Biotechnology in Functional Foods and Nutraceuticals

These functional foods or nutraceuticals have become increasingly important to consumers who are inter-ested in the health benefi ts of functional foods in the prevention of illness and

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—Debasis Bagchi

To my mother with gratitude and affection.

—Francis C Lau

To my wife, Sumita, and two sons, Rohit and Roneet, for their consistent

help and support.

—Dilip K Ghosh

Contents

Preface xiEditors xiiiContributors xv

PART I Biotechnology for the Enhancement of

Functional Foods and Nutraceuticals

1

Chapter Advances in Biotechnology for the Production of Functional Foods 3

Yun-Hwa Peggy Hsieh and Jack Appiah Ofori

2

Chapter Functional Foods and Biotechnology in Japan 29

Harukazu Fukami

3

Chapter Basic and Clinical Studies on Active Hexose Correlated Compound 51

Takehito Miura, Kentaro Kitadate, Hiroshi Nishioka, and Koji Wakame

Chapter Improving the Bioavailability of Polyphenols 81

Tetsuya Konishi and M Mamunur Rahman

6

Chapter The Function of the Next Generation Polyphenol, “Oligonol” 91

Takehito Miura, Kentaro Kitadate, and Hajime Fujii

7

Chapter Application of Biotechnology in the Development of a Healthy Oil Capable

of Suppressing Fat Accumulation in the Body 103

Hiroyuki Takeuchi

8

Chapter Effects of Nutraceutical Antioxidants on Age-Related Hearing Loss 113

Shinichi Someya, Tomas A Prolla, and Masaru Tanokura

PART II The Impact of Genetic Modifi cation on

Functional Foods

9

Chapter Increased Production of Nutriments by Genetically Engineered Bacteria 127

Kazuhiko Tabata and Satoshi Koizumi

10

Chapter Recent Advances in the Development of Transgenic Pulse Crops 139

Susan Eapen

11

Chapter The Improvement and Enhancement of Phyto-Ingredients Using

New Technology of Genetic Recombination 157

Chapter The Use of Biotechnology to Reduce the Dependency of Crop Plants

on Fertilizers, Pesticides, and Other Agrochemicals 197

Zeba F Alam

14

Chapter Animal Biotechnology: Applications and Potential Risks 219

Rama Shanker Verma, Abhilash, Sugapriya M.D., and Chithra R.

Chapter Microbial Production of Organic Acids and Its Improvement

by Genome Shuffl ing 265

Takashi Yamada

PART III New Frontier in Food Manufacturing Process

17

Chapter Microalgal Biotechnology in the Production of Nutraceuticals 279

Niels-Henrik Norsker, Maria Barbosa, and René Wijffels

18

Chapter The Innovation of Technology for Microalgae Cultivation and Its Application

for Functional Foods and the Nutraceutical Industry 313

Akira Satoh, Masaharu Ishikura, Nagisa Murakami, Kai Zhang, and Daisuke Sasaki

19

Chapter Production of Nattokinase as a Fibrinolytic Enzyme by

an Ingenious Fermentation Technology: Safety and Effi cacy Studies 331

Shinsaku Takaoka, Kazuya Ogasawara, and Hiroyoshi Moriyama

20

Chapter Synthesis of Antihypertensive GABA-Enriched Dairy Products Using

Lactic Acid Bacteria 349

Chapter Tracking the Careers of Grape and Wine Polymers Using Biotechnology

and Systems Biology 389

John P Moore and Benoit Divol

23

Chapter The Impact of Supercritical Extraction and Fractionation Technology on the

Functional Food and Nutraceutical Industry 407

Andrés Moure, Beatriz Díaz-Reinoso, Herminia Domínguez, and Juan Carlos Parajó

24

Chapter The Application of Nanotechnology to Functional Foods and Nutraceuticals

to Enhance Their Bioactivities 447

Ping-Chung Kuo

PART IV Quality Assurance and Safety: Design and

Implementation

25

Chapter Enhancing the Nutritional Quality of Fruit Juices: Advanced Technologies

for Juice Extraction and Pasteurization 465

Robert D Hancock and Derek Stewart

26

Chapter Probiotics: Health Benefi ts, Effi cacy, and Safety 485

Nagendra P Shah

27

Chapter Use of High Pressure Technology to Inactivate

Bacterial Spores in Foods 497

Noriyuki Igura, Seiji Noma, and Mitsuya Shimoda

PART V Legal, Social, and Regulatory Aspects of Food

Biotechnology

28

Chapter Regulations of Biotechnology: Generally Recognized as Safe (GRAS)

and Health Claims 507

Ryan R Simon, Earle R Nestmann, Kathy Musa-Veloso, and Ian C Munro

29

Chapter Global Food Biotechnology Regulations and Urgency for Harmonization 531

Dilip K Ghosh and Peter Williams

PART VI Future of Biotechnology

30

Chapter Future Strategies for the Development of Biotechnology-Enhanced

Functional Foods and Their Contribution to Human Nutrition 545

Dilip K Ghosh, Francis C Lau, and Debasis Bagchi

Index 549

Preface

Biotechnology has been used thousands of years ago in the manufacturing of food products The most ancient form of biotechnology, fermentation, involved the use of microorganisms such as yeasts for the production of wine, vinegar, and bread Dairy products such as yogurt and cheese were produced by lactic acid bacteria and molds Although these techniques are still used, the cul-tures that were used in ancient times have been modifi ed to provide high-quality products with increased yield Modern food biotechnology has evolved into a billion-dollar industry, with the promise of producing foods that provide functions beyond the basic nutrients they contain These functional foods or nutraceuticals have become increasingly important to consumers who are inter-ested in the health benefi ts of functional foods in the prevention of illness and chronic conditions.Biotechnology is a collection of biology-based technologies used mainly in agriculture, food science, and medicine Agricultural biotechnology may involve the use of molecular and/or bio-chemical techniques to produce desired traits, while eliminating many unwanted traits in plants, through the use and manipulation of genetic information In fact, agricultural biotechnology has been seriously affected by the new recombinant DNA technique that emerged in the 1970s Genetic modifi cation has signifi cantly improved the yield, quality, and nutritional value of crop plants and animal products It was estimated that approximately 13.3 million farmers in 25 countries were using agricultural biotechnology in 2009 This came at a time when the world sought science-based and consumer-focused approaches to solving the problem of feeding a growing population In this respect, agricultural biotechnology is able to deliver resilient crops with enhanced yield even when they are grown in harsh environments

Animal biotechnology also plays an important role in agriculture today Genetic modifi cation is used to improve livestock selection and breeding Moreover, animal genomics is utilized to provide optimal nutritional needs for animals to generate high-quality animal products such as meat, milk, and eggs Overall, biotechnology helps in enhancing food manufacturing processes, improving food preservation, and ensuring food safety Thus, biotechnology provides the necessary means for the development and improvement of bioactive components in functional foods and nutraceuticals.This book covers the various aspects of biotechnology in nutraceuticals and functional foods The goal of the book is to provide readers with comprehensive reviews, by a panel of experts from around the world, focusing on state-of-the-art topics that are broad in scope yet concise in structure This book is divided into six parts The fi rst part gives an overview of recent advances in bio-technology and their contribution to food science The second part examines the impact of genetic modifi cation on functional foods The third part explores food manufacturing technology The fourth part gives insight into quality assurance and safety of foods The fi fth part updates current views on legal, social, and regulatory aspects of food biotechnology A fi nal commentary concludes the book by offering an overview of future directions in the applications of biotechnology to func-tional foods and nutraceuticals

Editors

Debasis Bagchi received his PhD degree in medicinal chemistry in 1982 He is a professor in the

Department of Pharmacological and Pharmaceutical Sciences at University of Houston College of Pharmacy, Houston, Texas Dr Bagchi is also senior vice president of Research & Development

of InterHealth Nutraceuticals, Inc., Benicia, California Dr Bagchi is currently the president-elect

of American College of Nutrition, Clearwater, Florida, and also serves as a distinguished advisor

on the Japanese Institute for Health Food Standards, Tokyo, Japan and as chairperson on the Nutraceuticals and Functional Foods Division, Institute of Food Technologists, Chicago, Illinois

He serves as the vice-chair of the International Society for Nutraceuticals and Functional Foods (ISNFF) His research interests include free radicals, human diseases, carcinogenesis, pathophysiol-ogy, mechanistic aspects of cytoprotection by antioxidants, regulatory pathways in obesity and gene expression, and biotechnology

Dr Bagchi has 271 peer-reviewed publications and numerous books He has delivered invited lectures in various national and international scientifi c conferences, organized workshops, and group discussion sessions He is a fellow of the American College of Nutrition, member of the Society of Toxicology, member of the New York Academy of Sciences, fellow of the Nutrition Research Academy, and member of the trichloroethylene (TCE) stakeholder Committee of the Wright Patterson Air Force Base, Dayton, Ohio Dr Bagchi is a member of the Study Section and Peer Review Committee of the National Institutes of Health, Bethesda, Maryland He is also serving as editorial

board member of numerous peer reviewed journals, including Antioxidants and Redox Signaling, Journal of Functional Foods, Cancer Letters, Journal of American College of Nutrition, The Original Internist, and other scientifi c and medical journals He is currently serving as associate editor of the Journal of Functional Foods and Journal of American College of Nutrition.

Dr Bagchi received funding from various institutions and agencies, including the U.S Air Force Offi ce of Scientifi c Research, Nebraska State, Department of Health, Biomedical Research Support Grant from National Institutes of Health, National Cancer Institute, Health Future Foundation, The Procter & Gamble Company, and Abbott Laboratories

Francis C Lau is currently a scientist at InterHealth Nutraceuticals Inc., Benicia, California He

obtained his BS degree in biochemistry from the University of Alberta, Edmonton, Canada He went on to pursue his MS degrees in molecular biology and computer information systems from the University of San Francisco and the University of Houston, respectively Dr Lau obtained his PhD degree in neuroscience from the College of Veterinary Medicine at Texas A&M University

He then held a postdoctoral fellowship at the National Institutes of Health, where he was granted an Intramural Research Training Award Prior to joining InterHealth Nutraceuticals, Dr Lau held a position at the United States Department of Agriculture (USDA) Human Nutrition Research Center

on Aging, where he studied the benefi ts of nutraceuticals and functional foods such as dietary oxidants in memory and aging He has published numerous scientifi c papers, invited reviews, and book chapters He is a fellow of the American College of Nutrition His recent research interests focus on the effects of nutraceuticals and functional foods on cardiovascular health, joint health, diabetes health, and on promoting healthy body weight

anti-Dilip K Ghosh received his PhD in biomedical science from the University of Calcutta (UoC),

India Previously, he held positions in Organon (India) Ltd., a division of Organon International, BV and AKZO-NOBEL, The Netherlands; HortResearch, New Zealand; USDA-ARS, HNRCA at Tufts University, Boston; and The Smart Foods Centre, University of Wollongong, Australia He has been

involved for a long time in drug development and functional food research & development and its commercialization in both academic and industry domains He is a fellow of the American College

of Nutrition and is also a member of several editorial boards Currently, Dr Ghosh is a director at nutriConnect (http://www.nutriconnect.com.au) His research interests include oxidative stress, bio-active, functional foods and their relationship with human health, and regulatory and scientifi c aspects of functional foods and nutraceuticals

Contributors

Abhilash

Department of Biotechnology

Stem Cell and Molecular Biology Laboratory

Indian Institute of Technology Madras

Bioprocess Engineering group

Wageningen University and Research

Wageningen, The Netherlands

Chithra R.

Department of Biotechnology

Stem Cell and Molecular Biology Laboratory

Indian Institute of Technology Madras

Chennai, India

Kevin M Davies

The New Zealand Institute for Plant

and Food Research Limited

Palmerston North, New Zealand

Beatriz Díaz-Reinoso

Departamento Enxeñería Química

Universidade de Vigo, Edifi cio Politécnico

Departamento Enxeñería Química

Universidade de Vigo, Edifi cio Politécnico

Franck Grattepanche

Laboratory of Food BiotechnologyInstitute of Food Science and NutritionZürich, Switzerland

Yun-Hwa Peggy Hsieh

Department of Nutrition, Food and Exercise Sciences

Florida State UniversityTallahassee, Florida

Noriyuki Igura

Laboratory of Food Process EngineeringKyushu University

Fukuoka, Japan

Masaharu Ishikura

Life Science Business Division

Yamaha Motor Co., Ltd

Shizuoka, Japan

Kentaro Kitadate

Bio Chemical Branch

and

Scientifi c and Research Advisory Unit

Research and Development Division

Amino Up Chemical Co., Ltd

Sapporo, Japan

Satoshi Koizumi

Bioprocess Development Center

Kyowa Hakko Bio Co., Ltd

Ibaraki, Japan

Tetsuya Konishi

Department of Functional and Analytical

Food Sciences

Niigata University of Pharmacy and

Applied Life Sciences

Niigata, Japan

Ping-Chung Kuo

Department of Biotechnology

National Formosa University

Taiwan, Republic of China

Christophe Lacroix

Laboratory of Food Biotechnology

Institute of Food Science and Nutrition

Seiji Noma

Laboratory of Food Process EngineeringKyushu University

Fukuoka, Japan

Niels-Henrik Norsker

Bioprocess Engineering group

Wageningen University and Research

Wageningen, The Netherlands

Jack Appiah Ofori

Department of Nutrition, Food and

Exercise Sciences

Florida State University

Tallahassee, Florida

Kazuya Ogasawara

Japan Bio Science Laboratory Co., Ltd

Ibaraki-shi, Osaka, Japan

Juan Carlos Parajó

Departamento Enxeñería Química

Universidade de Vigo, Edifi cio Politécnico

Niigata University of Pharmacy and

Applied Life Sciences

Niigata, Japan

Daisuke Sasaki

Life Science Business Division

Yamaha Motor Co., Ltd

Shizuoka, Japan

Akira Satoh

Life Science Business Division

Yamaha Motor Co., Ltd

Shizuoka, Japan

Jessica Scalzo

New Zealand Institute for Plant and Food

Research Limited

Hawke’s Bay Research Centre

Havelock North, New Zealand

Department of Applied Biological ChemistryUniversity of Tokyo

Kazuhiko Tabata

Bioprocess Development CenterKyowa Hakko Bio Co., LtdIbaraki, Japan

Rama Shanker Verma

Department of Biotechnology

Stem Cell and Molecular Biology Laboratory

Indian Institute of Technology Madras

Chennai, India

Koji Wakame

R&D Division, Bio Chemical Branch

Amino Up Chemical Co., Ltd

Kiyota-ku, Sapporo, Japan

René Wijffels

Bioprocess Engineering group

Wageningen University and Research

Wageningen, The Netherlands

School of Life Science and Biotechnology

Shanghai Jiao Tong University

Tokyo, Japan

Kai Zhang

Life Science Business DivisionYamaha Motor Co., LtdShizuoka, Japan

Part I

Biotechnology for the Enhancement of Functional Foods and Nutraceuticals

The old adage “you are what you eat” derives from the idea that you must eat good food in order to

be fi t and healthy The role of diet in health promotion and disease prevention has been edged for centuries, based on experience and epidemiological data Recent research has revealed the important role that diet plays in preventing and/or slowing the progression of major chronic diseases such as cancer, diabetes, and cardiovascular diseases (CVDs) This has heightened popular awareness of the importance of diet in well-being (Barnes and Prasain, 2005) Diet has thus become

acknowl-a core component of public heacknowl-alth placknowl-ans geacknowl-ared towacknowl-ard preventing premacknowl-ature chronic diseacknowl-ases, promoting healthier aging, and maintaining optimum health throughout life

CONTENTS

1.1 Introduction 31.2 Biotechnology for the Production of Plant-Based Functional Foods 51.2.1 Biofortifi cation with Essential Micronutrients 51.2.1.1 Vitamin A 81.2.1.2 Iron 81.2.1.3 Zinc 101.2.2 Biofortifi cation with Phytochemicals 101.2.3 Modifi cation of Macronutrients 111.2.3.1 Oils 111.2.3.2 Proteins 141.2.4 Production of Hypoallergenic Foods 161.2.5 Reduction of Antinutrients 171.3 Biotechnology for the Production of Animal-Based Functional Foods 171.3.1 Meat Products 18

1.3.1.1 In Vitro Meat 18

1.3.1.2 Meat with a Modifi ed FA Profi le 181.3.2 Dairy Foods 191.3.2.1 Milk for the Lactose-Intolerant Population 191.3.2.2 Milk with Enriched Antimicrobial Protein, Lysozyme 201.3.2.3 Milk with an Improved FA Profi le 211.4 Final Remarks 22References 22

This belief in the health benefi ts of foodstuffs is the basis for the current surge in interest in nutraceuticals and functional foods (Milner, 2002) Food has been used as a pharmaceutical agent

to treat diseases since time immemorial and the pharmaceutical use of food is the basis of the concept of nutraceuticals (Klein et al., 2000) The term nutraceutical, a combination of “nutrition” and “pharmaceutical,” is generally defi ned as “a food or part of a food that provides medicinal and health benefi ts, including the prevention and/or treatment of a disease” (Ramaa et al., 2006) There

is less agreement about a defi nition of functional foods; they have been defi ned in various ways, but

at present there is no universally accepted defi nition Nor is there likely to be, as foods which are not currently considered to be functional foods may come to be regarded as such in the future and functional food is therefore technically only a concept (Roberfroid, 2002) Functional foods have been broadly defi ned as “foods similar in appearance to conventional foods, which are consumed

as part of a normal diet and have demonstrated physiological benefi ts and/or reduce the risk of chronic disease beyond basic nutritional functions” (Clydesdale, 1997) Although there is no clear-cut distinction between functional foods and nutraceuticals, the basic difference between them is in the form in which they are presented Functional foods are “foods” similar in appearance to conven-tional foods, or they may even be conventional foods, consumed as part of a usual diet, demonstrated

to have physiological benefi ts and/or to reduce the risk of chronic disease beyond basic nutritional functions (Health Canada, 2002) Nutraceuticals, on the other hand, are dietary supplements that supply a concentrated version of a postulated bioactive agent extracted from a food, and are presented

in a “nonfood matrix,” often in the form of capsules or tablets and utilized with the aim of promoting health in dosages that exceed those naturally present in foods (Espin et al., 2007)

Largely as a result of consumers’ growing awareness of the health benefi ts of food, there has been a huge increase in the demand for nutraceuticals and functional foods Modern agricultural and food manufacturing practices are orientated toward the production of value-added crops and manufactured food products that are not only nutritious, wholesome, and palatable, but which also have health enhancing and disease preventing benefi ts (Hsieh and Ofori, 2007) However, beyond this demand for foods with health enhancing qualities, consumers are looking for natural products Consequently, not only are the ingredients themselves used to enhance food required to be natural but also the processes from which they are derived Among the processes currently utilized for food production, biotechnology seems to offer a powerful way to produce all kinds of desired food materials while at the same time fulfi lling the criteria of being natural (Senorans et al., 2003).The Convention on Biological Diversity (CBD) defi nes biotechnology as “any technological application that uses biological systems, living organisms, or derivatives thereof, to make or modify products or processes for specifi c use” (CBD, 2000) Traditionally, biotechnology has been used to manufacture food products for more than 8000 years: examples include bread, alcoholic beverages, cheese, yoghurt, vinegar, and other foodstuffs produced using the enzymes inherent in various microorganisms In recent years new techniques have become available and these forms of modern biotechnology are commonly referred to as “genetic engineering” or, from the scientifi c perspec-tive, “recombinant DNA technology.” The CBD defi nes modern biotechnology as “the application

of (a) in vitro nucleic acid techniques, including recombinant DNA and direct injection of nucleic

acid into cells or organelles or (b) fusion of cells beyond the taxonomic family that overcome natural physiological reproductive or recombination barriers and that are not techniques used in traditional breeding and selection” (CBD, 2000) As in food production, a major revolution has resulted from the use of modern biotechnology in agriculture In plant breeding, biotechnology can be used to introduce new characteristics with specifi c benefi ts into plants in a far more selective, controlled, and precise manner than is possible using traditional plant breeding techniques

Early applications focused on the production of high yielding crop varieties to meet the demands

of an ever-growing world population through the provision of crop varieties with improved nomic qualities such as drought, disease, and insect resistance, but there may also be additional benefi ts Insect-resistant corn (Bt corn) sustains relatively minimal insect damage and is therefore much less prone to infection by fungi and molds compared to non-insect-resistant (non-Bt) corn

agro-As a consequence, the level of toxins such as afl atoxin, which is carcinogenic for both humans and livestock, produced by these pathogens is much lower in Bt corn than in non-Bt corn These crops with improved agronomic qualities are referred to as the fi rst generation of genetically modifi ed (GM) crops, the benefi ts of which are not always immediately apparent to consumers More recent advances in modern biotechnology have been concerned with the production of crops and food ani-mals endowed with additional nutritional and health benefi ts that are more obvious to the consumer Even conventional breeding programs have begun to shift towards the production of crops with enhanced health benefi ts, partly as a result of the reservations that certain individuals and popula-tions have regarding GM foods These benefi ts involve improvements in food quality and safety,

as well as providing consumers with foods that are specifi cally designed to be more nutritious and benefi cial to health (Chassy et al., 2004)

As this chapter will demonstrate, functional foods not only address the serious problem of global malnutrition but can also provide consumers with a wide range of palatable food options on grocery shelves for the treatment or prevention of ailments Examples of selected crops genetically engineered to enhance their nutritional content are listed in Table 1.1 A representative selection of the functional food materials that have been developed to date will be examined in turn, including the technologies, the rationale behind their development, and their benefi ts, as well as future goals

OF PLANT-BASED FUNCTIONAL FOODS

1.2.1 BIOFORTIFICATION WITH ESSENTIAL MICRONUTRIENTS

Micronutrient defi ciency is a major global health problem, with over two billion people in the world today estimated to be lacking in key vitamins and minerals, particularly vitamin A, zinc, iron, and iodine (WHO/WFP/UNICEF, 2001) Biofortifi cation of staple foods, which involves the genetic engineering of foods to provide varieties that contain higher than normal amounts of health- promoting nutrients, is seen as a new and better approach to combating nutritional defi ciencies, and

is likely eventually to replace traditional techniques such as supplementation and fortifi cation (Haas

et al., 2005) Biofortifi cation is an attractive option, as traditional methods suffer from serious comings, including the high costs involved and the diffi culty of enabling individuals in remote areas

short-to gain access short-to these improved foods An effective supplementation and fortifi cation program is dependent upon political stability, which is often lacking in the areas where such nutritional inter-vention programs are most needed Also, although fertilization of crops to increase mineral con-centrations in the edible portions does indeed lead to increases in leaf mineral concentrations and improved yield, this does not always appreciably increase the mineral concentrations in the fruit, seed, or grain In addition, fertilizers can be costly, both economically and environmentally, and must be reapplied at intervals (White and Broadley, 2005) Another advantage of biofortifi cation is that it does not require a change in behavior by either farmers or consumers (IFPRI, 2002) While concerted international efforts have been made for decades to alleviate micronutrient malnutrition, transgenic approaches can complement ongoing breeding efforts and provide urgently needed bio-fortifi ed crops to feed the burgeoning world population with nutritious food (Mayer et al., 2008).Rice is unquestionably the most signifi cant food crop in the world, with over 50% of the world’s population depending on rice as their daily staple food (Zimmermann and Hurrell, 2002) A great deal of work has gone into breeding better varieties of rice to keep pace with growing demand Since rice is the staple food for most of the developing world, improving its nutritional value through bio-fortifi cation can also help address the problem of malnutrition (Bajaj and Mohanty, 2005) Efforts

to improve the nutritional value of other staple food crops such as wheat, corn, and cassava through biofortifi cation continue as a part of programs to address global malnutrition, which predominantly affects the poorest regions of the world where these crops are consumed as staples Biofortifi cation

of these staple crops has mainly focused on increasing vitamin A, zinc, and iron levels, because

Examples of Selected Staple Crops Genetically Engineered to Enhance Their Nutritional Content

Rice

Provitamin A Accumulation of carotenoids in the endosperm of rice Introduction of the entire b-carotene biosynthetic pathway into the

rice endosperm through the insertion of two genes, phytoene synthesis (psy) and phytoene desaturase (crt 1), using Agrobacterium-mediated transformation

Ye et al (2000), Paine et al

(2005)

Iron Increase iron content of the seeds by 2-fold compared to

normal rice coupled with increased bioavailability of iron

in the seeds through enhanced iron absorption

Insertion of ferritin (pfe), metallothioneinlike (rgMT) and phytase

(phyA) genes into rice embryos through Agrobacterium-mediated

transformations Pfe gene promotes accumulation of iron in the gene whereas the rgMT gene and phyA gene enhance the absorption of the iron and reduce the amount of the iron absorption inhibitor (phytic acid), respectively, thereby increasing bioavailability of the iron

Lucca et al (2002)

Zinc Increase in the zinc content of IR68144 (a conventionally

bred high iron variety) from 34 ppm to amounts ranging from 36.2 to 55.5 ppm High zinc content even after polishing

A biolistic-mediated method using a construct of the plasmid pGPTV bar/Fer which encodes for the soybean ferritin protein

from Glycine max L, and the endosperm-specifi c promoter Glu

Insertion of a chimeric gene pGlu2S, encoding a precursor polypeptide of the sulfur-rich seed storage protein, sesame 2S

albumin (S2SA), into rice using Agrobacterium-mediated

transformation

Lee et al (2003)

Lysine Increase in lysine content of seeds by 0.9–6.6% Plasmid DNAs with tRNA lys genes coding lysine instead of

glutamine (Gln), glutamic acid (Glu), and asparagine (Asn); or coding lysine at the chain termination codon, was introduced into rice callus through particle bombardment

Wu et al (2003)

Maize

Protein Increase in protein content and decrease in starch content Insertion of a construct SAG12-IPT gene consisting of the

cytokinin-synthesizing isopentenyl transferase (IPT) gene and the cysteine protease gene SAG (senescence associated gene) 12, into maize

Naqvi et al (2009)

Iron Simultaneous expression of ferritin and phytase led to both

an increase in the overall iron content (20–70%) and its bioavailability as a result of an increase in phytase expression (up to 3 IU/g of seed) which reduced levels of phytic acid

Insertion of the plasmids pSF2 (expressing the soybean ferritin gene and pLPL–phyA (expressing the phytase gene) into maize callus by particle bombardment

Drakakaki et al (2005)

Potato

Provitamin A A total carotenoid increase of up to 2.5-fold and an increase

in β-carotene levels up to a maximum of 14-fold in potato tubers

Tuber-specifi c silencing of a key gene, lycopene ε-cyclase (LYC-e)

in a branched competitive pathway in the biosynthesis of carotenoids which is responsible for the production of lutein instead of carotenoids The plasmid pBI33:As-e was introduced

into potato (Desiree variety) through Agrobacterium-mediated

transformation

Diretto et al (2006)

Protein Increase in total protein content by 35–45% and an increase

in all essential amino acids by 2.5- to 10-fold.

Plasmids were constructed from a gene that encodes the

seed-specifi c protein, amaranth seed albumin (AmA1) from Amaranthus

hypochondriacus, and the GBSS tuber-specifi c promoter gene

Plasmids were then inserted into potato shoots using

Agrobacterium-mediated transformation

Chakraborty et al (2000)

Methionine Increase in the content of free and bound methionine by

2- to 6-fold Also an increase in the amounts of soluble isoleucine and serine

Cotransferring cystathionine γ-synthase (CgS Δ90 ) and rich storage protein (15-kD β-zein) genes in potato (Desiree

methionine-variety) using Agrobacterium strains

Dancs et al (2008)

Wheat

Protein, zinc, and Iron Increase in the amount of protein, zinc, and iron in the grain

by 10–15%

Insertion of the gene, dubbed gpc-B1, from a wild emmer wheat

into conventional wheat plants

defi ciencies in these nutrients account for the majority of the problems in micronutrient-defi cient individuals (WHO/WFP/UNICEF, 2001) Biofortifi cation of staple foods with iodine is virtually nonexistent, as consumption of iodized salt is easily the best way to prevent iodine defi ciency disor ders The following sections will discuss examples of biofortifi cation with the micronutrients vitamin A, zinc, and iron, focusing primarily on rice More information on rice biotechnology can

be found in the recent review by Bajaj and Mohanty (2005) Other staple crops have also been the target of biofortifi cation with micronutrients HarvestPlus, a nongovernment organization commit-ted to the development of biofortifi ed crops for malnourished populations, is biofortifying seven key staple crops that will have the greatest impact in alleviating micronutrient malnutrition or hidden hunger in Asia and Africa These crops are beans, cassava, maize, pearl millet, rice, sweet potato, and wheat The countries that their current projects are targeting include D R Congo, Rwanda, Nigeria, Zambia, Uganda, Mozambique, India, Bangladesh, and Pakistan (HarvestPlus, 2009)

1.2.1.1 Vitamin A

Although rice plants do possess carotenoids in their photosynthetic tissues they are absent in the endosperm, which is the edible part remaining after rice has been milled to remove the oil-rich aleurone layer that turns rancid during storage Because the endosperm is lacking in carotenoids, vitamin A defi ciency (VAD) tends to be a serious health issue in those parts of the world where rice

is consumed as the staple food, namely Asia, Africa, and Latin America In Asia, for example, more than 180 million children and women suffer from VAD (Chong, 2003) The development of golden rice (GR) was motivated by the need to alleviate VAD, which represents a major global health problem GR is the generic name given to types of GM rice that produce β-carotene (a precursor

of vitamin A) in the endosperm It is so called because the yellow color of the grain, which can

be ascribed to the high content of carotenoid, is easily discernible after milling and polishing GR was initially produced by modifying the Japonica variety Taipei 309 β-Carotene is naturally syn-thesized in the vegetative tissues of rice rather than in the endosperm, which lacks two of the steps

in the biosynthetic pathway These two steps are controlled by two genes, namely phytoene synthase (psy) and phytoene desaturase (crt 1) GR technology is based on introducing the entire β-carotene bio-synthetic pathway into the rice endosperm through the insertion of these two genes (psy and crt 1) using

an Agrobacterium-mediated transformation (Ye et al., 2000) This technology was later also shown

to work with other rice cultivars (Datta et al., 2003) The archetypal technology, however, needed improvement in order to increase the amount of carotenoid available per gram of rice, as it could only produce a maximum total carotenoid level of 1.6 μg per gram dry weight of rice (Al-Babili and Beyer, 2005; Al-Babili et al., 2006; Grusak, 2005), of which about half was in the form of β-carotene (Grusak, 2005) The children most at risk of VAD would therefore need to consume unrealistic amounts of GR in order to meet their recommended daily intakes of vitamin A equiva-lents Further research led to the development of a new generation of GR (referred to as GR 2) using

an enzyme from maize which increased grain carotenoid levels more than 23-fold The use of this enzyme overcomes a bottleneck in β-carotene synthesis GR 2 contains levels of total carotenoids of

up to 37 μg per gram, of which a very high proportion is β-carotene GR 2 development involved the replacement of the daffodil psy gene originally used with the equivalent gene from maize because psy, one of the two genes used in the development of GR, was found to be the limiting factor in β-carotene accumulation in the endosperm of rice Psy from maize was selected after systematic testing of psy from various other plants in a model plant system because of its ability to substantially increase carotenoid accumulation (Paine et al., 2005) GR 2 has the potential to provide 50% of the Recommended Dietary Allowance (RDA) for vitamin A, though the overall bioavailability would depend on the presence of dietary oils and proteins (Anonymous, 2005)

1.2.1.2 Iron

Unlike VAD, iron defi ciency, which is the most widespread nutritional defi ciency in the world, is a major problem both in developed and developing countries, though it is 3–4 times more prevalent in

developing countries (HarvestPlus, 2007) As with β-carotene, the removal of the outer layer (which includes the aleurone layer) of the rice seed during commercial milling decreases the iron content

of rice (Doesthale et al., 1979), because most of the iron in rice accumulates in the aleurone layer The average iron content of polished rice is 2 ppm, although some varieties naturally have high iron content (IRRI, 2006) Through the use of biotechnology, signifi cant progress has been made to enhance the iron content of rice

Ferritin, an iron-storage protein found in both plants and animals, has been found to provide a good source of iron when orally administered to rats suffering from anemia (Beard et al., 1996) This fi nding suggests that increasing the ferritin content of cereals through genetic engineering may

be a good way to solve iron defi ciency problems Based on this hypothesis, Goto and coworkers

(1999), introduced the soybean ferritin gene into rice plants by Agrobacterium-mediated

transfor-mation This was done under the control of a rice seed storage protein glutelin promoter, GluB-1 (−1302/ +18), to ensure that the iron accumulated specifi cally in the grain Transgenic seeds that accumulated the soybean ferritin in the endosperm could store up to three times more iron than nor-mal seeds The International Rice Research Institute (IRRI) based in the Philippines has produced an iron dense rice, IR68144, through conventional breeding by crossing a high-yielding variety (IR72) with a tall traditional variety (Zawa Bonday) containing a relatively high iron content (4.1 ppm) in the grain (IRRI, 2006) The IRRI whole grain had an iron content of 21 ppm, which is about double the normal level in rice Scientists have also found that after polishing for 15 min, IR68144 had approximately 80% more iron than a well known but low-iron commercial variety (ISIS, 2004) Human studies using women with low iron stores proved that the consumption of IR68144 increased body iron by 20% (IRRI, 2006), indicating that increased iron content does indeed translate into enhanced iron status in the consumer IR68144 was also found to have a high zinc content (34 ppm), which is not surprising because research has shown that zinc and iron densities are positively cor-related It has also been reported that IR68144 combines a high vitamin A content and a high yield with good fl avor, texture, and cooking qualities (ISIS, 2004) Vasconcelos and coworkers (2003) were able to transform IR68144 with the soybean ferritin gene driven by the glutelin promoter to obtain transgenic rice plants with an even higher content of iron and zinc, even after polishing Transformation of IR68144 was achieved through a biolistic-mediated method using a construct of

the plasmid pGPTV bar/Fer (which encodes for the soybean ferritin protein from Glycine max L.)

and the endosperm-specifi c promoter Glu B-1

The amount of iron that is bioavailable depends both on iron intake and absorption, and hence increasing the iron content of foods will not necessarily translate into a proportional increase

in absorbed iron In the developing world, dietary iron mainly comes from nonheme sources such as grains and legumes, which are high in phytic acid, a recognized potent inhibitor of iron absorption (Hurrell et al., 1992) Thus, increasing iron intake through the provision of iron-en-riched crop varieties will not solve iron defi ciency problems unless the diet is also low in iron absorption inhibitors such as phytic acid or contains components that enhance the absorption and utilization of iron Lucca and coworkers (2002) addressed this problem through the inser-tion of ferritin (pfe), metallothionein-like (rgMT), and phytase (phyA) genes into rice embryos

through Agrobacterium-mediated transformations Regenerated plants expressing these three

genes showed a 2-fold increase in the iron content of seeds compared to negative controls The plants also showed an increase in phytase activity of as much as 130-fold for some of the plant lines compared to controls About 90% of phytase activity was retained after incubating the trans-genic ground rice under stomach conditions, and the phytic acid content of the seed was greatly reduced Other studies have shown that cysteine and cysteine-containing peptides obtained from meat have the capacity to enhance the absorption of nonheme iron by binding the iron through its thiol group (Layrisse et al 1984; Taylor et al., 1986) Thus, by overexpressing a cysteine-rich protein, metallothionein, through the insertion of the rgMT gene, the cysteine content of the seed protein in the endosperm could be increased to levels that would further enhance iron bioavail-ability in the transgenic rice (Lucca et al., 2002)

Micronutrients are as essential for plant life as they are for humans, and abnormalities in plant growth and development occur when they are not available in optimum quantities Globally, about 30% of the cultivated soils are considered calcareous, with low iron availability, because the iron present is only sparingly soluble in the soil solution Crops grown on these soils are therefore also defi cient in iron (Takahashi, 2003) Hence, another approach to tackling the iron defi ciency problem

is to produce crops that are tolerant to low iron availability, thus ensuring that crops grown on careous soils are not iron defi cient Plants have naturally developed mechanisms to cope with iron defi ciency stress One such mechanism adopted by graminaceous plants (which include rice) is to release phytosiderophores, which form a soluble complex with the inorganic iron in the soil by chela-tion which can then be absorbed by the roots (Curie et al., 2001) The level of tolerance exhibited

cal-by crops under iron defi ciency stress is directly proportional to the amount of phytosiderophores produced and secreted into the soil (Takagi et al., 1984) Compared to other members of the grami-naceous family (wheat, barley, rye, oats, sorghum, and maize), rice has the least tolerance in iron defi cient situations because it produces only small amounts of phytosiderophores due to low activity

of the nicotianamine aminotransferase (NAAT) gene responsible for the production and secretion

of phytosiderophores Barley has the highest tolerance among plants in this group (Römheld and Marschner, 1990) Takahashi (2003) produced GM rice with enhanced NAAT activity by purify-ing the NAAT protein from barley roots and inserting it into rice using the vector pBIGRZ1, which permits the expression of inserted genes under regulation by their own promoters The transgenic rice plants harboring the NAAT gene from barley exhibited enhanced phytosiderophore release and consequently greater tolerance to soils with low iron availability

1.2.1.3 Zinc

Zinc defi ciency occurs both in crops and humans, causing decreased crop yields and predisposing humans to diseases (Hotz and Brown, 2004; Welch and Graham, 2004) It is prevalent in regions whose soils are defi cient in zinc, resulting in crop plants that are zinc defi cient (Cakmak et al., 1999; Hotz and Brown, 2004) Fifty per cent of cereal growing areas suffer from soils with low plant availability of zinc (Cakmak, 2002) As with other micronutrients, most of the zinc present in seed

is located in the embryo and aleurone layers, leaving the endosperm with a very low zinc tion (Ozturk et al., 2006) Thus, milling to remove the embryo and aleurone layers further reduces the concentration of zinc in cereals At present, polished rice has an average zinc content of 12 ppm (IRRI, 2006)

concentra-Enrichment of cereal grains with zinc has become a high-priority area for research as part of the effort to minimize zinc defi ciency and its associated health problems Although originally devel-oped to boost iron content, as discussed in the previous section, IR68144 (IRRI, 2006) and its improved transgenic variety (Vasconcelos et al., 2003) are also examples of GM zinc-enriched rice varieties The IR68144 improved variety was found to have a zinc content of 34 ppm (ISIS, 2004) and the four lines obtained from the GM IR68144 have zinc contents ranging from 36.2 to 55.5 ppm, which compare favorably to the average rice zinc content of 20 ppm (Vasconcelos et al., 2003) Because iron and zinc concentrations are positively correlated, selecting for high-iron varieties will also tend to select for high-zinc varieties, as seen in the examples above

1.2.2 B IOFORTIFICATION WITH P HYTOCHEMICALS

Phytochemicals, also referred to as phytonutrients, are a group of plant-based chemicals that have been identifi ed as being active in disease prevention Among their health benefi ts are maintaining bone and joint health (Cerhan et al., 2003; Wattanapenpaiboon et al., 2003), cancer prevention (Manson et al., 2007), and lowering serum cholesterol levels (Lerman et al., 2008) Phytochemicals are found notably in fruits and vegetables and fall under several categories, such as terpenoids or isoprenoids, which include: the carotenoids found in carrots and tomatoes, respectively; polypheno-lics, which include the isofl avones and anthocyanins found in soybeans and green tea, respectively;

and glucosinolates, which include the indoles and isothiocyanates found in broccoli and mustard, respectively The amount of these phytochemicals varies greatly in plants, and levels in some plants may be either low or nonexistent Much research has been done in this area to improve the phy-tochemical composition of certain food crops by either increasing their levels or preventing their degradation and/or conversion to nonbiologically active compounds Efforts have also been made

to ensure phytochemicals are available in widely consumed plants that do not naturally accumulate them Biofortifi cation is, therefore, not only used to increase levels of essential micronutrients in staple foods to address global malnutrition related defi ciencies, but also to provide varieties that contain higher than normal amounts of phytochemicals to address particular ailments

Epidemiologic studies have demonstrated that the high consumption of foods derived from beans is linked to a low incidence of hormone-related cancers, menopausal symptoms, osteoporosis, and CVDs (Cornwell et al., 2004; Dixon and Ferreira, 2002; Watanabe et al., 2002) This protective action of soybean-derived food products is ascribed to the isofl avonoid content of soybean This has led to an extensive research effort to biofortify more widely consumed foods such as vegetables, grains, and fruits with isofl avonoids in order to enhance the dietary intake of these compounds and thus their associated health benefi ts Genistein, which is the backbone of all isofl avonoids, is pro-duced from the dehydration of 2-hydroxyisofl avanone, which may occur spontaneously or through the catalytic action of dehydratase Formation of 2-hydroxyisofl avanone, which represents the fi rst step in the biosynthesis of isofl avonoids, is catalyzed by the enzyme 2-hydroxyisofl avanone syn-thase, commonly referred to as IFS Cloning of genes encoding IFS from several leguminous plants, including red clover, licorice, and soybeans, has made it possible to synthesize isofl avones in plants that do not ordinarily accumulate such metabolites (Liu et al., 2007)

soy-The consumption of onions and related alliums such as garlic and leeks is associated with the lowering of serum cholesterol (Gorinstein et al., 2006; Vidyashankar et al., 2008) and is also known to have a protective effect on platelet aggregation (Chang et al., 2004; Hubbard et al., 2006), which translates into a reduced risk of CVDs Garlic and onion extracts have also been reported

to have antibiotic (Tsao and Yin, 2001; Yin and Tsao, 1999), antitumor (Li et al., 2002), dant (Campos et al., 2003, Drobiova et al., 2009), hypoglycemic (El-Demerdash et al., 2005), and antithrombotic properties (Fukao et al., 2007; Yamada et al., 2004) The health-promoting proper-ties of alliums have been linked to several organosulfur containing compounds For example, the hypocholesterolemic effect of onion and garlic is attributed to S-methylcysteine sulfoxide (SMCS) (Augusti and Mathew, 1974; Sainani et al., 1979) When onion is broken through smashing or cut-ting, amino acid sulfoxides and a particular enzyme (lachrymatory factor synthase) are released This enzyme catalyzes the conversion of the sulfoxides into a vapor form, which enters the eyes and causes the tears associated with the cutting of onions The discovery of the gene responsible for triggering this tear production has led to the production of a GM onion that does not produce tears,

antioxi-by turning off the gene that expresses the enzyme The GM onion was created antioxi-by Dr Colin Eady at the New Zealand Crop & Food Research Laboratory Shutting down the gene was achieved using the process of RNA interference By turning off the lachrymatory factor synthase gene, valuable sulfur compounds which otherwise would have been converted to the tear-causing agent and lost are instead made available for redirection into compounds that are known for their fl avor and health properties (Environmental Graffi ti, 2008) Individuals who avoid cooking with onions as a result of the tear factor now have an alternative that will enable them to reap the health benefi ts that onions have to offer

1.2.3 MODIFICATION OF MACRONUTRIENTS

1.2.3.1 Oils

CVD is the leading cause of death in the United States, with statistics indicating that CVD causes approximately 1.4 million deaths annually (Johnson et al., 2007) The fatty acid (FA) profi le of the

diet is the predisposing risk factor for CVD, with the main culprits being saturated FAs (SFAs), trans-FAs, and cholesterol Unlike SFAs, monounsaturated FAs (MUFAs) and polyunsaturated FAs (PUFAs), especially omega-3 PUFAs, convey several health benefi ts Consequently, there is a steadily increasing demand for unsaturated FAs (UFAs) to be incorporated in the diet and in addi-tion to the search for alternative sources, the FA profi les of certain oils are being modifi ed to increase their levels of unsaturated fats and decrease their saturated and transfat content in order to produce even healthier oils and products made from them.

1.2.3.1.1 Oils with Healthier FA Profi le

The essential FAs linoleic and α-linolenic acid, which are C18 PUFAs, may be metabolized into very long chain PUFAs (VLCPUFAs) (C20 and C22) in the body once consumed, although the conversion process is slow and ineffi cient compared to the direct consumption of these VLCPUFAs

in the form of fi sh oils (Domergue et al., 2005) Nutritionally, the most important VLCPUFAs include arachidonic acid (AA, C20H32O2), eicosapentanoic acid (EPA, C20H30O2), and docosa-hexaenoic acid (DHA, C22H32O2) At present fi sh oil is the major source of EPA and DHA The machinery and materials necessary for the biosynthesis of a wide range of FAs from conventional oilseed crops is already in place, but they lack several additional enzymes (certain fatty-acyl desatu-rases and elongases) that are necessary for the biosynthesis of VLCPUFAs (Abbadi et al., 2004) Various genes responsible for the biosynthesis of PUFAs have been cloned from organisms such as fungi, algae, mosses, higher plants, and mammals (Warude et al., 2006)

The use of these isolated genes allows the manipulation of plants to enhance their PUFA profi le Because of the continuing decrease in marine resources as a result of overfi shing, coupled with the environmental impact of fi sh farming, neither farmed nor wild fi sh represent a sustainable source

of the VLCPUFAs necessary to ensure healthy nutrition for the ever growing world population (Abbadi et al., 2004) In addition, it has been recommended that the consumption of many types

of fi sh be limited because of widespread contamination with pollutants such as heavy metals and dioxins (America.gov, 2004) Making VLCPUFAs available in nonfi sh sources (notably in the form

of annual oilseed crops) would be a sustainable solution to this imminent depletion of our supply

of VLCPUFAs from fi sh, and would also avoid exposure to toxic environmental contaminants such

as dioxin Abbadi and coworkers (2004) have produced linseed and tobacco plants that synthesize VLCPUFA in their seed by introducing genes for fatty-acyl desaturases and elongases obtained from a variety of organisms that produce VLCPUFAs The best results were obtained using plant and algal gene sequences The transgenic plants accumulated signifi cant levels of the VLCPUFAs (5% of C20 VLCPUFA including AA and EPA) in their seed The researchers were able to identify bottlenecks in the accumulation of desirable PUFAs, which paves the way for further research aimed at improving the levels of VLCPUFAs (Abbadi et al., 2004) This achievement holds promise for the production of healthier and more nutritious oils for human consumption Also, the use of such oils in the production of animal feed could improve the PUFA content of animal products such

as eggs, meat, and dairy food

In a further development, scientists at Rothamsted Research Institute in Hertfordshire, UK have genetically engineered plants to produce fi sh oils Key genes were fi rst isolated from a species of

microscopic single-celled marine algae known as Thalassiosira pseudonana The isolated genes

were then inserted into crops such as linseed and oil seed rape (canola) and the transgenic plants were found to synthesize omega-3 PUFAs in their seed oils The researchers are currently working

to optimize and improve the levels of omega-3 PUFAs produced by these transgenic plants (BBC News, 2007)

1.2.3.1.2 Stearic Acid Rich Oils

Although SFAs have been shown to raise serum total cholesterol and consequently increase the risk

of CVD, recent studies have indicated that stearic acid (C18H36O2) has no effect on total cholesterol, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol (Yu et al., 1995), when

compared to other SFAs such as palmitic (C16H32O2), lauric (C12H24O2), and myristic (C14H28O2) FAs (Muller et al., 2001) Bakery products usually require the use of solid fat to provide the desired functionality To date, the choices of solid fats have been largely limited to animal fats such as lard and butter and tropical fats from palm kernel and coconut, both of which are hyper-cholesterolemic due to their high contents of palmitic, lauric, and myristc FAs The other solid fat options, namely partially hydrogenated oils, are also harmful due to the transfats they con-tain Food manufacturers are, therefore, faced with the challenge of fi nding healthier alternatives capable of replacing animal fats and tropical oils without compromising quality and functionality (Dirienzo et al., 2008).

Stearic acid rich oils are one possible alternative being considered (Kris-Etherton et al., 2005) Facciotti and coworkers (1999) have reported the development of a type of canola oil that con-tains almost 40% stearic acid compared to the 1% of naturally occurring canola oil Their success was based on earlier work by Hawkins and Kridl (1998), who isolated an enzyme that helps make stearic acid from the tropical plant mangosteen, whose seeds contain large amounts of stearic acid They then inserted the gene for this enzyme, which belongs to a family of enzymes known as thioesterases, into the canola plant The enzyme allows stearic acid to accrue in the plant by dis-rupting the biosynthesis of oleic acid, which accounts for most of the fat available in commercial canola oil Both oleic and stearic acids have 18 carbon atoms, but the former contains a double bond whereas the latter has no double bond In the course of the biosynthesis of these two FAs,

an enzyme called desaturase creates the double bond to form oleic acid when the carbon chain reaches 18 atoms The lengthening of the carbon chain is accomplished by various thioesterases; the thioesterase obtained from the mangosteen releases stearic acid before desaturase can convert it into oleic acid, resulting in the accumulation of stearic acid However, although the amount of stearic acid produced increased, the content was still insuffi cient Facciotti and coworkers (1999) created mutants of this gene (Garm FatA1) and tested them to determine which of their enzymes were most active in producing stearic acid The best performing genes were then introduced into canola plants, resulting in oils that contain almost 40% of stearic acid

1.2.3.1.3 Healthier and More Stable Cooking Oils

Though PUFAs are nutritionally highly valuable, they are easily oxidized and readily break down (turn rancid) in storage and under extreme heat, rendering them unsuitable for cooking, especially in deep frying situations To enhance their thermal stability, oils are partially hydrogenated to reduce the level of unsaturation In the process, however, transfats are formed Because of the deleterious health effect of transfats, the Food and Drug Administration (FDA) makes it mandatory for food manufacturers to list all transfat content on product labels (21 CFR Part 101) (FDA, 2003) There is, therefore, a trend toward producing transfat free but thermally stable cooking oils to increase their acceptability and utility

Until relatively recently, rapeseed was a fairly minor crop due to its natural content of erucic acid (C22H42O2) and glucosinolates The bitter taste of erucic acid prevented its use in food and the toxic effect of the glucosinolates that remain after the pressing of rapeseed meal prevented its use in ani-mal feed However, modern plant breeding has led to improved rapeseed cultivars free of erucic acid and glucosinolates, and these new cultivars are referred to as “double zero.” “Double zero” rapeseed

was developed in Canada and was renamed “canola” (Canadian oil, low acid) to distinguish it from

nonedible rapeseed As a result of its high-level content of MUFAs (60–70%) and PUFAs (about 22%), coupled with its low-level content of SFAs (about 7%) (Canola Council, 2008; GMOCompass, 2008), rapeseed oil has become signifi cant as a healthy cooking oil Canola oil is now seen to have the potential to help consumers achieve dietary recommendations and hence reduce the risk

of CVD Compared to the other oils commonly consumed in the United States, namely soybean, peanut, sunfl ower, corn, cottonseed, palm, and olive oils, canola oil has the lowest concentration of SFAs In addition, canola oil is abundant in MUFAs and is also the richest source of the omega-3 essential PUFA, α-linolenic acid, of any of these oils (Johnson et al., 2007)

The oil extracted from traditional varieties of canola has only limited cooking applications because of its relatively high proportion of PUFAs Through genetic engineering, canola oil with increased oleic acid (C18H34O2) content and/or reduced PUFA content has been developed Oleic acid is a MUFA which is thought to confer lower heart disease and cancer risk benefi ts on olive oil Pioneer High-Bred International, Inc has developed two novel canola lines, 45A37 and 46A40, which have high oleic acid content and low linolenic acid content These were produced by fi rst inducing mutagenesis by exposing canola varieties to a solution 8 mM ethylnitrosourea in dimeth-ylsulfoxide to achieve the high oleic acid trait, followed by traditional breeding with canola varieties such as Apollo and Stellar to achieve the low linolenic acid trait Oils processed from these novel lines are referred to as P6 canola oil and 45A37 and 46A40 have 24% higher levels of oleic acid and 40% or 75% lower levels of linoleic and linolenic acid, respectively, compared to traditional canola oil (Health Canada, 2000).

On June 6, 2004, at the BIO conference in San Francisco, Dow AgroSciences LLC showcased its improved canola oil, dubbed NatreonTM This new canola oil variety was produced using the latest tools in plant breeding technology (Dow AgroSciences, 2004) Natreon canola oil contains 7% satu-rated fats, 75% oleic acid, 3% linolenic acid, and 15% linoleic acid, and is virtually transfat free In addition to its nutritional and stability qualities, Natreon canola oil has a neutral fl avor and therefore does not interfere with the natural fl avor of food (Dow AgroSciences, 2004; NRCC, 2002) Monola oil, produced by Nutrihealth Pty Limited in Melbourne, Australia is another example of high oleic canola oil This was obtained from normal canola modifi ed by traditional plant breeding, with no genetic engineering involved Monola oil contains 6% SFAs (1% less than traditional canola oil), 70% oleic acid, 20% linoleic acid, and 2.6% linolenic acid (GRDC, 2007) Lowering the levels of PUFAs through an increase in the oleic acid content eliminates the need for partial hydrogenation as the oil is less liquid and less prone to rancidity, and results in the production of few or no trans-FAs

In this way a healthier, and at the same time thermally stable, cooking oil is produced At present,

as a result of its superior stability, high oleic acid canola oil is being used by manufacturers instead

of hydrogenated vegetable oils in products such as breads, cakes, and potato chips, which results in more healthful and better quality products (AFIC, 2004) Other vegetable cooking oils including cottonseed, sunfl ower, saffl ower, soybean, peanut, and palm oils have also been produced through biotechnology to improve thermal stability as well as to boost health-enhancing qualities Detailed information on the compositions and applications of commercially available GM oils are available and can be found in the book by Richard D O’Brian (2008)

One high-protein corn variety known as quality maize protein (QMP) was developed at The International Maize and Wheat Improvement Center (CIMMYT) in Mexico This protein-rich corn variety was developed using traditional breeding techniques to incorporate a series of special genes

to offset the undesirable side effects of the opaque 2 (o2) mutation without jeopardizing the added protein trait (CIMMYT, 2000) o2 is a previously discovered mutation that endowed grain

value-proteins in the endosperm with twice the nutritional qualities of normal maize owing to a 2-fold increase in the levels of lysine and tryptophan (Mertz et al., 1964) Utilization of o2 in breeding programs in the past, however, was fraught with problems such as a soft endosperm that resulted

in damaged kernels, increased susceptibility to pests and fungal diseases, lower yields, and inferior food processing qualities, none of which were easily overcome (Bjarnason and Vasal, 1992) QMP has a similar yield and agronomic performance to regular corn, but with 30% more lysine, 55% more tryptophan, and 38% less leucine The higher tryptophan and lower leucine content leads to higher niacin availability Only 34% of regular corn protein intake is utilized compared to 74% of the same amount of QMP Comparing the nitrogen balance of QMP to skimmed milk indicates that the protein quality of QMP is 90% of that of milk (Graham et al., 1980) Other nutritional benefi ts of QMP include higher calcium and carbohydrate content and increased carotene utilization (Prasanna

et al., 2001) In a related development, a maize breeder (Raman Babu) working at the Indian Council for Agricultural Research (ICAR) used a combination of biotechnology and conventional methods

to further improve the quality of QMP This was achieved by crossing lines of QMP with the parents

of a popular normal hybrid known as Vivek Hybrid-9 Molecular markers were then used to quickly select the offspring that contained both the desirable parentage of the original hybrid and the quality protein trait Using this procedure, they were able to develop the QMP hybrid in less than half the time it would have taken if only conventional selection methods had been employed The qualities

of this new hybrid, in addition to the high protein content it derives from the QMP line, are that it is early maturing compared to both QMP and Vivek Hybrid-9 and out-yields both parents This is of particular importance, as years of research to develop a variety that could out-yield Vivek Hybrid-9 had previously been unsuccessful (cgiarNews, 2005)

Other biotechnological means have also been employed to increase the protein content of maize Programmed cell death is widely utilized during plant development to discard tissues or organs that are no longer needed, to adjust to environmental changes or to alter existing organs (Young et al., 2004) Every corn kernel results from a fl ower on an ear of a corn During the developmental pro-cess the ear produces a pair of fl owers for every kernel However, through programmed cell death

one of the pair of fl owers is aborted, leaving a single fl ower for each group (ScienceDaily, 2005)

By introducing the cytokinin synthesizing isopentenyl transferase (IPT) gene under the control of

the Arabidopsis senescence-inducible promoter from the cysteine protease gene SAG (senescence

associated gene) 12 in tobacco leaves, it has been shown that cytokinin plays a role in regulating entry into a senescence program (Gan and Amasino, 1995) Although programmed cell death can occur at both early and later stages of development whereas senescence occurs only at later stages

of development and is thus developmentally separate, similar hormones may regulate entry into either program (Young et al., 2004) Based on this information, Young and coworkers (2004) set out

to investigate whether cytokinin might affect the abortion of corn fl oral organs through the duction of the SAG12-IPT construct into maize The insertion of the construct essentially rescues the aborted fl ower, resulting in the fusion of two embryos in one kernel This hinders the growth

intro-of the endosperm, resulting in kernels with an increased ratio intro-of embryo to endosperm content Because much of the oil and protein reserves available in the maize grain are stored in the embryo, this fusion event, generating kernels composed of two embryos with diminished endosperm content, increases the contribution of these storage reserves, thereby improving the nutritional value (i.e., high protein and low starch) of this corn variety The reduced starch content that results from the decreased endosperm in this corn variety, if applied to sweet corn, would appeal to the large number

of weight-watchers who are interested in low-carbohydrate diets and hence normally avoid corn in their diets Despite the fusion the kernels are no bigger than normal and look just like ordinary corn,

but are nutritionally superior (ScienceDaily, 2005).

As noted above, the poor nutritional value of corn arises due to its low content of the tial amino acids lysine, tryptophan, and methionine The poultry industry spends over $1 bil-

essen-lion annually on synthetic methionine as a dietary supplement in feed (ScienceDaily, 2002) It is

therefore imperative that the levels of methionine in corn be increased, not only to address global

malnutrition problems in areas that are dependent on corn as a staple, but also to reduce the costs involved in breeding and raising livestock and poultry where maize is used as a major component

of animal feed Two researchers (Jinsheng Lai and Joachim Messing) at the Waksman Institute of Microbiology, at Rutgers University in New Jersey, found a way to increase the methionine content

of corn This was achieved without the addition of foreign DNA by fi rst isolating the gene for onine and then adjusting the genetic signals that control the synthesis of methionine Through this manipulation the researchers were able to increase the plant’s ability to produce more of its own

methi-naturally occurring protein (Lai and Messing, 2002; ScienceDaily, 2002).

1.2.4 PRODUCTION OF HYPOALLERGENIC FOODS

Soy offers several health benefi ts such as the potential to reduce the incidence of coronary heart diseases (CHD), prostate cancer and breast cancer, as well as the ability to decrease and increase LDL and HDL cholesterol levels, respectively The antioxidant properties of soy isofl avones have benefi cial effects on the function of blood vessels and also protect LDL from oxidation (Chema et al., 2006) Thus, on 26 October 1999, the FDA published a fi nal rule (64 FR 57700) which approves the health claim for soy protein as reducing the risk of CHD (FDA, 1999) Increased awareness of the health benefi ts of soy consumption, as well as its functional properties as a food ingredient, has led

to widespread utilization of soybean products in a diverse range of food products However, some individuals are allergic to soy proteins The increasing use of soybean products in processed food products poses a potential threat to such allergic individuals Therefore, recent research interests have been directed toward producing hypoallergenic soybeans and soy products The development

of hypoallergenic soybeans, and hence soy products, would not only protect sensitive als from allergic reactions, but would also enable allergic individuals to gain the aforementioned benefi ts of soy proteins, as at present avoidance of the food containing the allergenic moiety is the only treatment for individuals with allergy The soybean proteins Gly m Bd 60K, Gly m Bd 30K, and Gly m Bd 28K are the main seed allergens in soybean-allergic individuals Gly m Bd 30K (also referred to as P34), although accounting for only 1% of total seed protein, represents the major soy protein allergen Biotechnology provides a way to eliminate undesirable proteins such as Gly m Bd 30K in order to enhance food safety and allows allergic individuals to avail themselves of the health benefi ts that such food items have to offer

individu-The use of transgene-induced gene silencing to prevent the accumulation of Gly m Bd 30K in soybean seeds has been achieved The resultant transgenic seeds do not accrue Gly m Bd 30K protein and the property and quality of the seed remain unchanged compared to controls despite the removal of this allergenic protein, indicating that it does not play a role in either seed protein processing or maturation (Herman et al., 2003) In a related development, Song and coworkers (2008) at the University of Illinois employed liquid and solid fermentation processes using benign microorganisms to produce hypoallergenic soy fl our They reported that allergenic protein produc-tion was lowered by as much as 97%, depending on the type of microorganism used The bacterial

species Lactobacillus plantarum, commonly found in sauerkraut, achieved the best hypoallergenic

result The fermentation process also had an added benefi t of increasing the content of some tial amino acids, thereby improving the nutritional quality of the product The product did, however, have a fl avor typical of fermented oriental soy products such as soy sauce and miso soup This may not pose a problem as Americans are fast becoming accustomed to these fermented soy sauce prod-ucts already on the market In other parts of the world, particularly Europe where GM foods are strongly opposed, removal of soy allergens without genetic engineering presents a viable alternative process (Song et al., 2008)

essen-Another area of research has been to reduce the allergenicity of other food varieties such as wheat and products derived from them, such as wheat fl our Protecting individuals who are allergic

to wheat proteins is an enormous task, as many processed foods which are consumed daily contain wheat fl our as an ingredient The availability of hypoallergenic fl our would thus greatly benefi t

these individuals A great deal of research has therefore been devoted to this effort in order to meet consumer demand for hypoallergenic fl our Watanabe and coworkers (2000) have produced hypoal-lergenic fl our by enzymatic fragmentation using the enzymes cellulase and actinase Subsequent research proved that the allergy suppressive effect of the new allergenic wheat fl our acted by hinder-ing allergen absorption from the intestinal tract and inducing oral tolerance (Watanabe et al., 2004) Unfortunately this product has a consistency that makes processing by usual methods diffi cult and gelatinizing of the starch in the product and the addition of surfactants are necessary to make the hypoallergenic wheat fl our suitable for food processing (Watanabe et al., 2000).

1.2.5 REDUCTION OF ANTINUTRIENTS

Antinutrients are plant compounds that decrease the nutritional value of plant food by making an essential nutrient unavailable or indigestible when consumed by humans or animals They achieve this either by binding nutrients to make them unavailable or by inhibiting the enzymes needed for digestion Phytic acid, the principal storage form of phosphorus in mature seeds or grains, is a major antinutritive factor in whole legume seeds and cereal grains It is considered antinutritive because it limits the bioavailability of minerals such as zinc, iron, and calcium by forming indigestible chelates with these metals (Saha et al., 1994) It would therefore be useful to reduce the phytic acid content of foods in order to improve their nutritional value In April 2002, the ARS–USDA and the Arkansas

Agricultural Experiment Station released a low phytic acid mutant of rice known as KBNT lpa1-1

The mutation was induced by γ radiation of the Arkansas rice cultivar, Kaybonnet (KBNT) (Wells

et al., 1995) The proportion of seed phosphorous tied up as phytic acid in KBNT lpa1-1 is reduced

from 71% to 39% and the inorganic seed phosphorous is increased from 5% to 32%, with little effect

on the total seed phosphorous (Larson et al., 2000) Rutger et al (2004) produced the goldhull low

phytic acid (GLPA) rice by hybridizing the low phytic acid mutant (KBNT lpa1-1) with the goldhull

color cultivar “Bluebelle” (Bollich et al., 1968) This was followed by selection for recombinants

that possess the recessive gene lpa1-1 (responsible for low phytic acid) from the fi rst parent and the recessive gene gh (responsible for the goldhull color) from the second parent Although the original low phytic acid mutant KBNT lpa1-1 is phenotypically the same as the original parent, GLPA is set

apart by the goldhull color, allowing for identity preservation of the line

Considerable effort has gone into the enrichment of rice and other staples with metals like zinc and iron to address global malnutrition associated with their defi ciencies, but these efforts will be futile if increasing the concentration of these metals in staple food crops is offset by the chelating effect of phytic acid Low phytic acid foods that increase the bioavailability of these essential met-als will serve as a necessary complementary measure to biofortifi cation efforts Attempts to reduce the phytic acid content have not been limited to staple crops but have also included products made from these crops In recent times, brown rice has been prized as a healthy food ingredient for its high nutritive value This is, however, limited by its relatively large amount of phytic acid Akiko

et al (2005) used enzyme hydrolysis to reduce the phytic acid content of brown-rice bread They

added 0.2 and 1.0 g of phytase (Aspergillus niger phytase) to 521 g of dough material before mixing,

fermenting, and baking They reported that the addition of 0.2 g of the phytase to the dough material before baking reduced the phytic acid content of brown-rice bread with less negative effect on bread appearance compared to the addition of 1.0 g of the phytase Starting with a raw material that is low

in phytic acid and implementing this enzyme hydrolysis would lead to a fi nal product with a very low phytic acid content and hence a more nutritious product

OF ANIMAL-BASED FUNCTIONAL FOODS

As with plant-based foods, biotechnology has also been used in the production of animal-based functional foods to provide specifi c nutritional and health benefi ts Currently these biotechnological

approaches have focused mainly on reducing the fat tissue and improving the FA profi le of meat and dairy products with their naturally high saturated fat content Although most consumers generally resist the idea of eating food derived from bioengineered animals, the FDA permits meat and milk from clones of adult cattle, pigs, goats, and their offspring and they are deemed to be as safe to eat

as food from conventionally bred animals, according to three documents released by the FDA on 15 January 2008 (Osborne, 2009) However, little has been done with animal biotechnology compared

to plant biotechnology because the animal genome is larger and more complex than that of plants and hence genetic modifi cation of animals is more diffi cult and costly (Montaldo, 2006) This sec-tion will review some of the meat- and dairy product-based foods that have been developed with improved health values

1.3.1 MEAT PRODUCTS

1.3.1.1 In Vitro Meat

In vitro meat is effectively animal fl esh that has never been part of a complete living animal It is

different from synthetic and artifi cial meat, both of which have the taste and texture of meat but do not consist of meat (Innovation Watch, 2007) The technology works by taking from a live animal stem cells, or myoblasts, that are preprogrammed to grow into muscle, placing them in a growth medium, and supplying the necessary nutrients for growth, such as glucose, minerals, and amino acids The stem cells are then poured into a three-dimensional sponge-like scaffolding made of protein to which they can attach themselves; growth is stimulated by fi ring electrical impulses into the muscle cells, ultimately forming muscle fi bers that can be harvested to produce a minced-meat

product (The New York Times, 2005; The Times, 2008) It is predicted that 20 years from now it will be possible to use this technology to grow a whole beef or pork loin The production of in vitro

meat would circumvent many of the problems, especially pollution, associated with conventional farming For example, in the United States livestock farming produces 1.4 billion tons of animal waste annually Once a meat-cell culture has been produced it will be equivalent to regular yeast

or yoghurt cultures; meat growers would not need to use a new animal for each set of starter cells

and hence the meat industry would no longer be dependent on slaughtering animals (The New York Times, 2005).

This technology holds great promise in the area of meat-based functional foods owing to the fact that the nutrients can be controlled For example, most meats have a high content of omega-6 FAs,

which can cause high cholesterol and other health problems With in vitro meat, omega-6 FAs can

be replaced by the healthier omega-3 FAs (Edelman et al., 2005) In vitro meat could also lead to

easy control of the fat content of meat and reduce the incidence of food-borne diseases associated

with the consumption of contaminated meat (The Times, 2008).

1.3.1.2 Meat with a Modifi ed FA Profi le

It has been well documented that the consumption of omega-3 FAs offers some protection against CVD However, meat from pigs, cows, and other food mammals typically has higher levels of omega-6 FAs as a result of an animal diet of grains that are rich in such FAs, as well as the inabil-ity to transform it into its healthier version of omega-3 FAs For example, pork generally contains approximately 15% omega-6 FAs and 1% omega-3 FAs (Pig Progress.net, 2008) High levels of omega-6 FAs translate into a high omega-6 to omega-3 FA ratio associated with CVD, cancer, and infl ammatory and autoimmune diseases On the other hand, increased levels of omega-3 FAs, and hence a low omega-6 to omega-3 FA ratio, are known to exert a protective effect (Simopoulos, 2002) It has been demonstrated that the greatest risk factor for ischemic heart disease and arterio-sclerosis is not high cholesterol intake but a high omega-6 to omega-3 FA ratio in the diet (Okuyama and Ikemoto, 1999) As such it has been recommended that the omega-6 to mega-3 FA ratio should not exceed 4 (Wood et al., 2004) Western diets typically have an omega-6 to omega-3 FA ratio

of 15–20, well above the recommended range of 1–4 (Simopoulos, 2002) Consumption trends for

omega-3 FAs are either static or declining (Lee et al., 2006) Professional organizations and health agencies are therefore recommending an increase in the consumption of omega-3 FA rich foods in order to promote a reduction in the omega-6 to omega-3 FA ratio (Garg et al., 2006; Kolanowski and Laufenberg, 2006).

There has therefore been a great deal of research devoted to producing meat products with a lower omega-6 to omega-3 FA ratio to improve meat quality and counteract the popular belief that meat products are inherently unhealthy One such effort has resulted in the production of healthier meat from pork through an increase in the omega-3 FA content This achievement was based on prior research where mice capable of transforming omega-6 FAs into omega-3 FAs were created by

transplanting a gene from the roundworm Caenorhabditis elegans into normal mice (Kang et al.,

2004) This raised the possibility of genetically engineering livestock with higher levels of omega-3 FAs, as livestock do not normally have the ability to convert omega-6 to omega-3 FAs because they lack an omega-3 FA desaturase gene (Lai et al., 2006) The roundworm gene (fat 1 gene) was fi rst transferred into fetal pig cells, after which the cells were cloned and transferred into 14 mother pigs Six of the 12 offspring produced tested positive for the gene and its ability to synthesize omega-3 FAs The omega-3 FA content of the engineered pigs was 8% of the total muscle fat compared to 1% for their unmodifi ed counterparts (Lai et al., 2006)

1.3.2 D AIRY F OODS

Milk is a high quality food source that is rich in protein, fat, carbohydrate, minerals, vitamins, and growth factors The supply of human milk is not continuous and this has led to the use of livestock milk as a substitute However, livestock milk is not a perfect substitute for human milk and is associated with problems such as lactose intolerance and allergy Thus, in addition to engineering milk products to improve their FA profi le as previously mentioned, biotechnology has been used to engineer livestock milk that is as similar as possible to human breast milk in order to address some

of the problems associated with the consumption of livestock milk

1.3.2.1 Milk for the Lactose-Intolerant Population

About 70% of adults are denied the nutritional benefi ts of milk because they suffer from lactose intolerance, an intestinal disorder that arises due to the lack of the enzyme lactase Lactose remains unabsorbed in the intestines of sufferers, causing severe intestinal discomfort characterized by abdominal pain and diarrhea (FoodReactions, 2005) In western societies, where milk represents a major component of the diet, lactose intolerance effectively restricts the use of this precious nutri-tional source for many people Since milk can provide much of the calcium necessary to maintain bone structure, lactose intolerance has been associated with osteopenia in the later stages of life (Saltzman and Russell, 1998) The nutritional value of milk, coupled with its widespread utilization

in many food formulations, means that low-lactose milk offers immense benefi ts to a large centage of the adult population Several biotechnological methods have been developed to produce low-lactose milk The following paragraphs summarize the techniques utilized in the production of lactose-free or reduced lactose milk

per-Popular methods involve postharvest treatment of milk with lactose hydrolyzing enzymes obtained from microbiological sources to produce low-lactose milk These techniques involve the use of enzymes (free or immobilized) from various sources to hydrolyze lactose into galactose and glucose For example, the Organic Valley Family of Farms in LaFarge, Wisconsin, market a lactose-free milk produced by the hydrolysis of the lactose in milk using lactase enzyme derived from the

dairy yeast, Kluyveromyces The company fi rst skims organic whole milk obtained from

pasture-fed cows to the desired fat content The enzyme lactase is then added to the milk and the mixture stirred slowly for 24 h, the time required for the lactase enzyme to break down the lactose The milk

is then tested to ensure it is lactose free before it is pasteurized to inactivate the enzyme (Organic Valley, 2008) This treatment is, however, very expensive and the end products of the enzymatic

hydrolysis (glucose and galactose) render the milk much sweeter, which appears unnatural to most consumers (Jelen and Tossavainen, 2003).

Fermentation using various enzymes has also been employed to produce low-lactose milk Lins and Leao (2002) used fermentation of skim milk to convert lactose into ethanol and carbon dioxide

using the enzyme Kluyveromyces marxianus CBS 6164 (free or immobilized in Ca-alginate (2%)

beads) obtained from yeast According to the researchers, it is possible to remove all the lactose in skimmed milk in less than 4 h using 30 or 50 g cells per liter of milk The carbon dioxide, ethanol, water, and other volatile products resulting from the fermentation are removed when the fermented milk without cells is subjected to spray drying This procedure makes it possible to obtain a pow-dered skim milk free of any kind of sugar and without signifi cant alterations in the lipid and protein profi le that can be consumed not only by the lactose intolerant, but also by diabetics and obese individuals, as well as individuals suffering from galactosaemia

Genetic engineering to produce low-lactose milk offers a low cost alternative to these approaches

So far, research has tended to focus on the use of mice as convenient stepping stones for ultimate applications in dairy cattle Jost et al (1999) reported the development of transgenic mice that expressed intestinal lactase in the mammary gland and produced low-lactose milk This was achieved by introducing a DNA construct into the mice that contained the rat intestinal lactase-phlorizin hydrolase cDNA under the control of the mammary-specifi c α lactalbumin promoter The transgenic mice expressed the foreign lactase construct during lactation, as evidenced by the presence of lactase in the milk secreted Lactase synthesis led to a 50% and 85% reduction in milk lactose in milk collected at 0 and 8 h after milking, respectively Both cases were associated with a corresponding increase in galactose and glucose content, thus demonstrating the enzymatic hydro-lysis of lactose during storage in the mammary gland Newborn mice suckling low-lactose milk from these transgenic mice displayed growth patterns akin to mice suckling milk from nontrans-genic control mice, indicating that the nutritional quality of the milk from these transgenic mice was not signifi cantly changed

1.3.2.2 Milk with Enriched Antimicrobial Protein, Lysozyme

Lysozyme is a widespread antimicrobial protein that is found in the saliva, tears, and milk of all mammals (Jolles and Jolles, 1984) It restricts the growth of bacteria that cause intestinal infections and diarrhea, but stimulates the growth of benefi cial intestinal bacteria Lysozyme is therefore con-sidered to be one of the major components of human milk responsible for the health and well-being

of breast-fed infants (Lonnerdal, 2003) Lactation, and hence the supply of these benefi cial proteins from human milk, is not permanent, requiring milk from livestock to be substituted for human milk Although milk from livestock can be easily and continuously obtained, its content of antimi-crobial proteins such as lysozyme is much lower than the levels found in human milk Enriching the milk of cows and goats with lysozyme is thus expected to be benefi cial in protecting infants and children from diarrheal diseases which, according to the Institute for One World Health (iOWH), kill two million children worldwide annually (iOWH, 2006)

Maga and coworkers (2006a) reported the generation of transgenic goats expressing human lysozyme (HLZ) in the mammary gland Dairy goats were chosen as the experimental model because they are small ruminants with relatively short gestation periods and generation intervals compared to cattle, allowing results to be obtained faster and more economically The transgenic goats were created by standard pronuclear microinjection with a DNA construct made up of 23 kb

of the promoter and the 3′ regulatory elements of the bovine αS1-casein gene coupled to the 540-bp cDNA for HLZ This gene was previously expressed in the milk of transgenic mice (Maga et al., 1995) Expression of HLZ in the transgenic goats was confi rmed by Northern blot analysis of RNA isolated from sloughed somatic cells in milk Expression of the transgene did not affect the gross composition of the milk The transgenic goats exhibited a healthier udder based on fewer sloughed mammary epithelial cells and leukocytes, which are indicators of lower levels of intramammary bac-terial infection However the presence of HLZ in transgenic milk affected several of the processing

characteristics of milk For example, the rennet clotting time of HLZ milk was signifi cantly lower than that of nontransgenic controls (Maga et al., 2006a) To test the effi cacy of the HLZ milk, the researchers fed pasteurized HLZ milk to young goats (ruminants) and pigs (nonruminants) Because pigs have a digestive system similar to humans, they were chosen as a model to provide an idea of how the HLZ milk would impact the digestive tract of humans In both animal models, the results

of the study indicated that HLZ milk had an impact on the growth of bacteria in the gastrointestinal tract, though in opposite ways Piglets fed HLZ milk had lower levels of coliform bacteria in the

small intestine, including fewer Escherichia coli compared to the control group that were fed milk

from nontransgenic goats On the other hand, kid goats fed HLZ milk had higher levels of coliform

bacteria and about the same level of E coli compared to their control group However, both sets

of young animals were healthy and exhibited normal growth patterns The researchers concluded that the differences observed in the two species were as a result of the fact that goats, being rumi-nants, have a different digestive system and as such a different collection of bacteria compared to pigs, which have a single stomach Such transgenic HLZ dairy herds hold a great deal of promise

in developing countries where intestinal diseases threaten the lives of infants and children The researchers argued that the benefi t of their research would be more pronounced if the technology was applied to dairy cattle rather than goats, because the amount of milk produced by cows is much greater than is possible with goats (Maga et al., 2006b)

1.3.2.3 Milk with an Improved FA Profi le

As already pointed out, high quantities of dietary fat, especially saturated fats, have been associated with an increase in blood cholesterol and consequently an increased risk for CHD and atherosclerosis

In contrast, unsaturated fats (poly- and monounsaturated) have a benefi cial effect on the heart by reducing serum cholesterol In the United States approximately 33% of saturated fats in the diet are obtained from the consumption of dairy products (Havel, 1997) Changing the FA content of milk through a reduction in its SFA content and an increase in its UFA content promises to be

a worthwhile approach towards reducing the risk of CHD Based on dietary recommendations, the nutritionally ideal milk should possess a FA composition of 10% PUFA, 8% SFA, and 82% MUFA This differs markedly from the typical cows’ milk FA composition of 5% PUFA, 70% SFA, and 25% MUFA (Grummer, 1991) Researchers have sought to improve the FA profi le of milk

by feeding livestock diets such as linseed oil or fi sh oil rich in these heart-healthy UFAs, with the hope that milk from livestock raised on such diets will have higher levels of these UFAs However, the presence of healthy FAs in the diet does not necessarily transfer into the milk produced by these animals (NUTRAingredients, 2005) Consumed UFAs are substantially biohydrogenated in the rumen before absorption in the small intestine, which results in the milk FAs becoming more saturated (Doreau et al., 1997)

In ruminants, high quantities of long-chain SFAs absorbed in the intestine are not refl ected

in the milk FA composition This is because the activity of the enzyme stearoyl-CoA desaturase (SCD) present in the epithelial cells of the mammary gland converts SFA to MUFA (Tocher et al., 1998) Increasing the SCD activity in the mammary gland would therefore not only increase the MUFA content but also decrease the SFA content of milk Reh and coworkers (2004) tested this hypothesis by generating transgenic dairy goats using a DNA construct designed to express rat SCD cDNA in the mammary gland under the control of the bovine β-lactoglobulin promoter through a standard pronuclear microinjection procedure The FA composition of milk from four female transgenic goats was analyzed on days 7, 14, and 30 of their fi rst lactation In two of the ani-mals, the expression of the transgene altered the overall FA composition of the resulting milk such that at day 7 the transgenic milk had less saturated and more MUFA content compared to milk from nontransgenic controls However, this effect diminished by day 30 of lactation The percent-ages of protein and fat in milk from the four transgenic goats were within the same range as milk from nontransgenic goats, indicating that expression of the transgene had no effect on the gross composition of the milk