, 152, 250, 265 dairy sector, 251 dandelion, 42 data collection, 48 database, 230 de novo, 7, 32, 59 death, ix, 12, 17, 28, 105, 106, 107, 147, 148, 214 deaths, 132, 140, 235, 238, 243 decay, 78, 126, 127, 135 decisions, 48, 51, 188, 257 decomposition, 196 deduction, 118 defects, 28, 154, 292 defense, 7, 15, 16, 37, 38, 81, 97, 208 defenses, 16 deficiency, 4, 28, 32, 47, 61, 63, 70, 210, 226, 227, 233, 268, 269, 270, 278, 279 deficit, 20, 25, 70 definition, 136, 157 deforestation, 234 degradation, 8, 18, 39, 82, 87, 94, 179, 232 degrading, 87 dehydration, 19, 23, 60, 67, 171, 173, 174, 186 dehydrogenase, 20, 21, 25, 29, 36, 55, 64, 82 delivery, 69, 154, 156, 192 demand, xi, 45, 85, 162, 187, 189, 233, 243, 244, 253, 254, 255 demographics, 117 denaturation, 173, 179 denial, 106 Denmark, 255 density, 150, 156, 261 Department of Agriculture, 109, 124, 256 deposition, 30, 189, 206, 213, 227 deposits, 10, 210 deprivation, viii, 99, 101, 104, 107 deregulation, 68 derivatives, 81, 93, 192, 197, 221 desiccation, 179 desire, 101, 248 detachment, 101 detection, xii, 6, 283, 284, 285, 291, 292, 294, 295, 301 detoxification, 15, 17, 81 91 detoxifying, 17, 18 developed countries, 5, 48, 50, 74, 108, 142, 268, 276 developing countries, x, 3, 4, 8, 27, 30, 39, 42, 44, 45, 47, 48, 49, 50, 54, 60, 69, 74, 231, 232, 233, 239, 242, 247, 254, 258, 265, 268, 278 Development Assistance, 248 developmental process, 27 deviation, 273, 295, 300 dexamethasone, 227 diabetes, 148, 151, 158, 268, 277, 278, 280 diabetes mellitus, 158 diarrhea, 30, 40, 87 diastolic blood pressure, 149, 151, 154

Index diet, viii, ix, 4, 15, 28, 32, 73, 75, 88, 89, 91, 110, 147, 148, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 188, 189, 192, 196, 197, 202, 203, 206, 207, 208, 209, 210, 212, 214, 219, 225, 226, 227, 243, 268, 269, 277, 278, 286, 288, 289 dietary, ix, x, 75, 83, 89, 111, 147, 148, 150, 152, 154, 155, 156, 158, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 200, 201, 202, 203, 204, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 220, 221, 222, 224, 225, 226, 227, 228, 229, 230, 276, 277, 278, 279, 280, 293, 296 dietary fat, 187, 188, 212, 213, 214, 217, 229 dietary habits, 148 dietary intake, 158, 190, 194, 207, 208, 215, 229, 279 dietary supplementation, 188, 191, 193, 198, 202, 203, 204, 206, 207, 210, 211, 230 diets, ix, x, 28, 30, 89, 90, 93, 108, 114, 115, 147, 159, 188, 189, 197, 202, 203, 206, 207, 208, 211, 214, 216, 217, 218, 219, 220, 222, 223, 224, 228, 229, 268, 277 differentiation, xi, 159, 253, 262, 263, 264 diffusion, 42 digestibility, 36, 55, 86, 94, 203, 222, 223, 230 digestion, xii, 30, 223, 295, 296, 298, 299, 300, 301 dioxin, 53 dipeptides, 210 direct measure, 285 disappointment, 101 discourse, 51 discrimination, 192 discriminatory, 44 diseases, 4, 12, 16, 24, 30, 39, 54, 86, 141, 145, 148, 150, 156, 238, 250, 251 disinhibition, 111 dissatisfaction, 107, 264 distillation, 293, 301 distortions, 43 distribution, 4, 42, 54, 55, 93, 99, 108, 128, 130, 192, 202, 204, 225, 227, 255, 256, 257, 277 diterpenoids, 37 divergence, 255 diversification, 9 diversity, 5, 49, 62, 90, 296 division, 26 DNA, 6, 7, 17, 82 docosahexaenoic acid, 152, 155, 156, 189, 190 domestication, 232 donkey, x, 231, 233, 236 dorsi, 259 down-regulation, 66 dressings, 61 drinking, 51, 275, 296 drinking water, 51, 296 drought, 4, 17, 18, 19, 20, 21, 22, 23, 24, 56, 60, 68, 70, 237, 249 droughts, 236 drug treatment, 269

311

drugs, 10, 36, 39, 52, 63 dry matter, 87 drying, 93, 296 durability, 16 duration, ix, 125, 131, 137, 139, 140, 179, 217 dwarfism, 27 dyes, 36 dyslipidemia, ix, 147, 149

E earth, 78 eating, 100, 105, 107, 109, 111, 236, 262, 263 ecological, 93 Ecological Economics, 250 ecological systems, 93 ecology, 251 economic activity, 245 economic development, 126, 250 economic growth, 254, 268 economic losses, 45 Economic Research Service, 110 economics, 89, 100, 265 ecosystems, 243 edema, 87 Education, 116, 117, 119, 149, 158, 260, 269, 271, 272 effluent, 284, 286, 292 effluents, 86, 290 egg, 157, 204, 206, 213, 214, 216, 217, 218, 221, 222, 224, 226, 227 Egypt, 234 eicosapentaenoic acid, 189, 190 elastin, 39 elderly, 128, 160, 238, 280 elders, 234 electrophoresis, 6 elongation, 26 EM, 66 emigration, 132, 140 emission, xii, 295, 296 emotional, 52, 100, 101, 105, 106, 107, 248 emotions, 101, 106, 107 employment, 129, 130, 131, 132, 233, 237, 242, 245, 246 encephalopathy, 44 encoding, 14, 15, 17, 20, 21, 25, 27, 29, 36, 39, 62, 152 endocrine, 296 endogeneity, viii, 113, 118, 120, 121 endonuclease, 81, 93 endosperm, 28, 29, 30, 31, 35, 64, 68, 71 endothelial cell, 158, 160 endothelial cells, 158 endotoxins, 79 energy, viii, ix, 10, 24, 35, 64, 73, 74, 77, 78, 83, 87, 89, 114, 115, 147, 151, 153, 157, 159, 211, 232, 233, 239, 241, 268, 270, 275, 276

312

Index

energy consumption, 78 England, 251 enterprise, x, 231, 233 environment, ix, x, 7, 10, 12, 24, 27, 30, 48, 50, 54, 125, 135, 137, 173, 231, 232, 239, 240, 242, 248, 250, 257, 261, 263 environmental conditions, 6, 17, 24 environmental degradation, 232 environmental factors, 268 environmental impact, 8, 56 environmental issues, 232 environmental predictors, 124 environmental protection, 251 enzymatic, 17, 38, 79, 91, 202, 287 enzyme inhibitors, 13 enzymes, 10, 15, 17, 20, 21, 24, 26, 29, 35, 36, 37, 38, 39, 60, 62, 66, 79, 86, 96, 152, 153, 154, 156, 187, 230 EPA, 33, 34, 152, 153, 154, 155, 156, 189, 190, 208 epidemic, ix, 125, 126, 132, 136, 137, 139, 140, 141, 143, 157, 249, 250 epidemics, 131, 236 epithelial cell, 12 epithelial cells, 12 epithelium, 12 epitope, 41 epitopes, 65, 69 equating, 28 equilibrium, 136 equipment, 11, 171 equity, 238 Eritrea, 235, 236 erythrocytes, 196, 218, 226 Escherichia coli, 20, 25, 31, 32, 51, 61, 83, 84, 94 essential fatty acids, 34 essential oils, 197, 201, 202, 211, 219, 220, 221, 222 ester, 207 esters, 10, 197 ethanol, 10, 35, 40, 64, 67, 68, 83, 85, 91, 93, 202 ethers, 197 ethics, 265 Ethiopia, 101, 233, 236, 239, 249, 250 Ethiopian, 248 ethnicity, 117 ethylene, 27 etiology, 277, 278, 280 Euro, 143, 144, 261 Europe, 44, 45, 46, 47, 50, 51, 52, 53, 69, 150, 157, 235 European Commission, 44, 45, 254, 255, 265 European Regional Development Fund, 166 European Union, 10, 43, 44, 45, 48, 51, 52, 54, 150, 162, 166, 185, 242, 254, 255, 256, 257 Europeans, 53 evaporation, 202 evolution, xi, 13, 249, 253, 268, 278 exclusion, 19, 118 excretion, ix, 125 exercise, 158, 297

exotoxins, 79 experimental design, 89, 260 expert, 70, 262 exploitation, 27, 48, 89, 184, 232 export subsidies, 46 exports, 233, 256 exposure, 12, 52, 56, 150, 295, 296 extraction, xii, 35, 39, 42, 78, 81, 197, 202, 230, 290, 293, 295, 297, 298, 299, 301 extraction process, 42, 197

F failure, 9, 54, 152 family, 23, 101, 103, 116, 117, 129, 132, 197, 237, 244, 245 family income, 117, 244 famine, 45, 235, 236 FAO, 3, 10, 30, 49, 58, 70, 77, 238, 265, 278 farm land, 254 farmers, 3, 4, 5, 8, 9, 10, 11, 12, 27, 39, 42, 45, 47, 48, 54, 233, 240, 243, 250, 254, 255, 256, 259 farming, x, xi, 47, 53, 63, 162, 231, 232, 233, 236, 240, 243, 248, 249, 253, 258, 260, 261, 262, 263, 264 farming techniques, 263 farmland, 256 farms, xi, 243, 249, 253, 259, 264 FAS, 43, 69 fasting, 149, 227, 269 Fasting, xi, 267 fat, x, 104, 148, 150, 151, 152, 153, 156, 157, 158, 159, 163, 187, 188, 189, 190, 206, 208, 210, 211, 212, 213, 214, 216, 221, 225, 229, 234, 257, 265, 266, 268, 270, 275, 276 fat soluble, 190 fats, 5, 76, 79, 189, 213, 214, 224 fatty acid, 7, 10, 34, 36, 54, 57, 61, 150, 151, 152, 156, 157, 159, 160, 187, 188, 189, 190, 191, 193, 207, 208, 210, 211, 212, 213, 214, 215, 216, 217, 222, 225, 226, 229, 290 fatty acids, 7, 10, 36, 54, 57, 150, 151, 152, 156, 157, 159, 160, 187, 188, 189, 190, 191, 193, 211, 212, 213, 214, 216, 226, 229, 290 fauna, 233 fax, 253 FDT, 285, 286, 288, 289 fear, 45, 52, 54, 77, 100, 106 fears, 45, 50, 54 February, 66, 236, 238 feed additives, 96 feedback, 24, 33, 35, 83 feedback inhibition, 83 feeding, 15, 40, 41, 69, 81, 83, 85, 87, 92, 101, 160, 189, 190, 193, 196, 217, 222, 224, 225, 226, 227, 228, 245, 249 feedstock, viii, 10, 35, 73 feelings, 105, 106, 108, 115

Index fees, 246 feet, 87 females, 117, 120, 121, 149, 244 fermentation, 10, 35, 74, 82, 83, 87, 90, 91, 92, 93, 94, 95, 96 ferric ion, 196 ferritin, 30, 32, 58, 59, 60, 65, 69, 196, 269, 271, 274, 275, 276, 277, 279, 280 fertility, 74, 140, 233, 256 fertility rate, 140 fertilization, 4, 5 fertilizer, 5, 8, 78, 243 fertilizers, vii, 4, 5, 8, 27, 243 ferulic acid, 55 fever, 51, 79 fiber, 6, 9, 54, 211 fiber content, 6 fibrinogen, 149 fibrosis, 40 FID, 283, 284 film, 171, 177 financial capital, 246 financial resources, 115 financial support, 49, 156 financing, 246 Finland, 5, 85, 255, 279 fire, 237 firms, 43 first generation, 10, 11 fish, ix, x, xii, 33, 85, 90, 104, 136, 148, 152, 153, 154, 155, 156, 157, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 189, 190, 211, 213, 214, 243, 287, 291, 293, 295, 296, 297, 300, 301 fish meal, 85, 189, 190, 214 fish oil, 33, 152, 153, 154, 155, 157, 159, 160, 189, 190, 211, 213, 214 fishing, x, 161, 170, 171, 176, 184 fitness, 16, 151 fixation, 91 flame, 283 flatulence, 87 flavonoid, 38, 70 flavonoids, 38, 58, 64, 188, 203, 204, 219, 223 flavor, 63, 188, 205, 206, 210, 229, 293, 294 flavors, 190, 195, 206 flexibility, 11, 171 flooding, 17 flora, 233 flora and fauna, 233 flow, 36, 85, 133, 296 flow rate, 296 fluctuations, 9 fluid, 198 flushing, 186 focusing, 10, 47, 215 folate, 28, 29, 57, 209, 228 Folate, 57

313

folic acid, 28, 228 food, vii, viii, ix, x, xi, xii, 3, 4, 8, 9, 10, 11, 17, 18, 24, 25, 27, 28, 29, 35, 38, 39, 42, 43, 44, 45, 47, 48, 49, 50, 51, 52, 53, 54, 56, 57, 58, 60, 61, 62, 66, 69, 70, 73, 74, 76, 77, 78, 81, 83, 86, 87, 89, 90, 93, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 113, 114, 115, 116, 117, 118, 119, 120, 121, 123, 124, 125, 126, 132, 147, 150, 151, 152, 159, 162, 170, 171, 173, 175, 176, 187, 188, 189, 206, 215, 221, 223, 231, 232, 233, 234, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 253, 254, 255, 256, 257, 258, 259, 262, 265, 266, 267, 268, 269, 273, 277, 278, 279, 283, 286, 289, 290, 291, 296 food additives, 187, 188 food aid, 45, 51 Food and Drug Administration, 43 food industry, 86 food intake, 151 food production, vii, 3, 9, 17, 73, 89, 232, 249, 251 food products, 4, 43, 173, 187 food programs, 123 food safety, 44, 49, 51, 53, 56, 259, 265, 266 foodstuffs, 236 foreign exchange, x, 231, 233, 242 forests, 251 formaldehyde, 79 fortification, 4, 5, 29, 59, 64, 278 fossil, x, 231, 232 fossil fuel, x, 231, 232 fossil fuels, x, 231, 232 fowl, 234, 239, 244 Fox, 266 fragmentation, 47 France, 10, 45, 60, 85, 177, 236, 248, 254, 255, 258, 283 free radical, 17, 192, 196, 207, 209, 210, 219, 229 free radical scavenger, 192 free radicals, 17, 196, 209, 210 free trade, 44, 63, 257 free trade agreement, 44 freedom, 47, 51, 54 freezing, 20, 68, 163, 170, 176, 177, 185 fresh water, vii, 4 freshwater, 287, 289, 290 fructose, 24, 26, 42, 64 fruits, 26, 27, 29, 38, 57, 63, 64, 108, 152, 203, 230 frustration, 101 frying, 166 fuel, 35, 36, 67, 239 funding, 4 fungal, 15, 30, 80 fungi, 74, 76, 77, 78, 79, 86, 87, 89, 91, 95, 96, 219 fungus, 69, 87, 94 Fusarium, 78, 80, 84, 92 Fusarium oxysporum, 84 fusion, 13, 14, 20, 60, 61 fusion proteins, 13

314

Index

G Gadus morhua, 184, 186, 292 Gallus gallus domestic, 220 garbage, 240 gas, xii, 82, 85, 90, 179, 186, 283, 284, 289, 290, 291, 292, 293, 294, 296 gas chromatograph, xii, 283, 284, 289, 291, 292, 293, 294 gastroenteritis, 40 gastrointestinal, 87, 210, 268 gastrointestinal tract, 210 gauge, 104 GDP, 233, 245, 269 gel, 164, 165, 166, 183 gels, x, 161, 162, 163, 164, 165, 166, 183 gender, viii, xi, 113, 114, 115, 117, 118, 119, 121, 232, 238, 247, 267, 270, 275, 276 gender differences, viii, 113, 114, 115, 121 gender equality, 232 gender equity, 238 gene, 5, 6, 7, 12, 13, 14, 15, 16, 17, 19, 20, 21, 23, 25, 26, 27, 28, 29, 30, 31, 32, 33, 38, 39, 40, 56, 57, 59, 60, 62, 63, 64, 65, 67, 68, 69, 70, 71, 81, 82, 92, 97, 152, 214 gene expression, 17, 23, 57, 60 gene pool, 5 gene silencing, 7, 15 gene transfer, 29, 60 generation, xii, 6, 9, 10, 36, 39, 54, 233, 248, 294, 295, 296, 298 genes, 5, 6, 7, 8, 12, 13, 14, 15, 16, 17, 18, 19, 21, 23, 24, 25, 28, 29, 30, 32, 33, 34, 36, 37, 39, 42, 55, 58, 60, 61, 62, 64, 65, 66, 67, 69, 70, 79, 81, 82, 83, 157 genetic diversity, 5 genetics, 6, 41, 91 Geneva, 157, 278 genocide, 237 genome, 7 genomics, 68 genotypes, 37 Germany, 10, 45, 53, 85, 95, 97, 251, 254, 255, 296 gestation, 244 gibberellin, 27, 35, 59, 65, 67 gifts, 243 glucose, 25, 64, 66, 149, 151, 152, 154, 160, 215 glutamate, 29, 82, 210 glutamic acid, 92 glutathione, 17, 21, 194, 210, 226 glutathione peroxidase, 194, 226 glycemic index, 159 glycine, 67 glycolysis, 25 Glycoprotein, 40 glycoside, 15 GMO, 52, 59, 261 GNP, 108

goal setting, 110 gold, 159 gossypol, 15, 63 gout, 78, 81 gouty arthritis, 81 governance, 4 government, 9, 43, 44, 45, 51, 53, 54, 99, 247, 256 GPP, 38 gracilis, 34, 91 grades, 224 grain, 8, 14, 24, 26, 27, 54, 68 grains, vii, 4, 11, 24, 26, 28, 30, 32, 86, 241, 243 gram-negative bacteria, 79 grants, 157 grass, 23, 61 grasses, 23, 78 gravity, 129, 131, 178, 179 grazing, 238, 240, 261 Great Britain, 144 Great Lakes, 292 Greece, 255 Green Revolution, 8, 24, 26, 60, 65 green tea, 203, 219, 222 Greenland, x, 65, 161, 164, 170, 184 Gross Domestic Product (GDP), 245, 269 groundwater, 8 groups, xii, 52, 80, 127, 128, 129, 132, 155, 197, 210, 238, 245, 246, 270, 283, 297 growth, vii, viii, 16, 17, 20, 23, 24, 26, 38, 51, 57, 61, 63, 64, 66, 67, 73, 74, 76, 77, 78, 83, 85, 86, 89, 93, 100, 108, 150, 173, 188, 196, 197, 207, 213, 216, 218, 220, 222, 223, 228, 243, 245, 251, 254, 255, 265, 268 growth factor, 38 growth factors, 38 growth hormone, 51 growth inhibition, 67 growth rate, viii, 66, 73, 74, 76, 77, 78, 89, 254, 255 GST, 22 guard cell, 25 guava, 204 guidance, 48 guidelines, 48, 49 Guinea, 244 Gulf War, 238 gut, 12, 14, 30, 151

H H. pylori, 277 haemoglobin, 279 halibut, x, 161, 164, 170, 184 halophytes, 18, 19 handling, 44, 50, 85, 301 hands, 48, 54 happiness, 100, 109 haptoglobin, 149 hard currency, 233

Index hardships, vii, viii, 113, 114, 115, 118, 120, 121 harm, 10, 65 harmonization, 43, 48 harmony, 65 Harvard, 110 harvest, 11, 26, 42 harvesting, 77 hazards, 50, 68 healing, 41 health, vii, viii, xii, 8, 33, 38, 39, 43, 52, 53, 67, 87, 92, 104, 109, 110, 113, 114, 115, 116, 117, 118, 119, 120, 121, 123, 124, 148, 149, 150, 151, 152, 153, 155, 173, 187, 188, 191, 192, 206, 208, 211, 212, 214, 215, 226, 228, 230, 238, 245, 251, 257, 266, 268, 269, 277, 278, 295, 296, 301 Health and Human Services, 143 health care, 245 health education, 109 health effects, 295, 296 health problems, 149, 152 health status, 110, 148 heart, 151, 152, 155, 158, 159, 160, 173, 209, 219, 220, 221 heart disease, 148, 151, 159, 219 heart failure, 151, 152 heat, 17, 21, 35, 81, 170, 196, 200, 207, 209, 215, 226, 228 heat shock protein, 21 heating, 85, 191 heavy metal, 19, 209 heavy metals, 19, 209 hedonic, 258, 259, 261, 262, 264, 265 height, 6, 104, 165, 269 Helicobacter pylori, 268, 277, 279 helix, 39, 66 hematology, 89 hematopoietic, 80 heme, 207, 277 hemicellulose, 79 hemisphere, 256 hemoglobin, xi, 267, 269, 270, 271, 274, 280 hepatitis, 65, 126, 142, 143, 144, 145, 277, 279 Hepatitis A, viii, ix, 125, 126, 127, 129, 131, 132, 133, 135, 136, 137, 139, 140, 141, 142, 143, 144, 145 hepatitis B, 41, 65, 277, 279 herbal, 219, 220, 221 herbicide, 11 herbicides, 4, 11, 53 herbivores, 37 herbs, 197, 201, 202, 206, 219, 220, 234 herring, 287, 291 heterogeneous, 140 heterotrophic, 26, 77, 80 high density lipoprotein, ix, 147, 149, 150, 151 high school, 116, 117, 119, 120, 122 high temperature, 21, 69 higher quality, 8, 188 highlands, 248, 249

315

high-level, 104, 107 hips, 114 Hispanic, 114, 119 histidine, 210 histidine-containing dipeptides, 210 HIV, 39, 52, 59, 66 HIV/AIDS, 52, 59 Hoechst, 85 hogs, 256 holistic, 238 Holland, 93 homeostasis, 17, 152, 154, 157 homogeneity, 301 homogenized, 297 Honda, 70, 85, 92 hookworm, 268 hormone, 23 hormones, 38, 51, 74, 256, 278 horses, 235 hospital, 137, 246 host, 7, 79, 100, 101 House, 94, 123, 231 household, xi, 9, 42, 104, 109, 113, 114, 115, 128, 129, 130, 131, 132, 133, 134, 234, 238, 241, 242, 243, 244, 245, 246, 254, 267 household income, 9, 238, 241, 242 household waste, 244 households, 108, 111, 113, 114, 115, 117, 124, 127, 128, 129, 130, 132, 133, 134, 171, 237, 238, 239, 242, 246, 250, 251, 269 HSP, 22 human, viii, x, xii, 8, 28, 30, 38, 39, 48, 49, 53, 63, 66, 68, 70, 71, 73, 74, 75, 76, 77, 78, 79, 80, 81, 86, 87, 94, 101, 108, 131, 145, 153, 158, 160, 162, 187, 188, 190, 191, 208, 215, 216, 219, 220, 226, 230, 232, 235, 243, 245, 250, 251, 268, 278, 283, 284, 285, 291, 295, 301 human capital, 49 human subjects, 215 human values, 108 human welfare, 232, 251 humanitarian, 39, 47, 48, 62 humans, 12, 28, 29, 48, 83, 87, 141, 152, 153, 159, 188, 191, 196, 208, 215, 216, 219, 222, 223, 233 humidity, 144 husbandry, xi, 243, 251, 253, 264 hybrid, 135 hybridization, 5 hydride, xii, 295, 296, 298 hydro, 77, 86, 197 hydrocarbons, 77, 86 hydrochloric acid, 297 hydrogen, 15, 196, 203, 204, 210, 215, 298 hydrogen peroxide, 196, 298 hydrogenation, 191, 215 hydrolysis, 79, 81, 92, 202, 287 hydrolyzed, 86, 192, 210 hydroperoxides, 194, 210 hydrophilic, 197

316

Index

hydroxyproline, 39 hygiene, ix, 125, 135 hygienic, ix, 126, 127, 135, 136, 140 hypercholesterolemia, 208 hyperglycaemia, 151 hyperlipidemia, 148, 155 hypersensitive, 15, 21 hypertension, ix, 147, 148, 149, 150, 153 hypertriglyceridemia, 153, 159, 160 hypertrophy, 151, 152 hypothalamic, 115 hypothesis, 111

I IARC, 302 ice, 22, 170, 171, 176, 258, 294 IDA, 268, 269, 276 identification, 7, 17, 47, 108, 150, 215, 286, 296 identity, 39, 101, 120, 284 IgE, 69 IgG, 79 IL-1, 154 IL-6, 154, 155 illiteracy, 269 imbalances, 254 immersion, 172 immigration, 132, 140 immune response, 220 immune system, 29 immunity, 132, 133, 135, 151, 220, 228 immunization, 70, 79, 126, 142, 144 immunological, 60, 196, 225 impact assessment, 249 impairments, 152 implementation, 47, 89, 118 imports, 43, 44, 45, 233, 234, 255 impregnation, 171 in situ, 42 in vitro, 191, 197, 201, 203, 210, 211, 220, 222 in vivo, 192, 196, 202, 203, 211, 216, 218, 224, 228 inactive, 27 incentive, 262 incidence, ix, 4, 102, 126, 127, 141, 143, 147, 183 inclusion, 63, 88, 152, 153, 157, 171, 214 income, vii, x, xi, 4, 8, 9, 10, 27, 45, 56, 105, 108, 114, 115, 117, 121, 231, 232, 233, 234, 238, 240, 241, 242, 244, 245, 246, 247, 250, 251, 256, 257, 267 income inequality, 108, 115 incomes, 233, 238, 243, 244, 245 India, 10, 45, 50, 73, 74, 90, 93, 96, 235, 245, 249, 250, 251 Indian, 45, 91, 251 indication, 40, 41, 88, 180, 206, 234 indicators, xii, 108, 283, 290, 291, 292 indigenous, 241 indole, 36, 37

induction, 264 industrial, vii, viii, 10, 25, 30, 36, 39, 40, 49, 60, 73, 74, 85, 86, 89, 287, 290 industrialized countries, 10, 148 industry, 39, 52, 53, 78, 86, 87, 129, 254, 255, 256, 257 inert, 12 infancy, 100 infants, 75 infection, viii, ix, 15, 16, 125, 126, 127, 132, 133, 134, 136, 137, 141, 142, 197, 276, 277, 278, 279 infections, ix, 12, 16, 125, 126, 135, 142, 268 infectious, 70, 126, 127, 132, 133, 134, 135, 136, 140, 143, 145, 148 infectious disease, 126, 127, 136, 140, 143, 148 infectious diseases, 126, 127, 136, 140, 148 infertile, 20 infertility, 28 infestations, 11 inflammation, 148, 149, 151, 152, 154, 155, 157, 158, 159, 160, 219, 276, 277, 278, 279 inflammatory, 148, 149, 152, 154, 156, 159, 160, 187, 189, 277 inflammatory disease, 189 inflation, 240 influenza, 131, 142, 143 influenza a, 143 informal sector, 240 Information Technology, 73 infrastructure, 4, 9, 42, 48, 50, 238, 255, 256 infusions, 219 ingestion, ix, 125, 126, 132, 133, 134, 203 inhibition, 25, 56, 67, 69, 83, 154, 195, 201, 202 inhibitor, 14, 67 inhibitors, 13 inhibitory, 220, 223 inhibitory effect, 223 initiation, 48 innovation, x, 62, 231 inoculum, 83 inorganic, vii, xii, 4, 27, 74, 208, 227, 243, 295, 296, 297, 298, 299, 300, 301 inorganic salts, 4 insecticides, 12, 83 insects, 11, 12, 13, 15 insecurity, vii, viii, 3, 4, 8, 42, 47, 54, 56, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 110, 113, 114, 123, 124 insight, 158 inspection, 238 institutions, 47, 52, 149 instruments, 48, 49, 118 insulin, ix, 40, 147, 148, 149, 150, 151, 152, 153, 154, 156, 158, 159, 160 insulin resistance, ix, 147, 148, 149, 158, 160 insulin sensitivity, 151, 152, 153, 154, 159 insurance, 233, 251 intangible, 257 intangible attributes, 257

Index integration, 29, 55, 238, 241 intellectual property, 42, 46, 58, 62 intensity, xii, 171, 172, 283, 284, 285, 286, 294 intentions, 266 interaction, 63, 117, 120, 172, 173, 174, 175, 178, 179, 180, 196, 233, 277 interactions, xi, 25, 37, 47, 54, 97, 158, 207, 233, 253, 258 intercellular adhesion molecule, 149 interference, xii, 7, 37, 50, 55, 62, 63, 69, 295 interleukin-6, 155, 158, 160 interleukins, 149 intermediaries, 244 international markets, 42, 49, 256 international trade, 42, 43, 44 interpretation, 48 interval, 130, 141 intervention, ix, 115, 125, 126, 131, 137, 139, 141, 147, 152, 153, 154, 155, 219, 278 intracellular signaling, 17 intramuscular, 157, 210 intrinsic, 24 invasive, 10, 11 invasive species, 11 inventions, 46, 67 investment, 59, 100, 233, 234, 244, 247 iodine, 4, 5, 63 ions, viii, 17, 19, 21, 46, 117, 118, 120, 121, 172, 174, 175, 196, 200, 204, 207, 221 Iraq, 238, 248 Ireland, 161, 162, 164, 170, 171, 177, 185, 186, 255 iron, 5, 17, 30, 55, 57, 58, 59, 61, 63, 69, 187, 190, 196, 203, 207, 210, 212, 221, 225, 268, 270, 275, 276, 277, 278, 279, 280 iron deficiency, 61, 63, 268, 270, 278, 279 iron transport, 30 irrigation, vii, 4, 8 irritability, 101 ischaemia, 159 ischemic, 148 ischemic heart disease, 148 Islam, 248 isoelectric point, 181 isoflavones, 203, 221 isoforms, 26 isolation, 204 isomers, 191, 192, 215 Italian population, 127, 136 Italy, ix, 85, 125, 126, 127, 129, 131, 132, 133, 135, 136, 137, 139, 140, 141, 142, 143, 144, 145, 248, 253, 254, 255, 259

J JAMA, 123, 158 January, 43, 138, 238 Japan, 45, 48, 150, 254

317

Japanese, 9, 41, 200, 207, 209, 222, 224, 225, 226, 228 jobs, 246, 271 joints, 81 Jordan, 159 journalists, 50, 51, 52 judge, 284, 285, 286 judges, 284, 285 Jun, 219, 279 Jung, 215, 292 justification, 44

K Kenya, 47, 236, 240, 241, 245, 246, 247, 248, 249, 250, 251 kernel, 31 kidney, 28, 78, 81, 208, 209, 227 kidney stones, 78 kidneys, 80 killing, 12, 15, 237 kinase, 21, 25, 57, 68, 69 kinases, 21, 22 kindergarten, 134 kinetics, 95 King, 57, 218, 223, 230, 234 Kiribati, 243, 248 koji, 92 Korea, 254 Krebs cycle, 26

L labeling, 43, 44, 45, 48, 49, 56, 60, 66 labour, 11, 85, 243, 244, 245 laccase, 39 lactoferrin, 39, 41 lactose, 85 laminated, 170, 292 land, vii, x, 4, 8, 9, 10, 17, 35, 89, 101, 231, 232, 233, 235, 238, 239, 240, 243, 245, 246 land use, 10, 240, 246, 248 large-scale, 42, 78, 85 larvae, 13, 14 larval, 63 latex, 42, 70 Latin America, 231, 232, 255, 257, 278 Latino, 117 law, 68 laws, 49 L-carnitine, 210, 211, 228 LDL, 150 leaching, 171 lead, 15, 16, 17, 51, 52, 81, 87, 100, 101, 104, 106, 107, 117, 140, 149, 154, 189, 208, 209, 210, 233 leakage, 18 lectin, 14, 67

318

Index

left ventricular, 151 legislation, 44, 45, 261 legume, 11, 26, 32, 248 legumes, 14, 29, 152 lens, 51 leptin, 152, 159 lettuce, 29 leucine, 210, 229 liberalisation, 257 licenses, 47 licensing, 47, 48, 62 life satisfaction, 111 life span, 89 life-cycle, 110 lifestyle, vii, ix, 3, 147, 148, 157, 269, 276 lignin, 35, 36, 38, 55, 79, 87, 93, 96, 230 lignocellulose, 78, 79, 83, 96 likelihood, 10, 12, 13, 120 limitation, 87 limitations, 50 Lincoln, 58 linear, viii, 113, 118, 120, 121, 209, 276 linear regression, 209, 276 linkage, 6 linoleic acid, 154, 155, 189, 191, 215, 216, 228, 294 linolenic acid, 160, 189, 190, 191 lipase, 40, 154 lipid, 97, 149, 150, 151, 152, 153, 156, 159, 160, 188, 189, 190, 192, 193, 194, 195, 196, 198, 199, 200, 201, 202, 204, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 220, 221, 222, 223, 225, 226, 229, 286, 288, 289, 290, 292, 293 lipid metabolism, 149, 208, 215 lipid oxidation, 151, 153, 188, 192, 193, 195, 196, 198, 199, 201, 202, 206, 207, 209, 210, 211, 212, 213, 217, 220, 221, 222, 229, 290 lipid peroxidation, 213, 218, 225, 226 lipid peroxides, 196 lipid profile, 149, 156, 160 lipids, 24, 74, 152, 153, 154, 157, 159, 160, 188, 189, 210, 216, 222 lipolysis, 158 lipophilic, 202 lipopolysaccharides, 79 lipoprotein, ix, 147, 149, 150, 151, 152, 153, 154, 156, 226 lipoproteins, 220 lipoxygenase, 81, 97 liquefaction, 39 liquid fuels, 10 liquor, 96 liver, 75, 80, 150, 154, 156, 192, 196, 206, 208, 209, 217, 220, 221, 225, 227, 272, 297, 298 liver cancer, 80 livestock, x, xi, 45, 87, 89, 231, 232, 233, 234, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 256, 265 LMW, 31, 33 local authorities, 239

location, 18, 171 locus, 5 London, 55, 56, 57, 63, 92, 109, 110, 251 loneliness, 101 long-term, vii, 4, 53, 115, 143, 160, 186, 220, 221, 222, 239 loss of consciousness, 106 losses, 11, 42, 108, 173, 236 love, 234 low molecular weight, 17 low risk, 137, 141 low temperatures, 82 low-density, 152, 220 low-density lipoprotein, 152 lower prices, 233 low-income, 111, 114, 115, 116, 117, 120, 124 low-level, 104 LPO, 218, 226 LSD, 169, 170, 172, 173, 174, 175, 176, 177, 178, 180, 181, 182 lumen, 192 lupus, 185 lutein, 58, 206, 207, 220 Luxembourg, 255 Luxemburg, 45 lycopene, 28, 29, 57, 58, 60, 66, 206, 207, 224 lysine, 32, 33, 71 lysis, 79 lysozyme, 41

M mackerel, 166, 167, 176, 185, 196, 287, 289, 290 macronutrients, 211 mad cow disease, 44 Madison, 251 magazines, 52 magnesium, 277, 279 mainstream, ix, 161, 162 maintenance, 17, 74, 234, 240 maize, 5, 8, 9, 10, 11, 15, 21, 22, 25, 26, 28, 29, 30, 35, 39, 42, 43, 45, 51, 52, 58, 59, 60, 61, 62, 68, 71, 223, 241, 245, 246, 249 major depression, 124 malaria, 268 malate dehydrogenase, 25, 64 Malaysia, 73 males, 117, 120, 121, 149, 244, 279 malnutrition, 3, 42, 59, 108, 238, 243 malondialdehyde, 196 Mammalian, 32 mammalian cell, 219 mammalian cells, 219 management, vii, 4, 11, 13, 44, 47, 48, 52, 53, 62, 109, 243, 244, 245, 250 management practices, 250 manganese, 17, 70 manganese superoxide, 70

Index manganese superoxide dismutase, 70 mangoes, 204 manipulation, 19, 51, 59, 64, 67, 105, 106, 107, 209, 210 man-made, 188 mannitol, 19, 20 manufacturing, 93, 258, 259, 264 manure, 77, 233, 241, 242, 243, 248 MAPK, 21 mapping, 6, 7, 81 marine environment, 145 marital status, 117 market, xi, xii, 5, 9, 38, 42, 43, 44, 45, 49, 54, 69, 85, 136, 163, 237, 240, 242, 246, 253, 254, 255, 256, 258, 295, 296, 297 market access, 49 market disruption, 237 market share, xi, 253, 255, 258 marketing, 79, 239, 245, 246, 248 markets, 8, 42, 45, 49, 237, 239, 254, 255, 256, 258, 265, 266 marriages, 243 Maryland, 123 MAS, 6 mass media, 50, 51 mass spectrometry, 293 mass transfer, 173, 174, 175 Massachusetts, 74, 187 Massachusetts Institute of Technology, 74, 92, 93, 96, 124 materialism, 100, 101, 102, 104, 105, 106, 107, 108, 109, 111 maternal, 110, 214 matrix, 181, 183, 284, 287, 290, 297, 298 maximum price, 261 meals, 100, 103, 117, 155, 190 measles, 235 measurement, 94, 277, 285 measures, 12, 54, 104, 109, 114, 116, 117, 118, 120, 139, 141, 142 meat, vii, x, xi, 104, 108, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 234, 236, 237, 241, 242, 243, 245, 253, 254, 255, 256, 257, 258, 259, 260, 262, 263, 264, 265, 266, 267, 268, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 291, 292, 294, 297 media, 40, 41, 42, 50, 51, 52, 53, 54, 55, 76 median, 102, 154 mediators, 115 medicine, 52, 211, 251 Mediterranean, 160 melting, 189 membranes, 152, 195, 196, 207 memory, 236

319

men, viii, xi, 113, 114, 115, 118, 120, 121, 148, 149, 159, 234, 235, 267, 268, 269, 270, 271, 274, 275, 276 menstruation, 268 mental health, 108, 109, 111, 124, 214 mental state, 151 messages, 107 meta-analysis, 153, 154 metabolic, ix, 7, 8, 9, 19, 29, 33, 35, 37, 38, 42, 56, 60, 62, 63, 64, 67, 71, 81, 86, 96, 147, 148, 151, 152, 156, 157, 158, 160, 211, 280 metabolic changes, 151 metabolic pathways, 8, 9, 19, 29, 35, 64 metabolic rate, 151 metabolic syndrome, ix, 147, 148, 151, 156, 157, 158 metabolism, 8, 15, 17, 25, 26, 35, 36, 54, 60, 62, 63, 64, 67, 80, 93, 149, 154, 158, 188, 208, 209, 211, 212, 215, 225, 226 metabolite, 70, 81, 93 metabolites, 27, 36, 37, 76, 79, 80, 82, 87, 202, 203, 204, 296 metabolome, 62 metal ions, 19, 204 Metallothionein, 227 Metallothioneins, 227 metals, 20, 57, 69, 196, 296 metaphor, 111 metaphors, 52 methane, 77, 239 methanol, 92, 202, 219, 222, 297 methionine, 32, 33, 77, 82, 208, 211, 289, 293 metric, 78 Mexican, 114 Mexican Americans, 114 Mexico, 254 mice, 153, 160, 191 microalgae, 56, 77, 92 microbes, 81, 82, 83, 86 microbial, viii, 12, 15, 16, 35, 42, 60, 67, 73, 76, 81, 82, 86, 87, 89, 91, 94, 95, 96, 173, 220, 290 microbial cells, 81, 82, 86 microflora, 79, 85, 94 micronutrients, 4, 5, 70 microorganism, 89 microorganisms, vii, viii, 10, 73, 74, 75, 76, 77, 78, 84, 86, 89, 209, 210, 277 microwave, 298 Middle East, 45 middle-aged, 279, 280 military, 100 milk, 4, 39, 101, 191, 232, 234, 236, 241, 242, 243, 245, 246, 247 Millennium, 57 millet, 241 mimicking, 139 mineral cofactors, 187 mineral water, 142, 292 mineralization, 209, 228, 301

320

Index

minerals, 4, 5, 8, 28, 30, 54, 86, 89, 96, 151, 152, 187, 188, 211, 268 Ministry of Education, 157 minority, 236 misinterpretation, 50, 52 misleading, 52, 53 Missouri, 113 mitochondria, 17, 18 mitochondrial, 26, 29, 64, 153 mitogen, 21 mitogen-activated protein kinase, 61, 68, 71 mixing, 85, 190, 297 MMA, 296, 298 MnSOD, 17, 18 mobility, 5, 251 modeling, 135, 136, 140, 141 models, viii, 113, 114, 118, 120, 121, 126, 127, 131, 133, 136, 141, 145 moderates, 117 modernization, 110 moieties, 29 moisture, 78, 172, 173, 174, 175, 178, 179, 196 moisture content, 78, 173, 174, 175, 179 molasses, 86 molecular markers, 5, 6 molecular weight, 17 molecules, 7, 9, 15, 17, 19, 29, 33, 36, 38, 149, 150, 160, 202, 284 Møller, 62 money, 9, 102, 103, 105, 117, 235, 258 monocyte, 160 monomers, 42 monoterpenes, 202 monoterpenoids, 37 mood, 105, 111 morality, 111 moratorium, 43, 44 morbidity, viii, 125, 268 morphine, 37 morphological, 6, 93 morphology, 63 mortality, ix, 110, 132, 147, 148, 152 mortality rate, 132 motivation, 101, 107 mouth, 51 movement, 49 mRNA, 15, 152 multivariate, xi, 119, 267, 276, 277, 291 murder, 236 muscle, 149, 150, 154, 173, 188, 189, 191, 192, 196, 202, 203, 207, 208, 209, 210, 212, 213, 217, 218, 223, 224, 225, 226, 227, 229, 259, 293, 294, 297, 298 muscle extract, 229 muscle tissue, 149 muscles, 210, 224 mushrooms, 83, 97 Muslim, 240 mutagen, 7

mutagenesis, 6, 7, 14, 21, 82 mutant, 7, 22, 58, 65, 82, 83 mutants, 83, 90, 91, 94, 96 mutation, 5, 6, 11, 12, 13, 57, 83 mutation rate, 6 mutations, 6, 7, 12, 13, 16, 62, 83 MVA, 37 mycelium, 87 mycology, 93 myoglobin, 195

N NaCl, 19, 173 NAM, 23, 60 narcotic, 37 national, xi, 42, 43, 45, 47, 48, 49, 114, 115, 121, 127, 128, 233, 247, 256, 267, 268, 276, 278 National Academy of Sciences, 144 National Institutes of Health, 46 National Research Council, 217, 297 National Science Foundation, 46 natural, xi, 4, 5, 6, 9, 10, 16, 43, 85, 87, 90, 92, 127, 140, 188, 191, 192, 194, 197, 198, 202, 203, 206, 211, 216, 217, 223, 229, 231, 232, 248, 255, 256, 261, 297 natural disasters, 4 natural food, 255 natural gas, 85, 90 natural resources, xi, 231, 248 nausea, 87 NCA, 70 Near East, 238 necrosis, 149 negative relation, 173, 174, 175 neglect, 242 nematode, 14, 183 nematodes, 14 neonatal, 220 Netherlands, 51, 186, 249, 250, 255, 259 network, ix, 29, 125, 126, 127, 128, 129, 131, 132, 133, 135, 140, 245 neurotoxicity, 80 New England, 123 New York, 56, 59, 91, 92, 93, 95, 109, 110, 123, 157, 218, 225, 291, 302 New Zealand, 5, 256 news coverage, 53 newspapers, 50, 51, 52, 53, 54 next generation, 14 NGOs, 44, 53 nickel, 58 Nicotiana tabacum, 66 nicotine, 37 Nielsen, 265 Nigeria, 242, 243, 249 NIH, 46 nitric acid, xii, 295, 297, 298, 299

Index nitric oxide, 149 nitrogen, 25, 27, 36, 54, 68, 76, 78, 81, 82, 163, 177, 178, 179, 180, 210, 223 nodules, 66 noise, 285 non-native, 10, 11 non-native species, 10, 11 non-profit, 47 non-tariff barriers, 43 normal, 17, 19, 20, 26, 27, 29, 30, 34, 89, 104, 107, 158, 187, 196, 200, 237, 279 normal conditions, 26, 196 norms, 268 North America, 254, 255, 257, 266, 280 Norway, 267 n-type, 278 nuclear, 25, 50, 149 nuclear power, 50 nucleic acid, 74, 76, 77, 78, 80, 81, 82, 87, 89, 90, 93, 94 nucleus, 80 nursery school, 129 nutraceutical, 39 nutrient, 5, 6, 24, 27, 30, 54, 88, 107, 108, 114, 115, 154, 192, 196, 203, 207, 222, 238, 246, 248, 249, 251, 269, 273 nutrient cycling, 238, 246, 249, 251 nutrients, 3, 4, 5, 7, 76, 86, 87, 109, 115, 123, 148, 150, 188, 232, 233, 239 nutrition, viii, xi, 4, 7, 42, 59, 70, 86, 108, 109, 110, 113, 114, 210, 222, 226, 228, 243, 244, 245, 267, 268, 276, 278, 279 nutritional assessment, 92 nutritional deficiencies, 207 nuts, 152

O obese, 148, 149, 152, 154, 159, 160 obesity, ix, 101, 123, 147, 148, 149, 152, 153, 157, 158, 159, 216, 268, 271, 279, 280 objectivity, 248 obligations, 243 occupational, 92 odorants, 290, 292, 293 odors, 190, 206 OECD, 49 oil, vii, 4, 10, 34, 36, 45, 58, 66, 71, 78, 82, 86, 90, 152, 153, 154, 155, 157, 159, 160, 189, 190, 197, 201, 202, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222 oils, 34, 189, 191, 196, 197, 201, 202, 211, 213, 215, 219, 220, 221, 222 oilseed, 33, 45, 57, 61 olfaction, 285, 286, 290 olive, 152, 189, 213 olive oil, 152, 189, 213

321

omega-3, 65, 67, 157, 159, 160, 211, 213, 214, 226, 229 omega-6, 65, 229 omission, 51, 115 online, 59, 69 opposition, 43 optical, 296 optimization, 82 oral, 41, 69 organ, 115, 223, 270, 276, 277, 296 organic, vii, xi, 4, 10, 25, 51, 53, 78, 83, 96, 193, 208, 211, 227, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 296 organic food, 253, 254, 255, 256, 257, 258, 266 organism, 77, 82, 85 organization, 44, 47 organizations, 44, 45, 46, 47, 53 organoleptic, xii, 212, 283 osmosis, 19 osmotic, 12, 19, 20, 71, 74, 82, 90, 171, 173, 186 osmotic dehydration, 171, 186 osteoporosis, 151 overeating, viii, 99, 100, 107, 108, 111 overload, 277 overweight, 104, 105, 107, 108, 115, 148, 149, 152, 153, 154, 158, 159, 160, 268 ownership, 46, 47, 233 oxidation, 151, 152, 153, 156, 188, 190, 192, 193, 194, 195, 196, 198, 199, 200, 201, 202, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 217, 220, 221, 222, 224, 225, 229, 290 oxidation products, 188, 194, 195, 205 oxidative, x, 18, 19, 20, 21, 22, 23, 24, 36, 60, 61, 62, 67, 69, 148, 149, 152, 157, 159, 188, 190, 192, 194, 195, 196, 197, 198, 199, 200, 201, 202, 204, 205, 206, 207, 208, 209, 210, 211, 213, 216, 217, 218, 219, 222, 223, 224, 225, 226, 228, 229 oxidative agents, 69 oxidative damage, 223 oxidative stress, 18, 19, 20, 21, 60, 61, 67, 69, 148, 149, 157, 192, 209, 224, 226, 228 oxide, 149, 298 oxides, 229 oxygen, 17, 25, 177, 192, 203, 204, 206, 207, 224, 225 oyster, 286, 292 oysters, 287, 290, 293

P Pacific, 243, 257, 258, 279, 292 packaging, x, 161, 162, 176, 177, 184, 185, 213 pain, 87, 106 palm oil, 189 Pan1, 267 pancreas, 154, 209 pancreatic, 81, 82 pandemic, 131, 142, 143, 236

322

Index

paper, 40, 58, 62, 71, 78, 86, 113, 226, 231, 248, 249, 261 paradox, 280 Paraguay, 9 paralysis, 12 parameter, 136 parasites, 61, 183, 184, 268 parasympathetic, 37 parasympathetic nervous system, 37 parenthood, 109 parents, 101, 102, 103, 128 Paris, 248, 265 Partial Least Squares, 290 particles, 153 partnership, 62 pastoral, x, 231, 232, 234, 236 pasture, 235 pastures, 234 Patent and Trademark Office, 46 patents, 42, 46, 47 pathogenesis, 149 pathogenic, 219 pathogens, 15, 16, 39 pathology, 87, 89 pathways, 8, 9, 13, 15, 17, 19, 21, 25, 29, 34, 35, 36, 37, 38, 64, 81, 114, 115, 208 patients, 126, 142, 154, 158, 160, 270 pay off, 107 PCR, 7 penalty, 16 peptides, 15, 16, 210, 211 per capita, 108, 254 per capita income, 108 perception, xi, 16, 25, 44, 52, 61, 193, 253, 257, 258, 264, 265, 285, 286 perceptions, 48, 52, 110, 258, 264 performance, 6, 59, 64, 108, 110, 123, 151, 166, 178, 180, 188, 197, 202, 203, 208, 209, 216, 217, 219, 220, 221, 222, 223, 224, 225, 227, 228, 229, 246, 264, 265 perinatal, 279 permit, 48 peroxidation, 212 peroxide, 17, 18 personal, 100, 101 personal values, 101 personality, 100, 101, 109, 110 personality traits, 100 personality type, 109 pest control, 12, 56, 58 pest management, 13 pest populations, 13 pesticide, 12 pesticides, 4, 8, 12 pests, 4, 8, 11, 12, 13, 14, 15, 17, 24, 35, 54, 55, 62, 67 petrodiesel, 10 petroleum, 85, 89, 91 petroleum products, 89

pH, 79, 81, 171, 172, 173, 178, 179, 181, 182, 183, 204, 290 pH values, 171, 173, 179 pharmaceutical, 10, 36, 38, 39, 52 pharmaceuticals, 63, 68, 261 pharmacokinetics, 222 pharmacological, 226 phenol, 197 phenolic, 17, 38, 203, 209, 220, 223, 230 phenolic acid, 223 phenolic acids, 223 phenolic compounds, 17, 38 phenotype, 6, 22, 27 phenotypes, 6, 7, 8, 21, 71 phenotypic, 6, 69 phenylalanine, 38, 75, 82, 83, 91 Philadelphia, 145 philosophy, 100, 162 phloem, 12, 26 phone, 102, 104 phosphate, 20, 25, 90 phosphoenolpyruvate, 25 phospholipids, 154, 156 phosphorous, 81 phosphorus, 209, 228 phosphorylation, 21 photographs, 52 photoreceptor, 59 photosynthesis, 20, 24, 25, 26, 54, 56, 61, 62, 63, 64, 65 photosynthetic, 17, 21, 24, 25, 26, 68, 77 physical activity, ix, 147, 269 physical health, viii, 113, 114, 117, 118, 120 physical properties, 189 physical world, 100 physicochemical, 163, 168, 171, 176, 185, 212 physiological, 17, 19, 62, 82, 100, 115, 150, 191, 196, 203, 227 physiology, 7 phytates, 188 phytochemicals, 197, 201, 202, 203, 230 phytoremediation, 19 pig, 212, 218, 243, 244, 254, 255, 265, 278 pigments, 38, 202, 204, 206, 224 pigs, 51, 53, 83, 89, 188, 239, 243, 244, 254, 255, 256, 259 pilot study, 29 Pisum sativum, 14, 32 placebo, 155 plague, 11, 235, 249 plants, viii, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 28, 29, 30, 32, 33, 34, 35, 36, 37, 38, 39, 42, 48, 52, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 73, 76, 77, 197, 201, 202, 206, 209, 210, 211 plasma, xii, 19, 151, 152, 159, 160, 192, 202, 208, 209, 210, 216, 217, 222, 225, 228, 295, 296, 298 plasma membrane, 12, 19, 152

Index plasmid, 81 plastic, 85, 170, 177 plastics, 9, 42 plastid, 25 platelet, ix, 147, 149, 150, 152, 154 platelet aggregation, 152 play, viii, 10, 25, 42, 50, 99, 257, 290 ploughing, 234 point mutation, 6 Poisson, 270, 275 Poland, 185 policy makers, 42, 48, 247 poliovirus, 142 political aspects, 74 political crisis, 45 politicians, 50, 247 politics, 54, 69, 100 pollen, 41 pollution, 89, 92, 145, 233 polyamine, 56, 70 polymer, 42 polymerization, 42 polymers, 9, 39, 42, 54, 69 polynucleotide, 81 polyphenols, 197, 203, 211, 212, 219, 222, 223, 230 polysaccharides, 79, 223 polyunsaturated fat, x, 33, 36, 55, 61, 67, 69, 70, 159, 160, 188, 210, 211, 213 polyunsaturated fatty acid, x, 33, 36, 55, 61, 67, 69, 70, 159, 160, 188, 210, 211, 213 polyunsaturated fatty acids, x, 33, 36, 55, 61, 67, 69, 70, 159, 160, 188, 210, 211, 213 pomace, 83, 84, 90, 200, 201, 203, 222, 223, 230 pools, 66 poor, x, 4, 5, 8, 12, 17, 42, 53, 63, 64, 86, 108, 115, 117, 120, 163, 169, 188, 209, 231, 232, 233, 237, 239, 240, 243, 244, 245, 246, 249, 276 poor health, 120 population, vii, ix, 3, 4, 5, 6, 8, 29, 45, 73, 74, 91, 93, 108, 114, 121, 125, 127, 129, 132, 135, 136, 137, 139, 140, 141, 171, 236, 242, 243, 244, 245, 246, 247, 248, 268, 269, 270, 276, 277, 279 population density, 243 population growth, vii, 73, 74, 93 pores, 12 pork, xi, 216, 229, 254, 255, 258, 259, 264, 265, 266, 267, 270, 276, 277, 278 Portugal, 255 positive emotions, 106 positive relation, 173, 175 positive relationship, 173, 175 postmenopausal, 155, 160 postmortem, 202 potato, 9, 17, 21, 25, 26, 28, 33, 38, 39, 51, 56, 57, 58, 59, 63, 66, 67, 68, 69, 166, 204 potatoes, 26 pouches, 170 poultry, x, xi, 89, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 201, 202, 203, 204, 206, 207,

323

208, 209, 210, 211, 212, 214, 216, 218, 223, 225, 226, 228, 231, 233, 239, 243, 244, 255, 256, 266, 267, 270, 276, 277, 278 poverty, vii, viii, 3, 4, 42, 43, 50, 54, 99, 101, 107, 113, 114, 117, 232, 246, 249, 250, 251, 254 poverty alleviation, 232, 246, 250, 251 poverty line, 114, 254 poverty reduction, 42, 43 powder, 171, 200, 222, 225 powders, 171 power, x, 3, 50, 100, 231, 232, 233, 238, 239, 241, 242, 243, 248, 251, 254 prediction, ix predictors, 115, 121, 124 pre-existing, 46 preference, 13, 163, 210, 265 pregnancy, 28, 268 premium, 45, 51, 254, 255, 256 premiums, 255, 259, 266 preservatives, 187 pressure, ix, 12, 13, 44, 54, 74, 147, 149, 151, 152, 154, 156, 159, 160, 189, 215, 246, 257 prevention, 90, 214, 217, 278 preventive, 268, 277 prices, 8, 9, 10, 45, 46, 85, 86, 233, 236, 237, 246, 255, 256, 257, 261, 268 primary products, 256 primary school, 269 priorities, 48 private, 47, 245 private sector, 47 proactive, 49 probability, viii, 113, 118, 120, 121, 127, 128, 129, 132, 133, 135, 136 probe, 7, 259 producers, 10, 43, 50, 54, 233, 248, 255, 256, 262 product attributes, 262 product performance, 264 production, vii, viii, x, xi, 4, 5, 8, 9, 10, 17, 27, 35, 36, 38, 39, 40, 42, 54, 55, 56, 58, 61, 62, 63, 66, 67, 68, 73, 74, 76, 77, 78, 79, 81, 82, 83, 85, 86, 88, 89, 90, 91, 92, 93, 94, 95, 96, 123, 149, 150, 154, 164, 194, 196, 197, 224, 226, 229, 231, 232, 233, 234, 238, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 253, 254, 255, 256, 257, 258, 259, 261, 263, 264, 265 production costs, xi, 8, 27, 253, 255, 258, 259 productivity, vii, 4, 8, 9, 17, 27, 35, 96, 227, 233, 244, 247 proficiency testing, 297 profit, 244 progeny, 71, 227 program, 29, 89, 118, 121, 157, 264 proinflammatory, ix, 147, 154, 157 promote, 14, 47, 48, 148, 196, 207, 210, 296 promoter, 14, 16, 18, 20, 21, 22, 27, 29, 30, 34, 38 property, 42, 46, 57, 58, 62, 67, 83 prostaglandin, 154 proteases, 210

324

Index

protection, 12, 20, 46, 47, 52, 67, 70, 200, 202, 209, 211, 232, 251, 257 protective genes, 17 protein, viii, 9, 12, 13, 14, 15, 20, 21, 22, 23, 25, 30, 32, 33, 35, 38, 39, 41, 57, 60, 63, 64, 66, 68, 70, 71, 73, 74, 75, 76, 77, 78, 79, 81, 82, 83, 84, 85, 86, 87, 89, 90, 91, 92, 93, 94, 95, 96, 97, 104, 149, 153, 160, 164, 165, 173, 179, 194, 198, 209, 211, 216, 227, 229, 232, 233, 240, 243, 248, 270, 277, 278 protein kinases, 21, 22 protein oxidation, 194, 198, 229 protein sequence, 12 proteins, 5, 9, 10, 12, 13, 14, 15, 17, 21, 33, 35, 36, 38, 39, 40, 52, 54, 56, 64, 67, 74, 75, 82, 86, 149, 152, 153, 155, 187, 196, 208, 210, 268, 297, 298 protocol, 44 Pseudomonas, 79, 82 psychological functions, 150 psychological stress, 115 psychology, 100, 111 Psychosomatic, 111 public, ix, 16, 42, 43, 44, 46, 47, 48, 50, 51, 52, 53, 54, 58, 121, 125, 126, 135, 141, 148, 149, 236, 241, 245, 265, 296 public awareness, 48, 52 public health, ix, 125, 126, 135, 141, 148, 296 Public Health Service, 143 public opinion, 54 public sector, 46, 47, 58 public welfare, 52 PUFA, x, 155, 188, 189, 190, 192, 202, 210, 214, 221, 222 PUFAs, 34 pulps, 91 pumps, 296 purchasing power, 3, 254 purification, 82 pyrimidine, 26, 59 pyruvate, 25, 62 Pyszczynski, 105, 109

Q quail, 193, 200, 207, 209, 221, 224, 225, 227, 228 qualifications, 52 quality control, 269, 290 quality improvement, 66 quality of life, viii, 113, 114 quartile, 271, 273, 275, 276 quartz, 296 quercetin, 203 questionnaire, 111, 259, 269 questionnaires, 258 quinine, 209 quinone, 228 quotas, ix, 43, 161, 162

R race, 114, 117, 157, 236 racism, 107 radical, 194, 196, 204, 206, 224 radical formation, 204 rancid, 177, 290 random, 6, 29 range, vii, ix, x, 4, 13, 15, 16, 29, 36, 46, 62, 79, 82, 87, 153, 161, 162, 166, 171, 176, 179, 183, 184, 185, 243, 244, 255, 256, 257, 298, 300 rat, 31, 228 ratings, 107, 163, 169, 258, 263 rats, 88, 192, 202, 208, 209, 217, 221, 227 raw material, viii, 73, 76, 83, 86, 87, 289, 292 raw materials, viii, 73, 86, 87 reactive oxygen, 15, 17, 149, 187, 192 reactive oxygen species (ROS), 15, 17, 149, 187 reactivity, 196 reading, 141 reagents, 39, 297 real time, 286 reality, 247 reasoning, 53 receptors, 12, 13, 17 recognition, x, 45, 231 recombinant DNA, 5, 83 recombination, 82, 83 reconcile, 52 recovery, xii, 19, 82, 93, 295 recycling, 56, 78, 232, 248 red meat, 258, 268 redfish, x, 161, 170, 176, 178, 179, 180, 181, 182, 183 redistribution, 60 redox, 227 reduction, ix, 12, 15, 26, 35, 37, 42, 43, 74, 81, 82, 83, 85, 90, 92, 96, 120, 126, 127, 136, 147, 150, 151, 152, 153, 154, 166, 197, 204, 210, 239, 264, 296 redundancy, 62 refineries, 86 refining, 191, 215 regional, 126, 127, 254 regression, 102, 117, 118, 119, 120, 209, 270, 276, 291 regression analysis, 119 regular, vii, viii, 53, 73, 99, 100, 152, 154, 156, 234 regulation, 7, 17, 23, 25, 35, 37, 42, 45, 53, 148, 151, 152, 208 regulations, 42, 43, 44, 48, 49, 51, 56 regulators, 16, 52, 53, 54 regulatory requirements, 48 Reinhardtius hippoglossoides, 184 rejection, 51, 100, 189 relationship, viii, 100, 113, 114, 117, 118, 119, 120, 121, 173, 174, 175, 208, 214, 232, 277, 279, 292 relationships, 52, 90, 109, 223, 286, 288, 290, 294

Index relatives, 5 relativity, 265 relevance, 284 reliability, 284, 301 Reliability, 109 religion, 100 renewable resource, 239 renin, 149, 150 renin-angiotensin system, 158 repair, 74 repetitions, 284 replication, 39 representativeness, 292 reproduction, 16, 81, 100, 228 research, vii, viii, xi, 21, 27, 40, 45, 46, 47, 49, 54, 55, 69, 73, 75, 81, 89, 100, 113, 114, 115, 117, 118, 120, 121, 157, 170, 185, 186, 202, 203, 211, 242, 267 Research and Development, 242, 250 researchers, 14, 28, 52, 81, 121, 296 reservation, 261 reserves, vii, 4 reservoir, 188 residuals, 277, 278 residues, 10, 39, 78, 83, 86, 89, 91, 94, 96, 233, 239, 243, 245, 248 resistance, ix, 6, 11, 12, 13, 15, 16, 17, 21, 55, 57, 60, 62, 63, 64, 67, 68, 69, 71, 147, 148, 149, 153, 158, 160, 226 resistin, 158 resources, vii, xi, 9, 10, 11, 26, 35, 47, 48, 49, 57, 68, 73, 74, 93, 113, 115, 231, 233, 238, 239, 240, 248, 250 restaurant, 244 Resveratrol, 219 retail, 60, 176, 183, 254, 255, 257 retention, 202, 203, 228, 285 retinol, 209, 224 revenue, 234, 242 revolt, 237 Reynolds, 56, 65, 248, 250 ribonucleic acid, 82, 91 rice, 5, 8, 9, 12, 13, 14, 18, 20, 21, 23, 24, 25, 26, 27, 30, 31, 33, 35, 39, 46, 54, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 78, 84, 90, 95, 236, 241, 243 risk, viii, ix, xi, 13, 44, 48, 50, 52, 58, 61, 74, 87, 89, 108, 113, 114, 117, 120, 121, 125, 127, 133, 134, 135, 136, 140, 141, 144, 147, 148, 149, 150, 151, 152, 154, 156, 158, 159, 239, 241, 243, 267, 276, 277, 278, 279, 295, 296, 298 risk assessment, viii, 48, 74 risk factors, viii, ix, 113, 117, 125, 127, 134, 136, 140, 141, 147, 149, 158, 159, 279 risk management, 243 risks, 11, 52, 87, 136 Rita, 261, 265 RNA, 7, 37, 55, 62, 63, 69, 77, 81, 82 RNAi, 7, 15, 16, 21, 36, 59, 63, 69

325

rodent, 87, 89 rodents, 152, 296 Rome, 58, 70, 248, 265 ROS, 17, 18, 21 routines, 100 Royal Society, 212 rubber, 9, 42 rubbers, 9 ruminant, 191, 234, 246, 250 rural, vii, 3, 4, 5, 9, 10, 110, 233, 241, 242, 243, 244, 245, 246, 250, 251, 268, 270, 272, 275, 276 rural areas, 3, 241, 242, 243, 244, 245, 268, 276 rural population, 246 Russia, 254 Rwanda, 237, 251 RXR, 154

S Saccharomyces cerevisiae, 68, 84, 90, 93 sacrifice, 234 sadness, 106 safeguard, 45, 296 safety, 12, 43, 44, 48, 51, 61, 81, 100, 171, 186, 215, 216, 222, 237, 262, 263, 264 sales, 243, 244, 246, 253, 254, 255, 256 saline, 18 salinity, 8, 17, 19, 20, 21, 23, 24, 55, 70 salmon, 90, 153, 165, 170, 176, 185, 287, 288, 289, 290, 291, 292, 294 salt, 18, 19, 20, 21, 22, 23, 60, 64, 67, 71, 164, 166, 172, 173, 174, 175, 186, 229, 259 salts, 4 saltwater, 90 sample, 114, 116, 117, 120, 121, 163, 166, 168, 170, 175, 177, 180, 183, 260, 268, 269, 270, 271, 276, 296, 298, 300, 301 sampling, 268, 293 sanctions, 238 satisfaction, 105, 111 saturated fat, ix, 36, 104, 147, 151, 152, 187, 189, 208 saturated fatty acids, 36, 187, 189, 208 savings, 233, 241, 249 scalable, 39 scarcity, 109 school, 116, 117, 119, 120, 122, 123, 127, 128, 129, 130, 131, 132, 133, 134, 236, 246, 260, 269, 278 scientists, 47, 54 Scomber scombrus, 184, 290 scores, x, 161, 163, 164, 166, 168, 170, 175, 176, 178, 179, 181, 182, 183, 184, 194, 210, 261, 262, 263, 265, 285 SCP, viii, 73, 74, 76, 77, 78, 79, 80, 81, 82, 83, 85, 86, 87, 88, 89, 91, 92, 94, 95 seafood, vii, ix, xii, 104, 125, 126, 127, 133, 134, 136, 137, 138, 139, 141, 147, 152, 153, 156, 162,

326

Index

163, 164, 168, 170, 171, 173, 185, 186, 283, 286, 289, 290, 295, 296, 297, 298, 300, 301 search, 234 searching, 86 seasonal pattern, 137 seasonality, 137 seaweed, 86, 298 secretion, 150, 153, 154, 156 security, vii, viii, x, xi, 3, 4, 8, 10, 11, 17, 27, 28, 39, 42, 43, 44, 45, 53, 99, 100, 101, 102, 104, 105, 106, 107, 108, 109, 110, 113, 115, 121, 231, 232, 233, 234, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 247, 248, 250 seed, 26, 27, 30, 33, 36, 56, 61, 62, 66, 68, 70, 71, 223 seedlings, 67 seeds, 6, 14, 19, 27, 28, 30, 31, 32, 33, 58, 59, 65, 67, 70, 71, 190, 214, 222, 223 segmentation, 265 selectivity, 51 selenium, 5, 63, 187, 193, 208, 209, 211, 212, 214, 226, 227 Self, 109, 251 self-awareness, 109 self-concept, 100 self-esteem, 107, 109 semiarid, 242 sensation, 114 sensations, 206 sensitivity, 50, 120, 151, 152, 153, 154, 159 separation, 76, 202, 215, 284, 296 septicemia, 13, 14 sequencing, 81 series, viii, xi, 6, 8, 38, 50, 99, 101, 105, 117, 163, 168, 231 serum, xi, 38, 41, 68, 109, 115, 123, 153, 154, 160, 207, 226, 267, 269, 270, 276, 277, 278, 279, 280 serum albumin, 38, 68 serum ferritin, xi, 267, 269, 270, 276, 277, 278, 279, 280 services, 100, 107, 233, 238, 239 SES, 268, 270, 271, 272, 275 settlers, 236 severity, 27 sex, 105, 109, 118, 260, 271 sexuality, 100 shame, 115 Shanghai, 268 shape, 51, 100, 107 shaping, 50, 157 sharing, 109 shear, 166, 167, 172 sheep, 234, 238, 239, 242, 244, 245, 255, 256 shellfish, ix, 125, 126, 133, 134, 136 shelter, 12, 118 shock, 21, 82 shocks, 237 shores, 236 short period, 209

shortage, 241 short-term, 227, 239 shrimp, 291, 293 sign, 120 signal transduction, 15, 21 signaling, 21, 25, 27, 67 signaling pathway, 21, 27, 67 signaling pathways, 21, 67 signalling, 149, 150, 154, 156, 262, 264, 296 signals, 15, 65, 223, 284 silk, 9, 39, 67 silver, x, 161, 162, 163, 166, 169, 179, 183, 186 similarity, 12 simulation, 133, 139 simulations, 135, 136, 137, 138, 141 Singapore, 90 sites, 12, 19 skeletal muscle, 188, 189, 203, 209, 210, 217, 218, 223 skeleton, 37 skills, 123 skin, 80, 87, 168, 169, 191, 200, 206, 207, 208, 209, 211, 234 sleep, 106 smallpox, 131 smoke, x, 161, 168, 171, 172, 173, 174, 175, 176, 294 smoking, 269, 275, 276, 287 social class, 254, 268, 278 social environment, 110 social norms, 268 social obligations, 243 social order, 100 social roles, 107 social security, viii, 107 social skills, 123 social status, 245 social structure, vii, 3, 52 socioeconomic, viii, xi, 113, 114, 115, 143, 267, 268, 276, 278 socioeconomic conditions, 278 socioeconomic status, viii, xi, 113, 114, 267, 268, 276 sociology, 110 SOD, 17, 18, 187, 207, 208, 218 SOD1, 18 sodium, 19, 71, 104, 164, 165, 208, 222 soft drinks, 4 software, 291 soil, vii, 4, 5, 8, 11, 12, 17, 24, 78, 90, 225, 233, 256, 296 soils, 69, 208, 243 solar, 77, 96 solar energy, 77 solid state, 83, 87, 90, 91, 92, 94, 95 solutions, x, 42, 162, 174, 175, 302 solvent, 202, 297 solvents, 202 Somalia, 233

Index sorbitol, 26, 42, 56 South Africa, 9, 236, 254 South America, 83 South Pacific, 111 Southeast Asia, 10, 142, 233 soy, 58, 85, 221, 268, 278 soybean, 10, 28, 30, 33, 34, 45, 58, 59, 69, 76, 189, 223 soybeans, 42 Spain, 3, 147, 187, 254, 255, 258, 265, 277 speciation, 226, 300 species, ix, x, 5, 7, 10, 11, 12, 15, 17, 18, 26, 56, 64, 76, 77, 78, 79, 80, 87, 89, 91, 92, 96, 149, 150, 155, 156, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 176, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 189, 191, 192, 215, 219, 234, 242, 244, 245, 247, 250, 296, 297, 298, 300, 301 specificity, 12, 16 spectrophotometry, 229 spectrum, 5, 171 speed, 5, 7, 297 spermidine, 70 spina bifida, 28 spinach, 26, 51, 68 spiritual, 100 spleen, 208, 209 sporadic, 139 spouse, 101, 129 SRS, 192 stability, x, xii, 36, 39, 188, 194, 195, 196, 197, 198, 199, 200, 201, 202, 204, 205, 206, 207, 208, 209, 210, 213, 216, 217, 218, 219, 222, 224, 225, 229, 283 stabilization, 196 stabilize, 14, 17, 242 stages, 42, 88, 196 stainless steel, 85 standard deviation, xii, 117 standards, xi, 43, 48, 49, 126, 135, 253, 256, 257, 258, 261, 262, 263, 264, 297 Staphylococcus, 81 Staphylococcus aureus, 81 starch, 25, 26, 39, 56, 83, 84, 223 starvation, 45, 51, 52, 62, 108, 236 statistics, 117, 265, 279 steel, 85 sterilization, 85 stimulus, 284 stock, 85, 237, 248, 249, 251 storage, 24, 26, 27, 30, 32, 33, 42, 62, 162, 163, 165, 166, 170, 176, 177, 178, 179, 180, 181, 182, 185, 186, 188, 194, 196, 201, 205, 209, 213, 216, 217, 218, 220, 221, 229, 286, 287, 288, 289, 290, 292, 293, 294 strain, 54, 82, 83 strain improvement, 82 strains, 12, 78, 82, 83, 142, 191 strategies, vii, x, 4, 5, 7, 12, 13, 14, 15, 16, 19, 24, 28, 30, 35, 36, 38, 42, 48, 54, 55, 57, 71, 81, 94,

327

126, 127, 142, 143, 188, 190, 208, 226, 247, 251, 263 strawberries, 219 streams, 96 strength, 163, 166 streptavidin, 64 stress, 17, 18, 19, 20, 21, 22, 23, 24, 37, 54, 55, 56, 60, 61, 62, 64, 65, 66, 67, 68, 69, 70, 71, 115, 148, 149, 157, 158, 192, 196, 200, 207, 209, 224, 225, 226, 228 stroke, 151, 152 stromal, 25 structural changes, 115 structural gene, 21 structural protein, 17 students, 129, 185, 234 sub-cellular, 18, 46 subgroups, 127 subjectivity, 285, 286 sub-Saharan Africa, 47, 249 subsidies, 43, 46, 258 subsistence, 3, 8, 9, 11, 27, 47, 54, 232, 242, 245 substances, 74, 150, 195, 206, 220, 223 substitution, 190, 213 substrates, 77, 78, 83, 85, 86, 89 sucrose, 25, 26, 63, 66 suffering, 45, 148, 152 sugar, 9, 10, 18, 25, 26, 65, 92, 248, 265 sugar beet, 10, 18, 92 sugar cane, 10, 248 sugarcane, 78, 86, 91 sugars, 10, 19, 21, 25, 35 suicide, 236 sulfate, 225, 226 sulphate, 278 sulphur, 77, 83, 85, 210 Sun, 71, 279, 280 sunflower, 45, 153, 189, 215, 216, 217 sunlight, 78 superoxide, 17, 62, 65, 66, 69, 187, 204, 227 superoxide dismutase, 17, 62, 66, 69, 187, 204 supervision, 238 supplemental, 207, 210, 213, 224, 226, 227 supplements, viii, 73, 87, 154, 155, 159, 188, 195, 196, 197, 201, 202, 205, 207, 209, 212, 216, 229 suppliers, 171, 240 supply, vii, viii, 10, 42, 50, 77, 78, 86, 99, 101, 105, 107, 108, 113, 114, 148, 150, 186, 232, 239, 242, 245, 255, 257, 296 supply chain, 42 suppression, 15, 26, 29, 36, 37, 57, 65, 82, 152 surgical, 39 surprise, 21 surveillance, 127, 144, 238 survival, x, 9, 13, 15, 19, 100, 101, 104, 105, 107, 137, 142, 144, 231, 233 surviving, 28 susceptibility, 16, 67, 77, 199, 200, 201, 204, 205, 206, 207, 208, 209, 210, 213, 217, 222, 224

328

Index

sustainability, 10, 42, 247 sustainable development, x, 250 Switzerland, 254 symbiotic, 14 symbolic, 108, 109 symbols, 108 sympathetic, 52, 115 sympathetic nervous system, 115 symptom, vii, viii, 99 symptoms, 65, 87, 152, 227 syndrome, ix, 150, 153, 157, 158, 160, 280 synergistic, 199, 202, 205, 211 synergistic effect, 202 synthesis, 6, 15, 19, 20, 26, 35, 36, 37, 56, 57, 59, 65, 70, 75, 81, 152, 153, 154, 208, 209, 225 systems, xi, 46, 48, 69, 93, 115, 220, 232, 233, 243, 245, 246, 248, 249, 250, 251, 253, 256, 257, 260, 261, 263, 264, 284 systolic blood pressure, 154

T T cell, 41, 69 Taiwan, 77 tangible, 101 tannins, 83, 85, 203, 223 Tanzania, 240, 241, 249, 250 target population, 135 targets, 6, 17, 24 tariff, 43, 257 tariffs, 46 taste, x, 108, 161, 164, 166, 169, 172, 177, 183, 184, 199, 201, 206, 210, 260, 265 taxonomic, 12 tea, 9, 197, 200, 204, 205, 212, 219, 220, 236 teachers, 129 technology, 5, 9, 10, 11, 12, 13, 17, 19, 26, 27, 28, 35, 39, 42, 43, 47, 48, 49, 51, 52, 54, 55, 82, 86, 162, 170, 171, 176, 182, 247, 248, 283, 290 technology transfer, 42, 47, 48 television, 52 temperature, 18, 22, 67, 69, 85, 144, 259, 296, 297, 298, 299 temporal, 126, 131, 141 tension, 101 terpenes, 197 Terror Management Theory, 105, 109 Thailand, 45, 70, 258 thawing, 166, 176 theory, 93, 109, 110, 111 thermal stability, 39 thinking, 74, 106, 107 thiobarbituric acid, 195, 206, 223, 224 thioredoxin, 21 Third World, 249 Thomson, 226 threat, 12, 64, 105, 110, 111 threatened, viii, 99, 101, 107

threatening, 107 threats, viii, 99, 107 threonine, 32, 33, 83 thrombomodulin, 155 thrombosis, 149, 158 tiger, 46 time, ix, 3, 5, 8, 11, 12, 27, 35, 42, 51, 52, 59, 76, 79, 87, 101, 108, 113, 125, 131, 132, 133, 134, 135, 155, 157, 177, 178, 179, 180, 181, 182, 191, 209, 214, 236, 243, 246, 259, 285, 289, 293, 296, 297 timing, 118 Timmer, 4, 69 tissue, 15, 20, 26, 27, 39, 74, 148, 149, 152, 154, 156, 158, 159, 175, 188, 203, 208, 211, 212, 213, 227, 297 tobacco, 9, 14, 15, 21, 26, 36, 37, 38, 39, 45, 55, 57, 58, 62, 63, 64, 66, 67, 68 Tocopherol, 192 tocopherols, 28, 192, 193, 194, 213, 218 tocotrienols, 28, 192 tofu, 277 Tokyo, 160 tolerance, 11, 17, 18, 19, 20, 21, 22, 23, 24, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 tomato, 8, 18, 19, 23, 26, 27, 29, 38, 51, 56, 57, 59, 60, 63, 64, 65, 66, 70, 170, 200, 204, 224, 225 total energy, 270 total product, 233, 256 toxic, xii, 15, 16, 80, 82, 87, 295, 296, 301 toxicity, viii, 73, 76, 79, 80, 87, 89, 92, 207, 208, 209, 226 toxicological, 87, 89 toxicology, 92 toxin, 12, 13, 14, 41, 79 toxins, 12, 13, 14, 36, 58, 76, 77, 79, 80, 81, 89 trace elements, 218, 227 trade, 9, 42, 43, 44, 45, 48, 49, 50, 54, 55, 63, 69, 234, 244, 254, 257 trade war, 44 trading, xi, 49, 56, 66, 236, 253 trading partners, 66 tradition, 236 training, 261, 284 traits, 6, 7, 8, 17, 29, 63, 70, 206, 214, 223, 226 trans, 152, 154, 191 transcript, 57, 81 transcription, 17, 22, 23, 46, 60, 61, 64, 156 transcription factor, 22, 23, 60, 61, 64, 156 transcription factors, 22, 23, 60, 156 transcriptional, 149, 152, 153, 154 transduction, 22 transfer, 7, 15, 17, 19, 29, 39, 42, 47, 48, 60, 68, 172, 173, 174, 175, 227, 238 transformation, 7, 8, 25, 29, 38, 46, 55, 58, 71 transgene, 29, 39, 66 transgenesis, 5 transgenic, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 24, 25, 26, 27, 28, 29, 30, 32, 33,

Index 34, 36, 37, 38, 39, 40, 42, 46, 52, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 transgenic plants, 7, 11, 12, 14, 15, 16, 17, 18, 19, 24, 25, 27, 34, 38, 39, 52, 59, 63, 65, 66, 67, 68, 71 transition, xi, 148, 267, 268, 276, 279 transmission, ix, 66, 125, 126, 127, 128, 132, 133, 134, 135, 136, 144, 279 transparency, 48 transpiration, 22, 55 transport, 7, 26, 30, 60, 233, 237, 238, 240, 242, 249 transportation, 50 transposons, 7 travel, 129, 133, 135, 138 trawlers, 164 trees, 9, 19, 65 trend, xi, 257, 267, 275, 276, 279 triacylglycerols, 58 trial, 48, 153, 157, 164, 167 tribes, 236 Trichoderma viride, 84 triglyceride, ix, 147, 153, 154, 210 triglycerides, 149, 150, 151, 153, 156 trout, 287, 289, 292, 293 trust, 52, 53, 58, 68 trypsin, 39 tryptophan, 32, 33, 37, 70, 75, 82, 83 tubers, 26, 39, 58, 59 tumor, 296 tumour, 149 Turkey, 194, 195, 198, 205, 220, 222 turkeys, 198, 219, 221, 228, 239 turnover, 151 type 2 diabetes, 148, 149, 154, 158, 277, 280 type 2 diabetes mellitus, 148, 149, 154, 158 typology, 110 tyrosine, 15

U U.S. Department of Agriculture, 40, 41, 43, 69, 114, 117, 124, 256, 266 ubiquitin, 18, 68 Uganda, 45, 47, 250 uncertainty, 100, 106 UNEP, 232, 251 unfolded, 52 unhappiness, 101 United Kingdom, 3, 55, 56, 62, 66, 95, 96, 141, 170, 231, 254, 255, 258, 297 United Nations, 49, 238, 251 United Nations Environment Program, 251 United States, vii, viii, 42, 69, 109, 110, 111, 113, 114, 116, 122, 123, 124, 143, 144, 253, 256 univariate, 277 universities, 46, 48 updating, 132 urban areas, 254, 269

329

urban population, 243 urbanization, vii, 4, 74, 237, 239 urea, 75 uric acid, 81, 210 uridine, 26 urinary, 81 Uruguay, 256 Utah, 97

V vaccination, ix, 64, 125, 126, 127, 135, 137, 139, 140, 141, 142, 145 vaccine, 39, 40, 41, 64, 69, 133, 135, 137, 139, 140, 238 vacuole, 19 vacuum, 170, 171, 176 validation, 150, 296 validity, 104, 109 values, xii, 76, 86, 100, 101, 105, 108, 109, 110, 111, 136, 140, 154, 156, 163, 164, 165, 166, 167, 171, 172, 173, 174, 175, 178, 179, 180, 181, 183, 194, 195, 205, 218, 226, 250, 265, 270, 295, 299, 300, 301 variability, 182, 241, 255, 285, 299 variable, viii, 46, 113, 114, 117, 118, 120, 121, 135, 175, 296, 297 variables, 117, 118, 120, 178, 180, 181, 182, 183, 270, 273, 274, 276, 277 variation, 5, 6, 60, 77, 118, 191, 254 vascular cell adhesion molecule, 155 vasodilatation, 149 vector, 81, 117 vegetable oil, 190, 213 vegetables, 27, 29, 104, 108, 152, 189, 204, 230, 268, 270, 279 vegetation, 234 velvet, 167, 169, 184 village, 269 vinblastine, 36 vincristine, 36 violence, 237 violent, 251 viral diseases, 12 viral hepatitis, 144 virus, ix, 40, 41, 60, 65, 70, 125, 131, 133, 134, 135, 142, 143, 144, 235, 277, 279 virus infection, 142 viruses, 141 visceral adiposity, ix, 147 vitamin A, 4, 28, 47, 70, 104, 226 vitamin C, 28, 29, 56, 91, 104, 217, 218, 228, 270, 273 vitamin D, 209 vitamin E, 28, 56, 58, 192, 193, 213, 214, 216, 217, 218, 220, 222, 224, 226, 227, 228, 229, 230 vitamin K, 209, 228

330

Index

vitamins, 4, 5, 7, 8, 28, 29, 30, 54, 64, 74, 76, 83, 86, 152, 187, 192, 209, 218, 227, 268 volatility, 56 vomiting, 87

W walking, 81 war, 108 Warsaw, 185 waste products, 233 waste water, 85, 92, 145 wastes, 10, 76, 78, 83, 84, 86, 94 wastewater treatment, 92 water, vii, ix, 4, 8, 12, 19, 20, 22, 51, 55, 65, 66, 68, 70, 85, 125, 126, 133, 136, 142, 162, 163, 171, 173, 175, 176, 179, 181, 184, 186, 195, 196, 202, 208, 209, 211, 240, 292, 296, 297 water-holding capacity, 164 water-soluble, 171, 209, 210 wealth, vii, viii, 4, 36, 39, 54, 100, 101, 113, 234 wear, 115 weight gain, 26, 123, 148, 174 weight loss, ix, 147, 153, 158, 159, 174 weight reduction, ix, 152 Weinberg, 142 welfare, 45, 114, 115, 117, 121, 124, 225, 245, 251, 261, 263, 265 well-being, viii, 43, 113, 114, 116, 150 West Africa, 242, 249 western countries, 187, 258 Western Europe, 254 WHC, 164, 165, 166, 183 wheat, 8, 10, 19, 20, 24, 26, 27, 55, 58, 65, 68, 70, 78, 96, 268 whey, 85, 89, 92, 94, 95, 164, 165 WHO, 28, 30, 70, 157, 158, 278, 279 wild type, 13, 17, 19, 22, 25, 27, 30, 31, 32, 37, 83 wisdom, 141

women, viii, x, xi, 107, 110, 111, 113, 114, 115, 117, 118, 119, 120, 121, 124, 148, 149, 155, 159, 160, 231, 232, 235, 244, 245, 267, 268, 269, 270, 271, 274, 275, 276 wood, 78, 86, 203, 223 wood waste, 86 wool, 234 workers, 129, 269 workplace, 130, 132, 134 World Bank, 3, 70, 71, 249, 250 World Development Report, 70 World Food Program, 42 World Health Organization, 148, 157, 278 World Trade Organization, 43, 44, 49 worm, 15 WTP, 258, 260, 263

X xanthophyll, 207, 224 X-rays, 6

Y yeast, 25, 74, 76, 77, 81, 82, 83, 92, 93, 94, 96, 200, 206, 208, 224, 225 yield, 6, 8, 11, 16, 24, 25, 26, 27, 35, 37, 64, 66, 68, 71, 82, 83, 284 yield loss, 11 yogurt, viii, 73, 76, 263, 266 yolk, 204, 206, 214, 216, 217, 218, 224 young adults, 157, 158, 160

Z Zea mays, 13, 30, 32 Zimbabwe, 45, 236, 237, 239, 249, 250, 251 Zinc, 5, 17, 18, 23, 30, 59, 65, 69, 81, 187, 207, 208, 209, 212, 214, 225, 227, 228