Fundamentals of food biotechnology

Tai Lieu Chat Luong



Fundamentals of Food
Biotechnology



Fundamentals of Food
Biotechnology
Second Edition
Byong H. Lee
Distinguished Professor, School of Biotechnology
Jiangnan University, Wuxi, China
Invited Distinguished Professor, Department of Food Science &
Biotechnology, Kangwon
National University, Chuncheon, Korea
Adjunct Professor, Department of Food Science & Agric Chemistry McGill
University, Montreal, Quebec, Canada


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Library of Congress Cataloging-in-Publication Data
Lee, B. H. (Byong H.)
Fundamentals of food biotechnology / Byong H. Lee. – Second edition.
pages cm
Includes bibliographical references and index.
ISBN 978-1-118-38495-4 (cloth)
1. Food–Biotechnology. I. Title.
TP248.65.F66L44 2015
664 – dc23
2014032719
A catalogue record for this book is available from the British Library.
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Typeset in 9/11pt TimesTenLTStd by Laserwords Private Limited, Chennai, India
1

2015


Contents
Preface
What Is Biotechnology?

xiii

What Is Food Biotechnology?

xvii

Part I New Trends and Tools of Food Biotechnology
1 Fundamentals and New Aspects
1.1
1.2
1.3

xi

Biotechnological applications of animals, plants, and microbes
Cellular organization and membrane structure
Bacterial growth and fermentation tools
1.3.1 Classification and reproduction of biotechnologically important bacterial system
1.3.2 Bacterial growth
1.3.3 Environmental factors affecting bacterial growth
1.4 Fungal growth and fermentation tools
1.5 Classical strain improvement and tools
1.5.1 Natural selection and mutation
1.5.2 Recombination
Summary
1.6 Systems/synthetic biology and metabolic engineering
Summary
1.7 Bioengineering and scale-up process
1.7.1 Microbial and process engineering factors affecting performance and economics
1.7.2 Fermentor and bioreactor systems
1.7.3 Mass transfer concept
1.7.4 Heat transfer concept
1.7.5 Mass and heat transfer practice
1.7.6 Scale-up and scale-down of fermentations
1.7.7 Scale-up challenges
Summary
1.8 Molecular thermodynamics for biotechnology
1.8.1 Protein folding and stability
Summary
1.8.2 Downstream processes on crystallization and chromatography
Summary
1.9 Protein and enzyme engineering
Summary

1.10 Genomics, proteomics, and bioinformatics
Summary

1
3
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vi

CONTENTS

1.11 Biosensors and nanobiotechnology
1.11.1 Biosensor
1.11.2 Nanobiotechnology and nanobiosensor
Summary
1.12 Quorum sensing and quenching
Summary
1.13 Micro- and nano-encapsulations
1.13.1 Microencapsulation
1.13.2 Nanoencapsulation
Summary
Bibliography

2 Concepts and Tools for Recombinant DNA Technology
2.1 Concepts of macromolecules: function and synthesis
2.1.1 DNA replication
2.1.2 Roles of RNA
2.1.3 Detailed aspects of protein synthesis
2.2 Concepts of recombinant DNA technology

2.2.1 Restriction endonucleases
2.2.2 Plasmid vectors
2.2.3 Purpose of gene cloning
2.3 DNA sequencing
2.4 Polymerase chain reaction (PCR)
2.5 Manipulation techniques of DNA
2.5.1 Isolation and purification of nucleic acids
2.5.2 Agarose gel electrophoresis
2.5.3 Blotting and hybridization
2.6 Gene cloning and production of recombinant proteins
2.6.1 Cloning and expression of bacterial β-galactosidase in E. coli
2.6.2 Cloning, expression, and production of bovine chymosin (rennet) in yeast K. lactis
Summary
Bibliography

Part I

Questions and Answers

Part II Applications of Biotechnology to Food Products
3 Yeast-Based Processes and Products
3.1 Food yeasts and derivatives
3.1.1 Introduction
3.1.2 Industrial processes
Summary
3.2 Alcoholic beverages
3.2.1 Introduction
3.2.2 Production and sales of major alcoholic beverages
3.2.3 Production processes
Summary

3.3 Industrial alcohols
3.3.1 Introduction
3.3.2 Raw materials and microorganisms
3.3.3 Production processes
3.3.4 Economics
Summary
3.4 Bread and related products
3.4.1 Introduction

109
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232


CONTENTS

3.4.2 Ingredients and formulations
3.4.3 Production processes
3.4.4 New developments
Summary
Bibliography

4 Bacteria-Based Processes and Products
4.1

4.2

4.3

4.4

4.5

4.6

Dairy products
4.1.1 Introduction
4.1.2 Basic knowledge of manufacture of dairy products
4.1.3 Metabolic systems in lactic acid bacteria
4.1.4 Genetic modification of lactic acid bacteria

4.1.5 Applications of genetic engineering
Summary
Meat and fish products
4.2.1 Introduction
4.2.2 Fermented meat products
4.2.3 New developments
4.2.4 Fermented fish products
Summary
Vegetable products
4.3.1 Introduction
4.3.2 Fermented vegetable products
4.3.3 Fermented soy products
4.3.4 New developments
Summary
Vinegar and other organic acids
4.4.1 Introduction
4.4.2 Acetic acid
4.4.3 Citric acid
4.4.4 Lactic acid
4.4.5 Malic acid
4.4.6 Fumaric acid
Summary
Bacterial biomass
4.5.1 Introduction
4.5.2 Microorganisms for the production of biomass
4.5.3 Raw materials for the production of biomass
4.5.4 Production process
4.5.5 Nutritional aspects
4.5.6 Economics and new developments
Summary

Polysaccharides
4.6.1 Introduction
4.6.2 Microbial polysaccharides
4.6.3 Fermentation process
4.6.4 Bacterial polysaccharides
4.6.5 Other polysaccharides
Summary
Bibliography

5 Other Organism-Based Processes and Products
5.1

Enzymes
5.1.1 Introduction
5.1.2 Production of enzymes
5.1.3 Applications

vii

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viii

5.2

5.3

5.4

5.5

5.6

5.7

5.8

CONTENTS

5.1.4 New developments and protein engineering
5.1.5 Economics
Summary
Sweeteners
5.2.1 Introduction
5.2.2 Nutritive sweeteners
5.2.3 High-intensity sweeteners
5.2.4 Low calorie sweeteners
5.2.5 New developments
Summary
Flavors and amino acids
5.3.1 Introduction
5.3.2 Microbial flavors
5.3.3 Enzymatic flavor generation
5.3.4 Amino acids
5.3.5 Economics
Summary
Vitamins and pigments
5.4.1 Introduction
5.4.2 Production of vitamins
5.4.3 Production of pigments
5.4.4 Economics
Summary
Mushrooms
5.5.1 Introduction

5.5.2 Cultivation
5.5.3 Culture preservation
Summary
Cocoa, tea, and coffee fermentation
5.6.1 Introduction
5.6.2 Cocoa fermentation
5.6.3 Coffee fermentation
5.6.4 Tea fermentation
Summary
Bacteriocins
5.7.1 Introduction
5.7.2 Classification
5.7.3 Mode of action
5.7.4 Bioengineering of bacteriocins
5.7.5 Applications of bacteriocins
5.7.6 Commercial production of bacteriocins
Summary
Functional foods and nutraceuticals
5.8.1 Probiotics and prebiotics
5.8.2 Health claim regulation
Summary
Bibliography

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Part II Questions and Answers

411

Part III Other Potential Applications of the New Technology

431

6 Plant Biotechnology, Animal Biotechnology, and Safety Assessment
6.1 Plant biotechnology
6.1.1 Introduction
6.1.2 Plant cell and tissue cultivation

433
433
433
435

CONTENTS

6.2

6.3

6.1.3 Plant breeding
6.1.4 Application of plant cell and tissue culture
Summary
Animal biotechnology
6.2.1 Introduction
6.2.2 Transgenic animals
6.2.3 Animal cell culture
Summary
Food safety issues of new biotechnologies
6.3.1 Introduction
6.3.2 Safety evaluation of novel food products
6.3.3 Genetically modified microorganisms and their products
6.3.4 Genetically modified plants and their products
6.3.5 Genetically modified animals and their products
6.3.6 Detection methods of GM crops
6.3.7 Detection methods of transgenic animals and fish
6.3.8 Containment: physical and biological
6.3.9 Promises and limitations
Summary
Bibliography

Part III Questions and Answers
Index

ix

437
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Preface
In the past decade, major breakthroughs have happened and enormous progress has been

made in all aspects of genetic engineering and biotechnology. This is clearly reflected in the
voluminous publications of original research, patents, peer reviewed books, and symposia.
However, an exciting account of how this new biotechnology can affect traditional methods of producing foods and beverages is the need of the hour. Many professional reference
texts on food biotechnology are now available, but none of it is appropriate as classroom
text. Most such volumes are the work of multiple contributors and the normal didactic criteria required to explain terms, flowcharts and frames of reference are lacking. No attempt
has been made to explain the translation of basic scientific information into practical applications. Moreover, biotechnology has become a fashionable subject and, as one of the most
abused buzz words of the decade, it now comprises a huge body of information. The very
scope of this knowledge presents serious problems to instructors and students. Which facts
are the most important for them to learn and which are less important? How can they assess
the significance of food systems and food products? In writing this book, I have tried to
keep these problems at the forefront and have therefore aimed at making the treatment of
food biotechnology comprehensible rather than comprehensive. I see that separate pieces
of a puzzle eventually fit together to form a picture that is clearer and more readily etched
in memory than the design on the individual pieces. Experience in teaching this subject has
made clear to me the importance of explaining the basic concepts of biotechnology, which
is essentially multidisciplinary, to students who may have limited backgrounds in the scale
up of bioengineering and rapidly developing new tools.
I hope that this book will prove valuable to both students and instructors as well as
to research and industrial practitioners in specific aspects of the field who seek a broad
view on food biotechnology. This book aims to give readers, general science students,
and practicing researchers, an overview of the essential features of food biotechnology
not covered in other institutions as typical science curriculum. The treatment of subjects
is necessarily selective, but the volume seeks to balance the traditional biotechnologies
with the new, and science and engineering with their industrial applications and potential.
Because of the interdisciplinary nature of the subject and the overlapping nature of the
principles of biochemistry, microbiology, and biochemical engineering, the second edition
does not include this part. Instead, the New Trends and Tools of Food Biotechnology
section in Part I (Fundamentals and New Aspects) has included Systems/Synthetic Biology
and Metabolic Engineering, Bioengineering and Scale-Up Process, Molecular Thermodynamics for Biotechnology, Protein and Enzyme Engineering, Genomics, Proteomics and
Bioinformatics, Biosensors and Nanobiotechnology, Quorum Sensing and Quenching,

and Micro- and Nanoencapsulations. For the Concepts and Tools for Recombinant DNA
Technology (Chapter 2), examples of Gene Cloning and Production of Recombinant
Proteins have been included. In Chapter 5 on Other Microorganisms-Based Processes
and Products, a new section on Bacteriocins and Functional Foods and Nutraceuticals was


xii

PREFACE

supplemented and the Waste Management and Food Processing section was deleted; it
will be included in my forthcoming book entitled: “Advanced Fermentation Technology.”
In Part III, Chapter 6 included Plant Biotechnology, Animal Biotechnology, and Safety
Assessment and Detection Methods and other sections were detailed. Up-to-date reading
materials as well as questions and answers have been included in all parts.
I must, of course, thank all those students who have helped me by compiling materials used in the class to produce this book. I greatly appreciate the contribution of many
scientists who have embellished this book by permitting me to reproduce their tables and
figures, which have been illustrated in the pages of this book. I must accept my ignorance
and limitations naturally imposed on a book of this scope when it is written by a single
individual.
A special note of thanks also goes to my previous research associates and students for the
first edition at the McGill University, Dr. S. Y. Park, Dr. J. L. Berger who helped me in typing and drawing the figures, and other associates, G. Arora, M. Torres, M. B. Habibi-Najafi,
and graduate students, M. Bellem, M. Daga, J. James, and T. Wang who helped me in
many ways.
Most of all, my thanks go to Prof. Jian Chen, the President, and Prof. Guchang Du,
the Dean for their support during my stay in the School of Biotechnology at Jiangnan
University in China and the other staff in the 9th floor: Dr. F. Fang, Dr. Z. Kang, Dr. L. Song,
Dr. J. Zhang, Dr. J. Zhou, Dr. L. Liu, and Prof J. Li for their friendship during my absence.
I would like to specially thank Dr. Gazi Sakir for his comments on a part of the bioengineering/scale up section, as well as my students, Dr. Zixing Dong, Yousef Mahammad, and
Nestor Ishimwe, and all international students who took my courses on Food Biotechnology and Advanced Fermentation Technology at the Jiangnan University.

Last but not the least, I thank my wife Young for her love and encouragement; I also
thank and appreciate my sons, Edward in Toronto and David in New York, for their
patience and support during the preparation of this second manuscript.
December, 2013

Byong H. Lee
Wuxi, China


What Is Biotechnology?
We are in the middle of another industrial revolution in which biotechnology, depending
mainly on microbes, plays a major role in the production of exotic drugs, industrial chemicals, bioingredients, fuel, and even food. Although biotechnology involves the potential use
of all living forms, microorganisms have played a major role in the development of this discipline because of the ease of mass growth, the rapid growth that occurs in media consisting
of cheap waste materials, and the massive diversity of metabolic types. These characteristics
in turn allow for a diverse selection of potential products and facilitate genetic manipulation to improve strains for new products.
The bio in “biotechnology” means life and refers to microbes and other living cells
including animal and plant cells. The technology comprises the growth of living cells in vats
(fermentors or bioreactors) containing nutrients and oxygen (if needed) at the specified
conditions, and the processing of biological materials produced by the cells through process
integration and optimization at top efficiency for achieving commercialization. Biotechnology has arisen through the interaction between various parts of biology and engineering,
employing techniques derived from three well-recognized disciplines: biochemistry, microbiology, and biochemical engineering.
The term biotechnology is not a new one, although it has certainly become fashionable in
recent years. It had its origin in prebiblical times but was not widely used until the postwar
university boom in the 1950–1960s, when the volume of scientific and engineering research
output rose dramatically. New disciplines emerged out of increasing specialization. Thus in
the early 1960s, research groups and university departments as well as journals arose with
titles such as BioTechnology, Biochemical Engineering, and Bioengineering. “Biotechnology” is the term that has commonly survived. Table I.1 shows that prior to the twentieth
century, biotechnology consisted almost solely of spontaneous processes. The introduction
of the fed batch in the production of baker’s yeast is probably the starting point of controlled biological processes designated as biotechnological. Biotechnology thus includes
many traditional processes such as brewing, baking, wine making, and cheese making; and

the production of soy sauce, tempeh, many secondary metabolites (antibiotics, steroids,
polysaccharides, etc.), and numerous food ingredients (amino acids, flavors, vitamins, and
enzymes). Traditionally, the biotechnological process based on classical microbial fermentation has been augmented by simple genetic manipulation using a mutagenic agent to
improve microorganisms for food fermentation and to enhance the production of bioingredients. However, it is not possible to predetermine the gene that will be affected by a
given mutagen, and it is difficult to differentiate the few superior producers from the many
inferior producers found among the survivors of a mutation treatment.
The potential of fermentation techniques was dramatically increased in the late 1960s
and early 1970s through achievements in molecular genetics, cell fusion, and enzyme
technology. A new biotechnology was founded based on these methods. However,
additional completely novel, very powerful biotechnology techniques were developed


xiv

Table I.1

WHAT IS BIOTECHNOLOGY?

Biotechnology Milestones

Date

Before 6000 B.C.
Before fourteenth century
1650
1680
1857–1876
1881
1885
Nineteenth century

1940s

1953
1957
1955–60

1970–1972
1973
1974
1975
1978
1982
1983
1984
1985
1986
1987
1989
1989–1991
1990
1992
1994

2004

Milestone
Old biotechnology
Leavening of bread, alcoholic beverages, and vinegar from fermented juice
Beer and wine production, vinegar industry (Orleans)
Cultivation of mushrooms (France)

Yeast cells first seen by Anton van Leeuwenhoek
Fermentative ability of microbes demonstrated by Pasteur
Microbiological production of lactic acid
Artificial growth of mushrooms (U.S.A.)
Ethanol, acetic acid, butanol, acetone production, sewage treatment,
baker’s yeast, sulfite process for glycerol, citric acid
Introduction of sterility to the mass cultivation of microbes for antibiotics
(penicillin, streptomycin, chlorotetracycline) and bioingredients (amino
acid, enzymes, vitamins, steroids, polysaccharides) and vaccines
Discovery of the structure of DNA by Watson and Crick
Manufacture of glutamic acid by Kinoshita et al.
Manufacture of citric acid by the submerged culture process
New Biotechnology
Bacterial plasmid DNA and transformation of E. coli
Genetic barriers breached (restriction enzymes, ligase)
Expression of heterologous gene in E. coli
Hybridoma made (monoclonal antibody)
Somatostatin (first recombinant DNA product)
Recombinant human insulin (Humulin®)
Heterologous plant gene expression
Cohen/Boyer Patent
Recombinant human growth hormone (Protropin®)
Recombinant hepatitis B vaccine (Recombivax HB), Recombinant
∝−interferon (Roferon A®)
Recombinant tissue plasminogen activator (Activase®), Recombinant
tryptophan
Recombinant interleukin-2 (Proleukin®), Recombinant γ-interferon
(Immuneron®)
Recombinant rennet (Gist-Brocades, Genencor, and Pfizer)
Recombinant vitamin C (Genencor), bacteriophage-resistant lactic starters

Maltase-enhanced baker’s yeast (Gist-Brocades)
Lipase (Unilever), Amylase (Novomil®)
Flavr Savr tomato (Calgene), Recombinant bovine somatrotrophin, BST
(Eli Lilly; Monsanto), Brewing yeast (Carlsberg; British Brewing
Research Institute), Acetolactate decarboxylase (Novo Maturex)
47 genetically modified crops on market

out of experiments conducted in bacterial genetics and molecular biology: the field now
called genetic engineering. The discovery of genetic engineering via recombinant DNA
technology is responsible for the current biotechnology boom. Recombinant DNA technology was an outgrowth of basic research on restriction enzymes and enzymes involved
in DNA replication. Not only do these techniques offer the prospect of improving existing
processes and products, but also they enable us to develop totally new products and new


WHAT IS BIOTECHNOLOGY?

xv

processes that were not possible using standard mutation techniques. This new technology
has spawned a new industry and prompted a dramatic refocusing of the research directions
of established companies.
Biotechnology is not itself a product or range of products like microelectronics; rather,
it is a range of enabling technologies, which will find application in many industrial sectors.
It has been defined in many forms, but in essence it implies the use of microorganisms and
animal and plant cells:
• for the production of goods and services (Canadian definition)
• for the utilization of biologically derived molecules, structures, cells, or organisms to
carry out a specific process (U.S. definition)
• for the integrated use of biochemistry, microbiology, and chemical engineering
to achieve industrial application of microbes and cultured tissue cells (European

Federation definition).



What Is Food
Biotechnology?
Food biotechnology is the application of modern biotechnological techniques to the
manufacture and processing of food. Fermentation of food, which is the oldest biotechnological process, and food additives, as well as plant and animal cell cultures, are
included. New developments in fermentation and enzyme technological processes, genetic
engineering, protein engineering, bioengineering, and processes involving monoclonal
antibodies have introduced exciting dimensions to food biotechnology. Although traditional agriculture and crop breeding are not generally regarded as food biotechnology,
agricultural biotechnology, i.e., of animal and plant foods, is expected to become an
increasingly important “engine” of development for the agri-food industry. Nevertheless,
food biotechnology is a burgeoning field that transcends many scientific disciplines.
How do these new technologies ultimately affect our food supply? Biotechnology
will influence the production and preservation of raw materials and the planned alteration of their nutritional and functional properties. It also affects the development of
production/processing aids and direct additives that can improve the overall utilization
of raw materials. This illustrates the diverse nature of the field of food biotechnology.
The new aspects of modern biotechnology will not necessarily revolutionize the food
industry, but certainly they will play an increasingly useful and economic role. Techniques
such as enzyme/cell immobilization and genetic engineering are now beginning to have
a considerable impact on raw material processing. The potential for developing rapid,
inexpensive, and highly sensitive biosensor kits for food analysis is considerable. New
developments in biochemical engineering will also be of advantage to industries using
traditional mechanical or physical methods, which will be replaced by modern unit
operations in product recovery and advanced fermentation control. There are great
difficulties in precisely forecasting economic opportunities arising from technical progress.
The annual value of biotechnologically related products in the food and drink industries
is expected to reach U.S.$35 billion dollars by the year 2000, compared with that of the
pharmaceutical industries (U.S.$24 billion) and commodity chemicals (U.S.$12 billion).∗

The technology must be economically effective, yet preserve the capacity of the world’s
largest industry to generate wealth. It has also to meet the changing fashions in food, without disturbing the traditional virtues of wholesomeness and natural appeal. Thus clear and
rational policies are needed regarding the regulatory status of bioengineered products.
Regulatory provisions follow the same procedures used to establish the safety of conventionally derived food products but are still undergoing clarification with respect to the
safety of genetically cloned system. Because of the recognition that some rDNA products
∗

Throughout the text, all dollar amounts refer to U.S. dollars.


xviii

WHAT IS FOOD BIOTECHNOLOGY?

without any side effects are already on the market, the initial concerns over possible health
hazards have been relaxed, particularly for single constituents or defined chemical mixtures. The safety issue of whole foods is more difficult than that of single ingredient products, however. For example, recombinant chymosin produced by microorganisms is used to
replace calf rennet in cheesemaking. It has been used in 60% of all cheese manufactured
since 1990. Benefits include purity, reliable supply, a 50% cost reduction, and high cheese
yield. In 1994 the transgenic Flav Savr tomato was marketed by Calgene in the United
States after a lengthy regulatory process. The Flav Savr tomato offers improved flavor and
extended shelf life. Calgene argues that the use of biotechnology per se poses no specific
risks and that products should not be discriminated against on the grounds of their method
of production. On the other hand, a number of issues such as aller-genecity, labeling of all
recombinant foods, and consumer perception, as well as ethical and moral issues, will need
further regulatory clearances and public debate.


Part I
New Trends and Tools
of Food Biotechnology

1

Fundamentals and New
Aspects

1.1 Biotechnological applications of animals,
plants, and microbes
In transgenic biotechnology (also known as genetic engineering), a known gene is inserted
into an animal, plant, or microbial cell in order to achieve a desired trait. Biotechnology
involves the potential use of all living forms, but microorganisms have played a major role
in the development of biotechnology. This is because of the following reasons: (i) mass
growth of microorganisms is possible, (ii) cheap waste materials which act as the media for
the growth of microorganisms can be rapidly grown, and (iii) there is massive diversity in
the metabolic types, which in turn gives diverse potential products and results in the ease of
genetic manipulation to improve strains for new products. However, mass culture of animal
cell lines is also important to manufacture viral vaccines and other products of biotechnology. Currently, recombinant DNA (rDNA) products produced in animal cell cultures
include enzymes, synthetic hormones, immunobiologicals (monoclonal antibodies, interleukins (ILs), lymphokines), and anticancer agents. Although many simpler proteins can
be produced by recombinant bacterial cell cultures, more complex proteins that are glycosylated (carbohydrate-modified) currently must be made in animal cells. However, the
cost of growing mammalian cell cultures is high, and thus research is underway to produce
such complex proteins in insect cells or in higher plants. Single embryonic cell and somatic
embryos are used as a source for direct gene transfer via particle bombardment, and analyze transit gene expression. Mammarian cell-line products (expressed by CHO, BHK (baby
hamster kidney), NSO, meyloma cells, C127, HEK293) account for over 70% of the products in the biopharmaceutical markets including therapeutic monoclonal antibodies.
Biopharmaceuticals may be produced from microbial cells (e.g., recombinant
Escherichia coli or yeast cultures), mammalian cell culture, plant cell/tissue culture,
and moss plants in bioreactors of various configurations, including photo-bioreactors.
The important issues of cell culture are cost of production (a low-volume, high-purity
product is desirable) and microbial contamination by bacteria, viruses, mycoplasma, and

Fundamentals of Food Biotechnology, Second Edition. Byong H. Lee.
© 2015 John Wiley & Sons, Ltd. Published 2015 by John Wiley & Sons, Ltd.


4

CH1

FUNDAMENTALS AND NEW ASPECTS

so on. Alternative but potentially controversial platforms of production that are being
tested include whole plants and animals. The production of these organisms represents
a significant risk in terms of investment and the risk of nonacceptance by government
bodies due to safety and ethical issues.
The important animal cell culture products are monoclonal antibodies; it is possible for
these antibodies to fuse normal cells with an immortalized tumor cell line. In brief, lymphocytes isolated from the spleen (or possibly blood) of an immunized animal are fused with
an immortal myeloma cell line (B cell lineage) to produce a hybridoma, which has the antibody specificity of the primary lymphoctye and the immortality of the myeloma. Selective
growth medium (hyaluronic acid (HA) or hypoxanthine–aminopterin–thymidine (HAT))
is used to select against unfused myeloma cells; primary lymphoctyes die quickly during
culture but only the fused cells survive. These are screened for production of the required
antibody, generally in pools to start with and then after single cloning, the protein is purified. As mammals are also a good bioreactor to secrete the fully active proteins in milk,
several species since 1985 have been cloned including cow, goat, pig, horse, cat, and most
recently dog, but the most research has been on cloning of cattle. Genetically modified
(GM) pigs, sheep, cattle, goats, rabbits, chickens, and fish have all been reported.
The main potential commercial applications of cloned and GM animals include production of food, pharmaceuticals (“pharming”), xenotransplantation, pets, sporting animals
and endangered species. GM animals already on sale include cloned pet cats, GM ornamental fish, cloned horses, and at least one rodeo bull. Two pharmaceutical products from
the milk of GM animals have completed (Phase III and Phase II) clinical trials, respectively,
and may be on the market in the EU in the next few years. Cloned livestock (especially pigs
and cattle) are widely expected to be used within the food chain somewhere in the world,

though it would not be economical to use cloned animals directly for food or milk production, but clones would be used as parents of slaughter pigs, beef cattle, and possibly also
milk-producing dairy cows. The first drug manufactured from the milk of a GM goat was
ATryn (brand name of the anticoagulant antithrombin) by GTC Biotherapeutics in 2006.
It is produced from the milk of goats that have been GM to produce human antithrombin.
A goat that produces spider’s web protein, which is stronger and more flexible than steel
(BioSteel), was successfully produced by a Quebec-based Canadian company, Nixia.
Faster-growing GM salmon developed by a Canadian company is also awaiting regulatory approval, principally for direct sale to fish farming markets. Canada has also
approved the GM pig (trade named “Enviropig”) developed by University of Guelph and
it is designed to reduce phosphorus pollution of water and farmers’ feed costs. Enviropig
excretes less phosphorous manure and is a more environmentally friendly pig. It will
be years before meat from genetically engineered pigs could be available for human
consumption. Molecular pharming can also produce a range of proteins produced from
cloned cattle, goats, and chickens. An ornamental fish that glows in the dark is now
available in the market. It was created by cloning the deoxyribonucleic acid (DNA) of
jellyfish with that of a zebra fish. GM fish may escape and damage the current ecosystem
by colonizing waters. Some tropical fish, like piranhas, could be engineered to survive in
the cold and this could lead to major problems. These details will be covered in the section
on Animal Biotechnology.
Recently, the production of foreign proteins in transgenic plants has become a viable
alternative to conventional production systems such as microbial fermentation or mammalian cell culture. Transgenic plants are now used to produce pharmacologically active
proteins, including mammalian antibodies, blood product substitutes, vaccines, hormones,
cytokines, a variety of other therapeutic agents, and enzymes. Efficient biopharmaceutical


1.1

BIOTECHNOLOGICAL APPLICATIONS OF ANIMALS, PLANTS, AND MICROBES

5

production in plants involves the proper selection of a host plant and gene expression
system in a food crop or a nonfood crop. Genetically engineered plants, acting as
bioreactors, can efficiently produce recombinant proteins in larger quantities than mammalian cell systems. Plants offer the potential for efficient, large-scale production of
recombinant proteins with increased freedom from contaminating human pathogens.
During the last two decades, approximately 95 biopharmaceutical products have been
approved by one or more regulatory agencies for the treatment of various human diseases
including diabetes mellitus, growth disorders, neurological and genetic maladies, inflammatory conditions, and blood dyscrasias. None of the commercially available products are
currently produced using plants mainly because of the low yield and expensive purification
costs; however, DNA-based vaccines are potential candidates for plant-based production
in the future. After the cell is grown in tissue culture to develop a full plant, the transgenic
plant will express the new trait, such as an added nutritional value or resistance to a pest.
The transgenic process allows research to reach beyond closely related plants to find useful
traits in all of life’s vast resources. The details of transgenic plants will be covered in the
section on Plant Biotechnology.
All the biopharmaceutical products are mostly manufactured commercially through
various fermentation routes on using genetically engineered microorganisms like E. coli,
yeast, and fungi. Some of the biopharmaceutical products produced commercially
through fermentation routs are human insulin, streptokinase, erythropoietin, hepatitis
B vaccine, human growth hormone, IL, granulocyte-colony stimulating factor (GCSF),
granulocyte-macrophage colony stimulating factor (GMCSF), alfa-interferon, gamma
interferon, and so on. All three domains – animals, plants and microbes – are not only
involved in production of biopharmaceuticals but also find their application in manufacture of food products (Figure 1.1). Although there is a high level of public support for the
development of new biotech, that is, for the production of new medicines (insulin, interferon, hormone, etc.), diagnostics (cancer detection kits), and food enzymes (recombinant

Food biotechnology (old and new)
Animal foods

Plant foods

Microbial foods

1. Animal breeding
2. Transgenic animals
3. Animal cell culture

1. Crop breeding
2. Transgenic plants
3. Plant cell culture

1. Fermented foods
2. Microbial ingredients
3. Transgenic microbes
4. Nutraceuticals/functional foods
5. Diagnostic/biosensors
(food safety)

Agricultural biotechnology (animal & plant foods)
Figure 1.1 Concept of food biotechnology.