Bioprocesses and Biotechnology For Functional Foods and Nutraceuticals

Bioprocesses and
Bio technology
for Functional Foods
and Nutraceu ticaIs
edited by
Jean-Richard Neeser
Nestle' Research Center
Lausanne and
University of Geneva
Geneva, Switzerland
J. Bruce German
Nestle' Research Center
Lausanne, Switzerland, and
University of California, Davis
Davis, California, U.S.A.
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NUTRACEUTICAL SCIENCE AND TECHNOLOGY
Series Editor
FEREIDOON
SHAHIDI, PH.D., FACS, FCIC, FCIFST, FRSC
University Research Professor
Department of Biochemistry
Memorial University of Newfoundland
St. Johns, Newfoundland, Canada
1 . Phytosterols as Functional Food Components and Nutraceuti-

cals, edited b y Paresh C. Dutta
2. Bioprocesses and Biotechnology for Functional Foods and Nu-
traceuticals, edited b y Jean-Richard Neeser and Bruce J. Ger-
man
ADDITIONAL VOLUMES IN PREPARATION

Series Introduction
The Nutraceuticals Science and Technology series provides a comprehensive

and authoritative source of the most recent information for those interested in
the eld of nutraceuticals and functional foods. There is now a growing body
of knowledge, sometimes arising from epidemiological studies and often
substantiated by preclinical and clinical studies, demonstrating the relation-
ship between diet and health status. Many of the bioactives present in foods,
from both plant and animal sources, have been shown to be eective in disease
prevention and health promotion. The emerging ndings in the nutrigenomics
and proteomics areas further reect the importance of diet in a deeper sense,
and this, together with the increasing burden of prescription drugs in treat-
ment of chronic diseases such as cardiovascular ailments, certain types of
cancer, diabetes, and a variety of inammatory diseases, has raised interest in
functional foods and nutraceuticals to a new high. This interest is quite
widespread, from producers to consumers, regulatory agencies, and health
professionals.
In this series, particular attention is paid to the most recent and
emerging information on a range of topics covering the chemistry, biochem-
istry, epidemiology, nutrigenomics and proteomics, engineering, formula-
tion, and processing technologies related to nutraceuticals, functional foods,
and dietary supplements. Quality management, safety, and toxicology, as well
as disease prevention and health promotion aspects of products of interest,
are addressed. The series also covers relevant aspects of preclinical and
clinical trials, as well as regulatory and labeling issues.
This series provides much needed information on a variety of topics. It
addresses the needs of professionals, students, and practitioners in the elds

of food science, nutrition, pharmacy, and health, as well as leads conscious
consumers to the scientic origin of health-promoting substances in foods,
nutraceuticals, and dietary supplements. Each volume covers a specic topic
of related foods or prevention of certain types of diseases, including the
process of aging.
Fereidoon Shahidi

Preface
Science and its applications to biotechnology today are facing the greatest
opportunities in the history of mankind. Biological systems of virtually all
sorts can be controlled in ways not thought possible as recently as a decade
ago. The genomics revolution in the study of biological organisms is
empowering all the life sciences. The use of genomics and functional genomics
in disease target identication and drug discovery is propelling the pharma-
ceutical industry into a new era of successful intervention in human disease,
promising individual health through therapeutics. In the view of many
scientists and economists, innovation in agriculture will enrich virtually every
human activityfrom food and energy production to communication to
polymer design to human habitation. With such unprecedented knowledge of
living organisms, application of this knowledge to biological productivity can
begin to address the great challenges of modern societies: starvation and food
shortages, global energy, pollution, and safety. The inherent eciencies of
biology will continue to revolutionize and empower the lives of individuals by
enhancing quality of life, preventing disease, and extending human perform-
ance capabilities. In no eld is the promise of innovation in agriculture from
biotechnology so vivid as that of food.
Ironically, at the precise moment that biotechnology is poised to revo-
lutionize every aspect of food, the consuming public, including scientists, has
lost faith in modern science to improve our food supply. The world is turning its
back on science and the application of biotechnology to food at a time when
scientic knowledge has become most predictive and useful in food applica-
tions. With the challenges facing the worlds immense population, do we dare
slow the progress of science addressing our most essential human need?

The contributors to this book have taken on the challenge of addressing
this problem directly. An important underlying cause in this loss of condence
by the public is a real or perceived disconnection within the scientic
community and a sense that biotechnology as big business is leaving main-
stream scientists behind. Such a perception is incorrect but emerges from a
lack of knowledge and communication. This book is a clear statement of
clarication.
An international group of scientists from academic, governmental, and
industrial research settings have addressed the problem directly. These
individuals have shown unusual vision in their writing and the potential of
modern biological science to revolutionize the biotechnology of foods. Each
chapter articulates the contributors view of the possible future of food
biotechnology and how science will realize that promise within his or her
respective specialization. We are pleased to present a broad spectrum of
research perspectives that not only illustrate the power and safety of biotech-
nological research but should serve as a blueprint for the progress of the
science of foods.
Part I addresses biological organisms in which scientic research
illustrates how powerfully biotechnology can improve all aspects of tradi-
tional food commodity production. As the scope of the many agricultural
commodities is extremely wide, this book specically includes areas that have
not been well addressed in most other texts on biotechnology applications to
food and agriculture, which have focused solely or mostly on plant foods.
Consequently, the reader should refer to other reviews if specically interested
by plant foods.
In this rst parts, animal products are examined. Chapter 1 describes in
detail poultry and egg production. As a magnicent example of a modern
bioreactor, the laying hen represents an astonishingly productive organism
delivering one of agricultures most nutritious products. The poultry industry
has become one of the most successful and valuable contributors to the global
food supply. Chapter 2 addresses the dairy industry and its myriad product
oerings. Few commodities are more linked to food traditions around the
world, and dairy products are fast becoming the chosen carriers of innovative
nutritional values.
In Part II, microbial products are examined. Microbial systems provide
almost limitless potential for introducing biotechnology. From their origins
thousands of years ago, as perhaps the most primitive biological means to
process agricultural commodities, microbial systems are being re-examined
for producing specic food ingredients.
Two separate chapters provide an overview of the scientic strength and
application potential of microbiology to the future value of the food supply.
Finally, the health potential of probiotic organisms, bacteria that are ingested

for the purpose of directly aecting the consumers own intestinal health, is
the subject of a fascinating chapter. That biotechnology can impact the lives
and health of consumers through the consumption of bacteria designed
specically for this purpose is extremely exciting.
Part II also addresses (a) which health eects can be expected from
specic food ingredients and (b) how such ingredients can be produced for
further addition in food products. Both aspects are presented in some of the
chapters, whereas other authors have developed only one of these two ques-
tions, depending on the class of the functional ingredient.
Chapter 6 focuses on prebiotic carbohydrates from lactose and plant
polysaccharides. Here, health eects and production processes are equally
reported. The well-documented fructo-oligosaccharides (FOS) are not pre-
sented here due to the numerous reports already existing on these prebiotic
ingredients. Chapter 7 deals with dextrans and gluco-oligosaccharides, which
should be regarded more as colonic foods than as prebiotics. Both questions
of health eects and enzymatic technologies for production of these carbohy-
drates are discussed. These important reviews on non-digestible carbohy-
drates are followed by Chapter 8, which is entirely focused on human and
mechanistic studies aimed at measuring the eects of a prophylactic usage of
prebiotics to prevent gut disorders.
Chapter 9 deals with questions related to the addition of recombinant
milk proteins and peptides to infant formula. Such polypeptides may be
produced in transgenic animals or, alternatively, in microorganisms or plants.
These aspects are discussed in great detail, as are the questions related to
biochemical assessment, digestibility, and in vivo evaluation of these ingre-
dients. Chapter 10 is restricted to the use of enzymes as food ingredients,
especially for functional foods. Production guidelines are also presented.
In Chapters 11 and 12, the health eects of plant metabolites of two
classes are reported: isoavones and anandamides. Analytical aspects, bio-
logical eects, and intervention trials are thoroughly presented and discussed.
In Part IV, chapters address the vital issues that will promote or retard
the applications of biotechnology in our lifetime. How does the consumer
perceive biotechnology, its benets, and its risks? How can the consumer be
educated on the appropriate assessment of risk and benet? Legal implica-
tions of globalization of biotechnology remain important issues and have
been addressed from the perspective of the principles of both biotechnology
and international law.
It has been said that the nineteenth century saw the industrialization of
chemistry to produce chemicals, leading to the chemical industry and all the
improvements in the human condition that followed. Similarly, the twentieth
century saw the industrialization of chemistry to enhance biology; the com-
mercialization of everything from fertilizers and pesticides guided the human

condition through its greatest century ever, successfully addressing issues
from infectious diseases to agricultural productivity. The twenty-rst century
will see the industrialization of biology, which will drive another quantum
leap forward in the human condition. The scientic community has produced
the biological tools, and these tools are accelerating knowledge of biotech-
nology and its myriad applications. It is now up to the imaginations of
scientists and industrialists to create opportunities for utilization biotechno-
logical innovation throughout the human experience. This book provides a
glimpse into that future and how science will enable it.
Jean-Richard Neeser
Bruce J. German

Contents
Series Introduction Fereidoon Shahidi

Preface
Contributors
Introduction: The Role of Biotechnology in Functional Foods
Soichi Arai and Maseo Fujimaki
PART I. BIOTECHNOLOGICAL APPROACHES TO

MODIFYING AGRICULTURAL FOOD
SOURCES
1. Poultry, Eggs, and Biotechnology

Rosemary L. Walzem
2. Modern Biotechnology for the Production of Dairy Products

Pedro A. Prieto
3. Bacterial Food Additives and Dietary Supplements

Detlef Wilke
4. Genomics of Probiotic Lactic Acid Bacteria: Impacts on

Functional Foods
Todd R. Klaenhammer, Willem M. de Vos,
and Annick Mercenier

5. Biotechnological Modication of Saccharomyces cerevisiae:
Strategies for the Enhancement of Wine Quality
Linda F. Bisson
PART II. BIOTECHNOLOGY STRATEGIES FOR

PRODUCING SPECIFIC FOOD INGREDIENTS
6. Prebiotics from Lactose, Sucrose, Starch, and Plant

Polysaccharides
Martin J. Playne and Ross G. Crittenden
7. Dextran and Glucooligosaccharides

Pierre F. Monsan and Daniel Auriol
8. Prebiotics and the Prophylactic Management of Gut

Disorders: Mechanisms and Human Data
Robert A. Rastall and Glenn R. Gibson
9. Proteins and Peptides

Yuriko Adkins and Bo Lonnerdal
10. Enzymes
Jun Ogawa and Sakayu Shimizu
11. Chemical Analysis and Health Benets of Isoavones

Shaw Watanabe, Sayo Uesugi, and Ryota Haba
12. Anandamides and Diet: A New Pot of Nutritional Research

Is Simmering
A. Berger, G. Crozier Willi, and V. Di Marzo
PART III. PHYSIOLOGICAL TARGETS OF FUNCTIONAL

FOODS
13. Obesity and Energy Regulation

Kevin J. Acheson and Luc Tappy
14. Food, Fads, Foolishness, and the Future: Immune Function

and Functional Foods
Miriam H. Watson, M. Eric Gershwin,
and Judith S. Stern

15. Immunology and Inammation
Eduardo J. Schirin and Stephanie Blum
16. Inuence of Diet on Aging and Longevity

Katherine Mace and Barry Halliwell
17. Foods and Food Components in the Prevention of Cancer

Gary D. Stoner, Mark A. Morse, and Gerald N. Wogan
PART IV. CONSUMER ISSUES OF BIOTECHNOLOGY

AND FOOD PRODUCTS
18. Food Biotechnology and U.S. Products Liability Law:

The Search for Balance Between New Technologies and
Consumer Protection
Steven H. Yoshida
19. Scientic Concepts of Functional Foods in the Western

World
Steven H. Yoshida
20. Paradigm Shift: Harmonization of Eastern and Western

Food Systems
Cherl-Ho Lee and Chang Y. Lee
21. Consumer Attitudes Toward Biotechnology: Implications for

Functional Foods
Christine M. Bruhn

Contributors
Kevin J. Acheson Nestle Research Center, Lausanne, Switzerland
Yuriko Adkins Department of Nutrition, University of California, Davis,

Daniel Auriol Centre de Bioingenierie Gilbert Durand, INSA, Toulouse,

France
A. Berger Nestle Research Center, Lausanne, Switzerland
Linda F. Bisson Department of Viticulture and Enology, University of

California, Davis, Davis, California, U.S.A.
Stephanie Blum Nestle Research Center, Lausanne, Switzerland
Christine M. Bruhn Center for Consumer Studies, Department of Food

Science and Technology, University of California, Davis, Davis, California,
U.S.A.
Ross G. Crittenden Food Science Australia, Werribee, Victoria, Australia
Willem M. de Vos Wageningen University and Wageningen Center for

Food Sciences, Wageningen, The Netherlands

V. Di Marzo Istituto per la Chimica di Molecole di Interesse Biologico,
Naples, Italy
M. Eric Gershwin Division of Rheumatology, Allergy and Clinical Immu-

nology, University of California, Davis, Davis, California, U.S.A.
Glenn R. Gibson The University of Reading, Reading, England
Ryota Haba Tokyo University of Agriculture, Tokyo, Japan
Barry Halliwell National University of Singapore, Singapore
Todd R. Klaenhammer Department of Food Science, North Carolina State

University, Raleigh, North Carolina, U.S.A.
Chang Y. Lee Cornell University, Geneva, New York, U.S.A.
Cherl-Ho Lee Center for Advanced Food Science and Technology, The
Graduate School of Biotechnology, Korea University, Seoul, Korea
Bo Lonnerdal Department of Nutrition, University of California, Davis,

Katherine Mace Nestle Research Center, Lausanne, Switzerland
Annick Mercenier Nestle Research Center, Lausanne, Switzerland
Pierre F. Monsan Centre de Bioingenierie Gilbert Durand, INSA, Toulouse,

France
Mark A. Morse Division of Environmental Health Sciences, School of

Public Health and Comprehensive Cancer Center, The Ohio State University,
Columbus, Ohio, U.S.A.
Jun Ogawa Division of Applied Life Sciences, Kyoto University, Kyoto,

Japan
Martin J. Playne Melbourne Biotechnology, Hampton, and RMIT Uni-

versity, Melbourne, Victoria, Australia
Pedro A. Prieto Abbott Laboratories, Columbus, Ohio, U.S.A.

Robert A. Rastall School of Food Biosciences, The University of Reading,
Reading, England
Eduardo J. Schirin Nestle Research Center, Lausanne, Switzerland
Sakayu Shimizu Division of Applied Life Sciences, Kyoto University,

Kyoto, Japan
Judith S. Stern University of California, Davis, Davis, California, U.S.A.
Gary D. Stoner Division of Environmental Health Sciences, School of

Public Health and Comprehensive Cancer Center, The Ohio State University,
Columbus, Ohio, U.S.A.
Luc Tappy Physiology Institute, Lausanne, Switzerland
Sayo Uesugi Tokyo University of Agriculture, Tokyo, Japan
Rosemary L. Walzem Department of Poultry Science, Texas A&M Univer-

sity, College Station, Texas, U.S.A.
Shaw Watanabe Tokyo University of Agriculture, Tokyo, Japan
Miriam H. Watson University of California, Davis, Davis, California,

U.S.A.
Detlef Wilke Dr. Wilke & Partner Biotech Consulting GmbH, Wennigsen,
Germany
G. Crozier Willi Nestle Research Center, Lausanne, Switzerland
Gerald N. Wogan Biological Engineering Division, Massachusetts Institute

of Technology, Cambridge, Massachusetts, U.S.A.
Steven H. Yoshida Consultant, Davis, California, U.S.A.

1
Poultry, Eggs, and Biotechnology
Rosemary L. Walzem
Texas A&M University, College Station, Texas, U.S.A.
I. INTRODUCTION
The term poultry refers to domesticated species of birds valued for their meat
and eggs. The most frequently encountered examples, chickens and turkeys,
belong to the order Galliformes, as do pheasants, quail, and grouse. Notably,
two other orders of birds are included in the term poultry, Columbiformes,
doves and pigeons (e.g., squab), and Anseriformes, ducks and geese. Each of
the many species of birds within each order has been highly valued for many
thousands of years for both their beauty and their contribution to the diet (1).
Within each species there is a wide array of strains and types, varying in many
aspects of plumage, including feather color and shape. Body type and size,
growth rate, egg production, and disease resistance vary among individual
types of poultry. Thus the term encompasses a highly diverse group of birds of
broadly diering habits, genetic diversity, environmental requirements, and
nutritional needs.
It is a widely proposed that during prehistory, poultry consumption was
an adventitious event, the outcome of a successful hunt or fortunate discovery
of a nest of eggs. Poultry and eggs are noted sources of essential nutrients,
including energy, protein, fatty acids, vitamins, and minerals (2). Presumably,
an even more diverse collection of bird species was consumed in prehistory
essentially whatever could be caught. Humans evolved within this pattern of
food intake and inadvertently beneted from what is now termed biostream-
ing: namely, that certain desirable or essential nutrients consumed by birds,
retained and concentrated within their bodies, were made available, or
available in greater concentrations, to the humans who consumed those

birds. As such, poultry have made important nutritional contributions to
humans through biostreaming and through converting plant and insect foods
that are indigestible or unpalatable for humans to highly digestible and
nutritious food. An interesting possibility is that certain bioactive phyto-
chemicals or xenobiotics have only been consumed by humans as components
of poultry during our evolutionary history (36). Recognition that poultry
possessed the capability to acquire avor and texture attributes through their
diets and environment is likely the basis for traditional feeding strategies.
These strategies include the addition of herbs, particular juiciness (due to
enhanced subcutaneous and intramuscular fat deposits), promotion of feed
components such as corn, and other manipulations to alter the nal nutrient
and chemical composition to suit the consumer (7,8).
As huntergatherer societies transformed into agrarian-based societies,
plant and animal species that proved tractable to human cultivation were
encouraged. Food supplies stabilized and increased in abundance. Under
these conditions, sheer need or hunting and gathering skill inuenced human
dietary choices less while hedonistic, intellectual, and philosophical consid-
erations inuenced them more. These bits of sociological assumption are
noted to emphasize that our species has physiological and historical inclina-
tions to be omnivorous. Moreover, humans actively cultivate and fabricate
the foods they desire. Biotechnology provides another set of options to
improve food quality. Within this context, biotechnology is dened as the use
of microorganisms, plant cells, animal cells, or parts of cells such as enzymes,
immunoglobulins, or genes to make products or carry out processes (9).
One objective of this chapter is to provide factual information on
biotechnological approaches that can enhance the nutritional value of poultry
and eggs for use in human dietary supplement or contribute to other nonfood
products that enhance health or well-being (10). An example of a nonfood
application is the use of egg membranes to bandage ocular burn patients
(11). Another objective is to describe the types of enhancements that might
prove desirable within modern dietaries delivered by modern food supply
systems. This directed focus somewhat limits description of the benets that
biotechnology will confer to continued interactions between poultry and
humans. Table 1 provides a listing of many healthful components of eggs or
poultry that may be isolated, stabilized, or augmented by biotechnology.
Table 2 provides a generalized classication of modications or applications
according to groups most likely to benet. From this listing it is clear that
many of the biotechnological improvements that have the greatest priority at
present are those that improve the birds ability to digest and assimilate feed
and thus produce less waste. The environmental gains in water or soil quality
and sanitation realized through these eorts will improve human health in
indirect but meaningful ways (12,13). Similarly, biotechnological approaches

Table 1 Healthful Egg or Poultry Components That May Be Isolated,
Stabilized, or Augmented by Biotechnology
Components found in or suitable for delivery by poultry or eggs that have health or
biotechnological applications
Proteins
Native: immunoglobulins, lysoyzme, angiotensin-converting enzyme(ACE)-inhib-
itory oligopeptides, CCK-gastrin immunoreactive protein, phosvitin, transferrin,
ovomucoid, ovomucin, Cystatin, riboavin-binding protein, avidin
Engineered: insulin, growth hormone, human serum albumin, humanized immu-
noglobulins, monoclonal antibodies, a-interferon, spider silk
Lipids
Choline, lecithin, cephalin, betaine, cholesterol, sphingomyelin, a-tocopherol, ca-
rotenoids, xanthophylls, lutein, lycopene, n-3 fatty acids, vitamin K, vitamin D
Miscellaneous
Sialic acid, CaCO3, shell membranes
to enhance disease resistance and reduce mortality rate within the production
unit will improve human health through removal of antibiotics from feed and
absence of pathogenic organisms in poultry meat or eggs oered for sale.
Suggesting a fundamental change to the nutrient composition of poultry
meat or eggs is a more speculative endeavor. At present, there is a lack of
sucient physiological understanding to make unequivocal statements re-
garding what foods constitute an optimal human dietary. However, biotech-
nology provides additional tools to enhance nutritional value of foods as such
information becomes available. Moreover, nutritional optimization is likely
to be highly individual (14,15). In this regard, the inherent genetic diversity,
short generation time, and emerging cloning strategies for poultry provide the
exibility needed to provide consumers with eggs and meat specically
tailored to their physiological characteristics, organoleptic preferences, and
eating patterns.
Applicability is a concern in any scientic endeavor, and biotechnology
is no exception. Data from the Food for Thought II study conducted by the
International Food Information Council in 1997 showed that 70% of
consumers in Canada, Portugal, Japan, and the United States were likely to
purchase foods enhanced by biotechnology (16). In the Netherlands, United
Kingdom, Italy, and Sweden, at least half of consumers were so inclined. A
quarter to one-third of consumers in Austria and Germany indicated that
they would be likely to purchase such foods. In the fourth biannual tracking
survey of food and health news, Food for Thought IV, 2001, found that
biotechnology was the most reported single food and health issue, although

Table 2 Groups Beneting from Modications or Applications of Biotechnology
Types of modications and applications
Benet to the producer

Increased resistance to disease organisms
Enhanced feed digestion and assimilation capabilities
Improved control of food intake
Improved livability
Benet to the processor
Increased resistance to processing-related contamination
Reduced incidence of processing-sensitive phenotypes
Improved compatibility of starter materials for complementary approaches to
enhance safety, avor, texture, and stability
Reduced trimming waste through improved methods of further processing
Benet to the consumer
Improved sanitation
Enhanced vitamin or trace mineral content in whole products, or foods made from
those whole products
Enhanced bioavailability of nutrients, or redistribution of existing components
such as decreased total fat, and increased light to dark meat
Improved avor or texture of products including, or formulated with, poultry or eggs
Eggs containing protein or lipid functionalityavor, texture, therapeutics
(see Table 1)
Benet to society
Reduced fecal waste production through enhanced digestibility, leading to reduced
environmental impacts
Reduced medical costs due to malnutrition and conditions for which poultry or
eggs, or parts thereof, act as therapeutic agents or serve in the manufacture of
those agents
Reduced costs due to reduced food-borne illness as a result of improved live and
processing contamination resistance; improved bird welfare
much of the commentary was cautionary and not placed within a consumer
context (17). Despite these aspects of reporting, a poll of 1000 representative
American consumers above 18 years of age found that only 2% wanted to
know or were concerned about foods that were modied by biotechnology
(17). This same survey found that 33% of consumers believed modied foods
were currently in supermarkets, that more (65% compared with 52%) were
likely to purchase foods identied as modied by biotechnology for purposes
of reduced pesticides/antibiotics than for avor enhancement per se, and that
if foods of improved nutritional value were available through biotechnolog-
ical modication, that factor would encourage (36%) or have no eect (41%)

on the purchase of that product. Overall, these consumers believed that
biotechnology would improve their health and nutrition (39%) the quality,
taste, and variety of foods available to them (33%), but fewer expected
reduced food costs (9%). These data suggest that foods enhanced by
biotechnology are generally acceptable to consumers. The data also suggest
that consumers are becoming sophisticated about how and when biotechnol-
ogy can be used for particular purposes to their advantage.
II. ROLE OF POULTRY IN THE HUMAN DIET
Consumption of poultry and eggs is no longer an adventitious event. It has

also moved beyond being an infrequent luxury meal, such as the Sunday
dinner chicken, Thanksgiving turkey, or Christmas goose. Total per capita
poultry consumption in the year 2000 varied from a high of 57.4 kg per year in
Hong Kong to a low of 3.7 kg per year in Romania (18). Total poultry
production for all major countries was expected to be 59.6 million tons, and
total consumption of poultry meat to be 58.5 million tons in 2001. People in
the United States and China consume the most poultry. In the United States,
total poultry consumption increased from 15.6 kg per person per year in 1960
to 45.3 kg estimated for 2002 (19). Of this amount, 37.0 kg of chicken and 8.3
kg of turkey were consumed. This nearly threefold increase in consumption
was largely due to the vertical integration of the poultry industry, which has
made it capable of being exquisitely responsive to consumer demands in terms
of price, quality, and nal product form.
Consumers do demand poultry. Market data from 1997 showed that
85% of restaurants oered one or more poultry entrees, and that 12% of all
main-course entrees in 1997 contained chicken (20). Similarly, 12.3% of all
meals or snacks consumed in the United States contained chicken or poultry.
The top two appetizers in 2001 were chicken strips and chicken wings. In
addition to extremely popular nuggets, strips, ngers, or fried chicken, chicken
is increasingly used as a topping for salads. In 1997, chicken constituted 19%
of all main-dish salad, and 55% of all menus oered chicken on a salad. The
total value of U.S. poultry production in 2000 was $16.9 billion and clearly had
signicant impact on the spectrum of nutrients available within the diet
(3,5,2124).
Egg consumption in the United States followed a dierent path, de-
creasing from 403 per capita per year in 1945 to a low of 234 in 1991. That
decline has been widely attributed to concerns over cholesterol and changes in
breakfast meal habits (25). From 1970 to 1994, processed egg consumption
rose from 33 to 61 per person per year. In 2000, 198.4 million cases (360 eggs
per case) of shell eggs were produced in the United States, and approximately a

third were further processed to egg products (26). The term egg products refers
to processed or convenience forms of eggs obtained by breaking and process-
ing shell eggs. Egg products include any of various whole eggs, egg whites, and
egg yolks in frozen, refrigerated liquid, and dried forms, as well as specialty egg
products. Specialty egg products include prepeeled hard-cooked eggs, egg
rolls or long eggs, omelets, egg patties, quiches, quiche mixes, scrambled
eggs, and fried eggs. The newest category within the American Egg Boards
Egg Product Reference Guide is Eggs as Nutraceuticals (27). Several
publications now available (2830) describe novel uses and functionalities
for whole eggs and for egg components in particular. These emerging areas of
investigation suggest that potential is to develop novel health promoting
products with egg components is primarily limited by imagination of health
scientists, processors, and engineers working in the area (Table 2). Applica-
tions include food, cosmetics, and medical products. Thus, eggs and poultry
also provide starting materials for further processing that employs biotech-
nological methods, as well as being targets for direct modication by biotech-
nological techniques.
Several epidemiological studies or meta-analyses of dietary interven-
tion studies relating dietary fat and cholesterol to plasma cholesterol concen-
tration (3133) have led medical authorities such as the American Heart
Association to state that periodic egg consumption is unlikely to inuence
plasma cholesterol concentrations (34). This shift in dietary advice was among
the factors that caused egg consumption to increase to 258 per person per year
by 2000. Annual per capita egg consumption in a survey of 36 countries in
2000 varied from a low of 34 in India to a high of 320 in Japan. Despite the
decreasing condence in egg cholesterol risk, cholesterol reduction in eggs
remains as a much sought after target (3539), as does reduction of oxide
formation during processing (40).
Poultry meat and eggs are nutritious foods. Eggs in particular must
possess concentrated amounts of nutrients in order to support incubation and
hatch. Indeed, despite providing less than 1.3% of daily U.S. calories, eggs
provide 3.9% of daily protein, and similar or greater amounts of vitamin B12,
vitamin A, folate, vitamin E and riboavin (21). Egg protein is considered the
highest-quality protein and contains an ideal spectrum of essential amino
acids in a highly digestible form (41). Data from NHANES III showed that egg
consumers were better nourished than nonconsumers and had no higher
plasma cholesterol concentrations (21). Thus, even if cholesterol content
cannot be decreased (42), eggs remain a desirable vehicle for added nutritional
functionality.
Poultry meat is also nutritious (43), although the exact contribution it
makes to the diet is more variable (2,4449). Consumer selections, including
choice of white or dark meat and method of preparation, such as roasting or

deep fat frying, greatly inuence the net diet contribution. Biotechnology
oers options in food preparation that will support increasingly sophisti-
cated consumer demand for healthy functional foods even when consumed in
forms that are traditionally viewed as less healthy. For example, it was noted
that the industry must appreciate that its not going to be as easy as simply
reducing the fat percentage in a fried chicken patty, it will have to be made
with omega-3 enriched meat, using an estrogenic soy binder, and fried in a
non-absorbable fat that is fortied with antioxidants (50). Each component
mentioned in these chicken patties may be the product of biotechnological
processes.
III. HISTORY OF GENETIC AND ENVIRONMENTAL

MANIPULATION OF POULTRY
Because poultry and eggs are so nutritious and are such versatile components
of the diet, the goal of rst-generation biotechnological approaches was
primarily to produce more. The approaches used in these endeavors included
traditional methods of genetic selection for desired traits, controlled environ-
ments, nutritionally optimized diets, and feeding programs (51). At present,
poultry breeders oer strains of birds produced through traditional selection
methods that are highly suited to particular climates and rearing systems (52).
Poultry strains are also highly selected to achieve specic production goals;
thus birds that are grown for meat production are physically and physiolog-
ically distinct from those used for egg production (53). As a point of com-
parison, dogs are perhaps the only other commonly encountered species that
demonstrate equivalent diversity of body types, sizes, and colors inherent
within a given genome due to sustained selective breeding.
Whereas most poultry strains are quite handsome, extreme dierences
in external appearance (phenotype) do result in extreme preferences among
fanciers and raise practical issues for poultry breeders (5458). It is impor-
tant to note that all this diversity was obtained by traditional shuing of
genes by means of cross-mating. Table 2 provides a nonexhaustive list of
targets within selective breeding programs. Most traits are the result of co-
ordinate expression of multiple genes, and selection programs are directed
toward several simultaneous targets. Biotechnology will allow these complex
expression proles to be described and more readily manipulated by
traditional (5961) as well as nontraditional (6264) approaches. The ability
to manipulate phenotype-dening patterns of gene expression provides the
means to optimize bird biological characteristics. In contrast, traditional
breeding approaches are limited by coexpression of desirable and undesir-
able traits.

IV. HISTORY OF NUTRITIONAL MANIPULATION
OF POULTRY
Application of scientic methods to the study of nutrition started in the mid-

19th century. An interesting side note is that poultry, particularly chickens,
provided much of the data that shape our basic understanding of purpose and
metabolism of vitamins, (particularly thiamin, folate, and vitamin D), min-
erals (particularly calcium), lipotropes (choline and betaine), and macronu-
trients (65). Moreover, the widespread use of poultry throughout the world
has generated and continues to generate applied nutrition knowledge regard-
ing diet optimization in dierent environments and at dierent stages of the
life cycle. Many of the principles that underlie optimization of nutrition for
poultry health and production are implicit in eorts to develop similar
strategies for humans to combat chronic disease consumption of functional
foods.
Beginning in the early 1960s, health messages about eggs shifted from
wholly positive to cautionary on the basis of associations among plasma
cholesterol concentrations, the incidence of atherosclerotic cardiovascular
disease, and the cholesterol content of eggs (66). Messages regarding choles-
terol risk have grown equivocal as more has been learned about human
cholesterol metabolism (33,34,66,67), but the criticism spurred eorts to
describe and improve other health-building egg components. As a result, a
variety of specialty eggs have become available. These eggs typically are en-
riched in specic fatty acids, are reduced in cholesterol content, and possess
increased vitamin E, vitamin A, or iodine content (Table 3). Other nutritional
improvements include nonessential but perceived health-promoting nu-
trients such as phytoestrogens, catechins (also known as tea eggs), and ca-
rotenoids such as lutein and lycopene.
Further improvements in egg and poultry nutritional contents are
limited by the existing physiological traits of the birds. As such, they rep-
resent attractive targets for biotechnological improvement. For example, a
single egg from a hen reared to produce vitamin Eenriched eggs can cur-
rently provide 2025% of the daily value recommended, or about 35 mg
(68). In contrast, vitamin E prophylaxis doses are usually in the range of
200400 mg per day (69,70). Further improvements in egg vitamin E content
cannot be realized by dietary approaches as a result of the essential, but lim-
iting, role of tocopherol transport protein in directing vitamin E to egg yolk
(71). Overexpression of this protein by genetically modied birds could in-
crease yolk vitamin E concentration markedly. Moreover, the endogenous
stereospecicity of tocopherol transport protein for the alpha form of vi-
tamin E will ensure that the most bioactive form of the vitamin will be de-
posited into egg yolk. Increased tissue tocopherol content could stabilize

Table 3 Genetic Manipulation to Produce Specialty Birds
and Eggs
Meat-type birds
Growth rate
Feed eciency
Disease resistance
Frame functional properties, skeletal development, body shape
Carcass muscling distribution, proportion of light and dark meat
Carcass fat content and distribution
Market size
Feather color
Egg-type birds
Egg size
Shell quality and color
Feed eciency
Body size
Time to rst egg and duration of egg laying
Frame functional properties, skeletal development, body shape
Disease resistance
poultry meat during processing to prevent development of warmed over and

o-avors (72).
Among animal production systems, the poultry industry has been the
most proactive in coupling selective breeding to nutritional programs to
optimize bird performance in a variety of environments at each stage of the
life cycle. Through its use of complementary approaches, the poultry industry
has improved human nutrition and health through the provision of high-
quality animal protein rich in vitamins and minerals (zoonutrients). Biotech-
nology will further enable poultry and eggs to serve this role in human dietaries.
V. BIOTECHNOLOGY
A. Complementary Approaches
Biotechnology is dened as the use of microorganisms, plant cells, animal
cells, or parts of cells, such as enzymes, immunoglobulins, or genes, to make
products or carry out processes. Given the diverse opportunities aorded by
biotechnology to improve the food supply, multiple approaches to solve a
particular problem will be employed, with market forces driving most ecient
solutions. Because the poultry industry is highly vertically integrated, it has a
history of using complementary approaches to optimize production outcomes

(73). Thus, the full scope of potential for benet from biotechnology will likely
be rst realized in highly integrated production systems, such as are found
within the poultry industry.
Signicant concerns of consumers are food safety and sanitation.
Although the primary concern is microbial safety, the presence of pesticides,
hormones, and antibiotics also causes concern (74). These concerns are well
addressed by vertical integration of complementary approaches aorded by
biotechnology. For example, chicks and poults can be provided with neonatal
feed products containing probiotic bacterial cultures in combination with
prebiotic compounds in order to establish a healthy intestinal ora that
excludes pathogenic organisms from the intestine (75,76). Similar dietary
approaches are known to improve human health and well-being (77,78) and
are no less appropriate for poultry. Moreover, biotechnology can be used to
optimize the mixture of organisms, their ability to attach to the intestinal
mucosa and competitively exclude pathogens, and their ability to utilize spe-
cic prebiotic nutrients in the food and so synergize the protective eects.
These newly hatched birds may well be vaccinated by using recombinant
deoxyribonucleic acid (DNA) vaccines (79). The DNA vaccines are more
exible and allow a rapid response to changes in pathogens, and safety issues
with regard to use in food animals are being addressed (79).
Biotechnology allows the very eggs young birds start in to be selectively
enriched in protective immunoglobulins. These protective proteins are de-
posited into the yolk by the hen in response to maternal vaccination against
neonatal pathogens (80). Such responses occur spontaneously in nature in re-
sponse to pathogen exposure but are haphazard, and many hatchlings die
before the hen begins depositing eective amounts of antibodies. Biotechnol-
ogy allows this endogenous protective mechanism to be used proactively
(81,82). This same natural process is also used to develop high-quality anti-
bodies for use in various biotechnological applications (8388). When placed
in the grower house, these young birds can continue to be protected by
vaccines present in the corn they eat (89). These steps will decrease or eliminate
the need for antibiotics. Notably, the ora initiated in the birds at hatching
may be further engineered to contain strains that competitively exclude
pathogens from the skin or that produce proteins lethal to disease-causing
microbes such as Salmonella or Listeria species but that are harmless to
humans and other animals (9095). The bird itself may be modied to produce
such proteins in the skin or muscles. During processing, such meat would be
resistant to accidental contamination. Egg whites have long been used to
ne wines in order to remove undesirable compounds (96). Eggs containing
specic antibodies or binding proteins could provide biotechnological pro-
phylaxis to protect portions of the food supply from overt (terrorist) eorts to
contaminate it.

Poultry diets could contain other proteins or compounds produced with
biotechnological methods to enhance feed digestibility or ability to transport
and retain nutrients. For example, at present poultry diets must be formulated
with inorganic phosphorus to ensure an adequate intake of this essential
mineral element. The plant materials that constitute the bulk of poultry diets
also have phosphorus in the form of phytate. This organic form of phospho-
rus is indigestible by poultry, and as a result, it passes through the bird and is
excreted as waste, is broken down by microbes, and can ultimately enter the
water supply. At present, feed companies have products that contain micro-
bial enzymes called phytases that release phosphorus from phytate, thus
making it available to the bird (9799). This feed amendment decreases the
amount of inorganic phosphorus added to the diet and ultimately the amount
of waste phosphorus (100,101). The molecular biological attributes of phytase
enzymes from various sources have been studied extensively, and eective
isoforms have been cloned and expressed in cells and plants suitable for use as
feed ingredients (102). A second approach is to endow the birds themselves
with the ability to digest phytate; this has been done in pigs (103)and mice
(104). A third biotechnological approach to this topic is the selection of low-
phytate grains (105), or engineering of the plants to enhance the availability of
the minerals they contain (99,106,107). Biotechnological improvements in
poultry diets may also lead to the formulation of grains that contain
compounds used to enhance growth or promote lean muscle gains (108
110). Such nutritional strategies decrease the value of steroidal anabolic
agents and could increase the nutrient content of poultry and eggs. Similarly,
strategies such as these would support enhanced biostreaming of nutrients
into human dietaries. Modication of the birds themselves may also be used
to achieve production goals more eciently while diminishing the use of
exogenous anabolic agents. Market forces will inuence what becomes the
most persistent strategy.
1. Modifying the Bird Itself

Biotechnological approaches hold the potential to alter specic avian phys-
iological features that improve product quality or functionality. There are
two types of objectives sought after by methods that directly manipulate the
genome of birds. The rst type seeks targets identical with those of traditional
genetics. These include fundamental alterations in body composition to
reduce total fat, increase lean muscle mass, and increase the proportion of
white to dark meat (Table 3). As noted previously, poultry is a highly diverse
genetic population, and individual strains often possess certain desirable
traits to a great extent. The advantage that biotechnology oers is selectivity
in that desirable traits are usually present in combination with undesirable

traits and require many generations of selection to capture, if indeed the
undesirable and desirable traits will segregate while remaining highly herita-
ble. The second type of objective is the introduction of novel functions within
the bird. Overexpression of tocopherol transport protein to enhance vitamin
E content of meat and eggs is an example of this second type. The advantage
here is that objectives insurmountable by alternate approaches may be solved
by introduction of novel genes or novel patterns of gene expression within
existing genomes.
A number of examples that only begin to frame the enhancement in
nutritional content or functional properties aorded by altered gene expres-
sion can be suggested (Table 4). For example, as has been done in plants
(100,106), coordinate overexpression of metal transport and storage genes
could be used to enhance concentrations of iron, zinc, or copper in poultry
meat. Introduction of novel genes or reprogramming of existing genomes could
overcome functional defects such as the myopathy that underlies pale soft
exudates in turkeys (111). Novel ligand binding domains could be expressed to
add combinatorial capabilities in processing applications (112). Such capabil-
ity could allow, for example, stabilization of specic nutrients within foods,
addition or enhancement of avor or color components in poultry products,
or engineering capabilities to enhance texture in nal products. Altered
glycoprotein expression within muscle tissue could alter water-holding capac-
ity, pathogen resistance, browning ability, or fat penetration with frying (113
116). Each contemplated modication requires an exquisite understanding of
biology as well as functional aspects of poultry and eggs as biomaterials. Some
Table 4 Companies Dedicated to Transgenic Poultry Production
Company Focus
AviGenics, Athens, GA Biopharmaceutical protein production

http://www.avigenics.com Agronomic traits, including disease resistance
Improved feed eciency and muscle growth
Origen Therapeutics, Burlingame, Biopharmaceutical protein production
CA Somatic chimeras from elite stock
http://www.origentherapeutics.com
Sima Biotechnology, Minneapolis, Biopharmaceutical protein production
MN
TranXenoGen, Shrewsbury, MA Biopharmaceutical protein production
http://tranxenogen.com
Vivalis, Nantes, France Vaccine development with Aventis Pastuer
http://vivalis.com

of this knowledge will only be acquired through iterative attempts toward the
desired outcome.
2. Altering Gene Expression

As attractive as many of these changes are, there remains concern that altering
the genome or altering patterns of gene expression is somehow unsafethat
birds or eggs arising from transgenic strategies are unnatural. Careful studies
of the avian genome have found natural examples of ancient retroviral gene
insertions. These discoveries suggest that exogenous modication of endog-
enous genomes has been partially responsible for mutations that drive
evolutionary change (62). Hen feathering is a trait in some male birds that
arises from an approximately 40-fold increase in the enzyme aromatase within
the feather follicle of homozygous dominant mutants (117). Aromatase is one
of the enzymes responsible for the conversion of androgens to estrogens.
Elevated estrogen concentrations in the feather follicles of mutant males
change local gene expression such that feathers growing from these follicles
take on a rounded, characteristically hen shape. Matsumine and associates
(118) reported that retroviral insertion into a regulator sequence of the
aromatase gene removes the normal restriction of gene expression in extra-
gonadal tissues. The mutation is highly prized in Bantam birds because of its
eects on plumage phenotype. Another spontaneous retrovirally mediated
alteration in gene expression causes slow feathering (62). This gene has
practical utility within the poultry industry as a simple visual method to sex
chickens. Rapid feathering males (k+/k+) are bred to slow-feathering
females (K/) to produce slow feathering males (K/k+) and rapid feathering
females (K+/). As this method of sexing is widely used within the industry,
nearly everyone has safely consumed poultry containing retroviral elements.
Studies of these ancient gene insertions provide a pattern for the molecular
detail needed to create safe and stable constructs for intentional gene
insertions.
Traits such as egg production, growth, or body fatness result from
complex pattern expression from many genes (54,56,58). In such situations,
adding new genes may not be as desirable as controlling the pattern of
endogenous gene expression to positive eect. Although alteration of gene
expression is still in its infancy, small molecules capable of altering gene
expression proles are being created (119121). This approach holds much
promise but will require substantial expansion of our understanding of gene
interactions and development of appropriate delivery technologies to be
employed eectively. Early embryonic developmentinteractions between
developing tissue layershas inductive and suppressive eects that control
tissue phenotype (122124). Antibodies can be employed to alter such in-

teractions and thus the development of specic tissue types. Antibodies, mono-
clonal antibodies in particular, provide the means for quite specic and se-
lective knock-out of proteins involved in tissuetissue interactions or cell
surface receptors for signaling molecules. A patent has already been issued to
use antibodies to suppress adipose tissue development in poultry, and practical
application of the technique is being pursued (125,126).
Several approaches exist to insert novel genes into poultry. It is beyond
the scope of this chapter to describe these methods in detail, and the interested
reader is directed to relevant reviews (63,64,127130). Progress in method
development is ongoing as poultry provide unique challenges for transgenic
methodology (131). For the most part, methods are directed toward the germ
line in order that inserted genes are stably passed on to progeny. However,
given the extensive breeding programs that produce highly developed strains,
and the limited production interval (40 days for broilers and 60156 weeks for
layers), it seems that somatic transfer, such as is used in gene therapy for
human disease (132,133), could be developed as a viable alternative or adjunct
approach. Bird embryos develop atop a large fragile yolk that dees routine
microinjection techniques used in most mammalian systems (134). Thus,
manipulation and culture of modied embryos remain ongoing and active
areas of investigation (135).
Leading objectives in the eld of avian transgenesis are method
development for cultivation of embryonic stem cells and development and
testing of vector constructs that allow homologous recombination (64,136).
A third requirement, the ability to produce germ line chimeras, is established,
but not perfected. Somatic chimeras can be routinely produced by hand
(130), and mechanization of the process is being vigorously pursued (137). At
present, intense commercial interest in poultry transgenesis makes searches
of patent literature key to understanding advances in the eld (136,138,139),
although academic publications provide good general outlines (140). Because
methodologies in poultry lag behind those of plants and some other animal
species, the poultry industry is in a position to benet from the experience of
commodity groups in both strategy selection and consumer acceptance
issues.
There are several companies dedicated to transgenic poultry production
(Table 4). Most are engaged in producing therapeutic proteins or immuno-
globulin-rich eggs that can be further processed into diagnostics and thera-
peutic agents because of the premium price these productsas opposed to
food productscommand. Thus, the animal systems and methods to produce
nutritional and processing functionality are in active development but not yet
employed in food production per se. The proteins produced in eggs are high
quality and suitable for food grade and pharmaceutical uses. Egg-derived

proteins are likely to improve nutritional and organoleptic properties of
processed foods in a variety of products.
Other products that are likely to reach the market in the foreseeable
future are somatic chimeras produced from elite broiler stock, such as those
currently being developed by Origen Therapeutics (137). These birds are the
product of a biotechnological strategy similar to that used to propagate straw-
berries and grapes and, as a result, do not carry novel genes that could slow
approval of other nearly ready birds (Table 4). Briey, fertilized eggs are col-
lected from prolic egg laying strains and their development halted until blas-
todermal cells from donor embryos can be injected into the recipient embryo.
The donor embryos are derived from strains with superior growth but whose
egg production rates are typically low (58). The resulting hatchling has
heritage from both parental lines, but the bird itself typically exhibits superior
growth characteristics. This technology will ultimately be combined with em-
bryonic stem cell propagation and culture techniques to produce somatic
clones of modied animals.
VI. CONCLUSIONS
Increased knowledge about avian biological characterstics, coupled with var-

ious biotechnological methodologies, provides the means to supply consum-
ers with safe, wholesome poultry products. Current goals of improved disease
resistance and improved yield or growth rates will ultimately mature into
genuine improvements in utility and nutritional values of eggs and poultry
products. Such improvement will be further enhanced by complementary ap-
proaches that incorporate biotechnology at appropriate points in the pro-
duction system. These points will be found throughout the continuum of
animal rearing to food delivery or fabrication. Moreover, biotechnology is al-
ready supporting the enhancement and harvest of functional components
from eggs to improve human health through novel medicines, diagnostics,
and cosmetics (28,29). Great new and healthy poultry and egg foods and prod-
ucts that last longer and taste better can be expected.
The most likely immediate improvement in human health through
biotechnological improvements in poultry and eggs will be in decreased waste
and enhanced water quality and decreased reliance on antibiotics. These
eorts are already in use within the industry, and the ecacy of these methods
will continue to improve. However, as methods and understanding of avian
biological traits are advancing rapidly, biotechnological improvement of both
nutritional and functional properties of poultry and eggs should be realized
within the next several years. As the ability to inuence nutrient composition

in the whole bird through transgenic methods becomes practical, manipu-
lations will always be tempered by basic biological characteristics. Particular
changes that are incompatible with healthy, disease-resistant birds are un-
likely to be acceptable to consumers or producers.
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2
Modern Biotechnology for the
Production of Dairy Products
Pedro A. Prieto
Abbott Laboratories, Columbus, Ohio, U.S.A.
I. INTRODUCTION
A. Early and Modern Biotechnology
For practical reasons it is important to distinguish between the traditional or
early methods of biotechnology and the modern approaches and techniques
of this eld. This is particularly relevant in the case of dairy science and tech-
nology because regulations, riskbenet analyses, and perceptions of pro-
cesses involving the use of genetic engineering dier from those that do not
involve recombinant technology. Animal husbandry and food technology
have provided solutions to the challenges and problems encountered during
the production of milk and milk-derived products; the tools and methods of
these elds constitute early biotechnology. This is in contrast to modern
biotechnology, which is constituted by methods based on recombinant
deoxyribonucleic acid (DNA) techniques (1) and novel approaches for the
purication of materials, selection of microbial strains, fermentation and
manufacturing processes, and analysis of foods. The specialist who implants
embryos while aiming to expand a desirable characteristic in a herd of dairy
cows and the cheese maker who inoculates curd with a naturally occurring
starter culture are indeed using the tools and methods of traditional or early
biotechnology. On the other hand, the molecular biologist who attempts to
insert a gene fragment at a specic site of the bovine genome with the purpose
of producing dairy products containing human milk proteins is clearly using
modern methodologies. Likewise, the task of genetically modifying a micro-
bial strain to speed up the maturation process in a cheese requires modern

techniques. This review focuses on processes and technologies that utilize
genetic engineering and that, in most cases, produce genetically modied
organisms (GMOs) or their derivatives. The present account constitutes a
brief overview of modern biotechnology as it applies to the production and
modication of dairy products. Its purpose is to provide a catalogue of
techniques involving the use of recombinant nucleic acids in the context of
animal productivity and milk and milk-derived product remodeling or
improvement. Ancillary aspects are also discussed. The reader is referred to
previously published reviews that address general aspects of modern biotech-
nology (24).
Table 1 Biotechnology Applications That Aect the Production of Milk and

Milk-Derived Products
Impact on milk
Aspect of dairy production or
production Technology dairy product References
Feeding Use of GMOa Mostly Krishna 1998 (18),

to produce quantitative Crooker 1996 (5)
animal food
Improving Use of GMO Mostly Bera-Maillet et al.,
feed utilization, as probiotics quantitative, 2000 (6) Blum et
bioconversion or GMO-derived increase in feed al. 1999 (7) Gregg
enzymes eciency et al. 1998 (8)
Ziegelhogger
1999 (9)
Improvement of Use of recombinant Quantitative Bauman, 1999 (10)
growth rate, hormones
milk yields,
altering of
reproductive cycle
Improvement of milk Production of Qualitative and Prieto et al.
yields, synthesis transgenic animals quantitative 1999 (11)
of novel compounds and targeted mutants
in milk, alteration
of milk composition
Identication and Production and Qualitative Henriksen et al.,
production identication of 1999 (12)
of improved cultures microbial strains and
and production GMO-derived enzymes
of novel strains with to process milk and
custom designed milk-derived products
characteristics
a
GMO, genetically modied organism.

B. Scope of the Present Review
Table 1 lists some of the steps involved in the production of dairy products
and technologies that have been used or are being investigated for each step.
The table indicates whether the main target of a particular biotechnology is
focused on a quantitative eect, such as milk yield, or a qualitative eect, such
as the presence or increase of a particular compound in milk. Other technical
approaches that are closely related to those listed in Table 1, but are not
covered in this account are (a) the transgenic expression of hormones targeted
to the modication of carcass composition and (b) the use of animals as
bioreactors to produce biomolecules for pharmaceutical applications. These
subjects have been reviewed by Pursel and Solomon (13), Velander and
associates (14), and Ziomek (15). The technologies listed in Table 1 can also
be grouped in two categories: (a) those aimed at the dairy animal in regard to
its productivity and (b) those aimed at modifying milk or its derivatives. This
classication is useful to explain the aims and potential consequences of
particular technologies and the problems they intend to solve. The use of
genetically modied microorganisms in food products is analyzed in depth in
another chapter of the present volume; however, specic applications to dairy
products are briey discussed in Sec. V to illustrate novel technologies as they
pertain to fermented milk and cheese manufacturing.
II. USE OF GENETICALLY MODIFIED ORGANISMS TO

ENHANCE FEED EFFICIENCY FOR DAIRY COWS
Crooker (5) states, In animal agriculture, feed generally represents the major
input component while tissue gain (growth) or milk yield are the primary
useful outputs. In addition, Crooker indicates that approximately 30% of
the calories and proteins in the diet of a dairy cow nd their way into
productive functions such as milk synthesis. Biotechnology can impact the
manufacturing of animal feed in several ways, such as (a) improvement of
microbial strains for the synthesis of diet components, (b) production of
recombinant enzymes to improve digestion of diet components and improve
feed utilization, (c) production of recombinant microorganisms that act as
probiotics in the rumen of dairy cows, and (d) production of transgenic plants
as feed constituents.
A. Improved Microbial Strains

Microbial fermentation processes produce key amino acids used to supple-
ment feed. For example, the genera Corynebacterium and Brevibacterium are

used to synthesize lysine and threonine. Studies with these microbes eluci-
dated their metabolic pathways and key control points in amino acid
synthesis. This information was then applied to design and implement
strategies to circumvent metabolic bottlenecks such as mechanisms of feed-
back inhibition. An example of these studies and their applications is
described in a report by Malumbres and Martin (16) in which the genetic
control of the key enzymatic steps involved in amino acid synthesis is modied
by redirecting the ow of carbon skeletons. This was accomplished by
amplifying genes encoding feedback-resistant aspartokinase and homoserine
dehydrogenase.
B. Genetically Modified OrganismProduced Enzymes

and Genetically Modified Organisms as Probiotics
for Dairy Cows
Hydrolytic enzymes such as lactase (h-galactosidase) are used by lactose-
intolerant humans to aid in the digestion of lactose. Similarly, enzymes can be
used to increase feed eciency in dairy animals and ruminants in general.
Some enzymes used as feed additives or pretreatments are made naturally by
microbes. For instance, the white rot fungus Coprinus metarius enzymati-
cally degrades lignin, thus releasing cellulose and hemicellulose from plant-
based feed (17,18); the released polysaccharides become susceptible to the
action of other hydrolytic enzymes such as cellulases and hemicellulases, thus
providing glucose, which was unavailable before the fungal pretreatment.
Many naturally occurring enzymes have been cloned and are now produced
eciently through fermentation; examples of these are glycosidases such as
cellulases and xylanases (6,7). A more novel strategy is colonization of the
farm animals rumen with recombinant microorganisms that have been
modied to acquire new metabolic capabilities that in turn benet the host
animal (5). These probiotic organisms for ruminants are designed to support
specic functions that go beyond the overexpression of hydrolytic enzymes.
For example, Gregg and colleagues (8) developed strains of Butyrivibrio
brisolvens expressing a gene encoding uoroacetate dehalogenase. These
GMOs colonize the rumen of sheep and reduce symptoms of uoroacetate
poisoning.
C. Transgenic Manipulation of Plants for Enhancement

of Animal Feed
Another biotechnological option to aect the eciency of feed is the use of
transgenic plants. For instance, alfalfa- and tobacco-expressing cellulase
genes have been produced (9). These cultivars promote ecient use of feed

because their recombinant cellulases are aids in the hydrolysis of plant
components. Genetically modied plants can also be used to provide specic
nutrients; transgenic canola with increased methionine content exemplies
the potential of genetically modied plants as enhanced sources of amino
acids in the feed. This advantage is easily exploited because some of these
plants are already constituents of animal feed (19). Therefore, the expression
of nutrients in transgenic plants may aect both the quantity and the quality
of milk without costly purication of recombinant products (20). Another
example of transgenic synthesis of targeted nutrients in plants is the produc-
tion of specic fatty acids. Enzymes that participate in the biosynthesis of
polyunsaturated fatty acids (PUFAs) have been cloned by Knutzon and
coworkers (21,22) and Huang and associates (23), and transgenic seeds that
contain oils enriched in PUFA have been produced. On the other hand, it has
also been demonstrated that dairy cows fed sh oil increased, albeit ine-
ciently, the PUFA content of their milk (24). Feeding dairy cows with these
modied canola seeds or with PUFA-supplemented feed could result in large-
scale production of PUFA-enriched milk.
III. USES OF RECOMBINANT AND SYNTHETIC HORMONES

TO IMPROVE MILK YIELDS AND AFFECT
REPRODUCTIVE CYCLES
Hormone treatment of dairy cows is now used as an alternative method to

improve bioconversion of feed into milk. Since the 1930s scientists have
known that growth hormone or somatotropin from pituitary glands increases
milk yield in dairy cows (25). The commercial application of recombinant
bovine somatotropin (rBST) started in 1994 and today rBST is considered to
be the rst major biotech product applied to animal agriculture (26). Re-
ferring to productivity improvement attained by the use of rBST, Bauman
(10) states: The magnitude of the gain eciency of milk production was
equal to that normally achieved over a 10- to 20-year period with articial
insemination and genetic selection technologies. This statement illustrates
the potential for rapid impact that characterizes modern biotechnology.
Today the use of rBST to improve milk yields is common in the United
States and has been studied from several perspectives. Other hormones or
hormone analogues have been used in dairy cows to modify reproductive
cycles with particular aims. For example, a synthetic leuteinizing hormone
releasing hormone analogue is used to increase conception rates (27) and
estradiol and progesterone are used to induce lactation in prepubertal animals
(28). This application of hormones may or may not have commercial impact;
however, it unquestionably aids in the development of transgenic animal

technology targeted to synthesis of novel milk compounds in the dairy cow.
Hormones can be added exogenously as part of the management schedule of a
dairy farm, or they can also be produced in situ and in excess of their natural
concentrations by remodeling the genetic makeup of an animal (2931).
IV. TRANSGENIC ANIMALS
Transgenic animals (TAs) constitute perhaps one of the most tantalizing and
powerful modern technologies for the manipulation of quantitative and
qualitative aspects of dairy production. Most TAs and targeted mutants
(TMs) produced so far are experimental models in laboratory animals. These
are used to study the eects of gene expression in whole organisms or are
prototypes for the production of modied tissues or biological uids for
industrial purposes. The potential of genetically modied dairy cows for the
manufacture of food and specialized nutritional products resides mainly in
three aspects of the production of TAs and TMs: (a) technologies available for
the production of TAs, (b) current technical hurdles and limitations of these
technologies, and (c) applications of TAs and TMs to the elds of human
health and nutrition. Understanding these aspects of the technology is also
useful to evaluate the safety and potential environmental impact of TAs and
TMs.
A. Production of Transgenic Animals

Briey, a transgenic animal (TA) is a GMO that has acquired or lost a
function or functions with respect to the wild type (11). A function is gained in
a transgenic animal by inserting exogenous genetic information into its
genome, usually during its embryonic or proembryonic stage. Loss of
function is achieved by inserting a modied nonexpressible gene construct
in place of its homologous wild-type functioning gene. In these animals the
original gene is knocked out and the animals themselves are referred to as
targeted mutants (TMs), gene disrupted animals, or knockout animals. Partic-
ular aspects of the technologies and strategies for the production of TAs and
TMs for dairy production have been reviewed by Wall and colleagues (32),
Karatzas and Turner (33), Murray (34), and Pintado and Gutierrez Adan
(35). The present section focuses on modied or remodeled milk produced by
TAs or TMs. The production of TAs requires several steps: (a) design and
construction of the fusion gene (also called transgene) that is commonly
inserted into an embryo, (b) physical introduction of the fusion gene, and (c)
its incorporation into the genome.
The most common method to introduce fusion genes into cells for
generation of TAs or TMs is microinjection into the pronuclei of embryos

(36). Alternatively, the fusion gene can be electroporated directly into sperm
(37) or into the testis of an animal, thus propagating the transgene in sperm
(38). There are other technologies that are suitable for production of TAs and
TMs in which the fusion gene is not introduced into pronuclear stage
embryos; Chan and coworkers (39) describe the production of transgenic
cattle by injecting oocytes with replication-defective retroviral vectors con-
taining transgenes. Increasingly, multicellular embryos and somatic cells such
as broblasts are used as recipients of fusion genes to produce transgenic cows
(3942). These technologies are important because they result in the produc-
tion of cloned transgenic animals. Briey, if a transgene is incorporated into
the genome of a broblast and a cell line is established from this broblast,
cells can be tested for the presence and in some cases for the expression or
function of the transgene. As a last step, nuclei from this cell line can be
transferred to enucleated oocytes. In this fashion, several pseudopregnant
females can be implanted with cloned transgenic oocytes. Once a transgenic
embryo is produced, it is usually implanted into pseudopregnant cows. The
resulting calves are analyzed for the presence of the acquired transgene, and if
the transgene is found in the tissues of a calf, then it is allowed to mature for
further assessment. The resulting transgenic cow is then mated and its milk is
analyzed for the desired modications. Alternatively, as discussed previously,
induction of prepubertal lactation can abbreviate the time from implantation
of the embryo to the determination of successful production of remodeled
milk (28). This example illustrates how modern biotechnology methods and
tools combine to accelerate the development of prototype animals during the
feasibility stages of a biotechnology-based project.
All the mentioned steps are common in the production of targeted
mutants, with the exception of the rst step. The genetic element used to
produce a TM contains homologous elements that hybridize with endogenous
DNA, thus allowing its insertion into the precise site occupied by the gene that
is intended to be disrupted. It also contains fragments engineered to prevent
its transcription into a functional messenger ribonucleic acid (mRNA). An
additional method to control gene expression without obliterating endoge-
nous gene expression is transgenic expression of ribozymes, which are RNA
molecules that have the ability to hydrolyze RNA sequences in their midst
(43,44). When a ribozyme is expressed in a particular tissue it interferes with
the translation of mRNA transcripts, thus reducing the overall synthesis of a
given protein. This technology is useful in controlling the synthesis of a-
lactalbumin in lactating mammary glands (45) and enzymes involved in the
synthesis of lipids (46). Both instances are discussed later in the context of
targeted modications to the lactating mammary glands of dairy cows.
A key problem in the construction of a fusion gene is targeting its ex-
pression to selected tissues or organs. If the desired outcome is the modica-
tion of milk, the fusion gene requires a transcriptional regulatory element

(TRE) that promotes its expression in the lactating mammary gland. Lothar
Hennighausen and coworkers pioneered the development of such a TRE
(47,48). This group expressed human tissue plasminogen activator in the lac-
tating mammary glands of mice by using the TRE that directs the expression
of the gene encoding the most abundant whey protein in murine milk, the
whey acidic protein. Since then, several other lactation-specic TREs have
been isolated and characterized. Examples of milk protein TREs are those of
bovine n-casein (49), goat h-casein (50), and bovine a-lactalbumin (51).
B. Technical Problems and Limitations in the Production

of Dairy Transgenic Animals and Targeted Mutants
and Approaches to Their Solution
Transgenic dairy cows expressing exogenous components in their milk have
been produced by microinjection as described (41). However, the introduc-
tion of fusion genes into embryos, gametes, or gonads has several constraints
that limit the applications of this method for large-scale production of
transgenic cows and goats. Examples of these hurdles are summarized in
Table 2. Perhaps the most frequent problem experienced in generating
transgenic animals is the lack of specic insertion of the fusion gene into
the genome. For instance, a fusion gene can be integrated into a transcrip-
tionally inactive region of a chromosome, thus severely limiting the expres-
sion of a transgene. Piedrahita has reviewed the consequences of nonspecic
insertion of fusion genes and points out that the technologies for targeted
genome modication are still in the early developmental stage (52). The
strategies to circumvent this random insertion problem have been either to
shield the transgene from the surrounding chromatin or to target insertions to
specic sites of the genome. A transgene can be shielded by dominant
regulatory sequences known as locus control regions (LCRs) or matrix
attachment regions (MARs) (53,54). Alternatively, a transgene can be directed
to a specic genomic site by homologous recombination, the most frequently
used approach for TM generation (55). Homologous recombination has not
yet been perfected for the production of TAs, and the technique yields a low
frequency of successful transgenic events (52).
Experimental animals such as rabbits and mice have short gestational
times, relatively large litters, and relatively low maintenance cost for large
quantities of animals. For these reasons it has been acceptable to use fusion
gene transfer techniques that are inecient and produce relative low numbers
of useful transgenic events while developing prototypes in laboratory animals.
In large domestic animals such as cattle, these ineciencies cause a signicant
increase in the cost and time necessary to produce useful transgenic founders.
Several techniques are being developed to increase transfer eciency. For

Table 2 Hurdles for the Production of Transgenic Dairy Animals and Examples
of Techniques Explored to Circumvent Thema
Desired technical
Problem Consequence outcome Approaches
Nonspecic Transgene expression Transgene expression Transgene shielding:

integration aected by controlled by Fujiwara et al. (53),
of fusion gene surroundings TRE in fusion McKnight et al. (54)
into genome unpredictable control gene or regulatory Homologous
of transgene elements present recombination:
expression in targeted site Riele et al. (55)
Piedrahita (52)
Low frequency of Reliance of eorts to Higher percentage Microinjection of
transgenic events produce transgenic of live births embryos at two- or
due to low founders and herds Higher percentage of four-cell stage:
number of on low-probability transgenic animal Echelard et al. (41)
live births events; transgenic production Transgenic production
Low transfer animals not through retroviral
eciency inherent always obtained transfection:
in microinjection Chan (39)
technique
Long reproductive Necessity for Shorter periods to Lactaction induction:
cycle in cattle; long-term determine useful Ball et al. (28);
signicant lag maintenance of transgenic founders retroviral transfer
time between animals before through teat canal:
fusion gene their usefulness Archer et al. (56)
transfer and is determined
verication of
success
Low frequency Many nontransgenic Determination Embryo genotyping:
of transgenic embryos implanted of embryo Hyttinen et al. (57),
events due to and later potential before Jura et al. (58),
lack of ability nontransgenic implantation and Saberivand
to express animals maintained et al. (59)
gene products
Slow transgenic Wait time required for Fast establishment Cloning of TA
propagation commercialization of transgenic herd through nuclear
into herd of acquired or lost transfer: Cibelli
trait to establish et al. (42)
productive herd
a
TRE, transcriptional regulatory elements; TA, transgenic animal.

example, Chan and associates (39) have used viral vectors to transfer fusion
genes via reverse transcription to produce transgenic cattle. Also, Echelard
and colleagues (41) have microinjected cow embryos at the two- and four-cell
stages and compared, favorably, the number of live births obtained by this
technique with those resulting from microinjection into pronuclear stage
embryos. Another limitation inherent to the relatively long reproductive cycle
of cattle is the lag time between the fusion gene transfer and the production of
milk from a transgenic animal. An animal can have detectable elements of a
fusion gene in its genome, but their presence does not guarantee that the gene
product will be expressed in its milk; therefore, the true usefulness of a
transgenic animal cannot be determined until the animal produces milk. As
mentioned, one technique that shortens this wait period is to induce lactation
in prepuber heifers (28); another is to produce adult transgenic animals that
have been exposed to retroviral particles containing fusion genes that are
injected through the teat canal (56). This technique has the added advantage
that the fusion gene is not incorporated into the germ line, thus providing
short-term verication of the ecacy of a particular fusion gene. Several
groups have also developed methods to determine whether transfected
embryos can express transgene-encoded elements before implanting the
embryos into pseudopregnant animals (5759).
Finally, perhaps the most obvious limitation for commercialization of
milk and milk derived products for massive consumption is the establishment
of productive transgenic herds. Cibelli and coworkers (42) have produced
cloned transgenic calves by transferring the nuclei of stable transgenic fetal
broblasts into enucleated oocytes. The fusion gene contained elements
encoding h-galactosidase and neomycin resistance; this allowed for selection
of neomycin-resistant broblasts that were also determined to express h-ga-
lactosidase activity. Nuclei from this cell line were used to generate transgenic
oocyte clones. Although the techniques to obtain transgenic animals are rap-
idly evolving, commercial applications for the production of dairy products
have mostly been demonstrated in laboratory animals, and there are only a
few reports of the actual production of transgenic dairy cows. However, the
prototypes produced in the laboratory and the fast changing environment of
functional foods and dietary supplements are providing a basis to postulate
potentially useful transgenic animals that produce remodeled milk.
C. Applications of Transgenic Animals and Targeted

Mutants to the Production of Dairy Products
Bovine milk and its constituents are mostly used for general nutrition for the
population at large. For this reason, remodeled milk with added value is a
plausible commercial target. On the other hand, transgenically remodeled

milk is an attractive target for applications in special nutrition such as
products for nutritional disease management or for preterm infants and the
elderly. Production of transgenic animals that yield modied milk may also
benet the manufacturer of dairy products such as cheese, cream, and yogurt
or the dairy farm entrepreneur without an apparent direct advantage for the
consumer other than potential reductions in cost and increased availability.
Several reviews have listed potential modications to milk through transgenic
technology (32,34,60). Table 3 is a summary of proposed modications for
commercial applications of TAs and TMs to the production of dairy
products. It is important to reiterate that the use of the lactating mammary
gland as a bioreactor for the production of pharmaceutical compounds is not
being considered here. Only potential applications that are relevant for the
production of dairy products or other nutritional products such as dietary
supplements or medical foods are listed.
Perhaps the easiest way to obtain commercially viable dairy products
from genetically modied dairy cows is by eliminating native milk constitu-
ents, such as bovine h-lactoglobulin and lactose, that cause adverse reactions
in consumers. A TM that does not produce h-lactoglobulin is desirable for at
least two reasons: (a) its milk is more suitable for cheese manufacturing, and
(b) it is devoid of a major allergen of bovine milk (61).
Lactose-free and low-lactose products are now occupying a niche in
functional foods markets to satisfy the needs of consumers with actual or
perceived lactose intolerance. For a 1998 account on lactose intolerance the
reader is referred to de Vrese and associates (62). The extent to which lactose
intolerance plays an actual role in the commercial success of low-lactose or
lactose-free products in dierent markets is debatable. However, evidence
indicates that non-lactose-intolerant individuals are being driven away from
milk and dairy products by the perception that gastric symptoms of discom-
fort are attributable to lactose. This occurs even when lactose concentrations
in these products would not be enough to cause illness or discomfort due to
lactose intolerance or maldigestion (63,64). Nevertheless true lactose-intol-
erants and lactose maldigesters require low-lactose milk and dairy products as
sources of good quality protein and calcium. The reduction or elimination of
lactose is currently being achieved by treating milk or whey with h-galacto-
sidase. On the other hand, several strategies have been explored to produce
low-lactose milk in TAs or TMs. LHuillier and colleagues (45) reported the
reduction of a-lactalbumin, which is a component of the lactose synthetase
complex and is necessary for lactose synthesis. Other methods to eliminate
lactose or its eects are transgenic synthesis of h-galactosidase to hydrolyze
the disaccharide after its synthesis (65) and utilization of lactose as a raw
material to build larger sugars that have lower osmotic values and, therefore,
are less prone to cause some of the symptoms of lactose intolerance. The latter

Table 3 Examples of Desirable Acquired or Lost Traits in Milk Produced by Transgenic
Animals and Targeted Mutantsa
Example of Potential Potential commercial
Trait desired outcome approaches application
Lactose synthesis Reduced lactose Deletion or down- Low-lactose milk,

regulation of formulas for
a-lactalbumin or lactose intolerance
glactosyltransferase
gene TA expression
of glycosidases; Building
of larger, less osmotic
lactose-based
carbohydrates
Synthesis of h-Lactoglobulin- Knockout lactoglobulin Hypoallergenic milk
immunogenic free milk gene, introduction and formulas,
proteins of rybozymes to improved cheese
reduce h-lactoglobulin manufacturing
synthesis
Lipid synthesis Reduced fat milk, Transgenic expression Milk and dairy
modied fatty of rybozymes against products for weight
acid prole acetyl-CoA-carboxylase management
Synthesis of Milk with human TA expression of exogenous Infant formulas,
human milk oligosaccharides glycosyltransferases supplements with
glycoconjugates prebiotics
Synthesis of Milk containing Expression of transgenes Milk and dairy
oligo- and ber or slow- encoding glycosidases products for weight
polysaccharides digest starches and glycosyltransferases management
from plants and microbes
Immunoglobulin Milk containing TA expression of Milk products to
synthesis antibodies against antibodies or fragments prevent infectious
childhood diseases of antibodies diseases
Synthesis of Milk containing TA expression of Increased product
bacteriostatic lysozyme, antibacterial peptides shelf life
compounds defensins or proteins
Synthesis of Milk containing TA expression of Formulas that emulate
human milk human caseins, human milk proteins human milk
proteins lactoferrin
a
TA, transgenic animal; acetyl-CoA, acetyl-coenzyme A.
is achieved by expressing glycosyltransferases that utilize lactose as substrate

for the synthesis of elongating oligosaccharides (66). These two strategies may
have the advantage of allowing for normal expression of milk because lactose
is present at some point in the lactating mammary gland, thus osmotically
driving the movement of water from the plasma compartment (65). Lipid
content in milk can also be reduced. Ha and Kim (46) demonstrated that the

synthesis of lipids can be down-regulated by coexpression with ribozymes
directed against mRNA sequences encoding acetyl-coenzyme A (acetyl-CoA)
carboxylase, which catalyzes the key step in fatty acid synthesis. In a trans-
genic cow this inhibition has at least two potential eects: (a) production of
low-fat milk and (b) increase in milk production eciency. Because the reac-
tion catalyzed by the enzyme consumes energy, its inhibition prevents the
consumption of adenosine triphosphate (ATP) molecules that are then avail-
able to drive other synthetic reactions.
Another potential application of transgenic technology is the modi-
cation of the fatty acid prole of milk. Medical foods that contain specic
proles of long-chain polyunsaturated fatty acids (PUFAs) are used as
adjunct therapy to treat inammatory processes (67), and the literature is
replete with reports on the health benets derived from the consumption of
PUFAs [examples found in Kenler and coworkers (68) and Whelan and
associates (69)]. As stated, Knutzon and colleagues, and Huang and co-
workers (2123) have cloned and expressed enzymes involved in the reduction
and elongation of fatty acids and have produced transgenic plants that
synthesize high levels of PUFA. It is conceivable that through transgenic
expression of the enzymes identied by this group cow milk containing a
benecial PUFA prole could become available. This would be an example of
the transgenic synthesis of secondary gene products in which the aim is the
expression of a transgene-encoded enzyme, which in turn acts on substrates
available in the lactating mammary gland.
Another example is the synthesis of human milk oligosaccharides. We
have expressed several glycosyltransferases in the lactating mammary glands
of mice (11,66) and pigs and rabbits (70). The expression of these enzymes
results in the synthesis of oligosaccharides by elongating lactose, thus
simulating the process that in human mammary glands leads to the synthesis
of the varied repertoire of oligosaccharides that can be found in breast milk
(71). Several biological activities have been associated with these molecules;
Kunz and Rudlo and Kunz and associates (72,73) reviewed their potential
function, and most recently Zopf and Roth (74) have discussed their anti-
infective properties. The presence of these structures could add value to milk-
derived dietary supplements and medical foods. In addition, nonmammalian
glycosyltransferases, glycosidases, or enzymes such as epimerases can be ex-
pressed in the lactating mammary gland. Raw materials such as phospho-
rylated monosaccharides are pluripotent building blocks, which are used by
plants to synthesize bers and starches. Milk with such molecules would
provide novel benets for consumers who already have dairy products in their
diets.
An additional strategy to supplement milk for massive consumption or
for specic applications is to express transgenically antibodies against either

known human pathogens or microbes that cause spoilage in dairy products.
Cordle and his group (75,76) have produced antibodies against the human
rotavirus in milk of hyperimmunized cows. However, these antibodies had to
be concentrated because their relatively low concentration in the milk was not
sucient for human applications. It is conceivable to overexpress transgene-
encoded antibodies directly into cows milk, thus providing a nutritional
product with activity against pathogens that are common in day care centers
or nursing homes. In this fashion the milk itself has anti-infective properties
but does not generate typical antibiotic resistance. Although this application
may be limited to pathogens that act on gastrointestinal (GI) mucosal sur-
faces, it may be of value especially for persons with compromised immune
systems. In addition, cytokines have been transgenically produced in rabbit
milk (77). Such milk could be used in medical foods to attenuate the immune
response in patients with chronic inammatory processes or to boost immu-
nity in persons with immature or depressed immune systems such as preterm
infants and the elderly. Antibacterial proteins and peptides such as lysozyme
and defensins (7880) not only may help the consumer of dairy products to
ght mucosal pathogens but may have a signicant eect in increasing shelf
life of milk and its derivatives.
Finally, an area that has been reviewed by several workers in the eld is
the transgenic expression of human milk proteins in the milk of transgenic
animals (31,81,82). This research has been focused on simulating human milk,
which is considered the gold standard of infant nutrition. Particular proteins
of breast milk have been associated with dierent biological activities (83).
For example, a variant of human a-lactalbumin has antibacterial activity (84)
and h-casein generates, upon proteolytic hydrolysis, peptides that have neu-
rotropic eects and potential immunostimulatory properties (85,86). Al-
though the transgenic expression of breast milk proteins in animal milk
seem to be a natural target for companies that manufacture infant formula,
the applications are not limited to pediatric nutrition. These functional hu-
man-milk proteins may also be less allergenic than those naturally present in
bovine milk. The use of these proteins in the manufacturing of dairy products
could make accessible cheeses, fermented milks, and other products for those
who are allergic to bovine milk proteins. Conceivably, any protein including
enzymes can be expressed in the lactating mammary glands of dairy cows.
The examples described are only a few for which some level of technical fea-
sibility has been demonstrated in transgenic experimental animals or plants.
Modern biotechnology can contribute to the health of farm animals and the
qualitative and quantitative improvement of milk and dairy products through
the application of additional technologies briey mentioned in the next
section.

V. ADDITIONAL TECHNOLOGIES
There are additional recombinant-DNA technologies being developed that

could have a signicant impact in the manufacturing of dairy products. For
example, DNA vaccines are being explored for the treatment and prevention
of diseases in farm animals, including cattle (87). Additionally, signicant
developments have taken place in the design and use of DNA-based diag-
nostics for cattle, specically for dairy cows, as exemplied by the work of
Zimmer and Tegtmeier (88,89). Other technologies that have developed
signicantly since 1995 are the design and implementation of polymerase
chain reaction (PCR)-based determinations of microbial contamination in
dairy products. The assay developed by Nogva and coworkers for the
detection of Listeria monocytogenes in milk (90) is a prime example. Modern
techniques that do not involve recombinant DNA and have relied on the
development of advanced instrumentation are currently used to monitor
biochemical parameters of dairy herds. These techniques are also becoming
tools for DNA-based technologies. For example, biosensors have been
developed for the measurement of progesterone in milk and other uids
(91). Real-time measures obtained with these instruments can be used to
optimize the delivery of recombinant hormones or measure the impact of
transgenic expression of hormones and other factors that aect physiological
parameters of lactation. The convergence of powerful analytical biochemistry
methods and production-oriented biotechnology is also illustrated by the
selection and use of microbial strains for the production of fermented milks
and cheese. Although the topic of food microbes is discussed in depth
elsewhere in this volume, it is worthwhile to exemplify this marriage of
technologies with the work of Desmasures and colleagues (92) and Fortina
and Carminati (93). The rst group characterized Lactococcus spp. strains
from raw milk on the basis of biochemical parameters and genotyped and
classied them by using gene restriction analysis, thus providing an objective
basis for identifying a strain and predicting its eect on milk processing.
Fortina and his group (93) embarked on a systematic and comprehensive
study of the biochemical parameters of Lactobacillus helveticus strains in
natural cheese starters. They determined the extent of DNA homology by
DNA hybridization and several biochemical parameters such as proteolytic
and peptidase activity, lactic acid production, antibiotic and lysozyme
resistance, acidication properties, and cell surface protein synthesis. These
analyses, which involve characterization of bio- and genotypes, provide the
basis for quality control of strains and for appropriate selection and mixing of
strains to obtain specic organoleptic and biochemical outcomes. Reviews of
the uses of biotechnology in the selection, biochemical characterization,

production and development of milk-processing microbes can be found in
Henriksen and associates (12) and Palva (94).
The production and use of enzymes to process milk products are also
beneting from the tools provided by biotechnology. Techniques for the
immobilization of enzymes and entire microbes are being employed to
hydrolyze lactose from milk or whey streams (95). Likewise, several naturally
occurring and recombinant sources of milk clotting enzymes are being de-
veloped (9698). Another case of converging modern technologies is pre-
sented by Kim and colleagues (99); because the hydrolysis of lactose results in
the release of monosaccharides, milk treated with lactase acquires a sweet
taste that is objectionable to some consumers. This group cleverly enclosed
lactase in liposomes that can be dried and reconstituted in water or milk. The
liposomes were able to withstand a simulated gastric hydrolytic environment
but released their contents upon contact with bile salts. This technology rep-
resents a novel approach because health and organoleptic eects of a milk
containing liposomes would not be realized until consumption; the bile salts
of the consumer carry out the last phase of the lactose reduction process by
releasing lactase from the liposomes. Enzymes can also be used as process aids
to modify endogenous milk components. An example of the use of recombi-
nant enzymes to alter physicalchemical properties of milk proteins is the
overphosphorylation of caseins using recombinant protein phosphorylases
(100). This process may be desirable because additional phosphate in proteins
enhances calcium binding and because the properties of caseins (such as curd
formation) for dairy product manufacturing can be modulated by their degree
of phosphorylation. Though all the examples cited oer an overview of what
modern biotechnology has to oer for the improvement of dairy products and
their manufacturing processes, this analysis of applications is an incomplete
description of the technical eld. Better understanding of a technology re-
quires that it be studied from the standpoint not only of its potential benets
but also of its historical and social contexts.
VI. CONCLUSIONS
Regulatory, safety, and perceptual aspects of modern biotechnology are

being discussed in a polarized debate. A published account of this debate,
although slanted toward transgenic crops, can be found in the April 2001 issue
of Scientic American (101103) Salient features of this issue are two inter-
views that present metaphorical mirror images of the same topic; one
interview is with Robert Horsch (vice president of Product and Technology
Cooperation of Monsanto) and the other with Margaret Mellon (director of
the Agricultural and Biotechnology Program of the Union of Concerned

Scientists). A theme that permeates Margaret Mellons statements is the need
for study and analysis of genetically modied foods in order to assess their
risks, benets, and safety adequately. The rational fundament of her views
stems from a knowledgeable assessment of the current situation of biotech-
nology. She points out the urgent need of workers in the eld of biotechnology
to inform peers of the progress and risk assessments of nascent technologies
and their applications. Furthermore, accounts such as the present review have
the intrinsic purpose of discussing the future and evolving strengths and
weaknesses of biotechnology in addition to the current realities. Similarly,
some detractors of biotechnology point to its potential harm and not to sci-
entically established risks. Both are views of the future, and to some extent
both are missing a third important stream of ideas, which is precisely a dis-
cussion of methods needed to determine the safety and risk-versus-benet
analysis of biotechnological applications. The extent to which a particular
application of biotechnology is close to or distant from the consumer is a
consideration that already has practical consequences. For example, trans-
genic corn is approved in the United States for animal feed but not for human
consumption (102). Fig. 1 is a diagrammatic representation of the way
technologies aect the discrete units of the manufacturing process for dairy
products. Certainly, transgenically expressed PUFA in a plant component of
Figure 1 Eects of GMO and GMO-derived products on dierent elements and

dierent stages of dairy product manufacturing. TE, transgenic expression; TM, tar-
geted mutant; GMO, genetically modied organism.

animal feed that eventually nds its way into milk is far removed from the
consumer as compared with a transgenic tomato. This distance does not
address the potential ecological impact of any of the two transgenic cultivars,
but it illustrates a practical approach, already in use, to design operational
regulations. Mollet (104) provides another set of considerations to ascertain
the extent to which organisms are genetically modied. A rst group
constitutes the genetic modications that can occur spontaneously in nature
such as mutations; a second group represents the introduction of recombinant
DNA of a dierent strain of the same species; and the third group involves the
transfer of genetic information from one species to the other. Yet another
consideration is the permanence of a genetic change in the food chain or in
nature at large. Although not devoid of conceivable risks, a transgenic cow
producing remodeled milk as a result of the expression of a fusion gene
inserted through the teat canal will not transmit the genetic modication
through the germ line, and, theoretically, the resulting GMO cannot expand
the acquired or lost traits encoded by the transgene. It appears that an
important challenge for biotechnologists is to confront in an open and self-
scrutinizing manner the challenges posed by legitimate safety concerns and
perceptual distortions of the current nature and potential of biotechnology.
However, current biological problems and forces of the marketplace present
other challenges. In certain countries the future of the entire dairy industry is
in jeopardy as a result of epidemics such as bovine spongiform encephalop-
athy (BSE) and hoof and mouth disease. Piedrahita (52) suggests that a
targeted mutant, in which the expression of a gene encoding a normal prion
protein is impeded, could be less susceptible to contract BSE. Similarly, the
receptor sites for viruses and bacteria can be removed by targeted mutation,
thus generating animals that are resistant to viral infection through the loss of
a function. Last, the reader is referred to two volumes that discuss in depth
several aspects of biotechnology and agriculture: Transgenic Animals in
Agriculture, edited by Murray and coworkers (105), and Agricultural Bio-
technology, edited by Altman (106). Both publications contain chapters that
deal with ethical, regulatory, and strategic issues of transgenic technologies.
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3
Bacterial Food Additives and Dietary
Supplements
Detlef Wilke
Dr. Wilke & Partner Biotech Consulting GmbH, Wennigsen, Germany
I. INTRODUCTION
Microbes and microbial biomass are typically not considered as food

although they signicantly contribute to food properties and food quality.
Traditional spontaneous or controlled fermentations by lactic and acetic acid
bacteria of vegetables as well as milk strongly modify the taste, texture, and
consistency of such food raw materials, but foremost are preservation
measures: removal of fermentable sugars and concomitant acidication are
the preferred technological methods for natural conservation of noncooked
foodstu with high water content. Similarily alcoholic fermentation of bev-
erages improves both organoleptic properties as well as shelf life of sugar con-
taining fruit juices and starch containing crop mashes. In any case food
processing is the underlying reason for such microbial fermentation process-
es, and not the nutritional or dietary value of the food bacteria and metab-
olites that inevitably remain in the nal product.
However, it has long been recognized that fermented milk products such
as yogurt and ker exhibit some health benets that are supposed to be due to
a positive direct or indirect inuence on the composition of the human and
animal bacterial gut ora. This recognition also led to the concept of
probiotics, predominantly lactic acid bacteria, which are consumed as living
organisms in fermented milk products and are considered to pass the stomach
in order to colonize the gut transiently (see Chapter 4).
Beyond food bacteria and probiotics all other contributions to food
properties, taste, and nutritional qualities are provided by isolated bacterial

primary metabolites, enzymes, and other secretion products that are manu-
factured by industrial fermentation processes.
II. REGULATORY AND SAFETY ISSUES
Conceptually, ignoring any specic national legislation, food manufacturing

and distribution are subject to the responsibility and liability of the manu-
facturer and distributor, who must ensure that the consumption of such food
products does not risk or harm the health of the consumer. In other words,
there is no manufacturing or marketing approval policy for food products
comparable to a worldwide standard for pharmaceuticals. The administrative
measure used to ensure food safety is rigid governmental inspection of
manufacturing plants and products in the marketplace for general conformity
of raw materials, process technologies, and nished products, and particu-
larly hygiene. Among food control health issues is the search for any harmful
contamination, such as that by heavy metals, pesticides, or toxins. The latter
refers, for example, to aatoxins and other mycotoxins carried forward from
contaminated nut and cereal raw materials to processed foods, the negative
aspects of microbiological contributions to food production: health damag-
ing contamination and spoilage. The lack of regulatory approval policies in
food production reects the historically proven safety of food products but
also reects practical considerations, since food production is still mainly
performed by local small and midsized manufacturers with a virtually innite
number of dierent consumer end products.
Food bacteria used as starter cultures are exempted from approval and
labeling requirements, as are enzymes that are intra- or extracellular compo-
nents of such food bacteria. The underlying logic is clear: Traditionally
applied food bacteria and their enzymatic components used as processing
aids, including single isolates or improved mixed cultures, which are typically
used in starter cultures, have been proved safe. The use of any recombinant
food microorganism, however, depends on specic national product appro-
vals since they fall under not only the respective food safety directives but also
the more general issue of deliberate environmental release of genetically
modied organisms (GMOs).
No specic approval under the respective genetic engineering directives
is typically needed for microbial enzymes that are manufactured with
recombinant production strains as their manufacture is state of the art now
in the enzyme industry, provided that the commercial enzyme product is free
of GMOs and replicative nucleic acids. Enzyme production has a proven
safety record for both industrial manufacturing and enzyme application for
food, household, and technical purposes. In the past enzyme yields of

microbial production strains have been optimized by multiple mutation and
selection programs, and since 1990 the majority of all production strains are
further improved by specic overexpression of the respective enzyme gene
through genetic engineering. Most enzymes commercially available are
secreted from the microbial cell and can be easily puried to a cell-free
homogeneous protein concentrate by large-scale industrial downstream
processing technology.
The approval exemption for food microorganisms and enzymes applies
only to traditionally used known organisms, such as lactic acid bacteria and
yeast. Any introduction of unknown organisms or their enzyme products into
the food chain depends on extended safety tests that have to be presented to
the national authorities as part of the approval procedure. This would also
refer, for example, to a bacterial strain isolated from a local fermented food
not currently used in Western countries, as has been generally stipulated in the
novel food legislation that was part of the approval policies for transgenic
food.
Indeed many food processing enzymes are derived from and manufac-
tured with bacterial and fungal strains not particularly known as food
microorganisms. The introduction and use of such enzymes for food process-
ing purposes have required detailed safety and toxicity study of the enzyme
itself as well as of the microbial host. In case of the U.S. approval procedure
the Food and Drug Administration (FDA) may subsequently grant so-called
GRAS (generally recognized as safe) status. The certicate issued refers to the
particular product, e.g., an enzyme, manufactured with the particular strain
and does not automatically allow the use in food processing of the same
product derived from or manufactured in another microbial strain, species, or
genus. On the other hand, GRAS status of a compound manufactured with a
particular strain signicantly eases the subsequent approval of other fermen-
tation products manufactured with the same strain or species.
Besides enzymes, other microbial fermentation products are used as
processing aids for food production, typically referred to as food additives, or
for nutritional purposes, often referred to as food or dietary supplements. The
regulatory status of such ingredients as organic acids, amino acids, vitamins,
and polymers depends on the specic national food and dietary legislation.
The use of these ingredients, though absolutely natural and in most cases part
of regular food and even a human biochemical constituent, depends on the
particular compound approval, which may be restricted in dosage or even to
certain kinds of food, such as bakery goods, milk products, or wine only, and
must be labeled in convenience products.
Although certain fermentation products exhibit both functionalities
technological as well as nutritionaltheir use is not at the discretion of the
manufacturer: Approved dosages of additives for processing purposes, such

as vitamin C as an antioxidant, are typically too low to exhibit nutritional
benets, and higher dosages and health claims are only allowed if such
nutraceuticals are approved by the respective food supplement or dietary
food directives.
The introduction of any new chemical, biochemical, or semisynthetic
compound, whether natural or synthetic, into the food chain depends on
previous safety analysis of the compound and, in the case of biotechnological
manufacturing processes, on the safety analysis of the production strain.
Some manufacturers intentions to get approval for pharmacologically active
compounds, even natural compounds, under food additive or food supple-
ment regulations are highly questionable since the authorities are forced to
impede any circumvention of the rigid drug approval and prescription
legislation by launching health food products with pharmacologically active
ingredients.
The principle of current drug approval legislation is to provide mar-
keting licenses to each individual applicant per single nished dosage form of
a respective drug active ingredient. This very tough safety concept wouldfor
the reasons outlinednot t food products or food additives and dietary
supplements. Here the general use of a compound by any food processor is
approved but is specied for dosage range, food categories, and labeling
requirements. Current drug legislation, however, goes far beyond the mar-
keting approval of nished products with dened compositions: The drug
approval process also concerns the detailed process description of primary
and secondary manufacturing that leads to the drug ingredient and the
nished dosage form. The objective that the pharmaceutical manufacturer
has to attain is dened as good manufacturing practice (GMP). The GMP
directives basically describe how a manufacturing process must be developed,
validated, operated, controlled, and documented in order to exclude any
voluntary or unintended deviation from the manufacturing protocol, the drug
master le, that could lead to any unknown toxic contamination or any
change in product specication. Consequently the drug master le is part of
the marketing license, and any modication of the process by the manufac-
turer requires a notication or even a formal approval by the authorities in
advance of its commercial implementation.
In the early 1980s a new Bacillus subtilis fermentation process for the
amino acid L-tryptophan as a dietary supplement was introduced. Subse-
quently the production strain has been improved by genetic engineering of the
tryptophan operon, leading to a further yield improvement of L-tryptophan
synthesized from an externally fed metabolic precursor. This material regret-
tably caused a number of severe and even lethal side eects and had to be
withdrawn from the market. Upon inquiry it was discovered that because of
the improved fermentor yield with the genetically modied production strain

the recovery and purication process of the nal product was shortened.
However, the manufacturer did not check the safety of the product that
resulted from this revised process and overlooked a minor, but highly toxic
by-product that under the original purication regimen was safely removed
from the product. This regrettable experience, which was due to a lack of
process validation and general GMP compliance, but by no means a result
of genetic engineering, drew to the attention of the industry the urgent need
for strict process control, for change validation and change documentation of
any compound targeted to human or animal use regardless of whether it falls
under the formal GMP requirements of drug legislation: The process is the
product (McCormick, 1992).
III. AMINO ACIDS AND DERIVATIVES
L-Amino acids are the largest bacterial biotech products serving the nutrition
marketsfood, feed, and clinical nutrition. Fermentative amino acids as
single compounds have broadly substituted amino acids puried from hydro-
chloric acid hydrolysates of animal and vegetable proteins or from other
agroindustrial by-products, such as molasses. L-cystein, which is applied as a
dough conditioner in the baking industry, as an antioxidant in cosmetic
products, and as the starting material for S-carboxymethyl- and N-acetyl-L-
cystein, leading mucolytic ingredients in cough medicines, is manufactured by
protein hydrolysis and extraction from hair, bristles, and feathers. Meanwhile
recombinant Escherichia coli strains have been described with deregulated L-
cystein biosynthesis and overproduction of the amino acid (Leinfelder and
Heinrich, 1997), and commercial production has been commenced. The
industrial fermentation product will strongly compete against extracted L-
cystein because of the common complaint that the proteinaceous feedstocks
are not in line with the application of L-cystein in food products and
cosmetics. Indeed, a recent directive by the European Commission (2000/
63/EC) has banned use of L-cystein of human origin in the latter two market
segments.
L-Glutamic acid, or more precisely its salt, monosodium glutamate
(MSG), is the leading amino acid by volume. More than 800,000 tons/year of
MSG are manufactured worldwide by fermentation of Corynebacterium
glutamicum and related organisms. The bulk of this material is used as a
avor enhancer. Monosodium glutamate has partially replaced soya and
other vegetable hydrolysates as a seasoning and has strongly beneted from
the development of convenience food in Western countries. The compound is
manufactured by a few industrial biochemical companies in dedicated plants.
Scale of operation and advanced fermentation and recovery technology result

in production costs typical for semicommodities in the chemical marketplace
(Wilke, 1999).
The second largest L-amino acid is L-lysine, which is used predominant-
ly as a feed additive for fortifying the amino acid composition of feed grains
and other low- or suboptimal protein sources. L-Lysine strongly competes
with soya protein, which is a high-protein feed crop well balanced with respect
to L-lysine and other essential amino acids such as L-threonine and L-
tryptophan. L-Lysine is manufactured by fermentation of Corynebacterium
glutamicum and Escherichia coli. Its market volume is in the range of 400,000
tons/year worldwide, and the recent ban of animal protein feeds in the
European Union (2001/999/EC) which must be compensated by domestic
and imported feed crops, will certainly support the use of fermentative feed
amino acids.
All other L-amino acids are considerably smaller by volume (50200
tons/year each) and are used as food supplements, in enteral and parenteral
clinical nutrition formulations, and as building blocks for drug synthesis. Two
amino acids, L-phenylalanine and L-aspartic acid, are the building blocks of
N-L-aspartyl-L-phenylalanine methyl ester, aspartame, a widely used low-
caloric sweetener. Aspartame is an excellent example of a biotech food
additive since not only are the two amino acids derived from industrial
biotechnology processes but the coupling reaction can be performed enzy-
matically (Cheetham, 1995).
IV. NUCLEOTIDES
The two nucleotides disodium inosine monophosphate (IMP) and disodium

guanosine monophosphate (GMP) are used in processed meat products as
avor enhancers in combination with monosodium glutamate. The nucleo-
tides can be obtained by enzymatic hydrolysis and chemical modication of
yeast nucleic acids, or preferentially by direct fermentation with Bacillus or
Brevibacterium species.
V. VITAMINS
Vitamin C, ascorbic acid, is the most important compound of its class

manufactured in a combination of synthetic and biotechnological steps.
The annual production volume of vitamin C exceeds 60,000 tons worldwide
and clearly reects its signicance as a food additive (antioxidant) as well as a
dietary ingredient (mono- and multivitamin formulations). The Reichstein
process, which starts from glucose, includes a microbial transformation step,
the oxidation of sorbitol to sorbose with Acetobacter suboxidans. In this well-

established semisynthetic route a further chemical oxidation reaction has now
been replaced by a bacterial fermentation step, the oxidation of sorbose to L-
ketogulonic acid with Gluconobacter species. Some research programs are
focused on entirely biotechnological synthesis of ketogulonic acid from
glucose. Such concepts are getting more and more realizable as a result of
the genetic rearrangement of enzymes from dierent organisms in one
optimized production strain and the various kinds of targeted genetic
improvements of enzyme functionality, which together result in metabolic
pathway engineering of microbial (and plant) production systems.
Vitamin B2, riboavin, is manufactured by fungal as well as bacterial
fermentation (Ashbya gossypii and Bacillus subtilis, respectively). The world-
wide annual production volume of ca. 3,000 tons is predominantly used in the
feed sector, followed by dietary formulations. Vitamin B12, cyanocobalamin,
is derived from Propionibacterium species or Pseudomonas denitricans
fermentation. Its annual production volume of only 10 tons indicates that
vitamin B12 is exclusively used as a dietary supplement and in pharmaceutical
vitamin formulations.
VI. ORGANIC ACIDS
Industrial lactic acid fermentation with lactobacilli is signicantly growing

since new applications of this organic acid beyond its important use as food
acidulant are emerging. Highly puried thermostable lactic acid is the
building block for a novel generation of polymers, polylactides, allowing
the entry of this renewable feedstock into food packaging and various
technical markets. Its present production volume of approximately 150,000
tons/year therefore has disproportionate growth potential.
VII. ENZYMES
The largest class of bacterial enzymes by production volume, alkaline Bacillus

spp. proteases, is used outside the food industry, as a performance additive in
household detergents. Bacillus spp. and fungal proteases, however, also have
a small application in the manufacturing of vegetable, whey, meat, and sh
protein hydrolysates as seasonings or nonallergenic and readily resorbable
peptide sources in infant formulas, sports foods, etc.
Bacillus licheniformis heat-stable a-amylase is a bulk enzyme used in
industrial starch liquefaction, the rst step of glucose production from corn
and other starch feedstocks. Glucose is subsequently isomerized into a
glucosefructose mixture, high-fructose corn sirup, which is a major alterna-
tive to sucrose as a caloric sweetener in soft drinks since the 1970s. The

isomerization reaction is performed with immobilized enzymes, glucose
isomerases, derived from Bacillus coagulans, Streptomyces rubiginosus, Acti-
noplanes missouriensis, and other bacterial strains.
The majority of microbial enzymes used in food processing (dairy
products, bakery, fruit juices, etc.) are of fungal origin.
VIII. POLYMERS
Xanthan, a bacterial exopolysaccharide manufactured with Xanthomonas

campestris, has established a rm position as a thickener in dressings and
convenience food products in general (Becker et al., 1998). Xanthans wide
application in the food industry is based on its outstanding rheological pro-
perties and broad temperature, pH, and salt tolerance, which result in an
annual production volume for food and technical markets of ca. 40,000 tons.
Outside nutritional applications another bacterial polysaccharide may
be mentioned, namely, hyaluronic acid derived from Streptococcus equi
fermentation. This hydrocolloid is broadly used as a moisturizing agent in
cosmetic products and as a pharmaceutical ingredient and medical device,
respectively, for reconstitution of human synovial uids in ophthalmic and
joint disease indications. Originally hyaluronic acid was extracted from
animal tissue, cockscombs, but the fermentative material has higher molec-
ular weight (performance), is free of allergenic proteinaceous impurities
(safety), and can easily and reproducibly be manufactured by an industrial
process (economics). Hyaluronic acid is therefore a good example of the
benets of industrial biotech processes compared to natural extraction,
particularly if animal-derived compounds are considered for food, feed,
pharmaceutical, and cosmetic applications.
IX. MISCELLANEOUS BACTERIAL COMPOUNDS
Isomaltit, the sugar alcohol of isomaltulose, has been established as a non-

cariogenic, low-calorie sweetener in food, dietetic, and confectionary prod-
ucts. Isomaltulose is derived from sucrose by enzymatic conversion with
sucrose mutase found in various gram-negative bacteria. The commercial
conversion of sucrose into isomaltulose is performed with immobilized
Protaminobacter rubrum cells (Vandamme and Soetaert, 1995).
L-Carnitine is a vitaminlike cofactor of fatty acid metabolism that can
be isolated from meat and liver. The compound facilitates the transport of
long-chain fatty acids through the mitochondrial membrane and is therefore
considered to speed oxidation and energy generation from fat. Since its

industrial production has been achieved, it has gained a signicant position as
a food and feed supplement. The annual production volume is in the range of
300 tons. The chemical synthesis includes as a nal step a biotransformation
reaction that can be performed with a variety of soil bacteria and special
isolates (Yokozeki and Kubota, 1984; Kulla and Lehky, 1985) and that
introduces the h-hydroxy group in the stereochemical L-conguration, the
critical step in the entire synthesis. Alternatively L-carnitine can be obtained
from racemic D,L-carnitine by optical resolution, generally a less attractive
processing route. L-Carnitine may be considered a nutraceutical par excel-
lence: It is a natural compound, but its concentration in regular diets is too
low to eectuate any nutritional, degestive, or metabolic contributions. Its
dietary and health supporting potential can only be utilized if infant formulas,
sports drinks, and health foods (as well as animal feed formulations) are
supplemented with the respective nutraceutical in the appropriate and
regulatorily dened dosage. As has been mentioned, any pharmacological
ecacy is strictly excluded from food ingredients, supplements, and additives.
The natural carnitine derivative acetyl-L-carnitine is considered a neuro-
supportive compound, for which the industry seeks approval as a food
supplement with memory enhancing functionality, but also as a clinically
documented antiAlzheimers disease drug. How the regulatory authorities
will judge such strategies is open.
Trehalose, an a,a-disaccharide that is either extracted from yeast or
derived by fermentative production as a secreted metabolite of Brevibacterium
spp. can be used as an additive for cryopreservation of proteins and cellular
systems and for improvement of freeze stability of processed food (Van-
damme and Soetaert, 1995).
Cyclodextrins are cyclic oligosaccharides derived from starch by enzy-
matic degradation with Bacillus or Klebsiella spp. cyclodextrin glucosyl
transferases. Because of the toruslike structure of cyclodextrins they can
form inclusion complexes with a variety of compounds (Duchene and
Wouessidjewe, 1993). Their application ranges from stabilization of volatile,
light-, or oxygen-sensitive molecules to solubilization of hydrophobic
compounds such as vitamin E. In the United States, cyclodextrins are already
approved for food applications.
X. CONCLUSIONS
Bacterial and fungal production of biochemicals has estabished a rm

position in speciality chemical synthesis for food, feed, pharmaceutical,
and technical applications. Its competitive position compared to those of
chemical synthesis and natural product extraction has a clear technological

basis: The molecular structure of such biochemicals and secondary metab-
olites such as amino acids and antibiotics is rather complicated and often
characterized by one or more asymmetrical carbon centers, as a result,
chemical synthesis is dicult or even impossible with respect to reasonable
manufacturing costs. For well known natural compounds, such as ascorbic
or citric acid, there is no agricultural feedstock available for direct extraction
of bulk quantities that would meet present worldwide demands. Production
feasibility and production economy are therefore the underlying principle for
using microbial compounds for nutritional purposes, and only a few
products, such as xanthan gum, are unique microbial constituents without
an alternative production source and are used because of their unique
functional characteristics.
During the second half of the 20th century some 40 bacterial fermen-
tation and biotransformation products were launched for nutritional appli-
cations (Table 1), an average less than one new compound per year.
Meanwhile many biotechnological manufacturing routes are based on re-
combinant production organisms; however, despite its high potential for
generating new products by enzyme or metabolic pathway design, genetic
engineering has not really increased product ow. Certainly manufacturing
technology is no longer the bottleneck to launching new nutritional com-
pounds of biotechnological origin; rather, the market itself and the signicant
investments in basic and applied research as well as regulatory requirements
that are specied for introducing a new food additive or supplement are. At
Table 1 Bacterial Biotech Products for the Nutrition Markets
L-Glutamic acid Corynebacterium glutamicum

L-Lysin C. glutamicum, Escherichia coli
L-Cystein E. coli
Other L-Amino acids Various bacterial strains
Aspartame Various bacterial strains
Guanosine monophosphate Bacillus spp., Brevibacterium spp.
Inosine monophosphate Bacillus spp., Brevibacterium spp.
Ascorbic acid Acetobacter suboxidans, Gluconobacter spp.
Riboavin Bacillus subtilis
Lactic acid Lactobacillus spp.
Numerous food enzymes Various bacterial strains
Xanthan Xanthomonas campestris
Isomaltulose Protaminobacter rubrum
L-Carnitine Various bacterial strains
Trehalose Brevibacterium spp.
Cyclodextrins Bacillus spp., Klebsiella spp.

least the dierence in attitude compared to that of the pharmaceutical or even
the agrochemical industry is striking: Those industries have performed
intensive and very successful microbial compound screenings. As an example
we may consider microbial enzymes as digestive aids and as neutraceuticals
with further unexpected health benets. Such screening work is nowadays
strongly facilitated by microbial genomics data and expression libraries.
There are established neutraceuticals, such as vitamin E and polyun-
saturated fatty acids, for which improved manufacturing technologies would
be desirable. Vitamin E is a good example: A strong preference exists for the
natural compound in food and dietary products versus the less expensive and
unrestrictedly available synthetic material. Microbial production routes as
potential technological alternatives are now facing competition from im-
proved agricultural processes: Molecular plant genetics will soon give access
to conventional and transgenic crops with signicantly increased vitamin E
content or specialty oil content that cut the manufacturing costs of such
nutraceuticals (Wilke 1999). Presently advanced breeding technologies will
also provide new plant ingredients with particular nutritional and health
supporting properties, such as steroids or polysaccharides that may be further
modied by enzymatic and other biotechnological techniques.
REFERENCES
A Becker, F Katzen, A Puhler, L Ielpi. Xanthan gum biosythesis and application:

A biochemical/genetic perspective. Appl Microbiol Biotechnol 50:145152,
1998.
PSJ Cheetham. Biotransformations: New routes to food ingredients. Chem & Ind 3
April 1995: 265268.
D Duchene, D Wouessidjewe. New possibilities for the pharmaceutical use of cyclo-
dextrins and their derivatives. Chimicaoggi 1,2: 1724, 1993.
H Kulla, P Lehky. Verfahren zur Herstellung von L-Carnitin auf mikrobiellem Wege.
EP 0158194, 1985.
W Leinfelder, P Heinrich. Process for preparing O-acetylserine, L-cystein and L-
cystein-related products. WO 97/15673, 1997.
D McCormick. L-Trps lesson: The process is the product. Biotechnology 10:5, 1992.
EJ Vandamme, W Soetaert. Biotechnical modication of carbohydrates. FEMS
Microbiol Rev 16: 163186, 1995.
D Wilke. Chemicals from biotechnology: Molecular plant genetics will challenge the
chemical and the fermentation industry. Appl Microbiol Biotechnol 52:135
145, 1999.
K Yokozeki, K Kubota. Method for producing L-carnitine. EP 0122794, 1984.

4
Genomics of Probiotic Lactic Acid
Bacteria: Impacts on Functional
Foods
Todd R. Klaenhammer
North Carolina State University, Raleigh, North Carolina, U.S.A.
Willem M. de Vos
Wageningen University and Wageningen Center for Food Sciences,
Wageningen, The Netherlands
Annick Mercenier
Nestle Research Center, Lausanne, Switzerland
I. INTRODUCTION
The period since the early 1990s has seen numerous developments aimed at
improving the functionality of foods. This can be realized by appropriate
selection of raw materials, specic physicochemical processing, or the addi-
tion of ingredients (including health-benecial microorganisms). However, in
many cases food functionality can also be enhanced via biological conversions
such as by exploiting the activity of lactic acid bacteria or other microbes with
a long history of safe use in the food industry. Lactic acid bacteria are used for
the industrial production of fermented dairy, vegetable, and meat products
and form a group of evolutionarily related low-GC-content gram-positive
bacteria, comprising species of Lactobacillus, Lactococcus, or Leuconostoc.
Related gram-positive bacteria such as strains of Bacillus subtilis, Enterococ-
cus faecalis, or the somewhat more distantly related (high GC content)

Bidobacterium or Propionibacterium spp. are used for specic food fermen-
tations and can be applied similarly (Fig. 1). Whereas traditionally lactic acid
and related bacteria have been applied as starter bacteria, most food
innovations of lactic acid and related bacteria are correlated to their use in
fermentations with a specic function or the production of ingredients that
have potential as nutraceuticals. Yet another important application of these
microorganisms is their inclusion as live cells in food and feed matrixes or
nutritional complements for the design of health-promotingprobiotic or
functionalfood products.
The completion of many genomic sequences of lactic acid and related
bacteria and the application of functional genomics approaches are greatly
advancing the knowledge base for innovations (13). Moreover, food-grade
approaches for high-throughput screening or specic genetic improvements
such as self-cloning are continually being sophisticated while acceptable
selection systems are being perfected (4). Hence, this chapter summarizes
the recent advances in genomics, focusing on the innovations relating to pro-
biotic lactic acid bacteria.
Figure 1 Phylogenetic tree based on 16S recombinant deoxyribonucleic acid (rDNA)

sequences of lactic acid and related bacteria that have been completely or partially
analyzed at the genomic level. Left, low GC bacteria; right, high GC bacteria. The size
of the completely known genomes (underlined) is indicated in Mb. Bsu, Bacillus subtilis
(21); Lla, Lactococcus lactis (20); Blo, Bidobacterium longum (18); Lpl, Lactobacillus
plantarum (19); Efa, Enterococcus faecalis (22); Sth, Streptococcus thermophilus; Lac,
Lactobacillus acidophilus; Ljo, Lactobacillus johnsonii; Lga, Lactobacillus gasseri; Lbr,
Lactobacillus breve; Lhe, Lactobacillus helveticus; Lde, Lactobacillus delbrueckii; Lsa,
Lactobacillus sake; Lca, Lactobacillus casei; Lrh, Lactobacillus rhamnosus; Ppe,
Pediococcus pentosaeus; Lme, Leuconostoc mesenteroides; Ooe, Oenococcus oenus; Pfe,
Propionibacterium freudenreichii; Bbr, Bidobacterium breve; Bli, Brevibacteriujm
linens (2).

II. EVOLUTION OF THE PROBIOTIC CONCEPT
Since Eli Metchniko published The Prolongation of Life: Optimistic Studies

the eld of probiotics has become nearly 100 years old (5). However, today it
is a predominantly empirical science that remains to be deciphered scientif-
ically and mechanistically. In the rst generation of probiotic cultures (1904 to
1970), there was little accurate information about the identity of the cultures
or the specic health benets that could be attributed to them. Strains were
often chosen on the basis of poorly controlled screening experiments. Also,
products reputed to contain probiotic cultures often lacked quality control
over the identity of the culture(s) and their viability and levels (6,7).
Since the 1980s, major eorts have been invested in the proper identi-
cation and selection of probiotic strains. The evolution of molecular taxono-
my, deoxyribonucleic acid (DNA) ngerprinting, and ribosomal DNA
sequence analysis has allowed unequivocal identication of strains being used
in both clinical trials and commercial products (8). Armed with dened strains,
eorts began to correlate specic health traits (e.g., lactose tolerance, immu-
nomodulation, antimicrobial activity, anticarcinogenic activity) with specic
strains or strain combinations (7,911). However, most of these health-
benecial properties were studied primarily in vitro and in a variety of animal
models. Even though the number of well-controlled human intervention trials
is steadily increasing, there is still a lack of correlation between the dierent me-
thods used to select probiotic strains (7). Nevertheless, the end result of these
eorts is a rapidly accumulating database of well-conducted studies that are
beginning to unravel the clinical manifestations of probiotic cultures (12). With
the rapid progress over the past decade on the genetics of lactic acid bacteria
and pending release of complete genome sequence information for major
probiotic species, the eld is now positioned to progress on a number of fronts.
The genomic information available on both host and bacterial partners is
expected to reveal the contributions of probiotics and commensals to general
health and well-being and explicitly identify the mechanisms and corre-
sponding hostmicrobe cross-talk that provide the basis for their positive roles.
This platform of information is currently leading to the selection of
second-generation probiotics, those identied by their genomic content and
expression levels for specic traits with known probiotic functionality.
Rapidly growing insight into the structure and function of the target
ecosystem, and the intestinal microbiota that include endogenous lactic acid
bacteria (13), will also contribute to understanding and prediction of the
functionality of probiotics (1,14).
Last, third-generation probioticsgenetically modied cultures that
serve as delivery vehicles or production for biologicals such as antigens, en-
zymes, or complex carbohydratesare on the immediate horizon (15,16).

III. GENOMIC DEVELOPMENTS OF PROBIOTIC LACTIC
ACID BACTERIA
Exploration into the genomes of probiotic cultures is now well under way
(2) and promises to contribute signicantly to our understanding of the eco-
logical and phylogenetic characteristics, metabolic capacity, and safety of
probiotics. Hence genomics approaches are expected to lead the way to the
development of even newer generations of probiotics or functional cultures.
The International Scientic Association for Probiotics and Prebiotics
(ISAPP) (http://www.isapp.net/) has recommended that the genomes for all
commercial probiotic cultures be sequenced to ensure their identity, potential
functionalities, and, most importantly, safety (17).
The genomes of two probiotic species, Bidobacterium longum (18) and
Lactobacillus plantarum (19), have been completed, and those of at least eight
more are rapidly nearing completion: L. johnsonii (Pridmore et al., in prepa-
ration), L. acidophilus, L. gasseri, L. casei (two strains), L. rhamnosus, B.
longum (a second strain), and B. breve. Draft genome information also be-
came available in the public domain in 2002 with the publication and ap-
pearance of genome sequences for lactic acid bacteria (LAB) provided by the
Joint Genome Institute (http://www.jgi.doe.gov/JGI_microbial/html/index.
html) in collaboration with the Lactic Acid Bacteria Genomics Consortium
(see also Fig. 1).
The complete size of the present paradigm genomes of Lactococcus
lactis (20), B. longum (18), and L. plantarum (19) varies somewhat between 2
and 3 Mb, in line with their fastidious lifestyle and incapacity to live without
specic medium additions. In contrast, the genome of B. subtilis is somewhat
double that size and contains many genes for auxotrophic growth (21).
However, in several cases the genome size of these food-grade bacteria is
considerably enlarged by the presence of plasmids and other extrachromo-
somal elements that contribute to the genomic exibility of the host (22).
Notably, Lactococcus spp. are known to carry up to 20% of their DNA as
plasmids, and this characteristic may provide these starters with additional
exibility. Moreover, even though bacteriophages are a threat to industrial
fermentations, prophages correspond to a natural and diverse component of
many genomes that may contain up to half a dozen dierent copies, including
both complete and incomplete versions (23).
A. Functional Genomics and High-Throughput Approaches

Approximately one-third of the genes in a genome encode functions that have
not been described yet and cannot be annotated. Moreover, several genes

have multiple functions and many annotations are incorrect because they
refer to circumstantial evidence and not to an experimentally veried func-
tion. Hence, substantial eorts are focusing on elucidating genome functions
by a variety of approaches that include targeted or random gene inactivation,
overexpression analysis, and high-throughput expression proling by tran-
scriptome or proteome analysis. In the case of probiotic bacteria, this ap-
proach remains rather dicult as a consequence of the lack of validated in
vitro bioassays. Nevertheless, ongoing research aims at assigning a probiotic
function to specic genes by conducting head-to-head comparison of selected
sets of strains by a series of in vitro or in vivo models. Appropriate selection of
strains for these studies can be based either on comparative genomic analyses
or on isogenic pairs of strains corresponding to targeted mutants and their
wild-type counterparts.
Noteworthily, the rst proteomic analysis of a lactic acid bacterium, L.
lactis, was completed in 2003 (24). Even though this species is generally not
considered as a probiotic microorganism per se, similar studies are expected
to be reported soon for Lactobacillus and/or Bidobacterium spp.
B. Comparative Genomics and Strain Diversity

Comparative analysis of genomes may provide insight into evolutionary rel-
ations, reveal specic functions of genes within their context, and allow for the
analysis of genome diversity. A detailed study of L. plantarum using DNA
microarrays revealed high genomic diversity and identied specic lifestyle
islands that are characterized by aberrant codon usage or GC content and at
the same time seem to dier between strains (Molenaar et al., personal com-
munication). Whereas some of these islands in fact are lysogenic bacterio-
phages or remnants thereof (Brussow et al., personal communication), many
appear to code for relevant functions such as the production of antimicrobial
peptides or sugar transport and utilization systems (Molenaar et al., personal
communication). Equivalent studies conducted with L. johnsonii strains
revealed that specic chromosomal regions are restricted to the probiotic
strain La1 and a few closely related isolates. These regions include prophages
and genes that could reect the adaptation of La1 to the upper gastrointes-
tinal tract. Typically, the lack of amino acid biosynthetic pathways appears to
be compensated for by the existence of a wealth of peptidases and monomeric
nutrient uptake systems (Pridmore et al., in preparation).
The high diversity within species indicates that specic approaches are
needed to exploit fully the genomic potential of food-grade and probiotic
bacteria. One such approach is subtractive hybridization that aims to reveal
new sequences that are unique to a specic strain in comparison with another

(sequenced or well-studied) strain. A specic form of this approach is re-
presentational display analysis, which has been used to dierentiate natural
isolates of Bacillus spp. and is expected to be useful to capitalize on the di-
versity of other food-grade bacteria (25).
Although the completely sequenced genomes serve as the basis for
understanding genome organization, evolution, and expression, it is evident
that these and most of the unnished genomes (discussed previously) will
also be instrumental in the screening for target functions including the
health-promoting properties attributed to probiotic organisms. An exciting
set of discoveries are already apparent, revealing, for example, mbriae-like
structures on B. longum as well as the presence of a serpin-like gene, which
had not been identied before in prokaryotes and may play a role in im-
munomodulation (18). In L. plantarum gene regions that appear to promote
a lifestyle that is exible in habitats ranging from green plants to the human
gastrointestinal tract have been identied (19). This may seem a trivial way
to exploit a genomic sequence, but it is undoubtedly very straightforward,
eective, and immediate. A specic example is that before the publication of
the complete genome sequence of B. longum, predicted to encode approxi-
mately 2200 genes, only a handful of bidobacterial genes had been dis-
covered (18). Timely public availability of genome information for various
LAB species will catapult the elds collective eorts to carry on comparative
and functional genomic analyses of probiotic species within the lactic acid
bacteria. Features predicted by ISAPP (17) to be important for transient
persistence, survival, functionality, and safety of probiotic bacteria are sum-
marized in Table 1.
Genomics will help to decipher the mechanistic elements of these fea-
tures and their probable roles in probiotic functionality. Soon, second-gen-
eration probiotics will be completely characterized for genetic content and
potential functionality, on the basis of expression (transcriptomics, proteo-
mics, metabolomics) of these traits and many more likely to be discovered
through genomic analysis. The concomitant progress in the design of more
relevant bioassays will contribute to dening rational probiotic selection cri-
teria. Rather than remaining largely empirical and generic as they are at
present, specic health-benecial traits will be selectable, keeping the nal
product and specic consumer populations in mind. In addition, unique ge-
netic signatures for probiotic strains are likely to be found in the genomes
and could provide unequivocal and rapid strain identication through
RAPDs, patterned gene content in microarrays, and transcriptional prol-
ing. Last but not least, holistic approaches that can best be taken when the
complete genome of the microorganism of interest is known could be applied
to verify how processing and formulation aect the function of a selected
probiotic bacterium, a eld that remains virtually unexplored at present.

Table 1 Genetic Features Predicted to Be Important for
Probiotic Bacteria
Acid, bile, and stress tolerance

Surface and extracellular proteins
Lipoteichoic acid biosynthesis
Exopolysaccharides
Adherence and aggregation factors
Bacteriocin production
Carbohydrate (prebiotic) utilization and metabolism
Quorum sensors and response regulators
Biolm formation
Immune modulation
Putative virulence factor homologs
Siderophores, scavengers of Fe++
Antibiotic resistance/sensitivity
Gene transfer potential
Mobile genetic elements
Prophages, prophage remnants, lysogenic conversion characters
Source: Adapted from Ref. 17.
IV. BIOTECHNOLOGY APPLICATIONS:

THIRD-GENERATION PROBIOTIC STRAINS
Global views of probiotic genomes and knowledge of the elements and con-
ditions that regulate gene expression within these microbes will establish the
platform to isolate new probiotics. As mentioned, since the early 1990s,
biotechnology applications for lactic acid bacteria have supported the de-
velopment of genetic tools (e.g., transformation systems, cloning and expres-
sion vectors, food-grade expression systems, integration vectors, and systems
for gene inactivation) in a selected number of probiotic cultures (26). Genetic
accessibility is vital to establish the genotypephenotype relationships that
will ultimately dene the mechanisms underlying probiotic eects.
Beyond the use of genetic approaches to investigate probiotic cultures,
there is an exciting new eld developing that will exploit probiotic cultures in
new or improved applications. One application is the use of genetic modi-
cation to improve their inherent performance or activity. The rst example
of this approach was targeted inactivation of the D-lactate dehydrogenase
(D-ldh) gene in L. johnsonii La1, a probiotic culture bacterium that ferments
lactose to D- and L-lactate in a 60%:40% ratio. It has been reported that in
rare cases, D-lactate may accumulate in the blood of patients suering from
some intestinal disorders. The Codex Alimentarius also prevents the addition

of D-lactate-producing lactic acid bacteria to infant formulas. In order to
minimize D-lactate production, the gene (D-ldh) of La1 was isolated and an 8-
base-pair (8-bp) deletion was generated to inactivate its function. The
truncated copy of that gene was then used to replace the native D-ldh, via
homologous recombination within the genome. The resulting genetically
modied (GM) probiotic culture produced only L-lactate, at increased levels
(27). Even though the present legislation in some countries would not allow
the use of GM strains in food and feed products, such a strain certainly
corresponds to a probiotic with improved health properties.
Second, metabolic engineering allows for the rerouting of cellular
pathways and the production of specic metabolites. Although not yet
applied to probiotic cultures, metabolic engineering has allowed generation
of Lactococcus lactis with increased potential health benets that overpro-
duce the antioxidants mannitol and sorbitol (28), immunostimulatory exo-
polysaccharides (29), or vitamins such as folic acid (30). Moreover, lactic acid
bacteria that produce alanine instead of lactic acid that sequester ammonia, a
potential toxic intestinal metabolite, have been constructed (31). Similarly,
the construction of strains producing other compounds than lactic acid, such
as the presumed benecial butyrate, may be feasible.
Third, the use of lactic acid bacteria as delivery vehicles for biological
compounds has been considered and actively investigated for a number of
years (15,16,3235). The generally recognized as safe (GRAS) status of the
lactic acid bacteria and their suitability for oral consumption at levels as high
as 109 cfu/g make them attractive candidates for this application in both
human and animals. Probiotic cultures may oer additional advantages for
enhanced delivery of biologicals to specic locations in the gastrointestinal
tract, mouth, vagina, or other selected tissues.
The potential of lactic acid bacteria to act as a live mucosal delivery
system was rst investigated with protective antigens. The use of lactic acid
bacteria represents an attractive alternative to the use of attenuated patho-
genic microorganisms as vaccine carriers. Mucosal vaccines based on lactic
acid bacteria would be particularly suited to alleviate the drawbacks linked to
injectable vaccines: ease of administration, reduced side eects, no risk of
cross-contamination by needles, and limited side eects. By nature, lactic acid
bacteria are best adapted to passage through the stomach and would be ideal
carriers for vaccination of less immunocompetent individuals such as infants
and elderly. The most complete immunization studies have been carried out
with the C subunit of the tetanus toxin (TTFC). Both persistingi.e.,
Streptococcus gordonii, L. plantarum, and L. caseiand nonpersistingi.e.,
L. lactisspecies have been used as carriers. The strains producing sucient
levels of the antigen were shown to induce high levels of serum immunoglob-
ulin G (IgG) after nasal or intragastric administration. In the best cases, these

immune responses were shown to be protective against a tetanus toxin lethal
challenge. In addition, it was demonstrated that local TTFC-specic IgA was
elicited as well (for recent reviews see Refs. 15,16,3235). A series of other
antigens, including capsular polysaccharides (36), were successfully produced
in lactic acid bacteria. Immunizations in mouse models mimicking the
targeted mucosal infection are in progress in dierent laboratories (for
reviews see Refs. 33,35). Interestingly, the mechanisms by which these
recombinant lactic acid bacteria interact with the host immune cells have
begun to be analyzed (37,38). It is foreseen that these immunization studies
will shed a new light on the probiotic eld, as the readouts in these studies are
well dened and relatively easy to follow.
The concept of targeted mucosal delivery by lactic acid bacteria was
extended from antigens to interleukins by Steidler and associates, who rst
demonstrated that immune responses could be enhanced by the codelivery of
interleukin 2 II-2 or IL-6 and TTFC (39). This approach was next applied to
the design of new anti-inammatory treatments targeting inammatory
bowel disease. These authors constructed a recombinant L. lactis strain se-
creting murine IL-10 and successfully demonstrated that, upon intragastric
administration of repeated doses, this strain was able to prevent or treat
inammation in murine colitis models. Notably, this eect was obtained
with much lower (10,000-fold) doses of IL-10 than those required when
the interleukin was used as a single therapeutic agent (40). Steidler and
associates (41) further constructed a safe (no antibioresistance marker and
chromosomally integrated transgene) biologically contained strain secreting
the human IL-10 that should allow completion of a phase 1 clinical trial in
humans.
In the recent years, production and mucosal delivery of dierent types of
health-promoting molecules such as ScFv antibodies, allergens, or digestive
enzymes have been achieved with lactic acid bacteria. Targeted diseases in-
cluded microbial infections such as vaginal candidiosis (42) and dental caries
(43), allergies (44,45), autoimmune diseases (46), and metabolic defects such
as pancreatic insuciency (47). In most cases, the targeted biological eect
was indeed demonstrated in animal models mimicking the corresponding
human disease. Notably, this process often relied on the use of live bacterial
cells. Eorts are presently being devoted to improve the eciency of lacto-
cocci or lactobacilli as a delivery system. Therefore, mutants that release in-
tracellular compounds more eciently (48) or that present improved delivery
properties have been generated (Grangette, Hols, Delcour, and Mercenier,
unpublished).
Even though recombinant bacterial strains would not be accepted today
in functional foods, their future use in therapeutic approaches can be fore-
seen, provided that the benet/risk balance is positive. This novel approach is

rather promising as grafting the gene encoding for a therapeutic molecule
could enhance the natural health-benecial properties of a strain (49).
Genomic information and genetic tools will be critically important to
furthering the development of these applications. Systems for tailored gene
expression (regulated promoters; intracellular, anchored, secreted proteins)
have already been used for targeted and regulated delivery of specic
biological compounds. It is anticipated that these systems will become more
sophisticated in the near term and promote an explosion of biotechnological
applications with probiotic cultures.
V. CONCLUSIONS
Genomics is revolutionizing our view of microorganisms and the benecial

processes that they carry out. Global and detailed analyses of hostmicrobial
interactions are becoming feasible (50). Concurrently, the probiotic eld is
gaining increased scientic attention and research eorts are beginning to
unravel the mechanistic basis of the ways probiotic cultures contribute to
health and well-being. Genomic analysis of probiotic cultures will now play a
pivotal role in scientically understanding the 100-year-old concepts of
Metchniko. The impact of the genomic revolution on probiotic cultures
will be staggering, allowing a full view of their genetic composition, and an
understanding of the molecular mechanisms by which they interact with the
host and providing the knowledge base and tools to allow them to be
exploited in novel biotechnological applications.
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5
Biotechnological Modification of
Saccharomyces cerevisiae: Strategies
for the Enhancement of Wine
Quality
Linda F. Bisson
University of California, Davis, Davis, California, U.S.A.
I. INTRODUCTION
Saccharomyces cerevisiae is a premier research organism, serving as a genet-

ically tractable model system in which to dissect the biological properties of all
eukaryotes. Indeed, a battery of biotechnological tools has been developed for
the genetic manipulation and analysis of this yeast. Saccharomyces cerevisiae
is also a major participant in the bioconversion of foods and beverages in-
tended for human consumption. It is viewed as the rst true domestic or-
ganism, and its uses in the production of bread and fermented beverages
predate recorded history.
Throughout the millennia S. cerevisiae and its fungal cousins have been
the salvation of the human race during periods of disease and famine. This
yeast was an important source of micronutrients in many early human diets.
Fermented beverages produced by this organism were essential in numerous
cultures to assure a safe water supply and to preserve other foods. S. cerevisiae
is still used today to modify avor and to boost the nutritional content of
many modern foods in addition to lling its traditional roles in bread, beer
and wine production. Thus, this organism has had and continues to have a
crucial role in the provision of safe foods and beverages for humankind.

It is only natural to ask whether contemporary molecular strategies can
be used to engineer S. cerevisiae strains better suited for the bioconversion
processes in which this organism is involved or that can extend the range of
uses of this yeast, not only in the food system, but in the production of en-
zymes, proteins, and renewable energy sources. What are the risks of release
of genetically engineered strains of an organism so central to our food supply?
What are the benets? Do the benets outweigh any risks? These questions are
addressed in this chapter. In particular, the goals, strategies, and risks for the
genetic engineering of yeast strains involved in wine production are discussed.
The bioconversion of grape juice to wine represents the yeast natural
environment and poses many unique challenges to the genetic engineer.
II. PERCEIVED AND REAL RISKS OF GENETIC

ENGINEERING
In the physical world, for every action there is a reaction. The same premise
holds true for the biological world: For every genetic action there are thou-
sands of possible genetic reactions. It is our inability to predict all possible
outcomes of a directed genetic manipulation that is the underlying cause of
anxiety over genetic engineering of organisms and their subsequent use in our
food system. Will it be possible to predict the genetic responses to genomic
modication of S. cerevisiae? The answer to this question is yes. The com-
pletion of the sequence of the yeast genome has led to the development of tools
to monitor global changes in gene expression patterns and protein proles in
response to environmental or genetic manipulation (14). The application of
these genomic and proteomic tools is leading to an unprecedented under-
standing of the physiological characteristics and biological responses of this
organism (58). Well-designed studies will indeed allow the determination of
the possible biological consequences of a directed genetic change.
Sophisticated molecular tools that have been developed permit analysis
of the extent of lateral transfer of any modied genetic trait across a
population of dierent strains of S. cerevisiae and to other yeasts and bacteria
with which this organism shares an environmental niche (9). Rapid screening
techniques exist and continue to be optimized that negate the need to use and
maintain selectable markers to detect desired recombinant strains (10). This
eliminates the need to use drug resistance or any heterologous dioxyribonu-
cleic acid (DNA) as a marker in the development of industrial strains of S.
cerevisiae and thereby prevents the transfer of such genes to other less benign
organisms in the environment. These kinds of strategies can and should be
developed and used for other organisms as well. Scientists are well aware of
the risks imposed by release of drug, pesticide, fungicide, and herbicide
resistance genes to a wild population and the potential for lateral gene

transfer. Convenience and expediency do not and need not outweigh valid
concerns of risk of lateral transfer, especially when those concerns can be
addressed experimentally.
Consumers raise another issue, that the process of genetic exchange and
mutation is unnatural and therefore undesired. This view is of course not
correct. Lateral gene transfer occurs in yeast as in most microbial popula-
tions. Indeed, the yeast propensity for incorporating native as well as alien
DNA into its genome and the resulting ease of genetic manipulation are the
reasons this organism has gained such prominence as an experimental system.
The process of sexual reproduction and haploid spore formation in fungi
serves the sole purpose of reassortment of the genome. Reassortment also
occurs during vegetative growth, though at a lower frequency. Spontaneous
mutation frequencies are high in native Saccharomyces populations (1116),
suggesting that this organism, like many others, is a natural tinkerer of its own
genome. Lateral gene transfer has been documented among dierent Sac-
charomyces species (9). The role of transposable elements such as the yeast Ty
element in host genomic evolution is only now becoming appreciated (12,17,
18). It is imperative that this native tendency to exchange genetic information
be anticipated in the design strategy for any engineered organism destined to
be released into the food chain. Although Saccharomyces is not pathogenic to
the general human population, strains causing potentially lethal infections of
severely immunocompromised individuals have been identied (19,20). These
strains appear to have several dierent genes that lead to enhanced patho-
genesis not found in the native Saccharomyces population (21,22). This
demonstrates that this organism can indeed obtain and stabilize genes via
natural processes from other microbes in an environment, becoming more t
for that environment. The potential to enhance pathogenicity of a normally
nonpathogenic organism must also be assessed in light of the ever-growing
immunocompromised subpopulation. Thus, Saccharomyces engages in fre-
quent genetic exchange and modication of existing traits. Rates of mutation
and genomic stability need to be evaluated across the wild or native
populations of Saccharomyces in order to understand the true potential risks
of release of a recombinant organism.
III. STRATEGIES FOR THE IMPROVEMENT OF

SACCHAROMYCES CEREVISIAE FOR WINE
PRODUCTION
Saccharomyces cerevisiae and S. bayanus are responsible for the conversion of

grape juice into wine. It has been argued that given their long association with
humans and the fact that wine has been produced for more than 7000 years,
Saccharomyces evolved along with the practice of the production of wine to

the modern species in existence today (23). Thus anaerobic fermentation of
grape juice is the environmental niche to which Saccharomyces has adapted
over the millennia. Credence for this view arises from the lack of dominance of
this organism on the surface of fruit (2325) and its relatively high level of
sensitivity to ultraviolet irradiation and extremes of temperature when com-
pared to those of other yeast residents of fruit surfaces. Heavy inoculation of
vineyards with specic Saccharomyces strains has demonstrated the poor
survivability of this organism in the wild. This trait greatly reduces the risk of
environmental contamination by a recombinant Saccharomyces strain. How-
ever, because of the close association of yeast strains with humans, popula-
tions may serve as vectors of transfer of yeast from one production facility to
another. In numerous examples in the wine industry the source of a yeast
contaminant has been an employee rather than any biologically active
materials entering the winery.
There are several properties of yeast that, if modied, would enhance
the quality of the wine produced or eliminate processing problems leading to
economic losses for producers (Fig. 1). Yeast metabolism is responsible for
the appearance of several key organoletpic components of wine. The impor-
tance of the yeast contribution depends upon the style of wine to be produced.
Yeast generate numerous detectable compounds in wine: higher alcohols and
their esters, volatile fatty acids, aldehydes, sulfur-containing volatiles, esters
derived from amino acid metabolism, and volatile phenols generated from the
enzymatic modication of plant phenolic compounds (10,26,27). Depending
upon the wine produced, enhancement or reduction of these activities may be
desired.
Researchers are also generating recombinant Saccharomyces strains
that would facilitate wine processing or the achievement of wine stability.
Protein present in wine post fermentation can denature, leading to the for-
mation of a visible haze. Yeast strains optimized for acid protease production
can reduce wine protein content, thereby eliminating haze (28). This will only
be a viable process if the peptides produced do not confer any unwanted
characters, such as bitterness, on the wine. Similarly, wine lterability is im-
proved by degradation of grape polysaccharides. Saccharomyces strains with
pectinase or cellulase activity that have been constructed oer an alternative
to the use of commercial enzyme preparations (2836). The advantage of a
recombinant strain would be the elimination of any potentially undesired side
activity that is present in the commercial preparations. However, an enzyme
addition may be easier to control and monitor than a recombinant strain.
Finally, yeast strains that possess enzymatic activities enhancing the
release of grape avor and aroma characters are also being developed and
tested. These strains express higher levels of native enzymes that impact avor
release, contain mutations of native genes leading to the release of metabolic

Figure 1 Objectives of genetic engineering of Saccharomyces cerevisiae for wine
production.
intermediates with aromatic character, or produce heterologous enzymes or

pathways for the synthesis of grape components (3741). Whether avor-
modifying strains gain consumer acceptance is another matter. Surveys that
have been conducted to date suggest that consumers regard such innovations
as correcting problems associated with poor starting quality of fruit or
incompetent processing decisions. In this case it is not the fact that the strain

is recombinant that is objectionable, but rather why such a strain is needed in
the rst place.
More important genetic changes are those that will make wine safer for
human consumption or improve its nutritional content. An example are our
eorts to engineer genetically strains that reduce the appearance of ethyl
carbamate in the fermentation environment. Ethyl carbamate is a naturally
occurring carcinogen formed spontaneously from the reaction of carbamyl
groupcontaining compounds such as urea and ethanol and found in virtually
all fermented foods and beverages (4244). Although ethyl carbamate has
been a part of the human diet for centuries, the current view is that every step
possible should be taken to reduce the level of this and other naturally
occurring carcinogens in our food supply.
It is also possible to engineer strains that would elevate the nutritional
value of wine by increasing the content of vitamins and cofactors in limited
supply in a typical unhealthy diet of developed countries. Nutritional en-
richment of a dietary staple would be quite benecial in the reduction of the
incidence of disease. Finally, eorts to generate recombinant yeast strains that
will increase the levels of important phytochemicals such as resveratrol are
under way (45). The aim of this work is to increase the level of bioactive
phenolic compounds that have been shown to have positive dietary outcomes
in the prevention of disease. Such engineered strains may be of more value to
the population as a whole than those leading to subtle dierences in wine
aroma and avor or the elimination of economic loss for producers.
A. The S. cerevisiae Native Environment

S. cerevisiae can be found in nature on grape surfaces (2325). Although only
a minor member of the ora of the grape surface, this organism dominates the
fermentation of crushed grapes. Initial levels of Saccharomyces in freshly
prepared grape juice are in the range of 1 to 100 cells/mL, vastly outnumbered
by other yeasts and bacteria present on the order of 106 to 108 cfu/mL. By the
end of fermentation, these numbers are reversed, and a nearly pure culture of
Saccharomyces exists (24). Saccharomyces is so well adapted to this environ-
mental niche that even when outnumbered millions to one in the initial juice, it
rapidly becomes the predominant organism present. Several biological prop-
erties are thought to allow the dominance of Saccharomyces. Principal among
these are the production of and resistance to ethanol. Concentrations of 6%
to 7% (v/v) of ethanol are inhibitory to most organisms, wild and commercial
Saccharomyces strains tolerate concentrations as high as 12% to 17%.
Sensitivity to ethanol is enhanced at low pH, and the pH of grape juice is
typically between 3.0 and 4.0. Sensitivity is also enhanced at higher temper-
atures, and rapid fermentation leads to elevation of juice temperature,

contributing to the toxicity of ethanol. Saccharomyces is also adept at
sequestration of micronutrients from the environment. This appears to be a
more pronounced trait in other yeasts with which Saccharomyces must
compete (46). This yeast also releases sulte at levels inhibitory to bacteria.
Saccharomyces evolved to resist the inhibitory factors, such as acetic acid, that
are the end products of metabolism of other organisms. As a consequence,
any genetically engineered organism has to be fully competitive with other
microbes.
The grape juice environment is high in osmolarity, ranging from 20% to
28% or higher in sugar concentration, an equimolar mixture of glucose and
fructose. Saccharomyces tolerates broad changes in medium specic gravity
as sugar is consumed and ethanol produced. The environment is most
frequently limiting in nitrogen, and functional genomic analyses that we have
conducted have shown that commercial strains are ecient nitrogen recyclers
(47). Wine strains of Saccharomyces rapidly strip grape juice of amino acids;
actively growing cells display large vacuoles typically associated only with
stationary phase in laboratory strains. In most cases, however, nutrients are in
sucient supply and net yeast cell division ceases as a result of the attainment
of terminal cell density. Nongrowing cells of Saccharomyces conduct most of
the conversion of sugar to ethanol during a typical fermentation (Fig. 2). A
sustained rate of fermentation requires the presence of specic nutrients called
survival factors (48). The survival factors are required for maintenance of
fermentative rates at high population densities and in the presence of high
Figure 2 Fermentation of a Chardonnay grape juice by S. cerevisiae. (.), specic

gravity; (n), log absorbance, 580 nm.

concentrations of ethanol. Most of the survival factors have been associated
with the generation of an ethanol-tolerant cell surface and the maintenance of
protein function in a high-ethanol environment. Tolerance of ethanol poses
great constraints on a biological system. Yeast persists under conditions not
conducive to the growth or survival of most organisms. Any genetic alteration
that impacts tness for the environment will be of little practical value.
B. Challenges Impacting Genetic Engineering Strategies of

Wine Strains of Saccharomyces
The production of wine is a natural fermentative process dependent upon the
activity of bacteria and non-Saccharomyces yeasts in addition to Saccharo-
myces. Delicate avors and aromas must be retained and protected at all
stages of the process. The limitations of the environment noted and the in-
ability to impact strain tness pose substantial challenges for the genetic
modication of wine strains. Further, since the production of wine does not
occur under sterile conditions, any introduced strain may become an estab-
lished winery resident and undergo genetic exchange with other members of
the same species. Thus, any strategy for the genetic construction of a wine
strain must consider the risk of lateral gene transfer and cross-contamination
of fermentation lots. Changes made must not alter the ability of the strain to
dominate the native environment, maintain ethanol tolerance, or complete
the fermentation. Several issues in addition to lateral gene transfer must
therefore be taken into account in the development of genetically altered wine
strains of Saccharomyces (Fig. 3).
The persistence of the modied organism in the winery environment is a
very crucial issue. The ideal strain would be one that is able to dominate a
fermentation when used as an inoculum but not dominate the winery ora.
This may be quite dicult to achieve in practice. Persistence in this case means
not only presence during the entire course of the fermentation but also the
ability to form biolms and displace other winery resident species. If the
modied organism is only desired in a subset of the wines being produced,
biolm establishment can be a serious problem for the winery. As discussed
Figure 3 Issues in the genetic engineering of wine yeast.

later, several genetic modications that have already been constructed in wine
strains are suitable only for a subset of wines being produced. The risk of
problems associated with cross-contamination of other fermentations where
these novel biological activities are not desired will prevent the widespread use
of these organisms. Success of persistence can be as much of a problem as
failure of persistence. The engineered Saccharomyces strain will have to be
able to compete with native organisms of the same species, as these will be
unpreventably present in the fermentation, but not compete with them for the
dominance of winery biolms. More information is clearly needed on the
ability of yeast strains to form and displace biolms to elucidate fully the risks
of persistence.
The engineered strain will also need to maintain all of the biological
features that permit dominance of the native environment over non- Saccha-
romyces yeasts and bacteria. We have found that a seemingly inconsequential
change in expression of a single glucose transporter gene (one of eight ex-
pressed during fermentation) that displayed no phenotype in pure culture
studies led to the loss of dominance of the altered strain when cocultivated
with the lactic acid bacteria commonly associated with grapes (49). Subtle
changes in the ability to maintain energy production therefore can have a
drastic impact on competitiveness of the strain.
Another critical challenge to the genetic modication of a yeast stain for
wine production is the need to be certain that the alteration does not lead to
the generation of any unwanted characters in the wine, particularly those that
would impart an o-odor or o-avor or result in the absence of important
organoleptic compounds. Saccharomyces produces hydrogen sulde and
several other types of objectionable sulfur containing compounds with low
thresholds of detection. These odiferous compounds are considered defects or
faults in the wine and are undesirable. The production of most of these
characters increases with increased nutritional or environmental stress. Any
genetic change that directly or indirectly increased the level of production of
these compounds would be useless commercially. Further, the biological
activities of Saccharomyces impact those of the other yeasts and bacteria
present in the fermenting medium. It is important that any change in the
genetic properties of Saccharomyces not lead to o-character production by
or otherwise inhibit the other microbes present.
It is also important to understand all of the possible side activities of any
heterologous gene used for genetic enhancement of wine strains. Grape juice
is a complex mixture of numerous classes of chemical compounds, the
majority of which have not been identied. Any novel biochemical activity
might lead to undesirable modications of other wine components that would
impact the nal characters or their stability in the wine. Finally, if the novel
trait poses any selective disadvantage to the yeast there will be strong selective

pressure against its maintenance. This may mean the appearance of secondary
mutations or simple loss of gene function. Both processes may negate the
benets of the initial change.
The limitations imposed by the process of wine production on the
construction of recombinant Saccharomyces strains may seem insurmount-
able. Success will be attained and risks be inconsequential only if the bio-
logical characteristics and responses of the organism and other residents of
the same environmental niche are well understood and predictable. In the case
of wine, while much work remains to be done; the capacity to anticipate the
impact of a directed change will be possible once the genomes of the major
species have been sequenced. Lessons learned from the biotechnological
manipulation of Saccharomyces to alter the expression of traits in the natural
environment will serve as a paradigm for alteration of the genomes of other
important food organisms.
C. Goals and Strategies for the Genetic Engineering of

Wine Strains
Reviews in 1996 and 2000 catalogued the many ways in which wine strains of
Saccharomyces might be modied to eliminate production of o-odors,
reduce incidence of arrest of fermentation, improve avorant properties of
wine, or facilitate wine processing (10,50). Therefore, this chapter takes a
dierent approach: analyzing the existing and proposed biotechnological
modications from the perspective of the type of recombinant DNA tech-
nology involved. Molecular tools can be used to modify the existing native
genomic DNA and thereby aect properties of the yeast or to create truly
recombinant organisms expressing nonnative sequences.
There are two broad types of goals for the construction of wine strains
of Saccharomyces: alteration of existing traits and generation of novel
characteristics. The genes used may be naturally occurring in other strains
of Saccharomyces, may be alterations of a native gene, or may be articial
genes constructed from native genes. Alteration of regulation via promoter
exchange and construction of novel enzymes by fusion of functional and
regulatory domains of existing genes are examples of constructs from native
genetic material. Most consumers would consider alteration of alleles (allele
replacement) or generation of a novel allele from native sequences to be fairly
benign and of low risk for the food supply. Indeed, given appropriate selective
pressure, such changes can be expected to occur in the population naturally.
However, this characteristic highlights the hazards of banning a technology
altogether rather than its specic uses.
Of greater concern are the cases of heterologous gene transfer, or the
incorporation of DNA that encodes a unique metabolic activity from a

dierent or foreign organism into the yeast genome. But here also the source
of the DNA needs to be considered. If it is derived from another Saccharo-
myces species or a closely related genus, such a change can be expected to pose
a low risk for the use of the recombinant organism, again because such
exchanges occur readily in nature. The DNA from more distantly related
organisms may present greater risks, but this is dependent upon the specic
gene that is being incorporated. Allele replacement, development of engi-
neered alleles, and heterologous gene expression strategies have all been
employed or are under active development for the generation of improved
wine strains. Examples of the uses of each of these strategies, the risks and
benets, are discussed in the following sections.
1. Allele Replacement
Allele replacement has several uses for the construction of improved wine
strains. We are using allele replacement strategies to eliminate the production
of two undesired compounds by wine yeasts: ethyl carbamate and hydrogen
sulde. Naturally occurring species of S. cerevisiae have that produce little to
no ethyl carbamate been identied. Our work has shown that the amount of
ethyl carbamate formed by yeast during fermentation is directly correlated
with the amount of urea released (51,52). Urea that is not immediately me-
tabolized can be released from the cells into the surrounding medium. The
amount of urea released is a function of the relative activities of arginase and
urea amidolyase (52). Urea amidolyase is a bifunctional enzyme containing
urea carboxylase and allophanate hydrolase activities on a single polypeptide.
Strains with a relatively higher ratio of urea amidolyase to arginase degrade
rather than release urea to the environment, whereas those with a lower ratio
appear to have a limited capacity for urea catabolism and do release this
compound (Fig. 4). Allele replacement strategies can be used to adjust the
ratio of these two enzymes in any commercial strain, thus reducing the
amount of ethyl carbamate formed.
A similar strategy is being employed to dene the enzymatic activities
leading to a release of hydrogen sulde. Hydrogen sulde is made from the
reduction of sulfate in the biosynthesis of the sulfur containing amino acids.
Low-hydrogen-suldereleasing strains appear to have higher activity of the
enzymes downstream of sulte reductase, particularly of O-acetylserine O-
acetylhomoserine sulfhydrolase, that x reduced sulfur in organic molecules
(Fig. 5). The technique of allele replacement can therefore be used to reduce
the production level of hydrogen sulde, by adjusting the ratio of activities of
sulde production and consumption. Allele replacement strategies can be
used to address other comparable goals either to increase production of a
desirable compound or to reduce production of the undesirable.

Figure 4 Ethyl carbamate production by yeast strains. Urea produced from the deg-
radation of arginine is excreted from the cells to the medium, whereupon a spon-
taneous chemical reaction with ethanol occurs, forming ethyl carbamate.
In these cases the biotechnological tools being used result in benign

genomic modications. The specic examples given rely on naturally occur-
ring alleles in the population to eliminate the production of a carcinogen or an
objectionable o-odor. Each requires a thorough biochemical understanding
of the source of the compound and the regulation of the enzymatic steps
involved. The ability to change a single allele or a small set of alleles will have
minimal impact on the biological activities of the organism, especially if those
changes already occur in nature. Biotechnological modication with the goal
of allele replacement can lead to commercial strains with improved properties.
Such strains pose little to no risk to the consumer.

Figure 5 Hydrogen sulde production by yeast. Sulde is produced from sulte via
the sulfate reduction pathway. Sulde not incorporated into homocysteine can be
released into the medium.
2. Engineered Alleles
There are also potential benets of the modication of alleles to forms that
currently do not exist in nature or that have not yet been identied. Such
changes may alter the regulation of a gene or pathway or modify the coding
sequence directly to change the properties of an enzyme or pathway. Since
natural processes are being modied, these changes can also be considered
relatively harmless to the environment as a whole. Strategies for strain
improvement that involve generation of null or knockout mutations are also
being employed, but it is important to know whether the loss of function will
lead to selection of suppressor mutations in the strain. The appearance of
suppressors may not pose an environmental threat but may eliminate the

eectiveness of the initial genomic modication. Specic examples of null
mutations are elimination of the decarboxylase responsible for a phenolic o-
avor (10,53), mutation of farnesyl-diphosphate synthetase (ERG20) to
introduce terpene production to wine yeast (54), deletion of ALD6 (acetalde-
hyde dehydrogenase) and concomitant reduction of acetic acid production
(55). Null mutation of sulte reductase has been posed as a strategy to
eliminate the appearance of hydrogen sulde (56), but this then requires that
the cells be provided with methionine in the medium. Methionine can itself be
degraded to objectionable sulfur containing compounds, so this is not a viable
strategy for the elimination of o-aromas. Alteration of regulation of nitrogen
catabolism by mutation of transcriptional repressors has been shown to
improve nitrogen consumption of wine strains (57). This can lead to sustained
or elevated fermentation rates under limiting nitrogen conditions.
Swapping a native promoter for one leading to a higher level of ex-
pression of the genes involved in glycerol production has also been under-
taken (58,59). However, in this case large amounts of undesired components,
succinate, acetate, 2,3-butanediol, were produced. The high acetaldehyde
level noted during the growth phase led to a reduction of maximal biomass
(58,59). It is not clear whether such a strain would be suciently competi-
tive with wild strains of Saccharomyces and be able to dominate a wine
fermentation.
An interesting example of the problems of tailoring a strain for a specic
style of wine concerns yeast glycosidase activity. Strains with enhanced
activity of this enzyme are being generated for white wine production and
the enhancement of wine avor as a result of release of volatile components
from bound sugar moieties (60). At the same time strains with reduced activity
of this enzyme are being produced to stabilize wine color losses as the enzymes
also catalyze the loss of sugar moieties from anthocyanins (61). However, in
the case of Sparkling wine production, color loss is desired (62). If cross-
contamination and persistence are problems, wineries attempting to use both
strains could suer from reduced avor evolution in white wines or excessive
color loss in red wines.
As with allele replacement, allele engineering creates no risk other than
the problem of cross-contamination of wine production lots. Such cross-con-
tamination, if it negatively impacts the organoleptic character (aroma, avor,
color, or mouth feel) of the wine, may lead to economic losses for the winery
but not pose a health or environmental hazard.
3. Engineering Novel Traits

The ease of stable incorporation of heterologous genetic material into the
genome of Saccharomyces has resulted in tremendous research interest in
modifying the metabolic properties of this organism. Indeed, much is being

learned about the ability to engineer nonnative biochemical pathways in this
yeast (63). What has emerged from these attempts is an appreciation of the
diculty of making a stable change in an organism highly adapted to a
specic environment. Yeast strains that possess novel or heterologous traits
important in wine production are also being developed.
The earliest attempts to engineer a wine strain genetically focused on
generation of a strain stably producing the bacterial enzyme that catalyzes the
conversion of malic acid to lactate. Malate and tartrate represent the two
major acid species of grape. In cooler growing regions, malate levels may be
unacceptably high at the time of harvest, leading to excessively tart wines. The
conversion of malate to lactate and the accompanying reduction in the tart-
ness of wines are caused by members of the lactic acid bacteria. If such a
reduction in malic acid is desired, proliferation of the lactic acid bacteria must
be encouraged. Yeast and the lactic acid bacteria compete for nutrients during
fermentation and produce compounds inhibitory toward the other species. It
is frequently quite challenging to obtain both alcoholic fermentation and
malolactic conversion in wines. The lactic acid bacteria produce a spectrum of
organoleptic characters in addition to the reduction in tartness. Some of these
traits, such as the cream and buttery notes, are desired in some wines, whereas
other traits (the mousy and other animal characters) are not desired.
Therefore, the incentive to obtain a strain of Saccharomyces capable of
reducing malic acid levels exists.
The rst approach taken by Williams and associates (64), was the
construction of a strain of Saccharomyces expressing the bacterial malolactic
enzyme (Fig. 6). However, the level of activity obtained was insucient to
lead to signicant metabolism of malate. The ecacy of use of the malolactic
enzyme from dierent species of lactic acid bacteria was also evaluated with
limited success (65,66). Many of these attempts have led to undesirable side
reactions such as increases in the level of acetic acid produced by the yeast. A
novel strategy was employed by van Vuuren and colleagues (67,68). The yeast
Schizosaccharomcyes pombe degrades malate to ethanol in a multistep
process. Malate is converted to pyruvate, which is subsequently degraded
to ethanol and CO2 (67). Although S. cerevisiae possesses the same pathway,
the substrate anity of the Saccharomyces malic enzyme for malate is so low
as to preclude ecient conversion of malate to pyruvate under enological
conditions. Further, Saccharomyces does not possess an ecient malate per-
mease. Instead malate is transported into the cell by passive diusion. The
cytoplasmic level of malate is well below the Km of the enzyme. The genes
encoding the S. pombe malate permease (mae1) and the malic enzyme (mae2)
were both used to transform wine strains of S. cerevisiae, resulting in more
ecient catabolism of malate (67). Strains expressing the bacterial mleS gene
were also transformed with the mae1 transporter gene; in much higher
eciency of conversion of malate to lactate by the recombinant yeast resulted

Figure 6 Strategies for the construction of malate utilizing strains of Saccha-
romyces cerevisiae. Panel A, Introduction of the bacterial malolactic (mleS) gene,
resulting in the production of lactate and CO2 from malate; panel B, use of the S.
pombe mae1 and mae2 genes, resulting in the production of ethanol and carbon
dioxide from malate; panel C, addition of the mae1 malate transporter to a strain
expressing the mleS gene.

(68). Ecient metabolism of malate was only obtained in strains able to
transport this compound into the cell eciently. Although these eorts have
been successful in the generation of Saccharomyces strains capable of
degrading malate, other concerns have been raised. In addition to reducing
tartness and sourness, the lactic acid bacteria consume many nutrients left by
the yeast that support bacterial growth. The activities and metabolites of the
lactic acid bacteria are relatively benign when compared to those of other
spoilage organisms. The degradation of malate by yeast may lead to an
increased risk of microbial spoilage of the wine.
Wine strains of Saccharomyces that produce bacteriocins have also been
constructed (69). Bacteriocin production by yeast strains oers many advan-
tages over the current practice of use of sulfur dioxide. Individuals decient in
sulte oxidase display a strong hypersensitivity to sulte. In severe cases,
exposure can lead to death. The role of sulte in elimination of unwanted
bacteria can be eectively replaced by bacteriocins. In addition, bacteriocins
show more selectivity than sulte and can be used to inhibit specic classes of
bacteria, not harming those that are desired in the wine.
Finally, much attention has been paid to the generation of yeast strains
possessing glycosidase activity. Many grape aroma compounds are present in
the fruit in the form of bound glycoside precursors. The terpenes are a prime
example. Unbound or free terpenes impart oral and intense fruity notes to
grapes and grape juice. The attachment of a glycoside group prevents
volatilization and therefore detection of these compounds. Bound terpenes
are hydrolyzed over time, thereby maintaining the aroma characteristics of
the fruit. The grape glycosidase is inhibited by glucose and is therefore not
active in juice or during the early stages of fermentation. Once fermentation
has been completed and the sugar content decreased, these enzymes can
slowly hydrolyze the precursor compounds over time. Yeast strains express-
ing a fungal h(1-4) endoglucanase have been generated (60,7072). These
strains result in the release of more intense fruity character in the wine.
However, the use of these strains may impact the long-term aging potential of
the wine and lead to the loss of too many characters too quickly.
The traits selected for heterologous expression in wine strains of
Saccharomyces are likewise expected to be environmentally benign. None
of these yeasts is being used commercially largely because of concerns about
cross-contamination of fermentations where such activity is not desired.
There are also concerns among winemakers of potentially unanticipated side
eects of such strains, such as a decrease in aging potential of the wine or
impacts on wine microbial stability. Finally, if heterologous enzymes are
used, it is important to understand any potential allergenic reactions to the
protein that might exist in the population.

IV. CONCLUSIONS
Biotechnological modication of Saccharomyces can be used to produce

strains with a better combination of native characteristics, with altered traits
leading to the enhancement of the nutritional quality of the product or to the
removal of naturally occurring carcinogens. The introduction of specic
heterologous genes can lead to improved organoleptic properties of the wine
or facilitate downstream processing. Lateral gene transfer and mutational
change of genomic information are processes that occur naturally in yeast
populations that must be taken into account in the risk assessment of any
genetic alteration of a population to be released. The risks to human health
and well-being of the generation of such modied strains of Saccharomyces
for food and beverage production are minimal, however. Risks to processing
and desired product composition are more real but can be adequately
evaluated. Thus, astute biotechnological modication of Saccharomyces
can indeed result in improved strains for the beverage industries that pose
little risk to the consumer and that may provide a more wholesome product.
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6
Prebiotics from Lactose, Sucrose,
Starch, and Plant Polysaccharides
Martin J. Playne
Melbourne Biotechnology, Hampton, and RMIT University,
Melbourne, Victoria, Australia
Ross G. Crittenden
Food Science Australia, Werribee, Victoria, Australia
I. INTRODUCTION
The human gastrointestinal tract contains a complex ecosystem of micro-

organisms with more than 400 dierent bacterial species, and potentially
thousands of dierent strains. These bacteria are highly important to our
health. They provide us with a barrier to infection by exogenous bacteria (1)
and much of the metabolic fuel for our colonic epithelial cells (2) and con-
tribute to normal immune function (3). Disturbances to this ecosystem leave
us more vulnerable to exogenous and endogenous intestinal infections.
Intestinal bacteria have also been implicated in causation of some chronic
diseases of the gut such as Crohns disease and ulcerative colitis (4,5). As we
age, changes occur in the composition of the intestinal microbiota that may
contribute to an increased level of undesirable microbial metabolic activity
and subsequent degenerative diseases of the intestinal tract (6,7). Manipulat-
ing the intestinal microbiota to restore or maintain a benecial population of
microorganisms is a reasonable approach to maintaining intestinal health in
cases when a deleterious or less than optimal population of microorganisms
has colonized the gut.
In this chapter, we consider the role that oligosaccharides and poly-
saccharides play as prebiotics and as ingredients in functional foods. We also
assess their ability to modulate gut microbial populations and activity, and

their ability to improve human health. Fructooligosaccharides and inulin are
reviewed in other chapters, as are human milk oligosaccharides.
A. Probiotics
Probiotics are microbial cell preparations that have a benecial eect on
the health and well-being of the host, by modulating mucosal and sys-
temic immunity, as well as improving the nutritional and microbial
balance in the intestinal tract (104).
Since being proposed as a health promoting technique by Metchniko in 1907
(8), the oldest and still most widely used method to increase the numbers of
advantageous bacteria in the intestinal tract is direct consumption of these live
bacteria in foods. These bacteria, called probiotics (9), have to date been
predominantly selected from the genera Lactobacillus and Bidobacterium,
both of which form part of the normal human intestinal microbiota. In the
probiotic approach, the ingested bacteria are selected to survive gastrointes-
tinal transit and to contribute positively to the activity of the intestinal
microbiota. Proposed mechanisms of health action include increasing colo-
nization resistance to exogenous and endogenous intestinal pathogens,
reducing putrefactive and genotoxic intestinal reactions, modulating the
innate immune system, contributing to lactose hydrolysis, and producing
short-chain fatty acids (SCFAs) and micronutrients such as vitamins.
B. Prebiotics
Prebiotics are nondigestible food ingredients that benecially aect the
host by selectively stimulating the growth and/or activity of one or a
number of bacteria in the colon, thus improving health (12).
These ingredients are normally restricted to certain carbohydrates (particu-
larly oligosaccharides) but could include certain proteins, peptides, and lipids.
Oligosaccharides are dened as glycosides composed of between 3 and 10
monomer sugar units. Nondigestible oligosaccharides (NDOs) can be distin-
guished from other carbohydrates on the basis of being resistant to digestion
in the stomach and small intestine. Not all oligosaccharides are NDOs.
Prebiotics represent a second strategy to improve the balance of
intestinal bacteria. Rather than introducing exogenous strains into an indi-
viduals intestinal tract, prebiotics aim to selectively stimulate the prolifer-
ation and/or activity of advantageous groups of bacteria already present in
the intestinal microbiota. To date, prebiotics have primarily been oligosac-
charides and other indigestible carbohydrates that increase the population of
strains of Bidobacterium spp. in humans and animals. Hence, they are often
referred to as bidogenic or bidus factors. The mechanism(s) by which

prebiotics promote the relatively specic proliferation of bidobacteria
among the other major genera of intestinal bacteria remains speculative. It
is possibly due to the relatively ecient utilization of these carbohydrates as
carbon and energy sources by bidobacteria and tolerance of these bacteria
to the SCFAs and acidication resulting from fermentation. Inulin and fruc-
tooligosaccharides remain the most intensively studied prebiotics. However,
there are a range of other indigestible di-, oligo-, and polysaccharides that
have been shown to have potential to act as prebiotics, and on which this
chapter focuses.
The prebiotic approach oers some advantages over the probiotic
strategy. Technologically, prebiotics are stable and can be incorporated into
a wider range of foods and beverages with longer shelf lives than probiotics.
They also promote the proliferation of indigenous microbiota, therefore
eliminating concerns about host specicity and colonization by exogenous
strains. Studies by Tannock and coworkers (10,11) have indicated that indi
viduals may carry their own unique complement of dominant strains of
bidobacteria. Prebiotics may therefore overcome the diculty facing exog-
enous probiotics of trying to persist in an environment in which there is
already an established microbiota and possibly host factors working against
them. Prebiotics also modify the activity of the microbiota by providing a
source of readily fermentable carbohydrate. Indeed, it may be this dietary
berlike characteristic of modifying the fermentative activity of the existing
microbiota that is the important factor in providing a number of health
benets to consumers.
C. Synbiotics
A synbiotic is a mixture of a probiotic bacterium and a prebiotic car-
bohydrate that benecially aects the host by improving the survival
and persistence of live microbial dietary supplements in the gut, by se-
lectively stimulating the growth and or metabolic activity of one or a
limited number of health-promoting bacteria, but especially the added
probiotic bacterial strain or strains.
There is an obvious potential for synergy between prebiotics and pro-
biotics. Hence, foods containing both prebiotic and probiotic ingredients have
been termed synbiotics (12). The prebiotics used in synbiotic combinations are
often currently selected on the basis of food technology issues and may not in
all cases be the optimal complement for the probiotic strains. However, if
synergy between the probiotic and prebiotic ingredients is to be exploited, the
probiotic strain should be able to utilize the prebiotic as a carbon substrate in
the gut eciently. Not all probiotic species and strains can utilize fructans as a
carbon and energy source. Other nondigestible carbohydrates may be the

most appropriate choice to include with many probiotics, to provide the best
synergistic combination to benet the probiotics survival and/or functional-
ity in the gastrointestine. Since it is unlikely a particular prebiotic carbohy-
drate will be fermented solely by the added probiotic strain in the intestinal
tract, it is desirable that the prebiotic selectively promote the growth and
activity only of benecial populations within the intestinal microbiota.
Synbiotic technology is relatively new and continues to evolve (Fig. 1).
Ultimately, manufacturers want prebiotics that can provide a health benet
to the consumer and complement their probiotic strains in the product as well
as in the gastrointestinal tract by promoting stability, viability, and function-
ality. The prebiotic should also add to the taste and physicochemical
properties of food products (14) (see Sec. VIII).
D. Why Induce Higher Numbers of Bifidobacteria

and Lactobacilli in the Intestinal Microbiota?
The answer to the question of why to induce higher numbers of bidobacteria
and lactobacilli in the intestinal microbiota is still largely theoretical since
studies conclusively linking naturally high levels of intestinal bidobacteria or
lactobacilli with improved health are yet to be published. The idea that
increasing the number of these particular genera in the intestinal tract might
be benecial to health stems largely from studies of the changes in the
composition of the intestinal microbiota with age. Bidobacteria dominate
Figure 1 Evolution of the development of synbiotics. GI, gastrointestinal; SCFA,

short-chain fatty acid.

the microbiota of breast-fed infants and, after remaining relatively stable as
one of the dominant intestinal genera throughout adulthood, reduce in
numbers in the elderly (6,13). In the elderly, the decrease in bidobacterial
numbers is concurrent with an increase in potentially putrefactive organisms
such as clostridia (6). Intestinal lactobacilli and bidobacteria are nonpatho-
genic, nonputrefactive, nontoxigenic, saccharolytic organisms that appear
from available knowledge to provide little opportunity for deleterious activity
in the intestinal tract. As such, they are reasonable candidates to target in
terms of restoring a favourable balance of intestinal species in cases when the
intestinal microbiota of the host contains a high proportion of potentially
harmful bacteria. The safe use of these bacteria as probiotics and emerging
evidence of benets from consumption of probiotic lactobacilli and bido-
bacteria add weight to the idea of enhancing these populations in the in-
testinal tract by using prebiotics. However, little is known of the role of many
genera and species residing within the intestinal tract in health and disease, or
how the microbiota composition and activity change with age, lifestyle, and
culture, and how the changes inuence human health. Other genera may also
be important targets for prebiotics, in terms of both promoting benecial
bacteria and suppressing populations and activity of harmful bacteria.
Further studies of colonic microecology and interactions between the micro-
biota and the host in health and disease are essential prerequisites to designing
appropriate prebiotic strategies.
II. TYPES OF NEW PREBIOTICS AND THEIR BIFIDOGENIC

EFFECTS
In terms of prebiotic eects on the composition of the colonic microbiota,

studies of inulin and fructooligosaccharides (108) currently dominate the
scientic literature. A number of studies have demonstrated that these
fructans can increase the proportion of bidobacteria in the fecal ora. On
the strength of these studies, inulin and fructooligosaccharides are now the
most commonly used prebiotics in functional foods in Europe, Japan, and
Australia (14). However, fructans are not the only carbohydrates that have
attracted the interest of researchers studying the eects of dietary components
on the intestinal microbiotas activity and composition. A number of other
oligo- and polysaccharides have been reported to act as bidogenic factors
and have been produced and marketed commercially for this purpose since
1985 (15). These include lactulose, galacto-, xylo-, isomalto-, and soybean
oligosaccharides; lactosucrose; and a range of indigestible cereal polysac-
charides and resistant starches. Carbohydrates (other than fructooligosac-
charides) marketed for their bidogenic potential are listed in Table 1. In most

cases, the amount of published research is limited thus far to in vitro or animal
studies, or small and limited human pilot trials. In many cases, too, feeding
trials have been conducted without adequate monitoring of uctuations in the
baseline levels of microbial populations before intervention. However, all
these carbohydrates have shown at least some potential to act as prebiotics
that may be borne out in future, well-designed human feeding studies.
A. Galactooligosaccharides
Next to fructooligosaccharides and inulin, galactooligosaccharides are per-
haps the most studied NDOs for prebiotic eects of the commercially
available oligosaccharides. Galactooligosaccharides are fermented by a range
of human Bidobacterium species that utilize these sugars relatively well
compared to many other intestinal bacteria when grown in vitro (16,17).
However, in terms of eliciting signicant bidogenic eects in humans, the
results of feeding trials have been mixed. Some trials have indicated that
galactooligosaccharides can induce increases in fecal Bidobacterium sp.
levels (1820), whereas others have observed no signicant bidogenic eect
(2123). This dierence in the observed responses between the trials was not
due to a dose eect since relatively low levels of galactooligosaccharides (2.5
g/day) were used in the trial by Ito and associates (1993) (19), whereas doses
up to 15 g/day produced no signicant response in the trial of Alles and col-
leagues (1999) (22). A range of factors may inuence the size of any increase in
Bidobacterium sp. numbers, one of which is the initial size of the population.
In comparing dierent trials conducted using fructooligosaccharides, Rao
(1999) (24) observed that the size of the bidogenic response was inversely
proportional to the size of the initial Bidobacterium sp. population rather
than there being a strong dose response. Prebiotics produce bidogenic
responses only in individuals with relatively low initial populations of these
bacteria, where there is room for an eect to occur. In the case of galactoo-
ligosaccharides, a consistently strong bidogenic response in humans remains
to be demonstrated.
B. Soybean Oligosaccharides
Composed mainly of ranose and stachyose, soybean oligosaccharides are
fermented well and relatively selectively by many human species of bidobac-
teria (25). Soybean oligosaccharides have been trialed in a number of small
human feeding studies that have demonstrated increases in fecal bidobacte-
rial populations (2629). Hence, they are promising bidogenic candidates
that warrant further investigation to determine their impact on the wider
intestinal microbiota population in well-designed human feeding trials.

Table 1 Typical Compositions of Commercial Oligosaccharidesa
Soybean oligosaccharide
SOYAOLIGO Stachyose 24%; ranose 8%; sucrose 39%,
glucose/fructose 16%; other saccharides 13%
[Calpis Food Industry, Japan]
Galactooligosaccharide
CUP OLIGO Minimum 70% of solids as galactooligosac-
charides (available as syrup and powder)
type: 4V-galactosyl lactose
[Nissin Sugar, Japan]
TOS 85 POWDER 85% Galactooligosaccharides; 0.3% galactose;

3.3% glucose; 10.7% lactose
TOS Elixor SYRUP 58.5% galactooligosaccharides of solids, 0.12%
galactose; 19.7% glucose; 20.65 lactose; dry
weight 73.9%
[Borculo-Domo, Netherlands]
OLIGOMATE 55 SYRUP >55% Galactooligosaccharides of solids;

<45% monosaccharides and lactose;
75% solids
OLIGOMATE 55 POWDER >55% Galactooligosaccharides; <45%
monosaccharides and lactose
TOS pure POWDER 99% Galactooligosaccharides (<5% moisture)
[Yakult Honsha, Japan]
Lactosucrose
NYUKA ORIGO LS-40L 43% Lactosucrose of solids; 42% sucrose; 6%
lactose; 3% monosaccharides; 6% other
oligosaccharides
NYUKA ORIGO LS-55L 58% Lactosucrose of solids; 23% sucrose; 8%
lactose; 3% monosaccharides; 8% other
oligosaccharides
NYUKA ORIGO LS-55P 57% Lactosucrose; 7% sucrose; 22% lactose;
4% monosaccharides; other oligosaccharides
10%; <5% moisture
PET-OLIGO L55 45% Lactofructose;b 10% sucrose; 10%
lactose; 3% monosaccharides; 6.5%
other oligosaccharides; 24% moisture
PET-OLIGO P55 57% lactofructose;b 7% sucrose; 20%
lactose; 3% monosaccharide; 7% other
oligosaccharide
NEWKA OLIGO LS-35 >35% Lactosucrose of solids; moisture <28%
[Ensuiko Sugar Rening
Company, Japan and
Hayashibara Shoji, Japan]

Table 1 Continued
Lactulose
CONCENTRATE (Duphalac, 67% Lactulose; <6% lactose; <11%
Biteral, Chronulac, Cephalac) galactose; <5% epilactose; <1.4%
tagatose; <0.7% fructose
CRYSTALLINE FORM >95% Lactulose; <2% lactose; <2.5%
galactose; <3% tagatose; <1.5% epilactose
[Solvay, Germany]
MLS-50 SYRUP 52% Lactulose; 19% mono- and disaccharides;

31% moisture
MLS-40 POWDER 41% Lactulose
MLC-A CRYSTALLINE 98% Lactulose
ANHYDRATE
MLC-H CRYSTALLINE 86% Lactulose
HYDRATE
[Morinaga, Japan]
Xylooligosaccharide
XYLO-OLIGO 95 POWDER >95% Oligosaccharide
XYLO-OLIGO 70 SYRUP >70% Oligosaccharide;
<30% monosaccharide; 25% moisture
[Suntory Ltd, Japan]
Isomaltooligosaccharide
ISOMALTO-500 SYRUP <25% Moisture; 40% monosaccharides;
29% disaccharide;18% trisaccharide;
13% tetrasaccharide plus (>50%
isomaltooligosaccharides)
ISOMALTO-900 SYRUP <25% Moisture; 4% monosaccharides;
47% disaccharide; 27% trisaccharide;
22% tetrasaccharide plus (>85% IMOs)
ISOMALTO-900 POWDER <5% Moisture; saccharides similar to the
ISOMALTO-900 syrup
[Showa Sangyo Co Ltd, Japan]
PANORUP SYRUP <25% Moisture; 4% monosaccharides;

47% disaccharide; 27% trisaccharide;
22% tetrasaccharide plus (>85% IMOs)
[Hayashibara Group, Japan]
BIOTOSE 50 SYRUP <25% Moisture; 41% glucose; 7% maltose;

20% isomaltose; 9% panose;
9% isomaltotriose; 14% others

Table 1 Continued
PANORICH SYRUP <26% Moisture; 23% glucose; 17% maltose;
9% isomaltose; 30% panose; 3% maltotriose;
2% isomaltotriose; 16% branched others
[Nihon Shokuhin Kako Ltd, Japan]
GENTOSE 50 SYRUP <30% Moisture; 2% fructose; 51% glucose;

30% gentiobiose; 11% gentiotriose;
5% gentiotetraose
GENTOSE 80 SYRUP <28% Moisture; 2% fructose; 6% glucose;
51% gentiobiose; 28% gentiotriose;
14% gentiotetraose
GENTOSE 80 POWDER <5% Moisture; <5% saccharides
[Nihon Shokuhin Kako Ltd, Japan]
PALATINOSE/ MALTULOSE >90% Gentio-oligosaccharides

ICP 100% Palatinose powder
ICO 45% Palatinose powder;
45% palatin-oligosaccharides;
10% saccharides
[Mitsui Sugar Co, Japan]
IOS Mixed syrup
a
Saccharides are expressed as percentage of total anhydrous saccharides present in all cases.
b
Lactofructose is structurally identical to lactosucrose.
C. Xylooligosaccharides
Xylooligosaccharides (XOS) are also fermented well by many bidobacteria
(3033) and have been proposed as potential prebiotics. Suntory Ltd, who are
the main producers of xylooligosaccharide products, claim that xylooligo-
saccharides are very stable at high temperatures and in acidic conditions com-
pared to other oligosaccharides. They also found that their eect on the
intestinal ora is three to four times greater, and consequently lower doses can
be used. In feeding trials in rodents (31) and in a small linear uncontrolled trial
in humans (30), feeding of XOS appeared to increase the numbers of
bidobacteria in feces relatively selectively. Larger and more rigorously
controlled crossover trials are required to conrm whether xylooligosaccha-
rides act as selective prebiotics in humans.
D. Isomaltooligosaccharides
Unlike the other NDOs discussed, isomaltooligosaccharides are partially
digested and absorbed in the small intestine. However, breath hydrogen tests

indicate that a proportion of isomaltooligosaccharides fed to adult humans
(25g dose) remains available for fermentation by intestinal bacteria (34).
Isomaltooligosaccharides are well fermented by bidobacteria, but also by
many species from other major intestinal genera, including bacteroides and
clostridia (35). In three sequential human feeding trials, isomaltooligosac-
charide consumption resulted in small increases in fecal Bidobacterium sp.
numbers at doses of 1020 g/day (3537). In the two trials that examined
eects on other groups of intestinal bacteria (35,36), the bidogenic eect
appeared to be relatively selective. However, further controlled human
crossover studies are required to conrm the prebiotic nature of isomal-
tooligosaccharides.
E. Lactulose
Lactulose was rst recognized to be bidogenic in 1957 (38). It was not until
1971 that other oligosaccharides were also found to be able to promote the
growth of bidobacteria. Lactulose has been shown to be a relatively selective
bidogenic factor in two studies using healthy adult subjects (39,40) and
promoted the development of a microbiota rich in bidobacteria in a
controlled study of an infant milk formula containing this disaccharide (41).
Hence, lactulose has found commercial applications as a bidogenic factor in
infant milk formulas and foods in addition to its use as a therapeutic to relieve
chronic constipation and hepatic encephalopathy (42).
F. Lactosucrose
Together with lactulose, and fructo-, soybean, isomalto-, and galactooligo-
saccharides, lactosucrose (h-D-galactopyranosyl-(1!4)-a-D-glucopyranosyl
h-D-fructofuranoside) is recognized by the Japanese Foods for Specied
Health Use (FOSHU) regulatory system as a bidogenic factor. This oligo-
saccharide is sold in Japan in a range of drinks, confectionaries, and pet foods
and as a table sugar. Bidobacteria ferment lactosucrose well in vitro relative
to most enterobacteriaceae (43) and have been observed to increase the size of
the fecal Bidobacterium sp. population in small trials on healthy adults (44
46) and patients with inammatory bowel disease (47). As with the other
oligosaccharides described in this chapter, lactosucrose shows good potential
as a prebiotic that may be borne out in further human studies.
III. EMERGING PREBIOTICS
In addition to lactulose and the oligosaccharides discussed thus far, a number

of other saccharides have been investigated for their eects on the human

intestinal microbiota. These include lactitol, which produced similar eects to
lactulose but was slightly less potent (40), and glucono-y-lactone, which
induced a selective increase in fecal Bidobacterium sp. numbers in humans
at a dosage of 3 or 9 g/day in a small sequential study (48). The prebiotic
eects of lactitol were summarized in 2000 (49). Depolymerized pyrodextrin
(50), partially hydrolysed guar gum (51), palatinose polycondensates (52),
and a-glucooligosaccharides (53) have also been demonstrated to have
potential as bidogenic factors, although all require further research to
conrm these eects.
Bacterial exopolysaccharides have to date remained largely unexplored
as a source of prebiotic substrates. A polysaccharide produced by a strain of
Streptococcus macedonicus was reported in 2001 to possess a structure with
repeated oligosaccharide units that form the backbone to human milk
oligosaccharides (54) and could potentially nd applications in infant milk
formulas. In the future, transglycosylation activity of glycosidases from
specic probiotic bacterial strains might be exploited to produce oligosac-
charides for specic synbiotic combinations.
A. Cereal Polysaccharides
The eect of cereal polysaccharides on microbial population dynamics in the
intestinal tract has not been intensively studied, but a few reports have
indicated that they may have potential to act as prebiotics. Arabinoxylans
and h-glucans are the major indigestible cereal ber constituents that are
fermentable by bacteria in the human gastrointestinal tract. Cereal h-glucans
are not fermented by most lactobacilli and bidobacteria (55), but h-gluco-
oligosaccharides produced by controlled hydrolysis of cereal h-glucans can be
utilized by some bidobacteria and lactobacilli (33,56), although studies of
their selectivity and prebiotic eect in vivo have not yet been reported.
Arabinoxylan and arabinoxylooligosaccharides appear to be good candidates
for further study to assess their prebiotic potential. In vitro, these carbohy-
drates are relatively selectively fermented by Bidobacterium longum and
Bidobacterium adolescentis and are poorly fermented by other intestinal
species, including bacteroides, enterococci, Clostridium dicile, Clostridium
perfringens, and Escherichia coli (31,33,55).
B. Resistant Starches as Prebiotics

In recent years, it has been recognized that resistant starches have the po-
tential to act as prebiotics, enhancing counts of bidobacteria in fecal samples
in animal studies (5761). Additionally, resistant starch can provide protec-
tion for probiotic bacteria during transit to the colon (60) and may act as an
adhesion site in the colon for the bacteria (62). This physical association

between probiotic bacterium and prebiotic substrate may be able to be ex-
ploited for strain-specic synbiotics. Furthermore, resistant starches can be
consumed in greater daily quantities (e.g., 40 g/day/adult human) without
bowel distension by gas production or laxative eects.
Resistant starches dier from NDOs in the rate of colonic fermentation.
They show a more sustained period of fermentation, whereas most NDOs are
rapidly fermented. Sustained fermentation results in colonic fermentation ac-
tivitys extending into the transverse and distal segments of the colon, rather
than being largely restricted to the proximal colon. There is, therefore, a
theoretical advantage in supplying the host with a combination of short-chain
oligosaccharides with a longer-chain complex carbohydrate, such as inulin or
resistant starch, to gain maximal prebiotic eects including suppression of
protein metabolism in the distal colon. Preliminary data in pigs from our
research indicate that the total pool of colonic bidobacteria increases with
diets containing both resistant starch and fructooligosaccharide (58).
IV. SUSTAINED DOSAGE AND WASHOUT EFFECTS
The ability of sustained prebiotic intake to minimize the frequency of dosage

of a probiotic bacteria needed to maintain a desired colonic population is not
well documented. The commercial culture suppliers view is usually that daily
dosage of a probiotic is necessary to maintain adequate viable numbers in the
colon. A few experiments have examined washout of probiotics after ces-
sation of daily dosage (6365). Probiotic strains usually persist in high num-
bers in the intestinal tract for little longer than the normal intestinal transit
period. We have examined such eects in pigs and mice, in the presence and
absence of continued dosage of an oligosaccharide (Oligofructose) and a re-
sistant starch (high-amylose HiMaize) during the washout period. The
studies were conducted using Bidobacterium animalis CSCC 1941 as the
dosed probiotic strain. Continued ingestion of the prebiotics after the cessa-
tion of probiotic dosage increased the time in which the Bidobacterium spp.
persisted in high numbers in the intestinal tract by several days. This result
demonstrates that synbiotic eects are possible and can promote enhanced
functionality (in this case, persistence) of complementary probiotic strains.
V. HEALTH EFFECTS OF PREBIOTICS
Studies on the health eects of prebiotics are in their infancy, and most of the
proposed benets remain to be thoroughly studied, in terms of both under-
standing mechanisms of action and demonstrating clinical ecacy. Although

Figure 2 Potential health benets proposed for prebiotics. SCFA, short-chain fatty
acid.

largely conceptual at this early stage of development and our understanding
of dietmicrobiotahost interactions, there are a range of benets to human
health that could potentially be supplied by prebiotics (Fig. 2).
A. Management of the Intestinal Microbiota: Improvement

of the Gut Barrier
Aiding the development of a bidus intestinal microbiota in milk formula
fed infants to mimic microbiota development in breast milkfed infants is one
application for prebiotics that has already gained medical and commercial
acceptance. Rapidly restoring a healthy microbiota in individuals with a
perturbed intestinal ora to prevent exogenous and endogenous intestinal
infections is another potential application that shows promise (66,67). Pre-
biotics may be especially applicable to the elderly, in whom Bidobacterium
sp. numbers decrease and are replaced by higher levels of putrefying bacteria.
Use of prebiotics to treat inammatory bowel diseases and irritable bowel
syndrome through improvement of intestinal microbiota composition and
activity has also been proposed (47,68,69) but not yet intensively studied.
Through their action in fecal bulking, prebiotic oligosaccharides are eective
in relieving constipation and maintaining normal stool frequency (70).
Immunomodulation eects, as observed for probiotics (7174), have thus
far been scarcely investigated for prebiotics. Prebiotics may have applications
in the prevention of food allergy in infants, as observed for probiotics (74), by
contributing to the development of a normal microbiota.
Bidogenic eects are the most discernable impact of prebiotics. Con-
sequently, even in the absence of synbiotic treatments where an exogenous
probiotic Bidobacterium sp. strain is added together with a prebiotic, the
results from any prebiotic addition will always be confounded in a physio-
logical or health sense by the increases in numbers of resident bidobacteria.
Thus, the physiological and health eects shown by prebiotics will often
overlap those found for bidobacteria.
B. Prevention of Colon Cancer

Since they supply a source of fermentable carbohydrate to the colon, dietary
berlike, anticarcinogenic eects have been proposed for prebiotics. Pro-
posed mechanisms include the supply of the colonic epithelium with SCFA,
particularly butyrate, and suppression of microbial protein metabolism, bile
acid conversion, and other toxigenic bacterial reactions. A number of rodent
studies [summarized by van Loo and associates 1999 (70)] have demonstrated
reduced cancer risk in animals consuming prebiotics. However, in these
studies, the animals often consumed oligosaccharide doses far in excess of

those tolerable by humans (in some cases greater than 10% of the diet).
Additionally, the rapid intestinal transit time in rodents compared to that in
humans means that eects observed with rapidly fermented oligosaccharides
in these models may not translate well to the distal colon and rectum in hu-
mans, the primary sites for colon carcinomas. Prebiotics that can supply a sus-
tained source of fermentable carbohydrate to intestinal microbiota may prove
the most eective in reducing the risk of colon cancer by extending SCFA
production and suppression of toxigenic bacterial reactions through to the
distal colon. Larger polysaccharides would have the added advantage of being
tolerated in higher doses than smaller, rapidly fermented oligosaccharides.
The levels of toxigenic and mutagenic enzymes (azoreductase, h-glucu-
ronidase, nitroreductase, etc.) and metabolites are often measured as an
indication of the eectiveness of prebiotics in improving the intestinal
environment. A number of studies have indicated that NDOs can reduce
the levels of these enzymes and metabolites in feces, although conclusive links
between these end points and reduced cancer risk remain to be established
(105107).
C. Improving Mineral Adsorption

Prebiotics have also been investigated for their eects on dietary mineral
adsorption in a number of animal and human studies. Although some human
studies have been neutral in their ndings (75,76), a number have reported
benecial eects on calcium absorption (7779). Further research is war-
ranted to investigate links between long-term prebiotic consumption and
improved bone density in humans at risk of development of osteoporosis.
D. Effects on Serum Lipid and Cholesterol Concentrations

A relatively large number of human studies have focused on the eects of
oligosaccharide and inulin intake on lipid metabolism. These include eight
human trials summarized by van Loo and colleagues (70) and more recent
trials (8084). Although some positive data have been reported, the results of
these studies have not consistently demonstrated favorable eects of NDO
and inulin consumption on serum triacylglycerol and cholesterol levels,
although no deleterious eects have been reported.
VI. SAFETY AND RECOMMENDED DOSAGES
Galactooligosaccharides are considered safe food ingredients as they are

minor constituents of human milk and can also be produced in the gut by

intestinal bacteria from ingested lactose. Acute toxicity tests have shown in
rats that ingestion of more than 15 g/kg body weight of b1-4 galactooligo-
saccharide was needed for LD50. No adverse symptoms were recorded in a
chronic toxicity test when 1.5 g/kg body weight was fed for 6 months.
Nonmutagenicity was conrmed by AMES-Salmonella and Rec assay.
Excessive intake (>30 g/day in adults) of most oligosaccharides and
lactulose causes osmotic diarrhea. Recommended intake of total oligosac-
charides from all sources is therefore 10 to 15 g/day for adult humans (0.14
0.21 g/kg body weight). Naturally occurring fructooligosaccharides in foods
such as onions are estimated to contribute between 1 and 9 g/day to most
Western diets. Generally NDOs have laxative eects or cause atulence and
bowel discomfort when daily intakes exceed 25 g/day in a 70-kg human.
VII. QUANTITATIVE MODELING OF CARBON FLOWS

IN THE HUMAN GUT
The contribution of NDOs to energy metabolism and carbon ow in humans

has not received much attention. Calculations of this type in the human
gastrointestinal tract have been made for ruminants and clearly would have
value in improving our understanding of fermentation in the human gastro
intestinal tract. For example, on active adult human of 70 kg body weight has
an energy expenditure of about 12 MJ per day. One would normally expect
a Western diet to contain 70 g fat (contributing 2772 kJ), 100 g protein
(contributing 1860 kJ), and 420 g carbohydrate (contributing 7350 kJ) to
make up the required 12 MJ. Up to 10% (or 42 g) of the carbohydrate sources
supplied would be expected to be resistant to digestion and be available for
fermentation in the colon. If this were all fermented, it would give rise to about
30 g or 428 mmole of SCFA. Clearly the more cellulosic and lignied
components would not be fermented. An additional 15 g/day of NDOs would
probably be completely fermented and theoretically give rise 143 mmole of
SCFA. The amount of carbon needed for the synthesis of bacterial cells
depends on the bacterial cell turnover rate and transit time in the colon. This
diversion of carbon reduces the amount of SCFA produced from carbohy-
drates in the colon.
Questions to be answered include the following:
The volume of microbial biomass produced
The contribution of prebiotics to total available carbon pool in the
colon
The contribution of epithelial cell material and mucins to the carbon
pool

The site of prebiotic fermentation in the colon (proximal vs. transverse
vs. distal)
The site and eects of carbon and other nutrient limitations on
microbial fermentative activity
Production and absorption rates of SCFA (as opposed to concen-
trations of SCFA)
VIII. FUNCTIONAL PROPERTIES OF NONDIGESTIBLE

OLIGOSACCHARIDES
There are a number of functional properties possessed by food grade

oligosaccharides that although they do not confer direct physiological eects
on the host are nevertheless important and make NDOs attractive for use in
foods. Examples are sweetness, low cariogenicity, and low caloric value, all
of which make them useful as a replacement for sugars and fats in foods. Their
indigestibility and subsequent eect on glucose and insulin responses also
make them suitable for diabetics. Obviously, there are factors aecting
functionality of the oligosaccharides in a foodstu. These include viscosity,
water activity, moisture retention, color, osmolality, and storage stability.
Technological properties of galactooligosaccharides are the following:
Appearance: translucent/colorless
Sweetness: usually less than half that of sucrose
Water activity: similar to that of sucrose
Viscosity: similar to that of high-fructose glucose syrup
Heat stability (e.g., galactooligosaccharides are stable to 160jC for 10
min at pH 7 and to 100jC for 10 min at pH 2)
Acid stability: xylo- and galactooligosaccharides found to be more acid-
stable than fructooligosaccharides (over the temperature span 5jC to
37jC)
Indigestibility: resistant to salivary and pancreatic alpha amylase and
other carbohydrases and gastric juice
Energy value: about 50% of that of sucrose; suitable for diabetics
Cariogenicity: low
Other food technology advantages: increased viscosity, reduced
Malliard reactions and crystal formation, and alteration of freezing
points
Applications of NDOs in foods include the following:
Dairy products: milk, yogurt, infant milk powders, fermented milk
drinks
Drinks: coee drinks, soft drinks, soybean milk

Confectionery: candy, muesli bars, sports bars, energy bars, cakes,
chocolate
Desserts: ice cream, pudding, jelly, sherbet
Bakery products: bread, buns
Jam: chilled jam, chocolate paste
Other products: health foods, diet sugar, processed meat products,
processed marine products, honey products
Feed additive: animal feeds for cattle, pigs, poultry, companion animals
IX. PRODUCTION OF OLIGOSACCHARIDES
Food-grade oligosaccharides are made by three processes: (a) extraction and

purication from plants, e.g., soybean oligosaccharides, inulin from chicory;
(b) controlled enzymic degradation of polysaccharides, e.g., xylooligosac-
charides, isomaltooligosaccharides, and some fructooligosaccharides; (c)
enzymic synthesis from sugars, e.g., some fructooligosaccharides, galactoo-
ligosaccharides, and lactosucrose.
A. Enzymic Synthesis of Oligosaccharides

Much research has been conducted on the enzymic synthesis of oligosacchar-
ides (8587). Simple, cheap methods have been developed for production of
food-grade oligosaccharide mixtures using enzymes. More complex and ex-
pensive methods requiring cofactors, such as uridine diphosphate (UDP),
and expensive glycosyl transferase enzymes have been developed for produc-
tion of structurally specic and longer-chain oligosaccharides. Lactose has
been the substrate used for enzymic production of galactooligosaccharides,
for isomerization to lactulose, and for chemical conversion to lactitol.
Lactosucrose is produced by enzymic synthesis from lactose and sucrose.
Consequently, much of this section concentrates on these processes using
lactose.
1. Galactooligosaccharides
Galactooligosaccharides are formed by the enzyme h-galactosidase (EC
3.2.1.23) from lactose in a transgalactosylation reaction. This synthetic
reaction occurs simultaneously with hydrolytic degradation of the lactose.
Thus, a mixture of glucose, galactose, lactose, and tri-, tetra-, and pentaoli-
gosaccharides are usually formed.
h-Galactosidases from dierent biological sources and even from dif-
ferent genera of bacteria have markedly dierent abilities to synthesize

galactooligosaccharides (88) and show variation in their ability to produce
particular chain lengths and structures. For example, several structures of
trisaccharide can be formed (see Fig. 3). Both 4V-galactosyl lactose (Galh1-
4Galh1-4Glc) and 6V-galactosyl lactose (Galh1-6Galh1-4Glc) predominate in
the commercial products. However, 3V-galactosyl lactose is also commonly
formed. A wide range of organisms have been studied for the suitability of
their h-galactosidases, and organisms such as Sterigmatomyces elviae have
given high oligosaccharide yields from lactose at 200 g/L at 85jC (89).
Manufacturing Process. Galactooligosaccharides are formed from
lactose in a batch reactor, using one or more h-galactosidase enzymes
(lactases) in sequence or together. Elevated temperatures are used (50jC
80jC). The principal reason for this method is to achieve a high solubility of
lactose and thus reduce water activity and push the enzymic reaction toward
synthesis of oligosaccharides rather than hydrolysis to monomer sugars.
Lactose concentrations of up to 400 g/L are used. The choice of enzymes is
important as some sources produce h1-4 linkages and others h1-6 linkages.
Some enzymes also tend to have stronger hydrolytic activity than others; e.g.,
h-galactosidase from Aspergillus niger has a strong hydrolytic activity. The
composition of the mixture of oligosaccharides produced by dierent en-
zymes varies; e.g., some produce more tetra-, penta- and hexasaccharides.
Figure 3 Chemical structures of the two predominant galatotrisaccharides present

in commercial galactooligosaccharide products: a-glu(1-4)-h-gal-(1-4)-h-gal and a-
glu(1-4)-h-gal-(1-6)-h-gal.

Generally, production of trisaccharides is dominant, forming more than 80%
of the total oligosaccharides in the mixture. Enzyme derived from Crypto-
coccus laurentii and Bacillus circulans tend to produce h1-4 linked galactoo-
ligosaccharide, whereas Aspergillus oryzae and Streptococcus thermophilus
produce predominantly h1-6 linked galactooligosaccharide (90). The former
enzymes are used by Nissin to produce their Cup-Oligo product, which is a
h1-4 galactosyl lactose, whereas Yakult and Snow Brand have used the latter
enzymes for their products, which have predominantly h1-6 linkages. Yakult
now also uses Bacillus circulans to produce h1-4 linked product. The Yakult
products are named Oligomate 55 and TOS 100. Typical mixtures for the
commercial products are shown in Table 1. Enzyme costs are an important
consideration in these processes. Hence reuse and immobilization of enzymes
have been researched. It is not thought that such techniques are yet used
commercially.
After batch reaction, the product mix is decolorized and demineralized,
ltered, and concentrated to produce either a syrup or a powder. When highly
concentrated product is required, such as for Yakults TOS100 product, then
further processing is required and this usually involves chromatographic
separation of the mono- and disaccharides from the longer-chain oligosac-
charides (see Fig. 4).
Food-grade galactooligosaccharides, although mixtures, are well de-
ned and consistent in composition. These characteristics are achieved by
good quality control of the manufacturing process at each stage, namely, in
choice of enzyme(s) source, temperature of process, lactose concentration,
purication steps, and concentration procedures. The production, properties,
eects, and uses of galactooligosaccharides were reviewed in 2000 (91).
2. Lactosucrose (h-D-Fructofuranosyl
4-O-h-D-Galactopyranosyl-a-D-Glucopyranoside)
Lactosucrose is a trisaccharide, the chemical structure of which is shown in
Fig. 5. Ensuiko Sugar Rening Company is the main producer of lactosu-
crose. It is produced from lactose and sucrose feedstocks by an enzymic
transfructosylation reaction to form a structure containing galactose-1-4-
glucose-1-2-fructose. The compound is also called lactosyl-fructoside. The
enzyme used is h-fructofuranosidase (invertase) (EC 3.2.1.26) from Arthro-
bacter species. Conditions required to produce high yields of lactosucrose
product are similar to those required for galactooligosaccharide production,
as the enzyme also hydrolyses the substrates.
Manufacturing Process. A mixture of sucrose and lactose (45:55) as a
38%50% solution, at pH 5.86.2, is reacted in the presence of the enzyme h-

Figure 4 Commercial process for the production of galactooligosaccharides.

Figure 5 The chemical structure of lactosucrose.
fructofuranosidase at 55jC60jC. After reaction, the enzyme is inactivated,

and the solution decolorized with activated carbon, ltered, concentrated,
puried, reltered, deionized with ion-exchange resins, and concentrated (92).
3. Isomaltooligosaccharides
Isomaltooligosaccharides (IMOs), such as isomaltose, panose, isomalto-
triose, and isomaltotetraose, have a-1-6 glucosidic linkages. Isomalto mix-
tures, such as Isomalto-900 produced by Showa Sangyo Co Ltd, are prepared
from corn starch by the actions of alpha-amylase, pullulanase, and alpha-
glucosidase. The IMOs occur naturally in foods such as miso, soy sauce, sake,
and honey. These isomaltooligosaccharide mixtures have also been called
anomalously linked oligosaccharides mixtures (ALOMs) (93).
Manufacturing Process. Yatake has described the process used by
Showa Sangyo (93). A 30% slurry of starch at pH 6.0 is dextrinized (liqueed)
using a thermostable bacterial a-amylase. The degree of hydrolysis expressed
as a dextrose equivalent is kept between 6 and 10. Simultaneous sacchari-
cation and transglucosidation of the solution of dextrin take place using
soybean h-amylase and fungal a-glucosidase (60jC, pH 5.0). The solution is
then ltered, decolorized, demineralized, and concentrated. The concentrate
is then separated into an oligosacchariderich fraction by moving bed liquid
chromatography using Na-form cation-exchange resins, to produce IMO-
900, and a sugarrich fraction to produce IMO-500. A powdered version of
IMO-900 is produced by spray drying.
Hayashibara Group produces Panorup, which is an isomaltooligosac-
charide mixture with similar properties. The Mitsui Sugar Company produces
palatinose (maltulose) products, including pure crystalline palatinose, and
mixtures of palatinose oligosaccharides, which also contain palatinose poly-
condensates, other saccharides, trehalulose, and monosaccharides. It has

been demonstrated to have a bidogenic eect (52). Nihon Shokuhin Kako
Co. Ltd produces Biotose 50, which is composed of isomaltose, panose,
isomaltotriose, and glucose. This product is produced by a similar process.
Nihon also produces Panorich, which is a panose syrup, containing 25%
panose, maltose, glucose, and some IMOs. It is also claimed to have some
bidogenic eect, although like other maltooligosaccharides, it exhibits
partial digestion in the stomach and small intestine. Along with maltooligo-
saccharides and glycosyl sucrose (coupling sugar), such oligosaccharide
mixtures cannot be regarded as true prebiotics. The Nihon company produces
Nisshoku Gentose, which because of the enzymes used results in a dierent
product of transglucosidation , and the resulting gentiooligosaccharide has a
h-1-6-glucosidic linkage, and not the usual a-1-4 and a-1-6 linkages. The
product is claimed to be bidogenic. Its composition is given in Table 1.
Nakakuki and coauthors (94,103) have reviewed oligosaccharides derived
from starch in comprehensive papers. The chemical structures of malto-,
isomalto-, and gentiooligosaccharides are shown in Fig. 6.
4. Specific Long-Chain Oligosaccharides

Although food-grade oligosaccharides are invariably mixtures of oligosac-
charides (both in chain length and in chemical structure), the pharmaceutical
and chemical industries have extensively studied enzymic routes for the
formation of specic oligosaccharides. Glycosidases (such as h-galactosidase)
are less selective and consequently less regiospecic than glycosyltransferases.
Glycosidases are widely available from plant cells, animal cells, and bacterial
cells. Glycosyltransferases, however, require expensive cofactors (UDP), and
their use can only be justied when longer-chain specic oligosaccharides are
required. One application of the use of glycosyltransferases is the production
of synthetic human milk oligosaccharides. A 1998 report (95) shows that it is
possible to produce UDP-galactose and globotriose on a large scale by
coupling metabolically engineered bacteria. This work could lead to the
development of new methods to produce oligosaccharides by coupling sugar
nucleotide production systems with glycosyltransferases.
B. Extraction from Plants and Controlled Enzymic

Hydrolysis
1. Xylooligosaccharides
The structure of xylan is variable, ranging from a linear 1,4-h-linked poly-
xylose chain to highly branched heteropolysaccharides. The main chains
consist of D-xylose, whereas the branches contain arabinofuranose and

Figure 6 Chemical structures of malto-, isomalto-, and gentiooligosaccharides.
glucuronic acid residues. Xylan is a major component of hemicellulose and

consequently is readily available from a range of lignocellulosic waste streams
(e.g., corncob, sugarcane bagasse).
Manufacturing Process. Xylooligosaccharides (Fig. 7), produced by
controlled hydrolysis of relatively unsubstituted xylan, are also well fer-
mented by many bidobacteria (30). The Suntory process uses pretreatment
with alkali of corncobs to release hemicelluloses. After washing, sacchari-
cation takes place, using endo-1,4-h-xylanase to produce a solution of 75%
80% oligosaccharide. This is then ultraltered, followed by reverse osmosis
(RO), which splits the product into two streams. The permeate stream, which
contains 60%70% oligosaccharide, receives a second RO treatment. The
permeate from this is discarded. The concentrate, which contains 70% or

Figure 7 Chemical structure of xylooligosaccharides.
more oligosaccharide, is puried to yield the product Xylooligo 70. The

concentrate from the rst RO treatment, which contains more than 95%
oligosaccharide, is puried and produces the Xylooligo 95 product.
2. Soybean Oligosaccharides
The Calpis Food Industry Company Ltd of Japan is the major producer of
soybean oligosaccharides. The oligosaccharides produced are a mixture of
stachyose, ranose, and sucrose. Stachyose (four monomers) and ranose
(three monomers) are a-galactooligosaccharides. The chemical structures of
ranose and stachyose are shown in Fig. 8. The mixture produced by Calpis
contains more than 26% galactooligosaccharides and less than 74% sucrose
and other saccharides. A typical composition is listed in Table 1.
Manufacturing Process. Soybean oligosaccharides are produced by
purication of soybean whey. The protein is removed by precipitation, and
the ltrate is decolorized and demineralized and then concentrated.
Figure 8 Structures of ranose and stachyose, the predominant oligosaccharides

in commercial soybean oligosaccharide products.

C. Chemical Conversion of Lactose
1. Lactulose
Lactulose is not a naturally occurring compound, although small amounts are
found in some processed milk lines when heat has been applied. It was rst
synthesized in 1930 using calcium hydroxide as the alkali for the isomerization
from lactose (see Fig. 9). Since then, many other alkalis have been tested,
including alkaline borate reagents. The conversions have been reviewed by
Mizota and associates (1987) (96). Lactulose is very soluble in water (76% at
30jC) and melts at 169jC.
Manufacturing Process. Puried lactose is dissolved, an alkali such as
sodium hydroxide is added with or without a catalyst such as borate, and the
mixture is heated to 100jC for the isomerization of lactose to lactulose. The
solution is then deionized and decolorized, and unreacted lactose is removed
as a precipitate after crystallization. After pasteurization, the product is
concentrated and syrups, powders, or crystals are produced.
2. Lactitol (4-h-D-Galactopyranosyl-D-Sorbitol)
Lactitol is a sugar alcohol with very high water solubility (206% at 25jC) but
is insoluble in ethanol (0.75% at 25jC). Lactitol is chemically derived from
Figure 9 Conversion of lactose to lactulose and lactitol.

lactose by catalytic hydrogenation at high temperature and pressure. It does
not occur naturally or in processed milk products.
Manufacturing Process. Lactitol is prepared from lactose (Fig. 9). The
conversion of a sugar to a sugar alcohol involves the reduction of a carbonyl
group. This can be achieved by catalytic hydrogenation. A typical manufac-
turing process converts a 30%40% solution of lactose using Raney nickel as
catalyst and hydrogen at 100jC to 200jC and 10,000- to 15,000-kPa pressure.
The sediment produced is ltered, decolorized with activated carbon, demin-
eralized by ion exchange, evaporated, and puried by crystallization. The
reduction of the carbonyl group can also be achieved with sodium borohy-
dride. The properties, production, and physiological eects of lactitol were
summarized by Booy (1987) (97).
X. FUTURE DIRECTIONS AND CHALLENGES

A. Lectin Ligands, Soluble Adhesion Receptor Analogues,
and Human Milk Oligosaccharides
The term chemical probiosis has been coined by workers at the Rowett Re-
search Institute in Scotland to characterize the ability of specic oligosac-
charides found in human milk and colostrum to block the adhesion of
Escherichia coli K88. A Canadian company, Synsorb Biotech Inc., has
developed oligosaccharides (Synsorb) with an inert carrier, which act as
alternative receptors to absorb lectinlike toxins from toxigenic bacteria
(verotoxigenic Escherichia coli, enterohaemorrhagic Escherichia coli, Clos-
tridium dicile). Zopf and Roth (98) discussed the use of oligosaccharides as
anti-infective agents, using a decoy oligosaccharide in the mucous layer to
bind the microorganisms carbohydrate binding proteins. They claim that
such oligosaccharides (as found in human milk) prevent pathogen attach-
ment. An example is given by the Neose Technologys (USA) anti-Helico-
bacter pylori oligosaccharide, NE 0080. Idota and colleagues (99) have
examined the utilization by bidobacteria and by lactobacilli of certain
human milk oligosaccharides. Sialyl lactose was only used by Bidobacterium
infantis, whereas N-acetyl-neuraminic acid was used by Bidobacterium
longum, Lactobacillus casei, and Lactobacillus salivarius, but not by Lacto-
bacillus acidophilus. Human milk contains 3 to 6 g/L of oligosaccharide (100),
whereas cows milk contains very little (0.030.06 g/L) and most of that is
sialyl lactose. The major oligosaccharides in human milk are lacto-N-tetraose
and lacto-N-fucopentaose. Kunz and Radlo (100) regard the evidence as
strong as these compounds are potent inhibitors of bacterial adhesion to
epithelial cells: Human milk oligosaccharides are considered to be soluble

receptor analogues of epithelial cell surfaces, participating in the nonimmu-
nological defence system of human milk-fed infants. In the development of
any oligosaccharide to compete with pathogen adhesion and in the gut to
impair colonization, care should to be taken to ensure that the oligosaccha-
ride does not also interfere with colonization of the normal intestinal micro-
biota.
B. Synbiotics
The concept of increasing the survival, persistence, and/or activity of a
probiotic strain with a complementary prebiotic is yet to be well demonstrated
in humans. Certainly, the concept has merit and the molecular tools now exist
to monitor individual strains and their activity in the intestinal microbiota
(101). Exploiting physical associations between probiotics and particulate
indigestible carbohydrates, such as between bidobacteria and resistant
starch (62), may provide a means to ensure probiotics can specically benet
from an added prebiotic. Prebiotics to complement specic lactobacilli
require development. The ultimate in synbiotic combinations will include
prebiotics that can not only benet the proliferation and activity of the
probiotic strains in the colon, but also protect the strains during gastrointes-
tinal transit and during manufacture, formulation, and storage.
C. Management of Microbiota Composition and Activity

Little is currently known of the subgenus changes in bidobacterial popula-
tions that can be induced by prebiotics or of the importance of these changes
in a health context. New molecular tools to investigate changes in population
levels at the genus level, such as reported in 2001 by Satokari and coworkers
(102), now allow an opportunity to investigate the eect of dierent prebiotics
on individual Bidobacterium species. These techniques can equally be
applied at the genus, species, and strain levels and will allow a better under-
standing of how prebiotics aect microbial population dynamics in the in-
testinal tract.
Increasing the numbers of bidobacteria or lactobacilli in the intestinal
microbiota of individuals with an unfavorable intestinal microbial balance
appears with our current understanding of the human intestinal microbiota to
be a reasonable approach to promoting intestinal health. However, basic
research into the composition and role of dierent microbial populations
within the intestinal microbiota in health and disease is an essential prereq-
uisite for the development of appropriate prebiotic strategies. A more
profound understanding of what constitutes a healthy intestinal micro-
biota composition, and which microbial groups and activities are denitively

involved in health and disease, will allow the development of prebiotics with
specically targeted health eects in the future.
D. Improving Human Health

The challenge remains to elucidate links between increased numbers of
bidobacteria within the intestinal microbiota induced by prebiotics and
benecial eects on the health of the host. Prophylaxis of intestinal infections
through the rapid restoration of colonization resistance after perturbations to
the intestinal microbiota is perhaps one of the simpler health benets to
examine and is being targeted in a number of research laboratories in Europe.
Prebiotics may have a positive role to play in protection against colorectal
cancer and in dietary mineral absorption and lipid metabolism. Further
studies to elucidate mechanisms by which prebiotics act in these cases are
certainly warranted.
XI. CONCLUSIONS
A range of nondigestible oligosaccharides, already commercially available,

show strong potential as prebiotics and can increase the populations of
bidobacteria within the intestinal microbiota. New prebiotic carbohydrates
continue to be developed with an emphasis on specic synbiotic interactions
and on extension of fermentation to intestinal sites beyond the proximal
colon. Synbiotic combinations will become more frequently adopted in pro-
biotic functional foods, not only with added bidobacteria, but also with new
prebiotics designed to complement specic lactobacilli. The development of
molecular methods to examine the composition and activity of the intestinal
microbiota and interactions with the host is central to the future progression
of prebiotic research.
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7
Dextran and Glucooligosaccharides
Pierre F. Monsan and Daniel Auriol
Centre de Bioingenierie Gilbert Durand, INSA, Toulouse, France
I. INTRODUCTION
Very recently, the European Commission authorized the use of a dextran

preparation as a new food ingredient for bakery applications (1). This clearly
illustrates the renewed interest in dextran as a food ingredient, in addition to
the nutritional applications of glucooligosaccharides. In fact, dextran oligo-
saccharides and isomaltooligosaccharides, which are closely related in struc-
ture as they present a high proportion of a-1,6 glucosidic linkages, are partly
or totally resistent to attack by humans and animals digestive enzymes. They
are not absorbed in the small intestine. In the large intestine, they are then me-
tabolized by the colonic bacterial ora and fermented into short-chain fatty
acids (2,3). Such nondigestible glucooligosaccharides are regarded as prebio-
tics or colonic foods. A prebiotic is a nondigestible food ingredient that
benecially aects the host by selectively stimulating the growth and/or
activity of one or a limited number of bacteria in the colon, and thus improves
host health (4). On the other hand, a colonic food is a food ingredient
enterring the colon and serving as substrate for the endogenous bacteria, thus
indirectly providing the host with energy, metabolic substrates, and essential
micronutrients.
II. DEXTRAN
Dextran is a D-glucosyl homopolysaccharide produced by lactic acid bacteria

of the genera Leuconostoc, Streptococcus, Lactococcus, and Lactobacillus (5
7). It contains more than 50% a-1,6 glucosidic linkages, with dierent addi-

tional branching through a-1,2, a-1,3, and a-1,4 linkages (8). This branching
is the basis of the classication of dextrans into three groups, A, B, and C,
corresponding to a-1,2, a-1,3, and a-1,4 branching, respectively (9). Dextran
biosynthesis does not involve the usual glycosyltransferase scheme with
nucleotideglucose substrate, as is the case for starch or cellulose biosyn-
thesis, for example. In fact, it was demonstrated by Hehre that dextran is
synthesized from sucrose by a cell-free ltrate (10). The corresponding extra-
cellular enzyme was named dextransucrase (E.C. 2.4.1.5) by Hestrin, Averini-
Shapiro, and Aschner (11).
Dextransucrase is able to use the energy of the glucosefructose osidic
bond of sucrose to catalyze the transfer of the D-glucosyl moiety through the
formation of a covalent glucosyl-enzyme intermediate (12,13). A dextran
polysaccharide is obtained, with a molecular weight ranging from 500 to 6,000
kda (14,15). D-fructose is released by the enzyme as a coproduct. In addition,
in the presence of an ecient acceptor molecule, which is generally a
carbohydrate, dextransucrase catalyzes the transfer of the D-glucosyl residue
onto the acceptor to yield low-molecular-weight oligosaccharides (1619).
Maltose is among the most ecient acceptors tested (20). When D-fructose
accumulates in the reaction medium, its pyranosyl form acts as an acceptor to
yield the disaccharide leucrose (21).
The most widely known dextran is produced by the strain Leuc. mesen-
teroides NRRL B-512F. It contains 95% a-1,6 osidic linkages and 5% a-1,3
branching. The products obtained by controlled chemical hydrolysis are used
for the production of chromatography supports (Sephadex) and blood
plasma substitutes or iron carriers (22).
A. Dextransucrase Structure
Dextransucrases have a common structure, similar to the structure of strep-
tococcal glucosyltransferases (13,2325), which consists of the following
(see Fig. 1):
An N-terminal signal peptide involved in protein excretion (26). The
gene encoding the dextransucrase DSR-A from Leuc. mesenteroides
NRRL B-1299 is the only known exception (27);
Figure 1 General structure of dextransucrases. A, N-terminal signal peptide; B,

variable region; C, catalytic domain; D, glucan binding domain, containing repeated
units. Average amino acid number in the dierent domains is given.

A variable region of unknown role (28), which is not present in DSR-A
(27);
A highly conserved N-terminal catalytic domain, which contains the ca-
talytic triad consisting of two aspartic acids and one glutamic acid re-
sidue (29,30) and presents a permutated (h/a)8 barrel structure (13);
A C-terminal glucan binding domain involved in both dextran and glu-
cooligosaccharide synthesis in the case of the dextransucrase from
Leuc. mesenteroides NRRL B-512F (31); this domain contains a se-
ries of repeating units (28,32,33).
The only exception to this general structure is the dextransucrase from
Leuc. mesenteroides NRRL B-1299, which catalyzes the synthesis of a-1,2
osidic linkages: This enzyme contains an additional catalytic domain at the C-
terminal end, which is specically involved in the synthesis of such a-1,2
branching (34).
The availability of an increasing number of dextransucrase genes, and
more broadly of glucansucrase genes, associated with a deeper characteriza-
tion of the structurefunction relationships of the corresponding enzymes at
the molecular level, suggests the potential to design new enzymes with con-
trolled specicity toward both substrates and acceptors, and controlled
regioselectivity in D-glucopyranosyl unit transfer (35). In fact, it is possible
to combine site-directed and random mutagenesis and/or molecular evolution
approaches. The single substitution of the threonine amino acid residue at
position 667 in the dextransucrase DSR-S from Leuc. mesenteroides NRRL
B-512F by an arginine residue, for example, results in the synthesis of a
dextran polymer that contains 13% a-1,3 glucosidic bonds, instead of 5%
with the native enzyme (35). In the case of glucosyltransferase GTF-S from
Streptococcus mutans, the substitution of the threonine residue 589 by an
aspartic acid residue results in a 30% increase of the amount of a-1,3 osidic
linkage in the synthesized glucan (36).
B. Prebiotic Colonic Food Dextran Oligosaccharides

The strain Leuc. mesenteroides NRRL B-1299 produces a dextran polymer
containing 27% to 35% a-1,2 osidic branching linkages, besides a limited
amount of a-1,3 osidic branching linkages (3740). The dextransucrase ac-
tivity is mostly associated with the cell fraction (4146). Both cell-associated
and soluble dextransucrase fractions catalyze the synthesis of a-1,2 branched
dextran polymers. In addition, these enzyme preparations keep their selec-
tivity in acceptor reactions, in the presence of maltose as D-glucopyranosyl
residue acceptor, for example (47,48). This reaction yields a mixture of three
families of glucooligosaccharides, with a maltose residue at the reducing end
(49) and containing in addition (Fig. 2)

Figure 2 Structure of the glucooligosaccharides obtained by the acceptor reaction
catalyzed by the dextransucrase from Leuconostoc mesenteroides NRRL B-1299 in
the presence of sucrose (D-glucosyl donor) and maltose (D-glucosyl acceptor). Series
OD, containing only a-1,6 osidic linkages in addition to the maltosyl residue located
at the reducing end; Series R, containing an additional a-1,2 osidic linkage at the
nonreducing end; Series RV, containing an additional a-1,2 osidic branching linkage
on the penultimate glucosyl residue at the nonreducing end.

Only a-1,6 linkages (series OD), which are closely related in structure to
isomaltooligosaccharides
a-1,6 Linkages and one a-1,2 linkage at the no-reducing end (series R)
a-1,6 Linkages and one a-1,2 linkage on the penultimate D-glucosyl
residue (series RV)
Such a-1,2 osidic linkages present at or near the no-reducing end result
in a very high resistance to hydrolysis by digestive enzymes in both humans
and animals (50). In fact, these glucooligosaccharides were initially designed
as low-calorie food-bulking agents to be used in combination with intense
sweeteners (47). But these glucooligosaccharides are metabolized by certain
species of benecial intestinal ora, e.g., bidobacteria, lactobacilli, and
particularly bacteroides, and they are poorly metabolized by potentially
detrimental strains (51,52). In fact, they are not signicantly metabolized
by germ-free rats that have no intestinal ora (50).
Such glucooligosaccharides have thus been developed as prebiotic
colonic foods for both animal (52) and human applications (BioEcolians,
BioEurope-Solabia). They are fermented by the intestinal ora to short-chain
fatty acids (SCFAs). In heteroxenic rats inoculated with complex human
intestinal ora, the short-chain fatty acid prole in the cecum changes (50),
with a decrease in butyric, isobutyric, and isovaleric acid proportions ( p <
0.01) and an increase in the proportion of caproic acid ( p < 0.05). When
compared to fructooligosaccharides and galactooligosaccharides, glucooli-
gosaccharides are not bidogenic, but they promote the growth of the cellu-
lolytic intestinal ora. More important is the fact that they induce a broader
range of glycolytic enzymes, without any signicantly increased side produc-
tion of gases, and thus without detrimental eects, as demonstrated by using
rats inoculated with human cecal ora (53). These glucooligosaccharides are
used in human food complement formulations, in combination with pro-
biotics (benecial living microorganisms) and B vitamins, to regulate the
intestinal transit. Their indigestibility has been compared to that of several
commercially available oligosaccharides and polysaccharides (54). Short-
chain fatty acid production for glucooligosaccharides is similar to that of
fructooligosaccharides, guar gum, guar hydrolysate, and gum arabic. Glu-
cooligosaccharides and maltooligosaccharide-like oligosaccharides were ad-
ded to an enteral formula control diet and fed to ileal-cannulated dogs. Ileal
digestibility was lower for both products when compared to that of the
control diet. Total fecal weights were higher and fecal concentration of bido-
bacteria was numerically increased. These products can thus be regarded as
dietary berlike ingredients (54).
The production of dextransucrase by fed-batch cultivation of Leuc.
mesenteroides NRRL B-1299 was optimized, using sucrose as both carbon

source and enzyme inducer (55,56). The addition of D-glucose to the
sucrose fed-batch feed prevents the repressor eect of D-fructose released
by dextransucrase activity (57). This results in a 100% increase in dex-
transucrase production, up to 9.7 U/mL culture medium. This dextransu-
crase activity is strongly associated with the insoluble phase comprising the
bacterial cells and the surrounding dextran slime (4146). It is thus easily
recovered by centrifugation and immobilized by entrapment in alginate gel
beads for developing a continuous process for glucooligosaccharide syn-
thesis from a sucrose and maltose mixture (58). The optimal operational
conditions have been determined, stressing the key eect of the sucrose/
maltose concentration ratio on the glucooligosaccharide yield and size
distribution (59).
The in vitro utilization by human gut microora of (a) industrial-grade
dextran (very probably produced by Leuc. mesenteroides NRRL B-512F
dextransucrase), (b) oligodextran fractions produced by controlled enzymatic
hydrolysis of dextran (60), and (c) maltodextrin was studied by using
anaerobic batch culture fermenters (61). Glucose and fructooligosaccharides
were used as reference carbohydrates. Fructooligosaccharides selectively
increased numbers of bidobacteria in the early stages of the fermentation.
Dextran and oligodextran resulted in an enrichment of bacteria with high
levels of persistence up to 48 hr, with production of elevated levels of butyrate
ranging from 5 to 14.85 mmol/L. A three-stage continuous culture cascade
system was used for more eective simulation of the conditions that prevail in
dierent regions of the large intestine. A low-molecular-mass oligodextran
fraction was then shown to be the best substrate for bidobacteria and lacto-
bacilli, when compared to dextran and maltodextrin. In addition, dextran and
oligodextran stimulate butyrate production more eciently, a characteristic
that has been shown to present very interesting potential antineoplastic
properties (61). These results underline the interest of oligodextrans as modu-
lators of the gut microora. As the dextran polymers produced by the dex-
transucrase from Leuc. mesenteroides NRRL B-512F contain about 95%
a-1,6 glucosidic linkages (8), the oligodextran molecules resulting from their
controlled enzymatic hydrolysis present a molecular structure very similar to
that of isomaltooligosaccharides (Fig. 3).
III. ISOMALTOOLIGOSACCHARIDES
Isomaltooligosaccharides (Fig. 3) are produced on the industrial scale (ap-

proximately 10,000 tons per year) in Japan, which is the market leader in the
eld of nondigestible oligosaccharides (61). They are obtained by the enzy-
matic transformation of starch, using a combination of a-amylase, h-amy-

Figure 3 Structure of the glucooligosaccharides found in commercial isomaltoo-
ligosaccharides. A, Isomaltooligosaccharides, containing a-1,6 osidic linkages (n = 0,
isomaltose; n = 1, isomaltotriose; n = 2, isomaltotetraose); B, maltooligosaccharides,
containing a-1,4 osidic linkages (n = 0, maltose; n = 1, maltotriose; n = 2,
maltotetraose); C, panose (a-D-Glc-1,6-maltose); D, nigerose (a-D-Glc-1,3-D-Glc); E,
kojibiose (a-D-Glc-1,2-D-Glc).

lase, pullulanase, and a-glucosidase (62). Commercial preparations contain
(6065) a mixture of isomaltose (DP 2, 23%), isomaltotriose (DP 3, 17%),
oligosaccharides (DP 46, 26%), and nonisomaltooligosaccharides (panose,
maltose, maltotriose, nigerose, kojibiose, 30%).
But isomaltooligosaccharides are partly digestible in the small intestine.
The degree of digestion decreases when the oligosaccharide DP is higher than
3 (64,65). At high dosages, they selectively promote the growth of bidobac-
teria in the human intestine. A minimal dosage of isomaltooligosaccharides of
810 g per day was necessary to increase fecal bidobacteria (62). By
comparison, a minimal dosage of fructooligosaccharides of 12 g per day
results in a similar eect. This dierence is attributed to the fact that
fructooligosaccharides are not digested in the small intestine, whereas iso-
maltooligosaccharides are partially digested (62). A commercially available
mixture of isomaltooligosaccharides was fractionated by preparative high-
performance liquid chromatography (HPLC). Two fractions were obtained,
IMO2, containing mainly disaccharides (86.4%), and IMO3, containing
trisaccharides and higher-DP oligosaccharides (89.9%). The administration
of IMO2 or IMO3 to humans in amounts ranging from 5 to 20 g per day
resulted in an increase in bidobacteria in a dose-dependent manner: An
IMO2 intake of 10 g per day and an IMO3 intake of 5 g per day both produced
a signicant increase of bidobacterial number in feces and an enhanced ratio
in fecal microora within 12 days (64). The rat jejunal loop method was used
to characterize the luminal clearance of isomaltooligosaccharides and their
hydrogenated derivatives, as an indication of their digestibility (65). They
were compared with isomaltooligosaccharide fractions IMO2 and IMO3 (see
previous discussion) with typical digestible saccharides and with typical
nondigestible saccharides. The clearance of isomaltooligosaccharides was sig-
nicantly smaller than that of the IMO2 fraction and digestible saccharides. It
was larger than that of the IMO3 fraction and signicantly larger than that of
nondigestible saccharides. Hydrogenated isomaltooligosaccharides presented
a clearance similar to that of maltitol and signicantly smaller than that of
isomaltooligosaccharides. These results show that isomaltooligosaccharides
are slowly digested in the rat jejunum. The components having higher molec-
ular mass are less digestible, and hydrogenated isomaltooligosaccharides are
nondigestible (65).
The metabolic fate of isomaltooligosaccharides in healthy men was
investigated by using carbon-13-labeled products (66). The expiration rates of
excess 13CO2 and hydrogen of six men were measured while sedentary and
while taking physical exercise after the carbon-13-labeled isomaltooligosac-
charide intakes. Breath hydrogen excretion remained constant after isomal-
tooligosaccharide ingestion in the sedentary state and increased in the exercise
test. Serum glucose and serum insulin increased 30 min after the oligosac-

charide ingestion. The 13CO2 recovery levels were 28.7% in the sedentary test
and 60.9% in the exercise test, versus 7080% in the case of maltose. This
indicates that isomaltooligosaccharides were partially digested and partially
fermented by the intestinal ora. The energy value of isomaltooligosacchar-
ides was about 75% of that of maltose (66).
Closely related in structure to isomaltooligosaccharides are the
branched maltooligosaccharides, which are oligomers of D-glucose linked
primarily by a-1,4 osidic linkages but containing at least one a-1,6 bond, with
a DP ranging from 2 to 5 (67): isomaltose, isomaltotriose, panose, and several
higher-molecular-mass oligosaccharides. They are generally produced by
serial reactions of starch with a-amylase and transglucosidase (67). They
can also be obtained from a 15% starch suspension using a Bacillus lichen-
iformis maltogenic amylase presenting both hydrolyzing and transglycosyla-
tion activities (68). The nonbranched carbohydrate content of the resulting
products can be greatly decreased by successive fermentation with immobi-
lized yeast cells (69). The functional properties of such branched oligosac-
charide mixtures have been determined and compared with those of
commercial isomaltooligosaccharide preparations, maltodextrin, and sucrose
(70). In addition, branched oligosaccharides present interesting prebiotic
properties (7172).
IV. CONCLUSIONS
It is increasingly clear that the resident bacterial ora of the colon plays a
major role in human nutrition and health, both directly, through metabolite
production and occupation of epithelial adhesion sites, and indirectly,
through the immunomodulatory response of the digestive tract. From this
point of view, prebiotic compounds are of outstanding interest, as they are
specically fermented and thus allow the control and modulation of the large
gut ora. Unfortunately, the individual roles of the roughly 500 dierent
bacterial species present in the colon are not at all understood as yet. Bi-
dobacterium and Lactobacillus species are the two main bacterial groups
identied as presenting health-promoting eects. Molecular biological tools
now introduce the potential to identify precisely the microbial strains present
at any site of the digestive tract, even if they cannot be grown on Petri dishes.
This will allow a more precise determination of the composition of the in-
testinal microbial ora than the simple counting of colony-forming units from
feces samples. In this context, it is essential to be able to design the broadest
possible range of nondigestible oligosaccharides, built from simple and safe
carbohydrate units. Such oligosaccharides will help to obtain precise control
of the colonic ora, and, for example, to control the nature and the level of

short-chain fatty acidsing produced in the colon. The main eects targeted are
better resistance to pathogens, decreased gut tumor risks, and reduced levels
of blood lipids.
From this point of view, glucooligosaccharides and glucansucrases are
very good candidates. Several glucooligosaccharide structures have been
proved to be partly or totally resistant to digestive enzymes, and particularly
the structures containing a-1,2 osidic linkages produced by the dextran-
sucrase from Leuc. mesenteroides NRRL B-1299. They present interesting
properties, with very limited detrimental eects (abdominal distension,
atulence, diarrhea). In addition, the availability of several dextransucrase
genes encoding enzymes catalyzing the synthesis of mostly a-1,6 osidic
linkages, but with dierent branching selectivities (a-1,2, a-1,3, and a-1,4
osidic linkages), opens the way to the generation of a broad diversity of
glucooligosaccharide structures, through the generation of enzyme variants
by site-directed mutagenesis, random mutagenesis, and directed evolution
[deoxyribonucleic acid (DNA) and family shuing]. The corresponding
glucooligosaccharides will have to be screened to identify the best functional
candidates.
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8
Prebiotics and the Prophylactic
Management of Gut Disorders:
Mechanisms and Human Data
Robert A. Rastall and Glenn R. Gibson

The University of Reading, Reading, England
I. INTRODUCTION
In recent times, the activities of the human gut microbiota have been more
fully elucidated. Although it is apparent that certain species may be involved
in gut disorders such as ulcerative colitis, bowel cancer, acute enteritis, and
pseudomembranous colitis (1), there has been much momentum for dietary
approaches that modulate the gut ora composition toward improved health
(2). Mainly historical associations have been made between probiotics and
gastrointestinal improvements, but there is a very rapidly developing func-
tional food sector in Europe based on food ingredients containing prebiotics.
These are nonviable food components that are selectively fermented in the
colon (by a benecial and not detrimental ora). In many cases, literature on
the eects of these ingredients in human studies is sparse, and our mechanistic
understanding inadequate. However, the prebiotic approach may be a very
straightforward route for preventative management of gut disease. The aim of
this chapter is to take a critical view of the proposed mechanisms for the
health promoting eects of prebiotics and an overview of recent human
studies in the area. It is not our aim to provide a comprehensive review of the
literature, but rather to focus on relevant recent research reports, concentrat-
ing on human trials and mechanisms of eect.

II. BACKGROUND TO THE PREBIOTIC CONCEPT
The human colon is an immensely complex microbial ecosystem containing

several hundred bacterial species (3). Within this diversity it is possible to
recognize bacteria with potentially positive or negative eects upon human
health (Fig. 1). The concept that diet can shift the balance away from
undesirable microorganisms toward physiologically positive species is not
new (Fig. 1). To this end, there is a thriving functional food industry based on
the concept of including lactic acid bacteria (probiotics) in foods such as
yogurt and other fermented milks. There are, however, concerns about the
survival of such microorganisms during processing, storage, and passage
through the alimentary system. For example, gastric acid, bile salts, and
pancreatic secretions are all barriers to long-term probiotic persistence in the
gut. Moreover, in the colon they then face a huge indigenous microora with
which they have to compete eectively if any advantageous properties are to
ensue.
Although certain probiotic strains may be robust enough to overcome
some or all of these confrontations, they are bound to be few in number and
Figure 1 Properties of bacterial genera in the colon.

perhaps compromised in terms of activity. An alternative approach to
improved microora modulation is therefore to intake carbohydrates that
resist digestion in the stomach and small intestine, reach the colon intact, and
are then selectively metabolized by (benecial) bidobacteria and/or lacto-
bacilli. This process is known as the prebiotic approach (4). In Europe, there
are four prebiotics commonly used in foods: inulin, fructooligosaccharides
(FOSs), lactulose, and galactooligosaccharides (GOSs). In Japan, the situa-
tion is more widespread with many oligosaccharides being incorporated into
foods for their prebiotic eects (5). For a summary of in vitro and in vivo trials
with prebiotics see Ref. (6). It can be said with some condence that good
prebiotics exist, and are being developed, that have selective eects on the gut
microbiota composition. A key question remains about the health bonuses,
however. Four promising areas of importance have arisen. They are discussed
in Sections IIIVI.
III. THE BARRIER EFFECT AGAINST GASTROINTESTINAL

PATHOGENS
One of the most mechanistically strong health benets proposed for prebiotics
is the barrier function against invading gastrointestinal pathogens. If partic-
ular, this may be a successful way of prophylactically addressing the burden of
microbial food safety through tackling problems such as those caused by
campylobacters, salmonellae, and Escherichia coli.
A. Proposed Mechanisms
There are several prospective mechanisms for the inhibition of pathogens by
bidobacteria and lactobacilli (Fig. 2). Fermentation of carbohydrates results
in the production of short-chain fatty acids (SCFAs) (4). These reduce
luminal pH in the colon to reach levels below those at which pathogens such
as E. coli can eectively multiply. In addition, increased populations of
bidobacteria and lactobacilli can compete with other organisms for nutrients
and receptors on the gut wall (7). Probiotics (the target microorganisms for
prebiotic intake) can also inhibit pathogens via a more direct mechanism.
They are known to produce antimicrobial agents active against a range of
pathogens (8). Although it is not known whether such metabolites function
eectively in the human gut, their powerful eects have led to their wide-
spread use as food preservatives (9).
Probiotics are also reputed to modulate the activities of the immune
system, resulting in nonspecic enhancement of immune function. In partic-
ular, encouraging results have been obtained with both lactobacilli (10) and

Figure 2 The probiotic barrier to gastrointestinal infections.
bidobacteria (11) against rotaviral infection in infants. Fortication of

indigenous probiotics, by ecient prebiotic use, should have similar eects.
B. Human Studies
It is, of course, impossible to collect human data on the probiotic barrier
against infection with pathogens through challenge-type testing. Most fer-
mentation studies in this area are alternatively carried out using in vitro
models of the human gut (12) or animals (13). Both have limitations but do
generate useful mechanistic data relevant to the situation for humans.
Although bacterial pathogenesis in humans is dicult to predict, certain
situations, such as antibiotic-associated diarrhea and gastrointestinal prob-
lems suered by frequent travelers, seem good avenues for prebiotic use.
Moreover, our own recent data have exploited the use of a rhesus monkey
colony to infect animals with enteropathogenic E. coli (14). The experiments
were carried out using placebo and blind control, with genotypic probes for
the bacteriological characteristics. In essence, some protection against diar-
rhea was seen in the presence of bidogenic substrates. These types of studies
need to be taken further through human trials that apply sound genomic
principles to the bacteriological characteristics (15).
IV. PROTECTION AGAINST COLON CANCER

A. Possible Mechanisms
The most likely means by which prebiotics could inuence the development of
bowel cancer is by modulation of the colonic ora. Prebiotics are fermented to

organic acids, and in some cases this includes butyrate (4,16). Butyrate
inhibits apoptosis (17) and is thought to be protective against colon cancer.
Many fecal microorganisms produce carcinogens and tumor promoters
from dietary and other components entering the colon (Fig. 3). In addition,
several enzymic activities, associated with fecal bacteria produce toxic or
carcinogenic products from substrates entering the colon (Table 1). The
microbial species responsible for these activities have not been unambigu-
ously identied beyond certain Bacteroides spp. and Clostridium spp. It is
known, however, that bidobacteria and lactobacilli do not have such
capabilities and a reduction in these activities can be demonstrated in feces
from humans or rats fed lactic acid bacteria or prebiotics (18,19).
B. Human Studies
Many in vivo data on the protective eects of prebiotics on colon cancer are
obtained from animal studies, in which, for example, inulin has been shown to
inhibit formation of aberrant crypt foci (20). Human studies are few in
number and tend to focus on fecal markers of carcinogenesis rather than
being epidemiological in nature. Human data from healthy volunteers are
summarized in Table 2. In three of the four trials, a decrease in several
markers of carcinogenesis was seen. In two of these, signicant increases in
bidobacteria were also observed; no microbiological analysis was carried out
in the third.
Figure 3 Production of carcinogens and tumor promoters by fecal bacterial

metabolism.

Table 1 Fecal Bacterial Enzymes Producing
Carcinogenic or Toxic Products
Enzyme Substrate
h-Glucosidase Plant glycosides

(e.g., rutin, cycasin)
Azoreductase Azocompounds
(e.g., benzidines)
Nitroreductase Nitrocompounds
(e.g., nitrochrysene)
h-Glucuronidase Biliary glucuronides
(e.g., benzidine)
IQ hydratase-dehydrogenase 2-Amino-3-methyl-
3H-imidazo (4,5-f )
quinoline (IQ)
Nitrate/nitrite reductase Nitrate, nitrite
A 1999 study, however (24), found no signicant changes in bidobac-

teria or in markers of carcinogenesis. These results might at rst sight seem
anomalous, as the test substrate (GOSs) has been found to be prebiotic in
many studies in the past (25). However, starting populations of bidobacteria
in the volunteers were rather high (9.29.4 log). It has been noted (26,27) that
the magnitude of the response to prebiotics by bidobacteria depends upon
the starting levels. It appears that there is a maximal level of bidobacteria
(about 10 log values) achievable in the human gut. If populations are already
at or near this level, then little or no further increase in numbers is generally
seen. This is an important point and is currently the subject of further
research. The implication is that a healthy diet supporting a balanced
gastrointestinal microora may not further benet from prebiotic functional
foods (24).
V. IMPROVED CALCIUM ABSORPTION
There has been increasing interest in recent years in the possibility of

increasing mineral (particularly calcium) absorption through the consump-
tion of prebiotics. Although the small intestine is the principal site of calcium
absorption in humans, signicant amounts are also thought to be absorbed
throughout the length of the gut; consequently, a maximizing of colonic
eects is desirable.

Table 2 Human Studies Investigating the Anticancer Eects of Prebioticsa
Subjects and Increase in

Prebiotic study design Dose, g/day bidobacteria Markers of carcinogenesis Reference
FOS 20, Placebo-controlled, 12.5 1.2 log No change in bile acids, 21

12 days neutral sterols,
nitroreductase,
azoreductase, or
h-glucuronidase
FOS 12, Controlled diet, 4 0.5 log Signicant decreases in 22
control and h-glucuronidase (75%)
treatment periods, and glycocholic acid
26 days hydroxylase (90%)
Resistant 12, Controlled diet, 55.2 F 3.5 Not investigated Signicant decreases in 23
starch (RS) high- and low-RS and 7.7 F 0.3 neutral sterols (30%),
periods, 28 days 4-cholesten-3-one (36%),
total bile acids (30%),
secondary bile acids (32%),
and h-glucosidase activity
(26%) in high-RS phase
compared to low RS-phase.
GOS 40, Placebo-controlled, 7.5 or 15 No signicant No signicant changes in 24
21 days increase SCFA, bile acids,
ammonia, or skatoles
a
FOS, fructooligosaccharide; RS, resistant starch; GOS, galactooligosaccharide; SCFA, short-chain fatty acid.

Several mechanisms have been postulated for increased calcium absorption as
induced by prebiotics (28) (Fig. 4), although it is far from clear at the present
time which (if any) actually operate in vivo.
1. Fermentation of prebiotics such as inulin results in a signicant
production of SCFA, leading to a reduction in luminal colonic pH. This is
likely to increase calcium solubility and overall levels in the gut.
2. Phytate (myoinositol hexaphosphate) is a component of plants that
reaches the colon largely intact (29). It also forms stable, insoluble complexes
with divalent cations, such as calcium, rendering them unavailable for trans-
port. Fermentation results in the bacterial metabolism of phytate, thereby
liberating calcium.
3. It is postulated that a calcium exchange mechanism operates in the
colon. In this system, SCFAs enter the colon in a protonated form and then
dissociate in the intracellular environment. The liberated proton is then
secreted into the lumen in exchange for a calcium ion.
B. Human Studies
Numerous animal studies have indicated that prebiotics increase absorption
of calcium from the colon, thereby decreasing losses from bone tissue (30).
Very few human studies have been carried out, however. In one such study,
the feeding of 40 g inulin/day for 28 days to nine healthy subjects resulted in a
signicant increase in calcium absorption (31). A more realistic 15-g inulin
Figure 4 Possible mechanisms of enhanced calcium uptake as a result of prebiotics.

FOS or GOS/day, when fed to 12 healthy subjects for 21 days, resulted in no
signicant eect on the absorption of calcium or iron (32).
In a more recent study, 12 adolescent boys (aged 1416) were fed 15 g
FOS/day for 9 days in a placebo-controlled trial against sucrose (33). The
data showed a 10.8% increase in calcium balance with no signicant eect on
urinary excretion.
VI. EFFECTS ON BLOOD LIPIDS
There is intense interest in the food industry in developing functional foods to

modulate concentrations of blood lipids such as cholesterol and triglycerides.
The mechanisms suggested by which prebiotics may inuence blood lipids are
summarized in Fig. 5. There is evidence that FOSs decrease the de novo
synthesis of triglycerides by the liver. The means by which this occurs is not
Figure 5 Possible mechanisms for the modulation of blood lipids by prebiotics.

fully understood, but the eect appears to be exerted at the transcriptional
level. It is also possible that prebiotics (such as inulin) can modulate insulin-
induced inhibition of triglyceride synthesis (34).
Eects on serum cholesterol levels have also been postulated for pre-
biotics, although the mechanism is more dicult to envisage. It has been
suggested (for reviews, see 34 and 35) that propionate produced by the bac-
terial fermentation of prebiotics inhibits the formation of serum low-density
lipoprotein (LDL) cholesterol. The diculty with this hypothesis is that
bacterial fermentation of prebiotics generally produces much more acetate
than propionate. Moreover, acetate is a metabolic precursor of cholesterol
and may therefore tend to increase, not decrease, serum levels. A more likely
role for the gut microora in the reduction of cholesterol is direct metabo-
lism of cholesterol by colonic bacteria, or its conversion to other metabolites
such as coprostanol (36). The evidence for this is not presently well claried,
however.
B. Human Studies
Human studies on the lipid lowering properties of prebiotics when consumed
at a realistic (tolerable) dose are not clear-cut (37). These can be divided into
trials carried out on subjects with hyperlipidemia and on normal subjects
(Table 3). Hitherto, data indicated that there may be a signicant eect on
Table 3 Eects of Prebiotics on Blood Lipidsa
Effect
Reference Date Prebiotic Dose Duration TG LDL-Ch Ch
Hyperlipidemic subjects
38 1984 FOS 8 g/day 14 days NS 10% 8%
39 1998 Inulin 18 g/day 6 weeks NS 14% 8.7%
Normal subjects
40 1995 Inulin 9 g/day 4 weeks 27% 7% 5%
41 1996 FOS 20 g/day 4 weeks NS NS NS
42 1997 Inulin 14 g/day 4 weeks NS NS NS
43 1998 Inulin 10 g/day 8 weeks 19% NS NS
44 1999 Inulin 15 g/day 3 weeks NS NS NS
44 1999 FOS 15 g/day 3 weeks NS NS NS
44 1999 GOS 15 g/day 3 weeks NS NS NS
a
NS, no signicant; TG, triglyceride; LDL Ch, LDL cholesterol; Ch, cholesterol; FOS, fructooligo-
saccharide; NS, not signicant; GOS, galactooligosaccharide.

blood triglycerides but not cholesterol in normal subjects, although three of
the ve studies did not show any eect on concentrations. For subjects with
elevated lipid levels, however, there does seem to be a useful decrease in
cholesterol levels. It would seem to be of high priority to carry out more
research in this areaespecially now that reliable methods for tracking
microbiota changes through molecular procedures are available (45).
VII. CONCLUSIONS
Prebiotics have a long history of use in Japan, but the market for prebiotics as
food ingredients in Europe is also now established (46). There has been a
tendency in the past for unsubstantiated health claims to be made for
functional foods, and it is essential, if we are to have condence in the pro-
tective eects of prebiotics, for any claims to be based on rigorous science
preferably carried out in humans. To date, there have been a few well-
designed volunteer studies, and these have sometimes yielded contradictory
conclusions. One problem with evaluation of the eects of prebiotics lies is the
diculty of identifying fecal microorganisms by using conventional culture-
based approaches. It is estimated that a large percentage of the total fecal
microora has yet to be described and is probably unculturable (47). The
advent of molecular methods of bacterial identication has undoubtedly
improved this situation (45).
Given the signicance of the human gut microbiota and its activities [the
colon is the bodys most metabolically active organ (48), it seems a very
reasonable approach to advocate dietary modulation by prebiotics. At pre-
sent, we are at the stage at which ecacious forms exist and can be made to
operate in the food matrix (49). The health benets that have been suggested
are varied but also very important. In addition to good human volunteer
studies we need enhanced mechanistic understanding of the health eects of
prebiotics. Progress is being made in this area, and it is to be expected that the
prebiotic approach to prevention of disease will have a much stronger
foundation. This will lead to better informed decisions by clinicians, nutri-
tionists, and consumers.
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Bioprocesses and Biotechnology For Functional Foods and Nutraceuticals

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Bioprocesses and

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1 . Phytosterols as Functional Food Components and Nutraceuti-

ADDITIONAL VOLUMES IN PREPARATION

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

The Nutraceuticals Science and Technology series provides a comprehensive

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

Series Introduction Fereidoon Shahidi

PART I. BIOTECHNOLOGICAL APPROACHES TO

1. Poultry, Eggs, and Biotechnology

2. Modern Biotechnology for the Production of Dairy Products

3. Bacterial Food Additives and Dietary Supplements

4. Genomics of Probiotic Lactic Acid Bacteria: Impacts on

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

PART II. BIOTECHNOLOGY STRATEGIES FOR

6. Prebiotics from Lactose, Sucrose, Starch, and Plant

7. Dextran and Glucooligosaccharides

8. Prebiotics and the Prophylactic Management of Gut

9. Proteins and Peptides

11. Chemical Analysis and Health Benets of Isoavones

12. Anandamides and Diet: A New Pot of Nutritional Research

PART III. PHYSIOLOGICAL TARGETS OF FUNCTIONAL

13. Obesity and Energy Regulation

14. Food, Fads, Foolishness, and the Future: Immune Function

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

16. Inuence of Diet on Aging and Longevity

17. Foods and Food Components in the Prevention of Cancer

PART IV. CONSUMER ISSUES OF BIOTECHNOLOGY

18. Food Biotechnology and U.S. Products Liability Law:

19. Scientic Concepts of Functional Foods in the Western

20. Paradigm Shift: Harmonization of Eastern and Western

21. Consumer Attitudes Toward Biotechnology: Implications for

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

Kevin J. Acheson Nestle Research Center, Lausanne, Switzerland

Yuriko Adkins Department of Nutrition, University of California, Davis,

Daniel Auriol Centre de Bioingenierie Gilbert Durand, INSA, Toulouse,

A. Berger Nestle Research Center, Lausanne, Switzerland

Linda F. Bisson Department of Viticulture and Enology, University of

Stephanie Blum Nestle Research Center, Lausanne, Switzerland

Christine M. Bruhn Center for Consumer Studies, Department of Food

Ross G. Crittenden Food Science Australia, Werribee, Victoria, Australia

Willem M. de Vos Wageningen University and Wageningen Center for

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

M. Eric Gershwin Division of Rheumatology, Allergy and Clinical Immu-

Glenn R. Gibson The University of Reading, Reading, England

Ryota Haba Tokyo University of Agriculture, Tokyo, Japan

Barry Halliwell National University of Singapore, Singapore

Todd R. Klaenhammer Department of Food Science, North Carolina State